Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
A POLYPEPTIDE STRUCTURE FOR USE AS A SCAFFOLD
FIELD OF THE INVENTION
The present invention relates to a polypeptide chain having a ~i sandwich
architecture which can be used as a scaffold, i.e. a supporting framework,
carrying
antigen- or receptor-binding fragments. More specifically, the present
invention relates
to a ~3 sandwich derived from the naturally occuring extracellular domain of
CTLA-4.
BACKGROUND OF THE INVENTION
The primary amino acid sequence of a protein determines its three-dimensional
(3D) structure, which in turn determines protein function. In most cases the
3D structure
is essentially uneffected by amino acids at loci where the amino acid side
chain is
directed towards the solvent. Loci where limited variety is allowed have the
side group
directed toward other parts of the protein. This general rule, however, does
not hold for
those proteins whose function consists of, or relies upon binding with other
biomolecules.
Binding of such proteins to other molecules is non-covalently but often very
tightly and
specifically. In such instance, the identity of residues of the interacting
domains that point
towards the solvent becomes very important as well as the 3D-structure that
they adopt,
because binding results from complementarity of the surfaces that come into
contact:
bumps fit into holes, dipoles align and hydrophobic residues contact other
hydrophobic
residues. Individual water molecules eventually can fill a well determined
space in
intermolecular interfaces, and then usually form hydrogen bonds to one or more
atoms
of the protein or other biomolecules or to other bound water.
The 3D structure can often be predicted on the basis of the primary amino acid
sequence. A major obstacle towards the correct prediction of the entire
protein structure
is the modelling of "loops". These are regions of the polypeptide chain that
have a lower
degree of structural and sequential regularity and where insertions and
deletions often
occur during evolution. Yet, this diversity does not fundamentally alter the
core structure
1
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
of the remaining polypeptide chain. Often such loops, which are on the outside
of the
protein structure, will determine the interaction with other biomolecules. The
conformation of loop fragments is largely controlled by the attachment of
their ends to
fragments that constitute the framework of the molecule, for instance
secondary structural
units. This is also the basic idea when grafting such polypeptide chains onto
the core of
the protein structure which functions as a scaffold. Scaffolds, which are able
to restrain
the 3D structure of a polypeptide chain within a loop, can thus be of utmost
importance.
Immunoglobulins are mufti domain proteins consisting of a heavy and a light
chain. The variable domain of immunoglobulins is composed of an antigen
binding site
formed by six loops clustered in space and carned by two domains that adopt a
so called-
(3 sandwich structure (one (3 domain from the heavy chain carrying 3 loops and
one from
the light chain carrying 3 loops) (Amit et al., 1986). The high sequence
variability of
these loops allows immunoglobulins to recognise a variety of antigens. Though
this
sequence variation does determine in part the structure of the loops and the
antigen
specificity, the gross structure of the immunoglobulin variable domain's
framework is
hereby not altered. Reversely, the latter (3-domains, consisting of anti-
parallel strands that
are connected by the loops, function as a scaffold that strongly determines
the three
dimensional structure of the loops by constraining their structure and
providing a carrier
function (Amzel,L.M. and Poljak,R.J. , 1979; Williams A.F.,1987). Furthermore,
the ~3-
sandwich architecture of both the heavy and light chains have a roughly half-
cylindrical
structure, with a more or less open side. In functional immunoglobulins, both
domains
(heavy and light chain) are packed onto eachother thereby forming a barrel-
like fold.
A serious drawback in using the immunoglobulin scaffold to restrain random
polypeptide sequences, however, is that the core (3-sandwich is composed of
two chains
(the heavy and light chains) which interact, thereby bringing the variable
loops of both
chains (6 in total) in close proximity. This complicates the use of
immunoglobulins for
restraining the conformation of randomized polypeptide sequences such as in
antibody
libraries on filamentous phage. Large peptide libraries on phage are described
in US
5223409 to Ladner et al. and US 5571698 to Ladner et al. In the latter system,
each
randomized peptide is fused to the gene III protein of the M13 phage. In this
regard, it is
2
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
clear that a large, mufti-domain protein is not ideal in phage-derived
selection of peptidic
leads. Consequently, efforts have been devoted to design novel, smaller and
simpler
proteins, based on the structure of the variable domain of an antibody. One of
these
efforts is the construction of a protein, named minibody, which consists of a
fusion of the
variable regions of an antibody and the CH3 domain of an immunoglobulin, all
in a
single polypeptide. Minibodies spontaneously dimerize to form an antibody
fragment that
is bivalent. The minibody scaffold, which contains a number of residue
modifications
with respect to the original immunoglobulin sequence, can be obtained both by
solid-
phase synthesis and by expression in bacteria (Bianchi et al, 1993; Bianchi et
al ,1994).
Further modifications of the latter molecule were successful in improving some
important
properties, for example its solubility in aqueous media (Bianchi et al ,1994).
Other
attempts to simplify the existing mufti domain immunoglobulin molecule have
resulted
in recombinant fragments, made of two forms: Fv fragments and Fab's. Fv
fragments are
heterodimers composed of a variable heavy chain (VH) and a variable light
chain (VL )
domain and are the smallest functional fragments of antibodies that maintain
the binding
and specificity of the whole antibody. In these constructs, the immunoglobulin
domains
forming the characteristic ~i-barrel can still interact with each other.
However, Fv
fragments are unstable. Stable Fv's have been produced by making recombinant
molecules in which the VH and VL domains are connected by a peptide linker so
that the
antigen-binding site is regenerated in a single molecule. These recombinant
molecules
are termed single chain Fv's (scFv's; Raag and Whitlow, 1995). The VH-VL
heterodimer
can alternatively be stabilized by an interchain disulfide bond and is then
termed dsFvs
(Reiter et al, 1994). Fab fragments, on the other hand, are composed of the
light chain and
the heavy chain Fd fragment (VH and CH1) connected to each other via the
interchain
disulfide bond between CL and CH1.
Taken together, each simplification of the otherwise complex multidomain
immunoglobulin molecule still results in a unit wherein two immunoglobulin
folds
interact with each other thereby reconstituting the antigen binding site which
consists of
two pairs of three loops each grafted onto a separate ~3-sandwich framework.
Another drawback for using immunoglobulins to restrain randomized polypeptide
3
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
sequences is the fact that the antigen-binding site of the latter molecules,
which is
constituted of six clustering loops, may not be able to bind small and/or
cryptic sites
within molecules. In other words, steric hindrance during the screening or
selection for
proteins that bind small and/or cryptic sites may occur when using an
immunoglobulin
framework. Solutions to the latter problem have been offered by restraining
randomized
polypeptide sequences of variable length by means of cyclization upon for
instance
incorporation of cysteine residues at fixed positions forming a disulfide
bond, or, by
means of incorporating a metal binding motif or a conserved hydrophobic core
into the
polypeptide, or, by means of embedding peptide segments in "small proteins"
with a
defined 3D structure such as endothelin, trefoil proteins, guanylin and the
like (Ladner,
1995; Cannon et al. 1996). While these solutions have been very elegant, they
result in
small peptides which may rather quickly be cleared from the bloodstream, thus
posing
problems for certain therapeutic purposes.
The present invention relates to an alternative, simpler version of a
monomeric
polypeptide, compared to antibody-derived and small protein scaffolds, which
can, to our
surprise, efficiently be used as a scaffold.
AIMS OF THE INVENTION
The more tightly a polypeptide segment is constrained, the more likely this
segment will bind to its target with high specificity and affinity (Ladner,
1995). In other
words, there is a need to design scaffolds which are able to carry, or are
embedded with,
polypeptides efficiently binding to an antigen or receptor. Already existing
scaffolds,
such as antibody-derived scaffolds and "small proteins", are still faced with
particular
limitations. Indeed, antibody-derived scaffolds containing 6 loops
constituting the
antigen-binding site, are composed of rather large and complex dimers which,
due to
steric hindrance, may not be usefull for carrying polypeptides binding small
and/or
cryptic sites. Furthermore, large molecules containing an antigen binding site
composed
of six loops have a relatively higher chance to bind in a non-specific manner,
or to cross-
react, compared to smaller and simpler molecules containing an antigen binding
site
4
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
composed of less than six loops (Maclennan, 1995). Moreover, antibody-derived
scaffolds have the tendency to denature more easily during the aggressive
purification
protocols required for biotherapeutic production (Maclennan, 1995). "Small
proteins",
on the other hand, may be quickly cleared from the bloodstream which is not
desirable
for certain therapeutic purposes. Furthermore, both antibody-derived -and
"small protein"
scaffolds, which are used to generate large peptide libraries using phage-
display
technology, are limited by the fact that phage display is not able to display
active forms
of proteins that require eukaryotic-specific posttranslational modifications
(such ~as
glycosylation) for activity (Cannon et al., 1996). Also Metzler et al. (1997)
indicated that
glycosylation is important for binding activity, structural integrity and
solubility of
CTLA-4.
In order to overcome the above-indicated limitations, it is an aim of the
present
invention to provide an alternative, simplier and preferably unglycosylated
scaffold which
is not easily cleared from the blood stream.
It is also an aim of the present invention to provide a scaffold structure
which is
sufficiently stable to allow grafting of polypeptide chains as loops onto said
scaffold
without substantial alteration of the scaffold structure.
It is further an aim of the present invention to provide a scaffold structure
that can
restrain randomized polypeptide sequences thereby determining the 3D-structure
of said
polypeptide sequences.
It is also an aim of the present invention to provide a scaffold structure
wherein
at least one, preferably two and more preferably three of the restrained
polypeptide loops
are constituting a binding domain for other molecules.
It is also an aim of the present invention to provide a scaffold structure
wherein
at least two, preferably three, more preferably four, more preferably five and
even more
preferably six of the restrained polypeptide loops are constituting at least
two binding
domains for other molecules. This aim more particularly relates to providing a
scaffold
structure that allows for the design of bispecific molecules wherein both
binding domains
are located on the opposite sides of the molecule.
It is also an aim of the present invention to provide a scaffold structure
which is
5
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
soluble under physiological conditions.
It is also an aim of the present invention to provide an unglycosylated
scaffold
structure that remains soluble and/or functional in a bacterial background. In
this regard,
it should be clear, in view of the prior art ( i.e. see Cannon et al., 1996,
and, Metzler et
al., 1997) which indicates that unglycosylated molecules may aggregate and/or
may have
a reduced bioactivity, that a person skilled in the art is guided away to use
an
unglycosylated scaffold.
It is also an aim of the present invention to provide a scaffold structure
that-is
essentially functional as a monomeric protein.
All these aims are met by the following embodiments of the present invention.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows a schematic representation of the scaffold of the present
invention. The
six basic ~i-strands are named S l, S2, S3, S4, SS and S6; the three
additional or optional
~3-strands are named A1, A2 and A3. The loops connecting the strands are
indicated by
a thin, black, curving line.
Figure 2 shows the sequence coding for SCA, which is based on the
extracellular part
of human CTLA-4 gene as described by Metzler et al. (1997) but without the C-
terminal
cysteine residue (Cys 123). (3-sheets are underlined, Cysteine residues and
glycosylation
sites are shown. Mutations and there effect on CD80/86 binding are also
indicated.
Figure 3 indicates the specific primers used to clone SCA as a ApaLllNotl
fragment.
Figure 4 shows a shematic representation of the Phagemid ACES 1 construct.
Antibody
genes : VL CL, variable (V) and constant (C) region of the light chain; VH-
CH,, variable
and first constant region of the heavy chain; PlacZ, promoter; rbs, ribosome
binding site;
S, signal sequence; H6, six histidines stretch for IMAC purification; tag, c-
myc-derived
tag; amber, amber codon that allows production of soluble Fab fragments in non-
6
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
suppressor strains, gllI, gene encoding one of the minor coat proteins of
filamentous
phage. Restriction sites used for cloning are indicated.
Figure 5 demonstrates the expected patterns of the BstNl fingerprints of
clones with the
correct sized insert.
Figure 6 demonstrates a strong signal showing binding to both B7.1-Ig and B7.2-
Ig and
not to BSA or plastic. The latter signal also decreases proportionately with
the amount
of phage present.
Figure 7 shows an SDS-page analysis with DTT (lanes 1 and 3) or without DTT
(lanes
2 and 4) followed by detection with anti-CTLA-4 (lanes 1 and 2) or with anti-
myc (lanes
3 and 4). which demonstrates that part of the expressed 16 kD SCA protein is
present in
the medium. The 83 kD band is probably due to a fusion of the SCA protein with
the
gene 3 coat protein of the phage.
Figure 8 indicates the specific primers used for amplification (oligol and
oligo 2) and
reamplification (oligo 1 and oligo 3) of the SCA.
Figure 9 schematic representation of the cloning strategy as described in
example 6.
Figure l0a Diagrammic presentation of selection of phage on av(33 integrin
using a
polyclonal phage ELISA (see also figure lOb for the enrichment factor of the
several
selection rounds).
Figure 11 Diagrammic presentation of phage ELISA of selected clones on av~33
integrin
Figure 12 Alignment of the wild type CTLA-4 CDR3 loop and border sequences
with
the peptides obtained after selection and showing the RGD sequence and its
selected
borders
7
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
Figure 13 Diagrammic presentation of soluble ELISA of selected RGD clones on
av(33
integrin
Figure 14 Diagrammic presentation of binding of anti CTLA-4 antibodies with
the
$ scaffold derivatives
Figure 15 Facs analysis of binding to HLTVECS cells
Figure 16 Diagrammic presentation of phage ELISA of scaffold derivatives with
altered
sequences in loops A1/S1 and S2/A2
DETAILED DESCRIPTION OF THE INVENTION
The invention described herein draws on previously published work and pending
patent applications. By way of example, such work consists of scientific
papers, patents
or pending patent applications. All these publications and applications, cited
previously
or below are hereby incorporated by reference.
The present invention relates to a scaffold composed of a single-chain
polypeptide having the following structural properties:
- it contains at least two cysteine residues which form at least one
disulphide
bond, and
- it possesses less than 10°Io of alpha helical conformation, and
- it contains at least six ~3-strands (S1, S2, S3, S4, S5, S6) which:
- are connected by amino acid loops of variable conformation and lenght
according to the following topology: S1-S2-S3-S4-SS-S6, and wherein at
least one of said loops is different for at least three contiguous amino acid
residues by way of substitution, deletion or insertion when compared to
the naturally occurring CTLA-4 molecule,
8
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
- form two (3-sheets (one formed by S 1/S4/S3 and one formed by
S6/S5/S2 wherein the symbol "/" denotes the hydrogen bonding
interactions between two spatially adjacent (3 strands) which are each
characterized by an anti-parallel arrangement of said ~i strands and which
are packed onto eachother so that they form a ~i sandwich architecture.
Furthermore, the present invention regards a scaffold as defined above which
may
contain a maximum of three additional (3 strands (A1, A2 and A3), and, which
has the
following topology: Al-S1-S2-A2-A3-S3-S4-S5-S6, and wherein at least one of
said
loops is different for at least three contiguous amino acid residues by way of
substitution,
deletion or insertion when compared to the naturally occurnng CTLA-4 molecule,
and,
which possesses the following ~i-sheets: A1/S1/S4/S3 and S6/S5/S2/A2/A3.
The present invention also relates to a scaffold as defined above wherein
-A1 is the amino acid sequence AQPAVVLA (SEQ lD 1) or any functionally
equivalent derivative of said sequence,
-S 1 is the amino acid sequence ASFPVEY (SEQ >D 2) or any functionally
equivalent derivative of said sequence,
-S2 is the amino acid sequence EVRVTVLRQA (SEQ >D 3) or any functionally
equivalent derivative of said sequence,
-A2 is the amino acid sequence QVTEVCAA (SEQ ID 4) or any functionally
equivalent derivative of said sequence,
-A3 is the amino acid sequence TYMMGNELTFLDDS (SED >D 5) or any
functionally equivalent derivative of said sequence,
-S3 is the amino acid sequence ICTGTSS (SEQ ID 6) or any functionally
equivalent derivative of said sequence,
-S4 is the amino acid sequence QVNLTIQ (SEQ ID 7) or any functionally
equivalent derivative of said sequence,
- S5 is the amino acid sequence GLYICKVE ( SEQ >D 8) or any functionally
equivalent derivative of said sequence,
- S6 is the amino acid sequence GIGNGTQIY (SEQ ID 9) or any functionally
equivalent derivative of said sequence.
9
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
The applicability of a scaffold lies in the ability to introduce diversity
without
destroying the tertiary structure of the protein fold and the ability to
recover binding
molecules from a diverse repertoire. The latter can be achieved by phage
display and
affinity selection on the ligand of choice. Although the properties of a
scaffold are largely
determined by the nature of the application, a list of important properties is
shown
below:
-the scaffold should be a small, globular protein,
-it should be a single domain (thus easier to produce, purify and engineer
into multivalent
or multispecific reagents),
-it should fold correctly and the tertiary structure should not be perturbed
by the
introduction of diversity,
-it should be stable, preferably in vivo if that is of relevance for the
chosen application,
-it should have permissive loops , patches and/or surfaces for introducing
diversity at a
number of chosen sites wherein the nature of the required binding surface
depends on the
application,
-it should have a large accessible binding surface which has the potential to
be affinity
matured,
-it should be engineerable to make monospecific/bispecific/trispecific or
multispecific
molecules -soluble and expressed well in e.g bacteria,
-it should allow fusion at the N-and/or C-terminus,
-it should should be preferably non-immunogenic and human if to be used
therapeutically
-and it should resistant to proteolysis.
A number of novel scaffolds have already been described. Perhaps the one in
the
most advanced state of development is the Z domain (Nord et al., 1997). Others
include,
the minibody (Pessi et al., 1993; Martin et al. 1994), tendamistat (McConnell
and Hoess.,
1995) , zinc finger (Choo et al., 1995; Hamers-Casterman et al., 1993),
cytochrome b56z
(Ku and Schultz, 1995), trypsin inhibitor (Rottgen and Collins, 1995),
synthetic coiled
coil (Houston et al., 1996), conotoxins, thioredoxin (Colas et al., 1996),
knottins (Smith
et al., 1998), green fluorescent protein (Abedi et al., 1998), and fibronectin
(Koide et al.,
1998). For the generation of binding ligands there is probably no ideal
scaffold for all
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
potential applications, the choice being governed by a changing number of the
parameters
cited above. It can be easily envisaged that the final application should have
the most
prominent role in this choice.
The disadvantages when antibodies are used as scaffolds are that:
-the binding surface is composed of two domains,
-the structure can not easily be inferred from the primary sequence,
-the construction of multimeric and multispecific reagents is complex and is
limited by
the two domain nature of an antibody molecule,
-the in vivo stability is not optimal,
-the performance of the antibody depends on the interactions between the light
and heavy
chains which cannot be predicted,
The disadvantages cited above are not encountered when the molecule of the
present
invention is used as a scaffold. Moreover, the molecule of the present
invention has, in
comparison to antibodies, the following advantages for use as a scaffold:
-it has monomeric nature which facilitates easier engineering of therapeutic
reagents and
allows engineering into a multivalent and/or multispecific molecule,
-it has improved stability and pharmacokinetics in comparison to recombinant
antibody
fragments due to the second non-Ig disulphide bridge,
-it has a binding domain consisting of at most three loops (which is half the
amount of
loops that constitute the binding domains of human antibodies), which enables
it to bind
to cryptic sites that are otherwise not accessible by antibodies. The latter
"smaller"
binding domain of the current invention also decreases the possibility of
binding to other
sites thereby decreasing the possibility of aspecific binding, and in addition
it also helps
in the design of small molecule mimetics, which is not the case for
antibodies.
-it allows for replacement of loops on the other side of the molecule (the
opposite site of
the (3-sandwich of where the CDR loops are located) allowing an easy design of
bispecific monomeric molecules.
-Furthermore, analogous to antibodies, the basic design of the molecule of the
present
invention is of human origin (the scaffold of the present invention can be
considered to
be derived from the naturally occurring extracellular domain of the molecule
CTLA-4),
11
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
so that therapeutic applications can be envisaged, reducing the risk of
immunogenicity.
-The molecule of the present invention also has been shown to function as a Fc
molecule
which suggests it can be engineered into a dimeric molecule that can recruit
effector
function,
-Moreover, the molecule of the present invention is a very small molecule when
compared to the bispecific molecules that have been described in the art and
that have
been designed to bind two different molecules or substances. This small size
results in
the advantage that if the molecule of the current invention is used to design
a bispecific
molecule that the two sites to which this molecule will bind will be brought
into a very
close proximity which has advantages in for instance drug targetting.
It should be clear that the scaffold of the present invention can be prepared
by any
method known in the art such as classical chemical synthesis, as described by
Houbenweyl (1974) and Atherton & Shepard (1989), or by means of recombinant
DNA
techniques as described by Maniatis et al. (1982).
The term "alpha helical conformation" refers to the conformation of a
polypeptide
segment where the main chain adopts a fragment of a right-handed helix (phi-
and psi-
angles both near 60°), wherein all of the C=O and N-H groups of the
peptide linkages lay
roughly parallel to the axis of the helix, each carbonyl group being hydrogen
bonded to
the fourth N-H group on up the chain. The number of amino acid per turn of the
helix is
3.61. The pitch (repeat distance) of the helix is 0.541 nm.
The term "beta ((3) strand" refers to the conformation of a polypeptide
segment
where the main chain adopts an extended conformation (phi- and psi-angles
around -120°
and +120°, respectively) leading to a relatively linear structure. A
beta strand can be
interrupted by one or two non-extended residues (for instance a beta bulge),
while still
be considered as a single strand, as long as the principal axes of the two
consecutive sub-
fragments do not form an angle greater than 90°.
The term "beta ((3) sandwich architecture" refers to two beta-sheets that pack
against eachother and in which the strands of both sheets form a so-called
relative 'twist
angle' of around 30° (within the range 20-50°).
The terms "any functionally equivalent derivative of said sequences" refer to
any
12
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
variant or fragment of the amino acid (aa) sequences represented by SEQ ID 1
to 9 which
does not result in a significant alteration of the global structural and
functional properties
of the scaffold of the present invention. The latter terms include, post-
translational
modifications of the as sequences represented by SEQ ID 1 to 9 such as
glycosylation,
acetylation, phosphorylation, modifications with fatty acids and the like.
Included within
the definition are, for example, amino acid sequences containing one or more
analogues
of an as (including unnatural aa's), amino acid sequences with substituted
linkages,
mutated versions of the amino acid sequences, peptides containing disulfide
bonds
between cysteine residues, biotinylated amino acid sequences as well as other
modifications known in the art. Included within the definition are also those
single amino
acid substitutions which can further improve the solubility of the
unglycosylated protein.
Examples of equivalent derivatives, which are not intended to limit the scope
of the
present invention but are purely illustrative, are:
-for Al, the amino acid sequences TQPSVVLA (SEQ ID 10), TQPPVVLA (SEQ ID 11),
SQPAVVLA (SEQ 1D 12), KQSPLLVVD (SEQ ID 13), KQSPMLVVN (SEQ 1D 14),
KQSPMLVAY (SEQ 1D 15), KQLPRLVVY (SEQ ID 16), AQRPLLIVA (SEQ ID 17),
TQSPAIMSA (SEQ ID 18), QESGPGLV (SEQ ID 19) and the like,
-for S1, the amino acid sequences ASFPCEY (SEQ ll~ 20), ASFSCEY (SEQ ID 21),
VSLSCRY (SEQ ID 22), VNLSCKY (SEQ ID 23), ATLVCNY (SEQ ID 24, VTMTCSA
(SEQ ID 25), VTMTCKS (SEQ ID 26), VTITCKA (SEQ ID 27), VTMSCKS (SEQ ID
28), MKLSCVA (SEQ ID 29), LRLSCAT (SEQ ID 30), LKLSCAA (SEQ ID 31),
RKLSCAA (SEQ ID 32), LSTTCTV (SEQ ID 33), VKLSCTA (SEQ ID 34), VKISCKA
(SEQ ID 35), LSITCTV (SEQ ID 36), VKISCKA (SEQ ID 37), VQISCKA (SEQ ID 38)
and the like,
-for S2, the amino acid sequences EVRVTVLRQT (SEQ ID 39), EVRVTVLREA (SEQ
ID 40), EFRASLYKGV (SEQ ID 41), EFRASLYKGA (SEQ ID 42), EFRASLHKGL
(SEQ ID 43), EFRASLHKGT (SEQ ID 44), EVRMYMYQQK (SEQ ID 45),
KNYLTWYQQK (SEQ ID 46), EVRVVWYQQK (SEQ ID 47), NFRLAWYQQK (SEQ
ID 48), KNFLAWYQQK (SEQ ID 49), EVRMSWVRQS (SEQ 117 50),
EVRMEWVRQP (SEQ 1D 51), EVRMSWVRHT (SEQ m 52), EVRMHWVRQA (SEQ
13
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
ID 53), EVRVNWVRQP (SEQ ID 54), E'RMNWVKQR (SEQ ID 55), EVRIEWVKQR
(SEQ ID 56), EVRVHWVRQS (SEQ >D 57), EVRIEWVKER (SEQ ID 58) and the like,
-for A2, the amino acid sequences QVTEVCAT (SEQ ID 59), QMTEVCAT (SEQ ID
60), QVTEVCAG (SEQ ID 61), QMTEVCAM (SEQ >D 62), SDVEVCVG (SEQ ID 63),
SAVEVCVV (SEQ ID 64), SAVEVCAV (SEQ ID 65), SAVEVCFI (SEQ >D 66),
SPRLLIYD (SEQ >D 67), SPKLLIYW (SEQ >D 68), PPKLLIYG (SEQ ID 69),
GLEWVAEI (SEQ ID 70), RLEWIAAS (SEQ ID 71), RLEWVATI (SEQ >D 72),
GLEWVAYI (SEQ 117 73), GLEWLGMI (SEQ ID 74), GLEWIGRI (SEQ >D 75),
GLEWIGWI (SEQ ID 76), GLEWLGMI (SEQ ID 77), GLEWIGEI (SEQ ID 78),
GLEWIGEI (SEQ ID 79) and the like,
-for A3, the amino acid sequences TFTVKNTLGFLDDP (SEQ >D 80),
TFTEKNTVGFLDYP (SEQ ID 81), TYMVEDELTFLDDS (SEQ >D 82),
TYTVENELTFIDDS (SEQ )D 83), NGNFTYQPQFRSNAEF (SEQ ID 84),
NGNFTYQPQFRPNVGF (SEQ >D 85), NGNFSHPHQFHSTTGF (SEQ 117 86),
NGNHSHPLQSHTNKEF (SEQ ID 87), YGNYSQQLQVYSKTGF (SEQ ID 88),
NGNYSHQPQFYSSTGF (SEQ >D 89), SWNMTHKINSNSNKEF (SEQ )D 90),
TSNLASGVPV (SEQ ID 91), ASTRESGVPD (SEQ ID 92), ASTRHIGVPD (SEQ ID
93), RLNSDNFATHYAESVKG (SEQ ID 94), RNKGNKYTTEYSASVKG (SEQ ID
95), SNGGGYTYYQDSVKG (SEQ ID 96), SSGSSTLHYADTVKG (SEQ >D 97),
WGDGNTDYNSALKS (SEQ ID 98), DPANGNIQYDPKFRG (SEQ >D 99),
YPGSGNTKYNEKFKG (SEQ >D 100), LPGSGSTNYNEKFKG (SEQ >D 101),
WGGGSIEYNPALKS (SEQ ID 102), LPGSGRTNYREKFKG (SEQ ID 103) and the
like,
for S3, the amino acid sequences FCSGTFN (SEQ >D 104), TCIGTSR (SEQ ID 105),
TCTGISH (SEQ >D 106), NCDGDFD (SEQ ID 107), NCDGNFD (SEQ >D 108),
NCDGKLG (SEQ >D 109), NCTVKVG (SEQ ID 110), NCDGKLG (SEQ ID 111),
DCDGKLG (SEQ ID 112), NCRGIHD (SEQ >D 113), RFSGSGS (SEQ >D 114),
RFTGSGS (SEQ ll~ 115), RFAGSGS (SEQ ll~ 116), KFIISRD (SEQ ID 117), RFIVSRD
(SEQ ID 118), RFTISRD (SEQ ID 119), RFTISRD (SEQ ID 120), RLSISFD (SEQ >D
121), KATITAD (SEQ ID 122), KATLTVD (SEQ ID 123), RLSISKD (SEQ ID 124),
14
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
KATFTAD (SEQ >D 125), and the like,
-for S4, the amino acid sequences RVNLTIQ (SEQ ID 126), KVNLTIQ (SEQ >D 127),
TVTFRLW (SEQ 1D 128), TVTFYLK (SEQ >D 129), TVTFYLQ (SEQ m 130),
SVTFYLQ (SEQ m 131), TVTFYLR (SEQ ID 132), KVIFNLW (SEQ ID 133),
SYSLTIS (SEQ >D 134), DFTLSIS (SEQ >D 135), DYTLTIS (SEQ >D 136), DFTLTIS
(SEQ >D 137), RLYLQMN (SEQ )D 138), ILYLQMN (SEQ >D 139), TLFLEMT (SEQ
>D 140), TLFLQMT (SEQ ID 141), QVFLKMN (SEQ ID 142), TAYLQL (SEQ ID 143),
TAYMQLS (SEQ )D 144), QVFLKMN (SEQ ID 145), TATMQLS (SEQ ID 146),
QIFLKMN (SEQ ID 147) and the like,
-for S5, the amino acid sequences GLYFCKVE (SEQ >D 148), GLYLCKVE (SEQ ID
149), GLYVCKVE (SEQ ID 150), DIYFCKIE (SEQ >D 151), DIYFCLKE (SEQ ID
152), ATYYCQQW (SEQ >D 153), AVYYCQNN (SEQ ID 154), ALYYCQQH (SEQ
ID 155), AVYVCQND (SEQ ID 156), GIYYCVLR (SEQ ID 157), AIYYCARN (SEQ
ID 158), GLYYCARR (SEQ >D 159), GMYYCARW (SEQ >D 160), ARYYCARE (SEQ
ID 161), AVYYCATK (SEQ ID 162), AVYFCARG (SEQ ID 163), AVYYCARH (SEQ
ID 164), AXYYCVSY (SEQ ID 165), AVYVCTRG (SEQ ID 166) and the like,
-for S6, the amino acid sequence GMGNGTQIY (SEQ ID 167), ERSNGTIIH (SEQ ID
168), EKSNGTII>=I (SEQ ID 169), EKSNGTVIH (SEQ ID 170), TFGVGTKLE (SEQ ID
171), TFGAGTKLE (SEQ ID 172), TFGGGTKLE (SEQ ID 173), YWGQGTSVT (SEQ
ID 174), VWGAGTTVT (SEQ ID 175), YWGRGTLVT (SEQ ID 176), YWGRGTLVT
(SEQ ID 177), YWGQGTTLT (SEQ ID 178), YWGQGTTLT (SEQ >D 179),
YWGQGTLVT (SEQ ID 180) and the like.
It should be clear that the scaffold of the present invention contains at
least six ~3-
strands (S1 to S6) and may, but does not have to, comprise one (A1 or A2 or
A3),
preferably two (A1 and A2, or, Al and A3, or A2 and A3), and most preferably
three (A1
and A2 and A3) additional ~i-strands (A1 to A3).
The terms "amino acid loops of variable conformation and length" refer to any
as sequence of any lenght and any conformation which is able to connect the ~i-
strands
of the current invention. The latter as loops preferably contain at least one
as fragment
which binds to a receptor or antigen (see further). In this regard, it should
be clear that
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
not all loops need to contain (a) receptor/antigen binding fragment (s).
Indeed, one out
of the 6 loops may contain an above-indicated fragment; preferably, two out of
the 6
loops contain an above-indicated fragment and more preferably, three out of
the 6 loops
contain an above-indicated fragment. However, also four, five and six out of
the 6 loops
may contain an above-indicated fragment. In the latter regard, the scaffold of
the present
invention may contain sets of fragments which are able to bind to more than
one,
preferably two, different receptors/antigens. For example, the loops which
connect S5 and
S6, S 1 and S2, S3 and S4, and, A2 and A3 contain fragments binding to a tumor
antigen
or B7.1B7.2 expressing cells and the loops connecting A3 and S3, S2 and A2, S4
and
S5, and, A1 and B contain fragments binding to a toxin able to kill the tumor
cell
expressing said tumor antigen or said B7.1B7.2 expressing cells in a manner
similar as
the one described in W091/07437 to Pfreundschuh and W096/40260 to De Boer & De
Gast, respectively. Other specific examples of such amino acid loops, which
are not
intended to limit the scope of the present invention but are purely
illustrative, are:
- loops connecting said (3-strands A1 and S1: SSHGV (SEQ ID 181), SSRGI (SEQ
ID
182), SSRGV (SEQ ID 183), SSRGV (SEQ ID 184), SNE (SEQ ID 185), NNE (SEQ ID
186), DNE (SEQ ID 187), DNA (SEQ ID 188), SPGEK (SEQ >D 189), QPGGS (SEQ
ID 190) and the like,
- loops connecting said (3-strands S 1 and S2: SPSI~1TD (SEQ ID 191), ASSI~1TD
(SEQ
ID 192), ASPGKAT (SEQ ID 193), ESSGKAD (SEQ >D 194), ASHGKAT (SEQ ID
195), SYNLLAK (SEQ >D 196), TYNLFSK (SEQ ID 197), SYNLFSR (SEQ >D 198),
TYNGTGK (SEQ ID 199), and the like,
- loops connecting said ~i-strands S2 and A2: ND (SEQ ID 200), DS (SEQ ID
201), GS
(SEQ ID 202), NS (SEQ ID 203), PGS (SEQ ID 204), PGQ (SEQ ID 205), PEK (SEQ
ID 206), PGK (SEQ JD 207), PDK (SEQ ID 208), and the like,
- loops connecting said (3-strands S3 and S4: ES (SEQ ID 209), GN (SEQ ID
210), NE
(SEQ >D 211), KD (SEQ >17 212), GT (SEQ ID 213), DSKS (SEQ ID 214), TSQS (SEQ
ID 215), NAKN (SEQ ID 216), NPKN (SEQ ID 217), and the like,
- loops connecting said ~3-strands S4 and S5: GLRAVDT (SEQ ID 218), GLRAADT
(SEQ ID 219), GLRAMDT (SEQ ID 220), GLSAMDT (SEQ >D 221), NLHVNHT (SEQ
16
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
>D 222), NLDVNHT (SEQ )D 223), NLYVNQT (SEQ ID 224), DLYVNQT (SEQ ID
225), NLFVNQT (SEQ 1D 226), NMSASQT (SEQ ID 227), and the like,
- loops connecting said ~3-strands S5 and S6: LMYPPPYFV (SEQ >D 228),
LMYPPPYYL (SEQ ID 229), LMYPPPYYV (SEQ ID 230), FMYPPPYLDN (SEQ 117
231), VMYPPPYLDN (SEQ fD 232), VLYPPPYIDN (SEQ ID 233), VMYPPPYIGN
(SEQ ID 234), AMYPPPYVYN (SEQ ID 235), and the like.
The terms "homologous" and "homology" are used in the current invention as
synonyms for "identical" and "identity"; this means that the amino acid
sequences which
are e.g. said to be 95 % homologous show 95 % identical amino acids in the
same
position upon alignment of the sequences.
It has to be understood that it is not an aim of the present invention to
provide
CTLA-4 molecules as such, nor molecules derived from CTLA-4 by means of single
amino acid substitutions with the intention to increase solubility or alter
the binding
affinity and/or binding specificity of a CTLA-4 molecule for B7-molecules, the
latter
being present on the outer surface of antigen presenting cells. The structure
as set out
above therefore should have less then 98% homology with the amino acid
sequence of
the naturally occurring CTLA-4-molecule, preferably less then 97%, more
preferably less
then 95% and even more preferably less then 90% homology with the amino acid
sequence of the naturally occurring CTLA-4-molecule. This is due to
alterations within
the loops which connect the several ~i-strands of the scaffold as set out
above, and does
not result from amino acid substitutions within the ~3-strands which could
increase
solubility or stability of the scaffold molecule without substantially
altering the concept
of the invention.
It is a preferred embodiment of the present invention that the amino acid
sequence
of at least one of the loops is different as compared to the amino acid
sequence of the
naturally occurnng CTLA-4 molecule, preferably two loops and even more
preferably
three loops. A loop is considered different if at least three, more preferably
four, and even
more preferably five contiguous amino acid residues of the loop are absent
(deletion),
additionally present (insertion), or altered (substitution) when compared to
the naturally
occurring CTLA-4 molecule. Preferably the loop which is different as compared
to the
17
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
naturally occurring CTLA-4 molecule is one of the loops which is not CDR1 nor
CDR2
nor CDR3 when one would take the naturally occurring CTLA-4 molecule as a
reference.
This preferred embodiment therefore refers to the replacement of at least one
of the loops
which are connecting the A1 and S 1, or S2 and A2, or A3 and S3, or S4 and S5
(3-strands,
or which are connecting the A1 and Sl, or S2 and S3, or S4 and S5 ~i-strands
of the
scaffold if the additional (3-strands A2 and A3 are ommitted. Replacement of
these loops
with randomized peptide sequences can not be considered to be analogous to the
replacement of loops in Ig-like proteins as described in the art. The molecule
of the
present invention is not only different, moreover, the loops being referred to
are located
on the other side of the molecule as the CDR1, CDR2 and CDR3 loops which can
be
related to the antigen determining region of Ig-like proteins.
In an even more preferred embodiment the loop which is different as compared
to the naturally occurring CTLA-4 molecule is one of the loops which is CDR1
or CDR2
or CDR3 when one would take CTLA-4 as a reference. This preferred embodiment
more
precisely refers to the replacement of at least one of the loops which are
connecting the
Sl and S2, or A2 and A3, or S3 and S4, or S5 and S6 ~3-strands, or which are
connecting
the Sl and S2, or S3 and S4, or S5 and S6 (3-strands of the scaffold if the
additional ~3-
strands A2 and A3 are ommitted. Such molecules therefore do not display
binding
affinity altogether for B7 molecules present on the surface of antigen
presenting cells.
The present invention further relates to a scaffold as defined above wherein
said
amino acid loops comprise fragments binding to a receptor or antigen.
The expression "fragments binding to a receptor or antigen" refers to any
possible
as sequence which is part of the said loops (i.e. said fragments comprise
maximally as
many aa's as the loop itself and comprise, more frequently, less aa's than the
loop
wherefrom they are derived) and which binds any receptor or antigen. In this
regard, it
should be clear that the scaffold of the present invention can carry any
randomized as
sequence.
The present invention concerns a single-chain polypeptide as defined above for
use as a scaffold. In other words, the scaffold of the present invention can
be used to
18
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
generate large peptide libraries which can be screened for peptides with
desired binding
characteristics as described in US 5223409 to Ladner et al. and US 5571698 to
Ladner
et al. In the two latter patents, a phage based system is used in which each
randomized
peptide is fused to the gene III protein of the M13 phage. However, also non-
phage based
systems such as screening peptides on polysomes or on the surface of E. coli
(Tuerk &
Gold, 1990, Science 249:505-510), or, using a system wherein each randomized
peptide
is fused to a DNA binding protein as described in US 5498530 to Schatz et al.
can be
used.
The scaffold of the present invention carrying carrying peptides selected from
randomized as sequences can be used for therapeutic, diagnostic, and related
purposes.
The man skilled in the art will appreciate that the targets or ligands bound
by the
recombinant proteins of the present invention can be proteins, nucleic acids,
lipids and
carbohydrates, or combinations thereof. It has to be understood that
'combinations
thereof ' refers to glycoproteins, lipoproteins, and the like but also refers
to dimeric
(homo- or heterodimeric) biomolecules that are bound to each other by non-
covalent
means. Therefore, the term 'antigen' as used throughout the specification, has
to be
interpreted in the broadest sense. Because one can use an artificial screening
system, the
man skilled in the art will also appreciate that recombinant proteins can also
be selected
or screened for binding to xenobiotic molecules, that would otherwise be
highly toxic for
a biological system.
It has to be understood that the invention also relates to those recombinant
proteins that are able to bind, as already indicated above, two entirely
unrelated
biomolecules or substances, by means of two binding surfaces, e.g. one surface
consisting of the loops connecting the ~3-strands on one side of the ~i-
sandwich and
comprising the CDR loops in the natural protein, and one surface consisting of
the loops
that connect the (3-strands on the other side of the ~3-sandwich. As such,
both binding
surfaces can operate independent from each other and selection or screening
for such
proteins can be achieved in two separate rounds:
- in the first round, the biomolecule that binds to one partner is retrieved
from a library
wherein the amino acid sequence of one set of loops has been randomized while
the other
19
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
set of loops is untouched e.g. as in the naturally occurring protein,
- in the second round, the biomolecule that binds to a second partner is
retrieved from a
library wherein the amino acid sequence of the other set of loops has been
randomized.
In the second round, the randomized library may already contain those loops
that bind for
the first partner or, alternatively, remain untouched as in the naturally
occurnng protein.
In the latter case, both sets of binding loops as retrieved which bind
separate partners
have to be recombined within a single scaffolding protein.The known and
potential
advantages of bispecific molecules is well known in the art. They provide key
tools-in
therapeutic and diagnostic procedures such as immunohistochemistry (see
example 11)
and enzyme immunoassays, radiotherapy and immunotherapy, drug targetting, and
for
redirecting biomolecules or certain cell types to new sites. One binding
partner can be
used to target for certain cell types such as cancer cells inducing
intracellular uptake,
while the other binding partner can be a component of a life sustaining
cellular
mechanism of which the function is inhibited upon binding. One of the binding
sites can
also be a cytotoxic molecule, thereby forcing uptake of the cytotoxic molecule
into the
targeted cells. This strategy can also be used to force uptake of endogeneous
molecules,
such as auto-immune antibodies, toward cell types, such as liver cells. One
binding
partner can be used to target for certain cell types, while the other binding
partner can be
used to target for other cell types thereby forcing both cell types to stay in
close
proximity, such as any lymphocytes with for instance lymphocytes of a
different type or
with for instance cancer cells. For many aspects the molecule of the current
invention has
the additional advantage that it is a much smaller molecule then the
bispecific antibodies
already described in the prior art. This results in a much closer contact
between for
instance cancer cells and a radioactive isotope, or between one cell type and
another, as
for instance will be the case if cytotoxic T lymphocytes are redirected
towards other cells
by the molecule of the present invention. In such case the molecule of the
present
invention has been provided with a binding domain specific for a site on the
targeted cells
and a second CD3 domain or a CD3 like domain that recruits and activates the
cytotoxic
T lymphocytes.
In another preferred embodiment one of the binding sites of the scaffold
itself can
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
also be used to function as a spacer or anchor in cases wherein immobilisation
of the
entire protein is desired, such as on membranes or on any carrier for
chromatographic
purposes, thereby allowing for a directed immobilisation. One of the loops can
for
instance comprise a repeat of Histidine residues or a His-tag thereby allowing
anchorage
or immobilisation to a carrier or membrane by means of a metal-chelate or by
means of
covalent linkage as for instance is described in patent application PCT/EP
98/03883. The
man skilled in the art will understand that it is not a prerequisite that the
two binding
domains bind to different molecules. Both binding domains might as well bind
identical
molecules, thereby providing for instance a device for use in agglutination
tests. One of
the binding domains can be selected for recognizing red blood cells, while the
other
domain can be selected for binding to disease-specific antibodies, thereby
providing a
device for agglutination tests. It has to be understood that whenever the term
'His-tag'
is used throughout the specification, a broad definition can be given to it,
meaning any
sequence that has an amount of histidine residues that is sufficiently high to
allow
appropriate application. In principle a 10 amino acids long sequence that has
two
histidine residues, and is repeated at least twice should suffice. More
typical, said 'His-
tag' contains at least three histidines within a stretch of 6 amino acids. The
term 'His-tag'
can also include any repetition of a histidine immediately followed by at
least one
residue. Preferably the term His-tag refers to at least three consecutive
histidine residues,
more preferably to at least four histidine residues, or even more preferably
to at least five
or at least six consecutive histidine residues.
In another preferred embodiment the present invention relates to the
introduction
of an RGD sequence, embedded within a randomized peptide sequence, within one
of the
loops of the scaffold, thereby making up a peptide library of RGD displaying
scaffold
molecules that can be selected for binding to integrin. Integrins are involved
in a number
of pathological processes that involve angiogenesis e.g thrombosis,
osteoporosis,
rheumatoid artheritus, diabetic retinopathy, cancer and atherosclerosis. av~33
integrins are
unique markers for activated angiogenic endothelium. As such they are ideal
targets for
antibody-based endothelial cell destruction. Specific targeting of the
integrins on the
tumor endothelial cell surface will have application as both imaging reagents,
as
21
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
antitumor reagents and targets for gene therapy vectors which can readily
access the
tumor via the tumor vasculature. A number of peptides which can specifically
target the
tumor endothelium have been discovered (for example RGD peptides which bind to
the
av(33 integrins). Previous studies have clearly shown that peptides containing
the RGD
motif can specifically target av(33 integrins (Pasqualini et al., 1995;
Pasqualini et al.,
1997). The specificity, affinity and pharmacokinetics of these peptide ligands
is
sub-optimal and would greatly benefit from being constrained and presented in
a larger
scaffold. High affinity binding should require the positioning of the RGD
motif at the
apex of an extended loop with flanking residues which conformationally
optimise the
display of the motif. The RGD binding motif has previously been introduced
into the
CDR3 loop of an antibody scaffold with demonstrated high affinity binding
(lOpM) for
av~33 integrin (Barbas et al., 1993)(Smith et al., 1994). Previous studies
have also
engineered Hirudisins as thrombin inhibitors with disintegrin activity by
introducing an
RGD sequence at the tip of the hirudins finger like structure. The
antiplatelet activity that
was observed was due to the introduced integrin directed RGD motif (Knapp et
al.,
1992). Due to the ubiquitous nature of integrins and related molecules, the
key to the
therapeutic potential of targeting integrins is to target them specifically.
By presenting the
RGD binding motif as a constrained specificity as part of a larger binder
surface will
allow the maturation of these molecules to have fine specificity to for
example av~33
integrin. By sequentially introducing diversity and selecting for binding
using phage
display for example such binding molecules may be further matured for affinity
and
subtype specificity.
The present invention also regards, as stated above, a scaffold as defined
above
wherein said single-chain polypeptide is unglycosylated. However, it has to be
understood that the scaffold of the present invention can also be
glycosylated. Though
one of the major advantages of the claimed scaffolds is the fact that they
remain
functional without glycosylation, allowing expression in bacterial systems,
the fact of
being unglycosylated is not a prerequisite for falling under the protected
matter. Once a
recombinant scaffold has been selected in a bacterial background for binding a
specific
molecule and thus as an unglycosylated protein, one can decide to glycosylate
the scaffold
22
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
by expression in a eukaryotic background. This can have distinct advantages
over the
non-glycosylated scaffold for therapeutic purposes.
The present invention will now be illustrated by reference to the following
examples which prove the concept of the invention and set forth particularly
advantageous embodiments. However, it should be noted that these embodiments
are
illustrative and can not be construed as to restrict the invention in any way.
EXAMPLES
Example 1: Cloning of a recombinant scaffold, named SCA, for display on the
surface of filamentous phage
The sequence coding for SCA, which is based on the extracellular part of human
CTLA-4 gene as described by Metzler et al. (1997) but without the C-terminal
cysteine
residue (Cys 123), is cloned into the phage display vector pCES-1 (figure 4).
The cysteine
residue is not included because it may present some problems with the correct
folding and
presentation of the SCA- p3 fusion on filamentous phage. This cloning creates
a
translational fusion of SCA to the N-terminus of p3 for display on filamentous
phage.
SCA is cloned as a ApaLllNotl fragment (neither of these restriction sites are
present
in human CTLA-4) using specific primers (figure 3). Human CTLA-4 is amplified
from
O.lng vector pBSK(+)hCTLA-4 using primers CTLA-4 (Front) and CTLA-4 (Back) at
IOpM, 10,1 PCR buffer plus magnesium at 2.5mM, 5~1 of dNTP's and 1~1 of taq
polymerise (Boerhinger Manheim EXPAND TM). Amplification is done with a hot
start
of 10 minutes at 94°C and then for 30 cycles lmin 94 ~, 1 min 50 ~ and
2 min 72 ~. The
correct sized PCR product is then purified (402bp) and digested with both
ApaLl and
Notl. Vector pCES 1 is also digested with ApaLl and Notl. Both digested vector
and
digested SCA- insert are purified. Ligation is performed overnight at
16°C and is
transformed into TG1. The BstNl fingerprints of clones with the correct sized
insert, as
assessed by PCR, demonstrate the expected patterns (Figure 5).
23
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
Example 2: Display of a recombinant, non-glycosylated and monomeric SCA-
protein on the surface of filamentous phage
Phage displaying non-glycosylated SCA proteins on their surface are prepared
as
follows. Clones 2, 4 and 5 from figure 4 are grown to an OD of 0.5 in the
presence of
ampicillin at 100~.g/ml and 2% glucose. Five mls of culture is infected with
helper phage
M13K07 at a multiplicity of infection of 20:1 and incubated at 37°C for
30 minutes.
Cells are resuspended in TY medium plus Amplicillin and Kanamycin (25~,g/ml)
and
grown overnight at 30°C. Phages are recovered by precipitation by PEG
and resuspended
in PBS. Because the SCA protein is derived from the human CTLA-4 protein
wherein
the original CDR loops are retained, a binding is expected with B7.1 and B7.2
proteins.
To demonstrate that such binding occurs, phages are titrated and a serial
dilution is made
to test for binding in ELISA to B7.1-Ig and B7.2-Ig. A strong signal is
obtained showing
binding to both B7.1-Ig and B7.2-Ig and not to BSA or plastic (Figure 6). The
latter
signal decreases proportionately with the amount of phage present. This
demonstrates that
un-glycosylated SCA has the proper conformation and remains functional as a
monomer.
There is no difference in binding behavior for B7-1 or B7-2 of SCA displaying
phage.
Example 3: Soluble SCA - His6/myc expressed in the non-suppressor strain
HB2151
The gene coding for SCA, preceded by the pelB signal sequence, is cloned into
the phage display vector pCES-1 (figure 4) under control of the IPTG inducible
lac-
promoter. The coding sequence is then fused C-terminally to a His6 followed
with a
myc-tag. An amber stopcodon is located at the junction between this coding
sequence and
the sequence coding for the phage coat protein gap. This allows one to choose
whether
the protein will be exposed on the surface of the viral coat through fusion
with the gap
coat protein, or expressed as a soluble protein , without the need for a
supplementary
cloning step. When a suppressor type of strain is transformed with this
construct,
translation will continue through the stopcodon resulting in a fused SCA-gap
protein
exposed on the surface of a tip of the phage. In a non-suppressor strain the
amber
stopcodon is recognized and translation stops. Subsequently, the His6- or myc-
tagged
24
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
protein is transported through the cell membrane and accumulates in the
periplasm. Non-
suppressor cells HB2151 are transformed with the His6/myc- tagged construct
and grown
in liquid LB medium with ampicilline for selection and 1 % glucose for
metabolic
repression of the lac-promoter at 28 °C until saturation is reached.
This saturated culture
is subsequently diluted 20 times in 20 ml LB+A+G and grown at 28 °C
until an OD600
is reached of 0.5 to 0.6, which takes about three hours. The glucose
containing medium
is then removed and replaced by LB+A and 0.1 mM IPT'G. The induced culture is
further
incubated for about 20 hours. The- next day the OD600 is measured and the
cells are
fractionated.
Example 4: Fractionation of transformed cells
To examine whether the expressed SCA protein is actually secreted and
accumulated in the periplasm, and possibly leaking out into the medium, a
periplasmic
fraction is prepared using a modified protocol as according to the osmotic
shock
procedure described by Neu and Heppel (1965).
Sixteen ml of cell culture is centrifuged en resuspended in 1 ml of icecold
TES-buffer
(200 mM Tris pHB, 20 % sucrose; 50 mM EDTA). The cells are then incubated for
10
min on ice and vortexed regularly. Subsequently, the mixture is centrifuged at
10,000 rpm
for 1.5 min and the supernatant is disgarded. The pellet is quickly taken up
in 1 ml ice
cold distilled water. After 10 min on ice and regular vortexing the mixture is
centrifuged
at 14,000 rpm for 2 min and the resulting supernatant is recuperated as a
periplasmic
fraction. This material was compared to pelleted cells and material leaking
into the
medium, by means of SDS-page and detection with anti-His or anti-myc. This
demonstrates that part of the expressed 16 kD SCA protein is indeed present in
the
periplasm and that part is leaking out into the medium (results not shown).
The material
present in the medium was dialysed against PBS and analyzed by means of SDS-
page
and detection with anti-CTLA4 or anti -myc (see figure 7) . The 16 kD SCA-
protein is
clearly present. The 83 kD band is probably due to a fusion of the SCA protein
with the
gene 3 coat protein of the phage.
25
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
Example 5: The soluble, bacterially expressed, non-glycosylated SCA-molecule
binds B7.1 and B7.2 on the membrane
The soluble SCA-molecule is tested for binding capacity to the human B7.1 and
B7.2 molecules. The EBV-transformed B cell line (RPMI 8866, 0.5-lx 105
cells/sample), which strongly expresses B7.1 and B7.2, is incubated for 20 min
at 4°C
with the soluble SCA-molecule. After washing twice in RPMI 1640 supplemented
with
10% FCS, the cells are incubated for another 20 min at 4°C with mouse
anti-cMyc mAb
( l p g/cell pellet) conjugated to biotine. After washing twice in RPMI 1640
supplemented
with 10% FCS, the cells are incubated for another 20 min at 4°C with
Streptavidin
conjugated to PE (Phycoerythrine). The cells are then washed twice in RPMI
1640 sup-
plemented with 10% FCS and finally suspended in PBS supplemented with 1 % BSA
and
0.1% NaN3 and analyzed with a FACScan flow cytometer (Becton Dickinson). The
specific binding of the SCA-molecule is expressed as the mean fluorescent
intensity in
arbitrary units. The results show that soluble, non-glycosylated SCA binds, in
a dose-
dependent way, to the RPMI 8866 cells which express B7.1 and B7.2 (results not
shown).
Example 6 : Construction of SCA- RGD repertoire for display on filamentous
phage
The well characterised RGD containing sequence that binds to integrins is
inserted such that it replaces the CDR3 loop of CTLA-4. Sequences are also
introduced
such that they replace the Al/Sl loop and the S2/A2 loops of CTLA-4 (see
example 16
and 17). These loops are situated at the side of the molecule opposite to the
CDR
sequences which mediate the binding of CTLA-4 to its natural ligands.
A fragment corresponding to CTLA-4 (Figure 9) is digested from pCES 1 as an
ApaLl
/Notl fragment. The template CTLA-4 DNA is then subjected to PCR with the
primers
oligo#1 and oligo#2 (Figure 8). The PCR product is then purified and
reamplified with
oligo#1 and oligo#3. Fragment DNA is recovered and digested with ApaLl and
Notl and
cloned into pCES 1 for phage display (Figure 9). In this way, a library of 1.3
x 10'
independent clones can be obtained.
Example 7 : Selection of repertoire on recombinant human av~i3 integrin
26
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
Three rounds of selection are performed on recombinant av(33 integrin coated
onto immunotubes at 10~g/ml (Figure 10 a and b). Polyclonal phage ELISA is
then
performed to monitor the selection using input phage for 1 2 and 3 rounds of
selection.
As can be seen initially the repertoire has reactivity to B7-lIg representing
the probable
presence of wild-type CTLA-4 in the repertoire. As the selection progresses
this
disappears and gives way to a positive signal on integrin (Figure 11).
Selected clones are tested for binding to antigen in phage ELISA as in (Marks
et al.,
1991). Phage ELISA is performed on av~33 integrin (lpg/ml), B7-lIg (lpg/ml)
and BSA
(l~g/ml). In all cases all clones are negative on B7-lIg and BSA. (Figure 10).
Examples 8 : Sequences of selected clones positive in phage ELISA
Positive clones are sequenced and some sequences are shown in (Figure 12) by
way of example. Also, another pointmutation is shown to be present in the CDR2
loop
if the CTLA-4 molecule is taken as a reference, further demonstrating another
permissive
site.
Example 9 : Binding of CTLA-4 RGD molecules in soluble ELISA
Soluble CTLA-4 RGD fragments are prepared as in Marks et al. (1991) and tested
for binding to av(33 integrin (l~g/ml) either as monomeric reagent or cross
linked via
9E10 to generate avidity. As can be seen, clones are positive in soluble ELISA
(Figure
13). 14 out of the 26 clones that are positive in phage ELISA (Figure 10) are
also positive
insoluble ELISA. A positive control is the mouse monoclonal antibody to av(33
integrin,
LM609 (Brooks et al., 1994). A negative control is a non binding clone as
identified by
phage ELISA.
27
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
Example 10 : ELISA with antibodies to CTLA-4
To test if the integrity of the immunoglobulin fold of the Sca molecule is
maintained after introduction of the RGD sequence into the CDR3, an ELISA can
be
performed using mouse polyclonal anti CTLA-4 serum and a monoclonal antibody
to
CTLA-4 (IGH310). This antibody is supposed to compete with B7-1B7-2 so it has
partial epitope overlap with the MYPPPY sequence of wild-type CTLA-4.
5 x 10" phage are coated directly in PBS overnight at 4~C and are challenged
with either
mouse polyclonal anti-CTLA-4 or mouse monoclonal anti-CTLA-4. As can be seen,
clones 7B, 8H and 11C and wild-type CTLA-4 are all positive with both
antibodies. The
signal obtained with the monoclonal antibody is greater than that obtained
with the
polyclonal serum. Furthermore the signal obtained with the RGD presenting
clones in
CDR3 is greater than that obtained with CTLA-4. This indicates that the
general structure
of SCA molecule remains intact after introduction of foreign sequences into
CDR3 due
to the positive signal obtained with the conformation dependant monoclonal
anti-CTLA-4. (Figure 14)
Example 11 : FACS analysis of binding to HLTVECS cells
Approximately 80,000 HUVECS cells/sample are cultured and are tested in FACS
for binding. As can be seen a positive signal is seen with the positive clone
. Whereas
with both empty pCES 1 vector and wild-type CTLA-4 phage no shift can be
observed.
(Figure 15). This example demonstrates that the selected RGD-containing
scaffolds not
only bind to integrin but also exhibit substantial specificity.
Example 12 : Construction of SCA-E
The gene coding for SCA, preceded by the pelB signal sequence and followed by
a combined His6/c-myc tag, is available in the phage display vector pCES
1CTLA4 (see
example 3). This vector is cut with NotI and ScaI and a 2183 by fragment is
isolated and
ligated with a 2654 by NotI/ScaI fragment originating from vector pCANTABSE
(Amersham Pharmacia, Uppsala, Sweden). The resulting vector pCESICTLA4E
differs
28
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
from the previously described vector pCESICTLA4 in that the CTLA4 molecule now
carries a C-terminal E-tag in stead of a double c-myc/His6 tag. The correct
sequence of
the vector is then verified by DNA sequencing.
Example 13 : Display of the recombinant SCA-E protein on the surface of
filamentous phage
Phage displaying SCA-E protein on their surface are prepared as described in
example 2 (Marks et al., 1991). To demonstrate binding of the SCA-E protein to
B7.lIg,
B7.2Ig and an anti E-tag antibody (Amersham Pharmacia, Uppsala, Sweden),
phages are
titrated and a serial dilution is made to test for binding in ELISA to these
molecules.
Strong signals are obtained showing binding to both B7.lIg and B7.2Ig and the
anti E-tag
antibody. This demonstrates that SCA-E has the proper conformation and remains
functional as a monomer.
Example 14 : Insertion of an integrin binding sequence (RGD) in the loop
formed
by ~3-sheets A1 and S1 in SCA-E
Using plasmid pCESICTLA4E (see example 3) as a template, the DNA coding
for the integrin binding sequence (RGD) is inserted in the loop formed by (3-
sheets A1
and S 1 using overlap extension PCR (Ho et al., 1989). In the A1/S 1 loop,
consisting of
residues SSRGI (see figure 2), the RGD sequence is inserted between the SS and
the RGI
residues. On both sides, the RGD sequence is flanked by a G4S linker sequence.
This
insertion can be accomplished by performing two PCR reactions using
pCESICTLA4E
plasmid DNA as a template. A first PCR, using primers Nos. 11209 and 11071,
amplifies the 5' end of the CTLA4E gene including the RGD sequence. The second
PCR,
using primers Nos. 11072 and 11210, amplifies the 3' end of the CTLA4E gene
including
the RGD sequence. Both PCR fragments are subsequently gelpurified and an
additional
PCR is performed on the mixture of both PCR fragments using primers Nos. 11209
and
11210. The resulting PCR fragment is gelpurified, cut with ApaLI and NotI, and
inserted
into the ApaLI/NotI opened vector pCES 1 CTLA4E. The resulting vector
pCESICTLA4E-Al/S1RGD is verified by DNA sequencing.
29
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
Primer sequences:
No. 11071 (SEQ ID 236):
5'-GTCACCACGAGAGCCACCGCCACCGCTGCTGGCCAGTACCACAG-3'
No. 11072 (SEQ >D 237):
5'-CGTGGTGACGGTGGCGGTGGCTCTCGAGGCATCGCCAGCTTTG-3'
No. 11209 (SEQ ID 238):
5'-GATTACGCCAAGCTTTGGAGC-3'
No. 11210 (SEQ 117 239):
5'-ATGCGGCCCCATTCAGATC-3'
Example 15 : Insertion of a His6 tag (HHHHHH) in the loop formed by ~3-sheets
A1
and S1 in SCA-E
Using plasmid pCESICTLA4E (see example 3) as a template, the DNA coding
for a His6 tag (H) is inserted in the loop formed by ~3-sheets A1 and S1 using
overlap extension PCR (Ho et al., 1989). In the Al/S1 loop, consisting of
residues
SSRGI (see figure 2), the HIsequence is inserted between the SS and the RGI
residues. On both sides, the ICI sequence is flanked by a G4S linker sequence.
This insertion can be accomplished by performing two PCR reactions using
pCES 1CTLA4E plasmid DNA as a template. A first PCR, using primers Nos. 11209
and
11069, amplifies the 5' end of the CTLA4E gene including the Isequence. The
second PCR, using primers Nos. 11070 and 11210, amplifies the 3' end of the
CTLA4E
gene including the HIsequence. Both PCR fragments are subsequently
gelpurified and an additional PCR is performed on the mixture of both PCR
fragments
using primers Nos. 11209 and 11210. The resulting PCR fragment is gelpurified,
cut
with ApaLI and NotI, and inserted into the ApaLI/NotI opened vector
pCESICTLA4E.
The resulting vector pCESICTLA4E-Al/SlHis6 is verified by DNA sequencing.
Primer sequences:
30
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
No. 11069 (SEQ ID 240):
5'-GTGGTGATGGTGATGGTGAGAGCCACCGCCACCGCTGCTGGCCAGTACC
ACAG-3'
No. 11070 (SEQ ID 241 ):
5'-CACCATCACCATCACCACGGTGGCGGTGGCTCTCGAGGCATCGCCAGCT
TTG-3'
No. 11209 (SEQ 117 242):
5'-GATTACGCCAAGCTTTGGAGC-3'
No. 11210 (SEQ ID 243):
5'-ATGCGGCCCCATTCAGATC-3'
Example 16 : Insertion of an integrin binding sequence (RGD) in the loop
formed
by (3-sheets S2 and A2 in SCA-E
Using plasmid pCESICTLA4E (see example 3) as a template, the DNA coding
for the integrin binding sequence (RGD) is inserted in the loop formed by ~3-
sheets S2
and A21 using overlap extension PCR (Ho et al., 1989). In the S2/A2 loop,
consisting
of residues DS (see figure 2), the RGD sequence is inserted between the D and
the S
residue. No flanking sequences are present. This insertion can be accomplished
by
performing two PCR reactions using pCESICTLA4E plasmid DNA as a template. A
first PCR, using primers Nos. 11209 and 11079, amplifies the 5' end of the
CTLA4E gene
including the RGD sequence. The second PCR, using primers Nos. 11080 and
11210,
amplifies the 3' end of the CTLA4E gene including the RGD sequence. Both PCR
fragments are subsequently gelpurified and an additional PCR is performed on
the
mixture of both PCR fragments using primers Nos. 11209 and 11210. The
resulting PCR
fragment is gelpurified, cut with ApaLI and NotI, and inserted into the
ApaLI/NotI
opened vector pCESICTLA4E. The resulting vector pCESICTLA4E-S2/A2RGD is
verified by DNA sequencing.
Primer sequences:
31
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
No. 11079 (SEQ ID 244):
5'-CACCTGGCTGTCACCACGGTCAGCCTGCCGAAGCACTG-3'
No. 11080 (SEQ )D 245):
5'-CAGGCTGACCGTGGTGACAGCCAGGTGACTGAAGTCTGTGC-3'
No. 11209 (SEQ ID 246):
5'-GATTACGCCAAGCTTTGGAGC-3'
No. 11210 (SEQ 1D 247):
5'-ATGCGGCCCCATTCAGATC-3'
Example 17 : Insertion of a His6 tag (HHHHHH) in the loop formed by ~3-sheets
S2
and A1 in SCA-E
Using plasmid pCES 1CTLA4E (see example 3) as a template, the DNA coding
for a His6 tag (F~IIi) is inserted in the loop formed by ~i-sheets S2 and A2
using
overlap extension PCR (Ho et al., 1989). In the S2/A2 loop, consisting of
residues DS
(see figure 2), the ~ sequence is inserted between the D and the S residue. No
flanking sequences are present. This insertion can be accomplished by
performing two
PCR reactions using pCESICTLA4E plasmid DNA as a template. A first PCR, using
primers Nos. 11209 and 11075, amplifies the 5' end of the CTLA4E gene
including the
ICI sequence. The second PCR, using primers Nos. 11077 and 11210, amplifies
the 3' end of the CTLA4E gene including the HI~I sequence. Both PCR fragments
are subsequently gelpurified and an additional PCR is performed on the mixture
of both
PCR fragments using primers Nos. 11209 and 11210. The resulting PCR fragment
is
gelpurified, cut with ApaLI and NotI, and inserted into the ApaLI/NotI opened
vector
pCESICTLA4E. The resulting vector pCESICTLA4E-S2/A2His6 is verified by DNA
sequencing.
Primer sequences:
32
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
No. 11075 (SEQ >D 248):
5'-GTGGTGATGGTGATGGTGGTCAGCCTGCCGAAGCACTG-3'
No. 11077 (SEQ )D 249):
5'-CACCATCACCATCACCACAGCCAGGTGACTGAAGTCTGTGC-3'
No. 11209 (SEQ 1D 250):
5'-GATTACGCCAAGCTTTGGAGC-3'
No. 11210 (SEQ 1D 251 ):
5'-ATGCGGCCCCATTCAGATC-3'
Example 18 : SCA-E A1/S1 His6 and SCA-E S2/A2 His6 displayed on phage are
able to bind coated anti His6-tag antibodies in ELISA
Phage displaying SCA-E Al/S1 His6 or SCA-E S2/A2 His6 protein on their
surface were prepared as described by Marks et al. (1991). To demonstrate
binding of
the SCA-E A1/Sl His6 or SCA-E S2/A2 His6 protein to an anti His6-tag antibody
(BabCO, Richmond, CA, USA), phages are titrated and a serial dilution is made
to test
for binding in ELISA to these molecules. Significant signals were obtained
showing
binding to the anti His6-tag antibody.
Example 19 : SCA-E A1/S1 His6 and SCA-E S2/A2 His6 displayed on phage are
able to bind Ni 2+ coated plates in ELISA
Phage displaying SCA-E A1/S1 His6 or SCA-E S2/A2 His6 protein on their
surface were prepared as described by Marks et al. (1991). To demonstrate
binding of
the SCA-E A1/S1 His6 or SCA-E S2/A2 His6 protein to Ni-NTA HisSorbTM Plates
(Qiagen GmbH, Hilden, Germany), phages are titrated and a serial dilution is
made to test
for binding to these plates. Significant signals were obtained showing binding
to the
Ni-NTA coated plates.
Example 20 : SCA-E A1/S1 His6 and SCA-E S2/A2 His6 displayed on phage are
still
able to bind coated recombinant B7.lIg and B7.2Ig in ELISA
Phage displaying SCA-E protein on their surface are prepared as described by
33
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
Marks et al. (1991). To demonstrate binding of the SCA-E A1/S1 His6 or SCA-E
S2/A2
His6 proteins to B7.lIg and B7.2Ig, phages are titrated and a serial dilution
is made to test
for binding in ELISA to these molecules. Strong signals can be obtained
demonstrating
binding to both B7.lIg and B7.2Ig. This demonstrates that the SCA-E E Al/S1
His6 or
SCA-E S2/A2 His6 proteins have the proper conformation and remain functional
as a
monomer. Furthermore, it demonstrates that both the A1/S1 and S2/A2 loops are
permissive sites for insertion of peptide sequences without destroying the
overall scaffold
conformation.
Example 21 : SCA-E A1/S1 RGD and SCA-E S2/A2 RGD displayed on phage are
still able to bind coated recombinant B7.lIg and B7.2Ig in ELISA
To test for the integrity of CTLA-4 derivatives in which foreign sequences are
introduced into the loops A1/S1 and S2/A2, a phage ELISA is performed on
antigens
av~33 integrin (l~g/ml), B7-lIg and B7-2Ig (l~g/ml) and BSA (l~g/ml). As can
be seen
AB' KL10 shows a positive signal on B7-lIg and B7-2Ig suggesting that despite
the
introduction of the sequence SGGGS into the A1/S1 loop, the functional
integrity of the
molecule is maintained (Figure 14), proving the concept of the invention that
the
molecule of the presdent invention can function as a scaffold. The integrity
of these
molecules is further verified as a weak binding in phage ELISA as can be seen
on anti
CTLA-4 (Figure 16).
34
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
LIST OF REFERENCES
-Abedi, M. R., Caponigro, G., and Kamb, A. (1998) Green fluorescent protein as
a
scaffold for intracellular presentation of peptides, Nucleic Acids Res. 26:
623-30
-Amit,A.G., Mariuzza,R.A.,Phillips,S.E.V., and Poljak,R.J. (1986) Three
dimensional
structure of an antigen-antibody complex at 2.8A resolution. Science 233: 747
-Amzel,L.M. and Poljak,R.J. (1979) Three dimensional structure of
immunoglobulins.
Ann.Rev.Biochem. 48: 961
-Atherton & Shepard (1989) Solid phase peptide synthesis. IRL Press, Oxford.
-Barbas, C. F. d., Languino, L. R., and Smith, J. W. (1993) High-affinity self-
reactive
human antibodies by design and selection: targeting the integrin ligand
binding site, Proc
Natl Acad Sci U S A. 90: 10003-7
-Bianchi,E.,Sollazzo,M.,Tramontano,A.,Pessi,A. (1993) Affinity purification of
a
difficult sequence protein. Int.J.Peptide Protein Res. 42: 93
-Bianchi,E,Venturini,S.,Pessi,A.,Tramontano,A.,Sollazzo,M. (1994) High level
expression and rational mutagenesis of a designed protein, the minibody: from
an
insoluble to a soluble protein.
-Brooks, P. C., Montgomery, A. M., Rosenfeld, M., Reisfeld, R. A., Hu, T.,
Klier, G.,
and Cheresh, D. A. (1994) Integrin alpha v beta 3 antagonists promote tumor
regression
by inducing apoptosis of angiogenic blood vessels, Cell. 79: 1157-64
-Cannon E.L., Ladner R.C., McCoy D. (1996) Phage-display technology. IVD
Technology Nov./Dec., Canon Communications.
-Choo, Y. and Klug, A. (1995) Designing DNA-binding proteins on the surface of
filamentous phage, Curr Opin Biotechnol. 6: 431-6
-Colas, P., Cohen, B., Jessen, T., Grishina, L, McCoy, J., and Brent, R.
(1996)
Genetic selection of peptide aptamers that recognize and inhibit cyclin-
dependent kinase
2, Nature. 380: 548-50
-Hamers-Casterman C., Atarhouch T., Muyldermans S., Robinson G., Hamers C.,
Songa E.B., Bendahman N., Hamers R. (1993) Naturally occuring antibodies
devoid
of light chains. Nature 363: 446-448
-Ho. S., Hunt H., Horton H., Pullen J. & Pease L. (1989) Site-directed
mutagenesis by
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
overlap extension using the polymerase chain reaction. Gene, 77, pp. 51-59.
- Houbenweyl (1974) Methode der organischen chemie, vol. 15, I & II (ed. Wunch
E).
Thieme, Stuttgart.
-Houston, M. E., Jr., Wallace, A., Bianchi, E., Pessi, A., and Hodges, R. S. (
1996)
Use of a conformationally restricted secondary structural element to display
peptide
libraries: a two-stranded alpha-helical coiled-coil stabilized by lactam
bridges, J Mol
Biol. 262: 270-82,
-Knapp, A., Degenhardt, T., and Dodt, J. Hirudisins. (1992) Hirudin-derived
thrombin inhibitors with disintegrin activity, J Biol Chem. 267: 24230-4
-Koide, A., Bailey, C. W., Huang, X., and Koide, S. (1998) The fibronectin
type III
domain as a scaffold for novel binding proteins, J Mol Biol. 284: 1141-51
-Ku, J. and Schultz, P. G. (1995) Alternate protein frameworks for molecular
recognition, Proc Natl Acad Sci U S A. 92: 6552-6
-Kubinyi H. (1995). Strategies and recent technologies in drug discovery.
Pharmazia
50:647-662.
-Ladner R. (1995) Constrained peptides as binding entities.TibTech 13:426-430.
-Maclennan J. (1995) Engineering microprotein ligands for large-scale affinity
purification. Biotechnology 13: 1181-1183.
-Maniatis T., Fritsch E., Sambrook J. (1982) Molecular cloning: a laboratory
manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
-Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G
(1991) By-passing immunisation. Human antibodies from V-gene libraries
displayed on
phage, J. Mol. Biol.; 222(3), pp. 581-97
-Martin, F., Toniatti, C., Salvati, A. L., Venturini, S., Ciliberto, G.,
Cortese, R., and
Sollazzo, M. (1994) The affinity-selection of a minibody polypeptide inhibitor
of human
interleukin-6, Embo J. 13: 5303-9
-McConnell, S. J. and Hoess, R. H. (1995) Tendamistat as a scaffold for
conformationally constrained phage peptide libraries, J Mol Biol. 250: 460-70
-Metzler W.J., Bajorath J., Fenderson W., Shaw S., Constantine K., Naemura J.,
Leytze G., Peach R., Lavoie T., Mueller L., Linsley P. (1997) Solution
structure of
36
CA 02367212 2001-09-27
WO 00/60070 PCT/EP99/02283
human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28.
Nat.
Struct. Biol. 4: 527-531.
-Neu,H.C. and Heppel,L.A. (1965) J.Biol.Chem. 240: 3685
-Nord, K., Gunneriusson, E., Ringdahl, J., Stahl, S., Uhlen, M., and Nygren,
P.
(1997) A. Binding proteins selected from combinatorial libraries of an alpha-
helical
bacterial receptor domain, Nat Biotechnol. 15: 772-7
-Pasqualini, R., Koivunen, E., and Ruoslahti, E. (1997) Alpha v integrins as
receptors
for tumor targeting by circulating ligands [see comments], Nat Biotechnol. 15:
542-6
-Pasqualini, R., Koivunen, E., and Ruoslahti, E. (1995) A peptide isolated
from phage
display libraries is a structural and functional mimic of an RGD-binding site
on integrins,
J Cell Biol. 130: 1189-96
-Pessi, A., Bianchi, E., Crameri, A., Venturini, S., Tramontano, A., and
Sollazzo, M.
(1993) A designed metal-binding protein with a novel fold [see comments],
Nature. 362:
367-9
-Raag,R., and Whitlow,M. (1995) Single-chain Fvs. FASEB J. 9:73
-Reiter,Y.,Brinkmann,U.,Webber,K.,Jung,S.-H.,Lee,B.K. and Pastan,I (1994)
Engineering interchain disulfide bonds into conserved framework regions of Fv
fragments: improved biochemical characteristics of recombinant immunotoxins
containing disulfide- stabilized Fv. Protein Eng. 7: 697
-Rottgen, P. and Collins, J. (1995) A human pancreatic secretory trypsin
inhibitor
presenting a hypervariable highly constrained epitope via monovalent phagemid
display,
Gene. 164: 243-50
-Smith, J. W., Hu, D., Satterthwait, A., Pinz-Sweeney, S., and Barbas, C. F.,
3rd
(1994) Building synthetic antibodies as adhesive ligands for integrins, J Biol
Chem. 269:
32788-95
-Smith, G. P., Patel, S. U., Windass, J. D., Thornton, J. M., Winter, G., and
Griffiths, A. D. (1998) Small binding proteins selected from a combinatorial
repertoire
of knottins displayed on phage, J Mol Biol. 277: 317-32
-Williams A.F. (1987) A year in the life of the immunoglobulin superfamily.
Immunol.Today 8: 298
37
