U.S. patent application number 15/013773 was filed with the patent office on 2016-09-01 for high affinity adaptor molecules for redirecting antibody specifity.
The applicant listed for this patent is Albert Collinson, Robert Kamen, Matti Sallberg, Gregor Schurmann, Anders Vahlne, Peter Wagner. Invention is credited to Albert Collinson, Robert Kamen, Matti Sallberg, Gregor Schurmann, Anders Vahlne, Peter Wagner.
Application Number | 20160251400 15/013773 |
Document ID | / |
Family ID | 43857355 |
Filed Date | 2016-09-01 |
United States Patent
Application |
20160251400 |
Kind Code |
A1 |
Collinson; Albert ; et
al. |
September 1, 2016 |
HIGH AFFINITY ADAPTOR MOLECULES FOR REDIRECTING ANTIBODY
SPECIFITY
Abstract
Disclosed are methods for identifying high affinity adaptor
molecules that bind to both a circulating antibody and a target
molecule and redirect the specificity of the circulating antibody
to the target molecule. Exemplary high affinity adaptor molecules
are also provided.
Inventors: |
Collinson; Albert;
(Marlborough, MA) ; Wagner; Peter; (Braunschweig,
DE) ; Sallberg; Matti; (Stockholm, SE) ;
Vahlne; Anders; (Stockholm, SE) ; Schurmann;
Gregor; (Hannover, DE) ; Kamen; Robert;
(Sudbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Collinson; Albert
Wagner; Peter
Sallberg; Matti
Vahlne; Anders
Schurmann; Gregor
Kamen; Robert |
Marlborough
Braunschweig
Stockholm
Stockholm
Hannover
Sudbury |
MA
MA |
US
DE
SE
SE
DE
US |
|
|
Family ID: |
43857355 |
Appl. No.: |
15/013773 |
Filed: |
February 2, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13497332 |
Jul 10, 2012 |
9284548 |
|
|
PCT/US2010/051484 |
Oct 5, 2010 |
|
|
|
15013773 |
|
|
|
|
61248778 |
Oct 5, 2009 |
|
|
|
61257351 |
Nov 2, 2009 |
|
|
|
Current U.S.
Class: |
530/322 |
Current CPC
Class: |
C40B 30/04 20130101;
C40B 50/06 20130101; C07K 14/00 20130101; C12N 15/1062 20130101;
C40B 40/08 20130101 |
International
Class: |
C07K 14/00 20060101
C07K014/00; C12N 15/10 20060101 C12N015/10 |
Claims
1. A method for identifying a high affinity adaptor molecule
capable of redirecting antibody specificity, the method comprising:
(a) providing a randomized library encoding a population of
candidate targeting peptides; (b) selecting a targeting peptide
from the display library which binds with high affinity and/or
selectivity to a target molecule; (c) linking the targeting peptide
to a ligand moiety via a linking moiety to form a candidate adaptor
molecule; and (d) evaluating the ability of the candidate adaptor
molecule to redirect the specificity of the circulating antibody to
the target molecule; thereby identifying the adaptor molecule.
2. The method of claim 1, wherein steps (a)-(d) are performed
consecutively.
3. The method of claim 1, wherein linking step (c) is performed
prior to step (b).
4. The method of claim 1, wherein the library is an mRNA display,
ribosome display, yeast display, phage display or synthetic peptide
library.
5. The method of claim 1, wherein the targeting peptide binds to
the target molecule with a binding affinity of 1 nM or lower.
6. The method of claim 1, wherein the ligand moiety comprises a
glycan moiety.
7. The method of claim 1, wherein the ligand moiety is a blood
group antigen.
8. The method of claim 1, wherein the ligand moiety is a gal
antigen or epitope thereof.
9. The method of claim 8, wherein the ligand moiety consists of one
or more gal-.alpha.-1-3-gal disaccharide units.
10. The method of claim 8, wherein the ligand moiety is a modified
gal antigen having modifications which reduce competitive binding
by interfering molecules.
11. The method of claim 8, wherein the ligand moiety is a modified
gal antigen having modifications which reduce enzymatic or chemical
degradation.
12. The method of claim 10, wherein the modified gal antigen
comprises a protecting group at a C6' position of a terminal
galactose residue.
13. The method of claim 1, wherein the ligand moiety is a
peptidomimetic of a gal antigen.
14. The method of claim 1, wherein the ligand moiety is a peptide
ligand moiety.
15. The method of claim 14, wherein the peptide ligand moiety
comprises an epitope that is selectively bound by an antigen
binding site of the circulating antibody.
16. The method of claim 15, wherein the peptide ligand moiety
comprises an idiotope of an antibody, wherein the idiotope is
selectively bound by a circulating anti-idiotypic antibody.
17. The method of claim 16, wherein the peptide ligand moiety
comprises a binding site portion of an Fc binding protein.
18. The method of claim 1, wherein the peptide ligand moiety is
selected by (i) providing a randomized mRNA display library
encoding a population of candidate peptide ligand moieties; and
(ii) selecting a peptide ligand moiety from the display library of
step (i) which binds with high affinity and/or selectivity to a
circulating antibody.
19. The method of claim 18, wherein the candidate peptide ligand
moieties are fused to targeting peptides prior to selection step
(ii).
20. The method of claim 18, wherein the candidate peptide ligand
moieties are fused to targeting peptides following selection step
(ii).
21. The method of claim 1, wherein the target molecule is a soluble
disease-associated molecule.
22. The method of claim 21, wherein the redirected antibody
specificity is evaluated by measuring opsonization or
neutralization of the soluble molecule.
23. The method of claim 1, wherein the ligand moiety is linked to
the targeting moiety with a bifunctional linker moiety.
24. The method of claim 23, wherein the bifunctional linker moiety
links the targeting moiety and the ligand moiety via an amino group
in the targeting moiety and a thiol moiety in the ligand
moiety.
25. The method of claim 1, wherein the target molecule is a present
on the surface of an infected or neoplastic cell.
26. The method of claim 25, wherein the redirected antibody
specificity is evaluated by measuring ADCC or CDC-dependent killing
of the cell.
27. A high affinity adaptor molecule identified according to the
method of any one of the preceding claims, the adaptor molecule
comprising (i) a targeting moiety which binds with high affinity or
selectivity to a target molecule, (ii) a ligand moiety which
specifically binds to a circulating antibody; and (iii) a linker
moiety linking the targeting moiety to the ligand moiety, wherein
the adaptor molecule facilitates a functional interaction between
the antibody and the target molecule.
28. The high affinity adaptor molecule of claim 27, wherein the
targeting moiety is a peptide targeting moiety.
29. The adaptor molecule of claim 27, wherein the targeting moiety
binds with high affinity or selectivity to VEGF ligand.
30. The adaptor molecule of any one of claims 27, wherein the
targeting moiety comprises one or more sequences selected from SEQ
ID NOs 1, 2, 3 and 4.
31. The adaptor molecule of any one of claims 27, wherein the
targeting moiety is PEGylated.
32. The adaptor molecule of any one of claims 27, wherein the
ligand moiety comprises a Gal antigen which specifically binds to a
circulating anti-Gal antibody.
33. A high affinity adaptor molecule selected from the group
consisting of: TABLE-US-00003 (a) (SEQ ID NO: 1)-X-Y, comprising:
H-Gly-D-Val-D-Gln-D-Glu-D-Asp-D-Val-D-Ser-D-Ser-D-
Thr-D-Leu-Gly-D-Ser-D-Trp-D-Val-D-Leu-D-Leu-D-Pro-
D-Phe-D-His-D-Arg-Gly-D-Thr-D-Arg-D-Leu-D-Ser-D-
Val-D-Trp-D-Val-D-Thr-PEG2-Cys-X-Y; (b) (SEQ ID NO: 2)-X-Y,
comprising: H-Gly-Gly-D-Phe-D-Glu-Gly-D-Leu-D-Ser-D-Gln-D-Ala-
D-Arg-D-Lys-D-Asp-D-Gln-D-Leu-D-Trp-D-Leu-D-Phe-D-
Leu-D-Met-D-Gln-D-His-D-Ile-D-Arg-D-Ser-D-Tyr-D-
Arg-D-Thr-D-Ile-D-Thr-PEG2-Cys-X-Y; (c) (SEQ ID NO: 3)-X-Y,
comprising: H-Gly-D-Val-Gly-Gly-D-Ser-D-Arg-D-Leu-D-Glu-D-Ala-
D-Tyr-D-Lys-D-Lys-D-Asp-D-His-D-Arg-D-Val-D-Phe-D-
Gln-D-Met-D-Ala-D-Trp-D-Leu-D-Gln-D-Tyr-D-Tyr-D-
Trp-D-Ser-D-Thr-D-Thr-PEG2-Cys-X-Y; and (d) (SEQ ID NO: 4)-X-Y,
comprising: H-Gly-D-Ser-Gly-D-Ser-Gly-D-Asn-D-Ala-D-Leu-D-His-
D-Trp-D-Val-D-Cys-D-Ala-D-Ser-D-Asn-D-Ile-D-Cys-D-
Trp-D-Arg-D-Thr-D-Pro-D-Trp-D-Ala-Gly-D-Gln-D-Leu-
D-Trp-Gly-D-Leu-D-Val-D-Arg-D-Leu-D-Thr-PEG2-Cys- X-Y;
wherein, X is bifunctional chemical linker with maleimide
functionality; and Y is an amino modified Gal-1-3-Gal disaccharide.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
Nonprovisional application Ser. No. 13/497,332 filed Jul. 10, 2012,
which issued as U.S. Pat. No. 9,284,548 on Mar. 15, 2016, which
claims priority to International Application No. PCT/US2010/051484
filed Oct. 5, 2010, which claims priority to U.S. Provisional
Application Ser. No. 61/248,778, filed Oct. 5, 2009, and U.S.
Provisional Application Ser. No. 61/257,351, filed Nov. 2, 2009.
The entire contents of the aforementioned applications are hereby
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The concept of redirecting the immune system to attack new
targets has long interested scientists as an appealing strategy for
targeted immunotherapy. By redirecting naturally circulating human
antibodies to attack desired targets in disease areas such as
cancer, autoimmune disease and infectious disease, one can avoid
the need for a special immunization. While this strategy has shown
early signs of success, most studies have not progressed beyond in
vitro demonstrations. For example, this strategy would be
particularly valuable if it made use of antibody already present in
the general population and could be made amenable to oral
administration. Thus, there is a need in the art for improved
methods of redirecting antibody specificity.
SUMMARY OF THE INVENTION
[0003] The present invention provides isolated adaptor molecules,
particularly bispecific adaptor peptides, which bind to both an
antibody and a desired target molecule with high binding affinity
and selectivity. Due to their high binding affinity and
selectivity, the adaptor molecules of the invention are capable of
efficiently redirecting circulating antibodies to a target molecule
not normally bound by the antibody molecule. Moreover, the adaptor
molecules disclosed herein provide one or more of the following
advantages over traditional antibody-mediated therapeutics: 1)
enable the simultaneous recruitment of multiple antibody-class
effector functions; 2) can be developed rapidly using the methods
of the invention; 3) have a low cost of goods; and 4) do not cause
an IgE-mediated hypersensitivity reaction when administered to a
subject.
[0004] The adaptor molecules generally comprise one or more
targeting moietics linked to one or more ligand moietics. In
certain embodiments, the ligand moiety comprises one or more Gal
antigen (e.g., Gal-.alpha.-1-3-Gal) or mimetic thereof. In one
embodiment, the targeting moiety comprises a peptide, e.g., a VEGF
or TNF.alpha.-binding peptide. Exemplary VEGF-binding peptides have
one or more of the amino acid sequences set forth in SEQ ID NO. 1,
2, 3, and/or 4. In another preferred embodiment, the peptide (e.g.,
the peptide sequence set forth in SEQ ID NO. 1, 2, 3, and/or 4) is
linked to a ligand moiety comprising one or more Gal antigens
(e.g., Gal-.alpha.-1-3-Gal disachharide).
[0005] In other embodiments, the targeting moiety comprises one or
more antibody, or antigen binding fragment thereof. Suitable
antibodies include, without limitation, Abciximab, Adalimumab,
Alemtuzumab, Basiliximab, Bevacizumab, Cetuximab, Certolizumab
pegol, Daclizumab, Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab
tiuxetan, Infliximab, Muromonab-CD3, Natalizumab, Omalizumab,
Palivizumab, Panitumumab, Ranibizumab, Rituximab, Tositumomab,
Trastuzumab, and/or Golimumab, or antigen binding fragments
thereof. In a preferred embodiment, the antibody (e.g., one or more
of the antibodies disclosed supra) or antigen binding fragments
thereof, is linked to a ligand moiety comprising one or more Gal
antigens (e.g., Gal-.alpha.-1-3-Gal). In another preferred
embodiment, the antibody (e.g., one or more of the antibodies
disclosed supra) or antigen binding fragments thereof, is linked to
a ligand moiety comprising one Gal-.alpha.-1-3-Gal disaccharide. In
another preferred embodiment, a ligand moiety comprising one or
more Gal antigens (e.g., Gal-.alpha.-1-3-Gal) is linked to one or
more variable regions of the antibody.
[0006] In other embodiments, the targeting moiety comprises an
antibody-like molecule. Suitable antibody-like molecules include,
without limitation, Adnectins, Affibodies, DARPins, Anticalins,
Avimers, and Versabodies, or antigen binding fragment thereof. In a
preferred embodiment, the antibody-like molecule is linked to a
ligand moiety comprising one or more Gal antigens (e.g.,
Gal-.alpha.-1-3-Gal).
[0007] In other embodiments, the targeting moiety of the invention
comprises an extracellular portion of a cell surface receptor, or
fragment thereof. Suitable cell surface receptors include, without
limitation, a TNF family receptor (e.g., a TNF.alpha. receptor,
e.g., a human TNF.alpha. receptor) and growth factor receptors of
the tyrosine kinase family, (e.g, p185HER2). In a preferred
embodiment, the extracellular portion of a cell surface receptor
molecule is linked to a ligand moiety comprising one or more Gal
antigens (e.g., Gal-.alpha.-1-3-Gal). In another preferred
embodiment, the extracellular portion of a cell surface receptor
molecule is linked to a ligand moiety comprising one
Gal-.alpha.-1-3-Gal disaccharide.
[0008] In other embodiments, the targeting moiety comprises a
ligand for a cell surface receptor. In a preferred embodiment, the
ligand is linked to a ligand moiety comprising one or more Gal
antigens (e.g., Gal-.alpha.-1-3-Gal). In another preferred
embodiment, the ligand is linked to a ligand moiety comprising one
Gal-.alpha.-1-3-Gal disaccharide.
[0009] In another aspect, the invention also provides methods for
identifying isolated adaptor molecules, such methods comprising:
providing a randomized library encoding a population of candidate
targeting moietics; selecting a targeting moiety from the display
library which binds with high affinity and/or selectivity to a
target molecule; linking the targeting moiety to a ligand moiety
via a linking moiety to form a candidate adaptor molecule; and
evaluating the ability of the candidate adaptor molecule to
redirect the specificity of the circulating antibody to the target
molecule. Suitable screening methods for use in the methods of the
invention include an mRNA display, ribosome display, yeast display,
phage display or screening of synthetic peptide library. In one
preferred embodiment, an mRNA display library is used to select a
targeting peptide. In another preferred embodiment, a phage display
library is used to select a targeting peptide.
[0010] A further aspect of the invention provides a method of
treating a disease (e.g., a cancer, an infectious disease, or an
autoimmune disease) in a subject, comprising administering to the
subject an effective amount of an isolated adaptor molecule of the
invention, thereby treating the disease. In specific embodiments
the disease is at least one of macular degeneration, diabetic
retinopathy, psoriasis, diabetes, cardiovascular ischemia,
rheumatoid arthritis, and osteoarthritis.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 is a schematic overview of an exemplary adaptor
molecule of the invention. The adaptor molecule is a bispecific
peptide comprising a high-affinity peptide targeting moiety
directed against a target of interest and a ligand moiety
comprising the glycopeptide epitope of an anti-gal antibody. By
binding to both the target molecule and a naturally-existing
anti-gal antibody, the adaptor molecule is capable of redirecting
the effector functions of the antibody to act on the target
molecule.
[0012] FIG. 2 provides an overview of the various method steps
conducted during mRNA display.
[0013] FIG. 3 depicts exemplary peptide libraries that may be used
in mRNA display for selection of high affinity adaptor
peptides.
[0014] FIG. 4 depicts four exemplary high affinity VEGF targeting
peptides of the invention.
[0015] FIG. 5 depicts exemplary methods and linkers for coupling a
target moiety (e.g., a VEGF targeting moiety of the invention) to a
ligand moiety.
[0016] FIG. 6 depicts HPLC and Mass spectrometry analyses of a
target moiety/ligand moiety coupling reaction.
[0017] FIG. 7 depicts HPLC and Mass spectrometry analyses of an
optimized target moiety/ligand moiety coupling reaction.
[0018] FIG. 8 depicts the results of in vitro assays demonstrating
the efficacy of VEGF-binding adaptors of the invention at
redirecting naturally occurring anti-gal antibodies to bind
VEGF.
DETAILED DESCRIPTION
[0019] This specification describes, inter alia, the identification
and production of novel, adaptor molecules that bind to both
antibodies and target molecules with high binding affinity and
selectivity. As used herein, the term "adaptor" refers to the
ability of the peptide to facilitate a functional interaction
between an antibody and a target molecule to which the antibody
does not normally bind. For example, the adaptor molecules of the
invention are capable of binding to both an antibody and a target
molecule such that the antibody and target are brought into close
proximity with each other. In certain embodiments, the antibody is
capable of facilitating an antibody response against the target
molecule. Exemplary antibody responses may include neutralization
or opsonization of a target molecule (e.g., a soluble factor such
as a cytokine or growth factor). Alternatively, where the target
molecule is present on the surface of virus, the antibody may
facilitate neutralization or opsonization of the virus. In other
embodiments, the target molecule may present on the surface of a
cell (e.g., an infected cell or tumor cell) and recruitment of the
antibody to the cell by the adaptor peptide facilitates the
induction of an antibody-mediated effector response (e.g.,
induction of a complement cascase or antibody-dependent cellular
cytotoxicity (ADCC)). The adaptor molecules disclosed herein are
particularly advantageous in that they do not appreciably activate
basophils, and therefore do not cause an IgE hypersensitivity
reaction when administered to a subject.
[0020] Adaptor molecules of the invention comprise at least three
moieties: (a) a targeting moiety, (b) a ligand moiety and (c) a
linker moiety. The targeting moiety (a) is a moiety which binds
with high affinity and/or selectivity to a target molecule. The
ligand moiety (b) is a moiety to which a circulating antibody binds
with high affinity and/or selectivity. The targeting moiety and
ligand moiety are operably linked via the intervening linker moiety
(c). Said linker moiety may be a covalent bond, chemical linker, or
peptide amino acid sequence, or any other moiety capable of linking
the targeting and ligand moieties of the adaptor peptide.
(a) Targeting Moiety
[0021] A targeting moiety of the invention has been selected for
its ability to bind with high affinity and/or selectivity to a
target molecule. In particular embodiments, the targeting moiety
specifically binds to the target molecule with a dissociation
constant (KD) of 100 nanomolar or less (e.g., 10 nM or less, 1 nM
or less, 100 pM or less, 10 pM or less, or 1 pM or less). In other
embodiments, the targeting moiety exhibits high selectivity. By
"specifically binds" is meant that the moiety recognizes and
interacts with a target molecule but that does not substantially
recognize and interact with other molecules in a sample, e.g., a
biological sample. In particular embodiments, the binding affinity
of the targeting moiety for the target molecule is at least 1000
fold higher than its binding affinity for a non-target molecule
(e.g., 10.sup.3 fold, 10.sup.4 fold, 10.sup.5 fold, 10.sup.6 fold,
or 10.sup.7 fold higher).
[0022] Targeting moieties can be selected for the ability to bind
with high selectivity and/or affinity to virtually any target
molecule. In certain embodiments, the targeting moiety binds to a
pathogen-associated target molecule, including, but not limited to,
surface proteins or antigens from a virus (e.g., HAV, HBV, or HCV,
HIV, influenza virus) , bacteria, yeast, parasites, or fungus. In
other embodiments, the target molecule is a cell surface protein,
including but not limited to, a cell surface antigen or receptor
from an infected host cell or a tumor cell. Exemplary
tumor-associated antigens include the growth factor receptor of the
tyrosine kinase family, p185HER2. In other embodiments, the target
molecule is a hormone or growth factor. Exemplary hormones or
growth factors include tumor necrosis factor alpha (TNF.alpha.) or
Vascular endothelial growth factor (VEGF). In other embodiments,
the target molecule is an antibody (e.g., an auto-antibody).
[0023] In one preferred embodiment, the targeting moiety of the
invention comprises a peptide moiety. The length of the peptide
targeting moiety is desirably between at least 3-200 amino acids,
preferably between at least 3-100 amino acids, more preferably
between 3-50 amino acids, and still more preferably between 3-30
amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino
acids). Exemplary peptides for use as targeting moieties are
described in WO/2010/014830, which is herein incorporated by
reference in its entirety. In a preferred embodiment, the peptide
is a VEGF-binding peptide comprising or consisting of any one or
more of the following amino acid sequences, or VEGF-binding
portions thereof:
TABLE-US-00001 SEQ ID NO. 1
H-Gly-Val-Gln-Glu-Asp-Val-Ser-Ser-Thr-Leu-Gly-Ser-
Trp-Val-Leu-Leu-Pro-Phe-His-Arg-Gly-Thr-Arg-Leu-
Ser-Val-Trp-Val-Thr SEQ ID NO. 2
H-Gly-Gly-Phe-Glu-Gly-Leu-Ser-Gln-Ala-Arg-Lys-Asp-
Gln-Leu-Trp-Leu-Phe-Leu-Met-Gln-His-Ile-Arg-Ser-
Tyr-Arg-Thr-Ile-Thr SEQ ID NO. 3
H-Gly-Val-Gly-Gly-Ser-Arg-Leu-Glu-Ala-Tyr-Lys-Lys-
Asp-His-Arg-Val-Phe-Gln-Met-Ala-Trp-Leu-Gln-Tyr-
Tyr-Trp-Ser-Thr-Thr; and/or SEQ ID NO. 4
H-Gly-Ser-Gly-Ser-Gly-Asn-Ala-Leu-His-Trp-Val-Cys-
Ala-Ser-Asn-Ile-Cys-Trp-Arg-Thr-Pro-Trp-Ala-Gly-
Gln-Leu-Trp-Gly-Leu-Val-Arg-Leu-Thr.
[0024] In other embodiments, the targeting moiety of the invention
comprises an antibody, or binding fragment thereof (e.g., a CDR
(e.g., CDRH3), a variable domain (VH or VL), or a Fab fragment).
Any antibody, or fragment thereof, from any animal species, is
contemplated for use in the methods and compositions described
herein. Suitable antibodies and antibody fragments include, without
limitation, single chain antibodies (see e.g., Bird et al. (1988)
Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad.
Sci. U.S.A 85:5879-5883, each of which is herein incorporated by
reference in its entirety), domain antibodies (see, e.g., U.S. Pat.
No. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245, each of
which is herein incorporated by reference in its entirety),
Nanobodies (see, e.g., U.S. Pat. No. 6,765,087, which is herein
incorporated by reference in its entirety), and UniBodies (see,
e.g., WO2007/059782, which is herein incorporated by reference in
its entirety In certain embodiments, the antibody is Abciximab,
Adalimumab, Alemtuzumab , Basiliximab, Bevacizumab, Cetuximab,
Certolizumab pegol, Daclizumab, Eculizumab, Efalizumab, Gemtuzumab,
Ibritumomab tiuxetan, Infliximab, Muromonab-CD3, Natalizumab,
Omalizumab, Palivizumab, Panitumumab, Ranibizumab, Rituximab,
Tositumomab, Trastuzumab, and/or Golimumab, or antigen binding
fragments thereof.
[0025] In other embodiments, the targeting moiety of the invention
comprises an antibody-like molecule. Suitable antibody-like
molecules include, without limitation, Adnectins (see, e.g., WO
2009/083804, which is herein incorporated by reference in its
entirety), Affibodies (see, e.g., U.S. Pat. No. 5,831,012, which is
herein incorporated by reference in its entirety), DARPins (see,
e.g., U.S. Patent Application Publication No. 2004/0132028, which
is herein incorporated by reference in its entirety), Anticalins
(see, e.g., U.S. Pat. No. 7,250,297, which is herein incorporated
by reference in its entirety), Avimers (see, e.g., U.S. Patent
Application Publication Nos. 200610286603, which is herein
incorporated by reference in its entirety), and Versabodies (see,
e.g., U.S. Patent Application Publication No. 2007/0191272, which
is hereby incorporated by reference in its entirety).
[0026] In other embodiments, the targeting moiety of the invention
comprises a ligand for a cell surface receptor, wherein the ligand
is capable of recruiting the adaptor molecule to cells that express
said cell surface receptor.
[0027] In other embodiments, the targeting moiety of the invention
comprises the extracellular portion of a cell surface receptor, or
fragment thereof, wherein the cell surface receptor, or fragment
thereof, is capable of recruiting the adaptor molecule to the
cognate ligand of said cell surface receptor. Suitable cell surface
receptors include, without limitation, TNF family receptors (e.g.,
a TNF.alpha. receptor, e.g., a human TNF.alpha. receptor) and
growth factor receptors of the tyrosine kinase family, (e.g,
p185HER2).
[0028] In certain exemplary embodiments, the targeting moiety of
the invention comprises an Fc fusion protein or immunoadhesin
(e.g., a TNF receptor-Fc fusion such as Etaneracept).
(b) Ligand Moiety
[0029] Ligand moieties of the invention comprise antigenic domains
which are bound by Immunoadhesions (Fc fusions proteins) present in
a subject. In some embodiments, the ligand moiety is bound by a
circulating antibody. Circulating antibodies may be present in the
subject due to naturally acquired immunity. Alternatively, the
circulating antibodies are present as a result of prior vaccination
of the subject. For example, the circulating antibodies may be
present as a result of childhood vaccination against small pox,
measles, mumps, rubella, herpes, hepatitis and polio. Accordingly,
a ligand moiety may comprise one or more epitopes that is
recognized by these circulating antibodies.
[0030] In some embodiments, however, a ligand moiety interacts with
an antibody that has been administered to the subject. For example,
an antibody that interacts with the ligand moiety of an adaptor
molecule of the invention can be co-administered with the adaptor
molecule. Further, the antibody that interacts with ligand moiety
may not normally exist in a subject but the subject has acquired
the antibody by introduction of a biologic material or antigen
(e.g., serum, blood, or tissue) so as to generate a high titer of
antibodies in the subject. For example, subjects that undergo blood
transfusion acquire numerous antibodies, some of which can interact
with a ligand moiety of the adaptor peptide.
[0031] A ligand moiety can comprise any compound capable of binding
to an antibody, including, without limitation, a peptide,
carbohydrate, lipid, antibody, or antibody-like molecule. In some
embodiments, a ligand moiety (e.g., a peptide, antibody or
antibody-like molecule) can comprise one or more non-natural amino
acids. Preferably, the ligand moiety comprises an epitope that
binds to a "high-titer antibody." The term "high-titer antibody" as
used herein, refers to an antibody that has high affinity for an
antigen (e.g., an epitope on an antigenic domain). For example, in
a solid-phase enzyme linked immunosorbent assay (ELISA), a high
titer antibody corresponds to an antibody present in a serum sample
that remains positive in the assay after a dilution of the serum to
approximately the range of 1:100-1:1000 in an appropriate dilution
buffer. Other dilution ranges include 1:200-1:1000, 1:200-1:900,
1:300-1:900, 1:300-1:800, 1:400-1:800, 1:400-1:700, 1:400-1:600,
and the like. In certain embodiments, the ratio between the serum
and dilution buffer is approximately: 1:100, 1:150, 1:200, 1:250,
1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700,
1:750, 1:800, 1:850, 1:900, 1:950, 1:1000.
[0032] In certain embodiments, the ligand moieties are antigenic
peptides obtained from a known target molecule (e.g., a surface
protein from a pathogen, tumor cell, or infected host cell) of the
antibody. The length of the peptide ligand moiety is desirably
between at least 3-200 amino acids, preferably between at least
3-100 amino acids, more preferably between 3-50 amino acids, and
still more preferably between 10-25 amino acids (e.g., 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, or 30 amino acids). In some embodiments,
the peptides are comprised of natural amino acids. In other
embodiments, the peptides include one or more non-natural amino
acids (e.g., D-amino acids).
[0033] In certain embodiments, the ligand moiety is a glycosylated
ligand moiety. Glycosylated ligand moietics can comprise or consist
of an antigenic saccharide or glycan moiety recognized by an
antibody in a subject. In some embodiments, the glycosylated ligand
moiety is linked directly or indirectly (i.e., via a linker moiety)
to the targeting moiety of the adaptor molecule. Such ligand
moieties lack an antigenic peptide moiety. In other embodiments,
the glycosylated ligand moiety is a glycopeptide comprising
additional antigenic peptide elements (e.g., an antigenic domain
comprising a peptide or epitope of a pathogen).
[0034] Exemplary glycosylated ligand moieties are derived from
blood group antigens. These antigens are generally surface markers
located on the outside of red blood cell membranes. Most of these
surface markers are proteins, however, some are carbohydrates
attached to lipids or proteins. Structurally, the blood group
determinants that can be used with the embodiments described herein
fall into two basic categories known as type I and type II. Type I
comprises a backbone comprised of a galactose 1-3 .beta. linked to
N-acetyl glucosamine while type II comprises, instead, a 1-4 .beta.
linkage between the same building blocks. The position and extent
of fucosylation of these backbone structures gives rise to the
Lewis-type and H-type specificities. For example, the presence of
an a-monofucosyl branch, solely at the C2-hydroxyl in the galactose
moiety in the backbone, constitutes the H-type specifity (Types I
and II), while further permutation by substitution of a-linked
galactose or a-linked N-acetylgalactosamine provides the molecular
basis of the familiar serological blood group classifications A, B,
and O. By first determining a patient's particular set of blood
group antigens, one can select a ligand moiety comprising one or
more blood group antigens that are outside of the repertoire of the
patient so as to generate a potent response to an adaptor molecule
comprising this ligand moiety and thereby redirecting the
antibodies present in the patient to target molecule bound by the
targeting moiety of said adaptor molecule. Exemplary blood group
antigens are set forth in detail in Table 2 of U.S. Pat. No.
7,318,926, which is hereby incorporated by reference in its
entirety.
[0035] In certain preferred embodiments, the glycosylated ligand
moiety comprises one or more gal-.alpha.-1-3 gal disaccharide sugar
units of the gal antigen. The gal antigen is produced in large
amounts on the cells of pigs, mice and New World monkeys by the
glycosylation enzyme galactosyltransferase (.alpha.(1,3)GT). Since
humans and Old World primates lack the gal antigen, they are not
immunotolerant to it and produce anti-gal antigen antibodies
(anti-Gal) throughout life in response to antigenic stimulation by
gastrointestinal bacteria. It has been estimated that anti-gal
antibodies represent more than 2% of circulating IgG and 1-8% of
circulating IgM in humans. The binding of anti-Gal to gal antigens
expressed on glycolipids and glycoproteins on the surface of
endothelial cells in donor organs leads to activation of the
complement cascade and hyperacute rejection, and also plays an
important role in occurrence of complement-independent delayed
xenograft rejection. Accordingly, the gal antigen has the ability
to generate a potent immune response.
[0036] In certain preferred embodiments, the glycosylated ligand
moiety to be joined or incorporated into the adaptor molecule
consists essentially of one or more Gal-.alpha.(1-3)-Gal
disaccharide sugar units and lacks any of the remaining portions of
the Gal antigen (e.g., GlcNac or Glc). For example, one or more
Gal-.alpha.(1-3)-Gal disaccharides may be linked to a targeting
moiety of an adaptor molecule via a free hydroxyl group at the
reducing end of a disaccharide unit (e.g., the hydroxyl group at
the C1 carbon of that is not involved in a glycosidic bond). In
certain embodiments the Gal disaccharide is linked to the targeting
moiety via a spacer moiety (e.g., C1-C6 alkyl spacer) at the free
hydroxyl group. Without being limited to any particular theory, it
is thought that the Gal-.alpha.(1-3)-Gal disaccharide portion of
the Gal antigen binds preferentially to anti-Gal antibodies but
exhibits limited binding to lectins (e.g., Galectin-3) or other
molecules that bind to other portions of the Gal antigen (e.g., the
Gal-.beta.(1-4)-GlcNAc moiety). In other embodiments, the
glycosylated ligand moiety to be joined or incorporated into
adaptor molecule comprises additional sugar residues of the Gal
antigen. For example, one or more trisaccharide
(Gal-.alpha.(1-3)-Gal-.beta.(1-4)-GlcNAc or
Gal-.alpha.(1-3)-Gal-.beta.(1-4)-Glc), tetrasccharide
(Gal-.alpha.(1-3)-Gal-.beta.(1-4)-GlcNAc-.beta.(1-3)-Gal) or
pentasaccharide
(Gal-.alpha.(1-3)-Gal-.beta.(1-4)-GlcNAc-.beta.(1-3)-Gal-.beta.(1-4)-Glc)
units of the gal antigen may incorporated.
[0037] In certain embodiments the gal antigen to be joined or
incorporated into an adaptor molecule is selected from
gal-.alpha.-(1,3) gal series of neoglycoproteins and can include:
Gal .alpha. 1-3Gal-BSA (3-atom spacer), Gal .alpha. 1-3Gal-BSA
(14-atom spacer), Gal .alpha. 1-3Gal-HSA (3-atom spacer), Gal
.alpha. 1-3Gal-HSA (14-atom spacer), Gal .alpha.
1-3Gal.beta.1-4GlcNAc-BSA (3-atom spacer),
Gal.alpha.1-3Gal.beta.1-4GlcNAc-BSA (14-atom spacer), Gal .alpha.
1-3Gal.beta.1-4GlcNAc-HSA (3-atom spacer), Gal .alpha.
1-3Gal.beta.1-4GlcNAc-HSA (14-atom spacer), Gal
.alpha.1-3Gal-Pentasaccharide-BSA (3-atom spacer), and the like. In
other embodiments the gal antigen can be selected from gal .alpha.
(1,3) gal analogue neoglycoproteins, including Gal .alpha.
1-3Gal.beta.1-4Glc-BSA (3-atom spacer), Gal .alpha. 1-3
Gal.beta.1-4Glc-HSA (3-atom spacer), Gala1-3Gal.beta.1-3G1cNAc-BSA
(3-atom spacer), Gal .alpha. 1-3Gal.beta.1-3GlcNAc-HSA (3-atom
spacer), Gal .alpha. 1-3Gal.beta.1-4(3-deoxyGlcNAc)-HSA (3-atom
spacer), Gal .alpha. 1-3Gal.beta.1-4(6-deoxyGlcNAc)-HSA, and the
like.
[0038] In yet other embodiments, a peptidomimetic of a Gal antigen
can be incorporated into an adaptor molecule of the invention.
Exemplary peptidomimetics include the .alpha.Gal-linked
glycopeptides Gal-.alpha.-YWRY (SEQ ID NO: 5), Gal-.alpha.-TWRY
(SEQ ID NO: 6) and Gal-.alpha.-RWRY (SEQ ID NO: 7). Other
peptidomimetics can be identified by screening a randomized library
of .alpha.Gal glycopeptides for anti-Gal antibody binding activity
using the methods of Xian et al. (see J. Comb. Chem., 6:126-134
(2004), which is incorporated by reference herein).
[0039] A Gal antigen, or peptidomimetic thereof, can be linked to a
targeting moiety (covalently and/or non-covalently) using any
art-recognized means. Art-recognized non-covalent linkages include
biotin-avidin or biotin-streptavidin linkages and other high
affinity binding partners (e.g., leucine zippers and the like).
Suitable means for covalent attachment include, without limitation,
those set forth in FIG. 4 and US Patent Application number
20100183635, which is hereby incorporated by reference in its
entirety. For example the gal antigen (or peptidomimeitc) may be
linked directly to the polypeptide backbone of a polypeptide
targeting moiety via a synthetic chemical linker. Exemplary
synthetic chemical linkers include bifunctional linker moieties,
e.g., linkers with maleimide functionality For example, the
bifunctional linker moiety may link the targeting moiety and the
ligand moiety via an amino group in the targeting moiety and a
thiol moiety in the ligand moiety, or vice versa. In one exemplary
emobodiment, a maleimide linker (e.g., Sulfo-SMCC) is used to link
a cysteine residue of a polypeptide targeting moiety to the amino
group of an amino modified Gal antigen (e.g.,
B.sub.di--(CH.sub.2).sub.3--NH.sub.2of FIG. 5). Additionally or
alternatively, the gal antigen may be linked to N-linked
oligosaccharide of a glycoprotein targeting moiety. In some
embodiments, one or more Gal antigens (e.g., gal-.alpha.-(1,3)
gal), or peptidomimetics thereof, are linked to a single site on a
target moiety. In other embodiments, one or more Gal antigens
(e.g., gal-.alpha.-(1,3) gal), or peptidomimetic thereof, are
linked to multiple sites on a target moiety. In certain
embodiments, the linker moiety is chemically-modified to reduce
enzymatic or chemical degradation.
[0040] Adaptor molecules comprising Gal antigen as disclosed herein
are particularly advantageous in that they do not appreciably
activate basophils, and therefore do not cause an IgE-mediated
hypersensistivity reaction when administered to a subject.
Notwithstanding, in some embodiments the adaptor molecules of the
invention will be assayed for their ability to activate basophils.
Suitable assays for measuring basophil activation are known in the
art, (see, e.g., J. Allergy Clin Immunol (2002) 110102-9, which is
hereby incorporated by reference in its entirety).
[0041] In certain embodiments, the ligand moiety comprises
polymeric binding molecules wherein the monomers are not amino
acids.
[0042] In certain exemplary embodiments, the high affinity adaptor
molecule is selected from the group consisting of:
TABLE-US-00002 (a) (SEQ ID NO: 1)-X-Y, comprising:
H-Gly-D-Val-D-Gln-D-Glu-D-Asp-D-Val-D-Ser-D-Ser-D-
Thr-D-Leu-Gly-D-Ser-D-Trp-D-Val-D-Leu-D-Leu-D-Pro-
D-Phe-D-His-D-Arg-Gly-D-Thr-D-Arg-D-Leu-D-Ser-D-
Val-D-Trp-D-Val-D-Thr-PEG2-Cys-X-Y; (b) (SEQ ID NO: 2)-X-Y,
comprising: H-Gly-Gly-D-Phe-D-Glu-Gly-D-Leu-D-Ser-D-Gln-D-Ala-
D-Arg-D-Lys-D-Asp-D-Gln-D-Leu-D-Trp-D-Leu-D-Phe-D-
Leu-D-Met-D-Gln-D-His-D-Ile-D-Arg-D-Ser-D-Tyr-D-
Arg-D-Thr-D-Ile-D-Thr-PEG2-Cys-X-Y; (c) (SEQ ID NO: 3)-X-Y,
comprising: H-Gly-D-Val-Gly-Gly-D-Ser-D-Arg-D-Leu-D-Glu-D-Ala-
D-Tyr-D-Lys-D-Lys-D-Asp-D-His-D-Arg-D-Val-D-Phe-D-
Gln-D-Met-D-Ala-D-Trp-D-Leu-D-Gln-D-Tyr-D-Tyr-D-
Trp-D-Ser-D-Thr-D-Thr-PEG2-Cys-X-Y; and (d) (SEQ ID NO: 4)-X-Y,
comprising: H-Gly-D-Ser-Gly-D-Ser-Gly-D-Asn-D-Ala-D-Leu-D-His-
D-Trp-D-Val-D-Cys-D-Ala-D-Ser-D-Asn-D-Ile-D-Cys-D-
Trp-D-Arg-D-Thr-D-Pro-D-Trp-D-Ala-Gly-D-Gln-D-Leu-
D-Trp-Gly-D-Leu-D-Val-D-Arg-D-Leu-D-Thr-PEG2-Cys- X-Y;
[0043] wherein, X is bifunctional chemical linker with maleimide
functionality; and Y is an amino modified Gal-1-3-Gal
disaccharide.
(c) Modified Adaptor Molecules
[0044] One or more moieties of the adaptor molecules of the
invention may be modified. In certain embodiments, a peptide moiety
of the adaptor molecule is modified. For example, peptide moieties
of an adaptor molecule can be modified to include non-natural amino
acids such as those described in U.S. Pat. No. 6,559,126,
incorporated herein by reference. For example, the peptides of the
invention may be composed of one or more, or most preferably all,
amino acids which are D-type optical isomers. These D-peptides have
several advantages with respect to antibodies and other protein
therapeutics. The smaller size and greater stability of the
D-peptides makes them simpler to formulate for pulmonary, topical
and oral delivery. D-peptides are also known to be poor immunogens
(Dintzis et al. (1993) PROTEINS: Structure, Function, and Genetics
16, 306-308). Furthermore, their resistance to enzymatic
degradation, and their ability to be combined with polymers,
results in enhanced pharmacokinetics compared to other peptide
drugs. Also, D-peptides have reduced manufacturing costs that could
be passed on to the consumer.
[0045] The peptide component of an adaptor molecule can also be
modified by any variety of standard chemical methods (e.g. , an
amino acid can be modified with a protecting group; the
carboxy-terminal amino acid can be made into a terminal amide
group; the amino-terminal residue can be modified with groups to,
e.g., enhance lipophilicity; or the polypeptide can be chemically
glycosylated or otherwise modified to increase stability or in vivo
half-life). Adaptor molecules of the invention may be designed to
include chemical modifications or particular amino acid sequences
which promote solubility. For example, in some embodiments peptide
moieties may be synthesized to include the amino acids DDD or KKK
in the N-terminal or C-terminal regions. Additionally or
alternatively, peptides and other targeting moieties may be
synthesized to include a PEGylation moiety at, for example, the
N-terminal and/or --C terminal regions. Exemplary PEGylation
moieties include PEG.sub.2-NH2 and PEG.sub.2-Cys-NH2 moieties.
[0046] The present invention also encompasses "conservative
sequence modifications" or "conservative amino acid modifications"
of the sequences described herein, i.e., amino acid sequence
modifications which do not significantly affect or alter the
binding characteristics of the peptide encoded by the nucleotide
sequence or containing the amino acid sequence. Such conservative
sequence modifications include nucleotide and amino acid
substitutions, additions and deletions. Modifications can be
introduced into sequences by standard techniques known in the art,
such as site-directed mutagenesis and PCR-mediated mutagenesis. In
some embodiments, the modifications are chosen by rational design,
and the designed peptides are generated by chemical synthesis as
described herein. "Conservative amino acid modifications" includes
conservative amino acid substitutions which are substitutions in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain (e.g., similar size, shape, electric
charge, chemical properties including the ability to form covalent
or hydrogen bonds, or the like). Families of amino acid residues
having similar side chains have been defined in the art. These
families include amino acids with basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine,
tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0047] A peptide or mimetic thereof of the invention may be
modified by one or more substitutions, particularly in portions of
the protein that are not expected to interact with a target
protein. It is expected that as many as 5%, 10%, 20%, 30%, 40%,
50%, or even 50% or more of the amino acids in peptide may be
altered by a conservative substitution without substantially
altering the affinity of the protein for target. It may be that
such changes will alter the immunogenicity of the polypeptide in
vivo, and where the immunogenicity is decreased, such changes will
be desirable. Further non-limiting examples of homologous
substitutions that can be made in the structures of the peptidic
molecules of the invention include substitution of D-phenylalanine
with D-tyrosine, D-pyridylalanine or D-homophenylalanine,
substitution of D-leucine with D-valine or other natural or
non-natural amino acid having an aliphatic side chain and/or
substitution of D-valine with D-leucine or other natural or
non-natural amino acid having an aliphatic side chain. In some
embodiments, conservative amino acid substitutions alone, i.e.,
without amino acid deletions or additions are the preferred type of
amino acid modification. One of skill in the art will appreciate
that such modifications or substitutions may be made at the DNA
level, thus encoding the altered or substituted peptide, or they
may be made at the protein level, e.g., by direct chemical
synthesis.
[0048] In some embodiments a peptide or peptide moiety of an
adaptor molecule may be made cyclic. Such "cyclic peptides" have
intramolecular links which connect two amino acids. Cyclic peptides
are often resistant to proteolytic degradation and are thus good
candidates for oral administration. The intramolecular linkage may
encompass intermediate linkage groups or may involve direct
covalent bonding between amino acid residues. In some embodiments,
the N-terminal and C-terminal amino acids are linked. In other
embodiments, one or more internal amino acids participate in the
cyclization. Other methods known in the art may be employed to
cyclize peptides of the invention. For example, cyclic peptides may
be formed via side-chain Azide-Alkyne 1,3-dipolar cycloaddition
(Cantel et al. J. Org. Chem., 73 (15), 5663-5674, 2008,
incorporated herein by reference). Cyclization of peptides may also
be achieved, e.g., by the methods disclosed in U.S. Pat. Nos.
5,596,078; 4,033,940; 4,216,141; 4,271,068; 5,726,287; 5,922,680;
5,990,273; 6,242,565; and Scott et al. PNAS. 1999. vol. 96 no. 24
P. 13638-13643, which are all incorporated herein by reference. In
some embodiments the intramolecular link is a disulfide bond mimic
or disulfide bond mimetic which preserves the structure that would
be otherwise be created by a disulfide bond.
[0049] In some particularly preferred embodiments, the cyclization
of peptides occurs via intramolecular disulfide bonds. In some
preferred embodiments, the formation of an intramolecular disulfide
bond increases the affinity of the peptide. Accordingly, the
methodology used to select and/or affinity mature the peptides or
mimetics thereof of the invention may be performed under conditions
which allow disulfide bond formation prior to and during selection
(e.g., oxidizing conditions). In some particularly preferred
embodiments the disulfide bonds may form between cysteine residues
which naturally exist in the library or peptide, or which are
introduced by the mutation process during one or more rounds of
selection. In other embodiments the peptides may be designed to
contain cysteine residues at particular positions such that it is
known which residues participate in the disulfide bond.
Intramolecular disulfide bonding between cysteine residues may be
induced by methods known in the art (e.g., U.S. Pat. Nos.
4,572,798; 6,083,715; 6,027,888, and WIPO Publication
WO/2002/103024 which are incorporated herein by reference).
[0050] In some embodiments, the formation of a disulfide bond (or
the formation of a cyclized or intramolecularly linked structure in
general) imparts a particular structure onto the peptide which is
important for target binding. Accordingly, the disulfide bonds
and/or cyclization preferably form prior to peptide selection such
that the potentially favorable structure created by bond formation
may be selected for. In some embodiments the peptides or mimetics
thereof of the invention may have more than one, two, three, or
more disulfide bonds. Further methods known in the art to generate,
and select peptides with intramolecular di-sulfide bonds,
intramolecular di-sulfide bond substitutes, and other
intramolecular links may be employed. For example, the methods
described in WO03040168, incorporated herein by reference, describe
methods to generate and select peptide apatamers, conotides, and
other cyclic peptides which, in some embodiments, may be employed
with the methods of the present invention.
[0051] In related embodiments a peptide conformation or structure
which is beneficial to binding (e.g., it increases binding
affinity) may be preserved or mimicked by chemical crosslinking or
other methods of peptide stabilization. For example, a beneficial
peptide conformation or structure which is formed by disulfide
bonds may be stabilized by chemical treatment or reaction, thus
allowing the preservation of the structure without a disulfide
bond. Indeed, peptide stabilization techniques may be employed to
stabilize peptides of the invention whether or not a disulfide bond
was originally present. For example, the techniques described in
Jackson, et al. J. Am. Chem. Soc. 1991, 113, 9391-9392; Phelan, et
al. J. Am. Chem. Soc. 1997, 119, 455-460; Bracken, et al. J. Am.
Chem. Soc. 1994, 116, 6431-6432, which are incorporated herein by
reference, may be used to stabilize peptides or peptide moieties of
the invention.
[0052] Other methods to stabilize peptides and peptide structures
may be used, e.g., olefinic cross-linking of helices through
O-allyl serine residues (Blackwell, H. E.; Grubbs, R. H. Angew.
Chem., Int. Ed. 1998, 37, 3281-3284, incorporated herein by
reference), all-hydrocarbon cross-linking (Schafmeister and Verdine
J. Am. Chem. Soc. 2000, 122 (24), 5891 -5892, incorporated herein
by reference) and the methods disclosed in U.S. Pat. No. 7183059
(incorporated herein by reference). The methods disclosed in
Blackwell et al. and Schafmeister et al. may be described as
producing "stapled" peptides, i.e., peptides which are covalently
locked into a particular conformational state or secondary
structure, or peptides which have a particular intramolecular
covalent linkage which predisposes them to form a particular
conformation or structure. If a peptide thus treated is predisposed
to, e.g., form an alpha-helix which is important for target
binding, then the energetic threshold for binding will be lowered.
Such "stapled" peptides have been shown to be resistant to
proteases and may also be designed to cross the cellular membrane
more effectively (also see Walensky et al. Science 2004:Vol. 305.
no. 5689, pp. 1466-1470; Bernal et al. J Am Chem Soc. 2007,
129(9):2456-7 which are incorporated herein by reference).
Accordingly, peptides or peptide moieties of the invention may be
thus stapled or otherwise modified to lock them into a specific
conformational shape or they may be modified to be predisposed to
particular conformation or secondary structure which is beneficial
for binding. It is contemplated that such peptide modifications may
occur prior to peptide selection such that the benefit of any
conformational constraints may also be selected for. Alternatively,
in some embodiments, the modifications may be made after selection
to preserve a conformation known to be beneficial to binding or to
further enhance a peptide candidate.
[0053] In other embodiments, the ligand moiety of an adaptor
molecule can be modified. Said modifications can be made, for
example, to minimize competitive binding by interfering molecules
or reduce enzymatic or chemical degradation of the ligand moiety
(e.g., under physiological conditions). As used herein, the term
"interfering molecule" refers to a binding molecule (e.g., a
circulating or cell surface receptor) that competes with
circulating antibodies for binding to an adaptor molecule and
prevents it from exerting its intended therapeutic effect (e.g., by
rapidly clearing the adaptor molecule from circulation). For
example, a glycosylated ligand moiety may comprise a gal antigen or
mimetic that has been chemically-modified to enhance preferential
binding by anti-Gal antibodies while minimizing undesirable binding
by a lectin (e.g., Galectin-3) or other interfering molecule.
Additionally or alternatively, a glycosylated ligand moiety may
comprise a gal antigen or mimetic that has been
chemically-modified, for example, to reduce enzymatic or chemical
degradation or facilitate covalent linkage. Exemplary modifications
include the addition of biologically inert protecting groups to
reactive hydroxyl groups on the sugar residues of a gal antigen,
e.g., via dehydrative coupling, reductive amination, or enzymatic
oxidation, e.g., with galactose oxidase. Protecting groups may be
added to the C-6' OH on the terminal Gal residue of a Gal epitope
(see, e.g., Andreana et al., Glycoconjugate J., 20: 107-118
(2004)). Exemplary protecting groups include amine (e g ,
aminopyridine) and oxime subsituents (e.g., O-Me-oxime, O-Et-oxime,
O-tBu-oxime, O-Bn-oxime and O-allyl-oxime). Alternatively, the
polar C-6' OH group can be replaced with a nonpolar hydrogen to
form a 6-deoxy-.alpha.-Gal derivative (see Janczuk et al.,
Carbohydrate Research, 337: 1247-1259 (2002), incorporated by
reference herein). In certain exemplary embodiments, the Gal
epitope can be modified (e.g., at the C-1 OH) with an amino
modifier (e.g., alkyl-NH2 substituent) to facilitate linkage to a
targeting moiety. Binding of anti-Gal antibodies to the modified
gal antigen can then be evaluated using art-recognized
methodologies (e.g., ELISA).
(d) Multivalent Adaptor Molecules
[0054] It is contemplated that a plurality of peptides or peptide
moieties of the sort disclosed herein could be connected to create
a composite adaptor molecule with increased avidity or valency.
Likewise, a peptide or peptide moiety of an adaptor molecule may be
attached to any number of other polypeptides, such as fluorescent
polypeptides, targeting polypeptides and polypeptides having a
distinct therapeutic effect.
II. Methods for Identifying High Affinity Adaptor Molecules
[0055] In certain aspects, the invention provides methods for
identifying an adaptor molecule with high binding affinity or
selectivity. The methods of the invention comprise (i) at least one
selection step to identify a high affinity targeting moiety (e.g.,
a targeting peptide moiety and/or ligand peptide moiety), and (ii)
and a linking step wherein the targeting and ligand moieties of the
adaptor molecule are linked to form the adaptor molecule.
[0056] In certain embodiments, the methods of the invention employ
ribosome or mRNA display as a selection step to identify one or
more targeting moieties of an adaptor molecule. A general overview
of ribosome and mRNA display methods is provided by Lipovsek and
Pluckthun (J. Immunological Methods, 290: 51-67 (2004)), hereby
incorporated by reference in its entirety. In preferred
embodiments, the targeting moiety (e.g., a peptide targeting
moiety) of the adapator molecule is identified using mRNA display.
An exemplary mRNA display methodology is depicted in FIG. 2.
Briefly, a starting library is obtained by, e.g., direct DNA
synthesis or through in-vitro or in-vivo mutagenesis. The double
stranded DNA library is then transcribed in-vitro (e.g., using T7
polymerase) and attached to a puromycin-like linker. In vitro
translation is carried out wherein the puromycin-like linker reacts
with the nascent translation product. The result, after
purification, is a highly diverse (.about.10.sup.13) library of
peptide-RNA fusion molecules. Reverse transcription generates a
cDNA/RNA hybrid, covalently linked to the transcribed peptide. This
complex is then selected for by using the target molecule (in the
case of a targeting peptide moiety) or an antibody (in the case of
a ligand peptide moiety). Peptides that bind the target or antibody
molecule (e.g., under stringent wash conditions) will be selected,
and the cDNA is easily eluted to identify the selected peptides.
The selection can be performed multiple times to identify high
affinity binders. It should be noted that the selection methodology
may be carried out under conditions such that intramolecular
disulfide bonds are present in the peptides during selections. In
other embodiments, the formation of disulfide bonds may be
prevented, if desired.
[0057] In additional or alternative embodiments, the methods of the
invention employ phage display and/or yeast display techniques as a
selection step to identify one or more targeting (e.g., peptide)
moieties of an adaptor molecule. Non-limiting examples of such
library screening methods are described, for example, in U.S. Pat.
Nos. 7,195,880; 6,951,725; 7,078,197; 7,022,479; 5,922,545;
5,830,721; 5,605,793, 5,830,650; 6,194,550; 6,699,658, each of
which is herein incorporated by reference in its entirety.
[0058] In other embodiments, the methods of the invention employ
libraries of synthetic peptides. Such synthetic peptides can be
chemically synthesized or enzymatically produced (e.g., by in vitro
translation from RNA or by enzymatic digestion of preexisting
proteins). In some embodiments, a library of synthetic peptides is
arrayed on a solid substrate (e.g., a glass slide).
[0059] In certain embodiments, the methods of the invention employ
mRNA libraries (e.g, mRNA display libraries) that encode randomized
peptides corresponding to a portion of a larger polypeptide that is
known to interact with a particular target or antibody molecule.
Exemplary mRNA display libraries are depicted in FIG. 3. For
example, a peptide library may comprise a population of linear
peptide molecules where the amino acid sequence of the peptides are
randomized at one or more amino acid positions (preferably at least
10 or more amino acid positions) within the molecule. This
randomized portion of the peptide sequence may be flanked by one or
more constant regions from the parent polypeptide.
[0060] In certain embodiments, the methods of the invention include
providing an mRNA display library that encodes a randomized
population of candidate targeting moieties (e.g., or peptides or
polypeptides). By way of example the targeting peptides may be
randomized peptides derived from a soluble ligand, e.g., VEGF. High
affinity targeting peptides are selected by screening
peptide-RNA-cDNA fusions from the library. Multiple selection
cycles are preferably performed to enrich the population of
molecules for those that bind to the target molecule (e.g., VEGF
receptor). By decreasing the concentration of target molecule in
each selection step, the peptides which bind to the target molecule
with highest affinity can be further enriched in the population.
Additional selection procedures that can be employed in each
selection step include: (1) contra-selection to eliminate
non-specific peptides; (2) competitive elution to identify
site-specific peptides; (3) and selection under specific solution
conditions (e.g., highly stringent wash conditions) to identify
stable peptides.
[0061] In certain embodiments, the members of a library are
modified prior to selection to include a coupling moiety which is
capable of reacting with a linker (e.g., a bifunctional linker) to
form a linking moiety. In other embodiments, the members of the
library are modified with a coupling moiety after the selection
step to facilitate linkage to the linking moiety. Examplary
coupling moieties include terminal amino acids (e.g., C- or
N-terminal cysteines or cysteine analogs) or amino acid side chains
(e.g., cysteine or cysteine analog side chains) which are capable
of reacting with a bifunctional linker of maleimide
functionality.
[0062] Once a high-affinity targeting moiety has been identified,
the peptide can then be linked via a linking moiety to a
pre-selected ligand moiety, thereby creating an adaptor molecule.
In certain embodiments, the ligand moiety has been pre-selected
using mRNA display methods. Alternatively, the targeting moiety can
be inserted as a constant region within a second mRNA display
library that encodes a randomized population of candidate ligands.
This second mRNA display library can then be subjected to further
selection steps wherein the library members are screened against an
antibody to identify an adaptor molecule. Thus, the candidate
ligands preferably correspond to a portion (e.g., an epitope) of an
antibody ligand. In one embodiment, an antibody ligand portion can
be an epitope of an antigen to which an antibody binds. In another
embodiment, the antibody ligand portion can be an idiotope of a
first antibody to which a second, anti-idiotypic, antibody binds.
In yet another embodiment, the antibody ligand portion can be an Fc
binding portion of an Fc binding protein (e.g., an Fc
receptor).
[0063] Having selected an adaptor molecule that binds with high
affinity and/or selectivity to both a target molecule and an
antibody molecule, the adaptor molecules can be evaluated for
ability to redirect antibody specificity to the target molecule.
For example, wherein the target molecule is cell surface molecule,
the ability of the adaptor molecule to induce an effector function
(e.g., antibody-dependent cellular cytotoxicity (ADCC) or
complement-dependent cytotoxicity (CDC) and cell killing can be
evaluated using art-recognized techniques.
[0064] In yet other embodiments, the members of a library are
linked to a ligand moiety prior to the selection step. For example,
each member of the library to be screened can be derivatized with a
Gal antigen and then subjected to a screening step to identify a
high affinity adaptor molecule. Said Gal antigens may be linked to
terminal amino acids or amino acid side chains of each peptide.
[0065] In yet other embodiments, an iterative selection process may
be employed wherein a targeting moiety is selected in a first
selection step (or first series of selection steps) and the
sequence of the targeting moiety is incorporated into the constant
region of an mRNA-peptide fusion to facilitate the selection of a
ligand moiety in a second selection step (or second series of
selection steps). For example, the first and second selection steps
(or series of selection steps) can be alternated in consecutive
rounds of selection to identify high affinity adaptor
molecules.
III. Methods for Synthesizing a High Affinity Adaptor Molecule.
[0066] Once the components of a high affinity adaptor molecule have
been identified using the methods described herein, they may be
produced using standard methods known in the art. For example,
peptides may be produced by recombinant DNA methods, inserting a
nucleic acid sequence (e.g., a cDNA) encoding the polypeptide into
a recombinant expression vector and expressing the DNA sequence
under conditions promoting expression. General techniques for
nucleic acid manipulation are described for example in Sambrook et
al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring
Harbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., Current
Protocols in Molecular Biology (Green Publishing and
Wiley-Interscience: New York, 1987) and periodic updates, herein
incorporated by reference. Appropriate cloning and expression
vectors for use with bacterial, fungal, yeast, and mammalian
cellular hosts can be found in Cloning Vectors: A Laboratory
Manual, (Elsevier, New York, 1985), the relevant disclosure of
which is hereby incorporated by reference. Other recombinant DNA
methods are described in U.S. Pat. Nos. 4,356,270 4,399,216,
4,506,013, 4,503,142, 4,952,682, 5,618,676, 5,854,018, 5,856,123,
5,919,651, and 6,455,275, which are all incorporated herein by
reference.
[0067] Adaptor molecules and their components may also be made by
chemical synthesis, using techniques that are well-known in the
art. For example, D-peptides can be synthesized using stepwise
addition of D-amino acids in a solid-phase synthesis method
involving the use of appropriate protective groups. Solid phase
peptide synthesis techniques commonly used for L-peptides are
described by Meinhofer, Hormonal Proteins and Peptides, vol. 2,
(New York 1983); Kent, et al., Ann. Rev. Biochem., 57:957 (1988);
Bodanszky et al., Peptide Synthesis, (2d ed. 1976); Atherton et al.
(1989) Oxford, England: IRL Press. ISBN 0199630674; Stewart et al.
(1984). 2nd edition, Rockford: Pierce Chemical Company, 91. ISBN
0935940030; and Merrifield (1963) J. Am. Chem. Soc. 85: 2149-2154
all of these references are incorporated herein by reference.
D-amino acids for use in the solid-phase synthesis of D-peptides
can be obtained from a number of commercial sources. D-peptides and
peptides that contain mixed L- and D-amino acids are known in the
art. Also, peptides containing exclusively D-amino acids
(D-peptides) have been synthesized. See Zawadzke et al., J. Am.
Chem. Soc., 114:4002-4003 (1992); Milton et al., Science 256:
1445-1448 (1992). Additional methods to make D-peptides have been
described in the art and can be found at least in WIPO Publication
No. WO/1997/013522, and U.S. Application No. 60/005,508, which are
both incorporated herein by reference.
[0068] The peptide of the present invention can be purified by
isolation/purification methods for proteins generally known in the
field of protein chemistry. Non-limiting examples include
extraction, recrystallization, salting out (e.g., with ammonium
sulfate or sodium sulfate), centrifugation, dialysis,
ultrafiltration, adsorption chromatography, ion exchange
chromatography, hydrophobic chromatography, normal phase
chromatography, reversed-phase chromatography, gel filtration, gel
permeation chromatography, affinity chromatography,
electrophoresis, countercurrent distribution or any combinations of
these. After purification, the peptides may be exchanged into
different buffers and/or concentrated by any of a variety of
methods known to the art, including, but not limited to, filtration
and dialysis. The purified polypeptide is preferably at least 85%
or 90% pure, more preferably at least 93% or 95% pure, and most
preferably at least 97%, 98%, or 99% pure. Regardless of the exact
numerical value of the purity, the peptide is sufficiently pure for
use as a pharmaceutical product.
EXEMPLIFICATION
[0069] The present disclosure is further illustrated by the figures
and the following examples, which should not be construed as
further limiting. The contents of all figures and all references,
patents and published patent applications cited throughout this
application are expressly incorporated herein by reference in their
entireties.
Example 1
Selection of High Affinity Anti-VEGF Peptides as Targeting Moiety
and Conjugation to an .alpha.Gal Ligand Moiety
[0070] Using the mRNA display method depicted in FIG. 2, a library
of randomized peptide sequence were screened for high affinity
binding to VEGF. Four high affinity anti-VEGF peptide sequences
(SEQ ID NOs 1-4) were selected. Each peptide was PEGylated at the
C-terminus with PEG.sub.2-NH2 or PEG.sub.2-Cys-NH2 moiety (see FIG.
4) to facilitate chemical coupling to Gal antigen (Bdi-(CH2)-NH2,
disacchride).
[0071] To facilitate chemical conjugation of each peptide to the
Gal disaccharide, a bifunctional linker with maleimide and
sulfo-NHS functionalities (Sulfo-SMCC, PIERCE) was employed. The
linker was reacted with the amino group present in the disaccharide
and a maleimide functionality in a sulfhydroxyl group of the
C-terminal cysteine residues of the peptide. To obtain the desired
compound and avoid any further reaction and formation of
by-products, the reaction was subjected to RP-HPLC purification
right after incubation. Ratios for amounts of reactants were
optimized to avoid formation of by-products. In an exemplary
synthesis, 6 mg Disaccharide compund B.sub.di--(CH.sub.2)--NH.sub.2
(.about.15 .mu.mol) was dissolved in 100 .mu.l 0.1 M Hepes buffer
containg 20% MeCN, pH 6.0; 1.3 mg Sulfo-SMCC Linker (.about.3
.mu.mol) was dissolved in 100 .mu.l 0.1 M Hepes buffer containing
20% MeCN, pH 6.0; and both solutions were mixed and incubated at
room temperature for 30 min under continuous rotation of the
reaction tube. 1 mg peptide 07-090 (07-090; lyophilisate, TFA
adduct, M.W. 3474 g/mol; .about.320 nmol) was dissolved in 1 ml
H.sub.2O/MeCN 80:20% Immediately after preparation the clear
peptide solution was added to the Linker-Disaccharide solution and
incubated at room temperature for additional 90 min under
continuous rotation of the reaction tube. The reaction mixture was
put on ice and an aliquot was analyzed on a RP-HPLC-C18 column. The
desired compound (B.sub.di--(CH.sub.2)--NH-Linker-S-07-090-peptide)
was purified by running a HPLC gradient from 0 to 50%; buffer A:
H.sub.2O/5% MeCN, 0.1% TFA, buffer B: H.sub.2O/5% MeCN, 0.1% TFA.
HPLC fractions were analyzed after dilution with 65% Methanol, 0.5%
formic acid by ESI-TOF-MS analysis and product fractions
identified. Relevant product peak fractions were frozen at
-80.degree. C. and subsequently lyophilized to complete dryness to
harbor the desired compound.
Example 2
Anti-VEGF-Specific Adaptor Molecules can Redirect Natural
Anti-.alpha.Gal Antibodies to VEGF
[0072] An assay was designed to test the ability of
.alpha.Gal-linked anti-VEGF peptides to redirect natural
antibodies, specific for .alpha.Gal, to bind to VEGF. The assay was
designed with recombinant VEGF on the solid phase. A dilution
series of ocGal-linked anti-VEGF peptides was then incubated with
the rVEGF followed by an incubation with sera from mice containing
high levels of anti-ocGal. The amount of bound anti-ocGal was then
indicated by an enzyme conjugated anti-mouse antibody. The presence
of enzyme was indicated by the addition of a colorometric substrate
and the increase in color was measured by determining the optical
density at 490 nm The data, set forth in FIG. 8, clearly shows that
the .alpha.Gal-linked anti-VEGF peptides could redirect natural
anti-.alpha.Gal antibodies to VEGF since a decrease in the amount
of peptide or antisera resulted in a decrease in the optical
density.
Sequence CWU 1
1
7129PRTArtificial SequenceSynthetic VEGF-binding peptide 1Gly Val
Gln Glu Asp Val Ser Ser Thr Leu Gly Ser Trp Val Leu Leu 1 5 10 15
Pro Phe His Arg Gly Thr Arg Leu Ser Val Trp Val Thr 20 25
229PRTArtificial SequenceSynthetic VEGF-binding peptide 2Gly Gly
Phe Glu Gly Leu Ser Gln Ala Arg Lys Asp Gln Leu Trp Leu 1 5 10 15
Phe Leu Met Gln His Ile Arg Ser Tyr Arg Thr Ile Thr 20 25
329PRTArtificial SequenceSynthetic VEGF-binding peptide 3Gly Val
Gly Gly Ser Arg Leu Glu Ala Tyr Lys Lys Asp His Arg Val 1 5 10 15
Phe Gln Met Ala Trp Leu Gln Tyr Tyr Trp Ser Thr Thr 20 25
433PRTArtificial SequenceSynthetic VEGF-binding peptide 4Gly Ser
Gly Ser Gly Asn Ala Leu His Trp Val Cys Ala Ser Asn Ile 1 5 10 15
Cys Trp Arg Thr Pro Trp Ala Gly Gln Leu Trp Gly Leu Val Arg Leu 20
25 30 Thr 54PRTArtificial SequenceSynthetic peptide 5Tyr Trp Arg
Tyr 1 64PRTArtificial SequenceSynthetic peptide 6Thr Trp Arg Tyr 1
74PRTArtificial SequenceSynthetic peptide 7Arg Trp Arg Tyr 1
* * * * *