U.S. patent application number 12/445923 was filed with the patent office on 2011-06-16 for synthetic antibodies.
This patent application is currently assigned to ARIZONA BOARD OF REGENTS, A BODY CORPORATE OF THE STATE OF ARIZONA. Invention is credited to John C. Chaput, Chris W. Diehnelt, Stephen A. Johnston, Neal Woodbury, Hao Yan.
Application Number | 20110143953 12/445923 |
Document ID | / |
Family ID | 39314793 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110143953 |
Kind Code |
A1 |
Johnston; Stephen A. ; et
al. |
June 16, 2011 |
Synthetic Antibodies
Abstract
The present invention provides methods for synthetic antibodies,
methods for making synthetic antibodies, methods for identifying
ligands, and related methods and reagents.
Inventors: |
Johnston; Stephen A.;
(Tempe, AZ) ; Woodbury; Neal; (Tempe, AZ) ;
Chaput; John C.; (Phoenix, AZ) ; Diehnelt; Chris
W.; (Maricopa, AZ) ; Yan; Hao; (Chandler,
AZ) |
Assignee: |
ARIZONA BOARD OF REGENTS, A BODY
CORPORATE OF THE STATE OF ARIZONA
|
Family ID: |
39314793 |
Appl. No.: |
12/445923 |
Filed: |
October 16, 2007 |
PCT Filed: |
October 16, 2007 |
PCT NO: |
PCT/US07/81536 |
371 Date: |
November 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60852040 |
Oct 16, 2006 |
|
|
|
60975442 |
Sep 26, 2007 |
|
|
|
Current U.S.
Class: |
506/9 ; 506/18;
530/387.1 |
Current CPC
Class: |
C07K 16/00 20130101;
C07K 2317/31 20130101; G01N 33/54306 20130101; C07K 16/14 20130101;
C07K 16/2881 20130101; C40B 40/10 20130101 |
Class at
Publication: |
506/9 ;
530/387.1; 506/18 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C07K 16/00 20060101 C07K016/00; C40B 40/10 20060101
C40B040/10 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This application was supported in part by U.S. government
funding under NIAID grant number 5 U54 A1057156 and NCI grant
number 5 U54 CA112952, and thus the U.S. government has certain
rights in the invention.
Claims
1. A method for identifying affinity elements to a target of
interest, comprising (a) contacting a substrate surface comprising
an array of between 10.sup.2 and 10.sup.7 different test compounds
of known composition with a target of interest under conditions
suitable for moderate affinity binding of the target to target
affinity elements if present on the substrate, wherein the target
is not an Fv portion of an antibody, and wherein the different test
compounds are not derived from the target; and (b) identifying test
compounds that bind to the target with at least moderate affinity,
wherein such compounds comprise target affinity elements.
2. The method of claim 1 wherein the substrate surface is
addressable.
3. The method of claim 1 or 2, further comprising (c) identifying
test compounds that do not bind to the target with at least
moderate affinity.
4. The method of any one of claims 1-3, wherein the test compounds
have a molecular weight of between 1000 Daltons and 10,000
Daltons.
5. The method of any one of claims 1-4, wherein the test compounds
are polypeptides.
6. The method of any one of claims 1-5, further comprising
contacting the same substrate surface or a separate substrate
surface with competitor, and determining a ratio of test compound
binding to target versus test compound binding to competitor.
7. The method of any one of claims 1-6 further comprising
identifying combinations of target affinity elements that bind to
different sites on the same target.
8. The method of 7, further comprising determining an appropriate
spacing between the target affinity elements in an affinity element
combination to increases a binding affinity and/or specificity for
the target of the affinity element combination relative to a
binding affinity and/or specificity of the target affinity elements
alone for the target.
9. The method of claim 7 or 8, further comprising linking a
combination of affinity elements, wherein the linker provides a
spacing of between about 0.5 nm and about 30 nm between a first
affinity element and a second affinity element.
10. The method of any one of claims 1-9 wherein one or both of the
first affinity element and the second affinity element have a
dissociation constant for binding to the first target of between
about 1 .mu.M and 500 .mu.M.
11. The method of any one of claims 8-10, further comprising
optimizing binding affinity of one or both of the first affinity
element and the second affinity element to the target.
12. A synthetic antibody made by the method of any one of claims
9-11.
13. A synthetic antibody comprising: (a) a first affinity element
that can bind a first target; (b) a second affinity element that
can bind the first target, and which can bind to the first target
in the presence of the first affinity element bound to the first
target; and (c) a linker connecting the first affinity element and
the second affinity element, wherein one or both of the first
affinity element and the second affinity element have a molecular
weight of at least 1000 Daltons; wherein at least one of the first
affinity element and the second affinity element are not derived
from the first target; wherein the synthetic antibody has an
increased binding affinity and/or specificity for the first target
relative to a binding affinity and/or specificity of the first
affinity element for the first target and relative to a binding
affinity and/or specificity of the second affinity element for the
target; and wherein the first target is not the Fv of an
antibody.
14. The synthetic antibody of claim 13 wherein both the first
affinity element and the second affinity element have a molecular
weight of between about 1000 Daltons and 10,000 Daltons.
15. The synthetic antibody of claim 13 or 14, wherein the linker
provides a spacing of between about 0.5 nm and about 30 nm between
the first affinity element and the second affinity element.
16. The synthetic antibody of any one of claims 13-15 wherein
neither the first affinity element nor the second affinity element
are derived from an Fv region of an antibody.
17. The synthetic antibody of any one of claims 13-16, wherein
neither the first affinity element nor the second affinity element
are derived from the first target.
18. The synthetic antibody of any one of claims 13-17, wherein the
first affinity element and the second affinity element comprise
polypeptides.
19. The synthetic antibody of any one of claims 13-17, wherein the
first affinity element and the second affinity element comprise
nucleic acids.
20. The synthetic antibody of claim 18, wherein a net charge of the
synthetic antibody at a pH 7 is between +2 and -2.
21. The synthetic antibody of any one of claims 13-20 wherein at
least one of the first and second affinity elements is a
non-naturally occurring compound.
22. The synthetic antibody of any one of claims 13-21, wherein the
linker is an amino acid linker.
23. The synthetic antibody of any one of claims 13-21 wherein the
linker is a nucleic acid linker.
24. The synthetic antibody of claim 23, wherein one or both of the
first affinity element and the second affinity element are not
nucleic acids.
25. The synthetic antibody of any one of claims 13-24 wherein the
first affinity element and the second affinity element are
different and bind to separate regions of the first target.
26. The synthetic antibody of any one of claims 13-25 further
comprising a third affinity element connected to the first affinity
element and the second affinity element.
27. The synthetic antibody of claim 26 wherein the third affinity
element can bind to a second target different than the first
target.
28. The synthetic antibody of claim 27, wherein the second affinity
element also binds to the second target, and wherein a spatial
arrangement of the first affinity element, the second affinity
element, and the third affinity element permits only one of the
first target and the second target to be bound by the synthetic
antibody.
29. The synthetic antibody of claim 26, wherein the synthetic
antibody further comprises a fourth affinity element connected to
the first affinity element, the second affinity element, and the
third affinity element, wherein the third and fourth affinity
elements are spatially arranged relative to the first affinity
element and the second affinity element to provide binding of a
further target in the presence of the first target bound to the
synthetic antibody.
30. The synthetic antibody of any one of claims 13-29 further
comprises a first signaling element and a second signaling element,
wherein a spatial relationship of the first signaling element and
the second signaling element are altered to produce a detectable
signal upon target binding to the synthetic antibody.
31. The synthetic antibody of any one of claims 13-30, bound to a
substrate.
32. A substrate comprising: (a) a surface; and (b) a plurality of
synthetic antibodies according to any one of claims 13-30 attached
to the surface.
33. The substrate of claim 32 wherein the plurality of synthetic
antibodies comprises a plurality of different synthetic
antibodies.
34. A method for making a synthetic antibody, comprising connecting
at least a first affinity element and a second affinity element for
a given target via a linker; wherein one or both of the first
affinity element and the second affinity element have a molecular
weight of at least 1000 Daltons; wherein at least one of the first
affinity element and the second affinity element are not derived
from the first target; wherein the synthetic antibody has an
increased binding affinity and/or specificity for the first target
relative to a binding affinity and/or specificity of the first
affinity element for the first target and relative to a binding
affinity and/or specificity of the second affinity element for the
target; and wherein the first target is not the Fv of an
antibody.
35. The method of claim 34 wherein both the first affinity element
and the second affinity element have a molecular weight of between
1000 Daltons and 10,000 Daltons.
36. The method of claim 34 or 35, wherein the linker provides a
spacing of between about 0.5 nm and about 30 nm between the first
affinity element and the second affinity element.
37. The method of any one of claims 34-36 wherein neither the first
affinity element nor the second affinity element are Fv regions of
an antibody.
38. The method of any one of claims 34-37, wherein at least one of
the first affinity element and the second affinity element
comprises a nucleic acid.
39. The method of any one of claims 34-37, wherein at least one of
the first affinity element and the second affinity element
comprises a polypeptide.
40. The method of claim 39, wherein a net charge of the synthetic
antibody at pH 7 is between +2 and -2.
41. A method for ligand identification, comprising: (a) contacting
a substrate surface comprising a target array with one or more
potential ligands, wherein the contacting is done under conditions
suitable for moderate to high affinity binding of the one or more
ligands to suitable targets present on the substrate; and (b)
identifying targets that bind to one or more of the ligands with at
least moderate affinity.
42. The method of claim 41, wherein the one or more potential
ligands are selected from the group consisting of antibodies and
synthetic antibodies according to any one of claims 13-31.
43. The method of claim 41 or 42, wherein the array of targets is
mounted in a flow chamber, wherein (i) a first buffer comprising
the one or more potential ligands is flowed over the addressable
array, (ii) wherein identifying targets that bind to one or more of
the ligands with at least moderate affinity comprises analyzing
real-time affinity data gathered by an array reader; (iii) the
first buffer flow over the addressable array is stopped after at
least moderate binding to the array is detected; (iv) repeating
steps (i)-(iii) a desired number of times using a further buffer
comprising one or more further potential ligands.
44. A method for identifying a synthetic antibody profile for a
test sample of interest, comprising contacting a substrate
comprising a plurality of synthetic antibodies according to the
present invention with a test sample and comparing synthetic
antibody binding to the test sample with synthetic antibody binding
to a control sample, wherein synthetic antibodies that
differentially bind to targets in the test sample relative to the
control sample comprise a synthetic antibody profile for the test
sample
45. The method of claim 44, wherein the control sample is contacted
with the same substrate as the test sample.
46. The method of claim 44 or 45, wherein the test sample is a
disease state test sample.
47. The method of claim 44 or 45, wherein the test sample is a
research test sample.
48. A composition, comprising: (a) a first affinity element bound
to a template nucleic acid strand; (b) a second affinity element
bound to a complementary nucleic acid strand, wherein the first
affinity element and the second affinity element non-competitively
bind to a common target; wherein the template nucleic acid strand
and the complementary nucleic acid strand are annealed via base
pairing to form an assembly; wherein the first affinity element and
the second affinity element are separated in the assembly; and
wherein either the template nucleic acid strand, the complementary
nucleic acid strand, or both, are bound to a surface of a
substrate.
49. An array, comprising a plurality of the compositions of claim
48 bound to a substrate surface, wherein the plurality of
compositions comprises one or both of: (a) a plurality of
compositions wherein the first ligand and the second ligand are the
same for each composition, but wherein the separation of the first
ligand from the second ligand in the assembly differs; and (b) a
plurality of compositions wherein the first ligand and/or the
second ligand are different for each composition.
Description
CROSS REFERENCE
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. Nos. 60/852,040 filed Oct. 16, 2006 and 60/975,442
filed Sep. 26, 2007, incorporated by reference herein in their
entirety.
BACKGROUND OF THE INVENTION
[0003] The basic use of antibodies or ligands is that they can
distinguish one component from others in a complex mixture. The
level of distinction required varies by use. The fundamental
problem in antibody (ligand) development is to find some entity
that can structurally complement a region or regions on the surface
of the target, and that complementation is higher to a necessary
degree above that of other components in the mixture.
[0004] Traditional antibodies are produced by injection of a
protein or genes encoding proteins into an animal, usually multiple
times over 1-4 months. Polyclonal antibodies are directly used from
the serum. They can be affinity purified if a sufficient amount of
the target protein is available. Using hybridoma technology,
individual clones producing one element of the polyclonal
population can be identified and the antibody propagated
indefinitely. This procedure is generally erratic in the quality of
the product, slow, low through put, suffers from contaminants and
is expensive. It also requires killing animals. The most advanced
form of this approach uses genetic immunization.sup.1. For each
antibody the gene corresponding to the protein sequence is
chemically synthesized and injected into the animal's skin with a
gene gun. In parallel a small amount of protein is in vitro
transcribed/translated using the same gene fragment. This protein
is attached to beads for a direct assessment of reactivity. This
system avoids the necessity of protein production for immunization,
contaminants and is relatively high through-put. The quality of the
antibodies is generally higher. However, this system still requires
labor intensive animal handling.sup.2. To produce replenishable
antibody, this system must be coupled to traditional monoclonal
production.sup.3.
[0005] Alternatives to direct production of antibodies in animals
generally involve recurrent selection processes which are
expensive, but more importantly not adaptable to high throughput
methods. Antibodies used clinically have affinities (Kd) for their
targets of 10.sup.-12 to 5.times.10.sup.-8 M/l. This affinity is
generated biologically by selecting mutations in the variable
region of the antibody. The variable region is basically a flexible
peptide held at the N and C-termini. By selecting from the
.about.10.sup.7 variants in any individual and mutationally
improving the sequence, antibody maturation can produce a good
binder to almost any target. The common approach to replicating
this process is to create a very large library (10.sup.9-10.sup.14
members) of molecules with variable nucleic acids or polypeptides
and panning against the target to find the one or few best binders.
A selection process is applied where strong binders out compete
weaker binders.
[0006] This basic approach of panning large libraries is the most
commonly used to find antibody-like elements. However, such panning
has severe limitations. First, since one is looking for a very good
match in interaction using a relatively short peptide or nucleic
acid one has to generate and search large libraries. This is both
time consuming and does not lend it self to high through put. In
most cases, recurrent selection (panning) must be used to find the
perfect match so only the best binding area on a target is found.
It is difficult to find binders to multiple areas on the target.
Other approaches have utilized meticulous application of chemistry
and structural determinations to produce a molecule in which two
small organic molecules were bound by a short rigid linker.
However, this approach demands exquisite chemistry and structural
biology, and the small molecules must be perfectly positioned for
binding, thus putting severe restrictions on the nature of the
linker. Furthermore, the nature of the binding elements, small
organic molecules, is inherently limiting. It has proven very
difficult to find a second site on a given protein that will
sufficiently bind a small organic molecule. On reflection this
makes perfect sense. Since the protein concentration in a cell is
60-100 mg/ml most exposed surfaces of a protein must be non-binding
or all proteins would agglomerate. Therefore, small molecules will
generally only bind in deep pockets on the protein.
[0007] Thus, new methods for ligand discovery and resulting ligands
for use in constructing, for example, synthetic antibodies are
needed in the art.
SUMMARY OF THE INVENTION
[0008] In a first aspect, the present invention provides methods
for identifying affinity elements to a target of interest,
comprising
[0009] (a) contacting a substrate surface comprising an array of
between 10.sup.2 and 10.sup.7 different test compounds of known
composition with a target of interest under conditions suitable for
moderate affinity binding of the target to target affinity elements
if present on the substrate, wherein the target is not an Fv
portion of an antibody, and wherein the different test compounds
are not derived from the target; and
[0010] (b) identifying test compounds that bind to the target with
at least moderate affinity, wherein such compounds comprise target
affinity elements. In one embodiment of the methods of this first
aspect of the invention, the substrate surface is addressable. In
another embodiment, the methods further comprise identifying test
compounds that do not bind to the target with at least moderate
affinity. In a further embodiment, the test compounds have a
molecular weight of between 1000 Daltons and 10,000 Daltons. In a
further embodiment, the test compounds are polypeptides. In another
embodiment, the methods further comprise contacting the same
substrate surface or a separate substrate surface with competitor,
and determining a ratio of test compound binding to target versus
test compound binding to competitor. In a further embodiment, the
methods further comprise identifying combinations of target
affinity elements that bind to different sites on the same target.
The methods may further comprise determining an appropriate spacing
between the target affinity elements in an affinity element
combination to increases a binding affinity and/or specificity for
the target of the affinity element combination relative to a
binding affinity and/or specificity of the target affinity elements
alone for the target. In a further embodiment, the methods comprise
linking a combination of affinity elements, wherein the linker
provides a spacing of between about 0.5 nm and about 30 nm between
a first affinity element and a second affinity element. The methods
may further comprise optimizing binding affinity of one or both of
the first affinity element and the second affinity element to the
target. In a further embodiment, the first aspect provides
synthetic antibodies made by the methods of the first aspect of the
invention.
[0011] In a second aspect, the present invention provides synthetic
antibodies comprising:
[0012] (a) a first affinity element that can bind a first
target;
[0013] (b) a second affinity element that can bind the first
target, and which can bind to the first target in the presence of
the first affinity element bound to the first target; and
[0014] (c) a linker connecting the first affinity element and the
second affinity element,
[0015] wherein one or both of the first affinity element and the
second affinity element have a molecular weight of at least 1000
Daltons;
[0016] wherein at least one of the first affinity element and the
second affinity element are not derived from the first target;
[0017] wherein the synthetic antibody has an increased binding
affinity and/or specificity for the first target relative to a
binding affinity and/or specificity of the first affinity element
for the first target and relative to a binding affinity and/or
specificity of the second affinity element for the target; and
[0018] wherein the first target is not the Fv of an antibody. In a
further embodiment, both the first affinity element and the second
affinity element have a molecular weight of between about 1000
Daltons and 10,000 Daltons. In another embodiment, the linker
provides a spacing of between about 0.5 nm and about 30 nm between
the first affinity element and the second affinity element. In a
further embodiment, neither the first affinity element nor the
second affinity element are derived from an Fv region of an
antibody. In another embodiment, neither the first affinity element
nor the second affinity element are derived from the first target.
In a still further embodiment, the first affinity element and the
second affinity element comprise polypeptides or nucleic acids. In
a further embodiment, the synthetic antibodies further comprise
third or further affinity elements connected to the first affinity
element and the second affinity element. In a further embodiment,
the synthetic antibodies are bound to a substrate.
[0019] In another embodiment, the present invention provides a
substrate comprising:
[0020] (a) a surface; and
[0021] (b) a plurality of synthetic antibodies according to the
second aspect of the invention attached to the surface.
[0022] In a third aspect, the present invention provides methods
for making a synthetic antibody, comprising connecting at least a
first affinity element and a second affinity element for a given
target via a linker;
[0023] wherein one or both of the first affinity element and the
second affinity element have a molecular weight of at least 1000
Daltons;
[0024] wherein at least one of the first affinity element and the
second affinity element are not derived from the first target;
[0025] wherein the synthetic antibody has an increased binding
affinity and/or specificity for the first target relative to a
binding affinity and/or specificity of the first affinity element
for the first target and relative to a binding affinity and/or
specificity of the second affinity element for the target; and
[0026] wherein the first target is not the Fv of an antibody. In
one embodiment, both the first affinity element and the second
affinity element have a molecular weight of between 1000 Daltons
and 10,000 Daltons. In another embodiment, the linker provides a
spacing of between about 0.5 nm and about 30 nm between the first
affinity element and the second affinity element. In further
embodiments, one or both of the first and second affinity elements
comprise a polypeptide or a nucleic acid.
[0027] In a further aspect, the present invention provides methods
for ligand identification, comprising:
[0028] (a) contacting a substrate surface comprising a target array
with one or more potential ligands, wherein the contacting is done
under conditions suitable for moderate to high affinity binding of
the one or more ligands to suitable targets present on the
substrate; and
[0029] (b) identifying targets that bind to one or more of the
ligands with at least moderate affinity. In one embodiment, the one
or more potential ligands are selected from the group consisting of
antibodies and synthetic antibodies according to the second aspect
of the invention. In a further embodiment, the array of targets is
mounted in a flow chamber, wherein
[0030] (i) a first buffer comprising the one or more potential
ligands is flowed over the addressable array,
[0031] (ii) wherein identifying targets that bind to one or more of
the ligands with at least moderate affinity comprises analyzing
real-time affinity data gathered by an array reader;
[0032] (iii) the first buffer flow over the addressable array is
stopped after at least moderate binding to the array is
detected;
[0033] (iv) repeating steps (i)-(iii) a desired number of times
using a further buffer comprising one or more further potential
ligands.
[0034] In another aspect, the present invention provides methods
for identifying a synthetic antibody profile for a test sample of
interest, comprising contacting a substrate comprising a plurality
of synthetic antibodies according to the present invention with a
test sample and comparing synthetic antibody binding to the test
sample with synthetic antibody binding to a control sample, wherein
synthetic antibodies that differentially bind to targets in the
test sample relative to the control sample comprise a synthetic
antibody profile for the test sample.
[0035] In a still further aspect, the present invention provides
compositions, comprising:
[0036] (a) a first affinity element bound to a template nucleic
acid strand;
[0037] (b) a second affinity element bound to a complementary
nucleic acid strand, wherein the first affinity element and the
second affinity element non-competitively bind to a common
target;
[0038] wherein the template nucleic acid strand and the
complementary nucleic acid strand are annealed via base pairing to
form an assembly;
[0039] wherein the first affinity element and the second affinity
element are separated in the assembly; and
[0040] wherein either the template nucleic acid strand, the
complementary nucleic acid strand, or both, are bound to a surface
of a substrate.
DESCRIPTION OF THE FIGURES
[0041] FIG. 1. Legend for conceptual drawings of synbody variations
shown FIGS. 2-8.
[0042] FIG. 2. Schematic of simple synbody.
[0043] FIG. 3. Schematic of synbodies specific for (a) homodimers
and (b) heterodimers.
[0044] FIG. 4(a-b). Schematic of synbodies that act as chemical OR
gates or switches.
[0045] FIG. 5. Schematic of synbodies that bind multiple A
molecules cooperatively (a.noteq.1, either positive or negative
cooperativity)
[0046] Figure. Schematic of synbodies that bind multiple different
molecules cooperatively (a.noteq.1, either positive or negative
cooperativity)
[0047] FIG. 7. Schematic of synbodies that act as signaling
molecular sensors; (a) two elements interact to form signal; (b)
two elements are displaced to form signal.
[0048] FIG. 8. Schematic of synbodies acting as actuators of enzyme
activity (homo or heteromultimer)
[0049] FIG. 9. (a) Representation of synthetic antibody. (b)
Construction of mini-library of synbodies with different
interpeptide distances. (c) One embodiment of a molecular slide
rule composition
[0050] FIG. 10. (a) Structure of maleimide sulfo-SMCC
(sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate)
(b) Conjugation of polypeptides to polylysine surface coating by
thiol attachment of a C-terminal cysteine of the polypeptide to
.epsilon. amine of a lysine monomer of the poly-lysine surface
coating using sulfo-SMCC.
[0051] FIG. 11. (a) Signal expected during attachment of protein
target to SPR chip surface. (b) Steps in attachment of protein
target to SPR chip surface.
[0052] FIG. 12. Expected SPR signal upon (a) interaction of a first
ligand alone with an immobilized target; (b) interaction of a
second ligand alone with an immobilized target; (c) interaction of
a first and second ligand with an immobilized target where the
ligands do not compete or interfere; (d) binding of two ligands
that do not bind distinct sites on the target, but instead compete
for the same binding site.
[0053] FIG. 13. Results of evaluation for binding to distinct
target sites, of a number of pairs of the polypeptides that were
identified as described in Example 2 (see Table 1).
[0054] FIG. 14.
5'-Dimethoxytrityl-N-dimethylformamidine-5-[N-(trifluoroacetylaminohexyl)-
-3-acrylimido]-2'-deoxyCytidine,
3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, used to
provide amine-modified cytosines in oligonucleotides.
[0055] FIG. 15. Schematic representation of a synbody specific for
gal80, comprising two polypeptide affinity elements identified as
described in Example 3 joined by a DNA linker.
[0056] FIG. 16. A synbody comprising polypeptide affinity
elements.
[0057] FIG. 17. Flow chart of the synthesis of a synbody comprising
polypeptide affinity elements.
[0058] FIG. 18. Relative SPR responses of BP1 and BP2-containing
synbodies with respect to gal80.
[0059] FIG. 19. Affinities (Kd) with respect to gal80 of affinity
elements BP1 and BP2 alone, BP1-BP2 containing synbody, and BP 1
and BP2 alone conjugated to DNA linker.
[0060] FIG. 20. Data derived from ELISA-type analyses confirming
the binding affinities of BP1 and BP2 alone for gal80 compared to
the BP1-BP2 containing synbody.
[0061] FIG. 21. Schematic of synbodies constructed by linking the
C-terminal glycines of two 20-mer polypeptides to the a and c amine
moieties of a lysine molecule, thereby providing a spacing of about
1 nm.
[0062] FIG. 22. Graph showing the 18 proteins to which 1C10 bound
with highest intensity, and relative intensities observed.
[0063] FIG. 23. Graph showing the 18 proteins to which SYN23-26
bound with highest intensity, and relative intensities
observed.
[0064] FIG. 24. Graph showing the 18 proteins to which SYN21-22
bound with highest intensity, and relative intensities
observed.
[0065] FIG. 25. Graph showing the 15 proteins to which the gal80
synbody bound with highest intensity, and relative intensities
observed.
[0066] FIG. 26. (a) Schematic of the 4-helix DNA tile linker
constructed from DNA oligonucleotides. (b) Location of aptamers
specific for thrombin incorporated into the single-stranded DNA
loops, providing a structure in which the aptamers extend from the
tile as shown schematically. (c) Structure having only a single
aptamer containing loop. (d) Another structure having only a single
aptamer containing loop.
[0067] FIG. 27. Graph showing results of thrombin-binding assays on
the DNA tile synbodies.
[0068] FIG. 28. Pairs of chemical moieties suitable for conjugation
by click-type chemistry.
[0069] FIG. 29. Four pairs of chemical moieties suitable for
conjugation by click-type chemistry that, when conjugations are
performed in the order indicated, provide four orthogonal
conjugations.
[0070] FIG. 30. Diagram of synthesis of a synbody comprising a
poly-(Gly-Ser) linker.
[0071] FIG. 31. Diagram showing conjugation of a maleimide
functionalized polypeptide with a thiol functionalized
oligonucleotide.
[0072] FIG. 32. Diagram of synthesis of a synbody comprising a
poly-(Gly-Hyp-Hyp) linker.
[0073] FIG. 33. Diagram of synthesis of a synbody comprising a
poly-(Gly-Hyp-Hyp) linker wherein both affinity elements are
attached by click-type chemistry conjugation.
[0074] FIG. 34. Schematic illustration of a concept underlying a
method for identification of optimized affinity elements and/or
linkers by allowing a synbody to self-assemble in association with
a target.
[0075] FIG. 35. Diagram showing three potentially reversible
conjugation chemistries.
[0076] FIG. 36. Diagram showing synthesis of a tetrapeptide
scaffold suitable for use as a synbody linker.
[0077] FIG. 37. Diagram illustrating orthogonal conjugation of up
to three affinity elements to tetrapeptide scaffold linker.
[0078] FIG. 38. Diagram showing synthesis of decapeptide scaffold
suitable for use as a synbody linker.
[0079] FIG. 39. Diagram illustrating orthogonal conjugation of
affinity elements to decapeptide scaffold linker.
DETAILED DESCRIPTION OF THE INVENTION
[0080] In a first aspect, the present invention provides methods
for identifying affinity elements to a target of interest,
comprising
[0081] (a) contacting a substrate surface comprising an array of
between 10.sup.2 and 10.sup.7 different test compounds of known
composition with a target of interest under conditions suitable for
moderate affinity binding of the target to target affinity elements
if present on the substrate, wherein the target is not an Fv
portion of an antibody, and wherein the different test compounds
are not derived from the target; and
[0082] (b) identifying test compounds that bind to the target with
at least moderate affinity, wherein such compounds comprise target
affinity elements.
[0083] The inventors have discovered that screening for affinity
elements to a target of interest using an array of different test
compounds of known composition permits a large amount of
chemical/structural space to be adequately sampled using only a
small fraction of the space. The resulting methods provide a rapid
and high throughput method for identifying affinity elements to
targets of interest. While not being bound by any specific
hypothesis, the inventors propose that the tremendously large
number of possible arrangements for a target of a given size
actually form a very limited number of structural forms or
combinations of patches of smaller sequences, providing the ability
to identify affinity elements to a target of interest by screening
a target of interest against a much smaller array of test compounds
(ie: potential affinity elements) than previously considered
possible. Since the composition of each test compound on the
substrate surface is known, the method is a screen for affinity
elements, not a selection. Screenable libraries as used in the
methods of the present invention are much smaller (.about.10.sup.2
to 10.sup.7) than selectable libraries (10.sup.9-10.sup.14). Thus,
the process of affinity element discovery is limited only by the
rate at which individual targets can be screened on test
compound-containing substrate surfaces. In this sense it is
distinct from current selection techniques, in which recurrent
selections using unknown sequences are required. Exemplary
substrate surfaces are described below.
[0084] In one embodiment, the substrate surface comprises an
addressable test compound array. "Addressable" means that test
compounds on the substrate surface are present at a specific
location on the substrate, and thus detection of binding events
serves to identify which test compound has bound target.
[0085] The "different test compounds of known composition" are of
known structure and/or composition. Thus, for example, if the test
compounds comprise or consist of nucleic acids or polypeptides,
their nucleic acid or amino acid sequence is known, while further
structural information may also be known (although this is not
required). Furthermore, the test compounds are not all related
based on minor variations of a core sequence or structure. Thus,
when the test compounds comprise nucleic acids or polypeptides, the
nucleic acid or polypeptide sequences are known, but the test
compounds are not simply a series of mutants/fragments of a known
sequence, nor a series of epitopes/possible epitopes from a given
antigen. The different test compounds may include variants of a
given test compound (such as polypeptide isoforms), but at least
10% of the test compounds on the array are structurally and/or
compositionally unrelated. In various embodiments, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 98%, or more of the test compounds on
the array are structurally and/or compositionally unrelated.
[0086] The different test compounds can comprise or consist of any
class of compounds capable of binding to a target of interest, but
the different test compounds are not derived from the target. As
used herein, "not derived from" means that the test compounds are
not fragments of the target to be screened. In this embodiment, for
example, if the target is a nucleic acid, the different test
compounds do not consist of a polynucleotide found within the
target (on its sense or antisense strand). Similarly, if the target
is a protein, the test compounds do not individually consist of a
polypeptide found within the target, or an "antisense" version
thereof (ie: polypeptides which are encoded on the opposite strands
of the DNA encoding the protein target in a given reading frame,
which can have an affinity to bind each other based on hydropathic
complementary of the polypeptides).
[0087] It will be understood by those of skill in the art that the
arrays may further comprise control compounds, and that such
control compounds may be of any type suitable to serve as
appropriate controls for target binding, including but not limited
to antibodies, Fv regions of antibodies, variable regions of an
antibody, or antigen binding regions of an antibody, and control
compounds derived from the target. In various embodiments, up to
25% of the compounds on the substrate surface may be control
compounds; in various further embodiments, 20%, 15%, 10%, 5%, 4%,
3%, 2%, 1%, 0.5%, 0.1% or less of the compounds on the substrate
surface are control compounds.
[0088] In another embodiment, the different test compounds on the
array are not antibodies, Fv regions of antibodies, variable
regions of an antibody, or antigen binding regions of an
antibody.
[0089] Classes of test compounds suitable for use in the present
invention include, but are not limited to, nucleic acids,
polypeptides, peptoids, polysaccharides, organic compounds,
inorganic compounds, polymers, lipids, and combinations thereof.
The test compounds can be natural or synthetic. The test compounds
can comprise or consist of linear or branched heteropolymeric
compounds based on any of a number of linkages or combinations of
linkages (e.g., amide, ester, ether, thiol, radical additions,
metal coordination, etc.), dendritic structures, circular
structures, cavity structures or other structures with multiple
nearby sites of attachment that serve as scaffolds upon which
specific additions are made. In various preferred embodiments, all
or a plurality of the test compounds are non-naturally occurring.
In other embodiments, the test compounds are selected from the
group consisting of nucleic acids and polypeptides. In one specific
embodiment, if the different test compounds consist of nucleic
acids, then the target is not a nucleic acid. In another
embodiment, the different test compounds are not nucleic acids. In
a further embodiment, the test target is not a nucleic acid.
[0090] In a further embodiment, the different test compounds on the
substrate are of the same class of compounds (ie: all polypeptides;
all nucleic acids, all polysaccharides, etc.) In other embodiments,
the test compounds comprise different classes of compounds in any
ratio desired. These test compounds can be spotted on the substrate
or synthesized in situ, using standard methods in the art. The test
compounds can be spotted or synthesized in situ in combinations in
order to detect useful interactions, such as cooperative
binding.
[0091] The substrates may further comprise control compounds or
elements as discussed above, as well as identifying features (RFID
tags, etc.) as suitable for any given purpose.
[0092] In one embodiment, the different test compounds are chosen
at random using any technique for making random selections. In a
further embodiment, an algorithmic approach for selecting different
test compounds is used.
[0093] In a further embodiment, all or a plurality of the test
compounds on the array do not naturally occur in an organism from
which the target is derived, where the target is a biological
molecule. In another embodiment, where the test compounds comprise
polypeptides, all or a plurality of the polypeptide test compounds
are not found in the SWISSPROT database (web site
ebi.ac.uk/swissprot/), either as a full length polypeptide or as a
fragment of a polypeptide found in the SWISSPROT database. In other
words, the test compounds are not derived from naturally occurring
proteins. In another embodiment, where the test compounds comprise
nucleic acids, all or a plurality of the nucleic acid test
compounds are not found in the GENBANK database (web site
ncbi.nlm.nih.gov/Genbank/), either as a full length nucleic acid or
as a fragment of a nucleic acid found in the GENBANK database.
There are at least two reasons to use such "non-naturally
occurring" test compounds. First, there is little known about what
potential binding space would be occupied by a particular
collection of elements. Arguments could be made for or against many
alternatives. Second, life space (ie: naturally occurring
compounds) has been selected to meet many requirements beyond
simply binding, and the binding is in very specific conditions in
life. Thus, naturally occurring compounds suffer from constraints
over many degrees of freedom and these constraints would handicap a
search for affinity elements to a large number or targets. An
unanticipated benefit of using non-naturally occurring different
test compounds (as discussed below) is that, overall, at least in
the case of polypeptides, the resulting test compounds tend to be
more soluble and well behaved in solution than a similarly sized
set of compounds derived from life space compounds, which provides
advantages in binding assays, such as in the array-based formats
disclosed herein. In various further embodiments, at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more of the
test compounds on the array do not naturally occur in an organism
from which the target is derived, where the target is a biological
molecule. Similar various further embodiments are contemplated for
the specific nucleic acid and polypeptide embodiments disclosed
above.
[0094] In a further embodiment, the test compounds have a molecular
weight of between about (ie: +/-5%) 1000 Daltons (D) and 10,000 D.
As discussed below, test compounds within this molecular weight
class are of particular utility in preparing synthetic antibodies
(also referred to herein as "synbodies") according to the present
invention. In one embodiment, polypeptide test compounds for use in
the methods of this aspect of the invention are between about 1000
Daltons and 4000 Daltons (up to approximately 30 amino acid
residues); in various further embodiments between 1100D-4000D;
1200D-4000D; 1300D-4000D; 1400D-4000D; 1500D-4000D; 1000D-3500D;
1100D-3500D; 1200D-3500D; 1300D-3500D; 1400D-3500D; 1500D-3500D;
1000D-2000D; 1100D-3000D; 1200D-3000D; 1300D-3000D; 1400D-3000D;
and 1500D-3000D. In another embodiment, nucleic acid aptamers of up
to 10,000 Daltons are used (ie: approximately 30 bases).
[0095] As used herein, "at least moderate affinity binding" of the
target to target affinity elements generally means a binding
affinity of at least about (ie: +/-5%) 500 .mu.M. In various
further embodiments, "at least moderate binding affinity" for the
target means at least about 250 .mu.M, 150 .mu.M; 100 .mu.M, 50
.mu.M, or 1 .mu.M. In various further embodiments, the target
affinity elements possess binding affinity for the target of
between about (ie: +/-5%) 1 .mu.M and 500 .mu.M. In various further
embodiments, moderate affinity binding of the target to target
affinity elements generally means a binding affinity of between
about 1 .mu.M-250 .mu.M; 1 .mu.M-150 .mu.M; 10 .mu.M-500 .mu.M; 25
.mu.M-500 .mu.M; 50 .mu.M-500 .mu.M; 100 .mu.M-500 .mu.M; 10
.mu.M-250 .mu.M; 50 .mu.M-250 .mu.M; and 100 .mu.M-250 .mu.M.
[0096] As used herein, "binding" of test compounds to a target
refers to selective binding in a complex mixture (ie: above
background), and does not require that the binding be specific for
a given target, as traditional antibodies often cross-react. The
extent of acceptable target cross-reactivity for a given affinity
element depends on how it is to be used and can be determined by
those of skill in the art based on the teachings herein. For
example, methods to modify the affinity and selectivity of the
synthetic antibodies produced using the binders identified in the
methods of the invention are described below. Such binding can be
of any type, including but not limited to covalent binding,
hydrophobic interactions, van der Waals interactions, the combined
effect of weak non-covalent interactions, etc.
[0097] Specific conditions suitable for moderate affinity binding
of the target to the test compounds will depend on the type of
target and test compounds (ie: polypeptide, nucleic acid, etc.), as
well as the specific structure of each (ie: length, sequence,
etc.). Determination of suitable conditions for moderate affinity
binding of a specific target to a specific collection of test
compounds is well within the level of skill in the art based on the
teachings herein. In various non-limiting embodiments, conditions
such as those described in the examples that follow can be
used.
[0098] For example, the screen can be done under non-biological
conditions, such as non-aqueous conditions. This is in contrast to
prior methods of selection mentioned above that use a living system
in some phase. Most antibodies do not function when applied to the
surface of arrays. In contrast, the binding agents developed here
are screened to function on surfaces.
[0099] The binding can be detected by many other methods, including
but not limited to direct labeling of the target, secondary
antibody labeling of the target or directly determined by SPR
electrochemical detection, micromechanical detection (e.g.,
frequency shifts in resonant oscillators), electronic detection
(changes in conductance or capacitance), mass spectrometry or other
methods. The target can also be pre-incubated with another control
compound (ie, protein, drug or antibody, etc.) to block the binding
of particular classes of affinity targets in order to focus the
search. The binding can be done in the presence of competitive
inhibitors (including but not limited to E. coli extract or serum)
to accentuate specificity.
[0100] In another embodiment, the methods comprise identifying
affinity elements for more than one target at a time. The methods
of the invention are easily amenable to multiplexing. In one
embodiment, each target is labeled with a different signaling
label, including but not limited to fluorophores, quantum dots, and
radioactive labels. Such multiplexing can be accomplished up to the
resolution capability of the labels. Targets that bound two or more
affinity elements would produce summed signals. Other techniques
for multiplexing of the assays can be used based on the teachings
herein.
[0101] In various embodiments, the substrate surface comprises an
array of between 100 and 100,000,000 different test compounds. Such
arrays may further comprise control compounds or elements as
discussed above. In various other embodiments, the substrate
surface comprises between 100-10,000,000; 100-2,000,000;
100-5,000,000; 100-1,000,000; 100-500,000; 100-100,000, 100-75,000;
100-50,000; 100-25,000; 100-10,000; 100-5,000, 100-4,000,
250-1,000,000, 250-500,000, 250-100,000, 250-75,000; 250-50,000;
250-25,000; 250-10,000; 250-5,000, 250-4,000; 500-1,000,000;
500-500,000, 500-100,000, 500-75,000; 500-50,000; 500-25,000;
500-10,000; 500-5,000, 500-4,000; 1,000-1,000,000; 1,000-500,000;
1,000-100,000, 1,000-75,000; 1,000-50,000; 1,000-25,000;
1,000-10,000; 1,000-8,000, 1,000-5,000 and 1,000-5,000 different
test compounds.
[0102] As used herein "nucleic acids" are any and all forms of
alternative nucleic acid containing modified bases, sugars, and
backbones. These include, but are not limited to DNA, RNA,
aptamers, peptide nucleic acids ("PNA"), 2'-5' DNA (a synthetic
material with a shortened backbone that has a base-spacing that
matches the A conformation of DNA; 2'-5' DNA will not normally
hybridize with DNA in the B form, but it will hybridize readily
with RNA), locked nucleic acids ("LNA"), Nucleic acid analogues
include known analogues of natural nucleotides which have similar
or improved binding properties. "Analogous" forms of purines and
pyrimidines are well known in the art, and include, but are not
limited to aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil,
5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine,
1-methyladenine, 1-methylpseudouracil, 1-methylguanine,
1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine,
N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid, and 2,6-diaminopurine. DNA
backbone analogues provided by the invention include
phosphodiester, phosphorothioate, phosphorodithioate,
methylphosphonate, phosphoramidate, alkyl phosphotriester,
sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate,
morpholino carbamate, and peptide nucleic acids (PNAs),
methylphosphonate linkages or alternating methylphosphonate and
phosphodiester linkages (Strauss-Soukup (1997) Biochemistry
36:8692-8698), and benzylphosphonate linkages, as discussed in U.S.
Pat. No. 6,664,057; see also Oligonucleotides and Analogues, a
Practical Approach, edited by F. Eckstein, IRL Press at Oxford
University Press (1991); Antisense Strategies, Annals of the New
York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt
(NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense
Research and Applications (1993, CRC Press).
[0103] The term "polypeptide" is used in its broadest sense to
refer to a sequence of subunit amino acids, amino acid analogs, or
peptidomimetics. The subunits are linked by peptide bonds, except
as noted. The polypeptides may be naturally occurring, processed
forms of naturally occurring polypeptides (such as by enzymatic
digestion), chemically synthesized or recombinantly expressed.
Preferably, the polypeptides for use in the methods of the present
invention are chemically synthesized using standard techniques. The
polypeptides may comprise D-amino acids (which are resistant to
L-amino acid-specific proteases), a combination of D- and L-amino
acids, .beta. amino acids, and various other "designer" amino acids
(e.g., .beta.-methyl amino acids, C.alpha.-methyl amino acids, and
N.alpha.-methyl amino acids, etc.) to convey special properties.
Synthetic amino acids include ornithine for lysine, and norleucine
for leucine or isoleucine. In addition, the polypeptides can have
peptidomimetic bonds, such as ester bonds, to prepare polypeptides
with novel properties. For example, a polypeptide may be generated
that incorporates a reduced peptide bond, i.e.,
R.sub.1--CH.sub.2--NH--R.sub.2, where R.sub.1 and R.sub.2 are amino
acid residues or sequences. A reduced peptide bond may be
introduced as a dipeptide subunit. Such a polypeptide would be
resistant to protease activity, and would possess an extended
half-live in vivo. The affinity elements can also be peptoids
(N-substituted glycines), in which the sidechains are appended to
nitrogen atoms along the molecule's backbone, rather than to the
.alpha.-carbons, as in amino acids.
[0104] The term "polysaccharide" means any polymer (homopolymer or
heteropolymer) made of subunit monosaccharides, oligimers or
modified monosaccharides. The linkages between sugars can include
but are not limited to acetal linkages (glycosidic bonds), ester
linkages (including phophodiester linkages), amide linkages, ether
linkages, etc. The lipids can be any nonpolar-comprising
hydrocarbon-based molecule, including amphipathic, amphiphilic,
aliphatic, straight chain, branched, aromatic, saturated, or
unstaturated lipds. Specific lipid types that can be used as
affinity elements here include, but are not limted to
phospholipids, fatty acids, glycerides (mono-, di-, tri-, etc.),
sphingolipids, and waxes. Similarly, any other suitable organic
compounds, inorganic compounds, therapeutic agents, and polymers
can be used as affinity elements according to the present
invention.
[0105] The target can be any structure that an affinity element can
bind to other than an Fv portion of an antibody (ie: the antigen
binding portion of an antibody), including but not limited to
nucleic acids, polypeptides, peptoids, polysaccharides, organic
compounds, inorganic compounds, metabolites, sugar oligomers, sugar
polymers, other synthetic polymers (plastics, fibers, etc.),
polypeptide complexes, polypeptide aggregates, polypeptide/nucleic
acid complexes, lipids, glycoproteins, lipoproteins,
polypeptide/carbohydrate structures (such as peptdidogycans),
chromatin structures, membrane fragments, cells, tissues, organs,
organelles, inorganic surfaces, electrodes, semiconductor
substrates including but not limited to silicon-based substrates,
dyes, nanoparticles, nanotubes, nanowires, quantum dots, and
medical devices. The target can be a single such structure, or a
multimer of the same or different such structure (ie: homodimers,
heterodimer, etc.), as discussed in more detail below. As is also
discussed in more detail below, when additional affinity elements
are used, the target(s) for the further affinity elements can be
the same as the target for the first and/or second affinity
elements, or different. In one embodiment, the target is not an
antibody, an antibody bearing cell, or an antibody-binding cell
surface receptor (or portion thereof suitable for antibody
binding). In another embodiment, the target does not comprise a
nucleic acid. In a further embodiment, the target comprises a
polypeptide.
[0106] Any suitable substrate surface can be used in the methods of
the invention, including but not limited to surfaces provided by
microarrays, beads, columns, optical fibers, wipes, nitrocellulose,
nylon, glass, quartz, mica, diazotized membranes (paper or nylon),
silicones, polyformaldehyde, cellulose, cellulose acetate, paper,
ceramics, metals, metalloids, semiconductive materials, quantum
dots, coated beads, other chromatographic materials, magnetic
particles; plastics and other organic polymers such as
polyethylene, polypropylene, and polystyrene; conducting polymers
such as polypyrole and polyindole; micro or nanostructured surfaces
such as nucleic acid tiling arrays, nanotube, nanowire, or
nanoparticulate decorated surfaces; or porous surfaces or gels such
as methacrylates, acrylamides, sugar polymers, cellulose,
silicates, and other fibrous or stranded polymers. In one exemplary
embodiment, the substrate comprises a substrate suitable for use in
a "dipstick" device, such as one or more of the substrates
disclosed above.
[0107] In one non-limiting embodiment of the methods of this first
aspect of the invention, the target is detectably labeled (as
discussed above) such as, in the case of peptides or proteins, a
tag that can be bound by a labeled antibody. This target is then
applied to a spotted array on a slide containing between 5,000 and
1,000,000 test polypeptides of 20 amino acids long. In this
example, the polypeptides can be attached to the surface through
the C-terminus. The sequence of the polypeptides was generated
randomly from 19 amino acids, excluding cysteine. When running this
type of experiment, typically 0.1% to 10% of polypeptides show some
binding to the target. The binding reaction can include, for
example, an excess of E. coli proteins (such as a 100 fold excess)
as non-specific competitor labeled with another dye so that the
specificity ratio for each polypeptide binding target can be
determined. The polypeptides with the highest specificity and
binding can be picked. The identity of the polypeptide on each spot
is known, and thus they can be readily identified for further use,
either through use of stocks of the selected polypeptides or
resynthesis of the polypeptides.
[0108] Thus, in another embodiment, the methods further comprise
contacting the same substrate surface or a separate substrate
surface with competitor, and determining a ratio of test compound
binding to target versus test compound binding to competitor. This
enables identification of test compounds that not only have high
affinity for the target but also relatively low affinity for
competitor. In one embodiment, the target is a polypeptide and the
competitor comprises a cell lysate or protein extract, including
but not limited to a bacterial cell lysate or protein extract. In
another embodiment, the competitor is differentially labeled from
the target for ease of detection and binding ratio determination.
In further embodiments, the target/competitor screen is conducted
on two or more separate substrate surfaces (for example, E coli
lysate as the competitor on one, salmon sperm on another, abundant
serum proteins on another), and binding ratios compared across the
different competitors (such as in a matrix format) to identify
probes that are reasonably specific. An exemplary embodiment (E
coli lysate competition) is described in detail below.
[0109] In one embodiment, the methods further comprise (c)
identifying test compounds that do not bind to the target with at
least moderate affinity. Since the composition of each test
compound on the substrate is known, the methods of this first
aspect provide information on the binding affinity of the arrayed
test compounds for each target tested. These data can be used for a
variety of purposes, including but not limited to creating a
database of test compounds and their binding affinity (or lack
thereof) to different targets. Thus, in a further embodiment, the
methods of any aspect or embodiment of the invention further
comprise storing in a database the data obtained using the methods
of the invention. Such data includes, but is not limited to,
affinity element binding affinity (including quantitative
measurements of dissociation constants, binding free energy
changes, binding enthalpy changes and binding entropy changes),
specificity, and structure/sequence, and non-affinity element (ie:
non-binder) structure/sequence. Data from these analyses can be
used to create a database that allows predicting which affinity
elements bind different structures. Polypeptides in different
groups tend to bind different surfaces of the same protein. This
information can also be used to design better affinity elements for
lead target analysis.
[0110] In another embodiment, the methods of the invention further
comprise identifying combinations of affinity elements that bind to
different sites on the same target. The affinity elements selected
using the methods of the invention typically have relatively
moderate affinity for the target (.about.uM). By linking two
affinity elements that bind the same target non-competitively, the
affinity and selectivity can be increased (see data below). Thus,
combinations of affinity elements that bind to different target
sites are first identified. Natural antibodies do this by selection
of light and heavy chain variants that bind to sites on the protein
with synergy. The space between light and heavy chains is largely
fixed so the optimal binding site/spacing combination is selected
among millions of antibody variants. The methods disclosed herein
have an advantage over the natural process of antibody production
by allowing essentially any spacing between sites. If the target is
a dimer or a multimer, one affinity element can bind multiple sites
on the target complex simultaneously (ie: affinity element binding
to each of the monomers). For example, it is estimated that
approximately 60% of soluble proteins are dimers or other
multimers. Therefore, in many cases joining two (or more) copies of
a single affinity element may provide increased affinity and/or
selectivity, though affinity and/specificity may be enhanced by
using two (or more) different affinity elements when the target
comprises a multimer.
[0111] Any suitable technique for identifying affinity elements
that bind to different sites on the same target can be used, and
many such techniques are known to those of skill in the art. In
some cases, particularly for homodimeric proteins, the same
affinity element can be used twice to create the synthetic antibody
(ie: the binding is still for different sites, one to each member
of the homodimeric pair). In one non-limiting example, affinity
elements that bind to different sites on the same target are
identified by pre-incubating the target with a first affinity
element, under conditions to promote binding of the first affinity
element to the target, and then contacting the target with one or
more further affinity elements, to see which further affinity
elements bind to the target in the presence of first affinity
element bound to the target. For example, one method to discover
polypeptides binding to different sites on the same protein is to
pre-incubate the protein target with one polypeptide affinity
element and observe which polypeptides on the array still bind. By
doing this in an iterative fashion one can classify all the binding
polypeptides as to target sites on a protein. Another method is to
combine all protein specific polypeptide affinity elements in a
pairwise manner and then spot them on the array to assess binding
to the original target. Two polypeptide affinity elements that bind
to two different areas of the protein should have more than
additive affinity. Even though the polypeptide affinity elements
are not spaced at a single distance, there is a random distribution
of polypeptide spacing. If the average spacing is around the
optimal distance, then enhanced binding can occur. This can also be
affected by the length and flexibility of the linker arm to the
surface. In this way the pairs of polypeptide affinity elements
that bind different sites on the target can be discovered in a high
through put fashion. Data supporting both approaches to finding
pairs is discussed below. The pairs of polypeptide affinity
elements can be affixed to a surface as a mixture to take advantage
of the cooperative binding. However, only a subset of the
polypeptides would be in the optimal spacing. An alternative is to
affix the pairs of polypeptides on a surface that has been
derivatized with orthogonal chemistries so that the polypeptides
can be distributed in a chosen spacing. Another embodiment involves
binding the target to a surface plasmon chip and each polypeptide
is flowed over to determine its binding to the target. Then the
same is done for each pair of polypeptide affinity elements. For
polypeptide affinity elements that occupy the same or overlapping
sites on the target, the response will be the average of the
individual polypeptide affinity elements. For those occupying
different sites the response will be the sum. As predicted by our
analysis of the effectiveness of screening versus selection, using
this technique we readily obtain several polypeptide affinity
elements binding two or more sites on the target.
[0112] The methods of the invention further comprise connecting two
or more affinity elements (for example, as described in any of the
synthetic antibody embodiments below) for a given target via a
linker to create a synthetic antibody, wherein an affinity and/or
specificity of the synthetic antibody for the target is increased
relative to an affinity and/or specificity of either affinity
element alone for the target, as discussed in more detail
below.
[0113] The methods of the invention do not try to make one high
affinity, perfect match synthetic antibody, but instead takes
advantage of it being easier to find two weak binders and link them
to produce a higher affinity binder. While not being bound by any
specific hypothesis, the inventors believe that since most of the
surfaces of proteins are not deeply pocketed, it will be beneficial
to use larger molecules to sufficiently bind (near micromolar) the
surface. This is difficult to do by selection in a library.
Therefore we have developed efficient methods to screen for binding
elements. However, screenable libraries are necessarily much
smaller than selectable libraries (10.sup.9-10.sup.14). These two
demands seem contradictory. We want to limit the library size but
search larger molecule space. For example, the sequence space of 20
amino acid polypeptides using all possible 20 amino acids is
.about.10.sup.26. Our surprising discovery was that these two
demands can be reconciled because the structural space represented
on the surface of proteins is covered by a small number of 20 amino
acid polypeptides. This allows using a small number of compounds to
cover enough space to give at least micromolar Kds on two or more
sites per target. In addition, since this system allows arriving at
the lead ligands by screening, it has the important implication
that these synbodies could be produced in a high through put
fashion.
[0114] In another embodiment, the method further comprises linking
two affinity elements at an appropriate distance to obtain an
increase in specificity and affinity. The linker can be any
molecule or structure that can connect the first and second
affinity elements, including but not limited to nucleic acid
linkers, amino acid linkers, any polymeric linker (heteropolymers
or homopolymer), PEG linkers, nucleic acid tiles, etc. In some
embodiments, the linker is a polymer comprising one or more
proline-glycine-proline subunits. In some embodiments the linker is
a polymer comprising one or more hydroxproline subunits. A variety
of polymers comprising proline and/or hydroxproline are capable of
forming helical structures having useful and potentially
optimizable rigidity and elasticity properties. Such linkers can be
naturally occurring compounds/structures or may be non-natural,
including but not limited to nucleic acid analogues, amino acid
analogues, etc. Connection between an affinity element and a linker
can be of any type, including but not limited to covalent binding,
hydrogen bonding, ionic bonding, base pairing, electrostatic
interaction, and metal coordination depending on the type of linker
and the types of affinity elements. Selection of an appropriate
linker for use in the synthetic antibodies of the invention is well
within the level of skill in the art based on the teachings herein.
The linker can be rigid or flexible, depending on the desired
characteristics of the linker, as described in more detail
below.
[0115] Ideal linking can produce an affinity the product of the two
individual binding constants of the affinity elements. One approach
to this is to make a collection of each pair of affinity elements,
such as polypeptides, that bind different sites bound at different
distances on one or more linkers and then measure the affinity of
each linked pair of affinity elements to the target (this is
discussed in more detail below). Those binding cooperatively will
have much higher affinity for the target. One could also mix the
different constructions, incubate them with the target and then
remove and wash the target (for example on nickel beads if the
target were histidine tagged). The synthetic antibodies binding
from the mixture would be the ones with the optimal spacing of the
individual affinity elements. The identity of the high affinity
binding synthetic antibody could be determined directly by mass
spectrometry or indirectly by including an identifying tag on each
construct.
[0116] In the process of carrying out this procedure we have noted
an unexpected phenomenon. Combinations of some affinity elements
will create a synthetic antibody that has an increase in affinity
and specificity of about 10 fold. However, this increase is not
distance sensitive, although polypeptide affinity elements do not
show the increase if they are less than 1 nm apart from each other
in the synthetic antibody. We interpret this type of response as a
"caging" of the target as opposed to true cooperative binding. The
increase in affinity is due we think basically to creating a high
local concentration of binding sites that the target bounces
between.
[0117] In one embodiment, an optimal linker distance provides a
spacing of between about (+/-5%) 0.5 nm and about 30 nm between a
first affinity element and a second affinity element. In various
further embodiments, the spacing is between about 0.5 nm-25 nm, 0.5
nm-20 nm, 0.5 nm-15 nm, 0.5 nm-10 nm, 1 nm-30 nm, 1 nm-25 nm, 1
nm-20 nm, 1 nm-15 nm, and 1 nm-10 nm.
[0118] In another embodiment, a net charge of the resulting
synthetic antibody at a pH 7 is between +2 and -2, particularly
when the affinity elements comprise or consist of polypeptides. The
inventors have discovered that synthetic antibodies with this
characteristic tend to work better than those without this
characteristic.
[0119] In another embodiment, the synthetic antibody binds to the
target non-specifically. The inventors have surprisingly discovered
that some synthetic antibodies developed through binding to a given
target show high affinity binding (ie: nM) to other targets as well
(see examples below). In this embodiment, the synthetic antibody
can be used to selectively target multiple targets, or target
specificity can be modified by techniques known to those of skill
in the art. For some applications it may be desirable to create
synbodies with even higher or otherwise altered affinity or
selectivity. Thus, in a further and completely optional embodiment
of the different aspects of the invention, the methods further
comprise optimizing binding affinity of one or both of the first
affinity element and the second affinity element for the target.
Such optimization may be desired to produce even higher affinity
binding or specificity synbodies or synbodies with specific
affinities or selectivities in any range tailored for a particular
application (e.g., reversible binding to a chromatographic
material). In one embodiment, the optimization is carried out on a
substrate, which is not possible with standard antibodies. Any
techniques for optimizing the affinity of the synthetic antibody
for the target can be used.
[0120] In one non-limiting example of a polypeptide-based synbody,
one or both of the polypeptides in the synbody is subjected to
array alanine scanning An array is synthesized such that each amino
acid in the starting sequence is changed to alanine (or any other
amino acid as suitable) one by one. The original target protein is
then bound to the array. If the particular amino acid is important
for binding, it will bind to the target less well when substituted
with alanine (assuming it was not alanine to begin with). This
procedure will identify the critical amino acids. The amino acids
that need to be optimized may or may not be the ones most strongly
affected by the alanine substitutions. Often the alanine
substitutions in combination with structural analysis suggest other
amino acids or regions of the polypeptide that could be optimized.
Once the critical amino acids are identified by this method, a new
set of polypeptides with substitutions of the 20 different amino
acids at the alanine critical or non-critical sites can be
synthesized. These sets of polypeptides can be assayed against the
target to find new ones with the improved characteristics. When
using larger arrays (30,000 or more) it is actually possible to use
a more sophisticated initial scan if desired. For example, all
possible pairs of amino acids within the 17 variable positions in
the polypeptide can be replaced with all combinations of 10 amino
acids (there are 27,200 such polypeptides). This allows one to
recognize amino acids that are in themselves important, and also to
find pairwise or compensatory interactions as well that can enhance
the binding. In many cases, this pairwise approach may alleviate
the need for subsequent optimization (by providing substantial
local optimization in itself). In other cases, it will simply
determine which amino acids should be included in the subsequent
optimization rounds as described below. It will be apparent to
those skilled in the art based on the teachings herein that there
are many variations of this approach possible for an initial screen
to locate important structure/function elements of the
polypeptides. This may include varying a different number of the
amino acid positions at a time (more than 2), changing the number
of amino acids tested per position, including non-natural amino
acids or amide linked monomers into the polypeptide, creating
truncations and deletions instead of substitutions, etc.
[0121] The optimization methods may further comprise constructing
an array that has a wide variety of amino acids (natural or
unnatural) substituted at each critical site. For example, if there
were 3 critical amino acids indicated by the alanine scanning, and
20 amino acids variants were used at each of these sites, an array
would consist of 8,000 polypeptides. The target protein is then
applied to this array. Binding relative to the original polypeptide
is compared. The selection on these arrays can be geared towards
improved affinity and or specificity. Once selected, the improved
polypeptides can be reinserted into the synbody to produce higher
or otherwise modified affinity, selectivity, and/or kinetics of
binding. For example, it may be desirable to set the affinity at a
specific value. This is particularly true for applications
associated with chromatography, staining of cells and sensor
systems where dynamic binding is useful, and it would thus be
desirable to generate synbodies that reversibly bind a target. In
fact, the key issue may be to adjust the on and off times rather
than the affinity. This can be done by kinetic studies of binding
and release. Such studies can be done on the arrays with the proper
equipment.
[0122] Those of skill in the art will recognize, based on the
teachings herein, alternative methods to optimize the synbody. For
example, a phage, mRNA display or yeast/bacterial display system
could be used to detect the better binders. As an example for mRNA
display, a chip with 4000 oligos can be purchased that would have
16 different amino acid encoded substitutions at 3 sensitive
positions. These would be primed with a T7 containing primer to
make fragments that can be in vitro transcribed/translated to make
the polypeptide attached to its encoding mRNA. This library can be
panned against the target protein to select the improved
binders.
[0123] In various embodiments, the methods further comprise
connecting to the synthetic antibody further affinity elements
(third affinity element, fourth affinity element, etc.) that bind
to the first target or other targets. In embodiments where one or
more further affinity elements bind to the same target as the first
and second affinity elements, the one or more further affinity
elements may be connected to the first and/or second affinity
element by the linker, or may be connected to the first and/or
second affinity element by a one or more further linkers (second
linker, third linker, etc.), which may be a further linker or may
comprise or consist of a different class of compound. Where
multiple linkers are used, the spatial arrangement between affinity
elements connected by different linkers can be the same or
different. In various further embodiments where the further
affinity elements bind to the same target as the first and second
affinity elements, the linker or further linker(s) provides a
spatial arrangement of the further affinity element(s) to the first
and the second affinity element that increases a binding affinity
and/or specificity of the synthetic antibody for the target
relative to a binding affinity and/or specificity of the further
affinity elements for the target.
[0124] Thus, the methods for making synbodies as disclosed herein
can be used to make, for example, any of the synbody embodiments
disclosed herein, including but not limited to those disclosed in
FIGS. 1-8, and which are discussed in detail below).
[0125] In another embodiment, the invention provides synthetic
antibodies made by the methods of this first aspect of the
invention. As discussed herein, the structural complexity of the
proteome surface space can be covered by .about.1000-10,000 or so
affinity elements (such as polypeptides or other polymers) that can
bind at .about.micromolar affinity, and linking them together leads
to high affinity and specificity synthetic antibodies, one could
make a stock of 1000 or so binders (ie: affinity elements) that
could be combined in pairs and linked to quickly make a ligand to
anything. Thus, the invention further comprises a pool of affinity
elements isolated according to the methods of the invention. The
stocks could be pre-made in at large quantities so production could
be immediately initiated. Recall that an antibody diversity of
.about.10.sup.7 per person is capable of binding to almost
anything. 1000 binders would represent 10.sup.6 pairs and if they
can be linked in 10 different ways this stock would represent
10.sup.7 ligands. The equivalent of antibody diversity could be
stored on the shelf for rapid, inexpensive production.
[0126] In a second aspect, the present invention provides synthetic
antibodies, comprising:
[0127] (a) a first affinity element that can bind a first
target;
[0128] (b) a second affinity element that can bind the first
target, and which can bind to the first target in the presence of
the first affinity element bound to the first target; and
[0129] (c) a linker connecting the first affinity element and the
second affinity element,
[0130] wherein one or both of the first affinity element and the
second affinity element have a molecular weight of at least 1000
Daltons;
[0131] wherein at least one of the first affinity element and the
second affinity element are not derived from the first target;
[0132] wherein the synthetic antibody has an increased binding
affinity and/or specificity for the first target relative to a
binding affinity and/or specificity of the first affinity element
for the first target and relative to a binding affinity and/or
specificity of the second affinity element for the target; and
[0133] wherein the first target is not an Fv region of an
antibody.
[0134] Synthetic antibodies according to this aspect of the
invention can be obtained against any target or targets of
interest, and can generally bind to the target(s) both in solution
and on surfaces, thus increasing the range of applications for
their use. The spatial arrangement (ie, specific spacing and/or
orientation) of the affinity elements in the synbodies improves
affinity for a target relative to the affinity of the individual
affinity elements for the target, and thus the synthetic antibodies
are suitable for a wide variety of uses, including but not limited
to ex-vivo diagnostics, for example in standard ELISA-like formats
or in multiplex arrays; in vivo as imaging agents or as
therapeutics for specific indications; as binding agents for
affinity separation techniques and reagents, including but not
limited to affinity columns and affinity beads; as detectors for
environmental or biological agents; and as catalysts for chemical
reactions. As therapeutics, the synthetic antibodies can be used to
bind a target or for mediating binding and uptake in specific cells
or as "smart drugs" for drug delivery.
[0135] As used herein, an "increased binding affinity and/or
specificity of the synthetic antibody" means any increase relative
to the binding affinity and/or specificity of the first affinity
element for the first target and relative to a binding affinity
and/or specificity of the second affinity element for the target.
In various embodiments, the increase is 10-fold, 100-fold,
1000-fold, or more over either individual element.
[0136] In a further embodiment, one or both of the first and second
affinity elements have a molecular weight of between about 1000
Daltons and 10,000 Daltons. In one embodiment, polypeptide
compounds for use in the methods of this aspect of the invention
are between about 1000 Daltons and 4000 Daltons (up to
approximately 30 amino acid residues). In another embodiment,
nucleic acid aptamers of up to 10,000 Daltons are used (ie:
approximately 30 bases).
[0137] Synbodies according to the present invention can be of any
suitable size, based on the sizes of the affinity elements and
linkers used.
[0138] Affinity elements (ie: compounds identified as being
affinity elements for a target of interest), targets, linkers, and
other terms used in this second aspect have the same meaning as
described above in the first aspect of the invention. Furthermore,
all embodiments disclosed in the first aspect of the invention can
be used in this second aspect of the invention.
[0139] In one embodiment, at least one of the first affinity
element and the second affinity element are not the Fv portion of
antibodies or antigen-binding portions thereof; in a further
embodiment, neither the first nor the second affinity elements are
the Fv of antibodies or antigen-binding portions thereof. The first
target is not the Fv of an antibody. In further embodiments, the
first target is not an antibody, an antibody bearing cell, or an
antibody-binding cell surface receptor (or portion thereof suitable
for antibody binding)
[0140] Within a given synthetic antibody, the first and second
affinity elements can be the same class of compound (ie: nucleic
acids, polypeptides, etc.), or they can be different types of
compounds. For example, the first affinity element can comprise or
consist of a nucleic acid and the second affinity element can
comprise or consist of a polypeptide. In one embodiment, one or
both of the first and second affinity elements comprise or consist
of polypeptides. Those of skill in the art will recognize a wide
variety of affinity element combinations according to the present
invention. In one embodiment, one or both of the first and second
affinity elements comprises or consists of a non-naturally
occurring compound, as discussed in the first aspect of the
invention. In further embodiments, one or both of the first and
second affinity elements does not comprise or consist of a nucleic
acid.
[0141] In one embodiment, one or both of the first and second
affinity elements, prior to inclusion in the synthetic antibodies
of this aspect have dissociation constant for binding to the first
target of between about 1 .mu.M and 500 .mu.M. Linkage of the first
and second affinity elements provides a synthetic antibody with an
increased affinity and/or specificity for the first target relative
to a binding affinity and/or specificity of the first affinity
element for the first target and relative to a binding affinity
and/or specificity of the second affinity element for the target.
Thus, the synthetic antibodies of the present invention combine two
weaker binders by linking them; as discussed above, one surprising
discovery herein is that the structural space represented on the
surface of proteins is covered by a small number of 20 amino acid
polypeptides. This allows using a small number of affinity elements
to cover enough space to give .about.micromolar Kds on two or more
sites per target. An added advantage is that using these relatively
larger molecules makes it less likely that the linker attachment
will disrupt the binding of the resulting synbody to the first
target.
[0142] In various embodiments, the first affinity element and the
second affinity element prior to inclusion in the synthetic
antibody have dissociation constant for binding to the first target
of between about 1 .mu.M-500 .mu.M; 1 .mu.M-150 .mu.M; 10 .mu.M-500
.mu.M; 25 .mu.M-500 .mu.M; 50 .mu.M-500 .mu.M; 100 .mu.M-500 .mu.M;
10 .mu.M-250 .mu.M; 50 .mu.M-250 .mu.M; and 100 .mu.M-250
.mu.M.
[0143] In one embodiment, an optimal linker distance provides a
spacing of between about 0.5 nm and about 30 nm between a first
affinity element and a second affinity element. In various further
embodiments, the spacing is between about 0.5 nm-25 nm, 0.5 nm-20
nm, 0.5 nm-15 nm, 0.5 nm-10 nm, 1 nm-30 nm, 1 nm-25 nm, 1 nm-20 nm,
1 nm-15 nm, and 1 nm-10 nm. Those of skill in the art can design
linkers for appropriate spacing based on the teachings herein.
[0144] In another embodiment, a net charge of the synthetic
antibody at a pH 7 is between +2 and -2, particularly when the
affinity elements comprise or consist of polypeptides. The
inventors have discovered that synthetic antibodies with this
characteristic tend to work better than those without this
characteristic.
[0145] While the synthetic antibodies of the invention comprise
first and second affinity elements, they can comprise further such
affinity elements (ie, third affinity element, fourth affinity
element, etc.), as discussed in more detail below.
[0146] As discussed above, the synthetic antibody has an increased
affinity and/or specificity for the first target relative to a
binding affinity and/or specificity of the first affinity element
for the first target and relative to a binding affinity and/or
specificity of the second affinity element for the target. For
example, the arrangement of the first and second affinity elements
may increase affinity of the resulting synthetic antibody for a
monomeric target (See, for example, FIG. 2). Alternatively, the
arrangement of the first and second affinity elements may increase
affinity and specificity of the synthetic antibody for a
homodimeric or heterodimeric target, where the individual affinity
elements would otherwise only be able to bind to a monomer (See,
for example, FIG. 3).
[0147] The first and second affinity element bind to the first
target, and their binding to the target is not exclusive, generally
by virtue of the first and second affinity elements binding to
different regions on the target. For example, where the target is a
single structure, the first and second affinity elements may bind
to different sites on the target (See, for example, FIG. 2).
Alternatively, where the target is a homodimer, the first and
second affinity elements may be identical and bind to the same
location but one to each monomer in the homodimer (See, for
example, FIG. 3, left panel). In a further example, where the
target is a heterodimer AB, the first affinity element can bind to
A and the second affinity element can bind to B (See, for example,
FIG. 3, right panel). Those of skill in the art will recognize many
variations based on the present disclosure. The targets for the
affinity elements can be at distances not attainable by
conventional antibodies. This distance can be to two different
targets, as noted.
[0148] As used herein, "binding" of affinity elements to a target
refers to selective binding in a complex mixture (ie: above
background), and does not require that the binding be specific for
a given target as traditional antibodies often cross-react. The
extent of acceptable target cross-reactivity for a given synthetic
antibody depends on how it is to be used and can be determined by
those of skill in the art based on the teachings herein. For
example, methods to modify the affinity and selectivity of the
synthetic antibodies are described herein.
[0149] In various embodiments, the synthetic antibodies of the
invention can comprise further affinity elements (third affinity
element, fourth affinity element, etc.) that bind to the first
target or other targets. The one or more further affinity elements
may be connected to the first and/or second affinity element by the
linker, or may be connected to the first and/or second affinity
element by a one or more further linkers (second linker, third
linker, etc.), which may comprise or consist of a different class
of linker compound. Where multiple linkers are used, the spatial
arrangement between affinity elements connected by different
linkers can be the same or different. In various further
embodiments the binding affinity and/or specificity of the
resulting synthetic antibody for any further is increased relative
to a binding affinity and/or specificity of the further affinity
elements for the target.
[0150] Various further embodiments of synthetic antibodies
according to this second aspect of the invention include, but are
not limited to those provided in the Figures as follows:
[0151] FIG. 4: In this example, the synthetic antibody comprises
affinity element 1 that binds to target A, affinity element 2 that
binds to targets A and B, and affinity element 3 that binds to
target B. The spatial arrangement of the 3 affinity elements by the
linker provides that only one of targets A and B can be bound by
the synthetic antibody. In one non-limiting embodiment, the K.sub.d
of binding of target A is decreased by the K.sub.d of binding of B.
In this particular example, the binding is competitive and a rigid
linker, such as a nucleic acid linker, can be used. This synbody
acts a chemical OR gate, or to control the binding of one target by
the presence of another. As will be clear to those of skill in the
art, this can be generalized to 3 or more targets, for example, by
using additional affinity elements.
[0152] FIG. 5: In this example, the synthetic antibody comprises
affinity elements 1 and 2 that bind to target A. Further affinity
elements 3 and 4 are spatially arranged by the linker to affinity
elements 1 and 2 to provide cooperative binding of a second target
molecule A. For example, the dissociation constant for binding of
the second target molecule A is less than or greater than that of
the dissociation constant for binding of the first target molecule
A--thus, positive or negative cooperativity is possible though only
positive cooperativity is shown in the figure. This allows one to
alter the binding curve for a particular target molecule, making it
super- or sub-linear at low concentrations. This can be used, for
example, to generate high contrast ratio measurements between low
and high concentrations of the target.
[0153] FIG. 6: In this example, the synthetic antibody comprises
affinity elements 1 and 2 that bind to target A. Further affinity
elements 3 and 4 are spatially arranged by the linker to affinity
elements 1 and 2 to provide cooperative binding of target molecule
B. This is similar to FIG. 5 except that the cooperative binding
(positive or negative) is between two different target molecules.
This is another way of allowing B to influence the binding curve of
A or the other way around. Unlike the case in FIG. 4, the
interaction is not competitive, but is more like an allosteric
affector in an enzyme system.
[0154] FIG. 7: The ability to design conformational or functional
changes in the synbodies of the present invention upon binding
and/or alter the environment of a sensor molecule upon binding is a
unique capability of synbodies that cannot easily be designed into
antibodies or individual ligand systems. In this example, the
synthetic antibody comprises affinity elements 1 and 2 that bind to
target A, and wherein binding of A to affinity elements 1 and 2
results in a spatial arrangement of two previously separated
signaling elements (depicted as a circle and a square in the
figure) that leads to a change in signal indicating presence of
target A. The signaling elements can, for example, comprise or
consist of two (or more) fluorophores that interact via
fluorescence resonant energy transfer or one fluorophore and a
quencher (acting either via energy transfer or electron transfer).
Other interactions between a fluorophore and a second molecule or
simply another part of the synbody can be designed that change the
emission intensity, wavelength, spectral distribution, polarization
or excited state dynamics of the fluorophores upon binding to the
target. It is also possible for such conformational changes to
alter the absorbance properties of the fluorophores. In other
embodiments, the signaling elements can comprise or consist of one
or two (or more) electrochemical sensor molecules that interact to
change the observed midpoint potential or other aspects of the
current voltage relationship of one or more of the molecules.
Conformational changes of this kind can be directly observed via
methods that measure the change in index of refraction (e.g.,
surface plasmon resonance) or change the surface properties of the
material and thus the optical behavior at the interface (nonlinear
methods such as second harmonic generation). In further
embodiments, the signaling elements can comprise or consist of a
series of donor and acceptor signaling molecules that are all too
far apart for energy transfer to occur initially, but upon binding
of multiple target molecules (can either be the same or different
targets) become close enough together to form an energy (or
electron) transfer network. This makes signal generation nonlinear
and correlated with binding of multiple molecules (either the same
or different).
[0155] FIG. 8: In this example, the synthetic antibody comprises
affinity elements 1 and 2 that bind to target A. Further affinity
elements 3 and 4 are spatially arranged by the linker to affinity
elements 1 and 2 to self-assemble a complex of Targets A and B.
This example demonstrates the ability of the synbodies of the
invention to organize multiple components to direct the assembly of
enzymes or other functional systems from component parts. There are
many variations on this theme. In this figure, two targets are
brought together to form an enzyme by binding to the synbody.
Variations include, but are not limited to, bringing two subunits
in close contact for some function other than catalysis, or where
binding decreased enzyme activity or other functional activity.
This system provides a flexible template for programming enzymatic
or other functional activity in the same sense that an operon
serves as a template for interactions between proteins that
ultimately control gene transcription. All the same kinds of
binding-based control approaches seen in transcription or other
enzymatic control systems can be used here. Such systems could be
used to amplify a binding signal (in the same sense as an ELISA),
or to control the activity of an enzyme using in a chemical,
biochemical or biomedical process.
[0156] The synthetic antibodies of the invention can be present in
solution, frozen, or attached to a substrate. For example, a
library of synthetic antibodies can be produced, and arrayed on a
suitable substrate for use in various types of detection assays.
This provides a distinct advantage over conventional antibodies,
most of which do not work in array based applications. Thus, in
another embodiment, one or more synthetic antibodies of the
invention are bound to a surface of a substrate, either directly or
indirectly. The substrate can comprise an addressable array, where
the identity and location of each synthetic antibody on the array
is known. Examples of such suitable substrates include, but are not
limited to, microarrays, beads, columns, optical fibers, wipes,
nitrocellulose, nylon, glass, quartz, mica, diazotized membranes
(paper or nylon), silicones, polyformaldehyde, cellulose, cellulose
acetate, paper, ceramics, metals, metalloids, semiconductive
materials, quantum dots, coated beads, other chromatographic
materials, magnetic particles; plastics and other organic polymers
such as polyethylene, polypropylene, and polystyrene; conducting
polymers such as polypyrole and polyindole; micro or nanostructured
surfaces such as nucleic acid tiling arrays, nanotube, nanowire, or
nanoparticulate decorated surfaces; or porous surfaces or gels such
as methacrylates, acrylamides, sugar polymers, cellulose,
silicates, and other fibrous or stranded polymers. In one exemplary
embodiment, the substrate comprises a substrate suitable for use in
a "dipstick" device, such as one or more of the substrates
disclosed above.
[0157] Thus, in a further embodiment, the second aspect of the
invention provides a substrate comprising:
[0158] (a) a surface; and
[0159] (b) one or more synthetic antibodies of the second aspect
attached to the surface.
[0160] The substrate surface can comprise a plurality of the same
synthetic antibody, or a plurality of different synthetic
antibodies (where each synthetic antibody may itself also be
present in multiple copies, and wherein the affinity elements in
the different synthetic antibodies may be of different compounds
classes (ie: some affinity elements nucleic acid-based; some
polypeptide-based, etc.) When bound to a solid support, the
synthetic antibodies can be directly linked to the support, or
attached to the surface via known chemical means. In a further
embodiment, the synthetic antibodies can be arrayed on the
substrate so that each synthetic antibody (or subset of synthetic
antibodies) are individually addressable on the array, as discussed
herein. Thus, the substrates and/or the synthetic antibodies can be
derivatized using methods known in the art to facilitate binding of
the synthetic antibodies to the solid support, so long as the
derivitization does not interfere with binding of the synthetic
antibody to its target. The substrates may further comprise
reference or control compounds or elements, as well as identifying
features (RFID tags, etc.) as suitable for any given purpose.
[0161] In a third aspect, the present invention provides methods
for making synthetic antibodies (according to any of the synbody
embodiments disclosed herein), comprising connecting at least a
first affinity element and a second affinity element for a given
target via a linker;
[0162] wherein the second affinity element can bind to the target
in the presence of the first affinity element bound to the
target;
[0163] wherein one or both of the first affinity element and the
second affinity element have a molecular weight of at least 1000
Daltons;
[0164] wherein one or both of the first affinity element and the
second affinity element are not derived from the first target;
[0165] wherein the synthetic antibody has an increased binding
affinity and/or specificity for the first target relative to a
binding affinity and/or specificity of the first affinity element
for the first target and relative to a binding affinity and/or
specificity of the second affinity element for the target; and
[0166] wherein the first target is not an Fv region of an
antibody.
[0167] All terms and embodiments disclosed above for the first and
second aspects of the invention apply to this third aspect of the
invention. Connections between the affinity elements can be of any
type, including but not limited to covalent binding, hydrogen
bonding, ionic bonding, base pairing, electrostatic interaction,
and metal coordination, depending on the type of linker and the
types of affinity elements. Selection of an appropriate linker for
use in the methods of making synthetic antibodies of the invention
is well within level of skill in the art based on the teachings
herein. In further embodiments, three, four, or more affinity
elements can be physically connected by one, two, or more linkers.
In each of these embodiments, the affinity elements may all be of
the same compound type (nucleic acid, protein, etc.), different, or
combinations thereof. In various further embodiments, the further
affinity elements may bind to the same target or to one or more
different targets than the target bound by the first and second
affinity elements. When more than one linker is used, the linkers
may all be of the same compound type (nucleic acid, protein, etc.),
different, or combinations thereof.
[0168] The advantages of synthetic antibodies made by the methods
disclosed herein are discussed above. In one embodiment, the
methods comprise determining an appropriate spacing between the
affinity elements (ie: first affinity element and second affinity
element; first-second-third affinity element, etc.) in the affinity
element combination. An appropriate linker distance is one that
optimizes the affinity and/or specificity of the resulting synbody.
Any suitable technique for determining an appropriate spacing can
be used. In one non-limiting example, a predetermined set of
linkers that cover increments up to 100 nm are generated, and the
affinity elements are connected to each linker and the optimal
distance determined using appropriate binding assays. The linker
could be a derivatized PEG for example, but can be of any suitable
type that can be used to determine optimal spacing, as discussed in
detail above and in the examples that follow.
[0169] In another embodiment, determining optimal spacing involves
systems in which in situ synthesis of linkers on a surface is used
such that a series of compounds, (for example, polyalanine
peptides) is made with two variably spaced lysines, differentially
blocked, such that subsequent bulk attachment of the two peptides
(unblocking one lysine and then the other) gives a whole range of
spacings. Many other variations on this theme are possible using
peptides, nucleic acids or a variety of non-natural polymers,
heteropolymers, macrocycles, cavities, other scaffolds, and DNA
tiling arrays.
[0170] A further method involves using the flexibility of DNA to
create a set of matching oligonucleotides to separate two affinity
elements at set distances (FIG. 9a). The cassette aspect of this
system (as discussed in more detail below) allows ready
determination of which affinity elements synergize and at what
distance. Detection can be accomplished by any suitable method,
including but not limited to SPR electrochemical detection,
micromechanical detection (e.g., frequency shifts in resonant
oscillators), electronic detection (changes in conductance or
capacitance), mass spectrometry or other methods, or by spotting on
a slide with florescent detection of the target. An exemplary
system for SPR determination is depicted in FIG. 9c. On one slide
multiple combinations of polypeptides and their distances can be
tested as seen in FIG. 9c. This system is cost effective, simple,
available to broad affinity element repertoire, and amenable to
high throughput.
[0171] Thus, in a fourth aspect, the present invention provides a
composition, comprising:
[0172] (a) a first affinity element bound to a template nucleic
acid strand;
[0173] (b) a second affinity element bound to a complementary
nucleic acid strand, wherein the first affinity element and the
second affinity element non-competitively bind to a common
target;
[0174] wherein the template nucleic acid strand and the
complementary nucleic acid strand are bound to form an
assembly;
[0175] wherein the first affinity element and the second affinity
element are separated in the assembly; and
[0176] wherein either the template nucleic acid strand, the
complementary nucleic acid strand, or both, are bound to a surface
of a substrate.
[0177] In a further embodiment of this aspect, the composition
further comprises the common target bound to the first affinity
element and to the second affinity element.
[0178] These compositions (also referred to as a "molecular
slide-rule") can be used, for example, in the methods of the first,
third, and fifth aspects of the invention for determining an
optimal spatial separation of affinity elements in a synbody for a
given application.
[0179] The template nucleic acid strand and the complementary
nucleic acid strand are bound to form an assembly; this binding can
be of any type, including but not limited to covalent binding and
base pairing. One or both of the template nucleic acid strand and
the complementary nucleic acid strand are also bound to the
substrate surface; this binding can be of any type as discussed
above, such as covalent binding, while the template and
complementary nucleic acid strands are single stranded nucleic
acid; preferably DNA.
[0180] Affinity elements and substrates are as disclosed above. As
used in this aspect, "separated" means that the affinity elements
do not bind each other, but are positioned to permit determination
of optimal spacing of the affinity elements to permit binding of
the first and the second affinity elements to the target
simultaneously. For example, the different versions of the
composition have the affinity elements separated by repetitive
turns of the DNA helix (ie: the double stranded nucleic acid in the
assembly formed by the template nucleic acid strand and the
complementary strand base pairing).
[0181] In a further embodiment of this fourth aspect, the invention
provides an array, comprising a plurality of the compositions
disclosed above bound to a substrate surface, wherein the plurality
of compositions comprises one or both of:
[0182] (a) a plurality of compositions wherein the first ligand and
the second ligand are the same for each composition, but wherein
the separation of the first ligand from the second ligand in the
assembly differs; and
[0183] (b) a plurality of compositions wherein the first ligand
and/or the second ligand are different for each composition.
[0184] As used in this aspect, a plurality is 2 or more; preferably
3, 4, 5, 6, 7, 8, 9, 10, or more. The compositions of option (a)
are preferred for determining optimal distance between the first
and second affinity elements in the synbody, while option (b) is
preferred to multiplex the assay
[0185] Binding of the compositions of the fourth aspect of the
invention to the substrate can be by any suitable technique, such
as those disclosed herein.
[0186] In this fourth aspect, the double stranded nucleic acid is
used to template-direct the assembly of different affinity element
pairs with programmed nanometer-scale spacing. DNA is an ideal
material for developing synthetic architectures due to the fact
that it is easy to engineer and self-assembles into highly
reproducible structures of known morphology. In one non-limiting
example, the template strand is conjugated to affinity element 1
and annealed to a complementary strand which is conjugated to
affinity element 2. The system is designed such that affinity
element 1 is separated from affinity element 2 by one additional
base separations and the repetitive turns of a DNA helix (FIG. 9b).
Each base can be used to separate the two affinity agents. For each
turn of the DNA helix corresponds to separation distances of
roughly 4 nm, 7.5 nm, 11 nm. Each affinity element-pair complex is
spotted at independent positions on a surface and the relative or
actual binding of the target to each complex is determined by any
suitable technique, including but not limited to fluorescence or
surface plasmon resonance (SPR).
[0187] The compositions of this fourth aspect can be attached to a
surface (FIG. 9(c)) in an array format using a psoralen
photocrosslinking strategy. This can be done using a psoralen-DNA
`linker` strand that is able to recognize a region of the template
downstream of the variable strand. Once the linker strand is
annealed to the template, exposure to UV light results in chemical
cross linking of the linker strand to the DNA helix containing
affinity element 1 and 2. Excess linker strand is then removed from
the reaction mixture by affinity separation, and target binding
activity and specificity is carried out. Screening can be achieved
by traditional fluorescence-based assays whereby the synthetic
antibody is attached to a glass slide or to a bead and then
screened with fluorescently labeled target. Additionally, the
synthetic antibody can be attached to a gold surface and screened
with a label-free technique such as SPR, electrochemical detection,
micromechanical detection (e.g., frequency shifts in resonant
oscillators), electronic detection (changes in conductance or
capacitance), mass spectrometry or other methods.
[0188] In a fifth aspect, the present invention provides methods
for ligand identification, comprising:
[0189] (a) contacting a substrate surface comprising a target array
with one or more potential ligands, wherein the contacting is done
under conditions suitable for moderate to high affinity binding of
the one or more ligands to suitable targets present on the
substrate; and
[0190] (b) identifying targets that bind to one or more of the
ligands with at least moderate affinity.
[0191] The target array can be any array of targets of interest as
disclosed herein. In various embodiments, the array may comprise
50, 100, 500, 1000, 2500, 5000, 10,000; 100,000; 1,000,000;
10,000,000 or more targets. In a further embodiment, the target
array is addressably arrayed (as disclosed above for compound
arrays) for ease in identifying targets that have been bound.
Detection of binding can be via any method known in the art,
including but not limited to those disclosed elsewhere herein.
[0192] The targets may comprise any target class as described
herein. In one embodiment, the targets are protein targets. In a
further embodiment, the target array comprises a range of different
protein targets, for protein targets not all related based on minor
variations of a core sequence. In a further embodiment, the targets
are not antibodies or Fv regions of antibodies. In further
embodiments, the first target is not an antibody, an antibody
bearing cell, or an antibody-binding cell surface receptor (or
portion thereof suitable for antibody binding)
[0193] Similarly, the potential ligands can be any suitable
potential ligand as disclosed herein (ie: compounds or affinity
elements). In various embodiments, the potential ligand comprises a
synthetic antibody according to any aspect or embodiment of the
present invention. In a further embodiment, the potential ligand
may be one for which a target specificity has not previously been
established.
[0194] All terms and embodiments disclosed above apply equally to
this aspect of the invention. In embodiments where the synthetic
antibodies of the invention are used, the one or more synthetic
antibodies to be screen as potential ligands comprise a first
affinity element and a second affinity element, wherein one or both
of the first affinity element and the second affinity element have
a molecular weight of at least about 1000 Daltons; in further such
embodiments, one or both of the first and second affinity elements
comprise or consist of polypeptides Alternatively, the candidates
could be constructed from rational design of the ligands or even
from random sequences.
[0195] For artificial antibodies the starting point is almost
always the protein or other target. A library of variants (single
chain antibody clones, phage display of peptides, aptamer
libraries, etc.) is screened against the protein target. A single
clone or consensus of sequences is isolated as the specific ligand
to a specific target. In all these types of examples, the starting
point is a particular target for which a ligand is isolated.
[0196] In contrast, this aspect of the invention turns this
standard procedure for creating ligands on its head. We first
create one, a few or a library of potential ligands. For example,
we create a synbody (using, for example, the methods disclosed
above) consisting of two 20 mer polypeptides of random
(non-natural) sequence linked by a linker. In one non-limiting
embodiment, the synbody has the two different polypeptides linked
about 1 nM apart. The synbody is labeled and then reacted with an
array with 8000 human proteins. A protein is identified that the
synbody binds with high affinity and specificity. In this way a
very good synthetic antibody is isolated for that particular
protein. A unique aspect of this invention is that the usual
process is reversed--a potential ligand is made and then a library
of targets is screened for a target that is appropriately
reactive.
[0197] This system is amenable to high throughput or even massively
parallel screening. For example, a large number of potential
ligands can be constructed by combining various binding elements,
linkages, and spacing distances using, for example, the methods and
synthetic antibodies disclosed above. These could be mixed (or
prepared by combinatorial methods) and reacted with a large number
of targets. The ligand on each target could be identified by any
suitable technique, including but not limited to mass spectrometry,
bar coding or mixed fluorescent tags. An advantage of this system
is that it not only determines the affinity of the ligand for a
particular target, but also the off-target reactivities to all the
other proteins on the array.
[0198] This approach defies conventional wisdom, which would
suggest that the space of possible target shapes is far too large
for a screening strategy of this kind to produce synbodies having
antibody-like affinities and specificities. While not being bound
by a specific mechanism, the inventors believe (as described above)
that there are a very limited number of distinct substructures on
the surface of proteins. That is, unlike sequence space, the
structural space represented on the surface of proteins is very
limited. Proteins have a limited number of shapes on their surface.
A second aspect of the hypothesis is that a small number of
appropriately chosen ligands can represent the structural
complements of all the shapes present on protein surfaces. For
example, 5,000 20-amino acid polypeptides of non-life sequence can
provide most complementary shapes. A third aspect is that if two of
these shape binding elements are held at a fixed distance, the
resulting synbody is likely to find, in a library of reasonable
size, some protein having complementary shapes at that distance,
and will bind that protein in a cooperative fashion and with high
specificity.
[0199] In various further embodiments of this aspect of the
invention are methods for screening the antibodies and synbodies on
a protein microarray in a manner that reduces the number of (very
expensive) microarrays required for screening a given number of
candidates. In one non-limiting example, affinity data is read
using a real-time microarray reader with the protein microarray
mounted in a flow chamber. Buffer containing a single antibody or
synbody in very low concentration is flowed over the microarray
until binding is detected on a small number of targets; these will
be the highest affinity targets for that antibody or synbody. Since
the antibody or synbody has very low affinity for all but the few
targets for which it is specific, and since the antibody or synbody
is applied at very low concentration and the flow stopped after
binding is detected, nearly all targets will remain unoccupied and
even the occupied targets will be far from saturation. The process
can then be repeated with a second antibody or synbody, thereby
obtaining maximum benefit from the protein array.
[0200] In another embodiment, the methods of this aspect of the
invention can be used to identify new targets for existing
antibodies, including therapeutic, diagnostic, and research
antibodies. As disclosed below, the methods provide valuable
information on the specificity of such antibodies in a high
throughput and low cost manner, and allow identification of
antibodies specific for targets for which antibodies are currently
unavailable.
[0201] In a sixth aspect, the present invention provides methods
for identifying a synthetic antibody profile for a test sample of
interest, comprising contacting a substrate comprising a plurality
of synthetic antibodies according to the present invention with a
test sample and comparing synthetic antibody binding to the test
sample with synthetic antibody binding to a control sample, wherein
synthetic antibodies that differentially bind to targets in the
test sample relative to the control sample comprise a synthetic
antibody profile for the test sample.
[0202] As used in this aspect, a plurality means 2 or more;
preferably 50, 100, 250, 500, 1000, 2500, 5000, or more. The test
sample can be any sample of interest, including but not limited to
a patient tissue sample (such as including but not limited to
blood, serum, bone marrow, saliva, sputum, throat washings, tears,
urine, semen, and vaginal secretions or surgical specimen such as
biopsy or tumor, or tissue removed for cytological examination),
research samples (including but not limited to cell extracts,
tissue extracts, organ extracts, etc.), or any other sample of
interest. Such a patient sample can be from any patient class of
interest. The control sample can be any suitable control, such as a
similar tissue sample from a known normal, or any other standard.
Thus, the methods can be used, for example, as a diagnostic,
prognostic, or research tool. In one embodiment, the control sample
is contacted with the same substrate as the test sample; in another
embodiment, the control sample is contacted with a different but
similar or identical substrate as the test sample.
[0203] In this aspect, a plurality of synthetic antibody candidates
(ie: 10, 20, 50, 100, 250, 500, 1000, 2500, 5000 or more) are
arrayed in an addressable fashion, for example on a printed slide.
The ligands in the candidates could be from pre-selected sequences,
rational design or random sequence. These arrays would then be used
to screen samples of interest. For example they could be serum from
normal and affected subjects. Synthetic antibodies that bound
components of the serum and ones that differentially bound
components between the two samples could be selected. The actual
target or targets bound by each synthetic antibody could be
determined directly from the array by mass spectrometry or by using
the synthetic antibody as and affinity agent to purify the
targets.
[0204] Any one or all of the steps of the methods of the different
aspects of the invention can be automated or semi-automated, using
automated synthesis methods, robotic handling of substrates,
microfluidics, and automated signal detection and analysis hardware
(such as fluorescence detection hardware) and software.
[0205] Thus, in another aspect, the invention provides computer
readable storage media comprising a set of instructions for causing
a signal detection device to execute procedures for carrying out
the methods of the invention. For example, the procedures comprise
the signal processing, target affinity element identification steps
and databasing of the second aspect of the invention, and any/all
embodiments thereof The computer readable storage medium can
include, but is not limited to, magnetic disks, optical disks,
organic memory, and any other volatile (e.g., Random Access Memory
("RAM")) or non-volatile (e.g., Read-Only Memory ("ROM")) mass
storage system readable by a central processing unit ("CPU"). The
computer readable storage medium includes cooperating or
interconnected computer readable medium, which can exist
exclusively on the processing system of the processing device or be
distributed among multiple interconnected processing systems that
may be local or remote to the processing device.
[0206] The invention further provides kits, comprising any one or
more of the reagents disclosed herein. Such kits can be used, for
example, for selecting affinity elements and making synbodies out
of them, using the methods disclosed herein.
EXAMPLE 1
[0207] In one non-limiting embodiment of this second aspect of the
invention, an array of 4,000 polypeptides is spotted on a slide.
Each polypeptide is 20 amino acids in length, and is spotted such
that its orientation is controlled to be through the C-terminus. A
large amount of sequence and chemical space can be adequately
sampled using only a small fraction of the possible space. For
example, in the case of this array, there are
19.sup.17=5.times.10.sup.21 possible polypeptide sequences (the
first 3 amino acids are held constant, but this is not necessary
and cysteine is used only at the C-terminus as attachment via a
thiol), but we sampled just 4.times.10.sup.3 sequences and can
identify polypeptides that show moderate binding affinity and
specificity to a number of proteins.
[0208] The target protein is labeled with a florescent dye and
incubated with the array. Polypeptides that bind the target protein
are determined. Alternatively, we have incubated unlabelled
affinity tagged form of the target protein and detected binding by
virtue of a secondary antibody against the tag. Each sequence of
the polypeptides on each spot is already known; thus, the process
is a screen for elements, not a selection. Thus, the process of
ligand discovery is limited only by the rate at which individual
targets can be screened on pre-printed polypeptide arrays. In this
sense it is distinct from aptamer, phage or other panning methods,
in which recurrent selections using unknown sequences are required,
and only those elements that do bind a target are determined, while
those that do not bind are not known.
[0209] Whether such a small sequence space can yield effective
binders depends on how the binding space is shaped. If the slope of
relative binding affinity is very steep around the optimal
polypeptides, it is unlikely that one of the 4,000 polypeptides
will be close to one of the optimal polypeptides. If however, the
slope of the binding space is gradual, one may find polypeptides
that are on the "side of the mountain." If the determination of the
optimal polypeptide is by virtue of sequence similarity, it is very
unlikely that in 4000 polypeptides ones with sequence similar to
the optimal would be found in the 10.sup.21 possibilities (for 17
mer polypeptides).
[0210] Most experts in this field thought this process would not
work--but it does. Consistent with the logic above, most of the
polypeptides that bind a particular site on a protein do not
resemble each other in sequence. Therefore, while not being bound
by any hypothesis, we suggest the following explanation, which
represents a new insight into peptide sequence space. We propose
that the 10.sup.21 possible 17 mer polypeptides actually form a
very limited number (.about.4000) of structural forms. This view
has several important predictions and implications. First, the
space dimension would be much smaller. Therefore, around each
optimal sequence would be structurally related polypeptides on the
side of the mountain that would not necessarily have any sequence
similarity. Second, several proteins may bind to a specific peptide
but that peptide could be varied to bind better to one or the
other. In other words, the same 4000 polypeptides may be all that
is needed to generate synbodies to virtually an unlimited number of
targets.
[0211] Once a set of affinity agents are isolated for a given
target we may use these directly or use them to create an
artificial antibody. For the latter we identify two or more
elements that bind different sites on the targets. To do so we can,
for example, block target binding with the target polypeptides or
co-spot them on slides or we can put pairs onto DNA linkers to
determine pairs and spacing simultaneously (FIG. 9c). The pairs of
affinity elements may be valuable in themselves.
[0212] We then create a synbody using the system for measuring as
described. A first affinity element is covalently attached to a DNA
template strand, and separately attaching affinity element two to
different nucleotide positions on a complementary strand. We anneal
the two strands of DNA and immobilize the complex to 400 different
sites on a surface plasmon resonance (SPR) Flexchip. We then flow
the target of interest over the surface to identify different
ligand pairs and ligand pair separation distances with enhanced
binding. Ligand pairs and ligand pair separation distances with the
greatest binding enhancement are either used directly or
reconstructed with synthetic tethers based on the distance
parameter determined in the SPR analysis. We have used this process
to generate a synbody to Gal80 that exhibits enhanced binding as
described in detail in Example 6 below. The Gal80 synbody functions
with high affinity and high specificity in solution (Elisa format)
and on a solid surface (see Example 8).
[0213] Synbodies developed with the techniques disclosed above in
the second, third, and/or fourth aspects of the invention function
when immobilized to a surface and also function as a solution phase
binding agent. The highest binding synbody candidate from one
experiment was used as the detection agent in an ELISA experiment
and the solution phase dissociation constant (K.sub.d) was
determined for the synbody, each polypeptide on the synbody and the
DNA backbone (see Example 8). This data demonstrates that a large
increase in binding affinity can be achieved through the use of the
synergistic polypeptides with the proper distance. An additional
advantage to this approach is that the synbody is discovered in a
single assay and then there is enough of the synbody available to
immediately use as the detection agent in a functional assay. This
in effect couples discovery and production into a single step,
dramatically shortening the synbody development time.
EXAMPLE 2
Microarray Selection of Affinity Elements for Synbody
[0214] This example demonstrates the identification of affinity
elements by screening a target on an array of random polypeptides.
A microarray was prepared by robotically spotting about 4,000
distinct polypeptide compositions, two replicate array features per
polypeptide composition, on a glass slide having a poly-lysine
surface coating. Each polypeptide was 20 residues in length, with
glycine-serine-cysteine as the three C-terminal residues and the
remaining residues determined by a pseudorandom computational
process in which each of the 20 naturally occurring amino acids
except cysteine had an equal probability of being chosen at each
position. Cysteine was not used except at the C-terminal position,
to facilitate correct conjugation to the surface. Polypeptides were
conjugated to the polylysine surface coating by thiol attachment of
a C-terminal cysteine of the polypeptide to a maleimide
(sulfo-SMCC, sulfosuccinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate, see FIG. 10(a)),
which is covalently bonded to the .epsilon. amine of a lysine
monomer of the poly-lysine surface coating, as shown in FIG. 10(b).
The polypeptides were synthesized by Alta Biosciences, Birmingham,
UK. Each polypeptide was first dissolved in dimethyl formamide
overnight and master stock plates prepared by adding an equal
volume of water so that the final polypeptide concentration was
about 2 mg/ml. Working spotting plates were prepared by diluting
equal volumes of the polypeptides from the master plates with
phosphate buffered saline for a final polypeptide concentration of
about 1 mg/ml. The polypeptides were spotted in duplicate using a
SpotArray 72 microarray printer (Perkin Elmer, Wellesley, Mass.)
and the printed slides stored under an argon atmosphere at
4.degree. C. until used. Any other spotting/immobilization
chemistry and/or method operable for immobilizing polypeptides on
an array surface in a manner compatible with the intended array
assay may be employed; by way of non-limiting examples,
polypeptides may be conjugated directly to a polylysine surface
coating via an amide bond between the C-terminal residue of the
polypeptide and the .epsilon. amine of a lysine, or may be
conjugated to an aminosilane or other functionalized surface
exposing free amines. Linkers other than or in addition to SMCC may
also be employed; by way of non-limiting example, a PEG linker may
be used to position the polypeptide away from the substrate.
Surface functionalizations other than amine can be employed,
coupled with conjugation chemistry appropriate for attachment of
the affinity elements to the surface moieties provided. In some
embodiments the surface immobilization may be non-covalent.
[0215] Several polypeptides were identified as candidate affinity
elements for synbodies against an arbitrarily chosen protein
target, transferrin, by incubating transferrin on the polypeptide
microarray in the presence of E. coli lysate competitor.
Transferrin was randomly direct-labeled at free amines with
Alexa.TM. 555, and E. coli lysate was randomly direct-labeled at
free amines with Alexa.TM. 647. Three replicate arrays were
passivized by applying a mixture of BSA and mercaptohexanol for one
hour. The arrays were blocked with unlabelled E. coli lysate for
one hour, then washed three times with TBST (0.05% Tween) followed
by three times with water. A mixture of labeled transferrin and
labeled E. coli lysate was applied to the three replicate arrays
and incubated for three hours. The arrays were again washed three
times with TBST (0.05% Tween) followed by three times with water,
and scanned at 555 nm and 647 nm using an array reader.
Polypeptides were ranked as candidates for inclusion as affinity
elements of synbodies by computing a score for each polypeptide
equal to the mean raw 555 nm intensity over the six replicate
features, squared, divided by the mean raw 647 nm intensity over
the six replicate features. This simple scoring function tends to
favor candidate polypeptides that bind at least moderate affinity,
since otherwise the 555 nm intensity would be relatively lower, and
that are relatively specific, since otherwise the 647 nm intensity
would be relatively higher and contribute to a relatively lower
score. Many variations of this ranking and identification process
can be used, such as, by way of non-limiting examples, two-color
comparisons against other competitors; comparisons with data taken
in separate experiments with respect to other targets; and use of
scoring functions taking into account other factors, employing
other functional relationships, and/or involving statistical
analysis and/or preprocessing of data and/or correcting for
background fluorescence and/or other factors affecting the accuracy
of the measured intensities. Ten polypeptides (Table 1) were
identified for further evaluation for use as affinity elements in
synbodies by choosing the polypeptides having the highest score
(one polypeptide was rejected as difficult to synthesize, so the
polypeptides chosen were ten of those having the eleven highest
scores).
TABLE-US-00001 TABLE 1 Transferrin binding affinity elements TRF19
KEDNPGYSSEQDYNKLDGSC (SEQ ID NO: 1) TRF20 GQTQFAMHRFQQWYKIKGSC (SEQ
ID NO: 2) TRF21 QYHHFMNLKRQGRAQAYGSC (SEQ ID NO: 3) TRF22
HAYKGPGDMRRFNHSGMGSC (SEQ ID NO: 4) TRF23 FRGWAHIFFGPHVIYRGGSC (SEQ
ID NO: 5) TRF24 SVKPWRPL1TGNRWLNSGSC (SEQ ID NO: 6) TRF25
APYAPQQIHYWSTLGFKGSC (SEQ ID NO: 7) TRF26 AHKVVPQRQIRHAYNRYGSC (SEQ
ID NO: 8) TRF27 LDPLFNTSIMVNWHRWMGSC (SEQ ID NO: 9) TRF27
LDPLFNTSIMVNWHRWMGSC (SEQ ID NO: 10) TRF28 RFQLTQHYAQFWGHYTWGSC
(SEQ ID NO: 11)
EXAMPLE 3
Microarray Selection of Affinity Elements for DNA Linked
Synbody
[0216] This example demonstrates another embodiment of a process
for identifying affinity elements for incorporation into a synbody.
15-mer polypeptide affinity elements for a DNA linked synbody
specific for Gal80 were identified by obtaining and analyzing data
from several polypeptide microarray experiments performed using
standard 4,000 feature polypeptide microarrays each of whose
features comprised a polypeptide 15 residues in length, terminating
in glycine-serine-cysteine at the C-terminus, with the other 12
residues selected from 8 of the 20 naturally occurring amino acids
according to a pseudorandom algorithm. Four fluorophore-labeled
protein targets--gal80, gal80 complexed with gal4 binding
polypeptide, transferrin, and .alpha.-antitrypsin--were supplied to
LC Sciences for array analysis according to LC Sciences'
proprietary protocol, and binding (fluorescence intensity) data
were obtained. For screening against the random peptide array,
Gal80 was labeled with Cy3 and Cy5 fluorescent dyes (GE Healthcare)
according to the manufacturer's protocol. The dye-to-protein ratio
was determined using the Proteins and Labels settings on a Nanodrop
ND-100 spectrophotometer (Nanodrop Technologies). The
dye-to-protein ratio for Cy3 and Cy5 labeled Gal80 was 3.4 and 5.0
respectively. The blocking solution used to block the peptide
arrays was composed of 1% bovine serum albumin (BSA), 0.5% non-fat
milk, 0.05% Tween-20 in 1.times. phosphate buffered saline (PBS) pH
7.4. After blocking, each array was then washed 3 times with a wash
buffer composed of 0.05% Tween-20 in 1.times. PBS, pH 7.4. The
incubation buffer was composed of 1% bovine serum albumin (BSA),
0.5% non-fat milk, in 1.times. phosphate buffered saline (PBS) pH
7.4. An Axon GenePix 400B Microarray Scanner (Molecular Devices,
Sunnyvale, Calif.) was used to acquire images of the peptide
arrays. An initial scan of the array was acquired to determine any
background fluorescence from each peptide on the array. Fluorescent
intensities obtained after protein incubation were subtracted from
the background fluorescence and exported into Microsoft Excel for
analysis.
[0217] Gal4 binding polypeptide is known to bind gal80 at a
specific binding site (the gal4 binding site). 142 of the array
polypeptides bound gal80 at above-threshold fluorescent
intensities, 29 of the array polypeptides bound gal80 complexed to
gal4 binding polypeptide at above-threshold fluorescent
intensities, and 10 of the array polypeptides bound both gal80 and
gal80 complexed to gal4 binding polypeptide at above-threshold
fluorescent intensities. Polypeptides that bound gal80 complexed to
gal4 binding polypeptide but that did not bind gal80 alone were
rejected as likely to be binding to the gal4 binding polypeptide.
Intensity data for polypeptides that bound gal80 alone but not
gal80 complexed to gal4 binding polypeptide (implying that these
polypeptides were binding to the gal4 binding site on gal80) were
compared with the intensity data for the same polypeptides with
respect to transferrin and .alpha.-antitrypsin; polypeptides
showing significant binding to either transferrin or
.alpha.-antitrypsin were excluded, and of the polypeptides
remaining, the polypeptide having the highest intensity binding for
gal80 was chosen as a first affinity element for incorporation in
the gal80 synbody. Intensity data for polypeptides that bound both
gal80 alone and gal80 complexed to gal4 binding peptide (implying
that these polypeptides were binding gal80 at a site other than the
gal4 binding site) were compared with intensity data for the same
polypeptides with respect to transferrin and .alpha.-antitrypsin;
again, polypeptides showing significant binding to either
transferrin or .alpha.-antitrypsin were excluded, and of the
polypeptides remaining, the polypeptide having the highest
intensity binding for gal80 was chosen as the second affinity
element for incorporation in the gal80 synbody. The sequences of
the chosen polypeptides were as shown in Table 2.
TABLE-US-00002 TABLE 2 Ga180 binding affinity elements BP1
NH.sub.2-GTEKGTSGWLKTGSC-CO.sub.2H (SEQ ID NO: 12) BP2
NH.sub.2-EGEWTEGKLSLRGSC-CO.sub.2H (SEQ ID NO: 13)
EXAMPLE 4
SPR Verification of Binding Characteristics of Transferrin Synbody
Affinity Elements
[0218] This example demonstrates SPR determination of the binding
characteristics of affinity elements. Transferrin was immobilized
by amine-coupling to the carboxyl-functionalized surface of a
Biacore T100 CMS Dextran SPR chip as illustrated in FIG. 11. A 1:1
mixture of EDC (0.4M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
in water) and NHS (0.1M N-hydroxysuccinimide in water) was applied
300 at a flow rate of 5 to 10 .mu.l/min for a contact time of about
6 to 10 minutes to activate the surface by conjugating a maleimide
306 to the surface-exposed carboxyl groups. Transferrin 25 .mu.g/ml
in immobilization buffer selected for correct pH was then applied
302 at a flow rate of 5 to 10 .mu.l/min for a contact time of about
5 to 10 minutes, allowing the amine functionality on the
transferrin 308 to displace the activated NHS ester and bond to the
surface via an amide bond. Finally, ethylene diamine (1M ethylene
diamine-HCl at pH 8.5) was applied 304 at a flow rate of 5 to 10
.mu.l/min for a contact time of about 6 to 7 minutes to deactivate
any remaining reactive groups on the dextran chip surface. Flow
rates and contact times are adjusted as necessary to provide the
surface concentration of target desired for the intended
application, and may vary by target. In general, for evaluating
whether binding occurs, it is preferable to immobilize a relatively
large quantity of target, and higher flow rates and/or longer
contact times may be used. For determining kinetics, it is
preferable to limit the amount of target immobilized so as to
minimize rebinding and avidity effects, and lower flow rates and/or
contact times may be used.
[0219] Candidate affinity elements for the transferrin synbody
TRF19, TRF21, TRF23, TRF24, TRF25, and TRF26 were individually
evaluated for solution phase K.sub.D with respect to transferrin by
SPR analysis. Because the off rates for these polypeptides were
very high, K.sub.D values were estimated by measuring steady-state
response for at least five concentrations in a two-fold dilution
series, each concentration tested in duplicate. For each
experiment, response data were processed using a reference surface
to correct for bulk refractive index changes and any non-specific
binding. Data were also double referenced using responses from
blank running buffer injections. Each experiment was conducted at
25.degree. C. using PBST (0.01M Phosphate Buffered Saline, 0.138M
NaCl, 0.0027M KCl, 0.05% surfactant Tween20, pH 7.4) as the running
buffer on a Biacore T100 instrument. Analytes were injected for 60
s at a flow rate of 30 .mu.l/min. The antigen surfaces were
regenerated with 30 s consecutive pulses of NaOH/NaCl (50 mM NaOH
in 1M NaCl) and Glycine (10 mM glycine-HCl, pH 2.5). Estimate
K.sub.D values are shown in Table 3.
TABLE-US-00003 TABLE 3 K.sub.D values for transferrin synbody
candidate affinity elements Solution Phase K.sub.D TRF19 ~150 uM
TRF21 ~60 uM TRF23 ~50 uM TRF24 ~50 uM TRF25 ~60 uM TRF26 ~100
uM
EXAMPLE 5
SPR Analysis of Affinity Element Binding to Distinct/Multiple Sites
on Target
[0220] This example demonstrates an SPR-based method for
identifying polypeptide affinity elements that bind distinct sites
on a protein target. The transferrin target was immobilized on a
Biacore T100 SPR chip, and candidate polypeptides were applied in
1:1 mixtures in pairs and response data obtained, in accordance
with the methods described in Example 4 above. As illustrated in
FIG. 12, upon flowing candidate polypeptides over the immobilized
target, ideally one polypeptide applied alone would bind to a first
binding site on the target and produce a first characteristic SPR
response level (FIG. 12(a)), the other polypeptide would bind to a
second, distinct binding site on the target, producing a second
characteristic response level (FIG. 12(b)), and a mixture of the
two polypeptides together (at the same concentrations as before)
would produce a response level approximating the sum of the
response levels produced by each polypeptide alone, as the
polypeptides bind to distinct binding sites (FIG. 12(c)). However,
it is also possible that the two polypeptides do not bind distinct
sites on the target, but instead compete for the same binding site
(FIG. 12(d)), in which case the expected SPR response would be
intermediate between the response level produced by either
polypeptide separately and the sum of the two. FIG. 13 shows the
results of evaluation of a number of pairs of the polypeptides that
were identified as described in Example 2 (see Table 1). Among
other pairs, TRF23 and TRF26 had solution phase affinities for
transferrin in a range of K.sub.D of about 50 to 100 .mu.M (see
Table 3) and were found to bind distinct sites on transferrin.
[0221] Analysis to determine ability to bind distinct binding sites
can be performed by any other method operable to assess whether two
affinity elements do or do not mutually interfere in binding to the
target. By way of non-limiting example, this may be done by
comparing, by array experiment, SPR, or any other suitable method,
a polypeptide's binding characteristics with respect to a target
with the target pre-bound to a target-specific antibody; it may be
inferred that polypeptides that bind the target with and without
the antibody present are likely binding to a site other than the
site that the antibody binds, and that polypeptides that bind the
target without the antibody present and do not bind with the
antibody present are likely binding to the site that the antibody
binds.
EXAMPLE 6
Synthesis of DNA-Linker Synbody
[0222] This example demonstrates the synthesis of a synbody
specific for gal80, comprising two 15-mer polypeptide affinity
elements identified as described in Example 3 joined by a DNA
linker. The structure is illustrated schematically in FIG. 15. The
DNA linker sequence was determined randomly, subject to the
constraints that the sequence should not result in predicted
formation of secondary structures, should not be similar or
identical to any naturally occurring sequence as determined by
BLAST search, and the variable strand should have cytosine residues
at the locations at which attachment of the affinity elements is
desired (although other attachment modalities could be used, for
convenience the attachment employed involved C6 amine modification
of the cytosine base). The template strand 314 was amine-modified
at the 5' terminal cytosine residue to allow attachment of the
polypeptide affinity element 330 via a maleimide linker 328. The
variable strand 316 was reverse complementary to the template
strand and was amine-modified at an internal cytosine residue to
allow attachment of the other polypeptide affinity element 334,
again via a maleimide linker 332. A library of variable strands
were obtained, each amine-modified at a different position, to
provide a range of attachment points corresponding to a range of
separation distances between the affinity elements. Determination
of attachment points also took into account the angular orientation
of residues along the DNA helix, so as to avoid positioning the
affinity elements on opposite sides of the DNA backbone. For B-DNA
in solution under physiological conditions, the double helix makes
a complete rotation in about 10.4 to 10.5 base pairs and has a
length of about 3.4 nm per 10 base pairs. To align the attachment
points of the affinity elements at approximately the same angular
position around the longitudinal axis of the helix, and keeping in
mind that the affinity elements are attached to opposite strands,
the bases comprising the attachment points may be chosen at a
separation of approximately an even multiple of about 10.5 (one
full rotation) plus about 4 (to account for the difference in
angular position between the strands), plus or minus about 2 or 3
(since affinity elements do not necessarily bind optimally to the
target by being perfectly aligned with each other). By screening
various attachment points, various separation distances and
relative orientations of the affinity elements can be tested. For
the example here described, variable strands having amine-modified
cytosines at positions 13, 15, 17, 24, 26, and 28 (counting from
the 3' end of the variable strand) were obtained. The
amine-modified cytosines (hereafter dC C6) were incorporated in the
oligonucleotides using
5'-Dimethoxytrityl-N-dimethylformamidine-5-[N-(trifluoroacetylaminohexyl)-
-3-acrylimido]-2'-deoxyCytidine,
3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, see FIG. 14,
and have a trifluoroacetylaminohexyl moiety 310 extending from the
5 carbon of the cytidine base.
[0223] The polypeptides were conjugated to synthetic DNA template
314 and variable 316 strands in accordance with methods described
in detail in Williams B A R, Lund K, Liu Y, Yan H, Chaput J C:
Self-Assembled Peptide Nanoarrays: An Approach to Studying
Protein--Protein Interactions. Angew Chem Int Ed 2007,
46:3051-3054. The two DNA oligonucleotides, template strand 314 (5'
(dC C6)CC GAA ACA ACC GCG AGA GGC ACG CGC GTA GCC GTC ACC GGC
TAT-3' (SEQ ID NO: 14), wherein the 5' terminal dC C6 is
amine-modified cytosine as described above) and variable strand 316
(5' GCT ACG CGC GTG CCT CTC G(dC C6)G GTT GTT TCG GG-3' (SEQ ID NO:
15), wherein the dC C6 appearing at the position 13 counting from
the 3' terminus is amine-modified cytosine) were purchased from
Keck Oligonucleotide Synthesis Facility (Yale University). These
were conjugated (at the trifluoroacetyl moiety (312, FIG. 14) of
the amine-modified cytosine to the bifunctional linker
4-(maleimidomethyl)-1-cyclohexane carboxylic acid
N-hydroxysuccinimide ester (SMCC, Sigma Aldrich) 328, 332 by
combining 200 .mu.L of SMCC (1 mg/mL) in acetonitrile with 200
.mu.L of DNA (20 nmol) in 0.1 M KHPO.sub.4 buffer (pH 7.2).
Following a 3 h incubation at room temperature, a second portion
(20 .mu.L) of SMCC (10 mg/ml) was added and the reaction was
allowed to continue overnight at room temperature. Excess SMCC was
removed from the SMCC conjugated DNA samples by size exclusion
chromatography on a Nap-5 column (Amersham Bioscience). To
construct the polypeptide-oligonucleotide conjugates, the Gal 80
binding polypeptide 330 (NH.sub.2-GTEKGTSGWLKTGSC--CO.sub.2H, (SEQ
ID NO: 12) 20 nmol) was incubated with the SMCC-conjugated template
strand 314 (2 nmol) in 200 .mu.L of 0.1 M KHPO.sub.4 buffer (pH
7.2) and the Gal 4 activation domain peptide 334
(NH.sub.2-EGEWTEGKLSLRGSC--CO.sub.2H, (SEQ ID NO: 13) 20 nmol) was
incubated with the SMCC-conjugated variable strand 316 (2 nmol) in
200 .mu.L of 0.1 M KHPO.sub.4 buffer (pH 7.2) for 3 h at room
temperature, resulting in conjugation of the C-terminal cysteine of
the polypeptides to the respective SMCC linkers 328, 332.
Polypeptide-oligonucleotide conjugates were HPLC purified. The two
polypeptide-oligonucleotide conjugates readily undergo
hybridization by Watson-Crick base pairing.
[0224] The Gal 80-template strand conjugate 314 was cross-linked
338 to a thiol containing DNA oligonucleotide 318 (5' (psoralen) TA
GCC GGT GTG AAG TTT CTG CTA GTA ATG (thiol modifier C3) 3') (SEQ ID
NO: 16) which is partially reverse complementary to part of the
3'-terminal region of the template strand 314 and able to partially
hybridize to the template strand (and was then crosslinked 338 to
the template strand 314 for stability), with the 3' end of the
thiol containing oligo 318 extending single-stranded from the
synbody construct and providing, via the thiol modifier 320, a
conjugation site for maleimide-modified biotin 322, which in turn
provides a site to which streptavidin 324 conjugated HRP 326 can be
attached, enabling use of the construct in an ELISA-type assay.
Inclusion of the third DNA strand 318 is optional. If the third DNA
strand 318 is used, any attachment chemistry operable to attach any
desired entity to the unhybridized portion of the strand may be
used; by way of non-limiting example, any maleimide may be
conjugated to the thiol modifier, and if maleimide-modified biotin
is used, any streptavidin-linked entity may be applied to the
biotin. Hybridization occurred with 40 .mu.L of Gal 80-template
conjugate (2 nmol) and 4.8 .mu.L of the psoralen containing strand
(4 nmol) in 20 .mu.L crosslinking buffer (100 mM KCL, 1 mM
spermidine, 200 mM Hepes pH 7.8, and 1 mM EDTA pH 8) at 90.degree.
C. for 5 min. then cooled on ice for 30 min. The sample was placed
in one well of a 96 well flat bottom, clear NUNC plate and radiated
with ultra violet light (366 nm) for 15 min. Unreacted crosslinking
DNA was purified on streptavidin magnetic beads which contained the
biotinylated complementary DNA strand. The flow-through was
collected as the crosslinked Gal 80-template conjugate and
hybridized with equal molar ratio of the Gal 4-variable strand by
incubating in the presence of 1 M NaCl at 90.degree. C. for 5 min.
and then chilled on ice for 30 min. The disulfide bond on the
crosslinked DNA was reduced 30 min. before use by incubating with
10 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) at room
temperature for 30 min. The mercaptopropane was removed by using a
microcon YM-10 molecular weight spin column (Millipore).
EXAMPLE 7
Synthesis of Synbody
[0225] This example demonstrates the synthesis of the synbody shown
in FIG. 16 using polypeptide affinity elements previously
identified (sequences as shown in FIG. 16). As shown in FIG. 17,
lysine, protected by an Fmoc protecting group at the a amine and by
an ivDde protecting group at the c amine, was conjugated to a
cysteine residue which was in turn attached to the resin support
via an acid labile linkage. The Fmoc protecting group was removed,
the first polypeptide affinity element was synthesized by
sequential addition of residues by standard solid phase peptide
synthesis techniques from the a amine of the lysine, and the
terminal Fmoc protecting group was converted to Boc. The ivDde
protecting group was then removed from the .epsilon. amine of the
lysine, and the second polypeptide affinity element was synthesized
by sequential addition of residues to the exposed .epsilon. amine
of the lysine. The acid labile linkage of the cysteine residue to
the resin was cleaved, freeing the completed synbody. The foregoing
steps were performed in accordance with standard solid phase
peptide synthesis techniques. See, e.g., Atherton E, Sheppard R C:
Solid Phase peptide synthesis: a practical approach. Oxford,
England: IRL Press; 1989, and Stewart J M, Young J D: Solid Phase
Peptide Synthesis, 2d Ed. Rockford: Pierce Chemical Company; 1984,
which are incorporated herein by reference. Any other technique
operable for synthesizing and/or assembling the structure may be
employed; by way of non-limiting example, either or both
polypeptide affinity elements may be synthesized in place by
sequential addition of residues using standard solid phase
synthesis techniques, or by assembly of presynthesized
substructures. The lysine linker provides a spacing of about 1 nm
between the attachment points of the two polypeptides as shown in
FIG. 16. The cysteine may be biotinylated to enable detection using
fluorescently labeled streptavidin, or used for any other desired
functionalization. Other C-terminal residues or structures may also
be used; synbodies were also prepared having C-terminal glycine or
alanine in lieu of cysteine.
[0226] The synbodies were purified on a C-18 semi-preparative
column using 0.1% TFA in water and 90% CH.sub.3CN in 0.1% TFA with
gradient of 10 to 95% in 25 minutes, at flow rate of 4 ml/min and
verified by MALDI-TOF.
EXAMPLE 8
SPR Analysis of DNA-Linked Synbody and Linker Distance/Orientation
Optimization
[0227] This example demonstrates the optimization of linker length
for a DNA synbody, and demonstrates that the joinder of two
affinity elements having moderate affinity for a target by an
appropriate linker produces a synbody having affinity for the same
target that is substantially improved over that of the individual
affinity elements. DNA-linked synbody constructs (prepared as
described in Example 6) were immobilized on a Flexchip, and gal80
in solution was flowed over the chip and response data obtained. 12
distinct synbody constructs were evaluated, each having the BP1
polypeptide as one affinity element and the BP2 polypeptide as the
other affinity element. Six of the constructs had the BP1
polypeptide attached to the template strand and the BP2 polypeptide
attached to the variable strand at each of six different positions
(positions 13, 15, 17, 24, 26, and 28, counting from the 3' end of
the variable strand); the other six constructs were identical to
the first six except that positions of the two polypeptides were
reversed (i.e. the BP2 polypeptide was attached to the template
strand and the BP1 polypeptide was attached to the variable
strand). Relative SPR responses of these synbodies with respect to
gal80 were determined and compared, with the results shown in FIG.
18. The configuration with BP1 on the template strand and BP2 on
the variable strand produced a higher response than the reverse
configuration, and affinity of the synbody for gal80 declined as
the linker was elongated, indicating that a linker length
corresponding to about 13 to 17 DNA bases, or about 5 nm, was
optimal for this configuration. This corresponds well to the known
dimensions of the gal80 homodimeric structure, which is
approximately cylindrical, about 10 nm in length and about 5 nm in
diameter.
[0228] From on and off rates determined by SPR using the methods
described in Example 4 with gal80 immobilized on the SPR chip,
dissociation constants were obtained and compared for the
linker-optimized synbody having the BP1 affinity element on the
template strand and the BP2 affinity element at position 13 from
the 3' end of the variable strand, for each affinity element alone,
and for each affinity element complexed by itself to the
double-stranded DNA linker. As shown in FIG. 19, the affinity
elements alone had affinities in a K.sub.d range on the order of a
few .mu.M (K.sub.d=1.5 for BP1 and Kd=5.6 for BP2). FIG. 20 shows
the results of the SPR analysis of the binding of the BP1/BP2
DNA-linked synbody in solution, in a concentration series ranging
from 1 .mu.M to 7.81 nm, to surface-bound Gal80, indicating a
K.sub.d value of 91 nM. A gel shift assay was performed, again
resulting in an estimated K.sub.d value of about 100 nM.
[0229] These data were confirmed by ELISA-type analysis, where
gal80 was immobilized in an ELISA well using standard methods, and
the linker-optimized synbody, functionalized with
streptavidin-conjugated HRP as described in Example 6, was applied
in a concentration series and bound synbody detected in accordance
with standard ELISA techniques. As shown in FIG. 20, the synbody
was again found to have low nanomolar affinity for gal80, as
compared to affinities in the K.sub.d range of about 25 to 50 .mu.M
for each of the affinity elements individually with respect to
gal80.
[0230] The specificity of the linker-optimized synbody was assessed
by SPR determination of the affinity of the synbody for three
protein targets other than gal80 (.alpha.1-antitrypsin, albumin,
and transferrin). In each case the affinities were in a K.sub.d
range more than 1000 times greater than the K.sub.d of the synbody
for gal80.
EXAMPLE 9
SPR Analysis of Synbody
[0231] This example demonstrates that synbodies comprising affinity
elements identified as described in Example 2 are capable of
binding the target used for their identification (here,
transferrin) with affinity that is significantly better than the
affinity for the same target of either affinity element alone.
Various synbodies comprising various pairings of affinity elements
TRF-19 through TRF-26 (see Table 3) were synthesized in accordance
with the methods described in Example 7 above, and their affinities
for transferrin were evaluated by SPR with transferrin immobilized
on the SPR chip in accordance with the methods described in Example
4 above, and with K.sub.d values determined from kinetics. All of
the pairings evaluated resulted in synbodies having Kd values less
than the K.sub.d values of their individual affinity elements alone
(i.e., all were lower than about 50 .mu.M). The synbody comprising
TRF-26 and TRF-23 had K.sub.d with respect to transferrin of
150.+-.50 nm.
EXAMPLE 10
[0232] Synbodies were constructed by synthesizing two 20-mer
polypeptides on the a and .epsilon. amine moieties, respectively,
of a lysine molecule as described in Example 7 above, thereby
providing a spacing of about 1 nm as shown in FIG. 21. The thiol
group of the cysteine is biotinylated to enable detection using
fluorescently labeled streptavidin.
[0233] The polypeptide sequences used as binding elements in the
synbodies were determined as described in Example 2. Several
polypeptides corresponding to the loci at which transferrin bound
were selected, synthesized (replacing the terminal cysteine with
glycine to facilitate conjugation to the lysine linker for assembly
of the synbody), and analyzed by SPR as described in Example 4 to
identify pairs of polypeptides capable of simultaneously and
non-competitively binding distinct loci on transferrin. Several
such pairs were selected for incorporation into synbodies.
[0234] Two biotinylated anti-TRF synbodies (SYN23-26 and SYN 21-22)
were applied to a protein microarray having 8,000 features
(Invitrogen Protoarray Human Protein Microarray v. 4.0 for immune
response biomarker profiling), each feature comprising a distinct
human protein (GST fusion) adsorbed to a nitrocellulose coated
slide. Application of the synbodies to the microarray was performed
in accordance with manufacturer instructions: (see ProtoArray Human
Protein Microarray, Invitrogen, Catalog no. PAH052401, Version B,
15 December 2006, 25-0970, Users Manual.) After blocking the array
with 1% BSA/PBS/0.1% Tween for 1 hour at 4 C with gentle shaking,
120 .mu.l of probing buffer (1.times. PBS, 5 mM mgCl2, 0.5 mM DTT,
0.05% Triton X-100, 5% glycerol, 1% BSA) with synbody was applied
to the array. The prescribed cover slip was placed over the array
and adjusted to remove air bubbles. The array was incubated in a 50
ml conical tube, printed side up, for 1.5 hours at 4 C without
shaking The array was then removed from the conical tube inserted
diagonally into the array chamber, kept on ice. 8 ml probing buffer
was added to the chamber wall. The cover slip was removed and the
array was incubated in probing buffer for 1 minute on ice. The
probing buffer was decanted and drained. Two further washings were
performed adding 8 ml probing buffer, incubating on ice for 1
minute, and decanting and draining 5 nM fluorescently labeled
streptavidin diluted in 6 ml probing buffer was incubated on the
array for 30 minutes on ice in the dark, after which the solution
was decanted and drained. Three wash steps were performed, each by
adding 8 ml probing buffer, incubating for 1 minute on ice,
decanting, and draining The array was removed from the chamber,
centrifuged at 800.times.g for 5 minutes at room temperature. The
array was dried in the dark for 60 minutes at room temperature,
after which it was scanned using a fluorescent microarray scanner
and data was taken and analyzed.
[0235] The binding pattern data for SYN23-26 were compared with
data obtained for a high quality anti-TRF monoclonal antibody, 1C10
(K.sub.d=1.5 pm), on the same array. The sequences of the
polypeptide binding elements of SYN21-22 were QYHHFMNLKRQGRAQAYGSG
(SEQ ID NO: 17) and HAYKGPGDMRRFNHSGMGSG (SEQ ID NO: 18) and the
sequences of SYN23-26 were FRGWAHIFFGPHVIYRGGSG (SEQ ID NO: 19) and
AHKVVPQRQIRHAYNRYGSG (SEQ ID NO: 20).
[0236] Comparisons of the measured fluorescence intensity values
exceeding background (which are a measure of occupancy and, by
extension, binding affinity) for SYN23-26 with those for the 1C10
antibody are shown in FIG. 22 for the 18 proteins to which 1C10
bound with highest intensity and in FIG. 23 for the 18 proteins to
which SYN23-26 bound with highest intensity. Data for SYN21-22 are
shown in FIG. 24. Binding of SYN23-26 to transferrin and AKT1 was
evaluated by SPR, indicating estimated Kd values of about 1 nM with
respect to AKT1 and about 141 nM with respect to transferrin.
[0237] As can be seen from the intensity plot for the highest
affinity targets for the 1C10 anti-TRF antibody (FIG. 22, light
bars), 1C10 bound ten other targets with intensity equal to or
greater than that for TRF, and bound one target, AKT1, with more
than ten-fold higher intensity. Similar results were obtained for
SYN21-22 (FIG. 24).
[0238] The monoclonal antibody 1C10 and both synbody constructs
exhibited high specificity, as indicated by high affinities for
only a few targets, with the plot of affinities for all targets,
ranked in descending order by affinity, appearing to decline
rapidly and approximately exponentially. The highest affinities
observed for the antibody and for both synbodies corresponded to
targets other than transferrin. This data illustrates that bivalent
synbodies (SYN23-26 and SYN21-22), each having binding elements
chosen on the basis of their affinity for distinct sites on an
arbitrarily chosen protein target (transferrin), each have, with
respect to one target from a library of 8,000 (PCCA for SYN23-26
and Ig kappa light chain for SYN21-22), affinity and specificity
characteristics essentially equivalent to those exhibited by the
monoclonal antibody 1C10 for its highest affinity target
(AKT1).
[0239] It is noteworthy that SYN23-26 bound to seven targets (FIG.
4, PCCA, CASZ1, GRP58, AKT1, LINT, Fbox-21, and Phosphodiesterase)
with intensities higher than that exhibited by 1C10 for its nominal
target (TRF), suggesting that SYN23-26 could be used as a synthetic
antibody against any of these seven protein targets with quality
equivalent to that of a high quality commercial monoclonal
antibody.
EXAMPLE 11
[0240] A bivalent synbody having binding elements selected for
affinity for Gal80 was assembled and linked via a nucleic acid
linker, providing spacing between binding elements of approximately
5 nm, as described in Example 6 above. Binding elements BP1 and BP2
were identified as described in Example 3 above.
[0241] The (biotinylated) synbody was screened on an array of 4,000
yeast proteins (Invitrogen Protoarray Yeast Protein Microarray for
immune response biomarker profiling), and detected using Alexa.TM.
555-labeled streptavidin. Fluorescence intensity data was obtained
as shown in FIG. 25 (adjusted for background fluorescence). The
distribution of affinities over the highest-binding protein targets
was again comparable to that characteristic of a high quality
monoclonal antibody, and, again, the protein targets for which the
synbody exhibited the highest affinity did not include the target
(Gal80) for which the binding elements were originally
screened.
EXAMPLE 12
DNA Tile Synbody
[0242] This example demonstrates the assembly of a synbody having
DNA aptamer affinity elements linked by a DNA tile linker, and
demonstrates that the synbody so constructed has, with respect to
the target used to identify the aptamer affinity elements, an
affinity significantly greater than that of either of the aptamer
affinity elements with respect to the same target. The 4-helix DNA
tile linker was constructed from DNA oligonucleotides as shown
schematically in FIG. 26 and described in detail in Ke Y G, Liu Y,
Zhang J P, Yan H: A study of DNA tube formation mechanisms using
4-, 8-, and 12-helix DNA nanostructures. Journal of the American
Chemical Society 2006, 128(13):4414-4421, which is incorporated by
reference herein. The spacing between affinity elements is
determined in part by the number of helices and the choice of loops
in which to incorporate the aptamer affinity elements; the number
of helices and choice of loops may be varied to achieve a desired
spacing. The sequences of aptamers specific for thrombin shown in
Table 4 were incorporated into the first 340 and fourth 342
single-stranded DNA loops, providing a structure in which the
aptamers extend from the tile as shown schematically in FIG. 26(b),
with a spacing between aptamers (for the 4-helix tile) of about 2
nm. For comparison and evaluation of binding properties of this
two-aptamer synbody structure with similar structures having only a
single affinity element, structures were also synthesized having
only Apt1 in the first loop 340 without the presence of Apt2 (see
FIG. 26(c)) and having only Apt2 in the fourth loop 342 without the
presence of Apt1 (see FIG. 26(d)).
TABLE-US-00004 TABLE 4 Aptamer sequences used in DNA tile synbody
Sequence Source Apt1 5'-AGTCCGTGGTAGGGCAG Tasset D M, Kubik M F,
GTTGGGGTGACT-3 Steiner W: Oligonucleotide (SEQ ID NO: 21)
inhibitors of human thrombin that bind distinct epitopes. Journal
of Molecular Biology 1997, 272(5): 688-698 Apt2
5'-GGTTGGTGTGGTTGG-3' Bock L C, Griffin L C, (SEQ ID NO: 22) Latham
J A, Vermaas E H, Toole J J: Selection Of Single-Stranded-DNA
Molecules That Bind And Inhibit Human Thrombin. Nature 1992,
355(6360): 564- 566)
[0243] By gel shift assay, binding of the DNA tile synbody (FIG.
26(b)) to thrombin was evaluated and compared with the binding to
thrombin of each aptamer incorporated into its loop of the DNA tile
without the other aptamer present (FIGS. 26(c) and (d)).
Non-denaturing (8% polyacrylamide) gel electrophoresis was
performed at 25.degree. C. with constant 200V for 5 hours with 1 nM
of pre-annealed Sybr-Gold stained tile/aptamer pre-incubated for 1
hr at room temperature with concentrations of human
.alpha.-thrombin ranging from 0 to 100 nM. In the gel shift assay,
the synbody was found to have a K.sub.d with respect to thrombin of
about 5 nM, the tile incorporating apt1 only or apt2 only had
K.sub.d values above 100 nM.
[0244] Binding to thrombin was evaluated in an ELISA-type assay.
Wells of a 96 well plate were coated with 100 .mu.L of 30 .mu.g/mL
human .alpha.-thrombin and incubated at 4 C overnight. The plate
was washed twice with DDI H2O and passivated with 3% BSA in
1.times. PBS buffer for 1 hour. The plate was shaken out and 50
.mu.L of varying concentrations of analyte (DNA tile synbody, DNA
tile with each aptamer with the other not present, and each aptamer
alone, respectively) were incubated at RT for 1 hour. DNA tiles
were biotin-modified at the 5' end of one of the distal DNA strands
346 (see FIG. 26(a)). The plate was rinsed 10 times in 1.times. PBS
and 50 .mu.L of 1:1000 dilution of streptavidin-HRP in 0.1% BSA in
1.times. PBS was pipetted and incubated for 1 hour at RT. The plate
was again rinsed and 50 .mu.L of TMB was added and incubated at RT
for 15 minutes. 50 .mu.L of 0.5M HCl was added and the plate was
read immediately. Results are shown in FIG. 27 for the DNA tile
synbody 350; the DNA tile with Apt1 but not Apt2 present 352; the
DNA tile with Apt2 but not Apt1 present 356; Apt1 alone 354; and
Apt2 alone 358. Dissociation constant values estimated from this
assay were about 1 nM for the DNA tile synbody, about 10 nM for
Apt1 alone, and more than 1 .mu.M for Apt2 alone.
[0245] DNA tiles of other widths were also constructed and aptamer
attachments at separation distances of about 2, 4, 6, and 8 nm were
evaluated by non-denaturing gel shift assay (6% polyacrylamide).
The 6 nm separation produced an approximately two-fold improvement
of estimated K.sub.d in comparison to the 2, 4, or 8 nm separation
(K.sub.d estimated about 2 nM for the 2 nm separation vs. about 1
nM for the 6 nm separation.
EXAMPLE 13
Linkers
[0246] The linker employed in the compositions and methods
disclosed herein may be any structure, comprising one or more
molecules, operable for associating two or more affinity elements
together in a manner such that the resulting synbody has, with
respect to a target of interest, affinity and/or specificity
superior to that of the affinity elements when not so associated.
In various embodiments, the linker may be a separate structure to
which each of the two or more affinity elements is joined, and in
other embodiments, the linker may be integral with one or both
affinity elements. In some embodiments, it is desirable to choose
linker structures that are stable and reasonably soluble in an
aqueous environment, and amenable to efficient and specific
chemistries for attaching affinity elements in a desired position
and/or conformation.
[0247] Without limiting the generality of the foregoing, this
prospective example demonstrates several linker compositions and
chemistries for attaching affinity elements thereto, in addition to
the DNA linkers and lysine linkers described in other examples.
[0248] Polyproline and variants thereof may be used as a linker in
some embodiments. Polyproline forms a relatively rigid and stable
helical structure with a three-fold symmetry, so that attachment
sites spaced at three residue intervals are approximately aligned
with respect to their angular relationship to the axial dimension.
The distance between such attachment sites (three residues apart)
is about 9.4 A for polyproline II, in which the peptide bonds are
in trans conformation, and about 5.6 A for polyproline I, in which
the peptide bonds are in cis conformation. Hydroxyproline may be
substituted for proline in these constructs, to provide a more
hydrophilic structure and improve solubility. See Schumacher M,
Mizuno K, Chinger H P B: The Crystal Structure of the Collagen-like
Polypeptide (Glycyl-4(R)-hydroxyprolyl-4(R)-hydroxyprolyl)9 at 1.55
.ANG. Resolution Shows Up-puckering of the Proline Ring in the Xaa
Position. Journal of Biological Chemistry 2005,
280(21):20397-20403, which is incorporated herein by reference.
[0249] In general, synbodies comprising affinity elements and
linkers that can be synthesized by standard solid phase synthesis
techniques can be synthesized either by addition of amino acids or
other monomers in a stepwise fashion, or by joining preassembled
affinity elements and linkers or other presynthesized subunits.
Techniques for stepwise synthesis of peptides and other
heteropolymers are well known to persons of skill in the art. See,
e.g., Atherton E, Sheppard R C: Solid Phase peptide synthesis: a
practical approach. Oxford, England: IRL Press; 1989, and Stewart J
M, Young J D: Solid Phase Peptide Synthesis, 2d Ed. Rockford:
Pierce Chemical Company; 1984, which are incorporated herein by
reference.
[0250] Where synbodies are constructed by joining presynthesized
entities, it may be desirable to employ conjugation chemistries and
methods that are orthogonal, so that conjugation points can be
deprotected and added to without risking inadvertent deprotection
or modification of other addition points, and that are rapid and
high yield, so that adequate product is produced. FIG. 38
enumerates a number of conjugation pairs (pairs are denoted by the
arrows in FIG. 38) each comprising a chemical moiety to be present
on a peptide or other affinity element and another chemical moiety
to be present on the oligonucleotide, peptide scaffold, or other
linker, where the two members of the pair will react to form a
covalent linkage under conditions that will be readily determinable
by persons of ordinary skill in the art guided by the disclosures
hereof. It will be seen that certain of the "click" moieties shown
in FIG. 38 are capable of conjugating with more than one other
moiety; where such moieties are employed, it may be necessary to
perform the desired conjugations in an appropriate order so that
the desired conjugation takes place at any moieties that are
susceptible to reaction with more than one other moiety before such
other moieties are applied. FIG. 39 shows an illustrative example
in which four orthogonal conjugations are achieved performing four
"click" reactions, which should preferably be performed in the
order shown (for example, the thiol moiety 360 is intended to react
with the aldehyde moiety 364, but can also react with the maleimide
moiety 362; this is prevented by reacting the maleimide 362 with
its intended click pair 366 first, so that when the thiol 360 is
applied no maleimide 362 remains to react with it. The use of
"click" chemistry to perform conjugations between biopolymers and
other heteropolymers is well within the capability of persons of
ordinary skill in the art guided by the disclosures hereof, and is
described in detail in various references such as Kolb H C, Finn M
G, Sharpless K B: Click chemistry: Diverse chemical function from a
few good reactions. Angewandte Chemie-International Edition 2001,
40(11):2004 and Evans R A: The rise of azide-alkyne 1,3-dipolar
`click` cycloaddition and its application to polymer science and
surface modification. Australian Journal of Chemistry 2007,
60(6):384-395, which are incorporated herein by reference.
[0251] FIG. 30 shows the synthesis of a synbody comprising two
peptide affinity elements (TRF26 and TRF23) joined by a poly
Gly-Ser linker and further comprising a cysteine, attached via a
miniPEG, for labeling with a suitable fluorescent label. The entity
shown in FIG. 30(1) is first synthesized in large quantity (i.e.
0.5 to 1.0 mmole) in a microwave synthesizer by standard methods.
The ivDDE protecting group is then removed and the deprotected
product is split into ten aliquots. Again by microwave synthesis,
to each aliquot is added a predetermined number of Gly-Ser, ranging
from 1 to 10, so that each aliquot now has a linker comprising
(Gly-Ser).sub.n where n is 1 for the first aliquot, 2 for the
second, and so on up to 10 (FIG. 30(3)). For each aliquot, the
second peptide affinity element, TRF23, is then synthesized by
stepwise addition of amino acids (FIG. 30(4)). The synbody is then
cleaved from the resin. The t-butyl thiol protecting group intact
on the miniPEG-linked cysteine may be removed and a fluorescent
label added if desired (FIG. 30(5)).
[0252] FIG. 31 shows the conjugation of a maleimide-functionalized
peptide to a thiol-modified oligonucleotide, producing a
peptide-oligonucleotide conjugate that may be used to enable the
use of peptide affinity elements with the DNA tile linkers of
Example 9 above. The oligonucleotide conjugated to the peptide is
reverse complementary to an exposed DNA strand of the DNA tile and
stably hybridizes thereto.
[0253] FIG. 32 shows the synthesis of a poly-(Gly-Hyp-Hyp)-linked
synbody and illustrates a method for improving the ivDDE
deprotection (ivDDE deprotection in the presence of a long peptide
may be suboptimal due to interference by the peptides with access
to an ivDDE that is close to the resin surface). The structure
shown in FIG. 32(1) is first synthesized using standard solid phase
synthesis techniques. The ivDDE 370 protected lysine is deprotected
(FIG. 32(2)) and the first peptide affinity element TFR26 is
synthesized by stepwise addition of amino acids (FIG. 32(3)). The
alloc protecting group 368 is removed and Fmoc-Gly-Hyp-Hyp-OH
subunits are added to the linker to the length desired (FIG.
32(4)). The structure is then cleaved from the resin, and TRF23,
which has been presynthesized with a maleimide functionalization
374 of the terminal lysine, is conjugated to the furanyl moiety 372
of the poly-(Gly-Hyp-Hyp) linker (FIG. 32(5)).
[0254] FIG. 33 shows the synthesis of synbodies using
poly-(Gly-Hyp-Hyp) linkers of varying lengths by attaching both
affinity elements using mutually orthogonal conjugations.
(Gly-Hyp-Hyp)n linkers of varying lengths from n=1 to n=10 are
presynthesized with a furanyl moiety 376 for conjugation of a first
affinity element and a benzaldehyde moiety 378 for conjugation of a
second affinity element. The first affinity element 380,
functionalized with a hydrazide moiety, is conjugated to the
benzaldehyde moiety of the poly-(Gly-Hyp-Hyp) linker (FIG. 33(a)).
The second affinity element 384, functionalized with a maleimide
moiety 386, is conjugated to the furanyl moiety of the linker (FIG.
33(b)). These conjugations can be performed in a reaction mixture
containing multiple different linker lengths and/or multiple
peptide sequences, enabling production of a combinatorial library
representing multiple linker lengths and affinity element
combinations, from which constructs that optimally bind the target
of interest are identified using an affinity column or other
suitable screening method.
[0255] FIG. 34 illustrates schematically a method for determining
suitable linker lengths and affinity element sequences by allowing
the desired synbody structures to self-assemble in the presence of
the target of interest 394 such as transferrin. To a solution
containing transferrin 394 are added a first library combining a
variety of distinct affinity elements 388 (shown as peptide 1 in
FIG. 34) with linkers 390 of a variety of lengths to which the
affinity elements are conjugated, each linker 390 being
functionalized (at its terminus opposite the attachment point of
the affinity element, or other attachment point providing a desired
separation and/or orientation) with a moiety 392 suitable for
conjugation of a second affinity element 396. A second library
comprising a variety of distinct affinity elements 396 (peptide 2
in FIG. 34), each functionalized with a moiety 398 suitable for
conjugation with the linker, is added. Affinity elements 388, 396
having affinity for loci on the target 394 will tend to associate
with the target in their preferred positions and/or orientations.
Where a pair comprising an affinity element 388 plus linker 390 and
an affinity element 396 plus conjugation moiety 398 associate with
a target molecule in such a way that the conjugation moiety 398 of
the affinity element 396 and the conjugation moiety 392 of the
linker are in close proximity and appropriately oriented, reaction
will occur and a bond 392 will form, linking the two affinity
elements into a synbody, whose position and orientation with
respect to the target has been determined by the target itself
Synbodies bound to the target are then identified and
characterized. The concentrations of affinity elements used should
preferably be low enough to prevent significant conjugation between
affinity elements and linkers that are not associated with a target
molecule, but should be high enough so that affinity elements will
associate with target for sufficient time to allow the desired
pairs to conjugate. Also, the conjugation chemistry should be
reversible so as to allow the conjugation process reach an
equilibrium that favors the most suitable combinations; several
conjugation chemistries that are potentially reversible under
appropriate conditions are shown in FIG. 35. (Many other reversible
conjugation chemistries are possible; in any, obtaining the desired
reversibility will depend upon suitable reaction conditions.)
EXAMPLE 14
Cyclic Tetrapeptide Linker Synbody
[0256] This example demonstrates the synthesis of a cyclic
tetrapeptide having three orthogonally protected conjugation sites
for attachment of peptide or other affinity elements.
[0257] The structure shown in FIG. 36 is synthesized from three
modified amino acids, and a fourth one that is commercially
available, as shown. The three amino acids are first synthesized,
and the resin modified; the synthesis of the tetrapeptide is then
carried out, and peptides or other affinity elements are added;
thus, the tetrapeptide serves as a linker for construction of a
synbody.
[0258] Synthesis of the modified amino acids.
1-Methyl-1-phenylethyl 3-aminopropanoate (FIG. 46(3)) was
synthesized as follows: Over a suspension of NaH (50 mg, 2.1 mmol)
in diethyl ether (2 mL), a solution of 2-phenyl-2-propanol (2.5 g,
18.36 mmol) in 2 mL of diethyl ether was added dropwise. The
mixture was stirred at room temperature for 20 min and then cooled
at 0.degree. C. Trichloroacetonitrile (1.9 mL) was slowly added
(for 15 min) and the mixture was allowed to reach room temperature.
After 1 hour of stirring, the mixture was concentrated to dryness
and the resultant oil was dissolved in pentane (2 mL) and the
solution was filtered. The filtrate was evaporated to dryness, to
get a very dark oil, that we use immediately in the next reaction.
The freshly prepared 1-methyl-1,1-phenylethyl trichloroacetimidate
(2.7 g, 6.424 mmol) was added over a solution of
Fmoc-.beta.-alanine, (FIG. 46(1)), (1 g, 3.212 mmol) in DCM (8 mL).
After overnight stirring, the precipitated trichloroacetamide was
removed by filtration, and the filtrate mixture was evaporated to
dryness and purified by flash chromatography CH.sub.2Cl.sub.2/MeOH
(0% to 1%) to yield 1.158 g (84%) of compound 2 as a colorless
oil.
[0259] In a flask, (FIG. 46(2)) (1.158 g, 2.698 mmol) was dissolved
in DCM (4 mL), and diethylamine (12 mL) was added. Inmediately, the
mixture becomes clear. The mixture was stirred for 2 hours. After
adding 20 mL of toluene, the mixture was concentrated to dryness
and the separation carried out by flash chromatography, using 10%
of CH.sub.2Cl.sub.2/MeOH and 2% of Et.sub.3N to yield 526 mg (94%)
of (FIG. 46(3)) as a colorless oil.
[0260]
N.sup.2-(allyloxycarbonyl)-N.sup.3-(9-fluorenylmethoxycarbonyl)-2,3-
-diaminopropanoic acid (7) was synthesized as follows: Over a
solution of 2 g of asparagine (FIG. 46(4), 15.138 mmol) in 3.78 mL
of 4M NaOH solution cooled in an ice-bath, 1.615 mL of allyl
chloroformate (15.138 mmol) and 3.78 mL of 4M NaOH solution in
portions were added. The reaction was kept alkaline and stirred for
15 minutes at room temperature. The mixture was extracted with
ether and acidified with concentrated HCl, so the product was
crystallized, filtrated, and lyophilized to afford (FIG. 46(5))
(2.816 g, 86%) as a white solid. [Bis(trifluoroacetoxy)iodo]benzene
(8.402 g, 19.539 mmol) was added to a mixture of (FIG. 46(5))
(2.816 g, 13.026 mmol) and aqueous DMF (140 mL, 1:1, v/v). The
mixture was stirred for 15 min, and DIEA (4.54 mL, 26.052 mmol) was
added. After 8 hours the reaction, only half of the reaction went.
So, the same quantities of [Bis(trifluoroacetoxy)iodo]benzene and
DIEA were added, and the reaction was stirred overnight. The next
day, the solution was concentrated to dryness, the residue solved
in 100 mL of water and the organic side products were removed by
repeated washings with diethyl ether (4.times.100 mL). The water
phase was evaporated to dryness to yield product (FIG. 46(6)) that
was used in the next reaction without further purification.
[0261] The oil previously obtained ((FIG. 46(6)) was redissolved in
water (20 mL), and DIEA (2.24 mL, 13.026 mmol) and FmocOSu (4.393
g, 13.026 mmol) in acetonitrile (15 mL) were added, and the
reaction was allowed to stir for 1.5 h. The mixture was acidified
(to pH 2.0) by addition of HCl, and the product was extracted in
DCM (5.times.40 mL). The organic phases were combined, dried with
Na.sub.2SO.sub.4, and evaporated to dryness. The crude product
mixture was purified by flash chromatography (10% MeOH in DCM).
Hexane was added to the combined product fractions, and the
precipitate formed was filtered and washed with hexane, and dried
to yield a white solid (FIG. 46(7)).
[0262] 2-azido-3-[(9-fluorenylmethyloxycarbonyl)amino]-propanoic
acid (10) was synthesized as follows: A solution of NaN.sub.3
(9.841 g, 151.38 mmol) in 25 mL of H.sub.2O was cooled in an ice
bath and treated with 50 mL of CH.sub.2Cl.sub.2. The biphasic
mixture was stirred vigorously and treated with Tf.sub.2O (8.542 g,
282.14 mmol) for over a period of 30 min. The reaction mixture was
stirred at ice bath temperature for 2 h. After quenching with
aqueous NaHCO.sub.3, the layers were separated, and the aqueous
layer was extracted twice with CH.sub.2Cl.sub.2 (2.times.50 mL).
The organic layers were combined to afford 100 mL of TfN.sub.3
solution that was washed once with Na.sub.2CO.sub.3 and used in the
next reaction without further purification.
[0263] To a solution of L-asparagine (FIG. 46(4)) (2 g, 15.138
mmol) in 50 mL of H.sub.2O and 100 mL of MeOH were added:
K.sub.2CO.sub.3 (3.138 g, 22.707 mmol), CuSO.sub.4 (38 mg, 0.151
mmol), and the solution of TfN.sub.3 in CH.sub.2Cl.sub.2 previously
prepared. The reaction was stirred at room temperature overnight.
Then, solid NaHCO.sub.3 (10 g) was added carefully, and the organic
solvents evaporated. Concentrated HCl was added to the aqueous
solution to obtain pH=6, and 100 mL of 0.25 M PBS was added. Then,
ethyl acetate (3.times.150 mL) was used to do extractions. Next,
more concentrated HCl was used to reach pH=2 and new extractions
were carried out with ethyl acetate (5.times.150 mL) and the
extract concentrated to dryness to afford a yellow oil (FIG.
46(8)), that was used in the next reaction without further
purification.
[0264] [Bis(trifluoroacetoxy)iodo]benzene (19.529 g, 45.414 mmol)
was added to a mixture of the crude (FIG. 46(8)) (15.138 mmol) and
aqueous DMF (120 mL, 1:1, v/v). The mixture was stirred for 15 min,
and DIEA (10.546 mL, 60.552 mmol) was added. The reaction continue
overnight. The next day, the solution was concentrated to dryness,
the residue dissolved in 100 mL of water and the organic products
were removed by repeated washings with diethyl ether (3.times.100
mL). The water phase was evaporated to dryness to yield product
(FIG. 46(9)) as a pale oil, that was used in the next reaction
without further purification.
[0265] The oil previously obtained (FIG. 46(9)) was redissolved in
water (20 mL), and DIEA (2.6 mL, 15.138 mmol) and FmocOSu (5.106 g,
15.138 mmol) in acetonitrile (15 mL) were added, and the reaction
was allowed to stir for 1.5 h. The mixture was acidified (to pH
2.0) by addition of HCl, and the product was extracted in DCM
(5.times.40 mL). The organic phases were combined, dried with
Na.sub.2SO.sub.4, and evaporated to dryness. The crude product
mixture was purified by flash chromatography (10% MeOH in DCM).
Hexane was added to the combined product fractions, and the
precipitate formed was filtered and washed with hexane, and dried
to yield a white solid (FIG. 46(10)).
[0266] Derivatization of the resin. mixture of Boc- and
Fmoc-.beta.-alanine (2.0 eq of both, 4.0 equiv of TBTU, 8 equiv of
DIEA in DMG, 1 h at 25.degree. C.) was coupled to aminomethyl
polystyrene resin (1.0 g, 0.5 mmol/g). 50% TFA in DCM was used to
remove the Boc groups, and the exposed amino groups were capped
with acetanhydride treatment. Thus, the loading of the resin was
reduced to 0.16 mmol/g. A treatment of 20% piperidine in DMF was
used to remove the Fmoc groups, and
4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid was attached by
HATU-promoted coupling to obtain the derivatized resin.
[0267] Synthesis of the scaffold on the resin. Previously
derivatized resin (1.0 g, a loading of 0.16 mmol/g) was treated for
1 h at room temperature with a mixture of 1-methyl-1-phenylethyl
3-aminopropanoate (FIG. 46(3), 160 mg, 4 equiv) and NaCNBH.sub.3
(48 mg, 4 equiv) in DMF, containing 1% (v/v) AcOH (16 mL). The
resin was washed with DMF, DCM, and MeOH and dried on a filter.
[0268] The secondary amine was acylated with Aloc-Dpr(Fmoc)-OH 7
(5.0 equiv), using 5 equiv of PyAOP and 10 equiv of DIEA in
DMF-DCM, 1:9, v/v for 2 h at 25.degree. C. The Fmoc group was
removed by treatment of piperidine-DMF, 1:4, v/v, for 20 min at
25.degree. C. Couplings of
2-azido-3-[(9-fluorenylmethyloxycarbonyl)amino]propanoic acid (FIG.
46(10)) and Fmoc-Dpr-(Mtt)-OH (11) were carried out in each case,
by treatment with 5 equiv of the amino acid, 5 equiv of HATU and 10
equiv of collidine in DMF for 1 h at 25.degree. C. to afford
product (FIG. 46(12)). The removal of Mtt and PhiPr protections was
carried out by treatment with a solution of TFA in DCM (1:99, v/v,
for 6 min at 25.degree. C.), followed by immediate neutralization
by washings with a mixture of Py in DCM (1:5, v/v). Cyclization of
the peptide (FIG. 46(13)) was then performed using PyAOP as an
activator (5 equiv of PyAOP, 5 equiv of DIEA in DMF for 2 h at
25.degree. C.). After each coupling (including the cyclization
step), potentially remaining free amino groups were capped by an
acetic anhydride treatment.
[0269] Then, the resin was treated with TFA in DCM (1:1, v/v, 30
min at 25.degree. C.) to release the final product (FIG.
46(14)).
[0270] Sequential addition of peptides to the scaffold. The three
amino acid residues can be sequentially deprotected, reacted with
sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(Sulfo-SMCC) or other heterobifunctional linker, and the
corresponding peptide added. Thus, this scaffold allows
incorporation of up to three same or different peptides as shown in
FIG. 37. Peptides are chosen based on screening of target on a
random peptide microarray as described in preceding examples.
EXAMPLE 15
Cyclic Decapeptide Linker Synbody
[0271] This example demonstrates the synthesis of a cyclic
decapeptide scaffold from commercial Fmoc amino acids by solid
phase synthesis, using Trt-Lys(Fmoc)OH as the N-terminal amino
acid, and SASRIN resin as shown in FIG. 38. The cyclization of the
decapeptide is carried out in high dilution. This decapeptide
structure provides orthogonally protected conjugation sites
enabling attachment of up to four distinct peptides or other
affinity elements, and thus serves as a linker for the synbody.
[0272] Synthesis of the decapeptide
H.sub.2NLys(Fmoc)ProGlyLys(pNz)Lys(Boc)ProGly-Lys(Aloc)AlaOH (FIG.
48(b)). Assembly of the protected peptide was carried out manually.
Fmoc-Ala-SASRIN (0.5 g, 0.75 equiv/g) was washed and swollen with
CH.sub.2Cl.sub.2 (2.times.10 mL.times.15 min) and DMF (2.times.50
mL.times.15 min). Coupling reactions were performed using, relative
to the resin loading, 4 equiv of N-.alpha.-Fmoc-protected amino
acid activated in situ with 4 equiv of PyBOP and 8 equiv of DIEA in
8 mL of DMF for 30 min. The completeness of each coupling was
confirmed by Kaiser tests. N-.alpha.-Fmoc protecting groups were
removed by treatment with piperidine:DMF 1:4 (10
mL.times.4.times.10 min), the completeness of each deprotection
being verified by the UV absorption of the piperidine washings at
299 nm.
[0273] Peptide resin was treated repeatedly with
TFA:CH.sub.2Cl.sub.2 1:99 until the resin beads became dark purple
(10.times.10 mL.times.3 min). Each washing solution was neutralized
with pyridine:MeOH 1:4 (5 mL). The combined washings were
concentrated under reduced pressure, and white solid was obtained
by precipitation from EtOAc/petroleum ether. This solid was
dissolved in EtOAc, and pyridinium salts were extracted with water.
The organic layer was dried over Na.sub.2SO.sub.4, filtered, and
concentrated to dryness. Precipitation from
CH.sub.2Cl.sub.2/Et.sub.2O afford white solid which was further
desalted by solid-phase extraction and lyophilized to afford the
linear peptide. This material was used in the next step without
further purification.
[0274] Cyclization in solution (FIG. 48(c)). The above linear
peptide was dissolved in DMF (100 mL), and the pH was adjusted to
8-9 by addition of DIEA. HATU (1.1 equiv) was added, and the
solution was stirred at room temperature for 3 h. Solvent was
removed in vacuo; the residue was dissolved in TFA:CH.sub.2Cl.sub.2
1:1 (15 mL) and allowed to stand for 45 min at room temperature.
The solution was then concentrated under reduced pressure and the
residue was triturated with Et.sub.2O and filtered to yield the
crude product shown in FIG. 38(c). The scaffold can be
functionalized in order to attach it to different surfaces, or to
add a dye that will help in the studies.
[0275] Addition of linker. The scaffold can be functionalized in
order to attach it to different surfaces, or to add a dye that will
help in the studies. Thus, the linker in can be engineered to have
a thiol (SH) group at a terminal position. This thiol can be
oxidized to yield a dimer of the scaffold with attached affinity
elements. Also, the thiol can be used to attach the structure to
various other scaffolds and surfaces. The functionalization takes
place at the free NH.sub.2 group as shown in FIG. 39. As an
example, this amino group can be acylated using tert-butylthio
protected thioglycolic acid. At this point, the scaffold is ready
for sequential addition of peptides of interest.
[0276] Sequential addition of peptides to the scaffold. The four
lysine residues can be orthogonally (without affecting each other)
deprotected, reacted with
sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(Sulfo-SMCC) or other similar heterobifunctional linker, and the
corresponding NH.sub.2-protected peptide added. Thus, this scaffold
allows incorporation of up to four different peptides as shown in
FIG. 39.
[0277] The linker shown in FIG. 39 can be engineered to have a
thiol (SH) group at a terminal position. This thiol can be oxidized
to yield a dimer of the scaffold with attached affinity elements.
Also, the thiol can be used to attach the structure to various
other scaffolds and surfaces.
EXAMPLE 16
PGP Linker Synbody
[0278] This example demonstrates the synthesis of a synbody having
polypeptide affinity elements joined by a poly-(Pro-Gly-Pro)
linker, whose length can be determined by inserting the desired
number of (Pro-Gly-Pro) subunits, and its assembly by click
conjugation. As shown in FIG. 40, standard solid phase peptide
synthesis methods were used to synthesize, on a Symphony peptide
synthesizer, the structure shown in FIG. 40, comprising a
polypeptide affinity element 400, a poly-(Pro-Gly-Pro) linker 410,
and an azide moiety attached to lysine 402 as shown. A second
structure, comprising a second polypeptide affinity element 406,
and having an alkyne moiety 404 as shown, was separately
synthesized. The two structures were reacted in solution in the
presence of vitamin C and CuSO4 to produce the linked synbody
structure 408. Synthesis of the correct synbody structure was
verified by MALDI.
[0279] In this method, any linker can be used that can be
incorporated in the affinity element/linker/azide structure during
solid phase synthesis; thus, this method provides a way of testing
a variety of linker compositions.
[0280] A poly-(Pro-Gly-Pro) linked synbody was also constructed by
the thiazolidine formation process shown in FIG. 41. In this
synthesis, a polypeptide affinity element TRF 26 (SEQ ID NO. 8) 412
was synthesized together with its poly-(Pro-Gly-Pro) linker 414 by
standard solid phase peptide synthesis methods, having a cysteine
residue 416 at or near the opposite end of the linker from the
polypeptide affinity element 412 as shown. A second polypeptide
affinity element TRF 23 (SEQ ID NO. 5) 418 was synthesized having a
serine residue 420 near its C terminus, which was modified as shown
424. The two entities were reacted in solution at pH 4.5 to produce
the thiazolidine ring linkage 422 shown. Synthesis of the correct
synbody structure 426 was verified by MALDI.
REFERENCES
[0281] 1. Tang, D. C., DeVit, M. & Johnston, S. A. Genetic
immunization is a simple method for eliciting an immune response.
Nature 356, 152-4 (1992). [0282] 2. Chambers, R. S. & Johnston,
S. A. High-level generation of polyclonal antibodies by genetic
immunization. Nat Biotechnol 21, 1088-92 (2003). [0283] 3. Barry,
M. A., Barry, M. E. & Johnston, S. A. Production of monoclonal
antibodies by genetic immunization. Biotechniques 16, 616-8, 620
(1994). [0284] 4. Hust, M. & Dubel, S. Phage display vectors
for the in vitro generation of human antibody fragments. Methods
Mol Biol 295, 71-96 (2005). [0285] 5. Ellington, A. D. &
Szostak, J. W. In vitro selection of RNA molecules that bind
specific affinity elements. Nature 346, 818-22 (1990). [0286] 6.
Binz, H. K., Amstutz, P. & Pluckthun, A. Engineering novel
binding proteins from nonimmunoglobulin domains. Nat Biotechnol 23,
1257-68 (2005). [0287] 7. Peng, L. et al. Combinatorial chemistry
identifies high-affinity peptidomimetics against alpha(4)beta(1)
integrin for in vivo tumor imaging. Nat Chem Biol 2, 381-9 (2006).
[0288] 8. Masip, I., Perez-Paya, E. & Messeguer, A. Peptoids as
source of compounds eliciting antibacterial activity. Comb Chem
High Throughput Screen 8, 235-9 (2005). [0289] Roque, A. C. A.,
Lowe, C. R., & Taipa, M. A. "Antibodies and Genetically
Engineered Related Molecules: Production and Purification."
Biotechnol. Prog. 20, 639-654 (2004). [0290] Silverman, J., et. al.
"Multivalent avimer proteins evolved by exon shuffling of a family
of human receptor domains" Nat. Biotechnol. 23, 1556-1561 (2005).
[0291] Bes, C., et. al. "PIN-bodies: A new class of antibody-like
proteins with CD4 specificity derived from the protein inhibitor of
neuronal nitric oxide synthase" Biochem. Biophys. Res. Comm. 343,
334-344 (2006)
Sequence CWU 1
1
23120PRTArtificial SequenceSynthetic peptide 1Lys Glu Asp Asn Pro
Gly Tyr Ser Ser Glu Gln Asp Tyr Asn Lys Leu1 5 10 15Asp Gly Ser Cys
20220PRTArtificial SequenceSynthetic peptide 2Gly Gln Thr Gln Phe
Ala Met His Arg Phe Gln Gln Trp Tyr Lys Ile1 5 10 15Lys Gly Ser Cys
20320PRTArtificial SequenceSynthetic peptide 3Gln Tyr His His Phe
Met Asn Leu Lys Arg Gln Gly Arg Ala Gln Ala1 5 10 15Tyr Gly Ser Cys
20420PRTArtificial SequenceSynthetic peptide 4His Ala Tyr Lys Gly
Pro Gly Asp Met Arg Arg Phe Asn His Ser Gly1 5 10 15Met Gly Ser Cys
20520PRTArtificial SequenceSynthetic peptide 5Phe Arg Gly Trp Ala
His Ile Phe Phe Gly Pro His Val Ile Tyr Arg1 5 10 15Gly Gly Ser Cys
20620PRTArtificial SequenceSynthetic peptide 6Ser Val Lys Pro Trp
Arg Pro Leu Leu Thr Gly Asn Arg Trp Leu Asn1 5 10 15Ser Gly Ser Cys
20720PRTArtificial SequenceSynthetic peptide 7Ala Pro Tyr Ala Pro
Gln Gln Ile His Tyr Trp Ser Thr Leu Gly Phe1 5 10 15Lys Gly Ser Cys
20820PRTArtificial SequenceSynthetic peptide 8Ala His Lys Val Val
Pro Gln Arg Gln Ile Arg His Ala Tyr Asn Arg1 5 10 15Tyr Gly Ser Cys
20920PRTArtificial SequenceSynthetic peptide 9Leu Asp Pro Leu Phe
Asn Thr Ser Ile Met Val Asn Trp His Arg Trp1 5 10 15Met Gly Ser Cys
201020PRTArtificial SequenceSynthetic peptide 10Arg Phe Gln Leu Thr
Gln His Tyr Ala Gln Phe Trp Gly His Tyr Thr1 5 10 15Trp Gly Ser Cys
201115PRTArtificial SequenceSynthetic peptide 11Gly Thr Glu Lys Gly
Thr Ser Gly Trp Leu Lys Thr Gly Ser Cys1 5 10 151215PRTArtificial
SequenceSynthetic peptide 12Glu Gly Glu Trp Thr Glu Gly Lys Leu Ser
Leu Arg Gly Ser Cys1 5 10 151351DNAArtificial SequenceSynthetic DNA
13cccccccccg aaacaaccgc gagaggcacg cgcgtagccg tcaccggcta t
511438DNAArtificial SequenceSynthetic DNA 14gctacgcgcg tgcctctcgc
ccccccggtt gtttcggg 381532DNAArtificial SequenceSynthetic DNA
15tagccggtgt gaagtttctg ctagtaatgc cc 321620PRTArtificial
SequenceSynthetic peptide 16Gln Tyr His His Phe Met Asn Leu Lys Arg
Gln Gly Arg Ala Gln Ala1 5 10 15Tyr Gly Ser Gly 201720PRTArtificial
SequenceSynthetic peptide 17His Ala Tyr Lys Gly Pro Gly Asp Met Arg
Arg Phe Asn His Ser Gly1 5 10 15Met Gly Ser Gly 201820PRTArtificial
SequenceSynthetic peptide 18Phe Arg Gly Trp Ala His Ile Phe Phe Gly
Pro His Val Ile Tyr Arg1 5 10 15Gly Gly Ser Gly 201920PRTArtificial
SequenceSynthetic peptide 19Ala His Lys Val Val Pro Gln Arg Gln Ile
Arg His Ala Tyr Asn Arg1 5 10 15Tyr Gly Ser Gly 202029DNAArtificial
SequenceSynthetic DNA 20agtccgtggt agggcaggtt ggggtgact
292115DNAArtificial SequenceSynthetic DNA 21ggttggtgtg gttgg
152220PRTArtificial SequenceSynthetic peptide 22Ser Val Lys Pro Trp
Arg Pro Leu Ile Thr Gly Asn Arg Trp Leu Asn1 5 10 15Ser Gly Ser Gly
202320PRTArtificial SequenceSynthetic peptide 23Phe Arg Gly Trp Ala
His Ile Phe Phe Gly Pro His Val Ile Tyr Arg1 5 10 15Gly Lys Ser Gly
20
* * * * *