U.S. patent application number 16/719103 was filed with the patent office on 2022-08-18 for inhibition of tcr signaling with peptide variants.
The applicant listed for this patent is Signablok, Inc.. Invention is credited to Alexander B Sigalov.
Application Number | 20220259267 16/719103 |
Document ID | / |
Family ID | 1000006504943 |
Filed Date | 2022-08-18 |
United States Patent
Application |
20220259267 |
Kind Code |
A9 |
Sigalov; Alexander B |
August 18, 2022 |
Inhibition Of TCR Signaling With Peptide Variants
Abstract
The present invention provides compositions comprising peptides
derived from amino acid sequences (or from combinations thereof) of
fusion and other protein regions of various viruses, including but
not limited to, severe acute respiratory syndrome coronavirus,
herpesvirus saimiri, human herpesvirus 6, Lassa virus, lymphocytic
choriomeningitis virus, Mopeia virus, Tacaribe virus, Friend murine
leukemia virus; human T lymphotropic virus type 1; herpesvirus
ateles; Marburg virus; Sudan Ebola virus; Zaire Ebola virus, and
comprising L- and/or D-amino acids and combinations thereof, which
affect T cells by acting on the T cell antigen receptor (TCR). More
specifically, the peptides act on the
TCR.alpha..beta.-CD3.delta..epsilon.-CD3.gamma..epsilon.-.zeta..zeta.
signaling complex. Yet more specifically, the peptides act on the
TCR.alpha./CD3.delta..epsilon./.zeta..zeta. signaling module of
TCR. The present invention further relates to the prevention and
therapy of various T cell-related disease states involving the use
of these compositions. Specifically, the compositions are useful in
the treatment and/or prevention of a disease or condition where T
cells are involved or recruited. The compositions of the present
invention also are useful in the production of medical devices
comprising peptide matrices (for example, medical implants and
implantable devices).
Inventors: |
Sigalov; Alexander B;
(Worcester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Signablok, Inc. |
Shrewsbury |
MA |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20200123205 A1 |
April 23, 2020 |
|
|
Family ID: |
1000006504943 |
Appl. No.: |
16/719103 |
Filed: |
December 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16166984 |
Oct 22, 2018 |
10538558 |
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16719103 |
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12895454 |
Sep 30, 2010 |
10138276 |
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16166984 |
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61247033 |
Sep 30, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2710/16522
20130101; C12N 2710/16422 20130101; C07K 14/005 20130101; A61K
38/00 20130101 |
International
Class: |
C07K 14/005 20060101
C07K014/005 |
Claims
1-11. (canceled)
12. A method, comprising: a) providing; i) a patient having at
least one symptom of a disease or a medical condition where T cells
are involved or recruited; and ii) a peptide inhibitor comprising a
peptide consisting of no more than 25 amino acids according to
formula: R1-Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-R2, wherein: R1 is
selected from the group consisting of N-terminal sugar conjugate
and N-terminal lipid conjugate; Y1 is selected from the group
consisting of Arg, Arg-Arg, Arg-Arg-Arg and Arg-Arg-Arg-Arg (SEQ ID
NO: 82); Y2 is selected from the group consisting of Lys, Lys-Lys,
Lys-Lys-Lys and Lys-Lys-Lys-Lys (SEQ ID NO: 83); Y3 is selected
from the group consisting of A1-A2-A3, A*1-A*2-A*3-A*4 and
A**1-A**2-A**3-A**4, wherein: A1 is absent or is selected from the
group consisting of Val, Ile, Leu and a two amino acid peptide,
said peptide consisting of Val, Ile and Leu in any combination; A2
is absent or is selected from the group consisting of Gly and Met;
A3 is absent or is selected from the group consisting of Phe, Leu,
Ile and Tyr; A*1 is absent or is selected from the group consisting
of Arg, Gly, Leu and Ile; A*2 is absent or is selected from the
group consisting of Asn, Gln and Ala; A*3 is absent or is selected
from the group consisting of Ile, Leu and Ser; A*4 is absent or is
selected from the group consisting of Val, Asn and Gly; A**1 is
absent or is selected from the group consisting of Ala, Pro, Cys,
Thr, Asn, Met, Glu and a two amino acid peptide, said peptide
consisting of Ala, Pro, Cys, Thr, Asn, Met and Glu in any
combination; A**2 is absent or is selected from the group
consisting of Thr, Ser, Gly, Ala, Tyr, Pro, Ile, a two amino acid
peptide and a three amino acid peptide, said peptide consisting of
Thr, Ser, Gly, Ala, Tyr, Pro and Ile in any combination; A**3 is
absent or is selected from the group consisting of Gly, Met, Cys,
Pro, Leu, Ile, Gln and a two amino acid peptide, said peptide
consisting of Gly, Met, Cys, Pro, Leu, Ile and Gln in any
combination; A**4 is absent or is selected from the group
consisting of Ala, Thr, Glu, Asn and Ser; Y4 is a positively
charged amino acid selected from the group comprising Arg, Lys and
His; Y5 is selected form the group consisting of C1, C*1 and C**,
wherein: C1 is a four amino acid peptide consisting of Leu, Ile,
Thr and Pro in any combination; C*1 is a three amino acid peptide
consisting of Asp, Leu, Ile, Arg, Lys, Ser, Val and Glu in any
combination; C** is an eight amino acid peptide; Y6 is a positively
charged amino acid selected from the group comprising Arg, Lys and
His; Y7 is selected from the group consisting of E1-E2-E3, E* 1 and
E** 1-E**2-E**3, wherein: E1 is Val or Tyr; E2 is absent or is
selected from the group consisting of Ala and Phe; E3 is absent or
Gly; E* 1 is a three amino acid peptide consisting of Asp, Asn,
Leu, Lys, Ile, Val and Glu in any combination; E**1 is absent or is
selected from the group consisting of Gin, Cys, Glu, Trp and Arg;
E**2 is absent or is selected from the group consisting of Leu,
Ile, Phe, Gly and Lys; E**3 is absent or is selected from the group
consisting of Gly and Thr; Y8 is selected form the group consisting
of F1-F2, F*1 and F**1, wherein: F1 is absent or is selected from
the group consisting of Gly, Phe, Asn, a two amino acid peptide and
a three amino acid peptide, said peptide consisting of Gly, Phe and
Asn in any combination; F2 is absent or is selected from the group
consisting of Leu, Phe, Ile, Met, Thr, Gln, a two amino acide
peptide, a three amino acide peptide, a four amino acide peptide
and a five amino acide peptide, said peptide consisting of Leu,
Phe, Ile, Met, Thr and Gln in any combination; F*1 is a positively
charged amino acid selected from the group comprising Arg, Lys and
His; F**1 is absent or is selected from the group consisting of
Asn, Leu, Ile, Thr, Phe, Val, a two amino acid peptide, a three
amino acid peptide and a four amino acid peptide said peptide
consisting of Asn, Leu, Ile, Thr, Phe and Val in any combination;
Y9 is absent or G*1-G*2, wherein: G* 1 is selected from the group
consisting of Ile, Leu and Asp; G*2 is absent or is selected from
the group consisting of Asn and Thr; Y10 is absent or is selected
from the group consisting of Arg, Arg-Arg, Arg-Arg-Arg and
Arg-Arg-Arg-Arg (SEQ ID NO: 82); Y11 is absent or is selected from
the group consisting of Lys, Lys-Lys, Lys-Lys-Lys and
Lys-Lys-Lys-Lys (SEQ ID NO: 83); and R2 is absent or is C-terminal
lipid conjugate; and b) administering said peptide inhibitor to
said patient under conditions such that said at least one symptom
is reduced.
13. The method of claim 12, wherein said disease or said medical
condition comprises an autoimmune disorder.
14. The method of claim 13, wherein said autoimmune disorder is
selected from the group consisting of systemic lupus erythematosus,
rheumatoid arthritis, multiple sclerosis, type I diabetes,
inflammatory bowel disease, Crohn's disease, primary biliary
cirrhosis, chronic active hepatitis, atopic dermatitis, psoriasis,
pemphigus vulgaris and autoimmune pericarditis.
15. The method of claim 12, wherein: said R1 is selected from one
of the group consisting of N-terminal sugar conjugate and
N-terminal lipid conjugate; said Y1 is selected from one of the
group consisting of Arg, Arg-Arg, Arg-Arg-Arg and Arg-Arg-Arg-Arg
(SEQ ID NO: 82); said Y2 is selected from one of the group
consisting of Lys, Lys-Lys, Lys-Lys-Lys and Lys-Lys-Lys-Lys (SEQ ID
NO: 83); said Y3 is said A1-A2-A3, wherein; A1 is absent or is
selected from one of the group consisting of Val, Ile, and Leu or a
peptide consisting of two amino acids, said two amino acids being
selected from the group consisting of Val, Ile and Leu in any
combination; A2 is absent or is selected from one of the group
consisting of Gly and Met; A3 is absent or is selected from one of
the group consisting of Phe, Leu, Ile and Tyr; said Y4 is a
positively charged amino acid selected from one of the group
consisting of Arg, Lys and His; said Y5 is a four amino acid
peptide consisting of Leu, Ile, Thr and Pro in any combination;
said Y6 is a positively charged amino acid selected from one of the
group consisting of Arg, Lys and His; said Y7 is said E1-E2-E3,
wherein E1 is Val or Tyr; E2 is absent or is Ala or Phe; E3 is
absent or Gly; said Y8 is said F1-F2, wherein; F1 is absent or is
selected from the group consisting of a single amino acid, a two
amino acid peptide and a three amino acid peptide, said amino acid
being selected from the group consisting of Gly, Phe and Asn in any
combination; F2 is absent or is selected from the group consisting
of a single amino acid, a two amino acid peptide, a three amino
acid peptide, a four amino acid peptide and a five amino acid
peptide, said amino acid being selected from the group consisting
of Leu, Phe, Ile, Met, Thr and Gln in any combination; said Y9 is
absent; said Y10 is absent or is selected from the group consisting
of Arg, Arg-Arg, Arg-Arg-Arg, and Arg-Arg-Arg-Arg (SEQ ID NO: 82);
said Y11 is absent or is selected from the group consisting of Lys,
Lys-Lys-, Lys-Lys-Lys and Lys-Lys-Lys-Lys (SEQ ID NO: 83); and said
R2 is absent or is said C-terminal lipid conjugate.
16. The method of claim 12, wherein said N-terminal sugar conjugate
is 1-amino-glucose succinate.
17. The method of claim 12, wherein said N-terminal lipid conjugate
is selected from the group consisting of 2-aminododecanoate and
myristoylate conjugates.
18. The method of claim 12, wherein said C-terminal lipid conjugate
is selected from the group consisting of Gly-Tris-monopalmitate,
Gly-Tris-dipalmitate and Gly-Tris-tripalmitate conjugates.
19. The method of claim 12, wherein said peptide inhibitor is
attached to a carrier molecule.
20. The method of claim 12, wherein said peptide inhibitor is
conjugated at a free amine group with a polyalkylene glycol.
21. The method of claim 20, wherein the polyalkylene glycol is
polyethylene glycol.
22. The method of claim 12, wherein one or more amino acids is a
D-amino acid.
23. The method of claim 12, wherein said peptide inhibitor
comprises at least one amino acid selected from the group
consisting of L-amino acids and D-amino acids.
24. The method of claim 12, wherein said peptide inhibitor has
immunosuppressive activity.
25. The method of claim 12, wherein said peptide inhibitor has an
amino acid sequence selected from the group consisting of
Myr-Gly-Arg-Lys-Leu-Gly-Tyr-Lys-Leu-Leu-Thr-Ile-Arg-Tyr-Ala-Asn-Leu
(SEQ ID NO: 96),
Myr-Gly-Arg-Lys-Gly-Tyr-Arg-Pro-Thr-Pro-Ile-Arg-Val-Ala-Phe-Gly-Asn-Leu
(SEQ ID NO: 97),
Myr-Gly-Arg-Lys-Leu-Val-Leu-Gly-Lys-Ala-Ser-Val-Pro-Ala-Thr-Gly-Ser-Arg-L-
eu-Val-Ser-Lys-Tyr-Lys (SEQ ID NO: 98) and
Myr-Gly-Arg-Lys-Gly-Tyr-Leu-Gly-Pro-Gly-Lys-Asp-Leu-Ser-Arg-Val-Asn-Val-L-
ys-Gly-Arg (SEQ ID NO: 99).
26. The method of claim 12, wherein said peptide inhibitor is a T
cell receptor inhibitor
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Divisional and claims priority
to U.S. non-provisional application Ser. No. 16/166,984, filed Oct.
22, 2018, entitled "Inhibition Of TCR Signaling With Peptide
Variants". This application is a Continuation of application Ser.
No. 12/895,454 filed on Sep. 30, 2010, now U.S. patent Ser. No.
10/138,276, issued on Nov. 27, 2018, which claims priority to and
the benefit of U.S. provisional application Ser. No. 61/247,033
filed Sep. 30, 2009. The entire content of the aforementioned
provisional application is incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
[0002] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted Sequence Listing
with a file named "seqlist", created on Apr. 11, 2019 consisting of
48,051 bytes. The sequence listing contained in this ASCII
formatted document is part of the specification and is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to peptides or fragments,
homologs and derivatives thereof, which are derived from amino acid
sequences of fusion and other protein regions of various viruses
and from combinations thereof, and affect T cells by action on the
activating T cell receptor. The present invention further relates
to the treatment or prevention of various inflammatory and
autoimmune disease states or other conditions where T cells are
involved or recruited. In one embodiment, T cell receptor is
inhibited by variant peptides binding to the transmembrane regions
of the TCR.zeta. and CD3.delta..epsilon. subunits.
BACKGROUND OF THE INVENTION
1. T Cells and T Cell-Related Pathologies
[0004] Immune cells respond to the presence of foreign antigens
with a wide range of responses, including the secretion of
preformed and newly formed mediators, phagocytosis of particles,
cells are a subgroup of cells which together with other immune cell
types (polymorphonuclear, eosinophils, basophils, mast cells, B
cells, and NK cells), constitute the cellular component of the
immune system (U.S. Pat. No. 6,057,294; US Pat. Appl. 20050070478).
Under physiological conditions T cells function in immune
surveillance and in the elimination of foreign antigen. However,
under pathological conditions there is compelling evidence that T
cells play a major role in the causation and propagation of
disease. In these disorders, breakdown of T cell immunological
tolerance, either central or peripheral is a fundamental process in
the causation of autoimmune disease.
[0005] Central tolerance involves thymic deletion of self reactive
cells (negative selection) and positive selection of T cells with
low affinity for self major histocompatibility complex antigens
(MHC). In contrast, there are four, non-mutually exclusive
hypotheses that have been proposed to explain peripheral T cell
tolerance which are involved in the prevention of tissue specific
autoimmune disease. These include: anergy (loss of co-stimulatory
signals, down regulation of receptors critical for T cell
activation), deletion of reactive T cells, ignorance of the antigen
by the immune system and suppression of autoreactive T cells.
Tolerance once induced does not necessarily persist indefinitely. A
breakdown in any of these mechanisms may lead to autoimmune disease
(Srinivasan et al. Cytokine. 2009; 46:147-59; Mescher et al. Semin
Immunol 2007; 19:153-61; US Pat. Appl. 20050070478).
[0006] Numerous diseases are believed to result from autoimmune
mechanisms (Allen et al. J Pept Res 2005; 65:591-604; WO
2006077601). A non-exhaustive list of autoimmune disorders include:
systemic lupus erythematosus, rheumatoid arthritis, multiple
sclerosis, type I diabetes, gastroenterological conditions e.g.
inflammatory bowel disease e.g. Crohn's disease, primary biliary
cirrhosis, chronic active hepatitis; skin problems e.g. atopic
dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems
e.g. autoimmune pericarditis. Autoimmune diseases affect millions
of individuals worldwide and the cost of these diseases, in terms
of actual treatment expenditures and lost productivity, is measured
in billions of dollars annually. Millions of individuals suffer
from or are affected by autoimmune diseases. Over 400 000 people in
the United States suffer from multiple sclerosis (MS), while
diabetes affects about 18.2 million people (6.3% of the
population). Of the 18.2 million people with diabetes, 5-10% have
type I (juvenile) diabetes. Over 16 000 new cases of lupus are
reported every year, with the total number of diagnosed cases
ranging between 500 000 and 1.5 million; and 2.1 million people
(almost 1% of the American population) are affected by rheumatoid
arthritis. Psoriasis affects 4.5 million people, with 23% of those
further diagnosed with psoriatic arthritis. Irritable bowel
syndrome/inflammatory bowel disease (IBS/IBD) affects approximately
10-20%, or up to one in five people in America. T cells also play a
major role in the rejection for organ transplantation or graft
versus host disease by bone marrow (hematopoietic stem cell)
transplantation. Regulation of such immune responses is therefore
therapeutically desired.
2. T Cell Receptor
[0007] Autoimmune disease and other T cell-related pathologies are
characterised by the recruitment of T cells to sites of
inflammation. At these sites, T cells, coupled with their ability
to produce and regulate cytokines and influence B cell function,
orchestrate the immune response and shape the final clinical
outcome.
[0008] T cells respond to antigen via a polypeptide complex
composed of the ligand-binding T cell receptor (TCR)
disulfide-linked .alpha. and .beta. subunits (or .gamma. and
.delta. subunits in .gamma..delta. T cells) that have single
transmembrane (TM) spans per subunit and small intracellular tails
and associate non-covalently with hetero- (CD3.gamma..epsilon. and
CD3.delta..epsilon.) and homodimeric (.zeta..zeta.) signaling
subunits (Cambier J. C. Curr Opin Immunol 1992; 4:257-64; A. B.
Sigalov, ed, Multichain Immune Recognition Receptor Signaling. From
Spatiotemporal Organization to Human Disease, Springer-Verlag, New
York, 2008). The CD3.epsilon., .delta., and .gamma. chains have
single Ig-family extracellular domains, single presumably
.alpha.-helical TM spans, and intrinsically disordered
intracellular domains of 40-60 residues, whereas each .zeta.
subunit has a small extracellular region (9 residues) carrying the
intersubunit disulfide bond, a single presumably .alpha.-helical TM
span per subunit, and a large, intrinsically disordered cytoplasmic
domain of .about.110 residues. An understanding of the process of
TCR-mediated TM signal transduction and subsequent T cell
activation, leading to T cell proliferation and differentiation, is
therefore pivotal to both health and disease. Disturbance in this
intricate structure-function relationship of TCR, harmonising
antigen recognition with T cell activation may provide the
therapeutic means to deal with inflammatory and other T
cell-related disorders.
3. Viral Modulation of T Cell Receptor Signaling
[0009] To successfully infect, replicate and persist in the host,
viruses have evolved numerous strategies to take control of
multiple cellular processes including those that target TM signal
transduction mediated by immune receptors including TCR (Jerome K.
R. J Virol 2008; 82:4194-204, Sigalov A. B. PLoS Pathog 2009; 5.
e1000404; Kim W. M. and Sigalov A. B. Adv Exp Med Biol 2008;
640:325-49; Sigalov A. B. Adv Exp Med Biol 2008; 640:268-311).
Recent breakthroughs in our improved understanding of the
TCR-targeted strategies used by the viruses to escape from the host
immune surveillance reveal new therapeutic targets for antiviral as
well as immunomodulatory therapy (Sigalov A. B. Adv Exp Med Biol
2007; 601:335-44; Sigalov A. B. PLoS Pathog 2009; 5. e1000404).
Therefore, further investigation of how viruses have adapted to
disarm the innate and adaptive immune system will prove invaluable
in rational drug design efforts aiming to reduce immune activation
or inflammation. In particular, viral T cell evasion strategies can
be transferred to therapeutic strategies to treat T cell-mediated
diseases that require similar functionalities. Viruses represent
years of evolution and the efficiency and optimization that come
along with it.
4. Treatment of T Cell-Related Pathologies
[0010] Traditional reagents and methods used to attempt to regulate
an immune response in a patient also result in unwanted side
effects and have limited effectiveness (WO 2006077601). For
example, immunosuppressive reagents (e. g., cyclosporin A,
azathioprine, and prednisone) used to treat patients with
autoimnnune diseases also suppress the patient's entire immune
response, thereby increasing the risk of infection, and can cause
toxic side effects to non-lymphoid tissues. Due to the medical
importance of immune regulation and the inadequacies of existing
immunopharmacological reagents, reagents and methods to regulate
specific parts of the immune system have been the subject of study
for many years.
[0011] Antibodies have been considered as clinically significant
therapeutic agents for various T cell-related diseases. Traditional
costimulatory blockade using antibodies or fragments of antibodies,
while promising, is not without drawbacks. Disadvantages to these
approaches include inherent immunogenicity, unwanted Fc signaling,
as well as poor tissue penetration. There have also been some
indications that immunosuppression can occur with long-term
treatment (Allen et al. J Pept Res 2005; 65:591-604). The use of
antibodies is suggested (U.S. Pat. No. 6,221,352) to treat
autoimmune disorders such as rheumatoid arthritis. Specifically,
this patent covers the administration of monoclonal antibodies,
alone and/or coupled to cytotoxic or cytostatic agents. However,
antibody therapy poses serious disadvantages. First, as antibodies
are natural products they must be produced in cell lines or other
live expression systems. This raises a that there could be
contamination of antibody preparations by infectious agents such as
prions or viruses. Although tight regulation and regulatory
vigilance and surveillance can reduce this concern, the need for
ongoing monitoring and testing for contamination contributes to the
high cost of developing and administering antibody therapies. In
addition, antibody-based therapies require considerable logistical
support. As antibodies are proteins, they cannot be given orally,
except for those used to treat certain types of mucosal infectious
diseases, and therefore, systemic administration is required.
Another serious disadvantage of antibody-based therapies is the
high costs of production, storage, and administration. Moreover,
long infusions (i.e., for example, an hour or longer) require a
hospital environment and are often associated with mild to very
severe side effects. For example,
(genengnews.com/articles/chitem_print.aspx? aid=1668& chid=2),
in one trial, in which four patients in the U.K. were given an
anticancer antibody reactive against an important T cell receptor
(CD28) severe and life-threatening responses were observed; the
cause is at present not understood. This makes large-scale clinical
applications of a number of monoclonal antibodies with demonstrated
therapeutic activity impossible or, at least, severely compromised.
Fast degradation of the administered antibodies is another drawback
of antibody-based therapy.
[0012] Peptides based on TCR-derived sequences for disrupting TCR
function presumably by interfering with assembly have also been
disclosed (WO 96/22306 WO 97/47644, US Pat. Appl. 20050070478).
Despite multiple advantages of these peptides as compared to
antibodies, they have relatively low efficacy in terms of
inhibiting TCR, thus having a high potential for toxicity and side
effects, while the primary criteria for rational design of these
immunomodulatory peptides and optimizing their immunomodulatory
activity have not been suggested.
[0013] Filoviral immunosuppressive peptides and modified
derivatives thereof have been also disclosed (US Pat. Appl.
20070185025). Similarly to the TCR derived peptide sequences, these
peptides demonstrate immunosuppressive activity at relatively high
peptide doses, thus having a high potential for toxicity and side
effects, while the primary criteria for rational design of these
immunomodulatory peptides and optimizing their immunomodulatory
activity have not been suggested.
[0014] Novel uses of peptides derived from the 33 amino acid
residues-long HIV gp41 fusion peptide (FP.sub.1-33) domain, in
methods for prevention or treatment of autoimmune and other T
cell-mediated pathologies, have also been disclosed (WO
2006077601). The FP.sub.1-33 peptide was stated to be effective at
concentrations about 100 fold lower than the peptides of the
invention by Manolios (U.S. Pat. No. 6,057,294; US Pat. Appl.
20050070478). However, published and disclosed experimental data on
immunomodulatoiy activity of this 33 amino acid residues-long
peptide and its fragments (Cohen et al. Biochemistry 2008;
47:4826-33; Bloch et al. Faseb J 2007; 21:393-401; A. B. Sigalov.
Faseb J2007; 21:1633-34; author reply 1635; WO 2006077601) are
discrepant (A. B. Sigalov. Faseb J2007; 21:1633-34; author reply
1635; WO 2006077601). While full length FP.sub.1-33 variants have
been disclosed as pharmaceutical compositions to treat T
cell-mediated pathologies (WO 2006077601), in other publications it
has been stated that in contrast to 16 amino acid residue-long
N-terminal region (FP.sub.1-16) of FP.sub.1-33 and its fragments
(FP.sub.1-8, FP.sub.5-13, and FP.sub.9-16), the C-terminal half of
FP.sub.1-33 (FP.sub.17-32) has been found to be inactive in all
assays used (A. B. Sigalov. Faseb J2007; 21:1633-34; author reply
1635).
5. Prior Art
[0015] Prior art (U.S. Pat. No. 6,221,352) suggests to use
antibodies to target specific TCRs to treat autoimmune disorders
such as rheumatoid arthritis. Specifically, this patent covers the
administration of monoclonal antibodies, alone and/or coupled to
cytotoxic or cytostatic agents. However, antibody therapy poses
serious disadvantages. First, as antibodies are natural products
they must be produced in cell lines or other live expression
systems. This raises a that there could be contamination of
antibody preparations by infectious agents such as prions or
viruses. Although tight regulation and regulatory vigilance and
surveillance can reduce this concern, the need for ongoing
monitoring and testing for contamination contributes to the high
cost of developing and administering antibody therapies. In
addition, antibody-based therapies require considerable logistical
support. As antibodies are proteins, they cannot be given orally,
except for those used to treat certain types of mucosal infectious
diseases, and therefore, systemic administration is required.
Another serious disadvantage of antibody-based therapies is the
high costs of production, storage, and administration. Moreover,
long infusions (i.e., for example, an hour or longer) require a
hospital environment and are often associated with mild to very
severe side effects. For example
(genengnews.com/articles/chitem_print.aspx?aid=1668&chid=2), in
one trial, in which four patients in the U.K. were given an
anticancer antibody reactive against an important T cell receptor
(CD28) severe and life-threatening responses were observed; the
cause is at present not understood. This makes large-scale clinical
applications of a number of monoclonal antibodies with demonstrated
therapeutic activity impossible or, at least, severely compromised.
Fast degradation of the administered antibodies is another drawback
of antibody-based therapy.
[0016] Another prior art (U.S. Pat. No. 6,057,294; US Pat. Appl.
20050070478) suggests to use TCR-derived peptide sequences to treat
autoimmune and other T cell-related disorders. Despite multiple
advantages of these peptides as compared to antibodies, they have
relatively low efficacy in terms of inhibiting TCR, thus having a
high potential for toxicity and side effects, while the primary
criteria for rational design of these immunomodulatory peptides and
optimizing their immunomodulatory activity have not been
suggested.
[0017] Yet another prior art (US Pat. Appl. 20070185025) provides
uses of filoviral immunosuppressive peptides and modified
derivatives thereof to treat T cell-mediated pathologies. Similarly
to the TCR-derived peptide sequences, these peptides demonstrate
immunosuppressive activity at relatively high peptide doses, thus
having a high potential for toxicity and side effects, while the
primary criteria for rational design of these immunomodulatory
peptides and optimizing their immunomodulatory activity have not
been suggested.
[0018] Yet another prior art (WO 2006077601) provides novel uses of
peptides derived from the HIV gp41 fusion peptide domain, in
methods for prevention or treatment of autoimmune and other T
cell-mediated pathologies. These peptides were demonstrated to be
effective at concentrations about 100 fold lower than the peptides
of the invention by Manolios (U.S. Pat. No. 6,057,294; US Pat.
Appl. 20050070478). However, the suggested peptide sequences of
this invention are based on the only amino acid sequences, the
primary sequence of the HIV gp41 fusion peptide domain, which in
addition to the lack of the primary criteria for rational design of
these immunomodulatory peptides and optimizing their
immunomodulatory activity, strongly limits further optimization of
efficacy and specificity of targeting and inhibiting TCR by peptide
variants.
[0019] Yet still another prior art (US Pat. Appl. 20080096809)
provides membrane binding diastereomeric peptides comprising amino
acid sequences corresponding to a fragment of a transmembrane
proteins, wherein at least two amino acid residues of the
diastereomeric peptides being in a D-isomer configuration. These
peptides are suggested to be useful in inhibiting fusion membrane
protein events, including specifically viral replication and
transmission. However, these peptides are not designed specifically
to treat T cell-related disorders.
[0020] What is needed in the art is a broad-based TCR-targeted
therapy rationally designed to disrupt protein-protein interactions
as specifically and effectively as viruses do that may be
administered to treat various diseases having an underlying T cell
etiology that is safe and effective.
SUMMARY OF THE INVENTION
[0021] Novel aspect of the present invention consists of peptides
derived from amino acid sequences of fusion and other protein
regions of various viruses, including but not limiting to, severe
acute respiratory syndrome coronavirus (SARS-CoV), herpesvirus
saimiri (HVS), human herpesvirus 6 (HHV-6), Lassa virus (LASV),
lymphocytic choriomeningitis virus (LCMV), Mopeia virus (MOPV),
Tacaribe virus (TACV), Friend murine leukemia virus (MLV); human T
lymphotropic virus type 1 (HTLV-1); herpesvirus ateles (HVA);
Marburg virus (MARV); Sudan Ebola virus (SEBOV); Zaire Ebola virus
(ZEBOV), which target and inhibit T cell receptor (TCR). In
addition, in this invention, the criteria of designing these
immunomodulatory peptides and a new approach to optimizing their
immunomodulatory activity are suggested. Novel uses of these
peptides in methods for prevention or treatment of autoimmune and
other T cell-mediated pathologies are also suggested.
[0022] The peptides and compositions of the present inventions are
derived from amino acid sequences of fusion and other protein
regions of various viruses, can be designed and formulated to be
delivered orally, optimized for their efficacy and specificity in
accordance to the suggested criteria, thus improving upon prior art
and overcoming current limitations in the prior art. It is
advantageous to transfer therapeutic strategies that target
redundant processes found among a number of viruses. Viruses
represent years of evolution and the efficiency and optimization
that come along with it.
[0023] Peptides and compositions of the present invention can be
used commercially as therapeutic agents to treat T cell-related
disorders. A non-exhaustive list of disorders in which T cells are
involved/recruited include: allergic diathesis e.g. delayed type
hypersensitivity, contact dermatitis; autoimmune disease e.g.
systemic lupus erythematosus, rheumatoid arthritis, multiple
sclerosis, diabetes, Guillain-Barre syndrome, Hashimotos disease,
pernicious anaemia; gastroenterological conditions e.g.
inflammatory bowel disease, Crohn's disease, primary biliary
cirrhosis, chronic active hepatitis; skin problems e.g. atopic
dermatitis, psoriasis, pemphigus vulgaris; infective disease;
respiratory conditions e.g. allergic alveolitis; cardiovascular
problems e.g. autoimmune pericarditis; organ transplantation;
inflammatory conditions e.g. myositis, ankylosing spondylitis; any
disorder where T cells are involved/recruited.
[0024] The present invention relates to peptides and compounds,
which affect T cells by action on the T cell antigen receptor
(TCR). The peptides and compositions of the present invention are
derived from amino acid sequences (or from combinations thereof) of
fusion and other protein regions of various viruses, including but
not limiting to, severe acute respiratory syndrome coronavirus
(SARS-CoV), herpesvirus saimiri (HVS), human herpesvirus 6 (HHV-6),
Lassa virus (LASV), lymphocytic choriomeningitis virus (LCMV),
Mopeia virus (MOPV), Tacaribe virus (TACV), Friend murine leukemia
virus (Fr-MLV); human T lymphotropic virus type 1 (HTLV-1);
herpesvirus ateles (HVA); Marburg virus (MARV); Sudan Ebola virus
(SEBOV); Zaire Ebola virus (ZEBOV), and consisting of L- and/or
D-amino acids and combinations thereof. The present invention
further relates to the prevention and therapy of various T
cell-related disease states involving the use of these peptides and
compounds. Specifically, the peptides and compounds are useful in
the treatment and/or prevention of a disease or condition where T
cells are involved or recruited. The peptides of the present
invention also are useful in the production of medical devices
comprising peptide matrices (for example, medical implants and
implantable devices). In one embodiment, TCR signaling is inhibited
by variant peptides binding to the transmembrane regions of the
CD3.delta., .epsilon. and TCR .zeta. subunits.
[0025] In one embodiment, the present invention contemplates a
variant SARS-CoV FP sequence-based TCR peptide inhibitor itself or
comprising at least one amino acid addition and/or substitution
that optimizes binding to CD3.delta.,.epsilon. and TCR .zeta.
(subunits relative to the TCR .alpha. subunit transmembrane domain
(TCR.alpha. TMD: V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L) (SEQ ID
NO: 78). In one embodiment, the peptide further comprises a
C-terminal and/or an N-terminal sugar conjugate. In one embodiment,
the sugar conjugate is 1-amino-glucose succinate. In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal lipid conjugate. In one embodiment, the lipid conjugate
is selected from the group comprising 2-aminododecanoate or
myristoylate. In one embodiment, the lipid conjugate is selected
from the group comprising Gly-Tris-monopalmitate,
Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment,
the peptide comprises a cyclic peptide. In one embodiment, the
peptide comprise a disulfide-linked dimer. In one embodiment, the
peptide inhibitor includes amino acids selected from the group
including, but not limited to, L-amino acids, or D-amino acids.
[0026] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant SARS-CoV FP sequence-based TCR peptide
inhibitor itself or comprising at least one amino acid addition
and/or substitution that optimizes binding to CD3.delta.,.epsilon.
and TCR .zeta. subunits relative to the TCR.alpha. TMD capable of
reducing said T cell activation; b) administering said inhibitor to
said patient under conditions such that said at least one symptom
is reduced. In one embodiment, the medical condition comprises an
autoimmune disorder. In one embodiment, the autoimmune disorder is
selected from the group consisting of systemic lupus erythematosus,
rheumatoid arthritis, multiple sclerosis, type I diabetes,
gastroenterological conditions e.g. inflammatory bowel disease e.g.
Crohn's disease, primary biliary cirrhosis, chronic active
hepatitis; skin problems e.g. atopic dermatitis, psoriasis,
pemphigus vulgaris; cardiovascular problems e.g. autoimmune
pericarditis.
[0027] In one embodiment, the present invention contemplates a
peptide inhibitor comprising an amino acid sequence consisting of
G-Y-X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub-
.9 (SEQ ID NO: 1), wherein X.sub.1 and X.sub.6 are selected from
the group consisting of R, K or H; X.sub.2, X.sub.3, X.sub.4 and
X.sub.5 are selected from the group consisting of L, I, T or P;
X.sub.7 is selected from the group consisting of V or Y; X.sub.8
consists of A or F or nothing; and X.sub.9 consists of G or
nothing. In one embodiment, the peptide further comprises a
C-terminal and/or an N-terminal sugar conjugate. In one embodiment,
the sugar conjugate is 1-amino-glucose succinate. In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal lipid conjugate. In one embodiment, the lipid conjugate
is selected from the group comprising 2-aminododecanoate or
myristoylate. In one embodiment, the lipid conjugate is selected
from the group comprising Gly-Tris-monopalmitate,
Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment,
the peptide comprises a cyclic peptide. In one embodiment, the
peptide comprise a disulfide-linked dimer. In one embodiment, the
peptide inhibitor includes amino acids selected from the group
including, but not limited to, L-amino acids, or D-amino acids.
[0028] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a peptide inhibitor comprising an amino acid
sequence consisting of
G-Y-X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub-
.9 (SEQ ID NO: 1), wherein X.sub.1 and X.sub.6 are selected from
the group consisting of R, K or H; X.sub.2, X.sub.3, X.sub.4 and
X.sub.5 are selected from the group consisting of L, I, T or P;
X.sub.7 is selected from the group consisting of V or Y; X.sub.8
consists of A or F or nothing; and X.sub.9 consists of G or nothing
capable of reducing said T cell activation; b) administering said
inhibitor to said patient under conditions such that said at least
one symptom is reduced. In one embodiment, the medical condition
comprises an autoimmune disorder. In one embodiment, the autoimmune
disorder is selected from the group consisting of systemic lupus
erythematosus, rheumatoid arthritis, multiple sclerosis, type I
diabetes, gastroenterological conditions e.g. inflammatory bowel
disease e.g. Crohn's disease, primary biliary cirrhosis, chronic
active hepatitis; skin problems e.g. atopic dermatitis, psoriasis,
pemphigus vulgaris; cardiovascular problems e.g. autoimmune
pericarditis.
[0029] In one embodiment, the present invention contemplates a
variant HTLV-1 gp21.sup.313-353 sequence-based TCR peptide
inhibitor itself or comprising at least one amino acid addition
and/or substitution that optimizes binding to CD3.delta.,.epsilon.
and TCR .zeta. subunits relative to the TCR .alpha. subunit
transmembrane domain (TCR.alpha. TMD:
V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L) (SEQ ID NO: 78). In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal sugar conjugate. In one embodiment, the sugar conjugate
is 1-amino-glucose succinate. In one embodiment, the peptide
further comprises a C-terminal and/or an N-terminal lipid
conjugate. In one embodiment, the lipid conjugate is selected from
the group comprising 2-aminododecanoate or myristoylate. In one
embodiment, the lipid conjugate is selected from the group
comprising Gly-Tris-monopalmitate, Gly-Tris-dipalmitate, or
Gly-Tris-tripalmitate. In one embodiment, the peptide comprises a
cyclic peptide. In one embodiment, the peptide comprise a
disulfide-linked dimer. In one embodiment, the peptide inhibitor
includes amino acids selected from the group including, but not
limited to, L-amino acids, or D-amino acids.
[0030] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant HTLV-1 gp21.sup.313-353 sequence-based
TCR peptide inhibitor itself or comprising at least one amino acid
addition and/or substitution that optimizes binding to
CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0031] In one embodiment, the present invention contemplates a
variant HVS tyrosine kinase interacting protein (Tip).sup.211-228
sequence-based TCR peptide inhibitor itself or comprising at least
one amino acid addition and/or substitution that optimizes binding
to CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the TCR
.alpha. subunit transmembrane domain (TCR.alpha. TMD:
V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L) (SEQ ID NO: 78). In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal sugar conjugate. In one embodiment, the sugar conjugate
is 1-amino-glucose succinate. In one embodiment, the peptide
further comprises a C-terminal and/or an N-terminal lipid
conjugate. In one embodiment, the lipid conjugate is selected from
the group comprising 2-aminododecanoate or myristoylate. In one
embodiment, the lipid conjugate is selected from the group
comprising Gly-Tris-monopalmitate, Gly-Tris-dipalmitate, or
Gly-Tris-tripalmitate. In one embodiment, the peptide comprises a
cyclic peptide. In one embodiment, the peptide comprise a
disulfide-linked dimer. In one embodiment, the peptide inhibitor
includes amino acids selected from the group including, but not
limited to, L-amino acids, or D-amino acids.
[0032] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant HVS Tip.sup.211-228 sequence-based TCR
peptide inhibitor itself or comprising at least one amino acid
addition and/or substitution that optimizes binding to
CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0033] In one embodiment, the present invention contemplates a
variant HVA two-in-one protein (Tio).sup.225-242 sequence-based TCR
peptide inhibitor itself or comprising at least one amino acid
addition and/or substitution that optimizes binding to
CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the TCR
.alpha. subunit transmembrane domain (TCR.alpha. TMD:
V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L) (SEQ ID NO: 78). In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal sugar conjugate. In one embodiment, the sugar conjugate
is 1-amino-glucose succinate. In one embodiment, the peptide
further comprises a C-terminal and/or an N-terminal lipid
conjugate. In one embodiment, the lipid conjugate is selected from
the group comprising 2-aminododecanoate or myristoylate. In one
embodiment, the lipid conjugate is selected from the group
comprising Gly-Tris-monopalmitate, Gly-Tris-dipalmitate, or
Gly-Tris-tripalmitate. In one embodiment, the peptide comprises a
cyclic peptide. In one embodiment, the peptide comprise a
disulfide-linked dimer. In one embodiment, the peptide inhibitor
includes amino acids selected from the group including, but not
limited to, L-amino acids, or D-amino acids.
[0034] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant HVA Tio.sup.225-242 sequence-based TCR
peptide inhibitor itself or comprising at least one amino acid
addition and/or substitution that optimizes binding to
CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0035] In one embodiment, the present invention contemplates a
peptide inhibitor comprising an amino acid sequence consisting of
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-L-X.sub.7-X.sub.8-X.sub.9-
-E-X.sub.10-X.sub.11-X.sub.12-X.sub.13 (SEQ ID NO: 2) wherein
X.sub.1 consists of R, G, I, L or nothing; X.sub.2 consists of N,
Q, A or nothing; X.sub.3 consists of L, I, S or nothing; X.sub.4
consists of V, N, G or nothing; X.sub.5, X.sub.8, and X.sub.11 are
selected from the group consisting of R, K or H; X.sub.6 consists
of D, R, S or nothing, X.sub.7 consists of K, E, L or nothing,
X.sub.9 consists of L, V, E or nothing; X.sub.10 consists of N, K,
D or nothing; X.sub.12 consists of I, L, D or nothing; and X.sub.13
consists of N, T or nothing. In one embodiment, the peptide further
comprises a C-terminal and/or an N-terminal sugar conjugate. In one
embodiment, the sugar conjugate is 1-amino-glucose succinate. In
one embodiment, the peptide further comprises a C-terminal and/or
an N-terminal lipid conjugate. In one embodiment, the lipid
conjugate is selected from the group comprising 2-aminododecanoate
or myristoylate. In one embodiment, the lipid conjugate is selected
from the group comprising Gly-Tris-monopalmitate,
Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment,
the peptide comprises a cyclic peptide. In one embodiment, the
peptide comprise a disulfide-linked dimer. In one embodiment, the
peptide inhibitor includes amino acids selected from the group
including, but not limited to, L-amino acids, or D-amino acids.
[0036] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a peptide inhibitor comprising an amino acid
sequence consisting of
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-L-X.sub.7-X.sub.8-X.sub.9-
-E-X.sub.10-X.sub.11-X.sub.12-X.sub.13 (SEQ ID NO: 2) wherein
X.sub.1 consists of R, G, I, L or nothing; X.sub.2 consists of N,
Q, A or nothing; X.sub.3 consists of L, I, S or nothing; X.sub.4
consists of V, N, G or nothing; X.sub.5, X.sub.8, and X.sub.11 are
selected from the group consisting of R, K or H; X.sub.6 consists
of D, R, S or nothing, X.sub.7 consists of K, E, L or nothing,
X.sub.9 consists of L, V, E or nothing; X.sub.10 consists of N, K,
D or nothing; X.sub.12 consists of I, L, D or nothing; and X.sub.13
consists of N, T or nothing capable of reducing said T cell
activation; b) administering said inhibitor to said patient under
conditions such that said at least one symptom is reduced. In one
embodiment, the medical condition comprises an autoimmune disorder.
In one embodiment, the autoimmune disorder is selected from the
group consisting of systemic lupus erythematosus, rheumatoid
arthritis, multiple sclerosis, type I diabetes, gastroenterological
conditions e.g. inflammatory bowel disease e.g. Crohn's disease,
primary biliary cirrhosis, chronic active hepatitis; skin problems
e.g. atopic dermatitis, psoriasis, pemphigus vulgaris;
cardiovascular problems e.g. autoimmune pericarditis.
[0037] In one embodiment, the present invention contemplates a
variant LASV FP (gp2.sup.260-298) sequence-based TCR peptide
inhibitor itself or comprising at least one amino acid addition
and/or substitution that optimizes binding to CD3.delta.,.epsilon.
and TCR .zeta. subunits relative to the TCR .alpha. subunit
transmembrane domain (TCR.alpha. TMD:
V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L) (SEQ ID NO: 78). In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal sugar conjugate. In one embodiment, the sugar conjugate
is 1-amino-glucose succinate. In one embodiment, the peptide
further comprises a C-terminal and/or an N-terminal lipid
conjugate. In one embodiment, the lipid conjugate is selected from
the group comprising 2-aminododecanoate or myristoylate. In one
embodiment, the lipid conjugate is selected from the group
comprising Gly-Tris-monopalmitate, Gly-Tris-dipalmitate, or
Gly-Tris-tripalmitate. In one embodiment, the peptide comprises a
cyclic peptide. In one embodiment, the peptide comprise a
disulfide-linked dimer. In one embodiment, the peptide inhibitor
includes amino acids selected from the group including, but not
limited to, L-amino acids, or D-amino acids.
[0038] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant LASV FP (gp2.sup.260-298)
sequence-based TCR peptide inhibitor itself or comprising at least
one amino acid addition and/or substitution that optimizes binding
to CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0039] In one embodiment, the present invention contemplates a
variant LCMV FP (gp2.sup.266-304) sequence-based TCR peptide
inhibitor itself or comprising at least one amino acid addition
and/or substitution that optimizes binding to CD3.delta.,.epsilon.
and TCR .zeta. subunits relative to the TCR .alpha. subunit
transmembrane domain (TCR.alpha. TMD:
V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L) (SEQ ID NO: 78). In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal sugar conjugate. In one embodiment, the sugar conjugate
is 1-amino-glucose succinate. In one embodiment, the peptide
further comprises a C-terminal and/or an N-terminal lipid
conjugate. In one embodiment, the lipid conjugate is selected from
the group comprising 2-aminododecanoate or myristoylate. In one
embodiment, the lipid conjugate is selected from the group
comprising Gly-Tris-monopalmitate. Gly-Tris-dipalmitate, or
Gly-Tris-tripalmitate. In one embodiment, the peptide comprises a
cyclic peptide. In one embodiment, the peptide comprise a
disulfide-linked dimer. In one embodiment, the peptide inhibitor
includes amino acids selected from the group including, but not
limited to, L-amino acids, or D-amino acids.
[0040] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant LCMV FP (gp2.sup.266-304)
sequence-based TCR peptide inhibitor itself or comprising at least
one amino acid addition and/or substitution that optimizes binding
to CD3.delta.,.epsilon., and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0041] In one embodiment, the present invention contemplates a
variant MOPV FP (gp2.sup.258-296) sequence-based TCR peptide
inhibitor itself or comprising at least one amino acid addition
and/or substitution that optimizes binding to CD3.delta.,.epsilon.
and TCR .zeta. subunits relative to the TCR .alpha. subunit
transmembrane domain (TCR.alpha. TMD:
V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L) (SEQ ID NO: 78). In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal sugar conjugate. In one embodiment, the sugar conjugate
is 1-amino-glucose succinate. In one embodiment, the peptide
further comprises a C-terminal and/or an N-terminal lipid
conjugate. In one embodiment, the lipid conjugate is selected from
the group comprising 2-aminododecanoate or myristoylate. In one
embodiment, the lipid conjugate is selected from the group
comprising Gly-Tris-monopalmitate, Gly-Tris-dipalmitate, or
Gly-Tris-tripalmitate. In one embodiment, the peptide comprises a
cyclic peptide. In one embodiment, the peptide comprise a
disulfide-linked dimer. In one embodiment, the peptide inhibitor
includes amino acids selected from the group including, but not
limited to, L-amino acids, or D-amino acids.
[0042] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant MOPV FP (gp2.sup.258-296)
sequence-based TCR peptide inhibitor itself or comprising at least
one amino acid addition and/or substitution that optimizes binding
to CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0043] In one embodiment, the present invention contemplates a
variant TACV FP (gp2.sup.262-300) sequence-based TCR peptide
inhibitor itself or comprising at least one amino acid addition
and/or substitution that optimizes binding to CD3.delta.,.epsilon.
and TCR .zeta. subunits relative to the TCR .alpha. subunit
transmembrane domain (TCR.alpha. TMD:
V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L) (SEQ ID NO: 78). In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal sugar conjugate. In one embodiment, the sugar conjugate
is 1-amino-glucose succinate. In one embodiment, the peptide
further comprises a C-terminal and/or an N-terminal lipid
conjugate. In one embodiment, the lipid conjugate is selected from
the group comprising 2-aminododecanoate or myristoylate. In one
embodiment, the lipid conjugate is selected from the group
comprising Gly-Tris-monopalmitate, Gly-Tris-dipalmitate, or
Gly-Tris-tripalmitate. In one embodiment, the peptide comprises a
cyclic peptide. In one embodiment, the peptide comprise a
disulfide-linked dimer. In one embodiment, the peptide inhibitor
includes amino acids selected from the group including, but not
limited to, L-amino acids, or D-amino acids.
[0044] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant TACV FP (gp2.sup.262-300)
sequence-based TCR peptide inhibitor itself or comprising at least
one amino acid addition and/or substitution that optimizes binding
to CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0045] In one embodiment, the present invention contemplates a
variant CKS-17 sequence-based TCR peptide inhibitor itself or
comprising at least one amino acid addition and/or substitution
that optimizes binding to CD3.delta.,.epsilon. and TCR .zeta.
subunits relative to the TCR .alpha. subunit transmembrane domain
(TCR.alpha. TMD: V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L) (SEQ ID
NO: 78). In one embodiment, the peptide further comprises a
C-terminal and/or an N-terminal sugar conjugate. In one embodiment,
the sugar conjugate is 1-amino-glucose succinate. In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal lipid conjugate. In one embodiment, the lipid conjugate
is selected from the group comprising 2-aminododecanoate or
myristoylate. In one embodiment, the lipid conjugate is selected
from the group comprising Gly-Tris-monopalmitate,
Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment,
the peptide comprises a cyclic peptide. In one embodiment, the
peptide comprise a disulfide-linked dimer. In one embodiment, the
peptide inhibitor includes amino acids selected from the group
including, but not limited to, L-amino acids, or D-amino acids.
[0046] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant CKS-17 sequence-based TCR peptide
inhibitor itself or comprising at least one amino acid addition
and/or substitution that optimizes binding to CD3.delta.,.epsilon.
and TCR .zeta. subunits relative to the TCR.alpha. TMD capable of
reducing said T cell activation; b) administering said inhibitor to
said patient under conditions such that said at least one symptom
is reduced. In one embodiment, the medical condition comprises an
autoimmune disorder. In one embodiment, the autoimmune disorder is
selected from the group consisting of systemic lupus erythematosus,
rheumatoid arthritis, multiple sclerosis, type I diabetes,
gastroenterological conditions e.g. inflammatory bowel disease e.g.
Crohn's disease, primary biliary cirrhosis, chronic active
hepatitis; skin problems e.g. atopic dermatitis, psoriasis,
pemphigus vulgaris; cardiovascular problems e.g. autoimmune
pericarditis.
[0047] In one embodiment, the present invention contemplates a
variant SEBOV gp2.sup.584-600 sequence-based TCR peptide inhibitor
itself or comprising at least one amino acid addition and/or
substitution that optimizes binding to CD3.delta.,.epsilon. and TCR
.zeta. subunits relative to the TCR .alpha. subunit transmembrane
domain (TCR.alpha. TMD: V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L)
(SEQ ID NO: 78). In one embodiment, the peptide further comprises a
C-terminal and/or an N-terminal sugar conjugate. In one embodiment,
the sugar conjugate is 1-amino-glucose succinate. In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal lipid conjugate. In one embodiment, the lipid conjugate
is selected from the group comprising 2-aminododecanoate or
myristoylate. In one embodiment, the lipid conjugate is selected
from the group comprising Gly-Tris-monopalmitate,
Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment,
the peptide comprises a cyclic peptide. In one embodiment, the
peptide comprise a disulfide-linked dimer. In one embodiment, the
peptide inhibitor includes amino acids selected from the group
including, but not limited to, L-amino acids, or D-amino acids.
[0048] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant SEBOV gp2.sup.584-600 sequence-based
TCR peptide inhibitor itself or comprising at least one amino acid
addition and/or substitution that optimizes binding to
CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0049] In one embodiment, the present invention contemplates a
variant ZEBOV gp2.sup.584-600 sequence-based TCR peptide inhibitor
itself or comprising at least one amino acid addition and/or
substitution that optimizes binding to CD3.delta.,.epsilon. and TCR
.zeta. subunits relative to the TCR .alpha. subunit transmembrane
domain (TCR.alpha. TMD: V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L)
(SEQ ID NO: 78). In one embodiment, the peptide further comprises a
C-terminal and/or an N-terminal sugar conjugate. In one embodiment,
the sugar conjugate is 1-amino-glucose succinate. In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal lipid conjugate. In one embodiment, the lipid conjugate
is selected from the group comprising 2-aminododecanoate or
myristoylate. In one embodiment, the lipid conjugate is selected
from the group comprising Gly-Tris-monopalmitate,
Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment,
the peptide comprises a cyclic peptide. In one embodiment, the
peptide comprise a disulfide-linked dimer. In one embodiment, the
peptide inhibitor includes amino acids selected from the group
including, but not limited to, L-amino acids, or D-amino acids.
[0050] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant ZEBOV gp2.sup.584-600 sequence-based
TCR peptide inhibitor itself or comprising at least one amino acid
addition and/or substitution that optimizes binding to
CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0051] In one embodiment, the present invention contemplates a
variant MARV gp2.sup.585-601 sequence-based TCR peptide inhibitor
itself or comprising at least one amino acid addition and/or
substitution that optimizes binding to CD3.delta.,.epsilon. and TCR
.zeta. subunits relative to the TCR .alpha. subunit transmembrane
domain (TCR.alpha. TMD: V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L)
(SEQ ID NO: 78). In one embodiment, the peptide further comprises a
C-terminal and/or an N-terminal sugar conjugate. In one embodiment,
the sugar conjugate is 1-amino-glucose succinate. In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal lipid conjugate. In one embodiment, the lipid conjugate
is selected from the group comprising 2-aminododecanoate or
myristoylate. In one embodiment, the lipid conjugate is selected
from the group comprising Gly-Tris-monopalmitate,
Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment,
the peptide comprises a cyclic peptide. In one embodiment, the
peptide comprise a disulfide-linked dimer. In one embodiment, the
peptide inhibitor includes amino acids selected from the group
including, but not limited to, L-amino acids, or D-amino acids.
[0052] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant MARV gp2.sup.585-601 sequence-based
TCR peptide inhibitor itself or comprising at least one amino acid
addition and/or substitution that optimizes binding to
CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0053] In one embodiment, the present invention contemplates a
variant Fr-MLV Env gp.sup.548-564 sequence-based TCR peptide
inhibitor itself or comprising at least one amino acid addition
and/or substitution that optimizes binding to CD3.delta.,.epsilon.
and TCR .zeta. subunits relative to the TCR .alpha. subunit
transmembrane domain (TCR.alpha. TMD:
V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L) (SEQ ID NO: 78). In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal sugar conjugate. In one embodiment, the sugar conjugate
is 1-amino-glucose succinate. In one embodiment, the peptide
further comprises a C-terminal and/or an N-terminal lipid
conjugate. In one embodiment, the lipid conjugate is selected from
the group comprising 2-aminododecanoate or myristoylate. In one
embodiment, the lipid conjugate is selected from the group
comprising Gly-Tris-monopalmitate, Gly-Tris-dipalmitate, or
Gly-Tris-tripalmitate. In one embodiment, the peptide comprises a
cyclic peptide. In one embodiment, the peptide comprise a
disulfide-linked dimer. In one embodiment, the peptide inhibitor
includes amino acids selected from the group including, but not
limited to, L-amino acids, or D-amino acids.
[0054] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant Fr-MLV Env gp.sup.548-564
sequence-based TCR peptide inhibitor itself or comprising at least
one amino acid addition and/or substitution that optimizes binding
to CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0055] In one embodiment, the present invention contemplates a
variant HHV-6 U2.sup.428-60 sequence-based TCR peptide inhibitor
itself or comprising at least one amino acid addition and/or
substitution that optimizes binding to CD3.delta.,.epsilon. and TCR
.zeta. subunits relative to the TCR .alpha. subunit transmembrane
domain (TCR.alpha. TMD: V-I-G-F-R-I-L-L-L-K-V-A-G-F-N-L-L-M-T-L)
(SEQ ID NO: 78). In one embodiment, the peptide further comprises a
C-terminal and/or an N-terminal sugar conjugate. In one embodiment,
the sugar conjugate is 1-amino-glucose succinate. In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal lipid conjugate. In one embodiment, the lipid conjugate
is selected from the group comprising 2-aminododecanoate or
myristoylate. In one embodiment, the lipid conjugate is selected
from the group comprising Gly-Tris-monopalmitate,
Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment,
the peptide comprises a cyclic peptide. In one embodiment, the
peptide comprise a disulfide-linked dimer. In one embodiment, the
peptide inhibitor includes amino acids selected from the group
including, but not limited to, L-amino acids, or D-amino acids.
[0056] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a variant HHV-6 U24.sup.28-60 sequence-based TCR
peptide inhibitor itself or comprising at least one amino acid
addition and/or substitution that optimizes binding to
CD3.delta.,.epsilon. and TCR .zeta. subunits relative to the
TCR.alpha. TMD capable of reducing said T cell activation; b)
administering said inhibitor to said patient under conditions such
that said at least one symptom is reduced. In one embodiment, the
medical condition comprises an autoimmune disorder. In one
embodiment, the autoimmune disorder is selected from the group
consisting of systemic lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, type I diabetes, gastroenterological conditions
e.g. inflammatory bowel disease e.g. Crohn's disease, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular
problems e.g. autoimmune pericarditis.
[0057] In one embodiment, the present invention contemplates a
peptide inhibitor comprising an amino acid sequence consisting of
L-N-X.sub.1-X.sub.2-X.sub.3-L-X.sub.4-X.sub.5-L-X.sub.6-L-X.sub.7-X.sub.8-
-G-G-X.sub.9 (SEQ ID NO: 3) wherein X.sub.1 and X.sub.7 are
selected from the group consisting of R, K or H; X.sub.2 consists
of S, R, K, H, P or W; X.sub.3 consists of M, G or A; X.sub.4
consists of L, I, V, N or D; X.sub.5 consists of L, I, F, T, E, A
or G; X.sub.6 consists of E, Q, D, L, F, N or I, X.sub.8 consists
of Q, C, E, W or R, and X.sub.9 consists of L, I, F, T, N or
nothing. In one embodiment, the peptide further comprises a
C-terminal and/or an N-terminal sugar conjugate. In one embodiment,
the sugar conjugate is 1-amino-glucose succinate. In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal lipid conjugate. In one embodiment, the lipid conjugate
is selected from the group comprising 2-aminododecanoate or
myristoylate. In one embodiment, the lipid conjugate is selected
from the group comprising Gly-Tris-monopalmitate,
Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment,
the peptide comprises a cyclic peptide. In one embodiment, the
peptide comprise a disulfide-linked dimer. In one embodiment, the
peptide inhibitor includes amino acids selected from the group
including, but not limited to, L-amino acids, or D-amino acids.
[0058] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a peptide inhibitor comprising an amino acid
sequence consisting of
L-N-X.sub.1-X.sub.2-X.sub.3-L-X.sub.4-X.sub.5-L-X.sub.6-L-X-L-X-L-X-G-G-X-
.sub.9 (SEQ ID NO: 3) wherein X.sub.1 and X.sub.7 are selected from
the group consisting of R, K or H; X.sub.2 consists of S, R, K, H,
P or W; X.sub.3 consists of M, G or A; X.sub.4 consists of L, I, V,
N or D; X.sub.5 consists of L, I, F, T, E, A or G; X.sub.6 consists
of E, Q, D, L, F, N or I, X.sub.8 consists of Q, C, E, W or R, and
X.sub.9 consists of L, I, F, T, N or nothing capable of reducing
said T cell activation; b) administering said inhibitor to said
patient under conditions such that said at least one symptom is
reduced. In one embodiment, the medical condition comprises an
autoimmune disorder. In one embodiment, the autoimmune disorder is
selected from the group consisting of systemic lupus erythematosus,
rheumatoid arthritis, multiple sclerosis, type I diabetes,
gastroenterological conditions e.g. inflammatory bowel disease e.g.
Crohn's disease, primary biliary cirrhosis, chronic active
hepatitis; skin problems e.g. atopic dermatitis, psoriasis,
pemphigus vulgaris; cardiovascular problems e.g. autoimmune
pericarditis.
[0059] In one embodiment, the present invention contemplates a
peptide inhibitor comprising an amino acid sequence consisting of
L-Q-N-X.sub.1-X.sub.2-L-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-L-X.sub.8-
-X.sub.9-X.sub.10-X.sub.11-X.sub.12 (SEQ ID NO: 4) wherein X.sub.1,
X.sub.4 and X.sub.5 are selected from the group consisting of R, K
or H; X.sub.2 consists of D, R or S; X.sub.3 consists of E, K or L;
X.sub.5 and X.sub.7 consist of L, I, or T; X.sub.6 consists of L,
I, or P; X.sub.9 consists of Q, C, E, W or R, X.sub.10 consists of
K, G, F, L, I or nothing; X.sub.11 consists of T, G or nothing; and
X.sub.12 consists of F, L, I, T, N or nothing. In one embodiment,
the peptide further comprises a C-terminal and/or an N-terminal
sugar conjugate. In one embodiment, the sugar conjugate is
1-amino-glucose succinate. In one embodiment, the peptide further
comprises a C-terminal and/or an N-terminal lipid conjugate. In one
embodiment, the lipid conjugate is selected from the group
comprising 2-aminododecanoate or myristoylate. In one embodiment,
the lipid conjugate is selected from the group comprising
Gly-Tris-monopalmitate, Gly-Tris-dipalmitate, or
Gly-Tris-tripalmitate. In one embodiment, the peptide comprises a
cyclic peptide. In one embodiment, the peptide comprise a
disulfide-linked dimer. In one embodiment, the peptide inhibitor
includes amino acids selected from the group including, but not
limited to, L-amino acids, or D-amino acids.
[0060] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a peptide inhibitor comprising an amino acid
sequence consisting of
L-Q-N-X.sub.1-X.sub.2-L-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-L-X-L-X.s-
ub.8-X.sub.9-X-X.sub.11-X.sub.12 (SEQ ID NO: 4) wherein X.sub.1,
X.sub.4 and X.sub.8 are selected from the group consisting of R, K
or H; X.sub.2 consists of D, R or S; X.sub.3 consists of E, K or L;
X.sub.5 and X.sub.7 consist of L, I, or T; X.sub.6 consists of L,
I, or P; X.sub.9 consists of Q, C, E, W or R, X.sub.10 consists of
K, G, F, L, I or nothing; X.sub.11 consists of T, G or nothing; and
X.sub.12 consists of F, L, I, T, N or nothing capable of reducing
said T cell activation; b) administering said inhibitor to said
patient under conditions such that said at least one symptom is
reduced. In one embodiment, the medical condition comprises an
autoimmune disorder. In one embodiment, the autoimmune disorder is
selected from the group consisting of systemic lupus erythematosus,
rheumatoid arthritis, multiple sclerosis, type I diabetes,
gastroenterological conditions e.g. inflammatory bowel disease e.g.
Crohn's disease, primary biliary cirrhosis, chronic active
hepatitis; skin problems e.g. atopic dermatitis, psoriasis,
pemphigus vulgaris; cardiovascular problems e.g. autoimmune
pericarditis.
[0061] In one embodiment, the present invention contemplates a
peptide inhibitor comprising an amino acid sequence consisting of
L-Q-N-X.sub.1-X.sub.2-X.sub.3-X.sub.4-L-X.sub.5-X.sub.6-L-X.sub.7-X.sub.8-
-X.sub.9-X.sub.10-X.sub.11-X.sub.12 (SEQ ID NO: 5) wherein X.sub.1,
X.sub.5 and X.sub.8 are selected from the group consisting of R, K
or H; X.sub.2 and X.sub.4 consist of L, I, or T; X.sub.3 consists
of L, I, or P; X.sub.6 consists of D, R or S; X.sub.7 consists of
E, K or L; X.sub.9 consists of Q, C, E, W or R, X.sub.10 consists
of K, G, F, L, I or nothing; X.sub.11 consists of T, G or nothing;
and X.sub.12 consists of F, L, I, T, N or nothing. In one
embodiment, the peptide further comprises a C-terminal and/or an
N-terminal sugar conjugate. In one embodiment, the sugar conjugate
is 1-amino-glucose succinate. In one embodiment, the peptide
further comprises a C-terminal and/or an N-terminal lipid
conjugate. In one embodiment, the lipid conjugate is selected from
the group comprising 2-aminododecanoate or myristoylate. In one
embodiment, the lipid conjugate is selected from the group
comprising Gly-Tris-monopalmitate, Gly-Tris-dipalmitate, or
Gly-Tris-tripalmitate. In one embodiment, the peptide comprises a
cyclic peptide. In one embodiment, the peptide comprise a
disulfide-linked dimer. In one embodiment, the peptide inhibitor
includes amino acids selected from the group including, but not
limited to, L-amino acids, or D-amino acids.
[0062] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a patient having at least one
symptom of a disease or condition where T cells are involved or
recruited; and ii) a peptide inhibitor comprising an amino acid
sequence consisting of
L-Q-N-X.sub.1-X.sub.2-X.sub.3-X.sub.4-L-X.sub.5-X.sub.6-L-X.sub.7-X.sub.8-
-X.sub.9-X.sub.10-X.sub.11-X.sub.12 (SEQ ID NO: 5) wherein X.sub.1,
X.sub.5 and X.sub.8 are selected from the group consisting of R, K
or H; X.sub.2 and X.sub.4 consist of L, I, or T; X.sub.3 consists
of L, I, or P; X.sub.6 consists of D, R or S; X.sub.7 consists of
E, K or L; X.sub.9 consists of Q, C, E, W or R, X.sub.10 consists
of K, G, F, L, I or nothing; X.sub.11 consists of T, G or nothing;
and X.sub.12 consists of F, L, I, T, N or nothing capable of
reducing said T cell activation; b) administering said inhibitor to
said patient under conditions such that said at least one symptom
is reduced. In one embodiment, the medical condition comprises an
autoimmune disorder. In one embodiment, the autoimmune disorder is
selected from the group consisting of systemic lupus erythematosus,
rheumatoid arthritis, multiple sclerosis, type I diabetes,
gastroenterological conditions e.g. inflammatory bowel disease e.g.
Crohn's disease, primary biliary cirrhosis, chronic active
hepatitis; skin problems e.g. atopic dermatitis, psoriasis,
pemphigus vulgaris; cardiovascular problems e.g. autoimmune
pericarditis.
[0063] In one embodiment, the present invention contemplates a
protease-resistance immunotherapeutic peptide comprising a variant
transmembrane peptide derived from amino acid sequences (or from
combinations thereof) of fusion and other protein regions of
various viruses, including but not limiting to, SARS-CoV, HVS,
HHV-6, LASV, LCMV, MOPV, TACV, Fr-MLV; HTLV-1; HVA; MARV; SEBOV;
ZEBOV. In one embodiment, the variant peptide comprises at least
one D-amino acid.
[0064] In one embodiment, the present invention contemplates a
disulfide-linked dimer of an immunotherapeutic peptide comprising a
variant transmembrane peptide derived from amino acid sequences (or
from combinations thereof) of fusion and other protein regions of
various viruses, including but not limiting to, SARS-CoV, HVS,
HHV-6, LASV, LCMV, MOPV, TACV, Fr-MLV; HTLV-1; HVA; MARV; SEBOV;
ZEBOV.
[0065] In one embodiment, the present invention contemplates a
cyclic immunotherapeutic peptide comprising a variant transmembrane
peptide derived from amino acid sequences (or from combinations
thereof) of fusion and other protein regions of various viruses,
including but not limiting to, SARS-CoV, HVS, HHV-6, LASV, LCMV,
MOPV, TACV, Fr-MLV; HTLV-1; HVA; MARV; SEBOV; ZEBOV.
[0066] In one embodiment, the present invention contemplates a
medical device comprising a coating, wherein said coating comprises
the peptide derivative of Claim 1. In one embodiment, the coating
further comprises a polymer. In one embodiment, the polymer is
selected from the group including, but not limited to,
phosphorylcholine, polyvinyl pyrrolidone, poly(acrylic acid),
poly(vinyl acetamide), poly(propylene glycol), poly(ethylene
co-vinyl acetate), poly(n-butyl methacrylate) or
poly(styrene-b-isobutylene-b-styrene). In one embodiment, the
medical device is selected from the group including, but not
limited to, stents, grafts, implantable devices, catheters,
endoscopes (i.e., for example, laparoscopes), atrial/venous
fistulas, or cannulae.
BRIEF DESCRIPTION OF THE FIGURES
[0067] The following figures form part of the present specification
and are included to further illustrate aspects of the present
invention. The invention may be better understood by reference to
the figures in combination with the detailed description of the
specific embodiments presented herein.
[0068] FIG. 1A presents a schematic representation of one
embodiment of Multichain Immune Recognition Receptors (MIRRs)
expressed on many different immune cells--including T and B cells,
natural killer cells, mast cells, macrophages, basophils,
neutrophils, eosinophils and dendritic cells--and on platelets.
Position of MIRRs relative to the cell membrane is indicated by
blue lines. Cytoplasmic domains of the MIRR signaling subunits
represent a novel class of intrinsically disordered proteins and
are shown to be dimeric. Abbreviations: BCR, B cell receptor;
DAP-10 and DAP-12, DNAX adapter proteins of 10 and 12 kD,
respectively; DCAR, dendritic cell immunoactivating receptor; GPVI,
glycoprotein VI; ILT, Ig-like transcript; KIR, killer cell Ig-like
receptor; LIR, leukocyte Ig-like receptor; MAIR-II,
myeloid-associated Ig-like receptor; MDL-1, myeloid DAP
12-associating lectin 1; NITR, novel immune-type receptor; NK,
natural killer cells; SIRP, signal regulatory protein, TCR, T cell
receptor; TREM receptors, triggering receptors expressed on myeloid
cells.
[0069] FIG. 1B presents a schematic representation of one possible
structural and functional organization of MIRRs. Although it is not
necessary to understand the mechanism of an invention, it is
believed that transmembrane intersubunit heterointeractions between
MIRR recognition and signaling components (shown by arrows) have a
role in receptor assembly and integrity on resting cells. Curved
lines depict disorder of the cytoplasmic domains of MIRR signaling
subunits.
[0070] FIG. 1C illustrates one embodiment of a MIRR-mediated
transmembrane signal transduction utilizing the Signaling Chain
HOmoOLigomerization (SCHOOL) model. The model proposes that the
homooligomerization of signaling subunits in the cytoplasmic milieu
plays a key role in triggering MIRRs. Although it is not necessary
to understand the mechanism of an invention, it is believed that
ligand-induced MIRR clustering and reorientation (and/or receptor
reorientation in preexisting MIRR clusters) lead to formation of a
dimeric/oligomeric intermediate. It is further believed that in
this intermediate, receptors are in sufficient proximity and adopt
the correct (permissive) relative orientation and geometry to
promote trans-homointeractions between cytoplasmic domains of
signaling subunits resulting in formation of competent signaling
oligomers. It still further believed that in these oligomers,
protein tyrosine kinases phosphorylate the tyrosine residues in the
ITAMs (green rectangles) or the YxxM motif of DAP-10 (blue
rectangles), leading to the generation of activation signal(s),
dissociation of signaling oligomers and internalization of the
engaged MIRR ligand-binding subunits. Circular arrows indicate
ligand-induced receptor reorientation. All interchain interactions
in a dimeric intermediate are shown by dotted black arrows
reflecting their transition state. Curved lines depict disorder of
the cytoplasmic domains of MIRR signaling subunits. Phosphate
groups are shown as dark circles. A similar general scheme can be
considered for the pathway induced by receptor crosslinking, using
antibodies to signaling subunits (e.g. anti-CD3e or anti-Igb
antibodies for TCRs and BCRs, respectively). Abbreviations: ITAM,
immunoreceptor tyrosine-based activation motif.
[0071] FIG. 1D illustrates one embodiment of a specific blockade or
disruption of transmembrane interactions between recognition and
signaling subunits resulting in a physical and functional
disconnection of the MIRR subunits and "pre-dissociation" of the
receptor complex, thus preventing formation of signaling oligomers
and inhibiting ligand- (right panel) but not antibody (left
panel)-dependent immune cell activation.
[0072] FIG. 2 illustrates one embodiment of the SCHOOL-based
molecular mechanisms of T cell receptor (TCR) signaling.
Immunoreceptor tyrosine-based activation motifs (ITAMs) are shown
as gray rectangles. TCR-CD3-.zeta. components are represented as
whole polypeptides and as a simplified axial view. All interchain
interactions in intermediate complexes are shown by dotted arrows
reflecting their transition state. Circular arrow indicates
ligand-induced receptor reorientation. Interaction with multivalent
ligand (not shown) clusters the receptors and pushes them to
reorientate (I) and bring signaling subunits into a correct
relative orientation and in sufficient proximity in the formed
receptor oligomer (for illustrative purposes, receptor dimer is
shown), thus starting the trans-homointeractions between molecules
(II). Then, two alternative pathways can take a place depending on
the nature of activating stimuli. First is going through a stage IV
resulting in formation of 42 dimer (dimer of dimers) and
phosphorylation of the ITAM tyrosines, thus triggering downstream
signaling events. Then, the signaling 4 oligomers formed
subsequently dissociate from the TCR-CD3 complex, resulting in
internalization of the remaining engaged TCR-CD3 complexes (VII).
This pathway leads to partial (or incomplete) T cell activation.
Alternatively, the intermediate complex formed at the stage II can
undergo further rearrangements, starting trans-homointeractions
between CD3 proteins (III) and resulting in formation of an
oligomeric intermediate. Again, the stages I, II and III can be
reversible or irreversible depending on interreceptor proximity and
relative orientation of the receptors in TCR dimers/oligomers as
well as on time duration of the TCR-ligand contact and lifetime of
the receptor in TCR dimers/oligomers that generally correlate with
the nature of the stimulus and its specificity and
affinity/avidity. Next, in the signaling oligomers formed (III),
the ITAM tyrosines undergo phosphorylation by PTKs that leads to
generation of the activation signal, dissociation of signaling
oligomers and internalization of the remaining engaged
TCR.alpha..beta. chains (VIII, XI). This pathway provides at least
two different activation signals from the .zeta. and CD3 signaling
oligomers (signals A and B), respectively, and results in full T
cell activation. The distinct signaling through .zeta. and CD3
oligomers (or through various combinations of signaling chains in
CD3 oligomeric structures) might be also responsible for distinct
functions such as T cell proliferation, effector functions, T cell
survival, pathogen clearance, TCR anergy, etc. In addition, the
signaling oligomers formed can sequentially interact with the
signaling subunits of nonengaged TCRs resulting in formation of
higher-order signaling oligomers, thus amplifying and propagating
the activation signal (not shown). Also, this leads to the release
and subsequent internalization of the remaining nonengaged TCR
complexes and/or TCR.alpha..beta. chains (not shown).
Abbreviations: PTK, protein tyrosine kinase. Phosphate groups are
shown as filled gray circles.
[0073] FIG. 3 illustrates one embodiment of the SCHOOL-based
mechanisms of action of T cell receptor transmembrane inhibitors
such as the T cell receptor core peptide (CP) and HIV-1 gp41 fusion
peptide (FP). Considering the close similarity in patterns of
inhibition of T cell activation and immunosuppressive activity
observed for CP and FP, the SCHOOL model reasonably suggests a
similar molecular mechanism of action for both peptides. Within the
SCHOOL model, these peptides compete with the TCR.alpha. chain for
binding to the CD3.delta..epsilon. and .zeta. signaling subunits,
thus disrupting the transmembrane (TM) interactions between these
subunits and resulting in disconnection and predissociation of the
relevant signaling subunits from the remaining receptor complex
(also shown in the inset as a simplified axial view). This prevents
formation of signaling oligomers upon multivalent antigen
stimulation, thus inhibiting antigen-mediated T cell activation. In
contrast, stimulation of these "predissociated" MIRRs with
cross-linking antibodies to signaling subunit should still lead to
receptor triggering and cell activation. The model predicts that
the same mechanisms of inhibitory action can be applied to TCR TM
peptides corresponding to the TM regions of not only the
TCR.alpha..beta. recognition subunits but the corresponding
CD3.epsilon., CD3.delta., CD3.gamma. and .zeta. signaling subunits
as well. Abbreviations: TMP, transmembrane peptide.
[0074] FIG. 4 illustrates one embodiment of the SCHOOL-based
mechanisms of action of different T cell receptor transmembrane
inhibitors. Within the SCHOOL model, upon antigen stimulation of T
cells, T cell receptor .alpha.-chain (TCR.alpha.) transmembrane
peptide (TMP) prevents formation of all signaling oligomers,
including .zeta., CD3.epsilon., CD3.delta., and CD3.gamma.. This
inhibits T cell activation in both in vitro and in vivo. In
contrast, other TMPs prevent formation of signaling oligomers (and
therefore signaling) of selected signaling subunits. This inhibits
T cell activation in vivo whereas inhibition in vitro depends on
the evaluation method used. Abbreviations: AD, atopic dermatitis;
AIA, adjuvant-induced arthritis; IL-2, interleukin 2.
[0075] FIG. 5 (SEQ ID NOS: 78, 76, 6, 7, 28, 26, 77, 8, 41, 43, 45,
47, 48, 49, 50, 47, and 51, respectively) illustrates one
embodiment of similarities in the charge distribution patterns of
different immunomodulatory viral sequences. Primary sequence
analysis of proven and predicted immunomodulatory sequences of
viral fusion protein regions and other domains shows a similarity
in charge distribution pattern with two essential positively
charged residues spaced apart by 4 (class I) or 8 (class III) amino
acids or with three essential positively charged residues spaced
apart by 3 amino acids (class II), suggesting a similarity of the
SCHOOL-based mechanisms used by diverse viruses in their
pathogenesis to modulate the host immune response. Abbreviations:
TCR, T cell receptor; CP, core peptide, HIV, human immunodeficiency
virus; gp, glycoprotein; FP, fusion peptide/protein; TMD,
transmembrane domain; CKS-17, a synthetic retroviral envelope
heptadecapeptide; Fr-MLV, Friend murine leukemia virus; gp,
glycoprotein; HHV-6 U24, human herpesvirus 6 U24 protein; HTLV-1,
human T lymphotropic virus type 1; HVA, herpesvirus ateles; HVS,
herpesvirus saimiri; ITAM, immunoreceptor tyrosine-based activation
motif; LASV, Lassa virus; LCMV, lymphocytic choriomeningitis virus;
MARV, Marburg virus; MOPV, Mopeia virus; SARS-CoV, severe acute
respiratory syndrome coronavirus; SEBOV, Sudan Ebola virus; TACV,
Tacaribe virus; Tip, tyrosine kinase interacting protein; Tio,
two-in-one protein; TMD, transmembrane domain; ZEBOV, Zaire Ebola
virus.
[0076] FIG. 6A illustrates one embodiment of the SCHOOL-based
mechanisms of action of the Class I of TCR peptide inhibitors in
the transmembrane milieu. Helices of the transmembrane domains of T
cell receptor (TCR) .alpha. and .beta. chains, TCR chain, CD3
.epsilon., .gamma., and .delta. chains, as well as of the
transmembrane peptide inhibitors of the present invention are shown
as a simplified axial view of helical wheels. Although the
three-dimensional structures of the inhibitors of the invention
within the cell membrane are not known, it might be assumed that
these sequences may adopt a helical conformation upon membrane
binding. Abbreviations: VP, viral peptide.
[0077] FIG. 6B illustrates one embodiment of the SCHOOL-based
mechanisms of action of the Class II of TCR peptide inhibitors in
the transmembrane milieu. Helices of the transmembrane domains of T
cell receptor (TCR) .alpha. and .beta. chains, TCR .zeta. chain,
CD3 .epsilon., .gamma., and .delta. chains, as well as of the
transmembrane peptide inhibitors of the present invention are shown
as a simplified axial view of helical wheels. Although the
three-dimensional structures of the inhibitors of the invention
within the cell membrane are not known, it might be assumed that
these sequences may adopt a helical conformation upon membrane
binding. Abbreviations: VP, viral peptide.
[0078] FIG. 6C illustrates one embodiment of the SCHOOL-based
mechanisms of action of the Class III of TCR peptide inhibitors in
the transmembrane milieu. Helices of the transmembrane domains of T
cell receptor (TCR) .alpha. and .beta. chains, TCR .zeta. chain,
CD3 .epsilon., .gamma., and .delta. chains, as well as of the
transmembrane peptide inhibitors of the present invention are shown
as a simplified axial view of helical wheels. Although the
three-dimensional structures of the inhibitors of the invention
within the cell membrane are not known, it might be assumed that
these sequences may adopt a helical conformation upon membrane
binding. Abbreviations: VP, viral peptide.
[0079] FIG. 7 presents various embodiments of the Class I of TCR
peptide inhibitor sequences based upon a general formula, wherein
in the general formula describes variants of the parent sequence of
SARS-CoV FP.
[0080] FIG. 8 presents various embodiments of the Class II of TCR
peptide inhibitor sequences based upon a general formula, wherein
in the general formula describes variants of the parent sequences
of HTLV-1 gp21.sup.313-353, HVA Tio.sup.225-242, and HVS
Tip.sup.211-228.
[0081] FIG. 9 presents various embodiments of the Class III of TCR
peptide inhibitor sequences based upon a general formula, wherein
in the general formula describes variants of the parent sequences
of LASV FP (gp2.sup.260-298), LCMV FP (gp2.sup.266-304), MOPV FP
(gp2.sup.258-296), TACV FP (gp2.sup.262-300), CKS-17, SEBOV
gp2.sup.584-600, ZEBOV gp2.sup.584-600, MARV gp2.sup.585-601,
Fr-MLV Env gp.sup.548-564 and HHV-6 U24.sup.28-60.
[0082] FIG. 10 presents various embodiments of the combinatorial
TCR peptide inhibitor sequences based upon a combination of general
formulas, wherein in the general formula describes variants of the
parent sequences of SARS-CoV FP, HTLV-1 gp21.sup.313-353, HVA
Tio.sup.225-242, HVS Tip.sup.211-228, LASV FP (gp2.sup.260-298),
LCMV FP (gp2.sup.266-304), MOPV FP (gp2.sup.258-296), TACV FP
(gp2.sup.262-300), CKS-17, SEBOV gp2.sup.584-600, ZEBOV
gp2.sup.584-600, MARV gp2.sup.585-601, Fr-MLV Env gp.sup.548-564
and HHV-6 U24.sup.28-60.
DEFINITIONS
[0083] The term "T cell-mediated pathology" (or "T cell-related
pathologies", or "T cell-mediated disorder, or "T cell-related
disease"), as used herein, refers to any condition in which an
inappropriate T cell response is a component of the pathology. The
term is intended to include both diseases directly mediated by T
cells, and also diseases in which an inappropriate T cell response
contributes to the production of abnormal antibodies, as well as
graft rejection.
[0084] The term "ligand-induced T cell activation", as used herein,
refers to T cell activation in response to the stimulation by the
specific ligand.
[0085] The term "stimulation", as used herein, refers to a primary
response induced by ligation of a cell surface moiety. For example,
in the context of receptors, such stimulation entails the ligation
of a receptor and a subsequent signal transduction event. With
respect to stimulation of a T cell, such stimulation refers to the
ligation of a T cell surface moiety that in one embodiment
subsequently induces a signal transduction event, such as binding
the TCR/CD3 complex. Further, the stimulation event may activate a
cell and upregulate or downregulate expression or secretion of a
molecule.
[0086] The term "ligand", or "antigen", as used herein, refers to a
stimulating molecule that binds to a defined population of cells.
The ligand may bind any cell surface moiety, such as a receptor, an
antigenic determinant, or other binding site present on the target
cell population. The ligand may be a protein, peptide, antibody and
antibody fragments thereof, fusion proteins, synthetic molecule, an
organic molecule (e.g., a small molecule), or the like. Within the
specification and in the context of T cell stimulation, the ligand
(or antigen) binds the T cell antigen receptor and this binding
activates the T cell.
[0087] The term "activation", as used herein, refers to the state
of a cell following sufficient cell surface moiety ligation to
induce a noticeable biochemical or morphological change. Within the
context of T cells, such activation, refers to the state of a T
cell that has been sufficiently stimulated to induce cellular
proliferation. Activation of a T cell may also induce cytokine
production and performance of regulatory or cytolytic effector
functions. Within the context of other cells, this term infers
either up or down regulation of a particular physico-chemical
process.
[0088] The term "inhibiting T cell activation", as used herein,
refers to the slowing of T cell activation, as well as completely
eliminating and/or preventing T cell activation.
[0089] The term, "treating a disease or condition", as used herein,
refers to modulating T cell activation including, but not limited
to, decreasing cellular proliferation, cytokine production and
performance of regulatory or cytolytic effector functions and/or
slowing T cell activation, as well as completely eliminating and/or
preventing T cell activation. T cell-related diseases and/or
conditions treatable by modulating T cell activation include, but
are not limited to, systemic lupus erythematosus, rheumatoid
arthritis, multiple sclerosis, type I diabetes, gastroenterological
conditions e.g. inflammatory bowel disease e.g. Crohn's disease,
Guillain-Barre syndrome, Hashimotos disease, pernicious anaemia,
primary biliary cirrhosis, chronic active hepatitis; skin problems
e.g. atopic dermatitis, psoriasis, pemphigus vulgaris;
cardiovascular problems e.g. autoimmune pericarditis, allergic
diathesis e.g. Delayed type hypersensitivity, contact dermatitis,
AIDS virus, herpes simplex/zoster, respiratory conditions e.g.
allergic alveolitis, inflammatory conditions e.g. myositis,
ankylosing spondylitis, tissue/organ rejection.
[0090] The term, "subject" or "patient", as used herein, refers to
any individual organism. For example, the organism may be a mammal
such as a primate (i.e., for example, a human). Further, the
organism may be a domesticated animal (i.e., for example, cats,
dogs, etc.), livestock (i.e., for example, cattle, horses, pigs,
sheep, goats, etc.), or a laboratory animal (i.e., for example,
mouse, rabbit, rat, guinea pig, etc.).
[0091] The term, "therapeutically effective amount",
"therapeutically effective dose" or "effective amount", as used
herein, refers to an amount needed to achieve a desired clinical
result or results (inhibiting TCR-mediated cell activation) based
upon trained medical observation and/or quantitative test results.
The potency of any administered peptide or compound determines the
"effective amount" which can vary for the various compounds that
inhibit T cell activation (i.e., for example, compounds inhibiting
antigen-induced T cell activation). Additionally, the "effective
amount" of a compound may vary depending on the desired result, for
example, the level of T cell activation inhibition desired. The
"therapeutically effective amount" necessary for inhibiting T cell
proliferation may differ from the "therapeutically effective
amount" necessary for preventing cytokine production.
[0092] The term, "agent", as used herein, refers to any natural or
synthetic compound (i.e., for example, a peptide, a peptide
variant, or a small molecule).
[0093] The term, "composition", as used herein, refers to any
mixture of substances comprising a peptide and/or compound
contemplated by the present invention. Such a composition may
include the substances individually or in any combination.
[0094] The term, "intrinsic helicity", as used herein, refers to
the helicity which is adopted by a peptide in an aqueous
solution.
[0095] The term, "induced helicity", as used herein, refers to the
helicity which is adopted by a peptide when in the presence of a
helicity inducer, including, but not limited to, trifluoroethanol
(TFE), detergents (i.e., for example, sodium dodecyl sulfate
(SDS)), or lipids (i.e., for example, lipid vesicles (small lamilar
vesicles (SUVs) and/or large lamilar vesicles (LUVs) as described
herein).
[0096] The term "therapeutic drug", as used herein, refers to any
pharmacologically active substance capable of being administered
which achieves a desired effect. Drugs or compounds can be
synthetic or naturally occurring, non-peptide, proteins or
peptides, oligonucleotides or nucleotides, polysaccharides or
sugars. Drugs or compounds may have any of a variety of activities,
which may be stimulatory or inhibitory, such as antibiotic
activity, antiviral activity, antifungal activity, steroidal
activity, cytotoxic, cytostatic, anti-proliferative,
anti-inflammatory, analgesic or anesthetic activity, or can be
useful as contrast or other diagnostic agents.
[0097] The term "effective dose" as used herein refers to the
concentration of any compound or drug contemplated herein that
results in a favorable clinical response. In solution, an effective
dose may range between approximately 1 ng/ml-100 mg/ml, preferably
between 100 ng/ml-10 mg/ml, but more preferably between 500 ng/ml-1
mg/ml.
[0098] The term "administered" or "administering" a drug or
compound, as used herein, refers to any method of providing a drug
or compound to a patient such that the drug or compound has its
intended effect on the patient. For example, one method of
administering is by an indirect mechanism using a medical device
such as, but not limited to a catheter, syringe etc. A second
exemplary method of administering is by a direct mechanism such as,
local tissue administration (i.e., for example, extravascular
placement), oral ingestion, transdermal patch, topical, inhalation,
suppository etc.
[0099] The term "anti-inflammatory drug" means any compound,
composition, or drug useful for preventing or treating inflammatory
disease.
[0100] The term "medical device", as used herein, refers broadly to
any apparatus used in relation to a medical procedure.
Specifically, any apparatus that contacts a patient during a
medical procedure or therapy is contemplated herein as a medical
device. Similarly, any apparatus that administers a drug or
compound to a patient during a medical procedure or therapy is
contemplated herein as a medical device. "Direct medical implants"
include, but are not limited to, urinary and intravascular
catheters, dialysis catheters, wound drain tubes, skin sutures,
vascular grafts and implantable meshes, intraocular devices,
implantable drug delivery systems and heart valves, and the like.
"Wound care devices" include, but are not limited to, general wound
dressings, non-adherent dressings, burn dressings, biological graft
materials, tape closures and dressings, surgical drapes, sponges
and absorbable hemostats. "Surgical devices" include, but are not
limited to, surgical instruments, endoscope systems (i.e.,
catheters, vascular catheters, surgical tools such as scalpels,
retractors, and the like) and temporary drug delivery devices such
as drug ports, injection needles etc. to administer the medium. A
medical device is "coated" when a medium comprising an
anti-inflammatory drug (i.e., for example, a variant SARS-CoV
fusion peptide) becomes attached to the surface of the medical
device. This attachment may be permanent or temporary. When
temporary, the attachment may result in a controlled release of a
variant SARS-CoV fusion peptide.
[0101] The term "endoscope" refers to any medical device that is
capable of being inserted into a living body and used for tasks
including, but not limited to, observing surgical procedures,
performing surgical procedures, or applying medium to a surgical
site. An endoscope is illustrated by instruments including, but not
limited to, an arthroscope, a laparoscope, hysteroscope, cytoscope,
etc. It is not intended to limit the use of an endoscope to hollow
organs. It is specifically contemplated that endoscopes, such as an
arthroscope or a laparoscope is inserted through the skin and
courses to a closed surgical site.
[0102] The term "vascular access site" is defined herein as
referring to any percutaneous insertion of a medical device into
the vasculature. For example, a hemodialysis catheter placement
comprises a vascular access site. Such sites may be temporary
(i.e., placed for a matter of hours) or permanent (i.e., placed for
days, months or years).
[0103] The term "vascular graft" as used herein, refers to any
conduit or portion thereof intended as a prosthetic device for
conveying blood and, therefore, having a blood contacting surface
(i.e., "luminal"). While usually in a tubular form, the graft may
also be a sheet of material useful for patching portions of the
circumference of living blood vessels (these materials are
generally referred to as surgical wraps). Likewise, the term
vascular graft includes intraluminal grafts for use within living
blood vessels. The inventive grafts as such may also be used as a
stent covering on the exterior, luminal or both surfaces of an
implantable vascular stent.
[0104] The term "synthetic vascular graft" as used herein, refers
to any artificial tube or cannula designed for insertion into a
blood vessel. Such grafts may be constructed from
polytetrafluoroethylene (PTFE).
[0105] The term "syringe" or "catheter" as used herein, refers to
any device or apparatus designed for liquid administration, as
defined herein. A syringe or catheter may comprise at least one
storage vessel (i.e., for example, a barrel) wherein a single
medium resides prior to administration. A syringe or catheter
comprising two or more barrels, each containing a separate medium,
may mix the media from each barrel prior to administration or the
media of each barrel may be administered separately. One of skill
in the art will recognize that any catheter designed to perform
dialysis, as defined herein, may also administer liquids.
[0106] The term "dialysis/apheresis catheter" as used herein,
refers to any multi-lumen catheter (i.e., for example, a triple
lumen catheter) capable of providing a simultaneous withdrawal and
return of blood to a patient undergoing a blood treatment process.
Apheresis (called also pheresis) comprises a blood treatment
process involving separation of blood elements that can remove
soluble drugs or cellular elements from the circulation (Deisseroth
et al., "Use Of Blood And Blood Products" in Cancer: Principles And
Practice Of Oncology, Devita V. T. Jr. et al. Editors,
Philadelphia: J. B. Lippincott Company 1989, 2045-59). For example,
blood is withdrawn from a donor, some blood elements (i.e., for
example, plasma, leukocytes, platelets, etc.) are separated and
retained. The unretained blood elements are then retransfused into
the donor.
[0107] The term "dialysis catheter" as used herein, refers to any
device capable of removing toxic substances (impurities or wastes)
from the body when the kidneys are unable to do so. A dialysis
catheter may comprise a single catheter having at least a dual
lumen (i.e., one lumen withdraws arterial blood and a second lumen
returns the dialyzed blood to the venous system) or involve placing
two catheters--one that is placed in an artery, and one in an
adjacent vein. Dialysis catheters are most frequently used for
patients who have kidney failure, but may also be used to quickly
remove drugs or poisons in acute situations.
[0108] The term "peritoneal dialysis catheter" as used herein,
refers to any continuous flow catheters with at least two lumens,
one of which is a short lumen (used to infuse a dialysis solution
into the peritoneum), and the other of which is a long coiled lumen
having a plurality of openings, generally located on the inside of
the coil. It is believed that peritoneal solutes enter into the
coiled lumen openings and are thereby removed from the peritoneum.
One hypothesis suggests that peritoneal dialysis works by using the
peritoneal membrane inside the abdomen as the semipermeable
membrane. Special solutions that facilitate removal of toxins may
be infused in, remain in the abdomen for a time, and then drained
out.
[0109] The term "fixed split-tip dialysis catheter" as used herein,
refers to any catheter having at least two distinct elongated end
portions that extend substantially parallel to the longitudinal
axis of the catheter and are flexible to the lateral displacement
of an infused fluid. It is believed that this flexibility prevents
a permanent catheter tip splay that is known to injure tissue.
Usually a fixed-tip dialysis catheter provides indwelling vascular
access for patients undergoing long-term renal dialysis care (i.e.,
for example, end-stage renal disease).
[0110] The term "femoral catheter" as used herein, refers to any
catheter that is inserted into the femoral vein. Femoral catheters
are typically provided for intermediate term blood access because
the superior vena cava is relatively close to the right atrium of
the heart, the minimal range of shape changes of these veins with
natural movements of the patient (to minimize the damage to the
vessel intima), and because of good acceptance by the patients of
the skin exit on the thoracic wall. Further, the femoral veins are
easy to cannulate, so that catheters of this invention may be
inserted into the femoral veins at the bed side.
[0111] The term "attached" as used herein, refers to any
interaction between a medium (or carrier) and a therapeutic drug.
Attachment may be reversible or irreversible. Such attachment
includes, but is not limited to, covalent bonding, and non-covalent
bonding including, but not limited to, ionic bonding, Van der Waals
forces or friction, and the like. A drug is attached to a medium
(or carrier) if it is impregnated, incorporated, coated, in
suspension with, in solution with, mixed with, etc. The term
"covalent bonding" as used herein, refers to an attachment between
two compounds (I.e., for example, a medium and a drug) that
comprising a sharing of electrons.
[0112] As used herein, the term "peptide" refers to linear or
cyclic or branched compounds containing amino acids, amino acid
equivalents or other non-amino groups, while still retaining the
desired functional activity of a peptide. Peptide equivalents can
differ from conventional peptides by the replacement of one or more
amino acids with related organic acids such as p-aminobenzoic acid
(PABA), amino acid analogs, or the substitution or modification of
side chains or functional groups. Peptide equivalents encompass
peptide mimetics or peptidomimetics, which are organic molecules
that retain similar peptide chain pharmacophore groups as are
present in the corresponding peptide. The term "peptide" refers to
peptide equivalents as well as peptides. The amino acids can be in
the L or D form so long as the binding function of the peptide is
maintained.
[0113] As used herein, the term "cyclic peptide" refers to a
peptide having an intramolecular bond between two non-adjacent
amino acids. The cyclization can be effected through a covalent or
non-covalent bond. Intramolecular bonds include, but are not
limited to, backbone to backbone, side-chain to backbone and
side-chain to side-chain bonds. As used herein, the term "dimer" as
applied to peptides refers to molecules having two peptide chains
associated covalently or non-covalently, with or without linkers.
Peptide dimers wherein the peptides are linked C-terminus to
N-terminus may also be referred to as "tandem repeats" or "tandem
dimers." Peptide dimers wherein the peptides are linked C- to
C-terminus, or N- to N-terminus may also be referred to as
"parallel repeats" or "parallel dimers."
[0114] The term "placing" as used herein, refers to any physical
relationship (i.e., secured or unsecured) between a patient's
biological tissue and a surgical material, wherein the surgical
material comprises a pharmaceutical drug that may be, optionally,
attached to a medium. Such a physical relationship may be secured
by methods such as, but not limited to, gluing, suturing, stapling,
spraying, laying, impregnating, and the like. The term "parts by
weight", as used herein, when used in reference to a particular
component in a composition denotes the weight relationship between
the component and any other components in the composition for which
a pan by weight is expressed.
[0115] The term "protecting groups", as used herein, refer to those
groups which prevent undesirable reactions (such as proteolysis)
involving unprotected functional groups. In one embodiment, the
present invention contemplates that the protecting group is an acyl
or an amide. In one embodiment, the acyl is acetate. In another
embodiment, the protecting group is a benzyl group. In another
embodiment, the protecting group is a benzoyl group. The present
invention also contemplates combinations of such protecting
groups.
[0116] The term "protein", as used herein, refers to compounds
comprising amino acids joined via peptide bonds and includes
proteins and polypeptides; and may be an intact molecule, a
fragment thereof, or multimers or aggregates of intact molecules
and/or fragments; and may occur in nature or be produced, e.g., by
synthesis (including chemical and/or enzymatic) or genetic
engineering. The terms "protein" and "polypeptide" are used herein
interchangeably.
[0117] As used herein, where "amino acid sequence" is recited
herein to refer to an amino acid sequence of a protein molecule. An
"amino acid sequence" can be deduced from the nucleic acid sequence
encoding the protein. However, terms such as "polypeptide" or
"protein" are not meant to limit the amino acid sequence to the
deduced amino acid sequence, but include post-translational
modifications of the deduced amino acid sequences, such as amino
acid deletions, additions, and modifications such as
glycolsylations and addition of lipid moieties.
[0118] The term "portion" when used in reference to a protein (as
in "a portion of a given protein") refers to fragments of that
protein. The fragments may range in size from four amino acid
residues to the entire amino sequence minus one amino acid.
[0119] The term "analog", as used herein, includes any peptide
having an amino acid sequence substantially identical to one of the
sequences specifically shown herein in which one or more residues
have been conservatively substituted with a functionally similar
residue and which displays the abilities as described herein.
Examples of conservative substitutions include the substitution of
one non-polar (hydrophobic) residue such as isoleucine, valine,
leucine or methionine for another, the substitution of one polar
(hydrophilic) residue for another such as between arginine and
lysine, between glutamine and asparagine, between glycine and
serine, the substitution of one basic residue such as lysine,
arginine or histidine for another, or the substitution of one
acidic residue, such as aspartic acid or glutamic acid for
another.
[0120] The term "conservative substitution", as used herein, also
includes the use of a chemically derivatized residue in place of a
non-derivatized residue provided that such peptide displays the
requisite inhibitory function on T cells as specified herein. The
term derivative includes any chemical derivative of the peptide of
the invention having one or more residues chemically derivatized by
reaction of side chains or functional groups.
[0121] The term "homolog" or "homologous" when used in reference to
a polypeptide refers to a high degree of sequence identity between
two polypeptides, or to a high degree of similarity between the
three-dimensional structure or to a high degree of similarity
between the active site and the mechanism of action. In a preferred
embodiment, a homolog has a greater than 60% sequence identity, and
more preferably greater than 75% sequence identity, and still more
preferably greater than 90% sequence identity, with a reference
sequence.
[0122] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as,
for example, by the programs SIM+LALNVIEW, LALIGN and DIALIGN
(expasy.ch/tools) using default gap weights, share at least 80
percent sequence identity, preferably at least 90 percent sequence
identity, more preferably at least 95 percent sequence identity or
more (e.g., 99 percent sequence identity). Preferably, residue
positions which are not identical differ by conservative amino acid
substitutions.
[0123] The terms "variant" and "mutant" when used in reference to a
polypeptide refer to an amino acid sequence that differs by one or
more amino acids from another, usually related polypeptide. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties. One type
of conservative amino acid substitutions refers to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Preferred conservative
amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine. More rarely, a variant may have
"non-conservative" changes (e.g., replacement of a glycine with a
tryptophan). Similar minor variations may also include amino acid
deletions or insertions (in other words, additions), or both.
Guidance in determining which and how many amino acid residues may
be substituted, inserted or deleted without abolishing biological
activity may be found using computer programs well known in the
art, for example, DNAStar software. Variants can be tested in
functional assays. Preferred variants have less than 10%, and
preferably less than 5%, and still more preferably less than 2%
changes (whether substitutions, deletions, and so on).
[0124] It is understood by the person of ordinary skill in the art
that the terms "CD3Z_HUMAN", "T cell receptor .zeta. subunit", "CD
antigen CD247", "TCR .zeta.", ".zeta. signaling subunit" and "TCR
.zeta. signaling chain" refer to the naturally occurring human
protein listed in the UniProt Knowledgebase (UniProtKB,
www.uniprot.org) under the name "CD3Z_HUMAN". The protein amino
acid sequence can be found under the entry UniProt KB/Swiss-Prot
P20963. It is further understood that the terms "CD3D_HUMAN", "CD
antigen CD3d", "CD3 .delta. subunit", "CD3.delta.", "CD3.delta.
signaling subunit" and "CD3.delta. signaling chain" refer to the
naturally occurring human protein listed in the UniProt
Knowledgebase (UniProtKB, www.uniprot.org) under the name
"CD3D_HUMAN". The protein amino acid sequence can be found under
the entry UniProt KB/Swiss-Prot P04234. It is still further
understood that the terms "CD3G_HUMAN", "CD antigen CD3g", "CD3
.gamma. subunit", "CD3.gamma.", "CD3.gamma. signaling subunit" and
"CD3.gamma. signaling chain" refer to the naturally occurring human
protein listed in the UniProt Knowledgebase (UniProtKB,
www.uniprot.org) under the name "CD3G_HUMAN". The protein amino
acid sequence can be found under the entry UniProt KB/Swiss-Prot
P09693. It is still further understood that the terms "CD3E_HUMAN",
"CD antigen CD3e", "CD3 .epsilon. subunit", "CD3.epsilon.",
"CD3.epsilon. signaling subunit" and "CD3.epsilon. signaling chain"
refer to the naturally occurring human protein listed in the
UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the name
"CD3E_HUMAN". The protein amino acid sequence can be found under
the entry UniProt KB/Swiss-Prot P07766. It is still further
understood that the terms "TCA_HUMAN", "T cell receptor alpha chain
C region", "TCR .alpha. subunit", "TCR.alpha.", "TCR.alpha.
ligand-binding subunit" and "TCR.alpha. ligand-binding chain" refer
to the naturally occurring human protein listed in the UniProt
Knowledgebase (UniProtKB, www.uniprot.org) under the name
"TCA_HUMAN". The protein amino acid sequence can be found under the
entry UniProt KB/Swiss-Prot P01848. It is still further understood
that the terms "TCB_HUMAN", "T cell receptor beta chain C region",
"TCR .beta. subunit", "TCR.beta.", "TCR.beta. ligand-binding
subunit" and "TCR.beta. ligand-binding chain" refer to the
naturally occurring human protein listed in the UniProt
Knowledgebase (UniProtKB, www.uniprot.org) under the name
"TCB_HUMAN". The protein amino acid sequence can be found under the
entry UniProt KB/Swiss-Prot P01850.
[0125] It is understood by the person of ordinary skill in the art
that the terms "PP65_HCMVA", "65 kDa phosphoprotein", "65 kDa
matrix phosphoprotein", "Tegument protein UL83", "Phosphoprotein
UL83", "Cytomegalovirus pp 65 tegument protein", and "CMV pp65"
refer to the naturally occurring protein of human cytomegalovirus
listed in the UniProt Knowledgebase (UniProtKB, www.uniprot.org)
under the name "PP65_HCMVA". The protein amino acid sequence can be
found under the entry UniProt KB/Swiss-Prot P06725. It is further
understood that the terms "SPIKE_CVHSA", "Spike glycoprotein",
"human severe acute respiratory syndrome coronavirus", and "SARS
CoV" refer to the naturally occurring spike glycoprotein of human
human severe acute respiratory syndrome coronavirus listed in the
UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the name
"SPIKE_CVHSA". The protein amino acid sequence can be found under
the entry UniProt KB/Swiss-Prot P59594. It is still further
understood that the terms "TIP_SHV2C", "Tyrosine-protein
kinase-interacting protein", "tip", "herpesvirus saimiri", and "HVS
Tip" refer to the naturally occurring tyrosine-protein
kinase-interacting protein of herpesvirus saimiri listed in the
UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the name
"TIP_SHV2C". The protein amino acid sequence can be found under the
entry UniProt KB/Swiss-Prot P22575. It is still further understood
that the terms "TIO_ATHV3", "Two-in-one protein", "protein tio",
"herpesvirus ateles", and "HVA Tip" refer to the naturally
occurring two-in-one protein of herpesvirus ateles listed in the
UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the name
"TIO_ATHV3". The protein amino acid sequence can be found under the
entry UniProt KB/Swiss-Prot Q9YJQ8. It is still further understood
that the terms "ENV_HTL1A", "Envelope glycoprotein gp62", "Env
polyprotein", "Human T cell leukemia virus 1 gp62", "Human T cell
leukemia virus 1 gp21", and "HTLV-1 gp21" refer to the naturally
occurring glycoprotein 21 of human T cell leukemia virus 1 listed
in the UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the
name "ENV_HTL1A". The protein amino acid sequence can be found
under the entry UniProt KB/Swiss-Prot P03381. It is still further
understood that the terms "ENV_HV1H2", "Envelope glycoprotein
gp160", "Env polyprotein", "Human immunodeficiency virus type 1
gp41", "Human immunodeficiency virus type 1 gp41", "HIV-1 gp41",
and "HIV gp41" refer to the naturally occurring glycoprotein 41 of
human immunodeficiency virus type 1 listed in the UniProt
Knowledgebase (UniProtKB, www.uniprot org) under the name
"ENV_HV1H2". The protein amino acid sequence can be found under the
entry UniProt KB/Swiss-Prot P04578. It is further understood that
the terms "GLYC_LASSJ", "Pre-glycoprotein polyprotein GP complex",
"Lassa virus glycoprotein G2", and "LASV gp2" refer to the
naturally occurring glycoprotein G2 of Lassa virus listed in the
UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the name
"GLYC_LASSJ". The protein amino acid sequence can be found under
the entry UniProt KB/Swiss-Prot P08669. It is still further
understood that the terms "GLYC_LYCVW", "Pre-glycoprotein
polyprotein GP complex", "Lymphocytic choriomeningitis virus
glycoprotein G2", and "LCMV gp2" refer to the naturally occurring
glycoprotein G2 of Lymphocytic choriomeningitis virus listed in the
UniProt Knowledgebase (UniProtKB, www.uniprot org) under the name
"GLYC_LYCVW". The protein amino acid sequence can be found under
the entry UniProt KB/Swiss-Prot P07399. It is still further
understood that the terms "GLYC_MOPEI", "Pre-glycoprotein
polyprotein GP complex", "Mopeia virus glycoprotein G2", and "MOPV
gp2" refer to the naturally occurring glycoprotein G2 of Mopeia
virus listed in the UniProt Knowledgebase (UniProtKB,
www.uniprot.org) under the name "GLYC_MOPEI". The protein amino
acid sequence can be found under the entry UniProt KB/Swiss-Prot
P19240. It is still further understood that the terms "GLYC_TACV",
"Pre-glycoprotein polyprotein GP complex", "Tacaribe virus
glycoprotein G2", "TCRV gp2", and "TACV gp2" refer to the naturally
occurring glycoprotein G2 of Tacaribe virus listed in the UniProt
Knowledgebase (UniProtKB, www.uniprot.org) under the name
"GLYC_TACV". The protein amino acid sequence can be found under the
entry UniProt KB/Swiss-Prot P18141. It is still further understood
that the terms "CKS-17", "CKS-17 immunosuppressive domain",
"Cas-Br-E murine leukemia virus immunosuppression region",
"Envelope protein 15E", and "CKS-17, an mulv-related
heptadecapeptide" refer to the naturally occurring
immunosuppressive domain of murine leukemia virus listed in the
UniProt Knowledgebase (UniProtKB, www.uniprot org) under the name
"ENV_MLVCB". The protein amino acid sequence can be found under the
entry UniProt KB/Swiss-Prot P08360. It is still further understood
that the terms "VGP_EBOSB", "Envelope glycoprotein", "Sudan Ebola
virus glycoprotein 2", and "SEBOV gp2" refer to the naturally
occurring glycoprotein 2 of Sudan Ebola virus listed in the UniProt
Knowledgebase (UniProtKB, www.uniprot org) under the name
"VGP_EBOSB". The protein amino acid sequence can be found under the
entry UniProt KB/Swiss-Prot Q66814. It is still further understood
that the terms "VGP_EBOZM", "Envelope glycoprotein", "Zaire Ebola
virus glycoprotein 2", and "ZEBOV gp2" refer to the naturally
occurring glycoprotein 2 of Zaire Ebola virus listed in the UniProt
Knowledgebase (UniProtKB, www.uniprot.org) under the name
"VGP_EBOZM". The protein amino acid sequence can be found under the
entry UniProt KB/Swiss-Prot Q05320. It is still further understood
that the terms "VGP_MABVM", "Envelope glycoprotein", "Lake Victoria
marburgvirus glycoprotein 2", "Marburg virus gp2", and "MARV gp2"
refer to the naturally occurring glycoprotein 2 of Marburg virus
listed in the UniProt Knowledgebase (UniProtKB, www.uniprot.org)
under the name "VGP_MABVM". The protein amino acid sequence can be
found under the entry UniProt KB/Swiss-Prot P35253. It is still
further understood that the terms "ENV_MLVF5", "Envelope
glycoprotein", "Envelope protein 15E", "Friend murine leukemia
virus envelope glycoprotein", "FrMLV gp", and "Fr-MLV gp" refer to
the naturally occurring envelope glycoprotein of Friend murine
leukemia virus listed in the UniProt Knowledgebase (UniProtKB,
www.uniprot org) under the name "ENV_MLVF5". The protein amino acid
sequence can be found under the entry UniProt KB/Swiss-Prot P03390.
It is still further understood that the terms "U24_HHV6U",
"Glycoprotein U24", "Human herpesvirus 6A glycoprotein U24", "HHV-6
glycoprotein U24", "HHV-6 EoLF", and "HHV-6 U24" refer to the
naturally occurring glycoprotein U24 of human herpesvirus 6A listed
in the UniProt Knowledgebase (UniProtKB, www.uniprot org) under the
name "U24_HHV6U". The protein amino acid sequence can be found
under the entry UniProt KB/Swiss-Prot Q69559.
DETAILED DESCRIPTION OF THE INVENTION
[0126] The present invention relates to peptides or fragments,
homologs and derivatives thereof, which are derived from amino acid
sequences of fusion and other protein regions of various viruses
and from combinations thereof, and affect T cells by action on the
activating T cell receptor (TCR). The present invention further
relates to the treatment or prevention of various inflammatory and
autoimmune disease states or other conditions where T cells are
involved or recruited. In one embodiment, T cell receptor is
inhibited by variant peptides binding to the transmembrane regions
of the TCR.zeta. and CD3.delta..epsilon. subunits.
[0127] Various methods of application are proposed to use these
protein variants including, but not limited to; i) treating T
cell-related diseases or other medical conditions where T cells are
involved or recruited; ii) drug delivery systems; iii) a
sequence-based rational drug design method; iv) protease-resistance
immunotherapeutic peptides; v) coatings of medical devices, such as
implants and implantable devices.
[0128] The present invention contemplates constructing a series of
variant peptides capable of reducing said T cell activation by
action on the activating T cell receptor. TCR is a member of family
of multichain immune recognition receptors (MIRRs) which are
characterized by a common and distinct receptor architectural
feature--their ligand-binding subunits and signaling subunits
represent separate transmembrane protein chains that are
noncovalently bound in the transmembrane milieu (A. B. Sigalov.
Trends Immunol. 2004; 25:583-9; A. B. Sigalov. Adv Exp Med Biol
2008; 640:268-311; A. B. Sigalov. Adv Exp Med Biol 2008;
640:121-63; A. D. Keegan and Paul W. E. Immunol Today 1992;
13:63-68). The invariant TCR signaling chains, namely,
CD3.epsilon., .delta., .gamma., and TCR .zeta., all have a
conserved single negative charge in their TM domains, while TM
domains of the variant TCR .alpha. and .beta. chains contain one
(TCR.beta.) or two (TCR.alpha.) positive charges. Studies on the
TCR assembly (Manolios et al. Science 1990; 249:274-7; Call et al.
Cell 2002:111:967-79) showed that the integrity and functionally of
the receptor is provided by TM electrostatic interactions. The
positively charged amino acid residues (Lys and Arg) in the TM
region of the TCR.alpha. chain interact with the negatively charged
amino acid residues (Asp) of the TM domains of .zeta. homodimer
(Asp) and CD3.delta..epsilon. heterodimer whereas Lys in the TM
domain of the TCR.beta. chain interacts with the negatively charged
amino acid residues in the TM domains of CD3.gamma. (Glu) and
CD3.epsilon. (Asp) chains of the CD3.gamma..epsilon. heterodimer.
Recently, these interactions have been suggested as universal
therapeutic targets for a diverse variety of T cell-related
pathologies (A. B. Sigalov. Trends Imnmunol. 2004; 25:583-9; A. B.
Sigalov. Adv Exp Med Biol 2007; 601:335-44). It has been also
suggested that the molecular mechanisms targeting the TCR TM
interactions underlie ability of different human viruses such as
human immunodeficiency virus (HIV), cytomegalovirus (CMV), severe
acute respiratory syndrome coronavirus (SARS-CoV) to modulate
and/or escape the host immune response (A. B. Sigalov. Trends
Pharmnacol Sci 2006; 27:518-24; A. B. Sigalov. Faseb J 2007;
21:1633-34; A. B. Sigalov. Adv Exp Med Biol 2008; 640:268-311; T.
M. Kim and A. B. Sigalov. Adv Exp Med Biol 2008; 640:325-49; A. B.
Sigalov. PLoS Pathog 2009; 5:e1000404).
[0129] The
TCR/CD3.epsilon..delta./CD3.epsilon..gamma./.zeta..zeta.-couple- d
antigen receptor signaling pathway resident within T cell membranes
represents but one mechanism responsible for antigen-mediated T
cell activation. Although it is not necessary to understand the
mechanism of an invention, it is believed that these variant
peptides insert themselves into the T cell membrane and act as a
"receptor decoy" for antigen molecules. It is further believed that
TCR triggering and subsequent cell activation requires the
antigen-induced bridging of multiple TCRs that generates an
intracellular activation signal by bringing membrane-embedded
CD3.epsilon., .delta., .gamma., and TCR .zeta. signaling subunits
into close proximity and proper (permissive orientation) to trigger
the receptor. These peptide variants may prevent antigen-mediated T
cell activation by reducing CD3.epsilon., .delta., .gamma., and TCR
aggregation by generating TCR.zeta./CD3.delta..epsilon./peptide
variant bridges. It is further believed that the molecular basis
for the prevention of T cell activation is based upon
protein-protein interactions. It is still further believed that the
molecular mechanisms of action of the peptides of the invention
derived from amino acid sequences (or from combinations thereof) of
fusion and other protein regions of various viruses, including but
not limiting to, SARS-CoV, HTLV-1, HVA, HVS, LASV, LCMV, MOPV,
TACV, CKS-17, SEBOV, ZEBOV, MARV, Fr-MLV and HHV-6, are used by the
viruses in vivo to enter the T cell without triggering the
self-defense response. It is further believed that viral immune
evasion strategies can be transferred to therapeutic strategies
that require similar functionalities. Viruses represent years of
evolution and the efficiency and optimization that come along with
it. It is still further believed that the viruses use their fusion
and other immunosuppressive sequences to disrupt the
protein-protein interactions the
TCR.alpha./CD3.delta..epsilon./.zeta..zeta. signaling module of the
T cell receptor responsible for alarming the cell of the presence
of the intruder and, by doing so, blocks the receptor's ability to
send the distress signal.
[0130] Protein-protein interactions are involved in most biological
processes and thus represent an appealing target for innovative
drug development. These interactions can be targeted by small
molecule inhibitors, peptides, and peptidomimetics. Consequently,
indirect protein therapy that alters protein-protein interactions
represents an alternative to direct protein therapeutics (i.e., for
example, immunotherapy) and avoids dangerous side effects.
Indirectly acting peptides may serve as active regulators and
participate in molecular cross talk, which drives metabolic
processes. These indirectly acting peptides are also extremely
potent, showing high specificity, and have few toxicological
problems. Moreover, these indirectly acting peptides do not
accumulate in organs or suffer from drug-drug interactions as many
small molecules do. They can be used as therapeutic agents, or as a
starting point for developing peptidomimetics and small molecular
weight inhibitors.
I. T Cell Receptor-Mediated T Cell Activation
[0131] Immune responses rely upon the detection of specific
antigens by T cells, antigen exposure activating a T cell which
possibly leads to cell proliferation and differentiation. The TCR
is the central signaling pathway regulating T cell biology (K. R.
Jerome J Virol 2008; 82:4194-204). This receptor allows the T cell
to recognize antigen presented in the context of major
histocompatibility complex (MHC) class I or class II molecules
expressed on infected cells or professional antigen-presenting
cells. TCR signaling in naive T cells drives their activation and
expansion. In effector or memory T cells, TcR signaling drives
expansion and triggering of effector functions, such as cytokine
synthesis and cytotoxicity. The TCR-mediated activation process is
complex and multifaceted, and despite decades of research remains
controversial (Wedagedera et al. Biophys J 2006; 91:1604-18; A. B.
Sigalov, ed, Multichain Immune Recognition Receptor Signaling: From
Spatiotemporal Organization to Human Disease, Springer-Verlag, New
York, 2008).
[0132] T cell receptor is an attractive target for therapy of a
variety of T cell-related pathologies (A. B. Sigalov, ed,
Multichain Immune Recognition Receptor Signaling: From
Spatiotemporal Organization to Human Disease, Springer-Verlag, New
York, 2008). TCR is also critically important for the orchestration
of the antiviral response and also for the direct killing of
infected cells. Till recently, antibodies or fragments of
antibodies have been considered as clinically significant
therapeutic agents for various T cell-related diseases. For
example, the use of antibodies is suggested (U.S. Pat. No.
6,221,352) to treat autoimmune disorders such as rheumatoid
arthritis. However, antibody therapy poses serious disadvantages
such as a potential contamination of antibody preparations by
infectious agents such as prions or viruses, the high cost of
developing and administering antibody therapies, inherent
immunogenicity and other side effects. Other approach includes
peptides based on TCR-derived sequences for disrupting TCR function
(WO 96/22306; WO 97/47644; US Pat. Appl. 20050070478). Despite
multiple advantages of these peptides as compared to antibodies,
they have relatively low efficacy in terms of inhibiting TCR, thus
having a high potential for toxicity and side effects.
[0133] Since among the cells of the immune system, T cells are
critically important for the orchestration of the antiviral
response and pose a threat to the successful evasion of viruses,
and since TCR signaling is central to the development and function
of T cells, it is not surprising that many viruses have evolved
mechanisms to modulate TCR signaling. In this context, the ability
viruses have developed over centuries of evolution to specifically
affect TCR activation plays a crucial role in viral pathogenesis.
Considering the efficiency and optimization of the viral T cell
evasion strategy, it is very attractive to successfully transfer
these highly effective and specific TCR-targeted viral approaches
to therapeutic strategies that require similar functionalities.
[0134] The TCR-mediated activation signal can be divided into four
parts: 1) the extracellular recognition of a multivalent antigen
resulting in the aggregation, or clustering, of the TCRs, 2) TCR
triggering and transmembrane (TM) signal transduction, 3)
phosphorylation of the CD3.epsilon., .delta., .gamma., and TCR
.zeta. Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) by
protein tyrosine kinases (PTKs) and activation of specific
intracellular pathways, and 4) the activation of genes in the
nucleus. The extracellular recognition of an antigen, the
TCR-triggered biochemical cascades and the mechanisms of gene
activation are understood in significant detail. However, despite
extensive studies, the mechanism by which the TCR transduces
ordered information such as antigen recognition from outside the
cell via receptor TM and juxtamembrane (JM) regions into
intracellular biochemical events (part 2), the mode of action of
this clinically relevant peptide had not been elucidated until 2004
when a novel model of TCR signaling, the Signaling Chain
HOmoOLigomerization (SCHOOL) model, was first introduced (A. B.
Sigalov. Trends Immunol. 2004; 25:583-89), for the first time
considering the TCR triggering and subsequent cell activation as a
result of interplay between specific extracellular, transmembrane
and intracellular protein-protein interactions.
II. SCHOOL Model of TCR Signaling
[0135] Multichain immune recognition receptors (MIRRs) expressed on
various cells (See FIG. 1A) are believed to recognize foreign
antigens and initiate many biological responses. Members of the
MIRR family are believed to be multisubunit complexes that are
formed by the noncovalent transmembrane association of
recognition/binding subunits with signaling subunits (See, FIG.
1B). Therapeutic strategies contemplated herein involve MIRR
triggering and subsequent transmembrane signaling. MIRR-mediated
signal transduction, its role in health and disease, and the use of
these receptors as attractive targets for rational drug design
efforts in the treatment of several immune disorders are described
in (US Pat. Appl. 20090075899; A. Sigalov. Semin. Immunol. 2005;
17:51-64; A. B. Sigalov. Trends Immunol. 2004; 25:583-89; A. B.
Sigalov. Trends Pharmacol Sci 2006; 27:518-24; A. B. Sigalov. Adv
Exp Med Biol 2007; 601:335-44; A. B. Sigalov. Adv Exp Med Biol
2008; 640:268-311) which are incorporated herein by reference in
their entirety.
[0136] In one embodiment, the present invention contemplates
therapeutic targets compatible with a novel model of MIRR
triggering and subsequent transmembrane signal transduction; the
Signaling Chain HOmoOLigomerization (SCHOOL) model (See FIG. 1C)
(A. B. Sigalov. Adv Exp Med Biol 2008; 640:268-311; A. B. Sigalov.
Adv Exp Med Biol 2008; 640:121-63). Although it is not necessary to
understand the mechanism of an invention, it is believed that the
structural similarity of the MIRRs provides the basis for the
similarity in the mechanisms of MIRR-mediated signaling. It is also
believed that the model reveals MIRR transmembrane interactions as
new therapeutic targets (See FIG. 1D). It is further believed that
a general pharmaceutical approach based upon this SCHOOL model can
be used to treat diverse immune-mediated diseases.
[0137] Application of the SCHOOL model to the transmembrane signal
transduction mediated by a T cell antigen receptor (TCR) (See FIG.
2) reveals the TCR.alpha./CD3.epsilon..delta./.zeta..zeta.
transmembrane interactions as therapeutic targets and suggests that
an inhibition of TCR signaling may be achieved by using
transmembrane-targeted agents which specifically disrupt
transmembrane interactions between the TCR.alpha. binding subunit
and the CD3.epsilon..delta. and .zeta..zeta. signaling subunits.
For example, the simplest agents would be synthetic transmembrane
peptides (TMPs) corresponding to the TCR.alpha. transmembrane
domain (See FIG. 3). This mechanism explains surprising
similarities in characteristics and immunomodulatory activities of
the T cell receptor core peptide (CP) and HIV-1 gp41 fusion peptide
(See TABLE 1) (Amon et al. Biochim Biophys Acta 2006; 1763:879-88;
Wang et al. Cell Immunol 2002:215:12-19; Wang et al. Clin Inmmunol
2002; 105:199-207; Quintana et al. J Clin Invest 2005;
115:2149-58). The SCHOOL mechanism of TCR signaling also suggest
the molecular explanation of apparent discrepancies in the observed
apparent discrepancy in inhibitory activity of different CD3
transmembrane peptides between in vitro and in vivo T cell unbutton
(See FIG. 4) (Collier et al. Scand J Immunol 2006; 64:388-91; A. B.
Sigalov. Adv Exp Med Biol 2007; 601:335-44; A. B. Sigalov. Adv Exp
Med Biol 2008; 640:121-63).
TABLE-US-00001 TABLE 1 Similarities in characteristics and
immunomodulatory activites of the T cell receptor core peptide and
HIV-1 gp41 fusion peptide Characteristics/Activation Model CP FP
Colocalization with TCR + + Coprecipitation with TCR + +
Immunosuppressive activity in vivo + + Inhibition of in vitro
activation: antigen + + anti-TCR.beta. - ND anti-CD3 antibody - -
PMA/ionomycin Abbreviations: TCR, T cell receptor; CP, core
peptide; FP, fusion peptide; PMA, phorbol 12-myristate 13-acetate;
ND, not determined.
[0138] Without being limited by a particular theory, the basic
principles of one proposed mechanism by which peptides and other
compound of the present invention may work by TCR-mediated
transmembrane signaling. See, FIGS. 2 & 3.
[0139] It is believed that multivalent antigen-induced clustering
of a
TCR.alpha..beta./CD3.epsilon..delta./CD3.epsilon..gamma./.zeta..zeta.
receptor complex leads to formation of the relevant CD3.epsilon.,
CD3.delta., CD3 .gamma. and .zeta. signaling oligomers with
subsequent phosphorylation of the ITAM-Tyr residues and
transmembrane transduction of the T cell activation signal. See,
FIG. 2. This hypothesis suggests that a TCR Core Peptide (TCR-CP),
a peptide corresponding to the transmembrane region of TCR.alpha.,
penetrates the cell membrane and competitively binds to the
transmembrane domain of heterodimeric CD3.epsilon..delta. and
homodimeric .zeta..zeta. signaling signaling subunits, thus
displacing a TCR.alpha. chain from interacting with these signaling
subunits, thereby resulting in a "pre-dissociation" of a
TCR.alpha..beta./CD3.epsilon..delta./CD3.epsilon..gamma./.zeta..zeta.
receptor complex. As a consequence, antigen (but not
antibody)-induced TCR clustering does not lead to formation of
CD3.epsilon..delta., CD3.epsilon..gamma. and homodimeric
.zeta..zeta. signaling oligomers and subsequent T cell activation.
See, FIG. 3. This is the only mechanism that explains the observed
characteristics and immunomodulatory activity features of the TCR
CP and HIV-1 gp41 FP. See, TABLE 1.
[0140] Normal transmembrane (TM) interactions between the
ligand-binding TCR.alpha. subunit and the CD3.epsilon..delta. and
signaling subunits forming a functional
TCR.alpha..beta./CD3.epsilon..delta./CD3.epsilon..gamma./.zeta..zeta.
receptor complex comprise two positively charged amino acid
residues (Lys and Arg) within the TCR.alpha. transmembrane portion
and negatively charged aspartic acid pairs in the transmembrane
domains of the CD3.epsilon..delta. heterodimer and homodimer,
thereby allowing subunit association (Call et al. Cell 2002;
111:967-79). Although it is not necessary to understand the
mechanism of an invention, it is believed that interactions between
positively charged amino acids of a viral sequence-based peptide
inhibitors of the invention and aspartic acid residues of the
CD3.epsilon..delta. heterodimer and .zeta..zeta. homodimer disrupt
the transmembrane interactions between the TCR.alpha. subunit and
the the CD3.epsilon..delta. and .zeta..zeta. signaling subunits,
thereby "disconnecting" TCR.alpha. and result in a non-functioning
T cell receptor. See FIGS. 3, 6A, 6B, and 6C.
III. T Cell Receptor Inhibitory Peptides of Viral Origin and
Variants Thereof
[0141] Although it is not necessary to understand the mechanism of
an invention, it is believed that a hydrophobic/polar/charged amino
acid sequence patterning, rather than sequence similarity, within a
TCR inhibitory viral sequence plays a dominant role in the
development of effective peptide-based inhibitors of T cell
activation. For example, despite the lack of sequence similarity
(See FIGS. 3 and 5), the fusion peptide (FP) in the N terminus of
the HIV envelope glycoprotein, gp41, has been shown to inhibit T
cell antigen receptor (TCR)-mediated T cell activation in vitro and
in vivo more effectively than the transmembrane TCR core peptide
(CP) with 100-fold lower the 50% inhibitory concentration (IC50)
values for FP than those observed for CP.
[0142] In some embodiments, as contemplated by the present
invention, optimal peptide inhibitors and peptide inhibitor
analogues are designed using hydrophobic/polar/charged sequence
pattern criteria and associated evaluation techniques. These
criteria and techniques are described in (US Pat. Appl.
20090075899) and incorporated herein by reference in their
entirety. The peptide inhibitors of the present invention may then
be synthesized and tested in T cell function inhibition assays.
[0143] Listed in FIG. 5 are viral fusion and other sequences with
known or unknown immunomodulatory activity. As surprisingly found,
charge distribution patterns for fusion and other protein regions
of various viruses are conserved in many unrelated viruses and show
similarities to those for TCR CP and HIV FP which are known to
exhibit TCR inhibitory activity. Exploratory investigation of the
sequences listed reveals three major classes of the charge
distribution patterns. Class I and Class III are characterized by
two positively charged residues spaced apart by 4 and 8 amino
acids, respectively, whereas Class II is characterized by three
positively charged residues spaced apart by 3 amino acids. Within
the SCHOOL model of TCR signaling, a striking similarity in the
charge distribution patterns suggests a similarity in their mode of
action (A. B. Sigalov. PLoS Pathog 2009; 5:e1000404). See FIGS. 6A,
6B, and 6C. This clearly demonstrates that different viruses have
adopted similar mechanisms of specifically targeting TCR,
disrupting receptor architecture and suppressing the immune system.
Importantly, by virtue of the acquired insight into this conserved
structural motif, expanded predictions, hypotheses and conclusions
can be derived to being answering the question of if shared
TCR-targeted strategies represents a conserved function or if it
represents a convergent tactic of divergent viruses.
[0144] The transmembrane regions of the TCR.alpha., CD3.epsilon.,
CD3.delta., and .zeta. chains are highly conserved and the
substitutions between species are very conservative. This suggests
a functional role for the transmembrane regions of TCR.alpha.,
CD3.epsilon., CD3.delta., and .zeta. constituents of the TCR
complex. These regions strongly interact between themselves, thus
maintaining the integrity of the TCR signaling complex in resting
cells. These transmembrane domains are short and should be easily
mimicked by synthetic peptides and compounds of the invention
derived from the listed fusion and other proteins of various
viruses. Based on these features, and taking advantage of the
SCHOOL model of TCR signaling to explain TCR-mediated cell
activation, the present invention contemplates a new approach of
intervening and modulating TCR function using a billion year-long
drug development process of nature. In some embodiments, synthetic
peptides and compounds are contemplated that may provide successful
treatment options in the clinical setting.
[0145] In one embodiment, the present invention contemplates a
series of peptides that are inhibitors of a TCR. Although it is not
necessary to understand the mechanism of an invention, it is
believed that this inhibition is mediated by disrupting the
transmembrane interactions between the recognition, TCR.alpha., and
signaling subunits, namely CD3.epsilon., CD3.delta., and .zeta.. In
other embodiments, these peptide inhibitors treat and/or prevent
diseases and/or conditions comprising T cell activation. In one
embodiment, the peptide inhibitors mediate antigen-induced T cell
activation. In another embodiment, the present invention
contemplates a drug delivery system comprising peptide inhibitors
of the present invention. Although it is not necessary to
understand the mechanism of an invention, it is believed that the
peptide inhibitor drug delivery system functions by penetrating the
T cell membrane.
[0146] Sequence-based rational design can be used as a tool in
order to increase the effectiveness of the peptides to inhibit the
function of the
TCR.alpha..beta./CD3.epsilon..delta./CD3.epsilon..gamma./.zeta..zeta.
receptor complex. Principles and techniques of a sequence-based
rational design are described in (US Pat. Appl. 20090075899) and
incorporated herein by reference in their entirety. For example, a
conservative amino acid substitution of arginine for lysine or
insertion of at least one supplemental positively charged amino
acid residue (i.e., for example, arginine and/or lysine) may be
made in certain locations on alpha-helixes of the peptides of the
present inventions. Although it is not necessary to understand the
mechanism of an invention, it is believed that these changes should
result in increased binding activity to the transmembrane domains
of the CD3.epsilon..delta. and .zeta..zeta. receptor complex
signaling subunit dimers, thus enhancing the effectiveness of the
peptides to inhibit the function of TCR.
[0147] TCR peptide inhibitors and variants thereof contemplated
herein may be modified at the carboxy terminal without loss of
activity. Accordingly, it is intended that the present invention
includes within its scope, peptides which include additional amino
acids to the "core" sequence of the peptide of the present
invention and which affect the interaction of TCR.alpha..beta.,
CD3.delta. and .zeta..zeta. subunits of the TCR signaling
complex.
[0148] In some embodiments, the peptide inhibitors comprise
D-stereoisomeric amino acids, thereby allowing the formation of
immunotherapeutic peptides with increased resistance to protease
degradation. In one embodiment, the D-amino acid peptide inhibitors
are used for the clinical treatment in T cell-mediated disorders.
Although it is not necessary to understand the mechanism of an
invention, it is believed that these peptide inhibitors prevent T
cell activation.
[0149] In some embodiments, the present invention contemplate
peptide inhibitors that are protease resistant. In one embodiment,
such protease-resistant peptide inhibitors are peptides comprising
protecting groups. For example, a peptide peptide may be protected
from exoproteinase degradation by N-terminal acetylation ("Ac")
and/or C-terminal amidation.
[0150] In some embodiments, the peptide inhibitors comprise
conjugated lipids and/or sugars. In other embodiments, the peptide
inhibitors comprise hydrophobic amino acid motifs, wherein said
motifs are known to increase the membrane penetrating ability of
peptides and proteins. Although it is not necessary to understand
the mechanism of an invention, it is believed that either
lipid/sugar conjugation and/or hydrophobic amino acid motifs
increase the efficacy TCR antigen receptor complex inhibition using
the peptides and compositions of the invention.
[0151] In some embodiment, the peptides and compounds contemplated
by the present invention may be used for production of
peptide/compound-containing medical devices for local
anti-inflammatory therapy and/or for the prevention of immune
response.
IV. Classes of Transmembrane Peptide Variant TCR Inhibitors
[0152] The present invention described herein relates to synthetic
peptides of viral origin and derivatives thereof, which may be
useful in the clinical treatment and/or prevention of T
cell-mediated disorders.
[0153] In one embodiment, the present invention contemplates a
Class I peptide derivative having the general formula
R.sub.1-A-B-C-D-E-F-R.sub.2 (SEQ ID NO: 84) (See FIG. 7), or a
di-sulfide bridged, linear dimer thereof, or a cyclic dimer
thereof, wherein;
[0154] A is absent, or 1-4 D- or L-amino acids selected from the
group including, but not limited to, Val, Ile, Leu, Gly, Met, Tyr,
and Phe;
[0155] B is a positively charged D- or L-amino acid;
[0156] C is a peptide comprising 4 hydrophobic D- or L-amino acids,
or Thr and 3 hydrophobic D- or L-amino acids, including D- or
L-cysteine or a D- or L-cysteine homologue;
[0157] D is a positively charged D- or L-amino acid;
[0158] E is Val or Tyr, and 1-2 D- or L-amino acids selected from
the group including, but not limited to, Ala, Phe, and Gly
following Val or Tyr;
[0159] F is absent or 0 to 8 D- and L-amino acids selected from the
group including, but not limited to, Gly, Phe, Asn, Leu, Ile, Met,
Thr and Gln;
[0160] R.sub.1 is absent (i.e., for example, --H) or
1-amino-glucose succinate, 2-aminododecanoate, or myristoylate;
and
[0161] R.sub.2 is absent (i.e, for example, --H) or
Gly-Tris-monopalmitate, -dipalmitate and -tripalmitate.
[0162] In some embodiments, peptide derivatives are created wherein
(SEQ ID NO: 85);
[0163] A is a peptide comprising 1-4 amino acids selected from the
group comprising Gly, Tyr, Cys, Val, Leu, Ile, and Met;
[0164] B is selected from Arg, His or Lys;
[0165] C is a peptide comprising 4 amino acids selected from the
group comprising Leu, Ile, Thr, and Pro;
[0166] D is selected from from Arg, His or Lys;
[0167] E is a peptide comprising 3 amino acids selected from the
group comprising Val, Tyr, Ala, Phe, and Gly; and
[0168] F is a peptide comprising 8 amino acids selected from the
group comprising Gly, Phe, Asn, Leu, Ile, Met, Thr and Gln.
[0169] In one embodiment, the present invention contemplates a
peptide derivative having the general formula R.sub.1-[Arg and/or
Lys].sub.n=0-4-A-B-C-D-E-F-[Arg and/or Lys].sub.n=0-4-R.sub.2 or a
di-sulfide bridged, linear dimer thereof, or a cyclic dimer
thereof, wherein (SEQ ID NO: 86);
[0170] A may be i) absent; or ii) 1-4 amino acids selected from the
group including, but not limited to, Val, Ile, Leu, Gly, Met, Phe,
or Tyr;
[0171] B may be selected from the group including, but not limited
to, Arg, Lys, or His;
[0172] C is 4 amino acids selected from the group including, but
not limited to, Leu, Ile, Thr, or Pro;
[0173] D may be selected from the group including, but not limited
to, Arg, Lys, or His;
[0174] E may be i) Val or Tyr; or ii) Val or Tyr and 1-2 amino
acids selected from the group including, but not limited to, Ala,
Phe, or Gly;
[0175] F may be i) absent or ii) 1-8 amino acids selected from the
group including, but not limited to, Gly, Phe, Asn, Leu, Ile, Met,
Thr and Gln.
[0176] R.sub.1 and R.sub.2 may be either i) absent; ii) a
conjugated lipid selected from the group including, but not limited
to, Gly-Tris-monopalmitate, -dipalmitate and -tripalmitate; or iii)
a conjugated sugar selected from the group including, but not
limited to, 1-amino-glucose succinate, 2-aminododecanoate, or
myristoylate. See, FIG. 7.
[0177] As referred to herein, hydrophobic amino acids include, but
are not limited to, Ala, Val, Leu, Ile, Pro, Phe, Trp, and Met;
positively charged amino acids include, but are not limited to,
Lys, Arg and His; and negatively charged amino acids include, but
are not limited to, Asp and Glu.
[0178] The above general formula represents one embodiment of Class
I of TCR peptide inhibitory sequences of viral origin (for example,
SARS-CoV fusion peptide, FP; See FIG. 5) comprising at least one
conserved domain that contains a highly homologous sequence between
this sequence and the TCR.alpha. transmembrane domain. In one
embodiment, a SARS-CoV FP-derived TCR inhibitory peptide comprises
MYKTPTLKYFGGFNFSQIL (SEQ ID NO: 6) along or with various lipid
and/or sugar derivatives that may, or may not, have a disulfide
bridged dimer, or represent a cyclic dimer.
[0179] In one embodiment, the present invention contemplates a
Class II peptide derivative having the general formula
R.sub.1-A*-B*-C*-D*-E*-F*-G*-R.sub.2 (See FIG. 8) (SEQ ID NO: 87),
or a di-sulfide bridged, linear dimer thereof, or a cyclic dimer
thereof, wherein;
[0180] A* is absent, or 1-4 D- or L-amino acids selected from the
group including, but not limited to, Arg, Gly, Leu, Ile, Asn, Gln,
Ala, Ser, and Val;
[0181] B* is a positively charged D- or L-amino acid;
[0182] C* is a peptide comprising 3 amino acids selected from the
group including, but not limited to, Asp, Leu, Ile, Arg, Lys, Ser,
Val, and Glu;
[0183] D* is a positively charged D- or L-amino acid;
[0184] E* is a peptide comprising 3 amino acids selected from the
group including, but not limited to, Asp, Asn, Leu, Lys, Ile, Val,
and Glu;
[0185] F* is a positively charged D- or L-amino acid;
[0186] G* is Ile, Leu or Asp, or a peptide comprising 2 amino acids
selected from the group including, but not limited to, Ile, Leu,
Asp, Asn, and Thr;
[0187] R.sub.1 is absent (i.e., for example, --H) or
1-amino-glucose succinate, 2-aminododecanoate, or myristoylate;
and
[0188] R.sub.2 is absent (i.e, for example, --H) or
Gly-Tris-monopalmitate, -dipalmitate and -tripalmitate.
[0189] In some embodiments, peptide derivatives are created wherein
(SEQ ID NO: 88);
[0190] A* is a peptide comprising 1-4 amino acids selected from the
group comprising Arg, Gly, Leu, Ile, Asn, Gln, Ala, Ser, and
Val;
[0191] B* is selected from Arg, His or Lys;
[0192] C* is a peptide comprising 3 amino acids selected from the
group comprising Asp, Leu, Ile, Arg, Lys, Ser, Val, and Glu;
[0193] D* is selected from from Arg, His or Lys;
[0194] E* is a peptide comprising 3 amino acids selected from the
group comprising Asn, Asp, Leu, Lys, Ile, Val, and Glu;
[0195] F* is selected from from Arg, His or Lys; and
[0196] G* is a peptide comprising 2 amino acids selected from the
group comprising Ile, Leu, Asp, Asn, and Thr.
[0197] In one embodiment, the present invention contemplates a
peptide derivative having the general formula R.sub.1-[Arg and/or
Lys].sub.n=0-4-A*-B*-C*-D*-E*-F*-G*-[Arg and/or
Lys].sub.n=0-4-R.sub.2 or a di-sulfide bridged, linear dimer
thereof, or a cyclic dimer thereof, wherein (SEQ ID NO: 89);
[0198] A* may be i) absent; or ii) 1-4 D- or L-amino acids selected
from the group including, but not limited to, Arg, Gly, Leu, Ile,
Asn, Gln, Ala, Ser, and Val;
[0199] B* may be selected from the group including, but not limited
to, Arg, Lys, or His;
[0200] C* is 3 amino acids selected from the group including, but
not limited to, Asp, Leu, Ile, Arg, Lys, Ser, Val, and Glu;
[0201] D* may be selected from the group including, but not limited
to, Arg, Lys, or His;
[0202] E* is 3 amino acids selected from the group including, but
not limited to, Ala, Phe, or Gly;
[0203] F* may be selected from the group including, but not limited
to, Arg, Lys, or His; and
[0204] G* may be i) Ile, Leu, or Asp, or ii) 2 amino acids selected
from the group including, but not limited to, Leu, Ile, Asn, and
Thr;
[0205] R.sub.1 and R.sub.2 may be either i) absent; ii) a
conjugated lipid selected from the group including, but not limited
to, Gly-Tris-monopalmitate, -dipalmitate and -tripalmitate; or iii)
a conjugated sugar selected from the group including, but not
limited to, 1-amino-glucose succinate, 2-aminododecanoate, or
myristoylate. See, FIG. 8.
[0206] The above general formula represents one embodiment of Class
II of TCR peptide inhibitory sequences of viral origin (for
example, HVS Tip.sup.211-228; See FIG. 5) comprising at least one
conserved domain that contains a highly homologous sequence between
this sequence and the HTLV-1 gp21.sup.313-353. In one embodiment, a
HVS Tip.sup.211-228-derived TCR inhibitory peptide comprises
ANERNIVKDLKRLENKIN (SEQ ID NO: 7) along or with various lipid
and/or sugar derivatives that may, or may not, have a disulfide
bridged dimer, or represent a cyclic dimer.
[0207] In one embodiment, the present invention contemplates a
Class III peptide derivative having the general formula
R.sub.1-A**-B**-C**-D**-E**-F**-R.sub.2 (See FIG. 8), or a
di-sulfide bridged, linear dimer thereof, or a cyclic dimer
thereof, wherein (SEQ ID NO: 90);
[0208] A** is absent, or 1-8 D- or L-amino acids selected from the
group including, but not limited to, Ala, Pro, Cys, Thr, Asn, Met,
Glu, Ser, Gly, Tyr, Leu, Ile, and Gln;
[0209] B** is a positively charged D- or L-amino acid;
[0210] C** is a peptide comprising 8 amino acids selected from the
group including, but not limited to, Ser, Trp, Arg, Lys, His, Pro,
Met, Gly, Ala, Thr, Leu, Ile, Val, Asp, Asn, Thr, Glu, Phe, and
Gln;
[0211] D** is a positively charged D- or L-amino acid;
[0212] E** is absent, or a peptide comprising 1-3 amino acids
selected from the group including, but not limited to, Gln, Cys,
Glu, Trp, Arg, Leu, Ile, Phe, Gly, and Lys;
[0213] F** is absent, or a peptide comprising 1-4 amino acids
selected from the group including, but not limited to, Asn, Leu,
Ile, Thr, Phe, and Val;
[0214] R.sub.1 is absent (i.e., for example, --H) or
1-amino-glucose succinate, 2-aminododecanoate, or myristoylate;
and
[0215] R.sub.2 is absent (i.e, for example, --H) or
Gly-Tris-monopalmitate, -dipalmitate and -tripalmitate.
[0216] In some embodiments, peptide derivatives are created wherein
(SEQ ID NO: 91);
[0217] A** is a peptide comprising 1-8 amino acids selected from
the group comprising Ala, Pro, Cys, Thr, Asn, Met, Glu, Ser, Gly,
Tyr, Leu, Ile, and Gln;
[0218] B** is selected from Arg, His or Lys;
[0219] C** is a peptide comprising 8 amino acids selected from the
group comprising Ser, Trp, Arg, Lys, His, Pro, Met, Gly, Ala, Thr,
Leu, Ile, Val, Asp, Asn, Thr, Glu, Phe, and Gln;
[0220] D** is selected from from Arg, His or Lys;
[0221] E** is a peptide comprising 3 amino acids selected from the
group comprising Gln, Cys, Glu, Trp, Arg, Leu, Ile, Phe, Gly, and
Lys;
[0222] F** is a peptide comprising 4 amino acids selected from the
group comprising Asn, Leu, Ile, Thr, Phe, and Val.
[0223] In one embodiment, the present invention contemplates a
peptide derivative having the general formula R.sub.1-[Arg and/or
Lys].sub.n=0-4-A**-B**-C**-D**-E**-F**-[Arg and/or
Lys].sub.n=0-4-R.sub.2 or a di-sulfide bridged, linear dimer
thereof, or a cyclic dimer thereof, wherein (SEQ ID NO: 92);
[0224] A** may be i) absent; or ii) 1-8 D- or L-amino acids
selected from the group including, but not limited to, Ala, Pro,
Cys, Thr, Asn, Met, Glu. Ser, Gly, Tyr, Leu, Ile, and Gin;
[0225] B** may be selected from the group including, but not
limited to, Arg, Lys, or His;
[0226] C** is 8 amino acids selected from the group including, but
not limited to, Ser, Trp, Arg, Lys, His, Pro, Met, Gly, Ala, Thr,
Leu, Ile, Val, Asp, Asn, Thr, Glu, Phe, and Gln;
[0227] D** may be selected from the group including, but not
limited to, Arg, Lys, or His;
[0228] E** may be i) absent, or ii) 1-3 amino acids selected from
the group including, but not limited to, Gln, Cys, Glu, Trp, Arg,
Leu, Ile, Phe, Gly, and Lys;
[0229] F** may be i) absent, or ii) 1-4 amino acids selected from
the group including, but not limited to, Asn, Leu, Ile, Thr, Phe,
and Val;
[0230] R.sub.1 and R.sub.2 may be either i) absent; ii) a
conjugated lipid selected from the group including, but not limited
to, Gly-Tris-monopalmitate, -dipalmitate and -tripalmitate; or iii)
a conjugated sugar selected from the group including, but not
limited to, 1-amino-glucose succinate, 2-aminododecanoate, or
myristoylate. See, FIG. 9.
[0231] The above general formula represents one embodiment of Class
III of TCR peptide inhibitory sequences of viral origin (for
example, LASV FP (gp2.sup.260-298); See FIG. 5) comprising at least
one conserved domain that contains a highly homologous sequence
between this sequence and the HIV-1 gp41 FP. In one embodiment, a
LASV FP (gp2.sup.260-298)-derived TCR inhibitory peptide comprises
GTFTWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAV (SEQ ID NO: 8) along or
with various lipid and/or sugar derivatives that may, or may not,
have a disulfide bridged dimer, or represent a cyclic dimer.
[0232] In one embodiment, the present invention contemplates a
method of rational designing of linear or cyclic peptides and
lipid- and/or sugar-conjugated peptides consisting of L- or
D-stereoisomeric amino acids in order to increase effectiveness of
the peptides in inhibiting the function of the
TCR.alpha..beta./CD3.epsilon..delta./CD3.epsilon..gamma./.zeta..zeta.
receptor complex. This method is described in (US Pat. Appl.
20090075899) and incorporated herein by reference in their
entirety. In one embodiment, the method comprises using rational
combinations of the peptide blocks from different classes of TCR
peptide inhibitor sequences of viral origin, namely A, A*, A**, B,
B*, B**, C, C*, C**, D, D*, D**, E, E*, E**, F, F*, F**, and G*, as
designated in FIGS. 7, 8, and 9, thereby optimizing and increasing
binding to the transmembrane domains of CD3.epsilon., .delta. and
TCR.zeta. chains. See, FIG. 10.
[0233] In one embodiment, the present invention contemplates a
combinatorial TCR peptide inhibitor sequence derivative having the
general formula R.sub.1-A**-B*-C*-D*-C-D-E**-R.sub.2 (See FIG. 10),
or a di-sulfide bridged, linear dimer thereof, or a cyclic dimer
thereof, wherein (SEQ ID NO: 93);
[0234] A** is absent, or 1-8 D- or L-amino acids selected from the
group including, but not limited to, Ala, Pro, Cys, Thr, Asn, Met,
Glu, Ser, Gly, Tyr, Leu, Ile, and Gln;
[0235] B* is a positively charged D- or L-amino acid;
[0236] C is a peptide comprising 4 hydrophobic D- or L-amino acids,
or Thr and 3 hydrophobic D- or L-amino acids, including D- or
L-cysteine or a D- or L-cysteine homologue;
[0237] C* is a peptide comprising 3 amino acids selected from the
group comprising Asp, Leu, Ile, Arg, Lys, Ser, Val, and Glu;
[0238] D is a positively charged D- or L-amino acid;
[0239] D* is a positively charged D- or L-amino acid;
[0240] E** is absent, or a peptide comprising 1-3 amino acids
selected from the group including, but not limited to, Gln, Cys,
Glu, Trp, Arg, Leu, Ile, Phe, Gly, and Lys;
[0241] R.sub.1 is absent (i.e., for example, --H) or
1-amino-glucose succinate, 2-aminododecanoate, or myristoylate;
and
[0242] R.sub.2 is absent (i.e, for example, --H) or
Gly-Tris-monopalmitate, -dipalmitate and -tripalmitate.
[0243] In some embodiments, peptide derivatives are created wherein
(SEQ ID NO: 94);
[0244] A** is a peptide comprising 1-8 amino acids selected from
the group comprising Ala, Pro, Cys, Thr, Asn, Met, Glu, Ser, Gly,
Tyr, Leu, Ile, and Gln;
[0245] B* is selected from Arg, His or Lys;
[0246] C is a peptide comprising 4 amino acids selected from the
group comprising Leu, Ile, Thr, and Pro;
[0247] C* is a peptide comprising 3 amino acids selected from the
group comprising Asp, Leu, Ile, Arg, Lys, Ser, Val, and Glu;
[0248] D is selected from from Arg, His or Lys;
[0249] D* is selected from from Arg, His or Lys; and
[0250] E** is a peptide comprising 3 amino acids selected from the
group comprising Gln, Cys, Glu, Trp, Arg, Leu, Ile, Phe, Gly, and
Lys.
[0251] In one embodiment, the present invention contemplates a
peptide derivative having the general formula R.sub.1-[Arg and/or
Lys]n=.sub.0-4-A**-B*-C*-D*-C-D-E**-[Arg and/or
Lys].sub.n=0-4-R.sub.2 or a di-sulfide bridged, linear dimer
thereof, or a cyclic dimer thereof, wherein (SEQ ID NO: 95);
[0252] A** may be i) absent; or ii) 1-8 D- or L-amino acids
selected from the group including, but not limited to, Ala, Pro,
Cys, Thr, Asn, Met, Glu, Ser, Gly, Tyr, Leu, Ile, and Gln;
[0253] B* may be selected from the group including, but not limited
to, Arg, Lys, or His;
[0254] C is 4 amino acids selected from the group including, but
not limited to, Leu, Ile, Thr, and Pro;
[0255] C* is 3 amino acids selected from the group including, but
not limited to, Asp, Leu, Ile, Arg, Lys, Ser, Val, and Glu;
[0256] D may be selected from the group including, but not limited
to, Arg, Lys, or His;
[0257] D* may be selected from the group including, but not limited
to, Arg, Lys, or His;
[0258] E** may be i) absent, or ii) 1-3 amino acids selected from
the group including, but not limited to, Gln, Cys, Glu, Trp, Arg,
Leu, Ile, Phe, Gly, and Lys;
[0259] R.sub.1 and R.sub.2 may be either i) absent; ii) a
conjugated lipid selected from the group including, but not limited
to, Gly-Tris-monopalmitate, -dipalmitate and -tripalmitate; or iii)
a conjugated sugar selected from the group including, but not
limited to, 1-amino-glucose succinate, 2-aminododecanoate, or
myristoylate. See, FIG. 10.
[0260] The above general formula represents one embodiment of
combinatorial TCR peptide inhibitory sequences of viral origin (See
FIG. 10) comprising at least one conserved domain that contains a
highly homologous sequence between this sequence and the TCR.alpha.
transmembrane domain, HVS Tip.sup.211-228, HVA Tio.sup.225-242,
HTLV gp21.sup.313-353, and/or HIV-1 gp41 FP. In one embodiment, a
combinatorial TCR inhibitory peptide of viral origin comprises
LQNRDLKRLLFLKRKT (SEQ ID NO: 9) along or with various lipid and/or
sugar derivatives that may, or may not, have a disulfide bridged
dimer, or represent a cyclic dimer.
[0261] In another embodiment, the method comprises conjugating at
least one lipid and/or at least one sugar to the C- and/or
N-termini of the peptide, thereby increasing binding to the
transmembrane domains of CD3.epsilon., .delta. and TCR.zeta. chains
and/or improving the penetration of the peptide variant into the T
cell membrane. In one embodiment, the lipid- and/or
sugar-conjugated peptide variants comprise D-amino acids, thereby
increasing resistance to protease degradation. In one embodiment, a
protease resistant peptide variant is useful clinically for
inhibiting T cell activation in T cell-mediated disorders.
[0262] In some embodiments, conjugated peptide variants are
synthesized using the standard procedures (Amon et al. BBA Mol Cell
Res 2006; 1763:879-88; Whittaker et al. Pept Res 1993; 6:125-8;
Chemistry of Peptide Synthesis, N. Leo Benoiton (ed.), CRC, 2005;
Gerber et al. FASEB J 12005; 19:1190-2; Kliger et al. J Biol Chem
1997; 272:13496-505, Merrifield et al. Biochemistry 1982;
21:5020-31).
[0263] In one embodiment, the rational design method comprises
inserting at least one polyarginine and/or polylysine sequence into
a TCR peptide inhibitor of the present invention, thereby
increasing binding to the transmembrane domains of the
CD3.epsilon., .delta. and TCR.zeta. chains and/or improving the
penetration of the peptide variant into the platelet membrane.
Other modifications of the peptides contemplated herein include,
but are not limited to, modifications to side chains, incorporation
of unnatural amino acids and/or their derivatives during peptide
synthesis and the use of crosslinkers and other methods which
impose conformational constraints on the peptides. It may also be
possible to add various groups to the peptide of the present
invention to confer advantages such as increased potency or
extended half life in vivo without substantially decreasing the
biological activity of the peptide. It is intended that such
modifications to the peptide of the present invention which do not
result in a decrease in biological activity are within the scope of
the present invention.
[0264] Any combination of the above embodiments may be used
together in order to increase effectiveness of the peptide variants
to inhibit the function of the
TCR.alpha..beta./CD3.epsilon..delta./CD3.epsilon..gamma./.zeta..zeta.
receptor complex. The most effective inhibitory peptides and
derivatives thereof may be identified by typical screening assay
procedures for evaluation of inhibition of T cell activation (Drug
Discovery And Evaluation. Pharmacological Assays, Hans G, Vogel
(ed.) Springer Berlin Heidelberg, 2007).
V. Peptide-Based Inhibitors
[0265] Although it is not necessary to understand the mechanism of
an invention, it is believed that inhibition of a T cell receptor
(TCR) signaling can be achieved by binding of a peptide-based
inhibitor of the present invention to the transmembrane (TM) domain
of the CD3.epsilon., .delta. and TCR.zeta. chains, thus
substituting the TCR.alpha. subunit and abolishing the interactions
between the TM domains of the TCR.alpha. and the CD3.epsilon.,
.delta. and TCR.zeta. chains. One possible result is the inhibition
of TCR-mediated transmembrane signaling because antigen binding to
the extracellular domains of the disulfide-linked TCR.alpha..beta.
antigen recognition subunits does not lead to clustering (i.e., for
example, oligomerization) of CD3.epsilon., .delta. and TCR.zeta.
chains. It is further believed that this clustering induces the
phosphorylation of tyrosine residues in the intracellular ITAM
domains of these signaling subunits and initiates downstream
signaling.
[0266] The TM domains of the TCR.alpha., CD3.epsilon., .delta. and
TCR.zeta. chains comprise hydrophobic sequences that may adopt a
stable alpha-helical structure within a T cell membrane lipid
bilayer. It is believed that electrostatic interactions between
these TM domains maintain the integrity of the
TCR.alpha..beta./CD3.delta..epsilon./CD3.epsilon..gamma./.zeta..zeta.
receptor complex and are provided by the interactions between the
positively charged amino acid residues in the TM regions of the
TCR.alpha..beta. chains and the negatively charged amino acid
residues in the TM domains of .zeta..zeta. homodimer,
CD3.epsilon..gamma., and CD3.delta..epsilon. heterodimers.
[0267] As disclosed in (WO 96/22306; WO 97/47644; US Pat. Appl.
20050070478), the simplest and the most selective and effective
peptide inhibitor would be a synthetic peptide corresponding to the
TM domain of TCR.alpha. subunit. However, as disclosed in (US Pat.
Appl. 20090075899) and incorporated herein by reference in its
entirety, peptide inhibitor sequence, alone, is not the only
relevant consideration. In one embodiment, a peptide inhibitor
targeted to the transmembrane interactions should be optimized for
cell membrane binding. In one embodiment, a peptide inhibitor
should be optimized for membrane insertion, thereby attaining a
close spatial proximity and/or proper orientation to an interacting
partner (i.e., for example, the TM domains of the .zeta..zeta.
homodimer and CD368 heterodimer). In one embodiment, a peptide
inhibitor should be optimized for binding effectiveness to an
interacting partner.
[0268] Although it is not necessary to understand the mechanism of
an invention, it is believed that TCR peptide inhibitors disclosed
in (WO 96/22306, WO 97/47644; US Pat. Appl. 20050070478; US Pat.
Appl. 20070185025: WO 2006077601) comprising the wild type TM
domain of TCR.alpha., filoviral peptides or HIV gp41 fusion peptide
(FP) are not optimized for each of the above three factors. Other
embodiments, however, are contemplated by the present invention by
using extracellularly administered synthetic peptides with primary
amino acid sequences of viral origin of the present invention which
are optimized for at least one of the above three considerations.
Thus, the 30-40% inhibition of T cell activation observed for the
TCR core peptide (CP), filoviral peptides and HIV gp41 FP (WO
96/22306; WO 97/47644; US Pat. Appl. 20050070478; US Pat. Appl.
20070185025; WO 2006077601), can be significantly improved in terms
of efficiency by rational design of the peptide-based inhibitors of
the present invention. For example, the inhibition activity the TCR
CP has been reported to increase from 30 to 80% by lipidation of
the relevant peptide inhibitors.
[0269] In summary, the present invention contemplates optimizing
the effectiveness and selectivity of peptide inhibitors for
TCR-mediated signaling, by adhering to at least one of these
guidelines: 1) ability to effectively bind to the platelet plasma
membrane and insert into the membrane (i.e., for example,
increasing hydrophobicity); 2) ability to adopt helical
conformation upon membrane binding and penetration (i.e., for
example, increasing intrinsic helicity); 3) ability to selectively
and effectively bind to the TM domain of the .zeta..zeta. homodimer
and CD368 heterodimer, thus effectively competing with the
TCR.alpha. subunit for the binding to these signaling subunit
(i.e., for example, by increasing stable .alpha.-helixes). These
guidelines are described in (US Pat. Appl. 20090075899) and
incorporated herein by reference in its entirety.
[0270] These guidelines were used to develop a method of rational
designing of the peptides of the present invention in order to
increase effectiveness of the peptides in inhibition of function of
the
TCR.alpha..beta./CD3.epsilon..delta./CD3.epsilon..gamma./.zeta..zeta.
receptor complex.
1. Hydrophobicity
[0271] The hydrophobicity (or lipophilicity) of peptides and
peptide analogues may be increased by i) inserting hydrophobic
regions; ii) improving the ability of a peptide-based inhibitors to
bind the membrane; and/or iii) improving the ability of a
peptide-based inhibitor to insert into a membrane. In one
embodiment, hydrophobic regions may be inserted into a peptide
inhibitor sequence by using lipophilic groups including, but not
limited to, myristoylate-, 1-amino-glucose succinate,
2-aminododecanoate, or Gly-Tris-palmitate, -dipalmitate or
-tripalmitate, coupled to the N- and/or C-termini of a peptide. In
one embodiment, the membrane binding/insertion ability of a peptide
inhibitor may be improved by using highly positively charged
poly-Lys or poly-Arg sequences coupled to an N- and/or C-terminus.
A general formula summarizing many suggested inhibitory peptides
and/or compositions is presented that incorporates both approaches
that are expected to increase the effectiveness of the peptides to
inhibit the function of TCR. See, FIGS. 7, 8, 9 and 10.
[0272] Lipid-binding activity of the test peptide-based inhibitors
can be predicted using ProtParam.TM. software
(us.expasy.org/tools/protparam.html) and experimentally evaluated
by different techniques such as, for example, surface plasmon
resonance (SPR) or sucrose-loaded vesicle binding assay. Based on
the obtained results, a peptide-based inhibitor with optimal
membrane-binding activity can be chosen. For example, "Grand
Average Of Hydropathy" (GRAVY) scores for peptides can be obtained
using ProtParam.TM., in which a score >-0.4 (=mean score for
cytosolic proteins) indicates the probability for membrane
association (i.e., for example, the higher the score, the greater
the probability for membrane association).
2. Helicity
[0273] As discussed above, the primary sequence of the parent
inhibitory peptides of the present invention (See FIG. 5 and TABLE
2), can be modified to improve the ability of various peptide-based
inhibitors contemplated herein to adopt helical conformation upon
membrane binding and penetration. Overall protein folding may be
specified by hydrophobic-polar residue patterning, whereas the
bundle oligomerization state, detailed main-chain conformation, and
interior side-chain rotamers may be engineered by computational
enumerations of packing in alternate backbone structures.
Main-chain flexibility is incorporated through an algebraic
parameterization of the backbone (Harbury et al. Science 1998;
282:1462-7).
[0274] Peptide helicity of the designed primary sequences of
various peptide-based inhibitors of the invention contemplated
herein can be first evaluated computationally using secondary
structure prediction programs. (i.e., for example, Expasy
Proteomics Tools; expasy.org/tools). The most promising inhibitors
can be measured experimentally for intrinsic and/or induced
helicity using circular dichroism (CD) spectroscopy. Circular
dicroism spectroscopy is used to analyze the secondary structure of
a protein and/or peptide. Specifically, CD spectroscopy measures
differences in the absorption of left-handed polarized light versus
right-handed polarized light which arise due to structural
asymmetry. The absence of regular structure results in zero CD
intensity, while an ordered structure results in a spectrum which
can contain both positive and negative signals. .alpha.-helix,
.beta.-sheet, and random coil structures each give rise to a
characteristic shape and magnitude of CD spectrum. The approximate
fraction of each secondary structure type that is present in any
peptide or protein can thus be determined by analyzing its far-UV
CD spectrum as a sum of fractional multiples of such reference
spectra for each structural type. Like all spectroscopic
techniques, the CD signal reflects an average of the entire
molecular population. Thus, while CD can determine that a peptide
or protein contains about 50% .alpha.-helix, it cannot determine
which specific residues are involved in the .alpha.-helical
portion. Based on the obtained results, a peptide-based inhibitor
optimized with the predicted and/or observed, intrinsic and/or
induced optimal helicity can be chosen.
[0275] Alternatively, secondary structure prediction programs (for
example, expasy.org/tools/) may be used to accurately predict the
peptide helicity based on primary sequence of the computationally
designed peptide-based inhibitors. A few of the available programs
include, but are not limited to: a) AGADIR--An algorithm to predict
the helical content of peptides; b) APSSP--Advanced Protein
Secondary Structure Prediction Server; c) GOR (Garnier et al.
Methods Enzymol 1996; 266:540-53); d) HNN--Hierarchical Neural
Network method (Guermneur et al. Bioinformatics 1999; 15:413-21);
e) Jpred--A consensus method for protein secondary structure
prediction at University of Dundee; f) JUFO--Protein secondary
structure prediction from sequence (neural network); g)
nnPredict--University of California at San Francisco (UCSF); h)
Porter--University College Dublin; i) PredictProtein--PHDsec,
PHDacc, PHDhtm, PHDtopology, PHDthreader, MaxHom, EvalSec from
Columbia University; j) Prof--Cascaded Multiple Classifiers for
Secondary Structure Prediction; k) PSA--BioMolecular Engineering
Research Center (BMERC)/Boston; 1) PSIpred--Various protein
structure prediction methods at Brunel University; m)
SOPMA--Geourjon and Deleage, 1995; n) Sspro--Secondary structure
prediction using bidirectional recurrent neural networks at
University of California; and o) DLP--Domain linker prediction at
RIKEN.
3. .alpha.-Helix Stability
[0276] Although it is not necessary to understand the mechanism of
an invention, it is believed that the TM domains of the TCR.alpha.,
CD3.delta., CD3.epsilon. and .zeta. chains represent stable
.alpha.-helixes and, thus, the interactions can be presented using
helix-wheel diagrams. See, FIGS. 2, 3, 5, 6A, 6B, and 6C. As
described in (US Pat. Appl. 20090075899) and incorporated herein by
reference in its entirety, these diagrams are based on the primary
peptide/protein sequence and can be created using commercially and
publicly available programs (i.e., including, but not limited to,
Antheprot v.6.0; antheprotpbil.ibcp.fr; or Helical Wheel Custom
Images and Interactive Java Applets;
cti.itc.virginia.edu/.about.cmg/Demo/wheel/wheelApp.html and
(net/helical.htm). These diagrams can be used for evaluation of
close proximity and/or proper orientation of positively charged
amino acid residue(s) of the peptide or peptide analogue of the
present invention towards an interacting partner (i.e., for
example, negatively charged TM residues of the .zeta..zeta.
homodimer and CD3.delta..epsilon. heterodimer).
[0277] The electrostatic interactions between the positively
charged amino acid residues in the TM regions of the TCR.alpha.
chain and the negatively charged amino acid residues in the TM
domains of .zeta..zeta. homodimer and CD3.delta..epsilon.
heterodimer stabilize the association of these respective subunits,
thereby playing a role in antigen-induced TCR-mediated T cell
activation. Some embodiments of peptide-based inhibitors
contemplated by the present invention aim to interrupt these
interactions and replace the TCR.alpha. subunit (See FIGS. 3, 6A,
6B, and 6C). In one embodiment, peptide-based inhibitors can be
computationally designed to increase their competitiveness with the
TCR.alpha. subunit. In one embodiment, competitiveness may be
increased by using a conservative amino acid substitution of
arginine for lysine. In another embodiment, competitiveness may be
increased by inserting a positively charged amino acid residue
(i.e., for example, arginine and/or lysine). In one embodiment, the
insertion and/or substitution is located within an .alpha.-helix of
the peptide-based inhibitors of the invention, thereby increasing
the binding activity to the TM domains of .zeta..zeta. homodimer
and CD3.delta..epsilon. heterodimer and enhancing the effectiveness
of the peptides to inhibit the function of TCR.
4. Peptide-Based Inhibitor Sequence Listing
[0278] A list of the sequences of the peptides and peptide
analogues shown below includes, but is not limited to, the
peptide-based inhibitors predicted to be effective in inhibiting
the TCR signaling mechanism. See, Table 2.
[0279] Accordingly, it is intended that the present invention
includes within its scope peptides which include additional amino
acids to the "core" sequence of the peptide of the present
invention and which affect the transmembrane interactions between
the TCR .alpha. subunit and CD3.epsilon., .delta. and .zeta.
subunits.
TABLE-US-00002 TABLE 2 Exemplary Peptide-Based
TCR.alpha..beta./CD3.delta..epsilon./CD3.gamma..epsilon./.zeta..zeta.
Complex Sequences Sequence (the "core" sequence of SEQ the peptide
of the present ID ## R.sub.1.sup.a invention is underlined)
R.sub.2.sup.b NO Class I 1 -- MYKTPTLKYFGGFNFSQIL (parent) - 6
(SARS-CoV FP) 2 -- MYKTPTLKYFG - 10 (SARS-CoV FP core peptide) 3 --
MYKIPTLKYFG - 11 4 -- MYKILTLKYFG - 12 5 -- GYKILTLKYFG - 13 6 --
GYRTPTLKVFG - 14 7 -- GFRIPLLKYFG - 15 8 --
(GFRIPLLKYFG).sub.2.sup.c - 16 9 LA GYRTPTLKVFG - 14 10 Myr
GYRTPTLKVFG - 14 11 - GYRTPTLKVFG + 14 12 Myr GYRTPTLKVFG + 14 13
-- GYRTPTLKVFGGFNFSQIL - 17 14 -- (GYRTPTLKVFGGFNFSQIL).sub.2 - 18
15 -- (C*GYRTPTLKVFGGFNFSQIL).sub.2.sup.d - 19 16 --
(C*GYRTPTLKVFGGFNFSQILC*).sub.2 - 20 17 -- GYRTPTLKVFGGFNFSQIL - 17
18 -- GYKTPTLKYFGGFNFSQIL - 21 19 LA GYKTPTLKYFGGFNFSQIL - 21 20 --
GYKTPTLKYFGGFNFSQIL + 21 21 Myr GYKTPTLKYFGGFNFSQIL - 21 22 Myr
GYKTPTLKYFGGFNFSQIL + 21 23 -- KKKRGYKTPTLKYFGGFNFSQILKR - 22 24 --
(KKKRGYKTPTLKYFGGFNFSQILKR).sub.2 - 23 25 -- KKRGYKTPTLKVFGKR - 24
26 -- (KKRGYKTPTLKVFGKR).sub.2 - 25 Class 2 27 --
AVPVAVWLVSALAMGAGVAGGITGSMSL - 26 ASGKSLLHEVDKD (parent) (HTLV-1
gp21.sup.313-353) 28 -- LASGKSLLHEVDKD - 27 (HTLV-1
gp21.sup.313-353 core peptide) 29 -- ATDGQLNHRVEKVEKKLT (parent) -
28 (HVA Tio.sup.225-242) 30 -- LNHRVEKVEKKLT - 29 (HVA
Tio.sup.225-242 core peptide) 31 -- ANERNIVKDLKRLENKIN (parent) - 7
(HVS Tip.sup.211-228) 32 -- IVKDLKRLENKIN - 30 (HVS Tip.sup.211-228
core peptide) 33 -- LASGKSLLHVEKKD - 31 34 -- LASGKRVEHEVDKD - 32
35 -- LASGKDLKHVEKKD - 33 36 -- LASGKSLLHLENKD - 34 37 --
ATDGQLNHRVEKLENKLT - 35 38 -- ANERNIVKRVERLENKIN - 36 39 Myr
LASGKDLKHVEKKD - 33 40 -- LASGKDLKHVEKKD + 33 41 Myr LASGKDLKHVEKKD
+ 33 42 -- (LASGKDLKHVEKKD).sub.2 - 79 43 --
(C*LASGKDLKHVEKKD).sub.2 - 80 44 -- LNHRVEKLENKLT - 81 45 --
KKRLNHRVEKLENKLTKR - 37 46 -- KKRLNHRVEKLENKLTKR + 37 47 LA
KKRLNHRVEKLENKLTKR - 37 48 Myr KKRLNHRVEKLENKLTKR - 37 49 --
RNIVKRVEKVEKKLT - 38 50 -- (RNIVKRVEKVEKKLT).sub.2 - 39 Class III
51 -- GTFTWTLSDSEGKDTPGGYCLTRWMLIEA - 8 ELKCFGNTAV (parent) (LASV
FP gp2.sup.260-298) 52 -- LTRWMLIEAELKCFG - 40 (LASV FP core
peptide) 53 -- GTFTWTLSDSSGVENPGGYCLTKWMILAA - 41 ELKCFGNTAV
(parent) (LCMV FP gp2.sup.266-304) 54 -- LTKWMILAAELKCFG - 42 (LCMV
FP core peptide) 55 -- GLFTWTLSDSEGNDMPGGYCLTRSMLIGL - 43
DLKCFGNTAI (parent) (MOPV FP gp2.sup.258-296) 56 -- LTRSMLIGLDLKCFG
- 44 (MOPV FP core peptide) 57 -- AFFSWSLTDPLGNEAPGGYCLEKWMLVAS -
45 ELKCFGNTAI (parent) (TACV FP gp2.sup.262-300) 58 --
LEKWMLVASELKCFG - 46 (TACV FP core peptide) 59 -- LQNRRGLDLLFLKEGGL
- 47 (parent) (CKS-17) 60 -- ILNRKAIDFLLRRWGGT - 48 (parent) (SEBOV
gp2.sup.584-600) 61 -- ILNRKAIDFLLQRWGGT - 49 (parent) (ZEBOV
gp2.sup.584-600) 62 -- LINRHAIDFLLTRWGGT - 50 (parent) (MARV
gp2.sup.585-601) 63 -- LQNRRGLDLLFLKEGGL - 47 (parent) (Fr-MLV Env
gp.sup.548-564) 64 -- VINDTSFVECIPPPQSRPAWNLWNNRRKT - 51 FSFL
(parent) (HHV-6 U24.sup.28-60) 65 -- QSRPAWNLWNNRRKT - 52 (HHV-6
U24.sup.28-60 core peptide) 66 -- GYCLTRWMLIEAELKCFGNTAV - 53 67 --
(GYC*LTRWMLIEAELKCFGNTAV).sub.2 - 54 68 --
(GYGLTRWMLIEAELKCFGNTAV).sub.2 - 55 69 LA GYCLTRWMLIEAELKCFGNTAV -
56 70 -- GYCLTRWMLIEAELKCFGNTAV + 56 71 Myr GYCLTRWMLIEAELKCFGNTAV
- 56 72 -- KKRGYCLTRWMLIEAELKCFGNTAVKR - 57 73 -- LQNRKAIDLWNNKEGG
+ 58 74 -- (LQNRKAIDLWNNKEGG).sub.2 - 59 75 -- LQNRKAIDLWNNKEGG +
58 76 Myr LQNRKAIDLWNNKEGG - 58 77 -- ILNRRGLDGLDLKEGG - 60 78 --
(ILNRRGLDGLDLKEGG).sub.2 - 61 79 -- KKRILNRRGLDGLDLKEGGKR - 62 80
-- CLTKPAWNLLFLKRKTF - 63 Combinatorial sequences 81 --
LQNRDLKRLLFLKRKT - 9 82 -- (LQNRDLKRLLFLKRKT).sub.2 - 64 83 LA
LQNRDLKRLLFLKRKT - 9 84 -- LQNRDLKRLLFLKRKT + 9 85 Myr
LQNRDLKRLLFLKRKT - 9 86 -- KKLQNRDLKRLLFLKRKTKR - 65 87 --
KKLQNRDLKRLLFLKRKTKR + 65 88 -- GQLNKTPTLKEGGL - 66 89 --
GYCLTRRGLKEVDKEGG - 67 90 -- (GYCLTRRGLKEVDKEGG).sub.2 - 68 91 --
(GYC*LTRRGLKEVDKEGG).sub.2 - 69 92 Myr GYCLTRRGLKEVDKEGG - 70 93 --
GYCLTRRGLKEVDKEGG + 70 94 -- KKGYCLTRRGLKEVDKEGGKR - 71 95 LA
KKGYCLTRRGLKEVDKEGGKR - 71 96 -- KKGYCLTRRGLKEVDKEGGKR + 71 97 LA
KKGYCLTRRGLKEVDKEGGKR + 71 98 -- IPPPQSRTPTLKVFGG - 72 99 --
(IPPPQSRTPTLKVFGG).sub.2 - 73 100 -- KKRIPPPQSRTPTLKVFGGKR - 74
.sup.aN-terminal group: LA, lipoamino acid, 2-aminododecanoate;
Myr,
myristoylate. .sup.bC-terminal group: Gly-Tris-tripalmitate.
.sup.cCyclic peptide. .sup.dDisulfide-linked dimer (or
disulfide-linked cyclic dimer). .sup.*Cys involved in disulfide
bond formation.
Abbreviations
[0280] TCR, T cell receptor; CP, core peptide, HIV, human
immunodeficiency virus; FP, fusion peptide/protein; gp,
glycoprotein; TMD, transmembrane domain; CKS-17, a synthetic
retroviral envelope heptadecapeptide; Fr-MLV, Friend murine
leukemia virus; gp, glycoprotein; HHV-6 U24, human herpesvirus 6
U24 protein; HTLV-1, human T lymphotropic virus type 1; HVA,
herpesvirus ateles; HVS, herpesvirus saimiri; ITAM, immunoreceptor
tyrosine-based activation motif; LASV, Lassa virus; LCMV,
lymphocytic choriomeningitis virus; MARV, Marburg virus; MOPV,
Mopeia virus; SARS-CoV, severe acute respiratory syndrome
coronavirus; SEBOV, Sudan Ebola virus; TACV, Tacaribe virus; Tip,
tyrosine kinase interacting protein; Tio, two-in-one protein; TMD,
transmembrane domain; ZEBOV, Zaire Ebola virus.
5. Peptide Variant Consensus Sequences
[0281] Based upon the specific sequences contemplated in Table 2,
the following consensus sequences may be constructed:
[0282] SEQ ID NO: 1:
G-Y-X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub-
.9, wherein X.sub.1 and X.sub.6 are selected from the group
consisting of R. K or H; X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are
selected from the group consisting of L, I, T or P; X.sub.7 is
selected from the group consisting of V or Y; X.sub.8 consists of A
or F or nothing; and X.sub.9 consists of G or nothing.
[0283] SEQ ID NO: 2:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-L-X-L-X-X.sub.9-E-X.sub.1-
0-X.sub.11-X.sub.12-X.sub.13 wherein X.sub.1 consists of R, G, I, L
or nothing; X.sub.2 consists of N, Q, A or nothing; X.sub.3
consists of L, I, S or nothing; X.sub.4 consists of V, N, G or
nothing; X.sub.5, X.sub.8, and X.sub.11 are selected from the group
consisting of R, K or H; X.sub.6 consists of D, R, S or nothing,
X.sub.7 consists of K, E, L or nothing, X.sub.9 consists of L, V, E
or nothing; X.sub.10 consists of N, K, D or nothing; X.sub.12
consists of I, L, D or nothing; and X.sub.13 consists of N, T or
nothing.
[0284] SEQ ID NO: 3:
L-N-X.sub.1-X.sub.2-X.sub.3-L-X.sub.4-X.sub.5-L-X.sub.6-L-X.sub.7-X.sub.8-
-G-G-X.sub.9 wherein X.sub.1 and X.sub.7 are selected from the
group consisting of R, K or H; X.sub.2 consists of S, R, K, H, P or
W; X.sub.3 consists of M, G or A; X.sub.4 consists of L, I, V, N or
D; X.sub.5 consists of L, I, F, T, E, A or G; X.sub.6 consists of
E, Q, D, L, F, N or I, X.sub.8 consists of Q, C, E, W or R, and
X.sub.9 consists of L, I, F, T, N or nothing.
[0285] SEQ ID NO: 4:
L-Q-N-X.sub.1-X.sub.2-L-X.sub.3-X.sub.4-X.sub.5-X.sub.6-L-X.sub.7-L-X.sub-
.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12 wherein X.sub.1, X.sub.4 and
X.sub.8 are selected from the group consisting of R, K or H;
X.sub.2 consists of D, R or S; X.sub.3 consists of E, K or L;
X.sub.5 and X.sub.7 consist of L, I, or T; X.sub.6 consists of L,
I, or P; X.sub.9 consists of Q, C, E, W or R, X.sub.10 consists of
K, G, F, L, I or nothing; X.sub.11 consists of T, G or nothing; and
X.sub.12 consists of F, L, I, T, N or nothing.
[0286] SEQ ID NO: 5:
L-Q-N-X.sub.1-X.sub.2-X.sub.3-X.sub.4-L-X.sub.5-X.sub.6-L-X.sub.7-X.sub.8-
-X.sub.9-X.sub.10-X.sub.11-X.sub.12 wherein X.sub.1, X.sub.5 and
X.sub.8 are selected from the group consisting of R, K or H;
X.sub.2 and X.sub.4 consist of L, I, or T; X.sub.3 consists of L,
I, or P; X.sub.6 consists of D, R or S; X.sub.7 consists of E, K or
L; X.sub.9 consists of Q, C, E, W or R, X.sub.10 consists of K, G,
F, L, I or nothing; X.sub.11 consists of T, G or nothing; and
X.sub.12 consists of F, L, I, T, N or nothing.
VI. Therapeutic Applications of TCR Peptide Variant Inhibitors of
Viral Origin
[0287] The invention further provides clinically therapeutic
methods of intervening and modulating TCR function comprising using
an agent selected from the group of agents or compositions of the
present invention that block/inhibit/prevent/disrupt interactions
between the TCR.alpha. chain and the homodimeric .zeta..zeta. and
heterodimeric CD3.delta..epsilon. subunits of TCR.
[0288] Targeting MIRRs including TCR; HIV therapy, and
high-throughput screening methods for screening and optimizing the
effective peptide variant TCR inhibitors of the present invention
that block/inhibit/prevent/disrupt interactions between the
TCR.alpha. chain and the homodimeric and heterodimeric
CD3.delta..epsilon. subunits of TCR are described in (US Pat. Appl.
20090075899) and incorporated herein by reference in its entirety.
See also FIGS. 1A, 1B, 1C, 1D, 2, and 3.
[0289] Various therapeutic applications of TCR inhibitors are
described in (U.S. Pat. No. 6,057,294; US Pat. Appl. 20050070478,
WO 96/22306; WO 97/47644; US Pat. Appl. 20070185025; WO 2006077601)
and incorporated herein by reference in their entirety.
[0290] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
EXAMPLES
[0291] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention.
[0292] The following non-limiting Examples are put forth so as to
provide those of ordinary skill in the art with illustrative
embodiments as to how the compounds, compositions, articles,
devices, and/or methods claimed herein are made and evaluated. The
Examples are intended to be purely exemplary of the invention and
are not intended to limit the scope of what the inventor regard as
his invention. Efforts have been made to ensure accuracy with
respect to numbers (e.g., amounts, temperature, etc.) but some
errors and deviations should be accounted for. Unless indicated
otherwise, parts are parts by weight, temperature is in .degree.
C., or is at ambient temperature, and pressure is at or near
atmospheric.
Example 1: Synthesis of Peptides
[0293] This example demonstrates one embodiment of a synthesized
SARS-CoV fusion protein-related peptide.
[0294] The first step is to synthesize the short hydrophobic
peptide corresponding to the SARS-CoV fusion peptide sequence.
Although it is not necessary to understand the mechanism of an
invention, it is believed that this peptide affects T cell receptor
assembly and may interact with the .zeta..zeta. homodimer and
CD3.delta..epsilon. heterodimer in a competitive fashion.
[0295] The synthesis of peptides may involve the use of protecting
groups. Peptides can be synthesized by linking an amino group to a
carboxyl group that has been activated by reaction with a coupling
agent, such as dicyclohexylcarbodiimide (DCC). The attack of a free
amino group on the activated carboxyl leads to the formation of a
peptide bond and the release of dicyclohexylurea. It can be
necessary to protect potentially reactive groups other than the
amino and carboxyl groups intended to react. For example, the
.alpha.-amino group of the component containing the activated
carboxyl group can be blocked with a tertbutyloxycarbonyl group.
This protecting group can be subsequently removed by exposing the
peptide to dilute acid, which leaves peptide bonds intact.
[0296] In one embodiment, the amino acid sequence of a competitive
peptide comprises
NH.sub.2-Met-Tyr-Lys-Thr-Pro-Thr-Leu-Lys-Tyr-Phe-Gly-Gly-Phe-As-
n-Phe-Ser-Gln-Ile-Leu-OH (i.e., MYKTPTLKYFGGFNFSQIL (SEQ ID NO:
6)), hereafter referred to as "SARS CoV FP". In another embodiment,
the amino acid sequence of a competitive peptide comprises
NH.sub.2-Met-Tyr-Ala-Thr-Pro-Thr-Leu-Ala-Tyr-Phe-Gly-Gly-Phe-Asn-Phe-Ser--
Gln-Ile-Leu-OH (i.e., MYATPTLAYFGGFNFSQIL) (SEQ ID NO: 75))
wherein, Lys.sub.3 and Lys.sub.5 of SARS CoV FP substituted with
Ala.sub.3 and Ala.sub.8, hereafter referred to as "SARS CoV
FP-AA".
[0297] Although it is not necessary to understand the mechanism of
an invention, it is believed that the positively charged Lys.sub.3
and Lys.sub.8 in SARS CoV FP form a salt bridge to an aspartic acid
residues in the transmembrane (TM) domains of the of the
.zeta..zeta. homodimer and CD3.delta..epsilon. heterodimer (A. B.
Sigalov. PLoS Pathog 2009; 5: e1000404) (See also 5 and 6A). Thus,
SARS CoV FP-AA may be considered a "control peptide" because of the
Ala.sub.3 and Alas substitutions.
[0298] Unprotected peptides can be purchased from specialized
companies (i.e., Sigma-Genosys, Woodlands, Tex., USA) with greater
than 95% purity as assessed by HPLC. Peptide molecular mass can be
checked by matrix-assisted laser desorption ionization mass
spectrometry.
Example 2: Solubility
[0299] This example demonstrates that the hydrophobic properties of
SARS CoV FP peptides and other peptides and compositions of the
present invention may be overcome without risking cell
toxicity.
[0300] The SARS CoV FP and SARS CoV FP-AA peptides can be noted to
be hydrophobic and insoluble in aqueous solutions. A variety of
solvents and carriers can be tested to improve their solubility.
Solvents and/or carriers that improve solubility of CP and CP-A
include, but not limited to, ethanol, dimethylsulphoxide (DMSO),
dimethyl formamide (DMF), and trifluoracetic acid (TFA). When using
DMSO as a solvent, the final concentration used in the platelet
function experiments can range from 0.063%-0.250%. DMSO
concentrations greater than 1% is believed to be toxic to cells.
Stock solutions of SARS CoV FP and SARS CoV FP-AA can be prepared
in DMSO and used at a 1:2000, 1:1000, or 1:400 dilution.
Example 3: Effect of T Cell Receptor Inhibitory Peptides of Viral
Origin on Antigen-Stimulated Proliferation on Rat Primed Lymph Node
Cells (PLNC) and T Cell Lines
A. Cells
[0301] The following cell lines can be used as described in (US
Pat. Appl. 20050070478) and incorporated herein by reference:
2B4.11, a murine T cell hybridoma that expresses a complete antigen
receptor on the cell surface and produces IL-2 following antigen
recognition (cytochrome-c); an interleukin-2 (IL-2) dependent T
cell line (CTLL) for conventional biological IL-2 assays; and the
B-cell hybridoma cell line LK 35.2 (LK, I-E.sup.k bearing) which
acts as the antigen presenting cell. The hybridomas is grown in T
cell medium (RPMI-1640 media containing 10% fetal calf serum (FCS),
gentamycin (80 .mu.g/ml), glutamine (2 mM) and mercaptoethanol
(0.002%)). The African green monkey kidney fibroblast cell line
(COS) is grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% FCS.
B. Antigen Presentation Assay
[0302] (Samelson et al. J Immunol 1987; 139:2708-14; US Pat. Appl.
20050070478)
[0303] The mouse T cell 2B4.11 hybridoma (2.times.10.sup.4) is
cultured in microtitre wells with LK35.2 antigen presenting B cells
(2.times.10.sup.4) and 50 .mu.M pigeon cytochrome-c. After 16 hr 50
microlitres of assay supernatant is removed and assayed for the
presence of IL-2. Serial twofold dilutions of the supernatant in
media are cultured with the IL-2 dependent T cell line CTLL. After
16 hr the CTLL cells are pulsed with .sup.3H-thymidine for 4 hr and
IL-2 measurements (IU/ml) determined. This assay can be performed
demonstrating that the "TCR inhibitor" is effective in inhibiting
TCR-mediated cell activation.
C. Primed Lymph Node Cells (PLNC)
[0304] Male Wistar rats are injected intradermally at the base of
the tail with 1 mg of heat-killed Mycobacterium tuberculosis (MTB)
are suspended in 0.2 ml of squalane. When acute arthritis is well
developed, after 10 to 16 days, rats are killed and the swollen
popliteal lymph nodes are removed and a single cell suspension is
made by pressing the tissue through a fine sieve under aseptic
conditions. Cells are washed in complete medium, resuspended and
counted. Approximately 3.5.times.10.sup.8 viable cells can be
obtained from two rats. The medium is RPMI 1640 supplemented with
25 mM Hepes, penicillin (100 .mu.g/ml), streptomycin (80 .mu.g/ml),
2.5.times.10.sup.-5 M 2-mercaptoethanol and 2% pooled normal rat
serum. The cells are pipetted into the wells of flat-bottom, 96
well microtitre plates at 2.times.10.sup.5/well and a suspension of
MTB is added to a final concentration of 100 .mu.g/ml. "TCR
inhibitors" or "control" peptides of the invention are delivered to
the wells in 20 .mu.l volume giving final concentrations of 1-100
.mu.g/ml peptides (or 1-100 .mu.M) and 0.01% acetic acid (or 0.1%
dimethylsulfoxide, DMSO), and a total of 200 .mu.l per well. The
plates are incubated at 37.degree. C. in a humidified incubator at
5% CO.sub.2 for 3 days and then are pulsed with 1 .mu.Ci per well
of .sup.3H-thymidine in 25 ml of medium. After a further overnight
incubation, the cultures are harvested using an automated cell
harvester, and counted in a 3-scintillation spectrometer.
D. T Cell Lines
[0305] (Sedgwick et al. J Immunol Methods 1989; 121:185-96; US Pat.
Appl. 20050070478)
[0306] PLNCs from MTB-immunised rats are cultured in 75.sup.2
culture flasks at 5.times.10.sup.6 per ml in a total of 50 ml
containing 100-.mu.g/ml MTB. After three days the cells are spun
down and resuspended in 2 ml medium in a 15 ml centrifuge tube and
are underlayered with 3 ml of Ficoll diatrizoate (9.9% Ficoll 400;
9.6% sodium diatrizoate), and centrifuged at 800 g for 20 minutes.
The T cell blasts are recovered from the interface, washed twice
and resuspended at 2.times.10.sup.5 per ml in medium supplemented
with 10% FCS and 15% con A-stimulated spleen cell supernatant, as a
source of IL-2. After four days culture in the rest phase,
2.times.10.sup.5 T cells per ml are restimulated with antigen and
10.sup.7 syngeneic rat thymocytes per ml to act as antigen
presenting cells. The latter are inactivated by incubation with 25
.mu.g/ml mitomycin C for 20 minutes at 37.degree. C. and carefully
washed three times. Cultures are in 75 cm.sup.2 flasks containing
50 ml and the antigen, MTB, is added at 100 .mu.g/ml. Flasks are
stood up vertically and cultured for 3 days. Again T cell blasts
are recovered by separation on Ficoll/diatrizoate, and the cycle is
repeated. After 2-4 cycles, the cells are set in 96-well plates at
10.sup.4 T cells/well and 10.sup.6 mitomycin-C-inactivated
thymocytes, in 200 ml medium containing 100 .mu.g/ml MTB and 2% rat
serum. To test the peptides and composition of the invention for
the ability to inhibit antigen-stimulated T lymphocyte
proliferation, additions of 200 .mu.l are made to the wells
containing "TCR inhibitors" or "control" peptides of the invention
in 0.1% acetic acid or 0.1% DMSO. Cultures are incubated for three
days, then .sup.3H-thymidine (1 .mu.Ci in 25 ml medium) is added
and the incubation continues overnight after which it is harvested
and counted in the .beta.-counter.
Example 4: Effect of T Cell Receptor Inhibitory Peptides of Viral
Origin on Adjuvant-Induced Arthritis in Rats
[0307] Method A. Arthritis in rats is induced by a single
intradermal injection of heat killed MTB in 200 .mu.l squalane
(adjuvant) at the base of the tail. To test the peptides and
composition of the invention in adjuvant-induced arthritis (AIA) in
rats, "TCR inhibitors" or "control" peptides of the invention (1-30
mg) are suspended in one millilitre squalane containing 5 mg of
MTB. That is, there is 1 mg MTB and 0.2-6 mg peptide in 0.2 ml of
squalane injected intradermally. At regular intervals for up to 28
days, animals are weighed and their arthritic condition is assessed
by measurement of ankle thickness and rear paw thickness (with a
micrometer) and recording the number of arthritic joints involved.
Rats are housed in standard cages after the initial tail injection
and allowed access to unlimited water and pellet food. Rats
generally develop arthritis 12-14 days after the injection. On day
29, the animals are sacrificed.
[0308] Method B. Three-month old female Lewis rats are raised and
maintained under pathogen-free conditions. To test the effect of
the peptides and composition of the invention on T cell activation
in vivo, AIA is used as a model system. AIA is induced by injecting
50 .mu.l of MTB suspended in incomplete Freund's adjuvant (IFA)
(0.5 mg/ml) at the base of the tail. At the time of AIA induction,
each rat also receives 100 .mu.g of "TCR inhibitors" or "control"
peptides of the invention, or PBS dissolved in 50 .mu.l of IFA and
mixed with MTB/IFA used to induce AIA. The day of AIA induction is
designated as day 0. Disease severity is assessed by direct
observation of all 4 limbs in each animal. A relative score between
0 and 4 is assigned to each limb, based on the degree of joint
inflammation, redness and deformity; thus the maximum possible
score for an individual animal is 16 (WO 2006077601). Arthritis is
also quantified by measuring hind limb diameter with a caliper.
Measurements are taken on the day of the induction of AIA and 26
days later (at the peak of AIA); the results are presented as the
mean.+-.SEM of the difference between the two values for all the
animals in each group. The person who scores the disease should be
blinded to the identity of the groups.
Example 5: Effect of T Cell Receptor Inhibitory Peptides of Viral
Origin on T Cell Proliferation and Cytokine Production
A. Cell Lines and Antigens
(WO 2006077601)
[0309] The CD4.sup.+ T cell clone A2b 21 reacts with the 180-188
epitope of the 65 kDa heat shock protein (HSP65) of M. tuberculosis
(MTB), this epitope is contained in the peptide MTB 176-190. MTB
Strain H37Ra and incomplete Freund's adjuvant (IFA) can be
purchased from Difco (Detroit, Mich., USA). Tuberculin purified
protein derivative (PPD) can be provided by the Statens Serum
institute (Copenhagen, Denmark). PMA, ionomycin, ovalbumin (OVA)
and Concanavalin A (Con A) can be purchased from Sigma (USA).
B. T Cell Proliferation
[0310] T cell proliferation assays are performed using either lymph
node cells (LNC) or the A2b T cell line, which reacts with the MtB
176-190 peptide. Popliteal and inguinal LNC are removed 26 days
after the injection of MTB in IFA, when strong T cell responses to
PPD and MTB 176-190 are detectable. LNC are cultured at a
concentration of 2.times.10.sup.5 cells per well; 5.times.10.sup.4
A2b T cells are stimulated in the presence of irradiated
5.times.10.sup.5 thymic antigen presenting cells (APC) per well.
The cells are plated in quadruplicates in 200 .mu.l round bottom
microtiter wells (Costar Corp., Cambridge, USA), with or without
antigen, in the presence of various concentrations of the "TCR
inhibitors" or "control" peptides of the invention. For some
experiments, the cells are activated with immobilized anti-CD3
antibodies or PMA/ionomycin. Cultures are incubated for 72 hr at
37.degree. C. in a humidified atmosphere of 7.5% CO.sub.2. T cell
responses are detected by the incorporation of
[methyl-3H]-thymidine (Amersham, Buckinghamshire, UK; 1
.mu.Ci/well), added during the last 18 hr of incubation. The
results of T cell proliferation experiments can be presented as the
% of inhibition of the T cell proliferation triggered by the
antigen in the absence of the "TCR inhibitors" or "control"
peptides of the invention.
C. Cytokine Assays
[0311] Supernatants are collected after 72 hr of stimulation, and
rat IL-10 and IFN.gamma. are quantified by enzyme-linked
immunosorbent assay (ELISA) using, for example, Pharmingen's OPTEIA
kit (Pharmingen, San Diego, USA). When needed, cytokine levels can
be expressed as percentage of cytokine inhibition relative to
cytokine levels when no "TCR inhibitors" or "control" peptides of
the invention are present. Otherwise, the cytokines can be shown as
pg/ml. The lower limits of detection for these experiments can be
15 pg/ml for IL-10 and IFN.gamma.. Cytokine amounts are calculated
based on calibration curves constructed using recombinant cytokines
as standards.
INCORPORATION BY REFERENCE
[0312] All of the patents and publications cited herein are hereby
incorporated by reference. Each of the applications and patents
cited in this text, as well as each document or reference cited in
each of the applications and patents (including during the
prosecution of each issued patent; "application cited documents"),
and each of the PCT and foreign applications or patents
corresponding to and/or paragraphing priority from any of these
applications and patents, and each of the documents cited or
referenced in each of the application cited documents, are hereby
expressly incorporated herein by reference. More generally,
documents or references are cited in this text, either in a
Reference List, or in the text itself; and, each of these documents
or references ("herein-cited references"), as well as each document
or reference cited in each of the herein-cited references
(including any manufacturer's specifications, instructions, etc.),
is hereby expressly incorporated herein by reference.
[0313] The references cited herein throughout, to the extent that
they provide exemplary procedural or other details supplementary to
those set forth herein, are all specifically incorporated herein by
reference.
EQUIVALENTS
[0314] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
99111PRTArtificial SequenceSyntheticmisc_feature(3)..(3)Xaa is R, K
or Hmisc_feature(4)..(7)Xaa is L, I, T or Pmisc_feature(8)..(8)Xaa
is R, K or Hmisc_feature(9)..(9)Xaa is V or
Ymisc_feature(10)..(10)Xaa is A or F or
nothingmisc_feature(11)..(11)Xaa is G or nothing 1Gly Tyr Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10215PRTArtificial
SequenceSyntheticmisc_feature(1)..(1)Xaa is R, G, I, L or
nothingmisc_feature(2)..(2)Xaa is N, Q, A or
nothingmisc_feature(3)..(3)Xaa is L, I, S or
nothingmisc_feature(4)..(4)Xaa is V, N, G or
nothingmisc_feature(5)..(5)Xaa is R, K or Hmisc_feature(6)..(6)Xaa
is D, R, S or nothingmisc_feature(8)..(8)Xaa is K, E, L or
nothingmisc_feature(9)..(9)Xaa is R, K or
Hmisc_feature(10)..(10)Xaa is L, V, E or
nothingmisc_feature(12)..(12)Xaa is N, K, D or
nothingmisc_feature(13)..(13)Xaa is R, K or
Hmisc_feature(14)..(14)Xaa is I, L, D or
nothingmisc_feature(15)..(15)Xaa is N, T or nothing 2Xaa Xaa Xaa
Xaa Xaa Xaa Leu Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa1 5 10
15316PRTArtificial SequenceSyntheticmisc_feature(3)..(3)Xaa is R, K
or Hmisc_feature(4)..(4)Xaa is S, R, K, H, P or
Wmisc_feature(5)..(5)Xaa is M, G or Amisc_feature(7)..(7)Xaa is L,
I, V, N or Dmisc_feature(8)..(8)Xaa is L, I, F, T, E, A or
Gmisc_feature(10)..(10)Xaa is E, Q, D, L, F, N or
Imisc_feature(12)..(12)Xaa is R, K or Hmisc_feature(13)..(13)Xaa is
Q, C, E, W or Rmisc_feature(16)..(16)Xaa is L, I, F, T, N or
nothing 3Leu Asn Xaa Xaa Xaa Leu Xaa Xaa Leu Xaa Leu Xaa Xaa Gly
Gly Xaa1 5 10 15417PRTArtificial
SequenceSyntheticmisc_feature(4)..(4)Xaa is R, K or
Hmisc_feature(5)..(5)Xaa is D, R or Smisc_feature(7)..(7)Xaa is E,
K or Lmisc_feature(8)..(8)Xaa is R, K or Hmisc_feature(9)..(9)Xaa
is L, I, or Tmisc_feature(10)..(10)Xaa is L, I, or
Pmisc_feature(11)..(11)Xaa is L, I, or Tmisc_feature(13)..(13)Xaa
is R, K or Hmisc_feature(14)..(14)Xaa is Q, C, E, W or
Rmisc_feature(15)..(15)Xaa is K, G, F, L, I or
nothingmisc_feature(16)..(16)Xaa is T, G or
nothingmisc_feature(17)..(17)Xaa is F, L, I, T, N or nothing 4Leu
Gln Asn Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa1 5 10
15Xaa517PRTArtificial SequenceSyntheticmisc_feature(4)..(4)Xaa is
R, K or Hmisc_feature(5)..(5)Xaa is L, I, or
Tmisc_feature(6)..(6)Xaa is L, I, or Pmisc_feature(7)..(7)Xaa is L,
I, or Tmisc_feature(9)..(9)Xaa is R, K or
Hmisc_feature(10)..(10)Xaa is D, R or Smisc_feature(12)..(12)Xaa is
E, K or Lmisc_feature(13)..(13)Xaa is R, K or
Hmisc_feature(14)..(14)Xaa is Q, C, E, W or
Rmisc_feature(15)..(15)Xaa is K, G, F, L, I or
nothingmisc_feature(16)..(16)Xaa is T, G or
nothingmisc_feature(17)..(17)Xaa is F, L, I, T, N or nothing 5Leu
Gln Asn Xaa Xaa Xaa Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa1 5 10
15Xaa619PRTArtificial SequenceSynthetic 6Met Tyr Lys Thr Pro Thr
Leu Lys Tyr Phe Gly Gly Phe Asn Phe Ser1 5 10 15Gln Ile
Leu718PRTArtificial SequenceSynthetic 7Ala Asn Glu Arg Asn Ile Val
Lys Asp Leu Lys Arg Leu Glu Asn Lys1 5 10 15Ile Asn839PRTArtificial
SequenceSynthetic 8Gly Thr Phe Thr Trp Thr Leu Ser Asp Ser Glu Gly
Lys Asp Thr Pro1 5 10 15Gly Gly Tyr Cys Leu Thr Arg Trp Met Leu Ile
Glu Ala Glu Leu Lys 20 25 30Cys Phe Gly Asn Thr Ala Val
35916PRTArtificial SequenceSynthetic 9Leu Gln Asn Arg Asp Leu Lys
Arg Leu Leu Phe Leu Lys Arg Lys Thr1 5 10 151011PRTArtificial
SequenceSynthetic 10Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly1 5
101111PRTArtificial SequenceSynthetic 11Met Tyr Lys Ile Pro Thr Leu
Lys Tyr Phe Gly1 5 101211PRTArtificial SequenceSynthetic 12Met Tyr
Lys Ile Leu Thr Leu Lys Tyr Phe Gly1 5 101311PRTArtificial
SequenceSynthetic 13Gly Tyr Lys Ile Leu Thr Leu Lys Tyr Phe Gly1 5
101411PRTArtificial SequenceSynthetic 14Gly Tyr Arg Thr Pro Thr Leu
Lys Val Phe Gly1 5 101511PRTArtificial SequenceSynthetic 15Gly Phe
Arg Ile Pro Leu Leu Lys Tyr Phe Gly1 5 101622PRTArtificial
SequenceSynthetic 16Gly Phe Arg Ile Pro Leu Leu Lys Tyr Phe Gly Gly
Phe Arg Ile Pro1 5 10 15Leu Leu Lys Tyr Phe Gly 201719PRTArtificial
SequenceSynthetic 17Gly Tyr Arg Thr Pro Thr Leu Lys Val Phe Gly Gly
Phe Asn Phe Ser1 5 10 15Gln Ile Leu1838PRTArtificial
SequenceSynthetic 18Gly Tyr Arg Thr Pro Thr Leu Lys Val Phe Gly Gly
Phe Asn Phe Ser1 5 10 15Gln Ile Leu Gly Tyr Arg Thr Pro Thr Leu Lys
Val Phe Gly Gly Phe 20 25 30Asn Phe Ser Gln Ile Leu
351940PRTArtificial SequenceSynthetic 19Cys Gly Tyr Arg Thr Pro Thr
Leu Lys Val Phe Gly Gly Phe Asn Phe1 5 10 15Ser Gln Ile Leu Cys Gly
Tyr Arg Thr Pro Thr Leu Lys Val Phe Gly 20 25 30Gly Phe Asn Phe Ser
Gln Ile Leu 35 402042PRTArtificial SequenceSynthetic 20Cys Gly Tyr
Arg Thr Pro Thr Leu Lys Val Phe Gly Gly Phe Asn Phe1 5 10 15Ser Gln
Ile Leu Cys Cys Gly Tyr Arg Thr Pro Thr Leu Lys Val Phe 20 25 30Gly
Gly Phe Asn Phe Ser Gln Ile Leu Cys 35 402119PRTArtificial
SequenceSynthetic 21Gly Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly
Phe Asn Phe Ser1 5 10 15Gln Ile Leu2225PRTArtificial
SequenceSynthetic 22Lys Lys Lys Arg Gly Tyr Lys Thr Pro Thr Leu Lys
Tyr Phe Gly Gly1 5 10 15Phe Asn Phe Ser Gln Ile Leu Lys Arg 20
252350PRTArtificial SequenceSynthetic 23Lys Lys Lys Arg Gly Tyr Lys
Thr Pro Thr Leu Lys Tyr Phe Gly Gly1 5 10 15Phe Asn Phe Ser Gln Ile
Leu Lys Arg Lys Lys Lys Arg Gly Tyr Lys 20 25 30Thr Pro Thr Leu Lys
Tyr Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu 35 40 45Lys Arg
502416PRTArtificial SequenceSynthetic 24Lys Lys Arg Gly Tyr Lys Thr
Pro Thr Leu Lys Val Phe Gly Lys Arg1 5 10 152532PRTArtificial
SequenceSynthetic 25Lys Lys Arg Gly Tyr Lys Thr Pro Thr Leu Lys Val
Phe Gly Lys Arg1 5 10 15Lys Lys Arg Gly Tyr Lys Thr Pro Thr Leu Lys
Val Phe Gly Lys Arg 20 25 302641PRTArtificial SequenceSynthetic
26Ala Val Pro Val Ala Val Trp Leu Val Ser Ala Leu Ala Met Gly Ala1
5 10 15Gly Val Ala Gly Gly Ile Thr Gly Ser Met Ser Leu Ala Ser Gly
Lys 20 25 30Ser Leu Leu His Glu Val Asp Lys Asp 35
402714PRTArtificial SequenceSynthetic 27Leu Ala Ser Gly Lys Ser Leu
Leu His Glu Val Asp Lys Asp1 5 102818PRTArtificial
SequenceSynthetic 28Ala Thr Asp Gly Gln Leu Asn His Arg Val Glu Lys
Val Glu Lys Lys1 5 10 15Leu Thr2913PRTArtificial SequenceSynthetic
29Leu Asn His Arg Val Glu Lys Val Glu Lys Lys Leu Thr1 5
103013PRTArtificial SequenceSynthetic 30Ile Val Lys Asp Leu Lys Arg
Leu Glu Asn Lys Ile Asn1 5 103114PRTArtificial SequenceSynthetic
31Leu Ala Ser Gly Lys Ser Leu Leu His Val Glu Lys Lys Asp1 5
103214PRTArtificial SequenceSynthetic 32Leu Ala Ser Gly Lys Arg Val
Glu His Glu Val Asp Lys Asp1 5 103314PRTArtificial
SequenceSynthetic 33Leu Ala Ser Gly Lys Asp Leu Lys His Val Glu Lys
Lys Asp1 5 103414PRTArtificial SequenceSynthetic 34Leu Ala Ser Gly
Lys Ser Leu Leu His Leu Glu Asn Lys Asp1 5 103518PRTArtificial
SequenceSynthetic 35Ala Thr Asp Gly Gln Leu Asn His Arg Val Glu Lys
Leu Glu Asn Lys1 5 10 15Leu Thr3618PRTArtificial SequenceSynthetic
36Ala Asn Glu Arg Asn Ile Val Lys Arg Val Glu Arg Leu Glu Asn Lys1
5 10 15Ile Asn3718PRTArtificial SequenceSynthetic 37Lys Lys Arg Leu
Asn His Arg Val Glu Lys Leu Glu Asn Lys Leu Thr1 5 10 15Lys
Arg3815PRTArtificial SequenceSynthetic 38Arg Asn Ile Val Lys Arg
Val Glu Lys Val Glu Lys Lys Leu Thr1 5 10 153930PRTArtificial
SequenceSynthetic 39Arg Asn Ile Val Lys Arg Val Glu Lys Val Glu Lys
Lys Leu Thr Arg1 5 10 15Asn Ile Val Lys Arg Val Glu Lys Val Glu Lys
Lys Leu Thr 20 25 304015PRTArtificial SequenceSynthetic 40Leu Thr
Arg Trp Met Leu Ile Glu Ala Glu Leu Lys Cys Phe Gly1 5 10
154139PRTArtificial SequenceSynthetic 41Gly Thr Phe Thr Trp Thr Leu
Ser Asp Ser Ser Gly Val Glu Asn Pro1 5 10 15Gly Gly Tyr Cys Leu Thr
Lys Trp Met Ile Leu Ala Ala Glu Leu Lys 20 25 30Cys Phe Gly Asn Thr
Ala Val 354215PRTArtificial SequenceSynthetic 42Leu Thr Lys Trp Met
Ile Leu Ala Ala Glu Leu Lys Cys Phe Gly1 5 10 154339PRTArtificial
SequenceSynthetic 43Gly Leu Phe Thr Trp Thr Leu Ser Asp Ser Glu Gly
Asn Asp Met Pro1 5 10 15Gly Gly Tyr Cys Leu Thr Arg Ser Met Leu Ile
Gly Leu Asp Leu Lys 20 25 30Cys Phe Gly Asn Thr Ala Ile
354415PRTArtificial SequenceSynthetic 44Leu Thr Arg Ser Met Leu Ile
Gly Leu Asp Leu Lys Cys Phe Gly1 5 10 154539PRTArtificial
SequenceSynthetic 45Ala Phe Phe Ser Trp Ser Leu Thr Asp Pro Leu Gly
Asn Glu Ala Pro1 5 10 15Gly Gly Tyr Cys Leu Glu Lys Trp Met Leu Val
Ala Ser Glu Leu Lys 20 25 30Cys Phe Gly Asn Thr Ala Ile
354615PRTArtificial SequenceSynthetic 46Leu Glu Lys Trp Met Leu Val
Ala Ser Glu Leu Lys Cys Phe Gly1 5 10 154717PRTArtificial
SequenceSynthetic 47Leu Gln Asn Arg Arg Gly Leu Asp Leu Leu Phe Leu
Lys Glu Gly Gly1 5 10 15Leu4817PRTArtificial SequenceSynthetic
48Ile Leu Asn Arg Lys Ala Ile Asp Phe Leu Leu Arg Arg Trp Gly Gly1
5 10 15Thr4917PRTArtificial SequenceSynthetic 49Ile Leu Asn Arg Lys
Ala Ile Asp Phe Leu Leu Gln Arg Trp Gly Gly1 5 10
15Thr5017PRTArtificial SequenceSynthetic 50Leu Ile Asn Arg His Ala
Ile Asp Phe Leu Leu Thr Arg Trp Gly Gly1 5 10
15Thr5133PRTArtificial SequenceSynthetic 51Val Ile Asn Asp Thr Ser
Phe Val Glu Cys Ile Pro Pro Pro Gln Ser1 5 10 15Arg Pro Ala Trp Asn
Leu Trp Asn Asn Arg Arg Lys Thr Phe Ser Phe 20 25
30Leu5215PRTArtificial SequenceSynthetic 52Gln Ser Arg Pro Ala Trp
Asn Leu Trp Asn Asn Arg Arg Lys Thr1 5 10 155322PRTArtificial
SequenceSynthetic 53Gly Tyr Cys Leu Thr Arg Trp Met Leu Ile Glu Ala
Glu Leu Lys Cys1 5 10 15Phe Gly Asn Thr Ala Val 205444PRTArtificial
SequenceSynthetic 54Gly Tyr Cys Leu Thr Arg Trp Met Leu Ile Glu Ala
Glu Leu Lys Cys1 5 10 15Phe Gly Asn Thr Ala Val Gly Tyr Cys Leu Thr
Arg Trp Met Leu Ile 20 25 30Glu Ala Glu Leu Lys Cys Phe Gly Asn Thr
Ala Val 35 405544PRTArtificial SequenceSynthetic 55Gly Tyr Gly Leu
Thr Arg Trp Met Leu Ile Glu Ala Glu Leu Lys Cys1 5 10 15Phe Gly Asn
Thr Ala Val Gly Tyr Gly Leu Thr Arg Trp Met Leu Ile 20 25 30Glu Ala
Glu Leu Lys Cys Phe Gly Asn Thr Ala Val 35 405622PRTArtificial
SequenceSynthetic 56Gly Tyr Cys Leu Thr Arg Trp Met Leu Ile Glu Ala
Glu Leu Lys Cys1 5 10 15Phe Gly Asn Thr Ala Val 205727PRTArtificial
SequenceSynthetic 57Lys Lys Arg Gly Tyr Cys Leu Thr Arg Trp Met Leu
Ile Glu Ala Glu1 5 10 15Leu Lys Cys Phe Gly Asn Thr Ala Val Lys Arg
20 255816PRTArtificial SequenceSynthetic 58Leu Gln Asn Arg Lys Ala
Ile Asp Leu Trp Asn Asn Lys Glu Gly Gly1 5 10 155932PRTArtificial
SequenceSynthetic 59Leu Gln Asn Arg Lys Ala Ile Asp Leu Trp Asn Asn
Lys Glu Gly Gly1 5 10 15Leu Gln Asn Arg Lys Ala Ile Asp Leu Trp Asn
Asn Lys Glu Gly Gly 20 25 306016PRTArtificial SequenceSynthetic
60Ile Leu Asn Arg Arg Gly Leu Asp Gly Leu Asp Leu Lys Glu Gly Gly1
5 10 156132PRTArtificial SequenceSynthetic 61Ile Leu Asn Arg Arg
Gly Leu Asp Gly Leu Asp Leu Lys Glu Gly Gly1 5 10 15Ile Leu Asn Arg
Arg Gly Leu Asp Gly Leu Asp Leu Lys Glu Gly Gly 20 25
306221PRTArtificial SequenceSynthetic 62Lys Lys Arg Ile Leu Asn Arg
Arg Gly Leu Asp Gly Leu Asp Leu Lys1 5 10 15Glu Gly Gly Lys Arg
206317PRTArtificial SequenceSynthetic 63Cys Leu Thr Lys Pro Ala Trp
Asn Leu Leu Phe Leu Lys Arg Lys Thr1 5 10 15Phe6432PRTArtificial
SequenceSynthetic 64Leu Gln Asn Arg Asp Leu Lys Arg Leu Leu Phe Leu
Lys Arg Lys Thr1 5 10 15Leu Gln Asn Arg Asp Leu Lys Arg Leu Leu Phe
Leu Lys Arg Lys Thr 20 25 306520PRTArtificial SequenceSynthetic
65Lys Lys Leu Gln Asn Arg Asp Leu Lys Arg Leu Leu Phe Leu Lys Arg1
5 10 15Lys Thr Lys Arg 206614PRTArtificial SequenceSynthetic 66Gly
Gln Leu Asn Lys Thr Pro Thr Leu Lys Glu Gly Gly Leu1 5
106717PRTArtificial SequenceSynthetic 67Gly Tyr Cys Leu Thr Arg Arg
Gly Leu Lys Glu Val Asp Lys Glu Gly1 5 10 15Gly6834PRTArtificial
SequenceSynthetic 68Gly Tyr Cys Leu Thr Arg Arg Gly Leu Lys Glu Val
Asp Lys Glu Gly1 5 10 15Gly Gly Tyr Cys Leu Thr Arg Arg Gly Leu Lys
Glu Val Asp Lys Glu 20 25 30Gly Gly6934PRTArtificial
SequenceSynthetic 69Gly Tyr Cys Leu Thr Arg Arg Gly Leu Lys Glu Val
Asp Lys Glu Gly1 5 10 15Gly Gly Tyr Cys Leu Thr Arg Arg Gly Leu Lys
Glu Val Asp Lys Glu 20 25 30Gly Gly7017PRTArtificial
SequenceSynthetic 70Gly Tyr Cys Leu Thr Arg Arg Gly Leu Lys Glu Val
Asp Lys Glu Gly1 5 10 15Gly7121PRTArtificial SequenceSynthetic
71Lys Lys Gly Tyr Cys Leu Thr Arg Arg Gly Leu Lys Glu Val Asp Lys1
5 10 15Glu Gly Gly Lys Arg 207216PRTArtificial SequenceSynthetic
72Ile Pro Pro Pro Gln Ser Arg Thr Pro Thr Leu Lys Val Phe Gly Gly1
5 10 157332PRTArtificial SequenceSynthetic 73Ile Pro Pro Pro Gln
Ser Arg Thr Pro Thr Leu Lys Val Phe Gly Gly1 5 10 15Ile Pro Pro Pro
Gln Ser Arg Thr Pro Thr Leu Lys Val Phe Gly Gly 20 25
307421PRTArtificial SequenceSynthetic 74Lys Lys Arg Ile Pro Pro Pro
Gln Ser Arg Thr Pro Thr Leu Lys Val1 5 10 15Phe Gly Gly Lys Arg
207519PRTArtificial SequenceSynthetic 75Met Tyr Ala Thr Pro Thr Leu
Ala Tyr Phe Gly Gly Phe Asn Phe Ser1 5 10 15Gln Ile
Leu769PRTArtificial SequenceSynthetic 76Gly Leu Arg Ile Leu Leu Leu
Lys Val1 57733PRTArtificial SequenceSynthetic 77Ala Val Gly Ile Gly
Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly1 5 10 15Ser Thr Met Gly
Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln 20 25
30Leu7820PRTArtificial SequenceSynthetic 78Val Ile Gly Phe Arg
Ile
Leu Leu Leu Lys Val Ala Gly Phe Asn Leu1 5 10 15Leu Met Thr Leu
207928PRTArtificial SequenceSynthetic 79Leu Ala Ser Gly Lys Asp Leu
Lys His Val Glu Lys Lys Asp Leu Ala1 5 10 15Ser Gly Lys Asp Leu Lys
His Val Glu Lys Lys Asp 20 258030PRTArtificial SequenceSynthetic
80Cys Leu Ala Ser Gly Lys Asp Leu Lys His Val Glu Lys Lys Asp Cys1
5 10 15Leu Ala Ser Gly Lys Asp Leu Lys His Val Glu Lys Lys Asp 20
25 308113PRTArtificial SequenceSynthetic 81Leu Asn His Arg Val Glu
Lys Leu Glu Asn Lys Leu Thr1 5 10824PRTArtificial SequenceSynthetic
82Arg Arg Arg Arg1834PRTArtificial SequenceSynthetic 83Lys Lys Lys
Lys18423PRTArtificial SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is
absent or 1-amino-glucose succinate, 2- aminododecanoate, or
myristoylateMISC_FEATURE(2)..(2)Xaa is absent or D- or L-amino acid
Val, Ile, Leu, Gly, Met, Tyr, or PheMISC_FEATURE(3)..(3)Xaa is
absent or D- or L-amino acid Val, Ile, Leu, Gly, Met, Tyr, or
PheMISC_FEATURE(4)..(4)Xaa is absent or D- or L-amino acid Val,
Ile, Leu, Gly, Met, Tyr, or PheMISC_FEATURE(5)..(5)Xaa is absent or
D- or L-amino acid Val, Ile, Leu, Gly, Met, Tyr, or
PheMISC_FEATURE(6)..(6)Xaa is a positively charged D- or L-amino
acidMISC_FEATURE(7)..(10)Xaa is Thr or hydrophobic D- or L-amino
acid, including D- or L-cysteine or a D- or L-cysteine
homologueMISC_FEATURE(11)..(11)Xaa is a positively charged D- or
L-amino acidMISC_FEATURE(12)..(12)Xaa is Val or
TyrMISC_FEATURE(13)..(13)Xaa is D- or L-amino acid Ala, Phe, or
GlyMISC_FEATURE(14)..(14)Xaa is absent or a D- or L-amino acid Ala,
Phe, or GlyMISC_FEATURE(15)..(22)Xaa is absent or D- or L-amino
acid Gly, Phe, Asn, Leu, Ile, Met, Thr or
GlnMISC_FEATURE(23)..(23)Xaa is absent or Gly-Tris-monopalmitate,
Gly- Tris-dipalmitate or Gly-Tris-tripalmitate 84Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 208521PRTArtificial
SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is Gly, Tyr, Cys, Val,
Leu, Ile, or MetMISC_FEATURE(2)..(4)Xaa is absent or Gly, Tyr, Cys,
Val, Leu, Ile, or MetMISC_FEATURE(5)..(5)Xaa is Arg, His or
LysMISC_FEATURE(6)..(9)Xaa is Leu, Ile, Thr, or
ProMISC_FEATURE(10)..(10)Xaa is Arg, His or
LysMISC_FEATURE(11)..(13)Xaa is Val, Tyr, Ala, Phe, or
GlyMISC_FEATURE(14)..(21)Xaa is Gly, Phe, Asn, Leu, Ile, Met, Thr
or Gln 85Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa 208631PRTArtificial
SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is absent; or
Gly-Tris-monopalmitate, Gly- Tris-dipalmitate or
Gly-Tris-tripalmitate; or a conjugated sugar selected from the
group including 1-amino-glucose succinate, 2- aminododecanoate, or
myristoylateMISC_FEATURE(2)..(5)Xaa is absent or Arg or
LysMISC_FEATURE(6)..(9)Xaa is absent or Val, Ile, Leu, Gly, Met,
Phe, or TyrMISC_FEATURE(10)..(10)Xaa is Arg, Lys, or
HisMISC_FEATURE(11)..(14)Xaa is Leu, Ile, Thr, or
ProMISC_FEATURE(15)..(15)Xaa is Arg, His or
LysMISC_FEATURE(16)..(16)Xaa is Val or TyrMISC_FEATURE(17)..(18)Xaa
is absent or Ala, Phe, or GlyMISC_FEATURE(19)..(26)Xaa is absent or
Gly, Phe, Asn, Leu, Ile, Met, Thr or GlnMISC_FEATURE(27)..(30)Xaa
is absent or Arg or LysMISC_FEATURE(31)..(31)Xaa is absent; or
Gly-Tris-monopalmitate, Gly- Tris-dipalmitate or
Gly-Tris-tripalmitate; or a conjugated sugar selected from the
group including 1-amino-glucose succinate, 2- aminododecanoate, or
myristoylate 86Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 308717PRTArtificial
SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is absent; or
1-amino-glucose succinate, 2- aminododecanoate, or
myristoylateMISC_FEATURE(2)..(5)Xaa is absent or D- or L-amino acid
Arg, Gly, Leu, Ile, Asn, Gln, Ala, Ser, or
ValMISC_FEATURE(6)..(6)Xaa is a positively charged D- or L-amino
acidMISC_FEATURE(7)..(9)Xaa is Asp, Leu, Ile, Arg, Lys, Ser, Val,
or GluMISC_FEATURE(10)..(10)Xaa is a positively charged D- or
L-amino acidMISC_FEATURE(11)..(13)Xaa is Asp, Asn, Leu, Lys, Ile,
Val, or GluMISC_FEATURE(14)..(14)Xaa is a positively charged D- or
L-amino acidMISC_FEATURE(15)..(15)Xaa is Ile, Leu, Asp, Asn, or
ThrMISC_FEATURE(16)..(16)Xaa is absent or Ile, Leu, Asp, Asn, or
ThrMISC_FEATURE(17)..(17)Xaa is absent; or Gly-Tris-monopalmitate,
Gly- Tris-dipalmitate or Gly-Tris-tripalmitate 87Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10
15Xaa8815PRTArtificial SequenceSyntheticMISC_FEATURE(1)..(4)Xaa is
Arg, Gly, Leu, Ile, Asn, Gln, Ala, Ser, or
ValMISC_FEATURE(5)..(5)Xaa is Arg, His or
LysMISC_FEATURE(6)..(8)Xaa is Asp, Leu, Ile, Arg, Lys, Ser, Val, or
GluMISC_FEATURE(9)..(9)Xaa is Arg, His or
LysMISC_FEATURE(10)..(12)Xaa is Asp, Asn, Leu, Lys, Ile, Val, or
GluMISC_FEATURE(13)..(13)Xaa is Arg, His or
LysMISC_FEATURE(14)..(15)Xaa is Ile, Leu, Asp, Asn, or Thr 88Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10
158925PRTArtificial SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is
absent; or Gly-Tris-monopalmitate, Gly- Tris-dipalmitate or
Gly-Tris-tripalmitate; or a conjugated sugar selected from the
group including 1-amino-glucose succinate, 2- aminododecanoate, or
myristoylateMISC_FEATURE(2)..(5)Xaa is absent or Arg or
LysMISC_FEATURE(6)..(9)Xaa is absent or D- or L-amino acid Arg,
Gly, Leu, Ile, Asn, Gln, Ala, Ser, or ValMISC_FEATURE(10)..(10)Xaa
is Arg, Lys, or HisMISC_FEATURE(11)..(13)Xaa is Asp, Leu, Ile, Arg,
Lys, Ser, Val, or GluMISC_FEATURE(14)..(14)Xaa is Arg, Lys, or
HisMISC_FEATURE(15)..(17)Xaa is Ala, Phe, or
GlyMISC_FEATURE(18)..(18)Xaa is Arg, Lys, or
HisMISC_FEATURE(19)..(19)Xaa is Ile, Leu, Asp, Asn, or
ThrMISC_FEATURE(20)..(20)Xaa is absent or Ile, Leu, Asp, Asn, or
ThrMISC_FEATURE(21)..(24)Xaa is absent or Arg or
LysMISC_FEATURE(25)..(25)Xaa is absent; or Gly-Tris-monopalmitate,
Gly- Tris-dipalmitate or Gly-Tris-tripalmitate; or a conjugated
sugar selected from the group including 1-amino-glucose succinate,
2- aminododecanoate, or myristoylate 89Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 259027PRTArtificial
SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is absent; or
1-amino-glucose succinate, 2- aminododecanoate, or
myristoylateMISC_FEATURE(2)..(9)Xaa is absent or D- or L-amino acid
Ala, Pro, Cys, Thr, Asn, Met, Glu, Ser, Gly, Tyr, Leu, Ile, or
GlnMISC_FEATURE(10)..(10)Xaa is a positively charged D- or L-amino
acidMISC_FEATURE(11)..(18)Xaa is Ser, Trp, Arg, Lys, His, Pro, Met,
Gly, Ala, Thr, Leu, Ile, Val, Asp, Asn, Thr, Glu, Phe, or
GlnMISC_FEATURE(19)..(19)Xaa is a positively charged D- or L-amino
acidMISC_FEATURE(20)..(22)Xaa is absent or Gln, Cys, Glu, Trp, Arg,
Leu, Ile, Phe, Gly, or LysMISC_FEATURE(23)..(26)Xaa is absent or
Asn, Leu, Ile, Thr, Phe, or ValMISC_FEATURE(27)..(27)Xaa is absent;
or Gly-Tris-monopalmitate, Gly- Tris-dipalmitate or
Gly-Tris-tripalmitate 90Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 259125PRTArtificial SequenceSyntheticMISC_FEATURE(1)..(8)Xaa
is Ala, Pro, Cys, Thr, Asn, Met, Glu, Ser, Gly, Tyr, Leu, Ile, or
GlnMISC_FEATURE(9)..(9)Xaa is Arg, His or
LysMISC_FEATURE(10)..(17)Xaa is Ser, Trp, Arg, Lys, His, Pro, Met,
Gly, Ala, Thr, Leu, Ile, Val, Asp, Asn, Thr, Glu, Phe, or
GlnMISC_FEATURE(18)..(18)Xaa is Arg, His or
LysMISC_FEATURE(19)..(21)Xaa is Gln, Cys, Glu, Trp, Arg, Leu, Ile,
Phe, Gly, or LysMISC_FEATURE(22)..(25)Xaa is Asn, Leu, Ile, Thr,
Phe, or Val 91Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20
259235PRTArtificial SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is
absent; or Gly-Tris-monopalmitate, Gly- Tris-dipalmitate or
Gly-Tris-tripalmitate; or a conjugated sugar selected from the
group including 1-amino-glucose succinate, 2- aminododecanoate, or
myristoylateMISC_FEATURE(2)..(5)Xaa is absent or Arg or
LysMISC_FEATURE(6)..(13)Xaa is absent or D- or L-amino acid Ala,
Pro, Cys, Thr, Asn, Met, Glu, Ser, Gly, Tyr, Leu, Ile, or
GlnMISC_FEATURE(14)..(14)Xaa is Arg, Lys, or
HisMISC_FEATURE(15)..(22)Xaa is Ser, Trp, Arg, Lys, His, Pro, Met,
Gly, Ala, Thr, Leu, Ile, Val, Asp, Asn, Thr, Glu, Phe, or
GlnMISC_FEATURE(23)..(23)Xaa is Arg, His or
LysMISC_FEATURE(24)..(26)Xaa is absent or Gln, Cys, Glu, Trp, Arg,
Leu, Ile, Phe, Gly, or LysMISC_FEATURE(27)..(30)Xaa is absent or
Asn, Leu, Ile, Thr, Phe, or ValMISC_FEATURE(31)..(31)Xaa is absent;
or Gly-Tris-monopalmitate, Gly- Tris-dipalmitate or
Gly-Tris-tripalmitate; or a conjugated sugar selected from the
group including 1-amino-glucose succinate, 2- aminododecanoate, or
myristoylateMISC_FEATURE(32)..(35)Xaa is absent or Arg or Lys 92Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10
15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30Xaa Xaa Xaa 359324PRTArtificial
SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is absent or
1-amino-glucose succinate, 2- aminododecanoate, or
myristoylateMISC_FEATURE(2)..(9)Xaa is absent or Ala, Pro, Cys,
Thr, Asn, Met, Glu, Ser, Gly, Tyr, Leu, Ile, or
GlnMISC_FEATURE(10)..(10)Xaa is a positively charged D- or L-amino
acidMISC_FEATURE(11)..(14)Thr or hydrophobic D- or L-amino acids,
including D- or L-cysteine or a D- or L-cysteine
homologueMISC_FEATURE(15)..(15)Xaa is a positively charged D- or
L-amino acidMISC_FEATURE(16)..(16)Xaa is Thr and 3 hydrophobic D-
or L-amino acid, including D- or L-cysteine or a D- or L-cysteine
homologueMISC_FEATURE(17)..(19)Xaa is hydrophobic D- or L-amino
acid, including D- or L-cysteine or a D- or L-cysteine
homologueMISC_FEATURE(20)..(20)Xaa is a positively charged D- or
L-amino acidMISC_FEATURE(21)..(21)Xaa is Gln, Cys, Glu, Trp, Arg,
Leu, Ile, Phe, Gly, and LysMISC_FEATURE(22)..(23)Xaa is absent or
Gln, Cys, Glu, Trp, Arg, Leu, Ile, Phe, Gly, or
LysMISC_FEATURE(24)..(24)Xaa is absent or Gly-Tris-monopalmitate,
Gly- Tris-dipalmitate or Gly-Tris-tripalmitate 93Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 209420PRTArtificial
SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is Ala, Pro, Cys, Thr,
Asn, Met, Glu, Ser, Gly, Tyr, Leu, Ile, or
Gln.MISC_FEATURE(2)..(8)Xaa is absent or Ala, Pro, Cys, Thr, Asn,
Met, Glu, Ser, Gly, Tyr, Leu, Ile, or GlnMISC_FEATURE(9)..(9)Xaa is
Arg, His or LysMISC_FEATURE(10)..(12)Xaa is Asp, Leu, Ile, Arg,
Lys, Ser, Val, or GluMISC_FEATURE(13)..(13)Xaa is Arg, His or
LysMISC_FEATURE(14)..(17)Xaa is Leu, Ile, Thr, or
ProMISC_FEATURE(18)..(18)Xaa is Arg, His or
LysMISC_FEATURE(19)..(19)Xaa is Gln, Cys, Glu, Trp, Arg, Leu, Ile,
Phe, Gly, or LysMISC_FEATURE(20)..(21)Xaa is absent or Gln, Cys,
Glu, Trp, Arg, Leu, Ile, Phe, Gly, and Lys 94Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa
209531PRTArtificial SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is
absent; or Gly-Tris-monopalmitate, Gly- Tris-dipalmitate or
Gly-Tris-tripalmitate; or a conjugated sugar selected from the
group including 1-amino-glucose succinate, 2- aminododecanoate, or
myristoylateMISC_FEATURE(2)..(5)Xaa is absent or Arg or
LysMISC_FEATURE(6)..(13)Xaa is absent or D- or L-amino acid Ala,
Pro, Cys, Thr, Asn, Met, Glu, Ser, Gly, Tyr, Leu, Ile, or
GlnMISC_FEATURE(14)..(14)Xaa is Arg, Lys, or
HisMISC_FEATURE(15)..(17)Xaa is Asp, Leu, Ile, Arg, Lys, Ser, Val,
or GluMISC_FEATURE(18)..(18)Xaa is Arg, His or
LysMISC_FEATURE(19)..(22)Xaa is Leu, Ile, Thr, or
ProMISC_FEATURE(23)..(23)Xaa is Arg, His or
LysMISC_FEATURE(24)..(26)Xaa is absent or Gln, Cys, Glu, Trp, Arg,
Leu, Ile, Phe, Gly, or LysMISC_FEATURE(27)..(30)Xaa is absent or
Arg or LysMISC_FEATURE(31)..(31)Xaa is absent; or
Gly-Tris-monopalmitate, Gly- Tris-dipalmitate or
Gly-Tris-tripalmitate; or a conjugated sugar selected from the
group including 1-amino-glucose succinate, 2- aminododecanoate, or
myristoylate 95Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 309616PRTArtificial
SequenceSyntheticMISC_FEATURE(1)..(1)A terminal conjugate moiety
affects G in this position. 96Gly Arg Lys Leu Gly Tyr Lys Leu Leu
Thr Ile Arg Tyr Ala Asn Leu1 5 10 159717PRTArtificial
SequenceSyntheticMISC_FEATURE(1)..(1)A terminal conjugate moiety
affects G in this position. 97Gly Arg Lys Gly Tyr Arg Pro Thr Pro
Ile Arg Val Ala Phe Gly Asn1 5 10 15Leu9823PRTArtificial
SequenceSyntheticMISC_FEATURE(1)..(1)A terminal conjugate moiety
affects G in this position. 98Gly Arg Lys Leu Val Leu Gly Lys Ala
Ser Val Pro Ala Thr Gly Ser1 5 10 15Arg Leu Val Ser Lys Tyr Lys
209920PRTArtificial SequenceSyntheticMISC_FEATURE(1)..(1)A terminal
conjugate moiety affects G in this position. 99Gly Arg Lys Gly Tyr
Leu Gly Pro Gly Lys Asp Leu Ser Arg Val Asn1 5 10 15Val Lys Gly Arg
20
* * * * *
References