U.S. patent application number 13/101894 was filed with the patent office on 2012-07-26 for identification of protein binding sites.
Invention is credited to Robert Hans Meloen, Wouter Cornelis Puijk, Jelle Wouter Slootstra, Evert van Dijk, Pieter van Dijken.
Application Number | 20120190564 13/101894 |
Document ID | / |
Family ID | 8172123 |
Filed Date | 2012-07-26 |
United States Patent
Application |
20120190564 |
Kind Code |
A1 |
Slootstra; Jelle Wouter ; et
al. |
July 26, 2012 |
IDENTIFICATION OF PROTEIN BINDING SITES
Abstract
The disclosure relates to the field of molecular recognition or
detection of discontinuous or conformational binding sites or
epitopes corresponding to a binding molecule, in particular, in
relation to protein-protein, protein-nucleic acid, nucleic
acid-nucleic acid or biomolecule-ligand interactions. Provided is a
synthetic molecular library allowing testing for, identification,
characterization, or detection of a discontinuous binding site able
to interact with a binding molecule, the library having been
provided with a plurality of test entities, each test entity
comprising at least one first segment spotted next to a second
segment, each segment having the capacity of being a potential
single part of a discontinuous binding site.
Inventors: |
Slootstra; Jelle Wouter;
(Lelystad, NL) ; Puijk; Wouter Cornelis;
(Lelystad, NL) ; Meloen; Robert Hans; (Lelystad,
NL) ; van Dijk; Evert; (Giethoorn, NL) ; van
Dijken; Pieter; (Harderwijk, NL) |
Family ID: |
8172123 |
Appl. No.: |
13/101894 |
Filed: |
May 5, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10411869 |
Apr 10, 2003 |
7972993 |
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13101894 |
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PCT/NL01/00744 |
Oct 10, 2001 |
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10411869 |
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Current U.S.
Class: |
506/9 ; 506/18;
506/30; 530/300 |
Current CPC
Class: |
C40B 30/04 20130101;
C07K 1/047 20130101; G01N 33/6845 20130101 |
Class at
Publication: |
506/9 ; 506/30;
506/18; 530/300 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 40/10 20060101 C40B040/10; C07K 2/00 20060101
C07K002/00; C40B 50/14 20060101 C40B050/14 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 11, 2000 |
EP |
00203518.6 |
Claims
1.-10. (canceled)
11. A method of screening for a binding site able to interact with
a binding molecule, the method comprising: screening a peptide
library with at least one potential binding molecule, wherein the
peptide library comprises a plurality of test entities, wherein the
plurality of test entities comprises at least a first segment and a
second segment, and wherein at least one of the test entities is
produced by spotting, at a solid phase, at least a first segment in
close proximity to a second segment; and detecting binding between
a test entity of the library and the potential binding
molecule.
12. The method according to claim 11, wherein the binding site is a
discontinuous binding site.
13. A synthetic molecule comprising a binding site identifiable by
the method according to claim 11.
14. A binding molecule comprising a binding site identifiable by
the method according to claim 11.
15. The synthetic molecule of claim 13 wherein the binding site
comprises a discontinuous binding site.
16. The binding molecule of claim 14 wherein the binding site
comprises a discontinuous binding site.
17.-20. (canceled)
21. A method for interfering with or effecting binding to a binding
molecule, the method comprising interacting the binding molecule
with the synthetic molecule of claim 13.
22. A method for interfering with or effecting binding to a binding
molecule, the method comprising interacting the binding molecule
with the binding molecule of claim 14.
23. A method for interfering with or effecting binding to a binding
molecule, the method comprising interacting the binding molecule
with the synthetic molecule of claim 15.
24. A method for interfering with or effecting binding to a binding
molecule, the method comprising interacting the binding molecule
with the binding molecule of claim 16.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending application
Ser. No. 10/411,869, filed on Apr. 10, 2003, now U.S. Pat. No.
______, which is a continuation of International Application
PCT/NL01/00744, filed Oct. 10, 2001, designating the United States,
published in English Apr. 18, 2002, as WO 02/31510 A1, which claims
priority from EP 00203518.6 filed Oct. 11, 2000, the contents of
each of which are incorporated herein by this reference.
FIELD OF THE INVENTION
[0002] The invention relates to the field of molecular recognition
or detection of discontinuous or conformational binding sites or
epitopes corresponding to or interacting with a binding molecule,
in particular, in relation to protein-protein or protein-ligand
interactions.
BACKGROUND OF THE INVENTION
[0003] Interactions between binding molecules, which in general are
biomolecules and their corresponding ligands, are central to life.
Cells often bear or contain receptor molecules that interact or
bind with a hormone, a peptide, a drug, an antigen, an effector
molecule or with another receptor molecule; enzymes bind with their
substrate; antibody molecules bind with an antigen, nucleic acid
with protein, and so on. By "interact or bind" it is meant that the
binding molecule and ligand approach each other within the range of
molecular forces and may influence each other's properties. This
approach takes the binding molecule and its ligand through various
stages of molecular recognition comprising increasing degrees of
intimacy and mutual effect: they bind.
[0004] Binding molecules have this binding ability because they
comprise distinct binding sites allowing for the recognition of the
ligand in question. The ligand, in turn, has a corresponding
binding site, and only when the two binding sites can interact
by--essentially spatial--complementarity, the two molecules can
bind. Needless to say, molecules having three dimensions have
binding sites that are of a three dimensional nature, often one or
more surface projections or protuberances of one binding site
correspond to one or more pockets or depressions in the other, a
three-dimensional lock-and-key arrangement, sometimes in an
induced-fit variety.
[0005] Sometimes, such a protuberance comprises a single loop of
the molecule in question, and it is only this protuberance that
essentially forms the binding site. In that case, one often terms
these binding sites as comprising a linear or continuous binding
site, wherein a mere linear part of the molecule in question is
essentially responsible for the binding interaction. This
terminology is widely used to describe, for example,
antibody-antigen reactions wherein the antigen comprises part of a
protein sequence, a linear peptide. One then often speaks about a
linear or continuous epitope, wherein the binding site (epitope) of
the antigenic molecule is formed by a loop of consecutively bound
amino acids. However, similar continuous binding sites (the terms
"epitope" and "binding site" are used interchangeably herein) can
be found with receptor-antigen interactions (such as with a T-cell
receptor), with receptor-ligand interactions such as with hormone
receptors and agonists or antagonists thereof, with
receptor-cytokine interactions, or with, for example,
enzyme-substrate or receptor-drug interactions, wherein a linear
part of the molecule is recognized as the binding site, and so
on.
[0006] More often, however, such a protuberance or protuberances
and depressions comprise various, distinct parts of the molecule in
question, and the combined parts essentially form the binding site.
Commonly, one names such a binding site comprising distinct parts
of the molecule in question a discontinuous or conformational
binding site or epitope. For example, binding sites laying on
proteins having not only a primary structure (the amino acid
sequence of the protein molecule), but also secondary and tertiary
structure (the folding of the molecule into alpha-helices or
beta-sheets and its overall shape), and sometimes even quaternary
structure (the interaction with other protein molecules) may
comprise in their essential protuberances or depressions amino
acids or short peptide sequences that lay far apart in the primary
structure but are folded closely together in the binding site.
[0007] Due to the central role binding molecules and their ligands
play in life, there is an ever expanding interest in testing for or
identification of the nature or characteristics of the binding
site. Notably, the rapid developments in evolving biotechnology
fields such as proteomics will result in the near future in the
identification of more and more binding molecules and their
corresponding ligands. The detection of protein-protein
interactions and enzyme-substrate interactions (not only of protein
enzymes but certainly also of for example catalytic RNA-based
interactions), and the identification of protein-nucleic acid and
of nucleic acid-nucleic acid pairs of binding molecule and
corresponding ligand, will certainly result in generating more
interest in where the exact interacting (binding) sites between
these molecules lay and how one can develop compounds (agonists,
antagonists, drugs) modulating the specific interaction.
[0008] Not only is one interested in the exact nature of the
particular interaction between binding molecule and ligand in
question, for example, in order to replace or supplement binding
molecules or ligands when needed, but one is also interested in
knowing approximating characteristics of the interaction in order
to find or design analogues, agonists, antagonists or other
compounds mimicking a binding site or ligand involved.
[0009] Versatile and rapid methods to test for or identify
continuous epitopes or binding sites are known. Most, if not all,
nucleic acid detection techniques, and molecular libraries using
these, entail hybridization of an essentially continuous nucleic
acid stretch with a complementary nucleic acid strand, be it DNA,
RNA or PNA. Little attention has been paid to methods allowing
rapid and straightforward identification of discontinuous binding
sites of an essentially nucleic acid nature. Although plenty of
such sites exist, think only of the lack of understanding
surrounding ribosomal binding sites where ribosomal proteins bind
to tRNA, of regulatory sites in promoter sequences, of interactions
between polymerases and replicases between DNA and RNA, of
catalytic RNA reactions, and so on, no molecular libraries exist
that provide easy access to such sites.
[0010] An early work in the peptide field is disclosed in PCT
International Publication No. WO 84/03564, related to a method of
detecting or determining antigenically active amino acid sequences
or peptides in a protein. This work, providing the so-called
Pepscan technology, wherein a plurality of different peptides is
synthesized by linking with a peptide bond a first amino acid to a
second, and so on, and on a second position in the test format yet
another first amino acid is linked to a second, and so on, after
which the synthesized peptides are each tested with the binding
molecule in question, allows the determination of every continuous
antigenic determinant or continuous epitope of importance in a
protein or peptide sequence. Pepscan technology taken in a broad
sense also provides for the testing for or identification of
(albeit linear) peptides essentially identical with, analogous to
or mimicking binding sites or ligands of a various nature
(mimotopes, Geyssen et al., Mol. Immunol. 23:709-715, 1986).
[0011] Pepscan technology allows identification of linear peptide
sequences interacting with receptor molecules, enzymes, antibodies,
and so on, in a rapid and straightforward fashion, allowing testing
of a great many peptides for their reactivity with the binding
molecule in question with relatively little effort. The order of
magnitude of testing capability having been developed with Pepscan
technology (e.g., also due to miniaturization of test formats; see,
e.g., PCT International Publication No. WO 93/09872) furthermore
allows at-random testing of a multiplicity of peptides, leading to
automated combinatorial chemistry formats wherein a great many
binding molecules are tested in a (if so desired at-random) pattern
for their reactivity with a molecular library of synthetic peptides
representing potential continuous binding sites or ligands,
allowing the rapid detection of particularly relevant molecules out
of tens of thousands of combinations of molecules tested.
[0012] However, for the testing of discontinuous or conformational
binding sites to a binding molecule, no formats similar to or as
versatile as Pepscan technology exist. Attempts to identify
discontinuous epitopes by Pepscan technology are cumbersome. It
does, in general, not suffice to merely extend synthesis of the
test peptides by linking more amino acids to the existing peptide
and hoping that some of the thus formed longer peptides will fold
in such a way that at least two distinct parts are presented in a
discontinuous fashion and are recognized by a binding molecule. In
that case, there is no way of finding out in a rapid and
straightforward fashion that the binding is indeed through a
discontinuous binding site; it might be that just a longer single
loop is responsible for the binding.
[0013] Some additional possibilities are provided by testing
synthetic peptide sequences that have been designed to comprise two
previously identified parts of a binding site, each part in essence
being linear and being part of a larger linear peptide. Early work
herein was done by Atassi and Zablocki (J. Biol. Chem. 252:8784,
1977) who describe that spatially or conformationally contiguous
surface residues (which are otherwise distant in sequence) of an
antigenic site of egg white lysozyme were linked by peptide bonds
into a single peptide which does not exist in lysozyme but attempts
to simulate a surface region of it. However, their technique,
called surface simulation synthesis, requires detailed knowledge of
the three-dimensional structure of the protein under study and a
full chemical identification of the residues constituting the
binding site beforehand, as well as their accurate conformational
spacing and directional requirements.
[0014] In the same fashion, Dimarchi et al. (Science 232:339-641,
1986) describe a 38 to 40 amino acid-long synthetic peptide
consisting of two previously identified separate peptidyl regions
of a virus coat protein. The peptide was synthesized using common
peptide synthesis technology (Merrifield et al., Biochemistry 21,
5020, 1982) by adding subsequent amino acids with a peptide bond to
an ever growing peptide resulting in a peptide wherein the two
peptidyl regions were connected by a diproline spacer presumably
functioning as indication of a secondary structural turn, thereby
providing a two-part epitope or binding site.
[0015] However, it is clear that when one has to know beforehand
the sequence of the (in this case only) two relevant parts in order
to provide the desired discontinuous binding site, it excludes the
feasibility of providing (desirably in a random fashion) a whole
array of merely potential discontinuous binding sites for large
scale testing. Furthermore, a major drawback of the above-mentioned
strategies is that, again, only linear epitopes or dominant binding
regions of discontinuous epitopes can be mimicked adequately. For
the more complete synthesis of a discontinuous binding site, all
the contributing parts have to be arranged in the proper
conformation to achieve high-affinity binding. Therefore, single
parts of discontinuous binding sites have to be linked.
[0016] Fifteen years after Dimarchi, Reineke et al. (Nature
Biotechnology, 17:271-275, 1999) provided a synthetic mimic of a
discontinuous binding site on a cytokine and a method to find such
a discontinuous binding site that allowed for some flexibility and
somewhat larger scale testing, wherein positionally addressable
peptide collections derived from two separate regions of the
cytokine were displayed on continuous cellulose membranes and
substituted in the process to find the best binding peptide. After
selection of the "best reactors" from each region, these were
combined to give rise to another synthetic peptide collection
(comprising peptides named duotopes) that again underwent several
rounds of substitutions.
[0017] Reineke et al. thus provide synthesis of peptide chains
comprising duotopes, however, again selected after previous
identification of putative constituting parts with Pepscan
technology, thereby still not allowing testing discontinuous
binding sites in a rapid and straight forward fashion.
[0018] However, as indicated before, protein domains or small
molecules that mimic binding sites are playing an increasing role
in drug discovery, diagnostics and biotechnology. The search for
particular molecules that bind to a binding site and mimic or
antagonize the action of a natural ligand has been initiated in
many laboratories. As indicated before, attempts to find such
structures in synthetic molecular libraries often fail because of
the essentially discontinuous nature and spatial complementarity of
most binding sites.
[0019] Thus, for the many more cases where the binding site may
essentially be discontinuous, improved means and methods to
identify these sites are needed, and, in particular, means and
methods are needed that allow testing for discontinuous binding
sites wherein the parts need not necessarily first be selected by
previous identification as a putative or even only tentative
constituting part of the desired discontinuous binding site but
bear only the potentiality of being part of that site by being a
molecule with more or less distinct features per se.
BRIEF SUMMARY OF THE INVENTION
[0020] Provided is a method for producing a molecular library
comprising providing the library with a plurality of test entities
wherein the entities have essentially been produced by segment
spotting, that is, by spotting, placing, or attaching in close
proximity at least two (di-, tri-, oligo- or multimeric) segments
of, for example, nucleic acids or peptides directly or indirectly
to a solid phase, such as an array surface, instead of by
sequentially synthesizing test molecules and spotting one molecule,
or several replicas of the one molecule, as a single entity, which
is done traditionally. In theory, the segments can be sequentially
synthesized in close proximity to each other, wherein, in a
repetitive fashion, one monomer (e.g., a nucleotide or an amino
acid) to another until a (in essence polymeric) molecule (segment)
of the desired length has been obtained.
[0021] Essentially, existing nucleic acid libraries comprise
nucleic acids that are synthesized sequentially by adding one
nucleotide or nucleoside at a time to the growing stretch, and
existing peptide libraries comprise peptides that are synthesized
sequentially by adding one amino acid at the time to a growing
stretch until the desired length has been reached. However, with
existing libraries, no attention is given to synthesizing specific
segments in close proximity to each other so that they together can
represent a putative binding site. With nucleic acids, the monomers
are essentially selected from a limited set of well known
nucleotides. With peptides, the monomers are essentially selected
from a well known set of amino acids. Not only naturally occurring
monomers are used. Synthetic nucleotides, such as peptide nucleic
acid (PNA) molecules, non-naturally occurring amino acids, or even
D-amino acids, are routinely used as monomers by which the
essentially polymeric molecules are generated or produced using a
method that is essentially in conformity with the sequential
synthesis of polymers from monomeric molecules in nature.
Preferred, according to the invention, however, is synthesizing the
segments before they are attached to the solid phase in close
proximity, thereby it is easier to create the desired test entity,
the putative binding site composed of two or more segments located
in close proximity and attached to the solid phase, e.g., the array
surface.
[0022] In "close proximity" herein reflects the possibility that a
putative binding molecule can bind to at least two of the closely
spotted segments or parts thereof and is defined in angstrom units,
reflecting the generally molecular scale of the binding sites. It
is preferred to attach the two or more segments that form the
desired test entity at no more than 100 angstroms away from each
other, however, obviating the need of long linkers, or when small
segments are used, distances of smaller than 50, or preferably
smaller than 30, or even smaller than 15 angstroms, are preferred,
the smaller distances in general creating a better fit for binding
sites. Minimal proximity is 1-2 angstroms, wherein the segments
are, for example, linked to variously protected thiol groups only
1-2 atoms on the polymer away from each other. Furthermore, the
length of a flexible linker should preferably be 10-100 angstroms,
where the preferred length of segments is at about 5-100 angstroms
and where the preferred distance between the tops of segments
amounts to 0-30 angstroms.
[0023] For example, two segments can be coupled, preferably as
loops, onto a (polycarbon)-polymer surface. With extra spaced
building blocks (for example, phenylalanine amino acids) it is
provided to obtain extended loops. On the (polycarbon)-surface, for
example, two types (see FIG. 1 for suitable types) of protected
cysteines (e.g., cys (trt) and cys (mmt)) and, for example, one
spacing building block is coupled. The cys (mmt) is deprotected
with 1% TFA while the cys (trt) remains protected. The first
segment is coupled to the deprotected cys (mmt). Then, the second
cys (trt) is deprotected with 95% TFA. Then, the second segment is
coupled to the now deprotected cys (trt). If desired, segments can
also be linked together using appropriate chemistry.
[0024] Alternatively, instead of directly linking the segments to
the surface (albeit via linkage groups), the segments may be first
linked to a template that itself is linked to the surface. In a
preferred embodiment, such a template is, for example, a peptide.
For example, two segments can be coupled onto a cyclic template
that itself is coupled to the polymer surface. The cyclic template
is, for example, a cyclic flexible peptide. The cyclic peptide
contains, for example, reactive groups such as four lysines (mmt),
two cysteines (trt) and two cysteines (butyl). The template is, for
example coupled to the resin via a sulfur.
[0025] The invention thus provides a molecular library that, albeit
also suited for detecting or screening for continuous binding
sites, is now particularly well suited for detecting or screening
for discontinuous binding sites, in particular in relation to
binding molecule-ligand interactions such as, for example,
protein-protein, protein-nucleic acid, and nucleic acid-nucleic
acid interactions, now that at least two different segments, each
of which may represent a part of a discontinuous binding site, are
spotted as single entity, tentatively representing a possibly as
yet unknown discontinuous binding site, herein also called a
binding body.
[0026] As used herein, the term "binding body" is generally used
for essentially all-peptide segment constructs, however, the
technology, as described for all-peptide combinations, can of
course also be used for nucleic acid combinations or combinations
of an even more mixed nature. A binding body, which is in essence a
synthetic molecule comprising a binding site identifiable or
obtainable by a method according to the invention as described
herein, is essentially a combination of random peptide segments
(fixed into one molecule or represented as one molecule on a test
surface), which acts as a binding molecule such as an antibody.
Just as in the case of antibodies, the recognition may more or less
be "degenerate," i.e., the binding site on the target molecule need
not always be optimal. The binding body may in principle bind to
any part of the target molecule. For instance: to neutralize the
action of TNF-alpha, one might develop a small molecule that
specifically interacts with the receptor binding site on TNF-alpha;
alternatively, one might develop an antibody that interacts with
TNF-alpha at an as yet undefined place and neutralizes its action.
This shows that sometimes small molecules are the solution and
sometimes large antibodies. Unfortunately, both have their
disadvantages: small molecules are difficult or impossible to make
for large recognition sites and large molecules like antibodies are
much easier to develop but cannot be used intracellularly and have
all sorts of pharmacological disadvantages like their
immunogenicity and their inability to act inside the cell.
[0027] The advantageous properties of the binding body combine
those of small and large molecules: binding bodies share advantages
of both. A preferred binding body consists of random peptide
segments, for example, slightly biased or shuffled to resemble CDRs
or other binding domains. If needed or desired, CDRs may be
mimicked by using, for example, 6 segments, each representing one
possible CDR, however, combinations of 2, 3 or 4 segments will
already provide diversity. The peptide segments preferably are
linked at both sides to a scaffold or solid phase. Thus, binding
bodies are made up of molecules with one, two or more peptide
segments.
[0028] Highly diverse binding body libraries can be generated based
on systematic combination of relatively small numbers of random
peptide segments. A library of 100 binding bodies is easily
produced using positionally defined peptide segment arrays as
described herein. Screening of such a library with any given
molecule is simple, fast and straightforward. Hits can be
translated directly into the amino acid or segment make up of the
binding body due to the positionally defined array. A library of
10,000 binding bodies can be easily generated by combining all
peptides from smaller libraries with each other or by starting with
a larger solid support surface. A library of 1,000,000 binding
bodies can, for example, be easily generated by combining all
peptides of smaller libraries into binding bodies that contain
three segments. Thus, a large diversity of binding bodies can be
generated starting with relatively small numbers of random peptides
(for instance, 10) and multiple combinations of peptides combined
into a single binding body (for instance, 6) to arrive at a
diversity of 1,000,000 or even larger.
[0029] Alternatively, the same binding body diversity can be
obtained starting with, for example, 1000 random peptides and using
just two peptide segments for each binding body. Just like
antibodies, binding bodies can "mature." Based on hits obtained
with an initial set of random binding bodies (above), new dedicated
libraries can be generated that will contain a high number of
improved combinations. The best ones can be selected or improved in
an additional round using a second dedicated library, and so on.
Development of high affinity binding bodies is thus provided by
chemistry to bind peptides, preferably both ends, to a molecular
scaffold or solid phase by using an array system in which each
binding body is positionally defined, further by appropriate
miniaturization and/or by appropriate bioinformatics to analyze the
data and to design subsequent improved binding bodies or dedicated
libraries of binding bodies.
[0030] The two or more different segments can, of course, each be
selected at random from any set of di-, tri-, or oligomeric
sequences, such as from di-, tri,- or oligonucleotides, or di-tri-,
or oligopeptides, but sometimes, it may be preferred to include at
least one specific segment in the entity, specific in the sense
that it has been selected from among known segments or distinct
parts of biomolecules, such as parts of genes, proteins, enzymes,
nucleic acids or unique fragments thereof, proteins involved in up-
or down-regulation of translation, t-RNAs, SNRPs, antibodies,
complementarity determining regions (CDRs), antigens, receptors,
transport proteins, transcription factors or factors involved in
up- or down-regulation of transcription, promoter sequences such
as, but not necessarily restricted to, the well known TATA-box
elements, repressor sites, operator sites and other control
elements, polymerases, and replicases, in short, from among known
segments or distinct parts of binding molecules known or suspected
to be involved in binding via a discontinuous binding site.
[0031] Known segments or parts thereof spotted in close proximity
may, of course, be already known as parts constituting a
discontinuous binding site. However, previous identification as
such is essentially not necessary, since screening for such sites
with a molecular library according to the invention allows rapid
and straightforward identification of the constituting segments or
parts thereof.
[0032] Screening such a library can easily be envisioned when the
library's molecules differ only in that constituting segments are
chosen in an overlapping fashion, wherein a first segment from a
distinct biomolecule is spotted next to a second, and to a third,
and to a fourth segment, and a second is spotted next to a third,
and to a fourth, and so on, if so required, until all possible
segments of the biomolecule have been spotted in close proximity
two-by-two (or three-by-three, or even more) together, which allows
for a systematic screening of possible discontinuous binding sites
present on the biomolecule.
[0033] However, an overlapping fashion is, of course, not required.
Random segment combinations spotted in close proximity will provide
valuable information about binding sites as well.
[0034] The invention thus provides a method for producing a
molecular library for identification or detection of a binding site
capable of interacting with a binding molecule, and, thus, for the
identification of a molecule as a binding molecule, the method
comprising providing the library with a plurality of segments
derived from binding molecules or their ligands, further comprising
spotting at least two of the segments in a pair, or three in a
threesome, or more in the respective plurality, preferably a
greater part of the pairs, threesomes on pluralities, most
preferably essentially all of the pairs, threesomes or pluralities,
by at least spotting a first segment next to a second segment, for
example, a segment which comprises a dimer, trimer, oligomer or
multimer.
[0035] Existing libraries, be they of, for example, nucleic acid
(containing a repetitive back-bone of nucleotides, nucleosides or
peptide nucleic acid, or combinations of these) or amino acid
(containing a repetitive back-bone of amino acids) nature have in
general in common that single molecules (or single segments) or a
plurality of replicas of the single molecules are spotted and used
as the entity representing the binding site. Such libraries
comprise oligomeric or multimeric molecules, such as stretches of
nucleic acids or amino acids, that have been produced by
sequentially linking, in a repetitive fashion, one monomer (e.g., a
nucleotide or an amino acid) to another, until a (in essence
polymeric) molecule of the desired length has been obtained.
[0036] Essentially, existing nucleic acid libraries comprise
nucleic acids that are synthesized sequentially by adding one
nucleotide or nucleoside at a time to the growing stretch, and
existing peptide libraries comprise peptides that are synthesized
sequentially by adding one amino acid at the time to a growing
stretch, until the desired length has been reached. With nucleic
acids, the monomers are essentially selected from a limited set of
well known nucleotides. With peptides, the monomers are essentially
selected from a well known set of amino acids. Not only naturally
occurring monomers are used. Synthetic nucleotides, such as peptide
nucleic acid (PNA) molecules, non-naturally occurring amino acids,
or even D-amino acids, are routinely used as monomers by which the
essentially polymeric molecules are generated or produced using a
method that is essentially in conformity with the sequential
synthesis of polymers from monomeric molecules in nature. These
single monomers are then spotted in a single fashion, one monomer
thought to represent the full, or nearly the full, binding site,
without taking into consideration the multiple parts of a binding
site constituting a discontinuous binding site.
[0037] Provided is the recognition that essentially using dimeric
or even larger (tri-, oligo-, or multimeric) segments in
combination, thus in pairs or threesomes or even more, offers
distinct advantages. It not only provides a faster method to arrive
at or recognize a molecule composed of various segments, it also
provides for fast and efficient shuffling of segments to generate a
molecule or test entity repertoire for the desired library. The
invention for example provides a method wherein synthesis is
started with a monomer in close proximity to which a second segment
comprising a dimer, such as a dinucleotide or a dipeptide, is
spotted. Herein, a segment comprising a dimer at least consists of
a dimer but can also be, for example, a trimer or any other
multimer linking monomers of any nature, as required. Of course,
once two segments have been spotted in close proximity, further
segments can be added thereto.
[0038] In a preferred embodiment, to speed up further synthesis, or
to be able to select distinct desired segments, provided is a
method wherein the first segment also comprises a dimer, and in a
yet even more preferred method, further segments comprise dimers as
well. In a preferred embodiment, the dimer comprises a dinucleotide
or dipeptide, but of course other dimers can be made also. The
invention is further explained in the detailed description where
several of the examples relate to libraries comprising molecules
wherein each of the segments comprises a peptide, such as a tri-, a
penta-, an octa-, or nonapeptide. It is, however, also provided by
the invention to use longer segments, e.g., 10 to 15, 15 to 20, 20
to 30 or 30 to 40 amino acids or nucleic acids long or longer and
to use of a varied nature, e.g., wherein one comprises a nucleic
acid and another comprises a peptide, to better mimic binding sites
that are found, for example, on nucleic acid-protein complexes.
[0039] In a preferred embodiment, as, for example, shown in the
examples, provided is a method wherein the first segment is spotted
or attached to the solid phase by a thioether bond next to the
second segment; however, the invention is, of course, not limited
thereto. Nucleotide/side segments can, for example, be covalently
linked or ligated by splicing enzymes or ligases or by overlapping
a first segment and the second segment with an in essence
relatively short nucleotide strand that is partly complementary to
both segments.
[0040] The invention thus provides a molecular library allowing
testing for, identification, characterization or detection of a
continuous or discontinuous binding site capable of interacting
with a binding molecule, the library having been provided with
pluralities (pairs, threesomes, foursomes, fivesomes, sixsomes) of
segments, each plurality preferably comprising at least one first
segment spotted in close proximity to a second segment, wherein at
least the second segment previously existed as a dimer or a
multimer. Preferably, each segment or part thereof having the
capacity to be a potential single part of a discontinuous binding
site, preferably wherein each of at least a first and a second
segment or part thereof represents a potential single part of a
discontinuous binding site. Such a library can, for example,
comprise a synthetic molecular library made by chemical spotting of
segments.
[0041] Preferably, such segments have distinct features, for
example, by being in essence segments that are, comprise or mimic
molecular components of living organisms, such as (combinations of)
nucleotides, sugars, lipids, amino acids, nucleic acid molecules
(DNA or RNA), peptide nucleic acid molecules (PNA), carbohydrates,
fatty acids or fats.
[0042] Herewith, provided is synthesis of molecules comprising
separate segments potentially representing at least two distinct
parts of a discontinuous binding site, the parts not necessarily
first being selected after previous identification of potential
constituting parts, thereby allowing testing for discontinuous
binding sites in a rapid and straightforward fashion.
[0043] The invention thus now allows identifying discontinuous
binding sites of receptor molecules that interact or bind at a
contact site with a hormone, a peptide, a drug, an antigen, an
effector molecule or with another receptor molecule, of enzymes
that bind with their substrate, of antibody molecules that bind
with a binding site on an antigen, nucleic acid that binds with
protein, and so on. In a preferred embodiment of the invention, at
least one of the segments comprises a peptide, another segment
being, for example, DNA, RNA, PNA, carbohydrate, a fatty acid, a
peptide, a hormone or an organic molecule altogether. In one
embodiment of the invention, all segments comprise a peptide. In
this way, a plurality of different binding bodies is synthesized by
spotting a first segment next to a second, and so on, and on a
second position in the test or library format yet another first
segment is linked to a second, and so on, after which the
synthesized binding bodies are each tested with the binding
molecule in question, allowing the determination of a discontinuous
antigenic determinant or discontinuous epitope of importance in,
for example, a nucleic acid, a protein or peptide sequence.
[0044] The peptide segment comprises at least two amino acids and
can, in principle, be as long as desired, e.g., containing a
hundred amino acids or even more. In preferred practice, the
peptide segment comprises from 3 to 30, preferably from 4 to 20,
even more preferably from 5 or 6 to 12 to 15 amino acids, such as 9
or 12 amino acids. Separate segments do not necessarily have to be
of equal length.
[0045] Furthermore, peptide segments to be spotted together, or at
least in close proximity to each other, can be selected at random,
or under guidance of (a) known protein or peptide sequence(s).
Selection at random provides a random library according to the
invention. Selection from known protein or peptide sequences is,
for example, useful when it is desired to find out whether a
discontinuous binding site is composed of distinct sites or parts
present at distinct proteins or peptides, for example, in a protein
complex to which a particular binding molecule can bind. Selection
of various peptide segments from one known protein or peptide
sequence is useful when it is desired to find out whether a
discontinuous binding site is composed of distinct sites or parts
present at one protein or peptide, for example, at a folded protein
to which a particular binding molecule can bind. Selection of
peptide segments can be done by selecting overlapping peptides from
such a known sequence. Overlapping peptides can have, for example,
all but one or two amino acids in common, preferably overlapping in
a contiguous fashion, or can overlap with only one or several amino
acids. For a quick scan for discontinuous binding sites on a known
protein, it is, for example, useful to select nonapeptide segments
from the protein sequence, of which one has, for example, a 5-amino
acid-long overlap with another peptide segment. Equally useful,
however, is to select tripeptide segments from the sequence having
an overlap of only one amino acid and to use three, or even more,
segments in constructing the putative binding site molecule to
which the to-be-tested binding molecule can bind.
[0046] Of course, such selection strategies are equally applicable
to segments of a different nature, nucleic acid segments comprising
a certain number of nucleotides, such as 5, 7, 9, and so on, can be
selected from known nucleic acid sequences comprising putative or
sought-after discontinuous binding sites, each segment selected
from a certain position in the known nucleic acid sequence, if
desired also in an overlapping fashion. The nucleic acid segment
comprises at least 2 nucleotides (be it DNA, RNA or PNA, or
functional equivalents thereof), and can, in principle, be as long
as desired, e.g., containing a hundred nucleotides or even more. In
preferred practice, the nucleic segment comprises from 3 to 30,
preferably from 4 to 20, even more preferably from 5 or 6 to 12 to
15 nucleotides, such as 9 or 12 nucleotides. Separate segments, of
course, do not necessarily have to be of equal length, and, as the
before, can even be of a different nature, e.g., peptide with
DNA.
[0047] The segments can, for example, be chemically attached to the
solid phase by chemical links or bonds. The links or bonds can be
formed using many combinations of strategies of, for example,
peptide or nucleotide chemistry and selective ligation reactions,
as known in the art. Ligation chemistry has been published, for
instance, by groups of Kent (Ph.E. Dawson et al., "Synthesis of
Proteins by Native Chemical Ligation," Science 266 (1994) 776-779),
Tam (J. P. Tam et al., "Peptide Synthesis using Unprotected
Peptides through Orthogonal Coupling Methods," Proc. Natl. Acad.
Sci. USA 92 (1995) 12485-12489); C. F. Liu et al., ("Orthogonal
Ligation of Unprotected Peptide Segments through Pseudoproline
Formation for the Synthesis of HIV-1 Protease Analogs," J. Am.
Chem. Soc. 118 (1996) 307-312); L. Zhang & J. P. Tam,
("Thiazolidone Formation as a General and Site-specific Conjugation
Method for Synthetic Peptides and Proteins," Analytical
Biochemistry 233 (1996) 87-93), and Mutter (G. Tuchscherer & M.
Mutter, "Protein Design as a Challenge for Peptide Chemists," J.
Peptide Science 1 (1995) 3-10); S. E. Cervigni et al.,
("Template-assisted Protein Design: Chimeric TASP by Chemoselective
Ligation, Peptides: Chemistry, Structure and Biology," P. T. P
Kaumaya & R. S. Hodges eds, Mayflower (1996) 555-557).
Possible strategies for the formation of links as preferably
provided by the invention are, for example:
[0048] 1. The link of a segment or segments with a solid phase is
formed using a homo- or hetero-bifunctional linking agent (S. S.
Wong: Chemistry of Protein Conjugation and Cross-Linking, CRC Press
Inc, Boca Raton, Fla. USA 1991). In this construction, a reactive
group in a segment is used to react with one part of the
bifunctional linking agent, thus facilitating the second part of
the linking agent to react with a reactive group from a solid
phase, or visa versa. For instance, a linker like MBS
(m-maleinimidobenzoic acid N-hydroxysuccinimide ester) can be used
to react via its active ester (succinimide) with an amino group of
one segment and via its maleinimide group with a free thiol group
from a solid phase, or visa versa. In this strategy, when linking
preferably no other free amino or thiol groups should be present in
the segment. In order to accomplish this, the amino or thiol groups
that should be involved in the reaction can be deprotected
selectively, for instance, by using a side chain protecting group
that can be cleaved by a mild reagent like 1% trifluoroacetic acid,
which leaves other side chain protecting groups intact.
[0049] 2. The link is formed by introduction of a modified amino
acid in the synthesis of one or more segments. Amino acids can be
modified, for instance, by introduction of a special group at the
side-chain or at the alpha-amino group. A modification at the
alpha-amino group leads to an amide or backbone modified peptide
(see, e.g., Gillon et al., Biopolymers, 31:745-750, 1991). For
instance, this group can be a maleinimido group at the side chain
amino group of lysine. At the end of the peptide synthesis, this
group will react fast and selective with a thiol group of a solid
phase. Tam et al. (PNAS 92:12485-12489, 1995) described a synthesis
of a peptide with a lysine residue that was modified in the side
chain with a protected serine residue. After deprotection and
selective oxidation using periodate, the alpha-amino, beta-hydroxy
function of the serine is converted into an aldehyde function that
can be ligated selectively with another thiol-bearing surface.
Also, peptide backbone links, via groups attached to the amide
groups of the peptide, can be used to spot segments (Bitan et al.,
J. Chem. Soc. Perkin Trans. 1:1501-1510, 1997; Bitan and Gilon,
Tetrahedron, 51:10513-10522, 1995; Kaljuste and Unden, Int. J.
Pept. Prot. Res. 43:505-511, 1994).
[0050] 3. Yet another way to form the link is to synthesize a
segment, such as a peptide, with a modified N-terminus. For
instance, an N-terminal alpha-haloacetamido group can be introduced
at the end of the synthesis. This group reacts fast and selectively
with a solid phase which contains a thiol group. For instance, the
first segment is synthesized with an N-terminal bromoacetamide and
the solid phase is provided with a cysteine. Although most
alpha-haloacetamide groups, like chloro-, bromo-, or iodoacetamide,
will react with thiol groups, in those cases where speedy
assembling is required, the bromoacetamide group is preferred
because of its ease of introduction and fast and selective reaction
with thiol groups.
[0051] Furthermore, provided is the possibility to address the link
in every position of the first and/or the second or consecutive
segment. For instance, for peptide segments, sets of peptides are
synthesized in which a cysteine or a side-chain modified lysine
(both amino acid residues, in a preferred embodiment, being able to
ligate selectively with another segment) shifts from the N-terminal
amino acid position one by one to the C-terminal amino acid
position. Combinations of these possibilities will, again, lead to
libraries as provided by the invention.
[0052] In another preferred embodiment, the segments are linked at
least twice in close proximity to the solid phase, preferably by
linking the respective ends of the segments to the surface, so
that, so-to-speak, looped segments are attached to the solid phase.
In such a preferred embodiment, pairs (or larger pluralities) of
looped segments are attached to the solid phase, presenting
themselves as binding bodies.
[0053] In a preferred embodiment, provided is a library wherein the
pluralities are positionally or spatially addressable, e.g., in an
array fashion, if desired aided by computer directed localization
and/or recognition of a specific pair or threesome (or larger
plurality) or set of pluralities within the dimensions (e.g., plane
or surface) of the support or solid phase of the library used. In
an array, the pluralities are, for example, addressable by their
positions in a grid or matrix.
[0054] A preferred embodiment of the invention further allows
up-scaling of the synthesis concerning the number of constructs on,
for example, a solid support per square centimeter. To facilitate
generation of a great many possible constructs, containing, for
example, test entities (pairs, threesomes or larger pluralities)
comprising at least two peptide segments of a protein, many
thousands of peptide constructs are made. For instance, when all
constructs in which both segments are, for instance, twelve amino
acids long are derived from a small protein with a length of 100
amino acid residues are needed, already 89.times.89=7,921 peptide
constructs are made if the segments are only linked to the solid
phase, for instance, via the C-terminus for the first segment and
the N-terminus of the second segment, or visa versa, or both, using
only one type of link. For a protein with a length of 1,000 amino
acid residues, at least 989.times.989=978,121 constructs are made.
For efficient ELISA testing of these numbers of constructs, high
construct densities on the solid support are preferred. High
densities of constructs on a solid support are provided by the
invention, wherein, for instance, (a layer of) a first segment with
a bromoacetamide group at the N-terminus is synthesized on a
surface of, for instance, 1 cm.sup.2. On yet another part of the
surface, another first-segment may be applied. On each of such a
peptide-functionalized surface of the support, a set of, for
instance, 10, preferably 50, preferably 100, or more second,
peptide segments containing a free thiol group are spotted or
gridded in a positionally or spatially addressable way, giving,
after coupling, so many different peptide pairs. Spotting can, for
instance, be done using piezo drop-on-demand technology, or by
using miniature solenoid valves. Gridding can, for instance, be
done using a set of individual needles that pick up sub-microliter
amounts of segment solution from a microtiter plate containing
solutions comprising the second segments. After the linking
reaction, subsequent deprotection and extensive washing of the
support to remove uncoupled peptide gives at least a peptide
construct pair density as large as 10 to 50, or even 100 to 200, or
up to 50 to 1000 spotted pairs per square centimeter. This density
allows the screening of a great many possible peptide pairs or
binding bodies derived from the proteins for binding with an
antibody. For example: in a preferred embodiment 20,000 to 100,000
constructs are made on 1000 cm.sup.2. Typically, the surface is
then screened for binding in ELISA with 100 ml of antibody solution
containing 1-10 .mu.g of antibody/ml. For example, indirect or
direct fluorescence detection allocates antibody binding
constructs. Direct fluorescence detection with confocal scanning
detection methods, for example, allows antibody detection on spots
generated with droplets of peptide-solution in the sub-nanoliter
range, making even higher construct densities feasible. Of course,
nucleic acid libraries can be made in a similar fashion.
[0055] Furthermore, provided is a solid support comprising a
library according to the invention, the solid support allowing
presentation of a potential discontinuous or conformational binding
site or epitope to a binding molecule, the solid support having
been provided with a plurality of test entities, each pair or
threesome or larger plurality of the test entities or binding
bodies being a possible representative of the binding site or
epitope and, for example, comprising at least one first peptide or
nucleotide, for example, covalently linked to a solid phase and a
second peptide or nucleotide.
[0056] In a preferred embodiment, the solid support comprises at
least a spot or dot (e.g., putative binding site, test entity, or
pair of segments) density as large as 10, 20, or 50, or even 100,
200, or up to 500 or even 1,000 spots per cm.sup.2, preferably
wherein the spots or dots are positionally or spatially
addressable.
[0057] The invention further provides a method to screen for, i.e.,
test, identify, characterize or detect a discontinuous binding site
capable of interacting with a binding molecule, comprising
screening a library as provided by the invention with binding
molecules, such as there are, for instance, antibodies, soluble
receptors, which contain a Fc-tail or a tag for detection,
receptors on cells, biotinylated molecules or fluorescent
molecules.
[0058] Alternative segments could comprise, for instance,
carbohydrates, non-natural amino acids, PNAs, DNAs, lipids, or
molecules containing peptide bond mimetics. In particular, provided
is a method to screen for a discontinuous binding site capable of
interacting with a binding molecule, comprising screening a library
according to the invention with at least one test entity and
detecting binding between a member of the library and the test
entity. In a preferred embodiment, the binding is detected
immunologically, for example, by ELISA techniques.
[0059] By detecting binding to a specific test entity (herein also
called a binding body) of the library, provided is the member or
binding body a synthetic molecule comprising the binding body or
test entity or pair or larger plurality of (looped) segments
comprising a discontinuous binding site identifiable or identified
or obtainable or obtained by a method according to the invention.
Thus, provided is use of a library according to the invention, use
of a solid support or solid phase or array surface provided with
one or more binding bodies or test entities according to the
invention, or use of a method according to the invention for
identifying or obtaining a synthetic molecule comprising a
discontinuous binding site or a binding molecule capable of binding
therewith. Because discontinuous binding sites are now provided,
such a synthetic molecule can advantageously be used in vitro or in
vivo for finding a binding molecule and for effecting and/or
affecting binding to a binding molecule, for example, to interact
or bind with receptor molecules that normally interact or bind with
a hormone, a peptide, a drug, an antigen, with an effector
molecule, with an agonist, with an antagonist, or with another
receptor molecule, with enzymes that normally bind with their
substrate, with antibody molecules, with nucleic acid, with
protein--in short--with biomolecules. The invention is further
explained in the detailed description without limiting the
invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0060] FIG. 1: six different cysteines that can be used in coupling
of bromine under different conditions.
[0061] FIG. 2: (spotting with dark coloring) Analysis of two
different peptides for showing the advantageous effect of two-sided
linking and the formation of loops. On the left, the peptide has an
amino-terminal Br. On the right the peptide has an amino-terminal
Br and a C-terminal Lysine-Br (synthesized as described in legend
FIG. 4B). Test was carried out in a miniwell setup (3 ul each
well). Surface is functionalized with thiol groups (--SH groups).
Peptides were coupled to the surface using the bromine- (Br-)group
of the peptide. Different concentrations of peptide were used for
coupling to the surface. Two sets of peptides were used, one with
one Br-group and the other (differs only from the previous peptide
by an extra lysine +Br-acetyl moiety on the C-terminal site of the
peptide) with two Br-groups. Binding was determined using differed
antibody concentrations in an ELISA setup.
[0062] FIG. 3: Proximity of segments after coupling on
solid-support. On the left side: on a minimal distance of 2
angstrom, linkers of 15 angstrom are coupled. The segments are
coupled to these linkers. The flexibility of the linkers allows the
termini of the two segments to move within distances of 0 to 30
angstroms. On the right side: the distances between the linkers can
be varied from 2 to 50 or more. As an example, 9 angstrom is shown.
This allows the termini of the two segments to move within
distances of 0 to 40 angstroms.
[0063] FIG. 3B: Schematic representation of how the two segments
are linked as loops to the poly-carbon polymer surface. The
preferred distances, at least in the case of CDR-derived binding
bodies, between the tops of the loops are 0 to 30 angstroms, which
is similar to that of the CDRs in an antibody.
[0064] FIG. 4: Schematic representation of how two segments can be
coupled onto the (polycarbon)-polymer surface. The drawing shows
four examples. In Example-1, two linear segments are coupled. In
Example-2, two looped segments are coupled. In Example-3, two
segments are coupled as loops. In Example-4, two segments are
coupled as loops. With extra spaced building blocks (e.g.,
phenylalanine amino acids) two obtain extended loops. On the
(polycarbon)-surface, two types of protected cysteines (cys (trt)
and cys (mmt)) and, for example, one spacing building block is
coupled. The cys (mmt) is deprotected with 1% TFA, while the cys
(trt) remains protected. The first segment is coupled to the
deprotected cys (mmt). Then, the second cys (trt) is deprotected
with 95% TFA. Then, the second segment is coupled to the now
deprotected cys (trt).
[0065] FIG. 4B: Schematic representation of how two segments can be
coupled onto a cyclic template that itself is coupled to the
polymer surface. The cyclic template is a cyclic flexible peptide.
The cyclic peptide contains four lysines (mmt), two cysteines (trt)
and two cysteines (butyl). The peptide is coupled to the resin via
a sulfur that is sensitive to 1% TFA. At the amino-terminus, a
bromine is attached as described previously. The procedure is as
follows: The synthesized peptide is treated with 1% TFA. This
results in deprotection of the lysines and de-coupling of the
peptide from the resin. The cysteines remain protected. After
raising the pH to 8, the N- and C-terminus of the peptide are
linked through the S and Br. Then, the --NH.sub.2 on the
deprotected lysines is coupled to Br. The resulting cyclic peptide,
with four Br and still four protected cysteines, is coupled to the
linkers via the Br. To the cyclic template coupled to the
linker-cysteines, two peptide segments are coupled. First, the two
cysteines (trt) are deprotected with 95% TFA. Then, the first
segment is coupled. Second, the two cys (butyl) are deprotected
with NaBH.sub.4. Then, the second segment is coupled.
[0066] FIG. 4C: Schematic representation of how two segments can be
coupled onto two other segments that are coupled to the polymer
surface. With free --SH on the surface, two segments are coupled to
the surface via a N- and C-terminal Br. The N-terminal Br is
synthesized as described previously. The C-terminal Br is linked to
a C-terminal Lysine as described in FIG. 4B. Both segments contain
protected cysteines on which two other segments are also coupled,
as described in FIG. 4B.
[0067] FIG. 5: Schematic representation of matrix-scan with two
segments. On the polymer surface, a mixture of cys (mmt) and cys
(trt) are coupled. After 1% TFA, the cys (mmt) is deprotected.
Then, in each square one peptide is coupled via one or two terminal
Br. Thus, peptide-1 in square-1, peptide-2 in square-2, etc., to
peptide-100 in square-100. Then, the cys(trt) is deprotected with
95% TFA. Then, 100 different peptides are spotted in each square.
Thus, peptide-1 to 100 in square-1, peptide 1 to 100 in square-2,
etc., to peptide-1 to peptide-100 in square-100.
[0068] FIG. 6: Binding-assay of all overlapping 30-mers covering
the linear sequence of hFSHR with the biotinylated synthetic 40-mer
hFSH-peptide biotin-EKEEARFCISINTTWAAGYAYTRDLVYKDPARPKIQKTAT-CONH2
(SEQ ID NO: 1 of the accompanying SEQUENCE LISTING, the contents of
which are incorporated herein by this reference). The 30-mer
peptides were spotted as described, and the 40-mer peptides were
synthesized using standard FMOC-chemistry. The various 30-mer
peptides were incubated with 1 microgram/ml hFSH-peptide. After
washing, the peptides were incubated with streptavidin-peroxidase,
and subsequently after washing, with peroxidase substrate and
H.sub.2O.sub.2.
[0069] FIG. 7: Schematic representation of the development of
synthetic mimics of discontinuous binding sites on the hTSHR and
hTSH. On thyroid cells, the hTSH-receptor binds hTSH. The
autoimmune antibodies from Graves and Hashimoto patients also bind
the hTSH-receptor. Through screening of all overlapping 30-mers of
hTSH, segments of the discontinuous binding site for hTSHR are
identified (as described for FSH, see legend FIG. 6). Through
screening of all overlapping 30-mers of hTSHR, segments of the
discontinuous binding sites for Graves and Hashimoto antibodies are
identified. Through modeling and usage of synthetic templates, the
individual segments are combined into one discontinuous synthetic
mimic.
[0070] FIG. 8: Schematic representation of an array comprising
synthetic mimics of discontinuous binding sites or binding bodies.
Binding bodies are selected and improved by making arrays that
contain a multiplicity of spatially addressable binding bodies on
the solid surface (or, alternatively, on a separate molecular
scaffold). The arrays can be incubated with target to screen for
binding bodies that bind the target of interest. Lead binding
bodies can be improved by making follow-up arrays composed of
multiple variants of the lead binding bodies, e.g., by sequence
shuffling. If the desired specificity and/or affinity is reached,
the binding bodies can be produced onto a scaffold and produced and
used in bulk.
[0071] FIG. 9: Schematic representation of the development of
synthetic mimics of discontinuous binding sites or binding bodies
derived from CDR sequences. Binding bodies are constructed by
positioning on a solid phase or array surface (preferably a
(polycarbon)-polymer surface) or on predefined scaffolds or
templates. Binding bodies can be derived from the Complementarity
Determining Regions (CDRs) of antibodies or any other protein motif
that is known to bind other molecules, preferably with high
affinity.
[0072] FIG. 10: Standard linear Pepscan on all overlapping
synthetic 12-mers covering the linear sequence of hTNF with
monoclonal antibody 210 (R&D Systems, MAB210, clone 1825.12,
through ITK Diagnostics Uithoorn, The Netherlands). A small peak
with the sequence IKSPCQRETPEG (SEQ ID NO: 2) was identified. The
y-axis are optical density values (OD) obtained using a ccd-camera
system. Rampo, rabbit-anti-mouse peroxidase (DAKO).
[0073] FIG. 11: Partial listing of peptides synthesized for
loop-loop 15-mer Matrix-scan (1. +AVRSSSRTPSDKPVZ (SEQ ID NO: 3);
2. +VRSSSRTPSDKPVAZ (SEQ ID NO: 4); 3. +RSSSRTPSDKPVAHZ (SEQ ID NO:
5); 4. +SSSRTPSDKPVAHVZ (SEQ ID NO: 6); 5. +SSRTPSDKPVAHVVZ (SEQ ID
NO: 7); 145. +FAESGQVYFGIIALZ (SEQ ID NO: 8)). All overlapping
15-mer loop-peptides covering the linear sequence of human tumor
necrosis factor (hTNF) were synthesized, i.e., 145 hTNF
loop-peptides in total. Z is a Cys-butyl. The amino terminus of all
peptides contain a bromo-group (+).
[0074] FIG. 12: Configuration of the loop-loop 15-mer Matrix-scan.
Schematic representation of matrix-scan with two loop segments. On
the polymer surface, a mixture of cys (mmt) and cys (trt) are
coupled. After 1% TFA, the cys (mmt) is deprotected. Then, in each
square, one peptide is coupled via its N-terminal Bromo-group (+).
Thus, peptide-1 in square-1, peptide-2 in square-2, etc., until
peptide-145 in square-145. Then, the cys(trt) is deprotected with
95% TFA. Then, in each square, 145 different peptides are spotted
simultaneously. Thus, peptide-1 to 145 in square-1, peptide 1-145
in square-2, etc., to peptide-1 to peptide-145 in square-145. Some
extra squares were used for controls (linear epitopes).
[0075] FIG. 13: Result of the loop-loop 15-mer Matrix-scan with
anti-hTNF mAb 210 (10 ug/ml). The result obtained with all 145
squares is plotted. Squares 66, 67 and 92 to 96 are clearly labeled
(firstly coupled loop-peptides). On top of these and other squares
spots are labeled as well (spots represent first peptide coupled
next to second loop peptide). Identified squares and spots: Sq-65:
+FKGQGCPSTHVLLTZ (SEQ ID NO: 9); Sq-66: +KGQGCPSTHVLLTHZ (SEQ ID
NO: 10); Sq-67: +GQGCPSTHVLLTHTZ (SEQ ID NO: 11); Sq-87:
+SYQTKVNLLSAIKSZ (SEQ ID NO: 12); Sq-88: +YQTKVNLLSAIKSPZ (SEQ ID
NO: 13); Sq-94: +LLSAIKSPCQRETPZ (SEQ ID NO: 14); Sq-95:
+LSAIKSPCQRETPEZ (SEQ ID NO: 15); Sq-127: +LEKGDRLSAEINRPZ (SEQ ID
NO: 16); Sq-128: +EKGDRLSAEINRPDZ (SEQ ID NO: 17); Peptide-65:
+FKGQGCPSTHVLLTZ (SEQ ID NO: 18); Peptide-70: +CPSTHVLLTHTISRZ (SEQ
ID NO: 19); Peptide-72: +STHVLLTHTISRIAZ (SEQ ID NO: 20);
Peptide-77: +LTHTISRIAVSYQTZ (SEQ ID NO: 21); Peptide-94:
+LLSAIKSPCQRETPZ (SEQ ID NO: 22); Peptide-95: +LSAIKSPCQRETPEZ (SEQ
ID NO: 23); Peptide-99: +KSPCQRETPEGAEAZ (SEQ ID NO: 24);
Peptide-126: +QLEKGDRLSAEINRZ (SEQ ID NO: 25); Peptide-129:
+KGDRLSAEINRPDYZ (SEQ ID NO: 26). The y-axis is in arbitrary
units.
[0076] FIG. 14: Result of the loop-loop 15-mer Matrix-scan with mAb
210 (10 ug/m) with details of squares 65 and 127. Combination of
loop-peptide 65 with loop-peptides 94, 95, combinations of
loop-peptide 65 with 126-127, combinations of loop-peptide 127 with
loop-peptides 65-77 and combinations of loop-peptide 127 with
loop-peptides 94-96 are labeled. The y-axis is in arbitrary
units.
[0077] FIG. 15: Three dimensional representation of the identified
binding loop-loop peptides with mAb-210 (10 ug/ml). Shown are the
three regions identified (peptides 65-69, 94-96 and 126-127):
GQGCPSTHVLLTHTIS (SEQ ID NO: 27) (VLLT are labeled); SAIKSPCQRE
(SEQ ID NO: 28) (KSPC are labeled); KGDRLSAEINR (SEQ ID NO: 29) (SA
are labeled).
[0078] FIG. 16: Result of the loop-loop 15-mer Matrix-scan of
loop-loop CDR-regions of antibodies with lysozyme-biotin (100
ug/ml, in triplo). Using 1 .mu.g/ml lysozyme-biotin no binding is
observed (not shown). Controls of only streptavidin-peroxidase in
between the tests were negative (not shown). Peptides A, B, C, D, E
and F: Peptide-A: +ARERDYRLDYZ (SEQ ID NO: 30) (HCDR3 of 1
fdl.pdb); Peptide-B: +ARGDGNYGYZ (SEQ ID NO: 31) (HCDR3 of
1mlb.pdb); Peptide-C: +LHGNYDFDGZ (SEQ ID NO: 32) (HCDR3 of
3hfl.pdb); Peptide-D: +ANWDGDYZ (SEQ ID NO: 33) (HCDR3 of
3hfm.pdb); Peptide-E: +ARRYGNSFDYZ (SEQ ID NO: 34) (HCDR3 of
1qfw.pdb); Peptide-F: +ARQGTAAQPYWYZ (SEQ ID NO: 35) (HCDR3 of
1qfw.pdb) (1fdl.pdb, 1mlb.pdb, 3hfl.pdb and 3hfm.pdb are antibodies
that bind lysozyme; 1qfw.pdb are two antibodies that bind human
choriogonadotrophin). All peptides have an amino terminal
bromo-group (+) and a carboxyterminal lysine-mmt (Z).
[0079] Peptides 1 to 27: Peptide-1: +RASGNIHNYLAZ (SEQ ID NO: 36)
(LCDR1 of 1fdl.pdb); Peptide-2: +RASQSISNNLHZ (SEQ ID NO: 37)
(LCDR1 of 1mlb.pdb); Peptide-3: +SASSSVNYMYZ (SEQ ID NO: 38) (LCDR1
of 3hfl.pdb); Peptide-4: +RASQSIGNNLHZ (SEQ ID NO: 39) (LCDR1 of
3hfm.pdb); Peptide-5: +RASESVDSYGNSZ (SEQ ID NO: 40) (LCDR1 of
1qfw.pdb); Peptide-6: +ASESVDSYGNSFZ (SEQ ID NO: 41) (LCDR1 of
1qfw.pdb); Peptide-71: +SESVDSYGNSFMZ (SEQ ID NO: 42) (LCDR1 of
1qfw.pdb); Peptide-8: +ESVDSYGNSFMQZ (SEQ ID NO: 43) (LCDR1 of
1qfw.pdb); Peptide-9: +RASESVDSYGNSFZ (SEQ ID NO: 44) (LCDR1 of
1qfw.pdb); Peptide-10: +ASESVDSYGNSFMZ (SEQ ID NO: 45) (LCDR1 of
1qfw.pdb); Peptide-11: +SESVDSYGNSFMQZ (SEQ ID NO: 46) (LCDR1 of
1qfw.pdb); Peptide-12: +RASESVDSYGNSFMZ (SEQ ID NO: 47) (LCDR1 of
1qfw.pdb); Peptide-13: +ASESVDSYGNSFMQZ (SEQ ID NO: 48) (LCDR1 of
1qfw.pdb); Peptide-14: +RASESVDSYGNSFMQZ (SEQ ID NO: 49) (LCDR1 of
1qfw.pdb); Peptide-15: +KASETVDSFVSZ (SEQ ID NO: 50) (LCDR1 of
1qfw.pdb); Peptide-16: +LLVYYTTTLADGZ (SEQ ID NO: 51) (LCDR2 of
1fdl.pdb); Peptide-17: +LLIKYVSQSSSGZ (SEQ ID NO: 52) (LCDR2 of
1mlb.pdb); Peptide-18: +RWIYDTSKLASGZ (SEQ ID NO: 53) (LCDR2 of
3hfl.pdb); Peptide-19: +LLIKYASQSISGZ (SEQ ID NO: 54) (LCDR2 of
3hfm.pdb); Peptide-20: +LLIYRASNLESGZ (SEQ ID NO: 55) (LCDR2 of
1qfw.pdb); Peptide-21: LLIFGASNRESGZ (SEQ ID NO: 56) (LCDR2 of
1qfw.pdb); Peptide-22: +QHFWSTPRTZ (SEQ ID NO: 57) (LCDR3 of
1fdl.pdb); Peptide-23: +QQSNSWPRTZ (SEQ ID NO: 58) (LCDR3 of
1mlb.pdb); Peptide-24: +QQWGRNPTZ (SEQ ID NO: 59) (LCDR3 of
3hfl.pdb); Peptide-25: +QQSNSWPYTZ (SEQ ID NO: 60) (LCDR3 of
3hfm.pdb); Peptide-26: +QQSDEYPYMYTZ (SEQ ID NO: 61) (LCDR3 of
1qfw.pdb); Peptide-27: +GQTYNHPYTZ (SEQ ID NO: 62) (LCDR3 of
1qfw.pdb) (1 fdl.pdb, 1mlb.pdb, 3hfl.pdb and 3hfm.pdb are
antibodies that bind lysozyme; 1 qfw.pdb are two antibodies that
bind human choriogonadotrophin). All peptides have an amioterminal
bromo-group (+) and a carboxyterminal lysine-mmt (Z).
[0080] The loop-loop peptide pair, +LHGNYDFDGZ (SEQ ID NO: 32)
+SESVDSYGNSFMQZ (SEQ ID NO: 46) (loop of HCDR3 of 3hfl.pdb with
loop of LCDR1 of 1qfw.pdb) that has the highest binding activity is
indicated by arrow.
[0081] FIG. 17: Result of Pepscan ELISA with two different
antibodies on single or double peptide loops coupled to Pepscan
minicards, as described above. Coupled to square-A: Loop peptide-1;
Coupled to square-B: first Loop peptide-1 followed by Loop
peptide-2; Coupled to square-C: Loop peptide-2; Coupled to
square-D: first Loop peptide-2 followed by Loop peptide-1. Loop
peptide-1: +KSYNRVTVMGGFKVEZ-conh2 (SEQ ID NO: 63); Loop peptide-2:
+LQENPFFSQPGAPILZ-conh2 (SEQ ID NO: 64). The y-axis are optical
density values (OD) obtained using a ccd-camera system. Both
loop-peptides are derived from human Follicle-Stimulating Hormone
(hFSH).
DETAILED DESCRIPTION OF THE INVENTION SYNTHESIS OF PEPTIDE
CONSTRUCTS
[0082] A polypropylene or polyethylene support, or of other
suitable material, was grafted with, for instance, polyacrylic
acid. As an example: a polypropylene support in a 6% acrylic acid
solution in water containing CuSO.sub.4 was irradiated using gamma
radiation at a dose of 12 kGy. The grafted solid support containing
carboxylic acid groups was functionalized with amino groups via
coupling of t-butyloxycarbonyl-hexamethylenediamine (Boc-HMDA)
using dicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole
(HOBt) and subsequent cleavage of the Boc groups using
trifluoroacetic acid. Subsequently, the surface is functionalized
with (when preferred, a mixture of differently protected) Cys amino
acids using standard Fmoc chemistry. Examples of differently
protected Cys groups are Cys (Trt) and Cys (mmt). After removal of
the FMOC, the amino group is acetylated. Side chain deprotection
can be done as described. Standard Fmoc peptide synthesis chemistry
was used to link peptides (segments) on to the amino functionalized
solid support. After cleavage of the Fmoc group of the last amino
acid and washing, bromoacetic acid was coupled using DCC or
DCC/HOBt. A second bromoacetic acid (in the same step) can be
coupled to the surface when, for example, a lysine (Lys) residue is
present in the peptide: The side chain protection chemistry of Lys
(using FMOC-Lys(MTT)-OH) allows that only the amino group of the
Lys-side chain is liberated (with 1% trifluoracetic acid in
dichloromethane), while the other amino acids still stay protected.
Subsequently, if only DCC was used, the peptide did contain a thiol
reactive bromoacetamide group. However, if DCC/HOBt was used to
couple bromoacetic acid, the peptide essentially did not contain
the bromo group, but another reactive group capable of reacting
efficiently with thiol groups, thus forming the same thioether link
between the segments. Coupling/ligation of a second peptide next to
a peptide coupled or synthesized on a solid support: Bromo
functionalized peptides can be coupled to the solid support (when a
thiol is present) in an aqueous solution containing a sodium
bicarbonate buffer at about ph 7-8. Peptides were synthesized at
polyethylene pins grafted with poly-hydromethylmethacrylate
(poly-HEMA). This graft polymer was made by gamma irradiation of
polyethylene pins in a 20% HEMA solution in methanol/water 80/20 or
70/30 at a dose of 30-50 kGy. The functionalized support can be
used for the synthesis of 1 .mu.mol of peptide/cm.sup.2 after
coupling of .beta.-alanine and an acid labile
Fmoc-2,4-dimethoxy-4'-(carboxymethyloxy)-benzhydrylamine (Rink)
linker. The peptides were synthesized using standard Fmoc
chemistry, and the peptide was deprotected and cleaved from the
resin using trifluoroacetic acid with scavengers. The cleaved
peptide containing a cysteine residue at a concentration of about 1
mg/ml was reacted with the solid support described above in a
water/sodium bicarbonate buffer at about pH 7-8, thus forming a
partially protected construct of two peptides each at least once
covalently bound via a thioether bond to the solid support. The
construct described above was deprotected following standard
procedures using trifluoroacetic acid/scavenger combinations. The
deprotected constructs on the solid support were extensively washed
using disrupting buffers containing sodium dodecylsulfate and
.beta.-mercaptoethanol and ultrasonic cleaning and were used
directly in ELISA. Subsequent cleaning in the disrupt buffers
allows repeated testing against antibodies in ELISA.
[0083] According to these methods, a library of constructs, for
example, consisting of a dodecapeptide segment coupled via its
C-terminally added cysteine residue next to a N-terminally
bromoacetylated second segment, allowing scanning a protein
sequence, for example, by steps of a single amino acid residue. The
bromoacetamide peptide was covalently bound to a functionalized
polypropylene/polyacrylic acid solid support in 3 .mu.l wells, as
described above. The cysteine-containing sequences are synthesized
on and cleaved from functionalized polyethylene pins, as described
above. Peptides are synthesized on a surface of a solid support, as
described above. On this peptide functionalized support, a second
peptide segment containing a free thiol group was spotted using
piezo drop-on-demand technology using a micro-dosing apparatus and
piezo autopipette (Auto prop-Micropipette AD-K-501) (Microdrop
Gesellschaft fur Mikrodosier Systeme GmbH). Alternatively, spotting
or gridding was done using miniature solenoid valves (INKX
0502600A; the Ice Company) or hardened precision ground gridding
pins (Genomic Solutions, diameters 0.4, 0.6, 0.8 or 1.5 mm).
Subsequent deprotection of the construct and extensive washing to
remove uncoupled peptide gave binding body constructs at the
spotted area. Peptide constructs generated with peptide solution
droplets in the nanoliter-range bind enough antibody for detection,
in this case using indirect fluorescence detection. Spots generated
with 0.25 nl-50 nl are smaller than 1 mm.sup.2. Thus, in this
set-up, binding body density can be as large as 100-1000 spots per
square centimeter. When using smaller equipment, densities can even
be higher.
[0084] In short, a thiol function is introduced on an
amino-functionalized solid support. This can be made by a direct
reaction of the amino groups with, for instance, iminothiolane or
by coupling of Fmoc-Cys(Trt)-OH, followed by Fmoc cleavage using
piperidine, acetylation, and trityl deprotection using
TFA/scavenger mixtures. This thiol-functionalized solid support can
be reacted with, for instance, a bromoacetamide-peptide containing
a protected cysteine residue. After coupling of the first peptide,
the cysteine can be deprotected using, for instance, a
TFA/scavenger mixture. As yet unused free thiol groups can be used
to couple a second bromoacetamide-peptide, again containing a
protected cysteine. This procedure can be repeated to make segment
constructs. Several types of scans can be used in combination with
this multi-segment scan.
Examples of Use
[0085] Proteins and peptides can be screened using any type of
binding molecule, e.g., biomolecules such as antibodies, soluble
receptors (which contain a Fc-tail or a tag for detection),
biotinylated molecules or fluorescent molecules. Alternative
segments could be, for instance, carbohydrates, non-natural amino
acids, PNAs, DNAs, lipids, and molecules containing peptide bond
mimetics.
TSH Example
[0086] The design and synthesis of synthetic mimics of
discontinuous binding sites of large proteins such as TSH or TSHR
is currently desired. Toward this aim, template based mimics of
proteins have provided a powerful new tool for basic research.
Technology provided herein enables one to map discontinuous binding
sites, couple these onto a synthetic template and monitor in detail
the structural and functional characteristics.
[0087] Pivotal to this approach is the possibility of synthesizing
and testing of 100,000s of synthetic peptides in array-format. This
is possible with the technologies provided herein. These include
peptide-array synthesis and new methodology in template chemistry.
Through chemistry, all kinds of synthetic groups are coupled on two
or more different positions on these templates, allowing
reconstruction of the discontinuous binding sites and the synthesis
of mimics. The development of methods that allow mapping of
discontinuous binding sites between large proteins is a major
research target. Various strategies have been adopted with moderate
success. The most successful techniques to date include X-ray
crystallography, Combinatorial libraries and Mass-Spectrometry.
[0088] We provide a new approach involving peptide-arrays. Peptide
array technology has long been used to identify short linear
peptides involved in binding. All overlapping linear peptides
(12-15-mers) of a given protein are synthesized on a solid-support
such as plastic or paper and incubated with the target protein,
most often an antibody. Those peptides that are recognized are
so-called linear epitopes. Discontinuous epitopes could not be
detected. Nevertheless, the early peptide-array technology laid the
foundation for methods that identify discontinuous epitopes in a
systematic fashion. This made it possible to couple on an array
surface any part of a protein (for instance, a peptide of 15 amino
acids long) next to any other part of a protein (for instance, a
peptide of 15 amino acids long) in any orientation. These arrays,
with all possible combinations of peptides, showed in our hands to
allow accurate definition of discontinuous epitopes (FIG. 2).
[0089] We now focus on discontinuous epitopes involved in Graves
disease and Hashimoto disease, but others are as well within reach.
The thyroid diseases are autoimmune diseases against the thyroid.
The antibodies bind discontinuous epitopes on the thyrotropin
receptor on the thyroid gland. Over activation (Graves) or blockage
(Hashimoto) of the thyroid gland leads to serious health problems.
Mapping of both the antibody binding regions as well as the TSH
binding region greatly contributes to the understanding of both
diseases. Subsequently, hTSH and hTSHR mimics of these
discontinuous epitopes will be used in new diagnostic tools
allowing early discovery of Graves and Hashimoto disease. Studies
on human Follicle-Stimulating Hormone (hFSH) and its receptor
(hFSHR) have revealed discontinuous binding sites. Biotinylated
40-mers covering various regions of hFSH were tested in a
peptide-array binding-assay as herein provided on all overlapping
30-mers covering the linear sequence of hFSHR.
[0090] One of the 40-mers clearly bound to a receptor region (FIG.
1). Based on these results a similar study on the hTSH/hSHR couple
hTSHR, a hormone-receptor couple that is structurally very similar
to the hFSH/hFSHR couple, provides peptides that can be used as
diagnostic tools for Graves and/or Hashimoto disease. Patients with
Graves or Hashimoto disease develop antibodies against their own
thyroid receptors which leads to hyper- or hypothyroidism,
respectively. Although the population of antithyrotropin receptor
antibodies is heterogeneous, most Graves antibodies bind the
N-terminus of the receptor, whereas most Hashimoto antibodies bind
the C-terminus of the receptor. In our study, panels of Graves and
Hashimoto sera are tested a) for binding in a peptide-array to the
set of overlapping 30-mers covering the hTSH-receptor; b) in a
competition-assay in which the binding of biotinylated 40-mer
TSH-peptides to hTSH-receptor is competed with Graves and Hashimoto
sera. In this way, discontinuous binding sites are mapped.
[0091] After mapping the discontinuous binding sites, synthetic
mimics are designed and synthesized. A primary strategy for
synthesis of this kind of synthetic mimics is the synthesis of
templates onto which the discontinuous epitope can be
reconstructed. The use of templates facilitates the possibility to
add various parts of the discontinuous epitope. In this way, hardly
any specific binding information will be lost by a high flexibility
of the peptide constructs. Attachment of peptides to template
structures will closely mimic the native discontinuous epitopes.
Recently, much progress has been made in this field. By using
stable templates as a framework on which to couple recognition
fragments, peptides can be obtained with desired activity.
Further Examples
Examples of Use
[0092] Mapping discontinuous epitope on human Tumor Necrosis Factor
(hTNF) (FIGS. 10-15).
[0093] The monoclonal antibody mAb-210 raised against hTNF was
tested on linear and loop peptides (mAb-210 was bought from R&D
Systems, MAB210, clone 1825.12, through ITK Diagnostics Uithoorn,
The Netherlands). Firstly, it was tested in Pepscan on all
overlapping linear 12-mers covering hTNF. This resulted in a minor
peak around sequence IKSPCQRETPEG (SEQ ID NO: 2) (FIG. 10).
Secondly, it was tested in Pepscan matrix-scan on double 15-mer
loop-loop peptides (as described in FIGS. 3 and 4 and explained
through FIGS. 11-12). Two loop-regions were labeled: peptide
sequence GQGCPSTHVLLT (SEQ ID NO: 65) (squares 65 to 67) and
SAIKSPCQRE (SEQ ID NO: 28) (squares 92 to 96) (FIGS. 13, 14). In
addition in various squares loop peptide spots were identified
corresponding to sequence GQGCPSTHVLLT (SEQ ID NO: 65) (spots
65-67); SAIKSPCQRE (SEQ ID NO: 28) (spots 92-96) and KGDRLSAEINR
(SEQ ID NO: 29) (spots 126-129) (FIG. 14). These three regions,
illustrated in FIG. 15 on the three-dimensional model of hTNF, are
located on one side of the hTNF molecule and form one large
discontinuous epitope region.
Identification of synthetic mimics of antibodies (binding bodies)
(FIG. 16).
[0094] From six different antibodies, the HCDR3-region
(complementary determining region three of the antibody heavy
chain) was synthesized as synthetic loop-peptides. As an example,
four different anti-lysozyme antibodies and two different
anti-choriogonadotrophin antibodies were selected: 1fdl.pdb (D1.3),
1mlb.pdb (D44.1), 3hfl.pdb (HyHel-5), 3hfm.pdb (HyHel-10) all
anti-lysozyme, and 1qfw.pdb, two anti-human choriogonadotrophin,
one anti-alpha and one anti-beta. The synthetic loop peptides were
coupled to the minicards as described above. The three-dimensional
coordinates (pdb-files) were extracted from the Protein Data Bank
(PDB) at www.rcsb.org (RCSB, Research Collaboratory for Structural
Bioinformatics) (Berman et al., 2000, The Protein Data Bank.
Nucleic Acids Research, 28 pp. 235-242; Bernstein et al. 1977, The
protein data bank: A computer-based archival file for
macromolecular structures. J. Mol. Biol. 112:535-542).
[0095] Together with each of the six peptides, 27 different other
loop peptides were coupled to the minicard as described in FIG. 3B:
thus, group-1 was a loop of HCDR3 of 1fdl.pdb coupled next to 27
different loops covering LCDR1, LCDR2 or LCDR3, group-2 was a loop
of 1mlb.pdb coupled next to 27 different loops covering LCDR1,
LCDR2 or LCDR3, etc. (LCDR, complementary determining region three
of the antibody light chain). The 27 different loop peptides
represented LCDR1, LCDR2 or LCDR3 of the same antibodies described
above (1fdl.pdb, 1mlb.pdb, 3hfl.pdb, 3hfm.pdb or 1qfw.pdb).
[0096] The result is shown in FIG. 16 (6 groups with 27 loop-loop
coupled peptides). The six loop-loop coupled peptides with the
highest binding activity were: +LHGNYDFDGZ (SEQ ID NO:
32)+SESVDSYGNSFMQZ (SEQ ID NO: 46) (loop of HCDR3 of 3hfl.pdb and
loop of LCDR11 qfw.pdb, respectively) (see FIG. 16); +LHGNYDFDGZ
(SEQ ID NO: 32)+RASESVDSYGNSFMQZ (SEQ ID NO: 49) (loop of HCDR3 of
3hfl.pdb and loop of LCDR1 1qfw.pdb, respectively); +LHGNYDFDGZ
(SEQ ID NO: 32)+RASESVDSYGNSFZ (SEQ ID NO: 44) (loop of HCDR3 of
3hfl.pdb and loop of LCDR1 1qfw.pdb, respectively); +LHGNYDFDGZ
(SEQ ID NO: 32)+ASESVDSYGNSFMZ (SEQ ID NO: 45) (loop of HCDR3 of
3hfl.pdb and loop of LCDR1 1qfw.pdb, respectively); +LHGNYDFDGZ
(SEQ ID NO: 32)+ASESVDSYGNSFZ (SEQ ID NO: 41) (loop of HCDR3 of
3hfl.pdb and loop of LCDR11 qfw.pdb, respectively); +LHGNYDFDGZ
(SEQ ID NO: 32)+LLVYYTTTLADGZ (SEQ ID NO: 51) (loop of HCDR3 of
3hfl.pdb and loop of LCDR2 1fdl.pdb, respectively).
[0097] The loop-loop peptide pair, +LHGNYDFDGZ (SEQ ID NO:
32)+SESVDSYGNSFMQZ (SEQ ID NO: 46) (loop of HCDR3 of 3hfl.pdb with
loop of LCDR1 of 1 qfw.pdb, respectively) that has the highest
binding activity is indicated by an arrow (FIG. 16). This loop-loop
peptide pair is derived from an anti-lysozyme antibody and an
anti-human choriogonadotrophin antibody. The results shown in FIG.
16 shows that particular pairs of synthetic CDRs show better
binding to lysozyme than other pairs, especially group-C.
Therefore, loop-loop combinations of synthetic loops representing
different CDRs of (different) antibodies, not necessarily derived
from the original antibody which in this example is an
anti-lysozyme antibody, can be used to identify lead synthetic
compounds that mimic antibodies. Construction of a double-loop
mimic of a discontinuous epitope (FIG. 17).
[0098] Two peptides that constitute two separate parts of a
discontinuous epitope were coupled to the surface of a minicard as
described above in the legend of FIG. 12 (cf. FIG. 3A and FIG. 4
(example-4)). A cys(mmt) was coupled alone or in combination with a
cys(trt) (in a 1:1 ratio) and/or val(mmt) (the cys and val in a
1:1, 1:3, 1:9 etc. ratio). In this way one peptide was coupled
(squares A and C) or two peptides with increasing valines in
between the cysteines were coupled (squares B and D) (cf. FIG. 4B
(example-4), FIG. 17). These four configurations were incubated
with two different antibodies.
[0099] Antibody-1 recognized, when the individual loop peptides are
coupled as a single loop, only loop peptide-2. Antibody-2
recognized, when the individual loop peptides are coupled as a
single loops, only loop peptide-1. When the two loop peptides are
combined, antibody-1 showed a higher binding activity with
peptide-1 as coupled first. When the two loop peptides are
combined, antibody-2 showed not a higher binding activity.
[0100] The results shown in FIG. 17 show that particular pairs of
synthetic loops of a discontinuous epitope show improved binding to
a particular antibody. Therefore, combinations of synthetic loops
that are part of a discontinuous epitope can be used to identify
lead synthetic compounds that mimic discontinuous epitopes.
Sequence CWU 1
1
65140PRTArtificialSynthetic hFSH peptide 1Glu Lys Glu Glu Ala Arg
Phe Cys Ile Ser Ile Asn Thr Thr Trp Ala1 5 10 15Ala Gly Tyr Ala Tyr
Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg 20 25 30Pro Lys Ile Gln
Lys Thr Ala Thr 35 40212PRTArtificialSynthetic 12-mer from linear
sequence of hTNF 2Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly1
5 10315PRTArtificialSynthetic 15-mer loop-peptide from linear
sequence of hTNF 3Ala Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys
Pro Val Glx1 5 10 15415PRTArtificialSynthetic 15-mer loop-peptide
from linear sequence of hTNF 4Val Arg Ser Ser Ser Arg Thr Pro Ser
Asp Lys Pro Val Ala Glx1 5 10 15515PRTArtificialSynthetic 15-mer
loop-peptide from linear sequence of hTNF 5Arg Ser Ser Ser Arg Thr
Pro Ser Asp Lys Pro Val Ala His Glx1 5 10
15615PRTArtificialSynthetic 15-mer loop-peptide from linear
sequence of hTNF 6Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala
His Val Glx1 5 10 15715PRTArtificialSynthetic 15-mer loop-peptide
from linear sequence of hTNF 7Ser Ser Arg Thr Pro Ser Asp Lys Pro
Val Ala His Val Val Glx1 5 10 15815PRTArtificialSynthetic 15-mer
loop-peptide from linear sequence of hTNF 8Phe Ala Glu Ser Gly Gln
Val Tyr Phe Gly Ile Ile Ala Leu Glx1 5 10
15915PRTArtificialSynthetic 15-mer loop-peptide from linear
sequence of hTNF 9Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu
Leu Thr Glx1 5 10 151015PRTArtificialSynthetic 15-mer loop-peptide
from linear sequence of hTNF 10Lys Gly Gln Gly Cys Pro Ser Thr His
Val Leu Leu Thr His Glx1 5 10 151115PRTArtificialSynthetic 15-mer
loop-peptide from linear sequence of hTNF 11Gly Gln Gly Cys Pro Ser
Thr His Val Leu Leu Thr His Thr Glx1 5 10
151215PRTArtificialSynthetic 15-mer loop-peptide from linear
sequence of hTNF 12Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile
Lys Ser Glx1 5 10 151315PRTArtificialSynthetic 15-mer loop-peptide
from linear sequence of hTNF 13Tyr Gln Thr Lys Val Asn Leu Leu Ser
Ala Ile Lys Ser Pro Glx1 5 10 151415PRTArtificialSynthetic 15-mer
loop-peptide from linear sequence of hTNF 14Leu Leu Ser Ala Ile Lys
Ser Pro Cys Gln Arg Glu Thr Pro Glx1 5 10
151515PRTArtificialSynthetic 15-mer loop-peptide from linear
sequence of hTNF 15Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr
Pro Glu Glx1 5 10 151615PRTArtificialSynthetic 15-mer loop-peptide
from linear sequence of hTNF 16Leu Glu Lys Gly Asp Arg Leu Ser Ala
Glu Ile Asn Arg Pro Glx1 5 10 151715PRTArtificialSynthetic 15-mer
loop-peptide from linear sequence of hTNF 17Glu Lys Gly Asp Arg Leu
Ser Ala Glu Ile Asn Arg Pro Asp Glx1 5 10
151815PRTArtificialSynthetic 15-mer loop-peptide from linear
sequence of hTNF 18Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu
Leu Thr Glx1 5 10 151915PRTArtificialSynthetic 15-mer loop-peptide
from linear sequence of hTNF 19Cys Pro Ser Thr His Val Leu Leu Thr
His Thr Ile Ser Arg Glx1 5 10 152015PRTArtificialSynthetic 15-mer
loop-peptide from linear sequence of hTNF 20Ser Thr His Val Leu Leu
Thr His Thr Ile Ser Arg Ile Ala Glx1 5 10
152115PRTArtificialSynthetic 15-mer loop-peptide from linear
sequence of hTNF 21Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr
Gln Thr Glx1 5 10 152215PRTArtificialSynthetic 15-mer loop-peptide
from linear sequence of hTNF 22Leu Leu Ser Ala Ile Lys Ser Pro Cys
Gln Arg Glu Thr Pro Glx1 5 10 152315PRTArtificialSynthetic 15-mer
loop-peptide from linear sequence of hTNF 23Leu Ser Ala Ile Lys Ser
Pro Cys Gln Arg Glu Thr Pro Glu Glx1 5 10
152415PRTArtificialSynthetic 15-mer loop-peptide from linear
sequence of hTNF 24Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala
Glu Ala Glx1 5 10 152515PRTArtificialSynthetic 15-mer loop-peptide
from linear sequence of hTNF 25Gln Leu Glu Lys Gly Asp Arg Leu Ser
Ala Glu Ile Asn Arg Glx1 5 10 152615PRTArtificialSynthetic 15-mer
loop-peptide from linear sequence of hTNF 26Lys Gly Asp Arg Leu Ser
Ala Glu Ile Asn Arg Pro Asp Tyr Glx1 5 10 152716PRTHuman 27Gly Gln
Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser1 5 10
152810PRTHuman 28Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu1 5
102911PRTHuman 29Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg1 5
103011PRTArtificialHCDR3 of 1fld.pdb 30Ala Arg Glu Arg Asp Tyr Arg
Leu Asp Tyr Glx1 5 103110PRTArtificialHCDR3 of 1mlb.pdb 31Ala Arg
Gly Asp Gly Asn Tyr Gly Tyr Glx1 5 103210PRTArtificialHCDR3 of
3hfl.pdb 32Leu His Gly Asn Tyr Asp Phe Asp Gly Glx1 5
10338PRTArtificialHCDR3 of 3hfm.pdb 33Ala Asn Trp Asp Gly Asp Tyr
Glx1 53411PRTArtificialHCDR3 of 1qfw.pdb 34Ala Arg Arg Tyr Gly Asn
Ser Phe Asp Tyr Glx1 5 103513PRTArtificialHCDR3 of 1qfw.pdb 35Ala
Arg Gln Gly Thr Ala Ala Gln Pro Tyr Trp Tyr Glx1 5
103612PRTArtificialLCDR1 of 1fdl.pdb 36Arg Ala Ser Gly Asn Ile His
Asn Tyr Leu Ala Glx1 5 103712PRTArtificialLCDR1 of 1mlb.pdb 37Arg
Ala Ser Gln Ser Ile Ser Asn Asn Leu His Glx1 5
103811PRTArtificialLCDR1 of 3hfl.pdb 38Ser Ala Ser Ser Ser Val Asn
Tyr Met Tyr Glx1 5 103912PRTArtificialLCDR1 of 3hfm.pdb 39Arg Ala
Ser Gln Ser Ile Gly Asn Asn Leu His Glx1 5 104013PRTArtificialLCDR1
of 1qfw.pdb 40Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Glx1
5 104113PRTArtificialLCDR1 of 1qfw.pdb 41Ala Ser Glu Ser Val Asp
Ser Tyr Gly Asn Ser Phe Glx1 5 104213PRTArtificialLCDR1 of 1qfw.pdb
42Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met Glx1 5
104313PRTArtificialLCDR1 of 1qfw.pdb 43Glu Ser Val Asp Ser Tyr Gly
Asn Ser Phe Met Gln Glx1 5 104414PRTArtificialLCDR1 of 1qfw.pdb
44Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Glx1 5
104514PRTArtificialLCDR1 of 1qfw.pdb 45Ala Ser Glu Ser Val Asp Ser
Tyr Gly Asn Ser Phe Met Glx1 5 104614PRTArtificialLCDR1 of 1qfw.pdb
46Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met Gln Glx1 5
104715PRTArtificialLCDR1 of 1qfw.pdb 47Arg Ala Ser Glu Ser Val Asp
Ser Tyr Gly Asn Ser Phe Met Glx1 5 10 154815PRTArtificialLCDR1 of
1qfw.pdb 48Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met Gln
Glx1 5 10 154916PRTArtificialLCDR1 of 1qfw.pdb 49Arg Ala Ser Glu
Ser Val Asp Ser Tyr Gly Asn Ser Phe Met Gln Glx1 5 10
155012PRTArtificialLCDR1 of 1qfw.pdb 50Lys Ala Ser Glu Thr Val Asp
Ser Phe Val Ser Glx1 5 105113PRTArtificialLCDR2 of 1fdl.pdb 51Leu
Leu Val Tyr Tyr Thr Thr Thr Leu Ala Asp Gly Glx1 5
105213PRTArtificialLCDR2 of 1mlb.pdb 52Leu Leu Ile Lys Tyr Val Ser
Gln Ser Ser Ser Gly Glx1 5 105313PRTArtificialLCDR2 of 3hfl.pdb
53Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Glx1 5
105413PRTArtificialLCDR2 of 3hfm.pdb 54Leu Leu Ile Lys Tyr Ala Ser
Gln Ser Ile Ser Gly Glx1 5 105513PRTArtificialLCDR2 of 1qfw.pdb
55Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Glx1 5
105613PRTArtificialLCDR2 of 1qfw.pdb 56Leu Leu Ile Phe Gly Ala Ser
Asn Arg Glu Ser Gly Glx1 5 105710PRTArtificialLCDR3 of 1fdl.pdb
57Gln His Phe Trp Ser Thr Pro Arg Thr Glx1 5
105810PRTArtificialLCDR3 of 1mlb.pdb 58Gln Gln Ser Asn Ser Trp Pro
Arg Thr Glx1 5 10599PRTArtificialLCDR3 of 3hfl.pdb 59Gln Gln Trp
Gly Arg Asn Pro Thr Glx1 56010PRTArtificialLCDR3 of 3hfm.pdb 60Gln
Gln Ser Asn Ser Trp Pro Tyr Thr Glx1 5 106112PRTArtificialLCDR3 of
1qfw.pdb 61Gln Gln Ser Asp Glu Tyr Pro Tyr Met Tyr Thr Glx1 5
106210PRTArtificialLCDR3 of 1qfw.pdb 62Gly Gln Thr Tyr Asn His Pro
Tyr Thr Glx1 5 106316PRTHuman 63Lys Ser Tyr Asn Arg Val Thr Val Met
Gly Gly Phe Lys Val Glu Glx1 5 10 156416PRTHuman 64Leu Gln Glu Asn
Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Glx1 5 10
156512PRTHuman 65Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr1 5
10
* * * * *
References