a generic approach for the generation of stable humanized single … · fragments from rabbit...

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A generic approach for the generation of stable humanized single-chain Fv

fragments from rabbit monoclonal antibodies

Leo Borras

#, Tea Gunde

#, Julia Tietz, Ulrich Bauer, Valérie Hulmann-Cottier, John PA

Grimshaw and David M Urech*

# both authors contributed equally; *corresponding author

From ESBATech AG, an ALCON biomedical research unit, Schlieren, Switzerland

Running head: Humanization and stabilization of rabbit variable domains

Address correspondence to: David Urech, PhD, Wagistrasse 21, CH-8952 Schlieren,

Switzerland, FAX +41-44-733 49 90; E-mail: [email protected]

ABSTRACT:

Despite their favorable pharmacokinetic

properties single-chain Fv antibody

fragments (scFvs) are not commonly

used as therapeutics, mainly due to

generally low stabilities and poor

production yields. In this work we

describe the identification and

optimization of a human scFv scaffold,

termed FW1.4 that is suitable for

humanization and stabilization of a

broad variety of rabbit antibody variable

domains. A motif consisting of five

structurally relevant framework residues

that are highly conserved in rabbit

variable domains was introduced into

FW1.4 to generate a generically

applicable scFv scaffold, termed

FW1.4gen. Grafting of complementarity

determining regions (CDRs) from 15

different rabbit monoclonal antibodies

onto FW1.4 and derivatives thereof,

resulted in humanized scFvs with

binding affinities in the range from 4.7 x

10-9

M to 1.5 x 10-11

M. Interestingly,

minimalistic grafting of CDRs onto

FW1.4gen, without any substitutions in

the framework regions, resulted in

affinities ranging from 5.7 x 10-10

M to

<1.8 x 10-12

M. When compared to

progenitor rabbit scFvs, affinities of

most humanized scFvs were similar.

Moreover, in contrast to progenitor

scFvs, which were difficult to produce,

biophysical properties of the humanized

scFvs were significantly improved, as

exemplified by generally good

production yields in a generic refolding

process, and by apparent melting

temperatures between 53 and 86 °C.

Thus, minimalistic grafting of rabbit

CDRs on the FW1.4gen scaffold presents

a simple and reproducible approach to

humanize and stabilize rabbit variable

domains.

1. INTRODUCTION

Due to their favorable pharmacokinetic

properties single-chain Fv (scFv) antibody

fragments represent an attractive format for

therapeutic applications (1,2). scFvs are

often derived from monoclonal antibodies

isolated from animal or human

http://www.jbc.org/cgi/doi/10.1074/jbc.M109.072876The latest version is at JBC Papers in Press. Published on January 7, 2010 as Manuscript M109.072876

Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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lymphocytes. As an alternative to

hybridoma screening, in vitro display

technologies, e.g. phage and ribosome

display, enable the selection of high-

affinity binding variable domains from

natural or synthetic genetic libraries.

Despite successful use of in vitro

randomization and selection systems,

generation of antibodies by immunization

and subsequent screening of full-size

antibodies (e.g. hybridoma supernatants)

comprises conceptual advantages. For

example, in contrast to in vitro display

systems, in vivo methods are less prone to

preferential selection of well expressed

clones, which in many cases results in loss

of potentially interesting antibodies.

Moreover, in vivo methods are preferred in

particular for addressing complex antigens,

such as integral membrane proteins that are

notoriously difficult to purify. However,

reducing a full-length monoclonal antibody

to the scFv format frequently is challenging

particularly due to solubility and stability

problems, which often impair expression

and purification. Therefore, technologies to

humanize and stabilize the scFv format

following isolation of a monoclonal

antibody remain critical for the generation

of scFv therapeutics.

Numerous approaches have been described

to improve biophysical properties of the

scFv format (3), which can be grouped into

two categories. In the first category,

variable domains of pre-existing scFvs are

engineered for improved stability, either by

rationally altering specific positions in the

framework regions (4-8), or by random

mutagenesis of framework positions and

subsequent screening by genetic selection

methods that favor stable scFvs (9-13). In

the second category, stabilization of the

binding moiety is achieved by loop-

grafting, i.e. transplantation of the

complementarity determining regions

(CDRs) onto acceptor frameworks with

suitable biophysical properties. For

example, loop-grafting of rodent CDRs

onto a suitable consensus human variable

domain framework was shown to result in

superior stability of the resulting scFv

fragment (14). This approach is particularly

interesting for the generation of scFvs for

therapeutic applications, since it combines

stabilization and humanization in one step.

However, due to the high structural

diversity, particularly of rodent variable

domains, a relatively large repertoire of

human acceptor frameworks is required to

match the major subtypes (15). In addition,

further amino-acid substitutions in the

human framework regions are often

required to restore the conformation of

animal CDRs (16-20). As a consequence,

humanization of antibodies is frequently

subject to engineering strategies

specifically designed for every individual

donor sequence, and is particularly

challenging for the scFv format since these

fragments tend to aggregate and are

difficult to produce. As a result, the

outcome of such laborious efforts is

unpredictable in many cases and the overall

success rate is low when compared to

humanization of Fabs or IgGs.

In contrast to humans and rodents,

framework variability in rabbits is very

limited because one VH germline gene

segment is preferentially used and accounts

for 80 to 90% of VDJ genes, which are

combined with multiple but homologous

VJ genes coding for the light chain. This

apparent limitation of antibody diversity in

rabbits is compensated by a high degree of

N-nucleotide addition at VD and DJ

junctions. Further VDJ gene diversification

occurs by somatic hypermutation and gene

conversion-like mechanisms upon antigenic


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stimulation (reviewed by (21)). As a

consequence of i) preferential VH1 gene

segment usage, ii) high homology among

Vκ gene segments and iii) usage of gene

conversion during antibody diversification,

rabbit variable domain frameworks are very

homologous to each other. Furthermore,

following immunization rabbit antibodies

mostly show significantly higher affinities

when compared to rodent antibodies

(unpublished data). Thus, due to their high

affinities and the relatively low structural

diversity, rabbit antibodies present an ideal

starting point for the development of a

generally applicable protocol to generate

humanized scFv therapeutics.

In the work presented here, we used a

single human scFv-scaffold of the Vκ1-

VH3 subtype to generate a set of scFvs

with high-affinity by grafting of CDRs

from 15 different rabbit monoclonal

antibodies directed against tumor necrosis

factor-alpha (TNF-alpha) or vascular

endothelial growth factor (VEGF). This

scaffold was previously identified from a

human library using a whole genome

screening approach (22). We further

identified a motif consisting of five

rationally altered framework positions,

which, when introduced into the human

acceptor scaffold, improved protein

stability and supported functional

presentation of rabbit CDRs. Most resulting

antibody fragments exhibited excellent

solubility, thermal stability and affinity,

and were successfully produced with high

yields in a generic refolding process from

inclusion bodies in E.coli.

2. MATERIALS & METHODS

2.1 Generation of rabbit monoclonal

antibodies

Rabbit monoclonal antibodies (rabbit

mAbs) were generated in collaboration

with Epitomics Inc. Briefly, new Zealand

white rabbits were immunized with

recombinant human VEGF165 (PeproTech

EC Ltd., London, UK) and peptides

thereof, or with recombinant human TNF-

alpha. Spleen cells of the immunized

rabbits were fused with rabbit immortal B

cells (240E-W2) as described previously

(23).

For VEGF-binders, 23,040 hybridomas

were screened for the presence of rabbit

mAbs to human VEGF165 by an enzyme

linked immunosorbant assay (ELISA).

Neutralizing activity of the 248 positive

hybridomas was assessed using a VEGF

receptor 2 (VEGFR2) blocking ELISA. Out

of 92 hybridomas showing inhibition of

VEGF binding to VEGFR2, 23 hybridomas

were selected based on consistent results in

the VEGFR2 blocking ELISA and cloned

twice using limiting dilution technique.

Binding affinities of cloned hybridomas

towards human VEGF165 were measured by

surface plasmon resonance (SPR), using a

BIAcore T-100 instrument (Biacore Inc.

Uppsala, Sweden). Total RNA of the 7

hybridomas secreting the most potent rabbit

mAbs was isolated from hybridoma cells

and the cDNAs encoding the variable light

and variable heavy chain were amplified by

RT-PCR. After PCR amplification,

sequenced DNA fragments were ligated

into a mammalian expression vector

containing rabbit CL and CH domains.

Correctness of amplified VL and VH

domains was confirmed by transient

expression of the rabbit mAbs in human

293 cells and subsequent analysis of 293

cell supernatants using the VEGFR2

blocking assay and SPR measurements.


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For TNF binders, supernatants of 5,640

hybridomas were screened for binding to

human TNF-alpha in ELISA. Out of 142

hits, 44 neutralized TNF-alpha induced

cytotoxicity in murine L929 cells.

Following cloning of the 44 confirmed hits

binding affinity to human TNF-alpha was

determined by SPR measurements. Eight

antibodies were selected for humanization

and reformatting based on potency in the

L929 assay.

2.2 Sequence alignments:

Antibody variable domain sequences were

aligned using CLUSTALW (24) and were

analyzed using the BioEdit sequence

alignment editor version 7.0.9.0 (Ibis

Biosciences, California, USA). The Kabat

numbering scheme was used for

nomenclature of residue positions as well

as for the definition of complementarity

determining regions (CDRs), except CDR-

H1 (25). The boundaries of CDR-H1 vary

substantially depending on whether loop

structure or sequence variability criteria are

considered for the CDR definition.

Therefore, in this study CDR-H1 was

defined from residue VH 26 to VH 35,

which is a combination of Kabat and

Chotia definitions and takes into account

both structural and sequence variability.

Nomenclature and overview of the human

and rabbit immunoglobulin germ line

sequences was according to the

International ImMunoGeneTics

information system (IMGT Montpellier,

France). The most closely related V-gene

germline sequences were identified based

on homology of VH and VL segments of

each hybridoma clone to rabbit germline

sequences. Amino acid alignments were

evaluated from residue 1 to 88 (for VL) and

from 1 to 92 (for VH).

2.3 Framework selection:

A pool of 88 well folding and stable scFv

antibodies previously isolated from a

library generated by amplification and

random combination of human VH and VL

domains from a naive human spleen cDNA

(22) was analyzed to select a suitable

acceptor framework for rabbit CDR

grafting. Human scFv sequences were

ranked a) according to expression level of

the respective clone in yeast and b)

according to homology to rabbit consensus

at core residues. Core residues were

defined as residue positions with less than

10% average relative side-chain

accessibility to the solvent using

information published by Honegger and

Pluckthun (26). Each of the two domains

selected to generate the acceptor scFv

scaffold FW1.4 (clone kI27 assigned to

germline IGKV1-5 and a43 assigned to

germline IGHV3-23) corresponds to a

mature human scFv clone that showed high

levels of soluble expression in yeast. The

amino acid sequence of FW1.4 is as

follows: EIVMTQSPSTLSASVGDRVIITC*CDRL1

*WYQQKPGKAPKLLIY*CDRL2*GVPSRF

SGSGSGAEFTLTISSLQPDDFATYYC*CD

RL3*FGQGTKLTVLGGGGGSGGGGSGGGG

SGGGGSEVQLVESGGGLVQPGGSLRLSCA

AS*CDRH1*WVRQAPGKGLEWVS*CDRH2

*RFTISRDNSKNTLYLQMNSLRAEDTAVY

YCAK*CDRH3*WGQGTLVTVSS

2.4 Generation of humanized scFvs from

rabbit monoclonal antibodies:

A first minimalistic humanized version was

generated for each of the 15 rabbit

monoclonal antibodies by combining

sequences of their CDRs with framework

region sequences of the human scFv

acceptor scaffold FW1.4. A second series

of “optimized” grafts was generated by


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CDR transfer onto optimized derivatives of

FW1.4 (referred to as FW1.4opt). Human

framework residues were substituted a) at

positions that are relevant for CDR

conformation by the respective amino acids

used in rabbit sequences and b) by grafting

of donor amino acids that potentially are in

contact with the antigen. Due to the lack of

crystal structures of rabbit antibody

variable domains, selection of positions

relevant for CDR conformation were

identified based on literature data

(14,16,19,27-29,30). Resulting clones

contained such substitutions at selected

subsets of positions: L69, H23, H24, H49,

H67, H69, H71, H73, H78 and H94.

Framework residues that potentially

interact with the antigen were identified by

alignment of the rabbit variable domains

with the nearest germ line counterpart (for

VL) or the rabbit variable heavy domain

consensus sequence (for VH). Differences

between aligned sequences were

hypothesized to result from in vivo affinity

maturation. Such mutations at positions

predicted to be solvent exposed and in

proximity to the antigen binding site as

well as the rare mutations that were found

at the positions mentioned above involved

in CDR conformation, were transferred to

the acceptor framework. Information about

solvent exposure of residues was extracted

from a sequence analysis by Honegger et

al.(26)). Pro and Gly residues introduced as

result of the somatic hypermutation process

were substituted if such mutations were

found in the proximity of CDRs.

2.5 Molecular cloning of scFv expression

vectors

DNA sequences encoding CDR-grafted

scFvs were optimized for E.coli codon

usage, GC content, mRNA secondary

structure, codon and motif repeats and

restriction sites, using LETO software

package (Entelechon GmbH, Regensburg,

Germany). Overlapping oligonucleotides

matching the optimized DNA sequence

were synthesized and genes were generated

by overlap extension techniques (27). A

(Gly4Ser)4 linker was used to connect VL

and VH domains. All scFv genes contained

5’ and 3’ flanking NcoI and HindIII

restriction sites, respectively, that allowed

cloning into the proprietary E.coli inclusion

body expression vector. Additional AccIII

and BamHI sites were introduced in the

linker sequence to enable domain shuffling.

Two derivatives were made for the

humanized scFvs derived from each of the

rabbit monoclonal antibodies 34, 43, 511

and 578 by domain shuffling. For this,

domains containing back-mutations to

donor residues in the framework regions

(optimized grafts; FW1.4opt) were paired

with the domains lacking mutations in the

framework (minimalistic grafts; FW1.4)

using standard DNA cloning techniques.

2.6 Expression and protein purification of

scFv fragments

E. coli BL21(DE3) transformed with the

respective inclusion body expression

plasmids were grown at 37°C in dYT

medium containing the appropriate

antibiotics. Protein expression was initiated

by addition of 1 mM IPTG (final

concentration) at an optical density (OD600)

of about 2.0. Three hours after induction, E.

coli cells were harvested, disrupted by

sonication and inclusion bodies were

isolated by repeated washing and

centrifugation steps. Inclusion bodies were

solubilized at a concentration of 10 mg/ml

in the presence of 6 M Gdn-HCl and

reduced by addition of 20mM DTT. Basic

refolding screenings were performed to

select best pH, redox-system


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(cystine/cysteine) and salt concentrations

from the range of tested conditions. Best

conditions for each individual scFv were

then used for a lab-scale refolding process.

For this, the scFv proteins were renatured

by rapid dilution into a 50 fold volume of

refolding buffer. After up-concentration

and dialysis against PBS-buffer (pH 6.0),

proteins were purified using size exclusion

chromatography. Content and purity of

eluted fractions were assessed by SDS-

Page and SE-HPLC. Refolding yield was

expressed as amount (mg) of refolded

protein obtained out of 1L refolding

solution.

For surface plasmon resonance (SPR)

measurements, periplasmic fractions

containing anti-VEGF “wild type” (wt)

scFvs and anti-TNF “wild type” (wt) scFvs

were prepared. Overnight starter cultures

were made by inoculating single E. coli

BL21(DE3) colonies from LB plates into 2

ml cultures of dYT medium with suitable

antibiotics and 1% glucose in a 37°C

shaker. 1.5 ml expression medium (dYT

with 45mM K2HPO4, antibiotics, 0.1%

glucose) was inoculated with 150 µl of the

overnight cultures (in triplicates). The

bacterial cultures were incubated in a 30°C

shaker until OD595 reached 1.5-2.

Expression was induced by the addition of

IPTG (isopropyl β-D-thio-

galactopyranoside) to a final concentration

of 0.5mM. Three hours after induction,

cultures were harvested by centrifuging for

10 min at 4500 rpm. The pellets were

resuspended in 300 µl fractionation buffer

(200 mM Tris-HCl, 1 mM EDTA, 20%

sucrose, 500 µg/ml lysozyme) and each set

of triplicates was pooled. After a static

incubation at room temperature for 15 min,

an equal volume of cold water was added

and the suspension was incubated for a

further 15 min. The supernatant was then

recovered as periplasmic fraction by

centrifuging for 15 min in a benchtop

centrifuge (13000 rpm and 4°C).

2.7 Thermostability measurements

Thermostability measurements were

performed using a differential scanning

calorimeter (DSC) and a Fourier

transformed infrared (FTIR)

spectrophotometer. Samples were first

dialyzed against phosphate buffered saline

(50 mM Na2HPO4, 150 mM NaCl, pH 6.5).

DSC was performed using a MicroCal high

throughput VP-Capillary-DSC (Microcal,

Massachusetts, USA). Measurements of the

difference in heat capacity between the

scFv samples in solution and reference

buffer were performed using protein

concentrations of approximately 1.0 mg/ml.

Measurements were performed over a

temperature range from 25 to 95°C at a

scan rate of 3.3 °C/min. Data were

analyzed using the Origin plotting software

(OriginLab, Massachusetts, USA).

Apparent melting temperatures were

estimated from DSC scans after

concentration normalization and

subtraction of the buffer-buffer baseline.

FTIR spectra were obtained on a Bruker

Tensor 27 FTIR spectrometer equipped

with an attenuated total reflectance Bio-

ATR cell (Bruker Optics, Faellanden,

Switzerland). Changes in secondary

structure of the samples were assessed by

heating from 25 to 95°C using 5°C steps

and 2.5°C steps for the dynamic range of

unfolding. At each temperature a total of

200 scans were recorded for each spectrum

at a resolution of 1 cm-1

. All spectra

manipulations were performed using OPUS

spectroscopy software (Bruker Optics,

Faellanden, Switzerland). Buffer reference

and transient atmospheric (CO2 and H2O)


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background were subtracted from the

spectra. Second derivative spectra were

obtained for the amide I band using a third

degree polynomial function with

smoothing. Degree of unfolding was

assessed by multi factorial analysis of

second derivative amid I band shape. A

linear calibration curve was generated,

assuming zero percent unfolding for the

spectra at 25, 30 and 35°C and 100 percent

unfolding for the spectra at the three

highest temperatures (85, 90 and 95°C),

which is adequate for most scFvs. Thermal

unfolding curves were determined by

fitting the FTIR spectra to a linear

regression as a function of temperature

using the calibration curve. The reported

apparent melting temperatures (Tm) for the

various scFvs correspond to the

temperature of 50 percent unfolding.

2.8 Binding kinetics and affinity of VEGF

and TNF antagonists

For binding kinetics measurements, SPR

measurements with BIAcoreTM

-T100 were

employed. All measurements were

performed at 25°C. Carboxymethylated

dextran biosensor chips (CM4, GE

Healthcare, Uppsala, Sweden) were

activated with N-ethyl-N’-(3-

dimethylaminopropyl) carbodiimide

hydrochloride and N-hydroxysuccinimide

according to the supplier’s instructions and

recombinant human VEGF165 (PeproTech

EC Ltd., London, UK) was immobilized on

a CM4 sensor chip using a standard amine-

coupling procedure to achieve a response of

approximately 200 resonance units. 2-fold

serial dilutions of VEGF antagonists (20-

0.16 nM) in HBS-EP buffer (10mM

HEPES, 150mM NaCl, 3 mM EDTA, and

0.05% surfactant P20, pH 7.4) were

injected into the flow cells at a flow rate of

30 µl/min for 5 min. Dissociation of the

anti-VEGF scFv from the VEGF on the

CM4 chip was allowed to proceed for 10

min. After each injection cycle, surfaces

were regenerated with 2 injections of

100mM NaOH.

The binding kinetics of anti-TNF scFvs

were measured using a NTA

(Nitrilotriacetic acid) sensor chip (Series S

Sensor Chip NTA, GE Healthcare,

Uppsala, Sweden) and His-tagged human

TNF-alpha (produced in-house). The chip

was loaded with 500 µM NiCl2 diluted in

HBS-EP buffer (10 mM HEPES, 150 mM

NaCl, 50 µM EDTA and 0.05% surfactant

P20, pH 7.4). Human TNF-alpha (2 nM)

was captured through the N-terminal his-

tag via Ni2+

NTA chelation. Three-fold

serial dilutions of TNF antagonists (90 -

0.014 nM) diluted in HBS-EP buffer were

injected into the flow cells at a flow rate of

30 µl/min for 5 min. Dissociation of the

TNF-alpha antagonists was allowed to

proceed for 10 min. Regeneration of the

chip surface was performed by injection of

regeneration solution (10mM HEPES,

150mM NaCl, 350 mM EDTA and 0.05%

surfactant P20, pH 8.3) followed by

injection of 50 mM NaOH.

Binding kinetics measurements of anti-

VEGF wt-scFvs as well as for 578-wt-IgG

were performed as described above for

humanized VEGF antagonists, using a

CM4 sensor chip with immobilized human

VEGF165. Binding kinetics of anti-TNF wt-

scFvs were measured using a CM5 sensor

chip with immobilized human TNF-alpha.

Periplasmic fractions containing his-tagged

wt-scFvs (or the 578-wt-IgG hybridoma

supernatant) were serially diluted in two-

fold steps in HBS-EP buffer. For VEGF

binders, surfaces were regenerated with 2

injections of 100 mM or 75 mM NaOH

after each cycle,, depending on the wt-


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scFv. For TNF binders, single cycle kinetic

measurements were done by sequential

injections of a scFv concentration series

without any regeneration steps.

The apparent dissociation (kd) and

association (ka) rate constants and the

apparent dissociation equilibrium constant

(KD) were calculated with BIAcore T100

evaluation Software version 2.0.1 using

one-to-one Langmuir binding model

(Biacore Inc., Uppsala, Sweden). Since the

concentration of the scFvs in the

periplasmic fractions was unknown, only

the apparent dissociation rate constants

were calculated by fitting the dissociation

curves to a one-to-one dissociation model

(R =R0*exp (-kd *(t –t0) ) + offset).

2.9 VEGF receptor 2 blocking assay

Recombinant human VEGFR2-Fc chimera

(R&D Systems Inc., Mineapolis,

Minnesota), consisting of amino acid

residues 1-764 of the extracellular domain

of human VEGFR2 fused to a 6x histidine

tagged Fc domain of human IgG1, was

coated on a 96-well Maxisorp ELISA plate

(Nunc, Langenselbold, Germany) at 0.2

µg/ml in PBS and blocked using PBS with

0.01% BSA and 0.2% Tween 20 (PBST).

Biotinylated human VEGF165 (2.6 nM) was

first incubated with 2-fold serially diluted

anti-VEGF scFvs and ranibizumab (15 –

0.01 nM) in PBST. After 24 hours of

incubation at room temperature, the

mixtures were transferred to the VEGF

receptor-immobilized plate and incubated

for 1.5 h at room temperature. Binding of

unblocked human VEGF165 to the

immobilized VEGFR2 was detected with

streptavidin-HRP conjugates

(Stereospecific Detection Technologies,

Baesweiler, Germany) followed by addition

of substrate (BM Blue POD substrate,

Roche Diagnostics, Mannheim, Germany).

Optical density at 450 nm was measured

using a Sunrise microplate reader (Tecan,

Mannedorf, Switzerland). The dose-

response curves of the scFvs were fitted to

a 4-parameter logistic fit to calculate EC50

values.

2.10 HUVEC proliferation assay

Human umbilical vein endothelial (HUVE)

cells (Promo Cell, Heidelberg, Germany)

were maintained in supplemented

Endothelial Cell Growth Medium (ECGM,

Promo Cell, Heidelberg, Germany) with

1% penicillin-streptomycin (PS). HUVECs

were seeded in poly-D-Lysine coated 96-

well plates (Becton Dickinson GmbH,

Heidelberg, Germany) at a density of 2’000

cells per well and incubated for 24h at

37°C. 3 fold serial dilutions of anti-VEGF

scFvs or ranibizumab (150-0.007 nM) and

recombinant human VEGF165 (Peprotech,

London, UK) (0.16 nM final concentration)

were prepared in starving medium (ECGM

without supplement containing 0.5% heat

inactivated FCS and 1% PS) and

preincubated for 60 min at room

temperature. VEGF concentrations that

stimulate submaximal HUVEC

proliferation (EC90) were used. Cells were

then washed once with starving medium

and the agonist-antagonist mixtures were

added to the cells and incubated for 4 days

in a 37°C/5% CO2 humidified incubator.

Cell proliferation was assessed by

measuring absorbance at 450 nm as

described above after addition of WST-1

cell proliferation reagent (Roche

Diagnostics GmbH, Mannheim, Germany).

Data were analyzed using a 4-parameter

logistic curve-fit, and the molar

concentration of VEGF inhibitor required

to reduce HUVEC proliferation to 50%

(EC50) was derived from inhibition curves.


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2.11 TNF-alpha induced apoptosis in L929

fibroblasts

Mouse L929 fibroblasts (Deutsche

Sammlung von Mikroorganismen und

Zellkulturen GmbH, Braunschweig,

Germany) between passages 6 and 15 were

seeded in 96-well plates (Nunc,

Langensebold, Germany) at a density of

60’000 cells per well in assay medium

(phenol red-free RPMI with L-Glutamine +

5% FCS). Cells were sensitized to TNF

alpha induced apoptosis by addition of 1

µg/ml of actinomycin D (Sigma, Steinheim,

Germany). 2 fold Serial dilutions of anti-

TNF alpha scFvs (14.2-0.014 nM) and

recombinant human TNF alpha (Peprotech

EC Ltd, London, UK) (1000 pg/ml or 19.16

pM final concentration) were prepared in

assay medium and preincubated for 30 min

at room temperature. After addition of the

agonist-inhibitor mixtures, the cells were

incubated for 20 h in a 37°C/5% CO2

humidified incubator. Cell proliferation

was assessed by measuring absorbance at

450 nm using a microplate reader (Genios

TECAN, Mannedorf, Switzerland) after

addition of a solution containing 1 mg/ml

XTT (Applichem, GmbH, Darmstadt,

Germany) in phenol red free RPMI and 25

µM PMS (Sigma-Aldrich, Steinheim,

Germany). Data were analyzed using a 4-

parameter logistic curve-fit, and the

concentration (in mass units) of anti-TNF

alpha scFvs required to neutralize TNF

alpha induced apoptosis by 50% (EC50) was

calculated.

2.12 Temperature induced oligomerization

and degradation.

578-FW1.4, 578-FW1.4opt and 578-

FWgen were concentrated up to 20, 40 and

60 mg/mL in formulation buffer (20 mM

tri-sodium citrate, 125 mM Nacl) pH6.5

and incubated 2 weeks at 40°C. Samples

were analyzed before and after 14 days

incubation for degradation using 12.5 %

sodium dodecyl sulfate–polyacrylamide gel

electrophoresis (SDS-PAGE) under

reducing and non-reducing conditions.

Size-exclusion high-performance liquid

chromatography (SE-HPLC) was used to

determine monomer content and soluble

aggregates of the samples before and after

the incubation period. Monomers were

resolved from non-monomeric species on a

TSKgel Super SW2000 column (TOSOH

Bioscience) and the percentage of

monomeric protein was calculated as the

area of the monomer peak divided by the

total area of all product peaks.

3. RESULTS

3.1 Selection of rabbit monoclonal

antibodies from hybridomas:

Monoclonal antibodies binding either to

human VEGF165 or human TNF-alpha were

selected from rabbits either immunized

with recombinantly produced human

VEGF165 or TNF-alpha, respectively.

Hybridoma screening (see materials and

methods) resulted in the selection of 7

VEGF neutralizing antibodies (375, 435,

509, 511, 534, 567 and 578) that potently

blocked binding of VEGF to hVEGFR2,

and 8 TNF-alpha neutralizing antibodies (1,

6, 15, 19, 34, 35, 42 and 43) that potently

inhibited TNF-alpha induced apoptosis in

the murine L929 cell line. Sequence

analysis of the rabbit monoclonal

antibodies showed that 7 kappa germlines

(out of 65 functional VL-gene segments)

were represented in the selected binders

(table 1). Based on homology assessment,

no germline VH gene segments could be

assigned, probably due to homologous

recombination occurring in rabbit V-genes.

The fact that an additional disulfide-bond

linking CDRH1 and CDRH2 was present in


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six of the 15 antibodies, together with

results from sequence distance analysis

(data not shown) indicates that at least two

different germline VH gene segments have

been used.

3.2 Identification of a human scFv-scaffold

suitable for humanization of rabbit variable

domains:

A yeast based genetic screening of the

human variable domain repertoire

performed earlier resulted in a number of

mature human scFvs antibody fragments

that were well and solubly expressed in the

cytoplasm of yeast, mammalian cells and

E.coli (22). From this pool of stable human

scFv sequences, a variable light and

variable heavy chain combination was

chosen to serve as acceptor framework for

rabbit CDRs. Selection of this human

acceptor framework from the pool of stable

scFv clones was based a) on the level of

soluble expression in yeast, and b) on its

homology to the rabbit variable region

consensus sequence at specific core

positions (see materials and methods). In

contrast to rodents and humans, these core

and interface residues frequently involved

in CDR conformation and relative

disposition of VL an VH, respectively

(14,19,27-31) are highly conserved in

rabbits. Therefore, a stable human scaffold

sharing high homology to rabbit sequences

at such core positions was thought to

generally support functional conformation

of rabbit CDRs. The chosen framework,

termed FW1.4, is of the Vκ1-VH3 type. In

combination with human CDRs, FW1.4

was well characterized in terms of thermal

stability, in vitro folding and expression in

microbial and mammalian systems (data

not shown). At the abovementioned core

positions, this framework shares high

percentage of identity with the respective

consensus residues of rabbit light chains

(88%) and heavy chains (85%).

3.3 Generation of humanized scFvs from

rabbit monoclonal antibodies by loop

grafting onto the human scFv scaffold

FW1.4 or derivatives thereof:

In one series of “minimalistic” grafts,

CDRs (as defined in materials and

methods) from 15 independent rabbit

monoclonal antibodies against TNF-alpha

and VEGF were grafted onto the human

acceptor framework 1.4 (FW1.4). Resulting

clones were designated by the number of

their parental rabbit IgG and the human

acceptor framework FW1.4 (e.g. 578-

FW1.4). In a second series, “optimized”

grafts were designed (e.g. 578-FW1.4opt)

by additional substitution of specific

framework residues. Such substitutions

were introduced a) at positions conserved

in rabbits, which are involved in CDR

conformation, and b) at positions

potentially involved in direct contact to the

antigen (see materials and methods). Of all

framework positions considered mainly

L69, H23, H24, H49, H67, H68, H69, H71,

H73, H78 and H94 frequently differed

between the various rabbit antibodies and

FW1.4 (see table 3). The limited number of

such positions and, more importantly, their

relatively high degree of conservation in

rabbit sequences (table 5) suggests that

only a small set of highly conserved

framework substitutions is generally

required for the humanization of rabbit

antibodies. In fact the motif consisting of

Thr-H23, Gly-H49, Thr-H73, Val-H78 and

Arg-H94 is highly conserved and may thus

present a generic solution to prepare a

human acceptor framework (e.g. FW1.4)

for minimalistic CDR grafting. To test this

hypothesis a third set of humanized scFvs


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were generated and characterized as

detailed in section 3.7.

3.4 Improved production yields following

loop grafting onto FW1.4 and its

derivatives:

Purification of scFv antibody fragments is

often limited by their relatively low

expression yields and their tendency to

form aggregates. We earlier experienced

that stable scFvs can be efficiently

produced by refolding from purified

inclusion bodies. Humanized rabbit TNF-

alpha and VEGF inhibitory scFvs were

expressed as inclusion bodies in E.coli.

Inclusion body expression levels were very

similar between all molecules tested (data

not shown). Following harvesting,

inclusion bodies were subjected to a

generic refolding process and monomeric

proteins were subsequently purified by

preparative size-exclusion chromatography

(for details see materials and methods).

Most humanized scFvs based on FW1.4 or

its derivatives were well producible by

refolding in a generic lab scale process

(materials and methods), resulting in yields

of up to 60 mg of purified protein per liter

of refolding solution (see table 1). Only two

molecules (534-FW1.4 and 34-FW1.4)

could not be purified in significant

amounts. Optimization of framework

regions by substitution of residues L69,

H23, H49, H71, H73, H78 and H94 in 534-

FW1.4opt, and L15, L40, L72, H23, H49,

H68, H69, H71, H73, H78 and H94 in 34-

FW1.4opt, only slightly improved

production yield (see table 1). Although

production of some optimized grafts

resulted in higher yields when compared to

their minimalistic counterparts, the

observed differences were minor in most

cases. Indeed, the impact of the highly

diverse CDRs on refolding yield seems to

be more significant than the few

substitutions in the framework regions. In

contrast, attempts to purify the scFv

fragments consisting of the parent rabbit

variable domains were not successful.

Minor quantities of secreted His-tagged

scFv fragments were thus produced with

poor purity from E.coli culture supernatants

by Ni-NTA affinity chromatography (data

not shown). These results indicate that

refolding and purification of scFvs based

on FW1.4 and its derivatives is particularly

efficient.

3.5 CDR grafting onto the human scFv

scaffold family derived from FW1.4

reproducibly results in functional variable

domains:

Binding kinetics and potency of the

minimalistic and optimized grafts of anti-

VEGF and anti-TNF-alpha scFvs were

compared. Data are summarized in table 1.

When exclusively CDRs were grafted from

the rabbit sequence to the human scaffold,

affinities of the resulting scFv to their

target proteins were in the low nanomolar

to high picomolar range (2.89x10-8

M –

1.8x10-10

M) for VEGF inhibitors. One

minimalistic graft (375-FW1.4) did no

longer show binding to VEGF. Affinities of

the TNF-alpha binding molecules were

lower with dissociation constants (KD)

ranging from 2.62x10-7

M to 5.16x10-10

M.

Two minimalistic grafted molecules (1-

FW1.4 and 6-FW1.4) did not show

significant binding to TNF. In both groups,

mutation of further residues in FW1.4

significantly enhanced binding, resulting in

an improvement of KD by one to three

orders of magnitude for all molecules

tested. Only one molecule (1-FW1.4opt)

did still not bind to its target. In terms of

potency, optimized grafts were also clearly

better compared to the variants generated


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by minimalistic CDR-grafting. For

example, 34-FW1.4opt, the most potent

TNF-alpha antagonist, blocked TNF-alpha

induced apoptosis of mouse L929

fibroblasts 5.6-fold more efficiently than

infliximab (compared in mass units) while

none of the TNF-alpha binders generated

by minimalistic grafting was sufficiently

potent to rescue cell growth at the tested

concentrations (table 1). Among the VEGF-

inhibitors, 578-FW1.4opt exhibited highest

affinity in SPR experiments and best

potency in ELISA. This scFv also showed

1.3 fold stronger inhibition of VEGF

induced HUVEC proliferation compared to

ranibizumab (see figure 1). In line with

SPR analysis the minimalistic graft 578-

FW1.4 displayed a 14.8 fold lower potency

(EC50 of 2.614 nM versus EC50 of 0.177

nM) compared to 578-FW1.4opt (see figure

1 and data not shown). Similarly, the

potencies of the optimized grafts 511-

FW1.4opt and 567-FW1.4opt were 68-fold

(EC50 of 3.832 nM versus EC50 of 260.576

nM) and 5.7-fold (EC50 of 6.694 nM versus

EC50 of 38.156 nM) higher, when

compared to the respective minimalistic

counterparts (see figure 1 and data not

shown). Surprisingly, when comparing off-

rates between optimized grafts of VEGF-

and TNF-inhibitors and their progenitor

rabbit scFv in SPR, the apparent loss in

binding strength was only very moderate

for most scFvs tested. Indeed, off-rates of

humanized scFvs were equal or at most

seven-fold lower than their rabbit

precursors for VEGF-inhibitors indicating

almost complete retention of activity upon

optimized grafting on FW1.4 (table 2). For

TNF inhibitors two molecules, 1-FW1.4opt

and 15-FW1.4opt, showed significant loss

in binding strength while off-rates of the

remaining scFvs were equal or at most 14.6

fold lower compared to their rabbit

precursors (table 2). Comparison of off-

rates between the rabbit scFv of 578 and

the full-length rabbit IgG of the same

binder showed only a 4 fold decrease in the

off-rate (3.66x10-5

s-1

versus 9.18x10-6

s-1

).

This difference in binding strength can

most probably be attributed to avidity

effects.

3.6 The human variable domain scaffold

family derived from FW1.4 provides drug-

like properties to humanized scFvs:

Aggregation during scFv fragment

purification and storage often correlates

with low thermal stability of the protein. In

order to evaluate thermal stabilities of the

humanized scFv antibody fragments,

apparent melting temperatures were

assessed by differential scanning

calorimetry. Briefly, heat capacity changes

of the scFv formulations were measured

over a temperature gradient ranging from

25 to 95 °C. Apparent melting temperatures

(Tm) are summarized in table 1. For the

least stable molecule (6-FW1.4opt), Tm was

at 53.7 °C. Tm of most other humanized

fragments was above 60 °C. Best results

were obtained with the VEGF-inhibitory

scFvs 578-FW1.4opt, 509-FW1.4opt and

511-FW1.4opt with Tm of 77.7, 80.1 and

86.9 °C, and with the TNF inhibitory scFvs

15-FW1.4opt and 34-FW1.4opt with

apparent melting temperatures at 71.6 and

78.1 °C, respectively. For all scFvs thermal

stability of the optimized molecule was

higher than that of the minimalistic graft,

indicating that the grafted framework

positions are relevant not only for CDR

positioning but also for domain stability.

3.7 A motif consisting of a few conserved

framework residues in VH accounts for

stability and CDR structure of humanized

scFvs


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Framework positions at which human

residues were substituted by rabbit amino

acids to generate optimized derivatives of

FW1.4 are listed in table 3. In order to

assess the relative contribution of

framework substitutions in the light and the

heavy chain, domain shuffling experiments

were performed with the humanized

variable domains of clones 511, 578, 34

and 43. Therefore, heavy chains and light

chains of minimalistic and optimized grafts

were recombined. ScFv constructs

consisting of a minimalistic light chain and

an optimized heavy chain exhibited near

equal affinities and comparable or even

better thermal stabilities when compared to

the optimized grafts (compare tables 1 and

4). In the opposite situation, when

optimized light chains were combined with

minimalistic heavy chains resulting scFvs

showed lower thermal stabilities and

affinities, which were 6-, 5-, 293- and 4-

fold decreased for such derivatives of 578,

511, 43 and 34, respectively (see also table

4). These results suggest that affinity and

stability of the humanized scFvs mainly

depends on the few conserved rabbit amino

acids introduced into the human heavy

chain framework. For this reason, as

hypothesized earlier, the set of amino acid

motifs required to support rabbit CDR

structure could be further generalized and

possibly reduced to the five most conserved

positions (table 5). To test this hypothesis,

a new generically applicable acceptor

framework termed FW1.4gen was designed

based on FW1.4. In the heavy chain, five

residues were substituted with conserved

rabbit amino acids at positions H23, H49,

H73, H78 and H94. At the other “vernier

zone” positions H69 and H71, the most

frequently used amino acids in rabbit heavy

chains were already present in the human

FW1.4. At positions H24 and H67, valine

and serine, respectively, would correspond

to the consensus residues according to the

collection of rabbit antibody sequences in

the Kabat database. However, this positions

are less conserved in rabbits (table 5), and

moreover, in the majority of the rabbit

variable domain sequences identified in the

course of this study, alanine and

phenylalanine were more abundant and

were thus not changed (compare table 3

and table 5).

3.8 Generic minimalistic CDR grafting

onto a modified human scFv scaffold

Variable domains of the VEGF binding

monoclonal antibodies 511 and 578 as well

as of the TNF binding antibodies 34 and 43

were humanized and reformatted into a

scFv fragment by minimalistic grafting of

CDRs onto FW1.4gen. This framework

contains the rabbit amino acid motif Thr-

H23, Gly-H49, Thr-H73, Val-H78 and Arg-

H94. Affinities of the VEGF inhibitory

scFv fragments 511-FW1.4gen and 578-

FW1.4gen, as determined by SPR were

found to be 5.7x10-10

M and 3.9x10-11

M,

respectively (table 6). When compared to

their optimized counterparts 511-FW1.4opt

and 578-FW1.4opt, affinity dropped

slightly for 511-FW1.4gen by a factor of

2.7. A minor, if at all significant loss in

affinity was observed also for 578-

FW1.4gen, which was attributed to the

HG94R mutation in FW1.4gen, flanking

CDRH3 and possibly affecting loop

conformation. Following minimalistic

grafting of the TNF inhibitory rabbit

antibody 34 and 43, no loss in affinity was

observed. On the contrary, the affinity of

34-FW1.4gen was more than 8-fold higher

than that of 34-FW1.4opt (compare tables 1

and 6). This is in sharp contrast to

minimalistic grafting of antibody 43 onto

the original FW1.4, where loss in affinity

was about 217-fold (table 1). In line with


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results from affinity measurements, also

relative potencies of the scFv fragments

based on the FW1.4gen framework were in

the same range as their optimized variants

on FW1.4 (compare tables 1 and 6). In the

VEGFR2 blocking assay, relative potency

of 511-FW1.4gen slightly dropped when

compared to 511-FW1.4opt, but was

identical when assessed in the HUVEC

proliferation assay. In contrast, no

significant loss in potency was observed for

578-FW1.4gen in the VEGFR2 blocking

assays well as in the cell-based assay (see

figure 1 and table 6). Differences in relative

potencies between the cell-based and

ELISA-based assays can probably be

attributed to day to day variations in assay

performance. Thus, potencies of 511-

FW1.4gen and 578-FW1.4gen did not

significantly differ when compared to their

optimized counterparts. In comparison to

ranibizumab, a market approved VEGF-

inhibitory Fab fragment, the EC50 to block

VEGF-induced proliferation of HUVEC

cells for 511-FW1.4gen was 12.7-fold

higher than for the benchmark molecule,

while the potency of 578-FW1.4gen was

similar to ranibizumab in the same assay.

For the TNF-inhibitory scFv 34-FW1.4gen,

no loss in potency to block TNF-induced

apoptosis was observed, while only a slight

if at all significant loss in potency was seen

for 43-FW1.4gen. Potencies of 34-

FW1.4gen and 43-FW1.4gen were 5.7-fold

and 3.8-fold higher when compared to

infliximab in mass units. Infliximab is a

market approved TNF inhibitory IgG.

Alternatively, when compared on a molar

basis, the relative potency of 34-FW1.4gen

was identical, whereas the potency of 43-

FW1.4gen was roughly 1.5-fold lower than

that of the benchmark molecule. In any

case it remains difficult to compare the

potency of full-size bivalent IgG with that

of a monovalent scFv of only 27 kDa.

Thermal stabilities of the four molecules

were assessed by differential scanning

calorimetry (DSC) as well as by Fourier-

transformed infrared spectrometry (FT-IR).

In DSC apparent melting temperatures

were 85.6, 81.3, 81.6 and 69.2 °C for 511-

FW1.4gen, 578-FW1.4gen, 34-FW1.4gen

and 43-FW1.4gen, respectively (table 6).

Therefore, thermal stabilities were higher,

when compared to 511-FW1.4opt, 578-

FW1.4opt 34-FW1.4opt and 43-FW1.4opt

(see also figure 2). Temperature induced

unfolding experiments in FT-IR confirmed

the exceptionally high apparent melting

temperatures of the same clones on the

FW1.4gen framework, with 71.8, 75.8, 76.5

and 65.6 °C, respectively. In line with DSC

data, again, Tm was lower for the respective

optimized clones on FW1.4 (figure 2 and

table 6).

578-FW1.4, 578-FW1.4opt and 578-FW1.4gen

were compared in a stability study under

accelerated conditions. No degradation of

the molecules was observed after

incubation at 40°C for 2 weeks at a

concentration of 60 mg/ml as assessed by

SDS-PAGE (data not shown). Aggregation

was monitored using SE-HPLC. All scFv

samples showed a main peak corresponding

to the expected monomer of the scFv that

eluted from the column after ∼9.2 minutes.

The monomer content of the starting scFv

solutions was 98% for 578-FW1.4 and 578-

FW1.4opt, and 94% for 578-FW1.4gen.

Monomer loss in the 60 mg/ml samples

after 2 weeks incubation at 40°C was below

2% for 578-FW1.4 and 578-FW1.4gen and

at about 6% for 578-FW1.4opt. One

additional peak which could not be clearly

assigned to a defined molecular weight was

observed with 578-FW1.4opt indicating a

slightly lower stability for this variant when

compared to 578-FW1.4 and 578-

FW1.4gen (Fig 3).


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The results presented above demonstrate

that highly potent and highly stable

humanized scFv antibody fragments can be

generated in reproducible manner by

minimalistic grafting of CDRs from rabbit

monoclonal antibodies onto a single

optimized human acceptor framework.

4. DISCUSSION

Antibody fragments offer particular

advantages over full-size antibodies. Most

fragments can be produced in microbial

expression systems. Due to their low

molecular weight smaller fragments such as

single-chain Fv (scFv) antibody fragments

freely pass kidney filtration and are cleared

from the circulation with terminal half-lives

(t1/2) of a few hours, while t1/2 of full-size

antibodies ranges up to several weeks. In

contrast to IgGs, scFvs have excellent

tissue penetration properties and were

shown to efficiently penetrate into cartilage

and even across certain epithelial barriers

(1,2,32). Thus, from a pharmacokinetic

perspective, the scFv format meets

requirements for local and superficial

therapies to achieve high local

concentrations. Due to the short half-life in

the circulation, local application of scFvs

leads to low systemic exposures reducing

the risk for systemic side effects. Thus,

local therapies with scFv antibody

fragments represent a promising approach

to cope with side effects related to systemic

therapies with IgGs. As a consequence, a

superior efficacy/safety profile is expected.

However, in many cases scFvs do not

possess the required drug-like properties.

Low solubility and high aggregation rates

are considered major drawbacks of the scFv

format. Besides this, high affinity binding

of the progenitor variable domains is

required to compensate for the lack of

avidity of monovalent scFvs, a prerequisite

that is only rarely met with rodent

antibodies. Our results demonstrate that

humanization of rabbit variable domains by

simple grafting of antigen binding loops

onto the FW1.4gen scaffold reproducibly

results in humanized scFvs with drug-like

biophysical properties that bind with high

affinity to their targets.

The rabbit antibody repertoire represents an

attractive source for antibodies for several

reasons. First, rabbit antibodies mostly

show significantly higher affinities when

compared to rodent antibodies. Second,

generation of antibodies that are cross-

reactive towards mouse antigens is possible

in many cases. This is of particular interest

for the preclinical evaluation of therapeutic

antibodies in mouse models of human

diseases. Third, framework variability in

rabbits is very limited leading to the

assumption that a generic framework

suitable for generation of humanized scFvs

by simple grafting of CDRs could be

designed. Rabbit antibodies have been

humanized before. For example, Rader and

colleagues have applied phage display to

humanize rabbit antibodies (33,34). In their

work selection of successfully humanized

molecules was based on binding activity of

the Fab fragment presented on a phage.

Although biophysical properties of these

Fab fragments were not characterized in

detail, it is likely that the corresponding

scFv fragments would exhibit considerable

variability in terms of stability and

solubility, requiring further characterization

and possibly even engineering to identify

scFvs with drug-like properties. Moreover,

the use of in vitro display systems for the

humanization of larger numbers of variable

domains is time consuming; on one hand

due to the screening procedure and on the

other hand due to laborious characterization


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of the numerous positives resulting from

each individual progenitor antibody. In

contrast, the FW1.4gen framework offers a

technically simple humanization solution

that reproducibly results in humanized scFv

fragments with favorable biophysical

properties.

As an alternative to humanization of animal

antibodies, genetic libraries have been

generated in the past that contained diverse

sets of synthetic CDRs on drug-like human

scFv scaffolds (15). Although such libraries

have been applied to generate binders

against a variety of targets (35-37), this

approach is limited by the typical

shortcomings of in vitro display

technologies, such as a) potential loss of

weakly expressed high-affinity binders, due

to preferential selection of well expressed

molecules, and b) the need for purified

target molecules. Particularly for the

generation of antibodies against complex,

difficult to purify antigens, such as GPCRs

or ion channels, animal immunization may

still be advantageous. For example,

immunization with transfected host cells or

vaccination with cDNA allows to

specifically presenting integral membrane

proteins in their native conformation and

natural environment to the immune system.

In contrast to in vitro display systems, such

an approach does not co-select for

unspecific binding to other proteins on the

cellular surface. For these reasons, animal

immunization remains an important starting

point for the generation of antibodies.

In this study we demonstrated that a broad

spectrum of monoclonal antibodies derived

from rabbit immunization can be

successfully humanized and reformatted to

a scFv by simple transfer of antigen

binding loops to a human framework. This

framework was specifically selected and

optimized to a) provide a universal acceptor

scaffold for rabbit CDRs and b) confer

drug-like properties to the resulting scFv.

In a first stage, humanized scFvs were

generated from 15 independent rabbit

monoclonal antibodies directed against two

different protein targets, by exclusive

transplantation of CDRs onto the human

acceptor framework FW1.4. In a second,

optimized set, specific framework residues

were additionally substituted. These

residues were assumed to interact with the

antigen or to be involved in defining CDR

structures. While already the first set of

fragments showed relatively strong

interaction with the antigen, all but one

optimized scFv bound to their target with

very high affinities ranging from 4.7x10-9

M

to 1.5x10-11

M. More than 50% of them

displayed equilibrium dissociation

constants in the sub-nanomolar range.

These scFvs were well producible in a

generic production process and exhibited

excellent thermal stabilities.

A generically applicable acceptor

framework for rabbit CDRs, termed

FW1.4gen, was created by substituting a set

of five amino acids in FW1.4 with residues

that are conserved in rabbit heavy chains

(HT23, HG49, HT73, HV78, HR94).

Exclusive grafting of CDRs onto the

FW1.4gen-scaffold resulted in humanized

scFvs with binding strength similar to

optimized grafts, and superior biophysical

properties with apparent melting

temperatures between 69.2 and 85.6 °C in

DSC. Moreover, comparison of potencies

of these VEGF- and TNF-alpha inhibitory

antibody fragments to ranibizumab and

infliximab, showed that the VEGF-

inhibitory 578-FW1.4gen and the TNF-

inhibitors 34-FW1.4gen and 43-FW1.4gen

were as potent as the benchmark molecules.

Therefore, biochemical and biophysical


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properties of these scFvs demonstrate that

the method described here reproducibly

results in single-chain antibody fragments

that have the potential to be developed for

therapeutic applications.

Future perspective: More recent methods to

isolate cDNA sequences coding for target

specific monoclonal antibodies from animal

and human sources (e.g. B-cell isolation

techniques (38)) will possibly generate

significantly more hits as compared to the

hybridoma technique. Higher numbers of

clones would, in turn, increase the chance

to identify rare events, such as binders

against difficult targets, which would

broaden the application spectrum for

therapeutic antibodies. A challenge for

hybridoma independent methods, however,

is the reliable generation of sufficient

protein amounts to perform functional and

biophysical screenings, which is essential

for the identification of drug-like antibody

fragments. For this reason, methods that

allow fast cloning, reproducible production

and purification are required. The use of

generally applicable frameworks enabling

high throughput humanization of wild-type

scFvs, fast cloning, and generic production

of fragments with favorable biophysical

properties in microbial systems presents a

promising approach for the development of

future scFv based therapeutics

5. ACKNOWLEDGMENTS

The authors thank Raphael Berweger,

Daniela Binggeli, Nicole Germann, Juliane

Konrad, Anja Marold, Lea Noser, Monique

Oswald, Philip Richle, Viola Schlosser,

Nelly Schwer and Gwynneth Zimmermann

for their excellent technical assistance.


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6. References:

1. Furrer, E., Berdugo, M., Stella, C., Behar-Cohen, F., Gurny, R., Feige, U., Lichtlen, P., and Urech,

D. M. (2009) Investigative ophthalmology & visual science 50(2), 771-778

2. Ottiger, M., Thiel, M. A., Feige, U., Lichtlen, P., and Urech, D. M. (2009) Investigative

ophthalmology & visual science 50(2), 779-786

3. Wörn, A., and Plückthun, A. (2001) J. Mol. Biol. 305, 989-1010

4. Wörn, A., and Plückthun, A. (1998) Biochemistry 37(38), 13120-13127

5. Steipe, B. (2004) Methods in enzymology 388, 176-186

6. Steipe, B., Schiller, B., Plückthun, A., and Steinbacher, S. (1994) J. Mol. Biol. 240(3), 188-192

7. Ewert, S., Honegger, A., and Pluckthun, A. (2003) Biochemistry 42(6), 1517-1528

8. Monsellier, E., and Bedouelle, H. (2006) Journal of molecular biology 362(3), 580-593

9. Jermutus, L., Honegger, A., Schwesinger, F., Hanes, J., and Plückthun, A. (2001) Proc. Natl. Acad.

Sci. U.S.A. 98(1), 75-80

10. Jespers, L., Schon, O., Famm, K., and Winter, G. (2004) Nature biotechnology 22(9), 1161-1165

11. Jung, S., Honegger, A., and Pluckthun, A. (1999) Journal of molecular biology 294(1), 163-180

12. Proba, K., Worn, A., Honegger, A., and Pluckthun, A. (1998) Journal of molecular biology 275(2),

245-253

13. Demarest, S. J., Chen, G., Kimmel, B. E., Gustafson, D., Wu, J., Salbato, J., Poland, J., Elia, M., Tan,

X., Wong, K., Short, J., and Hansen, G. (2006) Protein Eng Des Sel 19(7), 325-336

14. Ewert, S., Honegger, A., and Pluckthun, A. (2004) Methods (San Diego, Calif 34(2), 184-199

15. Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wolle, J.,

Pluckthun, A., and Virnekas, B. (2000) Journal of molecular biology 296(1), 57-86

16. Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B., Henner, D., Wong, W. L., Rowland, A. M.,

Kotts, C., Carver, M. E., and Shepard, H. M. (1992) Proceedings of the National Academy of

Sciences of the United States of America 89(10), 4285-4289

17. Adair, J. R., Athwal, D. S., Bodmer, M. W., Bright, S. M., Collins, A. M., Pulito, V. L., Rao, P. E.,

Reedman, R., Rothermel, A. L., Xu, D., and et al. (1994) Hum Antibodies Hybridomas 5(1-2), 41-

47

18. Hurle, M. R., and Gross, M. (1994) Current opinion in biotechnology 5(4), 428-433

19. Presta, L. G., Chen, H., O'Connor, S. J., Chisholm, V., Meng, Y. G., Krummen, L., Winkler, M., and

Ferrara, N. (1997) Cancer research 57(20), 4593-4599

20. Kettleborough. (1991) Protein engineering, 773 - 783

21. Lanning, D., Zhu, X., Zhai, S. K., and Knight, K. L. (2000) Immunological reviews 175, 214-228

22. Auf der Maur, A., Tissot, K., and Barberis, A. (2004) Methods (San Diego, Calif 34(2), 215-224

23. Huang, Y., Gu, B., Wu, R., Zhang, J., Li, Y., and Zhang, M. (2007) Hybridoma (2005) 26(6), 387-391

24. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic acids research 22(22), 4673-4680

25. Kabat, E. A., and Wu, T. T. (1991) J Immunol 147(5), 1709-1719

26. Honegger, A., and Pluckthun, A. (2001) Journal of molecular biology 309(3), 657-670

27. Adair, J. R. (1992) Biotechnol Genet Eng Rev 10, 1-142

28. Eigenbrot, C., Randal, M., Presta, L., Carter, P., and Kossiakoff, A. A. (1993) Journal of molecular

biology 229(4), 969-995


ww

.jbc.org/D

ownloaded from

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19

29. Luo, G. X., Kohlstaedt, L. A., Charles, C. H., Gorfain, E., Morantte, I., Williams, J. H., and Fang, F.

(2003) Journal of immunological methods 275(1-2), 31-40

30. Chothia, C., and Lesk, A. M. (1987) Journal of molecular biology 196(4), 901-917

31. Willuda, J., Honegger, A., Waibel, R., Schubiger, P. A., Stahel, R., Zangemeister-Wittke, U., and

Pluckthun, A. (1999) Cancer research 59(22), 5758-5767

32. Urech, D. M., Feige, U., Ewert, S., Schlosser, V., Ottiger, M., Polzer, K., Schett, G., and Lichtlen, P.

(2009) Ann Rheum Dis

33. Rader, C., Ritter, G., Nathan, S., Elia, M., Gout, I., Jungbluth, A. A., Cohen, L. S., Welt, S., Old, L. J.,

and Barbas, C. F., 3rd. (2000) The Journal of biological chemistry 275(18), 13668-13676

34. Steinberger, P., Sutton, J. K., Rader, C., Elia, M., and Barbas, C. F., 3rd. (2000) The Journal of

biological chemistry 275(46), 36073-36078

35. Fuh, G., Wu, P., Liang, W. C., Ultsch, M., Lee, C. V., Moffat, B., and Wiesmann, C. (2005) The

Journal of biological chemistry 281(10), 6625-6631

36. Fellouse, F. A., Wiesmann, C., and Sidhu, S. S. (2004) Proceedings of the National Academy of

Sciences of the United States of America 101(34), 12467-12472

37. Sidhu, S. S., Li, B., Chen, Y., Fellouse, F. A., Eigenbrot, C., and Fuh, G. (2004) Journal of molecular

biology 338(2), 299-310

38. Kodituwakku, A. P., Jessup, C., Zola, H., and Roberton, D. M. (2003) Immunology and cell biology

81(3), 163-170


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7. FIGURES

Figure 1. Characterization of VEGF and TNF antagonists (A, B) Binding of unblocked

biotinylated human VEGF165 (hVEGF) to ELISA wells coated with VEGFR2/Fc was measured

in presence of a constant amount of biotinylated hVEGF (2.6 nM) and varying concentrations of

scFvs and ranibizumab. (C, D). Dose-dependent inhibition of hVEGF-induced human umbilical

vein endothelial cell (HUVEC) proliferation by ranibizumab and scFvs. Cells were incubated

with hVEGF (0.16 nM) and with increasing concentrations of scFv variants or ranibizumab.

Cell proliferation was measured and plotted as percentages of cells treated with VEGF alone.

(D, F) Neutralization of TNF-induced cytotoxicity by TNF antagonists. hTNF (19.16 pM) and

serial dilutions of scFvs or infliximab were premixed and L929 mouse fibroblast cell suspension

was added. Proliferation was expressed as percentages of untreated cells. Curves show four-

parameter fits to the data. All potency data were normalized to the standard. Triplicate data were

used and error bars represent standard deviations.


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E

A B

F

C D


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Figure. 2. Thermal stability of FW1.4gen and FW1.4opt constructs. Thermal denaturation

curves of 511 (A), 578 (C), 34(E) and 43(G) variants calculated from FTIR spectra as a function

of temperature. DSC analysis of the same scFv humanized variants of 511 (B), 578 (D), 34(F)

and 43(H). The respective Tm of each curve for FW1.4opt variants (open circles) and equivalent

FW1.4gen (filled circles) are listed in table 6.


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C

E

G

D

F

H

A B


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Figure. 3. Stability study under accelerated conditions. SE-HPLC analysis before (dotted

line) and after (continuos line) 2 weeks incubation at 40°C and 60mg/ml of A) 578-FW1.4, B)

578-FW1.4opt and C) 578-FW1.4gen.

A

B

C


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8. TABLES:

Table 1. Pharmacodynamic and biophysical characterization of “minimalistic” and

“optimized” grafts. Affinities of anti-VEGF and anti-TNF scFvs were measured with

BIAcore using sensor chips with immobilized human VEGF165 or human TNF-alpha,

respectively. Potencies of VEGF antagonists were measured in the VEGFR2 blocking assay

while the ability of TNF antagonists to neutralize TNF-alpha induced apoptosis was assessed

in mouse L929 fibroblasts. Potencies of VEGF antagonists are compared to ranibizumab

(relative potency = EC50, ranibizumab/EC50, scFv) and potencies of TNF antagonists are compared

to infliximab (relative potency = EC50, infliximab/EC50, scFv). Thermostability measurements

were performed by DSC. Refolding yield of the respective scFvs is expressed as amount

(mg) of refolded protein obtained out of 1l refolding solution. NB denotes no binding

detected; ND denotes analysis not done.


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relative potency thermal stability refolding yield Germline

scFv kon (M-1

s-1

) koff (s-1

) KD (M) Tm by DSC (°C) (mg/l)IGKV-IGHV

genes

VEGF antagonists

375-FW1.4 NB NB NB ND ND 4

375-FW1.4opt 5.09x107

2.42x10-1

4.74x10-9

no complete inhibition 73.5/82.3 3.3

435-FW1.4 4.95x105

1.43x10-2

2.89x10-8

ND 61.1 5.5

435-FW1.4opt 1.13x106

1.04x10-4

9.22x10-11

1.1 62.4 17

509-FW1.4 3.52x106

1.08x10-2

3.06x10-9

ND 65.9 13.5

509-FW1.4opt 1.42x106

5.37x10-4

3.78x10-10

0.87 80.1 1.1

511-FW1.4 6.75x105

8.85x10-4

1.31x10-9

ND 77.7 13.5

511-FW1.4opt 5.32x105

1.12x10-4

2.11x10-10

0.83 86.9 9.2

534-FW1.4 ND ND ND ND ND 0

534-FW1.4opt 1.06x106

2.62x10-3

2.47x10-9

0.2 61.7 1.5

567-FW1.4 1.11x106

7.00x10-4

6.31x10-10

ND 58.5 13.4

567-FW1.4opt 1.17x106

1.67x10-4

1.43x10-10

1.84 ND 8.5

578-FW1.4 1.11x106

2.02x10-4

1.81x10-10

ND 69.6/77.3 1.8

578-FW1.4opt 1.58x106

3.76x10-5

2.37x10-11

1.63 77.8 8.5

TNF antagonists

1-FW1.4 NB NB NB no inhibition ND 2

1-FW1.4opt NB NB NB no inhibition ND 28.8

6-FW1.4 NB NB NB no inhibition ND 40

6-FW1.4opt 2.18x105

8.57x10-5

3.90x10-10

1.1 53.7 16.8

15-FW1.4 1.57x105

4.10x10-2

2.62x10-7

no inhibition 60.7 41.5

15-FW1.4opt 1.53x106

2.26x10-3

1.48x10-9

0.39 71.6 63.6

19-FW1.4opt 2.25x104

6.54x10-5

2.91x10-9

0.6 60.9 52 IGKV1S61

34-FW1.4 ND ND ND ND ND 0

34-FW1.4opt 9.79x105

1.46x10-5

1.49x10-11

5.6 78.1 3.9

35-FW1.4 5.84x105

3.01x10-4

5.16x10-10

no inhibition ND 1

35-FW1.4opt 7.72x105

1.50x10-4

1.94x10-10

5.2 52.3/67.5/75 0.7

42-FW1.4 1.42x105

8.35x10-3

5.87x10-8

no inhibition ND 2.8

42-FW1.4opt 1.21x105

4.19x10-4

3.46x10-9

no inhibition 64.2 54.3

43-FW1.4 6.47x104

1.76x10-3

2.71x10-8

no inhibition ND 30

43-FW1.4opt 2.12x105

2.66x10-5

1.25x10-10

4.2 63.7 20.1IGKV1S4

IGKV1S2

IGKV1S4

IGKV1S5

IGKV1S37

IGKV1S37

IGKV1S5

IGKV1S49

IGKV1S49

BIAcore determinations

IGKV1S49

IGKV1S36

IGKV1S37

IGKV1S49

IGKV1S37


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Table 2. Dissociation rate constants of parent and humanized scFvs. Kinetic measurements

of wild-type VEGF- and wild-type TNF-inhibitory scFvs were measured with BIAcore using

sensor chips with immobilized human VEGF165 and human TNF-alpha respectively. Dissociation

rate constants for optimized grafts are taken from table 1. Relative off-rates were calculated as

indicated in the table. NB denotes no binding detected; ND denotes analysis not done.


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scFv koff (s-1) koff (FW1.4opt)/koff (wt)

VEGF antagonists

375-wt 3.48x10-2 7

375-FW1.4opt 2.42x10-1 435-wt 6.38x10-5

1.6 435-FW1.4opt 1.04x10-4 509-wt 2.30x10-4

2.3 509-FW1.4opt 5.37x10-4 511-wt 4.78x10-5

2.3 511-FW1.4opt 1.12x10-4 534-wt 1.88x10-3

1.4 534-FW1.4opt 2.62x10-3 567-wt 8.58x10-5

1.9 567-FW1.4opt 1.67x10-4 578-wt 3.66x10-5

1 578-FW1.4opt 3.76x10-5

TNF antagonists

1-wt 2.26x10-4 ND

1-FW1.4opt NB 6-wt 2.21x10-5

3.9 6-FW1.4opt 8.57x10-5 15-wt <1x10-6

>2600 15-FW1.4opt 2.26x10-3 19-wt 6.04x10-5

1.1 19-FW1.4opt 6.54x10-5 34-wt <1x10-6

>14.6 34-FW1.4opt 1.46x10-5 35-wt ND

ND 35-FW1.4opt 1.50x10-4 42-wt 1.17x10-4

3.6 42-FW1.4opt 4.19x10-4 43-wt 3.59x10-6

7.4 43-FW1.4opt 2.66x10-5


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Table 3. Sequence alignment of humanized VEGF and TNF-alpha scFvs at regions possibly

influencing binding activity. Rabbit residues introduced in the acceptor framework FW1.4 are

in bold letters.


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Table 4. Binding affinities and thermal stabilities of domain shuffled variants of selected

VEGF and TNF antagonists. Domain shuffling experiments were performed by recombining

heavy chains and light chains of “minimalistic” and “optimized” grafts. Kinetic measurements

were done with BIAcore using sensor chips with immobilized human VEGF165 or human TNF-

alpha, respectively. Thermal stability was determined using DSC.

BIAcore determinations thermal stability

scFv kon (M-1 s-1) koff (s

-1) KD (M) Tm by DSC (°C)

VEGF antagonists

511-VL1.4-VH1.4opt 5.71x105 9.20x10-5 1.61x10-10 87.3

511-VL1.4opt-VH1.4 6.00x105 6.57x10-4 1.10x10-9 77.3

578-VL1.4-VH1.4opt 2.11x106 3.33x10-5 1.58x10-11 76.2

578-VL1.4opt-VH1.4 9.57x105 1.38x10-4 1.44x10-10 77.9

TNF antagonists

34-VL1.4-VH1.4opt 8.62x105 1.69x10-5 2.00x10-11 80

34-VL1.4opt-VH1.4 3.67x105 2.11x10-5 5.70x10-11 53.4/68.4/81

43-VL1.4-VH1.4opt 1.65x105 3.66x10-5 2.22x10-10 70.1

43-VL1.4opt-VH1.4 1.46x105 5.33x10-3 3.66x10-8 56.4


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Table 5. Analysis of amino acid frequencies at positions considered relevant for CDR

conformation in rabbit and mouse VH. Percentages reflect the amino acid frequency of

occurrence of 505 rabbit and 1478 mouse non redundant antibody sequences taken from the

“Kabat Database of Sequences of Proteins of Immunological Interest” as of April 2007. SI:

Simpson's index as measure of calculated diversity for each species. “% agree”: Percentage of

agreement; indicates the percentages of sequences in the Kabat database containing the

consensus residue for the position indicated above.Cons: Specifies the consensus residue.

PositionRabbit Mouse Rabbit Mouse Rabbit Mouse Rabbit Mouse Rabbit Mouse Rabbit Mouse Rabbit Mouse Rabbit Mouse Rabbit Mouse

D 0.0 0.0 0.2 0.1 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.7 0.0 0.0 0.2 0.5

E 0.0 1.2 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 0.0 0.0 0.0 0.2

K 22.4 54.8 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 11.3 7.1 0.6 38.3 0.0 0.1 1.4 2.7

R 0.0 0.4 0.0 0.0 0.6 0.1 0.0 0.0 0.0 0.0 81.9 32.4 0.0 0.8 0.0 0.1 92.1 82.5

H 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.2 0.2

T 77.0 12.5 0.0 10.3 0.2 0.3 0.8 7.7 0.0 0.1 0.2 0.5 77.1 30.8 0.4 0.7 0.8 3.7

S 0.0 4.7 0.0 0.9 0.2 12.3 63.9 0.0 0.0 0.1 6.0 2.4 1.8 0.5 0.0 0.6 3.7 2.8

N 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.2 0.0 0.0 0.0 5.0 22.4 0.0 0.0 0.0 0.1

Q 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.1 0.0 0.3

G 0.0 0.1 1.0 2.1 95.2 60.2 0.2 1.9 0.0 0.0 0.6 0.3 0.0 0.0 14.3 0.1 0.6 2.4

A 0.2 23.9 33.1 72.4 3.4 26.2 0.4 47.6 0.0 0.0 0.0 18.4 0.6 0.3 0.6 50.2 0.2 0.9

C 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0

P 0.2 0.1 0.0 0.8 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 1.0

V 0.0 2.1 65.3 10.9 0.2 0.6 0.2 1.2 0.8 6.0 0.0 35.7 0.4 0.1 80.5 11.6 0.2 0.1

I 0.2 0.0 0.0 0.3 0.0 0.0 0.4 3.5 84.6 37.8 0.0 0.4 14.3 1.3 0.0 0.4 0.0 1.2

L 0.0 0.1 0.4 0.1 0.0 0.1 0.4 7.9 14.3 46.4 0.0 2.8 0.0 0.0 3.2 24.9 0.2 0.7

M 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.1 1.0 0.0 0.0 0.1

F 0.0 0.0 0.0 2.3 0.0 0.1 33.1 30.3 0.2 7.9 0.0 0.0 0.0 0.0 0.0 3.0 0.0 0.3

Y 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 8.2 0.0 0.3

W 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.1

SI 0.6 0.4 0.5 0.5 0.9 0.4 0.5 0.3 0.7 0.4 0.7 0.3 0.6 0.3 0.7 0.3 0.8 0.7

% agree 77 55 65 72 95 60 64 48 85 46 82 36 77 38 81 50 92 83

Cons T K V A G G S A I L R R T K V A R R

73 78 9423 24 49 67 69 71


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Table 6. Pharmacodynamic and biophysical characterization of VEGF and TNF

antagonists grafted onto the FW1.4gen framework. Affinities of anti-VEGF and anti-TNF

scFvs were measured with BIAcore using sensor chips with immobilized human VEGF165 or

human TNF-alpha, respectively. Potencies of VEGF antagonists were measured in the VEGFR2

blocking assay while the ability of TNF antagonists to neutralize TNF-alpha induced apoptosis

was assessed in mouse L929 fibroblasts. Potencies of VEGF antagonists are compared to

ranibizumab (relative potency = EC50, ranibizumab/EC50, scFv) and potencies of TNF antagonists are

compared to infliximab (relative potency = EC50, infliximab/EC50, scFv). Thermostability

measurements were performed by DSC and FTIR. Refolding yield of the respective scFvs is

expressed as amount (mg) of refolded protein obtained out of 1l refolding solution.

relative potency refolding yield

scFv kon (M-1

s-1

) koff (s-1

) KD (M) Tm by DSC (°C) Tm by FTIR (°C) mg/l

VEGF antagonists

511-FW1.4gen 5.41x105

3.09x10-4

5.72x10-10 0.56 85.6 71.8 8

578-FW1.4gen 1.41x106

5.46x10-5

3.87x10-11 1.7 81.3 75.8 12.5

TNF antagonists

34-FW1.4gen 5.6x105

<1x10-6

<1.79x10-12 5.7 81.6 76.6 21

43-FW1.4gen 2.15x105

2.16x10-5

1.00x10-10 3.8 69.2 65.6 54.3

BIAcore determinations thermal stability


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Grimshaw and David Max UrechLeo Borras, Tea Gunde, Julia Tietz, Ulrich Bauer, Valerie Hulmann-Cottier, John P. A.

from rabbit monoclonal antibodiesA generic approach for the generation of stable humanized single-chain Fv fragments

published online January 7, 2010J. Biol. Chem.

10.1074/jbc.M109.072876Access the most updated version of this article at doi:

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a generic approach for the generation of stable humanized single … · fragments from rabbit...

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