U.S. patent application number 13/923631 was filed with the patent office on 2013-10-31 for binding agent.
The applicant listed for this patent is ROCHE DIAGNOSTICS OPERATIONS, INC.. Invention is credited to Andreas Gallusser, Dieter Heindl, Michael Schraeml, Christoph Seidel, Herbert von der Eltz.
Application Number | 20130289251 13/923631 |
Document ID | / |
Family ID | 44041540 |
Filed Date | 2013-10-31 |
United States Patent
Application |
20130289251 |
Kind Code |
A1 |
Gallusser; Andreas ; et
al. |
October 31, 2013 |
BINDING AGENT
Abstract
A binding agent of the Formula A-a':a-S-b:b'-B:X(n), wherein A
as well as B is a monovalent binder, a':a as well as b:b' is a
binding pair wherein a' and a do not interfere with the binding of
b to b' and vice versa, S is a spacer of at least 1 nm in length,
:X denotes a functional moiety bound either covalently or via a
binding pair to at least one of a', a, b, b' or S, (n) is an
integer and at least 1, - represents a covalent bond, and the
linker a-S-b has a length of 6 to 100 nm. Also disclosed are
methods of producing such binding agent and certain uses
thereof.
Inventors: |
Gallusser; Andreas;
(Penzberg, DE) ; Heindl; Dieter; (Paehl, DE)
; Schraeml; Michael; (Penzberg, DE) ; Seidel;
Christoph; (Weilheim, DE) ; von der Eltz;
Herbert; (Weilheim, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ROCHE DIAGNOSTICS OPERATIONS, INC. |
Indianapolis |
IN |
US |
|
|
Family ID: |
44041540 |
Appl. No.: |
13/923631 |
Filed: |
June 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP2011/073633 |
Dec 21, 2011 |
|
|
|
13923631 |
|
|
|
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Current U.S.
Class: |
530/391.1 |
Current CPC
Class: |
C07K 16/32 20130101;
C07K 2317/626 20130101; C07K 16/2863 20130101; C07K 2317/34
20130101; C07K 2317/92 20130101; C07K 16/18 20130101; C07K 2317/55
20130101; C07K 2317/94 20130101; C07K 2317/31 20130101; C07K 19/00
20130101 |
Class at
Publication: |
530/391.1 |
International
Class: |
C07K 19/00 20060101
C07K019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 23, 2010 |
EP |
10196685.1 |
Claims
1. A binding agent comprising Formula: A-a':a-S-b:b'-B:X(n),
wherein A is a monovalent binder; B is a monovalent binder; S is a
spacer of at least 1 nm in length; (n) is an integer and at least
1; - represents a covalent bond; a':a is a binding pair; b:b' is a
binding pair, wherein a' and a do not interfere with the binding of
b to b' and b' and b do not interfere with the binding of a' to a;
(: X) denotes a functional moiety bound either covalently or via a
binding pair to at least one of a', a, b, b' or S; and linker a-S-b
has a length of 6 to 100 nm.
2. The binding agent of claim 1, wherein the spacer S is 1 to 95 nm
in length.
3. The binding agent of claim 1, wherein the a':a binding pair and
the b:b' binding pair are selected from the group consisting of
leucine zipper domain dimers and hybridizing nucleic acid
sequences.
4. The binding agent of claim 1, wherein the a':a binding pair and
the b:b' binding pair are hybridizing nucleic acid sequences and
wherein the different hybridizing nucleic acid sequences of the
a':a binding pair does not hybridize with the b:b' binding
pair.
5. The binding agent of claim 1, wherein the spacer S is a nucleic
acid.
6. The binding agent of claim 5, wherein the a':a binding pair and
the b:b' binding pair are nucleic acids.
7. The binding agent of claim 6, wherein the monovalent binders A
and B are nucleic acids.
8. The binding agent of claim 1, wherein X is a functional moiety
selected from the group consisting of a labeling group, a binding
group and an effector group.
9. The binding agent of claim 1, wherein the functional moiety X is
bound to a, b, or S.
10. The binding agent of claim 1, wherein the functional moiety X
is bound to the spacer S.
11. The binding agent of claim 1, wherein the functional moiety X
is covalently bound to the spacer S.
12. The binding agent of claim 1, wherein the functional moiety X
is bound to the spacer S via a hybridizing nucleic acid.
13. The binding agent of claim 1, wherein the monovalent binders A
and B are polypeptides.
14. The binding agent of claim 13, wherein the monovalent binders A
and B are Fab-fragments of monoclonal antibodies.
15. The binding agent of claim 1, wherein X is a functional moiety
selected from the group consisting of a labeling group, a binding
group and an effector group and the monovalent binders A and B are
polypeptides.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/EP2011/073633, filed Dec. 21, 2011, which
claims the benefit of European Patent Application No. 10196685.1,
filed Dec. 23, 2010, the disclosures of which are all hereby
incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 19, 2013, is named SEQUENCE_LISTING.sub.--27205US.txt, and
is seven thousand five hundred and nineteen bytes in size.
BACKGROUND OF THE DISCLOSURE
[0003] Bispecific antibodies, or bispecific binding agents in
general, are unique in the sense that they can bind simultaneously
to two different epitopes on one antigen or to two different
antigens. This property enables the development of novel
therapeutic and diagnostic strategies that are not possible with
conventional monoclonal antibodies. A large panel of bispecific
dual binders, e.g. of bispecific antibody formats has been
developed and reflects the strong scientific as well as commercial
interest in these molecules.
[0004] Monoclonal antibodies (mAbs), being directed towards single
epitopes on the antigen, usually bind with affinities which are
less than the avidity of polyclonal antisera. However, certain
pairs of mAbs directed towards different epitopes on the same
antigen can bind that antigen more effectively and with an avidity
greater than the sum of the affinities of the corresponding
individual mAb alone.
[0005] However, the avidity constants for synergizing pairs of mAb
or for a chemically cross-linked bispecific F(ab')2 is generally
only up to 15 times greater than the affinity constants for the
individual mAb, which is significantly less than the theoretical
avidity expected for ideal combination between the reactants
(Cheong, H. S., et al., Biochem. Biophys. Res. Commun. 173 (1990)
795-800). One reason for this might be that the individual
epitope/paratope interactions involved in a synergistic binding
(resulting in a high avidity) must be orientated in a particular
way relative to each other for optimal synergy.
BRIEF SUMMARY OF THE DISCLOSURE
[0006] The present disclosure relates to a binding agent of the
Formula A-a':a-S-b:b'-B:X(n), wherein A as well as B is a
monovalent binder, wherein a':a as well as b:b' is a binding pair
wherein a' and a do not interfere with the binding of b to b' and
vice versa, wherein S is a spacer of at least 1 nm in length,
wherein :X denotes a functional moiety bound either covalently or
via a binding pair to at least one of a', a, b, b' or S, wherein
(n) is an integer and at least 1, wherein - represents a covalent
bond, and wherein the linker a-S-b has a length of 6 to 100 nm.
Also disclosed are a method of producing such binding agent and
certain uses thereof.
[0007] The present disclosure relates to a binding agent of the
Formula A-a':a-S-b:b'-B:X(n), wherein A as well as B is a
monovalent binder, wherein a':a as well as b:b' is a binding pair
wherein a' and a do not interfere with the binding of b to b' and
vice versa, wherein S is a spacer of at least 1 nm in length,
wherein :X denotes a functional moiety bound either covalently or
via a binding pair to at least one of a', a, b, b' or S, wherein
(n) is an integer and at least 1, wherein - represents a covalent
bond, and wherein the linker a-S-b has a length of 6 to 100 nm.
[0008] Further disclosed is a method of making such binding agent
and the use of such agent. e.g. in an immunoassay procedure.
[0009] The use of the novel binding agent, especially in an
immunological detection procedure is also described and claimed
BRIEF DESCRIPTION OF THE FIGURES
[0010] The features of this disclosure, and the manner of attaining
them, will become more apparent and the disclosure itself will be
better understood by reference to the following description of
embodiments of the disclosure taken in conjunction with the
accompanying drawing.
[0011] FIG. 1A is an analytical gel filtration experiments
assessing efficiency of the anti-pIGF1-R dual binder assembly.
Diagrams A, B and C show the elution profile of the individual dual
binder components (flourescein-ssFab' 1.4.168, Cy5-ssFab' 8.1.2 and
linker DNA (T=0); Fab' denotes an Fab'-fragment conjugated to a
single-stranded oligonucleotide). Diagram D shows the elution
profile after the 3 components needed to form the bi-valent binding
agent had been mixed in a 1:1:1 molar ratio. The thicker (bottom)
curve represents absorbance measured at 280 nm indicating the
presence of the ssFab' proteins or the linker DNA, respectively.
The thinner top curve in B) and D) (absorbance at 495 nm) indicates
the presence of fluorescein and the thinner top curve in a) and the
middle curve in d) (absorbance at 635 nm) indicates the presence of
Cy5. Comparison of the elution volumes of the single dual binder
components (VE.sub.ssFab'1.4.168.about.15 ml;
VE.sub.ssFab'8.1.2.about.15 ml; VE.sub.linker.about.16 ml) with the
elution volume of the reaction mix (VE.sub.mix.about.12 ml)
demonstrates that the dual binder assembly reaction was successful
(rate of yield: .about.90%). The major 280 nm peak that represents
the eluted dual binder nicely overlaps with the major peaks in the
495 nm and 635 nm channel, proving the presence of both ssFab'
8.1.2 and ssFab'1.4.168 in the peak representing the bi-valent
binding agent.
[0012] FIG. 1B is an analytical gel filtration experiments
assessing efficiency of the anti-pIGF1-R dual binder assembly.
Diagrams A, B and C show the elution profile of the individual dual
binder components (flourescein-ssFab' 1.4.168, Cy5-ssFab' 8.1.2 and
linker DNA (T=0); Fab' denotes an Fab'-fragment conjugated to a
single-stranded oligonucleotide). Diagram D shows the elution
profile after the 3 components needed to form the bi-valent binding
agent had been mixed in a 1:1:1 molar ratio. The thicker (bottom)
curve represents absorbance measured at 280 nm indicating the
presence of the ssFab' proteins or the linker DNA, respectively.
The thinner top curve in B) and D) (absorbance at 495 nm) indicates
the presence of fluorescein and the thinner top curve in a) and the
middle curve in d) (absorbance at 635 nm) indicates the presence of
Cy5. Comparison of the elution volumes of the single dual binder
components (VE.sub.ssFab'1.4.168.about.15 ml;
VE.sub.ssFab'8.1.2.about.15 ml; VE.sub.linker.about.16 ml) with the
elution volume of the reaction mix (VE.sub.mix.about.12 ml)
demonstrates that the dual binder assembly reaction was successful
(rate of yield: .about.90%). The major 280 nm peak that represents
the eluted dual binder nicely overlaps with the major peaks in the
495 nm and 635 nm channel, proving the presence of both ssFab'
8.1.2 and ssFab'1.4.168 in the peak representing the bi-valent
binding agent.
[0013] FIG. 1C is an analytical gel filtration experiments
assessing efficiency of the anti-pIGF1-R dual binder assembly.
Diagrams A, B and C show the elution profile of the individual dual
binder components (flourescein-ssFab' 1.4.168, Cy5-ssFab' 8.1.2 and
linker DNA (T=0); Fab' denotes an Fab'-fragment conjugated to a
single-stranded oligonucleotide). Diagram D shows the elution
profile after the 3 components needed to form the bi-valent binding
agent had been mixed in a 1:1:1 molar ratio. The thicker (bottom)
curve represents absorbance measured at 280 nm indicating the
presence of the ssFab' proteins or the linker DNA, respectively.
The thinner top curve in B) and D) (absorbance at 495 nm) indicates
the presence of fluorescein and the thinner top curve in a) and the
middle curve in d) (absorbance at 635 nm) indicates the presence of
Cy5. Comparison of the elution volumes of the single dual binder
components (VE.sub.ssFab'1.4.168.about.15 ml;
VE.sub.ssFab'8.1.2.about.15 ml; VE.sub.linker.about.16 ml) with the
elution volume of the reaction mix (VE.sub.mix.about.12 ml)
demonstrates that the dual binder assembly reaction was successful
(rate of yield: .about.90%). The major 280 nm peak that represents
the eluted dual binder nicely overlaps with the major peaks in the
495 nm and 635 nm channel, proving the presence of both ssFab'
8.1.2 and ssFab'1.4.168 in the peak representing the bi-valent
binding agent.
[0014] FIG. 1D is an analytical gel filtration experiments
assessing efficiency of the anti-pIGF1-R dual binder assembly.
Diagrams A, B and C show the elution profile of the individual dual
binder components (flourescein-ssFab' 1.4.168, Cy5-ssFab' 8.1.2 and
linker DNA (T=0); Fab' denotes an Fab'-fragment conjugated to a
single-stranded oligonucleotide). Diagram D shows the elution
profile after the 3 components needed to form the bi-valent binding
agent had been mixed in a 1:1:1 molar ratio. The thicker (bottom)
curve represents absorbance measured at 280 nm indicating the
presence of the ssFab' proteins or the linker DNA, respectively.
The thinner top curve in B) and D) (absorbance at 495 nm) indicates
the presence of fluorescein and the thinner top curve in a) and the
middle curve in d) (absorbance at 635 nm) indicates the presence of
Cy5. Comparison of the elution volumes of the single dual binder
components (VE.sub.ssFab'1.4.168.about.15 ml;
VE.sub.ssFab'8.1.2.about.15 ml; VE.sub.linker.about.16 ml) with the
elution volume of the reaction mix (VE.sub.mix.about.12 ml)
demonstrates that the dual binder assembly reaction was successful
(rate of yield: .about.90%). The major 280 nm peak that represents
the eluted dual binder nicely overlaps with the major peaks in the
495 nm and 635 nm channel, proving the presence of both ssFab'
8.1.2 and ssFab'1.4.168 in the peak representing the bi-valent
binding agent.
[0015] FIG. 2 presents a scheme of the Biacore.TM. experiment.
Schematically and exemplarily, two binding molecules in solution
are shown: The T0-T-Dig (linker 16), bi-valent binding agent and
the T40-T-Dig (linker 15), bi-valent binding agent. Both these
bi-valent binding agents only differ in their linker-length (a
central digoxigenylated T with no additional T versus 40 additional
Ts (20 on each side of the central T-Dig), between the two
hybridizing nucleic acid sequences). Furthermore, ssFab' fragments
8.1.2 and 1.4.168 were used.
[0016] FIG. 3 presents a Biacore.TM. sensorgram with overlay plot
of three kinetics showing the interaction of 100 nM bi-valent
binding agent (consisting of ssFab' 8.1.2 and ssFab' 1.4.168
hybridized on the T40-T-Dig ssDNA-linker, i.e. linker 15) with the
immobilized peptide pIGF-1R compared to the binding characteristics
of 100 nM ssFab' 1.4.168 or 100 nM ssFab' 8.1.2 to the same
peptide. Highest binding performance is obtained with the Dual
Binder construct, clearly showing, that the cooperative binding
effect of the Dual Binder increases affinity versus the target
peptide pIGF-1R.
[0017] FIG. 4 presents a Biacore.TM. sensorgram with overlay plot
of three kinetics showing the interactions of the bi-valent binding
agent consisting of ssFab' 8.1.2 and ssFab' 1.4.168 hybridized on
the T40-T-Dig ssDNA-linker, i.e. linker 15, with immobilized
peptides pIGF-1R (phosphorylated IGF-1R), IGF-1R or pIR
(phosphorylated insulin receptor). Highest binding performance is
obtained with the pIGF-1R peptide, clearly showing, that the
cooperative binding effect of the Dual Binder increases specificity
versus the target peptide pIGF-1R as compared to e.g. the
phosphorylated insulin receptor peptide (pIR).
[0018] FIG. 5 presents a Biacore.TM. sensorgram with overlay plot
of two kinetics showing the interactions of 100 nM bi-valent
binding agent consisting of ssFab' 8.1.2 and ssFab' 1.4.168
hybridized on the T40-T-Dig ssDNA-linker, i.e. linker 15, and a
mixture of 100 nM ssFab' 8.1.2 and 100 nM ssFab' 1.4.168 without
linker DNA. Best binding performance is only obtained with the
bi-valent binding agent, whereas the mixture of the ssFab's without
linker doesn't show an observable cooperative binding effect,
despite the fact that the total concentration of these ssFab's had
been at 200 nM.
[0019] FIG. 6 presents a schematic drawing of a Biacore.TM.
sandwich assay. This assay has been used to investigate the epitope
accessibility for both antibodies on the phosphorylated IGF-1R
peptide. <MIgGFcy>R presents a rabbit anti-mouse antibody
used to capture the murine antibody M-1.4.168. M-1.4.168 then is
used to capture the pIGF-1R peptide. M-8.1.2 finally forms the
sandwich consisting of M-1.4.168, the peptide and M-8.1.2
[0020] FIG. 7 presents a Biacore.TM. sensorgram showing the binding
signal (thick line) of the secondary antibody 8.1.2. to the pIGF-1R
peptide after this was captured by antibody 1.4.168 on the
Biacore.TM. chip. The other signals (thin lines) are control
signals: given are the lines from top to bottom 500 nM 8.1.2, 500
nM 1.4.168; 500 nM target unrelated antibody <CKMM>M-33-IgG;
and 500 nM target unrelated control antibody <TSH>M-1.20-IgG,
respectively. No binding event could be detected in any of these
controls.
[0021] FIG. 8 presents a schematic drawing of the Biacore.TM.
assay, presenting the biotinylated dual binders on the sensor
surface. On Flow Cell 1 (=FC1) (not shown) amino-PEO-biotin was
captured. On FC2, FC3 and FC4 bi-valent binding agents with
increasing linker length were immobilized. (shown are the dual
binders on FC2 (T0-bi=only one central T-Bi) and FC4 (T40-bi=one
central T-Bi and 20 Ts each up- and downstream), respectively).
Analyte 1: IGF-1R-peptide containing the M-1.4.168 ssFab' epitope
at the right hand end of the peptide (top line)--the M-8.1.2 ssFab'
phospho-epitope is not present, because this peptide is not
phosphorylated; analyte 2: pIGF-1R peptide containing the M-8.1.2
ssFab' phospho-epitope (P) and the M-1.4.168 ssFab' epitope (second
line); analyte 3: pIR peptide, containing the cross reacting
M-8.1.2 ssFab' phospho-epitope, but not the epitope for M-1.4.168
(third line).
[0022] FIG. 9 presents kinetic data of the Dual Binder experiment.
T40-T-Bi (linker dual binder with ssFab' 8.1.2 and ssFab' 1.4.168
(=T40 in the Figure) shows a 1300-fold lower off-rate
(kd=2.79E-05/s) versus pIGF-1R when compared to pIR
(kd=3.70E-02/s).
[0023] FIG. 10 presents a Biacore.TM. sensorgram, showing
concentration dependent measurement of the T40-T-Bi dual binding
agent vs. the pIGF-1R peptide (the phosphorylated IGF-1R peptide).
The assay setup was as depicted in FIG. 8. A concentration series
of the pIGF-1R peptide was injected at 30 nM, 10 nM, 2.times.3.3
nM, 1.1 nM, 0.4 nM, 0 nM. The corresponding data are given in the
table of FIG. 9.
[0024] FIG. 11 presents a Biacore.TM. sensorgram, showing
concentration dependent measurement of the T40-T-Bi dual binding
agent vs. the IGF-1R peptide (the non-phosphorylated IGF-1R
peptide). The assay setup was as depicted in FIG. 8. A
concentration series of the IGF-1R peptide was injected at 300 nM,
100 nM, 2.times.33 nM, 11 nM, 4 nM, 0 nM. The corresponding data
are given in the table of FIG. 9.
[0025] FIG. 12 presents a Biacore.TM. sensorgram, showing
concentration dependent measurement of the T40-T-Bi dual binding
agent vs. the pIR peptide (the phosphorylated insulin receptor
peptide). The assay setup was as depicted in FIG. 8. A
concentration series of the pIR peptide was injected at 100 nM,
2.times.33 nM, 11 nM, 4 nM, 0 nM. The corresponding data are given
in the table depicted as FIG. 9.
[0026] Although the drawings represent embodiments of the present
disclosure, the drawings are not necessarily to scale and certain
features may be exaggerated in order to better illustrate and
explain the present disclosure. The exemplifications set out herein
illustrate an exemplary embodiment of the disclosure, in one form,
and such exemplifications are not to be construed as limiting the
scope of the disclosure in any manner.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
1. Antibody Fragments
SEQ ID NO:1 V.sub.H (mAb 1.4.168): QCDVKLVESG GGLVKPGGSL KLSCAASGFT
FSDYPMSWVR QTPEKRLEWV ATITTGGTYT YYPDSIKGRF TISRDNAKNT LYLQMGSLQS
EDAAMYYCTR VKTDLWWGLA YWGQGTLVTV SA
SEQ ID NO:2 V.sub.L (mAb 1.4.168): QLVLTQSSSA SFSLGASAKL TCTLSSQHST
YTIEWYQQQP LKPPKYVMEL KKDGSHTTGD GIPDRFSGSS SGADRYLSIS NIQPEDESIY
ICGVGDTIKE QFVYVFGGGT KVTVLG
SEQ ID NO:3 V.sub.H (mAb 8.1.2): EVQLQQSGPA LVKPGASVKM SCKASGFTFT
SYVIHWVKQK PGQGLEWIGY LNPYNDNTKY NEKFKGKATL TSDRSSSTVY MEFSSLTSED
SAVYFCARRG IYAYDHYFDY WGQGTSLTVS S
SEQ ID NO:4 V.sub.L (mAb 8.1.2): QIVLTQSPAI MSASPGEKVT LTCSASSSVN
YMYWYQQKPG SSPRLLIYDT SNLASGVPVR FSGSGSVTSY SLTISRMEAE DAATYYCQQW
STYPLTFGAG TKLELK
2. Sequences of ssDNA
[0027] a) 17mer ssDNA (covalently bound with 5' end to Fab' of
anti-TroponinT MAB a or Fab' 1.4.168 to IGF-1R, respectively):
5'-AGT TCT ATC GTC GTC CA-3'(SEQ ID NO:5) b) 19mer ssDNA
(covalently bound with 3' end to Fab' of anti-TroponinT MAB b or
Fab' 8.1.2 to phosphorylated IGF-1R, respectively): 5'-A GTC TAT
TAA TGC TTC TGC-3'(SEQ ID NO:6) c) complementary 19mer ssDNA (used
as part of a linker): 5'-G CAG AAG CAT TAA TAG ACT-3'(SEQ ID NO:7)
d) complementary 17mer ssDNA (used as part of a linker): 5'-TGG ACG
ACG ATA GAA CT-3'(SEQ ID NO:8)
3. Sequences of Troponin T Epitopes
[0028] SEQ ID NO:9=ERAEQQRIRAEREKEUUSLKDRIEKRRRAERAEamide, wherein
U represents .beta.-Alanin. (The epitope "A" for antibody
anti-Troponin antibody a.) SEQ ID
NO:10=SLKDRIERRRAERAEOOERAEQQRIRAEREKEamide, wherein O represents
Amino-trioxa-octanoic-acid. (The epitope "B" for antibody
anti-Troponin antibody b.)
4. Sequences of IGF-1R/IR Epitopes
SEQ ID NO:11=FDERQPYAHMNGGRKNERALPLPQSST; IGF-1R (1340-1366)
[0029] SEQ ID NO:12=YEEHIPYTHMNGGKKNGRILTLPRSNPS;
hIR(1355-1382)
5. Protein Linker and Tag-Sequences
[0030] SEQ ID NO:13=GGGGS (=G4S) motif (e.g. as part of a
polypeptide linker)
SEQ ID NO:14=YPYDVPDYA (HA-Tag)
SEQ ID NO:15=GLNDIFEAQKIEWHE (Avi-Tag)
[0031] Although the sequence listing represents an embodiment of
the present disclosure, the sequence listing is not to be construed
as limiting the scope of the disclosure in any manner and may be
modified in any manner as consistent with the instant disclosure
and as set forth herein.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0032] The embodiments disclosed herein are not intended to be
exhaustive or limit the disclosure to the precise form disclosed in
the following detailed description. Rather, the embodiments are
chosen and described so that others skilled in the art may utilize
their teachings.
[0033] The present disclosure relates to a binding agent of the
Formula: A-a':a-S-b:b'-B:X(n), wherein A as well as B is a
monovalent binder, wherein a':a as well as b:b' is a binding pair
wherein a' and a do not interfere with the binding of b to b' and
vice versa, wherein S is a spacer of at least 1 nm in length,
wherein :X denotes a functional moiety bound either covalently or
via a binding pair to at least one of a', a, b, b' or S, wherein
(n) is an integer and at least 1, wherein - represents a covalent
bond, and wherein the linker a-S-b has a length of 6 to 100 nm. As
obvious the binding agent according to the present disclosure is a
binding agent comprising at least two monovalent binders of
different specificity. In one embodiment the binding agent
according to the present disclosure comprises two monovalent
binders. In one embodiment the binding agent according to the
present disclosure is a bi-valent or dual binding agent.
[0034] The generation of bispecific antibodies is e.g. described in
WO 2004/081051. In this application a bispecific antibody (BAb)
comprising two antibodies, each of which has a binding specificity
to a different epitope situated on the surface of a target
structure are disclosed. In order to achieve the desired
improvement in specificity two MAbs are used each having a
relatively low binding affinity for its respective epitope. The
BAbs produced provide high avidity for target tissue due to the
cumulative nature of the binding interactions but have much lower
affinity for cross-reactive non-target tissue due to the lower
affinity of the individual MAbs used to produce them. Production of
these bispecific antibodies is quite complex and e.g. requires
sophisticated chemical coupling and purification steps.
[0035] Bispecific monoclonal antibodies also represent quite
interesting novel therapeutic modalities. A broad spectrum of
bispecific antibody formats has been designed and developed (see
e.g. Fischer, N. and Leger, O., Pathobiology 74 (2007) 3-14). Such
bispecific therapeutic monoclonals can e.g. be obtained by chemical
cross-linking, interaction of appropriately engineered protein
domains, completely recombinant, etc. Obviously, recombinant
engineering of each of the binders and careful purification of the
desired heterodimer from biochemically alike homo-dimers represent
some of the challenges encountered.
[0036] Chelating recombinant antibodies (CRAbs), originally
described by Neri, D. et al. (1995) represent a species of very
high affinity antibodies, where two scFvs specific for
non-overlapping epitopes on the same antigen molecule are connected
by a flexible linker polypeptide. The original modeled and designed
anti-hen egg lysozyme (HEL) CRAB employed an 18 amino acid linker
polypeptide to span the distance between the two scFv antibodies
and the resulting affinity enhancement was subsequently shown to be
up to 100-fold higher than the superior of the two scFvs as shown
by a variety of biophysical methods (Neri, D. et al., J. Mol. Biol.
246 (1995) 367-373).
[0037] Wright M. J. and Deonarain M. P., (Molecular Immunology 44
(2007) 2860-2869) developed a phage display library for generation
of chelating recombinant antibodies. The library described there
uses expression vectors construed in such way to provide for dual
binders having linker peptides of various length in between the two
binding entities. Selection of the best binder, i.e. the dual
binder with the optimal length of such linker, is thereby
facilitated. However, for each such chelating recombinant antibody
a full library of recombinant expression systems (allowing for
expression of a "binder 1-linker (of variable length)-binder 2"
polypeptide) has to be construed.
[0038] As outlined above in a cursory manner, the manufacturing of
bispecific dual binders remains quite challenging and requires
sophisticated techniques to identify, construe and produce
individually each of those bispecific binding agents. The frequent
need for derivatizing, e.g., labeling such a bispecific binding
agent even adds a further level of complexity.
[0039] The instant disclosure provides the surprising disclosure
and findings that at least some of the disadvantages known from the
prior art can be overcome by way of the novel bispecific binding
agents and methods disclosed in the present embodiment.
[0040] As the skilled artisan will appreciate the binding agent
described in the present disclosure can be isolated and purified as
desired. In one embodiment the present disclosure relates to an
isolated binding agent as disclosed herein. An "isolated" binding
agent is one which has been identified and separated and/or
recovered from e.g. the reagent mixture used in the synthesis of
such binding agent. Unwanted components of such reaction mixture
are e.g. monovalent binders that did not end up in the desired
binding agent. In one embodiments, the binding agent is purified to
greater than 80%. In some embodiments, the binding agent is
purified to greater than 90%, 95%, 98% or 99% by weight,
respectively. In case both monovalent binders of the binding agent
according to the present disclosure are polypeptides purity is e.g.
easily determined by SDS-PAGE under reducing or nonreducing
conditions using, for example, Coomassie blue or silver stain in
protein detection. In case purity is assessed on the nucleic acid
level, size chromatography is applied to separate the binding agent
from side products and the OD at 260 nm is monitored to assess its
purity.
[0041] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an antibody" means one antibody
or more than one antibody.
[0042] The terms "polypeptide" and "protein" are used
inter-changeably. A polypeptide in the sense of the present
disclosure consists of at least 5 amino acids linked by alpha amino
peptidic bonds.
[0043] A "target molecule" is a biomolecule of interest for which a
method for determination or measurement is sought. Exemplary target
molecules are lipoproteins, polypeptides, complexes of
polypeptides, secondarily modified polypeptides and complexes
between polypeptides and nucleic acids. In one embodiment a target
molecule is a polypeptide.
[0044] A "monovalent binder" (A and B, respectively, in Formula I)
according to the present disclosure is a molecule interacting with
a target molecule, e.g. with a target polypeptide at a single site
(i.e. the specific binding site). In case monovalent antibodies or
antibody fragments are used as a binder this site is called the
paratop.
[0045] As will be appreciated, the monovalent binders A and B,
respectively, each specifically bind their corresponding antigen.
In an exemplary embodiment the epitopes specifically bound by the
monovalent binders A and B do not overlap. As the skilled artisan
will appreciate the term specific is used to indicate that other
biomolecules present in the sample do not significantly bind to the
binding agent used. In some embodiments, for a specific binder the
level of binding affinity to a biomolecule other than the target
molecule results in a binding affinity which is only 10% or less,
for example, in some embodiments only 5% or less of the affinity it
has to the specifically bound target molecule.
[0046] Examples of monovalent binders are peptides, peptide
mimetics, aptamers, spiegelmers, darpins, ankyrin repeat proteins,
Kunitz type domains, single domain antibodies (see: Hey, T. and
Fiedler, E., et al., Trends Biotechnol. 23 (2005) 514-522), and
monovalent fragments of antibodies.
[0047] In certain embodiments the monovalent binder is a
polypeptide. In exemplary embodiments each of the monovalent
binders A and B, respectively is a polypeptide.
[0048] In certain embodiments the monovalent binder A and B,
respectively, is a monovalent antibody fragment, for example a
monovalent fragment derived from a monoclonal antibody.
[0049] Monovalent antibody fragments include, but are not limited
to Fab, Fab'-SH, single domain antibody, Fv, and scFv fragments, as
provided below.
[0050] In exemplary embodiments at least one of the monovalent
binders is a single domain antibody, an Fab-fragment or an
Fab'-fragment of a monoclonal antibody.
[0051] It also represents an exemplary embodiment that in the
binding agent disclosed herein both the monovalent binders are
derived from monoclonal antibodies and are Fab-fragments, or
Fab'-fragments or an Fab-fragment and an Fab'-fragment. Also, some
embodiments include the binding agent comprising two Fab-fragments
as the monovalent binders A and B.
[0052] Monoclonal antibody techniques allow for the production of
extremely specific binding agents in the form of specific
monoclonal antibodies or fragments thereof. Particularly well known
in the art are techniques for creating monoclonal antibodies, or
fragments thereof, by immunizing mice, rabbits, hamsters, or any
other mammal with a polypeptide of interest. Another method of
creating monoclonal antibodies, or fragments thereof, is the use of
phage libraries of sFv (single chain variable region), specifically
human sFv. (See e.g., Griffiths et al., U.S. Pat. No. 5,885,793;
McCafferty et al., WO 92/01047; Liming et al., WO 99/06587).
[0053] Antibody fragments may be generated by traditional means,
such as enzymatic digestion or by recombinant techniques. For a
review of certain antibody fragments, see Hudson, P. J. et al.,
Nat. Med. 9 (2003) 129-134.
[0054] An Fv is a minimum antibody fragment that contains a
complete antigen-binding site and is devoid of constant region. In
one embodiment, a two-chain Fv species consists of a dimer of one
heavy- and one light-chain variable domain in tight, non-covalent
association. In one embodiment of a single-chain Fv (scFv) species,
one heavy- and one light-chain variable domain can be covalently
linked by a flexible peptide linker such that the light and heavy
chains can associate in a dimeric structure analogous to that in a
two-chain Fv species. For a review of scFv, see, e.g., Plueckthun,
In: The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg
and Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315;
see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458.
Generally, six hyper variable regions (HVRs) confer antigen-binding
specificity to an antibody. However, even a single variable domain
(or half of an Fv comprising only three HVRs specific for an
antigen) has the ability to recognize and bind antigen.
[0055] An Fab fragment contains the heavy- and light-chain variable
domains and also contains the constant domain of the light chain
and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
[0056] Various techniques have been developed for the production of
antibody fragments. Traditionally, antibody fragments were derived
via proteolytic digestion of intact antibodies (see, e.g.,
Morimoto, K. et al., Journal of Biochemical and Biophysical Methods
24 (1992) 107-117; and Brennan, M. et al., Science 229 (1985)
81-83). For example, papain digestion of antibodies produces two
identical antigen-binding fragments, called "Fab" fragments, each
with a single antigen-binding site, and a residual "Fc" fragment,
whose name reflects its ability to crystallize readily.
[0057] Antibody fragments can also be produced directly by
recombinant host cells. Fab, Fv and scFv antibody fragments can all
be expressed in and secreted from E. coli, thus allowing the facile
production of large amounts of these fragments. Antibody fragments
can be isolated from the antibody phage libraries according to
standard procedures. Alternatively, Fab'-SH fragments can be
directly recovered from E. coli. (Carter, P. et al., Bio/Technology
10 (1992) 163-167). Mammalian cell systems can be also used to
express and, if desired, secrete antibody fragments.
[0058] In certain embodiments, a monovalent binder of the present
disclosure is a single-domain antibody. A single-domain antibody is
a single polypeptide chain comprising all or a portion of the heavy
chain variable domain or all or a portion of the light chain
variable domain of an antibody. In certain embodiments, a
single-domain antibody is a human single-domain antibody (Domantis,
Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). In
one embodiment, a single-domain antibody consists of all or a
portion of the heavy chain variable domain of an antibody.
[0059] The term "oligonucleotide" or "nucleic acid sequence" as
used herein, generally refers to short, generally single stranded,
polynucleotides that comprise at least 8 nucleotides and at most
about 1000 nucleotides. In an exemplary embodiment an
oligonucleotide will have a length of at least 9, 10, 11, 12, 15,
18, 21, 24, 27 or 30 nucleotides. In an exemplary embodiment an
oligonucleotide will have a length of no more than 200, 150, 100,
90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides. The description
given below for polynucleotides is equally and fully applicable to
oligonucleotides.
[0060] The term oligonucleotide is to be understood broadly and
includes DNA and RNA as well as analogs and modification
thereof.
[0061] An oligonucleotide may for example contain a substituted
nucleotide carrying a substituent at the standard bases
deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC),
deoxythymidine (dT), deoxyuracil (dU). Examples of such substituted
nucleobases are: 5-substituted pyrimidines like 5 methyl dC,
aminoallyl dU or dC, 5-(aminoethyl-3-acrylimido)-dU, 5 propinyl dU
or dC, 5 halogenated-dU or dC; N substituted pyrimidines like N4
ethyl dC; N substituted purines like N6 ethyl dA, N2 ethyl dG; 8
substituted purines like 8-[6-amino)-hex-1-yl]-8-amino-dG or dA, 8
halogenated dA or dG, 8-alkyl dG or dA; and 2 substituted dA like 2
amino dA.
[0062] An oligonucleotide may for example contain a substituted
nucleotide carrying a substituent at the standard bases
deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC),
deoxythymidine (dT), deoxyuracil (dU). Examples of such substituted
nucleobases are: 5-substituted pyrimidines like 5 methyl dC,
aminoallyl dU or dC, 5-(aminoethyl-3-acrylimido)-dU, 5-propinyl-dU
or -dC, 5 halogenated-dU or -dC; N substituted pyrimidines like
N4-ethyl-dC; N substituted purines like N6-ethyl-dA, N2-ethyl-dG; 8
substituted purines like 8-[6-amino)-hex-1-yl]-8-amino-dG or -dA, 8
halogenated dA or dG, 8-alkyl dG or dA; and 2 substituted dA like 2
amino dA.
[0063] An oligonucleotide may contain a nucleotide or a nucleoside
analog. I.e. the naturally occurring nucleobases can be exchanged
by using nucleobase analogs like 5-Nitroindol d riboside; 3 nitro
pyrrole d riboside, deoxyinosine (dI), deoyxanthosine (dX); 7
deaza-dG, -dA, -dI or -dX; 7-deaza-8-aza-dG, -dA, -dI or -dX;
8-aza-dA, -dG, -dI or -dX; d Formycin; pseudo dU; pseudo iso dC; 4
thio dT; 6 thio dG; 2 thio dT; iso dG; 5-methyl-iso-dC; N8-linked
8-aza-7-deaza-dA; 5,6-dihydro-5-aza-dC; and etheno-dA or
pyrollo-dC. As obvious to the skilled artisan, the nucleobase in
the complementary strand has to be selected in such manner that
duplex formation is specific. If, for example, 5-methyl-iso-dC is
used in one strand (e.g. (a)) iso dG has to be in the complementary
strand (e.g. (a')).
[0064] The oligonucleotide backbone may be modified to contain
substituted sugar residues, sugar analogs, modifications in the
internucleoside phosphate moiety, and/or be a PNA.
[0065] An oligonucleotide may for example contain a nucleotide with
a substituted deoxy ribose like 2'-methoxy, 2'-fluoro,
2'-methylseleno, 2'-allyloxy, 4'-methyl dN (wherein N is a
nucleobase, e.g., A, G, C, T or U).
[0066] Sugar analogs are for example Xylose; 2',4' bridged Ribose
like (2'-O, 4'-C methylene)- (oligomer known as LNA) or (2'-O, 4'-C
ethylene)- (oligomer known as ENA); L-ribose, L-d-ribose, hexitol
(oligomer known as HNA); cyclohexenyl (oligomer known as CeNA);
altritol (oligomer known as ANA); a tricyclic ribose analog where
C3' and C5' atoms are connected by an ethylene bridge that is fused
to a cyclopropane ring (oligomer known as tricycloDNA); glycerin
(oligomer known as GNA); Glucopyranose (oligomer known as Homo
DNA); carbaribose (with a cyclopentan instead of a tetrahydrofuran
subunit); hydroxymethyl-morpholin (oligomers known as morpholino
DNA).
[0067] A great number of modification of the internucleosidic
phosphate moiety are also known not to interfere with hybridization
properties and such backbone modifications can also be combined
with substituted nucleotides or nucleotide analogs. Examples are
phosphorthioate, phosphordithioate, phosphoramidate and
methylphosphonate oligonucleotides.
[0068] PNA (having a backbone without phosphate and d-ribose) can
also be used as a DNA analog.
[0069] The above mentioned modified nucleotides, nucleotide analogs
as well as oligonucleotide backbone modifications can be combined
as desired in an oligonucleotide in the sense of the present
disclosure.
[0070] The linker L consisting of a-S-b has a length of 6 to 100
nm. In some embodiments the linker L consisting of a-S-b has a
length of 6 to 80 nm. Also, in some cases the linker has a length
of 6 to 50 nm or of 6 to 40 nm. In some embodiments the linker will
have a length of 10 nm or longer or of 15 nm in length or longer.
In some embodiments the linker has between 10 nm and 50 nm in
length. In some embodiments a and b, respectively, are binding pair
members and have a length of at least 2.5 nm each.
[0071] The length of non-nucleosidic entities of a given linker
(a-S-b) in theory and by complex methods can be calculated by using
known bond distances and bond angles of compounds which are
chemically similar to the non-nucleosidic entities. Such bond
distances are summarized for some molecules in standard text books:
CRC Handbook of Chemistry and Physics, 91st edition, 2010-2011,
section 9. However, exact bond distances vary for each compound.
There is also variability in the bond angles.
[0072] It is therefore more practical to use an average parameter
(an easy to understand approximation) in such calculation.
[0073] In the calculation of a spacer or a linker length the
following approximations apply: a) for calculating lengths of
nonnucleosidic entities an average bond length of 130 pm with an
bond angle of 180.degree. independently of the nature of the linked
atoms is used; b) one nucleotide in a single strand is calculated
with 500 pm and c) one nucleotide in a double strand is calculated
with 330 pm.
[0074] The value of 130 pm is based on calculation of the distance
of the two terminal carbonatoms of a C(sp3)-C(sp3)-C(sp3) chain
with a bond angle of 109.degree. 28' and a distance of 153 pm
between two C(sp3) which is approx 250 pm which translates with an
assumed bond angle of 180.degree. to and bond distance between two
C(Sp3) with 125 pm. Taking in account that heteroatoms like P and S
and sp2 and sp1 C atoms could also be part of the spacer the value
130 pm is taken. If a spacer comprises a cyclic structure like
cycloalkyl or aryl the distance is calculated in analogous manner,
by counting the number of the bonds of said cyclic structure which
are part of the overall chain of atoms that are defining the
distance.
[0075] The spacer S can be construed as required to e.g. provide
for the desired length as well as for other desired properties. The
spacer can e.g. be fully or partially composed of naturally
occurring or non-naturally occurring amino acids, of
phosphate-sugar units e.g. a DNA like backbone without nucleobases,
of glyco-peptidic structures, or at least partially of saccharide
units or at least partially of polymerizable subunits like glycols
or acryl amide.
[0076] The length of spacer S in a binding agent according to the
present disclosure may be varied as desired. In order to easily
make available spacers of variable length, a library, some
embodiments may have a simple synthetic access to the spacers of
such library. A combinatorial solid phase synthesis of a spacer may
be present in some embodiments. Since spacers have to synthesized
up to a length of about 100 nm, the synthesis strategy is chosen in
such a manner that the monomeric synthetic building blocks are
assembled during solid phase synthesis with high efficiency. The
synthesis of deoxy oligonucleotides based on the assembly of
phosphoramidite as monomeric building blocks perfectly meet this
requirements. In such spacer monomeric units within a spacer are
linked in each case via a phosphate or phosphate analog moiety.
[0077] The spacer S can contain free positively or/and negatively
charged groups of polyfunctional amino-carboxylic acids, e.g.
amino, carboxylate or phosphate. For example the charge carriers
can be derived from trifunctional aminocarboxylic acids which
contain a) an amino group and two carboxylate groups or b) two
amino groups and one carboxylate group. Examples of such
trifunctional aminocarboxylic acids are lysine, ornithine,
hydroxylysine, .alpha.,.beta.-diamino propionic acid, arginine,
aspartic acid and glutamic acid, carboxy glutamic acid and
symmetric trifunctional carboxylic acids like those described in
EP-A-0 618 192 or U.S. Pat. No. 5,519,142. Alternatively one of the
carboxylate groups in the trifunctional aminocarboxylic acids a)
can be replaced by a phosphate, sulphonate or sulphate group. An
example of such a trifunctional amino acid is phosphoserine.
[0078] The spacer S can also contain uncharged hydrophilic groups.
Examples of uncharged hydrophilic groups include ethylene oxide or
polyethylene oxide groups, for example, with at least three
ethylene oxide units, sulphoxide, sulphone, carboxylic acid amide,
carboxylic acid ester, phosphonic acid amide, phosphonic acid
ester, phosphoric acid amide, phosphoric acid ester, sulphonic acid
amide, sulphonic acid ester, sulphuric acid amide and sulphuric
acid ester groups. The amide groups may be primary amide groups,
for example carboxylic acid amide residues in amino acid side
groups e.g. the amino acids asparagine and glutamine. The esters
may also be derived from hydrophilic alcohols, in particular C1-C3
alcohols or diols or triols.
[0079] In one embodiment the spacer S is composed of one type of
monomer. For example, the spacer is composed exclusively of amino
acids, of sugar residues, of diols, of phospho-sugar units or it
can be a nucleic acid, respectively.
[0080] In one embodiment, the spacer is DNA. In an exemplary
embodiment the spacer is the L-stereoisomer of DNA also known as
beta-L-DNA, L-DNA or mirror image DNA. L-DNA features advantages
like orthogonal hybridization behaviour, which means that a duplex
is formed only between two complementary single strands of L-DNA
but no duplex is formed between a single strand of L-DNA and the
complementary DNA strand, nuclease resistance and ease of synthesis
even of a long spacer. As pointed out ease of synthesis and
variability in spacer length are important for a spacer library.
Spacers of variable length are extremely utile in identifying the
binding agent according to the present disclosure having a spacer
of optimal length thus providing for the optimal distance between
the two monovalent binders.
[0081] Spacer building blocks, as the name says, can be used to
introduce a spacing moiety into the spacer S or to build the spacer
S of the linker a-S-b.
[0082] Different numbers and kinds of non-nucleotidic as well
nucleotidic spacer building blocks are at hand for introducing
spacing moieties.
[0083] Many different non nucleotidic bifunctional spacer building
blocks are known in literature and a great variety is commercially
available. The choice of the non nucleotidic bifunctional spacer
building is influencing the charge and flexibility of the spacer
molecule.
[0084] In bifunctional spacer building blocks a hydroxyl group
which is protected with an acid labile protecting group is
connected to a phosphoramidite group.
[0085] Bifunctional spacer building blocks in one embodiment are
non-nucleosidic compounds. For example, such spacers are C2-C18
alkyl, alkenyl, alkinyl carbon chains, whereas said alkyl, alkenyl,
alkinyl chains may be interrupted by additional ethyleneoxy and/or
amide moieties or quarternized cationic amine moieties in order to
increase hydrophilicity of the linker. Cyclic moieties like
C5-C6-cycloalkyl, C4N, C5N, C4O, C5O-heterocycloalkyl, phenyl which
are optionally substituted with one or two C1-C6 alkyl groups can
also be used as nonnucleosidic bifunctional spacer moieties.
Exemplary bifunctional building blocks comprise C3-C6 alkyl
moieties and tri- to hexa-ethyleneglycol chains. Table I shows some
examples of nucleotidic bifunctional spacer building blocks with
different hydrophilicity, different rigidity and different charges.
One oxygen atom is connected to an acid labile protecting group
such as dimethoxytrityl and the other is part of a
phosphoramidite.
TABLE-US-00001 TABLE I Examples of non-nucleotidic bifunctional
spacer building blocks Non-nucleotidic bifunctional spacer building
blocks Reference ##STR00001## Seela, F., Nucleic Acids Research 15
(1987) 3113-3129 ##STR00002## Iyer, R.P., Nucleic Acids Research 18
(1990) 2855-2859 ##STR00003## WO 89/02931 A1 ##STR00004## EP 1 538
221 ##STR00005## US 2004/224372 ##STR00006## WO 2007/069092
[0086] A simple way to build the spacer S or to introduce spacing
moieties into the spacer S is to use standard D or L nucleoside
phosphoramidite building blocks. In one embodiment a single strand
stretch of dT is used. This is advantageous, because dT does not
carry a base protecting group.
[0087] Hybridization can be used in order to vary the spacer length
(distance between the binding pair members a and b) and the
flexibility of the spacer, because the double strand length is
reduced compared to the single strand and the double strand is more
rigid than a single strand.
[0088] For hybridization in one embodiment oligonucleotides
modified with a functional moiety X are used. The oligonucleotide
used for hybridization can have one or two terminal extensions not
hybridizing with the spacer and/or is branched internally.
[0089] Such terminal extensions that are not hybridizing with the
spacer (and not interfering with the binding pairs a:a' and b:b')
can be used for further hybridization events. In one embodiment an
oligonucleotide hybridizing with a terminal extension is a labeled
oligonucleotide. This labeled oligonucleotide again may comprise
terminal extensions or being branched in order to allow for further
hybridization, thereby a polynucleotide aggregate or dendrimer can
be obtained. A poly-oligonucleic acid dendrimer may be used in
order to produce a polylabel. or in order to get a high local
concentration of X.
[0090] In one embodiment the spacer S has a backbone length of 1 to
100 nm. With other words here the groups a and b of Formula I are
between 1 and 100 nm apart. In one embodiment a and b,
respectively, each are a binding pair member and the spacer S has a
backbone length of 1 to 95 nm.
[0091] "a':a" as well as "b:b'" each independently represent a
binding pair. In one embodiment each of the binding pair members a
and b, respectively, has a length of at least 2.5 nm.
[0092] a and a' are the members of the binding pair a':a and b and
b' are the members of the binding pair b:b', respectively. Each
member of a binding pair may be of a molecular weight of 10 kD or
below, for example. In further embodiments the molecular weight of
each binder of such binding pair is 8, 7, 6, 5 or 4 kD or
below.
[0093] The binding affinity for (within) a binding pair, a:a' or
b':b, respectively, is at least 10.sup.8 l/mol. Both binding pairs
are different. For a binding pair difference is e.g. acknowledged
if the affinity for the reciprocal binding, e.g. binding of a as
well as a' to b or b' is 10% of the affinity within the pair a:a'
or lower. Also, the reciprocal binding, i.e. binding of a as well
as a' to b or b', respectively, may be 5% of the affinity within
the pair a:a' or lower, or if it is 2% of the affinity within the
pair a:a' or lower. In one embodiment the difference is so
pronounced that the reciprocal (cross-reactive) binding is 1% or
less as compared to the specific binding affinity within a binding
pair.
[0094] In one embodiment a':a and b:b' are binding pairs and the
members of the binding pairs a':a and b:b' are selected from the
group consisting of leucine zipper domain dimers and hybridizing
nucleic acid sequences. In one embodiment both binding pairs
represent leucine zipper domain dimers. In one embodiment both
binding pairs are hybridizing nucleic acid sequences.
[0095] The term "leucine zipper domain" is used to denote a
commonly recognized dimerization domain characterized by the
presence of a leucine residue at every seventh residue in a stretch
of approximately 35 residues. Leucine zipper domains are peptides
that promote oligomerization of the proteins in which they are
found. Leucine zippers were originally identified in several
DNA-binding proteins (Landschulz, H. W. et al., Science 240 (1988)
1759-1764), and have since been found in a variety of different
proteins. Among the known leucine zippers are naturally occurring
peptides and derivatives thereof that dimerize or trimerize.
Examples of leucine zipper domains suitable for producing soluble
multimeric proteins are described in PCT application WO 94/10308,
and the leucine zipper derived from lung surfactant protein D (SPD)
described in Hoppe, H. J. et al., FEBS Lett. 344 (1994)
191-195.
[0096] Leucine zipper domains form dimers (binding pairs) held
together by an alpha-helical coiled coil. A coiled coil has 3.5
residues per turn, which means that every seventh residue occupies
an equivalent position with respect to the helix axis. The regular
array of leucines inside the coiled coil stabilizes the structure
by hydrophobic and Van der Waals interactions.
[0097] If leucine zipper domains form the first binding pair (a':a)
and the second binding pair (b:b'), both leucine zipper sequences
are different, i.e. sequences a and a' do not bind to b and b'.
Leucine zipper domains may be isolated from natural proteins known
to contain such domains, such as transcription factors. One leucine
zipper domain may e.g. come from the transcription factor fos and a
second one from the transcription factor jun. Leucine zipper
domains may also be designed and synthesized artificially, using
standard techniques for synthesis and design known in the art.
[0098] In an exemplary embodiment both members of the binding pairs
a':a and b:b', i.e. a, a', b and b' represent leucine zipper
domains and the spacer S consists of amino acids. In this
embodiment production of the construct a-S-b is easily possible.
Varying the length of such spacer S as desired is straightforward
for a person skilled in the art. Such polypeptide can be
synthesized or recombinantly produced.
[0099] E.g., recombinant fusion proteins comprising a spacer
polypeptide fused to a leucine zipper peptide at the N-terminus and
to a leucine zipper peptide at the C-terminus can be expressed in
suitable host cells according to standard techniques. A DNA
sequence coding for a desired peptide spacer can be inserted
between a sequence coding for a member of a first leucine zipper
domain a and in the same reading frame a DNA sequence coding for a
member of a second leucine zipper domain b.
[0100] The spacer S, if the linker a-S-b is a polypeptide in one
embodiment comprises once or several times a GGGGS (SEQ ID NO:13)
amino acid sequence motif. The spacer S may also comprise a tag
sequence. The tag sequence may be selected from commonly used
protein recognition tags such as YPYDVPDYA (HA-Tag) (SEQ ID NO:14)
or GLNDIFEAQKIEWHE (Avi-Tag) (SEQ ID NO:15).
[0101] In an exemplary embodiment both binding pairs (a':a) and
(b:b') are hybridizing nucleic acid sequences.
[0102] As indicated already by nomenclature, a and a' as well as b
and b' hybridize to one another, respectively. The nucleic acid
sequences comprised in a and a' one the one hand and in b and b' on
the other hand are different. With other words the sequences of in
the binding pair a':a do not bind to the sequences of the binding
pair b:b', respectively, and vice versa. In one embodiment the
present disclosure relates to an at least dual binding agent of
Formula I, wherein the binding pairs a:a' and b:b', respectively,
both are hybridizing nucleic acid sequences and wherein the
hybridizing nucleic acid sequences of the different binding pairs
a':a and b:b' do not hybridize with one another. With other words a
and a' hybridize to each other but do not bind to any of b or b' or
interfere with their hybridization and vice versa. Hybridization
kinetics and hybridization specificity can easily be monitored by
melting point analyses. Specific hybridization of a binding pair
(e.g. a:a') and non-interference (e.g. with b or b') is
acknowledged, if the melting temperature for the pair a:a' as
compared to any possible combination with b or b', respectively,
(i.e. a:b; a:b'; a':b and a':b') is at least 20.degree. C.
higher.
[0103] The nucleic acid sequences forming a binding pair, e.g.
(a:a') or any other nucleic acid sequence-based binding pair, may
compromise any naturally occurring nucleobase or an analogue
thereto and may have a modified or an un-modified backbone as
described above, provided it is capable of forming a stable duplex
via multiple base pairing. Stable means that the melting
temperature of the duplex is higher than 37.degree. C. In some
cases, the double strand consists of two fully complementary single
strands. However mismatches or insertions are possible as long as
the a stability at 37.degree. C. is given.
[0104] As the skilled artisan will appreciate a nucleic acid duplex
can be further stabilized by interstrand crosslinking. Several
appropriate cross-linking methods are known to the skilled artisan,
e.g. methods using psoralen or based on thionucleosides.
[0105] The nucleic acid sequences representing the members of a
binding pair may consist of between 12 and 50 nucleotides. Also, in
some embodiments such nucleic acid sequences will consist of
between 15 and 35 nucleotides.
[0106] RNAses are ubiquitous and special care has to be taken to
avoid unwanted digestion of RNA-based binding pairs and/or spacer
sequences. While it certainly is possible to use, e.g. RNA-based
binding pairs and/or spacers, binding pairs and/or spacers based on
DNA represent an exemplary embodiment.
[0107] Appropriate hybridizing nucleic acid sequences can easily be
designed to provide for more than two pairs of orthogonal
complementary oligonucleotides, allowing for an easy generation and
use of more than two binding pairs. Another advantage of using
hybridizing nucleic acid sequences in a binding agent of the
present disclosure is that modifications can be easily introduced
into a nucleic acid sequences. Modified building blocks are
commercially available which e.g. allow for an easy synthesis of a
linker comprising a functional moiety. Such functional moiety can
be easily introduced at any desired position and in any of the
structures a and a' as well as b and b' and/or S, provided they
represent an oligonucleotide.
[0108] In some embodiments the spacer S comprised in a binding
agent according to Formula I is a nucleic acid. In some embodiments
both binding pairs are hybridizing nucleic acid sequences and the
spacer S also is a nucleic acid. In this embodiment the linker L
consisting of a-S-b is an oligonucleotide.
[0109] In case the spacer S as well as the sequences a, a', b and
b' all are oligonucleotide sequences it is easily possible to
provide for and synthesize a single oligonucleotide representing
the linker L comprising S and the members a and b of the binding
pairs a':a and b:b', respectively. In case the monovalent binders A
and B, respectively, are polypeptides, they can each be coupled
easily to the hybridizing nucleic acid sequences a' and b',
respectively. The length of the spacer S comprised in such
construct can easily be varied in any desired manner. Based on the
three constructs a-S-b, A-a' and b'-B the binding agent of Formula
I can be most easily obtained according to standard procedures by
hybridization between a':a and b:b', respectively. When spacers of
different length are used, the resulting constructs, provide for
otherwise identical binding agents, yet having a different distance
in between the monovalent binders A and B. This allows for optimal
distance and/or flexibility.
[0110] In some embodiments the spacer S as well as the sequences a,
a', b and b' are DNA.
[0111] The enantiomeric L-DNA, is known for its orthogonal
hybridization behavior, its nuclease resistance and for ease of
synthesis of oligonucleotides of variable length. This ease of
variability in linker length via designing appropriate spacers is
important for optimizing the binding of a binding agent as
disclosed herein to its antigen or antigens.
[0112] In some embodiments the linker L (=a-S-b) is enantiomeric
L-DNA or L-RNA. In an exemplary embodiment linker a-S-b is
enantiomeric L-DNA. In an exemplary embodiment a, a', b and b' as
well as the spacer S are enantiomeric L-DNA or L-RNA. In an
exemplary embodiment a, a', b and b' as well as the spacer S are
enantiomeric L-DNA.
[0113] In one embodiment the spacer S is an oligonucleotide and is
synthesized in two portions comprising ends hybridizable with each
other. In this case the spacer S can be simply constructed by
hybridization of these hybridizable ends with one another. The
resulting spacer construct comprises an oligonucleotide duplex
portion. As obvious, in case the spacer is construed that way, the
sequence of the hybridizable oligonucleotide entity forming said
duplex is chosen in such a manner that no hybridization or
interference with the binding pairs a:a' and b:b' can occur.
[0114] As already described above the monovalent specific binders A
and B of Formula I may be nucleic acids. In one embodiment of the
present disclosure a', a, b, b', A, B and s all are oligonucleotide
sequences. In this embodiment the sub-units A-a', a-S-b and b'-B of
Formula I can easily and independently be synthesized according to
standard procedures and combined by hybridization according to
convenient standard procedures. The functional moiety X may be
selected from the group consisting of a binding group, a labeling
group, an effector group and a reactive group.
[0115] If more than one functional moiety X is present, each such
functional moiety can in each case be independently a binding
group, a labeling group, an effector group or a reactive group.
[0116] In one embodiment the functional moiety X may be selected
from the group consisting of a binding group, a labeling group and
an effector group.
[0117] In one embodiment the group X is a binding group. As obvious
to a person skilled in the art, the binding group X will be
selected to have no interference with the pairs a':a and b:b'.
[0118] Examples of binding groups are the partners of a bioaffine
binding pair which can specifically interact with the other partner
of the bioaffine binding pair. Suitable bioaffine binding pairs are
hapten or antigen and antibody; biotin or biotin analogues such as
aminobiotin, iminobiotin or desthiobiotin and avidin or
streptavidin; sugar and lectin, oligonucleotide and complementary
oligonucleotide, receptor and ligand, e.g., steroid hormone
receptor and steroid hormone. In one embodiment X is a binding
group and is covalently bound to at least one of a', a, b, b' or S
of the compound of Formula I. According to some embodiments the
smaller partner of a bioaffine binding pair, e.g. biotin or an
analogue thereto, a receptor ligand, a hapten or an oligonucleotide
is covalently bound to at least one of a', a, S, b or b' as defined
above.
[0119] In one embodiment functional moiety X is a binding group
selected from hapten; biotin or biotin analogues such as
aminobiotin, iminobiotin or desthiobiotin; oligonucleotide and
steroid hormone.
[0120] In one embodiment the functional moiety X is a reactive
group. The reactive group can be selected from any known reactive
group, like Amino, Sulfhydryl, Carboxylate, Hydroxyl, Azido,
Alkinyl or Alkenyl. In one embodiment the reavtive group is
selected from Maleinimido, Succinimidyl, Dithiopyridyl,
Nitrophenylester, Hexafluorophenylester.
[0121] In one embodiment the functional moiety X is a labeling
group. The labeling group can be selected from any known detectable
group. The skilled artisan will choose the number of labels as
appropriate for best sensitivity with least quenching.
[0122] The labeling group can be selected from any known detectable
group. In one embodiment the labeling group is selected from dyes
like luminescent labeling groups such as chemiluminescent groups
e.g. acridinium esters or dioxetanes or fluorescent dyes e.g.
fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine and
derivatives thereof, luminescent metal complexes such as ruthenium
or europium complexes, enzymes as used for CEDIA (Cloned Enzyme
Donor Immunoassay, e.g. EP 0 061 888), microparticles or
nanoparticles e.g. latex particles or metal sols, and
radioisotopes.
[0123] In one embodiment the labeling group is a luminescent metal
complex and the compound has a structure of the general formula
(II):
[M(L.sub.1L.sub.2L.sub.3)].sub.n-Y-X.sub.mA (II)
[0124] in which M is a divalent or trivalent metal cation selected
from rare earth or transition metal ions, L.sub.1, L.sub.2 and
L.sub.3 are the same or different and denote ligands with at least
two nitrogen-containing heterocycles in which L.sub.1, L.sub.2 and
L.sub.3 are bound to the metal cation via nitrogen atoms, X is a
reactive functional group which is covalently bound to at least one
of the ligands L.sub.1, L.sub.2 and L.sub.3 via a linker Y, n is an
integer from 1 to 10, and in some illustrative embodiments is 1 to
4, m is 1 or 2 and in some illustrative embodiments is 1 and A
denotes the counter ion which may be required to equalize the
charge.
[0125] The metal complex may be a luminescent metal complex i.e. a
metal complex which undergoes a detectable luminescence reaction
after appropriate excitation. The luminescence reaction can for
example be detected by fluorescence or by electrochemiluminescence
measurement. The metal cation in this complex is for example a
transition metal or a rare earth metal. The metal may be ruthenium,
osmium, rhenium, iridium, rhodium, platinum, indium, palladium,
molybdenum, technetium, copper, chromium or tungsten. In some
illustrative embodiments ruthenium is used.
[0126] The ligands L.sub.1, L.sub.2 and L.sub.3 are ligands with at
least two nitrogen-containing heterocycles. Aromatic heterocycles
such as bipyridyl, bipyrazyl, terpyridyl and phenanthrolyl may be
used. The ligands L.sub.1, L.sub.2 and L.sub.3 may be selected from
bipyridine and phenanthroline ring systems.
[0127] The complex can additionally contain one or several counter
ions A to equalize the charge. Examples of suitable negatively
charged counter ions are halogenides, OH.sup.-, carbonate,
alkylcarboxylate, e.g. trifluoroacetate, sulphate,
hexafluorophosphate and tetrafluoroborate groups.
Hexafluorophosphate, trifluoroacetate and tetrafluoroborate groups
are used in some illustrative embodiments. Examples of suitable
positively charged counter ions are monovalent cations such as
alkaline metal and ammonium ions.
[0128] In further embodiments the functional moiety X is an
effector group. An exemplary effector group is a therapeutically
active substance.
[0129] Therapeutically active substances have different ways in
which they are effective, e.g. in inhibiting cancer. They can
damage the DNA template by alkylation, by cross-linking, or by
double-strand cleavage of DNA. Other therapeutically active
substances can block RNA synthesis by intercalation. Some agents
are spindle poisons, such as vinca alkaloids, or anti-metabolites
that inhibit enzyme activity, or hormonal and anti-hormonal agents.
The effector group X may be selected from alkylating agents,
antimetabolites, antitumor antibiotics, vinca alkaloids,
epipodophyllotoxins, nitrosoureas, hormonal and antihormonal
agents, and toxins.
[0130] Currently exemplary alkylating agents include
cyclophosphamide, chlorambucil, busulfan, Melphalan, Thiotepa,
ifosphamide, Nitrogen mustard.
[0131] Currently exemplary antimetabolites include methotrexate,
5-Fluorouracil, cytosine arabinoside, 6-thioguanine,
6-mercaptopurin.
[0132] Currently exemplary antitumor antibiotics include
doxorubicin, daunorubicin, idorubicin, nimitoxantron, dactinomycin,
bleomycin, mitomycin, and plicamycin.
[0133] Currently exemplary spindle poisons include maytansine and
maytansinoids, vinca alkaloids and epipodophyllotoxins include
vincristin, vinblastin, vindestin, Etoposide, Teniposide.
[0134] Currently exemplary nitrosoureas include carmustin,
lomustin, semustin, streptozocin.
[0135] Currently exemplary hormonal and antihormonal agents include
adrenocorticorticoids, estrogens, antiestrogens, progestins,
aromatase inhibitors, androgens, antiandrogens.
[0136] Additional exemplary random synthetic agents include
dacarbazin, hexamethylmelamine, hydroxyurea, mitotane,
procarbazide, cisplastin, carboplatin.
[0137] The functional moiety X is bound either covalently or via an
additional binding pair to at least one of (a'), (a), (b), (b') or
S. The functional moiety X can occur once or several (n) times. (n)
is an integer and 1 or more than one. In some embodiments (n) is
between 1 and 100. Also, (n) may be 1-50. In certain embodiments n
is 1 to 10, or 1 to 5. In further embodiments n is 1 or 2.
[0138] For covalent binding of the functional moiety X to at least
one of a', a, b, b' or S any appropriate coupling chemistry can be
used. The skilled artisan can easily select such coupling chemistry
from standard protocols. It is also possible to incorporate a
functional moiety by use of appropriate building blocks when
synthesizing a', a, b, b' or S.
[0139] In an exemplary embodiment functional moiety X is bound to
a, b, or S of the binding agent as defined by Formula I. In an
exemplary embodiment functional moiety X is bound to the spacer S
of the binding agent as defined by Formula I.
[0140] In some embodiments functional moiety X is covalently bound
to a, b, or S of the binding agent as defined by Formula I.
[0141] If a functional moiety X is located within the a hybridizing
oligonucleotide representing a, a', b or b', respectively, in some
cases such functional moiety is bound to a modified nucleotide or
is attached to the internucleosidic P atom (WO 2007/059816).
Modified nucleotides which do not interfere with the hybridization
of oligonucleotides are incorporated into those oligonucleotides.
Such modified nucleotides may be C5 substituted pyrimidines or C7
substituted 7deaza purines.
[0142] Oligonucleotides can be modified internally or at the 5' or
3' terminus with non-nucleotidic entities which are used for the
introduction of functional moiety. In some embodiments such
non-nucleotidic entities are located within the spacer S, i.e.
between the two binding pair members a and b.
[0143] Many different non-nucleotidic modifier building blocks for
construction of a spacer are known in literature and a great
variety is commercially available. For the introduction of a
functional moiety either non-nucleosidic bifunctional modifier
building blocks or non-nucleosidic trifunctional modified building
blocks are either used as CPG for terminal labeling or as
phosphroamidite for internal labeling (see: Wojczewski, C. et al.,
Synlett 10 (1999) 1667-1678).
[0144] Bifunctional Modifier Building Blocks
[0145] Bifunctional modifier building blocks connect a functional
moiety or a--if necessary--a protected functional moiety to a
phosphoramidite group for attaching the building block at the 5'
end (regular synthesis) or at the 3' end (inverted synthesis) to
the terminal hydroxyl group of a growing oligonucleotide chain.
[0146] Bifunctional modifier building blocks are, for example,
non-nucleosidic compounds. For example, such modified building
blocks are C2-C18 alkyl, alkenyl, alkynyl carbon chains, whereas
said alkyl, alkenyl, alkynyl chains may be interrupted by
additional ethyleneoxy and/or amide moieties in order to increase
hydrophilicity of the spacer and thereby of the whole linker
structure. Cyclic moieties like C5-C6-cycloalkyl, C4N, C5N, C4O,
C5O-heterocycloalkyl, phenyl which are optionally substituted with
one or two C1-C6 alkyl groups can also be used as non-nucleosidic
bifunctional modified building blocks. In some cases modified
bifunctional building blocks comprise C3-C6 alkyl moieties and tri-
to hexa-ethyleneglycol chains. Non-limiting examples of
bifunctional modifier building blocks are given in Table II
below.
TABLE-US-00002 TABLE II Bifunctional non-nucleosidic modifier
building block Introduction of Reference ##STR00007## ##STR00008##
##STR00009## Pon, R.T., Tetrahedron Letters 32 (1991) 1715- 1718
Theisen, P. et al., Nucleic Acids Symposium Series (1992), 27
(Nineteenth Symposium on Nucleic Acids Chemistry) 99-100 EP 0 292
128 ##STR00010## ##STR00011## EP 0 523 978 ##STR00012##
##STR00013## Meyer, A. et al., Journal of Organic Chemistry 75
(2010) 3927-3930 ##STR00014## ##STR00015## Morocho, A.M. et al.,
Nucleosides, Nucleotides & Nucleic Acids 22 (2003) 1439-1441
##STR00016## ##STR00017## Cocuzza, A.J., Tetrahedron Letters 30
(1989) 6287- 6290
[0147] Trifunctional Modifier Building Blocks
[0148] Trifunctional building blocks connect (i) a functional
moiety or a--if necessary--a protected functional moiety, (ii) a
phosphoramidite group for coupling the reporter or the functional
moiety or a--if necessary--a protected functional moiety, during
the oligonucleotide synthesis to a hydroxyl group of the growing
oligonucleotide chain and (iii) a hydroxyl group which is protected
with an acid labile protecting group, for example, with a
dimethoxytrityl protecting group. After removal of this acid labile
protecting group a hydroxyl group is liberated which can react with
further phosphoramidites. Therefore trifunctional building blocks
allow for positioning of a functional moiety to any location within
an oligonucleotide. Trifunctional building blocks are also a
prerequisite for synthesis using solid supports, e.g. controlled
pore glass (CPG), which are used for 3' terminal labeling of
oligonucleotides. In this case, the trifunctional building block is
connected to a functional moiety or a--if necessary--a protected
functional moiety via an C2-C18 alkyl, alkenyl, alkinyl carbon
chains, whereas said alkyl, alkenyl, alkylnyl chains may be
interrupted by additional ethyleneoxy and/or amide moieties in
order to increase hydrophilicity of the spacer and thereby of the
whole linker structure and comprises a hydroxyl group which is
attached via a cleavable spacer to a solid phase and a hydroxyl
group which is protected with an acid labile protecting group.
After removal of this protecting group a hydroxyl group is
liberated which could then react with a phosphoramidite.
[0149] Trifunctional Building Blocks May be Non-Nucleosidic or
Nucleosidic.
[0150] Non-nucleosidic trifunctional building blocks are C2-C18
alkyl, alkenyl, alkynyl carbon chains, whereas said alkyl, alkenyl,
alkynyl are optionally interrupted by additional ethyleneoxy and/or
amide moieties in order to increase hydrophilicity of the spacer
and thereby of the whole linker structure. Other trifunctional
building blocks are cyclic groups like C5-C6-cycloalkyl, C4N, C5N,
C4O, C5O heterocycloalkyl, phenyl which are optionally substituted
with one ore two C1-C6 alkyl groups. Cyclic and acyclic groups may
be substituted with one --(C1-C18)alkyl-O-PG group, whereas said
C1-C18 alkyl comprises (Ethyleneoxy)n, (Amide)m moieties with n and
m independently from each other=0-6 and PG is an acid labile
protecting group. Exemplary trifunctional building blocks are C3-C6
alkyl, cycloalkyl, C5O heterocycloalkyl moieties optionally
comprising one amide bond and substituted with a C1-C6 alkyl O-PG
group, wherein PG is an acid labile protecting group, such as
monomethoxytrityl, dimethoxytrityl, pixyl, and xanthyl.
[0151] Non-limiting examples for non-nucleosidic trifunctional
building blocks are e.g. summarized in Table III.
TABLE-US-00003 TABLE III Examples for non-nucleosidic trifunctional
modifier building Blocks Trifunctional Introduction of Reference
##STR00018## ##STR00019## ##STR00020## Nelson, P.S. et al., Nucleic
Acids Research 20 (1992) 6253- 6259 EP 0 313 219 U.S. Pat. No.
5,585,481 U.S. Pat. No. 5,451,463 EP 0 786 468 WO 92/11388 WO
89/02439 ##STR00021## ##STR00022## ##STR00023## Su, S.-H. et al.,
Bioorganic & Medicinal Chemistry Letters 7 (1997) 1639-1644 WO
97/43451 ##STR00024## ##STR00025## ##STR00026## Putnam, W.C. et
al., Nucleosides, Nucleotides & Nucleic Acids 24 (2005) 1309-
1323 US 2005/214833 EP 1 186 613 ##STR00027## ##STR00028##
##STR00029## EP 1 431 298 ##STR00030## ##STR00031## WO 94/04550 Vu,
H., et al., Nucleic Acids Symposium Series (1993), 29 (Second
International Symposium on Nucleic Acids Chemistry), 19- 20
##STR00032## ##STR00033## WO 2003/019145 ##STR00034## ##STR00035##
Behrens, C. and Dahl, O., Nucleosides & Nucleotides 18 (1999)
291-305 WO 97/05156 ##STR00036## ##STR00037## Prokhorenko, I.A. et
al., Bioorganic & Medicinal Chemistry Letters 5 (1995)
2081-2084 WO 2003/104249 ##STR00038## ##STR00039## U.S. Pat. No.
5,849,879
[0152] Nucleosidic Modifier Building Blocks:
[0153] Nucleosidic modifier building blocks are used for internal
labeling whenever it is necessary not to influence the
oligonucleotide hybridization properties compared to a non-modified
oligonucleotide. Therefore nucleosidic building blocks comprise a
base or a base analog which is still capable of hybridizing with a
complementary base. The general formula of a labeling compound for
labeling a nucleic acid sequence of one or more of a, a', b, b' or
S comprised in a binding agent according to Formula I of the
present disclosure is given in Formula II.
##STR00040##
[0154] wherein PG is an acid labile protecting group such as
monomethoxytrityl, dimethoxytrityl, pixyl, and xanthyl, wherein Y
is C2-C18 alkyl, alkenyl alkinyl, wherein said alkyl, alkenyl,
alkinyl may comprise ethyleneoxy and/or amide moieties, wherein Y
is C4-C18 alkyl, alkenyl or alkinyl and contains one amide moiety
and wherein X is a functional moiety.
[0155] Specific positions of the base may be chosen for such
substitution to minimize the influence on hybridization properties.
Therefore the following positions may be used for substitution: a)
with natural bases: Uracil substituted at C5; Cytosine substituted
at C5 or at N4; Adenine substituted at C8 or at N6 and Guanine
substituted at C8 or at N2 and b) with base analogs: 7 deaza A and
7 deaza G substituted at C7; 7 deaza 8 Aza A and 7 deaza 8 Aza G
substituted at C7; 7 deaza Aza 2 amino A substituted at C7;
Pseudouridine substituted at N1 and Formycin substituted at N2.
[0156] Non-limiting examples for nucleosidic trifunctional building
blocks are given in Table IV.
TABLE-US-00004 TABLE IV Trifunctional nucleosidic A Reference
##STR00041## ##STR00042## Roget, A. et al., Nucleic Acids Research
17 (1989) 7643- 7651 WO 89/12642 WO 90708156 WO 93705060
##STR00043## ##STR00044## Silva, J.A. et al., Biotecnologia
Aplicada 15 (1998) 154-158 ##STR00045## ##STR00046## U.S. Pat. No.
6,531,581 EP 0 423 839 ##STR00047## ##STR00048## U.S. Pat. No.
4,948,882 U.S. Pat. No. 5,541,313 U.S. Pat. No. 5,817,786
##STR00049## ##STR00050## WO 2001/042505 ##STR00051## ##STR00052##
McKeen, C.M. et al., Organic & Biomolecular Chemistry 1 (2003),
2267- 2275 ##STR00053## ##STR00054## Ramzaeva, N. et al., Helvetica
Chimica Acta 83 (2000) 1108- 1126
[0157] In Tables II, III and IV, one of the terminal oxygen atom of
a bifunctional moiety or one of the terminal oxygen atoms of a
trifunctional moiety is part of a phosphoramidite that is not shown
in full detail but obvious to the skilled artisan. The second
terminal oxygen atom of trifunctional building block is protected
with an acid labile protecting group PG, as defined for Formula II
above.
[0158] Post-synthetic modification is another strategy for
introducing a covalently bound functional moiety into a linker or a
spacer molecule. In this approach an amino group is introduced by
using bifunctional or trifunctional building block during solid
phase synthesis. After cleavage from the support and purification
of the amino modified oligonucleotide is reacted with an activated
ester of a functional moiety or with a bifunctional reagent wherein
one functional group is an active ester. Exemplary active esters
are NHS ester or pentafluor phenyl esters.
[0159] Post-synthetic modification is especially useful for
introducing a functional moiety which is not stable during solid
phase synthesis and deprotection. Examples are modification with
triphenylphosphincarboxymethyl ester for Staudinger ligation (Wang,
C. C. et al., Bioconjugate Chemistry 14 (2003) 697-701),
modification with digoxigenin or for introducing a maleinimido
group using commercial available sulfo SMCC.
[0160] The functional moiety X in one embodiment is bound to at
least one of a', a, b, b' or S via an additional binding pair.
[0161] The additional binding pair to which a functional moiety X
can be bound may be a leucine zipper domain or a hybridizing
nucleic acid. In case the functional moiety X is bound to at least
one of a', a, b, b' or S via an additional binding pair member, the
binding pair member to which X is bound and the binding pairs a':a
and b:b', respectively, all are selected to have different
specificity. The binding pairs a:a', b:b' and the binding pair to
which X is bound each bind to (e.g. hybridize with) their
respective partner without interfering with the binding of any of
the other binding pairs.
[0162] Covalent Coupling of a Member of a Binding Pair to a
Monovalent Binder
[0163] Depending on the biochemical nature of the binder different
conjugation strategies are at hand.
[0164] In case the binder is a naturally occurring protein or a
recombinant polypeptide of between 50 to 500 amino acids, there are
standard procedures in text books describing the chemistry for
synthesis of protein conjugates, which can be easily followed by
the skilled artisan. (Hackenberger, C. P. et al., Angew. Chem.,
Int. Ed., 47 (2008)10030-10074).
[0165] In one embodiment the reaction of a maleinimido moiety with
a cystein residue within the protein is used. This is an exemplary
coupling chemistry in case e.g. an Fab or Fab'-fragment of an
antibody is used a monovalent binder. Alternatively in one
embodiment coupling of a member of a binding pair (a' or b',
respectively, of Formula I) to the C-terminal end of the binder
polypeptide is performed. C-terminual modification of a protein,
e.g. of an Fab-fragment can e.g. be performed as described (Sunbul,
Murat and Yin, Jun, Organic & Biomolecular Chemistry 7 (2009)
3361-3371).
[0166] In general site specific reaction and covalent coupling of a
binding pair member to a monovalent polypeptidic binder is based on
transforming a natural amino acid into an amino acid with a
reactivity which is orthogonal to the reactivity of the other
functional groups present in a protein. For example, a specific
cystein within a rare sequence context can be enzymatically
converted in an aldehyde (see Frese, M.-A. et al., ChemBioChem 10
(2009) 425-427). It is also possible to obtain a desired amino acid
modification by utilizing the specific enzymatic reactivity of
certain enzymes with a natural amino acid in a given sequence
context (see e.g.: Taki, M. et al., Protein Engineering, Design
& Selection 17 (2004) 119-126; Gautier, A. et al., Chemistry
& Biology 15 (2008) 128-136; Protease-catalyzed formation of
C--N bonds is used by Bordusa, F., Highlights in Bioorganic
Chemistry (2004) 389-403 and Sortase-mediated protein ligation is
used by Mao, H. et al., in J. Am. Chem. Soc. 126 (2004) 2670-2671
and reviewed by Proft, T., in Biotechnol. Lett 32 (2010) 1-10).
[0167] Site specific reaction and covalent coupling of a binding
pair member to a monovalent polypeptidic binder can also be
achieved by the selective reaction of terminal amino acids with
appropriate modifying reagents.
[0168] The reactivity of an N-terminal cystein with benzonitrils
(Ren, Hongjun, Xiao, et al., Angewandte Chemie, International
Edition 48 (2009) 9658-9662) can be used to achieve a site-specific
covalent coupling.
[0169] Native chemical ligation can also rely on C-terminal cystein
residues (Taylor, E. Vogel, Imperiali, B., Nucleic Acids and
Molecular Biology 22 (2009) (Protein Engineering) 65-96).
[0170] EP 1 074 563 describes a conjugation method which is based
on the faster reaction of a cystein within a stretch of negatively
charged amino acids with a cystein located in a stretch of
positively charged amino acids.
[0171] The monovalent binder may also be a synthetic peptide or
peptide mimic. In case a polypeptide is chemically synthesized,
amino acids with orthogonal chemical reactivity can be incorporated
during such synthesis (de Graaf, A. J. et al., Bioconjugate
Chemistry 20 (2009) 1281-1295). Since a great variety of orthogonal
functional groups is at stake and can be introduced into a
synthetic peptide, conjugation of such peptide to a linker is
standard chemistry.
[0172] In order to obtain a mono-labeled protein the conjugate with
1:1 stoichiometry may be separated by chromatography from other
conjugation products. This procedure is facilitated by using a dye
labeled binding pair member and a charged spacer. By using this
kind of labeled and highly negatively charged binding pair member,
mono conjugated proteins are easily separated from non labeled
protein and proteins which carry more than one linker, since the
difference in charge and molecular weight can be used for
separation. The fluorescent dye is valuable for purifying the
binding agent from un-bound components, like a labeled monovalent
binder.
[0173] Therefore in one embodiment a binding pair member (a' and/or
b', respectively of Formula I) which is labeled with a fluorescent
dye (e.g. synthesized using a bifunctional or trifunctional
modifier building block in combination with bifunctional spacer
building blocks during synthesis) for forming the binding agent of
the present disclosure may be used. In an exemplary embodiment the
spacer S as well as the sequences a, a', b and b' are DNA and at
least one of a' or b', respectively, is labeled with a fluorescent
dye. In some embodiments the spacer S as well as the sequences a,
a', b and b' are DNA and both a' and b', respectively, are labeled
each with a different fluorescent dye.
[0174] In one embodiment the present disclosure relates to a
bispecific binding agent of the Formula I: A-a':a-S-b:b'-B:X(n);
wherein A as well as B is a monovalent specific binder, wherein
a':a as well as b:b' represent a binding pair with a':a and b:b'
having a different specificity, wherein S represents a spacer,
wherein (: X) denotes a functional moiety bound via a further
binding pair to at least one of a', a, b, b' or S, wherein (n) is
an integer and at least 1, wherein - represents a covalent bond,
and wherein the linker a-S-b has a length of 6 to 100 nm.
[0175] In some embodiments the binding pairs a':a and b:b' are
hybridizing nucleic acid sequences, the spacer S is a nucleic acid
and the further binding pair to which the functional moiety X is
bound is also a nucleic acid. In such embodiments the spacer S may
be construed to comprise, in addition to the two specifically
hybridizing sequences a and a' and b and b', respectively, one or
more further sequences also capable of hybridizing to its or their
complementary sequences. In this embodiment a functional moiety X
is bound to the spacer S via a further binding pair also consisting
of hybridizing nucleic acid sequences.
[0176] A monovalent binder for use in the construction of a binding
agent as disclosed herein has to have a Kdiss from 10.sup.-2/sec to
10.sup.-5/sec. Also, a monovalent binder for use in the
construction of a binding agent as disclosed herein has to have a
Kdiss from 10.sup.-3/sec to 10.sup.-5/sec.
[0177] According to some embodiments, in the binding agent
according to Formula I, each of the monovalent binders A and B,
respectively has a Kdiss from 10.sup.-2/sec to 10.sup.-5/sec and in
some illustrative embodiments from 10.sup.-3/sec to
10.sup.-5/sec.
[0178] In some embodiments, the binding agent according to Formula
I has a Kdiss of 10.sup.-5/sec or better, or may have a Kdiss of
10.sup.-6/sec or better. In some embodiments the binding agent
according to Formula I may have a Kdiss of 10.sup.-7/sec or
better.
[0179] As the skilled artisan will appreciate the Kdiss is a
temperature-dependent value. Logically, the Kdiss-values of a
binding agent according to the present disclosure are determined at
the same temperature. As will be appreciated, a Kdiss-value is
determined at the same temperature at which the binding agent shall
be used, e.g., an assay shall be performed. In one embodiment the
Kdiss-values are established at room temperature, i.e. at
20.degree. C., 21.degree. C., 22.degree. C., 23.degree. C.,
24.degree. C. or 25.degree. C., respectively. In one embodiment the
Kdiss-values are established at 4 or 8.degree. C., respectively. In
one embodiment the Kdiss-values are established at 25.degree. C. In
one embodiment the Kdiss-values are established at 37.degree.
C.
[0180] As mentioned already above, it is now possible and pretty
straightforward to produce a binding agent as defined in Formula I.
A full library of a binding agent according to Formula I can be
easily provided, analyzed and the most powerful binding agent out
of such library produced at large scale, as required.
[0181] The library mentioned above refers to a full set of binding
agents according to Formula I, wherein each A, a, a', b, b' and B
are identical and wherein in the length of the spacer S is adjusted
to best meet the requirements set out for the binding agent. It is
easily possible to first use a spacer ladder spanning the whole
spectrum of 1 to 100 nm and having steps that are about 10 nm
apart. The spacer length is then again easily further refined
around the most appropriate length identified in the first
round.
[0182] In one embodiment the present disclosure relates to a method
of producing a binding agent of the Formula I:
A-a':a-S-b:b'-B:X(n), wherein A as well as B is a monovalent
binder, wherein a':a as well as b:b' is a binding pair, wherein a'
and a and do not interfere with the binding of b to b' and vice
versa, wherein S is a spacer of at least 1 nm in length, wherein (:
X) denotes a functional moiety bound either covalently or via a
binding pair to at least one of a', a, b, b' or S, wherein (n) is
an integer and at least 1, wherein - represents a covalent bond,
and wherein the linker a-S-b has a length of 6 to 100 nm, the
method comprising the steps of: a) synthesizing A-a' and b'-B,
respectively, b) synthesizing the linker a-S-b and c) forming the
binding agent of Formula I, wherein the functional moiety X bound
to at least one of a', a, b, b' or S is bound in step a), b) or
c).
[0183] In some embodiments of this method several linker molecules
with spacers of various lengths are synthesized and used in the
formation of binding agents according to Formula I comprising
spacers of variable length and those binding agent(s) are selected
having an improvement in the Kdiss of at least 5-fold over the
better of the two monovalent binders. Selection of a binding agent
with the desired Kdiss in one embodiment is performed by
BiaCore-analysis as disclosed in Example 2.8.
[0184] The binding agent according to the present disclosure can
e.g. be used to strongly bind an analyte in an immunoassay. If e.g.
an analyte has at least two non-overlapping epitopes the binding
agent of the present disclosure is construed such that the spacer S
has the optimal length for synergistic binding of the monovalent
binders to these epitopes. This improvement can e.g. be of great
utility in a method for detection of an analyte employing such
binding agent. In one embodiment the present disclosure therefore
relates to the use of a binding agent as disclosed herein above in
the detection of an analyte of interest. In certain embodiments the
detection method used is an enzyme-linked immunosorbent assay
(ELISA), a direct, indirect, competitive or sandwich immuno assay
employing any appropriate way of signal detection, e.g.
electrochemiluminescense, or the binding agent is used in
immunohistochemistry.
[0185] The following examples, sequence listing, and figures are
provided for the purpose of demonstrating various embodiments of
the instant disclosure and aiding in an understanding of the
present disclosure, the true scope of which is set forth in the
appended claims. These examples are not intended to, and should not
be understood as, limiting the scope or spirit of the instant
disclosure in any way. It should also be understood that
modifications can be made in the procedures set forth without
departing from the spirit of the disclosure.
Illustrative Embodiments
[0186] The following comprises a list of illustrative embodiments
according to the instant disclosure which represent various
embodiments of the instant disclosure. These illustrative
embodiments are not intended to be exhaustive or limit the
disclosure to the precise forms disclosed, but rather, these
illustrative embodiments are provided to aide in further describing
the instant disclosure so that others skilled in the art may
utilize their teachings.
1. A binding agent of the Formula A-a':a-S-b:b'-B:X(n), wherein A
as well as B is a monovalent binder, wherein a':a as well as b:b'
is a binding pair wherein a' and a do not interfere with the
binding of b to b' and vice versa, wherein S is a spacer of at
least 1 nm in length, wherein (: X) denotes a functional moiety
bound either covalently or via a binding pair to at least one of
a', a, b, b' or S, wherein (n) is an integer and at least 1,
wherein - represents a covalent bond, and wherein the linker a-S-b
has a length of 6 to 100 nm. 2. The binding agent of embodiment 1,
wherein the spacer S is 1 to 95 nm in length. 3. The binding agent
of embodiments 1 or 2, wherein the binding pairs are selected from
the group consisting of leucine zipper domain dimers and
hybridizing nucleic acid sequences. 4. The binding agent of any of
embodiments 1 to 3, wherein the binding pairs both are hybridizing
nucleic acid sequences and wherein the different hybridizing
nucleic acid sequences of the binding pairs a':a and b:b' do not
hybridize with one another. 5. The binding agent of any of
embodiments 1 to 4, wherein the spacer S is a nucleic acid. 6. The
binding agent of any of embodiments 1 to 5, wherein the spacer S is
a nucleic acid and wherein both binding pairs a':a as well as b:b'
also are a nucleic acid. 7. The binding agent of any of embodiments
1 to 6, wherein the spacer S is a nucleic acid and wherein both
binding pairs a':a as well as b:b' are nucleic acids and wherein
the monovalent binders A and B are nucleic acids. 8. The binding
agent of any of embodiments 1 to 7, wherein X is a functional
moiety selected from the group consisting of a labeling group, a
binding group and an effector group 9. The binding agent of any of
embodiments 1 to 8, wherein the functional moiety X is bound to a,
b, or S. 10. The binding agent of any of embodiments 1 to 9,
wherein the functional moiety X is bound to the spacer S. 11. The
binding agent of any embodiments 1 to 10, wherein the functional
moiety X is covalently bound to the spacer S. 12. The binding agent
of any of embodiments 1 to 10, wherein the functional moiety X is
bound to the spacer S via a hybridizing nucleic acid. 13. The
binding agent of any of embodiments 1 to 3, or embodiments 8 to 12,
wherein the monovalent binders A and B are polypeptides such as
Fab-fragments of monoclonal antibodies. 14. Use of a binding agent
according to any of embodiments 1 to 13 in the detection of an
analyte of interest. 15. Use of a binding agent according to any of
embodiments 1 to 13 in an immuno assay.
EXAMPLES
Bi-Valent Binding Agent to Troponin T
[0187] 1.1 Monoclonal Antibodies and Fab'-Fragments
[0188] Two monoclonal antibodies binding to human cardiac Troponin
T at different, non-overlapping epitopes, epitope A' and epitope
B', respectively, were used. Both these antibodies are used in the
current Roche Elecsys.TM. Troponin T assay, wherein Troponin T is
detected in a sandwich immuno assay format.
[0189] Purification of the monoclonal antibodies from culture
supernatant was carried out using state of the art methods of
protein chemistry.
[0190] The purified monoclonal antibodies are protease digested
with either pre-activated papain (anti-epitope A' MAb) or pepsin
(anti-epitope B' MAb) yielding F(ab')2 fragments that are
subsequently reduced to Fab'-fragments, i.e. A and B, respectively,
in Formula I (A-a':a-S-b:b'-B:X.sub.n), with a low concentration of
cysteamin at 37.degree. C.,. The reaction is stopped by separating
the cysteamin on a Sephadex G-25 column (GE Healthcare) from the
polypeptide-containing part of the sample.
[0191] 1.2 Conjugation of Fab'-Fragments to
ssDNA-Oligonucleotides
[0192] The Fab'-fragments are conjugated with the below described
activated ssDNAa and ssDNAb oligonucleotides.
[0193] Preparation of the Fab'-fragment-ssDNA conjugates A'' and
B'', respectively:
[0194] a) Fab'-Anti-Troponin T<Epitope a'>-ssDNA-Conjugate
(=a'')
[0195] For preparation of the Fab'-anti-Troponin T<epitope
A'>-ssDNAa-conjugate A'' a derivative of SED ID NO:5 is used,
i.e. 5'-AGT CTA TTA ATG CTT CTG C(=SEQ ID NO:5)-XXX-Y-Z-3', wherein
X=propylene-phosphate introduced via Phosphoramidite C3
(3-(4,4'-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-p-
hosphoramidite (Glen Research), wherein Y=3''-Amino-Modifier C6
introduced via 3'-Amino Modifier TFA Amino C-6 Icaa CPG (ChemGenes)
and wherein Z=4[N-maleinimidomethyl]cyclohexane-1-carboxy
introduced via Sulfosuccinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate (ThermoFischer).
[0196] b) Fab'-Anti-Troponin T<Epitope B'>-ssDNAb-Conjugate
(=B'')
[0197] For the preparation of the Fab'-anti-Troponin T<epitope
B'>-ssDNA-conjugate (B'') a derivative of SEQ ID NO:6 is used,
i.e. 5'-Y-Z-XXX-AGT TCT ATC GTC GTC CA-3', wherein
X=propylene-phosphate introduced via Phosphoramidite C3
(3-(4,4'-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-p-
hosphoramidite (Glen Research), wherein Y=5'-Amino-Modifier C6
introduced via
(6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-p-
hosphoramidite (Glen Research), and wherein
Z=4[N-maleinimidomethyl]cyclohexane-1-carboxy introduced via
Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate
(ThermoFischer).
[0198] The oligonucleotides of SEQ ID NO:5 or 6, respectively, have
been synthesized by state of the art oligonucleotide synthesis
methods. The introduction of the maleinimido group was done via
reaction of the amino group of Y with the succinimidyl group of Z
which was incorporated during the solid phase oligonucleotide
synthesis process.
[0199] The single-stranded DNA constructs shown above bear a
thiol-reactive maleimido group that reacts with a cysteine of the
Fab' hinge region generated by the cysteamine treatment. In order
to obtain a high percentage of single-labeled Fab'-fragments the
relative molar ratio of ssDNA to Fab'-fragment is kept low.
Purification of single-labeled Fab'-fragments (ssDNA:Fab'=1:1)
occurs via anion exchange chromatography (column: MonoQ, GE
Healthcare). Verification of efficient labeling and purification is
achieved by analytical gel filtration chromatography and
SDS-PAGE.
[0200] 1.3 Biotinylated Linker Molecules
[0201] The oligonucleotides used in the ssDNA linkers L1, L2 and
L3, respectively, have been synthesized by state of the art
oligonucleotide synthesis methods and employing a biotinylated
phosphoramidite reagent for biotinylation.
[0202] Linker 1 (=L1), a biotinylated ssDNA linker 1 with no spacer
except biotinylated thymidine has the following composition: 5-GCA
GAA GCA TTA ATA GAC T (Biotin-dT)-TGG ACG ACG ATA GAA CT-3'. It
comprises ssDNA oligonucleotides of SEQ ID NO:7 and 8,
respectively, and was biotinylated by using Biotin-dT (=T-Bi)
(5'-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-a-
crylimido]-2'-deoxyUridine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphora-
midite (Glen Research) in the middle of the spacer.
[0203] Linker 2 (=L2), a biotinylated ssDNA linker 2 with a 11 mer
spacer has the following composition: 5-GCA GAA GCA TTA ATA GAC T
T5-(Biotin-dT)-T5 TGG ACG ACG ATA GAA CT-3'. It comprises ssDNA
oligonucleotides of SEQ ID NO:7 and 8, respectively, twice
oligonucleotide stretches of five thymidines each and was
biotinylated by using Biotin-dT
(5'-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-a-
crylimido]-2'-deoxyUridine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphora-
midite (Glen Research) in the middle of the spacer.
[0204] Linker 3 (=L3), a biotinylated ssDNA linker 3 with a 31 mer
spacer has the following composition: 5-GCA GAA GCA TTA ATA GAC T
T15-(Biotin-dT)-T15 TGG ACG ACG ATA GAA CT-3'. It comprises ssDNA
oligonucleotides of SEQ ID NO:7 and 8, respectively, twice
oligonucleotide stretches of fifteen thymidines each and was
biotinylated by using Biotin-dT
(5'-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-a-
crylimido]-2'-deoxyUridine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphora-
midite (Glen Research).
[0205] 1.4 Epitopes for Monovalent Troponin T Binders A and B,
Respectively
[0206] Synthetic peptides have been construed that individually
only have a moderate affinity to the corresponding Fab'-fragment
derived from the anti-Troponin T antibodies a and b,
respectively.
[0207] a) The Epitope a' for Antibody a is Comprised in:
SEQ ID NO:9=ERAEQQRIRAEREKEUUSLKDRIEKRRRAERAEamide, wherein U
represents .beta.-Alanin.
[0208] b) The Epitope B' for Antibody b is Comprised in:
SEQ ID NO:10=SLKDRIERRRAERAEOOERAEQQRIRAEREKEamide, wherein O
represents Amino-trioxa-octanoic-acid
[0209] As the skilled artisan will appreciate it is possible to
combine these two epitope-containing peptides in two ways and both
variants have been designed and prepared by linear combining the
epitopes A' and B'. The sequences of both variants, the linear
sequences of epitopes A'-B' (=TnT-1) and B'-A' (=TnT-2),
respectively have been prepared by state of the art peptide
synthesis methods.
[0210] The sequences for epitopes A' and B', respectively, had been
modified compared to the original epitopes on the human cardiac
Troponin T sequence (P45379/UniProtKB) in order to reduce the
binding affinity for each of the Fabs thereto. Under these
circumstances the dynamics of the effect of hetero-bi-valent
binding is better visible, e.g. by analyzing binding affinity with
the Biacore.TM. Technology.
[0211] 1.5 Biomolecular Interaction Analysis
[0212] For this experiment a Biacore.TM. 3000 instrument (GE
Healthcare) was used with a Biacore.TM. SA sensor mounted into the
system at T=25.degree. C. Preconditioning was done at 100 .mu.l/min
with 3.times.1 min injection of 1 M NaCl in 50 mM NaOH and 1 min 10
mM HCl.
[0213] HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05%
Tween.RTM. 20 was used as system buffer. The sample buffer was
identical to the system buffer.
[0214] The Biacore.TM. 3000 System was driven under the control
software V1.1.1. Flow cell 1 was saturated with 7 RU D-biotin. On
flow cell 2, 1063 RU biotinylated ssDNA linker L1 was immobilized.
On flow cell 3, 879 RU biotinylated ssDNA linker L2 was
immobilized. On flow cell 4, 674 RU biotinylated ssDNA linker L3
was captured.
[0215] Thereafter, Fab' fragment DNA conjugate A'' was injected at
600 nM. Fab' fragment DNA conjugate B'' was injected into the
system at 900 nM. The conjugates were injected for 3 min at a flow
rate of 2 .mu.l/min. The conjugates were consecutively injected to
monitor the respective saturation signal of each Fab' fragment DNA
conjugate on its respective linker. Fab' combinations were driven
with a single Fab' fragment DNA conjugate A'', a single Fab'
fragment DNA conjugate B'' and both Fab' fragment DNA conjugates
A'' and B'' present on the respective linker. Stable baselines were
generated after the linkers have been saturated by the Fab'
fragment DNA conjugates, which was a prerequisite for further
kinetic measurements.
[0216] The artificial peptidic analytes TnT-1 and TnT-2 were
injected as analytes in solution into the system in order to
interact with the surface presented Fab' fragments.
[0217] TnT-1 was injected at 500 nM, TnT-2 was injected at 900 nM
analyte concentration. Both peptides were injected at 50 .mu.l/min
for 4 min association time. The dissociation was monitored for 5
min. Regeneration was done by a 1 min injection at 50 .mu.l/min of
50 mM NaOH over all flow cells.
[0218] Kinetic data was determined using the Biaevaluation software
(V.4.1). The dissociation rate kd (1/s) of the TnT-1 and TnT-2
peptides from the respective surface presented Fab' fragment
combinations was determined according to a linear Langmuir 1:1
fitting model. The complex halftime in min were calculated
according to the solution of the first order kinetic equation:
In(2)/(60*kd).
[0219] Results:
[0220] The experimental data given in Tables 1 and 2, respectively
demonstrate an increase in complex stability between analyte (TnT-1
or TnT-2), respectively, and the various heterobi-valent Fab'-Fab'
dimers A''-B'' as compared to the monovalent dsDNA Fab' A'' or B''
conjugate, respectively. This effect is seen in each Table in line
1 compared to lines 2 and 3.
TABLE-US-00005 TABLE 1 Analysis data using TnT-1 with linkers of
various length Fab' fragment Fab' fragment kd t1/2 diss DNA
conjugate A'' DNA conjugate B'' (1/s) (min) a) Linker L1 x x
6.6E-03 1.7 x -- 3.2E-02 0.4 -- x 1.2E-01 0.1 b) Linker L2 x x
4.85E-03 2.4 x -- 2.8E-02 0.4 -- x 1.3E-01 0.1 c) linker L3 Fab'
fragment Fab' fragment kd t1/2 diss DNA conjugate A'' DNA conjugate
B'' (/1/s) (min) x x 2.0E-03 5.7 x -- 1.57E-02 0.7 -- x 1.56E-02
0.7
TABLE-US-00006 TABLE 2 Analysis data using TnT-2 with linkers of
various length Fab' fragment Fab' fragment kd t1/2 diss DNA
conjugate A'' DNA conjugate B'' (/1/s) (min) a) Linker L1 x x
1.4E-02 0.8 x -- 4.3E-02 0.3 -- x 1.4E-01 0.1 b) Linker L2 x x
4.9E-03 2.3 x -- 3.5E-02 0.3 -- x 1.3E-01 0.1 c) Linker L3 x x
8.0E-03 1.5 x -- 4.9E-02 0.2 -- x 3.2E-01 0.04
[0221] The avidity effect is further dependent on the length of the
linker. In the sub-tables shown under Table 1, i.e. for the
artificial analyte TnT-1, the linker L3 comprising a 31 mer
thymidine-based spacer shows the lowest dissociation rate or
highest complex stability.
[0222] In the sub-tables shown under Table 2 the linker L2
comprising an 11 mer thymidine-based spacer exhibits the lowest
dissociation rate or highest complex stability for the artificial
analyte TnT-2.
[0223] These data taken together demonstrate that the flexibility
in linker length as inherent to the approach given in the present
disclosure is of great utility and advantage.
Example 2
Bi-Valent Binding Agent to Phosphorylated IGF-1R
[0224] 2.1 Monoclonal Antibody Development (mAb 8.1.2 and mAb
1.4.168)
[0225] a) Immunization of Mice
[0226] BALB/C mice are immunized at week 0, 3, 6 and 9,
respectively. Per immunization 100 .mu.g of the conjugate
comprising the phosphorylated peptide pIGF-1R (1340-1366) (SEQ ID
NO:11) is used. This peptide had been phosphorylated at tyrosine
1346 (=1346-pTyr) and coupled to KLH via the C-terminal cysteine
(=Aoc-Cys-MP-KLH-1340) to yield the conjugate used for
immunization. At weeks 0 and 6, respectively, the immunization is
carried out intraperitoneally and at weeks 3 and 9, respectively,
subcutaneously at various parts of the mouse body.
[0227] b) Fusion and Cloning
[0228] Spleen cells of immunized mice are fused with myeloma cells
according to Galfre G., and Milstein C., Methods in Enzymology 73
(1981) 3-46. In this process ca 1.times.10.sup.8 spleen cells of an
immunized mouse are mixed with 2.times.10.sup.7 myeloma cells
a(P3.times.63-Ag8653, ATCC CRL1580) and centrifuged (10 min at 250
g and 37.degree. C.). The cells are then washed once with RPMI 1640
medium without fetal calf serum (FCS) and centrifuged again at 250
g in a 50 ml conical tube. The supernatant is discarded, the cell
sediment is gently loosened by tapping, 1 ml PEG (molecular weight
4000, Merck, Darmstadt) is added and mixed by pipetting. After 1
min incubation in a water bath at 37.degree. C., 5 ml RPMI 1640
without FCS is added drop-wise at room temperature within a period
of 4-5 min. This step is repeated with additional 10 ml RPMI 1640
without FCS. Afterwards 25 ml RPMI 1640 containing 10% FCS is added
followed by an incubation step at 37.degree. C., 5% CO.sub.2 for 30
minutes. After centrifugation for 10 min at 250 g and 4.degree. C.
the sedimented cells are taken up in RPMI 1640 medium containing
10% FCS and seeded out in hypoxanthine-azaserine selection medium
(100 mmol/l hypoxanthine, 1 pg/ml azaserine in RPMI 1640+10% FCS).
Interleukin 6 at 100 U/ml is added to the medium as a growth
factor. After 7 days the medium is exchanged with fresh medium. On
day 10, the primary cultures are tested for specific antibodies.
Positive primary cultures are cloned in 96-well cell culture plates
by means of a fluorescence activated cell sorter.
[0229] c) Immunoglobulin Isolation from the Cell Culture
Supernatants
[0230] The hybridoma cells obtained are seeded out at a density of
1.times.10.sup.7 cells in CELLine 1000 CL flasks (Integra).
Hybridoma cell supernatants containing IgGs are collected twice a
week. Yields typically range between 400 .mu.g and 2000 .mu.g of
monoclonal antibody per 1 ml supernatant. Purification of the
antibody from culture supernatant was carried out using
conventional methods of protein chemistry (e.g. according to Bruck,
C., Methods in Enzymology 121 (1986) 587-695).
[0231] 2.2 Synthesis of Hybridizable Oligonucleotides
[0232] The following amino modified precursors, comprising the
sequences given in SEQ ID NOs: 5 and 6, respectively, were
synthesized according to standard methods. The below given
oligonucleotides not only comprise the so-called aminolinker, but
also a fluorescent dye. As the skilled artisan will readily
appreciate, this fluorescent dye is very convenient to facilitate
purification of the oligonucleotide as such, as well as of
components comprising them.
a) 5'-Fluorescein-AGT CTA TTA ATG CTT CTG C-(Spacer
C3)3-C7-Aminolinker-;
b) 5-Cy5 AGT CTA TTA ATG CTT CTG C-(Spacer
C3)3-C7-Aminolinker-;
c) 5'-Aminolinker-(Spacer C3)3-AGT TCT ATC GTC GTC
CA-Fluorescein-3';
[0233] d) 5'-Fluorescein-(beta L AGT CTA TTA ATG CTT CTG C)-(Spacer
C3)3-C7-Aminolinker-; (beta L indicates that this is an L-DNA
oligonucleotide) and e) 5'-Aminolinker-(Spacer C3)3-(beta L-AGT TCT
ATC GTC GTC CA)-Fluorescein-3' (beta L indicates that this is an
L-DNA oligonucleotide).
[0234] Synthesis was performed on an ABI 394 synthesizer at a 10
.mu.mol scale in the trityl on (for 5' amino modification) or
trityl off mode (for 3' amino modification) using commercially
available CPGs as solid supports and standard dA(bz), dT, dG (iBu)
and dC(Bz) phosphoramidites (Sigma Aldrich).
[0235] The following amidites, amino modifiers and CPG supports
were used to introduce the C3-spacer, a dye and amino moieties,
respectively, during oligonucleotide synthesis:
[0236] Spacer Phosphoramidite C3
(3-(4,4'-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-p-
hosphoramidite (Glen Research);
[0237] 5' amino modifier is introduced by using 5'-Amino-Modifier
C6
(6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosp-
horamidite (Glen Research);
[0238] 5'-Fluorescein Phosphoramidite
6-(3',6'-dipivaloylfluoresceinyl-6-carboxamido)-hexyl-1-O-(2-cyanoethyl)--
(N,N-diisopropyl)-phosphoramidite (Glen Research);
[0239] Cy5.TM. Phosphoramidite
1-[3-(4-monomethoxytrityloxy)propyl]-1'-[3-[(2-cyanoethyl)-(N,N-diisoprop-
yl phosphoramidityl]propyl]-3,3,3',3'-tetramethylindodicarbocyanine
chloride (Glen Research);
[0240] LightCycler Fluoresceine CPG 500 A (Roche Applied Science);
and
[0241] 3'-Amino Modifier TFA Amino C-6 Icaa CPG 500 A
(Chemgenes),
[0242] For Cy5 labeled oligonucleotides, dA(tac), dT, dG(tac)
dC(tac) phosphoramidites, (Sigma Aldrich), were used and
deprotection with 33% ammonia was performed for 2 h at room
temperature.
[0243] L-DNA oligonucleotides were synthesized by using
beta-L-dA(bz), dT, dG (iBu) and dC(Bz) phosphoramidites
(Chemgenes)
[0244] Purification of fluorescein modified hybridizable
oligonucleotides was performed by a two step procedure: First the
oligonucleotides were purified on reversed-phase HPLC
(Merck-Hitachi-HPLC; RP-18 column; gradient system [A: 0.1 M
(Et3NH)OAc (pH 7.0)/MeCN 95:5; B: MeCN]: 3 min, 20% B in A, 12 min,
20-50% B in A and 25 min, 20% B in A with a flow rate of 1.0
ml/min, detection at 260 nm. The fractions (monitored by analytical
RP HPLC) containing the desired product were combined and
evaporated to dryness. (Oligonucleotides modified at the 5' end
with monomethoxytrityl protected alkylamino group are detriylated
by incubating with 20% acetic acid for 20 min). The oligomers
containing fluorescein as label were purified again by IEX
chromatography on a HPLC [Mono Q column: Buffer A: Sodium hydroxide
(10 mM/I; pH .about.12) Buffer B 1M Sodium chloride dissolved in
Sodium hydroxide (10 mM/I; pH .about.12) gradient: in 30 minutes
from 100% buffer A to 100% buffer B flow 1 ml/min detection at 260
nm]. The product was desalted via dialysis.
[0245] Cy5 labeled oligomers were used after the first purification
on reversed-phase HPLC (Merck-Hitachi-HPLC; RP-18 column; gradient
system [A: 0.1 M (Et3NH)OAc (pH 7.0)/MeCN 95:5; B: MeCN]: 3 min,
20% B in A, 12 min, 20-50% B in A and 25 min, 20% B in A with a
flow rate of 1.0 ml/min, detection at 260 nm. The oligomers were
desalted by dialysis and lyophilized on a Speed-Vac evaporator to
yield solids which were frozen at -24.degree. C.
[0246] 2.3 Activation of Hybridizable Oligonucleotides
[0247] The amino modified oligonucleotides from Example 2 were
dissolved in 0.1 M sodium borate buffer pH 8.5 buffer (c=600
.mu.mol) and reacted with a 18-fold molar excess of Sulfo SMCC
(Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate
dissolved in DMF (c=3 mg/100 .mu.l) from Thermo Scientific, The
reaction product was thoroughly dialyzed against water in order to
remove the hydrolysis product of sulfoSMCC
4-[N-maleimidomethyl]cyclohexane-1-carboxylate.
[0248] The dialysate was concentrated by evaporation and directly
used for conjugation with a monovalent binder comprising a thiol
group.
[0249] 2.4 Synthesis of Linker Oligonucleotides Comprising
Hybridizable Oligonucleotides at Both Ends
[0250] Oligonucleotides were synthesized by standard methods on an
ABI 394 synthesizer at a 10 .mu.mol scale in the trityl on mode
using commercially available dT-CPG as solid supports and using
standard dA(bz), dT, dG (iBu) and dC(Bz) phosphoramidites (Sigma
Aldrich).
[0251] L-DNA oligonucleotides were synthesized by using
commercially available beta L-dT-CPG as solid support and
beta-L-dA(bz), dT, dG (iBu) and dC(Bz) phosphoramidites
(Chemgenes)
[0252] Purification of the oligonucleotides was performed as
described under Example 3 on a reversed-phase HPLC. The fractions
(analyzed/monitored by analytical RP HPLC) containing the desired
product were combined and evaporated to dryness. Detriylation was
performed by incubating with 80% acetic acid for 15 min) The acetic
acid was removed by evaporation. The reminder was dissolved in
water and lyophilized
[0253] The following amidites and CPG supports were used to
introduce the C18 spacer, digoxigenin and biotin group during
oligonucleotide synthesis:
[0254] Spacer Phosphoramidite 18
(18-O-Dimethoxytritylhexaethyleneglycol,
1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen
Research);
[0255] Biotin-dT
(5'-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-a-
crylimido]-2'-deoxyUridine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphora-
midite (Glen Research);
[0256] Biotin
Phosphoramidite1-Dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3--
O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite and
[0257]
5'-Dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2-
'-deoxy uridine,
3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite for amino
modification and postlabeling with
Digoxigenin-N-Hydroxyl-succininimidyl ester.
[0258] The following bridging constructs or linkers were
synthesized:
TABLE-US-00007 Linker 1: 5'-G CAG AAG CAT TAA TAG ACT-TGG ACG ACG
ATA GAA CT-3' Linker 2: 5'-G CAG AAG CAT TAA TAG ACT-(T40)-TGG ACG
ACG ATA GAA CT-3' Linker 3: 5'-[B-L]G CAG AAG CAT TAA TAG
ACT-(Biotin-dT)-TGG ACG ACG ATA GAA CT-3' Linker 4: 5'-[B-L]G CAG
AAG CAT TAA TAG ACT-T5-(Biotin-dT)-T5-TGG ACG ACG ATA GAA CT-3'
Linker 5: 5'-[B-L]G CAG AAG CAT TAA TAG ACT-T20-(Biotin-dT)-T20-TGG
ACG ACG ATA GAA CT-3' Linker 6: 5'-[B-L] G CAG AAG CAT TAA TAG
ACT-T30-(Biotin-dT)-T30-TGG ACG ACG ATA GAA CT-3' Linker 7: 5'-GCA
GAA GCA TTA ATA GAC T T5-(Biotin-dT)-T5 TG GAC GAC GAT AGA ACT-3'
Linker 8: 5'-GCA GAA GCA TTA ATA GAC T T10-(Biotin-dT)-T10 TGG ACG
ACG ATA GAA CT-3' Linker 9: 5'-GCA GAA GCA TTA ATA GAC T
T15-(Biotin-dT)-T15 TGG ACG ACG ATA GAA CT-3' Linker 10: 5'-GCA GAA
GCA TTA ATA GAC T T20-(Biotin-dT)-T20 TGG ACG ACG ATA GAA CT-3'
Linker 11: 5'-G CAG AAG CAT TAA TAG ACT-Spacer C18-
(Biotin-dT)-Spacer C18- TGG ACG ACG ATA GAA CT-3' Linker 12: 5'-G
CAG AAG CAT TAA TAG ACT-(Spacer C18)2-(Biotin-dT)-(Spacer C18)2-TGG
ACG ACG ATA GAA CT-3' Linker 13: 5'-G CAG AAG CAT TAA TAG
ACT-(Spacer C18)3-(Biotin-dT)-(Spacer C18)3-TGG ACG ACG ATA GAA
CT-3' Linker 14: 5'-G CAG AAG CAT TAA TAG ACT-(Spacer
C18)4-(Biotin-dT)-(Spacer C18)4-TGG ACG ACG ATA GAA CT-3' Linker
15: 5'-G CAG AAG CAT TAA TAG ACT-T20-(Dig-dT)-T20-TGG ACG ACG ATA
GAA CT-3' Linker 16: 5'-G CAG AAG CAT TAA TAG ACT-(Dig-dT)-TGG ACG
ACG ATA GAA CT- 3' Linker 17: 5'-G CAG AAG CAT TAA TAG
ACT-(Biotin-dT)-TGG ACG ACG ATA GAA CT-3'
[0259] The above bridging construct examples comprise at least a
first hybridizable oligonucleotide and a second hybridizable
oligonucleotide. Linkers 3 to 17 in addition to the hybridizable
nucleic acid stretches comprise a central biotinylated or
digoxigenylated thymidine, respectively, or a spacer consisting of
thymidine units of the length given above.
[0260] The 5'-hybridizable oligonucleotide corresponds to SEQ ID
NO:7 and the 3'-hybridizable oligonucleotide corresponds to SEQ ID
NO:8, respectively. The oligonucleotide of SEQ ID NO:7 will readily
hybridize with the oligonucleotide of SED ID NO:5. The
oligonucleotide of SEQ ID NO:8 will readily hybridize with the
oligonucleotide of SED ID NO:6.
[0261] In the above bridging construct examples [B-L] indicates
that an L-DNA oligonucleotide sequence is given; spacer C 18,
Biotin and Biotin dT respectively, refer to the C18 spacer, the
Biotin and the Biotin-dT as derived from the above given building
blocks; and T with a number indicates the number of thymidine
residues incorporated into the linker at the position given.
[0262] 2.5 Assembly of Dual Binder Construct
[0263] A) Cleavage of IgGs and Labeling of Fab' Fragments with
ssDNA
[0264] Purified monoclonal antibodies were cleaved with the help of
pepsin protease yielding F(ab')2 fragments that are subsequently
reduced to Fab' fragments by treatment with low concentrations of
cysteamine at 37.degree. C. The reaction is stopped via separation
of cysteamine on a PD 10 column. The Fab' fragments are labeled
with an activated oligonucleotide as produced according to Example
3. This single-stranded DNA (=ssDNA) bears a thiol-reactive
maleimido group that reacts with the cysteines of the Fab' hinge
region. In order to obtain high percentages of single-labeled Fab'
fragments the relative molar ratio of ssDNA to Fab'-fragment is
kept low. Purification of single-labeled Fab' fragments (ssDNA:
Fab'=1:1) occurs via ion exchange chromatography (column: Source 15
Q PE 4.6/100, Pharmacia/GE). Verification of efficient purification
is achieved by analytical gel filtration and SDS-PAGE.
[0265] B) Assembly of an Anti-pIGF-1R Dual Binder. The anti-pIGF-1R
dual binder is based on two Fab' fragments that target different
epitopes of the intracellular domain of IGF-1R: Fab' 8.1.2 detects
a phosphorylation site (pTyr 1346) and Fab' 1.4.168 a non-phospho
site of the said target protein. The Fab' fragments have been
covalently linked to single-stranded DNA (ssDNA): Fab' 1.4.168 to a
17mer ssDNA comprising SEQ ID NO:6 and containing fluorescein as an
fluorescent marker and Fab' 8.1.2 to a 19mer ssDNA comprising SEQ
ID NO:5 and containing Cy5 as fluorescent marker. In the following,
these Fab's with covalently bound 17mer or 19mer ssDNA are named
ssFab' 1.4.168 and ssFab' 8.1.2 respectively. Dual binder assembly
is mediated by a linker (i.e. a bridging construct comprising two
complementary ssDNA oligonucleotides (SEQ ID NOs: 7 and 8,
respectively) that hybridize to the corresponding ssDNAs of the
ssFab' fragments. The distance between the two ssFab' fragments of
the dual binder can be modified by using spacers, e.g. C18-spacer
or DNAs of different length, respectively.
[0266] For assembly evaluation the dual binder components ssFab'
8.1.2, ssFab' 1.4.168 and the linker constructs (I) (=linker 17 of
example 2.4) 5'-G CAG AAG CAT TAA TAG ACT T(-Bi)-TGG ACG ACG ATA
GAA CT-3' and (II) (=linker 10 of example 2.4) 5'-G CAG AAG CAT TAA
TAG ACT-(T20)-T(-Bi)-(T20)-TGG ACG ACG ATA GAA CT-3' were mixed in
equimolar quantities at room temperature. After a 1 minute
incubation step the reaction mix was analyzed on an analytical gel
filtration column (Superdex.TM. 200, 10/300 GL, GE Healthcare).
Comparison of the elution volumes (V.sub.E) of the single dual
binder components with the V.sub.E of the reaction mix demonstrates
that the dual binder has been formed successfully (FIG. 1). (The
biotinylated thymidine (T-(Bi)) in the middle of both of the
linkers is without function in these experiments.)
[0267] 2.6 Biacore.TM. Experiment Assessing Binding of Anti-pIGF-1R
Dual Binder to Immobilized IGF-1R and IR Peptides
[0268] For this experiment a Biacore.TM. 2000 instrument (GE
Healthcare) was used with a Biacore.TM. SA sensor mounted into the
system at T=25.degree. C. Preconditioning occurred at 100 .mu.l/min
with 3.times.1 min injection of 1 M NaCl in 50 mM NaOH and 1 min 10
mM HCl.
[0269] HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05%
Tween.RTM. 20 was used as system buffer. The sample buffer was
identical with the system buffer. The Biacore.TM. 2000 System was
driven under the control software V1.1.1.
[0270] Subsequently biotinylated peptides were captured on the SA
surface in the respective flow cells. 16 RU of
IGF-1R(1340-1366)[1346-pTyr; Glu(Bi-PEG-1340]amid (i.e. the -1346
tyrosine phosphorylated-peptide of SEQ ID NO:11 comprising a
PEG-linker bound via glutamic acid corresponding to position 1340
and being biotinylated at the other end of the linker) was captured
on flow cell 2. 18 RU of IGF-1R(1340-1366); Glu(Bi-PEG-1340]amid
(i.e. the -1346 tyrosine non-phosphorylated-peptide of SEQ ID NO:11
comprising a PEG-linker bound via glutamic acid corresponding to
position 1340 and being biotinylated at the other end of the
linker) was captured on flow cell 3. RU of
hIR(1355-1382)[1361-pTyr; Glu(Bi-PEG-1355]amid (i.e. the -1361
tyrosine phosphorylated-peptide of SEQ ID NO:12 comprising a
PEG-linker bound via glutamic acid corresponding to position 1355
of human insulin receptor and being biotinylated at the other end
of the linker) was captured on flow cell 4. Finally all flow cells
were saturated with d-biotin.
[0271] For the Dual Binder formation the assembly protocol as
described in Example 2.5 was used. When individual runs with only
one of the two ssFab's were performed, the absence or presence of
linker DNA did not affect the association or dissociation curves
(data not shown).
[0272] 100 nM of analyte (i.e. in these experiments a bi-valent
dual binding agent) in solution was injected at 50 .mu.l/min for
240 sec association time and dissociation was monitored for 500
sec. Efficient regeneration was achieved by using a 1 min injection
step at 50 .mu.l/min with 80 mM NaOH. Flow cell 1 served as a
reference. A blank buffer injection was used instead of an antigen
injection to double reference the data by buffer signal
subtraction.
[0273] In each measurement cycle one of the following analytes in
solution was injected over all 4 flow cells: 100 nM ssFab' 8.1.2,
100 nM ssFab' 1.4.168, a mixture of 100 nM ssFab' 8.1.2 and 100 nM
ssFab', 100 nM bi-valent binding agent consisting of ssFab' 8.1.2
and ssFab' 1.4.168 hybridized on linker (III) (5'-G CAG AAG CAT TAA
TAG ACT-T(20)-T(-Dig)-(T20)-TGG ACG ACG ATA GAA CT-3'(=linker 15 of
example 2.4)), and 100 nM bi-valent binding agent consisting of
ssFab' 8.1.2 and ssFab' 1.4.168 hybridized on linker (IV) (5'-G CAG
AAG CAT TAA TAG ACT-T(-Dig)-TGG ACG ACG ATA GAA CT-3'(=linker 16 of
example 2.4)), respectively. (The digoxigenylation of the middle
thymidine (T(-Dig)) in the above linkers is without relevance to
these experiments.)
[0274] The signals were monitored as time-dependent BIAcore.TM.
sensorgrams.
[0275] Report points were set at the end of the analyte association
phase (Binding Late, BL) and at the end of the analyte dissociation
phase (Stability Late, SL) to monitor the response unit signal
heights of each interaction. The dissociation rates kd (1/s) were
calculated according to a linear 1:1 Langmuir fit using the
Biacore.TM. evaluation software 4.1. The complex halftimes in
minutes were calculated upon the formula ln(2)/(60*kd).
[0276] The sensorgrams (FIG. 2-5) show a gain in both specificity
and complex stability in pIGF-1R binding when ssFab' 1.4.168 and
ssFab' 1.4.168 are used in form of a dual binder (=bi-valent
binding agent), probably due to the underlying cooperative binding
effect. Fab' 1.4.168 alone shows no cross reactivity for the pIR
peptide but does not discriminate between the phosphorylated and
unphosphorylated form of IGF-1R (T1/2 dis=3 min in both cases).
Fab' 8.1.2, however, binds only to the phosphorylated version of
the IGF1-R peptide but exhibits some undesired cross reactivity
with phosphorylated Insulin Receptor. The Dual Binder discriminates
well between the pIGF-1R peptide and both other peptides (see FIG.
4) and thus helps to overcome issues of unspecific binding. Note
that the gain in specificity is lost when both Fab's are applied
without linker DNA (FIG. 5). The gain in affinity of the Dual
Binder towards the pIGF-1R peptide manifests in increased
dissociation half times compared to individual Fab's and the Fab'
mix omitting the linker DNA (FIG. 3 and FIG. 5). Although the
tested Dual Binders with two different DNA linker lengths share an
overall positive effect on target binding specificity and affinity,
the longer linker ((III) with T40-T-Dig as a spacer) (i.e. linker
15 of example 2.4) seems to be advantageous with respect to both
criteria.
[0277] 2.7 Biacore.TM. Assay Sandwich of M-1.4.168-IgG and
M-8.1.2-IgG
[0278] A Biacore.TM. T100 instrument (GE Healthcare) was used with
a Biacore.TM. CM5 sensor mounted into the system. The sensor was
preconditioned by a 1 min injection at 100 .mu.l/min of 0.1% SDS,
50 mM NaOH, 10 mM HCl and 100 mM H3PO4.
[0279] The system buffer was HBS-ET (10 mM HEPES pH 7.4, 150 mM
NaCl, 1 mM EDTA, 0.05% Tween.RTM. 20). The sample buffer was the
system buffer.
[0280] The Biacore.TM. T100 System was driven under the control
software V1.1.1. Polyclonal rabbit IgG antibody
<IgGFC.gamma.M>R (Jackson ImmunoResearch Laboratories Inc.)
at 30 pg/ml in 10 mM Na-Acetate pH 4.5 was immobilized at 10 000 RU
on the flow cells 1, 2, 3, and 4, respectively, via EDC/NHS
chemistry according to the manufacturer's instructions. Finally,
the sensor surface was blocked with 1M ethanolamine. The complete
experiment was driven at 13.degree. C.
[0281] 500 nM primary mAb M-1.004.168-IgG was captured for 1 min at
10 .mu.l/min on the <IgGFC.gamma.M>R surface. 3 .mu.M of an
IgG fragment mixture (of IgG classes IgG1, IgG2a, IgG2b, IgG3)
containing blocking solution was injected at 30 .mu.l/min for 5
min. The peptide IGF-1R(1340-1366)[1346-pTyr; Glu(Bi-PEG-1340]amid
was injected at 300 nM for 3 min at 30 .mu.l/min. 300 nM secondary
antibody M-8.1.2-IgG was injected at 30 .mu.l min. The sensor was
regenerated using 10 mM Glycine-HCl pH 1.7 at 50 .mu.l/min for 3
min.
[0282] FIG. 6 describes the assay setup. In FIG. 7. the measurement
results are given. The measurements clearly indicate, that both
monoclonal antibodies are able to simultaneously bind two distinct,
unrelated epitopes on their respective target peptide. This is a
prerequisite to any latter experiments with the goal to generate
cooperative binding events.
[0283] 2.8 Biacore.TM. Assay Dual Binder on Sensor Surface
[0284] A Biacore.TM. 3000 instrument (GE Healthcare) was used with
a Biacore.TM. SA sensor mounted into the system at T=25.degree. C.
The system was preconditioned at 100 .mu.l/min with 3.times.1 min
injection of 1 M NaCl in 50 mM NaOH and 1 min 10 mM HCl.
[0285] The system buffer was HBS-ET (10 mM HEPES pH 7.4, 150 mM
NaCl, 1 mM EDTA, 0.05% Tween.RTM. 20). The sample buffer was the
system buffer.
[0286] The Biacore.TM. 3000 System was driven under the control
software V4.1.
[0287] 124 RU amino-PEO-biotin were captured on the reference flow
cell 1. 1595 RU biotinylated 14.6 kDa T0-Bi 37-mer ssDNA-Linker (I)
(5'-G CAG AAG CAT TAA TAG ACT-T(-Bi)-TGG ACG ACG ATA GAA CT-3')
(=linker 17 of example 2.4) and 1042 RU biotinylated 23.7 kDa
T40-Bi 77-mer ssDNA-Linker (II) (5'-G CAG AAG CAT TAA TAG
ACT-T(20)-(Biotin-dT)-(T20)-TGG ACG ACG ATA GAA CT-3'=linker 10 of
example 2.4) were captured on different flow cells.
[0288] 300 nM ssFab' 8.1.2 and 300 nM ssFab' 1.004.168 were
injected into the system at 50 .mu.l/min for 3 min. As a control
only 300 nM ssFab' 8.1.2 or 300 nM ssFab' 1.004.168 was injected to
test the kinetic contribution of each ssFab. As a control, buffer
was injected instead of the ssFabs. The peptides
pIR(1355-1382)[1361-pTyr]amid and IGF-1R(1340-1366)amid,
respectively, were injected into system at 50 .mu.l/min for 4 min,
free in solution, in concentration steps of 0 nM, 4 nM, 11 nM, 33
nM (twice), 100 nM and 300 nM. In another set of experiments to
measure the affinities versus the peptide
pIGF-1R(1340-1366)[1346-pTyr]amid the concentration steps of 0 nM,
0.4 nM, 1.1 nM, 3.3 nM (twice), 10 nM and 30 nM were used.
[0289] The dissociation was monitored at 50 .mu.l/min for 5.3 min.
The system was regenerated after each concentration step with a 12
sec pulse of 250 mM NaOH and was reloaded with ssFab' ligand.
[0290] FIG. 8 schematically describes the assay setup on the
Biacore.TM. instrument. The table given in FIG. 9 shows the
quantification results from this approach. FIGS. 10, 11 and 12
depict exemplary Biacore.TM. results from this assay setup using
the T40 dual binding agent.
[0291] The table in FIG. 9 demonstrates the benefits of the dual
binder concept. The T40 dual binding agent (a dual binding agent
with linker 10 of example 2.4, i.e. a linker with a spacer of
T20-Biotin-dT-T20) results in a 2-fold improved antigen complex
halftime (414 min) and a 3-fold improved affinity (10 .mu.M) as
compared to the TO dual binding agent (i.e. a dual binding agent
with linker 16 of example 2.4) with 192 min and 30 .mu.M,
respectively. This underlines the necessity to optimize the linker
length to generate the optimal cooperative binding effect.
[0292] The T40 dual binding agent (i.e. the dual binding agent
comprising the T40-Bi linker (linker 10 of example 2.4)) exhibits a
10 .mu.M affinity versus the phosphorylated IGF-1R peptide (table
in FIG. 9, FIG. 10). This is a 2400-fold affinity improvement
versus the phosphorylated insulin receptor peptide (24 nM) and a
100-fold improvement versus the non-phosphorylated IGF-1R
peptide.
[0293] Therefore, the goal to increase specificity and affinity by
the combination of two distinct and separated binding events is
achieved.
[0294] The cooperative binding effect especially becomes obvious
from the dissociation rates against the phosphorylated IGF-1R
peptide, where the dual binder shows 414 min antigen complex
halftime, versus 0.5 min with the monovalent binder 8.1.2 alone and
versus 3 min with the monovalent binder 1.4.168 alone,
respectively.
[0295] Furthermore, the fully assembled construct roughly
multiplies its dissociation rates kd (1/s), when compared to the
singly Fab' hybridized constructs (FIGS. 10, 11, 12 and table in
FIG. 9). Interestingly, also the association rate ka (1/Ms)
slightly increases when compared to the single Fab' interaction
events, this may be due to an increase of the construct's molecular
flexibility.
[0296] A diagnostic system using an intense washing procedure
should definitely foster the high performance of the T40 dual
binding agent, in contrast to individual (monovalent) Fab'
molecules. The hybridized construct, i.e. a bi-valent binding agent
according to the present disclosure, generates a specific and quite
stable binding event, while the monovalent binders more rapidly
dissociate, e.g. they are more rapidly washed away.
Example 3
Bi-Valent Binding Agent to HER2
[0297] 3.1 Assembly of an Anti-HER2 Bi-Valent Binding Agent
[0298] Two monoclonal antibodies binding to human HER2 (ErbB2 or
p185.sup.neu) at different, non-overlapping epitopes A and B were
used. The first antibody is anti-HER2 antibody 4D5 (huMAb4D5-8,
rhuMAb HER2, trastuzumab or HERCEPTIN.RTM.; see U.S. Pat. No.
5,821,337 incorporated herein by reference in its entirety).
[0299] The "4D5 epitope" is the region in the extracellular domain
of ErbB2 to which the anti-HER2 antibody 4D5 (ATCC CRL 10463)
binds. This epitope is close to the transmembrane domain of
ErbB2.
[0300] The second antibody is anti-HER2 antibody 2C4
(pertuzumab.RTM.). The antibody 2C4 and in particular the humanized
variants thereof are described in detail in WO 01/00245
incorporated herein by reference in its entirety. 2C4 is produced
by the hybridoma cell line deposited with the American Type Culture
Collection, Manassass, Va., USA under ATCC HB-12697. Examples of
humanized 2C4 antibodies are provided in Example 3 of WO 01/00245
(incorporated herein by reference in its entirety). The humanized
anti-HER2 antibody 2C4 is also called Pertuzumab.
[0301] Pertuzumab (formerly 2C4) is the first of a new class of
agents known as HER dimerization inhibitors (HDIs). Pertuzumab
binds to HER2 at its dimerization domain, thereby inhibiting its
ability to form active dimer receptor complexes and thus blocking
the downstream signal cascade that ultimately results in cell
growth and division (see Franklin, M. C., Cancer Cell 5 (2004)
317-328). Pertuzumab is a fully humanized recombinant monoclonal
antibody directed against the extracellular domain of HER2.
[0302] Purification of the monoclonal antibodies from culture
supernatant can be carried out using state of the art methods of
protein chemistry.
[0303] The purified monoclonal antibodies are protease digested
with either pre-activated papain or pepsin yielding F(ab').sub.2
fragments. These are subsequently reduced to Fab'-fragments with a
low concentration of cysteamin at 37.degree. C. The reaction is
stopped by separating the cysteamin on a Sephadex G-25 column (GE
Healthcare) from the polypeptide-containing part of the sample.
[0304] The obtained Fab'-fragments are conjugated with the
activated ssDNA polynucleotides.
[0305] a) Anti-HER2 Antibody 4D5 Fab'-ssDNA-Conjugate
[0306] For preparation of the anti-HER2 antibody 4D5
Fab'-ssDNA-conjugate a derivative of SED ID NO:5 is used, i.e.
5'-AGT CTA TTA ATG CTT CTG C(=SEQ ID NO:5)-XXX-Y-Z-3', wherein
X=propylene-phosphate introduced via phosphoramidite C3
(3-(4,4'-dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-p-
hosphoramidite (Glen Research), wherein Y=5'-amino-modifier C6
introduced via
(6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-p-
hosphoramidite (Glen Research), and wherein
Z=4[N-maleinimidomethyl]cyclohexane-1-carboxy introduced via
Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate
(ThermoFischer).
[0307] b) Anti-HER2 Antibody 2C4 Fab'-ssDNA-Conjugate
[0308] For the preparation of the anti-HER2 antibody 2C4
Fab'-ssDNA-conjugate B a derivative of SEQ ID NO:6 is used, i.e.
5'-Y-Z-XXX-AGT TCT ATC GTC GTC CA-3', wherein X=propylene-phosphate
introduced via Phosphoramidite C3
(3-(4,4'-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-p-
hosphoramidite (Glen Research), wherein Y=5'-Amino-Modifier C6
introduced via
(6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-p-
hosphoramidite (Glen Research), and wherein
Z=4[N-maleinimidomethyl]cyclohexane-1-carboxy introduced via
Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate
(ThermoFischer).
[0309] The polynucleotides of SEQ ID NO:5 or SEQ ID NO:6,
respectively, have been synthesized by state of the art
polynucleotide synthesis methods. The introduction of the
maleinimido group was done via reaction of the amino group of Y
with the succinimidyl group of Z which was incorporated during the
solid phase polynucleotide synthesis process.
[0310] The single-stranded DNA constructs bear a thiol-reactive
maleimido group that reacts with a cysteine of the Fab' hinge
region generated by the cysteamine treatment. In order to obtain a
high percentage of single-labeled Fab'-fragments the relative molar
ratio of ssDNA to Fab'-fragment is kept low. Purification of
single-labeled Fab'-fragments (ssDNA:Fab'=1:1) occurs via anion
exchange chromatography (column: MonoQ, GE Healthcare).
Verification of efficient labeling and purification is achieved by
analytical gel filtration chromatography and SDS-PAGE.
[0311] 3.2 Biomolecular Interaction Analysis
[0312] For this experiment a Biacore T100 instrument (GE
Healthcare) was used with a Biacore SA sensor mounted into the
system at T=25.degree. C. Preconditioning occurred at 100 .mu.l/min
with 3.times.1 min injection of 1 M NaCl in 50 mM NaOH, pH 8.0
followed by a 1 min injection of 10 mM HCl. The system buffer was
HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% P 20).
The sample buffer was the system buffer supplemented with 1 mg/ml
CMD (Carboxymethyldextrane).
[0313] Biotinylated ss-L-DNA linkers were captured on the SA
surface in the respective flow cells. Flow cell 1 was saturated
with amino-PEO-Biotin (PIERCE).
[0314] 40 RU of the biotinylated 37mer oligonucleotide linker
(linker 3 of example 2.4) were captured on flow cell 2. 55 RU of
the biotinylated 77mer oligonucleotide linker (linker 5 of example
2.4) were captured on flow cell 3. 60 RU of biotinylated 97mer
oligonucleotide linker (linker 6 of example 2.4) were captured on
flow cell 4.
[0315] 250 nM anti-HER2 antibody 4D5-Fab'-ss-L-DNA was injected
into the system for 3 min. 300 nM anti-HER2 antibody
2C4-Fab'-ss-L-DNA was injected into the system at 2 .mu.l/min for 5
min. The DNA-labeled Fab fragments were injected alone or in
combination.
[0316] As a control only 250 nM anti-HER2 antibody
4D5-Fab'-ss-D-DNA and 300 nM anti-HER2 antibody 2C4-Fab'-ss-D-DNA
was injected into the system. As a further control, buffer was
injected instead of the DNA-labeled Fab fragments. After
hybridization of the ss-L-DNA-labeled Fab fragments on the
respective ss-L-DNA bi-linkers, the analyte in solution hHER2-ECD
was injected at different concentration series from 24 nM, 8 nM, 3
nM, 1 nM, 0.3 nM, 0 nM into the system for 3.5 min association
phase at 100 .mu.l/min. The dissociation phase was monitored at 100
.mu.l/min for 15 min. The system was regenerated by a 30 sec
injection at 20 .mu.l/min of 100 mM gycine buffer (Glycine pH 11,
150 mM NaCl), followed by a second 1 min injection of water at 30
.mu.l/min.
[0317] The signals were measured as analyte
concentration-dependent, time resolved sensorgrams. The data was
evaluated using the Biacore Biaevaluation software 4.1. As a
fitting model a standard Langmuir binary binding model was
used.
[0318] Results:
[0319] No HER2-ECD interaction could be observed when ss-D-DNA
labeled Fab fragments were injected into the system, because the
ss-D-DNA-labeled Fab fragments did not hybridize with spiegelmeric
ss-L-DNA linkers presented on the sensor surface.
[0320] Table 3.: Kinetic results of the dual binder experiment.
Linker: Surface presented biotinylated ss-L-DNA polynucleotide
linker, Oligo.sub.--37mer-Bi, Oligo.sub.--77mer-Bi and
Oligo.sub.--97mer-Bi differing in linker length as described above.
ss-L-DNA-Fab: 2C4-ss-L-DNA: anti-HER2 antibody 2C4-Fab'-ss-L-DNA
labeled with 19mer-Fluorescein. 4D5-ss-L-DNA: anti-HER2 antibody
4D5-Fab'-ss-L-DNA labeled with 17mer-Fluorescein. 4D5-+2C4-ss-L-DNA
relates to the surface bound dual binding agent comprising the
combination of both monovalent anti-HER2-antibody fragments.
[0321] In Table 3 the following abbreviations are used: LRU: mass
in response units, which is hybridized on the sensor surface.
Antigen: a 87 kDa HER2-ECD was used as analyte in solution. ka:
association rate in (1/Ms). kd: dissociation rate in (1/s). t1/2
diss: antigen complex halftime calculated in hours according to the
solution In(2)/kd*3600 of a first order kinetic equation. KD:
affinity in molar. KD: affinity calculated in picomolar. Rmax:
Maximum analyte response signal at saturation in response units
(RU). MR: Molar Ratio, indicating the stoichiometry of the
interaction. Chi2, U-value: quality indicator of the
measurements.
TABLE-US-00008 TABLE 3 k.sub.a k.sub.d t'.sub.2-diss K.sub.D
R.sub.max Chi.sup.2 Linker ss-L-DNA-Fab LRU Antigen 1/Ms 1/s hours
K.sub.D M pM RU MR RU.sup.2 Oligo_35mer-Bi 4D5- + 2C4-ss-L-DNA 84
Her2-ECD 5.9E+05 6.7E-05 3 1.1E-10 100 59 0.9 0.2 Oligo_35mer-Bi
4D5-ss-L-DNA 16 Her2-ECD 4.0E+05 3.4E-05 6 8.5E-11 100 29 1.2 0.1
Oligo_35mer-Bi 2C4-ss-L-DNA 31 Her2-ECD 3.3E+05 3.6E-05 5 1.1E-10
100 26 0.6 0.03 Oligo_75mer-Bi 4D5- + 2C4-ss-L-DNA 87 Her2-ECD
5.1E+05 4.6E-08 4164 9.1E-14 0.1 65 1.0 0.1 Oligo_75mer-Bi
4D5-ss-L-DNA 16 Her2-ECD 2.9E+05 6.1E-05 3 2.1E-10 200 31 1.3 0.04
Oligo_75mer-Bi 2C4-ss-L-DNA 29 Her2-ECD 3.8E+05 6.3E-05 3 1.6E-10
200 32 0.7 0.03 Oligo_95mer-Bi 4D5- + 2C4-ss-L-DNA 76 Her2-ECD
5.0E+05 4.9E-08 3942 9.9E-14 0.1 58 1.0 0.1 Oligo_95mer-Bi
4D5-ss-L-DNA 14 Her2-ECD 3.0E+05 9.5E-05 2 3.1E-10 300 28 1.3 0.03
Oligo_95mer-Bi 2C4-ss-L-DNA 28 Her2-ECD 3.8E+05 6.8E-05 3 1.8E-10
300 27 0.6 0.03
[0322] In the above Table 35mer, 75mer and 95mer, respectively
should read 37mer, 77mer and 97mer, respectively.
[0323] The biacore data for the 37mer dual binder HER2-ECD
interaction (i.e. for a binder with a linker consisting solely the
hybridization sequences motives attached to the binders and a
central biotinylated thymidin) indicate that this dual binding
agent shows no improvement in kinetic performance. This is most
likely due to the insufficient linker length and the lack in
flexibility of the 37mer linker.
[0324] The biacore data for the 77mer dual binder HER2-ECD
interaction (i.e. for a binder with a linker comprising twice 20
thymidines a central biotinylated thymidin to increase the linker
length) indicate, that this dual binding agent shows a dramatic
improvement in its kinetic performance. This is most likely due to
an optimal linker length and the flexibility of this 77mer
linker.
[0325] The biacore data for the 97mer dual binder HER2-ECD
interaction (i.e. for a binder with a linker comprising twice 30
thymidines a central biotinylated thymidin to increase the linker
length) indicate, that this dual binding agent shows a dramatic
improvement in its kinetic performance. This is most likely due to
an optimal linker length and the flexibility of this 97mer
linker.
[0326] The data in Table 3 provide evidence for the presence of a
cooperative binding event. Despite the Rmax values of the fully
established dual binders are roughly double the signal height of
the singly Fab-armed constructs, the Molar Ratio values are exactly
1 (MR=1). This is a clear evidence for the presence of a
simultaneous, cooperative binding event of both Fab fragments. The
dual binder counts is a single molecule with a 1:1 Langmuir binding
stoichiometry. Despite having 2 independently binding HER2
interfaces no inter molecule binding between one dual binder and
two HER2 domains can be detected.
[0327] The avidity constants for synergizing pairs of monoclonal
antibodies or for a chemically cross-linked bispecific F(ab')2 is
generally only up to 15 times greater than the affinity constants
for the individual monoclonal antibodies, which is significantly
less than the theoretical avidity expected for ideal combination
between the reactants (Cheong, H. S., et al., Biochem. Biophys.
Res. Commun. 173 (1990) 795-800). Without being bound by this
theory one reason for this might be that the individual
epitope/paratope interactions involved in a synergistic binding
(resulting in a high avidity) must be orientated in a particular
way relative to each other for optimal synergy.
[0328] Furthermore, the data presented in Table 3 provides
evidence, that the short 37mer linker, which consists just from the
ss-L-DNA hybridization motives doesn't show enough flexibility
or/and linker length to produce the cooperative binding effect. The
37mer linker is a rigid, double helix L-DNA construct. The
hybridization generates a double L-DNA helix, which is shorter and
less flexible than the ss-L-DNA sequence. The helix shows reduced
degrees of freedom and can be seen as a rigid linker construct.
Table 3 shows, that the 37mer linker isn'table to generate a
cooperative binding event. The fully established 37mer dual binder
shows the same affinity like only the singly hybridized
constructs.
[0329] Extending the linker length by a highly flexible poly-T
ss-L-DNA to form a 77mer and a 97mer, respectively, provides for an
increase in affinity and especially in antigen complex stability kd
(1/s).
[0330] The chi2 values indicate a high quality of the measurements.
All measurements show extremely small errors. The data can be
fitted to a Langmuir 1:1 fitting model residuals deviate only +/-1
RU, small chi2 values and only 10 iterative calculations were
necessary for obtaining the data.
[0331] A cooperative binding effect works according to the physical
law, that the free binding energies .DELTA.G1 and .DELTA.G2
summarize. The affinities multiply: Kdcoop=KD1.times.KD2.
Furthermore, the dissociation rates also multiply: kd
coop=kd1.times.kd 2. This is exactly observable in the 77mer and
97mer linker experiment. This results in very long complex
half-lifes of 4146 hours (173 days) and 3942 hours (164 days),
respectively. The affinities are in the range of 100 fmol/l. It is
obvious, that a cooperative binding event occurs.
[0332] The association rates of all dual binding agents are faster,
when compared to the singly hybridized constructs. Despite showing
a higher molecular weight the association rate increases.
[0333] Here we could show, that tratsuzumab and pertuzumab linked
together in a complex as reported herein simultaneously binds to
the HER-2 extracellular domain (ECD). Both Fab fragments bind to
genuine epitopes on the HER2-ECD. Additionally both Fab fragments
strongly differ in their binding angles. By using the optimal 77mer
linker (about 30 nm in length) ss-L-DNA and its beneficial
flexibility and length properties a cooperative binding event could
be shown.
[0334] Hence cooperative binding between Herceptin-Fab and
Pertuzumab-Fab linked together via a highly flexible ss-L-DNA
linker could be shown.
[0335] While this disclosure has been described as having an
exemplary design, the present disclosure may be further modified
within the spirit and scope of this disclosure. This application is
therefore intended to cover any variations, uses, or adaptations of
the disclosure using its general principles. Further, this
application is intended to cover such departures from the present
disclosure as come within the known or customary practice in the
art to which this disclosure pertains.
Sequence CWU 1
1
151122PRTMus musculus 1Gln Cys Asp Val Lys Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro 1 5 10 15 Gly Gly Ser Leu Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser 20 25 30 Asp Tyr Pro Met Ser Trp Val
Arg Gln Thr Pro Glu Lys Arg Leu Glu 35 40 45 Trp Val Ala Thr Ile
Thr Thr Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp 50 55 60 Ser Ile Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Leu
Tyr Leu Gln Met Gly Ser Leu Gln Ser Glu Asp Ala Ala Met Tyr 85 90
95 Tyr Cys Thr Arg Val Lys Thr Asp Leu Trp Trp Gly Leu Ala Tyr Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120
2116PRTMus musculus 2Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ser
Phe Ser Leu Gly Ala 1 5 10 15 Ser Ala Lys Leu Thr Cys Thr Leu Ser
Ser Gln His Ser Thr Tyr Thr 20 25 30 Ile Glu Trp Tyr Gln Gln Gln
Pro Leu Lys Pro Pro Lys Tyr Val Met 35 40 45 Glu Leu Lys Lys Asp
Gly Ser His Thr Thr Gly Asp Gly Ile Pro Asp 50 55 60 Arg Phe Ser
Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ser Ile Ser 65 70 75 80 Asn
Ile Gln Pro Glu Asp Glu Ser Ile Tyr Ile Cys Gly Val Gly Asp 85 90
95 Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Val
100 105 110 Thr Val Leu Gly 115 3121PRTMus musculus 3Glu Val Gln
Leu Gln Gln Ser Gly Pro Ala Leu Val Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Met Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser Tyr 20 25
30 Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 Gly Tyr Leu Asn Pro Tyr Asn Asp Asn Thr Lys Tyr Asn Glu
Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Arg Ser Ser
Ser Thr Val Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Ile Tyr Ala Tyr
Asp His Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Leu Thr
Val Ser Ser 115 120 4106PRTMus musculus 4Gln Ile Val Leu Thr Gln
Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr
Leu Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 Tyr Trp
Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45
Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50
55 60 Gly Ser Val Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala
Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Thr Tyr
Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
105 517DNAArtificial Sequence17mer ssDNA 5agttctatcg tcgtcca
17619DNAArtificial Sequence19mer ssDNA 6agtctattaa tgcttctgc
19719DNAArtificial Sequencecomplementary 19mer ssDNA 7gcagaagcat
taatagact 19817DNAArtificial Sequencecomplementary 17mer ssDNA
8tggacgacga tagaact 17933PRTArtificial SequenceSynthetic peptide
9Glu Arg Ala Glu Gln Gln Arg Ile Arg Ala Glu Arg Glu Lys Glu Xaa 1
5 10 15 Xaa Ser Leu Lys Asp Arg Ile Glu Lys Arg Arg Arg Ala Glu Arg
Ala 20 25 30 Glu 1032PRTArtificial SequenceSynthetic peptide 10Ser
Leu Lys Asp Arg Ile Glu Arg Arg Arg Ala Glu Arg Ala Glu Xaa 1 5 10
15 Xaa Glu Arg Ala Glu Gln Gln Arg Ile Arg Ala Glu Arg Glu Lys Glu
20 25 30 1127PRTArtificial SequenceSynthetic peptide 11Phe Asp Glu
Arg Gln Pro Tyr Ala His Met Asn Gly Gly Arg Lys Asn 1 5 10 15 Glu
Arg Ala Leu Pro Leu Pro Gln Ser Ser Thr 20 25 1228PRTArtificial
SequenceSynthetic peptide 12Tyr Glu Glu His Ile Pro Tyr Thr His Met
Asn Gly Gly Lys Lys Asn 1 5 10 15 Gly Arg Ile Leu Thr Leu Pro Arg
Ser Asn Pro Ser 20 25 135PRTArtificial SequenceSynthetic peptide
(Spacer) 13Gly Gly Gly Gly Ser 1 5 149PRTArtificial SequenceHA-Tag
14Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 1515PRTArtificial
SequenceAvi-Tag 15Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu
Trp His Glu 1 5 10 15
* * * * *