U.S. patent application number 16/951130 was filed with the patent office on 2021-08-19 for polypeptides and uses thereof for treatment of autoimmune disorders and infection.
The applicant listed for this patent is COMPUGEN, LTD.. Invention is credited to Iris HECHT, Zurit LEVINE, Avi Yeshah ROSENBERG, Galit ROTMAN, Amir TOPORIK.
Application Number | 20210253667 16/951130 |
Document ID | / |
Family ID | 1000005564827 |
Filed Date | 2021-08-19 |
United States Patent
Application |
20210253667 |
Kind Code |
A1 |
TOPORIK; Amir ; et
al. |
August 19, 2021 |
POLYPEPTIDES AND USES THEREOF FOR TREATMENT OF AUTOIMMUNE DISORDERS
AND INFECTION
Abstract
This invention relates to C10RF32 protein and its variants and
fragments and fusion proteins thereof, pharmaceutical composition
comprising same and methods of use therof for treatment of immune
related disorders and infections.
Inventors: |
TOPORIK; Amir; (Pardes Hanah
Carkur, IL) ; ROSENBERG; Avi Yeshah; (Kfar Saba,
IL) ; ROTMAN; Galit; (Ramat HaSharon, IL) ;
LEVINE; Zurit; (Herzeliya, IL) ; HECHT; Iris;
(Tel Aviv, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
COMPUGEN, LTD. |
Holon |
|
IL |
|
|
Family ID: |
1000005564827 |
Appl. No.: |
16/951130 |
Filed: |
November 18, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15966694 |
Apr 30, 2018 |
|
|
|
16951130 |
|
|
|
|
15055960 |
Feb 29, 2016 |
|
|
|
15966694 |
|
|
|
|
14129974 |
Dec 29, 2013 |
9428574 |
|
|
PCT/IB2012/000533 |
Jul 1, 2012 |
|
|
|
15055960 |
|
|
|
|
61581201 |
Dec 29, 2011 |
|
|
|
61502880 |
Jun 30, 2011 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/18 20130101;
A61K 45/06 20130101; C07K 14/70503 20130101; A61K 39/395 20130101;
A61K 38/00 20130101; C12N 5/0636 20130101; Y02A 50/30 20180101;
C07K 2319/30 20130101; C07K 2319/74 20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06; C07K 16/18 20060101 C07K016/18; C12N 5/0783 20060101
C12N005/0783 |
Claims
1. A fusion protein comprising an isolated polypeptide fused to a
heterologous sequence, directly or indirectly via a linker peptide,
a polypeptide sequence or a chemical linker wherein the amino acid
sequence of said fusion protein is set forth in SEQ ID NO: 38, 39,
108-112, 116-190.
2. The fusion protein of claim 2, wherein the amino acid sequence
is set forth in any of SEQ ID NOs: 118 or 122.
3. A pharmaceutical composition comprising a fusion protein of any
of claims 2-3, and a pharmaceutically acceptable diluent or
carrier, adapted for treatment of any immune related disorder and
infectious disorder.
4. A method for treating an immune related disorder or an
infectious disorder in a subject in need thereof, comprising
administering to the subject an effective amount of the fusion
protein of claim 1 or pharmaceutical composition thereof.
5. The method of 4, wherein administering an effective amount of
the fusion protein-or pharmaceutical composition thereof to the
subject inhibits or reduces differentiation of, proliferation of,
activity of, and/or cytokine production and/or secretion by an
immune cell selected from the group consisting of Thl, Thl7, and/or
Th22, other cells that secrete, or cells that cause other cells to
secrete, inflammatory molecules.
6. The method of 4, wherein the fusion protein or pharmaceutical
composition thereof is administered in an effective amount to
inhibit or reduce differentiation of, proliferation of, activity
of, and/or cytokine production and/or secretion by Thl, Thl7 and/or
Th22 cells.
7. The method of claim 4, wherein the fusion protein or
pharmaceutical composition thereof is administered in an effective
amount to enhance the suppressive or immunomodulatory effect of
Tregs and/or Th2 cells on Thl or Thl 7 cells.
8. The method of claim 4, wherein the fusion protein or
pharmaceutical composition thereof is administered in an effective
amount to promote or enhance IL-10 production.
9. The method of claim 4, wherein the fusion protein or
pharmaceutical composition thereof is administered in an effective
amount to increase cell numbers or increase populations of any of
Tregs and/or Th2 cells.
10. The method of claim 4, wherein the fusion protein or
pharmaceutical composition thereof is administered in an effective
amount to inhibit the Th1 and/or Th1 7 pathways and to enhance the
activity of Tregs and/or Th2 cells on the Thl and Thl 7 pathways
and/or to promote or enhance IL-10 secretion.
11. The method of claim 4, wherein the fusion protein or
pharmaceutical composition thereof is administered in an effective
amount for reducing proinflammatory molecule production in a
subject.
12. The method according to claim 4 further comprising
administering a second therapeutic agent effective for treatment of
said immune related disorder and/or infectious disorder.
13. The method according to claim 4, wherein the immune related
disorder is selected from the group consisting of autoimmune
disease and immune disorder associated with graft transplantation
rejection, and wherein the infectious disorder is selected from the
disease caused by bacterial infection, viral infection, fungal
infection and/or other parasite infection.
14. The method according to claim 13, wherein the immune disorder
associated with graft transplantation rejection is selected from
the group consisting of acute and chronic rejection of organ
transplantation, allogeneic stem cell transplantation, autologous
stem cell transplantation, bone marrow transplantation, and graft
versus host disease.
15. The method according to claim 13, wherein the autoimmune
disease is selected from the group consisting of multiple
sclerosis, rheumatoid arthritis; psoriatic arthritis, discoid lupus
erythematosus, systemic lupus erythematosus (SLE); ulcerative
colitis; Crohn's disease; benign lymphocytic angiitis, autoimmune
lymphoproliferative syndrome, sarcoidosis, autoimmune
thrombocytopenic purpura, idiopathic thrombocytopenic purpura, pure
red cell aplasia, Sjogren's syndrome, rheumatic disease,
polymyalgia rheumatica, mixed connective tissue disease,
inflammatory rheumatism, degenerative rheumatism, extra-articular
rheumatism, juvenile arthritis, juvenile rheumatoid arthritis,
systemic juvenile idiopathic arthritis, arthritis uratica, muscular
rheumatism, chronic polyarthritis, reactive arthritis, Reiter's
syndrome, rheumatic fever, relapsing polychondritis, Raynaud's
phenomenon, vasculitis, cryoglobulinemic vasculitis,
ANCA-associated vasculitis, temporal arteritis, giant cell
arteritis, Takayasu arteritis, Behcet's disease, antiphospholipid
syndrome, myasthenia gravis, autoimmune haemolytic anaemia,
Guillain-Barre syndrome, chronic immune polyneuropathy, chronic
inflammatory demyelinating polyneuropathy, autoimmune thyroiditis,
insulin dependent diabetes mellitus, type I diabetes, Addison's
disease, membranous glomerulonephropathy, polyglandular autoimmune
syndromes, Goodpasture's disease, autoimmune gastritis, autoimmune
atrophic gastritis, pernicious anaemia,pemphigus, pemphigus
vulgaris, cirrhosis, primary biliary cirrhosis, idiopathic
pulmonary fibrosis, myositis, dermatomyositis, juvenile
dermatomyositis, polymyositis, fibromyositis, myogelosis, celiac
disease, celiac sprue dermatitis, immunoglobulin A nephropathy,
Henoch-Schonlein purpura, Evans syndrome, atopic dermatitis,
psoriasis, psoriasis vulgaris, psoriasis arthropathica, Graves'
disease, Graves' ophthalmopathy, scleroderma, systemic scleroderma,
progressive systemic scleroderma, diffuse scleroderma, localized
scleroderma, Crest syndrome, asthma, allergic asthma, allergy,
primary biliary cirrhosis, Hashimoto's thyroiditis, fibromyalgia,
chronic fatigue and immune dysfunction syndrome (CFIDS), primary
myxedema, sympathetic ophthalmia, autoimmune inner ear disease,
autoimmune uveitis, autoimmune chronic active hepatitis, collagen
diseases, ankylosing spondylitis, periarthritis humeroscapularis,
panarteritis nodosa, polyarteritis nodosa, chondrocalcinosis,
Wegener's granulomatosis, microscopic polyangiitis, chronic
urticaria, bullous skin disorders, pemphigoid, bullous pemphigoid,
cicatricial pemphigoid, vitiligo, atopic eczema, eczema, chronic
urticaria, autoimmune urticaria, normocomplementemic urticaria!
vasculitis, hypocomplementemic urticaria! vasculitis, alopecia
areata, alopecia universalis, alopecia totalis, Devic's disease,
pernicious anemia, childhood autoimmune hemolytic anemia,
idiopathic autoimmune hemolytic anemia, refractory or chronic
Autoimmune Cytopenias, Prevention of development of Autoimmune
Anti-Factor VIII Antibodies in Acquired Hemophilia A, Cold
agglutinin disease, Neuromyelitis Optica, Stiff Person Syndrome,
gingivitis, periodontitis, pancreatitis, myocarditis, gastritis,
gout, gouty arthritis, idiopathic pericarditis, anti-synthetase
syndrome, scleritis, macrophage activation syndrome, PAPA Syndrome,
Blau's Syndrome, adult and juvenile Still's disease, cryopyrin
associated periodic syndrome, Muckle-Wells syndrome, familial cold
auto-inflammatory syndrome, neonatal onset multisystem inflammatory
disease, chronic infantile neurologic cutaneous and articular
syndrome, familial Mediterranean fever, Hyper IgD syndrome,
Schnitzler's syndrome, autoimmune retinopathy, age-related macular
degeneration, and TNF receptor-associated periodic syndrome
(TRAPS).
16. The method according to claim 13, wherein the autoimmune
disease is selected from the group consisting of multiple
sclerosis, rheumatoid arthritis, type I diabetes, psoriasis, atopic
dermatitis, psoriasis vulgaris, psoriasis arthropathica, systemic
lupus erythematosus, inflammatory bowel disease, uveitis,
ulcerative colitis; Crohn's disease, and Sjogren's syndrome.
17. The method according to claim 16, wherein the multiple
sclerosis is selected from the group consisting of benign multiple
sclerosis, relapsing remitting multiple sclerosis, secondary
progressive multiple sclerosis, primary progressive multiple
sclerosis, chronic progressive multiple sclerosis,
transitional/progressive multiple sclerosis, progressive relapsing
multiple sclerosis, rapidly worsening multiple sclerosis,
clinically-definite multiple sclerosis, malignant multiple
sclerosis, also known as Marburg's Variant, acute multiple
sclerosis and condition relating to multiple sclerosis, selected
from the group consisting of Devic's disease, also known as
Neuromyelitis Optica; acute disseminated encephalomyelitis, acute
demyelinating optic neuritis, demyelinative transverse myelitis,
Miller-Fisher syndrome, encephalomyelradiculoneuropathy, acute
demyelinative polyneuropathy, tumefactive multiple sclerosis and
Balo's concentric sclerosis.
18. The method according to claim 16, wherein the rheumatoid
arthritis is selected from the group consisting of rheumatoid
arthritis, gout and pseudo-gout, juvenile idiopathic arthritis,
juvenile rheumatoid arthritis, Still's disease, ankylosing
spondylitis, rheumatoid vasculitis, and conditions relating to
rheumatoid arthritis, selected from the group consisting of
osteoarthritis, sarcoidosis, Henoch-Schonlein purpura, Psoriatic
arthritis, Reactive arthritis, Spondyloarthropathy, septic
arthritis, Haemochromatosis, Hepatitis, vasculitis, Wegener's
granulomatosis, Lyme disease, Familial Mediterranean fever,
Hyperimmunoglobulinemia D with recurrent fever, TNF receptor
associated periodic syndrome, and Enteropathic arthritis associated
with inflammatory bowel disease.
19. The method according to claim 16, wherein the uveitis is
selected from the group consisting of anterior uveitis,
iridocyclitis, intermediate uveitis pars planitis, posterior
uveitis, chorioretinitis and the panuveitis form.
20. The method according to claim 16, wherein the inflammatory
bowel disease is selected from the group consisting of Crohn's
disease and ulcerative colitis (UC) and conditions relating to IBD
selected from the group consisting of Collagenous colitis,
Lymphocytic colitis, Ischaemic colitis, Diversion colitis, Bechet's
disease, Indeterminate colitis.
21. The method according to claim 16, wherein the psoriasis is
selected from the group consisting of Nonpustular Psoriasis
including Psoriasis vulgaris and Psoriatic erythroderma,
erythrodermic psoriasis, Pustular psoriasis including Generalized
pustular psoriasis, pustular psoriasis of von Zumbusch, Pustulosis
palmaris et plantaris, persistent palmoplantar pustulosis, pustular
psoriasis of the Barber type, pustular psoriasis of the
extremities, Annular pustular psoriasis, Acrodermatitis continua,
Impetigo herpetiformis, conditions relating to psoriasis include,
drug-induced psoriasis, Inverse psoriasis, Napkin psoriasis,
Seborrheic-like psoriasis, Guttate psoriasis, Nail psoriasis, and
Psoriatic arthritis.
22. The method according to claim 16, wherein the diabetes is
selected from the group consisting of insulin-dependent diabetes
mellitus, idiopathic diabetes, juvenile type 1 diabetes, maturity
onset diabetes of the young, latent autoimmune diabetes in adults,
gestational diabetes, and condition relating to type 1 diabetes
selected from the group consisting of neuropathy including
polyneuropathy, mononeuropathy, peripheral neuropathy and
autonomicneuropathy; eye complications: glaucoma, cataracts,
retinopathy.
23. The method according to claim 16, wherein the Sjogren's
syndrome is selected from the group consisting of Primary Sjogren's
syndrome and Secondary Sjogren's syndrome and condition relating to
Sjogren's syndrome selected from the group consisting of connective
tissue disease, such as rheumatoid arthritis, systemic lupus
erythematosus, or scleroderma.
24. The method according to claim 16, wherein the systemic lupus
erythematosus is selected from the group consisting of discoid
lupus, lupus arthritis, lupus pneumonitis, lupus nephritis, and
condition relating to systemic lupus erythematosus, selected from
the group consisting of osteoarticular tuberculosis,
antiphospholipid antibody syndrome, inflammation of various parts
of the heart, selected from the group pericarditis, myocarditis,
and endocarditis, Lung and pleura inflammation, pleuritis, pleural
effusion, chronic diffuse interstitial lung disease, pulmonary
hypertension, pulmonary emboli, pulmonary hemorrhage, and shrinking
lung syndrome, lupus headache, Guillain-Barre syndrome, aseptic
meningitis, demyelinating syndrome, mononeuropathy, mononeuritis
multiplex, myasthenia gravis, myelopathy, cranial neuropathy,
polyneuropathy, and vasculitis.
25. The method according to claim 4, wherein the treating comprises
treatment of immune related disorder without global
immunosuppression.
26. The method of claim 4, wherein the subject does not respond to
treatment with TNF blockers.
27. A method for treating an immune related disorder or an
infectious disorder in a subject in need thereof, comprising
administering to the subject an effective amount of the fusion
protein of claim 2 or pharmaceutical composition thereof.
28. The method of 28, wherein administering an effective amount of
the fusion protein to the subject inhibits or reduces
differentiation of, proliferation of, activity of, and/or cytokine
production and/or secretion by an immune cell selected from the
group consisting of Thl, Thl7, and/or Th22, other cells that
secrete, or cells that cause other cells to secrete, inflammatory
molecules.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to C1ORF32 protein, and its
variants, fragments and fusion proteins thereof, pharmaceutical
compositions comprising same and methods of use therof for
treatment of immune related disorders and infections.
BACKGROUND OF THE INVENTION
[0002] The balance between proinflammatory mechanisms and the
dampening of excessive immune activation is important for treatment
of infectious disease and also for treatment of autoimmune disease.
The former benefits from an activated immune system while the
latter requires reduce immune system activity. Thus, the immune
system has the reciprocal tasks to protect the host against
invading pathogens, but simultaneously to prevent damage resulting
from unwanted reactions to self antigens.
[0003] The latter part is known as immune tolerance and performed
by a complex set of interactive and complementary pathways, which
regulate immune responses. T cells have the ability to react to a
variety of antigens, both self and nonself. Therefore, there are
many mechanisms that exist naturally to eliminate potentially
self-reactive responses--this is known as natural tolerance. The
main mechanism for eliminating potential auto-reactive T cells
occurs in the thymus and is known as central tolerance. Some
potentially autoreactive T cells escape central tolerance and,
therefore, peripheral tolerance mechanisms also exist. Despite
these mechanisms, some self-reactive T cells may `escape` and be
present in the repertoire; it is believed that their activation may
lead to autoimmune disease.
[0004] Studies on therapeutic tolerance have attempted to induce
and amplify potent physiological mechanisms of tolerance in order
to eliminate or neutralize self-reactive T cells and prevent or
treat autoimmune diseases. One way to induce tolerance is by
manipulation of the interaction between costimulatory ligands and
receptors on antigen presenting cells (APCs) and lymphocytes.
[0005] CTLA-4 is the most extensively studied costimulatory
molecule which down-regulates immune responses. The attributes of
immunosuppressive qualities and capacity to induce tolerance have
made its recognition as a potential immuno-therapeutic agent for
autoimmune mediated inflammatory disorders. Abatacept (commercial
name: Orencia) is a fusion protein composed of the ECD
(extracellular domain) of CTLA-4 fused to the Fc fragment of hIgG1.
Abatacept is believed to induce costimulation blockade, which has
been approved for treating patients with rheumatoid arthritis, by
effectively interfering with the inflammatory cascade.
[0006] Induction of disease control with the current therapies,
followed by progressive withdrawal in parallel with re-establishing
immune tolerance, may be an attractive approach in the future of
autoimmune therapies. Furthermore, due to their immune specificity,
in the absence of global immunosuppression, such therapies should
be safer.
[0007] T helper type 1 (Th1) cells are induced by IL-12 and produce
IFN-.gamma., while T helper type 2 (Th2) cells secrete IL-4, IL-5
and IL-13. Th1 cells can mediate proinflammatory or cell-mediated
immune responses, whereas Th2 cells mainly promote certain types of
humoral immunity. Some immune related diseases, such as autoimmune
reactions, inflammation, and infection, are characterized by a
dysregulation of the pro-versus anti-inflammatory tendencies of the
immune system, as well as an imbalance in the Th1 versus Th2
cytokine balance. During inflammation, induction of a shift in the
balance from Th1 to Th2 protects the organism from systemic
`overshooting` with Th1/pro-inflammatory cytokines, by reducing the
inflammatory tendencies of the immune system Immunomodulatory
therapies that are associated with a Th1 to Th2 immune shift have
protective effects in Th1-mediated autoimmune diseases, such as
multiple sclerosis and rheumatoid arthritis. For example,
Laquinimod, which has demonstrated efficacy in animal models of
several autoimmune diseases including MS, shows immunomodulatory
effects through Th1/Th2 shift, and does not lead to
immunosuppression. Glatiramer acetate (Copaxone) also induces
Th1/Th2 shift with decreased secretion of proinflammatory
cytokines, and increased secretion of antiinflammatory cytokines.
Furtheremore, GA-specific Th2 cells are able to migrate across the
blood-brain barrier and cause in situ bystander suppression of
autoaggressive Th1 T cells.
[0008] The balance between proinflammatory mechanisms and the
dampening of excessive immune activation is also critical for
successful clearance of a pathogen without harm to the host.
Excessive immune activation may lead to autoimmune attacks, while
too little immune activation will not result in clearance of the
pathogen from the host. Chronic pathogens exploit co-inhibitory
pathways to attenuate Ag-specific T cell immunity. Emerging data
from a wide range of studies on acute and chronic infections
support an important role for negative costimulatory receptors in
controlling infection. Most notably, exhausted T cells,
functionally impaired T cells which are present during chronic
infection and are characterized by reduced proliferative and
cytotoxic abilities, express high levels of multiple co-inhibitory
receptors such as CTLA-4, PD-1, and LAG3 (Crawford et al., Curr
Opin Immunol. 2009; 21:179-186; Kaufmann et al., J Immunol 2009;
182:5891-5897, Sharpe et al., Nat Immunol 2007; 8:239-245).
Furthermore, the exhausted phenotype can be reversed by blocking
co-inhibitory pathways (Rivas et al., J Immunol. 2009 ;
183:4284-91; Golden-Mason et al., J Virol. 2009; 83:9122-30), thus
allowing restoration of anti viral immune function, supporting
therapeutic application of co-inhibitory blockade in viral
infection.
[0009] One potentially promising strategy to control chronic
infections such as human immunodeficiency virus, hepatitis B virus,
and hepatitis C virus is therapeutic vaccination, which aims to
reduce persisting virus by stimulating a patient's own antiviral
immune responses. However, this approach has fallen short of
expectations, because antiviral T cells generated during chronic
infections often become functionally exhausted, as explained above,
and thus do not respond properly to therapeutic vaccination.
Therefore, it is necessary to restore T cell effector functions and
effectively boost endogenous T-cell responses in order to develop
therapeutic vaccines against chronic viral infections. Blocking the
negative signaling pathways, PD-1 and CTLA-4, could restore the
host immune system, enabling it to respond to further stimulation.
Blockade of the PD-1/PD-L1 pathway, for example, is able to restore
functional capabilities to exhausted CTLs (Hofmeyer et al, J.
Biomed. & Biotech. Vol. 2011, Art. ID 451694). Combining
therapeutic vaccination along with the blockade of inhibitory
signals could synergistically enhance functional CD8(+) T-cell
responses and improve viral control in chronically infected
individuals, providing a promising strategy for the treatment of
chronic viral infections. (Ha et al, Immunol Rev. 2008 June;
223:317-33). Antibodies to PD-1 and CTLA-4 are currently in
clinical trials in chronic hepatitis C, as promising candidates for
combination with both prophylactic and therapeutic vaccines
(Diepolder and Obst, Expert Rev Vaccines. 2010 Mar.;
9(3):243-7).
[0010] The therapeutic potential of co-inhibition blockade for
treating viral infection was extensively studied by blocking the
PD-1/PD-L1. Blocking this pathway was shown to be efficacious in
several animal models of infection including acute and chronic
Simian immunodeficiency virus (SIV) infection in rhesus macaques
(Valu et al., Nature 2009; 458:206-210) and in mouse model of
chronic viral infection with lymphocytic choriomeningitis virus
(LCMV) (Barber et al., Nature. 2006; 439:682-7). Modulation of
costimulatory pathway has also been proven effective in optimizing
antiviral immunity by limiting the memory T cell response to its
protective capacities (Teij aro et al., J Immunol. 2009: 182;
5430-5438). This has been demonstrated in models of influenza
infection in which inhibiting CD28 costimulation with CTLA4-Ig
suppressed primary responses in naive mice infected with influenza,
but was remarkably curative for memory CD4 T cell-mediated
secondary responses to influenza leading to improved clinical
outcome and increased survival to influenza challenge. The curative
effect of CTLA4-Ig on secondary responses was accompanied by
inhibition of proliferation and egress of lymphoid naive and memory
T cells, while leaving lung resident memory CD4 T cell responses
intact thus maintaining enhanced and rapid lung viral clearance
mediated by memory CD4 T cells, yet reducing lung
immunopathology.
[0011] These data demonstrate an active and reversible role for
molecules of the B7:CD28 family, PD-1, CTLA-4, and their ligands,
in virus-specific T cell exhaustion associated with chronic viral
infection and point to promising potential for immunotherapeutic
interventions based on manipulation of these inhibitory
networks.
[0012] Regulating costimulation using agonists and antagonists to
various costimulatory proteins has been extensively studied as a
strategy for treating autoimmune diseases, graft rejection, allergy
and cancer. This field has been clinically pioneered by CTLA4-Ig
(Abatacept, Orencia.RTM.) that is approved for treatment of RA, and
by the anti-CTLA4 antibody (Ipilimumab, Yervoy.RTM.), recently
approved for the treatment of melanoma. Other costimulation
regulators are currently in advanced stages of clinical development
including anti PD-1 antibody (MDX-1106) which is in development for
treatment of advanced/metastatic clear-cell renal cell carcinoma
(RCC) and anti-CD40L Antibody (BG9588, Antova.RTM.) for treatment
of renal allograft transplantation. In addition, such agents are in
clinical development for viral infections, for example the anti
PD-1 Ab, MDX-1106, is being tested for treatment of hepatitis C.
Another example is CP-675,206 (tremelimumab) and anti-CTLA4 Ab
which is in a clinical trial in hepatitis C virus-infected patients
with hepatocellular carcinoma.
[0013] B cells play a critical role in recognition of foreign
antigens and subsequent production of antibodies in the specific
humoral adaptive immune responses that provide protection against
various types of infectious agents. B cells play a critical role in
recognition of foreign antigens and they produce the antibodies
necessary to provide protection against various type of infectious
agents. T cell help to B cells is a pivotal process of adaptive
immune responses. Follicular helper T (Tfh) cells are a subset of
CD4+ T cells specialized in B cell help (reviewed by Crotty, Annu.
Rev. Immunol. 29: 621-663, 2011). Tfh cells express the B cell
homing chemokine receptor, CXCRS, which drives Tfh cell migration
into B cell follicles within lymph nodes in a CXCL13-dependent
manner. The requirement of Tfh cells for B cell help and T
cell-dependent antibody responses, indicates that this cell type is
of great importance for protective immunity against various types
of infectious agents, as well as for rational vaccine design.
[0014] Tfh cells selectively express a wealth of surface proteins,
which are involved in their selective localization (such as CXCRS)
and in direct physical interactions with B cells to provide B cell
help. Among the latter group are several members of the
costimulatory proteins family which are highly expressed in Tfh
cells, including the inducible co-stimulatory receptor ICOS, and
the negative costimulators (inhibitory receptors) PD-1 and BTLA
(Crotty, Annu. Rev. Immunol. 29: 621-663, 2011).
BRIEF SUMMARY OF THE INVENTION
[0015] The background art fails to provide therapies that target
multiple cells and pathways involved in autoimmunity and
inflammation, such as Th1, Th17, Th22, Th2, Tregs, or other cells
that secrete, or influence other cells that secrete, inflammatory
molecules such as cytokines, metalloproteases, chemokines and other
molecules. The background art also does not teach such therapies
that are targeted with regard to providing a balance between
excessive immune activation and desirable pro-inflammatory immune
activation, for the treatment of autoimmune diseases and infectious
disease.
[0016] The present invention is of C1ORF32 protein, and its
variants, fragments and fusion proteins thereof, pharmaceutical
compositions comprising same and methods of use therof for
treatment of immune related disorders and infections.
[0017] With regard to treatment for immune related diseases, it
should be noted that such diseases may optionally relate to any
disease in which it is desirable to induce immune tolerance. With
regard to treatment of infections, it should be noted that such
diseases may optionally relate to any disease in which T cell
exhaustion to a foreign pathogenic antigen plays a role.
[0018] According to at least some embodiments of the present
invention, there are provided C1ORF32 polypeptides, as an isolated
polypeptide, comprising an amino acid sequence of C1ORF32 IgV
domain fragment, set forth in any one of SEQ ID NOs: 29, 30,
41-105, with the proviso that the amino acid sequence does not
include the complete, exact sequence of SEQ ID NO: 35. Optionally,
the isolated polypeptide has less than 90% identity with SEQ ID
NO:35, optionally has less than 85% identity with SEQ ID NO:35 and
optionally has less than 80% identity with SEQ ID NO:35. Also
optionally and preferably, the isolated polypeptide consists
essentially of the amino acid sequence as set forth in any one of
SEQ ID NOs: 29, 30, 41-105, or optionally up to 95% identical
thereof. It is further contemplated that optionally the C1ORF32 IgV
domain fragment may be extended beyond the border delimited by the
end of SEQ ID NO:35, as long as one or more of the above conditions
regarding sequence identity are met. A further description of such
fragments is provided with regard to Table 1 below.
[0019] Briefly, a sequence alignment and comparison is provided
between SEQ ID NO:35 and SEQ ID NO:29, as an example of the above
described inventive sequences:
TABLE-US-00001 Query: 1
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAWQWKFKSYCQDRMGESLGMSSTRAQSL 60
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAWQWKFKSYCQDRMGESLGMSSTRAQSL Sbjct:
1 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAWQWKFKSYCQDRMGESLGMSSTRAQSL 60
Query: 61
SKRNLEWDPYLDCLDSRRTVRWASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDS 120
SKRNLEWDPYLDCLDSRRTVRWASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDS Sbjct:
61 SKRNLEWDPYLDCLDSRRTVRWASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDS 120
Query: 121 GLYYCIITTPDDLEGKNEGSLGLLVLGRTGLLADLLPSFAVEIMPE 166
GLYYCIITTPDDLEGKNEDSVELLVLG Sbjct: 121
GLYYCIITTPDDLEGKNEDSVELLVLG................... 147
[0020] For this non-limiting example, SEQ ID NO:29 is shorter than
SEQ ID NO:35 but as noted above, for some inventive sequences, the
inventive amino acid sequence is longer than SEQ ID NO:35, but
still fulfills one or more of the above conditions regarding
sequence identity. An equivalent difference exists between the
inventive sequences and SEQ ID NO:36, such that the inventive
sequences are as described above and herein, the proviso that the
amino acid sequence does not include the complete, exact sequence
of SEQ ID NO: 36. Optionally, the isolated polypeptide has less
than 90% identity with SEQ ID NO:36, optionally has less than 85%
identity with SEQ ID NO:36 and optionally has less than 80%
identity with SEQ ID NO:36. It is further contemplated that
optionally the C1ORF32 IgV domain fragment may be extended beyond
the border delimited by the end of SEQ ID NO:36, as long as one or
more of the above conditions regarding sequence identity are
met.
[0021] According to at least some embodiments, there is provided an
isolated polypeptide comprising a soluble C1ORF32 polypeptide or
fragment or variant thereof, having an amino acid substitution
preventing a cleavage of the C1ORF32 ECD (SEQ ID NO:14) between
amino acids F and A at positions 179 and 180 of any of
H19011_1_P8_V1 or H19011_1_P8 (Seq ID NOs: 4 or 5). Optionally, the
amino acid substitution in the cleavage site of C1ORF32 ECD (SEQ ID
NO:14) at positions 179 and 180 of any of H19011_1_P8_V1 or
H19011_1_P8 (Seq ID NOs: 4 or 5) is selected from FA.fwdarw.GA;
FA.fwdarw.AA; and FA.fwdarw.GG. Optionally and preferably, the
polypeptide has an amino acid sequence as set forth in any one of
SEQ ID NOs:45, 64, and 96.
[0022] According to at least some embodiments of the present
invention, there are provided C1ORF32 polypeptides, optionally
provided as fusion proteins containing a C1ORF32 polypeptide.
C1ORF32 fusion polypeptides optionally have a first fusion partner
comprising part of a C1ORF32 soluble polypeptide, or a sequence
homologous thereto, and a second fusion partner composed of a
heterologous sequence (respectively non-C1ORF32), fused together
directly or indirectly via a peptide linker sequence or a chemical
linker.
[0023] Optionally, the fusion protein comprises the polypeptide as
described herein, fused to a heterologous sequence, directly or
indirectly via a linker peptide, a polypeptide sequence or a
chemical linker.
[0024] Optionally the heterologous sequence comprises at least a
portion of an immunoglobulin constant domain. Also optionally, the
constant domain comprises an immunoglobulin heavy chain constant
region corresponding to an antibody isotype selected from the group
consisting of an IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA and IgD.
[0025] Optionally the immunoglobulin constant domain comprises the
hinge, CH2 and CH3 regions of a human IgG immunoglobulin, selected
from the group consisting of C.gamma.1, C.gamma.2, C.gamma.3 and
C.gamma.4 chain.
[0026] Optionally, the fusion protein further comprises a domain
that mediates dimerization or multimerization of the fusion protein
to form homodimers, heterodimers, homomultimers, or
heteromultimers. Optionally, the domain that mediates dimerization
or multimerization is selected from the group consisting of one or
more cysteines that are capable of forming an intermolecular
disulfide bond with a cysteine on the partner fusion protein, a
coiled-coil domain, an acid patch, a zinc finger domain, a calcium
hand domain,a CHI region, a CL region, a leucine zipper domain, an
SH2 (src homology 2) domain, an SH3 (src Homology 3) domain, a PTB
(phosphotyrosine binding) domain, a WW domain, a PDZ domain, a
14-3-3 domain, a WD40 domain, an EH domain, a Lim domain, an
isoleucine zipper domain, and a dimerization domain of a receptor
dimer pair.
[0027] According to at least some embodiments of the present
invention, there is provided a dimeric protein comprising a first
and a second fusion protein, wherein the first and the second
fusion proteins comprise the fusion protein as described herein and
wherein the first and the second fusion proteins are bound to one
another by covalent or noncovalent bonds to form a dimer.
[0028] According to at least some embodiments, the isolated
polypeptide is at least 80, 90, 95, 96, 97, 98 or 99% homologous to
a polypeptide set forth in any one of SEQ ID NOs: 29, 30, 41-105.
The C1ORF32 polypeptide may be of any species of origin. In further
embodiments, the C1ORF32 polypeptide is of murine, non-human
primate or human origin.
[0029] Without wishing to be limited by a single hypothesis,
according to at least some embodiments the C1ORF32 protein, which
may optionally comprise a fusion protein, inhibits the inflammatory
activity of Th1, Th17, Th22, or other cells that secrete, or cause
other cells to secrete, inflammatory molecules, including, but not
limited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6,
IL-23, IL-22, IL-21, and MMPs. Again without wishing to be limited
by a single hypothesis, according to at least some embodiments the
C1ORF32 protein can also increase the suppressive capacity of Tregs
or the immunomodulatory activity of Th2 cells. The C1ORF32 fusion
protein can also increase the production of anti-inflammatory
molecules such as the cytokine IL-10.
[0030] Optionally, the fragment is of at least about 20 amino acids
of the extracellular domain of C1ORF32, but no more than 169 amino
acids of the extracellular domain, in order to fulfill the
conditions regarding sequence identity as described with regard to
SEQ ID NO:35 as described above. Optionally, the fragment is of at
least about 21, 22, 23, 24, 25 and so forth amino acids of the
extracellular domain of C1ORF32, but no more than 100, 101, 102 and
so forth, up to 168 amino acids of the extracellular domain, as
described above, optionally including any integral value between 20
and 169 amino acids in length, although as noted above, the
inventive sequence may optionally have more amino acids than the
ECD of SEQ ID NO:35. Optionally the polypeptide is attached to a
detectable or therapeutic moiety.
[0031] According to at least some embodiments of the present
invention, there is provided a method for prevention of damage to
the myelin coat of neural cells in the central nervous system in MS
(multiple sclerosis) patients comprising administering to a subject
in need thereof a pharmaceutical composition comprising: a soluble
molecule having the C1ORF32 polypeptide, selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof; optionally
provided as a pharmaceutical composition. Optionally, the fragment
is as described above.
[0032] Multiple sclerosis (MS) is a chronic, inflammatory,
demyelinating disorder of the central nervous system (CNS), which
involves autoimmune responses to myelin antigens. It is
characterized by lesions within the CNS and demyelination is a key
feature of these lesions. Autoreactive T cells are thought to
initiate an autoimmune response directed against components of CNS
myelin. The main targets of the autoimmune reactions are thought to
be myelin basic protein (MBP), proteolipid protein (PLP) and myelin
oligodendrocyte glycoprotein (MOG). Experimental autoimmune
encephalomyelitis (EAE), an animal model of MS induced by
immunization with myelin components in adjuvant, shows comparable
neuronal pathology. Without wishing to be limited by a single
hypothesis, studies in EAE have provided convincing evidence that T
cells specific for self-antigens mediate pathology in these
diseases.
[0033] According to at least some embodiments of the present
invention, there is provided an isolated soluble C1ORF32
polypeptide, selected from the group consisting of SEQ ID NOs: 29,
30, 41-105, fragment, variant, or homolog thereof; optionally as a
fusion protein or conjugate, wherein said polypeptide or said
fusion protein or conjugate is used for anti-immune related
condition immunotherapy for an immune related disorder and/or
infection as described herein, optionally provided as a
pharmaceutical composition.
[0034] Optionally treating comprises one or more of preventing,
curing, managing, reversing, attenuating, alleviating, minimizing,
suppressing, managing, or halting the deleterious effects of the
above-described diseases.
[0035] Optionally, managing comprises reducing the severity of the
disease, reducing the frequency of episodes of the disease,
reducing the duration of such episodes, or reducing the severity of
such episodes or a combination thereof.
[0036] In another embodiment, the C1ORF32 polypeptides, fragments
or variants or homologs thereof, fusion proteins or conjugates
comprising same, or pharmaceutical composition comprising same, can
be used to treat patients who do not respond to TNF blockers.
[0037] According to at least some embodiments, the present
invention provides a method for combining therapeutic vaccination
with an antigen along with administration of the foregoing
pharmaceutical composition for treatment of infection. According to
at least some embodiments of the present invention, the antigen is
a viral antigen, a bacterial antigen, fungal antigen and/or other
parasite antigen.
[0038] According to at least some embodiments, the present
invention further provides a method for combining the foregoing
pharmaceutical composition, used as adjuvant, along with an antigen
in a vaccine, in order to increase the immune response. According
to at least some embodiments of the present invention, the antigen
is a viral antigen, a bacterial antigen, fungal antigen, parasite
antigen, and/or other pathogen's antigen.
BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1 shows a schematic drawing of a full length soluble
C1ORF32 protein. Signal Peptide is shown in italic face font; two
potential start points of IgV are shown in underlined italic and
bold shape; IgV domain is in bold face font; amino acid region
deleted in H19011_1_P9 (SEQ ID NO:6) as compared to the H19011_1_P8
(SEQ ID NO:4) is shown in dashed underline; the locations of the
three SNPs (DSVE.fwdarw.GSLG) is shown in double underlined italic
and bold shape; the location of point mutations (FA.fwdarw.GA,
FA.fwdarw.AA, FA->GG) is shown in bold and dashed underline.
Transmembrane domain (TM) of H19011_1_P8 (SEQ ID NO:4) is shown in
light font with dotted underline; in H19011_1_P9 (SEQ ID NO:6) the
TM starts at amino acid residues "FVG . . . ", 2 amino acids
downstream to the start point of the H19011_1_P8 (SEQ ID NO:4)
transmembrane domain. Vertical bars represent five possible
C-terminal end points of the soluble C1ORF32 proteins from position
1 on the left (end of IgV) to position 5 on the right (end of
predicted ECD).
[0040] FIG. 2 shows SDS-PAGE results for produced proteins.
[0041] FIG. 3 shows inhibitory effect of Fc-fused C1ORF32 ECD (SEQ
ID NO:108), Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110),
and C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on mouse
CD4+ T cell IFN.gamma. secretion. Purified CD4+CD25- T cells,
1.times.10.sup.5 per well, were stimulated with anti-CD3 mAb (2
ug/mL) in the presence of test proteins or control mouse IgG2a at
0, 3 and 10 .mu.g/ml. Culture supernatants were collected 48 hrs
post stimulation and analyzed using mouse IFNy ELISA kit. Results
are shown as Mean.+-.SD of four wells per point (Student's t-test ,
***P<0.001, compared with control mouse IgG2a).
[0042] FIG. 4 shows inhibitory effect of Fc-fused C1ORF32 ECD (SEQ
ID NO:108), Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110),
and C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on
expression of the early TCR activation marker, CD69.
1.times.10.sup.5 CD4+CD25- T cells were stimulated with plate bound
anti-CD3 (2 .mu.g/ml) together with test proteins or control mIgG2a
(10 .mu.g/ml). Cells were analyzed at 48 hrs for the expression
CD69 by flow cytometry (**; P value<0.001 compare to control
mIgG2A, student's T test).
[0043] FIG. 5 shows inhibitory effect of Fc-fused C1ORF32 ECD (SEQ
ID NO:108), (two different batches produced in CHO-S And in
CHO-DG44), Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110),
and C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) (two
different batches produced in CHO-S And in CHO-DG44) on naive T
cell proliferation. No inhibition was observed with the IgG2a
isotype control.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention, in at least some embodiments, relates
to any one of the C1ORF32 proteins, selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, fragments, variants and
homologs thereof and fusion proteins and conjugates containing
same, and pharmaceutical compositions comprising same, and nucleic
acid sequences encoding same, and the use thereof as a therapeutic
agent for treatment of immune related disorder and/or infection,
and/or optionally the corresponding DNAs or vectors or cells
expressing same for use in immunotherapy.
[0045] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0046] As used herein the term "isolated" refers to a compound of
interest (for example a polynucleotide or a polypeptide) that is in
an environment different from that in which the compound naturally
occurs e.g. separated from its natural milieu such as by
concentrating a peptide to a concentration at which it is not found
in nature. "Isolated" includes compounds that are within samples
that are substantially enriched for the compound of interest and/or
in which the compound of interest is partially or substantially
purified.
[0047] An "immune cell" refers to any cell from the hemopoietic
origin including but not limited to T cells, B cells, monocytes,
dendritic cells, and macrophages.
[0048] As used herein, the term "polypeptide" refers to a chain of
amino acids of any length, regardless of modification (e.g.,
phosphorylation or glycosylation).
[0049] The term "immune related disease (or disorder or condition)"
as used herein should be understood to encompass any disease
disorder or condition selected from the group including but not
limited to autoimmune diseases, inflammatory disorders and immune
disorders associated with graft transplantation rejection, such as
acute and chronic rejection of organ transplantation, allogenic
stem cell transplantation, autologous stem cell transplantation,
bone marrow tranplantation, and graft versus host disease.
[0050] As used herein the term "inflammatory disorders" and/or
"inflammation", used interchangeably, includes inflammatory
abnormalities characterized by disregulated immune response to
harmful stimuli, such as pathogens, damaged cells, or irritants.
Inflammatory disorders underlie a vast variety of human diseases.
Non-immune diseases with etiological origins in inflammatory
processes include atherosclerosis, and ischaemic heart disease.
Examples of disorders associated with inflammation include: Chronic
prostatitis, Glomerulonephritis, Hypersensitivities, Pelvic
inflammatory disease, Reperfusion injury, Sarcoidosis, Vasculitis,
Interstitial cystitis, normocomplementemic urticarial vasculitis,
pericarditis, myositis, anti-synthetase syndrome, scleritis,
macrophage activation syndrome, Bechet's Syndrome, PAPA Syndrome,
Blau's Syndrome, gout, adult and juvenile Still's disease,
cryropyrinopathy, Muckle-Wells syndrome, familial cold-induced
auto-inflammatory syndrome, neonatal onset multisystemic
inflammatory disease, familial Mediterranean fever, chronic
infantile neurologic, cutaneous and articular syndrome, systemic
juvenile idiopathic arthritis, Hyper IgD syndrome, Schnitzler' s
syndrome, TNF receptor-associated periodic syndrome (TRAPSP),
gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis,
myocarditis, vasculitis, gastritis, gout, gouty arthritis, and
inflammatory skin disorders, selected from the group consisting of
psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and
acne.
[0051] The term "autoimmune disease" as used herein should be
understood to encompass any disease in which recognition of a
"self" antigen (self-reactivity) is at least a part of the disease
process. According to at least some embodiments of the invention,
the autoimmune diseases should be understood to encompass any
disease disorder or condition including one or more of, but not
limited to, multiple sclerosis, rheumatoid arthritis; psoriatic
arthritis, discoid lupus erythematosus, systemic lupus
erythematosus (SLE); ulcerative colitis; Crohn's disease; benign
lymphocytic angiitis, autoimmune lymphoproliferative syndrome,
sarcoidosis, autoimmune thrombocytopenic purpura, idiopathic
thrombocytopenic purpura, pure red cell aplasia, Sjogren's
syndrome, rheumatic disease, polymyalgia rheumatica, mixed
connective tissue disease, inflammatory rheumatism, degenerative
rheumatism, extra-articular rheumatism, juvenile arthritis,
juvenile rheumatoid arthritis, systemic juvenile idiopathic
arthritis, arthritis uratica, muscular rheumatism, chronic
polyarthritis, reactive arthritis, Reiter's syndrome, rheumatic
fever, relapsing polychondritis, Raynaud's phenomenon, vasculitis,
cryoglobulinemic vasculitis, ANCA-associated vasculitis, temporal
arteritis, giant cell arteritis, Takayasu arteritis, Behcet's
disease, antiphospholipid syndrome, myasthenia gravis, autoimmune
haemolytic anaemia, Guillain-Barre syndrome, chronic immune
polyneuropathy, chronic inflammatory demyelinating polyneuropathy,
autoimmune thyroiditis, insulin dependent diabetes mellitus, type I
diabetes, Addison's disease, membranous glomerulonephropathy,
polyglandular autoimmune syndromes, Goodpasture' s disease,
autoimmune gastritis, autoimmune atrophic gastritis, pernicious
anaemia, pemphigus, pemphigus vulgaris, cirrhosis, primary biliary
cirrhosis, idiopathic pulmonary fibrosis, myositis,
dermatomyositis, juvenile dermatomyositis, polymyositis,
fibromyositis, myogelosis, celiac disease, celiac sprue dermatitis,
immunoglobulin A nephropathy, Henoch-Schonlein purpura, Evans
syndrome, atopic dermatitis, psoriasis, psoriasis vulgaris,
psoriasis arthropathica, Graves' disease, Graves' ophthalmopathy,
scleroderma, systemic scleroderma, progressive systemic
scleroderma, diffuse scleroderma, localized scleroderma, Crest
syndrome, asthma, allergic asthma, allergy, primary biliary
cirrhosis, Hashimoto's thyroiditis, fibromyalgia, chronic fatigue
and immune dysfunction syndrome (CFIDS), primary myxedema,
sympathetic ophthalmia, autoimmune inner ear disease, autoimmune
uveitis, autoimmune chronic active hepatitis, collagen diseases,
ankylosing spondylitis, periarthritis humeroscapularis,
panarteritis nodosa, polyarteritis nodosa, chondrocalcinosis,
Wegener's granulomatosis, microscopic polyangiitis, chronic
urticaria, bullous skin disorders, pemphigoid, bullous pemphigoid,
cicatricial pemphigoid, vitiligo, atopic eczema, eczema, chronic
urticaria, autoimmune urticaria, normocomplementemic urticarial
vasculitis, hypocomplementemic urticarial vasculitis, alopecia
areata, alopecia universalis, alopecia totalis, Devic's disease,
pernicious anemia, childhood autoimmune hemolytic anemia,
idiopathic autoimmune hemolytic anemia, refractory or chronic
Autoimmune Cytopenias, Prevention of development of Autoimmune
Anti-Factor VIII Antibodies in Acquired Hemophilia A, Cold
agglutinin disease, Neuromyelitis Optica, Stiff Person Syndrome,
gingivitis, periodontitis, pancreatitis, myocarditis, gastritis,
gout, gouty arthritis, idiopathic pericarditis, anti-synthetase
syndrome, scleritis, macrophage activation syndrome, PAPA Syndrome,
Blau's Syndrome, adult and juvenile Still's disease, cryopyrin
associated periodic syndrome, Muckle-Wells syndrome, familial cold
auto-inflammatory syndrome, neonatal onset multisystem inflammatory
disease, chronic infantile neurologic cutaneous and articular
syndrome, familial Mediterranean fever, Hyper IgD syndrome,
Schnitzler' s syndrome, autoimmune retinopathy, age-related macular
degeneration, or TNF receptor-associated periodic syndrome
(TRAPS).
[0052] As used herein, "multiple sclerosis" comprises multiple
sclerosis or a related disease, and optionally refers to all types
and stages of multiple sclerosis, including, but not limited to:
benign multiple sclerosis, relapsing remitting multiple sclerosis,
secondary progressive multiple sclerosis, primary progressive
multiple sclerosis, progressive relapsing multiple sclerosis,
chronic progressive multiple sclerosis, transitional/progressive
multiple sclerosis, rapidly worsening multiple sclerosis,
clinically-definite multiple sclerosis, malignant multiple
sclerosis, also known as Marburg's Variant, and acute multiple
sclerosis. Optionally, "conditions relating to multiple sclerosis"
include, e.g., Devic's disease, also known as Neuromyelitis Optica;
acute disseminated encephalomyelitis, acute demyelinating optic
neuritis, demyelinative transverse myelitis, Miller-Fisher
syndrome, encephalomyelradiculoneuropathy, acute demyelinative
polyneuropathy, tumefactive multiple sclerosis and Balo's
concentric sclerosis.
[0053] As used herein, "rheumatoid arthritis" comprises rheumatoid
arthritis or a related disease and refers to all types and stages
of rheumatoid arthritis, including, but not limited to: rheumatoid
arthritis, gout and pseudo-gout, juvenile idiopathic arthritis,
juvenile rheumatoid arthritis, Still's disease, ankylosing
spondylitis, rheumatoid vasculitis. Optionally, conditions relating
to rheumatoid arthritis include, e.g., osteoarthritis, sarcoidosis,
Henoch-Schonlein purpura, Psoriatic arthritis, Reactive arthritis,
Spondyloarthropathy, septic arthritis, Haemochromatosis, Hepatitis,
vasculitis, Wegener's granulomatosis, Lyme disease, Familial
Mediterranean fever, Hyperimmunoglobulinemia D with recurrent
fever, TNF receptor associated periodic syndrome, and Enteropathic
arthritis associated with inflammatory bowel disease.
[0054] As used herein, "Uveitis" refers to all types and stages of
Uveitis, including, but not limited to: anterior uveitis (or
iridocyclitis), intermediate uveitis (pars planitis), posterior
uveitis (or chorioretinitis) and the panuveitic form.
[0055] As used herein, "inflammatory bowel disease" also refers to
a related disease and refers to all types and stages of
inflammatory bowel disease (IBD), including, but not limited to:
Crohn's disease and ulcerative colitis (UC). Optionally, conditions
relating to IBD include, e.g., Collagenous colitis, Lymphocytic
colitis, Ischaemic colitis, Diversion colitis, Behget's disease,
Indeterminate colitis.
[0056] As used herein, "psoriasis" also refers to a related disease
and refers to all types and stages of psoriasis, including, but not
limited to: Nonpustular Psoriasis including Psoriasis vulgaris and
Psoriatic erythroderma (erythrodermic psoriasis), Pustular
psoriasis including Generalized pustular psoriasis (pustular
psoriasis of von Zumbusch), Pustulosis palmaris et plantaris
(persistent palmoplantar pustulosis, pustular psoriasis of the
Barber type, pustular psoriasis of the extremities), Annular
pustular psoriasis, Acrodermatitis continua, Impetigo
herpetiformis. Optionally, conditions relating to psoriasis
include, e.g., drug-induced psoriasis, Inverse psoriasis, Napkin
psoriasis, Seborrheic-like psoriasis, Guttate psoriasis, Nail
psoriasis, Psoriatic arthritis.
[0057] As used herein, "type 1 diabetes" refers to all types and
stages of type 1 diabetes, including, but not limited to:
insulin-dependent diabetes mellitus, idiopathic diabetes, juvenile
type ldiabetes, maturity onset diabetes of the young, latent
autoimmune diabetes in adults, gestational diabetes. Conditions
relating to type 1 diabetes include, neuropathy including
polyneuropathy, mononeuropathy, peripheral neuropathy and
autonomicneuropathy; eye complications: glaucoma, cataracts,
retinopathy.
[0058] As used herein, "Sjogren's syndrome" refers to all types and
stages of Sjogren's syndrome, including, but not limited to:
Primary Sjogren's syndrome and Secondary Sjogren's syndrome.
Conditions relating to Sjogren's syndrome include connective tissue
disease, such as rheumatoid arthritis, systemic lupus
erythematosus, or scleroderma. Other complications include
pneumonia, polmunary fibrosis, interstitial nephritis, inflammation
of the tissue around the kidney's filters, glomerulonephritis,
renal tubular acidosis, carpal tunnel syndrome, peripheral
neuropathy, cranial neuropathy, primary biliary cirrhosis (PBC),
cirrhosis, Inflammation in the esophagus, stomach, pancreas, and
liver (including hepatitis), Polymyositis, Raynaud's phenomenon,
Vasculitis, Autoimmune thyroid problems, lymphoma.
[0059] As used herein, "systemic lupus erythematosus", refers to
all types and stages of systemic lupus erythematosus, including,
but not limited to discoid lupus, lupus arthritis, lupus
pneumonitis, lupus nephritis. Conditions relating to systemic lupus
erythematosus include osteoarticular tuberculosis, antiphospholipid
antibody syndrome, inflammation of various parts of the heart, such
as pericarditis, myocarditis, and endocarditis, Lung and pleura
inflammation, pleuritis, pleural effusion, chronic diffuse
interstitial lung disease, pulmonary hypertension, pulmonary
emboli, pulmonary hemorrhage, and shrinking lung syndrome, lupus
headache, Guillain-Barre syndrome, aseptic meningitis,
demyelinating syndrome, mononeuropathy, mononeuritis multiplex,
myasthenia gravis, myelopathy, cranial neuropathy, polyneuropathy,
vasculitis.
[0060] As used herein the term "infectious disorder and/or disease"
and/or "infection", used interchangeably, includes any disorder,
disease and/or condition caused by presence and/or growth of
pathogenic biological agent in an individual host organism. As used
herein the term "infection" comprises the disorder, disease and/or
condition as above, exhibiting clinically evident illness (i.e.,
characteristic medical signs and/or symptoms of disease) and/or
which is asymtomatic for much or all of it course. As used herein
the term "infection" also comprises disorder, disease and/or
condition caused by persistence of foreign antigen that lead to
exhaustion T cell phenotype characterized by impaired functionality
which is manifested as reduced proliferation and cytokine
production. As used herein the term "infectious disorder and/or
disease" and/or "infection", further includes any of the below
listed infectious disorders, diseases and/or conditions, caused by
a bacterial infection, viral infection, fungal infection and /or
parasite infection.
[0061] As used herein the term "viral infection" comprises any
infection caused by a virus, optionally including but not limited
to Retroviridae (e.g., human immunodeficiency viruses, such as
HIV-1 or HIV-2, acquired immune deficiency (AIDS) also referred to
as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates,
such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A
virus; enteroviruses, human coxsackie viruses, rhinoviruses,
echoviruses);
[0062] Calciviridae (e.g., strains that cause gastroenteritis);
Togaviridae (e.g., equine encephalitis viruses, rubella viruses);
Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow
fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae
(e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae
(e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza
viruses, mumps virus, measles virus, respiratory syncytial virus);
Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g.,
Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses);
Arena viridae (hemorrhagic fever virus); Reoviridae (e.g.,
reoviruses, orbiviruses and rotaviruses); Birnaviridae;
Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses);
Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae
(most adenoviruses); Herperviridae (herpes simplex virus (HSV) 1
and 2, varicella zoster virus, cytomegalovirus (CMV), herpes
viruses); Poxviridae (variola virsues, vaccinia viruses, pox
viruses); and Iridoviridae (e.g., African swine fever virus); and
unclassified viruses (e.g., the etiological agents of Spongiform
encephalopathies, the agent of delta hepatitides (thought to be a
defective satellite of hepatitis B virus), the agents of non-A,
non-B hepatitis (class 1-internally transmitted; class
2-parenterally transmitted (i.e., Hepatitis C); Norwalk and related
viruses, and astroviruses) as well as Severe acute respiratory
syndrome virus and respiratory syncytial virus (RSV).
[0063] As used herein the term "fungal infection" comprises any
infection caused by a fungi, optionally including but not limited
to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides
immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida
albicans. As used herein the term "parasite infection" comprises
any infection caused by a parasite, optionally including but not
limited to protozoa, such as Amebae, Flagellates, Plasmodium
falciparum, Toxoplasma gondii, Ciliates, Coccidia, Microsporidia,
Sporozoa; helminthes, Nematodes (Roundworms), Cestodes (Tapeworms),
Trematodes (Flukes), Arthropods, and aberrant proteins known as
prions.
[0064] An infectious disorder and/or disease caused by bacteria may
optionally comprise one or more of Sepsis, septic shock, sinusitis,
skin infections, pneumonia, bronchitis, meningitis, Bacterial
vaginosis, Urinary tract infection (UCI), Bacterial
gastroenteritis, Impetigo and erysipelas, Erysipelas, Cellulitis,
anthrax, whooping cough, lyme disease, Brucellosis, enteritis,
acute enteritis, Tetanus, diphtheria, Pseudomembranous colitis, Gas
gangrene, Acute food poisoning, Anaerobic cellulitis, Nosocomial
infections, Diarrhea, Meningitis in infants, Traveller's diarrhea,
Hemorrhagic colitis, Hemolytic-uremic syndrome, Tularemia, Peptic
ulcer, Gastric and Duodenal ulcers, Legionnaire's Disease, Pontiac
fever, Leptospirosis, Listeriosis, Leprosy (Hansen's disease),
Tuberculosis, Gonorrhea, Ophthalmia neonatorum, Septic arthritis,
Meningococcal disease including meningitis, Waterhouse-Friderichsen
syndrome, Pseudomonas infection, Rocky mountain spotted fever,
Typhoid fever type salmonellosis, Salmonellosis with
gastroenteritis and enterocolitis, Bacillary dysentery/Shigellosis,
Coagulase-positive staphylococcal infections: Localized skin
infections including Diffuse skin infection (Impetigo), Deep
localized infections, Acute infective endocarditis, Septicemia,
Necrotizing pneumonia, Toxinoses such as Toxic shock syndrome and
Staphylococcal food poisoning, Cystitis, Endometritis, Otitis
media, Streptococcal pharyngitis, Scarlet fever, Rheumatic fever,
Puerperal fever, Necrotizing fasciitis, Cholera, Plague (including
Bubonic plague and Pneumonic plague), as well as any infection
caused by a bacteria selected from but not limited to Helicobacter
pyloris, Boreliai burgdorferi, Legionella pneumophilia,
Mycobacteria sps (e.g., M. tuberculosis, M. avium, M.
Intracellulare, M. kansaii, M gordonae), Staphylococcus aureus,
Neisseria gonorrhoeae, Neisseria meningitidis, Listeria
monocytogenes, Streptococcus pyogenes (Group A Streptococcus),
Streptococcus agalactiae (Group B Streptococcus), Streptococcus
(viridans group), Streptococcus faecalis, Streptococcus bovis,
Streptococcus (anaerobic sps.), Streptococcus pneumoniae,
pathogenic Campylobacter sp., Enterococcus sp Haemophilus
influenzae, Bacillus antracis, corynebacterium diphtheriae,
corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium
perfringers, Clostridium tetani, Enterobacter erogenes, Klebsiella
pneuomiae, Pasturella multicoda, Bacteroides sp., Fusobacterium
nucleatum, Sreptobacillus moniliformis, Treponema pallidium,
Treponema pertenue, Leptospira, and Actinomeyces israelli.
[0065] Non limiting examples of infectious disorder and/or disease
caused by virus is selected from the group consisting of but not
limited to acquired immune deficiency (AIDS), West Nile
encephalitis, coronavirus infection, rhinovirus infection,
influenza, dengue, hemorrhagic fever; an otological infection;
severe acute respiratory syndrome (SARS), acute febrile
pharyngitis, pharyngoconjunctival fever, epidemic
keratoconjunctivitis, infantile gastroenteritis, infectious
mononucleosis, Burkitt lymphoma, acute hepatitis, chronic
hepatitis, hepatic cirrhosis, hepatocellular carcinoma, primary
HSV-1 infection, (gingivostomatitis in children, tonsillitis &
pharyngitis in adults, keratoconjunctivitis), latent HSV-1
infection (herpes labialis, cold sores), aseptic meningitis,
Cytomegalovirus infection, Cytomegalic inclusion disease, Kaposi
sarcoma, Castleman disease, primary effusion lymphoma, influenza,
measles, encephalitis, postinfectious encephalomyelitis, Mumps,
hyperplastic epithelial lesions (common, flat, plantar and
anogenital warts, laryngeal papillomas, epidermodysplasia
verruciformis), croup, pneumonia, bronchiolitis, Poliomyelitis,
Rabies, bronchiolitis, pneumonia, German measles, congenital
rubella, Hemorrhagic Fever, Chickenpox, Dengue, Ebola infection,
Echovirus infection, EBV infection, Fifth Disease, Filovirus,
Flavivirus, Hand, foot & mouth disease, Herpes Zoster Virus
(Shingles), Human Papilloma Virus Associated Epidermal Lesions,
Lassa Fever, Lymphocytic choriomeningitis, Parainfluenza Virus
Infection, Paramyxovirus, Parvovirus B19 Infection, Picornavirus ,
Poxviruses infection, Rotavirus diarrhea, Rubella, Rubeola,
Varicella, Variola infection. An infectious disorder and/or disease
caused by fungi optionally includes but is not limited to Allergic
bronchopulmonary aspergillosis, Aspergilloma, Aspergillosis,
Basidiobolomycosis, Blastomycosis, Candidiasis, Chronic pulmonary
aspergillosis, Chytridiomycosis, Coccidioidomycosis,
Conidiobolomycosis, Covered smut (barley), Cryptococcosis,
Dermatophyte, Dermatophytid, Dermatophytosis, Endothrix,
Entomopathogenic fungus, Epizootic lymphangitis, Epizootic
ulcerative syndrome, Esophageal candidiasis, Exothrix, Fungemia,
Histoplasmosis, Lobomycosis, Massospora cicadina, Mycosis,
Mycosphaerella fragariae, Myringomycosis, Paracoccidioidomycosis,
Pathogenic fungi, Penicilliosis, Thousand cankers disease, Tinea,
Zeaspora, Zygomycosis.Non limiting examples of infectious disorder
and/or disease caused by parasites is selected from the group
consisting of but not limited to Acanthamoeba, Amoebiasis,
Ascariasis, Ancylostomiasis, Anisakiasis, Babesiosis,
Balantidiasis, Baylisascariasis, Blastocystosis, Candiru, Chagas
disease, Clonorchiasis, Cochliomyia, Coccidia, Chinese Liver Fluke
Cryptosporidiosis, Dientamoebiasis, Diphyllobothriasis, Dioctophyme
renalis infection, Dracunculiasis, Echinococcosis, Elephantiasis,
Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis,
Giardiasis, Gnathostomiasis, Hymenolepiasis, Halzoun Syndrome,
Isosporiasis, Katayama fever, Leishmaniasis, lymphatic filariasis,
Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis,
Primary amoebic meningoencephalitis, Parasitic pneumonia,
Paragonimiasis, Scabies, Schistosomiasis, Sleeping sickness,
Strongyloidiasis, Sparganosis, Rhinosporidiosis, River blindness,
Taeniasis (cause of Cysticercosis), Toxocariasis, Toxoplasmosis,
Trichinosis, Trichomoniasis, Trichuriasis, Trypanosomiasis,
Tapeworm infection.
[0066] A preferred example of infectious disease is a disease
caused by any of hepatitis B, hepatitis C, infectious
mononucleosis, EBV, cytomegalovirus, AIDS, HIV-1, HIV-2,
tuberculosis, malaria and schistosomiasis.
[0067] As used herein, the term "vaccine" refers to a biological
preparation that improves immunity to a particular disease, wherein
the vaccine includes an antigen, such as weakened or killed forms
of pathogen, its toxins or one of its surface proteins, against
which immune responses are elicited. A vaccine typically includes
an adjuvant as immune potentiator to stimulate the immune system.
As used herein, the term "therapeutic vaccine" and/or "therapeutic
vaccination" refers to a vaccine used to treat ongoing disease,
such as infectious disease.
[0068] As used herein, the term "adjuvant" refers to an agent used
to stimulate the immune system and increase the response to a
vaccine, without having any specific antigenic effect in
itself.
[0069] As used herein, a "costimulatory polypeptide" or
"costimulatory molecule" is a polypeptide that, upon interaction
with a cell-surface molecule on T cells, modulates T cell
responses.
[0070] As used herein, a "costimulatory signaling" is the signaling
activity resulting from the interaction between costimulatory
polypeptides on antigen presenting cells and their receptors on T
cells during antigen-specific T cell responses. Without wishing to
be limited by a single hypothesis, the antigen-specific T cell
response is believed to be mediated by two signals: 1) engagement
of the T cell Receptor (TCR) with antigenic peptide presented in
the context of MHC (signal 1), and 2) a second antigen-independent
signal delivered by contact between different costimulatory
receptor/ligand pairs (signal 2). Without wishing to be limited by
a single hypothesis, this "second signal" is critical in
determining the type of T cell response (activation vs inhibition)
as well as the strength and duration of that response, and is
regulated by both positive and negative signals from costimulatory
molecules, such as the B7 family of proteins.
[0071] As used herein, the term "B7" polypeptide means a member of
the B7 family of proteins that costimulate T cells including, but
not limited to B7-1, B7-2, B7-DC, B7-H5, B7-H1, B7-H2, B7-H3,
B7-H4, B7-H6, B7-S3 and biologically active fragments and/or
variants thereof. Representative biologically active fragments
include the extracellular domain or fragments of the extracellular
domain that costimulate T cells.
[0072] As used herein, "inflammatory molecules" refers to molecules
that induce inflammatory responses (directly or indirectly)
including, but not limited to, cytokines and metalloproteases such
as including, but not limited to, IL-1beta, TNF-alpha, TGF-beta,
IFN-gamma, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.
[0073] As used herein, the term "vaccine" refers to a biological
preparation that improves immunity to a particular disease, wherein
the vaccine includes an antigen, such as weakened or killed forms
of pathogen, its toxins or one of its surface proteins, against
which immune responses are elicited. A vaccine typically includes
an adjuvant as immune potentiator to stimulate the immune system.
As used herein, the term "therapeutic vaccine" and/or "therapeutic
vaccination" refers to a vaccine used to treat ongoing disease,
such as infectious disease.
[0074] As used herein, the term "adjuvant" refers to an agent used
to stimulate the immune system and increase the response to a
vaccine, without having any specific antigenic effect in
itself.
[0075] As used herein, a "vector" is a replicon, such as a plasmid,
phage, or cosmid, into which another DNA segment may be inserted so
as to bring about the replication of the inserted segment. The
vectors described herein can be expression vectors. As used herein,
an "expression vector" is a vector that includes one or more
expression control sequences
[0076] As used herein, an "expression control sequence" is a DNA
sequence that controls and regulates the transcription and/or
translation of another DNA sequence.
[0077] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual or intended function. Thus, two different polypeptides
operably linked together retain their respective biological
functions while physically linked together.
[0078] As used herein, "valency" refers to the number of binding
sites available per molecule.
[0079] As used herein, a "variant" polypeptide contains at least
one amino acid sequence alteration as compared to the amino acid
sequence of the corresponding wild-type polypeptide.
[0080] As used herein, "conservative" amino acid substitutions are
substitutions wherein the substituted amino acid has similar
structural or chemical properties. As used herein, the term "host
cell" refers to prokaryotic and eukaryotic cells into which a
recombinant vector can be introduced.
[0081] As used herein, "transformed" and "transfected" encompass
the introduction of a nucleic acid (e.g. a vector) into a cell by a
number of techniques known in the art.
[0082] As used herein, the terms "immunologic", "immunological" or
"immune" response is the development of a beneficial humoral
(antibody mediated) and/or a cellular (mediated by antigen-specific
T cells or their secretion products) response directed against a
peptide in a recipient patient. Such a response can be an active
response induced by administration of immunogen or a passive
response induced by administration of antibody or primed T-cells.
Without wishing to be limited by a single hypothesis, a cellular
immune response is elicited by the presentation of polypeptide
epitopes in association with Class I or Class II MHC molecules to
activate antigen-specific CD4+ T helper cells and/or CD8+ cytotoxic
T cells. The response may also involve activation of monocytes,
macrophages, NK cells, basophils, dendritic cells, astrocytes,
microglia cells, eosinophils, activation or recruitment of
neutrophils or other components of innate immunity. The presence of
a cell-mediated immunological response can be determined by
proliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocyte)
assays. The relative contributions of humoral and cellular
responses to the protective or therapeutic effect of an immunogen
can be distinguished by separately isolating antibodies and T-cells
from an immunized syngeneic animal and measuring protective or
therapeutic effect in a second subject.
[0083] An "immunogenic agent" or "immunogen" is capable of inducing
an immunological response against itself on administration to a
mammal, optionally in conjunction with an adjuvant.
[0084] As used herein, the term "C1ORF32" refers to the protein
encoded by any one of the H19011_1_T8 (SEQ ID NO:1), H19011_1_T9
(SEQ ID NO:2) transcripts reported herein, particularly to proteins
as set forth in any one of H19011_1_P8 (SEQ ID NO:4),
H19011_1_P8_V1 (SEQ ID NO:5), H19011_1_P9 (SEQ ID NO:6) or
H19011_1_P9_V1 (SEQ ID NO:34), variants and fragments thereof,
which can have therapeutic effect on immune related disorder and/or
infection.
[0085] Fragments of C1ORF32 polypeptides
[0086] As used herein the term "soluble C1ORF32" or "soluble
C1ORF32 proteins/molecules" refers to fragments of C1ORF32 that
include some or all of the IgV domain of the C1ORF32 polypeptide,
and lack some or all of the intracellular and/or transmembrane
domains, wherein said fragments retain a biological activity of
inhibition of T cell activation.
[0087] The soluble C1ORF32 molecules used in the methods of the
invention may or may not include a signal (leader) peptide
sequence.
[0088] Various fragments are given in Table 1 below. "N-term"
refers to the N-terminus, so "first N-term" refers to the
N-terminus. Particular sequences of interest, according to at least
some embodiments of the present invention, include but are not
limited to SEQ ID NOs: 29, 30, 41-105, and/or 45, 64, or 96. It
should be noted that one or more activities and/or functions
ascribed herein to any of SEQ ID NOs: 29, 30, 41-105 are also
applicable to any of SEQ ID NOs: 45, 64 or 96.
[0089] The below table describes a number of sequences with
abbreviations, which are defined as follows. The terms "first
N-term" and "second N-term" refer to the two potential start points
of the IgV domain, as shown in FIG. 1. The terms "#1 option cut
point" (and "#2 option cut point") refer to different possible
C-terminal end points of the soluble protein--also as shown in FIG.
1. FIG. 1 shows five different possible C-terminal end points,
which are as follows: the end of the IgV domain, #1 option cut
point, #2 option cut point, the end of the produced protein, and
the position before the start of the predicted TM (transmembrane
region, which occurs two amino acids after the end of the produced
protein). Reference to "+1" or "-1" from a reference point indicate
one amino acid after or before that point, respectively, with
larger numbers indicating a greater number of amino acids after or
before that point as stated below.
[0090] The right-most column relates to the SEQ ID NO of the amino
acid sequence fused to an
[0091] Fc sequence (hIgG1 C220S-SEQ ID NO 115) to form a fusion
protein; for example, the amino acid sequence having SEQ ID NO:14,
given as fused the above Fc sequence, has SEQ ID NO:116 as a fusion
protein.
TABLE-US-00002 TABLE 1 amino acid sequences discussed in the
present application SEQ ID NO with Fc (hIgG1 C220S- SEQ SEQ ID ID
115) NO: Amino acid sequence Description fusion 14
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV residues 21-184 116
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE of
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG H19011_1_P8
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD LEGKNEGSLGLLVLGRTGLLADLLPSFAVEIM
15 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV residues 21-169 117
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE of
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG H19011_1_P9
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD LEGKNEGSLGLLVLEWV 19
MDRVLLRWISLFWLTAMVEGLQVTVPDKKKVAM residues 1-184 118
LFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRM f the sequence
GESLGMSSTRAQSLSKRNLEWDPYLDCLDSRRT H19011_1_P8_V1
VRVVASKQGSTVTLGDFYRGREITIVHDADLQI GKLMWGDSGLYYCIITTPDDLEGKNEDSVELLV
LGRTGLLADLLPSFAVEIM 28 MDRVLLRWISLFWLTAMVEGLQVTVPDKKKVAM residues
119 LFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRM 1-169 of
GESLGMSSTRAQSLSKRNLEWDPYLDCLDSRRT H19011_1_P9
VRVVASKQGSTVTLGDFYRGREITIVHDADLQI GKLMWGDSGLYYCIITTPDDLEGKNEGSLGLLV
LEW 29 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV residues 120
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE 21-167 of
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG HI9011_1_P8_V1
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD LEGKNEDSVELLVLG 30
CHFSTSSHQPAVVQWKFKSYCQDRMGESLGMSS residues 121
TRAQSLSKRNLEWDPYLDCLDSRRTVRVVASKQ 42-145 of
GSTVTLGDFYRGREITIVHDADLQIGKLMWGDS (H19011_1_P8 GLYYC from first C
residue to last C residue) 35 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV
H19011_1_P 8_V1_ 122 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE
from_21_to_184 WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to
LEGKNEDSVELLVLGRTGLLADLLPSFAVEIM end of produced ECD -2 from end of
predicted ECD) 36 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV residues 123
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE 21-169 of
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG H19011_1_P9_V1
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD LEGKNEDSVELLVLEWV 37
MDRVLLRWISLFWLTAMVEGLQVTVPDKKKVAM residues 1-184 of 124
LFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRM H19011_1_P8
GESLGMSSTRAQSLSKRNLEWDPYLDCLDSRRT VRVVASKQGSTVTLGDFYRGREITIVHDADLQI
GKLMWGDSGLYYCIITTPDDLEGKNEGSLGLLV LGRTGLLADLLPSFAVSIM 40
MDRVLLRWISLFWLTAMVEGLQVTVPDKKKVAM residues 1-169 of 125
LFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRM H19011_1_P9_V1
GESLGMSSTRAQSLSKRNLEWDPYLDCLDSRRT VRVVASKQGSTVTLGDFYRGREITIVHDADLQI
GKLMWGDSGLYYCIITTPDDLEGKNEDSVELLV LEWV 41
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV residues 126
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE 21-167 of
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG H19011_1_P8
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD (first N- LEGKNEGSLGLLVLG term to
end of IgV) 42 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV HI 9011_1_P8_ 127
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE V1_from_21_
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG to_164 (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to -3 LEGKNEDSVELL from end
of IgV) 43 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_ 128
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_165
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-term
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD to -2 from LEGKNEDSVELLV end of
IgV) 44 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_ 129
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_166
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to -1 LEGKNEDSVELLVL from
end of IgV) 45 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_
130 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_184
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to end of
LEGKNEDSVELLVLGRTGLLADLLPSGGVEIM produced ECD with FA- >GG
mutation) 46 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_ 131
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_168
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +1 LEGKNEDSVELLVLGR after
end of IgV) 47 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_
132 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_169
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +2 LEGKNEDSVELLVLGRT
after end of IgV) 48 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV
H19011_1_P8_V1_ 133 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE
from_21_to_170 WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +3 LEGKNEDSVELLVLGRTG
after end of IgV - 3 before #1 option cut point) 49
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_p8_V1_ 134
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_171
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to -2 LEGKNEDSVELLVLGRTGL
before #1 option cut point) 50 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV
H19011_1_P8_V1_ 135 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE
from_21_to_172 WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to -1 LEGKNEDSVELLVLGRTGLL
before #1 option cut point) 51 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV
H19011_1_P8_V1_ 136 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE
from_21_to_173 WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to #1 LEGKNEDSVELLVLGRTGLLA
option cut point) 52 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV
H19011_1_p8_V1_ 137 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE
from_21_to_174 WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +1 LEGKNEDSVELLVLGRTGLLAD
after #1 option cut point) 53 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV
H19011_1_P8_V1_ 138 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE
from_21_to_175 WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +2
LEGKNEDSVELLVLGRTGLLADL after #1 option cut point -3 before #2
option cut point) 54 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV
HI9011_1_P8_V1_ 139 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE
from_21_to_176 WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +3
LEGKNEDSVELLVLGRTGLLADLL after #1 option cut point -2 before #2
option cut point) 55 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV HI
9011_1_P8_V1_ 140 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_177
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to -1
LEGKNEDSVELLVLGRTGLLADLLP before #2 option cut point) 56
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV HI9011_1_P8_V1_ 141
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_178
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to #2
LEGKNEDSVELLVLGRTGLLADLLPS option cut point 57
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_ 142
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_179
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +1
LEGKNEDSVELLVLGRTGLLADLLPSF after #2 option cut point) 58
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_ 143
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_180
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +2
LEGKNEDSVELLVLGRTGLLADLLPSFA after #2 option cut point) 59
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_ 144
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_181
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first M-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +3
LEGKNEDSVELLVLGRTGLLADLLPSFAV after #2 option cut point -3 from end
of produced ECD) 60 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV
H19011_1_P8_V1_ 145 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE
from_21_to_182 WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-term
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD to -2 from
LEGKNEDSVELLVLGRTGLLADLLPSFAVE end of produced ECD) 61
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_ 146
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_183
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to -1
LEGKNEDSVELLVLGRTGLLADLLPSFAVEI from end of produced ECD -3 from
end of predicted
ECD) 62 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1 147
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from 21 to 186
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to end of
LEGKNEDSVELLVLGRTGLLADLLPSFAVEIMP predicted ECD) E 63
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_ 148
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_185
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +1
LEGKNEDSVELLVLGRTGLLADLLPSFAVEIMP after produced ECD -1 from end of
predicted ECD) 64 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV HI9011_1_P8_V1_
149 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_184
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to end of
LEGKNEDSVELLVLGRTGLLADLLPSGAVEIM produced ECD with FA- >GA
mutation) 65 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV HI9011_1_P8_V1_ 150
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_187
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-term
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD to +3 after
LEGKNEDSVELLVLGRTGLLADLLPSFAVEIMP produced EW ECD +1 in predicted
TM) 66 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_ 151
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_188
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +2 in
LEGKNEDSVELLVLGRTGLLADLLPSFAVEIMP predicted TM) EWV 67
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_ 152
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_2
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG 1_to_189
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD (first N-
LSGKNEDSVELLVLGRTGLLADLLPSFAVEIMP term to +3 in EWVF predicted TM)
68 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_ 153
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_2
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV 7_to_164
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE (second N- DSVELL term to -3 from
end of IgV) 69 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_
154 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_165
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to -2 DSVELLV from end of
IgV) 70 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_ 155
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_166
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to -1 DSVELLVL from end of
IgV) 71 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_ 156
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_167
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to end DSVELLVLG of IgV) 72
DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_ 157
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_168
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +1 DSVELLVLGR after end
of IgV) 73 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_ 158
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_169
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +2 DSVELLVLGRT after end
of IgV) 74 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_ 159
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_170
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +3 DSVELLVLGRTG after end
of IgV -3 before +190 1 option cut point) 75
DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_ 160
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_171
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to -2 DSVELLVLGRTGL before
+190 1 option cut point) 76 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H1
9011_1_P8_V1_ 161 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_172
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to -1 DSVELLVLGRTGLL before
+190 1 option cut point) 77 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H1
9011_1_P8_V1_ 162 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_173
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +190 1 DSVELLVLGRTGLLA
option cut point) 78 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK
H19011_1_P8_V1_ 163 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD
from_27_to_174 CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +1 DSVELLVLGRTGLLAD after
+190 1 option cut point) 79 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK
H19011_1_P8_V1_ 164 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD
from_27_to_175 CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +2 after
DSVELLVLGRTGLLADL #190 1 option cut point -3before #190 2 option
cut point) 80 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK HI9011_1_P8_V1_ 165
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_176
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +3 DSVELLVLGRTGLLADLL
after #190 1 option cut point -2 before #190 2 option cut point) 81
DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_ 166
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_177
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to -1 DSVELLVLGRTGLLADLLP
before #190 2 option cut point) 82
DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK HI9011_1_P8_V1_ 167
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_178
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to #190 2
DSVELLVLGKTGLLADLLPS option cut point) 83
DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK HI9011_1_P8_V1_ 168
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_179
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +1 DSVELLVLGRTGLLADLLPSF
after #190 2 option cut point) 84 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK
H19011_1_P8_V1_ 169 SYCQDRMGSSLGMSSTRAQSLSKRNLEWDPYLD
from_27_to_180 CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +2 DSVELLVLGRTGLLADLLPSFA
after #190 2 option cut point) 85 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK
H19011_1_P8_V1_ 170 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD
from_27_to_181 CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +3
DSVELLVLGRTGLLADLLPSFAV after #190 2 option cut point -3 from end
of produced ECD) 86 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK
H19011_1_P8_V1_ 171 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD
from_27_to_182 CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to -2
DSVELLVLGRTGLLADLLPSFAVE from end of produced ECD) 87
DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_ 172
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_183
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to -1
DSVELLVLGRTGLLADLLPSFAVEI from end of produced ECD -3 from end of
predicted ECD) 88 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H1901
1_1_P8_V1_ 173 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_184
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to end of
DSVELLVLGRTGLLADLLPSFAVEIM produced ECD -2 from end of predicted
ECD) 89 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK HI9011_1_P8_V1_ 174
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_185
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +1
DSVELLVLGRTGLLADLLPSFAVEIMP after end of produced ECD -1 from end
of predicted ECD) 90 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK
H19011_1_P8_V1_ 175 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD
from_27_to_186 CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +2
DSVELLVLGRTGLLADLLPSFAVEIMPE after end of produced ECD end of
predicted ECD) 91 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK HI9011_1_P8_V1_
176 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_187
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +2
DSVELLVLGRTGLLADLLPSFAVEIMPEW after end of produced ECD +1 in
predicted TM)
92 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_ 177
SYCQDRMGSSLGMSSTRAQSLSKRNLEWDPYLD from_27_to_188
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +2 in
DSVELLVLGRTGLLADLLPSFAVEIMPEWV predicted TM) 93
DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_ 178
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_2
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV 7_to_189
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE (second N-
DSVELLVLGRTGLLADLLPSFAVEIMPEWVF term to +3 in predicted TM) 94
LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P9_V1_ 179
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_167
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to -2 LEGKNEDSVELLVLE from
end of predicted ECD) 95 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV
H19011_1_P9_V1_ 180 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE
from_21_to_168 WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to -1 LEGKNEDSVELLVLEW from
end of predicted ECD) 96 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV
H19011_1_P8_V1_ 181 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE
from_21_to_184 WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to end of
LEGKNEDSVELLVLGRTGLLADLLPSAAVEIM produced ECD with FA- >AA
mutation) 97 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P9_V1_ 182
VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_170
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +1 LEGKNEDSVELLVLEWVF
predicted TM) 98 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P9_V1_
183 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_171
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +2 LEGKNEDSVELLVLEWVFV
predicted TM) 99 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P9_V1_
184 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE from_21_to_172
WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG (first N-
REITIVHDADLQIGKLMWGDSGLYYCIITTPDD term to +3 LEGKNEDSVELLVLEWVFVG
predicted TM) 100 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P9_V1_
185 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_167
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to -2 DSVELLVLE from end of
predicted ECD) 101 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK
H19011_1_P9_V1_ 186 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD
from_27_to_168 CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to -1 DSVELLVLEW from end of
predicted ECD) 102 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK
H19011_1_P9_V1_ 187 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD
from_27_to_169 CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to end of DSVELLVLEWV
predicted ECD) 103 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK
H19011_1_P9_V1_ 188 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD
from_27_to_170 CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +1 DSVELLVLEWVF predicted
TM) 104 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P9_V1_ 189
SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_171
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +2 DSVELLVLEWVFV
predicted TM) 105 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P9_V1_
190 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD from_27_to_172
CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV (second N-
HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE term to +3 DSVELLVLEWVFVG
predicted TM)
[0092] In particular, the fragments of the extracellular domain of
C1ORF32 can include any sequence corresponding to any portion of or
comprising the IgV domain of the extracellular domain of C1ORF32,
having any sequence corresponding to residues of H19011_1_P8 (SEQ
ID NO:4) starting from any position between 18 and 31 and ending at
any position between 157 and 175 or corresponding to residues of
H19011_1_P8_V1 (SEQ ID NO:5) starting from any position between 18
and 31 and ending at any position between 157 and 175, or
corresponding to residues of H19011_1_P9 (SEQ ID NO:6) starting
from any position between 18 and 31 and ending at any position
between 159 and 172, or corresponding to residues of H19011_1_P9_V1
(SEQ ID NO:34) starting from any position between 18 and 31 and
ending at any position between 159 and 172. The base sequences as
given above are without a signal peptide.
[0093] The C1ORF32 proteins contain an immunoglobulin domain within
the extracellular domain, the IgV domain (or V domain), which is
related to the variable domain of antibodies. The IgV domain may be
responsible for receptor binding, by analogy to the other B7 family
members. The Ig domain of the extracellular domain includes one
disulfide bond formed between intradomain cystein residues, as is
typical for this fold and may be important for structure-function.
In SEQ ID NO: 4 these cysteines are located at residues 42 and
145.
[0094] In one embodiment, the first fusion partner is a soluble
fragment of C1ORF32. Without wishing to be limited by a single
hypothesis, it is believed that useful fragments are those that
retain the ability to bind to their natural receptor or receptors
and/or retain the ability to inhibit T cell activation. A C1ORF32
polypeptide that is a fragment of full-length C1ORF32 typically has
at least 20 percent, 30 percent, 40 percent, 50 percent, 60
percent, 70 percent, 80 percent, 90 percent, 95 percent, 98
percent, 99 percent, 100 percent, or even more than 100 percent of
the ability to bind its natural receptor(s) and/or of the ability
to inhibit T cell activation as compared to full-length C1ORF32.
Soluble C1ORF32 polypeptide fragments are fragments of C1ORF32
polypeptides that may be shed, secreted or otherwise extracted from
the producing cells. In other embodiments, the soluble fragments of
C1ORF32 polypeptides include fragments of the C1ORF32 extracellular
domain that retain C1ORF32 biological activity, such as fragments
that retain the ability to bind to their natural receptor or
receptors and/or retain the ability to inhibit T cell activation.
The extracellular domain can include 1, 2, 3, 4, or 5 contiguous
amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5
contiguous amino acids from the signal sequence. Alternatively, the
extracellular domain can have 1, 2, 3, 4, 5 or more amino acids
removed from the C-terminus, N-terminus, or both.
[0095] In some embodiments the extracellular domain is only the IgV
domain as set forth in SEQ ID NO: 29, or fragments or variants
thereof, or the region between the conserved cysteines of the IgV
domain which are located at residues 42 and 145 of the full-length
protein SEQ ID NO:4, corresponding to the sequence set forth in SEQ
ID NO: 30:
CHFSTSSHQPAVVQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLDCLDSRR
TVRVVASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDSGLYYC. In particular, the
fragments of the IgV domain can include any sequence corresponding
to residues of H19011_1_P8 (SEQ ID NO:4) starting from any position
between 18 and 31 and ending at any position between 157 and 175 or
corresponding to residues of H19011_1_P8_V1 (SEQ ID NO:5) starting
from any position between 18 and 31 and ending at any position
between 157 and 175, or corresponding to residues of H19011_1_P9
(SEQ ID NO:6) starting from any position between 18 and 31 and
ending at any position between 159 and 172 , or corresponding to
residues of H19011_1_P9_V1 (SEQ ID NO:34) starting from any
position between 18 and 31 and ending at any position between 159
and 172.
[0096] Generally, the C1ORF32 polypeptide fragments are expressed
from nucleic acids that include sequences that encode a signal
sequence. The signal sequence is generally cleaved from the
immature polypeptide to produce the mature polypeptide lacking the
signal sequence. The signal sequence of C1ORF32 can be replaced by
the signal sequence of another polypeptide using standard molecule
biology techniques to affect the expression levels, secretion,
solubility, or other property of the polypeptide. The signal
sequence that is used to replace the C1ORF32 signal sequence can be
any known in the art.
[0097] In a further embodiment, the fusion protein includes the
extracellular domain of C1ORF32, or a fragment thereof fused to an
Ig Fc region. Recombinant IgC1ORF32 polypeptides, fragments or
fusion proteins thereof fusion proteins can be prepared by fusing
the coding region of the extracellular domain of C1ORF32 or a
fragment thereof to the Fc region of human IgG1 or mouse IgG2a, as
described previously (Chapoval, et al., Methods MoI. Med,
45:247-255 (2000)).
[0098] Variants of C1ORF32 polypeptides
[0099] Useful variants of such C1ORF32 polypeptides include those
that increase biological activity, as indicated by any of the
assays described herein, or that increase half life or stability of
the protein. Soluble C1ORF32 polypeptides and C1ORF32 fragments, or
fusions thereof having C1ORF32 activity, can be engineered to
increase biological activity. In a further embodiment, the C1ORF32
polypeptide or fusion protein has been modified with at least one
amino acid substitution, deletion, or insertion that increases the
binding of the molecule to an immune cell, for example a T cell,
and transmits an inhibitory signal into the T cell.
[0100] Other optional variants are those C1ORF32 polypetpides that
are engineered to selectively bind to one type of T cell versus
other immune cells. For example, the C1ORF32 polypeptide can be
engineered to bind optionally to Tregs, Th0, Th1, Th17, Th2 or Th22
cells. Preferential binding refers to binding that is at least 10%,
20%, 30%, 40%, 50%, 60%f 70%, 80%, 90%, 95%, or greater for one
type of cell over another type of cell.
[0101] Still other variants of C1ORF32 can be engineered to have
reduced binding to immune cells relative to wildtype C1ORF32. These
variants can be used in combination with variants having stronger
binding properties to modulate the immune response with a moderate
impact. Also optionally, soluble C1ORF32 polypeptides and C1ORF32
fragments, or fusions thereof having C1ORF32 activity, can be
engineered to have an increased half-life relative to wildtype.
These variants typically are modified to resist enzymatic
degradation. Exemplary modifications include modified amino acid
residues and modified peptide bonds that resist enzymatic
degradation. Various modifications to achieve this are known in the
art.
[0102] Also optionally, variant C1ORF32 polypeptides can be
engineered to prevent a cleavage of the full C1ORF32 ECD (SEQ ID
NO:14) between amino acids F and A at positions 179 and 180 of
H19011_1_P8_V1 or H19011_1_P8 (Seq ID NOs: 4 or 5). According to at
least some embodiments of the present invention there is provided
one or more amino acid insertions, deletions or substitutions that
prevent the cleavage of the C1ORF32 polypeptides, fusion proteins,
or fragments thereof. Suitable amino acid substitutions include
conservative and non-conservative substitutions, as described
above. According to at least some embodiments of the present
invention the amino acid substitution in the cleavage site of
C1ORF32 ECD at positions 179 and 180 of H19011_1_P8_V1 or
H19011_1_P8 (Seq ID NOs: 4 or 5) is selected from but not limited
to: FA.fwdarw.GA (as for example disclosed in SEQ ID NO:64);
FA.fwdarw.AA (as for example disclosed in SEQ ID NO:96); and
FA.fwdarw.GG (as for example disclosed in SEQ ID NO:45).
[0103] The terms "individual", "host", "subject", and "patient" are
used interchangeably herein, and refer any human or nonhuman
animal. The term "nonhuman animal" includes all vertebrates, e.g.,
mammals and non-mammals, such as nonhuman primates, sheep, dogs,
cats, horses, cows chickens, amphibians, reptiles, etc.
[0104] Various aspects of the invention are described in further
detail in the following subsections.
[0105] Nucleic Acids
[0106] A "nucleic acid fragment" or an "oligonucleotide" or a
"polynucleotide" are used herein interchangeably to refer to a
polymer of nucleic acid residues. A polynucleotide sequence of the
present invention refers to a single or double stranded nucleic
acid sequences which is isolated and provided in the form of an RNA
sequence, a complementary polynucleotide sequence (cDNA), a genomic
polynucleotide sequence and/or a composite polynucleotide sequences
(e.g., a combination of the above).
[0107] Thus, the present invention encompasses nucleic acid
sequences described hereinabove; fragments thereof, sequences
hybridizable therewith, sequences homologous thereto e.g., at least
90%, at least 95, 96, 97, 98 or 99% or more identical to the
nucleic acid sequences set forth herein, sequences encoding similar
polypeptides with different codon usage, altered sequences
characterized by mutations, such as deletion, insertion or
substitution of one or more nucleotides, either naturally occurring
or man induced, either randomly or in a targeted fashion. The
present invention also encompasses homologous nucleic acid
sequences (i.e., which form a part of a polynucleotide sequence of
the present invention), which include sequence regions unique to
the polynucleotides of the present invention.
[0108] In cases where the polynucleotide sequences of the present
invention encode previously unidentified polypeptides, the present
invention also encompasses novel polypeptides or portions thereof,
which are encoded by the isolated polynucleotide and respective
nucleic acid fragments thereof described hereinabove and/or
degenerative variants thereof.
[0109] Thus, the present invention also encompasses polypeptides
encoded by the polynucleotide sequences of the present invention.
The present invention also encompasses homologues of these
polypeptides, such homologues can be at least 90%, at least 95, 96,
97, 98 or 99% or more homologous to the amino acid sequences set
forth below, as can be determined using BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters. Finally, the present invention also encompasses
fragments of the above described polypeptides and polypeptides
having mutations, such as deletions, insertions or substitutions of
one or more amino acids, either naturally occurring or man induced,
either randomly or in a targeted fashion.
[0110] As mentioned hereinabove, biomolecular sequences of the
present invention can be efficiently utilized as tissue or
pathological markers and as putative drugs or drug targets for
treating or preventing a disease.
[0111] Oligonucleotides designed for carrying out the methods of
the present invention for any of the sequences provided herein
(designed as described above) can be generated according to any
oligonucleotide synthesis method known in the art such as enzymatic
synthesis or solid phase synthesis. Equipment and reagents for
executing solid-phase synthesis are commercially available from,
for example, Applied Biosystems. Any other means for such synthesis
may also be employed; the actual synthesis of the oligonucleotides
is well within the capabilities of one skilled in the art.
[0112] Oligonucleotides used according to this aspect of the
present invention are those having a length selected from a range
of about 10 to about 200 bases preferably about 15 to about 150
bases, more preferably about 20 to about 100 bases, most preferably
about 20 to about 50 bases.
[0113] The oligonucleotides of the present invention may comprise
heterocyclic nucleosides consisting of purines and the pyrimidines
bases, bonded in a 3' to 5' phosphodiester linkage.
[0114] Preferable oligonucleotides are those modified in either
backbone, internucleoside linkages or bases, as is broadly
described hereinunder. Such modifications can oftentimes facilitate
oligonucleotide uptake and resistivity to intracellular
conditions.
[0115] Specific examples of preferred oligonucleotides useful
according to this aspect of the present invention include
oligonucleotides containing modified backbones or non-natural
internucleoside linkages. Oligonucleotides having modified
backbones include those that retain a phosphorus atom in the
backbone, as disclosed in U.S. Pat. Nos: 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466, 677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; and 5,625,050.
[0116] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free acid forms can also be
used.
[0117] Alternatively, modified oligonucleotide backbones that do
not include a phosphorus atom therein have backbones that are
formed by short chain alkyl or cycloalkyl internucleoside linkages,
mixed heteroatom and alkyl or cycloalkyl internucleoside linkages,
or one or more short chain heteroatomic or heterocyclic
internucleoside linkages. These include those having morpholino
linkages (formed in part from the sugar portion of a nucleoside);
siloxane backbones; sulfide, sulfoxide and sulfone backbones;
formacetyl and thioformacetyl backbones; methylene formacetyl and
thioformacetyl backbones; alkene containing backbones; sulfamate
backbones; methyleneimino and methylenehydrazino backbones;
sulfonate and sulfonamide backbones; amide backbones; and others
having mixed N, O, S and CH2 component parts, as disclosed in U.S.
Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 5,264,562;
[0118] 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;
5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;
5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;
5,633,360; 5,677,437; and 5,677,439.
[0119] Other oligonucleotides which can be used according to the
present invention, are those modified in both sugar and the
internucleoside linkage, i.e., the backbone, of the nucleotide
units are replaced with novel groups. The base units are maintained
for complementation with the appropriate polynucleotide target. An
example for such an oligonucleotide mimetic, includes peptide
nucleic acid (PNA). A PNA oligonucleotide refers to an
oligonucleotide where the sugar-backbone is replaced with an amide
containing backbone, in particular an aminoethylglycine backbone.
The bases are retained and are bound directly or indirectly to aza
nitrogen atoms of the amide portion of the backbone. United States
patents that teach the preparation of PNA compounds include, but
are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and
5,719,262, each of which is herein incorporated by reference. Other
backbone modifications, which can be used in the present invention
are disclosed in U.S. Pat. No: 6,303,374.
[0120] Oligonucleotides of the present invention may also include
base modifications or substitutions. As used herein, "unmodified"
or "natural" bases include the purine bases adenine (A) and guanine
(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil
(U). Modified bases include but are not limited to other synthetic
and natural bases such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azouracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further bases
include those disclosed in U.S. Pat. No: 3,687,808, those disclosed
in The Concise Encyclopedia Of Polymer Science and Engineering,
pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990,
those disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613, and those disclosed by
Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,
pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993.
Such bases are particularly useful for increasing the binding
affinity of the oligomeric compounds of the invention. These
include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6
and 0-6 substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. [Sanghvi YS et al. (1993) Antisense
Research and Applications, CRC Press, Boca Raton 276-278] and are
presently preferred base substitutions, even more particularly when
combined with 2'-O-methoxyethyl sugar modifications.
[0121] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates, which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. Such
moieties include but are not limited to lipid moieties such as a
cholesterol moiety, cholic acid, a thioether, e.g.,
hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,
dodecandiol or undecyl residues, a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid, a palmityl
moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol
moiety, as disclosed in U.S. Pat. No: 6,303,374.
[0122] It is not necessary for all positions in a given
oligonucleotide molecule to be uniformly modified, and in fact more
than one of the aforementioned modifications may be incorporated in
a single compound or even at a single nucleoside within an
oligonucleotide.
[0123] Peptides
[0124] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an analog or mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers. Polypeptides can be modified, e.g., by the
addition of carbohydrate residues to form glycoproteins. The terms
"polypeptide," "peptide" and "protein" include glycoproteins, as
well as non-glycoproteins.
[0125] Polypeptide products can be biochemically synthesized such
as by employing standard solid phase techniques. Such methods
include exclusive solid phase synthesis, partial solid phase
synthesis methods, fragment condensation, classical solution
synthesis. These methods are preferably used when the peptide is
relatively short (i.e., 10 kDa) and/or when it cannot be produced
by recombinant techniques (i.e., not encoded by a nucleic acid
sequence) and therefore involves different chemistry.
[0126] Solid phase polypeptide synthesis procedures are well known
in the art and further described by John Morrow Stewart and Janis
Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce
Chemical Company, 1984).
[0127] Synthetic polypeptides can be purified by preparative high
performance liquid chromatography [Creighton T. (1983) Proteins,
structures and molecular principles. WH Freeman and Co. N.Y.] and
the composition of which can be confirmed via amino acid
sequencing.
[0128] In cases where large amounts of a polypeptide are desired,
it can be generated using recombinant techniques such as described
by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier
et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984)
Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311,
Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984)
Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol.
6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant
Molecular Biology, Academic Press, NY, Section VIII, pp
421-463.
[0129] It will be appreciated that peptides identified according to
the teachings of the present invention may be degradation products,
synthetic peptides or recombinant peptides as well as
peptidomimetics, typically, synthetic peptides and peptoids and
semipeptoids which are peptide analogs, which may have, for
example, modifications rendering the peptides more stable while in
a body or more capable of penetrating into cells. Such
modifications include, but are not limited to N terminus
modification, C terminus modification, peptide bond modification,
including, but not limited to, CH2--NH, CH2--S, CH2--S=O,
O.dbd.C--NH, CH2--O, CH2--CH2, S.dbd.C--NH, CH.dbd.CH or CF.dbd.CH,
backbone modifications, and residue modification. Methods for
preparing peptidomimetic compounds are well known in the art and
are specified, for example, in Quantitative Drug Design, C. A.
Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which
is incorporated by reference as if fully set forth herein. Further
details in this respect are provided hereinunder.
[0130] Peptide bonds (--CO--NH--) within the peptide may be
substituted, for example, by N-methylated bonds (--N(CH3)--CO--),
ester bonds (--C(R)H--C--O--O--C(R)--N--), ketomethylen bonds
(--CO--CH2--), .alpha.-aza bonds (--NH--N(R)--CO--), wherein R is
any alkyl, e.g., methyl, carba bonds (--CH2--NH--), hydroxyethylene
bonds (--CH(OH)--CH2--), thioamide bonds (--CS--NH--), olefinic
double bonds (--CH.dbd.CH--), retro amide bonds (--NH--CO--),
peptide derivatives (--N(R)--CH2--CO--), wherein R is the "normal"
side chain, naturally presented on the carbon atom.
[0131] These modifications can occur at any of the bonds along the
peptide chain and even at several (2-3) at the same time.
[0132] Natural aromatic amino acids, Trp, Tyr and Phe, may be
substituted by synthetic non-natural acid such as Phenylglycine,
TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe,
halogenated derivatives of Phe or o-methyl-Tyr.
[0133] In addition to the above, the peptides of the present
invention may also include one or more modified amino acids or one
or more non-amino acid monomers (e.g. fatty acids, complex
carbohydrates etc).
[0134] As used herein in the specification and in the claims
section below the term "amino acid" or "amino acids" is understood
to include the 20 naturally occurring amino acids; those amino
acids often modified post-translationally in vivo, including, for
example, hydroxyproline, phosphoserine and phosphothreonine; and
other unusual amino acids including, but not limited to,
2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and ornithine. Furthermore, the term "amino acid"
includes both D- and L-amino acids.
[0135] Since the peptides of the present invention are preferably
utilized in therapeutics which require the peptides to be in
soluble form, the peptides of the present invention preferably
include one or more non-natural or natural polar amino acids,
including but not limited to serine and threonine which are capable
of increasing peptide solubility due to their hydroxyl-containing
side chain.
[0136] In cases where large amounts of the peptides of the present
invention are desired, the peptides of the present invention can be
generated using recombinant techniques such as described by Bitter
et al., (1987) Methods in Enzymol. 153:516-544, Studier et al.
(1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature
310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et
al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science
224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and
Weissbach & Weissbach, 1988, Methods for Plant Molecular
Biology, Academic Press, NY, Section VIII, pp 421-463.
[0137] Expression Systems
[0138] To enable cellular expression of the polynucleotides of the
present invention, a nucleic acid construct according to the
present invention may be used, which includes at least a coding
region of one of the above nucleic acid sequences, and further
includes at least one cis acting regulatory element. As used
herein, the phrase "cis acting regulatory element" refers to a
polynucleotide sequence, preferably a promoter, which binds a trans
acting regulator and regulates the transcription of a coding
sequence located downstream thereto.
[0139] Any suitable promoter sequence can be used by the nucleic
acid construct of the present invention.
[0140] Preferably, the promoter utilized by the nucleic acid
construct of the present invention is active in the specific cell
population transformed. Examples of cell type-specific and/or
tissue-specific promoters include promoters such as albumin that is
liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277],
lymphoid specific promoters [Calame et al., (1988) Adv. Immunol.
43:235-275]; in particular promoters of T-cell receptors [Winoto et
al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al.
(1983) Cell 33729-740], neuron-specific promoters such as the
neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci.
USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.
(1985) Science 230:912-916] or mammary gland-specific promoters
such as the milk whey promoter (U.S. Pat. No. 4,873,316 and
European
[0141] Application Publication No. 264,166). The nucleic acid
construct of the present invention can further include an enhancer,
which can be adjacent or distant to the promoter sequence and can
function in up regulating the transcription therefrom.
[0142] The nucleic acid construct of the present invention
preferably further includes an appropriate selectable marker and/or
an origin of replication. Preferably, the nucleic acid construct
utilized is a shuttle vector, which can propagate both in E. coli
(wherein the construct comprises an appropriate selectable marker
and origin of replication) and be compatible for propagation in
cells, or integration in a gene and a tissue of choice. The
construct according to the present invention can be, for example, a
plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an
artificial chromosome.
[0143] Examples of suitable constructs include, but are not limited
to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay,
pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available
from Invitrogen Co. (www.invitrogen.com). Examples of retroviral
vector and packaging systems are those sold by Clontech, San Diego,
Calif., including Retro-X vectors pLNCX and pLXSN, which permit
cloning into multiple cloning sites and the transgene is
transcribed from CMV promoter. Vectors derived from Mo-MuLV are
also included such as pBabe, where the transgene will be
transcribed from the 5'LTR promoter.
[0144] Currently preferred in vivo nucleic acid transfer techniques
include transfection with viral or non-viral constructs, such as
adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated
virus (AAV) and lipid-based systems. Useful lipids for
lipid-mediated transfer of the gene are, for example, DOTMA, DOPE,
and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65
(1996)]. The most preferred constructs for use in gene therapy are
viruses, most preferably adenoviruses, AAV, lentiviruses, or
retroviruses. A viral construct such as a retroviral construct
includes at least one transcriptional promoter/enhancer or
locus-defining elements, or other elements that control gene
expression by other means such as alternate splicing, nuclear RNA
export, or post-translational modification of messenger. Such
vector constructs also include a packaging signal, long terminal
repeats (LTRs) or portions thereof, and positive and negative
strand primer binding sites appropriate to the virus used, unless
it is already present in the viral construct. In addition, such a
construct typically includes a signal sequence for secretion of the
peptide from a host cell in which it is placed. Preferably the
signal sequence for this purpose is a mammalian signal sequence or
the signal sequence of the polypeptides of the present invention.
Optionally, the construct may also include a signal that directs
polyadenylation, as well as one or more restriction sites and a
translation termination sequence. By way of example, such
constructs will typically include a 5' LTR, a tRNA binding site, a
packaging signal, an origin of second-strand DNA synthesis, and a
3' LTR or a portion thereof. Other vectors can be used that are
non-viral, such as cationic lipids, polylysine, and dendrimers.
[0145] Recombinant Expression Vectors and Host Cells
[0146] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
protein of the invention, or derivatives, fragments, analogs or
homologs thereof. As used herein, the term "vector" refers to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments can be ligated. Another type of vector is a
viral vector, wherein additional DNA segments can be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) are integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are
operatively-linked. Such vectors are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0147] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, that is operatively-linked to the nucleic acid sequence
to be expressed. Within a recombinant expression vector,
"operably-linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequences in a manner that
allows for expression of the nucleotide sequence (e.g., in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell).
[0148] The term "regulatory sequence" is intended to include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described,
for example, in Goeddel, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990).
Regulatory sequences include those that direct constitutive
expression of a nucleotide sequence in many types of host cell and
those that direct expression of the nucleotide sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. The expression vectors of the invention can be
introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by nucleic acids as
described herein.
[0149] The recombinant expression vectors of the invention can be
designed for production of variant proteins in prokaryotic or
eukaryotic cells. For example, proteins of the invention can be
expressed in bacterial cells such as Escherichia coli, insect cells
(using baculovirus expression vectors) yeast cells or mammalian
cells. Suitable host cells are discussed further in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[0150] Expression of proteins in prokaryotes is most often carried
out in Escherichia coli with vectors containing constitutive or
inducible promoters directing the expression of either fusion or
non-fusion proteins. Fusion vectors add a number of amino acids to
a protein encoded therein, to the amino or C terminus of the
recombinant protein. Such fusion vectors typically serve three
purposes: (i) to increase expression of recombinant protein; (ii)
to increase the solubility of the recombinant protein; and (iii) to
aid in the purification of the recombinant protein by acting as a
ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to enable
separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin,
PreScission, TEV and enterokinase. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson,
1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.)
and pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione
S-transferase (GST), maltose E binding protein, or protein A,
respectively, to the target recombinant protein.
[0151] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and
pET 11d (Studier et al., Gene Expression Technology:
[0152] Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990) 60-89)-not accurate, pET11a-d have N terminal T7 tag.
[0153] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacterium with an
impaired capacity to proteolytically cleave the recombinant
protein. See, e.g., Gottesman, Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. (1990)
119-128. Another strategy is to alter the nucleic acid sequence of
the nucleic acid to be inserted into an expression vector so that
the individual codons for each amino acid are those preferentially
utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids
Res. 20: 2111-2118). Such alteration of nucleic acid sequences of
the invention can be carried out by standard DNA synthesis
techniques. Another strategy to solve codon bias is by using
BL21-codon plus bacterial strains (Invitrogen) or Rosetta bacterial
strain (Novagen), these strains contain extra copies of rare E.coli
tRNA genes.
[0154] In another embodiment, the expression vector encoding for
the protein of the invention is a yeast expression vector. Examples
of vectors for expression in yeast Saccharomyces cerevisiae include
pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan
and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al.,
1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego,
Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
[0155] Alternatively, polypeptides of the present invention can be
produced in insect cells using baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., SF9 cells) include the pAc series
(Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL
series (Lucklow and Summers, 1989. Virology 170: 31-39).
[0156] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987.
EMBO J. 6: 187-195), pIRESpuro (Clontech), pUB6 (Invitrogen), pCEP4
(Invitrogen) pREP4 (Invitrogen), pcDNA3 (Invitrogen). When used in
mammalian cells, the expression vector's control functions are
often provided by viral regulatory elements. For example, commonly
used promoters are derived from polyoma, adenovirus 2,
cytomegalovirus, Rous Sarcoma Virus, and simian virus 40. For other
suitable expression systems for both prokaryotic and eukaryotic
cells see, e.g., Chapters 16 and 17 of Sambrook, et al., Molecular
Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0157] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes
Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton,
1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell
receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and
immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc.
Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters
(Edlund, et al., 1985. Science 230: 912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, e.g., the
murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379)
and the alpha-fetoprotein promoter (Campes and Tilghman, 1989.
Genes Dev. 3: 537-546).
[0158] The present invention in at least some embodiments further
provides a recombinant expression vector comprising a DNA molecule
of the invention cloned into the expression vector in an antisense
orientation. That is, the DNA molecule is operatively-linked to a
regulatory sequence in a manner that allows for expression (by
transcription of the DNA molecule) of an RNA molecule that is
antisense to mRNA encoding for protein of the invention. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen that direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see, e.g., Weintraub, et al.,
"Antisense RNA as a molecular tool for genetic analysis,"
Reviews-Trends in Genetics, Vol. 1(1) 1986.
[0159] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but also to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0160] A host cell can be any prokaryotic or eukaryotic cell. For
example, protein of the invention can be produced in bacterial
cells such as E. coli, insect cells, yeast, plant or mammalian
cells (such as Chinese hamster ovary cells (CHO) or COS or 293
cells). Other suitable host cells are known to those skilled in the
art.
[0161] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0162] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Various selectable markers
include those that confer resistance to drugs, such as G418,
hygromycin, puromycin, blasticidin and methotrexate. Nucleic acids
encoding a selectable marker can be introduced into a host cell on
the same vector as that encoding protein of the invention or can be
introduced on a separate vector. Cells stably transfected with the
introduced nucleic acid can be identified by drug selection (e.g.,
cells that have incorporated the selectable marker gene will
survive, while the other cells die).
[0163] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) protein of the invention. Accordingly, the present
invention in at least some embodiments further provides methods for
producing proteins of the invention using the host cells of the
invention. In one embodiment, the method comprises culturing the
host cell of the present invention (into which a recombinant
expression vector encoding protein of the invention has been
introduced) in a suitable medium such that the protein of the
invention is produced. In another embodiment, the method further
comprises isolating protein of the invention from the medium or the
host cell.
[0164] For efficient production of the protein, it is preferable to
place the nucleotide sequences encoding the protein of the
invention under the control of expression control sequences
optimized for expression in a desired host. For example, the
sequences may include optimized transcriptional and/or
translational regulatory sequences (such as altered Kozak
sequences).
[0165] It should be noted, that according to at least some
embodiments of the present invention the C1ORF32 polypeptides as
described herein may optionally be isolated as naturally-occurring
polypeptides, or from any source whether natural, synthetic,
semi-synthetic or recombinant Accordingly, the C1ORF32 proteins may
be isolated as naturally-occurring proteins from any species,
particularly mammalian, including bovine, ovine, porcine, murine,
equine, and preferably human. Alternatively, the C1ORF32 proteins
may be isolated as recombinant polypeptides that are expressed in
prokaryote or eukaryote host cells, or isolated as a chemically
synthesized polypeptide.
[0166] A skilled artisan can readily employ standard isolation
methods to obtain isolated C1ORF32 proteins. The nature and degree
of isolation will depend on the source and the intended use of the
isolated molecules.
[0167] Fusion Proteins
[0168] According to at least some embodiments, C1ORF32 fusion
polypeptides have a first fusion partner comprising all or a part
of a C1ORF32 protein fused to a second polypeptide directly or via
a linker peptide sequence or a chemical linker useful to connect
the two proteins. The C1ORF32 polypeptide may or may not contain
the native signal peptide. The C1ORF32 polypeptide may optionally
be fused to a second polypeptide to form a fusion protein as
described herein. The presence of the second polypeptide can alter
the solubility, stability, affinity and/or valency of the C1ORF32
fusion polypeptide. As used herein, "valency" refers to the number
of binding sites available per molecule. In one embodiment the
second polypeptide is a polypeptide from a different source or
different protein.
[0169] According to at least some embodiments, the C1ORF32 protein
or fragment is selected for its activity for the treatment of
immune related disorder and/or infection and/or according to one or
more in vitro biological activities as described herein.
[0170] In one embodiment, the second polypeptide contains one or
more domains of an immunoglobulin heavy chain constant region,
preferably having an amino acid sequence corresponding to the
hinge, CH2 and CH3 regions of a human immunoglobulin C.gamma.1,
C.gamma.2, C.gamma.3 or C.gamma.4, chains or to the hinge, CH2 and
CH3 regions of a murine immunoglobulin Cy2a chain. SEQ ID NO: 20
provides exemplary sequence for the hinge, CH2 and CH3 regions of a
human immunoglobulin Cy1.
[0171] According to at least some embodiments, the fusion protein
is a dimeric fusion protein. In an optional dimeric fusion protein,
the dimer results from the covalent bonding of Cys residue in the
hinge region of two of the Ig heavy chains that are the same Cys
residues that are disulfide linked in dimerized normal Ig heavy
chains. Such proteins are referred to as IgC1ORF32 polypeptides,
fragments or fusion proteins thereof.
[0172] In one embodiment, the immunoglobulin constant domain may
contain one or more amino acid insertions, deletions or
substitutions that enhance binding to specific cell types, increase
the bioavailablity, or increase the stability of the C1ORF32
polypeptides, fusion proteins, or fragments thereof. Suitable amino
acid substitutions include conservative and non-conservative
substitutions, as described above.
[0173] The fusion proteins optionally contain a domain that
functions to dimerize or multimerize two or more fusion proteins.
The peptide/polypeptide linker domain can either be a separate
domain, or alternatively can be contained within one of the other
domains (C1ORF32 polypeptide or second polypeptide) of the fusion
protein. Similarly, the domain that functions to dimerize or
multimerize the fusion proteins can either be a separate domain, or
alternatively can be contained within one of the other domains
(C1ORF32 polypeptide, second polypeptide or peptide/polypeptide
linker domain) of the fusion protein. In one embodiment, the
dimerization/multimerization domain and the peptide/polypeptide
linker domain are the same. Further specific, illustrative and
non-limiting examples of dimerization/multimerization domains and
linkers are given below.
[0174] Fusion proteins disclosed herein according to at least some
embodiments of the present invention are of formula I: N-R1-R2-R3-C
wherein "N" represents the N-terminus of the fusion protein, "C"
represents the C-terminus of the fusion protein. In the further
embodiment, "R1" is a C1ORF32 polypeptide, "R2" is an optional
peptide/polypeptide or chemical linker domain, and "R3" is a second
polypeptide. Alternatively, R3 may be a C1ORF32 polypeptide and R1
may be a second polypeptide. Various non-limiting examples of
linkers are described in greater detail below.
[0175] Optionally, the fusion protein comprises the C1ORF32
polypeptide fragments selected from the group consisting of SEQ ID
NOs: 29, 30, 41-105, fused, optionally by a linker peptide of one
or more amino acids (e.g. GS) to one or more "half-life extending
moieties". A "half-life extending moiety" is any moiety, for
example, a polypeptide, small molecule or polymer, that, when
appended to protein, extends the in vivo half-life of that protein
in the body of a subject (e.g., in the plasma of the subject). For
example, a half-life extending moiety is, in an embodiment of the
invention, polyethylene glycol (PEG), monomethoxy PEG (mPEG) or an
immunoglobulin (Ig). In an embodiment of the invention, PEG is a 5,
10, 12, 20, 30, 40 or 50 kDa moiety or larger or comprises about
12000 ethylene glycol units (PEG12000).
[0176] The fusion protein may also optionally be prepared by
chemical synthetic methods and the "join" effected chemically,
either during synthesis or post-synthesis. Cross-linking and other
such methods may optionally be used (optionally also with the above
described genetic level fusion methods), as described for example
in U.S. Pat. No. 5,547,853 to Wallner et al, which is hereby
incorporated by reference as if fully set forth herein as a
non-limiting example only.
[0177] According to the present invention, a fusion protein may be
prepared from a protein of the invention by fusion with a portion
of an immunoglobulin comprising a constant region of an
immunoglobulin. More preferably, the portion of the immunoglobulin
comprises a heavy chain constant region which is optionally and
more preferably a human heavy chain constant region. The heavy
chain constant region is most preferably an IgG heavy chain
constant region, and optionally and most preferably is an Fc chain,
most preferably an IgG Fc fragment that comprises the hinge, CH2
and CH3 domains. The Fc chain may optionally be a known or "wild
type" Fc chain, or alternatively may be mutated or truncated. The
Fc portion of the fusion protein may optionally be varied by
isotype or subclass, may be a chimeric or hybrid, and/or may be
modified, for example to improve effector functions, control of
half-life, tissue accessibility, augment biophysical
characteristics such as stability, and improve efficiency of
production (and less costly). Many modifications useful in
construction of disclosed fusion proteins and methods for making
them are known in the art, see for example Mueller, et al, MoI.
Immun , 34(6):441-452 (1997), Swann, et al., Cur. Opin. Immun ,
20:493-499 (2008), and Presta, Cur. Opin. Immun 20:460-470 (2008).
In some embodiments the Fc region is the native IgG1, IgG2, or IgG4
Fc region. In some embodiments the Fc region is a hybrid, for
example a chimeric consisting of IgG2/IgG4 Fc constant regions.
[0178] Modications to the Fc region include, but are not limited
to, IgG4 modified to prevent binding to Fc gamma receptors and
complement, IgG1 modified to improve binding to one or more Fc
gamma receptors, IgG1 modified to minimize effector function (amino
acid changes), IgG1 with altered/no glycan (typically by changing
expression host or substuting the Asn at position 297), and IgG1
with altered pH-dependent binding to FcRn. The Fc region may
include the entire hinge region, or less than the entire hinge
region.
[0179] In another embodiment, the Fc domain may contain one or more
amino acid insertions, deletions or substitutions that reduce
binding to the low affinity inhibitory Fc receptor CD32B
(Fc.gamma.RIIB) and retain wild-type levels of binding to or
enhance binding to the low affinity activating Fc receptor CD16A
(Fc.gamma.RIIIA)
[0180] Another embodiment includes IgG2-4 hybrids and IgG4 mutants
that have reduced binding to FcR (Fc receptor) which increase their
half life. Representative IgG2-4 hybrids and IgG4 mutants are
described in Angal, S. et al., Molecular Immunology, 30(1):105-108
(1993); Mueller, J. et al., Molecular Immunology, 34(6): 441-452
(1997); and U.S. Pat. No. 6,982,323 to Wang et al. In some
embodiments the IgG1 and/or IgG2 domain is deleted; for example,
Angal et al. describe IgG1 and IgG2 having serine 241 replaced with
a proline.
[0181] In a further embodiment, the Fc domain contains amino acid
insertions, deletions or substitutions that enhance binding to
CD16A. A large number of substitutions in the Fc domain of human
IgG1 that increase binding to CD16A and reduce binding to CD32B are
known in the art and are described in Stavenhagen, et al., Cancer
Res., 57(18):8882-90 (2007). Exemplary variants of human IgG1 Fc
domains with reduced binding to CD32B and/or increased binding to
CD16A contain F243L, R929P, Y300L, V3051 or P296L substitutions.
These amino acid substitutions may be present in a human IgG1 Fc
domain in any combination.
[0182] In one embodiment, the human IgG1 Fc domain variant contains
a F243L, R929P and Y300L substitution. In another embodiment, the
human IgG1 Fc domain variant contains a F243L, R929P, Y300L, V3051
and P296L substitution. In another embodiment, the human IgG1 Fc
domain variant contains an N297A/Q substitution, as these mutations
abolishFcyR binding. Non-limiting, illustrative, exemplary types of
mutations are described in US Patent Application No. 20060034852,
published on Feb. 16, 2006, hereby incorporated by reference as if
fully set forth herein. The term "Fc chain" also optionally
comprises any type of Fc fragment.
[0183] Several of the specific amino acid residues that are
important for antibody constant region-mediated activity in the IgG
subclass have been identified. Inclusion, substitution or exclusion
of these specific amino acids therefore allows for inclusion or
exclusion of specific immunoglobulin constant region-mediated
activity. Furthermore, specific changes may result in
aglycosylation for example and/or other desired changes to the Fc
chain. At least some changes may optionally be made to block a
function of Fc which is considered to be undesirable, such as an
undesirable immune system effect, as described in greater detail
below.
[0184] Non-limiting, illustrative examples of mutations to Fc which
may be made to modulate the activity of the fusion protein include
the following changes (given with regard to the Fc sequence
nomenclature as given by Kabat, from Kabat EA et al: Sequences of
Proteins of Immunological Interest. US Department of Health and
Human Services, NIH, 1991): 220C.fwdarw.S; 233-238
ELLGGP.fwdarw.EAEGAP; 265D.fwdarw.A, preferably in combination with
434N.fwdarw.A; 297N.fwdarw.A (for example to block
N-glycosylation); 318-322 EYKCK.fwdarw.AYACA; 330-331AP.fwdarw.SS;
or a combination thereof (see for example M. Clark, "Chemical
Immunol and Antibody Engineering", pp 1-31 for a description of
these mutations and their effect). The construct for the Fc chain
which features the above changes optionally and preferably
comprises a combination of the hinge region with the CH2 and CH3
domains.
[0185] The above mutations may optionally be implemented to enhance
desired properties or alternatively to block non-desired
properties. For example, aglycosylation of antibodies was shown to
maintain the desired binding functionality while blocking depletion
of T-cells or triggering cytokine release, which may optionally be
undesired functions (see M. Clark, "Chemical Immunol and Antibody
Engineering", pp 1-31). Substitution of 331proline for serine may
block the ability to activate complement, which may optionally be
considered an undesired function (see M. Clark, "Chemical Immunol
and Antibody Engineering", pp 1-31). Changing 330 alanine to serine
in combination with this change may also enhance the desired effect
of blocking the ability to activate complement.
[0186] Residues 235 and 237 were shown to be involved in
antibody-dependent cell-mediated cytotoxicity (ADCC), such that
changing the block of residues from 233-238 as described may also
block such activity if ADCC is considered to be an undesirable
function.
[0187] Residue 220 is normally a cysteine for Fc from IgG1, which
is the site at which the heavy chain forms a covalent linkage with
the light chain. Optionally, this residue may be changed to another
amino acid residue (e.g., serine), to avoid any type of covalent
linkage (see M. Clark, "Chemical Immunol and Antibody Engineering",
pp 1-31) or by deletion or truncation.
[0188] The above changes to residues 265 and 434 may optionally be
implemented to reduce or block binding to the Fc receptor, which
may optionally block undesired functionality of Fc related to its
immune system functions (see "Binding site on Human IgG1 for Fc
Receptors", Shields et al, Vol 276, pp 6591-6604, 2001).
[0189] The above changes are intended as illustrations only of
optional changes and are not meant to be limiting in any way.
Furthermore, the above explanation is provided for descriptive
purposes only, without wishing to be bound by a single hypothesis.
In a further embodiment, the fusion protein includes the C1ORF32
fragment fused to an Ig Fc region. Recombinant Ig-C1ORF32 fragment
polypeptides can be prepared by fusing the coding region of the
C1ORF32 fragment to the Fc region of human IgG1 or mouse IgG2a, as
described previously (Chapoval, et al., Methods MoI. Med,
45:247-255 (2000)). Optionally, C1ORF32 fusion protein, comprising
an amino acid sequence of human C1ORF32 ECD fragment fused to
murine or human immunoglobulin Fc. Optionally, said fusion protein
comprises the amino acid sequence set forth in anyone of SEQ ID
NOs: 29, 30, 41-105, and/or 45, 64, or 96, fused to human IgG1 Fc
set forth in any one of SEQ ID NOs: 20, 21, 115. Optionally, the
amino acid sequence of said fusion protein is set forth in SEQ ID
NO: 39, 108-112, 116-190; optionally and preferably, the amino acid
sequence is set forth in any of SEQ ID NOs:112, 120 or
alternatively in any one of SEQ ID NOs:110, 136.
[0190] The aforementioned exemplary fusion proteins can incorporate
any combination of the variants described herein. In another
embodiment the terminal lysine of the aforementioned exemplary
fusion proteins is deleted.
[0191] The disclosed fusion proteins can be isolated using standard
molecular biology techniques. For example, an expression vector
containing a DNA sequence encoding a C1ORF32
[0192] ECD fragments or fusion proteins thereof fusion protein is
transfected into 293 cells by calcium phosphate precipitation and
cultured in serum-free DMEM. The supernatant is collected at 72 h
and the fusion protein is purified by Protein G, or preferably
Protein A SEPHAROSE.RTM. columns (Pharmacia, Uppsala, Sweden).
Optionally, a DNA sequence encoding a C1ORF32 fragments fusion
protein is transfected into GPEx.RTM. retrovectors and expressed in
CHO-S cells following four rounds of retrovector transduction. The
protein is clarified from supernatants using protein A
chromatography.
[0193] In another embodiment the second polypeptide may have a
conjugation domain through which additional molecules can be bound
to the C1ORF32 fragments fusion proteins. In one such embodiment,
the conjugated molecule is capable of targeting the fusion protein
to a particular organ or tissue; further specific, illustrative,
non-limiting examples of such targeting domains and/or molecules
are given below.
[0194] In another such embodiment the conjugated molecule is
another immunomodulatory agent that can enhance or augment the
effects of the C1ORF32 fusion protein. In another embodiment the
conjugated molecule is Polyethylene Glycol (PEG).
[0195] Peptide or Polypeptide Linker Domain
[0196] The disclosed C1ORF32 fusion proteins optionally contain a
peptide or polypeptide linker domain that separates the C1ORF32
polypeptide from the second polypeptide. In one embodiment, the
linker domain contains the hinge region of an immunoglobulin. In a
further embodiment, the hinge region is derived from a human
immunoglobulin. Suitable human immunoglobulins that the hinge can
be derived from include IgG, IgD and IgA. In a further embodiment,
the hinge region is derived from human IgG. Amino acid sequences of
immunoglobulin hinge regions and other domains are well known in
the art. In one embodiment, C1ORF32 fusion polypeptides contain the
hinge, CH2 and CH3 regions of a human immunoglobulin C.gamma.1
chain, optionally with the Cys at position 220 (according to full
length human IgG1, position 5 in SEQ ID NO: 20) replaced with a Ser
(SEQ ID NO: 115) having at least 85%, 90%, 95%, 99% or 100%
sequence homology to amino acid sequence set forth in SEQ ID NO:
20:
TABLE-US-00003 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK
[0197] The hinge can be further shortened to remove amino acids 1,
2, 3, 4, 5, or combinations thereof of any one of SEQ ID NOs: 20 or
115. In one embodiment, amino acids 1-5 of any one of SEQ ID NOs:
20 or 115 are deleted.
[0198] In another embodiment, C1ORF32 fusion polypeptides contain
the CH2 and CH3 regions of a human immunoglobulin C.gamma.1 chain
having at least 85%, 90%, 95%, 99% or 100% sequence homology to
amino acid sequence set forth in SEQ ID NO: 21:
TABLE-US-00004 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK
[0199] In another embodiment, the C1ORF32 fusion polypeptides
contain the CH2 and CH3 regions of a murine immunoglobulin Cy2a
chain at least 85%, 90%, 95%, 99% or 100% sequence homology to
amino acid sequence set forth in SEQ ID NO:31:
TABLE-US-00005 EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLM
ISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQ
THREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDL
PAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLT
CMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY
FMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFS RTPGK.
[0200] In another embodiment, the linker domain contains a hinge
region of an immunoglobulin as described above, and further
includes one or more additional immunoglobulin domains.
[0201] Other suitable peptide/polypeptide linker domains include
naturally occurring or non-naturally occurring peptides or
polypeptides. Peptide linker sequences are at least 2 amino acids
in length. Optionally the peptide or polypeptide domains are
flexible peptides or polypeptides. A "flexible linker" herein
refers to a peptide or polypeptide containing two or more amino
acid residues joined by peptide bond(s) that provides increased
rotational freedom for two polypeptides linked thereby than the two
linked polypeptides would have in the absence of the flexible
linker. Such rotational freedom allows two or more antigen binding
sites joined by the flexible linker to each access target
antigen(s) more efficiently. Exemplary flexible
peptides/polypeptides include, but are not limited to, the amino
acid sequences Gly-Ser (SEQ ID NO: 24), Gly-Ser-Gly-Ser (SEQ ID NO:
25), Ala-Ser (SEQ ID NO:26), Gly-Gly-Gly-Ser (SEQ ID NO: 27),
Gly4-Ser (SEQ ID NO: 106), (Gly4-Ser)2 (SEQ ID NO: 107),
(Gly4-Ser)3 (SEQ ID NO: 32) and (Gly4-Ser)4 (SEQ ID NO: 33).
Additional flexible peptide/polypeptide sequences are well known in
the art. Other suitable peptide linker domains include helix
forming linkers such as Ala-(Glu-Ala-Ala-Ala-Lys)n-Ala (n=1-5) (for
n=1, SEQ ID NO:114 as a non-limiting example). Additional helix
forming peptide/polypeptide sequences are well known in the art.
Additional example of a cleavable linker is TEV-linker Gly Ser Glu
Asn Leu Tyr Phe Gln Gly Ser Gly (SEQ ID NO:113). Non-limiting
examples of C1ORF32 polypeptide fragments fused to Fc portion via a
linker are depicted in SEQ ID NOs: 8 and 108-112.
[0202] Dimerization, Multimerization and Targeting Domains
[0203] The fusion proteins disclosed herein optionally contain a
dimerization or multimerization domain that functions to dimerize
or multimerize two or more fusion proteins. The domain that
functions to dimerize or multimerize the fusion proteins can either
be a separate domain, or alternatively can be contained within one
of the other domains (C1ORF32 polypeptide, second polypeptide, or
peptide/polypeptide linker domain) of the fusion protein.
[0204] Dimerization or multinierization can occur between or among
two or more fusion proteins through dimerization or multimerization
domains. Alternatively, dimerization or multimerization of fusion
proteins can occur by chemical crosslinking. The dimers or
multimers that are formed can be homodimeric/homomultimeric or
heterodimeric/heteromultimeric.The second polypeptide "partner" in
the C1ORF32 fusion polypeptides may be comprised of one or more
other proteins, protein fragments or peptides as described herein,
including but not limited to any immunoglobulin (Ig) protein or
portion thereof, preferably the Fc region, or a portion of a
biologically or chemically active protein such as the
papillomavirus E7 gene product, melanoma-associated antigen p97),
and HIV env protein (gp120). The "partner" is optionally selected
to provide a soluble dimer/multimer and/or for one or more other
biological activities as described herein.
[0205] A "dimerization domain" is formed by the association of at
least two amino acid residues or of at least two peptides or
polypeptides (which may have the same, or different, amino acid
sequences). The peptides or polypeptides may interact with each
other through covalent and/or non-covalent associations). Optional
dimerization domains contain at least one cysteine that is capable
of forming an intermolecular disulfide bond with a cysteine on the
partner fusion protein. The dimerization domain can contain one or
more cysteine residues such that disulfide bond(s) can form between
the partner fusion proteins. In one embodiment, dimerization
domains contain one, two or three to about ten cysteine residues.
In a further embodiment, the dimerization domain is the hinge
region of an immunoglobulin.
[0206] Additional exemplary dimerization domains can be any known
in the art and include, but not limited to, coiled coils, acid
patches, zinc fingers, calcium hands, a C.sub.H1-C.sub.L pair, an
"interface" with an engineered "knob" and/or "protruberance" as
described in U.S. Pat. No. 5,821,333, leucine zippers (e.g., from
jun and/or fos) (U.S. Patent No. 5,932,448), and/or the yeast
transcriptional activator GCN4, SH2 (src homology 2), SH3 (src
Homology 3) (Vidal, et al, Biochemistry, 43, 7336-44 ((2004)),
phosphotyrosine binding (PTB) (Zhou, et al., Nature, 378:584-592
(1995)), WW (Sudol, Prog, Biochys. MoL Bio., 65:113-132 (1996)),
PDZ (Kim, et al., Nature, 378: 85-88 (1995); Komau, et al, Science,
269.1737-1740 (1995)) 14-3-3, WD40 (Hu5 et al., J Biol Chem., 273,
33489-33494 (1998)) EH, Lim, an isoleucine zipper, a receptor dimer
pair (e.g., interleukin-8 receptor (IL-8R); and integrin
heterodimers such as LFA-I and GPIIIb/IIIa), or the dimerization
region(s) thereof, dimeric ligand polypeptides (e.g. nerve growth
factor (NGF), neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular
endothelial growth factor (VEGF), VEGF-C, VEGF-D, PDGF members, and
brain-derived neurotrophic factor (BDNF) (Arakawa, et al., J Biol.
Chem., 269(45): 27833-27839 (1994) and Radziejewski, et al.,
Biochem., 32(48): 1350 (1993)) and can also be variants of these
domains in which the affinity is altered. The polypeptide pairs can
be identified by methods known in the art, including yeast two
hybrid screens. Yeast two hybrid screens are described in U.S. Pat.
Nos. 5,283,173 and 6,562,576. Affinities between a pair of
interacting domains can be determined using methods known in the
art, including as described in Katahira, et at, J. Biol Chem, 277,
9242-9246 (2002)). Alternatively, a library of peptide sequences
can be screened for heterodimerization, for example, using the
methods described in WO 01/00814. Useful methods for
protein-protein interactions are also described in U.S. Pat. No.
6,790,624.
[0207] A "multimerization domain" is a domain that causes three or
more peptides or polypeptides to interact with each other through
covalent and/or non-covalent association(s). Suitable
multimerization domains include, but are not limited to,
coiled-coil domains. A coiled-coil is a peptide sequence with a
contiguous pattern of mainly hydrophobic residues spaced 3 and 4
residues apart, usually in a sequence of seven amino acids (heptad
repeat) or eleven amino acids (undecad repeat), which assembles
(folds) to form a multimeric bundle of helices. Coiled-coils with
sequences including some irregular distribution of the 3 and 4
residues spacing are also contemplated. Hydrophobic residues are in
particular the hydrophobic amino acids Val, Ile, Leu, Met, Tyr, Phe
and Trp. "Mainly hydrophobic" means that at least 50% of the
residues must be selected from the mentioned hydrophobic amino
acids.
[0208] The coiled coil domain may be derived from laminin In the
extracellular space, the heterotrimeric coiled coil protein laminin
plays an important role in the formation of basement membranes.
Apparently, the multifunctional oligomeric structure is required
for laminin function. Coiled coil domains may also be derived from
the thrombospondins in which three (TSP-I and TSP-2) or five
(TSP-3, TSP-4 and TSP-5) chains are connected, or from COMP
(COMPcc) (Guo, et at., EMBO J, 1998, 17: 5265-5272) which folds
into a parallel five-stranded coiled coil (Malashkevich, et al.,
Science, 274: 761-765 (1996)). Additional non limiting examples of
coiled-coil domains derived from other proteins, and other domains
that mediate polypeptide multimerization are known in the art such
as the vasodialator-stimulated phosphoprotein (VASP) domain,
matrilin-1 (CMP), viral fusion peptides, soluble NSF
(N-ethylmaleimide-sensitive factor) Attachment Protein receptor
(SNARE) complexes, leucine-rich repeats, certain tRNA synthetases,
are suitable for use in the disclosed fusion proteins.
[0209] In another embodiment, C1ORF32 polypeptides, fusion
proteins, or fragments thereof can be induced to form multimers by
binding to a second multivalent polypeptide, such as an antibody.
Antibodies suitable for use to multimerize C1ORF32 polypeptides,
fusion proteins, or fragments thereof include, but are not limited
to, IgM antibodies and cross-linked, multivalent IgG, IgA, IgD, or
IgE complexes.
[0210] Dimerization or multimerization can occur between or among
two or more fusion proteins through dimerization or multimerization
domains, including those described above. Alternatively,
dimerization or multimerization of fusion proteins can occur by
chemical crosslinking. Fusion protein dimers can be homodimers or
heterodimers. Fusion protein multimers can be homomultimers or
heteromultimers. Fusion protein dimers as disclosed herein are of
formula II: N-R1-R2-R3-C N-R4-R5-R6-C or, alternatively, are of
formula N-R1-R2-R3-C C-R4-R5-R6-N wherein the fusion proteins of
the dimer provided by formula II are defined as being in a parallel
orientation and the fusion proteins of the dimer provided by
formula III are defined as being in an antiparallel orientation.
Parallel and antiparallel dimers are also referred to as cis and
trans dimers, respectively. "N" and "C" represent the N- and
C-termini of the fusion protein, respectively. The fusion protein
constituents "R1", "R2" and "R3" are as defined above with respect
to formula I. With respect to both formula II and formula III, "R4"
is a C1ORF32 polypeptide or a second polypeptide, "R5" is an
optional peptide/polypeptide linker domain, and "R6" is a C1ORF32
polypeptide or a second polypeptide, wherein "R6" is a C1ORF32
polypeptide when "R4" is a second polypeptide, and "R6'" is a
second polypeptide when "R4" is a C1ORF32 polypeptide. In one
embodiment, "R1" is a C1ORF32 polypeptide, "R4" is also a C1ORF32
polypeptide, and "R3" and "R6" are both second polypeptides.
[0211] Fusion protein dimers of formula II are defined as
homodimers when "R1"="R4", "R2"="R5" and "R3"="R6". Similarly,
fusion protein dimers of formula III are defined as homodimers when
"R1"="R6", "R2"="R5" and "R3"="R4". Fusion protein dimers are
defined as heterodimers when these conditions are not met for any
reason. For example, heterodimers may contain domain orientations
that meet these conditions (i.e., for a dimer according to formula
II, "R1" and "R4" are both C1ORF32 polypeptides, "R2" and "R5" are
both peptide/polypeptide linker domains and "R3" and "R6" are both
second polypeptides), however the species of one or more of these
domains is not identical. For example, although "R3" and "R6" may
both be C1ORF32 polypeptides, one polypeptide may contain a
wild-type C1ORF32 amino acid sequence while the other polypeptide
may be a variant C1ORF32 polypeptide. An exemplary variant C1ORF32
polypeptide is C1ORF32 polypeptide that has been modified to have
increased or decreased binding to a target cell, increased activity
on immune cells, increased or decreased half life or stability.
Dimers of fusion proteins that contain either a CHI or CL region of
an immunoglobulin as part of the polypeptide linker domain
preferably form heterodimers wherein one fusion protein of the
dimer contains a CHI region and the other fusion protein of the
dimer contains a CL region.
[0212] Fusion proteins can also be used to form multimers. As with
dimers, multimers may be parallel multimers, in which all fusion
proteins of the multimer are aligned in the same orientation with
respect to their N- and C-termini. Multimers may be antiparallel
multimers, in which the fusion proteins of the multimer are
alternatively aligned in opposite orientations with respect to
their N- and C-termini. Multimers (parallel or antiparallel) can be
either homomultimers or heteromultimers. The fusion protein is
optionally produced in dimeric form; more preferably, the fusion is
performed at the genetic level as described below, by joining
polynucleotide sequences corresponding to the two (or more)
proteins, portions of proteins and/or peptides, such that a joined
or fused protein is produced by a cell according to the joined
polynucleotide sequence. A description of preparation for such
fusion proteins is described with regard to U.S. Pat. No. 5,851,795
to Linsley et al, which is hereby incorporated by reference as if
fully set forth herein as a non-limiting example only.
[0213] Targeting Domains
[0214] The C1ORF32 polypeptides and fusion proteins can contain a
targeting domain to target the molecule to specific sites in the
body. Optional targeting domains target the molecule to areas of
inflammation. Exemplary targeting domains are antibodies, or
antigen binding fragments thereof that are specific for inflamed
tissue or to a proinflammatory cytokine including but not limited
to IL17, IL-4, IL-6, IL-12, IL-21, IL-22, and IL-23. In the case of
neurological disorders such as Multiple Sclerosis, the targeting
domain may target the molecule to the CNS or may bind to VCAM-I on
the vascular epithelium. Additional targeting domains can be
peptide aptamers specific for a proinflammatory molecule. In other
embodiments, the C1ORF32 fusion protein can include a binding
partner specific for a polypeptide displayed on the surface of an
immune cell, for example a T cell. In still other embodiments, the
targeting domain specifically targets activated immune cells.
Optional immune cells that are targeted include Th0, Th1, Th 17,
Th2 and Th22 T cells, other cells that secrete, or cause other
cells to secrete inflammatory molecules including, but not limited
to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6, IL-23,
IL-22, IL-21, and MMPs, and Tregs. For example, a targeting domain
for Tregs may bind specifically to CD25.
[0215] The above changes are intended as illustrations only of
optional changes and are not meant to be limiting in any way.
Furthermore, the above explanation is provided for descriptive
purposes only, without wishing to be bound by a single
hypothesis.
[0216] Dimerization or multimierization can occur between or among
two or more fusion proteins through dimerization or multimerization
domains Alternatively, dimerization or multimerization of fusion
proteins can occur by chemical crosslinking. The dimers or
multimers that are formed can be homodimeric/homomultimeric or
heterodimeric/heteromultimeric. The second polypeptide "partner" in
the C1ORF32 fusion polypeptides may be comprised of one or more
other proteins, protein fragments or peptides as described herein,
including but not limited to any immunoglobulin (Ig) protein or
portion thereof, preferably the Fc region, or a portion of a
biologically or chemically active protein such as the
papillomavirus E7 gene product, melanoma-associated antigen p97),
and HIV env protein (gp120). The "partner" is optionally selected
to provide a soluble dimer/multimer and/or for one or more other
biological activities as described herein.
[0217] Addition of Groups
[0218] If a protein according to the present invention is a linear
molecule, it is possible to place various functional groups at
various points on the linear molecule which are susceptible to or
suitable for chemical modification. Functional groups can be added
to the termini of linear forms of the protein according to at least
some embodiments of the invention. In some embodiments, the
functional groups improve the activity of the protein with regard
to one or more characteristics, including but not limited to,
improvement in stability, penetration (through cellular membranes
and/or tissue barriers), tissue localization, efficacy, decreased
clearance, decreased toxicity, improved selectivity, improved
resistance to expulsion by cellular pumps, and the like. For
convenience sake and without wishing to be limiting, the free
N-terminus of one of the sequences contained in the compositions
according to at least some embodiments of the invention will be
termed as the N-terminus of the composition, and the free
C-terminal of the sequence will be considered as the C-terminus of
the composition. Either the C-terminus or the N-terminus of the
sequences, or both, can be linked to a carboxylic acid functional
groups or an amine functional group, respectively.
[0219] Non-limiting examples of suitable functional groups are
described in Green and Wuts, "Protecting Groups in Organic
Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991, the
teachings of which are incorporated herein by reference. Preferred
protecting groups are those that facilitate transport of the active
ingredient attached thereto into a cell, for example, by reducing
the hydrophilicity and increasing the lipophilicity of the active
ingredient, these being an example for "a moiety for transport
across cellular membranes".
[0220] These moieties can optionally and preferably be cleaved in
vivo, either by hydrolysis or enzymatically, inside the cell.
(Ditter et al., J. Pharm. Sci. 57:783 (1968); Ditter et al., J.
Pharm. Sci. 57:828 (1968); Ditter et al., J. Pharm. Sci. 58:557
(1969); King et al., Biochemistry 26:2294 (1987); Lindberg et al.,
Drug Metabolism and Disposition 17:311 (1989); and Tunek et al.,
Biochem. Pharm. 37:3867 (1988), Anderson et al., Arch. Biochem.
Biophys. 239:538 (1985) and Singhal et al., FASEB J. 1:220 (1987)).
Hydroxyl protecting groups include esters, carbonates and carbamate
protecting groups Amine protecting groups include alkoxy and
aryloxy carbonyl groups, as described above for N-terminal
protecting groups. Carboxylic acid protecting groups include
aliphatic, benzylic and aryl esters, as described above for
C-terminal protecting groups. In one embodiment, the carboxylic
acid group in the side chain of one or more glutamic acid or
aspartic acid residue in a composition of the present invention is
protected, preferably with a methyl, ethyl, benzyl or substituted
benzyl ester, more preferably as a benzyl ester. Non-limiting,
illustrative examples of N-terminal protecting groups include acyl
groups (--CO--R1) and alkoxy carbonyl or aryloxy carbonyl groups
(--CO--O--R1), wherein R1 is an aliphatic, substituted aliphatic,
benzyl, substituted benzyl, aromatic or a substituted aromatic
group. Specific examples of acyl groups include but are not limited
to acetyl, (ethyl)-CO--, n-propyl-CO--, iso-propyl-CO--,
n-butyl-CO--, sec-butyl-CO--, t-butyl-CO--, hexyl, lauroyl,
palmitoyl, myristoyl, stearyl, oleoyl phenyl-CO--, substituted
phenyl-CO--, benzyl-CO-- and (substituted benzyl)-CO--. Examples of
alkoxy carbonyl and aryloxy carbonyl groups include CH3--O--CO--,
(ethyl)-O--CO--, n-propyl-O--CO--, iso-propyl-O--CO--,
n-butyl-O--CO--, sec-butyl-O--CO--, t-butyl-O--CO--,
phenyl-O--CO--, substituted phenyl-O--CO-- and benzyl-O--CO--,
(substituted benzyl)-O--CO--, Adamantan, naphtalen, myristoleyl,
toluen, biphenyl, cinnamoyl, nitrobenzoy, toluoyl, furoyl, benzoyl,
cyclohexane, norbornane, or Z-caproic. In order to facilitate the
N-acylation, one to four glycine residues can be present in the
N-terminus of the molecule.
[0221] The carboxyl group at the C-terminus of the compound can be
protected, for example, by a group including but not limited to an
amide (i.e., the hydroxyl group at the C-terminus is replaced with
--NH.sub.2, --NHR.sub.2 and --NR.sub.2R.sub.3) or ester (i.e. the
hydroxyl group at the C-terminus is replaced with --OR.sub.2).
R.sub.2 and R.sub.3 are optionally independently an aliphatic,
substituted aliphatic, benzyl, substituted benzyl, aryl or a
substituted aryl group. In addition, taken together with the
nitrogen atom, R.sub.2 and R.sub.3 can optionally form a C4 to C8
heterocyclic ring with from about 0-2 additional heteroatoms such
as nitrogen, oxygen or sulfur. Non-limiting suitable examples of
suitable heterocyclic rings include piperidinyl, pyrrolidinyl,
morpholino, thiomorpholino or piperazinyl. Examples of C-terminal
protecting groups include but are not limited to --NH.sub.2,
--NHCH.sub.3, --N(CH.sub.3).sub.2, --NH(ethyl), --N(ethyl).sub.2,
--N(methyl) (ethyl), --NH(benzyl), -N(C1-C4 alkyl)(benzyl),
--NH(phenyl), --N(C1-C4 alkyl) (phenyl), --OCH.sub.3, --O-(ethyl),
--O-(n-propyl), --O-(n-butyl), --O-(iso-propyl), --O-(sec-butyl),
--O-(t-butyl), --O-benz yl and --O-phenyl.
[0222] Substitution by Peptidomimetic Moieties
[0223] A "peptidomimetic organic moiety" can optionally be
substituted for amino acid residues in the composition of this
invention both as conservative and as non-conservative
substitutions. These moieties are also termed "non-natural amino
acids" and may optionally replace amino acid residues, amino acids
or act as spacer groups within the peptides in lieu of deleted
amino acids. The peptidomimetic organic moieties optionally and
preferably have steric, electronic or configurational properties
similar to the replaced amino acid and such peptidomimetics are
used to replace amino acids in the essential positions, and are
considered conservative substitutions. However such similarities
are not necessarily required. According to preferred embodiments of
the present invention, one or more peptidomimetics are selected
such that the composition at least substantially retains its
physiological activity as compared to the native protein according
to the present invention.
[0224] Peptidomimetics may optionally be used to inhibit
degradation of the peptides by enzymatic or other degradative
processes. The peptidomimetics can optionally and preferably be
produced by organic synthetic techniques. Non-limiting examples of
suitable peptidomimetics include D amino acids of the corresponding
L amino acids, tetrazol (Zabrocki et al., J. Am. Chem. Soc.
110:5875-5880 (1988)); isosteres of amide bonds (Jones et al.,
Tetrahedron Lett. 29: 3853-3856 (1988));
LL-3-amino-2-propenidone-6-carboxylic acid (LL-Acp) (Kemp et al.,
J. Org. Chem. 50:5834-5838 (1985)). Similar analogs are shown in
Kemp et al., Tetrahedron Lett. 29:5081-5082 (1988) as well as Kemp
et al., Tetrahedron Lett. 29:5057-5060 (1988), Kemp et al.,
Tetrahedron Lett. 29:4935-4938 (1988) and Kemp et al., J. Org.
Chem. 54:109-115 (1987). Other suitable but exemplary
peptidomimetics are shown in Nagai and Sato, Tetrahedron Lett.
26:647-650 (1985); Di Maio et al., J. Chem. Soc. Perkin Trans.,
1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317 (1989); Olson
et al., J. Am. Chem. Soc. 112:323-333 (1990); Garvey et al., J.
Org. Chem. 56:436 (1990). Further suitable exemplary
peptidomimetics include
hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et
al., J. Takeda Res. Labs 43:53-76 (1989));
1,2,3,4-tetrahydro-isoquinoline-3-carboxylate (Kazmierski et al.,
J. Am. Chem. Soc. 133:2275-2283 (1991)); histidine isoquinolone
carboxylic acid (HIC) (Zechel et al., Int. J. Pep. Protein Res. 43
(1991)); (2S, 3S)-methyl-phenylalanine, (2S,
3R)-methyl-phenylalanine, (2R, 3S)-methyl-phenylalanine and (2R,
3R)-methyl-phenylalanine (Kazmierski and Hruby, Tetrahedron Lett.
(1991)).
[0225] Exemplary, illustrative but non-limiting non-natural amino
acids include beta-amino acids (beta3 and beta2), homo-amino acids,
cyclic amino acids, aromatic amino acids, Pro and Pyr derivatives,
3-substituted Alanine derivatives, Glycine derivatives,
ring-substituted Phe and Tyr Derivatives, linear core amino acids
or diamino acids. They are available from a variety of suppliers,
such as Sigma-Aldrich (USA) for example.
[0226] Protein Chemical Modifications
[0227] In the present invention any part of a protein according to
at least some embodiments of the invention may optionally be
chemically modified, i.e. changed by addition of functional groups.
For example the side amino acid residues appearing in the native
sequence may optionally be modified, although as described below
alternatively other parts of the protein may optionally be
modified, in addition to or in place of the side amino acid
residues. The modification may optionally be performed during
synthesis of the molecule if a chemical synthetic process is
followed, for example by adding a chemically modified amino acid.
However, chemical modification of an amino acid when it is already
present in the molecule ("in situ" modification) is also
possible.
[0228] The amino acid of any of the sequence regions of the
molecule can optionally be modified according to any one of the
following exemplary types of modification (in the peptide
conceptually viewed as "chemically modified"). Non-limiting
exemplary types of modification include carboxymethylation,
acylation, phosphorylation, glycosylation or fatty acylation. Ether
bonds can optionally be used to join the serine or threonine
hydroxyl to the hydroxyl of a sugar. Amide bonds can optionally be
used to join the glutamate or aspartate carboxyl groups to an amino
group on a sugar (Garg and Jeanloz, Advances in Carbohydrate
Chemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz,
Ang. Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal
bonds can also optionally be formed between amino acids and
carbohydrates. Fatty acid acyl derivatives can optionally be made,
for example, by acylation of a free amino group (e.g., lysine)
(Toth et al., Peptides: Chemistry, Structure and Biology, Rivier
and Marshal, eds., ESCOM Publ., Leiden, 1078-1079 (1990)).
[0229] As used herein the term "chemical modification", when
referring to a protein or peptide according to the present
invention, refers to a protein or peptide where at least one of its
amino acid residues is modified either by natural processes, such
as processing or other post-translational modifications, or by
chemical modification techniques which are well known in the art.
Examples of the numerous known modifications typically include, but
are not limited to: acetylation, acylation, amidation,
ADP-ribosylation, glycosylation, GPI anchor formation, covalent
attachment of a lipid or lipid derivative, methylation,
myristylation, pegylation, prenylation, phosphorylation,
ubiquitination, or any similar process.
[0230] Other types of modifications optionally include the addition
of a cycloalkane moiety to a biological molecule, such as a
protein, as described in PCT Application No. WO 2006/050262, hereby
incorporated by reference as if fully set forth herein. These
moieties are designed for use with biomolecules and may optionally
be used to impart various properties to proteins.
[0231] Furthermore, optionally any point on a protein may be
modified. For example, pegylation of a glycosylation moiety on a
protein may optionally be performed, as described in PCT
Application No. WO 2006/050247, hereby incorporated by reference as
if fully set forth herein. One or more polyethylene glycol (PEG)
groups may optionally be added to O-linked and/or N-linked
glycosylation. The PEG group may optionally be branched or linear.
Optionally any type of water-soluble polymer may be attached to a
glycosylation site on a protein through a glycosyl linker.
[0232] Altered Glycosylation
[0233] Proteins according to at least some embodiments of the
invention may be modified to have an altered glycosylation pattern
(i.e., altered from the original or native glycosylation
pattern).
[0234] As used herein, "altered" means having one or more
carbohydrate moieties deleted, and/or having at least one
glycosylation site added to the original protein.
[0235] Glycosylation of proteins is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences, asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-acetylgalactosamine, galactose, or xylose to a
hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used.
[0236] Addition of glycosylation sites to proteins according to at
least some embodiments of the invention is conveniently
accomplished by altering the amino acid sequence of the protein
such that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues in the sequence of the original
protein (for O-linked glycosylation sites). The protein's amino
acid sequence may also be altered by introducing changes at the DNA
level.
[0237] Another means of increasing the number of carbohydrate
moieties on proteins is by chemical or enzymatic coupling of
glycosides to the amino acid residues of the protein.
[0238] Depending on the coupling mode used, the sugars may be
attached to (a) arginine and histidine, (b) free carboxyl groups,
(c) free sulfhydryl groups such as those of cysteine, (d) free
hydroxyl groups such as those of serine, threonine, or
hydroxyproline, (e) aromatic residues such as those of
phenylalanine, tyrosine, or tryptophan, or (f) the amide group of
glutamine. These methods are described in WO 87/05330, and in Aplin
and Wriston, CRC Crit. Rev. Biochem., 22: 259-306 (1981).
[0239] Removal of any carbohydrate moieties present on proteins
according to at least some embodiments of the invention may be
accomplished chemically or enzymatically. Chemical deglycosylation
requires exposure of the protein to trifluoromethanesulfonic acid,
or an equivalent compound. This treatment results in the cleavage
of most or all sugars except the linking sugar (N-acetylglucosamine
or N-acetylgalactosamine), leaving the amino acid sequence
intact.
[0240] Chemical deglycosylation is described by Hakimuddin et al.,
Arch. Biochem. Biophys., 259: 52 (1987); and Edge et al., Anal.
Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate
moieties on proteins can be achieved by the use of a variety of
endo- and exo-glycosidases as described by Thotakura et al., Meth.
Enzymol., 138: 350 (1987).
[0241] Methods of Treatment
[0242] As used herein "therapeutic agent" is any one of the C1ORF32
proteins and polypeptides fragments according to at least some
embodiments of the present invention, and/or fusion proteins and/or
multimeric proteins comprising same, and/or nucleic acid sequence
or fragments thereof encoding same.
[0243] As mentioned herein above, the therapeutic agents can be
used to treat immune related disorders as recited herein, and/or
autoimmune disorders as recited herein, and/or infectious disorders
as recited herein, and/or for blocking and/or promoting immune
costimulation mediated by the C1ORF32 polypeptides in a
subject.
[0244] According to an additional aspect of the present invention
the therapeutic agents can be used to prevent pathologic inhibition
of T cell activity, such as that directed against chronic
infections; and/or prevent pathologic stimulation of T cell
activity, such as that directed against autoantigens in autoimmune
diseases. For example, these molecules can be administered to cells
in culture, in vitro or ex vivo, or to human subjects, e.g., in
vivo, to treat, prevent and to diagnose a variety of disorders.
Preferred subjects include human patients, having disorders
mediated by cells expressing the C1ORF32 protein, and cells that
possess C1ORF32 activity.
[0245] According to an additional aspect of the present invention
the therapeutic agents can be used to inhibit T cell activation, as
can be manifested for example by T cell proliferation and cytokine
secretion.
[0246] Thus, according to an additional aspect of the present
invention there is provided a method of treating immune related
disorders as recited herein, and/or autoimmune disorders as recited
herein, and/or infectious disorders as recited herein, and/or for
blocking or promoting immune stimulation mediated by the C1ORF32
polypeptide in a subject by administering to a subject in need
thereof an effective amount of any one of the therapeutic agents
and/or a pharmaceutical composition comprising any of the
therapeutic agents and further comprising a pharmaceutically
acceptable diluent or carrier.
[0247] The subject according to the present invention is a mammal,
preferably a human which is diagnosed with one of the disease,
disorder or conditions described hereinabove, or alternatively is
predisposed to at least one infectious disorder, and/or immune
related disorder.
[0248] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented. Hence, the mammal to be treated
herein may have been diagnosed as having the disorder or may be
predisposed or susceptible to the disorder. "Mammal" for purposes
of treatment refers to any animal classified as a mammal, including
humans, domestic and farm animals, and zoo, sports, or pet animals,
such as dogs, horses, cats, cows, etc. Preferably, the mammal is
human.
[0249] As used herein the term "treating" refers to preventing,
delaying the onset of, curing, reversing, attenuating, alleviating,
minimizing, suppressing or halting the deleterious effects of the
above-described diseases, disorders or conditions. It also includes
managing the disease as described above. By "manage" it is meant
reducing the severity of the disease, reducing the frequency of
episodes of the disease, reducing the duration of such episodes,
reducing the severity of such episodes and the like.
[0250] Treating, according to the present invention, can be
effected by specifically upregulating the amount and/or the
expression of at least one of the polypeptides of the present
invention in the subject.
[0251] Optionally, upregulation may be effected by administering to
the subject at least one of the polypeptides of the present
invention (e.g., recombinant or synthetic) or an active portion
thereof, as described herein. However, since the bioavailability of
large polypeptides may potentially be relatively small due to high
degradation rate and low penetration rate, administration of
polypeptides is preferably confined to small peptide fragments
(e.g., about 100 amino acids). The polypeptide or peptide may
optionally be administered in as part of a pharmaceutical
composition, described in more detail below.
[0252] It will be appreciated that treatment of the above-described
diseases according to at least some embodiments of the present
invention may be combined with other treatment methods known in the
art (i.e., combination therapy), as described herein.
[0253] Immune System Related Disease Treatment
[0254] The therapeutic agents and/or a pharmaceutical composition
comprising same, as recited herein, according to at least some
embodiments of the present invention can also be used in
combination with one or more of the following agents to regulate an
immune response: soluble gp39 (also known as CD40 ligand (CD40L),
CD154, T-BAM, TRAP), soluble CD29, soluble CD40, soluble CD80 (e.g.
ATCC 68627), soluble CD86, soluble CD28 (e.g. 68628), soluble CD56,
soluble Thy-1, soluble CD3, soluble TCR, soluble VLA-4, soluble
VCAM-1, soluble LECAM-1, soluble ELAM-1, soluble CD44, antibodies
reactive with gp39 (e.g. ATCC HB-10916, ATCC HB-12055 and ATCC
HB-12056), antibodies reactive with CD40 (e.g. ATCC HB-9110),
antibodies reactive with B7 (e.g. ATCC HB-253, ATCC CRL-2223, ATCC
CRL-2226, ATCC HB-301, ATCC HB-11341, etc), antibodies reactive
with CD28 (e.g. ATCC HB-11944 or mAb 9.3), antibodies reactive with
LFA-1 (e.g. ATCC HB-9579 and ATCC TIB-213), antibodies reactive
with LFA-2, antibodies reactive with IL-2, antibodies reactive with
IL-12, antibodies reactive with IFN-gamma, antibodies reactive with
CD2, antibodies reactive with CD48, antibodies reactive with any
ICAM (e.g., ICAM-1 (ATCC CRL-2252), ICAM-2 and ICAM-3), antibodies
reactive with CTLA4 (e.g. ATCC HB-304), antibodies reactive with
Thy-1, antibodies reactive with CD56, antibodies reactive with CD3,
antibodies reactive with CD29, antibodies reactive with TCR,
antibodies reactive with VLA-4, antibodies reactive with VCAM-1,
antibodies reactive with LECAM-1, antibodies reactive with ELAM-1,
antibodies reactive with CD44; L104EA29YIg, CD80 monoclonal
antibodies (mAbs), CD86 mAbs, gp39 mAbs, CD40 mAbs, CD28 mAbs;
anti-LFA1 mAbs, antibodies or other agents targeting mechanisms of
the immune system such as CD52 (alemtuzumab), CD25 (daclizumab),
VLA-4 (natalizumab), CD20 (rituximab), IL2R (daclizumab) and MS4A1
(ocrelizumab); novel oral immunomodulating agents have shown to
prevent lymphocyte recirculation from lymphoid organs such as
fingolimod (FTY720) or leading to lymphocyte depletion such as
mylinax (oral cladribine) or teriflunomide; and agents that prevent
immunoactivation such as panaclar (dimethyl fumarate BG-12) or
laquinimod (ABR216062). Other combinations will be readily
appreciated and understood by persons skilled in the art. In some
embodiments, the therapeutic agents can be used to attenuate or
reverse the activity of a pro-inflammatory drug, and/or limit the
adverse effects of such drugs.
[0255] As persons skilled in the art will readily understand, the
combination can include the therapeutic agents and/or a
pharmaceutical composition comprising same, according to at least
some embodiments of the invention and one other immunosuppressive
agent; the therapeutic agents and/or a pharmaceutical composition
comprising same, as recited herein, with two other
immunosuppressive agents, the therapeutic agents and/or a
pharmaceutical composition comprising same, as recited herein, with
three other immunosuppressive agents, etc. The determination of the
optimal combination and dosages can be determined and optimized
using methods well known in the art.
[0256] The therapeutic agent according to the present invention and
one or more other therapeutic agents can be administered in either
order or simultaneously.
[0257] The invention also encompasses the use of the therapeutic
agents and/or a pharmaceutical composition comprising same
according to at least some embodiments of the invention in
combination with other pharmaceutical agents to treat immune system
diseases. For example, autoimmune disease may be treated with
molecules according to at least some embodiments of the invention
in conjunction with, immunosuppressants such as corticosteroids,
cyclosporin, cyclophosphamide, prednisone, azathioprine,
methotrexate, rapamycin, tacrolimus, biological agents such as
TNF-alpha blockers or antagonists, immunosuppressive agents (e.g.,
antibodies against other lymphocyte surface markers (e.g., CD40,
alpha-4 integrin) or against cytokines), other fusion proteins
(e.g., CTLA-4-Ig (Orencia.RTM.), TNFR-Ig (Enbrel.RTM.)), TNF-alpha
blockers such as Enbrel, Remicade, Cimzia and Humira,
cyclophosphamide (CTX) (i.e. Endoxan.RTM., Cytoxan.RTM.,
Neosar.RTM., Procytox.RTM., Revimmune.TM.), methotrexate (MTX)
(i.e. Rheumatrex.RTM., Trexall.RTM.), belimumab (i.e.
Benlysta.RTM.), or other immunosuppressive drugs (e.g., cyclosporin
A, FK506-like compounds, rapamycin compounds, or steroids),
anti-proliferatives, cytotoxic agents, or other compounds that may
assist in immunosuppression. or any other biological agent
targeting any inflammatory cytokine, nonsteroidal antiinflammatory
drugs/Cox-2 inhibitors, hydroxychloroquine, sulphasalazopryine,
gold salts, etanercept, infliximab, mycophenolate mofetil,
basiliximab, atacicept, rituximab, cytoxan, interferon beta-1a,
interferon beta-1b, glatiramer acetate, mitoxantrone hydrochloride,
anakinra and/or other biologics and/or intravenous immunoglobulin
(IVIG). Non-limiting examples of such known therapeutics include
interferons, such as IFN-beta-1a (REBIF.RTM.. AVONEX.RTM. and
CINNOVEX .RTM.) and IFN-beta-1b (BETASERON.RTM., EXTAVIA.RTM.,
BETAFERON.RTM., ZIFERON.RTM.); glatiramer acetate (COPAXONE.RTM.),
a polypeptide; natalizumab (TYSABRI.RTM.); and mitoxantrone
(NOVANTRONE.RTM.), a cytotoxic agent.
[0258] Thus, treatment of multiple sclerosis using the agents
according to at least some embodiments of the present invention may
be combined with, for example, any known therapeutic agent or
method for treating multiple sclerosis. Non-limiting examples of
such known therapeutic agent or method for treating multiple
sclerosis include interferon class, IFN-beta-1a (REBIF.RTM.,
AVONEX.RTM. and CINNOVEX.RTM.) and IFN-beta-1b (BETASERON.RTM.,
EXTAVIA.RTM., BETAFERON.RTM., ZIFERON.RTM.); glatiramer acetate
(COPAXONE.RTM.), a polypeptide; natalizumab (TYSABRI.RTM.); and
mitoxantrone (NOVANTRONE.RTM.), a cytotoxic agent, Fampridine
(AMPYRA.RTM.). Other drugs include corticosteroids, methotrexate,
cyclophosphamide, azathioprine, and intravenous immunoglobulin
(IVIG), inosine, Ocrelizumab (R1594), Mylinax (Caldribine),
alemtuzumab (Campath), daclizumab (Zenapax), Panaclar/dimethyl
fumarate (BG-12), Teriflunomide (HMR1726), fingolimod (FTY720),
laquinimod (ABR216062), as well as Haematopoietic stem cell
transplantation, Neurovax, Rituximab (Rituxan) BCG vaccine, low
dose naltrexone, helminthic therapy, angioplasty, venous stents,
and alternative therapy, such as vitamin D, polyunsaturated fats,
medical marijuana.
[0259] Thus, treatment of rheumatoid arthritis, using the agents
according to at least some embodiments of the present invention may
be combined with, for example, any known therapeutic agent or
method for treating rheumatoid arthritis. Non-limiting examples of
such known therapeutic agents or methods for treating rheumatoid
arthritis include glucocorticoids, nonsteroidal anti-inflammatory
drug (NSAID) such as salicylates, or cyclooxygenase-2 inhibitors,
ibuprofen and naproxen, diclofenac, indomethacin, etodolac
Disease-modifying antirheumatic drugs (DMARDs)-Oral DMARDs:
Auranofin (Ridaura), Azathioprine (Imuran), Cyclosporine
(Sandimmune, Gengraf, Neoral, generic), D-Penicillamine
(Cuprimine), Hydroxychloroquine (Plaquenil), IM gold Gold sodium
thiomalate (Myochrysine) Aurothioglucose (Solganal), Leflunomide
(Arava), Methotrexate (Rheumatrex), Minocycline (Minocin),
Staphylococcal protein A immunoadsorption (Prosorba column),
Sulfasalazine (Azulfidine). Biologic DMARDs: TNF-.alpha. blockers
including Adalimumab (Humira), Etanercept (Enbrel), Infliximab
(Remicade), golimumab (Simponi), certolizumab pegol (Cimzia), and
other Biological DMARDs, such as Anakinra (Kineret), Rituximab
(Rituxan), Tocilizumab (Actemra), CD28 inhibitor including
Abatacept (Orencia) and Belatacept.
[0260] Thus, treatment of IBD, using the agents according to at
least some embodiments of the present invention may be combined
with, for example, any known therapeutic agent or method for
treating IBD. Non-limiting examples of such known therapeutic
agents or methods for treating IBD include immunosuppression to
control the symptom, such as prednisone, Mesalazine (including
Asacol, Pentasa, Lialda, Aspiro),azathioprine (Imuran),
methotrexate, or 6-mercaptopurine, steroids, Ondansetron,
TNF-.alpha. blockers (including infliximab, adalimumab golimumab,
certolizumab pegol), Orencia (abatacept), ustekinumab
(Stelara.RTM.), Briakinumab (ABT-874), Certolizumab pegol
(Cimzia.RTM.), ITF2357 (givinostat), Natalizumab (Tysabri),
Firategrast (SB -683699), Remicade (infliximab), vedolizumab
(MLN0002), other drugs including GSK1605786 CCX282-B (Traficet-EN),
AJM300, Stelara (ustekinumab), Semapimod (CNI-1493) tasocitinib
(CP-690550), LMW Heparin MMX, Budesonide MMX, Simponi (golimumab),
MultiStem.RTM., Gardasil HPV vaccine, Epaxal Berna (virosomal
hepatitis A vaccine), surgery, such as bowel resection,
strictureplasty or a temporary or permanent colostomy or ileostomy;
antifungal drugs such as nystatin (a broad spectrum gut antifungal)
and either itraconazole (Sporanox) or fluconazole (Diflucan);
alternative medicine, prebiotics and probiotics, cannabis,
Helminthic therapy or ova of the Trichuris suis helminth.
[0261] Thus, treatment of psoriasis, using the agents according to
at least some embodiments of the present invention may be combined
with, for example, any known therapeutic agent or method for
treating psoriasis. Non-limiting examples of such known
therapeutics for treating psoriasis include topical agents,
typically used for mild disease, phototherapy for moderate disease,
and systemic agents for severe disease. Non-limiting examples of
topical agents: bath solutions and moisturizers, mineral oil, and
petroleum jelly; ointment and creams containing coal tar, dithranol
(anthralin), corticosteroids like desoximetasone (Topicort),
Betamethasone, fluocinonide, vitamin D3 analogues (for example,
calcipotriol), and retinoids. Non-limiting examples of
phototherapy: sunlight; wavelengths of 311-313 nm, psoralen and
ultraviolet A phototherapy (PUVA). Non-limiting examples of
systemic agents: Biologics, such as interleukin antagonists,
TNF-.alpha. blockers including antibodies such as infliximab
(Remicade), adalimumab (Humira), golimumab, certolizumab pegol, and
recombinant TNF-.alpha. decoy receptor, etanercept (Enbrel); drugs
that target T cells, such as efalizumab (Xannelim/Raptiva),
alefacept (Ameviv), dendritic cells such Efalizumab; monoclonal
antibodies (MAbs) targeting cytokines, including anti-IL-12/IL-23
(ustekinumab (brand name Stelara)) and anti-Interleukin-17;
Briakinumab (ABT-874); small molecules, including but not limited
to ISA247; Immunosuppressants, such as methotrexate, cyclosporine;
vitamin A and retinoids (synthetic forms of vitamin A); and
alternative therapy, such as changes in diet and lifestyle, fasting
periods, low energy diets and vegetarian diets, diets supplemented
with fish oil rich in Vitamin A and Vitamin D (such as cod liver
oil), Fish oils rich in the two omega-3 fatty acids
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and
contain Vitamin E Ichthyotherapy, Hypnotherapy, cannabis.
[0262] Thus, treatment of type 1 diabetes, using the agents
according to at least some embodiments of the present invention may
be combined with, for example, any known therapeutic agent or
method for treating type ldiabetes. Non-limiting examples of such
known therapeutics for treating type 1 diabetes include insulin,
insulin analogs, islet transplantation, stem cell therapy including
PROCHYMAL.RTM., non-insulin therapies such as il-1beta inhibitors
including Anakinra (Kineret.RTM.), Abatacept (Orencia.RTM.),
Diamyd, alefacept (Ameviv.RTM.), Otelixizumab, DiaPep277 (Hsp60
derived peptide), Alpha 1-Antitrypsin, Prednisone, azathioprine,
Ciclosporin, El-INT (an injectable islet neogenesis therapy
comprising an epidermal growth factor analog and a gastrin analog),
statins including Zocor.RTM., Simlup.RTM., Simcard.RTM.,
Simvacor.RTM., Sitagliptin (dipeptidyl peptidase (DPP-4)
inhibitor), Anti-CD3 mAb (e.g., Teplizumab); CTLA4-Ig (abatacept),
Anti IL-1Beta (Canakinumab), Anti-CD20 mAb (e.g, rituximab).
[0263] Thus, treatment of uveitis, using the agents according to at
least some embodiments of the present invention may be combined
with, for example, any known therapeutic agent or method for
treating uveitis. Non-limiting examples of such known therapeutics
for treating uveitis include corticosteroids, topical cycloplegics,
such as atropine or homatropine, or injection of PSTTA (posterior
subtenon triamcinolone acetate), antimetabolite medications, such
as methotrexate, TNF-.alpha. blockers (including infliximab,
adalimumab, etanercept, golimumab, certolizumab pegol).
[0264] Thus, treatment for Sjogren's syndrome, using the agents
according to at least some embodiments of the present invention may
be combined with, for example, any known therapeutic agent or
method for treating for Sjogren's syndrome. Non-limiting examples
of such known therapeutics for treating for Sjogren' s syndrome
include Cyclosporine, pilocarpine (Salagen) and cevimeline
(Evoxac), Hydroxychloroquine (Plaquenil), cortisone (prednisone and
others) and/or azathioprine (Imuran) or cyclophosphamide (Cytoxan),
Dexamethasone, Thalidomide, Dehydroepiandrosterone, NGX267,
Rebamipide, FID 114657, Etanercept, Raptiva, Belimumab, MabThera
(rituximab); Anakinra, intravenous immune globulin (IVIG),
Allogeneic Mesenchymal Stem Cells (AlloMSC), Automatic
neuro-electrostimulation by "Saliwell Crown".
[0265] Thus, treatment for systemic lupus erythematosus, using the
agents according to at least some embodiments of the present
invention may be combined with, for example, any known therapeutic
agent or method for treating for systemic lupus erythematosus.
Non-limiting examples of such known therapeutics for treating for
systemic lupus erythematosus include corticosteroids and
Disease-modifying antirheumatic drugs (DMARDs), commonly
anti-malarial drugs such as plaquenil and immunosuppressants (e.g.
methotrexate and azathioprine) Hydroxychloroquine, cytotoxic drugs
(e.g., cyclophosphamide and mycophenolate), Hydroxychloroquine
(HCQ), Benlysta (belimumab), nonsteroidal anti-inflammatory drugs,
Prednisone, Cellcept, Prograf, Atacicept, Lupuzor, Intravenous
Immunoglobulins (IVIGs), CellCept (mycophenolate mofetil), Orencia,
CTLA4-IgG4m (RG2077), rituximab, Ocrelizumab, Epratuzumab, CNTO
136, Sifalimumab (MEDI-545), A-623 (formerly AMG 623), AMG 557,
Rontalizumab, paquinimod (ABR-215757), LY2127399, CEP-33457,
Dehydroepiandrosterone, Levothyroxine, abetimus sodium (LIP 394),
Memantine, Opiates, Rapamycin, Renal transplantation, stem cell
transplantation.
[0266] The therapeutic agents and/or a pharmaceutical composition
comprising same, as recited herein, according to at least some
embodiments of the invention, may be administered as the sole
active ingredient or together with other drugs in immunomodulating
regimens or other anti-inflammatory agents e.g. for the treatment
or prevention of allo- or xenograft acute or chronic rejection or
inflammatory or autoimmune disorders, or to induce tolerance.
[0267] For example, it may be used in combination with a
calcineurin inhibitor, e.g. cyclosporin A or FK506; an
immunosuppressive macrolide, e.g. rapamycine or a derivative
thereof; e.g. 40-O-(2-hydroxy)ethyl-rapamycin, a lymphocyte homing
agent, e.g. FTY720 or an analog thereof, corticosteroids;
cyclophosphamide; azathioprene; methotrexate; leflunomide or an
analog thereof; mizoribine; mycophenolic acid; mycophenolate
mofetil; 15-deoxyspergualine or an analog thereof;
immunosuppressive monoclonal antibodies, e.g., monoclonal
antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD 1
la/CD18, CD7, CD25, CD 27, B7, CD40, CD45, CD58, CD 137, ICOS,
CD150 (SLAM), 0X40, 4-1BB or their ligands; or other
immunomodulatory compounds, e.g. CTLA4-Ig (abatacept, ORENCIA.RTM.
or belatacept), CD28-Ig, B7-H4-Ig, or other costimulatory agents,
or adhesion molecule inhibitors, e.g. mAbs or low molecular weight
inhibitors including LFA-1 antagonists, Selectin antagonists and
VLA-4 antagonists.
[0268] Where the therapeutic agents and/or a pharmaceutical
composition comprising same, as recited herein, according to at
least some embodiments of the invention are administered in
conjunction with other immunosuppressive/immunomodulatory or
anti-inflammatory therapy, e.g. as herein above specified, dosages
of the co-administered immunosuppressant, immunomodulatory or
anti-inflammatory compound will of course vary depending on the
type of co-drug employed, e.g. whether it is a steroid or a
cyclosporin, on the specific drug employed, on the condition being
treated and so forth.
[0269] According to at least some embodiments of the present
invention, there is provided use of a combination of the
therapeutic agents and/or a pharmaceutical composition comprising
same, as recited herein, and a known therapeutic agent effective
for treating infection.
[0270] The therapeutic agents and/or a pharmaceutical composition
comprising same, as recited herein, can be administered in
combination with one or more additional therapeutic agents used for
treatment of bacterial infections, including, but not limited to,
antibiotics including
[0271] Aminoglycosides, Carbapenems, Cephalosporins, Macrolides,
Lincosamides, Nitrofurans, penicillins, Polypeptides, Quinolones,
Sulfonamides, Tetracyclines, drugs against mycobacteria including
but not limited to Clofazimine, Cycloserine, Cycloserine,
Rifabutin, Rifapentine, Streptomycin and other antibacterial drugs
such as Chloramphenicol, Fosfomycin, Metronidazole, Mupirocin, and
Tinidazole.
[0272] The therapeutic agents and/or a pharmaceutical composition
comprising same, as recited herein, can be administered in
combination with one or more additional therapeutic agents used for
treatment of viral infections, including, but not limited to,
antiviral drugs such as oseltamivir (brand name Tamiflu) and
zanamivir (brand name Relenza) Arbidol--adamantane derivatives
(Amantadine, Rimantadine)--neuraminidase inhibitors (Oseltamivir,
Laninamivir, Peramivir, Zanamivir) nucleotide analog reverse
transcriptase inhibitor including Purine analogue guanine
(Aciclovir#/Valacyclovir, Ganciclovir/Valganciclovir,
Penciclovir/Famciclovir) and adenine (Vidarabine), Pyrimidine
analogue, uridine (Idoxuridine, Trifluridine, Edoxudine), thymine
(Brivudine), cytosine (Cytarabine); Foscarnet; Nucleoside
analogues/NARTIs: Entecavir, Lamivudine, Telbivudine, Clevudine;
Nucleotide analogues/NtRTIs: Adefovir , Tenofovir; Nucleic acid
inhibitors such as Cidofovir; InterferonInterferon alfa-2b,
Peginterferon alfa-2a; Ribavirin#/Taribavirin; antiretroviral drugs
including zidovudine, lamivudine, abacavir, lopinavir, ritonavir,
tenofovir/emtricitabine, efavirenz each of them alone or a various
combinations, gp41 (Enfuvirtide), Raltegravir, protease inhibitors
such as Fosamprenavir, Lopinavir and Atazanavir, Methisazone,
Docosanol, Fomivirsen, Tromantadine.
[0273] The therapeutic agents and/or a pharmaceutical composition
comprising same, as recited herein, can be administered in
combination with one or more additional therapeutic agents used for
treatment of fungal infections, including, but not limited to,
antifungal drugs of the Polyene antifungals, Imidazole, triazole,
and thiazole antifungals, Allylamines, Echinocandins or other anti
fungal drugs.
[0274] Alternatively or additionally, an upregulating method may
optionally be effected by specifically upregulating the amount
(optionally expression) in the subject of at least one of the
polypeptides of the present invention or active portions
thereof.
[0275] As is mentioned hereinabove and in the Examples section
which follows, the biomolecular sequences of this aspect of the
present invention may be used as valuable therapeutic tools in the
treatment of diseases, disorders or conditions in which altered
activity or expression of the wild-type gene product (known
protein) is known to contribute to disease, disorder or condition
onset or progression. For example, in case a disease is caused by
overexpression of a membrane bound-receptor, a soluble variant
thereof may be used as an antagonist which competes with the
receptor for binding the ligand, to thereby terminate signaling
from the receptor.
[0276] According to at least some embodiments, immune cells,
preferably T cells, can be contacted in vivo or ex vivo with the
therapeutic agents to modulate immune responses. The T cells
contacted with the therapeutic agents can be any cell which
expresses the T cell receptor, including .alpha./.beta. and
.gamma./.delta. T cell receptors. T-cells include all cells which
express CD3, including T-cell subsets which also express CD4 and
CDS. T-cells include both naive and memory cells and effector cells
such as CTL. T-cells also include cells such as Th1, Tc1, Th2, Tc2,
Th3, Th17, Th22, Treg, and Tr1 cells. T-cells also include
NKT-cells and similar unique classes of the T-cell lineage.
[0277] In a further embodiment, the additional therapeutic agent
functions to inhibit or reduce T cell activation through a separate
pathway. In one such embodiment, the additional therapeutic agent
is a CTL A-4 fusion protein, such as CTLA-4-Ig (abatacept).
CTLA-4-Ig fusion proteins compete with the co-stimulatory receptor,
CD28, on T cells for binding to CD80/CD86 (B7-1/B7-2) on antigen
presenting cells, and thus function to inhibit T cell activation.
In another embodiment, the additional therapeutic agent is a
CTLA-4-Ig fusion protein known as belatacept. Belatacept contains
two amino acid substitutions (L104E and A29Y) that markedly
increase its avidity to CD86 in vivo. In another embodiment, the
additional therapeutic agent is Maxy-4.
[0278] In another embodiment, the second therapeutic agent is
cyclophosphamide (CTX). Cyclophosphamide (the generic name for
Endoxan.RTM., Cytoxan.RTM., Neosar.RTM., Procytox.RTM.,
Revimmune.TM.), also known as cytophosphane, is a nitrogen mustard
alkylating agent from the oxazophorines group. It is used to treat
various types of cancer and some autoimmune disorders. In a further
embodiment, IgC1ORF32 polypeptides, fragments or fusion proteins
thereof and CTX are coadministered in effective amount to prevent
or treat a chronic autoimmune disease or disorder such as Systemic
lupus erythematosus (SLE). Cyclophosphamide (CTX) is the primary
drug used for diffuse proliferative glomerulonephritis in patients
with renal lupus. In some embodiments the combination therapy is
administered in an effective amount to reduce the blood or serum
levels of anti-double stranded DNA (anti-ds DNA) auto antibodies
and/or to reduce proteinuria in a patient in need thereof.
[0279] In another embodiment, the second therapeutic is Tysabri or
another therapeutic for MS. In a further embodiment, IgC1ORF32
polypeptides, fragments or fusion proteins thereof is cycled with
Tysabri or used during a drug holiday in order to allow less
frequent dosing with the second therapeutic and reduce the risk of
side effects such as PML and to prevent resistance to the second
therapeutic.
[0280] In another embodiment, the second therapeutic agent
preferentially treats chronic inflammation, whereby the treatment
regimen targets both acute and chronic inflammation. In a further
embodiment the second therapeutic is a TNF-alpha blocker.
[0281] In another embodiment, the second therapeutic agent is a
small molecule that inhibits or reduces differentiation,
proliferation, activity, and/or cytokine production and/or
secretion by Th1, Th17, Th22, and/or other cells that secrete, or
cause other cells to secrete, inflammatory molecules, including,
but not limited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma,
IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. In another embodiment,
the second therapeutic agent is a small molecule that interacts
with Tregs, enhances Treg activity, promotes or enhances IL-10
secretion by Tregs, increases the number of Tregs, increases the
suppressive capacity of Tregs, or combinations thereof.
[0282] Typically useful small molecules are organic molecules,
preferably small organic compounds having a molecular weight of
more than 100 and less than about 2,500 daltons, more preferably
between 100 and 2000, more preferably between about 100 and about
1250, more preferably between about 100 and about 1000, more
preferably between about 100 and about 750, more preferably between
about 200 and about 500 daltons. Small molecules comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and typically include at
least an amine, carbonyl, hydroxyl or carboxyl group, preferably at
least two of the functional chemical groups. The small molecules
often comprise cyclical carbon or heterocyclic structures andlor
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Small molecules also include
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. In one embodiment, the small molecule is
retinoic acid or a derivative thereof. The examples below
demonstrate that retinoic acid inhibits or reduces differentiation
and/or activity of ThI 7 cells. In a further embodiment, the
compositions are used in combination or succession with compounds
that increase Treg activity or production. Exemplary Treg enhancing
agents include but are not limited to glucocorticoid fluticasone,
salmeteroal, antibodies to IL-12, IFN-gamma, and IL-4; vitamin D3,
and dexamethasone, and combinations thereof. Antibodies to other
proinflammatory molecules can also be used in combination or
alternation with the disclosed C1ORF32 polypeptide selected from
the group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,
variant, a homolog, a fusion protein or a conjugate thereof.
Preferred antibodies bind to IL-6, IL-23, IL-22 or IL-21.
[0283] As used herein the term "rapamycin compound" includes the
neutral tricyclic compound rapamycin, rapamycin derivatives,
rapamycin analogs, and other macrolide compounds which are thought
to have the same mechanism of action as rapamycin (e.g., inhibition
of cytokine function). The language "rapamycin compounds" includes
compounds with structural similarity to rapamycin, e.g., compounds
with a similar macrocyclic structure, which have been modified to
enhance their therapeutic effectiveness. Exemplary Rapamycin
compounds are known in the art. The language "FK506-Hke compounds"
includes FK506, and FK506 derivatives and analogs, e.g., compounds
with structural similarity to FK506, e.g., compounds with a similar
macrocyclic structure which have been modified to enhance their
therapeutic effectiveness. Examples of FK506-like compounds
include, for example, those described in WO 00101385. Preferably,
the language "rapamycin compound" as used herein does not include
FK506-like compounds.
[0284] Other suitable therapeutics include, but are not limited to,
anti-inflammatory agents. The anti-inflammatory agent can be
non-steroidal, steroidal, or a combination thereof. One embodiment
provides oral compositions containing about 1% (w/w) to about 5%
(w/w), typically about 2.5 (w/w) or an anti-inflammatory agent.
Representative examples of non-steroidal anti-inflammatory agents
include, without limitation, oxicams, such as piroxicam, isoxicam,
tenoxicam, sudoxicam; salicylates, such as aspirin, disalcid,
benorylate, trilisate, safapryn, solprin, diflunisal, and fendosal;
acetic acid derivatives, such as diclofenac, fenclofenac,
indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac,
zidometacin, acematacin, fentiazac, zomepirac, clmdanac, oxepinac,
felbmac, and ketorolac; fenamates, such as mefenamic, meclofenamic,
flufenamic, niflumic, and tolfenamic acids; propionic acid
derivatives, such as ibuprofen, naproxen, benoxaprofen,
flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen,
pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen,
tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles,
such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone,
and trimethazone. Mixtures of these non-steroidal anti-inflammatory
agents may also be employed.
[0285] Representative examples of steroidal anti-inflammatory drugs
include, without limitation, corticosteroids such as
hydrocortisone, hydroxyl-triamcinolone, alpha-methyl dexamethasone,
dexamethasone-phosphate, beclomethasone dipropionates, clobetasol
valerate, desonide, desoxymethasone, desoxycorticosterone acetate,
dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone
valerate, fluadrenolone, fluclorolone acetonide, fludrocortisone,
flumethasone pivalate, fiuosinolone acetonide, fluocinonide,
flucortine butylesters, fluocortolone, fluprednidene
(fluprednylidene) acetate, flurandrenolone, halcinonide,
hydrocortisone acetate, hydrocortisone butyrate,
methylprednisolone, triamcinolone acetonide, cortisone,
cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate,
fluradrenolone, fludrocortisone, diflurosone diacetate,
fluradrenolone acetonide, medrysone, amcinafel, amcinafide,
betamethasone and the balance of its esters, chloroprednisone,
chlorprednisone acetate, clocortelone, clescinolone, dichlorisone,
diflurprednate, flucloronide, flunisolide, fluoromethalone,
fluperolone, fluprednisolone, hydrocortisone valerate,
hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone,
paramethasone, prednisolones prednisone, beclomethasone
dipropionate, triamcinolone, and mixtures thereof.
[0286] Methods of Therapeutic Use
[0287] The C1ORF32 polypeptides, fragments or fusion proteins
thereof are useful as therapeutic agents. According to at least
some embodiments, immune cells, preferably T cells, can be
contacted in vivo or ex vivo with C1ORF32 fusion polypeptides to
decrease or inhibit immune responses including, but not limited to
inflammation. According to at least some other embodiments, immune
cells, preferably T cells, can be contacted in vivo or ex vivo with
C1ORF32 fusion polypeptides to decrease or inhibit T cell
exhaustion, optionally in combination with another therapeutic
agent. In both cases, preferably the costimulatory pathway is
modulated to achieve a desired immune system balance.
[0288] In either case, the T cells contacted with C1ORF32 fusion
polypeptides can be any cell which expresses the T cell receptor,
including .alpha./.beta. and .gamma./.delta. T cell receptors.
T-cells include all cells which express CD3, including T-cell
subsets which also express CD4 and CDS. T-cells include both naive
and memory cells and effector cells such as CTL. T-cells also
include cells such as Th1, Tc1, Th2, Tc2, Th3, Th17, Th22, Treg,
and Trl cells. T-cells also include NKT-cells and similar unique
classes of the T-cell lineage. For example the compositions can be
used to modulate Th1, Th17, Th22, or other cells that secrete, or
cause other cells to secrete, inflammatory molecules, including,
but not limited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma,
IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. The compositions can
also be used to increase or promote the activity of Tregs, increase
the production of cytokines such as IL-10 from Tregs, increase the
differentiation of Tregs, increase the number of Tregs, or increase
the survival of Tregs. The compositions can also be used to
increase or promote the activity of Th2 cells, increase the
production of cytokines such as IL-10 or IL-4 from Th2 cells,
increase the differentiation of Th2 cells, increase the number of
Th2 cells, or increase the survival of Th2 cells.
[0289] Although the below discussion applies generally to the
reversal of T cell exhaustion, preferably for treatment of such
exhaustion memory T cells are treated. Optionally, additionally or
alternatively, treatment of T cell exhaustion encompasses the
modulation of cytokine secretion, or other cells that secrete, or
cause other cells to secrete, inflammatory molecules, including,
but not limited to, IL-2, TNF-alpha, IFN-gamma, 15 Granzyme B and
MMPs or modulate the expression of molecues of the
costimulatory/coinhibtory-family including, but not limited to
PD-1, Tim3, CTLA4 and LAGS.
[0290] In some embodiments, the disclosed C1ORF32 polypeptide,
selected from the group consisting of SEQ ID NOs: 29, 30, 41-105,
or a fragment, variant, a homolog, a fusion protein or a conjugate
thereof, are administered in combination with a second therapeutic.
Combination therapies may be useful in immune modulation. In some
embodiments, C1ORF32 polypeptide, selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof, can be used to
attenuate or reverse the activity of a pro-inflammatory drug,
and/or limit the adverse effects of such drugs.
[0291] Other immune cells that can be treated with the disclosed
C1ORF32 polypeptides, fragments or fusion thereof include T cell
precursors, antigen presenting cells such as dendritic cells and
monocytes or their precursors, B cells or combinations thereof. The
C1ORF32 compositions can be used to modulate the production of
antibodies by B cells by contacting the B cells with an effective
amount of the C1ORF32 composition to inhibit or reduce antibody
production by the B cell relative to a control. The C1ORF32
compositions can also modulate the production of cytokines by the B
cells.
[0292] Methods of Treating Inflammatory Responses
[0293] The C1ORF32 polypeptides, fragments or fusion proteins
thereof, selected from the group consisting of SEQ ID NOs: 29, 30,
41-105, or a fragment, variant, a homolog, a fusion protein or a
conjugate thereof, according to at least some embodiments of the
present invention inhibit T cell activation, as manifested by T
cell proliferation and cytokine secretion. Specifically, the
proteins inhibit T Th1 and Th17 responses, while promoting Th2
responses. The C1ORF32 polypeptides, fragments or fusion proteins
thereof, selected from the group consisting of SEQ ID NOs: 29, 30,
41-105, or a fragment, variant, a homolog, a fusion protein or a
conjugate thereof, according to at least some embodiments of the
present invention are potentially used for therapy of diseases that
require down-regulation of costimulatory pathways and or such that
require downregulation of Th1 and/or Th17 responses. A further
embodiment provides methods for treating or alleviating one or more
symptoms of inflammation. In a further embodiment, the compositions
and methods disclosed are useful for treating chronic and
persistent inflammation. Inflammation in general can be treated
using the disclosed C1ORF32 polypeptides or fragment or fusions
thereof.
[0294] According to at least some embodiments of the present
invention, there is provided use of an isolated C1ORF32 polypeptide
as described herein or a fusion protein comprising an isolated
C1ORF32 polypeptide as described herein, optionally in a
pharmaceutical composition comprising a pharmaceutically acceptable
diluent or carrier, for treatment of an immune related disorder
and/or infection.
[0295] An immune response including inflammation can be inhibited
or reduced in a subject, preferably a human, by administering an
effective amount of C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof, to inhibit or
reduce the biological activity of an immune cell or to reduce the
amounts of proinflammatory molecules at a site of inflammation.
Exemplary proinflammatory molecules include, but are not limited
to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6, IL-23,
IL-22, IL-21, and MMPs. Th1 and Th17 are exemplary T cells that can
be targeted for inhibition by C1ORF32 polypeptide selected from the
group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,
variant, a homolog, a fusion protein or a conjugate thereof, to
inhibit or reduce inflammation.
[0296] Without wishing to be limited by a single hypothesis for
this biological mechanism or any other biological mechanism
described herein, the C1ORF32 polypeptides, fragments or fusion
proteins thereof are useful for treating inflammation by any or all
of the following: inhibiting or reducing differentiation of Th1,
Thl7, Th22, and/or other cells that secrete, or cause other cells
to secrete, inflammatory molecules, including, but not limited to,
IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6, IL-23,
IL-22, IL-21, and MMPs; inhibiting or reducing activity of ThI, Th
17, Th22, and/or other cells that secrete, or cause other cells to
secrete, inflammatory molecules, including, but not limited to,
IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6, IL-23,
IL-22, IL-21, and MMPs; inhibiting or reducing the Th1 and/or Thl7
pathways; inhibiting or reducing cytokine production and/or
secretion by Th1, Th17, Th22, and/or other cells that secrete, or
cause other cells to secrete, inflammatory molecules, including,
but not limited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma,
IL-17, IL-6 IL-23, IL-22, IL-21, and MMPs; inhibiting or reducing
proliferation of Th1, Th17, Th22, and/or other cells that secrete,
or cause other cells to secrete, inflammatory molecules, including,
but not limited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma,
IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.
[0297] Additionally, C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof can also enhance
Th2 immune responses. C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof can also act
directly on Th2 cells to promote or enhance production of IL-4,
IL-5 or IL-10, or to increase the number of Th2 cells, resulting in
inhibition of Th1 and/or Th17, and in immune modulation via a
Th1/Th2 shift.
[0298] Additionally, C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof can cause Tregs to
have an enhanced suppressive effect on an immune response. Tregs
can suppress differentiation, proliferation, activity, and/or
cytokine production and/or secretion by Th1, Th17, Th22, and/or
other cells that secrete, or cause other cells to secrete,
inflammatory molecules, including, but not limited to, IL-1beta,
TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6, IL-23, IL-22, IL-21,
and MMPs. For example, C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof can cause Tregs to
have an enhanced suppressive effect on Th1 and/or Th17 cells to
reduce the level of IFN-gamma and IL-17 produced, respectively.
C1ORF32polypeptide selected from the group consisting of SEQ ID
NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusion
protein or a conjugate thereof can also act directly on Tregs to
promote or enhance production of IL-10 to suppress the Th1 and/or
Th17 pathway, and/or to increase the number of Tregs.
[0299] Additionally, C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof can cause Th2 to
have an enhanced modulatory effect on an immune response. Th2 cells
can modulate differentiation, proliferation, activity, and/or
cytokine production and/or secretion by Th1, Th17, Th22, and/or
other cells that secrete, or cause other cells to secrete,
inflammatory molecules, including, but not limited to, IL-1beta,
TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6, IL-23, IL-22, IL-21,
and MMPs. For example, C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof can cause Th2
cells to have an enhanced modulatory effect on Th1 and/or Th17
cells to reduce the level of IFN-gamma and IL-17 produced,
respectively. C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof can also act
directly on Th2 cells to promote or enhance production of IL-10 to
suppress the Th1 and/or Th17 pathway, and/or to increase the number
of Th2 cells.
[0300] Without wishing to be limited by a single hypothesis, it is
believed that C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof acts at multiple
points in multiple T cell pathways. For example, polypeptide
selected from the group consisting of SEQ ID NOs: 29, 30, 41-105,
or a fragment, variant, a homolog, a fusion protein or a conjugate
thereof can inhibit the differentiation of naive T cells into
either Th1 or Th17 cells. Alternatively, polypeptide selected from
the group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,
variant, a homolog, a fusion protein or a conjugate thereof can
interact with Th1 cells or Th17 cells, or both to inhibit or reduce
the production of proinflammatory molecules.
[0301] Additionally, C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof may increase the
differentiation of and/or promote Th2 responses resulting in an
immunomdulatory effect on the Th1 and/or Th17 pathways to reduce
the level of INF-gamma and/or IL-17 produced. C1ORF32 polypeptide
selected from the group consisting of SEQ ID NOs: 29, 30, 41-105,
or a fragment, variant, a homolog, a fusion protein or a conjugate
thereof enhances the production of IL-10 from cells such as Th2
and/or Tregs, which in turn inhibits the activity of Th1 and/or
Th17 cells.
[0302] Additionally, C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof can affect Tregs
to have an enhanced suppressive effect on Th1 and/or Th17 pathways
to reduce the level of INF-gamma and/or IL-17 produced.
Additionally, C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof can enhance the
production of IL-10 which inhibits the activity of Th1 and/or Th17
cells.
[0303] Inhibition of Th1 Responses
[0304] a. Inhibition of Th1 Development
[0305] One method for inhibiting or reducing inflammation includes
administering an effective amount of a C1ORF32 polypeptide selected
from the group consisting of SEQ ID NOs: 29, 30, 41-105, or a
fragment, variant, a homolog, a fusion protein or a conjugate
thereof to inhibit Th1 development in a subject in need thereof.
Inflammation can be inhibited or reduced by blocking naive T cells
from differentiating into Th1 cells by administering C1ORF32
polypeptide selected from the group consisting of SEQ ID NOs: 29,
30, 41-105, or a fragment, variant, a homolog, a fusion protein or
a conjugate thereof. In one embodiment, the C1ORF32 polypeptide
selected from the group consisting of SEQ ID NOs: 29, 30, 41-105,
or a fragment, variant, a homolog, a fusion protein or a conjugate
thereof may inhibit or reduce proliferation of Th1 cells. C1ORF32
polypeptide selected from the group consisting of SEQ ID NOs: 29,
30, 41-105, or a fragment, variant, a homolog, a fusion protein or
a conjugate thereof may also reduce naive T cells from
differentiating into Th1 cells, by blocking antigen presenting cell
maturation. Alternatively, C1ORF32 polypeptide selected from the
group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,
variant, a homolog, a fusion protein or a conjugate thereof
increase the differentiation of Th2 cells and thereby reduce the
number of Th1 cells in a subject. By restricting the number of Th1
cells that can develop in the subject, the amount of
proinflammatory molecules such as INF-gamma can be reduced or
contained. INF-gamma stimulates the production or release of other
proinflammatory molecules including IL-1beta, TNF-alpha, and MMPs.
Thus, by controlling the number of Th1 cells in a subject, the
levels of these other proinflammatory molecules can be controlled,
thereby reducing inflammatory responses.
[0306] b. Inhibition of Proinflammatory Molecules
[0307] Another embodiment provides a method of inhibiting or
reducing inflammation in a subject by administering to the subject
an effective amount of a C1ORF32 polypeptide selected from the
group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,
variant, a homolog, a fusion protein or a conjugate thereof to
inhibit or reduce production of proinflammatory molecules by Th1
cells.
[0308] Exemplary proinflammatory molecules produced by Th1 cells
includes IFN-gamma. In this embodiment the C1ORF32 polypeptide
selected from the group consisting of SEQ ID NOs: 29, 30, 41-105,
or a fragment, variant, a homolog, a fusion protein or a conjugate
thereof can interact directly with the Th1 cell and inhibit or
reduce IFN-gamma production by the Th1 cells. In this embodiment,
the amount of proinflammatory molecules is regulated rather than
the population of Th1 cells.
[0309] Inhibition of Th17 Responses
[0310] a. Inhibition of Th17 Development
[0311] Inflammation can also be inhibited or reduced in a subject
by administering an effective amount of a C1ORF32 polypeptide
selected from the group consisting of SEQ ID NOs: 29, 30, 41-105,
or a fragment, variant, a homolog, a fusion protein or a conjugate
thereof, to inhibit or block naive T cells from developing into
Th17 cells. In one embodiment, the C1ORF32 polypeptide selected
from the group consisting of SEQ ID NOs: 29, 30, 41-105, or a
fragment, variant, a homolog, a fusion protein or a conjugate
thereof increases the suppressive activity of Tregs on the
differentiation of naive T cells into Th17 cells by an amount
sufficient to reduce the number of Th17 cells in a subject.
Alternatively, the C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof inhibits or
reduces proliferation of Th17 cells. C1ORF32 polypeptide selected
from the group consisting of SEQ ID NOs: 29, 30, 41-105, or a
fragment, variant, a homolog, a fusion protein or a conjugate
thereof may also reduce naive T cells from differentiating into
Th17 cells, by blocking antigen presenting cell maturation. By
reducing the population of Th17 cells in a subject, the amount of
IL-17 can be reduced, as well as IL-22 and IL-21. IL-17 is a
proinflammatory cytokine that causes increases in other
proinflammatory molecules such as IL-1beta, TNF-alpha, and MMPs.
Thus, by reducing the amount of IL-17 these other proinflammatory
molecules can be reduced, thereby reducing or inhibiting
inflammation.
[0312] b. Inhibition of IL-17 Production
[0313] Still another embodiment provides a method for treating
inflammation in a subject by administering an effective amount of
C1ORF32 polypeptide selected from the group consisting of SEQ ID
NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusion
protein or a conjugate thereof, to inhibit production of IL-17 by
Th17 cells, as well as IL-22 and IL-21. In this embodiment, the
C1ORF32 polypeptide selected from the group consisting of SEQ ID
NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusion
protein or a conjugate thereof can act directly on Th17 cells, for
example by binding to Th17 cells resulting in inhibition of IL-17
(or IL-22 and IL-21) production by those Th17 cells. As noted
above, inhibition or reduction of IL-17 (and IL-22 or IL-21) leads
to the reduction of other proinflammatory molecules, thereby
reducing or inhibitng inflammation.
[0314] Inhibiting Th1 and Th17 Responses
[0315] The disclosed C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof can be used to
inhibit both the Th1 and Th17 pathways simultaneously. Using one
anti-inflammatory agent to inhibit two separate pathways provides
more robust inhibition or reduction of the immune response.
[0316] Promoting Th2 Responses and IL-10 Production.
[0317] Inflammation can also be treated by administering C1ORF32
polypeptide selected from the group consisting of SEQ ID NOs: 29,
30, 41-105, or a fragment, variant, a homolog, a fusion protein or
a conjugate thereof thereof to a subject in an amount effective to
enhance Th2 responses, and the suppressive activity of IL-10
producing cells, and to enhance suppressive or modulatory activity
on the Th1 and/or Th17 pathways. In this embodiment the disclosed
C1ORF32 polypeptide selected from the group consisting of SEQ ID
NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusion
protein or a conjugate thereof cause an increased suppressive
effect on IFN-gamma and/or IL-17 production. Another embodiment
provides a method for treating inflammation by administering an
effective amount of C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof to increase
production of IL-10 by Th2, Tregs or other immune cells.
[0318] Increased production of IL-10 results in the decreased
production of IL-17 by Th17 cells and deceased production of
IFN-gamma by Th1 cells. In this embodiment, the C1ORF32 polypeptide
selected from the group consisting of SEQ ID NOs: 29, 30, 41-105,
or a fragment, variant, a homolog, a fusion protein or a conjugate
thereof can interact directly with with immune cells to increase
IL-10 production.
[0319] Still another embodiment provides a method for treating
inflammation by administering an effective amount of C1ORF32
polypeptide selected from the group consisting of SEQ ID NOs: 29,
30, 41-105, or a fragment, variant, a homolog, a fusion protein or
a conjugate thereof to inhibit or interfere with the Th1 pathway
and Th17 pathway, and to enhance the suppressive effect on the Th1
and/or Th17 pathways by Th2 cells.
[0320] The C1ORF32 polypeptide selected from the group consisting
of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a
fusion protein or a conjugate thereof can also be administered to a
subject in an amount effective to increase Th2 cell populations or
numbers.
[0321] IL-10 production can be increased relative to a control by
contacting Th2 cells, Tregs or other immune cells with an effective
amount of C1ORF32 polypeptide selected from the group consisting of
SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a
fusion protein or a conjugate thereof having C1ORF32 activity. The
increase can occur in vitro or in vivo.
Inflammatory Disease to Be Treated
[0322] Representative inflammatory or autoimmune diseases and
disorders that may be treated using C1ORF32 polypeptide selected
from the group consisting of SEQ ID NOs: 29, 30, 41-105, or a
fragment, variant, a homolog, a fusion protein or a conjugate
thereof include, but are not limited to multiple sclerosis,
rheumatoid arthritis, type I diabetes, psoriasis, systemic lupus
erythematosus, inflammatory bowel disease, uveitis, and Sjogren's
syndrome.
[0323] C1ORF32 polypeptide selected from the group consisting of
SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a
fusion protein or a conjugate thereof acts at multiple points in
the inflammatory pathway master regulator to control the expression
and/or activity of effectory cytokines such as IFN-gamma and
TNF-alpha. Therefore, the C1ORF32 compositions described herein are
particularly useful for treating patients that do not respond to
TNF-alpha blockers such as Enbrel, Remicade, Cimzia and Humira, or
where TNF-alpha blockers are not safe or effective. In addition,
because of its activity as a master regulator in the inflammatory
pathway, the C1ORF32 compositions disclosed are particularly useful
for treating chronic and persistent inflammation.
[0324] In a further embodiment, the C1ORF32 compositions described
herein are used to treat relapsing and/or remitting multiple
sclerosis.
Inhibition of Epitope Spreading
[0325] Epitope spreading refers to the ability of B and T cell
immune response to diversify both at the level of specificity, from
a single determinant to many sites on an auto antigen, and at the
level of V gene usage (Monneaux, F. et al., Arthritis &
Rheumatism, 46(6): 1430-1438 (2002). Epitope spreading is not
restricted to systemic autoimmune disease. It has been described in
T cell dependent organ specific diseases such as Diabetes mellitus
type 1 and multiple sclerosis in humans, and EAE induced
experimental animals with a variety of myelin proteins.
[0326] Epitope spreading involves the acquired recognition of new
epitopes in the same self molecule as well as epitopes residing in
proteins that are associated in the same macromolecular complex.
Epitope spreading can be assessed by measuring delayed-type
hypersensitivity (DTH) responses, methods of which are known in the
art.
[0327] One embodiment provides a method for inhibiting or reducing
epitope spreading in a subject by administering to the subject an
effective amount of C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof. In a further
embodiment the C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof inhibits epitope
spreading in individuals with multiple sclerosis. Preferably, the
C1ORF32 polypeptide selected from the group consisting of SEQ ID
NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusion
protein or a conjugate thereof inhibits or blocks multiple points
of the inflammation pathway.
[0328] Yet another embodiment provides a method for inhibiting or
reducing epitope spreading in subjects with multiple sclerosis by
administering to a subject an effective amount of C1ORF32
polypeptide selected from the group consisting of SEQ ID NOs: 29,
30, 41-105, or a fragment, variant, a homolog, a fusion protein or
a conjugate thereof to inhibit or reduce differentiation of,
proliferation of, activity of, and/or cytokine production and/or
secretion by Th1, Th17, Th22, and/or other cells that secrete, or
cause other cells to secrete, inflammatory molecules, including,
but not limited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma,
IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. Another embodiment
provides a method for treating multiple sclerosis by administering
to a subject an effective amount of C1ORF32 polypeptide selected
from the group consisting of SEQ ID NOs: 29, 30, 41-105, or a
fragment, variant, a homolog, a fusion protein or a conjugate
thereof thereof to interact with Tregs, enhance Treg activity,
promote or enhances IL-10 secretion by Tregs, increase the number
of Tregs, increase the suppressive capacity of Tregs, or
combinations thereof. Another embodiment provides a method for
treating multiple sclerosis by administering to a subject an
effective amount of C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof to interact with
Th2 cells, enhance Th2 activity, promote or enhance IL-10 secretion
by Th2 cells, increase the number of Th2 cells, increase the
modulatory capacity of Th2 cells, or combinations thereof.
[0329] Induction of Immune Tolerance
[0330] In one embodiment, the present invention provides a method
for inducing or re-establishing immune tolerance in a subject by
administering to the subject an effective amount of C1ORF32
polypeptide selected from the group consisting of SEQ ID NOs: 29,
30, 41-105, or a fragment, variant, a homolog, a fusion protein or
a conjugate thereof. In a further embodiment the C1ORF32
polypeptide selected from the group consisting of SEQ ID NOs: 29,
30, 41-105, or a fragment, variant, a homolog, a fusion protein or
a conjugate thereof induces tolerance in individuals with immune
related diseases. In a specific embodiment the C1ORF32 polypeptide,
fragment or fusion protein thereof induces tolerance in individuals
with multiple sclerosis or any other immune related disease as
described herein. Preferably, the C1ORF32 polypeptide selected from
the group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,
variant, a homolog, a fusion protein or a conjugate thereof
inhibits or blocks multiple points of the inflammation pathway. In
another specific embodiment, the C1ORF32 polypeptide selected from
the group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,
variant, a homolog, a fusion protein or a conjugate thereof induces
tolerance in individuals with rheumatoid arthritis. Another
embodiment provides a method for treating immune related diseases
by administering to a subject an effective amount of C1ORF32
polypeptide selected from the group consisting of SEQ ID NOs: 29,
30, 41-105, or a fragment, variant, a homolog, a fusion protein or
a conjugate thereof to induce immune tolerance by interacting with
Tregs, enhancing Treg activity, increasing the number of Tregs,
increase the suppressive capacity of Tregs, or combinations
thereof. Another embodiment provides a method for treating immune
related diseases by administering to a subject an effective amount
of C1ORF32 polypeptide selected from the group consisting of SEQ ID
NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusion
protein or a conjugate thereof to promote or enhance IL-10
secretion by immune cells.
[0331] Use of the Therapeutic Agents According to at Least Some
Embodiments of the Invention for Adoptive Immunotherapy:
[0332] One of the cardinal features of some models of tolerance is
that once the tolerance state has been established, it can be
perpetuated to naive recipients by the adoptive transfer of
donor-specific regulatory cells. Such adoptive transfer studies
have also addressed the capacity of T-cell subpopulations and non-T
cells to transfer tolerance. Such tolerance can be induced by
blocking costimulation or upon engagement of a co-inhibitory B7
with its counter receptor. This approach, that have been
successfully applied in animals and is evaluated in clinical trials
in humans, (Scalapino K J and Daikh D I. PLoS One. 2009;
4(6):e6031; Riley et al., Immunity. 2009; 30(5): 656-665) provides
a promising treatment option for autoimmune disorders and
transplantation. Therapeutic agents according to at least some
embodiments of the invention, are used for_for adoptive
immunotherapy. Thus, in at least some embodiments, the invention
provides methods for in vivo or ex vivo tolerance induction,
comprising administering effective amount of the therapeutic agent
according to at least some embodiments, to a patient or to
leukocytes isolated from the patient, in order to induce
differentiation of tolerogenic regulatory cells; followed by
ex-vivo enrichment and expansion of said cells and reinfusion of
the tolerogenic regulatory cells to said patient.
[0333] Alternatively, immune responses can be enhanced in a patient
by removing immune cells from the patient, contacting immune cells
in vitro with an agent that inhibits C1ORF32 activity, and/or which
inhibits the interaction of C1ORF32 with its natural binding
partners, and reintroducing the in vitro stimulated immune cells
into the patient. In another embodiment, a method of modulating
immune responses involves isolating immune cells from a patient,
transfecting them with a nucleic acid molecule encoding a form of
C1ORF32, such that the cells express all or a portion of the
C1ORF32 polypeptide according to various embodiments of the present
invention on their surface, and reintroducing the transfected cells
into the patient. The transfected cells have the capacity to
modulate immune responses in the patient.
[0334] Pharmaceutical Compositions
[0335] The present invention, in some embodiments, features a
pharmaceutical composition comprising a therapeutically effective
amount of a therapeutic agent according to the present invention.
According to the present invention the therapeutic agent could be
any one of soluble C1ORF32 polypeptide selected from the group
consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a
homolog, a fusion protein or a conjugate thereof or a corresponding
nucleic acid sequence encoding. The pharmaceutical composition
according to the present invention is further used for the
treatment of immune related disorder and/or infection as described
herein. The therapeutic agents of the present invention can be
provided to the subject alone, or as part of a pharmaceutical
composition where they are mixed with a pharmaceutically acceptable
carrier.
[0336] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the active compound, i.e., soluble C1ORF32
polypeptide selected from the group consisting of SEQ ID NOs: 29,
30, 41-105, or a fragment, variant, a homolog, a fusion protein or
a conjugate thereof or a corresponding nucleic acid sequence
encoding. The pharmaceutical compounds according to at least some
embodiments of the present invention may include one or more
pharmaceutically acceptable salts. A "pharmaceutically acceptable
salt" refers to a salt that retains the desired biological activity
of the parent compound and does not impart any undesired
toxicological effects (see e.g., Berge, S. M., et al. (1977) J.
Pharm. Sci. 66: 1-19). Examples of such salts include acid addition
salts and base addition salts. Acid addition salts include those
derived from nontoxic inorganic acids, such as hydrochloric,
nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous
and the like, as well as from nontoxic organic acids such as
aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic
acids, hydroxy alkanoic acids, aromatic acids, aliphatic and
aromatic sulfonic acids and the like. Base addition salts include
those derived from alkaline earth metals, such as sodium,
potassium, magnesium, calcium and the like, as well as from
nontoxic organic amines, such as N,N'-dibenzylethylenediamine,
N-methylglucamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, procaine and the like.
[0337] A pharmaceutical composition according to at least some
embodiments of the present invention also may include one or more
pharmaceutically acceptable anti-oxidants. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like. A pharmaceutical composition according to at least some
embodiments of the present invention also may include additives
such as detergents and solubilizing agents (e.g., TWEEN 20
(polysorbate-20), TWEEN 80 (polysorbate-80)) and preservatives
(e.g., Thimersol, benzyl alcohol) and bulking substances (e.g.,
lactose, mannitol).
[0338] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions according to at
least some embodiments of the present invention include water,
buffered saline of various buffer content (e.g., Tris-HCl, acetate,
phosphate), pH and ionic strength, ethanol, polyols (such as
glycerol, propylene glycol, polyethylene glycol, and the like), and
suitable mixtures thereof, vegetable oils, such as olive oil, and
injectable organic esters, such as ethyl oleate.
[0339] Proper fluidity can be maintained, for example, by the use
of coating materials, such as lecithin, by the maintenance of the
required particle size in the case of dispersions, and by the use
of surfactants.
[0340] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0341] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions according to at least some embodiments
of the present invention is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
[0342] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
Sterile injectable solutions can be prepared by incorporating the
active compound in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by sterilization microfiltration. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying (lyophilization) that yield a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0343] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0344] The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred per cent, this
amount will range from about 0.01 per cent to about ninety-nine
percent of active ingredient, preferably from about 0.1 per cent to
about 70 per cent, most preferably from about I per cent to about
30 per cent of active ingredient in combination with a
pharmaceutically acceptable carrier.
[0345] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms according to at least some
embodiments of the present invention are dictated by and directly
dependent on (a) the unique characteristics of the active compound
and the particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
compound for the treatment of sensitivity in individuals.
[0346] A composition of the present invention can be administered
via one or more routes of administration using one or more of a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. Preferred routes of
administration for therapeutic agents according to at least some
embodiments of the present invention include intravascular delivery
(e.g. injection or infusion), intravenous, intramuscular,
intradermal, intraperitoneal, subcutaneous, spinal, oral, enteral,
rectal, pulmonary (e.g. inhalation), nasal, topical (including
transdermal, buccal and sublingual), intravesical, intravitreal,
intraperitoneal, vaginal, brain delivery (e.g.
intra-cerebroventricular, intra-cerebral, and convection enhanced
diffusion), CNS delivery (e.g. intrathecal, perispinal, and
intra-spinal) or parenteral (including subcutaneous, intramuscular,
intraperitoneal, intravenous (IV) and intradermal), transdermal
(either passively or using iontophoresis or electroporation),
transmucosal (e.g., sublingual administration, nasal, vaginal,
rectal, or sublingual), administration or administration via an
implant, or other parenteral routes of administration, for example
by injection or infusion, or other delivery routes and/or forms of
administration known in the art. The phrase "parenteral
administration" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrastemal injection and
infusion or using bioerodible inserts, and can be formulated in
dosage forms appropriate for each route of administration. In a
specific embodiment, a protein, a therapeutic agent or a
pharmaceutical composition according to at least some embodiments
of the present invention can be administered intraperitoneally or
intravenously.
[0347] Compositions of the present invention can be delivered to
the lungs while inhaling and traverse across the lung epithelial
lining to the blood stream when delivered either as an aerosol or
spray dried particles having an aerodynamic diameter of less than
about 5 microns. A wide range of mechanical devices designed for
pulmonary delivery of therapeutic products can be used, including
but not limited to nebulizers, metered dose inhalers, and powder
inhalers, all of which are familiar to those skilled in the art.
Some specific examples of commercially available devices are the
Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn
II nebulizer (Marquest Medical Products, Englewood, Colo.); the
Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park,
N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford,
Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin
powder preparations approved or in clinical trials where the
technology could be applied to the formulations described
herein.
[0348] In some in vivo approaches, the compositions disclosed
herein are administered to a subject in a therapeutically effective
amount. As used herein the term "effective amount" or
"therapeutically effective amount" means a dosage sufficient to
treat, inhibit, or alleviate one or more symptoms of the disorder
being treated or to otherwise provide a desired pharmacologic
and/or physiologic effect. The precise dosage will vary according
to a variety of factors such as subject-dependent variables (e.g.,
age, immune system health, etc.), the disease, and the treatment
being effected. For the polypeptide compositions disclosed herein
and nucleic acids encoding the same, as further studies are
conducted, information will emerge regarding appropriate dosage
levels for treatment of various conditions in various patients, and
the ordinary skilled worker, considering the therapeutic context,
age, and general health of the recipient, will be able to ascertain
proper dosing. The selected dosage depends upon the desired
therapeutic effect, on the route of administration, and on the
duration of the treatment desired. For polypeptide compositions,
generally dosage levels of 0.0001 to 100 mg/kg of body weight daily
are administered to mammals and more usually 0.001 to 20 mg/kg. For
example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight,
3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or
within the range of 1-10 mg/kg. An exemplary treatment regime
entails administration once per week, once every two weeks, once
every three weeks, once every four weeks, once a month, once every
3 months or once every three to 6 months. Generally, for
intravenous injection or infusion, dosage may be lower. Dosage
regimens are adjusted to provide the optimum desired response
(e.g., a therapeutic response). For example, a single bolus may be
administered, several divided doses may be administered over time
or the dose may be proportionally reduced or increased as indicated
by the exigencies of the therapeutic situation. It is especially
advantageous to formulate parenteral compositions in dosage unit
form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited
as unitary dosages for the subjects to be treated; each unit
contains a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms according to at least some embodiments of the present
invention are dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0349] Optionally the polypeptide formulation may be administered
in an amount between 0.0001 to 100 mg/kg weight of the patient/day,
preferably between 0.001 to 20.0 mg/kg/day, according to any
suitable timing regimen. A therapeutic composition according to at
least some embodiments according to at least some embodiments of
the present invention can be administered, for example, three times
a day, twice a day, once a day, three times weekly, twice weekly or
once weekly, once every two weeks or 3, 4, 5, 6, 7 or 8 weeks.
Moreover, the composition can be administered over a short or long
period of time (e.g., 1 week, 1 month, 1 year, 5 years).
[0350] Alternatively, therapeutic agent can be administered as a
sustained release formulation, in which case less frequent
administration is required. Dosage and frequency vary depending on
the half-life of the therapeutic agent in the patient. In general,
the half-life for fusion proteins may vary widely. The dosage and
frequency of administration can vary depending on whether the
treatment is prophylactic or therapeutic. In prophylactic
applications, a relatively low dosage is administered at relatively
infrequent intervals over a long period of time. Some patients
continue to receive treatment for the rest of their lives. In
therapeutic applications, a relatively high dosage at relatively
short intervals is sometimes required until progression of the
disease is reduced or terminated, and preferably until the patient
shows partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic regime.
Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, the route of administration, the time of administration,
the rate of excretion of the particular compound being employed,
the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compositions
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0351] A "therapeutically effective dosage" of C1ORF32 soluble
protein polypeptide selected from the group consisting of SEQ ID
NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusion
protein or a conjugate thereof, preferably results in a decrease in
severity of disease symptoms, an increase in frequency and duration
of disease symptom-free periods, an increase in lifepan, disease
remission, or a prevention or reduction of impairment or disability
due to the disease affliction.
[0352] One of ordinary skill in the art would be able to determine
a therapeutically effective amount based on such factors as the
subject's size, the severity of the subject's symptoms, and the
particular composition or route of administration selected. In
certain embodiments, the polypeptide compositions are administered
locally, for example by injection directly into a site to be
treated. Typically, the injection causes an increased localized
concentration of the polypeptide compositions which is greater than
that which can be achieved by systemic administration. For example,
in the case of a neurological disorder like Multiple Sclerosis, the
protein may be administered locally to a site near the CNS. In
another example, as in the case of an arthritic disorder like
Rheumatoid Arthritis, the protein may be administered locally to
the synovium in the affected joint. The polypeptide compositions
can be combined with a matrix as described above to assist in
creating a increased localized concentration of the polypeptide
compositions by reducing the passive diffusion of the polypeptides
out of the site to be treated.
[0353] Pharmaceutical compositions of the present invention may be
administered with medical devices known in the art. For example, in
an optional embodiment, a pharmaceutical composition according to
at least some embodiments of the present invention can be
administered with a needles hypodermic injection device, such as
the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851;
5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples
of well-known implants and modules useful in the present invention
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicaments through the skin; U.S. Pat. No.
4,447,233, which discloses a medication infusion pump for
delivering medication at a precise infusion rate; U.S. Pat. No.
4,447,224, which discloses a variable flow implantable infusion
apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196,
which discloses an osmotic drug delivery system having
multi-chamber compartments; and U.S. Pat. No. 4,475,196, which
discloses an osmotic drug delivery system. These patents are
incorporated herein by reference. Many other such implants,
delivery systems, and modules are known to those skilled in the
art.
[0354] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0355] Therapeutic compositions can be administered with medical
devices known in the art. For example, in an optional embodiment, a
therapeutic composition according to at least some embodiments of
the present invention can be administered with a needles hypodermic
injection device, such as the devices disclosed in U.S. Pat. Nos.
5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;
or 4,596,556. Examples of well-known implants and modules useful in
the present invention include: U.S. Pat. No. 4,487,603, which
discloses an implantable micro-infusion pump for dispensing
medication at a controlled rate; U.S. Pat. No. 4,486,194, which
discloses a therapeutic device for administering medicaments
through the skin; U.S. Pat. No. 4,447,233, which discloses a
medication infusion pump for delivering medication at a precise
infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable
flow implantable infusion apparatus for continuous drug delivery;
U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery
system having multi-chamber compartments; and U.S. Pat. No.
4,475,196, which discloses an osmotic drug delivery system. These
patents are incorporated herein by reference. Many other such
implants, delivery systems, and modules are known to those skilled
in the art.
[0356] In certain embodiments, C1ORF32 polypeptide selected from
the group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,
variant, a homolog, a fusion protein or a conjugate thereof
according to at least some embodiments of the present invention can
be formulated to ensure proper distribution in vivo. For example,
the blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds according to at
least some embodiments of the present invention cross the BBB (if
desired), they can be formulated, for example, in liposomes. For
methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos.
4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one
or more moieties which are selectively transported into specific
cells or organs, thus enhance targeted drug delivery (see, e.g., V.
V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting
moieties include folate or biotin (see, e.g., U.S. Pat. No.
5,416,016 to Low et al.); mannosides (Umezawa et al., (1988)
Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman
et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995)
Antimicrob. Agents Chemother. 39:180); surfactant protein A
receptor (Briscoe et al. (1995) Am. J Physiol. 1233:134); p120
(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.
Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion;
I. J. Fidler (1994) Immunomethods 4:273.
[0357] Formulations for Parenteral Administration
[0358] In a further embodiment, compositions disclosed herein,
including those containing peptides and polypeptides, are
administered in an aqueous solution, by parenteral injection. The
formulation may also be in the form of a suspension or emulsion. In
general, pharmaceutical compositions are provided including
effective amounts of a peptide or polypeptide, and optionally
include pharmaceutically acceptable diluents, preservatives,
solubilizers, emulsifiers, adjuvants and/or carriers. Such
compositions optionally include one or more for the following:
diluents, sterile water, buffered saline of various buffer content
(e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and
additives such as detergents and solubilizing agents (e.g., TWEEN
20 (polysorbate-20), TWEEN 80 (polysorbate-80)), anti-oxidants
(e.g., water soluble antioxidants such as ascorbic acid, sodium
metabisulfite, cysteine hydrochloride, sodium bisulfate, sodium
metabisulfite, sodium sulfite; oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol;
and metal chelating agents, such as citric acid, ethylenediamine
tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid),
and preservatives (e.g., Thimersol, benzyl alcohol) and bulking
substances (e.g., lactose, mannitol). Examples of non-aqueous
solvents or vehicles are ethanol, propylene glycol, polyethylene
glycol, vegetable oils, such as olive oil and corn oil, gelatin,
and injectable organic esters such as ethyl oleate. The
formulations may be freeze dried (lyophilized) or vacuum dried and
redissolved/resuspended immediately before use. The formulation may
be sterilized by, for example, filtration through a bacteria
retaining filter, by incorporating sterilizing agents into the
compositions, by irradiating the compositions, or by heating
thecompositions.
[0359] Formulations for Topical Administration
[0360] C1ORF32 polypeptide selected from the group consisting of
SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a
fusion protein or a conjugate thereof, nucleic acids, and vectors
disclosed herein can be applied topically. Topical administration
does not work well for most peptide formulations, although it can
be effective especially if applied to the lungs, nasal, oral
(sublingual, buccal), vaginal, or rectal mucosa.
[0361] Compositions can be delivered to the lungs while inhaling
and traverse across the lung epithelial lining to the blood stream
when delivered either as an aerosol or spray dried particles having
an aerodynamic diameter of less than about 5 microns.
[0362] A wide range of mechanical devices designed for pulmonary
delivery of therapeutic products can be used, including but not
limited to nebulizers, metered dose inhalers, and powder inhalers,
all of which are familiar to those skilled in the art. Some
specific examples of commercially available devices are the
Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn
II nebulizer (Marquest Medical Products, Englewood, Colo.); the
Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park,
N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford,
Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin
powder preparations approved or in clinical trials where the
technology could be applied to the formulations described
herein.
[0363] Formulations for administration to the mucosa will typically
be spray dried drug particles, which may be incorporated into a
tablet, gel, capsule, suspension or emulsion. Standard
pharmaceutical excipients are available from any formulator. Oral
formulations may be in the form of chewing gum, gel strips, tablets
or lozenges.
[0364] Transdermal formulations may also be prepared. These will
typically be ointments, lotions, sprays, or patches, all of which
can be prepared using standard technology. Transdermal formulations
will require the inclusion of penetration enhancers.
[0365] Controlled Delivery Polymeric Matrices
[0366] C1ORF32 polypeptide selected from the group consisting of
SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a
fusion protein or a conjugate thereof, nucleic acids, and vectors
disclosed herein may also be administered in controlled release
formulations. Controlled release polymeric devices can be made for
long term release systemically following implantation of a
polymeric device (rod, cylinder, film, disk) or injection
(microparticles). The matrix can be in the form of microparticles
such as microspheres, where peptides are dispersed within a solid
polymeric matrix or microcapsules, where the core is of a different
material than the polymeric shell, and the peptide is dispersed or
suspended in the core, which may be liquid or solid in nature.
Unless specifically defined herein, microparticles, microspheres,
and microcapsules are used interchangeably. Alternatively, the
polymer may be cast as a thin slab or film, ranging from nanometers
to four centimeters, a powder produced by grinding or other
standard techniques, or even a gel such as a hydrogel.
[0367] Either non-biodegradable or biodegradable matrices can be
used for delivery of polypeptides or nucleic acids encoding the
polypeptides, although biodegradable matrices are preferred. These
may be natural or synthetic polymers, although synthetic polymers
are preferred due to the better characterization of degradation and
release profiles. The polymer is selected based on the period over
which release is desired. In some cases linear release may be most
useful, although in others a pulse release or "bulk release" may
provide more effective results. The polymer may be in the form of a
hydrogel (typically in absorbing up to about 90% by weight of
water), and can optionally be crosslinked with multivalent ions or
polymers.
[0368] The matrices can be formed by solvent evaporation, spray
drying, solvent extraction and other methods known to those skilled
in the art. Bioerodible microspheres can be prepared using any of
the methods developed for making microspheres for drug delivery,
for example, as described by Mathiowitz and Langer, J. Controlled
Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers,
6:275-283 (1987); and Mathiowitz, et al., J. Appl Polymer ScL,
35:755-774 (1988). The devices can be formulated for local release
to treat the area of implantation or injection--which will
typically deliver a dosage that is much less than the dosage for
treatment of an entire body--or systemic delivery. These can be
implanted or injected subcutaneously, into the muscle, fat, or
swallowed.
Diagnostic Uses of C1ORF32
[0369] Soluble polypeptides according to at least some embodiments
of the present invention may also be modified with a label capable
of providing a detectable signal, either directly or indirectly,
including, but not limited to, radioisotopes and fluorescent
compounds. Such labeled polypeptides can be used for various uses,
including but not limited to, prognosis, prediction, screening,
early diagnosis, determination of progression, therapy selection
and treatment monitoring of disease and/or an indicative condition,
as detailed above.
[0370] According to at least some embodiments, the present
invention provides a method for imaging an organ or tissue, the
method comprising: (a) administering to a subject in need of such
imaging, a labeled polypeptide; and (b) detecting the labeled
polypeptide to determine where the labeled polypeptide is
concentrated in the subject. When used in imaging applications, the
labeled polypeptides according to at least some embodiments of the
present invention typically have an imaging agent covalently or
noncovalently attached thereto. Suitable imaging agents include,
but are not limited to, radionuclides, detectable tags,
fluorophores, fluorescent proteins, enzymatic proteins, and the
like. One of skill in the art will be familiar with other methods
for attaching imaging agents to polypeptides. For example, the
imaging agent can be attached via site-specific conjugation, e.g.,
covalent attachment of the imaging agent to a peptide linker such
as a polyarginine moiety having five to seven arginines present at
the carboxyl-terminus of and Fc fusion molecule. The imaging agent
can also be directly attached via non-site specific conjugation,
e.g., covalent attachment of the imaging agent to primary amine
groups present in the polypeptide. One of skill in the art will
appreciate that an imaging agent can also be bound to a protein via
noncovalent interactions (e.g., ionic bonds, hydrophobic
interactions, hydrogen bonds, Van der Waals forces, dipole-dipole
bonds, etc.).
[0371] In certain instances, the polypeptide is radiolabeled with a
radionuclide by directly attaching the radionuclide to the
polypeptide. In certain other instances, the radionuclide is bound
to a chelating agent or chelating agent-linker attached to the
polypeptide. Suitable radionuclides for direct conjugation include,
without limitation, 18 F, 124 I, 125 I, 131 I, and mixtures
thereof. Suitable radionuclides for use with a chelating agent
include, without limitation, 47 Sc, 64 Cu, 67 Cu, 89 Sr, 86 Y, 87
Y, 90 Y,105 Rh, 111 Ag, 111 In, 117m Sn, 149 Pm, 153 Sm, 166 Ho,
177 Lu, 186 Re, 188 Re, 211 At, 212 Bi, and mixtures thereof.
Preferably, the radionuclide bound to a chelating agent is 64 Cu,
90 Y, 111 In, or mixtures thereof. Suitable chelating agents
include, but are not limited to, DOTA, BAD, TETA, DTPA, EDTA, NTA,
HDTA, their phosphonate analogs, and mixtures thereof. One of skill
in the art will be familiar with methods for attaching
radionuclides, chelating agents, and chelating agent-linkers to
polypeptides of the present invention. In particular, attachment
can be conveniently accomplished using, for example, commercially
available bifunctional linking groups (generally heterobifunctional
linking groups) that can be attached to a functional group present
in a non-interfering position on the polypeptide and then further
linked to a radionuclide, chelating agent, or chelating
agent-linker.
[0372] Non-limiting examples of fluorophores or fluorescent dyes
suitable for use as imaging agents include Alexa Fluor.RTM. dyes
(Invitrogen Corp.; Carlsbad, Calif.), fluorescein, fluorescein
isothiocyanate (FITC), Oregon Green.TM.; rhodamine, Texas red,
tetrarhodamine isothiocynate (TRITC), CyDye.TM. fluors (e.g., Cy2,
Cy3, Cy5), and the like.
[0373] Examples of fluorescent proteins suitable for use as imaging
agents include, but are not limited to, green fluorescent protein,
red fluorescent protein (e.g., DsRed), yellow fluorescent protein,
cyan fluorescent protein, blue fluorescent protein, and variants
thereof (see, e.g., U.S. Pat. Nos. 6,403,374, 6,800,733, and
7,157,566). Specific examples of GFP variants include, but are not
limited to, enhanced GFP (EGFP), destabilized EGFP, the GFP
variants described in Doan et al., Mol. Microbiol., 55:1767-1781
(2005), the GFP variant described in Crameri et al., Nat.
Biotechnol., 14:315-319 (1996), the cerulean fluorescent proteins
described in Rizzo et al., Nat. Biotechnol, 22:445 (2004) and
Tsien,Annu. Rev. Biochem., 67:509 (1998), and the yellow
fluorescent protein described in Nagal et al., Nat. Biotechnol.,
20:87-90 (2002). DsRed variants are described in, e.g., Shaner et
al., Nat. Biotechnol., 22:1567-1572 (2004), and include
mStrawberry, mCherry, morange, mBanana, mHoneydew, and mTangerine.
Additional DsRed variants are described in, e.g., Wang et al.,
Proc. Natl. Acad. Sci. U.S.A., 101:16745-16749 (2004) and include
mRaspberry and mPlum. Further examples of DsRed variants include
mRFPmars described in Fischer et al., FEBS Lett.,577:227-232 (2004)
and mRFPruby described in Fischer et al., FEBS Lett., 580:2495-2502
(2006).
[0374] In other embodiments, the imaging agent that is bound to a
polypeptide according to at least some embodiments of the present
invention comprises a detectable tag such as, for example, biotin,
avidin, streptavidin, or neutravidin. In further embodiments, the
imaging agent comprises an enzymatic protein including, but not
limited to, luciferase, chloramphenicol acetyltransferase,
.beta.-galactosidase, .beta.-glucuronidase, horseradish peroxidase,
xylanase, alkaline phosphatase, and the like.
[0375] Any device or method known in the art for detecting the
radioactive emissions of radionuclides in a subject is suitable for
use in the present invention. For example, methods such as Single
Photon Emission Computerized Tomography (SPECT), which detects the
radiation from a single photon gamma-emitting radionuclide using a
rotating gamma camera, and radionuclide scintigraphy, which obtains
an image or series of sequential images of the distribution of a
radionuclide in tissues, organs, or body systems using a
scintillation gamma camera, may be used for detecting the radiation
emitted from a radiolabeled polypeptide of the present invention.
Positron emission tomography (PET) is another suitable technique
for detecting radiation in a subject. Miniature and flexible
radiation detectors intended for medical use are produced by
Intra-Medical LLC (Santa Monica, Calif.). Magnetic Resonance
Imaging (MRI) or any other imaging technique known to one of skill
in the art is also suitable for detecting the radioactive emissions
of radionuclides. Regardless of the method or device used, such
detection is aimed at determining where the labeled polypeptide is
concentrated in a subject, with such concentration being an
indicator of disease activity.
[0376] Non-invasive fluorescence imaging of animals and humans can
also provide in vivo diagnostic information and be used in a wide
variety of clinical specialties. For instance, techniques have been
developed over the years for simple ocular observations following
UV excitation to sophisticated spectroscopic imaging using advanced
equipment (see, e.g., Andersson-Engels et al., Phys. Med. Biol.,
42:815-824 (1997)). Specific devices or methods known in the art
for the in vivo detection of fluorescence, e.g., from fluorophores
or fluorescent proteins, include, but are not limited to, in vivo
near-infrared fluorescence (see, e.g., Frangioni, Curr. Opin. Chem.
Biol., 7:626-634 (2003)), the Maestro.TM. in vivo fluorescence
imaging system (Cambridge Research & Instrumentation, Inc.;
Woburn, Mass.), in vivo fluorescence imaging using a flying-spot
scanner (see, e.g., Ramanujam et al., IEEE Transactions on
Biomedical Engineering, 48:1034-1041 (2001), and the like.
[0377] Other methods or devices for detecting an optical response
include, without limitation, visual inspection, CCD cameras, video
cameras, photographic film, laser-scanning devices, fluorometers,
photodiodes, quantum counters, epifluorescence microscopes,
scanning microscopes, flow cytometers, fluorescence microplate
readers, or signal amplification using photomultiplier tubes.
[0378] The present invention is further illustrated by the below
examples related to C1ORF32 antigen, its domains and expression
data as well as prophetic examples describing the manufacture of
fully human antibodies thereto. This information and examples is
illustrative and should not be construed as further limiting. The
contents of all figures and all references, patents and published
patent applications cited throughout this application are expressly
incorporated herein by reference.
EXAMPLES
Example 1
Design and Production of Mutated C1ORF32 ECD Polypeptides
[0379] This Example relates to production of ECD fragments.
[0380] Materials and Methods
C1ORF32-P8-ECD-mFC Production in CHO Cells:
[0381] To produce C1ORF32-P8-ECD-mFC in CHO cells, retrovector
constructs containing cDNA that code for the protein were
transduced into CHO cells, and 3-4 rounds of transductions were
carried out. Cell pool of the last transduction was grown in shake
flasks by fed-batch production. Medium was harvested at 12-14 days
when cell viability dropped to .about.50%.
[0382] The harvested medium was clarified by depth filtration
followed by 0.2 .mu.m filter, and the clarified material was
subjected to Protein A chromatography. The eluate, pH .about.3.7,
was incubated with mixing for 60 minutes at room temperature to
facilitate viral inactivation, neutralized to pH .about.7.2 and
filtered through a 0.2 .mu.m filter. Finally, the solution was
buffer exchanged to PBS pH 7.2, concentrated to the desired protein
concentration and filtered.
[0383] Results
[0384] SDS-PAGE analysis, under non-reducing conditions, of
C1ORF32-P8-V1-ECD-mFC produced in CHO cells, as described below,
revealed three main bands: the expected MW of .about.100 kD, a band
at MW of .about.751(D, and a third one at MW of .about.55 kD.
SDS-PAGE analysis in reduced conditions revealed two main
bands--one at the expected MW of .about.50 kD and an additional of
.about.301(D.
[0385] Western Blot analyses using anti-C1ORF32-P8-V1-ECD and
anti--mFc demonstrated the presence of mainly three protein
species: the intact full-size dimer consisting of two chains each
with an C1ORF32 ECD and a Fc (homodimer); a one-arm cleaved dimer
consisting of one chain with an C1ORF32 ECD and a Fc and second
chain with mainly the Fc (heterodimer); and a two-armed cleaved
dimer composed of two chains with mainly the Fc (fully cleaved
dimer).
[0386] N-terminal sequencing revealed a major cleavage site towards
the C-terminus of the C1ORF32 ECD between amino acids F and A at
positions 179 and 180 of H19011_1_P8_V1 or H19011_1_P8 (Seq ID NOs:
4 or 5). To prevent this cleavage the amino acids F and A at
positions 179 and 180 of H19011_1_P8_V1 or H19011_1_P8 (SEQ ID NOs:
4 or 5) were mutated as follows: FA.fwdarw.GA, FA.fwdarw.AA, and
FA.fwdarw.GG. The resulted C1ORF32 ECD fragments are represented
by, for example, SEQ ID NOs : 64, 96, and 45, respectively.
Example 2
Production of Fc-Fused C1ORF32 Proteins
[0387] The Fc-fused C1ORF32 ECD proteins were produced as follows:
Fc-fused C1ORF32 ECD (SEQ ID NO:108), corresponds to C1ORF32 ECD
without signal peptide (SEQ ID NO: 64) fused to mouse mIgG2a Fc
(SEQ ID NO: 31) via GS_TEV_linker_SG (SEQ ID NO:113).
[0388] Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110)
corresponds to C1ORF32 ECD-Delta DLLPSFAVEIM fragment without
signal peptide (SEQ ID NO:51) fused to mouse mIgG2a Fc (SEQ ID NO:
31) via GS_TEV_linker_SG (SEQ ID NO:113). C1ORF32 ECD-Delta
RTGLLADLLPSFAVEIM (SEQ ID NO:112) corresponds to C1ORF32 ECD-Delta
RTGLLADLLPSFAVEIM fragment without signal peptide (SEQ ID. NO:29)
fused to mouse mIgG2a Fc (SEQ ID NO: 31) via GS_TEV_linker_SG (SEQ
ID NO:113).
[0389] All the fused proteins were produced using two service
providers; Catalent and ExcellGene, each using its own production
protocol as described below:
[0390] Production by Catalent (Middleton, Wis., USA):
Codon-optimized cDNA sequences encoding C1ORF32 ECD (SEQ ID
NO:108), Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110),
and C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) were
produced at Catalent in CHO cells, in pIRESpuro3 vector.
[0391] The cDNA sequence of the insert was verified by Catalent and
was used to construct GPEx.RTM. retrovectors, followed by four
rounds of retrovector transduction into Catalent's "in-house" CHO-S
cell line. A pooled population was produced and expanded and gene
copy index was 2.7. Cell culture supernatants were analyzed by
Catalent's Fc ELISA assay and relative productivity of the 4.times.
transduced pool was 28 .mu.g/ml.
[0392] The protein was produced in 5 L wave bioreactor, and
purified according to their in-house process. The level of purity
was estimated at >95%, by SDS PAGE and Coomassie staining (data
not shown). The concentration of purified protein was measured by
absorbance at A280 nm, and estimated at 1.60 mg/ml (using an
absorbance coefficient of 1.28 mg/ml). Endotoxin levels were
tested, and estimated at 0.25-0.5 EU/ml. A total of .about.400 mg
were obtained from .about.10L of cell pool.
[0393] Production by ExcellGene (Valais, Switzerland):
Codon-optimized cDNA sequences encoding C1ORF32 ECD (SEQ ID
NO:108), Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110),
and C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) were
synthesized and subcloned into ExcellGene's proprietary expression
vector (pXLG6), designed for high yield transient gene expression
vectors. Expression vectors were then transiently-transfected into
Exellgene's CHOExpress.TM. host cells, and cells were cultured for
12-14 days in TubeSpin50.RTM. bioreactors. Viability was determined
daily, and productivity was assessed at the end of the culture by
Western Blot analysis. A second transient transfection was then
performed, and the resultant cells were cultured in 1-2 L to obtain
the required amount of protein. Culture supernatants were clarified
and purified on Protein A columns. Purified proteins were analyzed
by a standard SDS-PAGE gel under standard conditions, for which
results are shown in FIG. 2, and concentration was determined by
A280. Proteins were tested for bioburden and endotoxin. In FIG. 2,
lane 12 relates to the protein having the sequence of SEQ ID
NO:110, while lane 13 relates to the protein having the sequence of
SEQ ID NO:112.
Example 3
The Effect of Fc-Fused C1ORF32 ECD (SEQ ID NO:108), Fc-Fused
C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32
ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on TCR-Mediated
Activation of Mouse CD4 T Cells
[0394] The effect of Fc-fused C1ORF32 ECD (SEQ ID NO:108), Fc-fused
C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32
ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112)on TCR-mediated
activation of mouse T cells, was evaluated by testing their effect
on IFN.gamma. secretion and on the expression of the activation
marker CD69 in CD4+CD25- purified T cells.
[0395] Methods
Mouse CD4 T Cells Isolation.
[0396] CD4+CD25- T cells were isolated from pools of spleens and
lymph nodes of BALB/C mice by one step negative selection using T
cell isolation Kit (Miltenyi Cat#130-093-227) according to the
manufacturer's instructions. The purity obtained was >95%. The
cells used in the functional assays, CD4+ T cells, were untouched
CD4+CD25- cells, which include CD4+CD62L.sup.high naive T cells
(85-90% of bulk CD4 population) and CD4+CD62L.sup.low memory
cells.
Activation of Mouse CD4 T Cells
[0397] Anti-CD3 mAb (clone 145-2C11) alone or together with
Fc-fused C1ORF32 ECD (SEQ ID NO:108), Fc-fused C1ORF32 ECD-Delta
DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32 ECD-Delta
RTGLLADLLPSFAVEIM (SEQ ID NO:112) or with control mIgG2a (BioXCell
Cat. #BE0085) were co-immobilized at the stated concentrations on
96-well flat bottom tissue culture plates (Sigma, Cat. #92096),
overnight at 4.degree. C. Wells were washed 3 times with PBS and
plated with 1.times.10.sup.5 CD4+ T cells per well at 37.degree. C.
Culture supernatants were collected 48 h post stimulation and
analyzed for IFNy secretion using mouse IFN.gamma. ELISA kit
(R&D Systems). The effect of immobilized Fc-fused C1ORF32 ECD
(SEQ ID NO:108), Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID
NO:110), and C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112)
(10 .mu.g/ml) on the activation marker CD69 was analyzed by FACS,
48 h post simulations with plate bound anti-CD3.
Results
[0398] Effect of Fc-Fused C1ORF32 ECD (SEQ ID NO:108), Fc-Fused
C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32
ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on Mouse CD4 T Cells
IFN.gamma. Secretion
[0399] In order to evaluate the effect of Fc-fused C1ORF32 ECD (SEQ
ID NO:108), Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110),
and C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on CD4 T
cell response, titrated amounts of the respective Fc-fusion C1ORF32
ECD protein or control mIgG2a were immobilized on 96-well plates
together with anti-CD3 mAb, and IFNy secretion from CD4+ T cells
was analyzed. Cells were activated for 48 hrs using full RPMI
containing 10% FBS, 1 mM sodium pyruvate, 100 IU/ml Pen-Strep
(without 2-ME). The results presented in FIG. 3 indicate potent
inhibitory effect Fc-fused C1ORF32 ECD (SEQ ID NO:108), Fc-fused
C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32
ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on CD4 T cells
IFN-.gamma. secretion.
[0400] Effect of Fc-fused C1ORF32 ECD (SEQ ID NO:108), Fc-fused
C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32
ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on the Early Activation
Marker CD69
[0401] In order to evaluate the effect of Fc-fused C1ORF32 ECD (SEQ
ID NO:108), Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110),
and C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on the
early activation marker CD69, an experiment similar to that
described in FIG. 3 herein was carried out, using 10 ug/ml of
Fc-fused C1ORF32 ECD (SEQ ID NO:108), Fc-fused C1ORF32 ECD-Delta
DLLPSFAVEIM (SEQ ID NO:110), or C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM
(SEQ ID NO:112), respectively. All Fc-fused C1ORF32 ECD proteins
tested had a suppressive effect on early activation markers CD69 at
48h post stimulation as manifested in a reduction of CD69
upregulation upon stimulation, as shown in FIG. 4.
[0402] Conclusion
[0403] Immobilized Fc-fused C1ORF32 ECD (SEQ ID NO:108), Fc-fused
C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), or C1ORF32 ECD-Delta
RTGLLADLLPSFAVEIM (SEQ ID NO:112) inhibit CD4 T cell activation to
a similar extent as manifested by reduced IFNy secretion and CD69
expression compare to control Ig upon TCR stimulation.
Example 4
The Effect of Fc-Fused C1ORF32 ECD (SEQ ID NO:108), Fc-Fused
C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), or C1ORF32 ECD-Delta
RTGLLADLLPSFAVEIM (SEQ ID NO:112) on Naive CD4+ T Cell
Proliferation
Procedure:
[0404] Naive CD4+ T cells were isolated from spleens of D011.10
mice (Jackson) via automax sort: CD4-negative sort (Miltenyi
Cat#130-095-248), including anti-CD25 (Miltenyi Cat#130-091-072) in
the negative sort cocktail, followed by CD62L-positive sort
(Miltenyi Cat #130-049-701). Balb/c total splenocytes were also
collected from one mouse, and irradiated with 3000rads to serve as
antigen presenting cells (APCs) for the D011.10 CD4+ T cells. Naive
CD4+ T cells were cultured at 2.5.times.10.sup.5 cells per well in
flat-bottom 96-well plates with irradiated APCs at a ratio of 1:1
(APCs to T cells) in 200u1 of HL-1 medium, and activated with 2
ug/ml OVA323-339 in the presence of one of Fc-fused C1ORF32 ECD
(SEQ ID NO:108), (produced in CHO-S and in CHO-DG44 as detailed in
Example 2) Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110),
(produced in CHO-DG44 as detailed in Example 2) and or C1ORF32
ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) (produced in CHO-S and
in CHO-DG44 as detailed in Example 3) or Isotype control Ig
(mIgG2a, BioXCell Cat. #BE0085) at the indicated concentrations
(0.1-10 ug/ml). The cells were pulsed with luCi of
tritiated-thymidine at 24 hours, and harvested at 72 hours.
[0405] Results and conclusions:
[0406] As demonstrated in FIG. 5, the Fc-fused C1ORF32 ECD (SEQ ID
NO:108), as well as Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID
NO:110), or C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112)
inhibit T cell proliferation in response to OVA323-339 activation
in a dose dependent manner A slightly reduced activity
(approximately 3 fold) was achieved by the proteins produced in
CHO-DG44 versus those produced in CHO-S. These differences reflect
batch to batch variation of these proteins.
Example 5
The Effect of Fc-Fused C1ORF32 ECD (SEQ ID NO:108), Fc-Fused
C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), or C1ORF32 ECD-Delta
RTGLLADLLPSFAVEIM (SEQ ID NO:112) on Autoimmune Disease or
Infectious Disease in a Subject
[0407] A plurality of animals, having a disease model appropriate
for testing of the autoimmune disease, receives each of the above
described Fc-fused ECD proteins in a plurality of doses. A dose
dependent response is seen.
[0408] Similarly, a plurality of animals infected with an
infectious disease in which T cell exhaustion is exhibited (for
example through chronic viral infection) and receives each of the
above Fc-fused ECD proteins in a plurality of doses, in combination
with an pharmaceutical agent in a pharmaceutically effective amount
against the infectious agent. A dose dependent response is
seen.
Example 6
[0409] Effect of C1ORF32 Protein Fragments and/or Fusion Proteins
on Activation of Naive CD4+ T Cells with Anti-CD3/ANTI-CD28 Coated
Beads
[0410] Naive CD4+ T cells are isolated from 5 SJL (Harlan, Israel)
mice via automax sort. Beads are coated with anti-CD3 (0.5 ug/m1;
clone 2C11) and anti-CD28 (2 ug/ml; clone 37.51 eBioscience)
following manufacturer's protocol (Dynabeads M-450 Epoxy Cat.
140.11, Invitrogen), and with increasing concentrations of any one
of the C1ORF32 protein fragments and/or fusion proteins thereof
(0.1-10 ug/ml). The total amount of protein used for beads coating
with any one of the C1ORF32 protein fragments and/or fusion
proteins thereof is completed to 10 ug/ml with Control Ig. Naive
CD4+ T cells (0.5.times.10.sup.6/well) are activated with the
coated beads at a ratio of 1:2 (beads to T cells). The cells are
pulsed with luCi of tritiated-thymidine after 24 hours, and
harvested after 72.
[0411] The ability of any one of the C1ORF32 protein fragments
and/or fusion proteins thereof to inhibite T cell proliferation and
elicit its effect in a dose dependent manner is checked. An
appropriate response is seen.
Example 7
[0412] Dose Response Effect of C1ORF32 Protein Fragments and/or
Fusion Proteins Thereof on Mouse CD4+ T Cell Activation With Plate
Bound Anti-CD3, as Manifested in Cytokine Production and Expression
of the Activation Marker CD69
[0413] Untouched CD4+CD25- T cells are isolated from pools of
spleen and lymph node cells of BALB/C mice by negative selection
using CD4+CD62L+ T cell isolation Kit (Miltenyi Cat#130-093-227)
according to the manufacturer's instructions. The desired purity
obtained is >95%.
[0414] Tissue culture 96-well plates are coated overnight at
4.degree. C. with 2 ug/ml anti-CD3 mAb (clone 145-2C11) in the
presence of any one of the C1ORF32 protein fragments and/or fusion
proteins thereof at 1, 5 and 10 .mu.g/ml. Control mIgG2a (Clone
C1.18.4 from BioXCell; Cat#BE0085) is added to each well in order
to complete a total protein concentration of 12 .mu.g/ml per well.
Wells are plated with 1.times.10.sup.5 CD4+CD25- T cells per well.
At 48 hrs post stimulation, culture supernatants are collected and
analyzed using mouse IFN.gamma. ELISA kit, and cells are analyzed
for expression of the activation marker CD69 by flow cytometry.
[0415] The inhibitory effects of any one of the C1ORF32 protein
fragments and/or fusion proteins thereof on CD4 T cell activation
is demonstrated by reduced IFN.gamma. secretion and reduced
expression of CD69 upon TCR stimulation, compared to control mIgG2a
and CTLA4-Ig.
Example 8
[0416] The Effect of C1ORF32 Protein Fragments and/or Fusion
Proteins Thereof on CD4+ T Cell Differentiation In Vitro
[0417] To test the ability of any one of the C1ORF32 protein
fragments and/or fusion proteins thereof to inhibit CD4+ T cell
differentiation, naive CD4+ T cells are isolated from D011.10 mice,
which are transgenic for a T cell receptor (TCR) that is specific
for OVA323-339 peptide. Using D011.10 T cells enables studying both
polyclonal (anti-CD3/anti-CD28 mAbs) and peptide-specific responses
on the same population of CD4+ T cells. Naive CD4+ T cells are
isolated from D011.10 mice and activated in culture in the presence
of anti-CD3/anti-CD28 coated beads or OVA323-339 peptide plus
irradiated BALB/c splenocytes, in the presence of any one of the
C1ORF32 protein fragments and/or fusion proteins thereof, control
Ig, or B7-H4 Ig. The cells are activated in the presence of Th
driving conditions as follows: Th0 cell-(IL-2), Th1 cell-(IL-2+
IL-12), Th2 cell-(IL-2+ IL-4), or Th17 cell-(TGF-.beta.+ IL-6+
IL-23+ anti-IL-2). The effects on T cell differentiation and
Th-specific responses are assessed by measuring cell proliferation
and subtype specific cytokine production: IL-4, IL-5, IL-10, IL-17,
IFN-.gamma.. An appropriate response is seen.
Example 9
[0418] Assesment of the Effect of Any One of the C1ORF32 Protein
Fragments and/or Fusion Proteins Thereof on Human T Cells
Activation
[0419] The effect of any one of the C1ORF32 protein fragments
and/or fusion proteins thereof on human T cell response is tested
by two different in vitro assays using purified human T cells. In
the first assay, human T cells are activated by anti-CD3 and
anti-CD28 coated beads, and in the other assay, activation is
carried out using anti-CD3 and anti-CD28 antibodies in the presence
of autologous, irradiated PBMCs. The regulatory activity of any one
of the C1ORF32 protein fragments and/or fusion proteins thereof on
human T cell activation, is evaluated by measuring cell
proliferation and cytokine release.
[0420] Study I--Activation of Human T Cells with Anti-CD3 and
Anti-CD28-Coated Beads is Inhibited by Fusion Proteins
[0421] Naive CD4+ T cells are isolated from 4 healthy human donors
and activated with anti-CD3 mAb/anti-CD28 mAb coated beads in the
presence of control mIgG2a, or any one of the C1ORF32 protein
fragments and/or fusion proteins thereof. Two side-by-side culture
sets are set up; one culture being pulsed at 24 hours with
tritiated-thymidine and harvested at 72 hours while the second
plate is harvested at 96 hours for cytokine production via
LiquiChip.
[0422] Study II--Activation of Human T Cells with Irradiated
Autologous PBMCs is Inhibited by Fusion Proteins
[0423] Total PBMCs are isolated from fresh blood of healthy human
donors using ficoll gradient. 10.times.10.sup.6 total PBMCs are
resuspended in Ex-Vivo 20 medium, and irradiated at 3000rad. These
cells are used to activate the isolated T cells in vitro, by
presenting the anti-CD3, anti-CD28 and either of the test proteins.
The rest of PBMCs are used for isolation of T cells using CD4+ T
cell Isolation Kit II from Miltenyi.
[0424] For activation, 5.times.10.sup.5 isolated T cells are
cultured in the presence of 5.times.10.sup.5 autologous irradiate
PBMCs. Anti-CD3 (0.5 .mu.g/ml), anti-CD28 (2 ug/ml) and either of
any one of the C1ORF32 protein fragments and/or fusion proteins
thereof or control Ig (mIgG2a) are added in a soluble form. The
cultures are pulsed with 1 uCi of triated thymidine at 24 hrs, and
proliferation is measured at 72 hours.
Example 10
The Effect of Any One of the C1ORF32 Protein Fragments Upon Ectopic
Expression in APC-Like Cells, on Human T Cell Responses
[0425] The effects of any one of the C1ORF32 protein fragments on
human T cell responses are evaluated following their ectopic
expression in `T cell stimulator` cells: a murine thymoma cell
line, Bw5147, which are engineered to express membrane-bound
anti-human CD3 antibody fragments, that can trigger the TCR-complex
on human T cells, with or without co-expression of putative
costimulatory or coinhibitory ligands.
[0426] Codon-optimized cDNAs encoding any one of the C1ORF32
protein fragments are gene-synthesized and directionally cloned
into a retroviral vector pCJK2 via Sfi-I sites. Monocistronic
expression constructs are generated. The constructs are validated
by agarose gel electrophoresis and were expressed in Bw5147 cells
displaying high levels of membrane bound anti-CD3 antibody (Bw-3/2)
(Leitner et al., 2010). As negative control Bw5147 cells transduced
with "empty" vector (pCJK2) are used. In addition, Bw-3/2 cells
expressing costimulatory molecules (ICOSL and CD70) and
Bw-3/2-cells expressing coinhibitory molecules (B7-H3 and
B7-H1/PD-L1) are also used as controls. Homogenously high
expression of the stimulating membrane-bound anti-CD3 antibody is
confirmed by FACS using a DyLight-649 anti-mouse IgG (H+L) antibody
that reacts with the murine single chain antibody expressed on the
stimulator cells. Presence and high level transcription of
expression monocistronic constructs in the respective stimulator
cells is confirmed by qPCR.
[0427] T cells are purified from buffy coats or heparinised blood
derived from healthy volunteer donors and the mononuclear fraction
is obtained by standard density centrifugation using Ficoll-Paque
(GE-Healthcare). Untouched bulk human T cells are obtained through
MACS-depletion of CD11b, CD14, CD16, CD19, CD33 and MHC-class
II-bearing cells with the respective biotinylated mAb in
conjunction with paramagnetic streptavidin beads (Leitner et al.,
2009).
[0428] Purified CD8 T cells and CD4 T cells are obtained by adding
biotinylated CD4 and CD8 mAb to the pools. Naive CD4 T cells are
isolated using the Naive CD4+T cell Isolation Kit II (Miltenyi
Biotec). Following isolation, cells are analyzed for purity by
FACS, and samples with sufficient purity (>90%) were used for
the experiments.
[0429] The stimulator cells are harvested, counted, irradiated
(2.times.3000 rad) and seeded in flat-bottom 96-well plates (20000
cells/well). Liquid nitrogen stored MACS-purified T cells are
thawed, counted and added to the wells at 100.000 cells per well;
total volume was 200 .mu.l/well. Triplicate wells are set up for
each condition. Following 48 hours of co-culture, .sup.3H-thymidine
(final concentration of 0.025 mCi; PerkinElmer/NewEngland Nuclear
Corporation, Wellesley, MA) are added to the wells. Following
further culturing for 18 hours, the plates were harvested on
filter-plates and incorporation of .sup.3H-Thymidine was determined
as described in Pfistershammer et al., 2004. In addition, a series
of experiments with MACS-purified T cell subsets (CD8 T cells, CD4
T cells, and naive CD45RA-positive CD4 T cells) are performed.
Additional controls in all experiments include wells with
stimulator cells alone to assess the cells microscopically and also
to determine basal .sup.3H-Thymidine incorporation of the
stimulator cell w/o T cells. Results with stimulator cells that
quickly disintegrated following irradiation are excluded from the
analysis.
[0430] Results show the effect of stimulator cells expressing any
one of the C1ORF32 protein fragments on the proliferation of human
bulk T cells, CD4+ T cells, CD8+ T cells, or naive CD4 CD45RA+ T
cells, compared to cells expressing control costimulatory molecules
(ICOSL and CD70), which results in a consistent and pronounced
stimulation of proliferation of all cell subtypes, and compared to
cells expressing control coinhibitory molecules (B7-H3 and
B7-H1/PD-L1), which results in a mild inhibition of proliferation
of different T cell subtypes.
Example 11
Characterizing the Target Cells for Any One of the C1ORF32 Protein
Fragments Proteins by Determining Their Binding Profile to Immune
Cells
[0431] Splenocytes from D011.10 mice (transgenic mice in which all
of the CD4+ T cells express a T cell receptor that is specific for
OVA323-339 peptide) are activated in the presence of OVA323-339
peptide, and cells are collected at t=0, 6, 12, 24, and 48 hours
following initial activation to determine which cell type is
expressing a receptor for any one of the C1ORF32 protein fragments
over time. Cells are then co-stained for CD3, CD4, CD8, B220, CD19,
CD11b, and CD11c.
Example 12
[0432] Assesment of the Effect of Any One of the C1ORF32 Protein
Fragments and/or Fusion Proteins Thereof on the Ability of B Cells
to Class-Switch and Secrete Antibody
[0433] Resting B cells are isolated from unprimed C57BL/6 mice and
activated in vitro in the presence of anti-CD40 plus (i) no
exogenous cytokine, (ii) IL-4, or (iii) IFN-.gamma.. The cell
cultures receive control Ig (mIgG2a), anti-CD86 mAb (as a positive
control for increased Ig production), or any one of any one of the
C1ORF32 protein fragments and/or fusion proteins thereof, at the
time of culture set up, and are cultured for 5 days. Any one of the
C1ORF32 protein fragments and/or fusion proteins thereof are tested
at three concentrations each. At the end of culture, supernatants
are tested for the presence of IgM, IgG1, and IgG2a via ELISA. If
there appears to be an alteration in the ability of the B cells to
class-switch to one isotype of antibody versus another, then the
number of B cells that have class switched is determined via
ELISPOT. If there is an alteration in the number of antibody
producing cells, then it is determined if there is an alteration in
the level of .gamma.1- and .gamma.2a-sterile transcripts versus the
mature transcripts for IgG1 and IgG2a. An appropriate response is
seen.
Assesment of the Therapeutic Effect of Any One of the C1ORF32
Protein Fragments and/or Fusion Proteins Thereof for Treatment of
Autoimmune Diseases
Example 13
[0434] Efficacy of Any One of the C1ORF32 Protein Fragments and/or
Fusion Proteins Thereof in Mouse R-EAE Model of Multiple
Sclerosis
[0435] The therapeutic effect of any one of the C1ORF32 protein
fragments and/or fusion proteins thereof for treatment of
autoimmune diseases is tested in a mouse model of Multiple
Sclerosis; Relapsing Remitting Experimental Autoimmune
Encephalomyelitis (R-EAE):
[0436] Female SJL mice 6 weeks old are purchased from Harlan and
maintained in the CCM facility for 1 week prior to beginning the
experiment. Mice are randomly assigned into groups of 10 animals
and primed with 50 .mu.g PLP139-151/CFA on day 0. Mice receive 6
i.p. injections of 100 ug/dose of any one of the C1ORF32 protein
fragments and/or fusion proteins thereof, mIgG2a isotype control,
or CTLA4-Ig (mouse ECD fused to mouse IgG2a Fc) as positive
control. Treatments begin at the time of onset of disease remission
and are given 3 times per week for 2 weeks. Mice are followed for
disease symptoms. On day 35, (during the peak of the disease
relapse) 5 mice of each group are assayed for DTH (delayed type
hypersensitivity) response to disease inducing epitope (PLP139-151)
and to relapse-associated myelin epitope (PLP178-191) via injection
of 10 .mu.g of PLP139-151 in one ear and PLP178-191 into the
opposite ear. The level of ear swelling is assayed at 24 hours post
challenge.
[0437] The decrease in disease severity of R-EAE-induced mice upon
treatment with any one of the C1ORF32 protein fragments and/or
fusion proteins thereof, in a therapeutic mode is tested and
compared to the level of inhibition of CTLA4-Ig.
[0438] In addition, inhibition of DTH responses to the disease
inducing epitope (PLP139-151) and to relapse-associated epitope
(PLP178-191). in ROEAE mice treated with any one of the C1ORF32
protein fragments and/or fusion proteins thereof is tested.
[0439] To test the dose dependency of the efficacy of any one of
the C1ORF32 protein fragments and/or fusion proteins thereof as
well as its mode of action in the PLP-induced R-EAE model, disease
is induced as described above and mice are treated from onset of
disease remission with 100, 30 or 10 ug/dose of any one of the
C1ORF32 protein fragments and/or fusion proteins thereof, 3 times
per week over two weeks. The ability of C1ORF32 protein fragments
and/or fusion proteins thereof to decrease the level of disease
severity in a dose dependent manner, as well as the ability to
inhibit DTH responses to spread epitopes PLP178-191 and MBP84-104,
to inhibit proliferation as well as reduction in IFN.gamma., IL-17,
IL-4 and IL-10 release is tested.
[0440] The beneficial effect of any one of the C1ORF32 protein
fragments and/or fusion proteins thereof in the R-EAE model on
reduction in the infiltration of immune cells to the CNS is also
tested.
Example 14
[0441] Efficacy of Any of the C1ORF32 Protein Fragments and/or
Fusion Proteins Thereof in Mouse CIA Models of Rheumatoid
Arthritis
[0442] Any one of the C1ORF32 protein fragments and/or fusion
proteins thereof is tested in mouse model of collagen-induced
arthritis (CIA) which is a model of rheumatoid arthritis. Male
DBA/1 mice are housed in groups of 8-10, and maintained at
21.degree. C..+-.2.degree. C. on a 12 h light/dark cycle with food
and water ad libitum. Arthritis is induced by immunisation with
type II collagen emulsified in complete Freund's adjuvant. Mice are
monitored on a daily basis for signs of arthritis. On the
appearance of arthritis (day 1) treatment with any one of the
C1ORF32 protein fragments and/or fusion proteins thereof, mIgG2a
isotype control or CTLA4-Ig (mouse ECD fused to mouse IgG2a Fc) as
positive control (100 ug/dose, each) is initiated and given 3 times
per week for 10 days. Hind footpad swelling is measured (using
microcalipers), as well as the number and degree of joint
involvement in all four limbs. This yields two measurements,
clinical score and footpad thickness that can be used for
statistical assessment.
[0443] At the end of the treatment period mice are bled and
sacrificed. For histological analysis, paws are removed at post
mortem, fixed in buffered formalin (10% v/v), then decalcified in
EDTA in buffered formalin (5.5% w/v). The tissues are then embedded
in paraffin, sectioned and stained with haematoxylin and eosin. The
scoring system is as follows:
[0444] 0=normal; 1 =synovitis but cartilage loss and bone erosions
absent or limited to discrete foci; 2=synovitis and significant
erosions present but normal joint architecture intact; 3=synovitis,
extensive erosions, joint architecture disrupted.
[0445] The ability of the treatment of mice with established CIA
with any one of the C1ORF32 protein fragments and/or fusion
proteins thereof to result in potent reduction of clinical score,
paw swelling and histological damage is tested and compared to the
efficacy obtained with CTLA4-Ig.
[0446] Effect of Any of the C1ORF32 Protein Fragments and/or Fusion
Proteins Thereof on Tolerance Induction in Transfer Model of
CIA
[0447] To further understand the effect of any one of the C1ORF32
protein fragments and/or fusion proteins thereof on immune
regulation, the ability of these proteins to induce tolerance in a
transfer model of arthritis is analyzed.
[0448] In brief, spleen and LN cells from arthritic DBA/1 mice
treated for 10 days with any one of the C1ORF32 protein fragments
and/or fusion proteins thereof or control Ig2a are removed and
injected i.p into T-cell deficient C.B-17 SCID recipients. The mice
then receive an injection of 100 .mu.g type II collagen (without
CFA), necessary for successful transfer of arthritis. Arthritis is
then monitored in the SCID mice. Histology is performed and
anti-collagen antibody levels are measured to determine that the
C1ORF32 protein fragments and/or fusion proteins thereof treatment
confers long-term disease protection.
Example 15
[0449] Assessment of the Effect of Any One of C1ORF32 Protein
Fragments and/or Fusion Proteins Thereof in a Viral Infection Model
of TMEV
[0450] Theiler's murine encephalomyelitis virus (TMEV) is a natural
endemic pathogen of mice that causes an induced demyelinating
disease (TMEV-IDD) in susceptible strains of mice (SJL/J, H-2KS)
that resembles the primary progressive form of MS (Munz et al., Nat
Rev Immunol 2009; 9:246-58). TMEV infection results in a life-long
persistent virus infection of the CNS leading to development of a
chronic T cell-mediated autoimmune demyelinating disease triggered
via de novo activation of CD4 T cell responses to endogenous myelin
epitopes in the inflamed CNS (i.e. epitope spreading) (Miller et
al., Nat Med 1997; 3:1133-6; Katz-Levy et al., J Clin Invest 1999;
104:599-610).
[0451] SJL mice clear the majority of the virus within 21 days post
infection, however a latent viral infection is maintained and
infect microglia, astrocytes, and neurons. Disease symptoms are
manifested around day 25-30 post infection.
[0452] The effect of treatment with any one of C1ORF32 protein
fragments and/or fusion proteins thereof on acute and chronic
phases of viral infection is studied in the TMEV-IDD model by
assessment of viral clearance and disease severity.
Method:
[0453] Female SJL/J mice (5-6 weeks) are infected with TMEV by
intracranial inoculation in the right cerebral hemisphere of
3.times.10.sup.7 plaque forming units (PFU) of the BeAn strain 8386
of TMEV in 30 ul serum-free medium. From day 2 post infection mice
are treated with Control Ig, or any one of C1ORF32 protein
fragments and/or fusion proteins thereof, at 100 ug/dose each; 3
doses/week for 2 weeks.
[0454] Mice are followed for clinical scoring. On day 7 and day 14
post infection (after 3 and 6 treatments respectively) brains and
spinal cords are collected from 5 mice in each treatment group for
plaque assays. The tissues are weighted so that the ratio of PFU/mg
of CNS tissue could be calculated after the plaque assay is
completed.
TMEV plaque assay:
[0455] Brains and spinal cords of mice treated with Control Ig
(mouse IgG2a), or with any one of C1ORF32 protein fragments and/or
fusion proteins thereof are collected at days 7 and 14
post-infection from non-perfused anesthetized mice. The Brains and
spinal cords are weighed, and homogenized. CNS homogenates are
serially diluted in DMEM and added to tissue culture-treated plates
of confluent BHK-21 cells for lh incubation at room temperature,
with periodic gentle rocking.
[0456] A media/agar solution is mixed 1:1 (volume:volume), added to
cells and allowed to solidify at room temperature. The plates are
then cultured at 34 deg C for 5 days. At the end of culture, 1 ml
of formalin is added and incubated at room temperature for 1 h to
fix the BHK monolayer. The formalin is poured off into a waste
container, and the agar is removed from the plates. Plaques are
visualized by staining with crystal violet for 5 min, and plates
are gently rinsed with diH2O. To determine PFU/ml homogenate, the
number of plaques on each plate is multiplied by the dilution
factor of the homogenate and divided by the amount of homogenate
added per plate. The PFU/ml is divided by the weight of the tissue
to calculate PFU/mg tissue.
Example 16
[0457] Assessment of the Effect of Any One of C1ORF32 Protein
Fragments and/or Fusion Proteins Thereof on Primary and Secondary
Immune Response to Viral Infection in a Mouse Model of
Influenza
[0458] To test the effect of any one of C1ORF32 protein fragments
and/or fusion proteins thereof on primary and secondary immune
responses to viral infection, BALB/c naive mice (for primary immune
responses) and `HA-memory mice`, is used, as well as `polyclonal
flu-memory mice` (to assess secondary responses mediated by memory
CD4 T cells), which are generated as detailed in Teijaro et al., J
Immunol. 2009: 182; 5430-5438, and described below.
[0459] To obtain `HA-memory mice`, first HA-specific memory CD4 T
cells are generated, naive CD4 T cells are purified from spleens of
HA-TCR mice [BALB/c-HA mice which express transgenic T cell
receptor (TCR) specific for influenza hemagglutinin (HA) peptide
(110-119)] and primed in vitro by culture with 5.0 microg/ml HA
peptide and mitomycin C-treated, T-depleted BALB/c splenocytes as
APCs for 3 days at 37.degree. C. The resultant activated
HA-specific effector cells are transferred into congenic BALB/c
(Thyl.1) hosts (5.times.10.sup.6 cells/mouse) to yield "HA-memory
mice" with a stable population of HA-specific memory CD4 T
cells.
[0460] To obtain `polyclonal-memory mice`, first polyclonal
influenza-specific memory CD4 T cells are generated, by infecting
BALB/c mice intranasally with a sublethal dose of PR8 influenza,
CD4 T cells are isolated 2-4 months postinfection, and the
frequency of influenza-specific memory CD4 T cells is determined by
ELISPOT. CD4 T cells from previously primed mice are transferred
into BALB/c hosts to generate "polyclonal flu-memory" mice with a
full complement of endogenous T cells.
[0461] Primary and secondary responses to influenza virus are
tested by infecting naive BALB/c mice or BALB/c-HA memory mice and
BALB/c `polyclonal flu-memory mice` with sublethal or lethal doses
of PR8 influenza virus by intranasal administration. Mice are
treated with any one of C1ORF32 protein fragments and/or fusion
proteins thereof or with mIgG2a control before and following
influenza challenge. Weight loss and mortality will be monitored
daily. Six days after the challenge, viral content in the
bronchoalveolar lavage (BAL) is analyzed by collecting lavage
liquid and testing the supernatant for viral content by determining
the tissue culture infectious dose 50% (TCID50) in MDCK cells. In
addition, lung tissue histopathology is performed.
[0462] To test the effect of any one of C1ORF32 protein fragments
and/or fusion proteins thereof on T cell expansion BALB/c or
BALB/c-HA memory mice or BALB/c `polyclonal flu-memory mice` are
infected as above and administered with BrdU (1 mg/dose) on days 3,
4 and 5 post infection. On day 6, spleen and lung are harvested and
BrdU incorporation is estimated. Cytokine production by lung memory
CD4 T cells during influenza challenge is also studied in
HA-specific memory CD4 T cells stimulated in vitro with HA peptide
in the presence of any one of C1ORF32 protein fragments and/or
fusion proteins thereof or IgG2a for 18 hours.
Example 17
[0463] Assessment of the Effect of Any One of C1ORF32 Protein
Fragments and/or Fusion Proteins Thereof on Primary and Secondary
CD8 T Cell Response to Viral Infection in a Mouse Model of
Influenza
[0464] The effect of any one of C1ORF32 protein fragments and/or
fusion proteins thereof on primary CD8 T cell responses to
influenza virus is studied according to methods as described in the
literature (Hendriks et al., J Immunol 2005; 175; 1665-1676;
Bertram et al., J Immunol. 2004; 172:981-8) using C57BL/6 mice
infected with influenza A HKx31 by intranasal or intraperitoneal
administration. Any one of C1ORF32 protein fragments and/or fusion
proteins thereof or mIgG2a control is administered during priming
Animal weight loss and mortality is monitored daily. To follow
virus-specific CD8+ T cells, MHC H-2Db tetramers loaded with the
major CD8 T cell epitope, the NP366-374 peptide are used.
Virus-specific H-2D.sup.b/NP366-374+ CD8+ T cells in the lung,
draining lymph nodes, and spleen are expected to reach a peak
around day 8-10 post infection and decline thereafter to only 1.5%
virus-specific CD8 T cells (Hendriks et al J Immunol 2005; 175;
1665-1676; Bertram et al., J Immunol. 2002; 168:3777-85; Bertram et
a., J Immunol. 2004; 172:981-8). Thus, mice are sacrificed at days
8 and 21 post infection, and virus-specific CD8 T cell numbers is
evaluated in the lung, draining lymph nodes and spleen. Viral
clearance is assessed. CD8 T cell responses are evaluated in spleen
cell suspensions, and include intracellular IFN-.gamma. staining
and CTL activity, as previously described (Bertram et a., J
Immunol. 2004; 172:981-8) and detailed below.
[0465] Cells are surface-stained with FITC-conjugated anti-mouse
CD62L, PE-conjugated anti-mouse CD8 to measure CD8+ activated T
cells (or anti-mouse CD4 to follow CD4+ cells). In addition to
these Abs, allophycocyanin-labeled tetramers consisting of murine
class I MHC molecule H-2D.sup.b, .beta..sub.2-microglobulin, and
influenza NP peptide, NP.sub.366-374 are used to measure
influenza-specific CD8 T cells. For intracellular IFN-.gamma.
staining, cell suspensions are restimulated in culture medium for 6
h at 37.degree. C. with 1 .mu.M NP366-374 peptide and GolgiStop (BD
PharMingen, San Diego, Calif.). Cells are then harvested,
resuspended in PBS/2% FCS/azide, and surface stained with
PE-anti-CD8 and FITC-anti-CD62L as described above. After surface
staining, cells will be fixed in Cytofix/Cytoperm solution (BD
PharMingen) and then stained with allophycocyanin-conjugated
antimouse IFN-.gamma. diluted in 1.times. perm/wash solution (BD
PharMingen). Samples are analyzed by Flow Cytometry.
[0466] For cytotoxicity assays (CTL responses) splenocytes from
influenza-infected mice are incubated for 2 h at 37.degree. C. to
remove adherent cells. Serial 3-fold dilutions of effectors are
assayed for anti-influenza NP.sub.366-374-specific CTL activity
against .sup.51Cr-labeled EL4 cells pulsed with 50 .mu.M NP366-374
peptide for 6 h as described by Bertram et al 2002 and Bertram et
al 2004.
[0467] At 3 weeks postinfection, some mice are rechallenged with
the serologically distinct influenza A/PR8/34 (PR8), which shares
the NP gene with influenza A HKx31, but differs in hemagglutinin
and neuraminidase, so that neutralizing Abs do not limit the
secondary CTL response. Mice are sacrificed at days 5 & 7
following virus rechallenge, and virus-specific CD8 T cell numbers
is evaluated in the lung, draining lymph nodes and spleen as
described by Hendriks et al and Bertram et al (Hendriks et al., J
Immunol 2005; 175; 1665-1676; Bertram et al., J Immunol. 2004;
172:981-8) and detailed above. Secondary CD8 T cell responses,
including intracellular IFN-.gamma. staining and CTL activity, are
evaluated in spleen cell suspensions of mice at days 5 & 7
following virus rechallenge, as described above.
[0468] To determine the effect of any one of C1ORF32 protein
fragments and/or fusion proteins thereof on expansion and
accumulation of memory CD8+ T cells during the secondary response,
adoptive transfer experiments are performed, according to methods
previously described (Hendriks et al., J Immunol 2005; 175;
1665-1676; Bertram et al., J Immunol. 2004; 172:981-8): mice are
immunized with influenza influenza A HKx31. Twenty-one days later,
T cells are purified from spleens on mouse T cell enrichment
immunocolumns (Cedarlane Laboratories, Hornsby, Ontario, Canada)
and labeled with CFSE (alternatively Thy1.1 congenic mice are used
as recipients). Equal numbers of tetramer-positive T cells are
injected through the tail vein of recipient mice. Mice are
rechallenged with influenza virus as described above, and 7 days
later splenocytes are evaluated for donor virus-specific CD8 T
cells, as detailed above.
Example 18
[0469] Assessment of Protein Expression in Exhausted T Cells, and
the Binding and Effect of Any One of C1ORF32 Protein Fragments
and/or Fusion Proteins Thereof on Reversing Exhausted T Cell
Phenotype
[0470] Memory CD8 T-cell differentiation proceeds along distinct
pathways after an acute versus a chronic viral infection (Klenerman
and HillNat Immunol 6, 873-879, 2005). Memory CD8 T cells generated
after an acute viral infection are highly functional and constitute
an important component of protective immunity. In contrast, chronic
infections are often characterized by varying degrees of functional
impairment of virus-specific T-cell responses, and this defect is a
principal reason for the inability of the host to eliminate the
persisting pathogen. Although functional effector T cells are
initially generated during the early stages of infection, they
gradually lose function during the course of the chronic infection
leading to exhausted phenotype characterized by impaired T cell
functionality.
[0471] Study I. The effect of any one of C1ORF32 protein fragments
and/or fusion proteins thereof on clearance of viral infection and
on T cell functions during acute and chronic viral infection.
[0472] The effect of any one of C1ORF32 protein fragments and/or
fusion proteins thereof on acute and chronic viral infection is
evaluated in a mouse model of infection with LCMV (lymphocytic
chroriomeningitis virus) according to methodology described by
Wherry et al J. Virol. 77: 4911-4927, 2003 and Barber et al Nature,
2006, and detailed below.
[0473] Two LCMV strains that can cause either acute or chronic
infections in adult mice are used; the Armstrong strain which is
cleared within a week, and the clone 13 strain which establishes a
persistent infection that can last for months. As these two strains
differ in only two amino acids, preserving all known T cell
epitopes, it is possible to track the same CD8 T cell responses
after an acute or chronic viral infection. In contrast to the
highly robust memory CD8 T cells generated after an acute Armstrong
infection, LCMV-specific CD8 T cells become exhausted during a
persistent clone 13 infection (Wherry et al J. Virol. 77:
4911-4927, 2003; Barber et al., Nature. 2006; 439:682-7).
[0474] Mice are infected with 2 .times.10.sup.5 PFU of Armstrong
strain of LCMV intraperitoneally to initiate acute infection or 2
.times.10.sup.6 PFU of C1-13 intravenously to initiate chronic
infection. Mice are treated i.p. with any one of C1ORF32 protein
fragments and/or fusion proteins thereof or with mIgG2a control,
and with specific anti-C1ORF32 protein--antibody, or an isotype
control.
[0475] The mice are monitored for numbers of virus specific CD8 T
cells in the spleen, using virus-specific MHC tetramer epitopes,
such as D.sup.bNP.sub.396-404 and D.sup.bGP.sub.33-41 which differ
in acute or chronic infections. CD8 T cell functional assays, such
as intracellular cytokines levels and CTL activity, are carried out
as described by Wherry et al J. Virol. 77: 4911-4927, 2003, and
similarly to those described in Example 40. Additional assays
include production by splenocytes after stimulation with virus
specific epitopes; and assessment of viral titers in the serum and
in the spleen, liver, lung and kidney (Wherry et al J. Virol. 77:
4911-4927, 2003; Barber et al., Nature. 2006; 439:682-7).
[0476] Study II. Assessment of C1ORF32 protein expression on
exhausted T cells and binding of any one of C1ORF32 protein
fragments and/or fusion proteins thereof to exhausted T cells in
order to evaluate regulation ofthese proteins or their counterpart
receptors during exhaustion of T cells:
[0477] T cells are isolated from mice with chronic LCMV infection
induced with C1-13 strain. The cells are co-stained with
fluorescently labeled anti-PD-1 Ab as positive control (PD-1 is
highly expressed by exhausted T cells) and biotinylated C1ORF32
protein fragments and/or fusion proteins thereof or biotinylated
anti-C1ORF32 protein antibodies, and respective isotype control.
Binding is detected by FACS analysis using fluorescently labeled
streptavidin.
Example 19
Assessment of Any One of C1ORF32 Protein Fragments Expression in
Follicular Helper T (TFH) Cells and the Binding of IG Fusion
Proteins to TFH Cells
[0478] Follicular helper T (Tfh) cells are a subset of CD4+ T cells
specialized in B cell help (reviewed by Crotty, Annu. Rev. Immunol.
29: 621-663, 2011). Tfh cells migrate into B cell follicles within
lymph nodes, and interact with cognate B cells at the T cell-B cell
border and subsequently induce germinal center B cell
differentiation and germinal center formation within the follicle
(Reviewed by Crotty, Annu. Rev. Immunol. 29: 621-663, 2011). The
requirement of Tfh cells for B cell help and T cell-dependent
antibody responses, indicates that this cell type is of great
importance for protective immunity against various types of
infectious agents, as well as for rational vaccine design.
[0479] Tfh cells are readily identifiable at the peak of the CD4+ T
cell response to an acute lymphocytic choriomeningitis virus (LCMV)
infection as
CXCR5.sup.hi'SLAM.sup.loBTLA.sup.hiPD1.sup.hiPD1.sup.hiBcl6.sup.+
virus-specific CD4+ T cells (Choi et al 2011, Immunity 34:
932-946). T cells are isolated from mice with acute LCMV infection
induced with 2 .times.10.sup.5 PFU of Armstrong strain of LCMV
administered intraperitoneally. The cells are co-stained with
fluorescently labeled antibodies for markers of Tfh (CXCRS, PD1,
BTLA, Bc16) which are highly expressed by Tfh cells, and
biotinylated C1ORF32 protein fragments and/or fusion proteins
thereof or biotinylated antibodies specific for C1ORF32 proteins,
and respective isotype controls. Binding of Fc fused protein or
antibody is detected by FACS analysis using fluorescently labeled
streptavidin. An appropriate response is seen.
Example 20
[0480] Assessment of the Effect of Any One of C1ORF32 Protein
Fragments and/or Fusion Proteins Thereof on Follicular Helper T
(TFH) Cells Generation and Activity
[0481] In order to investigate the effect of any one of C1ORF32
protein fragments and/or fusion proteins thereof on Tfh
differentiation and development of B cell immunity in vivo, C57BL/6
are treated with any one of C1ORF32 protein fragments and/or fusion
proteins thereof and an isotype control throughout the course of an
acute viral infection with Armstrong strain of LCMV (lymphocytic
choriomeningitis virus). Tfh differentiation and Bc16 protein
expression is assessed by FACS analysis as described by Eto et al
2011 (PLoS One 6: e17739). Splenocytes are analyzed 8 days
following LCMV infection, Tfh generation
(CD44.sup.hiCXCR5.sup.hiSLAM.sup.lo) and Bcl6 expression is
evaluated by FACS analysis. In addition, the effect of any one of
C1ORF32 protein fragments and/or fusion proteins thereof on
antigen-specific B cell responses is evaluated as described by Eto
et al 2011 (PLoS One 6: e17739), including titers of anti-LCMV IgG
in the serum at 8 days following LCMV infection, and quantitation
by FACS analysis of plasma cell (CD138.sup.+IgD.sup.-) development
at 8 days post-infection, gated on CD19+ splenocytes.
Example 21
[0482] The Effect of Any One of C1ORF32 Protein Fragments and/or
Fusion Proteins Thereof in Modulation of Type 1 Diabetes in NOD
Mice, CD28-KO NOD, and B7-2-KO NOD
[0483] The effect of any one of C1ORF32 protein fragments and/or
fusion proteins thereof is studied in a widely used mouse model of
type 1 diabetes: nonobese diabetic (NOD) mice which develop
spontaneous In NOD mice, spontaneous insulitis, the hallmark
pathologic lesion, evolves through several characteristic stages
that begin with peri-insulitis and end with with invading and
destructive insulitis and overt diabetes. Peri-insulitis is first
observed at 3-4 wk of age, invading insulitis at 8-10 wk, and
destructive insulitis appears just before the onset of clinical
diabetes, with the earliest cases at 10-12 wk. At 20 wk of age,
70-80% of female NOD mice become diabetic (Ansari et al 2003 J.
Exp. Med. 198: 63-69).
[0484] Two KO mice: CD-28-KO NOD mice and B7-1/B7-2 double KO NOD
mice, --which develop accelerated diabetes (Lenschow et al 1996
Immunity 5: 285-293; Salomon et al 2000 Immunity 12: 431-440), are
also used.
[0485] Study I: NOD mice are treated with any one of C1ORF32
protein fragments and/or fusion proteins thereof early and late
phases during the evolution of diabetes, before or after disease
onset, to examine the effects of these compounds on disease
pathogenesis and to demonstrate that such treatment reduces disease
onset and ameliorates pathogenesis. To study the effect on
insulitis, blood glucose levels are measured 3 times/week, for up
to 25 weeks (Ansari et al 2003 J. Exp. Med. 198: 63-69).
[0486] Mechanism of disease modification and mode of action is
studied by experimental evaluation of individual immune cell types:
pancreas, pancreatic LNs and spleen will be harvested to obtain
Tregs, Th subtypes and CD8 T cells, DCs and B cells. Effect on
cytokines secretion from cells isolated from pancreas, pancreatic
LN and spleen is analysed, focused on IFNg, IL-17, IL-4, IL-10 and
TGFb. Upon effect of the tested compounds, the mechanism of disease
modification is studied by examination of individual immune cell
types (including Tregs, Th subtypes and CD8 T cells, DCs and B
cells); cytokines (IFNg, IL-17, IL-4, IL-10 and TGFb) and
histology. Histologycal analysis of the pancreas is carried out to
compare the onset of insulitis, and the lymphocyte
infiltration.
[0487] Study II--The effect of any one of C1ORF32 protein fragments
and/or fusion proteins thereof in modulation of Type 1 Diabetes in
Adoptive transfer model
[0488] To further investigate the mode of action of the Ig fusion
proteins, an adoptive transfer model of diabetes is used. T cells
from diabetic or prediabetic NOD donors are transfered to NOD SCID
recipient mice. These mice are monitored for development of
diabetes. The urine glucose and blood glucose, and assess histology
of the pancreas, and T cell responses are monitored as described in
the previous example.
[0489] Study III--Diabetes is also induced by the transfer of
activated CD4+CD62L+CD25-BDC2.5 T cells (transgenic for TCR
recognizing islet specific peptide 1040-p31 activated by incubation
with 1040-p31) to NOD recipients. Mice are treated with any one of
C1ORF32 protein fragments and/or fusion proteins thereof, control
mIgG2a or positive control. Treatments begin 1 day following
transfer. Mice are followed for glucose levels 10-28 days post
transfer (Bour-Jordan et al., J Clin Invest. 2004;
114(7):979-87).
[0490] Seven days post treatment pancreas, spleen, pancreatic LN
and peripheral lymph node cells are extracted and examined for
different immune cell populations. In addition, recall responses
are measured by testing ex-vivo proliferation and cytokine
secretion in response to p31 peptide. C1ORF32 protein fragments
and/or fusion proteins thereof prevent or reduce disease onset or
the severity thereof in the above studies.
Example 22
[0491] The Effect of Any One Oof C1ORF32 Protein Fragments and/or
Fusion Proteins Thereof in Lupus Mouse Models
[0492] Study I: The lupus-prone mouse model, (NZB.times.NZW)F1
(B/W) is used. Cyclophosphamide (CTX) is the primary drug used for
diffuse proliferative glomerulonephritis in patients with renal
lupus, Daikh and Wofsy reported that combination treatment with CTX
and CTLA4-Ig was more effective than either agent alone in reducing
renal disease and prolonging survival of NZB/NZW Fl lupus mice with
advanced nephritis (Daikh and Wofsy, J Immunol, 166(5):2913-6
(2001)). In the proof-of-concept study, treatments with any one of
C1ORF32 protein fragments and/or fusion proteins thereof and CTX
either alone or in combination are tested.
[0493] Blood samples are collected 3 days before the protein
treatment and then every other week during and after treatments for
plasma anti-dsDNA autoantibody analysis by ELISA.
Glomerulonephritis is evaluated by histological analysis of
kidneys. Proteinuria is measured by testing fresh urine samples
using urinalysis dipsticks.
[0494] C1ORF32 protein fragments and/or fusion proteins thereof
have a beneficial effect in at least ameliorating lupus
nephtiris.
[0495] Study II: The NZM2410-derived B6.Slel.S1e2.S1e3 mouse model
of SLE is used. NZM2410 is a recombinant inbred strain produced
from NZB and NZW that develops a highly penetrant lupus-like
disease with an earlier onset of disease (Blenman et al 2006 Lab.
Invest. 86: 1136-1148). The effect of any one of C1ORF32 protein
fragments and/or fusion proteins thereof is studied in this model
by assessment of proteinuria and autoantibodies as described
above.
[0496] Study III: An induced lupus model is used.This model is
based on chronic graft-vs-host (cGVH) disease induced by the
transfer of Ia-incompatible spleen cells from one normal mouse
strain (such as B6.C-H2(bm12)/KhEg (bm12)) to another (such as
C57BL/6), which causes an autoimmune syndrome resembling systemic
lupus erythematosus (SLE), including anti-double-stranded DNA
(anti-dsDNA) autoantibodies and immune complex-type proliferative
glomerulonephritis (Appleby et al Clin. Exp. Immunol. 1989 78:
449-453); Eisenberg and Choudhury 2004 Methods Mol. Med.
102:273-284).
[0497] Lupus is induced in this model following injection of spleen
cells from bm12 mice into C57BL/6 recipients. The effect any one of
C1ORF32 protein fragments and/or fusion proteins thereof is studied
in this model by assessment of proteinuria and autoantibodies as
described above. T cell and responses B cell responses will also be
evaluated.
[0498] Study IV: The MRL/lpr lupus prone mouse model is used. The
effect of any one of C1ORF32 protein fragments and/or fusion
proteins thereof is studied in this model by assessment of
proteinuria and autoantibodies as described above.
Example 23
[0499] The Effect of Any of C1ORF32 Protein Fragments and/or Fusion
Proteins Thereof in the Control of Intestinal Inflammation
[0500] An adoptive transfer mouse model of colitis in mice is used,
whereby Transfer of CD45RB.sup.high-CD4+ naive T cells from BALB/c
mice to syngeneic SCID mice leads to the development of an IBD-like
syndrome by 6-10 wks after T cell reconstitution, similar to human
Crohn's disease.
[0501] SCID mice are reconstituted by i.p. injection of syngeneic
CD45RB.sup.high-CD4+ T cells either alone or cotransferred with
syngeneic CD45RB.sup.low-CD4+ or CD25.sup.+CD4+ cells
(4.times.10.sup.5/mouse of each cell population) (Liu et al., J
Immunol. 2001; 167(3): 1830-8). Colitic SCID mice, reconstituted
with syngeneic CD45RB.sup.highCD4+ T cells from spleen of normal
mice, are treated i.p. with any one of C1ORF32 protein fragments
and/or fusion proteins thereof or Ig isotype control, twice a week
starting at the beginning of T cell transfer up to 8 wk. All mice
are monitored weekly for weight, soft stool or diarrhea, and rectal
prolapse. All mice are sacrificed 8 wk after T cell transfer or
when they exhibit a loss of.20% of original body weight. Colonic
tissues are collected for histologic and cytologic examinations.
C1ORF32 protein fragments and/or fusion proteins thereof have a
beneficial effect in at least ameliorating inflammatory bowel
disease.
Example 24
[0502] The Effect of Any One of C1ORF32 Protein Fragments and/or
Fusion Proteins Thereof in Mouse Model of Psoriasis
Study I: Establishment of Psoriasis SCID Xenograft Model.
[0503] Human psoriasis plaques are transplanted on to the SCID
mice. Shave biopsies (2.5_2.5 cm) are taken from patients with
generalized plaque psoriasis involving 5-10% of the total skin that
did not receive any systemic treatment for psoriasis or
phototherapy for 6 months and did not receive any topical
preparations other than emollients for 6 weeks. The biopsies are
obtained from active plaques located on the thigh or arm. Each
piece of biopsy is divided into four equal parts of approximately 1
cm2 size. Each piece is transplanted to a separate mouse.
[0504] Under general anesthesia, a graft bed of approximately 1 cm2
is created on the shaved area of the back of a 7- to 8-week-old
CB17 SCID mouse by removing a full-thickness skin sample, keeping
the vessel plexus intact on the fascia covering the underlying back
muscles. The partial thickness human skin obtained by shave biopsy
is then orthotopically transferred onto the graft bed. Nexaband, a
liquid veterinary bandage (Veterinary Products Laboratories,
Phoenix, Ariz.) is used to attach the human skin to the mouse skin
and an antibiotic ointment (bacitracin) is applied. Mice are
treated intraperitoneally three times per week for 4 weeks with any
one of C1ORF32 protein fragments and/or fusion proteins thereof,
isotype control or CTLA4-Ig (positive control).
[0505] Punch biopsies (2 mm) are obtained on day 0 (before
treatment) and day 28 (after treatment) of the study period.
Biopsies are snap frozen and cryosections for histopathological and
immunohistochemical studies. Therapeutic efficacy is determined by
comparing pre- and post treatment data: (i) rete peg lengths to
determine the effect on epidermal thickness and (ii) the level of
lymphomononuclear cell infiltrates to determine the effect on
inflammatory cellular infiltrates. (Raychaudhuri et al. 2008, J
Invest Dermatol.;128(8):1969-76; Boehncke et al., 1999 Arch
Dermatol Res 291:104-6).
[0506] C1ORF32 protein fragments and/or fusion proteins thereof
have a beneficial effect in at least ameliorating psoriasis.
Study II: The Effect of Any One of C1ORF32 Protein Fragments and/or
Fusion Proteins Thereof in Psoriasis and Colitis Model by Adoptive
Transfer of CD45RB in CD4+ T Cells in SCID Mice
[0507] Immunocompromised mice are injected intraveneously (i.v.)
with 0.3_10.sup.6 CD4+ CD45RBhi cells. On the day following the
adoptive transfer of cells, mice are injected intraperitoneally
(i.p.) with 10 microg of staphylococcal enterotoxin B (Davenport et
al., Int Immunopharmacol. 2002 Apr.; 2(5):653-72). Recipient mice
are treated with any one of C1ORF32 protein fragments and/or fusion
proteins thereof, isotype control or CTLA4-Ig (positive control).
Mice are evaluated once a week for 8 weeks for weight loss and
presence of skin lesions.
Example 25
[0508] The Effect of Any One of C1ORF32 Protein Fragments and/or
Fusion Proteins Thereof in Modulating Transplant Rejection
[0509] Study I: THE EFFECT OF any one of C1ORF32 protein fragments
and/or fusion proteins thereof IN A MODEL OF ALLOGENEIC ISLET
TRANSPLANTATION IN DIABETIC MICE. To test the effect of any one of
C1ORF32 protein fragments and/or fusion proteins thereof on
transplant rejection, a model of allogeneic islet transplantation
is used. Diabetes is induced in C57BL/6 mice by treatment with
streptozotocin. Seven days later, the mice are transplanted under
the kidney capsule with pancreatic islets which are isolated from
BALB/c donor mice. Recipient mice are treated with any one of
C1ORF32 protein fragments and/or fusion proteins thereof or with
mIgG2a as a negative control. Tolerance with ECDI-fixed donor
splenocytes is used as the positive control for successful
modulation islet graft rejection. Recipient mice are monitored for
blood glucose levels as a measure of graft acceptance/rejection
(Luo et al., PNAS, Sep. 23, 2008_vol. 105_no. 38_14527-14532).
[0510] Study II: THE EFFECT OF any one of C1ORF32 protein fragments
and/or fusion proteins thereof IN THE HYA-MODEL OF SKIN GRAFT
REJECTION. In humans and certain strains of laboratory mice, male
tissue is recognized as non-self and destroyed by the female immune
system via recognition of histocompatibility-Y chromosome encoded
antigens (Hya). Male tissue destruction is thought to be
accomplished by cytotoxic T lymphocytes in a helper-dependent
manner
[0511] To test the effect of any one of C1ORF32 protein fragments
and/or fusion proteins thereof on transplanatation, the Hya model
system is used, in which female C57BL/6 mice receive tail skin
grafts from male C57BL/6 donors.
[0512] In this study, female C57BL/6 mice are engrafted with
orthotopic split-thickness tail skin from age matched male C57BL/6
mice. The mice are treated with any one of C1ORF32 protein
fragments and/or fusion proteins thereof, isotype control mIgG2a
Immunodominant Hya-encoded CD4 epitope (Dby) attached to female
splenic leukocytes (Dby-SP) serve as positive control for
successful modulation of graft rejection (Martin et al., J Immunol.
2010 Sep. 15; 185(6): 3326-3336). Skin grafts are scored daily for
edema, pigment loss and hair loss. Rejection is defined as complete
hair loss and more than 80% pigment loss.
[0513] In addition, T cell recall responses of cells isolated from
spleens and draining lymph nodes at different time points are
studied in response to CD4 specific epitope (Dby), CD8 epitopes
(Uty and Smcy) or irrelevant peptide (OVA 323-339) while anti CD3
stimulation is used as positive control for prolifereation and
cytokine secretion.
[0514] Study III: The effect of any one of C1ORF32 protein
fragments and/or fusion proteins thereof on graft rejection is
studied in a murine model of syngeneic bone marrow cells
transplantation using the Hya model system described above. Male
hematopoietic cells expressing the CD45.1 marker are transplanted
to female host mice which express the CD45.2 congenic marker.
Female hosts are treated with any one of C1ORF32 protein fragments
and/or fusion proteins thereof or with isotype control mIgG2a. The
female hosts are followed over time and the presence of CD45.1+
cells is monitored.
Example 26
[0515] The Effect of C1ORF32 Protein Fragments and/or Fusion
Proteins Thereof in Treatemnt of Alopecia Areata
[0516] To induce alopecia areata (AA), lesional skin from C3H/HeJ
mice that had developed spontaneous AA lesions is grafted onto the
back of non-affected C3H/HeJ mice, as described previously (McElwee
et al., 1998). Breifly, up to six grafts .about.1-1.5cm in diameter
are aseptically removed from each donor by pinch cutting. The
grafts are placed in sterile PBS while the recipient mice are
prepared for transplantation. Recipient mice are anestized and a
circular pice of skin .about.1.5 cm in diameter is aseptically
removed from the dorsal anto-posterior midline and replaced with a
donor skin graft. The graft is oriented 180.degree. from normal in
order to have the hair growing in opposite direction to the
reciepient hair for easy identification. Four to seven weeks after
grafting the recipients develop initial hair loss. Recipient mice
are treated with any one of C1ORF32 protein fragments and/or fusion
proteins thereof, isotype control or CTLA4-Ig (positive control).
Morphological changes are examined and documented daily.
Photographs are taken before treatment, and once per week during
and after treatment. After completion of treatment, all mice are
anesthesized , their dorsal skin is shaved and animals are
sacrificed by cervical dislocation. Skin samples are taken for
histopathological examination and immunohistochemistry.
[0517] It will be appreciated that various features of the
invention which are, for clarity, described in the contexts of
separate embodiments may also be provided in combination in a
single embodiment. Conversely, various features of the invention
which are, for brevity, described in the context of a single
embodiment may also be provided separately or in any suitable
sub-combination. It will also be appreciated by persons skilled in
the art that the present invention is not limited by what has been
particularly shown and described hereinabove.
Sequence CWU 1
1
19011407DNAHomo Sapiens 1ccggcggcgc gatccagccc ccggccccgc
ctgcgcggcc ggcccggcgg gcgctgcgcc 60cagggacgcc cggtgcccgc cgctccgccg
ccgcccgctg ccgcggggtg acagcgatcc 120ttctgttcca gccatttccc
actttcctca ctccgtaatt cggctgggaa gttggggaag 180atggataggg
tcttgctgag gtggatttct ctcttctggc taacagccat ggtcgaaggc
240cttcaggtca cagtgcccga caagaagaag gtggccatgc tcttccagcc
cactgtgctt 300cgctgccact tctcaacatc ctcccatcag cctgcagttg
tgcagtggaa gttcaagtcc 360tactgccagg atcgcatggg agaatccttg
ggcatgtcct ctacccgggc ccaatctctc 420agcaagagaa acctggaatg
ggacccctac ttggattgtt tggacagcag gaggactgtt 480cgagtagtag
cttcaaaaca gggctcgact gtcaccctgg gagatttcta caggggcaga
540gagatcacga ttgttcatga tgcagatctt caaattggaa agcttatgtg
gggagacagc 600ggactctatt actgtattat caccacccca gatgacctgg
aggggaaaaa tgagggctca 660ctgggactgc tggtgttggg caggacaggg
ctgcttgctg atctcttgcc cagttttgct 720gtggagatta tgccagagtg
ggtgtttgtt ggcctggtgc tcctgggcgt cttcctcttc 780ttcgtcctgg
tggggatctg ctggtgccag tgctgccctc acagctgctg ctgctatgtc
840cgctgcccat gctgcccaga ttcctgctgg tgccctcaag cctgtgagta
cagtgaccgc 900tggggagaca gagcgatcga gagaaatgtc tacctctcta
cctgacagct gtgtgcgctg 960ggttcctcct ccacctcctg tcctgccacc
cccaagattg gtcattccag actcttctcc 1020gctgggtgcc cctggcctca
gggatgacca ttctcatttg ccttttcacc tacatacacc 1080tctccacact
tcttatccat atctatcact ccatgcattt ggaattctca tggacactat
1140tgataaaatg gaagggcagg tttggcgtgg tgaggttgtg gtgtaagact
gttccctctc 1200cctggggcat tcaaactaga ggaaaccttc tctggtcgtt
cccttcccat gcagagaagt 1260tcctttttat atgagaagag tgtgcaaact
gtggcctttg ggcacccacc cagccacaga 1320tttgttttat ttactcccat
gatgacatgg gccacaatag ggcctagttc ttatttgagg 1380attcacaatt
tttaccttac tggccaa 140721350DNAHomo Sapiens 2ccggcggcgc gatccagccc
ccggccccgc ctgcgcggcc ggcccggcgg gcgctgcgcc 60cagggacgcc cggtgcccgc
cgctccgccg ccgcccgctg ccgcggggtg acagcgatcc 120ttctgttcca
gccatttccc actttcctca ctccgtaatt cggctgggaa gttggggaag
180atggataggg tcttgctgag gtggatttct ctcttctggc taacagccat
ggtcgaaggc 240cttcaggtca cagtgcccga caagaagaag gtggccatgc
tcttccagcc cactgtgctt 300cgctgccact tctcaacatc ctcccatcag
cctgcagttg tgcagtggaa gttcaagtcc 360tactgccagg atcgcatggg
agaatccttg ggcatgtcct ctacccgggc ccaatctctc 420agcaagagaa
acctggaatg ggacccctac ttggattgtt tggacagcag gaggactgtt
480cgagtagtag cttcaaaaca gggctcgact gtcaccctgg gagatttcta
caggggcaga 540gagatcacga ttgttcatga tgcagatctt caaattggaa
agcttatgtg gggagacagc 600ggactctatt actgtattat caccacccca
gatgacctgg aggggaaaaa tgagggctca 660ctgggactgc tggtgttgga
gtgggtgttt gttggcctgg tgctcctggg cgtcttcctc 720ttcttcgtcc
tggtggggat ctgctggtgc cagtgctgcc ctcacagctg ctgctgctat
780gtccgctgcc catgctgccc agattcctgc tggtgccctc aagcctgtga
gtacagtgac 840cgctggggag acagagcgat cgagagaaat gtctacctct
ctacctgaca gctgtgtgcg 900ctgggttcct cctccacctc ctgtcctgcc
acccccaaga ttggtcattc cagactcttc 960tccgctgggt gcccctggcc
tcagggatga ccattctcat ttgccttttc acctacatac 1020acctctccac
acttcttatc catatctatc actccatgca tttggaattc tcatggacac
1080tattgataaa atggaagggc aggtttggcg tggtgaggtt gtggtgtaag
actgttccct 1140ctccctgggg cattcaaact agaggaaacc ttctctggtc
gttcccttcc catgcagaga 1200agttcctttt tatatgagaa gagtgtgcaa
actgtggcct ttgggcaccc acccagccac 1260agatttgttt tatttactcc
catgatgaca tgggccacaa tagggcctag ttcttatttg 1320aggattcaca
atttttacct tactggccaa 13503639PRTHomo Sapiens 3Met Asp Arg Val Leu
Leu Arg Trp Ile Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met Val Glu Gly
Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala 20 25 30Met Leu Phe
Gln Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser 35 40 45His Gln
Pro Ala Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp 50 55 60Arg
Met Gly Glu Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu65 70 75
80Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
85 90 95Arg Arg Thr Val Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val
Thr 100 105 110Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala 115 120 125Asp Leu Gln Ile Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr 130 135 140Cys Ile Ile Thr Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser145 150 155 160Val Glu Leu Leu Val Leu
Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu 165 170 175Pro Ser Phe Ala
Val Glu Ile Met Pro Glu Trp Val Phe Val Gly Leu 180 185 190Val Leu
Leu Gly Val Phe Leu Phe Phe Val Leu Val Gly Ile Cys Trp 195 200
205Cys Gln Cys Cys Pro His Ser Cys Cys Cys Tyr Val Arg Cys Pro Cys
210 215 220Cys Pro Asp Ser Cys Cys Cys Pro Gln Ala Leu Tyr Glu Ala
Gly Lys225 230 235 240Ala Ala Lys Ala Gly Tyr Pro Pro Ser Val Ser
Gly Val Pro Gly Pro 245 250 255Tyr Ser Ile Pro Ser Val Pro Leu Gly
Gly Ala Pro Ser Ser Gly Met 260 265 270Leu Met Asp Lys Pro His Pro
Pro Pro Leu Ala Pro Ser Asp Ser Thr 275 280 285Gly Gly Ser His Ser
Val Arg Lys Gly Tyr Arg Ile Gln Ala Asp Lys 290 295 300Glu Arg Asp
Ser Met Lys Val Leu Tyr Tyr Val Glu Lys Glu Leu Ala305 310 315
320Gln Phe Asp Pro Ala Arg Arg Met Arg Gly Arg Tyr Asn Asn Thr Ile
325 330 335Ser Glu Leu Ser Ser Leu His Glu Glu Asp Ser Asn Phe Arg
Gln Ser 340 345 350Phe His Gln Met Arg Ser Lys Gln Phe Pro Val Ser
Gly Asp Leu Glu 355 360 365Ser Asn Pro Asp Tyr Trp Ser Gly Val Met
Gly Gly Ser Ser Gly Ala 370 375 380Ser Arg Gly Pro Ser Ala Met Glu
Tyr Asn Lys Glu Asp Arg Glu Ser385 390 395 400Phe Arg His Ser Gln
Pro Arg Ser Lys Ser Glu Met Leu Ser Arg Lys 405 410 415Asn Phe Ala
Thr Gly Val Pro Ala Val Ser Met Asp Glu Leu Ala Ala 420 425 430Phe
Ala Asp Ser Tyr Gly Gln Arg Pro Arg Arg Ala Asp Gly Asn Ser 435 440
445His Glu Ala Arg Gly Gly Ser Arg Phe Glu Arg Ser Glu Ser Arg Ala
450 455 460His Ser Gly Phe Tyr Gln Asp Asp Ser Leu Glu Glu Tyr Tyr
Gly Gln465 470 475 480Arg Ser Arg Ser Arg Glu Pro Leu Thr Asp Ala
Asp Arg Gly Trp Ala 485 490 495Phe Ser Pro Ala Arg Arg Arg Pro Ala
Glu Asp Ala His Leu Pro Arg 500 505 510Leu Val Ser Arg Thr Pro Gly
Thr Ala Pro Lys Tyr Asp His Ser Tyr 515 520 525Leu Gly Ser Ala Arg
Glu Arg Gln Ala Arg Pro Glu Gly Ala Ser Arg 530 535 540Gly Gly Ser
Leu Glu Thr Pro Ser Lys Arg Ser Ala Gln Leu Gly Pro545 550 555
560Arg Ser Ala Ser Tyr Tyr Ala Trp Ser Pro Pro Gly Thr Tyr Lys Ala
565 570 575Gly Ser Ser Gln Asp Asp Gln Glu Asp Ala Ser Asp Asp Ala
Leu Pro 580 585 590Pro Tyr Ser Glu Leu Glu Leu Thr Arg Gly Pro Ser
Tyr Arg Gly Arg 595 600 605Asp Leu Pro Tyr His Ser Asn Ser Glu Lys
Lys Arg Lys Lys Glu Pro 610 615 620Ala Lys Lys Thr Asn Asp Phe Pro
Thr Arg Met Ser Leu Val Val625 630 6354254PRTHomo Sapiens 4Met Asp
Arg Val Leu Leu Arg Trp Ile Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met
Val Glu Gly Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala 20 25
30Met Leu Phe Gln Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
35 40 45His Gln Pro Ala Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp 50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr Leu Asp
Cys Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr 100 105 110Leu Gly Asp Phe Tyr Arg Gly Arg Glu
Ile Thr Ile Val His Asp Ala 115 120 125Asp Leu Gln Ile Gly Lys Leu
Met Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135 140Cys Ile Ile Thr Thr
Pro Asp Asp Leu Glu Gly Lys Asn Glu Gly Ser145 150 155 160Leu Gly
Leu Leu Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu 165 170
175Pro Ser Phe Ala Val Glu Ile Met Pro Glu Trp Val Phe Val Gly Leu
180 185 190Val Leu Leu Gly Val Phe Leu Phe Phe Val Leu Val Gly Ile
Cys Trp 195 200 205Cys Gln Cys Cys Pro His Ser Cys Cys Cys Tyr Val
Arg Cys Pro Cys 210 215 220Cys Pro Asp Ser Cys Trp Cys Pro Gln Ala
Cys Glu Tyr Ser Asp Arg225 230 235 240Trp Gly Asp Arg Ala Ile Glu
Arg Asn Val Tyr Leu Ser Thr 245 2505254PRTHomo Sapiens 5Met Asp Arg
Val Leu Leu Arg Trp Ile Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met Val
Glu Gly Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala 20 25 30Met
Leu Phe Gln Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser 35 40
45His Gln Pro Ala Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp
50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser
Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val Val Ala Ser Lys Gln Gly
Ser Thr Val Thr 100 105 110Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala 115 120 125Asp Leu Gln Ile Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135 140Cys Ile Ile Thr Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser145 150 155 160Val Glu Leu
Leu Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu 165 170 175Pro
Ser Phe Ala Val Glu Ile Met Pro Glu Trp Val Phe Val Gly Leu 180 185
190Val Leu Leu Gly Val Phe Leu Phe Phe Val Leu Val Gly Ile Cys Trp
195 200 205Cys Gln Cys Cys Pro His Ser Cys Cys Cys Tyr Val Arg Cys
Pro Cys 210 215 220Cys Pro Asp Ser Cys Cys Cys Pro Gln Ala Cys Glu
Tyr Ser Asp Arg225 230 235 240Trp Gly Asp Arg Ala Ile Glu Arg Asn
Val Tyr Leu Ser Thr 245 2506235PRTHomo Sapiens 6Met Asp Arg Val Leu
Leu Arg Trp Ile Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met Val Glu Gly
Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala 20 25 30Met Leu Phe
Gln Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser 35 40 45His Gln
Pro Ala Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp 50 55 60Arg
Met Gly Glu Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu65 70 75
80Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
85 90 95Arg Arg Thr Val Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val
Thr 100 105 110Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala 115 120 125Asp Leu Gln Ile Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr 130 135 140Cys Ile Ile Thr Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Gly Ser145 150 155 160Leu Gly Leu Leu Val Leu
Glu Trp Val Phe Val Gly Leu Val Leu Leu 165 170 175Gly Val Phe Leu
Phe Phe Val Leu Val Gly Ile Cys Trp Cys Gln Cys 180 185 190Cys Pro
His Ser Cys Cys Cys Tyr Val Arg Cys Pro Cys Cys Pro Asp 195 200
205Ser Cys Trp Cys Pro Gln Ala Cys Glu Tyr Ser Asp Arg Trp Gly Asp
210 215 220Arg Ala Ile Glu Arg Asn Val Tyr Leu Ser Thr225 230
23571287DNAArtificial SequenceSynthetic polynucleotide 7atggataggg
tcttgctgag gtggatttct ctcttctggc taacagccat ggtcgaaggc 60cttcaggtca
cagtgcccga caagaagaag gtggccatgc tcttccagcc cactgtgctt
120cgctgccact tctcaacatc ctcccatcag cctgcagttg tgcagtggaa
gttcaagtcc 180tactgccagg atcgcatggg agaatccttg ggcatgtcct
ctacccgggc ccaatctctc 240agcaagagaa acctggaatg ggacccctac
ttggattgtt tggacagcag gaggactgtt 300cgagtagtag cttcaaaaca
gggctcgact gtcaccctgg gagatttcta caggggcaga 360gagatcacga
ttgttcatga tgcagatctt caaattggaa agcttatgtg gggagacagc
420ggactctatt actgtattat caccacccca gatgacctgg aggggaaaaa
tgaggactca 480gtggaactgc tggtgttggg caggacaggg ctgcttgctg
atctcttgcc cagttttgct 540gtggagatta tgggatccga gaacctgtac
tttcagggca gcggcgagcc cagaggcccc 600accatcaagc cctgcccccc
ctgcaagtgc ccagccccta acctgctggg cggacccagc 660gtgttcatct
tcccccccaa gatcaaggac gtgctgatga tcagcctgag ccccatcgtg
720acctgcgtgg tggtggacgt gagcgaggac gaccccgacg tgcagatcag
ctggttcgtg 780aacaacgtgg aggtgcacac cgcccagacc cagacccacc
gggaggacta caacagcacc 840ctgcgggtgg tgtccgccct gcccatccag
caccaggact ggatgagcgg caaagaattc 900aagtgcaagg tgaacaacaa
ggacctgcct gcccccatcg agcggaccat cagcaagccc 960aagggcagcg
tgagagcccc ccaggtgtac gtgctgcccc ctcccgagga agagatgacc
1020aagaaacagg tgaccctgac ctgcatggtg accgacttca tgcccgagga
catctacgtg 1080gagtggacca acaacggcaa gaccgagctg aactacaaga
acaccgagcc cgtgctggac 1140agcgacggca gctacttcat gtatagcaag
ctgagagtcg agaagaaaaa ctgggtggag 1200cggaacagct acagctgcag
cgtggtgcac gagggcctgc acaaccacca caccaccaag 1260agcttcagcc
ggacccccgg caagtga 12878428PRTArtificial SequenceSynthetic
polypeptide 8Met Asp Arg Val Leu Leu Arg Trp Ile Ser Leu Phe Trp
Leu Thr Ala1 5 10 15Met Val Glu Gly Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala 20 25 30Met Leu Phe Gln Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser 35 40 45His Gln Pro Ala Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp 50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp
Asp Pro Tyr Leu Asp Cys Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr 100 105 110Leu Gly Asp Phe
Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala 115 120 125Asp Leu
Gln Ile Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135
140Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp
Ser145 150 155 160Val Glu Leu Leu Val Leu Gly Arg Thr Gly Leu Leu
Ala Asp Leu Leu 165 170 175Pro Ser Phe Ala Val Glu Ile Met Gly Ser
Glu Asn Leu Tyr Phe Gln 180 185 190Gly Ser Gly Glu Pro Arg Gly Pro
Thr Ile Lys Pro Cys Pro Pro Cys 195 200 205Lys Cys Pro Ala Pro Asn
Leu Leu Gly Gly Pro Ser Val Phe Ile Phe 210 215 220Pro Pro Lys Ile
Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val225 230 235 240Thr
Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile 245 250
255Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
260 265 270His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala
Leu Pro 275 280 285Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe
Lys Cys Lys Val 290 295 300Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu
Arg Thr Ile Ser Lys Pro305 310 315 320Lys Gly Ser Val Arg Ala Pro
Gln Val Tyr Val Leu Pro Pro Pro Glu 325 330 335Glu Glu Met Thr Lys
Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp 340 345 350Phe Met Pro
Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr 355 360 365Glu
Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser 370 375
380Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu
Lys Lys Asn Trp Val Glu385 390 395 400Arg Asn Ser Tyr Ser Cys Ser
Val Val His Glu Gly Leu His Asn His 405 410 415His Thr Thr Lys Ser
Phe Ser Arg Thr Pro Gly Lys 420 4259524DNAArtificial
SequenceSynthetic polynucleotide 9gtgagtacag tgaccgctgg ggagacagag
cgatcgagag aaatgtctac ctctctacct 60gacagctgtg tgcgctgggt tcctcctcca
cctcctgtcc tgccaccccc aagattggtc 120attccagact cttctccgct
gggtgcccct ggcctcaggg atgaccattc tcatttgcct 180tttcacctac
atacacctct ccacacttct tatccatatc tatcactcca tgcatttgga
240attctcatgg acactattga taaaatggaa gggcaggttt ggcgtggtga
ggttgtggtg 300taagactgtt ccctctccct ggggcattca aactagagga
aaccttctct ggtcgttccc 360ttcccatgca gagaagttcc tttttatatg
agaagagtgt gcaaactgtg gcctttgggc 420acccacccag ccacagattt
gttttattta ctcccatgat gacatgggcc acaatagggc 480ctagttctta
tttgaggatt cacaattttt accttactgg ccaa 5241057DNAArtificial
SequenceSynthetic polynucleotide 10gcaggacagg gctgcttgct gatctcttgc
ccagttttgc tgtggagatt atgccag 571161DNAArtificial SequenceSynthetic
polynucleotide 11agtgggtgtt tgttggcctg gtgctcctgg gcgtcttcct
cttcttcgtc ctggtgggga 60t 611266DNAArtificial SequenceSynthetic
polynucleotide 12ctgctggtgc cagtgctgcc ctcacagctg ctgctgctat
gtccgctgcc catgctgccc 60agattc 661320DNAArtificial
SequenceSynthetic polynucleotide 13ctgctggtgc cctcaagcct
2014164PRTArtificial SequenceSynthetic polypeptide 14Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Gly Ser Leu Gly Leu Leu 130 135 140Val Leu Gly Arg Thr Gly
Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145 150 155 160Val Glu Ile
Met15149PRTArtificial SequenceSynthetic polypeptide 15Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Gly Ser Leu Gly Leu Leu 130 135 140Val Leu Glu Trp
Val1451633DNAArtificial SequenceSynthetic polynucleotide
16ctagctagcc accatggata gggtcttgct gag 331729DNAArtificial
SequenceSynthetic polynucleotide 17cgcggatccc ataatctcca cagcaaaac
291820PRTArtificial SequenceSynthetic polypeptide 18Cys Glu Tyr Ser
Asp Arg Trp Gly Asp Arg Ala Ile Glu Arg Asn Val1 5 10 15Tyr Leu Ser
Thr 2019184PRTArtificial SequenceSynthetic polypeptide 19Met Asp
Arg Val Leu Leu Arg Trp Ile Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met
Val Glu Gly Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala 20 25
30Met Leu Phe Gln Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
35 40 45His Gln Pro Ala Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp 50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr Leu Asp
Cys Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr 100 105 110Leu Gly Asp Phe Tyr Arg Gly Arg Glu
Ile Thr Ile Val His Asp Ala 115 120 125Asp Leu Gln Ile Gly Lys Leu
Met Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135 140Cys Ile Ile Thr Thr
Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser145 150 155 160Val Glu
Leu Leu Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu 165 170
175Pro Ser Phe Ala Val Glu Ile Met 18020232PRTArtificial
SequenceSynthetic polypeptide 20Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala1 5 10 15Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro 20 25 30Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105
110Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr 130 135 140Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser145 150 155 160Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220Ser
Leu Ser Leu Ser Pro Gly Lys225 23021217PRTArtificial
SequenceSynthetic polypeptide 21Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His65 70 75 80Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn145 150 155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205Lys Ser Leu
Ser Leu Ser Pro Gly Lys 210 21522416PRTArtificial SequenceSynthetic
polypeptide 22Met Asp Arg Val Leu Leu Arg Trp Ile Ser Leu Phe Trp
Leu Thr Ala1 5 10 15Met Val Glu Gly Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala 20 25 30Met Leu Phe Gln Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser 35 40 45His Gln Pro Ala Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp 50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp
Asp Pro Tyr Leu Asp Cys Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr 100 105 110Leu Gly Asp Phe
Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala 115 120 125Asp Leu
Gln Ile Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135
140Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp
Ser145 150 155 160Val Glu Leu Leu Val Leu Gly Arg Thr Gly Leu Leu
Ala Asp Leu Leu 165 170 175Pro Ser Phe Ala Val Glu Ile Met Glu Pro
Lys Ser Cys Asp Lys Thr 180 185 190His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser 195 200 205Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 210 215 220Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro225 230 235 240Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 245 250
255Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
260 265 270Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr 275 280 285Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 290 295 300Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu305 310 315 320Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys 325 330 335Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 340 345 350Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 355 360 365Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 370 375
380Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala385 390 395 400Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 405 410 41523411PRTArtificial SequenceSynthetic
polypeptide 23Met Asp Arg Val Leu Leu Arg Trp Ile Ser Leu Phe Trp
Leu Thr Ala1 5 10 15Met Val Glu Gly Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala 20 25 30Met Leu Phe Gln Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser 35 40 45His Gln Pro Ala Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp 50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp
Asp Pro Tyr Leu Asp Cys Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr 100 105 110Leu Gly Asp Phe
Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala 115 120 125Asp Leu
Gln Ile Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135
140Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp
Ser145 150 155 160Val Glu Leu Leu Val Leu Gly Arg Thr Gly Leu Leu
Ala Asp Leu Leu 165 170 175Pro Ser Phe Ala Val Glu Ile Met Asp Lys
Thr His Thr Cys Pro Pro 180 185 190Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro 195 200 205Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr 210 215 220Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn225 230 235 240Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 245 250
255Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
260 265 270Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser 275 280 285Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys 290 295 300Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp305 310 315 320Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe 325 330 335Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 340 345 350Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 355 360 365Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 370 375
380Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr385 390 395 400Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405
410242PRTArtificial SequenceSynthetic polypeptide 24Gly
Ser1254PRTArtificial SequenceSynthetic polypeptide 25Gly Ser Gly
Ser1262PRTArtificial SequenceSynthetic polypeptide 26Ala
Ser1274PRTArtificial SequenceSynthetic polypeptide 27Gly Gly Gly
Ser128169PRTArtificial SequenceSynthetic polypeptide 28Met Asp Arg
Val Leu Leu Arg Trp Ile Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met Val
Glu Gly Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala 20 25 30Met
Leu Phe Gln Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser 35 40
45His Gln Pro Ala Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp
50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser
Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val Val Ala Ser Lys Gln Gly
Ser Thr Val Thr 100 105 110Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala 115 120 125Asp Leu Gln Ile Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135 140Cys Ile Ile Thr Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Gly Ser145 150 155 160Leu Gly Leu
Leu Val Leu Glu Trp Val 16529147PRTArtificial SequenceSynthetic
polypeptide 29Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met
Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135
140Val Leu
Gly14530104PRTArtificial SequenceSynthetic polypeptide 30Cys His
Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys1 5 10 15Phe
Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser 20 25
30Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro
35 40 45Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala
Ser 50 55 60Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu65 70 75 80Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly
Lys Leu Met Trp 85 90 95Gly Asp Ser Gly Leu Tyr Tyr Cys
10031233PRTArtificial SequenceSynthetic polypeptide 31Glu Pro Arg
Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro1 5 10 15Ala Pro
Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys 20 25 30Ile
Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val 35 40
45Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe
50 55 60Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
Glu65 70 75 80Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro
Ile Gln His 85 90 95Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys
Val Asn Asn Lys 100 105 110Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile
Ser Lys Pro Lys Gly Ser 115 120 125Val Arg Ala Pro Gln Val Tyr Val
Leu Pro Pro Pro Glu Glu Glu Met 130 135 140Thr Lys Lys Gln Val Thr
Leu Thr Cys Met Val Thr Asp Phe Met Pro145 150 155 160Glu Asp Ile
Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn 165 170 175Tyr
Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met 180 185
190Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser
195 200 205Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His
Thr Thr 210 215 220Lys Ser Phe Ser Arg Thr Pro Gly Lys225
2303215PRTArtificial SequenceSynthetic polypeptide 32Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10
153320PRTArtificial SequenceSynthetic polypeptide 33Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly
Ser 2034235PRTArtificial SequenceSynthetic polypeptide 34Met Asp
Arg Val Leu Leu Arg Trp Ile Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met
Val Glu Gly Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala 20 25
30Met Leu Phe Gln Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
35 40 45His Gln Pro Ala Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp 50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr Leu Asp
Cys Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr 100 105 110Leu Gly Asp Phe Tyr Arg Gly Arg Glu
Ile Thr Ile Val His Asp Ala 115 120 125Asp Leu Gln Ile Gly Lys Leu
Met Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135 140Cys Ile Ile Thr Thr
Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser145 150 155 160Val Glu
Leu Leu Val Leu Glu Trp Val Phe Val Gly Leu Val Leu Leu 165 170
175Gly Val Phe Leu Phe Phe Val Leu Val Gly Ile Cys Trp Cys Gln Cys
180 185 190Cys Pro His Ser Cys Cys Cys Tyr Val Arg Cys Pro Cys Cys
Pro Asp 195 200 205Ser Cys Cys Cys Pro Gln Ala Cys Glu Tyr Ser Asp
Arg Trp Gly Asp 210 215 220Arg Ala Ile Glu Arg Asn Val Tyr Leu Ser
Thr225 230 23535164PRTArtificial SequenceSynthetic polypeptide
35Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly
Arg Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145 150 155
160Val Glu Ile Met36149PRTArtificial SequenceSynthetic polypeptide
36Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Glu
Trp Val14537184PRTArtificial SequenceSynthetic polypeptide 37Met
Asp Arg Val Leu Leu Arg Trp Ile Ser Leu Phe Trp Leu Thr Ala1 5 10
15Met Val Glu Gly Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala
20 25 30Met Leu Phe Gln Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser
Ser 35 40 45His Gln Pro Ala Val Val Gln Trp Lys Phe Lys Ser Tyr Cys
Gln Asp 50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser Ser Thr Arg Ala
Gln Ser Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr Leu
Asp Cys Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val Val Ala Ser Lys
Gln Gly Ser Thr Val Thr 100 105 110Leu Gly Asp Phe Tyr Arg Gly Arg
Glu Ile Thr Ile Val His Asp Ala 115 120 125Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135 140Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Gly Ser145 150 155 160Leu
Gly Leu Leu Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu 165 170
175Pro Ser Phe Ala Val Glu Ile Met 18038416PRTArtificial
SequenceSynthetic polypeptide 38Met Asp Arg Val Leu Leu Arg Trp Ile
Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met Val Glu Gly Leu Gln Val Thr
Val Pro Asp Lys Lys Lys Val Ala 20 25 30Met Leu Phe Gln Pro Thr Val
Leu Arg Cys His Phe Ser Thr Ser Ser 35 40 45His Gln Pro Ala Val Val
Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp 50 55 60Arg Met Gly Glu Ser
Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu65 70 75 80Ser Lys Arg
Asn Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser 85 90 95Arg Arg
Thr Val Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr 100 105
110Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
115 120 125Asp Leu Gln Ile Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr 130 135 140Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Gly Ser145 150 155 160Leu Gly Leu Leu Val Leu Gly Arg Thr
Gly Leu Leu Ala Asp Leu Leu 165 170 175Pro Ser Phe Ala Val Glu Ile
Met Glu Pro Lys Ser Cys Asp Lys Thr 180 185 190His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 195 200 205Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 210 215 220Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro225 230
235 240Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 245 250 255Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val 260 265 270Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr 275 280 285Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr 290 295 300Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu305 310 315 320Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 325 330 335Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 340 345
350Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
355 360 365Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser 370 375 380Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala385 390 395 400Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 405 410 41539411PRTArtificial
SequenceSynthetic polypeptide 39Met Asp Arg Val Leu Leu Arg Trp Ile
Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met Val Glu Gly Leu Gln Val Thr
Val Pro Asp Lys Lys Lys Val Ala 20 25 30Met Leu Phe Gln Pro Thr Val
Leu Arg Cys His Phe Ser Thr Ser Ser 35 40 45His Gln Pro Ala Val Val
Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp 50 55 60Arg Met Gly Glu Ser
Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu65 70 75 80Ser Lys Arg
Asn Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser 85 90 95Arg Arg
Thr Val Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr 100 105
110Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
115 120 125Asp Leu Gln Ile Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr 130 135 140Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Gly Ser145 150 155 160Leu Gly Leu Leu Val Leu Gly Arg Thr
Gly Leu Leu Ala Asp Leu Leu 165 170 175Pro Ser Phe Ala Val Glu Ile
Met Asp Lys Thr His Thr Cys Pro Pro 180 185 190Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 195 200 205Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 210 215 220Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn225 230
235 240Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg 245 250 255Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val 260 265 270Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser 275 280 285Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys 290 295 300Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp305 310 315 320Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 325 330 335Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 340 345
350Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
355 360 365Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly 370 375 380Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr385 390 395 400Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 405 41040169PRTArtificial SequenceSynthetic polypeptide
40Met Asp Arg Val Leu Leu Arg Trp Ile Ser Leu Phe Trp Leu Thr Ala1
5 10 15Met Val Glu Gly Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val
Ala 20 25 30Met Leu Phe Gln Pro Thr Val Leu Arg Cys His Phe Ser Thr
Ser Ser 35 40 45His Gln Pro Ala Val Val Gln Trp Lys Phe Lys Ser Tyr
Cys Gln Asp 50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser Ser Thr Arg
Ala Gln Ser Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
Leu Asp Cys Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val Val Ala Ser
Lys Gln Gly Ser Thr Val Thr 100 105 110Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile Thr Ile Val His Asp Ala 115 120 125Asp Leu Gln Ile Gly
Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135 140Cys Ile Ile
Thr Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser145 150 155
160Val Glu Leu Leu Val Leu Glu Trp Val 16541147PRTArtificial
SequenceSynthetic polypeptide 41Leu Gln Val Thr Val Pro Asp Lys Lys
Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe
Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser
Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro
Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val
Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg
Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Gly Ser Leu Gly
Leu Leu 130 135 140Val Leu Gly14542144PRTArtificial
SequenceSynthetic polypeptide 42Leu Gln Val Thr Val Pro Asp Lys Lys
Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe
Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser
Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro
Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val
Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg
Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 14043145PRTArtificial SequenceSynthetic
polypeptide 43Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met
Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135
140Val14544146PRTArtificial SequenceSynthetic polypeptide 44Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val
Leu14545164PRTArtificial SequenceSynthetic polypeptide 45Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr
Gly Leu Leu Ala Asp Leu Leu Pro Ser Gly Gly145 150 155 160Val Glu
Ile Met46148PRTArtificial SequenceSynthetic polypeptide 46Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly
Arg14547149PRTArtificial SequenceSynthetic polypeptide 47Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg
Thr14548150PRTArtificial SequenceSynthetic polypeptide 48Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr
Gly145 15049151PRTArtificial SequenceSynthetic polypeptide 49Leu
Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10
15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala
20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly
Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys
Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg
Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val
Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His
Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser
Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu
Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg
Thr Gly Leu145 15050152PRTArtificial SequenceSynthetic polypeptide
50Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly
Arg Thr Gly Leu Leu145 15051153PRTArtificial SequenceSynthetic
polypeptide 51Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met
Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135
140Val Leu Gly Arg Thr Gly Leu Leu Ala145 15052154PRTArtificial
SequenceSynthetic polypeptide 52Leu Gln Val Thr Val Pro Asp Lys Lys
Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe
Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser
Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro
Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val
Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg
Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp145
15053155PRTArtificial SequenceSynthetic polypeptide 53Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Gly
Leu Leu Ala Asp Leu145 150 15554156PRTArtificial SequenceSynthetic
polypeptide 54Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met
Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135
140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu145 150
15555157PRTArtificial SequenceSynthetic polypeptide 55Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Gly
Leu Leu Ala Asp Leu Leu Pro145 150 15556158PRTArtificial
SequenceSynthetic polypeptide 56Leu Gln Val Thr Val Pro Asp Lys Lys
Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe
Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser
Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro
Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val
Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg
Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu
Pro Ser145 150 15557159PRTArtificial SequenceSynthetic polypeptide
57Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly
Arg Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser Phe145 150
15558160PRTArtificial SequenceSynthetic polypeptide 58Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile
100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile
Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser
Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp
Leu Leu Pro Ser Phe Ala145 150 155 16059161PRTArtificial
SequenceSynthetic polypeptide 59Leu Gln Val Thr Val Pro Asp Lys Lys
Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe
Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser
Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro
Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val
Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg
Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala145 150 155 160Val60162PRTArtificial
SequenceSynthetic polypeptide 60Leu Gln Val Thr Val Pro Asp Lys Lys
Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe
Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser
Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro
Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val
Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg
Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala145 150 155 160Val Glu61163PRTArtificial
SequenceSynthetic polypeptide 61Leu Gln Val Thr Val Pro Asp Lys Lys
Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe
Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser
Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro
Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val
Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg
Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala145 150 155 160Val Glu Ile62166PRTArtificial
SequenceSynthetic polypeptide 62Leu Gln Val Thr Val Pro Asp Lys Lys
Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe
Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser
Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro
Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val
Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg
Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala145 150 155 160Val Glu Ile Met Pro Glu
16563165PRTArtificial SequenceSynthetic polypeptide 63Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Gly
Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145 150 155 160Val Glu Ile
Met Pro 16564164PRTArtificial SequenceSynthetic polypeptide 64Leu
Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10
15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala
20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly
Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys
Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg
Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val
Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His
Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser
Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu
Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg
Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser Gly Ala145 150 155 160Val
Glu Ile Met65167PRTArtificial SequenceSynthetic polypeptide 65Leu
Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10
15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala
20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly
Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys
Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg
Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val
Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His
Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser
Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu
Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg
Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145 150 155 160Val
Glu Ile Met Pro Glu Trp 16566168PRTArtificial SequenceSynthetic
polypeptide 66Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met
Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135
140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser Phe
Ala145 150 155 160Val Glu Ile Met Pro Glu Trp Val
16567169PRTArtificial SequenceSynthetic polypeptide 67Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Gly
Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145 150 155 160Val Glu Ile
Met Pro Glu Trp Val Phe 16568138PRTArtificial SequenceSynthetic
polypeptide 68Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 13569139PRTArtificial
SequenceSynthetic polypeptide 69Asp Lys Lys Lys Val Ala Met Leu Phe
Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln
Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg
Met Gly Glu Ser Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu
Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp
Ser Arg Arg Thr Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser
Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile
Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105
110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu
115 120 125Gly Lys Asn Glu Asp Ser Val Glu Leu Leu Val 130
13570140PRTArtificial SequenceSynthetic polypeptide 70Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu 130 135 14071141PRTArtificial SequenceSynthetic
polypeptide 71Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly 130 135
14072142PRTArtificial SequenceSynthetic polypeptide 72Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg 130 135 14073143PRTArtificial
SequenceSynthetic polypeptide 73Asp Lys Lys Lys Val Ala Met Leu Phe
Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln
Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg
Met Gly Glu Ser Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu
Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp
Ser Arg Arg Thr Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser
Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile
Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105
110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu
115 120 125Gly Lys Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg
Thr 130 135
14074144PRTArtificial SequenceSynthetic polypeptide 74Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 14075145PRTArtificial
SequenceSynthetic polypeptide 75Asp Lys Lys Lys Val Ala Met Leu Phe
Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln
Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg
Met Gly Glu Ser Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu
Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp
Ser Arg Arg Thr Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser
Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile
Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105
110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu
115 120 125Gly Lys Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg
Thr Gly 130 135 140Leu14576146PRTArtificial SequenceSynthetic
polypeptide 76Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135
140Leu Leu14577147PRTArtificial SequenceSynthetic polypeptide 77Asp
Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10
15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe
20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser
Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp
Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val
Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr
Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile
Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile
Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser
Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu
Ala14578148PRTArtificial SequenceSynthetic polypeptide 78Asp Lys
Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His
Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25
30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser
35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro
Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala
Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg
Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly
Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile
Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val
Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala
Asp14579149PRTArtificial SequenceSynthetic polypeptide 79Asp Lys
Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His
Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25
30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser
35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro
Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala
Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg
Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly
Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile
Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val
Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp
Leu14580150PRTArtificial SequenceSynthetic polypeptide 80Asp Lys
Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His
Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25
30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser
35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro
Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala
Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg
Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly
Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile
Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val
Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu
Leu145 15081151PRTArtificial SequenceSynthetic polypeptide 81Asp
Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10
15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe
20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser
Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp
Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val
Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr
Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile
Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile
Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser
Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp
Leu Leu Pro145 15082152PRTArtificial SequenceSynthetic polypeptide
82Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1
5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys
Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met
Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp
Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val
Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe
Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln
Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys
Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp
Ser Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala
Asp Leu Leu Pro Ser145 15083153PRTArtificial SequenceSynthetic
polypeptide 83Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135
140Leu Leu Ala Asp Leu Leu Pro Ser Phe145 15084154PRTArtificial
SequenceSynthetic polypeptide 84Asp Lys Lys Lys Val Ala Met Leu Phe
Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln
Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg
Met Gly Glu Ser Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu
Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp
Ser Arg Arg Thr Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser
Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile
Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105
110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu
115 120 125Gly Lys Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg
Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145
15085155PRTArtificial SequenceSynthetic polypeptide 85Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala Val145 150 15586156PRTArtificial SequenceSynthetic
polypeptide 86Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135
140Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala Val Glu145 150
15587157PRTArtificial SequenceSynthetic polypeptide 87Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala Val Glu Ile145 150 15588158PRTArtificial
SequenceSynthetic polypeptide 88Asp Lys Lys Lys Val Ala Met Leu Phe
Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln
Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg
Met Gly Glu Ser Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu
Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp
Ser Arg Arg Thr Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser
Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile
Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105
110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu
115 120 125Gly Lys Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg
Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala Val Glu
Ile Met145 150 15589159PRTArtificial SequenceSynthetic polypeptide
89Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1
5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys
Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met
Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp
Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val
Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe
Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln
Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys
Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu
Asp Ser Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu
Ala Asp Leu Leu Pro Ser Phe Ala Val Glu Ile Met Pro145 150
15590160PRTArtificial SequenceSynthetic polypeptide 90Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala Val Glu Ile Met Pro Glu145 150 155
16091161PRTArtificial SequenceSynthetic polypeptide 91Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala Val Glu Ile Met Pro Glu145 150 155
160Trp92162PRTArtificial SequenceSynthetic polypeptide 92Asp Lys
Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His
Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25
30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser
35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro
Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala
Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg
Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly
Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile
Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val
Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu
Leu Pro Ser Phe Ala Val Glu Ile Met Pro Glu145 150 155 160Trp
Val93163PRTArtificial SequenceSynthetic polypeptide 93Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala Val Glu Ile Met Pro Glu145 150 155 160Trp Val
Phe94147PRTArtificial SequenceSynthetic polypeptide 94Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu
Glu14595148PRTArtificial SequenceSynthetic polypeptide 95Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Glu
Trp14596164PRTArtificial SequenceSynthetic polypeptide 96Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr
Gly Leu Leu Ala Asp Leu Leu Pro Ser Ala Ala145 150 155 160Val Glu
Ile Met97150PRTArtificial SequenceSynthetic polypeptide 97Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Glu Trp Val
Phe145 15098151PRTArtificial SequenceSynthetic polypeptide 98Leu
Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10
15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala
20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly
Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys
Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg
Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val
Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His
Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser
Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu
Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Glu Trp
Val Phe Val145 15099152PRTArtificial SequenceSynthetic polypeptide
99Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Glu
Trp Val Phe Val Gly145 150100141PRTArtificial SequenceSynthetic
polypeptide 100Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Glu 130 135
140101142PRTArtificial SequenceSynthetic polypeptide 101Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Glu Trp 130 135 140102143PRTArtificial
SequenceSynthetic polypeptide 102Asp Lys Lys Lys Val Ala Met Leu
Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His
Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp
Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser
Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu
Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly
Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr
Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105
110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu
115 120 125Gly Lys Asn Glu Asp Ser Val Glu Leu Leu Val Leu Glu Trp
Val 130 135 140103144PRTArtificial SequenceSynthetic polypeptide
103Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1
5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys
Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met
Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp
Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val
Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe
Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln
Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys
Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp
Ser Val Glu Leu Leu Val Leu Glu Trp Val Phe 130 135
140104145PRTArtificial SequenceSynthetic polypeptide 104Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Glu Trp Val Phe 130 135
140Val145105146PRTArtificial SequenceSynthetic polypeptide 105Asp
Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10
15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe
20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser
Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp
Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val
Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr
Arg Gly Arg Glu Ile
85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp
Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr Thr Pro Asp
Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu Leu Leu Val
Leu Glu Trp Val Phe 130 135 140Val Gly1451065PRTArtificial
SequenceSynthetic polypeptide 106Gly Gly Gly Gly Ser1
510710PRTArtificial SequenceSynthetic polypeptide 107Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser1 5 10108408PRTArtificial
SequenceSynthetic polypeptide 108Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp
Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr
Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala145 150 155 160Val Glu Ile Met Gly Ser Glu Asn Leu
Tyr Phe Gln Gly Ser Gly Glu 165 170 175Pro Arg Gly Pro Thr Ile Lys
Pro Cys Pro Pro Cys Lys Cys Pro Ala 180 185 190Pro Asn Leu Leu Gly
Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile 195 200 205Lys Asp Val
Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val 210 215 220Val
Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val225 230
235 240Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu
Asp 245 250 255Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile
Gln His Gln 260 265 270Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys
Val Asn Asn Lys Asp 275 280 285Leu Pro Ala Pro Ile Glu Arg Thr Ile
Ser Lys Pro Lys Gly Ser Val 290 295 300Arg Ala Pro Gln Val Tyr Val
Leu Pro Pro Pro Glu Glu Glu Met Thr305 310 315 320Lys Lys Gln Val
Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu 325 330 335Asp Ile
Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr 340 345
350Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
355 360 365Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
Ser Tyr 370 375 380Ser Cys Ser Val Val His Glu Gly Leu His Asn His
His Thr Thr Lys385 390 395 400Ser Phe Ser Arg Thr Pro Gly Lys
405109417PRTArtificial SequenceSynthetic polypeptide 109Met Asp Arg
Val Leu Leu Arg Trp Ile Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met Val
Glu Gly Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala 20 25 30Met
Leu Phe Gln Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser 35 40
45His Gln Pro Ala Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp
50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser
Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val Val Ala Ser Lys Gln Gly
Ser Thr Val Thr 100 105 110Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala 115 120 125Asp Leu Gln Ile Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135 140Cys Ile Ile Thr Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser145 150 155 160Val Glu Leu
Leu Val Leu Gly Arg Thr Gly Leu Leu Ala Gly Ser Glu 165 170 175Asn
Leu Tyr Phe Gln Gly Ser Gly Glu Pro Arg Gly Pro Thr Ile Lys 180 185
190Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro
195 200 205Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met
Ile Ser 210 215 220Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val
Ser Glu Asp Asp225 230 235 240Pro Asp Val Gln Ile Ser Trp Phe Val
Asn Asn Val Glu Val His Thr 245 250 255Ala Gln Thr Gln Thr His Arg
Glu Asp Tyr Asn Ser Thr Leu Arg Val 260 265 270Val Ser Ala Leu Pro
Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu 275 280 285Phe Lys Cys
Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg 290 295 300Thr
Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val305 310
315 320Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu
Thr 325 330 335Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val
Glu Trp Thr 340 345 350Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn
Thr Glu Pro Val Leu 355 360 365Asp Ser Asp Gly Ser Tyr Phe Met Tyr
Ser Lys Leu Arg Val Glu Lys 370 375 380Lys Asn Trp Val Glu Arg Asn
Ser Tyr Ser Cys Ser Val Val His Glu385 390 395 400Gly Leu His Asn
His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 405 410
415Lys110397PRTArtificial SequenceSynthetic polypeptide 110Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr
Gly Leu Leu Ala Gly Ser Glu Asn Leu Tyr Phe145 150 155 160Gln Gly
Ser Gly Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro 165 170
175Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile
180 185 190Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser
Pro Ile 195 200 205Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp
Pro Asp Val Gln 210 215 220Ile Ser Trp Phe Val Asn Asn Val Glu Val
His Thr Ala Gln Thr Gln225 230 235 240Thr His Arg Glu Asp Tyr Asn
Ser Thr Leu Arg Val Val Ser Ala Leu 245 250 255Pro Ile Gln His Gln
Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys 260 265 270Val Asn Asn
Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys 275 280 285Pro
Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro 290 295
300Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val
Thr305 310 315 320Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr
Asn Asn Gly Lys 325 330 335Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro
Val Leu Asp Ser Asp Gly 340 345 350Ser Tyr Phe Met Tyr Ser Lys Leu
Arg Val Glu Lys Lys Asn Trp Val 355 360 365Glu Arg Asn Ser Tyr Ser
Cys Ser Val Val His Glu Gly Leu His Asn 370 375 380His His Thr Thr
Lys Ser Phe Ser Arg Thr Pro Gly Lys385 390 395111411PRTArtificial
SequenceSynthetic polypeptide 111Met Asp Arg Val Leu Leu Arg Trp
Ile Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met Val Glu Gly Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala 20 25 30Met Leu Phe Gln Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser 35 40 45His Gln Pro Ala Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp 50 55 60Arg Met Gly Glu
Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu65 70 75 80Ser Lys
Arg Asn Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser 85 90 95Arg
Arg Thr Val Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr 100 105
110Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
115 120 125Asp Leu Gln Ile Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr 130 135 140Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser145 150 155 160Val Glu Leu Leu Val Leu Gly Gly Ser
Glu Asn Leu Tyr Phe Gln Gly 165 170 175Ser Gly Glu Pro Arg Gly Pro
Thr Ile Lys Pro Cys Pro Pro Cys Lys 180 185 190Cys Pro Ala Pro Asn
Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro 195 200 205Pro Lys Ile
Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr 210 215 220Cys
Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser225 230
235 240Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
His 245 250 255Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala
Leu Pro Ile 260 265 270Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe
Lys Cys Lys Val Asn 275 280 285Asn Lys Asp Leu Pro Ala Pro Ile Glu
Arg Thr Ile Ser Lys Pro Lys 290 295 300Gly Ser Val Arg Ala Pro Gln
Val Tyr Val Leu Pro Pro Pro Glu Glu305 310 315 320Glu Met Thr Lys
Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe 325 330 335Met Pro
Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu 340 345
350Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr
355 360 365Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val
Glu Arg 370 375 380Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu
His Asn His His385 390 395 400Thr Thr Lys Ser Phe Ser Arg Thr Pro
Gly Lys 405 410112391PRTArtificial SequenceSynthetic polypeptide
112Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly
Gly Ser Glu Asn Leu Tyr Phe Gln Gly Ser Gly Glu Pro145 150 155
160Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro
165 170 175Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
Ile Lys 180 185 190Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr
Cys Val Val Val 195 200 205Asp Val Ser Glu Asp Asp Pro Asp Val Gln
Ile Ser Trp Phe Val Asn 210 215 220Asn Val Glu Val His Thr Ala Gln
Thr Gln Thr His Arg Glu Asp Tyr225 230 235 240Asn Ser Thr Leu Arg
Val Val Ser Ala Leu Pro Ile Gln His Gln Asp 245 250 255Trp Met Ser
Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu 260 265 270Pro
Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg 275 280
285Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys
290 295 300Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro
Glu Asp305 310 315 320Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr
Glu Leu Asn Tyr Lys 325 330 335Asn Thr Glu Pro Val Leu Asp Ser Asp
Gly Ser Tyr Phe Met Tyr Ser 340 345 350Lys Leu Arg Val Glu Lys Lys
Asn Trp Val Glu Arg Asn Ser Tyr Ser 355 360 365Cys Ser Val Val His
Glu Gly Leu His Asn His His Thr Thr Lys Ser 370 375 380Phe Ser Arg
Thr Pro Gly Lys385 39011311PRTArtificial SequenceSynthetic
polypeptide 113Gly Ser Glu Asn Leu Tyr Phe Gln Gly Ser Gly1 5
1011412PRTArtificial SequenceSynthetic polypeptide 114Ala Glu Ala
Ala Ala Lys Glu Ala Ala Ala Lys Ala1 5 10115232PRTArtificial
SequenceSynthetic polypeptide 115Glu Pro Lys Ser Ser Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala1 5 10 15Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105
110Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr 130 135 140Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser145 150 155 160Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220Ser
Leu Ser Leu Ser Pro Gly Lys225 230116396PRTArtificial
sequenceSynthetic polypeptide 116Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser
Ser Thr Arg
Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu
Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser
Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg
Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys
Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120
125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Gly Ser Leu Gly Leu Leu
130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser
Phe Ala145 150 155 160Val Glu Ile Met Glu Pro Lys Ser Ser Asp Lys
Thr His Thr Cys Pro 165 170 175Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe 180 185 190Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 195 200 205Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 210 215 220Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro225 230 235
240Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
245 250 255Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val 260 265 270Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala 275 280 285Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg 290 295 300Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly305 310 315 320Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 325 330 335Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 340 345 350Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 355 360
365Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
370 375 380Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys385 390
395117381PRTArtificial sequenceSynthetic polypeptide 117Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Gly Ser Leu Gly Leu Leu 130 135 140Val Leu Glu Trp Val Glu
Pro Lys Ser Ser Asp Lys Thr His Thr Cys145 150 155 160Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 165 170 175Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 180 185
190Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
195 200 205Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 210 215 220Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu225 230 235 240Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys 245 250 255Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 260 265 270Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 275 280 285Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 290 295 300Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln305 310
315 320Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly 325 330 335Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 340 345 350Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn 355 360 365His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 370 375 380118416PRTArtificial sequenceSynthetic
polypeptide 118Met Asp Arg Val Leu Leu Arg Trp Ile Ser Leu Phe Trp
Leu Thr Ala1 5 10 15Met Val Glu Gly Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala 20 25 30Met Leu Phe Gln Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser 35 40 45His Gln Pro Ala Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp 50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp
Asp Pro Tyr Leu Asp Cys Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr 100 105 110Leu Gly Asp Phe
Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala 115 120 125Asp Leu
Gln Ile Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135
140Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp
Ser145 150 155 160Val Glu Leu Leu Val Leu Gly Arg Thr Gly Leu Leu
Ala Asp Leu Leu 165 170 175Pro Ser Phe Ala Val Glu Ile Met Glu Pro
Lys Ser Ser Asp Lys Thr 180 185 190His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser 195 200 205Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 210 215 220Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro225 230 235 240Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 245 250
255Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
260 265 270Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr 275 280 285Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 290 295 300Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu305 310 315 320Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys 325 330 335Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 340 345 350Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 355 360 365Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 370 375
380Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala385 390 395 400Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 405 410 415119400PRTArtificial sequenceSynthetic
polypeptide 119Met Asp Arg Val Leu Leu Arg Trp Ile Ser Leu Phe Trp
Leu Thr Ala1 5 10 15Met Val Glu Gly Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala 20 25 30Met Leu Phe Gln Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser 35 40 45His Gln Pro Ala Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp 50 55 60Arg Met Gly Glu Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu65 70 75 80Ser Lys Arg Asn Leu Glu Trp
Asp Pro Tyr Leu Asp Cys Leu Asp Ser 85 90 95Arg Arg Thr Val Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr 100 105 110Leu Gly Asp Phe
Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala 115 120 125Asp Leu
Gln Ile Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr 130 135
140Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Gly
Ser145 150 155 160Leu Gly Leu Leu Val Leu Glu Trp Glu Pro Lys Ser
Ser Asp Lys Thr 165 170 175His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser 180 185 190Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg 195 200 205Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro 210 215 220Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala225 230 235 240Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 245 250
255Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
260 265 270Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr 275 280 285Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 290 295 300Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys305 310 315 320Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser 325 330 335Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 340 345 350Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 355 360 365Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 370 375
380Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys385 390 395 400120379PRTArtificial sequenceSynthetic polypeptide
120Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro145 150 155
160Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
165 170 175Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr 180 185 190Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn 195 200 205Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg 210 215 220Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val225 230 235 240Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 245 250 255Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 260 265 270Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 275 280
285Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
290 295 300Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu305 310 315 320Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe 325 330 335Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly 340 345 350Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr 355 360 365Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 370 375121336PRTArtificial
sequenceSynthetic polypeptide 121Cys His Phe Ser Thr Ser Ser His
Gln Pro Ala Val Val Gln Trp Lys1 5 10 15Phe Lys Ser Tyr Cys Gln Asp
Arg Met Gly Glu Ser Leu Gly Met Ser 20 25 30Ser Thr Arg Ala Gln Ser
Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro 35 40 45Tyr Leu Asp Cys Leu
Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser 50 55 60Lys Gln Gly Ser
Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly Arg Glu65 70 75 80Ile Thr
Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp 85 90 95Gly
Asp Ser Gly Leu Tyr Tyr Cys Glu Pro Lys Ser Ser Asp Lys Thr 100 105
110His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
115 120 125Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 130 135 140Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro145 150 155 160Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 165 170 175Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val 180 185 190Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 195 200 205Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 210 215 220Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu225 230
235 240Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys 245 250 255Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser 260 265 270Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp 275 280 285Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 290 295 300Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala305 310 315 320Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
335122396PRTArtificial sequenceSynthetic polypeptide 122Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Gly
Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145 150 155 160Val Glu Ile
Met Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 165 170 175Pro
Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe 180 185 190Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 195 200 205Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 210 215 220Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro225 230
235 240Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr 245 250 255Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val 260 265 270Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala 275 280 285Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg 290 295 300Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly305 310 315 320Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 325 330 335Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 340 345
350Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
355 360 365Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His 370 375 380Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys385 390 395123381PRTArtificial sequenceSynthetic polypeptide
123Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Glu
Trp Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys145 150 155
160Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
165 170 175Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu 180 185 190Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys 195 200 205Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys 210 215 220Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu225 230 235 240Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 245 250 255Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 260 265 270Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 275 280
285Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
290 295 300Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln305 310 315 320Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly 325 330 335Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln 340 345 350Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn 355 360 365His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380124416PRTArtificial
sequenceSynthetic polypeptide 124Met Asp Arg Val Leu Leu Arg Trp
Ile Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met Val Glu Gly Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala 20 25 30Met Leu Phe Gln Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser 35 40 45His Gln Pro Ala Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp 50 55 60Arg Met Gly Glu
Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu65 70 75 80Ser Lys
Arg Asn Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser 85 90 95Arg
Arg Thr Val Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr 100 105
110Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
115 120 125Asp Leu Gln Ile Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr 130 135 140Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Gly Ser145 150 155 160Leu Gly Leu Leu Val Leu Gly Arg Thr
Gly Leu Leu Ala Asp Leu Leu 165 170 175Pro Ser Phe Ala Val Glu Ile
Met Glu Pro Lys Ser Ser Asp Lys Thr 180 185 190His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 195 200 205Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 210 215 220Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro225 230
235 240Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 245 250 255Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val 260 265 270Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr 275 280 285Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr 290 295 300Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu305 310 315 320Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 325 330 335Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 340 345
350Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
355 360 365Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser 370 375 380Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala385 390 395 400Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 405 410 415125401PRTArtificial
sequenceSynthetic polypeptide 125Met Asp Arg Val Leu Leu Arg Trp
Ile Ser Leu Phe Trp Leu Thr Ala1 5 10 15Met Val Glu Gly Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala 20 25 30Met Leu Phe Gln Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser 35 40 45His Gln Pro Ala Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp 50 55 60Arg Met Gly Glu
Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu65 70 75 80Ser Lys
Arg Asn Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser 85 90 95Arg
Arg Thr Val Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr 100 105
110Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
115 120 125Asp Leu Gln Ile Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr 130 135 140Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser145 150 155 160Val Glu Leu Leu Val Leu Glu Trp Val
Glu Pro Lys Ser Ser Asp Lys 165 170 175Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro 180 185 190Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 195 200 205Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 210 215 220Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn225 230
235 240Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val 245 250 255Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu 260 265 270Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 275 280 285Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 290 295 300Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr305 310 315 320Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 325 330 335Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 340 345
350Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
355 360 365Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 370 375 380Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly385 390 395 400Lys126379PRTArtificial
sequenceSynthetic polypeptide 126Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp
Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr
Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Glu Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro145 150 155 160Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro 165 170 175Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr 180 185 190Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 195 200 205Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 210 215 220Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val225 230
235 240Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser 245 250 255Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys 260 265 270Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp 275 280 285Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe 290 295 300Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu305 310 315 320Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 325 330 335Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 340 345
350Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
355 360 365Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370
375127376PRTArtificial sequenceSynthetic polypeptide 127Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala145 150 155 160Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 165 170 175Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 180 185
190Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
195 200 205Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln 210 215 220Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln225 230 235 240Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala 245 250 255Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro 260 265 270Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 275 280 285Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 290 295 300Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr305 310
315 320Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr 325 330 335Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe 340 345 350Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 355 360 365Ser Leu Ser Leu Ser Pro Gly Lys 370
375128377PRTArtificial sequenceSynthetic polypeptide 128Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Glu Pro Lys Ser Ser
Asp Lys Thr His Thr Cys Pro Pro Cys Pro145 150 155 160Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185
190Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
195 200 205Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 210 215 220Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His225 230 235 240Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys 245 250 255Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln 260
265 270Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu 275 280 285Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn305 310 315 320Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 340 345 350Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 355 360 365Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375129378PRTArtificial
sequenceSynthetic polypeptide 129Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp
Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr
Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Cys Pro Pro Cys145 150 155 160Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 165 170 175Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 180 185 190Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 195 200 205Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 210 215 220Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu225 230
235 240His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 245 250 255Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 260 265 270Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu 275 280 285Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 290 295 300Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn305 310 315 320Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 325 330 335Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 340 345
350Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
355 360 365Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370
375130396PRTArtificial sequenceSynthetic polypeptide 130Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Gly
Leu Leu Ala Asp Leu Leu Pro Ser Gly Gly145 150 155 160Val Glu Ile
Met Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 165 170 175Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 180 185
190Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
195 200 205Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 210 215 220Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro225 230 235 240Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr 245 250 255Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val 260 265 270Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 275 280 285Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 290 295 300Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly305 310
315 320Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro 325 330 335Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser 340 345 350Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 355 360 365Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His 370 375 380Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys385 390 395131380PRTArtificial sequenceSynthetic
polypeptide 131Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met
Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135
140Val Leu Gly Arg Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
Pro145 150 155 160Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe 165 170 175Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val 180 185 190Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe 195 200 205Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr225 230 235 240Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250
255Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
260 265 270Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 275 280 285Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly 290 295 300Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro305 310 315 320Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
380132381PRTArtificial sequenceSynthetic polypeptide 132Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Glu
Pro Lys Ser Ser Asp Lys Thr His Thr Cys145 150 155 160Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 165 170 175Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 180 185
190Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
195 200 205Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 210 215 220Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu225 230 235 240Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys 245 250 255Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 260 265 270Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 275 280 285Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 290 295 300Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln305 310
315 320Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly 325 330 335Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 340 345 350Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn 355 360 365His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 370 375 380133382PRTArtificial sequenceSynthetic
polypeptide 133Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met
Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135
140Val Leu Gly Arg Thr Gly Glu Pro Lys Ser Ser Asp Lys Thr His
Thr145 150 155 160Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe 165 170 175Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro 180 185 190Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val 195 200 205Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr 210 215 220Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val225 230 235 240Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 245 250
255Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
260 265 270Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 275 280 285Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 290 295 300Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly305 310 315 320Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp 325 330 335Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 340 345 350Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 355 360 365Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
380134383PRTArtificial sequenceSynthetic polypeptide 134Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Gly
Leu Glu Pro Lys Ser Ser Asp Lys Thr His145 150 155 160Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 165 170 175Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 180 185
190Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
195 200 205Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 210 215 220Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser225 230 235 240Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys 245 250 255Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 260 265 270Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 275 280 285Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 290 295 300Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn305 310
315 320Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser 325 330 335Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg 340 345 350Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 355 360 365His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 370 375 380135384PRTArtificial
sequenceSynthetic polypeptide 135Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys
His
Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp
Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr
Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Glu Pro Lys Ser
Ser Asp Lys Thr145 150 155 160His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser 165 170 175Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 180 185 190Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 195 200 205Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 210 215 220Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val225 230
235 240Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr 245 250 255Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr 260 265 270Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu 275 280 285Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys 290 295 300Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser305 310 315 320Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 325 330 335Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 340 345
350Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
355 360 365Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 370 375 380136385PRTArtificial sequenceSynthetic
polypeptide 136Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met
Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135
140Val Leu Gly Arg Thr Gly Leu Leu Ala Glu Pro Lys Ser Ser Asp
Lys145 150 155 160Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 165 170 175Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser 180 185 190Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp 195 200 205Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn 210 215 220Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val225 230 235 240Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 245 250
255Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
260 265 270Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 275 280 285Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr 290 295 300Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu305 310 315 320Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 325 330 335Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 340 345 350Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 355 360 365Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 370 375
380Lys385137386PRTArtificial sequenceSynthetic polypeptide 137Leu
Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10
15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala
20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly
Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys
Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg
Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val
Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His
Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser
Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu
Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg
Thr Gly Leu Leu Ala Asp Glu Pro Lys Ser Ser Asp145 150 155 160Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 165 170
175Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
180 185 190Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu 195 200 205Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 210 215 220Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg225 230 235 240Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys 245 250 255Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 260 265 270Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 275 280 285Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 290 295
300Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp305 310 315 320Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val 325 330 335Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp 340 345 350Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His 355 360 365Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 370 375 380Gly
Lys385138387PRTArtificial sequenceSynthetic polypeptide 138Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr
Gly Leu Leu Ala Asp Leu Glu Pro Lys Ser Ser145 150 155 160Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 165 170
175Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
180 185 190Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 195 200 205Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 210 215 220His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr225 230 235 240Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 245 250 255Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 260 265 270Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 275 280 285Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 290 295
300Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu305 310 315 320Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 325 330 335Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 340 345 350Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 355 360 365His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 370 375 380Pro Gly
Lys385139388PRTArtificial sequenceSynthetic polypeptide 139Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr
Gly Leu Leu Ala Asp Leu Leu Glu Pro Lys Ser145 150 155 160Ser Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 165 170
175Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
180 185 190Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 195 200 205His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 210 215 220Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr225 230 235 240Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn 245 250 255Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 260 265 270Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 275 280 285Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 290 295
300Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val305 310 315 320Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro 325 330 335Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr 340 345 350Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val 355 360 365Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 370 375 380Ser Pro Gly
Lys385140389PRTArtificial sequenceSynthetic polypeptide 140Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr
Gly Leu Leu Ala Asp Leu Leu Pro Glu Pro Lys145 150 155 160Ser Ser
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 165 170
175Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
180 185 190Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val 195 200 205Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val 210 215 220Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser225 230 235 240Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 245 250 255Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 260 265 270Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 275 280 285Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 290 295
300Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala305 310 315 320Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 325 330 335Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu 340 345 350Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser 355 360 365Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 370 375 380Leu Ser Pro Gly
Lys385141390PRTArtificial sequenceSynthetic polypeptide 141Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly
Arg Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser Glu Pro145 150 155
160Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
165 170 175Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp 180 185 190Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp 195 200 205Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly 210 215 220Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn225 230 235 240Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp 245 250 255Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 260 265 270Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 275 280
285Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
290 295 300Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile305 310 315 320Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr 325 330 335Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys 340 345 350Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys 355 360 365Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 370 375 380Ser Leu Ser
Pro Gly Lys385 390142391PRTArtificial sequenceSynthetic polypeptide
142Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly
Arg Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser Phe Glu145 150 155
160Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
165 170 175Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 180 185 190Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 195 200 205Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp 210 215 220Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr225 230 235 240Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 245 250 255Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 260 265 270Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 275 280
285Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
290 295 300Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp305 310 315 320Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 325 330 335Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 340 345 350Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser 355 360 365Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 370 375 380Leu Ser Leu
Ser Pro Gly Lys385 390143392PRTArtificial sequenceSynthetic
polypeptide 143Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met
Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135
140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser Phe
Ala145 150 155 160Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala 165 170 175Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro 180 185 190Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val 195 200 205Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 210 215 220Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln225 230 235 240Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 245 250
255Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
260 265 270Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro 275 280 285Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr 290 295 300Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser305 310 315 320Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr 325 330 335Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 340 345 350Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 355 360 365Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 370 375
380Ser Leu Ser Leu Ser Pro Gly Lys385 390144393PRTArtificial
sequenceSynthetic polypeptide 144Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp
Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr
Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala145 150 155 160Val Glu Pro Lys Ser Ser Asp Lys Thr
His Thr Cys Pro Pro Cys Pro 165 170 175Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys 180 185 190Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 195 200 205Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 210 215 220Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu225 230
235 240Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His 245 250 255Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys 260 265 270Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 275 280 285Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu 290 295 300Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro305 310 315 320Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 325 330 335Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 340 345
350Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 370 375 380Lys Ser Leu Ser Leu Ser Pro Gly Lys385
390145394PRTArtificial sequenceSynthetic polypeptide 145Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Gly
Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145 150 155 160Val Glu Glu
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 165 170 175Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 180 185
190Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
195 200 205Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp 210 215 220Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu225 230 235 240Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 245 250 255His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 260 265 270Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 275 280 285Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 290 295 300Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr305 310
315 320Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn 325 330 335Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe 340 345 350Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn 355 360 365Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr 370 375 380Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys385 390146395PRTArtificial sequenceSynthetic polypeptide
146Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly
Arg Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145 150 155
160Val Glu Ile Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
165 170 175Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro 180 185 190Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr 195 200 205Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn 210 215 220Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg225 230 235 240Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 245 250 255Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 260 265 270Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 275 280
285Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
290 295 300Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe305 310 315 320Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu 325 330 335Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe 340 345 350Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly 355 360 365Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr 370 375 380Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys385 390 395147398PRTArtificial
sequenceSynthetic polypeptide 147Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp
Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr
Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala145 150 155 160Val Glu Ile Met Pro Glu Glu Pro Lys
Ser Ser Asp Lys Thr His Thr 165 170 175Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe 180 185 190Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 195 200 205Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 210 215 220Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr225
230 235 240Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val 245 250 255Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys 260 265 270Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser 275 280 285Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro 290 295 300Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val305 310 315 320Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 325 330 335Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 340 345
350Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
355 360 365Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His 370 375 380Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys385 390 395148397PRTArtificial sequenceSynthetic polypeptide
148Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly
Arg Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145 150 155
160Val Glu Ile Met Pro Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
165 170 175Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu 180 185 190Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu 195 200 205Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys 210 215 220Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys225 230 235 240Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 245 250 255Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 260 265 270Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 275 280
285Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
290 295 300Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys305 310 315 320Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln 325 330 335Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly 340 345 350Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln 355 360 365Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn 370 375 380His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys385 390
395149396PRTArtificial sequenceSynthetic polypeptide 149Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Gly
Leu Leu Ala Asp Leu Leu Pro Ser Gly Ala145 150 155 160Val Glu Ile
Met Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 165 170 175Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 180 185
190Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
195 200 205Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 210 215 220Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro225 230 235 240Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr 245 250 255Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val 260 265 270Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 275 280 285Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 290 295 300Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly305 310
315 320Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro 325 330 335Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser 340 345 350Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 355 360 365Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His 370 375 380Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys385 390 395150399PRTArtificial sequenceSynthetic
polypeptide 150Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met
Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln
Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile
Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met
Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro
Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135
140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu Pro Ser Phe
Ala145 150 155 160Val Glu Ile Met Pro Glu Trp Glu Pro Lys Ser Ser
Asp Lys Thr His 165 170 175Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val 180 185 190Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 195 200 205Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu 210 215 220Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys225 230 235 240Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 245 250
255Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
260 265 270Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile 275 280 285Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 290 295 300Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu305 310 315 320Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn 325 330 335Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 340 345 350Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 355 360 365Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 370 375
380His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys385
390 395151400PRTArtificial sequenceSynthetic polypeptide 151Leu Gln
Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro
Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25
30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu
35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg
Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg
Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr
Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp
Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly
Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly
Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr
Gly Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145 150 155 160Val Glu
Ile Met Pro Glu Trp Val Glu Pro Lys Ser Ser Asp Lys Thr 165 170
175His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
180 185 190Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 195 200 205Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro 210 215 220Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala225 230 235 240Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val 245 250 255Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 260 265 270Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 275 280 285Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 290 295
300Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys305 310 315 320Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 325 330 335Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp 340 345 350Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser 355 360 365Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala 370 375 380Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys385 390 395
400152401PRTArtificial sequenceSynthetic polypeptide 152Leu Gln Val
Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr
Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val
Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40
45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala
Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Gly Arg Thr Gly
Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala145 150 155 160Val Glu Ile
Met Pro Glu Trp Val Phe Glu Pro Lys Ser Ser Asp Lys 165 170 175Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 180 185
190Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
195 200 205Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 210 215 220Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn225 230 235 240Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 245 250 255Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu 260 265 270Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 275 280 285Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 290 295 300Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr305 310
315 320Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu 325 330 335Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu 340 345 350Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 355 360 365Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 370 375 380Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly385 390 395
400Lys153370PRTArtificial sequenceSynthetic polypeptide 153Asp Lys
Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His
Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25
30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser
35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro
Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala
Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg
Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly
Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile
Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val
Glu Leu Leu Glu Pro Lys Ser Ser Asp 130 135 140Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly145 150 155 160Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 165 170
175Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
180 185 190Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 195 200 205Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg 210 215 220Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys225 230 235 240Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu 245 250 255Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 260 265 270Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu 275 280 285Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp 290 295 300Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val305 310 315 320Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 325 330 335Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 340 345
350Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
355 360 365Gly Lys 370154371PRTArtificial sequenceSynthetic
polypeptide 154Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Glu Pro Lys Ser Ser 130 135
140Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly145 150 155 160Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 165 170 175Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 180 185 190Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 195 200 205His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 210 215 220Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly225 230 235 240Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 245 250
255Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
260 265 270Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 275 280 285Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 290 295 300Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro305 310 315 320Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 325 330 335Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 340 345 350His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 355 360 365Pro
Gly Lys 370155372PRTArtificial sequenceSynthetic polypeptide 155Asp
Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10
15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe
20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser
Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp
Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val
Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr
Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile
Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile
Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser
Val Glu Leu Leu Val Leu Glu Pro Lys Ser 130 135 140Ser Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu145 150 155 160Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 165 170
175Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
180 185 190His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 195 200 205Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 210 215 220Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn225 230 235 240Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 245 250 255Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 260 265 270Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 275 280 285Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 290 295
300Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro305 310 315 320Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 325 330 335Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 340 345 350Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 355 360 365Ser Pro Gly Lys
370156373PRTArtificial sequenceSynthetic polypeptide 156Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Glu Pro Lys 130 135 140Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu145 150 155 160Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 165 170 175Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 180 185
190Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
195 200 205Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser 210 215 220Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu225 230 235 240Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala 245 250 255Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro 260 265 270Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 275 280 285Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 290 295 300Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr305 310
315 320Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu 325 330 335Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser 340 345 350Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser 355 360 365Leu Ser Pro Gly Lys
370157374PRTArtificial sequenceSynthetic polypeptide 157Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Glu Pro 130 135 140Lys Ser Ser Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu145 150 155 160Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 165 170 175Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 180 185
190Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
195 200 205Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn 210 215 220Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp225 230 235 240Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro 245 250 255Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu 260 265 270Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 275 280 285Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 290 295 300Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr305 310
315 320Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 325 330 335Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 340 345 350Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu 355 360 365Ser Leu Ser Pro Gly Lys
370158375PRTArtificial sequenceSynthetic polypeptide 158Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Glu 130 135 140Pro Lys Ser Ser Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro145 150 155 160Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 165 170 175Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 180 185
190Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
195 200 205Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr 210 215 220Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp225 230 235 240Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu 245 250 255Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg 260 265 270Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 275 280 285Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 290 295 300Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys305 310
315 320Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser 325 330 335Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser 340 345 350Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser 355 360 365Leu Ser Leu Ser Pro Gly Lys 370
375159376PRTArtificial sequenceSynthetic polypeptide 159Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala145 150 155 160Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 165 170 175Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 180 185
190Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
195 200 205Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln 210 215 220Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln225 230 235 240Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala 245 250 255Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro 260 265 270Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 275 280 285Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 290 295 300Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr305 310
315 320Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr 325 330 335Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe 340 345 350Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 355 360 365Ser Leu Ser Leu Ser Pro Gly Lys 370
375160377PRTArtificial sequenceSynthetic polypeptide 160Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90
95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp Gly
100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp
Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu Leu Leu Val Leu
Gly Arg Thr Gly 130 135 140Leu Glu Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro145 150 155 160Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 195 200 205Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215
220Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His225 230 235 240Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 245 250 255Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln 260 265 270Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu 275 280 285Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn305 310 315 320Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330
335Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
340 345 350Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 355 360 365Lys Ser Leu Ser Leu Ser Pro Gly Lys 370
375161378PRTArtificial sequenceSynthetic polypeptide 161Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Glu Pro Lys Ser
Ser Asp Lys Thr His Thr Cys Pro Pro Cys145 150 155 160Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 165 170 175Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 180 185
190Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
195 200 205Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 210 215 220Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu225 230 235 240His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 245 250 255Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 260 265 270Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 275 280 285Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 290 295 300Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn305 310
315 320Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 325 330 335Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 340 345 350Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 355 360 365Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 370 375162379PRTArtificial sequenceSynthetic polypeptide 162Asp
Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10
15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe
20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser
Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp
Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val
Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr
Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile
Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile
Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser
Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Glu
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro145 150 155 160Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 165 170
175Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
180 185 190Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn 195 200 205Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg 210 215 220Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val225 230 235 240Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser 245 250 255Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 260 265 270Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 275 280 285Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 290 295
300Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu305 310 315 320Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe 325 330 335Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly 340 345 350Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr 355 360 365Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 370 375163380PRTArtificial sequenceSynthetic
polypeptide 163Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135
140Leu Leu Ala Asp Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
Pro145 150 155 160Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe 165 170 175Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val 180 185 190Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe 195 200 205Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr225 230 235 240Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250
255Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
260 265 270Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 275 280 285Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly 290 295 300Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro305 310 315 320Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
380164381PRTArtificial sequenceSynthetic polypeptide 164Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu Glu
Pro Lys Ser Ser Asp Lys Thr His Thr Cys145 150 155 160Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 165 170 175Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 180 185
190Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
195 200 205Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 210 215 220Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu225 230 235 240Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys 245 250 255Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 260 265 270Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 275 280 285Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 290 295 300Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln305 310
315 320Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly 325 330 335Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 340 345 350Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn 355 360 365His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 370 375 380165382PRTArtificial sequenceSynthetic
polypeptide 165Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135
140Leu Leu Ala Asp Leu Leu Glu Pro Lys Ser Ser Asp Lys Thr His
Thr145 150 155 160Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe 165 170 175Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro 180 185 190Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val 195 200 205Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr 210 215 220Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val225 230 235 240Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 245 250
255Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
260 265 270Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 275 280 285Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 290 295 300Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly305 310 315 320Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp 325 330 335Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 340 345 350Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 355 360 365Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
380166383PRTArtificial sequenceSynthetic polypeptide 166Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu
Pro Glu Pro Lys Ser Ser Asp Lys Thr His145 150 155 160Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 165 170 175Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 180 185
190Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
195 200 205Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 210 215 220Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser225 230 235 240Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys 245
250 255Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile 260 265 270Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro 275 280 285Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu 290 295 300Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn305 310 315 320Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 325 330 335Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 340 345 350Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 355 360
365His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370
375 380167384PRTArtificial sequenceSynthetic polypeptide 167Asp Lys
Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His
Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25
30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser
35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro
Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala
Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg
Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly
Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile
Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val
Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu
Leu Pro Ser Glu Pro Lys Ser Ser Asp Lys Thr145 150 155 160His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 165 170
175Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
180 185 190Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro 195 200 205Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 210 215 220Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val225 230 235 240Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr 245 250 255Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 260 265 270Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 275 280 285Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 290 295
300Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser305 310 315 320Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp 325 330 335Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser 340 345 350Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala 355 360 365Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
380168385PRTArtificial sequenceSynthetic polypeptide 168Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu
Pro Ser Phe Glu Pro Lys Ser Ser Asp Lys145 150 155 160Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 165 170 175Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 180 185
190Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
195 200 205Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 210 215 220Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val225 230 235 240Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu 245 250 255Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys 260 265 270Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 275 280 285Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 290 295 300Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu305 310
315 320Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu 325 330 335Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys 340 345 350Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu 355 360 365Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 370 375 380Lys385169386PRTArtificial
sequenceSynthetic polypeptide 169Asp Lys Lys Lys Val Ala Met Leu
Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His
Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp
Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser
Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu
Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly
Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr
Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105
110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu
115 120 125Gly Lys Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg
Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala Glu Pro
Lys Ser Ser Asp145 150 155 160Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly 165 170 175Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 180 185 190Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 195 200 205Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 210 215 220Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg225 230
235 240Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys 245 250 255Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu 260 265 270Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 275 280 285Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu 290 295 300Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp305 310 315 320Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 325 330 335Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 340 345
350Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
355 360 365Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 370 375 380Gly Lys385170387PRTArtificial sequenceSynthetic
polypeptide 170Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135
140Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala Val Glu Pro Lys Ser
Ser145 150 155 160Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly 165 170 175Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met 180 185 190Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 195 200 205Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val 210 215 220His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr225 230 235 240Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 245 250
255Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
260 265 270Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 275 280 285Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 290 295 300Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu305 310 315 320Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 325 330 335Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 340 345 350Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 355 360 365His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 370 375
380Pro Gly Lys385171388PRTArtificial sequenceSynthetic polypeptide
171Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1
5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys
Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met
Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp
Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val
Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe
Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln
Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys
Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp
Ser Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala
Asp Leu Leu Pro Ser Phe Ala Val Glu Glu Pro Lys Ser145 150 155
160Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
165 170 175Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu 180 185 190Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser 195 200 205His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu 210 215 220Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr225 230 235 240Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 245 250 255Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 260 265 270Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 275 280
285Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
290 295 300Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val305 310 315 320Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro 325 330 335Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr 340 345 350Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val 355 360 365Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 370 375 380Ser Pro Gly
Lys385172389PRTArtificial sequenceSynthetic polypeptide 172Asp Lys
Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His
Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25
30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser
35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro
Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala
Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg
Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly
Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile
Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val
Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu
Leu Pro Ser Phe Ala Val Glu Ile Glu Pro Lys145 150 155 160Ser Ser
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 165 170
175Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
180 185 190Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val 195 200 205Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val 210 215 220Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser225 230 235 240Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 245 250 255Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 260 265 270Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 275 280 285Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 290 295
300Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala305 310 315 320Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 325 330 335Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu 340 345 350Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser 355 360 365Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 370
375 380Leu Ser Pro Gly Lys385173390PRTArtificial sequenceSynthetic
polypeptide 173Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135
140Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala Val Glu Ile Met Glu
Pro145 150 155 160Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu 165 170 175Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp 180 185 190Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp 195 200 205Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 210 215 220Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn225 230 235 240Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 245 250
255Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
260 265 270Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 275 280 285Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn 290 295 300Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile305 310 315 320Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr 325 330 335Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 340 345 350Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 355 360 365Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 370 375
380Ser Leu Ser Pro Gly Lys385 390174391PRTArtificial
sequenceSynthetic polypeptide 174Asp Lys Lys Lys Val Ala Met Leu
Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His
Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp
Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser
Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu
Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly
Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr
Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105
110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu
115 120 125Gly Lys Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg
Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala Val Glu
Ile Met Pro Glu145 150 155 160Pro Lys Ser Ser Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro 165 170 175Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 180 185 190Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 195 200 205Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 210 215 220Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr225 230
235 240Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 245 250 255Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 260 265 270Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 275 280 285Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys 290 295 300Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp305 310 315 320Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 325 330 335Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 340 345
350Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
355 360 365Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 370 375 380Leu Ser Leu Ser Pro Gly Lys385
390175392PRTArtificial sequenceSynthetic polypeptide 175Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala Val Glu Ile Met Pro Glu145 150 155 160Glu Pro Lys
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 165 170 175Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 180 185
190Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
195 200 205Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val 210 215 220Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln225 230 235 240Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln 245 250 255Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala 260 265 270Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 275 280 285Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 290 295 300Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser305 310
315 320Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr 325 330 335Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr 340 345 350Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe 355 360 365Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys 370 375 380Ser Leu Ser Leu Ser Pro Gly
Lys385 390176393PRTArtificial sequenceSynthetic polypeptide 176Asp
Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10
15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe
20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser
Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp
Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val
Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr
Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile
Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile
Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser
Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp
Leu Leu Pro Ser Phe Ala Val Glu Ile Met Pro Glu145 150 155 160Trp
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 165 170
175Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val 195 200 205Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr 210 215 220Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu225 230 235 240Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His 245 250 255Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 260 265 270Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 275 280 285Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 290 295
300Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro305 310 315 320Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn 325 330 335Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 340 345 350Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val 355 360 365Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln 370 375 380Lys Ser Leu Ser
Leu Ser Pro Gly Lys385 390177394PRTArtificial sequenceSynthetic
polypeptide 177Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Gly Arg Thr Gly 130 135
140Leu Leu Ala Asp Leu Leu Pro Ser Phe Ala Val Glu Ile Met Pro
Glu145 150 155 160Trp Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Cys Pro Pro Cys 165 170 175Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 180 185 190Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 195 200 205Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 210 215 220Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu225 230 235 240Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 245 250
255His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
260 265 270Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 275 280 285Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu 290 295 300Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr305 310 315 320Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 325 330 335Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 340 345 350Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 355 360 365Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 370 375
380Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys385
390178395PRTArtificial sequenceSynthetic polypeptide 178Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Gly Arg Thr Gly 130 135 140Leu Leu Ala Asp Leu Leu
Pro Ser Phe Ala Val Glu Ile Met Pro Glu145 150 155 160Trp Val Phe
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 165 170 175Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 180 185
190Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
195 200 205Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn 210 215 220Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg225 230 235 240Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val 245 250 255Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser 260 265 270Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 275 280 285Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 290 295 300Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe305 310
315 320Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 325 330 335Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe 340 345 350Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly 355 360 365Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr 370 375 380Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys385 390 395179379PRTArtificial sequenceSynthetic
polypeptide 179Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met
Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser
His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln
Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln
Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys
Leu Asp Ser Arg Arg Thr Val65 70
75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp
Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu
Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr
Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu
Asp Ser Val Glu Leu Leu 130 135 140Val Leu Glu Glu Pro Lys Ser Ser
Asp Lys Thr His Thr Cys Pro Pro145 150 155 160Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 165 170 175Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 180 185 190Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 195 200
205Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
210 215 220Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val225 230 235 240Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser 245 250 255Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys 260 265 270Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp 275 280 285Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 290 295 300Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu305 310 315
320Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
325 330 335Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly 340 345 350Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr 355 360 365Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 370 375180380PRTArtificial sequenceSynthetic polypeptide 180Leu
Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1 5 10
15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro Ala
20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met Gly
Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser Lys
Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser Arg
Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr Val
Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val His
Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp Ser
Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu Glu
Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Glu Trp
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro145 150 155 160Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170
175Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
180 185 190Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 195 200 205Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 210 215 220Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr225 230 235 240Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295
300Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro305 310 315 320Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser 325 330 335Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln 340 345 350Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His 355 360 365Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 370 375 380181396PRTArtificial
sequenceSynthetic polypeptide 181Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp
Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr
Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Gly Arg Thr Gly Leu Leu Ala Asp Leu Leu
Pro Ser Ala Ala145 150 155 160Val Glu Ile Met Glu Pro Lys Ser Ser
Asp Lys Thr His Thr Cys Pro 165 170 175Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe 180 185 190Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 195 200 205Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 210 215 220Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro225 230
235 240Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr 245 250 255Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val 260 265 270Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala 275 280 285Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg 290 295 300Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly305 310 315 320Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 325 330 335Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 340 345
350Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
355 360 365Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His 370 375 380Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys385 390 395182382PRTArtificial sequenceSynthetic polypeptide
182Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Glu
Trp Val Phe Glu Pro Lys Ser Ser Asp Lys Thr His Thr145 150 155
160Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
165 170 175Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro 180 185 190Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val 195 200 205Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr 210 215 220Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val225 230 235 240Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 245 250 255Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 260 265 270Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 275 280
285Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
290 295 300Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly305 310 315 320Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp 325 330 335Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp 340 345 350Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 355 360 365Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380183383PRTArtificial
sequenceSynthetic polypeptide 183Leu Gln Val Thr Val Pro Asp Lys
Lys Lys Val Ala Met Leu Phe Gln1 5 10 15Pro Thr Val Leu Arg Cys His
Phe Ser Thr Ser Ser His Gln Pro Ala 20 25 30Val Val Gln Trp Lys Phe
Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu 35 40 45Ser Leu Gly Met Ser
Ser Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn 50 55 60Leu Glu Trp Asp
Pro Tyr Leu Asp Cys Leu Asp Ser Arg Arg Thr Val65 70 75 80Arg Val
Val Ala Ser Lys Gln Gly Ser Thr Val Thr Leu Gly Asp Phe 85 90 95Tyr
Arg Gly Arg Glu Ile Thr Ile Val His Asp Ala Asp Leu Gln Ile 100 105
110Gly Lys Leu Met Trp Gly Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
115 120 125Thr Pro Asp Asp Leu Glu Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu 130 135 140Val Leu Glu Trp Val Phe Val Glu Pro Lys Ser Ser
Asp Lys Thr His145 150 155 160Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val 165 170 175Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 180 185 190Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu 195 200 205Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 210 215 220Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser225 230
235 240Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys 245 250 255Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile 260 265 270Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro 275 280 285Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu 290 295 300Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn305 310 315 320Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 325 330 335Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 340 345
350Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
355 360 365His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 370 375 380184384PRTArtificial sequenceSynthetic polypeptide
184Leu Gln Val Thr Val Pro Asp Lys Lys Lys Val Ala Met Leu Phe Gln1
5 10 15Pro Thr Val Leu Arg Cys His Phe Ser Thr Ser Ser His Gln Pro
Ala 20 25 30Val Val Gln Trp Lys Phe Lys Ser Tyr Cys Gln Asp Arg Met
Gly Glu 35 40 45Ser Leu Gly Met Ser Ser Thr Arg Ala Gln Ser Leu Ser
Lys Arg Asn 50 55 60Leu Glu Trp Asp Pro Tyr Leu Asp Cys Leu Asp Ser
Arg Arg Thr Val65 70 75 80Arg Val Val Ala Ser Lys Gln Gly Ser Thr
Val Thr Leu Gly Asp Phe 85 90 95Tyr Arg Gly Arg Glu Ile Thr Ile Val
His Asp Ala Asp Leu Gln Ile 100 105 110Gly Lys Leu Met Trp Gly Asp
Ser Gly Leu Tyr Tyr Cys Ile Ile Thr 115 120 125Thr Pro Asp Asp Leu
Glu Gly Lys Asn Glu Asp Ser Val Glu Leu Leu 130 135 140Val Leu Glu
Trp Val Phe Val Gly Glu Pro Lys Ser Ser Asp Lys Thr145 150 155
160His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
165 170 175Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 180 185 190Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro 195 200 205Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 210 215 220Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val225 230 235 240Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 245 250 255Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 260 265 270Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 275 280
285Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
290 295 300Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser305 310 315 320Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp 325 330 335Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 340 345 350Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 355 360 365Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
380185373PRTArtificial sequenceSynthetic polypeptide 185Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Glu Glu Pro Lys 130 135 140Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu145 150 155 160Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 165 170 175Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 180 185
190Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
195 200 205Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 210 215 220Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu225 230 235
240Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
245 250 255Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro 260 265 270Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln 275 280 285Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala 290 295 300Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr305 310 315 320Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 325 330 335Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 340 345 350Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 355 360
365Leu Ser Pro Gly Lys 370186374PRTArtificial sequenceSynthetic
polypeptide 186Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Glu Trp Glu Pro 130 135
140Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu145 150 155 160Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp 165 170 175Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp 180 185 190Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly 195 200 205Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 210 215 220Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp225 230 235 240Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 245 250
255Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
260 265 270Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn 275 280 285Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile 290 295 300Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr305 310 315 320Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys 325 330 335Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 340 345 350Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 355 360 365Ser
Leu Ser Pro Gly Lys 370187375PRTArtificial sequenceSynthetic
polypeptide 187Asp Lys Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val
Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His Gln Pro Ala Val Val
Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser
Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn
Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr
Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu
Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala
Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu
Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys
Asn Glu Asp Ser Val Glu Leu Leu Val Leu Glu Trp Val Glu 130 135
140Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro145 150 155 160Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys 165 170 175Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val 180 185 190Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp 195 200 205Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 210 215 220Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp225 230 235 240Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 245 250
255Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
260 265 270Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys 275 280 285Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 290 295 300Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys305 310 315 320Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 325 330 335Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 340 345 350Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 355 360 365Leu
Ser Leu Ser Pro Gly Lys 370 375188376PRTArtificial
sequenceSynthetic polypeptide 188Asp Lys Lys Lys Val Ala Met Leu
Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe Ser Thr Ser Ser His
Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys Ser Tyr Cys Gln Asp
Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40 45Thr Arg Ala Gln Ser
Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr 50 55 60Leu Asp Cys Leu
Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser Lys65 70 75 80Gln Gly
Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly Arg Glu Ile 85 90 95Thr
Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys Leu Met Trp Gly 100 105
110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr Thr Pro Asp Asp Leu Glu
115 120 125Gly Lys Asn Glu Asp Ser Val Glu Leu Leu Val Leu Glu Trp
Val Phe 130 135 140Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala145 150 155 160Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro 165 170 175Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val 180 185 190Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 195 200 205Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 210 215 220Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln225 230
235 240Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala 245 250 255Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro 260 265 270Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr 275 280 285Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser 290 295 300Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr305 310 315 320Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 325 330 335Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 340 345
350Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
355 360 365Ser Leu Ser Leu Ser Pro Gly Lys 370
375189377PRTArtificial sequenceSynthetic polypeptide 189Asp Lys Lys
Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His Phe
Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25 30Lys
Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser 35 40
45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro Tyr
50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala Ser
Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg Gly
Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly Lys
Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile Thr
Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val Glu
Leu Leu Val Leu Glu Trp Val Phe 130 135 140Val Glu Pro Lys Ser Ser
Asp Lys Thr His Thr Cys Pro Pro Cys Pro145 150 155 160Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185
190Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
195 200 205Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 210 215 220Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His225 230 235 240Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys 245 250 255Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln 260 265 270Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 275 280 285Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn305 310
315 320Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu 325 330 335Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val 340 345 350Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln 355 360 365Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375190378PRTArtificial sequenceSynthetic polypeptide 190Asp Lys
Lys Lys Val Ala Met Leu Phe Gln Pro Thr Val Leu Arg Cys1 5 10 15His
Phe Ser Thr Ser Ser His Gln Pro Ala Val Val Gln Trp Lys Phe 20 25
30Lys Ser Tyr Cys Gln Asp Arg Met Gly Glu Ser Leu Gly Met Ser Ser
35 40 45Thr Arg Ala Gln Ser Leu Ser Lys Arg Asn Leu Glu Trp Asp Pro
Tyr 50 55 60Leu Asp Cys Leu Asp Ser Arg Arg Thr Val Arg Val Val Ala
Ser Lys65 70 75 80Gln Gly Ser Thr Val Thr Leu Gly Asp Phe Tyr Arg
Gly Arg Glu Ile 85 90 95Thr Ile Val His Asp Ala Asp Leu Gln Ile Gly
Lys Leu Met Trp Gly 100 105 110Asp Ser Gly Leu Tyr Tyr Cys Ile Ile
Thr Thr Pro Asp Asp Leu Glu 115 120 125Gly Lys Asn Glu Asp Ser Val
Glu Leu Leu Val Leu Glu Trp Val Phe 130 135 140Val Gly Glu Pro Lys
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys145 150 155 160Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 165 170
175Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
180 185 190Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp 195 200 205Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 210 215 220Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu225 230 235 240His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 245 250 255Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 260 265 270Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 275 280 285Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 290 295
300Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn305 310 315 320Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 325 330 335Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 340 345 350Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr 355 360 365Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 370 375
* * * * *