U.S. patent application number 15/937809 was filed with the patent office on 2018-08-02 for synthetic antibody mimic peptides.
The applicant listed for this patent is Formurex, Inc.. Invention is credited to Bhaskara Rao JASTI, Hyun JOO, Xiaoling LI, Sameer SACHDEVA, Dan SU, Jerry TSAI, Yu ZHENG.
Application Number | 20180215828 15/937809 |
Document ID | / |
Family ID | 58498897 |
Filed Date | 2018-08-02 |
United States Patent
Application |
20180215828 |
Kind Code |
A1 |
LI; Xiaoling ; et
al. |
August 2, 2018 |
SYNTHETIC ANTIBODY MIMIC PEPTIDES
Abstract
The present disclosure relates to compositions and methods
comprising peptide molecules that mimic the binding and functional
properties of native antibodies relative to their respective
targets. Some embodiments comprise peptide-drug conjugates (PDCs)
comprising the mimic peptides disclosed herein. The targets of
these mimic peptides include epidermal growth factor receptor
(EGFR), and human epidermal growth factor receptor 2 (HER2),
vascular endothelial growth factor (VEGF), programmed cell death
protein 1 (PD-1), and programmed death-ligand 1 (PD-L1). The
present disclosure comprises application of the knob-socket
computational model to design antibody mimics for proteins.
Inventors: |
LI; Xiaoling; (Dublin,
CA) ; TSAI; Jerry; (Stockton, CA) ; JASTI;
Bhaskara Rao; (Stockton, CA) ; JOO; Hyun;
(Stockton, CA) ; ZHENG; Yu; (Stockton, CA)
; SU; Dan; (Fremont, CA) ; SACHDEVA; Sameer;
(Piscataway, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Formurex, Inc. |
Stockton |
CA |
US |
|
|
Family ID: |
58498897 |
Appl. No.: |
15/937809 |
Filed: |
March 27, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14882436 |
Oct 13, 2015 |
9957325 |
|
|
15937809 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/74 20130101;
C07K 7/08 20130101; C07K 2318/00 20130101; A61K 38/00 20130101;
G01N 2333/71 20130101; C07K 16/2863 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 7/08 20060101 C07K007/08 |
Claims
1. An isolated antibody mimic peptide comprising at least one of a
motif sequence WX1EX2PX3FYX4YX5A (SEQ ID NO: 78) or the reverse of
said motif sequence, said motif sequence having N amino acids,
wherein the length of said motif sequence ranges from 7 to 25 amino
acids; wherein P is at amino acid position N/2 of the motif
sequence or next thereto; wherein each of X1, X2, X3, X4, and X5 is
any of 0 to 8 amino acids, and wherein the FY in the motif sequence
can be replaced with YW, YL, YI, YV, YF, WY, LY, IY, VY, YY, TYY,
TYW, TYL, TYI, TYV, TYF, TWY, TLY, TIY, TVY, or TFY.
2. The antibody mimic peptide of claim 1, wherein the X1 is SG.
3. The antibody mimic peptide of claim 2, wherein X2 is NG,
4. The antibody mimic peptide of claim 3, wherein X3 is G,
5. The antibody mimic peptide of claim 4, wherein X4 is D.
6. An isolated antibody mimic peptide comprising a reverse sequence
of SEQ ID NO: 1-22.
7. The antibody mimic peptide of claim 1, wherein said antibody
mimic peptide is conjugated to a substance.
8. The antibody mimic peptide of claim 2, wherein said antibody
mimic peptide is conjugated to a substance.
9. The antibody mimic peptide of claim 3, wherein said antibody
mimic peptide is conjugated to a substance.
10. The antibody mimic peptide of claim 4, wherein said antibody
mimic peptide is conjugated to a substance.
11. The antibody mimic peptide of claim 5, wherein said antibody
mimic peptide is conjugated to a substance.
12. The antibody mimic peptide of claim 6, wherein said antibody
mimic peptide is conjugated to a substance.
13. A pharmaceutical composition comprising the antibody mimic
peptide of claim 1 and a pharmaceutically acceptable carrier.
14. A method for treating a mammal comprising the step of
administering to the mammal an effective amount of the composition
of claim 13.
15. A kit for diagnosing a disease comprising an antibody mimic
peptide of claim 1 binding to EFGR.
16. A method for designing an antibody mimic peptide of claim 1
comprising identifying and mapping of a binding surface based on
interactions between antibody and epitope; and selecting the amino
acids with high interaction frequency to form a peptide.
17. The method of claim 16, wherein the antibody and epitope
interaction is based on crystal structure information.
18. The method of claim 16, wherein the peptide is 10-30 amino
acids.
19. The method of claim 16, wherein the interaction frequency is
determined by binding energy.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/882,436, filed Oct. 13, 2015, which is herein incorporated
by reference in its entirety.
BACKGROUND
Field of the Disclosure
[0002] The present disclosure relates to compositions and methods
comprising peptide molecules that mimic the binding and functional
properties of native antibodies relative to their respective
targets. These peptide molecules may be referred to herein
synonymously as antibody mimics, mimic peptides, or simply mimics.
Some embodiments comprise peptide-drug conjugates (PDCs) comprising
the mimic peptides disclosed herein. Certain embodiments comprise
methods of using the mimic peptides disclosed herein as targeting
moieties and/or binding ligands, alone or as part of a PDC, such as
in drug delivery or diagnostic assays. In various embodiments of
the present disclosure, the targets of these mimic peptides can
include but are not limited to epidermal growth factor receptor
(EGFR), and human epidermal growth factor receptor 2 (HER2),
vascular endothelial growth factor (VEGF), programmed cell death
protein 1 (PD-1), and programmed death-ligand 1 (PD-L1).
Sequence Listing
[0003] This application hereby incorporates by reference the
material of the electronic Sequence Listing filed concurrently
herewith. The material in the electronic Sequence Listing is
submitted as a text (.txt) file entitled "LXL002_ST25.txt" created
on Oct. 13, 2015, which has a file size of 20 KB, and is herein
incorporated by reference in its entirety.
Brief Description of Related Art
[0004] Antibodies, as well as their smaller constituent fragments,
have found wide application for use in diagnostics and
therapeutics. One example is the use of antibodies alone in
treatment of diseases such as cancer. Another example is the use of
an antibody as a composite part of an antibody-drug conjugate
(ADC), wherein the antibody can act as the targeting agent in
targeted drug delivery compositions and methods.
Antibodies/antibody fragments are especially amenable for use as
targeting agents because they are highly specific for their target
receptors and bind with high affinity. A particular type of
targeted drug delivery therapy is anti-cancer therapy. The primary
goal of anti-cancer therapy generally is of course to kill cancer
cells without affecting normal, healthy cells. Targeted drug
delivery methods are particularly effective in treating cancer
because they specifically target cancer cells which overexpress
certain antigen receptors, without harming or with less harm to
normal cells which do not express the antigens or do so to a lesser
extent. In these targeted drug delivery methods generally an
anti-cancer drug is attached to a targeting moiety, such as an
antibody or antibody fragment, thus creating an ADC which targets
and binds to the cell receptor and thus introduces the drug to the
cell.
[0005] A number of monoclonal antibodies are currently known and
approved for treating various diseases, such as cancer. These
include cetuximab (trade name ERBITUX.RTM., Bristol-Myers
Squibb/Merck KGaA/Eli Lilly), which targets EGFR; pertuzumab (also
known as 2C4, trade name PERJETA.RTM., Genentech), which targets
HER2; and, trastuzumab (trade names HERCLON.RTM. and
HERCEPTIN.RTM., Genentech), which also targets HER2. In the example
of cancer treatment, the antibody selectively binds a target
antigen on the cancer cell with high affinity, and exerts its
effects by various means including blocking cell signaling,
antibody-dependent cytotoxicity, or complement dependent
cytotoxicity.
[0006] Antibody treatment alone, however, has proven inadequate in
many cases, due to reasons including insufficient cell killing,
resistance to the antibodies, etc. For this reason antibody
treatment is frequently in conjunction with chemotherapy. One
method of treatment is to administer separately an antibody and a
drug. Another is the use of an antibody-drug conjugate (ADC), in
which an antibody is linked (conjugated) to a drug compound. This
brings together in one molecule the specificity and affinity of the
antibody for a particular cell type, and the toxicity for that cell
of a potent anti-cancer drug. In the case of cancer treatment,
generally the antibody of the ADC targets a cell receptor that is
overexpressed in tumor cells relative to normal, healthy cells.
Thus, tumor cells are preferentially targeted for the cytotoxic
effects of the drug.
[0007] At least two ADCs are currently in the market as therapeutic
agents: brentuximab-vedotin (trade name ADCETRIS.RTM., Seattle
Genetics), in which brentuximab which targets CD30-expressing cells
is conjugated to monomethyl auristatin E (MMAE), a potent
anti-microtubule agent; and, ado-trastuzumab-emtansine (trade name
KADCYLA.RTM., Genentech), in which trastuzumab is conjugated to one
or more DM1 molecules (DM1 being a derivative of maytansine
comprising a linker moiety, as described below), an inhibitor of
microtubule formation. Treatment of trastuzumab-resistant patients
with the ADC trastuzumab-emtansine, for example, results in
increased survival. See S. Verma et al., N. Engl. J. Med. (2012)
367:1783-1791.
[0008] ADCs generally comprise antibody, drug, and linker moieties,
and may additionally comprise spacer, attachment, and other
moieties. The choice of the drug and the linker that conjugates the
antibody to the drug are key determinants of the activity and
tolerability of the ADC. The linker should be such that it remains
stable in the blood and before the ADC attaches to the target cell.
Then, depending on the particular mode of action of the drug, it
may be preferable that the linker remain intact upon
antibody-receptor binding, or that it be cleaved at some point
after binding; e.g., immediately after binding and before
internalization of the ADC within the cell, or after
internalization to release the drug inside the cell. Thioether
linkers, for example, are relatively non-cleavable linkers--the
mode of action of ADCs comprising these linkers depends on the
degradation of the antibodies (not cleavage of the linker) to
release the drug, which remains attached to the linker. As an
example, the ADC ado-trastuzumab-emtansine uses a non-cleavable
thioether linker to conjugate the maytansinoid drug to the
antibody. As another example, linkers comprising hydrazone are
destabilized at low pH and are thus cleaved in the low-pH
environment of the cell lysosome, releasing drug from antibody.
Gemtuzumab-ozogamicin (trade name MYLOTARG.RTM., Wyeth), an ADC
comprising a monoclonal antibody to CD33 in combination with the
potent drug N-acetyl-.gamma.-calicheamicin, is conjugated via a
hydrazone linker which is selectively cleaved in the acidic
intracellular environment of the cancer cell. As another example,
linkers comprising disulphide bond(s) are destabilized in a
reducing environment and are thus cleaved in the reducing
environment of the cytosol, releasing drug from antibody.
[0009] Linkers that are cleaved by peptidases can also be used in
ADCs, such as for example dipeptide linkers. As an example, a
citrulline-valine linker is used in the ADC brentuximab-vedotin.
Cleavage of this linker is achieved only by proteases inside the
cancer cell lysosome, thus imparting a greater stability to this
ADC outside the cell.
[0010] Generally, the cytotoxic agents currently used as the
"drugs" in ADCs are highly potent, toxic and non-specific molecules
which thus cannot be used in therapy alone because of their
toxicity to normal, healthy cells. These cytotoxic agents include
but are not limited to auristatins, maytansinoids and
calcheamicins, with IC.sub.50 values in the picomolar range.
Monomethyl auristatin E (MMAE) is an auristatin which is a
component of brentuximab-vedotin, as well as a number of ADCs in
clinical trials. Mertansine, a maytansinoid, is a component of the
approved ADC ado-trastuzumab-emtansine. Auristatins and
maytansinoids exert their cytotoxic affect by binding to tubulin
and inhibiting its polymerization, which leads to apoptosis of the
target cell.
[0011] The mode of activity of ADCs generally is as follows. Upon
binding of the antibody moiety to the target antigen (usually a
cell receptor), the antigen and ADC undergo receptor-mediated
endocytosis. After internalization within the cell, the ADC is
degraded (which could occur in the lysosome or the cytosol,
depending on the ADC), and the drug is released and causes the
cytotoxic effect and hence cell death. The rate and extent of
internalization are important factors, as they impact the efficacy
of the ADC. Furthermore, the number of receptors on the cell is an
important factor in ADC efficacy--clearly, ADCs specific for
receptors that are overexpressed on certain cells, such as cancer
cells, will result in preferential targeting of these cells.
[0012] The extensive use of full-length antibodies for applications
such as treatment and targeted drug delivery, however, has been
limited by various factors, including the high cost and time
involved in producing antibodies, and their large molecular weight
which presents several drawbacks, including their limited ability
to penetrate tumor tissues, which is often a requirement in ADC
systems for the effective toxicity of drugs that act
intracellularly. Thus there is a need for alternatives to
antibodies/antibody fragments, which should have binding
specificity and affinity properties similar to the native antibody
but not require the lengthy and complicated processes involved in
generating antibodies, especially when they are to be used in
therapy and/or as targeting agents.
[0013] Some synthetic alternatives to antibodies/antibody fragments
have been developed. Although these have low molecular weight
compared to antibodies, they are based on conventional, full-sized
antibodies, and/or identified and derived through a long and
tedious in vitro screening process. Therefore, not only are
alternatives to antibodies/antibody fragments needed, but also
methods of rationally designing (such as in silico) molecules that
functionally mimic antibodies in their binding affinity for
target.
[0014] The present disclosure describes compositions and methods
comprising effective antibody mimic peptides, which overcome the
limitations outlined above. The use of the antibody mimic peptides
described herein include diagnostic agents, reagents, and targeting
moieties in peptide-drug conjugates (PDCs), which are the
equivalent of ADCs except that peptide mimics are used in place of
antibodies, and are as effective as their corresponding ADCs.
SUMMARY
[0015] The present disclosure comprise an isolated peptide
comprising at least one of a motif sequence
WX.sub.1EX.sub.2PX.sub.3FYX.sub.4YX.sub.5A or the reverse of said
motif sequence, said motif sequence having N.sub.N amino acids,
wherein P is proline. W is tryptophan. E is glutamic acid, F is
phenylalanine. Y is tyrosine, A is alanine, and each of X.sub.1,
X.sub.2, X.sub.3, X.sub.4 and X.sub.5 is any of 0 to 8 amino acids;
and, wherein P is at amino acid position N.sub.N/2 of the motif
sequence (i.e., in the middle) or next thereto.
[0016] Other embodiments comprise the peptide
WX.sub.1EX.sub.2PX.sub.3FYX.sub.4YX.sub.5A, but wherein the FY in
the motif sequence is replaced with YW, YL, YI, YV, YF, WY, LY, IY,
VY, YY, TYY, TYW, TYL, TYI, TYV, TYF, TWY, TLY, TIY, TVY, or TFY,
wherein T is threonine, L is leucine, I is isoleucine, and V is
valine. In some embodiments the length of the motif sequence ranges
from 7 to 25 amino acids. In some embodiments, the motif sequence
comprises 7, 10, 15, 20, or 25 amino acids.
[0017] Some embodiments of the present disclosure comprise an
isolated peptide comprising at least one motif sequence comprising
a first sequence QX.sub.1SNX.sub.2PARX.sub.3TDX.sub.4, a second
sequence QX.sub.5SNX.sub.6PARX.sub.7TDX.sub.8GP, the reverse of
said first sequence, or the reverse of said second sequence,
wherein the motif sequence has N.sub.N amino acids, wherein P is
proline, Q is glutamine, S is serine, N is asparagine, A is
alanine, R is arginine, T is threonine, D is aspartic acid, G is
glycine, and each of X.sub.1, X.sub.2, X.sub.3. X.sub.4. X.sub.5.
X.sub.6. X.sub.7, and X.sub.8 is any of 0 to 8 amino acids; and,
wherein P is at amino acid position N.sub.N/2 of the motif sequence
(i.e., in the middle) or next thereto. In some embodiments the
length of this motif sequence ranges from 8 to 25 amino acids. In
some embodiments, the motif sequence comprises 8, 10, 15, 20, or 25
amino acids.
[0018] Some embodiments comprise an isolated peptide comprising a
sequence selected from group consisting of the sequences listed in
Table 1; i.e., SEQ ID NO. 1, SEQ ID NO. 2. SEQ ID NO. 3, SEQ ID NO.
4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID
NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12. SEQ ID NO. 13.
SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16. SEQ ID NO. 17, SEQ ID
NO. 18, SEQ ID NO. 19. SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22,
SEQ ID NO. 23, and SEQ ID NO. 24.
[0019] Some embodiments comprise an isolated peptide comprising a
sequence selected from group consisting of the sequences listed in
Table 7; i.e., SEQ ID NO. 26. SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID
NO. 29, SEQ ID NO. 30. SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33,
SEQ ID NO. 34, SEQ ID NO. 35. SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID
NO. 38. SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41. SEQ ID NO. 42,
SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45 and SEQ ID NO. 46.
[0020] Some embodiments comprise an isolated peptide comprising a
sequence selected from group consisting of the sequences listed in
Tables 8, 9 and 10; i.e., SEQ ID NO. 48. SEQ ID NO. 49, SEQ ID NO.
50, SEQ ID NO. 51. SEQ ID NO. 52, SEQ ID NO. 53. SEQ ID NO. 54, SEQ
ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57. SEQ ID NO. 58, SEQ ID NO.
59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62. SEQ ID NO. 63, SEQ
ID NO. 64, SEQ ID NO. 65. SEQ ID NO. 66, SEQ ID NO. 67. SEQ ID NO.
68, SEQ ID NO. 69, SEQ ID NO. 70. SEQ ID NO. 71, SEQ ID NO. 72, SEQ
ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76 and SEQ ID
NO. 77.
[0021] Some embodiments comprise one or more of the peptides
described herein comprising a motif that effectively targets EGFR
or HER2. Some embodiments comprise a peptide as described above
conjugated to a substance wherein the substance is a therapeutical
or a detection moiety. In some embodiments the drug is an
anti-cancer drug. In some embodiments the peptide is conjugated to
MMAE. Some embodiments comprise a method for detecting a disease
associated with EGFR. HER2, VEGF, PD-1, or PD-L1 expression, or a
combination thereof, comprising the use of one or more of the
peptides described herein.
[0022] Some embodiments of the present disclosure comprise a kit
for research reagent or diagnosing a disease associated with EGFR,
HER2, VEGF. PD-1, or PD-L1 expression, using one or more of the
peptides described herein. Some embodiments comprise a method for
treating a disease associated with EGFR, HER2. VEGF. PD-1, or PD-L1
comprising the use of one or more of the peptides described herein
and/or their conjugates. Some embodiments comprise methods of cell
killing comprising a peptide conjugate as described herein.
[0023] Further embodiments of the present disclosure comprise
methods of using peptides disclosed herein as delivery vehicles. In
some embodiments, the methods comprise the use of peptides
disclosed herein in protein-protein interactions. Specific
embodiments comprise methods of targeted drug delivery,
diagnostics, and/or therapeutics comprising the peptides disclosed
herein. Some embodiments comprise methods of treating or preventing
cancer comprising the peptides disclosed herein. Certain
embodiments comprise peptide-drug conjugates (PDC) comprising one
or more of the peptides disclosed herein conjugated to drug.
Additional embodiments comprise targeted delivery agents comprising
said peptide-drug conjugates. Some embodiments comprise therapeutic
methods comprising said targeted delivery agents.
[0024] In another aspect, the present invention describes the
application of the knob-socket computational model to design
antibody mimics for proteins including EGFR, HER2, VGEF, PD-1,
podoplanin, etc. The present invention provide a method for
designing an antibody mimic peptide comprising identifying and
mapping of binding surface based on interactions between antibody
and epitope, and selecting the amino acids with high interaction
frequency to form a peptide wherein the peptide is 10-30 amino
acids in length. The antibody and epitope interaction is based on
crystal structure information.
BRIEF DESCRIPTION OF THE FIGURES
[0025] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0026] FIG. 1A shows a ribbon diagram of the cetuximab-EGFR epitope
interface, and FIG. 1B shows a schematic diagram of the
cetuximab-EGFR epitope interaction, mapping the various knobs and
sockets.
[0027] FIG. 2 shows a schematic diagram of an antibody mimic
(WX.sub.1EX.sub.2FYX.sub.3YX.sub.4A. SEQ ID NO: 82)-EGFR epitope
interaction, showing various knobs and sockets.
[0028] FIG. 3 shows a docking image of the Pep11 mimic peptide in
EGFR.
[0029] FIG. 4 shows confocal images of treated cells of cell lines
A431, MDA-MB-468 and HEK 293. The AlexaFluor column represents
cells treated with only membrane dye, FITC-Pep11 displays cells
treated with FITC-conjugated Pep11 peptide, and Overlay represents
a merge of the AlexaFluor and FITC-PEP11 images.
[0030] FIG. 5 shows confocal images of treated cells of cell lines
A431, MDA-MB-468 and HEK 293. The AlexaFluor column represents
cells treated with only membrane dye; FITC-Pep25 displays the cells
treated with FITC-conjugated peptide Pep25, the scrambled peptide;
and, Overlay column represent a merge of the AlexaFluor and
FITC-Pep25 images.
[0031] FIG. 6 shows confocal images of treated cells from cell
lines A431 and MDA-MB-468, demonstrating treatment with Pep11 after
treatment with cetuximab. The AlexaFluor column represents cells
treated with only membrane dye; cetuximab+FITC-Pep11 displays cells
treated with FITC-conjugated peptide Pep11 after blocking the EGFR
by cetuximab for 30 min.; and, Overlay represents a merge of the
AlexaFluor and cetuximab+FITC-Pep11 images.
[0032] FIG. 7 shows flow cytometry results displaying the mean
fluorescent intensity (MFI) of cells from cell lines A431,
MDA-MB-468 and HEK 293, treated with different mimic peptides (as
listed) or untreated ("Cells only").
[0033] FIG. 8A shows a typical surface plasmon resonance (SPR)
sensogram. FIG. 8B shows sensogram results of surface-immobilized
EGFR treated with Pep11 (RU=resonance units; s=seconds).
[0034] FIG. 9 shows sensogram results of surface-immobilized bovine
serum albumen (BSA) treated with scrambled peptide, Pep25.
[0035] FIG. 10 shows SPR sensogram results of surface-immobilized
EGFR treated with scrambled peptide, Pep25.
[0036] FIG. 11 shows the interaction of the first tyrosine residue
in Pep11 with EFGR (A), and the interaction of the first tyrosine
residue in Pep22 with EGFR (B).
[0037] FIG. 12 shows the optical density (OD) for total and
phosphorylated EGFR at varying concentrations, as analyzed by
ELISA.
[0038] FIG. 13 shows the percentage of inhibition of
phosphorylation by different antibody mimic peptides and cetuximab
after stimulation of cells by 50 ng/ml EGF.
[0039] FIG. 14 shows synthesis of auristatin E.
[0040] FIG. 15 shows synthesis of 5-benzoylpentanoic ester of
auristatin E (BPA).
[0041] FIG. 16 shows synthesis of a hydrazone intermediate.
[0042] FIG. 17 shows synthesis of a peptide (SEQ ID NO: 83) drug
conjugate.
[0043] FIG. 18 shows pH hydrolysis studies of PDC at pH 5 and
7.4.
[0044] FIG. 19 shows the IC.sub.50 value of AE, peptide and PDC on
A431 cell line.
[0045] FIG. 20 shows the IC.sub.50 value of AE, peptide and PDC on
cells of the MDA-MB-468 cell line.
[0046] FIG. 21 shows the IC.sub.50 value of AE, peptide and PDC on
HEK 293 cell line.
[0047] FIG. 22 shows percent viability after cetuximab treatment on
different cell lines.
[0048] FIG. 23 shows a 2-D plot of the binding interface between
pertuzumab and HER2 using the Knob-Socket model.
DETAILED DESCRIPTION
[0049] The present disclosure describes peptides that can serve as
synthetic alternatives to antibodies and antibody fragments; i.e.,
antibody mimic peptides, or peptides. These peptides can be used in
a wide variety of applications including, for example but without
limitation, as targeting or delivery agents, in imaging,
therapeutics and diagnostics, for various diseases or disorders,
and as reagents in research. These peptides can be used alone or in
conjunction with other compounds. As one example, they can be
conjugated to drugs in targeted drug delivery systems, such as in
peptide-drug conjugates (PDC) for treating cancer or other
diseases. As yet another example, they can be conjugated to probes
for diagnostic or research applications.
[0050] The present invention provides a rational and novel design
of peptide antibody mimics based on the crystal structure and
utilizing Knob-Socket computational design. This model represents
the complexities of packing between two molecules. There are three
types of sockets: (1) free socket, favoring only intra-molecule
packing; (2) filled socket, favoring interaction with knobs; and
(3) non-socket, disfavoring secondary structure. The amino acid
propensities in these three socket classes essentially represent an
amino acid for structure in packing. The Knob-Socket model was
applied to map and identify socket residues, and peptide sequences
were designed based on the best fitting knobs into the sockets
identified on the receptor epitopes.
[0051] Specifically, the invention dealt with the identification of
the binding surface based on the interactions between antibody and
epitope using crystal information from protein database or mapping
the binding surface by simulation/modeling. After the
identification and mapping of the binding surface, the surface is
reduced to a two dimensional map by using the knob and socket
model. Based on the probability of interactions between knobs and
sockets, the antibody mimic molecules are designed by selecting the
amino acids with high interaction frequency to form a peptide that
is composed of 10 to 30 amino acids. After screening using the
docking software Molecular Operating Environment (MOE), the
antibody mimic molecules are then synthesized using a solid phase
peptide synthesis approach. These molecules are characterized for
their binding specificity, affinity, and potential biological
functions.
[0052] Functional alternatives to full-length antibodies are known
and include antibody fragments, synthetic peptides, and synthetic
combinations of the two; e.g., Fab, F(ab')2, scFv, minibodies,
peptibodies and antibody mimic peptides. However, the methods of
identifying and designing these alternatives are still based on
conventional antibodies and/or through long, tedious and costly
screening techniques such as phage, ribosome and mRNA display.
[0053] The synthetic peptides disclosed herein have the
functionality of known antibodies, and thus can be used in
applications where currently an antibody would be used, with
equivalent or better efficacy, while circumventing the
above-described limitations. Two examples of such applications
where a peptide of the present disclosure can be utilized are where
an antibody itself is administered.
[0054] The invention also provides pharmaceutical compositions
comprising a peptide of the invention and a pharmaceutically
acceptable carrier. The pharmaceutical compositions comprising
peptide of the invention are for use in, but not limited to,
diagnosing, detecting, or monitoring a disorder; in preventing,
treating, managing, or ameliorating a disorder or one or more
symptoms thereof; and/or in research. In a specific embodiment, a
composition comprises one or more peptides of the invention.
[0055] In another embodiment, the pharmaceutical composition
comprises one or more peptides of the invention for treating a
disorder in peptide activity is detrimental. Preferably, the
prophylactic or therapeutic agents known to be useful for or having
been or currently being used in the prevention, treatment,
management, or amelioration of a disorder, such as cancer or a
tumor, or one or more symptoms thereof. In accordance with these
embodiments, the composition may further comprise of a carrier,
diluent, or excipient.
[0056] The peptide of the invention can be incorporated into
pharmaceutical compositions suitable for administration to a
subject. Typically, the pharmaceutical composition comprises a
peptide of the invention and a pharmaceutically acceptable carrier.
As used herein, "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. Examples of
pharmaceutically acceptable carriers include one or more of water,
saline, phosphate buffered saline, dextrose, glycerol, ethanol and
the like, as well as combinations thereof. In many cases, it will
be preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Pharmaceutically acceptable carriers may further
comprise minor amounts of auxiliary substances such as wetting or
emulsifying agents, preservatives or buffers, which enhance the
shelf life or effectiveness of the antibody or antibody
portion.
[0057] Various delivery systems are known and can be used to
administer one or more peptide of the invention or the combination
of one or more peptides of the invention and a prophylactic agent
or therapeutic agent useful for preventing, managing, treating, or
ameliorating a disorder or one or more symptoms thereof; e.g.,
reducing tumor angiogenesis, encapsulation in liposomes,
microparticles, microcapsules, etc. Methods of administering a
prophylactic or therapeutic agent of the invention include, but are
not limited to, parenteral administration (e.g., intradermal,
intramuscular, intraperitoneal, intravenous and subcutaneous),
epidural administration, intratumoral administration, and mucosal
administration (e.g., intranasal and oral routes). In addition,
pulmonary administration can be employed; e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309;
5,934,272; 5,874,064; 5,855,913; 5,290,540; and, 4,880,078; and PCT
Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO
98/31346, and WO 99/66903, each of which is incorporated herein by
reference in their entireties.
[0058] In one embodiment, a peptide of the invention, combination
therapy, or a composition of the invention is administered using
pulmonary drug delivery technology (see, e.g., Alkermes, Inc.,
Cambridge, Mass., US). In a specific embodiment, prophylactic or
therapeutic agents of the invention are administered
intramuscularly, intravenously, intratumorally, orally,
intranasally, pulmonary, or subcutaneously. The prophylactic or
therapeutic agents may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local.
[0059] In a specific embodiment, it may be desirable to administer
the prophylactic or therapeutic agents of the invention locally to
the area in need of treatment. This may be achieved by, for example
and not by way of limitation, local infusion, by injection, or by
means of an implant, said implant being of a porous or non-porous
material, including membranes and matrices, such as sialastic
membranes, polymers, fibrous matrices, or collagen matrices. In one
embodiment, an effective amount of one or more peptides of the
invention antagonists is administered locally to the affected area
to a subject to prevent, treat, manage, and/or ameliorate a
disorder or a symptom thereof. In another embodiment, an effective
amount of one or more peptides of the invention is administered
locally to the affected area in combination with an effective
amount of one or more therapies (e.g., one or more prophylactic or
therapeutic agents) other than a peptide of the invention of a
subject to prevent, treat, manage, and/or ameliorate a disorder or
one or more symptoms thereof.
[0060] In another embodiment, the prophylactic or therapeutic agent
can be delivered in a controlled release or sustained release
system. In one embodiment, a pump may be used to achieve controlled
or sustained release. See, e.g., Langer, Science, 249: 1527-1533
(1990); Sefton, CRC Crit. Rev. Biomed. Eng., 14: 201-240 (1987);
Buchwald et al., Surgery, 88: 507-516 (1980); and, Saudek et al.,
N. Engl. J. Med., 321: 574-579 (1989). In another embodiment,
polymeric materials can be used to achieve controlled or sustained
release of the therapies of the invention. See, e.g., Goodson, J.
M, Medical Applications of Controlled Release, Vol. II,
Applications and Evaluations (Langer and Wise, eds., CRC Press
Inc., Boca Raton, 1984), chapter 6, pages 115-138; Controlled Drug
Bioavailability, Drug Product Design and Performance (Smolen and
Ball, eds., Wiley, New York, 1984); and, Langer and Peppas, J.
Macromol. Sci. Rev. Macromol. Chem. Phys., C23:61-126 (1983). See
also, Levy et al., Science, 228:190-192 (1985); During et al., Ann.
Neurol., 25:351-356 (1989); Howard et al., J. Neurosurg.,
71:105-112 (1989); U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015;
5,989,463; and, 5,128,326; and, PCT Publication Nos. WO 99/15154
and WO 99/20253. Examples of polymers used in sustained release
formulations include, but are not limited to, poly(2-hydroxy ethyl
methacrylate), poly(methyl methacrylate), poly(acrylic acid),
poly(ethylene-co-vinyl acetate), poly(methacrylic acid),
polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),
poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol),
polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and
polyorthoesters. In a preferred embodiment, the polymer used in a
sustained release formulation is inert, free of leachable
impurities, stable on storage, sterile, and biodegradable. In yet
another embodiment, a controlled or sustained release system can be
placed in proximity of the prophylactic or therapeutic target, thus
requiring only a fraction of the systemic dose. See, e.g., Goodson,
Medical Applications of Controlled Release (1984), pages
115-138.
[0061] Controlled release systems are discussed in the review by
Langer (Science, 249:1527-1533 (1990)). Any technique known to one
of skill in the art can be used to produce sustained release
formulations comprising one or more therapeutic agents of the
invention. See, e.g., U.S. Pat. No. 4,526,938; PCT Publication Nos.
WO 91/05548 and WO 96/20698; Ning et al., "Intratumoral
Radioimmunotherapy of a Human Colon Cancer Xenograft Using a
Sustained-Release Gel," Radiother. Oncol., 39:179-189 (1996); Song
et al., "Antibody Mediated Lung Targeting of Long-Circulating
Emulsions," PDA J. Pharm. Sci. Tech., 50:372-377 (1996); Cleek et
al., "Biodegradable Polymeric Carriers for a bFGF Antibody for
Cardiovascular Application," Proceed. Intl. Symp. Control. Rel.
Bioact. Mater., 24:853-854 (1997); and, Lam et al.,
"Microencapsulation of Recombinant Humanized Monoclonal Antibody
for Local Delivery," Proceed. Intl. Symp. Control Rel. Bioact.
Mater., 24:759-760 (1997), each of which is incorporated herein by
reference in their entireties.
[0062] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include, but are not limited
to, parenteral (e.g., intravenous), intradermal, subcutaneous,
oral, intranasal (e.g., inhalation), transdermal (e.g., topical),
transmucosal, and rectal administration. In a specific embodiment,
the composition is formulated in accordance with routine procedures
as a pharmaceutical composition adapted for intravenous,
subcutaneous, intramuscular, oral, intranasal, or topical
administration to human beings. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lignocaine to
ease pain at the site of the injection.
[0063] If compositions of the invention are to be administered
topically, the compositions can be formulated in the form of an
ointment, cream, transdermal patch, lotion, gel, shampoo, spray,
aerosol, solution, emulsion, or other form well-known to one of
skill in the art. See, e.g., Remington's Pharmaceutical Sciences
and Introduction to Pharmaceutical Dosage Forms, 19th ed. (Mack
Publishing Co., Easton, Pa., 1995). For non-sprayable topical
dosage forms, viscous to semi-solid or solid forms comprising a
carrier or one or more excipients compatible with topical
application and having a dynamic viscosity preferably greater than
water are typically employed. Suitable formulations include,
without limitation, solutions, suspensions, emulsions, creams,
ointments, powders, liniments, salves, and the like, which are, if
desired, sterilized or mixed with auxiliary agents (e.g.,
preservatives, stabilizers, wetting agents, buffers, or salts) for
influencing various properties, such as, for example, osmotic
pressure. Other suitable topical dosage forms include sprayable
aerosol preparations wherein the active ingredient, preferably in
combination with a solid or liquid inert carrier, is packaged in a
mixture with a pressurized volatile (e.g., a gaseous propellant) or
in a squeeze bottle. Moisturizers or humectants can also be added
to pharmaceutical compositions and dosage forms if desired.
Examples of such additional ingredients are well known in the
art.
[0064] If a method of the invention comprises intranasal
administration of a composition, the composition can be formulated
in an aerosol form, spray, mist or in the form of drops. In
particular, prophylactic or therapeutic agents for use according to
the present invention can be conveniently delivered in the form of
an aerosol spray presentation from pressurized packs or a
nebulizer, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0065] If a method of the invention comprises oral administration,
compositions can be formulated orally in the form of tablets,
capsules, cachets, gelcaps, solutions, suspensions, and the like.
Tablets or capsules can be prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone, or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose, or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc, or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or,
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art. Liquid preparations for
oral administration may take the form of, but not limited to,
solutions, syrups or suspensions, or they may be presented as a dry
product for constitution with water or other suitable vehicle
before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives,
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol, or fractionated vegetable oils); and, preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, flavoring, coloring,
and sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated for slow release,
controlled release, or sustained release of a prophylactic or
therapeutic agent(s).
[0066] A method of the invention may comprise pulmonary
administration; e.g., by use of an inhaler or nebulizer, of a
composition formulated with an aerosolizing agent. See, e.g., U.S.
Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;
5,855,913; 5,290,540; and, 4,880,078; and, PCT Publication Nos. WO
92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903,
each of which is incorporated herein by reference in their
entireties. In a specific embodiment, an antibody of the invention,
combination therapy, and/or composition of the invention is
administered using Alkermes pulmonary drug delivery technology
(Alkermes, Inc., Cambridge, Mass., US).
[0067] A method of the invention may comprise administration of a
composition formulated for parenteral administration by injection
(e.g., by bolus injection or continuous infusion). Formulations for
injection may be presented in unit dosage form (e.g., in ampoules
or in multi-dose containers) with an added preservative. The
compositions may take such forms as suspensions, solutions, or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle (e.g., sterile pyrogen-free
water) before use.
[0068] A method of the invention may additionally comprise
administration of compositions formulated as depot preparations.
Such long acting formulations may be administered by implantation
(e.g., subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compositions may be formulated
with suitable polymeric or hydrophobic materials (e.g., as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives (e.g., as a sparingly soluble
salt).
[0069] Methods of the invention encompass administration of
compositions formulated as neutral or salt forms. Pharmaceutically
acceptable salts include those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0070] Generally, the ingredients of compositions are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the mode of
administration is infusion, a composition can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the mode of administration is by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0071] In particular, the invention also provides that one or more
of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is packaged in a hermetically sealed
container such as an ampoule or sachette indicating the quantity of
the agent. In one embodiment, one or more of the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
is supplied as a dry sterilized lyophilized powder or water free
concentrate in a hermetically sealed container and can be
reconstituted (e.g., with water or saline) to the appropriate
concentration for administration to a subject. Preferably, one or
more of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is supplied as a dry sterile
lyophilized powder in a hermetically sealed container at a unit
dosage of at least 5 mg, more preferably at least 10 mg, at least
15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50
mg, at least 75 mg, or at least 100 mg. The lyophilized
prophylactic or therapeutic agents or pharmaceutical compositions
of the invention should be stored at between 2.degree. C. and
8.degree. C. in its original container and the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
should be administered within 1 week, preferably within 5 days,
within 72 hours, within 48 hours, within 24 hours, within 12 hours,
within 6 hours, within 5 hours, within 3 hours, or within 1 hour
after being reconstituted. In an alternative embodiment, one or
more of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is supplied in liquid form in a
hermetically sealed container indicating the quantity and
concentration of the agent. Preferably, the liquid form of the
administered composition is supplied in a hermetically sealed
container at least 0.25 mg/ml, more preferably at least 0.5 mg/ml,
at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8
mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at
least 50 mg/ml, at least 75 mg/ml, or at least 100 mg/ml. The
liquid form should be stored at between 2.degree. C. and 8.degree.
C. in its original container.
[0072] The peptide of the invention can be incorporated into a
pharmaceutical composition suitable for parenteral administration.
Preferably, the peptide will be prepared as an injectable solution
containing 0.1-250 mg/ml. The injectable solution can be composed
of either a liquid or lyophilized dosage form in a flint or amber
vial, ampoule or pre-filled syringe. The buffer can be L-histidine
(1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0).
Other suitable buffers include but are not limited to, sodium
succinate, sodium citrate, sodium phosphate or potassium phosphate.
Sodium chloride can be used to modify the toxicity of the solution
at a concentration of 0-300 mM (optimally 150 mM for a liquid
dosage form). Cryoprotectants can be included for a lyophilized
dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other
suitable cryoprotectants include trehalose and lactose. Bulking
agents can be included for a lyophilized dosage form, principally
1-10% mannitol (optimally 2-4%). Stabilizers can be used in both
liquid and lyophilized dosage forms, principally 1-50 mM
L-methionine (optimally 5-10 mM). Other suitable bulking agents
include glycine, arginine, can be included as 0-0.05%
polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRU
surfactants.
[0073] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans with other antibodies. The preferred
mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In an embodiment, a
peptide described herein is administered by intravenous infusion or
injection. In another embodiment, the peptide is administered by
intramuscular or subcutaneous injection.
[0074] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., antibody or antibody
portion) in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile, lyophilized powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum drying and spray-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including, in the composition, an agent that delays absorption, for
example, monostearate salts and gelatin.
[0075] The peptide of the present invention can be administered by
a variety of methods known in the art, although for many
therapeutic applications, the preferred route/mode of
administration is subcutaneous injection, intravenous injection, or
infusion. As will be appreciated by the skilled artisan, the route
and/or mode of administration will vary depending upon the desired
results. In certain embodiments, the active compound may be
prepared with a carrier that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.
(Marcel Dekker, Inc., New York, 1978).
[0076] In certain embodiments, a binding protein of the invention
may be orally administered, for example, with an inert diluent or
an assimilable edible carrier. The compound (and other ingredients,
if desired) may also be enclosed in a hard or soft shell gelatin
capsule, compressed into tablets, or incorporated directly into the
subject's diet. For oral therapeutic administration, the compounds
may be incorporated with excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. To administer a compound
of the invention by other than parenteral administration, it may be
necessary to coat the compound with, or co-administer the compound
with, a material to prevent its inactivation.
[0077] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, a binding protein of the
invention is coformulated with and/or coadministered with one or
more additional therapeutic agents that are useful for treating
disorders in which peptide activity is detrimental. Furthermore,
one or more binding proteins of the invention may be used in
combination with two or more of the foregoing therapeutic agents.
Such combination therapies may advantageously utilize lower dosages
of the administered therapeutic agents, thus avoiding possible
toxicities or complications associated with the various
monotherapies.
[0078] In another aspect, this invention provides a method of
treating (e.g. curing, suppressing, ameliorating, delaying, or
preventing the onset of, or preventing recurrence or relapse of) or
preventing a tumor in a subject. The method includes administering
to a subject a peptide, in an amount sufficient to treat or prevent
the tumor or cancer.
[0079] It should be understood that peptides claimed in the
invention can also be used alone or in combination with an
additional agent; e.g., a therapeutic agent, said additional agent
being selected by the skilled practitioner for its intended
purpose. For example, the additional agent can be a therapeutic
agent that is recognized in the art as being useful to treat a
cancer, tumor, or other disease. The additional agent also can be
an agent that imparts a beneficial attribute to the therapeutic
composition; e.g., an agent which affects the viscosity of the
composition.
[0080] It should further be understood that the combinations which
are to be included within this invention are those combinations
useful for their intended purpose. The agents set forth below are
illustrative for purposes and not intended to be limited. The
combinations, which are part of this invention, can be the
antibodies of the present invention and at least one additional
agent selected from the lists below. The combination can also
include more than one additional agent, e.g., two or three
additional agents, if the combination is such that the formed
composition can perform its intended function.
[0081] Preferred combinations of therapeutic agents may interfere
at different points in the pro-tumorigenic or pro-angiogenic
signaling pathways. Preferred examples of therapeutic agents useful
in the methods and compositions of the invention include
antineoplastic agents, radiotherapy, and chemotherapy such as DNA
alkylating agents, cisplatin, carboplatin, anti-tubulin agents,
paclitaxel, docetaxel, taxol, doxorubicin, gemcitabine, gemzar,
anthracyclines, adriamycin, topoisomerase I inhibitors,
topoisomerase II inhibitors, 5-fluorouracil (5-FU), leucovorin,
irinotecan, receptor tyrosine kinase inhibitors (e.g., erlotinib,
gefitinib), COX-2 inhibitors (e.g., celecoxib), and kinase
inhibitors.
[0082] The peptide(s) of the invention may also be administered in
combination with agents, such as methotrexate, 6-MP, azathioprine
sulphasalazine, mesalazine, olsalazine
chloroquinine/hydroxychloroquine, penicillamine, aurothiomalate
(intramuscular and oral), azathioprine, colchicine, corticosteroids
(oral, inhaled and local injection), beta-2 adrenoreceptor agonists
(salbutamol, terbutaline, salmeteral), xanthines (theophylline,
aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium
and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adenosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signaling by
proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g., IRAK,
NIK, IKK, p38 or MAP kinase inhibitors), IL-130 converting enzyme
inhibitors, TNF.alpha. converting enzyme (TACE) inhibitors, T-cell
signaling inhibitors such as kinase inhibitors, metalloproteinase
inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines,
angiotensin converting enzyme inhibitors, soluble cytokine
receptors and derivatives thereof (e.g., soluble p55 or p75 TNF
receptors and the derivatives p75TNFRIgG (Enbrel.TM.) and
p55TNFRIgG (lenercept), sIL-1RI, sIL-1RII, and sIL-6R),
anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and
TGF.beta.), celecoxib, folic acid, hydroxychloroquine sulfate,
rofecoxib, etanercept, infliximab, naproxen, valdecoxib,
sulfasalazine, methylprednisolone, meloxicam, methylprednisolone
acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide,
propoxyphene napsylate/apap, folate, nabumetone, diclofenac,
piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone HCl,
hydrocodone bitartrate/apap, diclofenac sodium/misoprostol,
fentanyl, anakinra, human recombinant, tramadol HCl, salsalate,
sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate
sodium, prednisolone, morphine sulfate, lidocaine hydrochloride,
indomethacin, glucosamine sulf/chondroitin, amitriptyline HCl,
sulfadiazine, oxycodone HCl/acetaminophen, olopatadine HCl,
misoprostol, naproxen sodium, omeprazole, cyclophosphamide,
rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18,
anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740,
Roflumilast, IC-485, CDC-801, and Mesopram.
[0083] Non-limiting examples of therapeutic agents for cancers with
which a peptide of the invention can be co-administered or used in
combination include the following: budenoside; epidermal growth
factor; sulfasalazine; aminosalicylates; 6-mercaptopurine;
azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine;
olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1
receptor antagonists; anti-IL-1.beta. monoclonal antibodies;
anti-IL-6 monoclonal antibodies; growth factors; elastase
inhibitors; pyridinyl-imidazole compounds; and antibodies to or
antagonists of other human cytokines or growth factors, for
example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16,
IL-17, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the
invention, or antigen binding portions thereof, can be combined
with antibodies to cell surface molecules such as CD2, CD3, CD4,
CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90, or their
ligands.
[0084] Other examples of therapeutic agents with which a peptide of
the invention can be combined include the following: TNF
antagonists, for example, anti-TNF antibodies, D2E7 (PCT
Publication No. WO 97/29131), CA2, CDP 571, TNFR-Ig constructs
(p75TNFRIgG and p55TNFRIgG (lenercept)), and PDE4 inhibitors.
Binding proteins of the invention can be combined with, e.g.,
mesalamine, prednisone, azathioprine, mercaptopurine, infliximab,
methylprednisolone sodium succinate, diphenoxylate/atrop sulfate,
loperamide hydrochloride, methotrexate, omeprazole, folate,
ciprofloxacin/dextrose-water, hydrocodone bitartrate/apap,
tetracycline hydrochloride, fluocinonide, metronidazole,
thimerosal/boric acid, cholestyramine/sucrose, ciprofloxacin
hydrochloride, hyoscyamine sulfate, meperidine hydrochloride,
midazolam hydrochloride, oxycodone HCl/acetaminophen, promethazine
hydrochloride, sodium phosphate, sulfamethoxazole/trimethoprim,
celecoxib, polycarbophil, propoxyphene napsylate, hydrocortisone,
multivitamins, balsalazide disodium, codeine phosphate/apap,
colesevelam HCl, cyanocobalamin, folic acid, levofloxacin,
methylprednisolone, natalizumab, and interferon-gamma.
[0085] Non-limiting examples of therapeutic agents with which a
binding protein of the invention can be combined include the
following: aspirin, nitroglycerin, isosorbide mononitrate,
metoprolol succinate, atenolol, metoprolol tartrate, amlodipine
besylate, diltiazem hydrochloride, isosorbide dinitrate,
clopidogrel bisulfate, nifedipine, atorvastatin calcium, potassium
chloride, furosemide, simvastatin, verapamil HCl, digoxin,
propranolol hydrochloride, carvedilol, lisinopril, spironolactone,
hydrochlorothiazide, enalapril maleate, nadolol, ramipril,
enoxaparin sodium, heparin sodium, valsartan, sotalol
hydrochloride, fenofibrate, ezetimibe, bumetanide, losartan
potassium, lisinopril/hydrochlorothiazide, felodipine, captopril,
and bisoprolol fumarate.
[0086] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of a binding protein of the invention. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired therapeutic result. A therapeutically effective amount of
the binding protein may be determined by a person skilled in the
art and may vary according to factors such as the disease state,
age, sex, and weight of the individual, and the ability of the
binding protein to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the binding protein are outweighed by the
therapeutically beneficial effects. A "prophylactically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired prophylactic result.
Typically, since a prophylactic dose is used in subjects prior to
or at an earlier stage of disease, the prophylactically effective
amount will be less than the therapeutically effective amount.
[0087] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0088] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a peptide of the invention is
0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be noted that
dosage values may vary with the type and severity of the condition
to be alleviated. It is to be further understood that for any
particular subject, specific dosage regimens should be adjusted
over time according to the individual need and the professional
judgment of the person administering or supervising the
administration of the compositions, and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed composition.
[0089] Peptide-drug conjugates (PDCs) are targeted drug delivery
systems, which combine the targeting specificity of a peptide
(similar to that of the antibody in an ADC) with the cytotoxicity
of a drug (or toxin). PDCs generally comprise a targeting peptide
(which will be referred to in the discussion that follows simply as
"peptide") covalently bound (conjugated) via a linker to a
cytotoxic drug or toxin. The choice of specific peptide, linker and
drug in each PDC, in combination, determines that PDC's targeting
capability (specificity, binding affinity, etc.), pharmacokinetics,
and mechanism of cell killing.
[0090] The term "peptide-drug conjugate" refers to poly- or
oligo-amino acids having a specific sequence, such as an antibody
mimic peptide, chemically linked to a chemical moiety, such as a
therapeutic or cytotoxic agent (also referred as a substance). The
term "agent" is used herein to denote a chemical compound, a
mixture of chemical compounds, a biological macromolecule, or an
extract made from biological materials. Preferably, the therapeutic
or cytotoxic agents include, but are not limited to, pertussis
toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
l-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, maytansine, auristatins, puromycin and
analogs or homologs thereof.
[0091] It is of utmost importance that the peptide specifically
binds the desired target and with high affinity. Generally, the
higher the binding affinity of the peptide, the more effective is
the drug conjugate. As with ADCs, the choice of peptide-drug linker
and its optimization also play an important role in the design of
PDCs. At least two important aspects must be considered in
designing a PDC linker. First, the linker should be capable of
linking both the peptide and the drug without adversely affecting
properties and activities of either; i.e., targeting functionality
of the peptide and cytotoxic effects of the drug. Second, the
linker should be stable during storage and in the systemic
circulation until it reaches the target. Additionally, if a
cleavable linker is used, the linker should be relatively easily
cleaved (or otherwise permit separation of peptide and drug) after
reaching the target cell.
[0092] Both cleavable and non-cleavable linkers can be used in
conjugating peptides to drugs. Cleavable linkers can be cleaved
intracellularly or extracellularly, and non-cleavable linkers
cannot easily be enzymatically or chemically cleaved, if at all.
These non-cleavable linkers generally undergo complete degradation
in the lysosome along with their associated peptides. Again like
ADCs, described above, the most frequently used cleavable linkers
are hydrazone and disulphide linkers. Drugs used in PDCs are
generally the same as those in ADCs.
[0093] Peptides possess several advantages over antibodies/antibody
fragments in targeted drug delivery systems, including ease of
synthesis, better tolerance of a wider range of pH and temperature,
and flexibility in chemical modification. Perhaps most importantly,
due to the peptide's smaller size and molecular weight it
facilitates higher cell uptake as compared to antibodies. Moreover,
targeting peptides are generally less toxic and have lower
immunogenic potential than antibodies or their fragments. For at
least all of the foregoing reasons, peptides are ideal candidates
as targeting agents.
[0094] The antibodies, antibody fragments, peptides and
combinations thereof currently in use for targeting in ADC and/or
PDC systems are designed and identified from conventional
antibodies or through time-consuming screening techniques, such as
phage, ribosome and mRNA display. The present disclosure, by
contrast, presents synthetic peptides possessing the functionality
of known antibodies, thus bypassing the lengthy development and
screening techniques.
[0095] Each of the peptides of the present disclosure comprises a
motif sequence. These motif sequences comprise amino acid residues
corresponding to amino acids in the native antibody "knobs" which
pack the epitope "sockets" at the antibody-epitope interface, as is
known from the antibody-epitope crystal structure. For a
description of the knob-socket model, see H. Joo et al., J. Molec.
Biol. (2012) 419:234-254, which is incorporated herein by reference
for its teachings of the knob-socket model. Briefly, the
knob-socket model describes contacts in the "packing" of peptide
.alpha.-helices as a "knob" of one amino acid residue of a first
.alpha.-helix, which fits into a "socket" of three amino acid
residues of a second .alpha.-helix. Schematic diagrams of knob and
socket packing interface lattices are shown in FIGS. 1B and 2,
which represent the cetuximab-EGFR epitope interface, and a model
mimic peptide-EGFR peptide interface, respectively. See also the
Examples below. As shown in FIGS. 1B and 2, each epitope socket is
packed by a particular knob, and is represented in a shade of gray.
Each amino acid residue of the epitope socket is presented by its
single-letter code within a circle along the solid black lines. The
black lines represent covalent bonds between amino acids; socket
residues which "pack" with their side chains are represented by a
dashed black line; hydrogen bonds are represented by dashed red
lines. The amino acid residues of the epitope socket not involved
in packing are omitted or shown as a small gray dot in the diagram
(without a letter code). The amino acids of the knobs are also
within circles, inside the gray shaded regions.
[0096] The peptides described herein exhibit specificity, binding
affinity and functionality equivalent to or better than that of the
original/native antibodies, as demonstrated in the Examples below,
based on the results of in vitro cellular binding/uptake and flow
cytometry studies (specificity), surface plasmon resonance (SPR)
studies (binding affinity), and phosphorylation studies
(functionality--mimic peptide inhibition of phosphorylation).
[0097] The present disclosure also demonstrates effective
incorporation of the peptides described herein in PDCs, which PDCs
were synthesized, characterized and evaluated against various cell
lines, as described in the Examples.
[0098] In some embodiments herein, peptides are described which
particularly target EGFR or HER2. The overexpression of EGFR or
HER2 has been implicated in the development of a wide range of
epithelial cancers, including breast, colon, head and neck, kidney,
pancreas and prostate. In certain embodiments, PDCs were
synthesized comprising these peptides.
[0099] The compositions and methods described herein provide for
peptides which are highly effective in mimicking native antibodies,
while avoiding the limitations of using native antibodies such as
time-consuming screening methods. The peptides disclosed herein can
thus be used to target, treat, identify, bind, etc. a variety of
targets such as, for example but without limitation, those
implicated in the development of a wide range of epithelial
cancers, including breast, colon, head and neck, kidney, pancreas
and prostate cancers.
[0100] In some embodiment, the antibody mimic peptide can be used
for diagnosing a disease. The disease is tumor or cancer. The
disease is associated with abnormal expression of EGFR or HER2
proteins.
[0101] In one embodiment, the antibody mimic peptide further
comprises a conjugate structure having a substance, wherein the
substance is a detection reagent, such as a detector peptide
labeled with a reporter moiety.
[0102] In one embodiment, the detector peptide is a peptide, or
antigen-binding fragment thereof, that is specific for the EGFR or
HER2 or PD1 or PDL1.
[0103] The reporter moiety can be any of a wide range of
materials/reporter systems known in the art. In some embodiments,
the reporter moiety comprises a first member of a ligand-receptor
pair including, but not limited to, an enzyme (e.g., horseradish
peroxidase (HRP), alkaline phosphatase, luciferase,
beta.-galactosidase, glucose oxidase, lysozyme, malate
dehydrogenase, glucose-6-phosphate dehydrogenase); metal sol,
selenium sol, carbon sol, and the like; colored or colorable
particles (e.g., colored or colorable latex particles); colloidal
metal particles (e.g., colloidal gold, colloidal silver, colloidal
platinum, colloidal selenium) and isotopes of radioactive and
non-radioactive. Examples of methods known in the art for detecting
the reporter include, but are not limited to, detection methods by
visible inspection, ultraviolet (UV) and visible spectrophotometry,
fluorimetry and radiation counters.
[0104] The reporter moiety may be covalently or non-covalently
bound/coupled to the detector peptide. The binding/coupling can be
accomplished by any method known in the art. For example, reagents
used for binding/coupling include, but are not limited to,
glutaraldehyde, p-toluene diisocyanate, various carbodiimide
reagents, p-benzoquinone m-periodate, N,Ni-o-phenylenedimaleimide,
recombinant methods, and the like.
EXAMPLES
[0105] In the following Examples, peptides specific for EGFR, HER2,
PD1 and PDL1 were synthesized and characterized to determine their
effectiveness especially as components of PDCs in targeted drug
delivery.
Example Peptide Sequences for EGFR
[0106] Antibody mimics were designed utilizing the crystal
structure of Cetuximab-EGFR and knob-socket model. The crystal
structure (PDB 1YY9) was analyzed to determine the residues in
Cetuximab which have maximum interaction with EGFR epitopes. The
EGFR epitope surface was then analyzed and mapped to define sockets
in which knobs from Cetuximab bind. Each socket is formed by three
amino acid residues on the EGFR surface and binds with the amino
acid knob residues from the antibody's complementary binding region
loops. Antibody mimic molecules were designed by selecting the knob
residues with high probability of packing into sockets on the EGFR
surface.
[0107] Antibody mimics were designed by placing high-preference
knob residues like Trp (W), Glu (E), Phe (F), Tyr (Y), and Ala (A)
in the sockets, and were connected according to the distance
between them with other amino acids. Twenty five antibody mimics
(Pep1-Pep25) were designed having different knob residues based on
the frequency in the VSS and ISI sockets. Antibody mimics with
sequences, Pep1-Pep11 were designed using knobs YY, YW, YL, YI, YV,
YF, WY, LY, IY, VY, FY respectively in VSS and ISI sockets.
Pep12-Pep22 were designed in a similar way with an additional
threonine residue. Knobs in Pep12-Pep22 have TYY, TYW, TYL, TYI,
TYV, TYF, TWY, TLY, TIY, TVY, TFY respectively. Pep23 and Pep24
were designed to have the reverse sequence of Pep11 and Pep6 to
test the sequence requirement for binding, and Pep25, a scrambled
peptide, was created as a negative control.
[0108] Molecular modeling studies: The interactions between the
original antibody/designed peptides and EGFR were identified by
docking studies using Molecular Operating Environment (MOE)
software. The antibody mimic molecules were prepared by adding
protons and performing the stochastic confirmation approach to find
the lowest energy confirmation. The molecules were then
energetically minimized and docking was performed. Conformation of
the antibody mimic with lowest energy was then analyzed by MOE
software to determine the binding energy, which indicates better
binding. The total number of interactions and preserved
interactions between EGFR and designed molecules, which shows the
similarly in binding of the designed peptide with the original
antibody, were also analyzed. Antibody mimics having low binding
energy and a higher number of interactions and preserved
interactions were then selected for further experiments and
evaluation.
[0109] Design of antibody mimics was developed based on the
Cetuximab-EGFR crystal structure and Knob-Socket model. Using the
frequency data of knobs and sockets, the design of peptide
sequences was developed based on those knobs which best fit into
the sockets presented on the EGFR epitope. A ribbon diagram of the
interface and side-chains of the amino acids which form packing
sockets are shown in FIG. 1A. C-alpha positions of the knobs from
the antibody are represented as sphere. Orange color represents
knobs from CDR1 (the bottom ball), purple for CDR2 (top three
balls), and green for CDR3 amino acids (middle four balls).
[0110] A schematic of the packing interface lattice represented by
knobs and sockets is shown in FIGS. 1B, 2 and 23. Sockets packing
the same knob are represented in the gray scale. Residues forming
packing sockets are presented along the solid black line, and those
which are not involved with packing are omitted or shown as small
gray dots. Knobs are presented as circles, color-coded in the same
way as in the ribbon diagram. Molecular modeling and mapping data
suggested that the tyrosine 102 residue (Y102) is very important,
as it showed multiple interactions. The Y102 residue was found to
be a knob for the ISI, SVI, VAI, AFI, and AQF socket residues on
EGFR.
[0111] The drug auristatin E, cetuximab, the peptide Pep11, and a
Pep11-auristatin E PDC were studied and the IC.sub.50 value of each
molecule was obtained. For Pep11 see Table 1 (No. 11). As is known,
cetuximab is a chimeric mouse/human monoclonal antibody (mAb) which
binds the EGF receptor (EGFR), and is used; e.g., in cancer
treatment. The studies were conducted using two cancer cell lines,
A431 and MDA-MB-468, which overexpress EGFR, and a control cell
line, HEK 293, which does not overexpress EGFR.
[0112] Peptides specifically bound and were internalized by EGFR
overexpressing cell lines at levels three- to four-fold greater
than control cells. Peptides showed receptor binding affinity (as
provided by the calculated K.sub.D, described below), in the
nanomolar range, with Pep11 exhibiting a binding affinity of 252 nM
as shown by surface plasmon resonance (SPR) studies. The results of
EGFR phosphorylation studies also demonstrated that peptides
binding to EGFR would inhibit EGF binding, at a level similar to
that of cetuximab. The specificity, binding affinity, and
functional activity of the peptides disclosed herein, as
demonstrated in the Examples below, show that these peptides
possess the important characteristics of the corresponding native
antibodies.
[0113] The PDC was also shown to be approximately 10-fold more
potent than the drug alone in its toxicity towards EGFR
overexpressed cancer cells. The PDC also demonstrated a potency
more than 100-fold lower than drug alone in toxicity against
control cells.
[0114] A ribbon diagram of the cetuximab-EGFR binding interface and
side-chains of the amino acids which form packing sockets in the
epitope is shown in FIG. 1A. The C-alpha positions of the antibody
sequence knobs are represented in FIG. 1A as the spheres, each
corresponding to a knob sequence from cetuximab's complementarity
determining regions 1, 2 and 3, as shown. In FIG. 1B, the knob
amino acids are numbered according to their position in the
cetuximab sequence, and color coded to correspond to the CDRs shown
in the ribbon diagram of FIG. 1A. FIG. 2 illustrates the same
epitope sockets with the knobs from a peptide.
[0115] Twenty-five peptides (Pep1-Pep25; see Table 1) were
synthesized, with different knob residues associated with the VSS
and ISI sockets of the EGFR epitope. See FIG. 1B, in which the VSS
socket is packed, where "VSS" and "ISI" refer to the three amino
acids primarily forming the sockets (i.e., valine-serine-serine and
isoleucine-serine-isoleucine, respectively; see FIGS. 1a and 2).
The amino acid sequences represented by Pep1-Pep11 have knob
residues YY, YW, YL, YI, YV, YF, WY, LY, IY, VY, and FY,
respectively, which fit in the VSS and ISI sockets.
[0116] Pep12-Pep22 have an additional threonine residue (Thr or T)
relative to Pep1-Pep11. Pep12-Pep22 therefore have sequences TYY,
TYW, TYL, TYI, TYV, TYF, TWY, TLY, TIY, TVY, and TFY, respectively,
as knob residues. Pep23 and Pep24 have the reverse sequence of
Pep11 and Pep6, respectively. A peptide of scrambled, random
sequence was also created, Pep25, as a negative control.
[0117] For example, Pep6, Pep11, Pep22, and Pep24 demonstrated a
calculated binding energy in the range of -32 kcal/mol to -41
kcal/mol, and high number of calculated total and preserved
interactions with the EGFR epitope (in the range of 13-17 total and
4-8 preserved interactions). By comparison Pep25, the control
peptide of scrambled sequence, had a calculated docking energy of
-23.69 kcal/mol, 7 total interactions with the epitope and 2
preserved interactions. Molecular binding studies between the
peptides and EGFR indicated that the total number of interactions
and preserved interactions between EGFR and the peptides
demonstrate the similarity of peptide-epitope binding with that of
native antibody as shown in Table 3.
TABLE-US-00001 TABLE 1 Peptide sequences Energy Total Preserved No.
Molecule (kcal/mol) Interactions Interactions 1. WSGENGPGYYDYEA
(SEQ ID NO: 1) -35.80 13 7 2. WSGENGPGYWDYEA(SEQ ID NO: 2) -24.54
13 6 3. WSGENGPGYLDYEA(SEQ ID NO: 3) -33.82 13 5 4.
WSGENGPGYIDYEA(SEQ ID NO: 4) -28.95 13 7 5. WSGENGPGYVDYEA(SEQ ID
NO: 5) -28.42 13 3 6. WSGENGPGYFDYEA(SEQ ID NO: 6) -32.47 13 4 7.
WSGENGPGWYDYEA(SEQ ID NO: 7) -35.91 13 4 8. WSGENGPGLYDYEA(SEQ ID
NO: 8) -28.28 14 4 9. WSGENGPGIYDYEA(SEQ ID NO: 9) -26.00 14 3 10.
WSGENGPGVYDYEA(SEQ ID NO: 10) -28.39 13 6 11. WSGENGPGFYDYEA(SEQ ID
NO: 11) -40.43 17 8 12. WSGENGPGTYYDYEA(SEQ ID NO: -31.28 14 6 12)
13. WSGENGPGTYWDYEA(SEQ ID NO: -28.78 12 4 13) 14.
WSGENGPGTYLDYEA(SEQ ID NO: -35.62 14 5 14) 15. WSGENGPGTYIDYEA(SEQ
ID NO: 15) -31.25 10 2 16. WSGENGPGTYVDYEA(SEQ ID NO: -34.33 13 2
16) 17. WSGENGPGTYFDYEA(SEQ ID NO: -37.13 13 6 17) 18.
WSGENGPGTWYDYEA(SEQ ID NO: -37.94 13 7 18) 19. WSGENGPGTLYDYEA(SEQ
ID NO: -31.89 14 7 19) 20. WSGENGPGTIYDYEA(SEQ ID NO: 20) -29.32 12
4 21. WSGENGPGTVYDYEA(SEQ ID NO: -36.50 14 6 21) 22
WSGENGPGTFYDYEA(SEQ ID NO: -35.34 14 6 22) 23 AEYDYFGPGNEGSW(SEQ ID
NO: 23) -30.92 10 3 24 AEYDFYGPGNEGSW(SEQ ID NO: 24) -40.31 13 4
25. Control SGEWAYDGYEPNFG(SEQ ID NO: 25) -23.69 7 2
[0118] In FIG. 3, Pep11 is illustrated docking at the same site in
EGFR as cetuximab. Tyrosine residue (pink) from Pep11 is
representing a knob which is fitting into the socket formed by
alanine (orange), valine (green) and isoleucine (blue) in the EGFR
epitope.
Binding Specificity Studies of Peptides
[0119] A431, MDA-MB-468 and HEK 293 cells were cultured in 75
cm.sup.2 culture flasks in DMEM supplemented with 10% FBS. For
binding/internalization studies, 5.times.10.sup.5 cells were seeded
in cover slips in 6 well culture plates overnight. After the medium
was removed, the cells were washed with HBSS and then cells were
treated with FITC labeled antibody mimic and scrambled (control)
peptide in serum free DMEM for 30 minutes at 37.degree. C. Cells
were then washed with HBSS to remove any unbound peptides and
incubated with AlexaFluor 594 wheat germ agglutinin to stain cell
membranes. Cells were fixed with 4% paraformaldehyde and cover
slips were mounted on slides which contain a drop of SlowFade.RTM..
Cells were then imaged with an inverted Leica DMIRE2 fluorescence
microscope (Leica Biosystems Richmond Inc., Richmond, USA) using a
Yokogawa CSUX1 confocal scanner unit. For flow cytometry studies,
cells were washed with HBSS after incubation with antibody mimics,
control peptide for 30 minutes, and dissociated using trypsin-EDTA.
Cells were then centrifuged at 500 g for 5 min. Supernatant was
removed and cell pellets were re-suspended in FACS solution. The
cell suspension was then analyzed on a Beckman Coulter flow
cytometer and data were analyzed using the Cell Questpro. A total
of 10000 events were collected monitoring FITC.
Binding Affinity Studies of Peptides
[0120] Binding affinity constant of antibody mimics was calculated
by surface plasmon resonance (Reichert SR7000DC). A carboxymethyl
dextran gold sensor chip was activated by N-hydroxysuccinimide
(NHS) and N-ethyl-N''-[3-(diethylamino)propyl] carbodiimide (EDC)
at 25.degree. C. using phosphate buffered saline pH 7.4 with 0.05%
tween 20 as running buffer. Recombinant human EGFR (30 .mu.g/mL),
or BSA (20 .mu.g/mL) as control, was immobilized by flowing the
antigen over the activated chip in 10 mM sodium acetate buffer pH
4.5 at 15 .mu.L/min. for 6 minutes. Unreactive sites were blocked
by flowing over the chip 1 M ethanolamine pH 8.5 at 10 .mu.L/min
for 8 minutes. The real-time binding and dissociation rates of
different concentrations of mimic peptides over EGFR and BSA were
studied by flowing the peptides over the chip at 25 .mu.L/min. The
resulting sensograms were globally fitted using Scrubber2 software
(BioLogic Software, Campbell, Australia) in a 1:1 binding model to
determine the association rate (k.sub.on), dissociation rate
(k.sub.d or k.sub.off), and dissociation constant K.sub.D.
EGFR Phosphorylation Studies of Peptides
[0121] Cell based ELISA was performed to determine the inhibition
of EGFR phosphorylation. Briefly, 2.times.10.sup.4 A431 cells were
seeded in 96 well plates in DMEM medium supplemented with 10% FBS
for 24 hours. The cells were then treated with different peptides
and cetuximab 10 .mu.M in serum free medium for 45 minutes at
37.degree. C. After that, cells were stimulated by EGF 50 ng/mL for
20 minutes at 37.degree. C. After washing the cells, fixing
solution was added for 20 minutes at room temperature. Cells were
treated with Primary mouse anti EGFR and anti phospho EGFR
antibodies were added first, after washing cells were treated with
anti-mouse HRP secondary antibody for 1 hour at room temperature.
Phosphorylation inhibition was calculated using determined as
follows:
( OD Untreated ) - ( OD Treated ) ( OD Untreated ) * 100
##EQU00001##
[0122] In the present Examples, FITC-labeled peptides specifically
bound the cell membranes and were internalized into the cells which
overexpress EGFR within 30 minutes when incubated at 37.degree. C.
As observed from the images (see FIGS. 4 and 5), significantly
higher fluorescence was seen in A431 and MDA-MB-468 cell lines as
compared with HEK 293 cells, which demonstrated the binding
specificity of the peptides towards EGFR. The scrambled antibody
mimic (Pep25) showed negligible binding to the cell line
overexpressing EGFR demonstrating the specificity for EGFR of the
peptides disclosed herein.
[0123] Epitope specificity of the peptides was evaluated by
cellular uptake of the peptides, as an example cells treated with
Pep11 for 30 minutes at 37.degree. C. and fluorescence images with
cetuximab than from those not pre-incubated with antibody were
determined. As shown in FIG. 6, the results demonstrated that cell
receptors were blocked by cetuximab in the pre-incubated cells and
that binding of Pep11 to cells was inhibited/decreased following
cetuximab treatment.
[0124] Binding specificity of peptides was also studied using flow
cytometry. In this study, the binding specificity was verified by
differential cellular uptake of peptide by cells with different
levels of EGFR expression. Internalization of peptides was
significantly higher in cancer cell line A431 and MDA-MB-468, which
overexpress EGFR, as depicted by the increase in mean fluorescence
intensity (MFI). See FIG. 7. The uptake of the peptides was most
likely a result of receptor mediated endocytosis. Negligible uptake
of the peptides was observed in the HEK 293 cell line, which does
not overexpress EGFR, when compared to the fluorescent intensity of
cell only samples. These results demonstrated the specificity of
the peptides towards EGFR. The scrambled peptide (Pep25) showed
negligible binding to the cancer cell lines, which demonstrated the
specificity of the non-scrambled peptides. All the peptides
(non-scrambled) showed a 3- to 4-fold increase in fluorescent
intensity as compared with the control ("Cells only").
[0125] Antibody binds to its target antigen not only with
specificity, but also with high affinity. Binding affinity
indicates how quickly and with what strength the antibody/mimic
binds the target antigen. Binding affinity of any antibody/mimic
for target can be determined by SPR analysis. This results in a
sensogram, as shown in FIG. 8a, which is a typical sensogram (where
"YYYY" is immobilized antigen), which has an association,
equilibrium and dissociation phase. In the association phase the
antibody/mimic analyte begins binding (associating with) the
immobilized antigen and thus producing measurable SPR response,
until binding reaches equilibrium. At equilibrium, the number of
binding sites becomes saturated and binding of antibody/mimic to
antigen stops, until a dissociation phase is observed where
antibody/mimic begins to dissociate from antigen.
[0126] Binding affinity constants, as represented by K.sub.D (M),
were obtained by SPR analysis of peptides with EGFR by the methods
described above, and were in the range of 252 nM-4.7 .mu.M. See
Table 2, below. The sensograms showed concentration-dependent
binding, with an increase in binding corresponding to an increase
in mimic peptide concentration. See FIG. 8b (where the curves
represent various concentrations of Pep11). Antibody mimics with
K.sub.D values in the range of 23-800 nM are reported in the
literature. Binding affinity of previous antibody mimics have been
20- to 200-fold lower than the affinity of the original antibody.
By contrast, peptides in the present Example demonstrated a binding
affinity (K.sub.D) for EGFR approximately 50-fold greater than
cetuximab's (e.g., 252 nM for Pep11 vs. 5.2 nm for cetuximab).
These results clearly demonstrated that the peptides of the present
disclosure are have high affinity for EGFR, and will thus be very
effective and useful as targeting moieties for EGFR.
[0127] Scrambled peptide did not show binding to the EGFR and no
increase in response was seen with increase in concentration of the
control peptide. This shows the specificity of the [non-scrambled]
peptides. That scrambled peptide did not bind to EGFR could be
attributed to the fact that in scrambled peptide the packing of
knob and socket pairs is disturbed, and designed knobs can no
longer pack into their respective sockets. Moreover.
[non-scrambled] peptides did not show binding to BSA, thus
demonstrating the selectivity and specificity of these peptides
towards EGFR.
[0128] Pep24, having the reverse amino acid sequence of Pep6, still
demonstrated binding affinity similar to that of Pep6 with
comparable K.sub.D values, thus demonstrating that the peptide
confirmation remains the same as that of Pep6, even with reverse
amino acid sequence.
TABLE-US-00002 TABLE 2 SPR results on EGFR as immobilized surface
Peptide Kon M.sup.-1s.sup.-1 Koff s.sup.-1 K.sub.D M Pep6 1.21 *
10.sup.3 .+-. 0.01 * 10.sup.3 5.81 * 10.sup.-3 .+-. 0.03 *
10.sup.-3 4.80 * 10.sup.-6 .+-. 0.25 * 10.sup.-6 Pep11 1.00 *
10.sup.5 .+-. 0.03 * 10.sup.5 2.52 * 10.sup.-2 .+-. 0.05 *
10.sup.-2 2.52 * 10.sup.-7 .+-. 0.09 * 10.sup.-7 Pep22 4.35 *
10.sup.4 .+-. 0.02 * 10.sup.4 2.59 * 10.sup.-2 .+-. 0.04 *
10.sup.-2 5.95 * 10.sup.-7 .+-. 0.10 * 10.sup.-7 Pep24 1.43 *
10.sup.4 .+-. 0.01 * 10.sup.4 1.03 * 10.sup.-1 .+-. 0.03 *
10.sup.-1 7.20 * 10.sup.-6 .+-. 0.32 * 10.sup.-6 Pep25 NA NA NA
TABLE-US-00003 TABLE 3 Knob-socket frequency of binding for ISI
socket Knobs W P G Y F V I L A E K R Q D S T N M H C Frequency 0 1
1 7 0 3 2 1 2 2 1 0 4 0 1 3 4 2 2 0
[0129] In the EGFR phosphorylation study, A431 cells, which
overexpress EGFR, were used. Phosphorylation caused by the
different peptides and cetuximab was determined by cell-based
ELISA. Total EGFR protein content and phosphorylated EGFR were both
were calculated. Total EGFR content remained the same with and
without stimulation by EGF at 50 ng/mL when anti-EGFR antibody was
used as the primary antibody. EGF stimulation did not increase the
total EGFR content, but only caused an increase in phosphorylated
EGFR. Anti-phosphorylated EGFR antibody depicted the percent
inhibition of phosphorylation by different molecules and was in the
range of 4.40-8.13% as compared with 38% by cetuximab when
stimulated by EGF. See FIG. 12.
[0130] The percent inhibition by peptides was around 5-fold less
than that of cetuximab (see FIG. 13), which could be attributed to
the small size of the peptides--though these molecules bind to the
same site as that of cetuximab, they may not be able to prevent a
conformational change as cetuximab does, which conformational
change would inhibit the ligand from binding to EGFR.
Synthesis of Peptide Drug Conjugates
[0131] The following Example demonstrates a peptide as described
herein conjugated to a drug to create a mimic PDC. In the following
Example auristatin E (AE) is the drug. Auristatin E is a known
anti-neoplastic agent, and when used for treatment, for example of
cancer, is bound to antibody.
Synthesis of 5-Benzoylpentanoic Ester of Auristatin E
[0132] Monomethyl auristatin E (MMAE) was dissolved in
dioxane:water (1:1) to create a drug solution, and 0.42 mmol of 37%
aqueous formaldehyde and 0.13 mmol of sodium cyanobohydride were
added to the drug solution. The pH was adjusted to 6-7 with 0.1 N
HCl and the mixture was heated at 100.degree. C. for 2 hours. The
reaction mixture was then poured into a saturated sodium
bicarbonate aqueous solution and ethyl acetate. Ethyl acetate layer
was separated and dried by adding sodium sulphate. A white crystal
powder was obtained. See FIG. 14.
[0133] Auristatin E (0.07 mmol) was dissolved in anhydrous
methylene chloride (DCM), followed by the addition of 0.14 mmol
N,N'-Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine
(DMAP). Then, 5-benzoylpentanoic acid (0.14 mmol) was added to the
auristatin E solution. The reaction was performed overnight at room
temperature. The product was purified and separated using
preparative thin layer chromatography using 5% methanol in ethyl
acetate as developing solvent. See FIG. 15.
Synthesis of Hydrazone Intermediate
[0134] Benzoylpentanoic ester of auristatin E (1 eq) and
maleimidocaproyl hydrazide (5 eq) were added together in anhydrous
methanol with 0.01% acetic acid. The reaction mixture was stirred
overnight at room temperature. The reaction mixture was added into
DMSO and only methanol was evaporated under reduced pressure.
Finally, the product was purified using a semi-preparative column
in reversed-phase (RP) HPLC using a gradient of triethylammonium
acetate buffer pH 7 and acetonitrile. See FIG. 16.
Synthesis of Peptide Drug Conjugate
[0135] Peptide with a free thiol group (e.g., WSGENGPGTFYDYEAC-SH,
SEQ ID NO: 83) and the hydrazone intermediate were dissolved
separately in tert-butyl alcohol and 50 mM ammonium bicarbonate
buffer pH 7.4 (1:3). The pH of both solutions was adjusted to 7.4.
Peptide was added to the hydrazone solution dropwise, and the
reaction mixture was stirred for 10 minutes. The reaction mixture
was then directly purified through HPLC using a gradient of
triethyl ammonium acetate (TEAA) buffer pH 7 and acetonitrile.
Finally, the resulting product was lyophilized. See FIG. 17.
Peptide-Drug Conjugate pH Hydrolysis Studies
[0136] Peptide-drug conjugate was evaluated for half-life
determination at pH 5 and 7.4. For this, a 1 mg/mL concentration of
PDC was incubated in ammonium bicarbonate buffers, pH 5 and 7.4.
Degradation of PDC was evaluated by calculating the area under the
curve after 0, 0.5, 1, 2, 4, 6 and 48 hours using HPLC with a
gradient method, with TEAA buffer pH 7 and acetonitrile.
[0137] See Table 4 for MS and HPLC data for the MMAE, its
intermediates, and the PDC.
In Vitro Cytotoxicity of Peptide-Drug Conjugate
[0138] Cytotoxicity studies of auristatin E, peptide, peptide-drug
conjugate and cetuximab were performed on A431, MDA-MB-468 and HEK
293 cells. Cells were cultured to 80% confluency in T75 culture
flasks using Dulbecco's Modified Eagle's Medium (DMEM) as growth
medium. The cell density was determined using a Coulter Counter.
The cells were seeded onto 96 well plates and grown for 24 h. to
50% confluency. This was followed by treatment with various
concentrations of the drug auristatin E (AE), peptide-drug
conjugate, peptide, and cetuximab ranging from 0.00001 to 10000 nM
for 72 h. at 37.degree. C. At the end of the incubation period, the
cells were fixed using 10% trichloroacetic acid, followed by
washing with distilled water and drying. The cellular proteins were
stained using 50 .mu.L of 0.4% sulforhodamine B (SRB) in 1% acetic
acid. Unbound SRB was washed with 1% acetic acid and the plates
were dried overnight. The cell-bound SRB was then solubilized using
200 .mu.L of 10 mM unbuffered Tris base solution. SRB absorbance
was measured at 560 nm wavelength using a Plate Reader. The percent
viability of cells was plotted as a function of log concentration
and data was analyzed in GraphPad Prism Version 5.0 software
(GraphPad Software, Inc., California, USA) using a
nonlinear-regression curve fit (variable slope four parameter
equation).
TABLE-US-00004 TABLE 4 MMAE, intermediates and PDC MS and HPLC data
MS (g/mol) MS (g/mol) [M + H]+ [M + H]+ HPLC Molecule Calculated
Observed Purity (%) Monomethyl auristatin E 718.2 720.5 99 (MMAE)
Auristatin E (AE) 731.2 732.5 96 AE-benzoylpentanoicacid 920.6
922.2 93 (AEBPA) Hydrazone Intermediate 1127.7 1127.6 98 Pep11-MMAE
conjugate (PDC) 2922.9 2922.8 98
[0139] A pH hydrolysis study of the PDC was performed at pH 5 and
7.4. As the PDC has a hydrazone bond, it is important for the PDC
to be stable at pH 7.4 and hydrolyze at the acidic pH of 5.
Half-life of the PDC was calculated in ammonium bicarbonate
buffers, pH 5 and 7.4. The results show that the hydrazone bond was
successfully formed and then selectively cleaved at pH 5. The pH
can thus be used as a trigger to release the drug from the PDC; for
example, when in the low pH environment of cancer cells. The PDC
half-life was found to be 2.8 hours at pH 5. PDC was found to be
stable at pH 7.4 and its half-life was observed to be more than 48
hours. See FIG. 18 and Table 5.
TABLE-US-00005 TABLE 5 Half-life of PDC in pH 5 and 7.4 pH
Half-life (h) 5 2.8 7.4 >48
[0140] Cytotoxicity studies were performed to determine the potency
of drug, peptide, peptide-drug conjugate and cetuximab. Percentage
of viable cells were calculated using the sulphorhodamine
cytotoxicity assay. The drug auristatin E showed similar potency
and IC.sub.50 values across cancer and normal cells, which
indicated the non-specificity of the drug. This suggests that a
prodrug or a drug-conjugate approach is best suited for use of the
drug in treatment.
[0141] The peptide itself showed low potency across the cancer cell
lines that over-express EGFR (i.e., A431 and MDA-MB-468) as
compared with the drug. See FIGS. 19 and 20. The antibody mimic
peptide showed selectivity towards cancer cells over normal cells,
as it demonstrated 2- to 3-fold lower potency towards control cells
(HEK 293) as compared to cancer cells that over-express EGFR. See
FIGS. 19-21. Peptide-drug conjugate was found to be around 10-fold
more potent as compared to the drug itself towards EGFR
over-expressing cancer cells. PDC also showed significantly low
potency in HEK 293 cells, which do not overexpress EGFR, relative
to cells that overexpress EGFR. This clearly demonstrated that the
peptide-drug conjugate selectively bound to, and was internalized
via receptor-mediated endocytosis by, cells which over-express
EGFR. See FIGS. 19-21 and Table 6.
[0142] It is proposed that the cell killing mechanism of PDCs
starts upon binding of the targeting peptide to the EGFR, which is
overexpressed on the surface of the cancer cells. After binding,
the PDC-EGFR complex undergoes internalization through
receptor-mediated endocytosis. EGFR is known to be internalized
relatively quickly after binding to ligand. Following
internalization, the whole PDC-EGFR complex is degraded within the
lysosome, which is acidic in pH and rich in proteolytic enzymes.
This results in the release of the cytotoxic agent (in our
examples, AE), which then exerts its cytotoxic affect by binding to
intracellular tubulin and inhibiting its polymerization, which
leads to apoptosis of the target cell.
[0143] Finally, because cetuximab demonstrated virtually no
cytotoxic effects (FIG. 22) (i.e., is virtually harmless to cells),
cetuximab conjugated to the peptides described herein present an
excellent option for use in PDCs.
TABLE-US-00006 TABLE 6 IC.sub.50 value of AE, peptide, PDC and
cetuximab across different cell lines IC.sub.50 nM Peptide-Drug
Cell Line Auristatin E Peptide Conjugate Cetuximab A431 0.0246
387.7 0.0026 NA MDA-MB-468 0.0117 420.3 0.0013 NA HEK 293 0.06258
1037.0 5.807 NA
HER2
[0144] Peptides were synthesized with motif sequences comprising
amino acids corresponding to knob amino acids in pertuzumab which
fit sockets on the HER2 epitope in the antibody-epitope binding
interface. There were a total of eight knobs on pertuzumab,
including T30, D31, Y32, N53, S54, Q61, R73, and G97. See FIG. 23.
For peptide sequences, see Table 7.
TABLE-US-00007 TABLE 7 HER2-targeting peptide motif sequences Name
Sequence E Binding site on HER2 Pertuzumab 128 235 245 254 257 268
286 290 295 296 311 HER2-Pep1 VTYDTGDRAPNSVGSTTKQ -35.10 249 250
285 303 304 308 310 311 313 314 (SEQ ID NO: 26) HER2-Pep2
VTYDTGNRAPNSVGSLGKQ -39.56 252 258 259 285 286 290 292 293 294 295
(SEQ ID NO: 27) 296 297 311 313 325 326 327 329 HER2-Pep3
VGYDTGLRAPNSVGSTTKQ -35.84 252 257 258 259 285 286 287 308 311 (SEQ
ID NO: 28) HER2-Pep4 VGYDTGLRAPNSVGSLGKQ -37.51 8 83 84 235 236 245
252 257 258 259 270 (SEQ ID NO: 29) 285 286 290 291 292 294
HER2-Pep5 LTYDTGLRAPNSVGSIYKQ -41.33 12 13 236 245 246 248 249 268
286 288 (SEQ ID NO: 30) 324 329 330 332 387 389 413 436 HER2-Pep6
LGYDTGLRAPNSVGSTTKQ -34.60 81 127 234 235 236 244 245 252 257 258
(SEQ ID NO: 31) 259 286 288 294 295 296 297 311 HER2-Pep7
VGYDTGLRAPNSVGSTKQ -38.27 81 83 127 128 156 235 268 269 289 290
(SEQ ID NO: 32) 291 292 294 295 296 297 HER2-Pep8
LTYDTGLRAPNSVGSYKQ -34.91 236 245 252 254 255 257 268 270 286 287
(SEQ ID NO: 33) 288 294 295 297 311 HER2-Pep9 LGYDTGLRAPNSVGSTKQ
-34.46 238 243 245 246 247 248 251 262 286 292 (SEQ ID NO: 34) 293
294 295 321 326 HER2- QKTTSGVSNPARLGTDYGV -41.35 59 83 128 154 234
235 236 244 245 269 Pep10 (SEQ ID NO: 35) 270 288 289 290 291 295
326 329 HER2- QYTFGSNEPARSLGTDNGP -48.67 35 59 236 245 268 269 270
281 286 287 Pep 11 (SEQ ID NO: 36) 289 290 291 292 325 326 327 329
HER2- QNYTFGVPSEARLGTDVTL -35.97 466 467 465 279 306 304 276 266
275 274 Pep12 (SEQ ID NO: 37) 239 273 249 272 237 263 264 HER2-
QKTSGVSNPARLGTDYGV -37.66 5 6 8 417 414 413 324 325 329 326 291
Pep13 (SEQ ID NO: 38) 292 290 234 83 235 128 154 244 216 HER2-
QKTSGVSNPARLGTEYGV -34.09 258 312 298 311 285 268 286 259 287 251
Pep14 (SEQ ID NO: 39) 248 266 250 252 HER2- QKGLSGVSNPARNGTDYTV
-34.15 296 255 252 256 257 258 259 250 311 286 Pep15 (SEQ ID NO:
40) 285 262 308 HER2- QKGLSGVSNPARNGTEYTV -34.09 252 286 311 248
238 247 243 250 285 249 Pep16 (SEQ ID NO: 41) 246 245 227 244 HER2-
QKYISGVSNPARLGTDYTL -36.56 264 246 247 248 243 251 226 227 239 216
Pep17 (SEQ ID NO: 42) 251 252 253 254 255 HER2- LTKDTGLRAPNSVSYIFGQ
-40.62 245 248 249 250 252 257 258 259 268 285 Pep18 (SEQ ID NO:
43) 286 287 296 311 313 HER2- QNYISGVSPKARLGTDYTL -37.10 330 329
327 326 296 295 297 252 311 285 Pep19 (SEQ ID NO: 44) 308 303 HER2-
QNYIFSGVSKARLGTDYTV -33.74 315 314 312 313 298 300 301 281 466 278
Pep20 (SEQ ID NO: 45) 279 305 430 437 438 465 281 5 4 3 1 HER2-
PGNDTGLSRAPENSGFTYQ -45.72 295 316 317 293 349 297 299 300 201 307
Pep21 (SEQ ID NO: 46) 321 319 298 327 326 341 437 304 305 279 410
347 383 310 HER2- TSPQGYPNDAGFLGRESTN -29.66 2 57 231 233 237 278
301 304 306 410 438 Pep22 (SEQ ID NO: 47)
[0145] As shown in Table 7, peptide motif sequences HER2-Pep1 to
HER2-Pep10 had a pattern of *YDT*R*PNS*G*Q* (SEQ ID NO: 84), where
Y, D, T, R, N, S, G, Q correspond to knob-forming amino acids in
pertuzumab which pack sockets in the HER2 epitope. The P residue
was introduced in the sequences as a tertiary structure constraint.
The (*) here represents no amino acid and/or one or more amino
acids that can be used as spacers.
[0146] Peptide motif sequences HER2-Pep11 to HER2-Pep21 had a
pattern of QYTFGSNEPARSLGTDNGP (SEQ ID NO: 36), where Q, S, N, R, T
and D (underlined) correspond to knob-forming amino acids in
pertuzumab, which pack sockets in the HER2 epitope. The P residue
at the ninth position in these sequences was introduced as a
tertiary structure constraint. The residues not underlined are
amino acids which fill the spaces between the knobs.
[0147] FIG. 23 shows a 2-dimensional schematic of the packing
interface between pertuzumab and HER2. It is a simplified lattice
diagram represented by the Knob-Socket model. There are a total of
20 sockets formed by the HER2 epitope at the binding interface.
Sockets that are packed by the same knob are represented in the
same gray scale (see, e.g., the knob labeled D31). Residues forming
sockets are presented along the solid black line, and residues not
involved with packing are omitted or shown as small gray dot.
[0148] Twenty-two peptides were designed, including one control
HER2-PEP22, which has the scrambled sequence of HER2-PEP11. See
Table 7. The estimated docking energy for all (non-scrambled)
peptides ranged from -33.7 kcal/mol to -48.7 kcal/mol. By
comparison, the scrambled peptide had an estimated docking energy
of -29.7 kcal/mol. Peptides HER2-PEP7, HER2-PEP11, HER2-PEP16,
HER2-PEP20, HER2-PEP21 and HER2-PEP22 were synthesized and further
evaluated.
[0149] All HER2-PEPs were designed and synthesized by solid phase
peptide synthesis to be used for binding studies with HER2 protein
by Surface Plasmon Resonance (SPR). FITC labeled HER2-PEPs were
also synthesized for cellular binding and uptake studies by
confocal microscopy and flow cytometry.
In Vitro Binding Specificity
[0150] HER2 protein was known to be overexpressed on various cancer
cells. Since HER2 is located on cellular surface, the major
methodology for the determination of binding with HER2 is using
imaging studies. The binding of peptides to HER2-overexpressed
cells can be studied using fluorescence based imaging techniques.
The FITC tag helps visualize and localize the peptides after being
incubated with cells, when observed using high-resolution electron
microscopy such as confocal laser scanning microscopy. The
advantage of confocal microscopy is its point illumination, which
eliminates out-of-focus light rays, producing images with better
resolution and reduced background signal. Additionally, a confocal
laser scanning microscope slices a thick sample into thin sections
and scans the image at different focal planes or depths within the
sample. This helps in building a three dimensional image of the
sample.
[0151] In addition to confocal microscopy, which permits a
qualitative study of the binding between antibody mimics and cells,
flow cytometry can be used to quantify the fluorescence of cell
samples. Flow cytometry is a technology that analyzes the physical
and chemical characteristics of particles in a fluid when the
particles pass through flow chamber.
[0152] The binding specificity of peptides toward HER2 was
evaluated by incubating the peptides in solution with different
cell lines. MDA-MB-361 and ZR-75-1 are breast cancer cells that are
known to express HER2 at high and medium levels. These two HER2
positive cell lines and one HER2 negative cell line, HEK293 (human
embryonic kidney cell), were used for binding specificity studies.
By using confocal microscopy, peptides were visualized and located
with a FITC tag after incubation, to determine whether they bound
specifically to HER2 positive cells. With flow cytometry, the
fluorescence of cell samples after incubation with different
antibody mimics could be precisely determined and used to analyze
the extent of binding of the peptides.
[0153] HER2 positive cells MDA-MB-361 and ZR-75-1, and control cell
HEK293, were purchased from ATCC (Manassas, Va., USA). Cell culture
reagents including Dulbecco's modified eagle's medium (DMEM),
L-Glutamine, Trypsin (TrypLE Express), Hanks Balanced Salt Solution
(HBSS), Recovery cell culture freezing media, Alexa fluor 594 wheat
germ agglutinin and Slow fade gold mounting medium were purchased
from Invitrogen (Carlsbad, Calif., USA). Penicillin-Streptomycin
was obtained from Cellgro, Mediatech Inc. (VA, USA). Hyclone Fetal
Bovine Serum (FBS) was purchased from Thermo Scientific (Logan,
Utah, USA). All microscopy supplies were purchased from VWR (USA).
FACS Calibur.TM. flow cytometer and BD CellQuest.TM. Pro software
were purchased from BD biosciences (USA). Cell culture supplies
were purchased from Santa Cruz Biotechnology Inc. (TX, USA).
[0154] Cell culture media was prepared by adding 10% FBS, 1%
penicillin streptomycin (5000 I.U./mL) and 1% L-Glutamine (200 mM)
to the DMEM. The media was stored in a refrigerator until use.
MDA-MB-361 and ZR-75-1 cells were cultured and maintained in T75
flasks (growth area, 75 cm.sup.2) in the prepared DMEM at
37.degree. C. and 5% CO2, and were passed when the confluency
reached about 80%. The cells were frozen in recovery freezing media
and kept in nitrogen cans for future use.
Confocal Microscopy Studies
[0155] MDA-MB-361, ZR-75-1 and HER293 cells were grown in T75
flasks at 37.degree. C. and 5% CO2. Upon reaching 80% confluency,
the cells were counted and seeded onto coverslips placed inside
6-well culture plates at a density of 80,000 cells/well. The cells
were allowed to attach for 24 hours before the experiment was
performed. Upon attachment, the cells were washed twice with HBSS
and serum free media.
[0156] Stock solutions of FITC labeled HER2-PEPs and control
peptide were prepared with dimethyl sulfoxide (DMSO). Stock
solutions were diluted with serum free media medium several times
and less than 1% of DMSO was presented in the final solutions of
all peptides (10 .mu.M), which were added to the 6-well culture
plates.
[0157] After incubating 15 minutes at 37.degree. C., the medium was
removed and the cells were washed twice with HBSS. The cells were
then treated with a 2.5 .mu.g/mL solution of Alexa fluor 594 wheat
germ agglutinin in HBSS for 10 minutes to stain the cell plasma
membrane. The dye solution was removed and cells were washed twice
with HBSS. The cells were then fixed by performing a 15 minute
treatment using 4% paraformaldehyde solution prepared in HBSS. The
cells were washed with HBSS followed by a final wash of distilled
water. A 10 .mu.L drop of the mounting medium (slow fade gold) was
placed on the microscopic slides and the coverslips with the fixed
cells were placed on the slides. The coverslips were then sealed on
all four sides. The slides were imaged on a Leica DMIRE2 confocal
laser scanning microscope with Yokogawa spinning disk using
64.times. magnification and oil immersion. The FITC fluorescence
was visualized using 491 nm and the Alexa fluor 594 fluorescence
was visualized using 561 nm wavelength filter.
Flow Cytometry Studies
[0158] Flow cytometric studies were carried out to quantify the
fluorescence intensity of cells after being incubated with FITC
labeled mimics. MDA-MB-361, ZR-75-1 and HEK293 cells were seeded in
6-well culture plates at a density of 3.times.10.sup.5 cells per
well for 24 hours and then incubated with FITC labeled mimics at a
concentration of 10 .mu.M in HBSS at 37.degree. C. for 15 minutes.
After washing with HBSS twice, cells remaining on the plates were
trypsinized with Trpsin-EDTA and centrifuged at 1500 rpm for 5 min.
Supernatant was removed and cell pellets were resuspended in 1 ml
of HBSS. The suspension was transferred to a FACS Calibur.TM. flow
cytometer and cell fluorescence was measured with high dynamic
range photomultipliers with a 530 nm filter. Quantitative changes
of fluorescence in different cells samples were assessed by mean
fluorescence intensity (MFI), and FACS data were analyzed using BD
CellQuest.TM. Pro software. Paired t-tests were used to determine
statistical significance using the GraphPad Prism 5.RTM. Software.
P-values less than or equal to 0.05 were considered to be
statistically significant.
Results
[0159] Confocal microscopy was used to study the in vitro binding
specificity of antibody mimics to HER2 positive cancer cells. Cell
images (not shown) taken by confocal microscopy showed that FITC
labeled antibody mimics can bind to MDA-MB-361 and ZR-75-1 cells
but not HEK293 cells. After incubation with 10 .mu.M of FITC
labeled antibody mimics solution, significant fluorescence were
observed on cellular surfaces of MDA-MB-361 and ZR-75-1 cells while
no fluorescence was detected on HEK293 cells. In contrast, the FITC
labeled control peptide HER2-PEP22 showed no binding to all the
three cell lines. The significant fluorescence of MDA-MB-361 and
ZR-75-1 cells could be a result from specific interaction between
HER2 and antibody mimics, which was further confirmed by SPR
binding affinity results in the next section.
[0160] In the confocal microscopy studies, Alexa Flour 594 dye was
used to colorize cell plasma membrane to help us visualize the
cell. Results demonstrated that, except for binding on the cell
surface, antibody mimics showed partially internalization by cells
to different extents. A possible mechanism of internalization of
antibody mimics is receptor-mediated endocytosis upon binding onto
HER2 protein. Differing from antibodies, antibody mimics are less
than 1/50th of the mass of their corresponding antibodies, which
makes it much easier for their internalization by cells. This
characteristic of peptides may be further used for targeting
delivery of drugs into HER2 overexpressed cancer cells.
[0161] Results from the confocal microscopy studies suggested that
the newly designed antibody mimics could bind to HER2 positive cell
lines. For further quantitative analysis of the binding ability of
these antibody mimics towards HER2 overexpressed cell lines, flow
cytometry studies were carried out to quantify the fluorescence of
HER2 positive and negative cells before and after incubation with
different cells. FACS histograms were obtained for different
samples. Based on these histograms, significant fluorescence peak
shift were observed for HER2 positive cells incubated with antibody
mimics except control peptide HER2-PEP22. In contrast, HER2
negative cells only showed little increase of the fluorescence,
which may result from non-specific binding, and absorption of all
the antibody mimics.
[0162] Mean fluorescence intensity (MFI) for all samples were
analyzed based on histograms using BD CellQuest.TM. Pro software.
The MFI of MDA-MB-361, ZR-75-1, and HEK 293 cells in blank HBSS
were 4.5.+-.0.2, 3.2.+-.0.4 and 3.1.+-.1.0, respectively, and the
corresponding MFI after incubation with control peptide
FITC-HER2-PEP22 were 22.1.+-.0.5, 19.6.+-.3.2 and 14.7.+-.1.7,
respectively. In contrast, when incubated with FITC labeled
antibody mimics, the MFI of MDA-MB-361 and ZR-75-1 cells were 3- to
21-fold higher than that of FITC labeled control peptide HER2-PEP22
(P<0.001). Additionally, the MFI for MDA-MB-361 and ZR-75-1
cells incubated with antibody mimics were 4- to 60-fold higher than
HEK 293 cells after same incubation treatment (P<0.001). No
significant difference in MFI was observed between HEK293 cells
treated with FITC labeled antibody mimics and control peptide.
These results suggested that newly designed antibody mimics can
bind specifically to HER2 positive cells, which was consistent with
results from confocal microscopy studies.
Binding Affinity Studies Using SPR
[0163] Dual Channel SPR Spectrometer SPR7000DC and SR7000 Gold
Sensor chip (500,000 Da Carboxymethyl Dextran) were purchased from
Reichert Technologies (USA). HER2 recombinant human protein was
purchased from Life Technologies.TM. Thermo Fisher Scientific Inc.
Pertuzumab was a gift from Stockton Hematology Oncology Medical
Group. Scrubber 2.RTM. software was download from BioLogic
Software. N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC),
N-hydroxysuccinimide (NHS), Ethanolamine (EA), and glycine were
purchased from Thermo Scientific Inc. Bovine serum albumin (BSA)
was purchased from Sigma-Aldrich.
[0164] To confirm that antibody mimics have the anticipated binding
affinity towards HER2 protein, SPR studies were performed using the
Dual Channel SPR Spectrometer SPR7000DC. All experiments were
carried out at 25.degree. C. with a data collection rate of 2 Hz.
The running method was created using the Autolink control software.
PBS with 0.01% Tween 20 (pH7.4) buffers were filtered and
thoroughly degassed before use. HER2 recombinant human protein was
covalently attached to the left channel of a 500,000 Da
carboxymethylated dextran sensor chip using EDC and NHS
chemistries. Then, the chip was blocked by passing EA for 8 minutes
at a rate of 10 .mu.L/min. All antibody mimics and Pertuzumab were
injected as soluble analytes in PBS with 0.01% Tween 20 at
different concentrations with an injection speed of 15 .mu.L/min.
Triplicate determinations were done at each concentration. The
bound surface was regenerated by sequential injections of 10 mM
glycine in PBST (pH 2.5).
[0165] The sensorgrams from SPR were globally treated using
Scrubber 2.RTM. software where all traces were zeroed, cropped,
aligned, and referenced. Kinetic constants ka and kd were
determined by global fitting analyses of the titration curves using
the 1:1 Lagmurian interaction model taking into account the mass
transport. The equilibrium dissociation constant (KD) was
calculated from the kd/ka ratio.
[0166] To test the binding specificity of antibody mimics to HER2
protein, all peptides were injected through a carboxymethylated
dextran sensor chip that was immobilized with bovine serum albumin
(BSA). In addition to testing the control peptide HER2-PEP22 on
HER2 immobilized sensor chip, we also tested two peptides
EGFR-PEP11 and EGFR-PEP22 designed for binding with EGFR in the SPR
studies.
[0167] Additionally, specific peptides were used for competition
studies with Pertuzumab in SPR. For competition studies, two
methods were used: sequential injections of antibody mimic and
Pertuzumab, and co-injection of antibody mimic and Pertuzumab
(i.e., in a mixture).
[0168] In the sequential injection studies, Pertuzumab solution was
first injected onto the surface chip immobilized with HER2,
followed by regeneration of the surface using 10 mM glycine
solution (pH=2.5). Then, antibody mimic solution was injected,
followed by injection of Pertuzumab solution without regeneration
to see the competitive effect of antibody mimic remaining on the
surface chip for binding between HER2 and Pertuzuma In another
experiment, antibody mimic solution was first injected onto the
sensor chip, followed by regeneration of the surface using 10 mM
glycine solution (pH=2.5). Then, Pertuzumab solution was injected
onto the sensor chip followed by injection of antibody mimic
solution without regeneration to see the competitive effect of any
Pertuzumab remaining on the surface chip for binding between HER2
and the antibody mimic.
[0169] In the co-injection studies, solutions with different ratios
of Pertuzumab and antibody mimic mixture were injected onto sensor
chip to study competition between Pertuzumab and antibody mimic for
binding with HER2. The apparent response (RU) results from both
Pertuzumab and HER2-PEP11 binding to HER2 on the sensor chip. The
sensorgrams with different apparent responses (RU) were then used
to calculate the contribution of Pertuzumab and HER2-PEP11 based on
the response (RU) from individual sensorgrams of pure Pertuzumab
and HER2-PEP11 respectively. The percentage of replacement (X) of
Pertuzumab by HER2-PEP11 can be calculated by using following
equations:
R.sub.apparent=R.sub.pertuzumabX(1-X)+R.sub.HER2-Pep11XX,
X=((R.sub.apparent-R.sub.pertuzumab)/(R.sub.HER2-Pep11-R.sub.pertuzumab)-
).times.100%,
where R.sub.apparent is apparent response based on sensorgram from
co-injection studies, R.sub.pertuzumab is response based on
sensorgram from individual injection of Pertuzumab with same
concentration used in co-injection studies, and R.sub.HER2-Pep11 is
response based on sensorgram from individual injection of
HER2-PEP11 with same concentration used in co-injection
studies.
[0170] Sensorgrams were obtained from binding studies of different
antibody mimics and Pertuzumab with HER2. For kinetics analysis,
different concentrations of antibody mimics and Pertuzumab were
passed over sensor chip with HER2 in order of increasing slop from
lowest concentration to the highest concentration. For the
sensorgram of antibody mimics, the curves exhibited fast on and off
rates and reach equilibrium state rapidly which is very common in
the studies of peptides molecules. Both kinetic constants ka and kd
were determined by global fitting analyses of the titration curves
using the 1:1 Lagmurian interaction model in software Scrubber.
Results showed that the control peptide HER2-PEP22 had the lowest
ka (130.9 M.sup.-1s.sup.-1) and highest kd (0.0181 s.sup.-1),
resulting in a calculated K.sub.D of 138.297 .mu.M which is about
2000 fold higher than that of antibody mimics HER2-PEP11
(K.sub.D=55.4 nM) and HER2-PEP21 (K.sub.D=56.0 nM). Since
HER2-PEP22 is scrambled sequence of HER2-PEP11, the SPR results
proved the importance of design the proper amino acids as knobs to
fit into different sockets on HER2 for preserve binding specificity
and affinity. HER2-PEPDE and HER2-PEPDF, with sequences based on
the CDR sequences of pertuzumab, have K.sub.D of 323 nM and 410 nM
respectively, which are much higher than that of HER2-PEP7
(K.sub.D=93.0 nM), HER2-PEP11 (K.sub.D=55.4 nM), HER2-PEP16
(K.sub.D=247.0 nM) and HER2-PEP21 (K.sub.D=56.0 nM). This
demonstrates the peptides disclosed herein have improved binding of
HER2. By comparison the antibody pertuzumab has a K.sub.D of 54.9
pM as measured by SPR.
PD1 and PDL1
[0171] By using the same method for generation EGFR and HER2,
additional peptides for PD1 and PDL1 were generated as listed in
Tables 8-10 below.
TABLE-US-00008 TABLE 8 PD 1: Short peptides SEQ ID NO: 48 LSYEFLLF
SEQ ID NO: 49 LKFLLFAF SEQ ID NO: 50 LSIEFLPV SEQ ID NO: 51
LSEEFLIL SEQ ID NO: 52 HLFEILMF SEQ ID NO: 53 HSEFFLTL SEQ ID NO:
54 LKEYFFLL SEQ ID NO: 55 LKEFYFLL SEQ ID NO: 56 SKYFPMAF SEQ ID
NO: 57 LKEYFLPL
TABLE-US-00009 TABLE 9 PD1: Longer peptides SEQ ID NO: 58
GLKEPVGYLGFGLPLGLF SEQ ID NO: 59 GLKEIPDYLGFGLPLGLF SEQ ID NO: 60
GLKEIPDYAGFGIPLGVD SEQ ID NO: 61 GLSIPVGYLGFGIPLGLP SEQ ID NO: 62
QLSIPVGYAGFGLPLGAP SEQ ID NO: 63 QLKEIPDYVSFAPLGADF SEQ ID NO: 64
GLKEPVGYLGFGLPGADF SEQ ID NO: 65 NHSEIPDFLGYGLPGADF SEQ ID NO: 66
QLSIEPVFLGYGLPLGLD SEQ ID NO: 67 ALKISEPVYLGFAPLVGD
TABLE-US-00010 TABLE 10 PD-L1 SEQ ID NO: 68 IAPDYSQERDTIEGKTPVR SEQ
ID NO: 69 IAPDYSQELDPIEGKTPVR SEQ ID NO: 70 IAPDYSQERDTIAGKTPVR SEQ
ID NO: 71 IAPDYSQELDPIAGKTPVR SEQ ID NO: 72 IAPDYSQELDPIAGKTPVR SEQ
ID NO: 73 IASDYSQERDTIEGKTPVR SEQ ID NO: 74 ILPDYSQELDPIEGKTPVR SEQ
ID NO: 75 VAPDYSQERDTIAGKTPVR SEQ ID NO: 76 LAPDYKQELDPIAFKTPVR SEQ
ID NO: 77 IAPDYSQELDPIVNVTPVR
Sequence CWU 1
1
84114PRTArtificial SequenceSynthetic amino acid - EGFR 1Trp Ser Gly
Glu Asn Gly Pro Gly Tyr Tyr Asp Tyr Glu Ala 1 5 10 214PRTArtificial
SequenceSynthetic amino acid - EGFR 2Trp Ser Gly Glu Asn Gly Pro
Gly Tyr Trp Asp Tyr Glu Ala 1 5 10 314PRTArtificial
SequenceSynthetic amino acid - EGFR 3Trp Ser Gly Glu Asn Gly Pro
Gly Tyr Leu Asp Tyr Glu Ala 1 5 10 414PRTArtificial
SequenceSynthetic amino acid - EGFR 4Trp Ser Gly Glu Asn Gly Pro
Gly Tyr Ile Asp Tyr Glu Ala 1 5 10 514PRTArtificial
SequenceSynthetic amino acid - EGFR 5Trp Ser Gly Glu Asn Gly Pro
Gly Tyr Val Asp Tyr Glu Ala 1 5 10 614PRTArtificial
SequenceSynthetic amino acid - EGFR 6Trp Ser Gly Glu Asn Gly Pro
Gly Tyr Phe Asp Tyr Glu Ala 1 5 10 714PRTArtificial
SequenceSynthetic amino acid - EGFR 7Trp Ser Gly Glu Asn Gly Pro
Gly Trp Tyr Asp Tyr Glu Ala 1 5 10 814PRTArtificial
SequenceSynthetic amino acid - EGFR 8Trp Ser Gly Glu Asn Gly Pro
Gly Leu Tyr Asp Tyr Glu Ala 1 5 10 914PRTArtificial
SequenceSynthetic amino acid - EGFR 9Trp Ser Gly Glu Asn Gly Pro
Gly Ile Tyr Asp Tyr Glu Ala 1 5 10 1014PRTArtificial
SequenceSynthetic amino acid - EGFR 10Trp Ser Gly Glu Asn Gly Pro
Gly Val Tyr Asp Tyr Glu Ala 1 5 10 1114PRTArtificial
SequenceSynthetic amino acid - EGFR 11Trp Ser Gly Glu Asn Gly Pro
Gly Phe Tyr Asp Tyr Glu Ala 1 5 10 1215PRTArtificial
SequenceSynthetic amino acid - EGFR 12Trp Ser Gly Glu Asn Gly Pro
Gly Thr Tyr Tyr Asp Tyr Glu Ala 1 5 10 15 1315PRTArtificial
SequenceSynthetic amino acid - EGFR 13Trp Ser Gly Glu Asn Gly Pro
Gly Thr Tyr Trp Asp Tyr Glu Ala 1 5 10 15 1415PRTArtificial
SequenceSynthetic amino acid - EGFR 14Trp Ser Gly Glu Asn Gly Pro
Gly Thr Tyr Leu Asp Tyr Glu Ala 1 5 10 15 1515PRTArtificial
SequenceSynthetic amino acid - EGFR 15Trp Ser Gly Glu Asn Gly Pro
Gly Thr Tyr Ile Asp Tyr Glu Ala 1 5 10 15 1615PRTArtificial
SequenceSynthetic amino acid - EGFR 16Trp Ser Gly Glu Asn Gly Pro
Gly Thr Tyr Val Asp Tyr Glu Ala 1 5 10 15 1715PRTArtificial
SequenceSynthetic amino acid - EGFR 17Trp Ser Gly Glu Asn Gly Pro
Gly Thr Tyr Phe Asp Tyr Glu Ala 1 5 10 15 1815PRTArtificial
SequenceSynthetic amino acid - EGFR 18Trp Ser Gly Glu Asn Gly Pro
Gly Thr Trp Tyr Asp Tyr Glu Ala 1 5 10 15 1915PRTArtificial
SequenceSynthetic amino acid - EGFR 19Trp Ser Gly Glu Asn Gly Pro
Gly Thr Leu Tyr Asp Tyr Glu Ala 1 5 10 15 2015PRTArtificial
SequenceSynthetic amino acid - EGFR 20Trp Ser Gly Glu Asn Gly Pro
Gly Thr Ile Tyr Asp Tyr Glu Ala 1 5 10 15 2115PRTArtificial
SequenceSynthetic amino acid - EGFR 21Trp Ser Gly Glu Asn Gly Pro
Gly Thr Val Tyr Asp Tyr Glu Ala 1 5 10 15 2215PRTArtificial
SequenceSynthetic amino acid - EGFR 22Trp Ser Gly Glu Asn Gly Pro
Gly Thr Phe Tyr Asp Tyr Glu Ala 1 5 10 15 2314PRTArtificial
SequenceSynthetic amino acid - EGFR 23Ala Glu Tyr Asp Tyr Phe Gly
Pro Gly Asn Glu Gly Ser Trp 1 5 10 2414PRTArtificial
SequenceSynthetic amino acid - EGFR 24Ala Glu Tyr Asp Phe Tyr Gly
Pro Gly Asn Glu Gly Ser Trp 1 5 10 2514PRTArtificial
SequenceSynthetic amino acid - Control 25Ser Gly Glu Trp Ala Tyr
Asp Gly Tyr Glu Pro Asn Phe Gly 1 5 10 2619PRTArtificial
SequenceSynthetic amino acid - HER 2 26Val Thr Tyr Asp Thr Gly Asp
Arg Ala Pro Asn Ser Val Gly Ser Thr 1 5 10 15 Thr Lys Gln
2719PRTArtificial SequenceSynthetic amino acid - HER 2 27Val Thr
Tyr Asp Thr Gly Asn Arg Ala Pro Asn Ser Val Gly Ser Leu 1 5 10 15
Gly Lys Gln 2819PRTArtificial SequenceSynthetic amino acid - HER 2
28Val Gly Tyr Asp Thr Gly Leu Arg Ala Pro Asn Ser Val Gly Ser Thr 1
5 10 15 Thr Lys Gln 2919PRTArtificial SequenceSynthetic amino acid
- HER 2 29Val Gly Tyr Asp Thr Gly Leu Arg Ala Pro Asn Ser Val Gly
Ser Leu 1 5 10 15 Gly Lys Gln 3019PRTArtificial SequenceSynthetic
amino acid - HER 2 30Leu Thr Tyr Asp Thr Gly Leu Arg Ala Pro Asn
Ser Val Gly Ser Ile 1 5 10 15 Tyr Lys Gln 3119PRTArtificial
SequenceSynthetic amino acid - HER 2 31Leu Gly Tyr Asp Thr Gly Leu
Arg Ala Pro Asn Ser Val Gly Ser Thr 1 5 10 15 Thr Lys Gln
3218PRTArtificial SequenceSynthetic amino acid - HER 2 32Val Gly
Tyr Asp Thr Gly Leu Arg Ala Pro Asn Ser Val Gly Ser Thr 1 5 10 15
Lys Gln 3318PRTArtificial SequenceSynthetic amino acid - HER 2
33Leu Thr Tyr Asp Thr Gly Leu Arg Ala Pro Asn Ser Val Gly Ser Tyr 1
5 10 15 Lys Gln 3418PRTArtificial SequenceSynthetic amino acid -
HER 2 34Leu Gly Tyr Asp Thr Gly Leu Arg Ala Pro Asn Ser Val Gly Ser
Thr 1 5 10 15 Lys Gln 3519PRTArtificial SequenceSynthetic amino
acid - HER 2 35Gln Lys Thr Thr Ser Gly Val Ser Asn Pro Ala Arg Leu
Gly Thr Asp 1 5 10 15 Tyr Gly Val 3619PRTArtificial
SequenceSynthetic amino acid - HER 2 36Gln Tyr Thr Phe Gly Ser Asn
Glu Pro Ala Arg Ser Leu Gly Thr Asp 1 5 10 15 Asn Gly Pro
3719PRTArtificial SequenceSynthetic amino acid - HER 2 37Gln Asn
Tyr Thr Phe Gly Val Pro Ser Glu Ala Arg Leu Gly Thr Asp 1 5 10 15
Val Thr Leu 3818PRTArtificial SequenceSynthetic amino acid - HER 2
38Gln Lys Thr Ser Gly Val Ser Asn Pro Ala Arg Leu Gly Thr Asp Tyr 1
5 10 15 Gly Val 3918PRTArtificial SequenceSynthetic amino acid -
HER 2 39Gln Lys Thr Ser Gly Val Ser Asn Pro Ala Arg Leu Gly Thr Glu
Tyr 1 5 10 15 Gly Val 4019PRTArtificial SequenceSynthetic amino
acid - HER 2 40Gln Lys Gly Leu Ser Gly Val Ser Asn Pro Ala Arg Asn
Gly Thr Asp 1 5 10 15 Tyr Thr Val 4119PRTArtificial
SequenceSynthetic amino acid - HER 2 41Gln Lys Gly Leu Ser Gly Val
Ser Asn Pro Ala Arg Asn Gly Thr Glu 1 5 10 15 Tyr Thr Val
4219PRTArtificial SequenceSynthetic amino acid - HER 2 42Gln Lys
Tyr Ile Ser Gly Val Ser Asn Pro Ala Arg Leu Gly Thr Asp 1 5 10 15
Tyr Thr Leu 4319PRTArtificial SequenceSynthetic amino acid - HER 2
43Leu Thr Lys Asp Thr Gly Leu Arg Ala Pro Asn Ser Val Ser Tyr Ile 1
5 10 15 Phe Gly Gln 4419PRTArtificial SequenceSynthetic amino acid
- HER 2 44Gln Asn Tyr Ile Ser Gly Val Ser Pro Lys Ala Arg Leu Gly
Thr Asp 1 5 10 15 Tyr Thr Leu 4519PRTArtificial SequenceSynthetic
amino acid - HER 2 45Gln Asn Tyr Ile Phe Ser Gly Val Ser Lys Ala
Arg Leu Gly Thr Asp 1 5 10 15 Tyr Thr Val 4619PRTArtificial
SequenceSynthetic amino acid - HER 2 46Pro Gly Asn Asp Thr Gly Leu
Ser Arg Ala Pro Glu Asn Ser Gly Phe 1 5 10 15 Thr Tyr Gln
4719PRTArtificial SequenceSynthetic amino acid - HER 2 47Thr Ser
Pro Gln Gly Tyr Pro Asn Asp Ala Gly Phe Leu Gly Arg Glu 1 5 10 15
Ser Thr Asn 488PRTArtificial SequenceSynthetic amino acid - PD1
48Leu Ser Tyr Glu Phe Leu Leu Phe 1 5 498PRTArtificial
SequenceSynthetic amino acid - PD1 49Leu Lys Phe Leu Leu Phe Ala
Phe 1 5 508PRTArtificial SequenceSynthetic amino acid - PD1 50Leu
Ser Ile Glu Phe Leu Pro Val 1 5 518PRTArtificial SequenceSynthetic
amino acid - PD1 51Leu Ser Glu Glu Phe Leu Ile Leu 1 5
528PRTArtificial SequenceSynthetic amino acid - PD1 52His Leu Phe
Glu Ile Leu Met Phe 1 5 538PRTArtificial SequenceSynthetic amino
acid - PD1 53His Ser Glu Phe Phe Leu Thr Leu 1 5 548PRTArtificial
SequenceSynthetic amino acid - PD1 54Leu Lys Glu Tyr Phe Phe Leu
Leu 1 5 558PRTArtificial SequenceSynthetic amino acid - PD1 55Leu
Lys Glu Phe Tyr Phe Leu Leu 1 5 568PRTArtificial SequenceSynthetic
amino acid - PD1 56Ser Lys Tyr Phe Pro Met Ala Phe 1 5
578PRTArtificial SequenceSynthetic amino acid - PD1 57Leu Lys Glu
Tyr Phe Leu Pro Leu 1 5 5818PRTArtificial SequenceSynthetic amino
acid - PD1 58Gly Leu Lys Glu Pro Val Gly Tyr Leu Gly Phe Gly Leu
Pro Leu Gly 1 5 10 15 Leu Phe 5918PRTArtificial SequenceSynthetic
amino acid - PD1 59Gly Leu Lys Glu Ile Pro Asp Tyr Leu Gly Phe Gly
Leu Pro Leu Gly 1 5 10 15 Leu Phe 6018PRTArtificial
SequenceSynthetic amino acid - PD1 60Gly Leu Lys Glu Ile Pro Asp
Tyr Ala Gly Phe Gly Ile Pro Leu Gly 1 5 10 15 Val Asp
6118PRTArtificial SequenceSynthetic amino acid - PD1 61Gly Leu Ser
Ile Pro Val Gly Tyr Leu Gly Phe Gly Ile Pro Leu Gly 1 5 10 15 Leu
Pro 6218PRTArtificial SequenceSynthetic amino acid - PD1 62Gln Leu
Ser Ile Pro Val Gly Tyr Ala Gly Phe Gly Leu Pro Leu Gly 1 5 10 15
Ala Pro 6318PRTArtificial SequenceSynthetic amino acid - PD1 63Gln
Leu Lys Glu Ile Pro Asp Tyr Val Ser Phe Ala Pro Leu Gly Ala 1 5 10
15 Asp Phe 6418PRTArtificial SequenceSynthetic amino acid - PD1
64Gly Leu Lys Glu Pro Val Gly Tyr Leu Gly Phe Gly Leu Pro Gly Ala 1
5 10 15 Asp Phe 6518PRTArtificial SequenceSynthetic amino acid -
PD1 65Asn His Ser Glu Ile Pro Asp Phe Leu Gly Tyr Gly Leu Pro Gly
Ala 1 5 10 15 Asp Phe 6618PRTArtificial SequenceSynthetic amino
acid - PD1 66Gln Leu Ser Ile Glu Pro Val Phe Leu Gly Tyr Gly Leu
Pro Leu Gly 1 5 10 15 Leu Asp 6718PRTArtificial SequenceSynthetic
amino acid - PD1 67Ala Leu Lys Ile Ser Glu Pro Val Tyr Leu Gly Phe
Ala Pro Leu Val 1 5 10 15 Gly Asp 6819PRTArtificial
SequenceSynthetic amino acid - PD-L1 68Ile Ala Pro Asp Tyr Ser Gln
Glu Arg Asp Thr Ile Glu Gly Lys Thr 1 5 10 15 Pro Val Arg
6919PRTArtificial SequenceSynthetic amino acid - PD-L1 69Ile Ala
Pro Asp Tyr Ser Gln Glu Leu Asp Pro Ile Glu Gly Lys Thr 1 5 10 15
Pro Val Arg 7019PRTArtificial SequenceSynthetic amino acid - PD-L1
70Ile Ala Pro Asp Tyr Ser Gln Glu Arg Asp Thr Ile Ala Gly Lys Thr 1
5 10 15 Pro Val Arg 7119PRTArtificial SequenceSynthetic amino acid
- PD-L1 71Ile Ala Pro Asp Tyr Ser Gln Glu Leu Asp Pro Ile Ala Gly
Lys Thr 1 5 10 15 Pro Val Arg 7219PRTArtificial SequenceSynthetic
amino acid - PD-L1 72Ile Ala Pro Asp Tyr Ser Gln Glu Leu Asp Pro
Ile Ala Gly Lys Thr 1 5 10 15 Pro Val Arg 7319PRTArtificial
SequenceSynthetic amino acid - PD-L1 73Ile Ala Ser Asp Tyr Ser Gln
Glu Arg Asp Thr Ile Glu Gly Lys Thr 1 5 10 15 Pro Val Arg
7419PRTArtificial SequenceSynthetic amino acid - PD-L1 74Ile Leu
Pro Asp Tyr Ser Gln Glu Leu Asp Pro Ile Glu Gly Lys Thr 1 5 10 15
Pro Val Arg 7519PRTArtificial SequenceSynthetic amino acid - PD-L1
75Val Ala Pro Asp Tyr Ser Gln Glu Arg Asp Thr Ile Ala Gly Lys Thr 1
5 10 15 Pro Val Arg 7619PRTArtificial SequenceSynthetic amino acid
- PD-L1 76Leu Ala Pro Asp Tyr Lys Gln Glu Leu Asp Pro Ile Ala Phe
Lys Thr 1 5 10 15 Pro Val Arg 7719PRTArtificial SequenceSynthetic
amino acid - PD-L1 77Ile Ala Pro Asp Tyr Ser Gln Glu Leu Asp Pro
Ile Val Asn Val Thr 1 5 10 15 Pro Val Arg 7812PRTArtificial
SequenceSynthetic amino acidmisc_feature(2)..(2)Xaa can be any
naturally occurring amino acidmisc_feature(4)..(4)Xaa can be any
naturally occurring amino acidmisc_feature(6)..(6)Xaa can be any
naturally occurring amino acidmisc_feature(9)..(9)Xaa can be any
naturally occurring amino acidmisc_feature(11)..(11)Xaa can be any
naturally occurring amino acid 78Trp Xaa Glu Xaa Pro Xaa Phe Tyr
Xaa Tyr Xaa Ala 1 5 10 7912PRTArtificial SequenceSynthetic amino
acidmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(5)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(9)..(9)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(12)Xaa can be any naturally occurring amino
acid 79Gln Xaa Ser Asn Xaa Pro Ala Arg Xaa Thr Asp Xaa 1 5 10
8014PRTArtificial SequenceSynthetic amino
acidmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(5)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(9)..(9)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(12)Xaa can be any naturally occurring amino
acid 80Gln Xaa Ser Asn Xaa Pro Ala Arg Xaa Thr Asp Xaa Gly Pro 1 5
10 8119PRTArtificial SequenceSynthetic amino acid 81Gln Tyr Thr Phe
Gly Ser Asn Glu Pro Ala Arg Ser Leu Gly Thr Asp 1 5 10 15 Asn Gly
Pro 8210PRTArtificial SequenceSynthetic amino
acidmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(4)..(4)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(7)Xaa can be any naturally occurring amino
acidmisc_feature(9)..(9)Xaa can be any naturally occurring amino
acid 82Trp Xaa Glu Xaa Phe Tyr Xaa Tyr Xaa Ala 1 5 10
8316PRTArtificial SequenceSynthetic amino acid 83Trp Ser Gly Glu
Asn Gly Pro Gly Thr Phe Tyr Asp Tyr Glu Ala Cys 1 5 10 15
8415PRTArtificial SequenceSynthetic amino
acidmisc_feature(1)..(1)Xaa can be any naturally occurring amino
acidmisc_feature(5)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(7)Xaa can be any naturally occurring amino
acidmisc_feature(11)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(13)Xaa can be any naturally occurring amino
acidmisc_feature(15)..(15)Xaa can be any naturally occurring amino
acid 84Xaa Tyr Asp Thr Xaa Arg Xaa Pro Asn Ser Xaa Gly Xaa Gln Xaa
1 5 10 15
* * * * *