US2010233176A1PendingUtilityA1
Methods and systems for predicting misfolded protein epitopes
Est. expiryOct 6, 2028(~2.2 yrs left)· nominal 20-yr term from priority
A61P 43/00A61P 37/04A61P 35/04A61P 31/00A61P 35/00A61P 25/16A61P 25/28A61P 25/00A61P 35/02A61P 15/00A61P 21/00A61P 13/12A61P 17/00A61P 11/00G06N 3/126A61K 2039/505G16B 20/00C07K 16/2863C12Y 207/10001C07K 2317/34C07K 2317/73C12Y 115/01001C12N 9/0089C07K 16/2872C07K 16/40G16B 15/00C07K 14/70596C12N 9/12G06N 7/01G16B 5/00G16B 20/50G16B 20/30G16B 15/20G16B 5/30
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Claims
Abstract
A method and system to identify an epitope unique to a misfolded form of a protein is provided. Sets of one or more amino acid residues are selected from a model representing the structure of the protein; the free energy of unfolding of each set is determined; and the epitope is identified from the sets having a total probability of unfolding above a minimum probability or a free energy of unfolding below a minimum energy. In other aspects, the invention provides for the use of epitopes identified by the epitope prediction methods, and related antibodies, to diagnose and treat disease and to screen samples for the presence of such epitopes.
Claims
exact text as granted — not AI-modified1 . A method of operating a system to identify an epitope unique to a misfolded form of a protein of interest, the system comprising an input unit, a candidate selection unit, a free energy computation unit, and an epitope identification unit, the method comprising:
(a) receiving by the input unit a model representing at least a portion of the structure of the protein of interest; (b) selecting by the candidate selection unit one or more sets from the model, each set comprising one or more amino acid residues; (c) determining by the free energy computation unit the free energy of unfolding of each set; and (d) identifying by the epitope identification unit the epitope by:
(i) determining the total probability of unfolding of each set based on the free energy of unfolding of one or more of the sets in isolation, and identifying the epitope from the sets having a total probability of unfolding above a minimum probability; or
(ii) identifying the epitope from the sets having a free energy of unfolding below a minimum energy.
2 . The method of claim 1 , wherein:
(a) the method further comprises receiving by the input unit a selection of a region in the model; and (b) each of the one or more sets selected by the candidate selection unit comprise at least one residue in the region.
3 . The method of claim 1 , wherein selecting by the candidate selection unit one or more sets from the model comprises selecting all sets comprising a sequence of one or more contiguous amino acid residues in the model.
4 . The method of claim 1 , wherein selecting by the candidate selection unit one or more sets from the model comprises selecting all sets comprising two or more non-contiguous sequences of one or more contiguous amino acid residues in the model.
5 . The method of claim 3 , wherein each sequence comprises a length between a minimum length and a maximum length.
6 . The method of claim 1 , wherein determining by the free energy computation unit the free energy of unfolding of each set comprises determining the enthalpy of unfolding of the set by:
(a) determining the total number of pairs of polar atoms in the model that (i) are comprised of at least one polar atom in the set, (ii) are more than one amino acid residue apart in the amino acid sequence of the model, and (iii) are within a cutoff distance from one another; and (b) determining the enthalpy of unfolding of the set as the product of the total number of pairs determined in (a) by a fixed interaction energy.
7 . The method of claim 1 , wherein determining by the free energy computation unit the free energy of unfolding of each set comprises determining the enthalpy of unfolding of the set by summing the results of solving the following equation for each pair of polar atoms in the model that are comprised of at least one atom in the set:
Δ
H
=
ε
′
cos
4
(
θ
)
(
(
1
r
)
10
-
β
(
1
r
)
12
)
wherein:
ΔH is the enthalpy of unfolding of the pair of polar atoms;
{acute over (∈)} is a fitting parameter;
θ is the angle between covalent bonds to carbon involving the polar atoms;
r is the distance between the pair of polar atoms; and
β is a fitting parameter.
8 . The method of claim 1 , wherein determining by the free energy computation unit the free energy of unfolding of each set comprises determining the enthalpy of unfolding of the set by:
(a) determining the solvent accessible surface area of the polar, nonpolar and hydroxyl functional groups that comprise the amino acid residues in the model; (b) determining the solvent accessible surface area of the polar, nonpolar and hydroxyl functional groups that comprise the amino acid residues in the model with the set removed; (c) determining the solvent accessible surface area of the polar, nonpolar and hydroxyl functional groups that comprise the amino acid residues of the set in isolation; (d) determining the sum of the solvent accessible surface area of the polar functional groups determined in (b) and (c) minus the solvent accessible surface area of the polar functional groups determined in (a); (e) determining the sum of the solvent accessible surface area of the nonpolar functional groups determined in (b) and (c) minus the solvent accessible surface area of the nonpolar functional groups determined in (a); (f) determining the sum of the solvent accessible surface area of the hydroxyl functional groups determined in (b) and (c) minus the solvent accessible surface area of the hydroxyl functional groups determined in (a); (g) determining the enthalpy of unfolding of the set by solving the following equation:
Δ
H
(
T
)
=
(
-
8.44
Δ
ASA
nonpolar
+
31.4
(
Δ
ASA
polar
+
Δ
ASA
hydroxyl
)
-
(
60
-
T
)
(
0.45
Δ
ASA
nonpolar
-
0.26
Δ
ASA
polar
-
0.09
Δ
ASA
hydroxyl
)
)
cal
mol
·
K
wherein:
ΔH is the change in enthalpy of unfolding of the set;
T is the temperature;
ΔASA polar is the change in solvent accessible surface area of the nonpolar functional groups determined in (d);
ΔASA nonpolar is the change in solvent accessible surface area of the nonpolar functional groups determined in (e); and
ΔASA hydroxyl is the change in solvent accessible surface area of the hydroxyl functional groups determined in (f).
9 . The method of claim 1 , wherein determining by the free energy computation unit the free energy of unfolding of each set comprises determining the enthalpy of unfolding of the set by:
(a) simulating the motion of the model in a molecular dynamics simulation; (b) summing the interaction energies between all pairs of amino acid residues in the model, wherein each pair comprises at least one amino acid residue in the set; (c) simulating the motion of the model with the set in an unfolded state in a molecular dynamics simulation; (d) summing the interaction energies between all pairs of amino acid residues in the set in the unfolded state; and (e) subtracting the interaction energies determined in (d) from (b).
10 . The method of claim 1 , wherein determining by the free energy computation unit the free energy of unfolding of each set comprises determining the free energy of breaking one or more salt bridges in the model by unfolding the set by determining the sum of the Coulombic energy between each pair of amino acid residues in the model, wherein each pair comprises at least one amino acid residue in the set, the Coulombic energy determined assuming a fixed permittivity within the protein.
11 . The method of claim 1 , wherein determining the free energy of unfolding of each set comprises determining the effects of one or more glycans on the free energy by solving the following equation:
Δ
G
PTM
=
-
RT
ln
(
1
-
a
r
o
(
1
-
erf
(
a
-
r
o
b
(
2
/
3
)
n
)
)
)
wherein:
ΔG ptm is the effect of the glycans on the free energy of unfolding;
R is the ideal gas constant;
T is the temperature;
a is the radius of the glycans;
r o is the starting position of the set around the glycans as measured from the center of the glycans;
erf is the error function;
b is the persistence length of the unfolded set; and
n is the number of residues in the unfolded set.
12 . The method of claim 1 , wherein determining the total probability of unfolding of each set based on the free energy of unfolding of one or more of the sets comprises, for each set, determining the sum of the equilibrium probabilities of unfolding of all of the sets comprising the amino acid residue sequence of the set, the equilibrium probability of unfolding of each set determined based on the free energy of unfolding of the set.
13 . A method for obtaining an immunogen useful to produce an antibody that binds selectively to a misfolded form of a protein relative to the natively folded form of that protein, the method comprising:
(a) applying the method according to claim 1 to identify an epitope presented uniquely by a misfolded form of said protein; and (b) producing an immunogen comprising a peptide that comprises an epitope unique to the misfolded form of said protein.
14 . An immunogen, whenever produced by the method according to claim 13 .
15 . A method for obtaining an antibody that binds selectively to a misfolded form of a protein relative to the natively folded form of that protein, the method comprising:
(a) applying the method according to claim 1 to identify an epitope unique to a misfolded form of said protein; and (b) producing an antibody that binds selectively to said epitope, thereby obtaining an antibody that binds selectively to said misfolded form of said protein.
16 . An antibody, whenever prepared by the method according to claim 15 .
17 . A method for obtaining a pharmaceutical composition useful to treat a subject presenting with a disease associated with a misfolded form of a protein, the method comprising combining a pharmaceutically acceptable carrier with a medically useful amount of an antibody or binding fragment thereof that binds to an epitope that is unique to the misfolded form of the protein relative to the natively folded form thereof and that was identified using the method according to claim 1 .
18 . A method for treating a subject presenting with a disease associated with a misfolded form of a protein, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of an antibody or binding fragment thereof that binds selectively to an epitope unique to the misfolded form of the protein relative to the natively folded form of the protein, wherein the epitope was identified by applying the method of claim 1 .
19 . A method for treating a subject presenting with a disease associated with a misfolded form of a protein, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of a vaccine comprising an immunogen that incorporates a peptide comprising an epitope unique to the misfolded form of the protein relative to the natively folded form of that protein, wherein the epitope was identified by applying the method of claim 1 .
20 . A method for detecting a misfolded form of a protein in a sample, the method comprising:
(a) treating a sample suspected to contain a misfolded form of the protein with an antibody or binding fragment that binds selectively to an epitope presented uniquely by the misfolded protein relative to the natively folded form of that protein, wherein said epitope has been identified by applying the method according to claim 1 ; and (b) determining whether an antigen:antibody complex has formed, the formation thereof being indicative of the presence in the sample of a misfolded form of said protein.
21 . A method of diagnosing disease associated with a misfolded form of a protein, the method comprising:
(a) treating a sample that is obtained from a subject and is suspected to contain the misfolded form of the protein with an antibody or binding fragment that binds selectively to an epitope presented uniquely by the misfolded protein relative to the natively folded form of that protein, wherein said epitope has been identified by applying the method according to claim 1 ; and (b) determining whether an antigen:antibody complex has formed, the formation thereof being indicative of the presence in the sample of a misfolded form of said protein.
22 . A method for obtaining an antibody that binds selectively to a misfolded form of a protein relative to the natively folded form of that protein, the method comprising:
(a) obtaining an immunogen comprising any three contiguous amino acids resident in a peptide having one of SEQ ID Nos. 1-80, with the proviso that the peptide does not have the sequence of SEQ ID No. 5, 7, 27-40 or 76; and (b) using the immunogen to produce an antibody that binds selectively to said epitope, thereby obtaining an antibody that binds selectively to said misfolded form of said protein.
23 . An antibody that binds to an epitope that includes at least 3 contiguous amino acids resident in a peptide having SEQ ID Nos 1-4, 6, 8-26, 28, 29, 31-36, 41-75 and 77-80.
24 . A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to claim 23 and a pharmaceutically acceptable carrier.
25 . A method for treating a subject presenting with or at risk for a disease associated with a misfolded form of a protein, the method comprising the step of administering to a subject in need thereof an effective amount of pharmaceutical composition comprising an antibody or binding fragment thereof that binds selectively to an epitope unique to the misfolded form of the protein relative to the natively folded form of that protein, wherein the misfolded protein to which the antibody binds is selected from tau, Abeta, SOD1, α-synuclein, PrP C , TTR, β2-microglobulin, EGFR, CD20, CD38, CD44, FasR, and Notch1.
26 . The method according to claim 25 , wherein the epitope to which the antibody binds comprises at least 3 contiguous amino acids resident in a peptide having any one of SEQ ID Nos. 1-4, 6, 8-26, 28, 29, 31-36, 41-75 and 77-80.
27 . A method according to claim 26 , wherein the antibody and disease to be treated are selected from:
(a) an antibody that binds selectively to the epitope designated by SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and particularly 3, 4, 6, 7, 8, 9 or 10, for the treatment of subjects afflicted with CJD or a related prion disease; (b) an antibody that binds selectively to the epitope designated by SEQ ID No. 16, 17, 18, 19, 20, 21 or 22 for the treatment of subjects afflicted with familial amyloid polyneuropathy or senile systemic amyloidosis or a disease related by the presence of misfolded TTR; (c) an antibody that binds selectively to the epitope designated by SEQ ID No. 23, 24, 25, or 26 for the treatment of subjects afflicted with renal accumulation of β2 microglobulin amyloid deposits or a disease related by the presence of misfolded β2 microglobulin; (d) an antibody that binds selectively to the epitope designated by SEQ ID No. 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, and particularly by SEQ ID Nos. 28, 29, 31, 32, 33, 34, 35 and 36 for the treatment of subjects afflicted with amyotrophic lateral sclerosis (ALS) or a disease related by the presence of misfolded SOD1; (e) an antibody that binds selectively to the epitope designated by SEQ ID No. 41, 42, 43, 44, or 45 for the treatment of subjects afflicted with leukemias or myelomas or a disease related by the presence of misfolded CD38; (f) an antibody that binds selectively to the epitope designated by SEQ ID No. 46, 47, f8, 49, or 50 for the treatment of subjects afflicted with colon cancer metastasis and or a disease related by the presence of misfolded CD44; (g) an antibody that binds selectively to the epitope designated by SEQ ID No. 51, 52, 53, 54, or 55 for the treatment of subjects afflicted with cancer in which Fas receptor is implicated; (h) an antibody that binds selectively to the epitope designated by SEQ ID No. 56, 57, 58, 59, or 60 for the treatment of subjects afflicted with cancers including cervical, head and neck, endometrial, lung and breast carcinomas, pleural mesotheliomas, malignant melanomas, Hodgkin lymphomas, anaplastic large cell non-Hodgkin lymphomas, or a disease related by the presence of misfolded NOTCH1 including certain acute myeloid leukemias and B-cell chronic lymphoid leukemias; (i) an antibody that binds selectively to the epitope designated by SEQ ID No. 61, 62, 63, 64, or 65 for the treatment of subjects afflicted with cancer in which Fas receptor activation is implicated; and (j) an antibody that binds selectively to the epitope designated by SEQ ID No. 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80, with the optional proviso that SEQ ID No. 76 is excluded, for the treatment of subjects afflicted with certain cancers and related disorders in which misfolded EGFR is implicated.
28 . A method for treating a subject presenting with, or at risk for, a disease associated with a misfolded form of a protein, the method comprising the step of administering to the subject a vaccine comprising an immunogen that incorporates a peptide constituting an epitope unique to the misfolded form of the protein relative to the natively folded form of that protein, wherein the epitope comprises at least 3 contiguous amino acids resident in a peptide having any one of SEQ ID Nos. 1-4, 6, 8-26, 28, 29, 31-36, 41-75 and 77-80.
29 . A method according to claim 28 wherein the vaccine and the disease to be treated are selected from:
(a) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and particularly 3, 4, 6, 7, 8, 9 or 10, for the treatment of subjects afflicted with CJD or a related prion disease; (b) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 11, 12, 13, 14, or 15 for the prophylaxis in subjects, particularly cattle, at risk for BSE or a related prion disease; (c) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 16, 17, 18, 19, 20, 21 or 22 for the treatment of subjects afflicted with familial amyloid polyneuropathy or senile systemic amyloidosis or a disease related by the presence of misfolded TTR; (d) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 23, 24, 25, or 26 for the treatment of subjects afflicted with renal accumulation of β2 microglobulin amyloid deposits or a disease related by the presence of misfolded β2 microglobulin; (e) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, and particularly by SEQ ID Nos. 28, 29, 31, 32, 33, 34, 35 and 36 for the treatment of subjects afflicted with amyotrophic lateral sclerosis (ALS) or a disease related by the presence of misfolded SOD1; (f) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 41, 42, 43, 44, or 45 for the treatment of subjects afflicted with leukemias or myelomas or a disease related by the presence of misfolded CD38; (g) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 46, 47, 48, 49, or 50 for the treatment of subjects afflicted with colon cancer metastasis and or a disease related by the presence of misfolded CD44; (h) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 51, 52, 53, 54, or 55 for the treatment of subjects afflicted with cancer in which Fas receptor activation is implicated; (i) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 56, 57, 58, 59, or 60 for the treatment of subjects afflicted with cancers including cervical, head and neck, endometrial, lung and breast carcinomas, pleural mesotheliomas, malignant melanomas, Hodgkin lymphomas, anaplastic large cell non-Hodgkin lymphomas, or a disease related by the presence of misfolded NOTCH1 including certain acute myeloid leukemias and B-cell chronic lymphoid leukemias; (j) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 61, 62, 63, 64, or 65 for the treatment of subjects afflicted with cancer in which Fas receptor activation is implicated; and (k) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80, with the optional proviso that SEQ ID No. 76 is excluded, for the treatment of subjects afflicted with certain cancers and related disorders in which misfolded EGFR is implicated.
30 . A peptide having an amino acid sequence set out in any one of SEQ ID Nos. 1-80, in isolated form.
31 . A peptide according to claim 30 , wherein the peptide comprises an amino acid substitution.
32 . A peptide according to claim 30 , wherein the peptide is a variant that is truncated by amino acid deletion to a length not exceeding 7 residues.
33 . A peptide according to claim 30 , wherein the peptide is a variant that is extended by amino acid addition to a length of at least 7 residues.Cited by (0)
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