U.S. patent application number 16/006968 was filed with the patent office on 2018-10-11 for chimeric polypeptides, polynucleotides encoding same, cells expressing same and methods of producing same.
This patent application is currently assigned to Protalix Ltd.. The applicant listed for this patent is Protalix Ltd.. Invention is credited to Tami ARIEL, Svetlana GINGIS-VELITSKI, Myriam GOLEMBO, Uri HANANIA, Tali KIZHNER, Yoseph SHAALTIEL.
Application Number | 20180291084 16/006968 |
Document ID | / |
Family ID | 55454115 |
Filed Date | 2018-10-11 |
United States Patent
Application |
20180291084 |
Kind Code |
A1 |
SHAALTIEL; Yoseph ; et
al. |
October 11, 2018 |
CHIMERIC POLYPEPTIDES, POLYNUCLEOTIDES ENCODING SAME, CELLS
EXPRESSING SAME AND METHODS OF PRODUCING SAME
Abstract
A plant produced chimeric polypeptide is provided. The plant
produced chimeric polypeptide comprising: (i) a first domain which
comprises a TNF Alpha binding domain of a TNF receptor, and (ii) a
second domain which comprises an Fc domain of an immunoglobulin,
wherein the first domain and the second domain are N-terminally to
C-terminally respectively sequentially translationally fused and
wherein the chimeric polypeptide specifically binds TNF Alpha.
Inventors: |
SHAALTIEL; Yoseph; (Timrat,
IL) ; HANANIA; Uri; (Carmiel, IL) ; KIZHNER;
Tali; (Atzmon-Segev, IL) ; ARIEL; Tami;
(Manof, IL) ; GINGIS-VELITSKI; Svetlana;
(Kiryat-Motzkin, IL) ; GOLEMBO; Myriam; (Moshav
Netaim, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Protalix Ltd. |
Carmiel |
|
IL |
|
|
Assignee: |
Protalix Ltd.
Carmiel
IL
|
Family ID: |
55454115 |
Appl. No.: |
16/006968 |
Filed: |
June 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14849963 |
Sep 10, 2015 |
10000551 |
|
|
16006968 |
|
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62048932 |
Sep 11, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1793 20130101;
C07K 2319/30 20130101; C07K 2319/02 20130101; A61K 36/00 20130101;
C07K 14/7151 20130101; C12N 15/8257 20130101 |
International
Class: |
C07K 14/715 20060101
C07K014/715; C12N 15/82 20060101 C12N015/82; A61K 38/17 20060101
A61K038/17 |
Claims
1. An oral dosage form comprising the plant cells comprising a
nucleic acid construct having a nucleic acid sequence encoding a
plant produced chimeric polypeptide comprising: (i) a first domain
which comprises a TNF.alpha. binding domain of a TNF receptor, and
(ii) a second domain which comprises an Fc domain of an
immunoglobulin, wherein said first domain and said second domain
are N-terminally to C-terminally respectively sequentially
translationally fused and wherein the chimeric polypeptide
specifically binds TNF.alpha., the oral dosage form comprising 1-20
mg of said polypeptide.
2. The oral dosage form of claim 1 comprising 1-10 mg of said
polypeptide.
3. The oral dosage form of claim 1 comprising 10-20 mg of said
polypeptide.
4. The oral dosage form of claim 1, wherein said chimeric
polypeptide comprises an additional domain encoding an endoplasmic
reticulum signal peptide translationally fused N-terminally to said
first domain.
5. The oral dosage form of claim 4, wherein said signal peptide is
a plant signal peptide.
6. The oral dosage form of claim 5, wherein said plant signal
peptide is as set forth in SEQ ID NO: 4.
7. The oral dosage form of claim 1, wherein said first domain is
200-250 amino acids long.
8. The oral dosage form of claim 7, wherein said first domain
comprises the amino acid sequence LCAP (SEQ ID NO: 11) and VFCT
(SEQ ID NO: 12).
9. The oral dosage form of claim 8, wherein said first domain
further comprises the amino acid sequence LPAQVAFXPYAPEPGSTC (SEQ
ID NO: 13).
10. The oral dosage form of claim 9, wherein said first domain is
as set forth in SEQ ID NO: 2.
11. The oral dosage form of claim 1, wherein said immunoglobulin is
IgG.sub.1.
12. The oral dosage form of claim 1, wherein said second domain is
as set forth in SEQ ID NO: 9.
13. The oral dosage form of claim 1, wherein said chimeric
polypeptide is as set forth in SEQ ID NO: 6.
14. The oral dosage form of claim 1, wherein said chimeric
polypeptide is as set forth in SEQ ID NO: 7.
15. The oral dosage form of claim 1, wherein said chimeric
polypeptide comprises a plant-specific glycan.
16. A method of treating a TNF.alpha.-associated medical condition
in a subject in need thereof, the method comprising orally
administering to the subject a therapeutically effective amount of
the oral dosage form of claim 1.
17. The method of claim 16, wherein said medical condition is an
inflammatory disease.
18. The method of claim 16, wherein said medical condition is an
autoimmune disease.
19. The method of claim 16, wherein said medical condition is
selected from the group consisting of rheumatoid arthritis,
ankylosing spondyloarthritis, plaque psoriasis, rheumatoid
polyarthritis, psoriatic arthritis, ankylosing spondyloarthritis
and juvenile idiopathic arthritis.
20. The method of claim 16, wherein said medical condition is an
inflammatory bowel disease.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/849,963 filed on Sep. 10, 2015 which claims the benefit
of priority under 35 USC .sctn. 119(e) of U.S. Provisional Patent
Application No. 62/048,932 filed on Sep. 11, 2014, the contents of
which are incorporated herein by reference in their entirety.
SEQUENCE LISTING STATEMENT
[0002] The ASCII file, entitled 74194SequenceListing.txt, created
on Jun. 12, 2018, comprising 87,059 bytes, submitted concurrently
with the filing of this application is incorporated herein by
reference.
FIELD AND BACKGROUND OF THE INVENTION
[0003] The present invention, in some embodiments thereof, relates
to chimeric polypeptides, polynucleotides encoding same, cells
expressing same and methods of producing same.
[0004] Tumor necrosis factor alpha (TNF.alpha.) is an important,
pro-inflammatory cytokine mediating the regulation of diverse
inflammatory, infectious and immune-related processes and diseases,
TNF.alpha. being considered the most important mediator responsible
for inflammatory pathology.
[0005] TNF-alpha is a 17 kD molecular weight protein, initially
synthesized as a transmembrane protein arranged in stable trimers,
then cleaved by metalloprotease-TNF alpha converting enzyme (TACE)
to form the homotrimeric soluble TNF (sTNF) which engages to its
cognate receptors (TNFRI, p55 and TNFRII, p75), expressed
ubiquitously. The ubiquitous TNF receptors provide the basis for
the wide variety of TNF-alpha mediated cellular responses.
[0006] TNF-alpha induces a wide variety of cellular responses, many
of which result in deleterious consequences, such as cachexia (loss
of fat and whole body protein depletion, leading to anorexia,
common in cancer and AIDS patients) and septic shock. Elevated
secretion of TNF-alpha has been implicated in a variety of human
diseases including diabetes, allograft rejection, sepsis,
inflammatory bowel diseases, osteoporosis, in many autoimmune
diseases such as multiple sclerosis, rheumatoid arthritis,
psoriasis, psoriatic arthritis, hypersensitivity, immune complex
diseases, and even in malaria, cancer and lung fibrosis.
[0007] The biological effect of TNF.alpha. is mediated by the two
distinct receptors. TNF-alpha receptors, when shed from mononuclear
cells, lower the TNF-alpha levels by "mopping up" and acting as
natural inhibitors Neutralization of TNF.alpha. by specific
antibodies and decoy receptors has become a common strategy for
regulation of TNF.alpha. mediated toxicity.
[0008] To date, five protein-based TNF.alpha. antagonists have been
approved by the US FDA for clinical use: Cimzia (Certolizumab
pegol), a TNFmAb Fab' fragment--PEG conjugate; Remicade
(Infliximab), a TNF rmAB; Humira (Adalimumab), a TNF rmAB,
Simponi.TM. (Golimumab), aTNF human monoclonal antibody and
etanercept, a fusion protein of soluble 75 kDa TNF.alpha. receptors
fused to the Fc fragment of human IgG (registered as
Enbrel.TM.).
[0009] Etanercept is indicated for rheumatoid arthritis (RA) and
other arthritic indications such as juvenile idiopathic arthritis
(JIA), psoriasis and Ankylosing Spondylitis (AS). Rheumatoid
arthritis (RA) is a chronic disease that affects approximately five
million people World Wide. Nearly 500,000 patients worldwide across
indications are treated with Enbrel. Enbrel sales in 2010 were 7.8
billion dollars and the total anti-TNF market amounted to 24.04
Billion dollars. Clinical trials of Enbrel therapy, current or
completed, include such diverse indications as adult respiratory
distress syndrome, pemphigus, Alzheimer's disease, Behcet's
syndrome, HIV, myocardial infarct, knee joint synovitis, lupus
nephritis, lichen planus, systemic amyloidosis, sciatica, vitiligo,
chronic fatigue syndrome, anorexia, TMJ, asthma, bronchitis,
diabetes, myelodysplastic disease and others.
[0010] Enbrel is currently produced in mammalian cells. The safety
of biopharmaceuticals has recently come to the forefront for both
patients and health care providers due to outbreaks of emerging
pathogens, most notably HIV, HCV, Cruezfeld-Jacob's Disease, West
Nile Virus and SARS, in multiple regions of the world, emphasizing
the risk of pathogen transmission through the use of human-or
animal-derived raw materials, such as blood-derived products
(serum, plasma cell medium components, etc) in the manufacture of
biopharmaceuticals. For example, approximately half (!) of the
hemophilia population contracted HIV until identification and
screening for the virus became widespread.
[0011] Screening and testing have improved recently, reducing the
threat of pathogen transmission, but risks still remain from
plasma-derived additives during recombinant manufacturing
processes. In particular, the risk from unknown pathogens is
significant, as these agents may appear in the blood supply in the
future and could have a significant impact on safety of
mammalian-cell-based biopharmaceuticals. Of particular concern are
biopharmaceutical drugs which require repeated, regular
administrations, specifically via injection, increasing the
cumulative risk to the patient.
[0012] However, eliminating animal-derived components from media
can significantly alter culture performance as well as
post-translational protein modifications. The glycosylation pattern
of an antibody molecule can affect its structural integrity, thus
influencing its biological function, physicochemical properties and
pharmacokinetics, altering both efficacy and safety, particularly
immunogenicity. Although no major outbreaks have occurred in recent
years, it is still critical to reduce dependence on blood and
plasma components in the manufacture of biopharmaceuticals.
Conversely, recombinant protein production in mammalian cell
culture is unsafe due to xeno contaminations. In 2009 Genzyme was
forced to temporarily close its main factory because of viral
contamination. It did not restore full supplies of the drugs until
2011. Due to the shortage in the only approved drug for Fabry
patients in the US, some people with Fabry disease have suffered
heart or kidney problems and one or more may have even died because
of the shortage.
[0013] Biopharmaceuticals, including modified human proteins, can
be produced in transgenic plants in order to address problems of
safety, viral infections, immune reactions, production yield and
cost. U.S. Pat. No. 6,391,638 and PCT WO2008/135991 teach
bioreactor devices for commercial-scale production of recombinant
polypeptides from plant cell culture. U.S. Pat. No. 7,951,557, U.S.
patent application Ser. Nos. 10/554,387 and 11/790,991 teach
construction and expression of nucleic acid vectors for recombinant
expression of human proteins in plant cells. PCT WO2007/010533
teaches the expression of recombinant human polypeptides in plant
cells, for enteral administration.
[0014] Additional background art includes: U.S. Pat. No. 7,915,225
to Finck et al, U.S. patent application Ser. Nos. 13/021,545 and
10/853,479 to Finck et al, U.S. patent application Ser. No.
11/906,600 to Li et al, U.S. patent application Ser. No. 10/115,625
to Warren et al and U.S. patent application Ser. No. 11/784,538 to
Gombotz et al.
SUMMARY OF THE INVENTION
[0015] According to an aspect of some embodiments of the present
invention there is provided a plant produced chimeric polypeptide
comprising:
(i) a first domain which comprises a TNF.alpha. binding domain of a
TNF receptor, and (ii) a second domain which comprises an Fc domain
of an immunoglobulin, wherein the first domain and the second
domain are N-terminally to C-terminally respectively sequentially
translationally fused and wherein the chimeric polypeptide
specifically binds TNF.alpha..
[0016] According to an aspect of some embodiments of the present
invention there is provided a chimeric polypeptide comprising:
(i) a first domain which comprises a TNF.alpha. binding domain of a
TNF receptor; (ii) a second domain which comprises an Fc domain of
an immunoglobulin; and (iii) a third domain comprising an
endoplasmic reticulum retention signal; wherein the first domain,
second domain and third domain are N-terminally to C-terminally
respectively sequentially translationally fused and wherein the
chimeric polypeptide specifically binds TNF.alpha..
[0017] According to some embodiments of the invention, the
polypeptide comprises an additional domain encoding an endoplasmic
reticulum signal peptide translationally fused N-terminally to the
first domain.
[0018] According to some embodiments of the invention, the signal
peptide is a plant signal peptide.
[0019] According to some embodiments of the invention, the plant
signal peptide is as set forth in SEQ ID NO: 4.
[0020] According to some embodiments of the invention, the first
domain is 200-250 amino acids long.
[0021] According to some embodiments of the invention, the first
domain comprises the amino acid sequence LCAP (SEQ ID NO: 11) and
VFCT (SEQ ID NO: 12).
[0022] According to some embodiments of the invention, the first
domain further comprises the amino acid sequence LPAQVAFXPYAPEPGSTC
(SEQ ID NO: 13) or LPAQVAFTPYAPEPGSTC (SEQ ID NO: 17).
[0023] According to some embodiments of the invention, the first
domain is as set forth in SEQ ID NO: 2.
[0024] According to some embodiments of the invention, the
immunoglobulin is IgG.sub.1.
[0025] According to some embodiments of the invention, the second
domain is as set forth in SEQ ID NO: 9.
[0026] According to some embodiments of the invention, the
polypeptide is as set forth in SEQ ID NO: 6.
[0027] According to some embodiments of the invention, the
polypeptide is as set forth in SEQ ID NO: 7, 204 or 205.
[0028] According to some embodiments of the invention, the
polypeptide is purified to at least 98% homogeneity.
[0029] According to some embodiments of the invention, the
polypeptide is capable of inhibiting TNF.alpha.-induced
apoptosis.
[0030] According to some embodiments of the invention, the
polypeptide comprises a plant-specific glycan.
[0031] According to some embodiments of the invention, the
plant-specific glycan is selected from the group consisting of a
core xylose and a core .alpha.-(1,3) fucose.
[0032] According to an aspect of some embodiments of the present
invention there is provided an isolated polynucleotide comprising a
nucleic acid sequence encoding the polypeptide.
[0033] According to an aspect of some embodiments of the present
invention there is provided a codon usage of the nucleic acid
sequence is optimized for Nicotinia tabaccum.
[0034] According to an aspect of some embodiments of the present
invention there is provided the isolated polynucleotide as set
forth in SEQ ID NO: 5.
[0035] According to an aspect of some embodiments of the present
invention there is provided a nucleic acid expression construct
comprising a nucleic acid sequence encoding the polynucleotide and
a cis-acting regulatory element active in a plant cell.
[0036] According to an aspect of some embodiments of the present
invention there is provided the cis-acting regulatory element is a
promoter.
[0037] According to an aspect of some embodiments of the present
invention there is provided a plant cell comprising the nucleic
acid construct.
[0038] According to an aspect of some embodiments of the present
invention there is provided the plant cell is a Nicotiana tabacum
plant cell.
[0039] According to an aspect of some embodiments of the present
invention there is provided the Nicotiana tabacum L. cv plant cell
is a Bright Yellow (BY-2) cell.
[0040] According to an aspect of some embodiments of the present
invention there is provided the plant cell is lyophilized.
[0041] According to an aspect of some embodiments of the present
invention there is provided a plant cell suspension culture
comprising the plant cell.
[0042] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
as an active ingredient the polypeptide and a pharmaceutically
acceptable carrier.
[0043] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
as an active ingredient the plant cell and a pharmaceutically
acceptable carrier.
[0044] According to an aspect of some embodiments of the present
invention there is provided a method of treating a
TNF.alpha.-associated medical condition in a subject in need
thereof, the method comprising administering to the subject a
therapeutically effective amount of the polypeptide, thereby
treating the TNF.alpha.-associated medical condition in the
subject.
[0045] According to an aspect of some embodiments of the present
invention there is provided a method of treating a
TNF.alpha.-associated medical condition in a subject in need
thereof, the method comprising administering to the subject a
therapeutically effective amount of the plant cells, thereby
treating the TNF.alpha.-associated medical condition in the
subject.
[0046] According to an aspect of some embodiments of the present
invention there is provided the polypeptide for use in treating a
TNF.alpha.-associated medical condition in a subject.
[0047] According to some embodiments of the invention, the plant
cells are for use in treating a TNF.alpha.-associated medical
condition in a subject.
[0048] According to an aspect of some embodiments of the present
invention there is provided a use of the polypeptide in treating a
TNF.alpha.-associated medical condition in a subject.
[0049] According to an aspect of some embodiments of the present
invention there is provided a use of the plant cells in treating a
TNF.alpha.-associated medical condition in a subject.
[0050] According to some embodiments of the invention, the medical
condition is an inflammatory disease.
[0051] According to some embodiments of the invention, the medical
condition is an autoimmune disease.
[0052] According to some embodiments of the invention, the medical
condition is selected from the group consisting of rheumatoid
arthritis, ankylosing spondyloarthritis, plaque psoriasis and
juvenile idiopathic arthritis.
[0053] According to some embodiments of the invention, the medical
condition is selected from the group consisting of rheumatoid
arthritis, inflammatory bowel disease, short bowel syndrome,
sepsis, endotoxic shock, AIDS, endometriosis, psoriasis,
cardiovascular disease, cancer, vitiligo, arthritis, rheumatoid
polyarthritis, psoriatic rheumatism, ankylosing spondyloarthritis,
plaque psoriasis, juvenile idiopathic arthritis, polyarticular
juvenile idiopathic arthritis, psoriasis arthritis, Wegener's
disease (granulomatosis), Crohn's disease, short bowel syndrome,
ulcerative cholitis, chronic obstructive pulmonary disease (COPD),
Hepatitis C, asthma, cachexia, atopic dermatitis. Alzheimer's
disease, hepatic encephalopathy, ADHD, chronic fatigue syndrome
dermatitis herpetiformis (Duhring's disease), contact dermatitis,
urticaria (including chronic idiopathic urticaria), autoimmune
blistering diseases, including pemphigus vulgaris, bullous
pemphigoid, myesthenia gravis, sarcoidosis, including pulmonary
sarcoidosis, scleroderma, reactive arthritis, hyper IgE syndrome,
multiple sclerosis and idiopathic hypereosinophil syndrome, and
allergy.
[0054] According to some embodiments of the invention, the medical
condition is an inflammatory bowel disease.
[0055] According to some embodiments of the invention, the
inflammatory bowel disease is ulcerative colitis or Crohn's
disease.
[0056] According to some embodiments of the invention, the plant
cells are formulated for oral administration.
[0057] According to some embodiments of the invention, the
polypeptide is formulated for parenteral administration.
[0058] According to some embodiments of the invention, the plant
cells are formulated for enteral administration and wherein the
medical condition is not an obesity, metabolic syndrome, diabetes
and a liver disease or disorder.
[0059] According to an aspect of some embodiments of the present
invention there is provided a method of producing the polypeptide,
comprising:
[0060] providing a cell as described herein; and
[0061] culturing the cell so as to produce the polypeptide.
[0062] According to some embodiments of the invention, the method
further comprises isolating the polypeptide from the cell.
[0063] According to some embodiments of the invention, the cell is
an isolated cell cultured in a plant cell culture medium.
[0064] According to some embodiments of the invention, the
culturing is performed in a disposable bioreactor.
[0065] According to an aspect of some embodiments of the present
invention there is provided a method of treating a
TNF.alpha.-associated medical condition in a subject in need
thereof, the method comprising orally administering to the subject
a therapeutically effective amount of the plant cells of claim 23,
wherein the therapeutically effective amount of the polypeptide in
the plant cells comprises 0.02-0.27 mg/kg, thereby treating the
TNF.alpha.-associated medical condition in the subject.
[0066] According to an aspect of some embodiments of the present
invention there is provided use of the plant cells of claim 23 in
the manufacture of a medicament identified for the treatment of a
TNF.alpha.-associated medical condition, wherein a therapeutically
effective amount of the polypeptide in the plant cells is 0.02-0.27
mg/kg, wherein the plant cells are formulated for oral
administration.
[0067] According to some embodiments of the invention, the
therapeutically effective amount comprises 0.02-0.12 mg
polypeptide/kg.
[0068] According to some embodiments of the invention, the
therapeutically effective amount comprises 0.12-0.27 mg
polypeptide/kg.
[0069] According to an aspect of some embodiments of the present
invention there is provided an oral dosage form comprising the
plant cells of claim 23 comprising 1-20 mg of the polypeptide.
[0070] According to some embodiments of the invention, the oral
dosage form comprises 1-10 mg of the polypeptide.
[0071] According to some embodiments of the invention, the oral
dosage form comprises 10-20 mg of the polypeptide.
[0072] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0073] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0074] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0075] In the drawings:
[0076] FIG. 1 is a schematic illustration of the amino acid
sequence of plant recombinant human (prh) TNFR2:Fc (also termed
herein PRX-106, SEQ ID NO:6). prh TNFR2:Fc cDNA for expression in
BY2 cells was assembled with a signal peptide for targeting the
fusion polypeptide composed of the TNF-binding moiety of the TNF
receptor and FC protein to the secretory pathway. Colour code for
the amino acids sequence: the signal peptide is coloured in yellow;
the TNF receptor portion is coloured in black (green); the Fc
portion of IgG1 is in blue; ER retention signal in red.
[0077] FIGS. 2A-2B show comparison of PRH TNFR2:FC and Commercial
Enbrel by Western-blot. prh TNFR2:Fc (lane 1) and commercial Enbrel
(lane 2) were analyzed under reducing conditions (panel A) and
non-reducing conditions (panel B) by 12% and 8% Tris-Glycine
SDS-PAGE, respectively. Membranes were blotted with an anti FC
antibody (upper panel) and with an anti TNFR2 antibody (lower
panel). Molecular weight marker is shown in right lanes. Lane 1:
prh TNFR2:FC; Lane 2:commercial Enbrel.
[0078] FIG. 3 is a graph showing TNF.alpha. binding by prh TNFR2:Fc
and commercial Enbrel by quantitative non radioactive assay for prh
TNFR2:Fc binding activity and molecular integrity. An ELISA plate
pre-coated with antibodies against TNF.alpha., was incubated with
TNF.alpha. followed by exposure to commercial Enbrel and
supernatant from BY2 cells expressing prh TNFR2:Fc. Serial
dilutions of both tested items are shown in the X axis. Fc portion
of the molecule was detected with Goat anti human IgG Fc HRP.
[0079] FIG. 4 is an image showing screening of individual cell
lines for expression of prh TNFR2:Fc by Western blot analysis.
[0080] FIGS. 5A-5F are images showing TNF.alpha. cytotoxicity in
A375 cells in the presence of prh TNFR2:Fc or commercial Enbrel by
MTT viability assay. FIG. 5A--untreated Cultured A375 cells; FIG.
5B--treated with TNF.alpha.; FIG. 5C--TNF.alpha. exposed cells
treated with prh TNFR2:Fc (3.125 ng/ml); FIG. 5D--TNF.alpha.
exposed cells treated with commercial Enbrel (3.125 ng/ml); FIG.
5E--TNF.alpha. exposed cells treated with prh TNFR2:Fc (100 ng/ml);
FIG. 5F--TNF.alpha. exposed cells treated with commercial Enbrel
(100 ng/ml).
[0081] FIG. 5G is a bar graph showing TNF.alpha. cytotoxicity in
A375 cells in the presence of prh TNFR2:Fc or commercial Enbrel by
MTT viability assay.
[0082] FIGS. 6A-6F are images showing TNF.alpha. cytotoxicity in
L929 cells in the presence of prh TNFR2:Fc or commercial Enbrel by
MTT viability assay. FIG. 6A--untreated Cultured L929 cells; FIG.
6B--treated with TNF.alpha.; FIG. 6C--TNF.alpha. exposed cells
treated with prh TNFR2:Fc (3.125 ng/ml); FIG. 6D--TNF.alpha.
exposed cells treated with commercial Enbrel (3.125 ng/ml); FIG.
6E--TNF.alpha. exposed cells treated with prh TNFR2:Fc (100 ng/ml);
FIG. 6F--TNF.alpha. exposed cells treated with commercial Enbrel
(100 ng/ml).
[0083] FIG. 6G is a bar graph showing TNF.alpha. cytotoxicity in
L929 cells in the presence of prh TNFR2:Fc or commercial Enbrel by
MTT viability assay.
[0084] FIGS. 7A-7B are graphs showing body weight changes following
TNBS challenge. Mice were orally administered with prh TNFR2:Fc 6
hours after TNBS induction. For four consecutive days body weights
were determined daily (means.+-.SE). FIG. 7A--Average weight loss
at day four following treatment with TNBS, presented in % loss from
original weight. column 1-saline control (n=15); column
2--Mock-host plant control cells (BY2-; n=15); column
3-PRX-106-plant cells expressing recombinant TNFR2:Fc (doseI)
(n=15); column 4-PRX-106-plant cells expressing recombinant
TNFR2:Fc (dose II) (n=7); column 5-Dexametason treated mice (n=10);
column 6-control mice (n=5). FIG. 7B--Average weight loss during
four days following treatment with TNBS, presented in % loss from
original weight. Note oral treatment with plant cells expressing
recombinant TNFR2:Fc attenuated body weight reduction.
[0085] FIG. 8 is a bar graph showing that oral administration of
plant cells expressing TNFR2:Fc inhibits TNBS-induced colonic
shorting. The mice with colitis were orally administered with plant
cells expressing TNFR2:Fc for four consecutive days after TNBS
induction and the colon lengths were determined at day 4
(means.+-.SE). From left to right: Column 1--control mice (n=5);
column 2--saline control (n=15); column 3--Mock-host plant control
cells (BY2-; n=15); column 4--PRX-106-plant cells expressing
recombinant TNFR2:Fc (doseI) (n=15); column 5--PRX-106-plant cells
expressing recombinant TNFR2:Fc (dose II) (n=7); column
6--Dexametason treated mice (n=10).
[0086] FIG. 9 is a bar graph showing that oral administration of
plant cells expressing TNFR2:Fc improved the macroscopic futures of
TNBS-induced colitis. Total macroscopic inflammation scores
(Wallace score) in control and treated rats at the endpoint of the
experiment (means.+-.SE). Column 1--control mice (n=5); column
2--saline control (n=15); column 3--Mock-host plant control cells
(BY2-; n=15); column 4--PRX-106-plant cells expressing recombinant
TNFR2:Fc (doseI) (n=15); column 5--PRX-106-plant cells expressing
recombinant TNFR2:Fc (dose II) (n=7); column 6--Dexametason treated
mice (n=10).
[0087] FIGS. 10A-10C are bar graphs showing serum cytokine content
in mice treated by oral administration of plant cells expressing
TNFR2:Fc as measured by a cytokine antibody array. Sera from groups
treated with Mock-host plant control cells (BY2-) (n=15) and Plant
cells expressing recombinant TNFR2:Fc protein dose I (n=15), and
dose II (n=7) were collected and subjected to cytokine magnetic
Luminex assay. TNBS-saline control (n=15); Mock-host plant control
cells (BY2-; n=15); PRX-106-plant cells expressing recombinant
TNFR2:Fc (doseI) (n=15); PRX-106-plant cells expressing recombinant
TNFR2:Fc (dose II) (n=7); Dexametason treated mice (n=10); control
mice (n=5).
[0088] FIGS. 11A-11B show serum cytokine content by cytokine
Antibody array.
[0089] FIG. 11A--Sera from groups treated with Mock-host plant
control cells (BY2-) (n=15) and plant cells expressing recombinant
TNFR2:Fc protein dose I (n=15), and dose II (n=7) were pooled,
collected and subjected to cytokine antibody array analysis.
[0090] FIG. 11B--Cytokine quantification of array. Results indicate
that treatment with PRX-106 reduced level of inflammatory mediators
like granulocyte colony-stimulating factor G-CSF, macrophage
colony-stimulating factor (M-CSF), potentially indicating reduced
systemic inflammation by lowering systemic recruitment of bone
marrow derived cells from the bloodstream.
[0091] FIG. 12 is a bar graph showing expansion of splenic Treg
population in animals treated with plant cells expressing
recombinant TNFR2:Fc. Spleen of Balb/c mice, treated with PRX-106
during TNBS induced colitis were analyzed for the percentages of
CD4+CD25+Foxp3+, bars indicate SE. Column 1--saline control (n=15);
column 2--Mock-host plant control cells (BY2-; n=15); column
3--PRX-106-plant cells expressing recombinant TNFR2:Fc (doseI)
(n=15); column 4--PRX-106-plant cells expressing recombinant
TNFR2:Fc (dose II) (n=7); column 5--Dexametason treated mice
(n=10); column 6--control mice (n=5).
[0092] FIGS. 13A-13B are graphs showing body weight changes
following DSS challenge. The mice with colitis were orally
administered with plant cells expressing TNFR2:Fc for seven
consecutive days 24 hours after DSS induction and the body weights
of mice were determined (means.+-.SE). FIG. 13A--Average weight
loss at day ten following treatment with DSS, presented in % loss
from original weight. column 1--saline control (n=10); column
2--Mock-host plant control cells (BY2-; n=10); column 3--oral
administration of plant cells expressing recombinant TNFR2:Fc
(n=10); column 4--control mice (n=5). FIG. 13B--Average weight loss
during ten days following treatment with DSS, presented in % loss
from original weight. Note oral treatment with PRX-106-plant cells
expressing recombinant TNFR2:Fc attenuated body weight reduction. *
P<0.05, ** P<0.01, *** P<0.001.
[0093] FIGS. 14A-14B show that oral administration of TNFR2:Fc
inhibited DSS-induced colonic shorting. The mice with colitis were
orally administered with plant cells expressing recombinant
TNFR2:Fc for 7 consecutive days after DSS induction and the colon
lengths were determined at day 10 (means.+-.SE). FIG. 14A--Column
1--saline control (n=10); column 2--Mock-host plant control cells
(BY2-; n=10); column 3--plant cells expressing recombinant TNFR2:Fc
(n=10); column 4--control mice (n=5). FIG. 14B--Representative
photograph of colons, ten days after the induction of DSS
colitis.
[0094] FIGS. 15A-15C are graphic presentations of cytokine profile
in colons obtained from treated mice. Cytokine secretion by ex
vivo--cultured punch biopsies harvested from the colon of Column
1--DSS treated mice receiving saline control (n=10); column 2--DSS
treated mice receiving Mock-host plant control cells (BY2-; n=10);
column 3--DSS treated mice receiving plant cells expressing 30
.mu.g recombinant TNFR2:Fc (n=10); column 4--control untreated mice
(n=5).
[0095] FIGS. 16A-16C are graphic presentations of serum cytokine
content assayed by cytokine Antibody array. Sera from mice treated
with Mock-host plant control cells (BY2-) n=15) and Plant cells
expressing recombinant TNFR2:Fc protein (n=10), were collected and
subjected to cytokine magnetic Luminexas say. Column 1--saline
control (n=10); column 2--Mock-host plant control cells (BY2-;
n=10); column 3--oral administration of plant cells expressing
recombinant TNFR2:Fc (n=10); column 4--control mice (n=5). *
P<0.05.
[0096] FIGS. 17A-17B show that therapeutic treatment with orally
administered plant cells expressing recombinant TNFR2:Fc reduces
the severity of DSS-induced colitis. FIG. 17A--Representative
histological sections were examined microscopically after H&E
staining with magnification .times.40 and .times.100. The images
are representative of at least seven mice per group. FIG. 17B--The
effect of orally administered plant cells expressing recombinant
TNFR2:FC on histological colitis score was determined. White
square--plant cells expressing recombinant TNFR2:Fc (n=7), Gray
square--Mock-host plant control cells (BY2-; n=10), Black
square--saline control (n=8). ** P<0.01, *** P<0.001.
[0097] FIG. 18 is a bar graph showing pharmacokinetics of TNFR2:Fc
in rat sera. Oral administration of plant cells expressing
recombinant TNFR2:Fc was initiated by free feeding. Rats (n=6)
received plant cells expressing recombinant TNFR2:Fc. Negative
controls received the same volumes of host BY2(-) plant.
[0098] FIG. 19 is a bar graph showing pharmacokinetics of TNFR2:Fc
in rat sera following oral administration of plant cells expressing
recombinant TNFR2:Fc by gavage. Rats (n=6) received plant cells
expressing recombinant TNFR2:Fc. Negative controls received the
same volumes of host BY2(-) plant.
[0099] FIG. 20 shows the PRX-106 sequence (SEQ ID NO: 6) elucidated
by mass-spec (green shown 84.8% coverage).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0100] The present invention, in some embodiments thereof, relates
to chimeric polypeptides, polynucleotides encoding same, cells
expressing same and methods of producing same.
[0101] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0102] Etanercept is a tumor necrosis factor (TNF) blocker
indicated for a number of inflammatory conditions such as
rheumatoid arthritis, polyarticular juvenile idiopathic arthritis,
plaque psoriasis, psoriatic arthritis and ankylosing spondylitis.
Etanercept is produced by recombinant DNA technology in Chinese
hamster ovary mammalian cell expression system. The production of
recombinant proteins in mammalian cell systems is hampered by
cellular fragility and the complex nutritional requirements of
cells and the possible contamination of the final product with
virus or prions.
[0103] Whilst reducing the present invention to practice, the
present inventors have constructed an expression vector for
recombinant expression of Enteracept (hereinafter, prh TNFR2:Fc) in
plant cells, transformed tobacco cells with the vector, and have
isolated catalytically active protein from the cell cultures. The
expressed recombinant protein retains its TNF.alpha. binding
activity and has shown favorable catalytic activity as evidenced by
its apoptosis regulatory activity.
[0104] In-vivo studies in animal models for inflammation e.g.,
inflammatory bowel disease, support the efficacy of the protein and
specifically an oral formulation thereof in treatment of said
indications.
[0105] Specifically, the effect of oral administration of prh
TNFR2:Fc in plant cells on colitis was examined in two
chemically-induced mouse models for IBD: (i) induced by
intra-rectal administration of the covalently reactive reagents
TNBS/oxazolone; and (ii) induced by injections of dextran sodium
sulfate. The results shown in Examples 3A-B, below, illustrate that
oral administration of plant cells expressing prh TNFR2:Fc
ameliorated weight loss, significantly increased colon length,
reduced colon damage (as determined histopathologically), reduced
the level of secreted pro-inflammatory cytokines in-situ and in
sera, and elicited splenic Treg expansion. Oral administration of
plant cells expressing the chimeric polypeptide shifted the T cells
population profile following treatment. Accordingly, there is a
shift in the relative number of regulatory T cell and NKT cell
populations upon administration of plants cells expressing the
chimeric polypeptide as shown using various FACS analysis markers
e.g, CD4.sup.+CD25.sup.+CD8.sup.+CD25.sup.+.
CD4.sup.+CD25.sup.+Foxp3.sup.+ and CD3.sup.+CD56.sup.+ in the
treated subjects.
[0106] These results conclusively show that prh TNFR2 is
biologically active as an anti-inflammatory agent. The present
results further support a role for orally administered plant cells
expressing recombinant TNFR2:Fc as an anti-inflammatory agent with
the capacity to ameliorate IBD.
[0107] The present inventors have further performed a toxicology
study in animals. The results presented in Example 4 below, show
that oral administration of plant cells expressing prh TNFR2:Fc is
safe and well tolerated.
[0108] Thus, according to an aspect of the present invention, there
is provided a plant produced chimeric polypeptide comprising:
(i) a first domain which comprises a TNF.alpha. binding domain of a
TNF receptor; and (ii) a second domain which comprises an Fc domain
of an immunoglobulin, wherein the first domain and the second
domain are N-terminally to C-terminally respectively sequentially
translationally fused and wherein the chimeric polypeptide
specifically binds TNF.alpha..
[0109] As used herein the term "plant produced" refers to the
chemical signature associated with plant expression, including, but
not limited to, host cell impurities in the preparation which
comprises the chimeric polypeptide and glycosylation patterns on
the chimeric polypeptide per se.
[0110] As used herein the term "chimeric polypeptide" refers to a
protein created through the joining of two or more individual
coding sequences which originally code for separate proteins.
Translation of the synthetic (non-naturally occurring) nucleic acid
sequence results in a single chimeric polypeptide with functional
properties derived from each of the original proteins. Such
recombinant fusion proteins are created artificially by recombinant
DNA technology.
[0111] As used the term "TNF.alpha." refers to Tumor necrosis
factor-alpha (TNF, cachexin, or cachectin) that is a cytokine
involved in systemic inflammation and a member of a group of
cytokines that stimulate the acute phase reaction. TNF.alpha. is
produced primarily by activated macrophages (M1), although it can
be produced by many other cell types as CD4+ lymphocytes, NK cells
and neurons. The protein is encoded by TNFA gene and has the
Ref_seq number: NP_000585. The protein is known to stimulate an
inflammatory response (pro-inflammatory cytokine).
[0112] As used herein the term "TNF receptor" or "TNFR" refers to a
polypeptide which is capable of binding TNF.alpha. in a specific
manner e.g., Kd below 10.sup.-5 M. According to a specific
embodiment, the TNFR is membrane bound.
[0113] The first domain is thus composed of at least the TNF
binding domain of a TNF receptor (TNFR). The first domain is a
soluble protein. Thus according to a specific embodiment, the first
domain and even the entire chimeric polypeptide are soluble
proteins which are not membrane anchored.
[0114] Soluble forms of TNFRs may include monomers, fusion proteins
(also called "chimeric proteins), dimers, trimers or higher order
multimers. In certain embodiments of the invention, the soluble
TNFR derivative is one that mimics the 75 kDa TNFR or the 55 kDa
TNFR and that binds to TNF.alpha.. in vivo. The soluble TNFR mimics
of the present invention may be derived from TNFRs p55 or p75 or
fragments thereof. TNFRs other than p55 and p75 also are useful for
deriving soluble TNFR for treating the various medical disorders
described herein, such for example the TNFR that is described in WO
99/04001. Soluble TNFR molecules used to construct TNFR mimics
include, for example, analogs or fragments of native TNFRs having
at least 20 amino acids, that lack the transmembrane region of the
native TNFR, and that are capable of binding TNF.alpha.. Such
soluble forms of TNFR compete for TNF.alpha. with the receptors on
the cell surface, thus inhibiting TNF.alpha. from binding to cells,
thereby preventing it from manifesting its biological activities.
Binding of soluble TNFRs to TNF.alpha. can be assayed using ELISA
or any other convenient assay.
[0115] According to a specific embodiment, the first domain is
derived from TNFR2. (e.g., AAA36755).
[0116] According to an embodiment of the invention, the first
domain is 200-250 amino acids long.
[0117] According to a specific embodiment, the first domain
comprises the amino acid sequence LCAP (SEQ ID NO: 11) and VFCT
(SEQ ID NO: 12).
[0118] According to a specific embodiment, the first domain
comprises the amino acid sequence LPAQVAFXPYAPEPGSTC (SEQ ID NO:
13), or LPAQVAFTPYAPEPGSTC (SEQ ID NO: 17).
[0119] According to a specific embodiment, the first domain is as
set forth in SEQ ID NO: 2 (encoded by SEQ ID NO: 1).
[0120] As used herein "an Fc domain of an immunoglobulin" refers to
a region of a heavy chain of an antibody, typically comprising at
least 2 constant domains (e.g., CH2 and CH3 domains, as these terms
are defined in the art) of the heavy chain. The Fc domain may be
obtained, for example, in the form of a dimer, by digestion of an
antibody by papain. A dimer of Fc domain polypeptides, connected by
disulfide bonds, forms the "tail" region of an antibody. As is
known in the art, Fc domains of some classes of antibodies may be
in the form of multimers. Thus, the Fc domain is optionally
monomeric, optionally dimeric and optionally multimeric.
Optionally, the polypeptide described herein is in the form of a
dimer, the polypeptide comprising an Fc dimer, or in the form of a
multimer, the polypeptide comprising an Fc multimer.
[0121] The Fc domain may encompass modified forms of a native Fc
domain (i.e., a domain which occurs naturally in an antibody), for
example, polypeptides having at least 90% homology, optionally at
least 95% homology, and optionally at least 98% homology, to a
native Fc domain. Modified Fc domains are described, for example,
in International Patent Applications WO 97/34631 and WO
96/32478.
[0122] Optionally, a native Fc is modified so as to remove sites
which provide structural features or biological activity that are
not required for embodiments of the present invention. Examples of
such sites include residues that affect or are involved in
disulfide bond formation, incompatibility with a selected host
cell, N-terminal heterogeneity upon expression in a selected host
cell, glycosylation, interaction with complement, binding to an Fc
receptor (other than a neonatal Fc receptor), and/or
antibody-dependent cellular cytotoxicity.
[0123] The polypeptide according to embodiments of the present
invention may also comprise a fragment of an Fc domain. Optionally,
the fragment comprises at least 20%, optionally at least 50%, and
optionally at least 80% of an Fc domain, as defined
hereinabove.
[0124] The Fc domain or fragment thereof optionally includes a
binding site for a neonatal Fc receptor (FcRn). This is of
particular significance when administering the chimeric polypeptide
via an enteral route.
[0125] According to one embodiment, attachment of an Fc domain or a
fragment thereof to the first domain results in a polypeptide
having a longer half-life in vivo than the first domain per se.
This may be due to the long serum half-life of the Fc domain (which
may be due to salvage of the Fc via binding to FcRn) and/or due to
the greater size of the polypeptide in comparison to the first
domain per se, which reduces clearance from the bloodstream by
glomerular filtration. According to another embodiment, the
resulting polypeptides have reduced immunogenicity as compared to
the first domain per se.
[0126] According to optional embodiments, the Fc domain or fragment
thereof is a human Fc domain (e.g., derived from a human antibody)
or fragment thereof.
[0127] According to exemplary embodiments, the Fc domain (or
fragment thereof) is an IgG (e.g., IgG1) Fc domain (or fragment
thereof).
[0128] According to a specific embodiment, the second domain is as
set forth in SEQ ID NO: 9 (encoded by SEQ ID NO: 8).
[0129] Thus, the second domain of the chimeric polypeptide
comprises at least a portion of a constant immunoglobulin domain,
e.g. a constant heavy immunoglobulin domain or a constant light
immunoglobulin domain. Preferably, the second domain comprises at
least a portion of a constant heavy immunoglobulin domain. The
constant heavy immunoglobulin domain is preferably an Fc fragment
comprising the CH2 and CH3 domain and, optionally, at least a part
of the hinge region. The immunoglobulin domain may be an IgG, IgM,
IgD or IgE immunoglobulin domain or a modified immunoglobulin
domain derived, therefrom. Preferably, the second domain comprises
at least a portion of a constant IgG immunoglobulin domain. The IgG
immunoglobulin domain may be selected from IgG1, IgG2, IgG3 of IgG4
domains or from modified domains such as are described in U.S. Pat.
No. 5,925,734. The immunoglobulin domain may exhibit effector
functions. In some embodiments, however, modified immunoglobulin
domains having modified, e.g. at least partially deleted, effector
functions may be used. Thus for example, the receptor.
[0130] According to an embodiment of the invention the chimeric
fusion of the first domain and the second domain forms Etanercept
(Immunex) having SEQ ID NO: 10.
[0131] It will be appreciated that the species origin of the first
domain and the second domain is selected according to the treated
subject. Thus, according to a specific embodiment, the first domain
and the second domain are of human origin or modified such that
they don't incur immunogenic reaction when administered to human
subjects.
[0132] As used herein "Etanercept" and "Enbrel.TM." are
interchangeably used to designate the commercially available
TNFR2:Fc by Immunex Corporation. Etanercept is a dimeric fusion
polypeptide consisting of the extracellular ligand-binding portion
of the human 75 kilodalton (p75) tumor necrosis factor receptor
(TNFR) linked to the Fc portion of human IgG1. The Fc component of
etanercept contains the constant heavy 2 (CH2) domain, the constant
heavy 3 (CH3) domain and hinge region, but not the constant heavy 1
(CH1) domain of human IgG1.
[0133] According to another embodiment of the invention there is
provided a chimeric polypeptide comprising:
(i) a first domain which comprises a TNF.alpha. binding domain of a
TNF receptor; (ii) a second domain which comprises an Fc domain of
an immunoglobulin; and (iii) a third domain comprising an
endoplasmic reticulum retention signal; wherein the first domain,
second domain and third domain are N-terminally to C-terminally
respectively sequentially translationally fused and wherein the
chimeric polypeptide specifically binds TNF.alpha..
[0134] Thus, according to this aspect of the invention, the
chimeric protein is expressed such that it is retained in the
endoplasmic reticulum (ER). According to a specific embodiment, at
least a portion (e.g., 20% or more) of the TNFR2:Fc molecules in
the cell are retained in the ER.
[0135] As used herein, the term "endoplasmic reticulum retention
signal peptide" refers to a peptide sequence which, when present at
the N- or C-terminus of a polypeptide, causes the polypeptide to be
retrieved from the Golgi apparatus, and retained in the endoplasmic
reticulum (see Rayon et al. Journal of Experimental Botany, Vol.
49, No. 326, pp. 1463-1472, 1998; and Neumann, et al Annals of
Botany, 2003; 92:167-180). In one embodiment, the endoplasmic
reticulum retention signal peptide is HDEL (SEQ ID NO: 14), KDEL
(SEQ ID NO: 15) or SEKDEL (SEQ ID NO: 16).
[0136] As mentioned, the first domain and second domain (and third
domain when present) are N-terminally to C-terminally respectively
sequentially translationally fused. This means that the first
domain is located N-terminally to the second domain (the carboxy
terminus of the first domain is translationally fused to the
N-terminus of the second domain), and the second domain is located
N-terminally of the third domain (the carboxy terminus of the
second domain is translationally fused to the N-terminus of the
third domain). Thus, the second domain is practically sandwiched by
the first domain at the N-terminus and the third domain at the
C-teminus. Schematic presentation is as follows: first
domain>second domain(>third domain) are orderly oriented from
the N-terminus to the C-terminus (see FIG. 1). The linkage between
the domains may be direct or indirect by the use of linkers such as
peptide linkers.
[0137] The molecule may further comprise an additional domain which
encodes for an endoplasmic reticulum signal sequence which is
oriented upstream (N-terminally) of the first domain and
translationally fused thereto.
[0138] As used herein "an endoplasmic reticulum (ER) signal
peptide" refers to a signal sequence, leader sequence or leader
peptide that is a short (e.g., 5-30 amino acids long) peptide
present at the N-terminus of the majority of newly synthesized
proteins that are destined towards the secretory pathway.
[0139] According to a specific embodiment, the ER signal peptide is
derived (taken) from a plant protein.
[0140] According to a specific embodiment, the endoplasmic
reticulum signal peptide is from N. plumbaginifolia Calreticulin
protein.
[0141] According to a further specific embodiment, the signal
peptide from N. plumbaginifolia Calreticulin protein is as set
forth in SEQ ID NO: 4 and encoded by the nucleic acid sequence of
SEQ ID NO: 3.
[0142] As used herein the term "translationally fused at the
N-terminal" or "translationally fused at the C-terminal" refers to
covalent attachment of the indicated peptide via a peptide bond to
the N-terminal or C-terminal amino acid of the respective domain
typically as a result of recombinant expression.
[0143] According to a specific embodiment, the chimeric polypeptide
is as set forth in SEQ ID NO: 6.
[0144] According to a specific embodiment, the chimeric polypeptide
is as set forth in SEQ ID NO: 7, 204 or 205.
[0145] As mentioned the recombinant chimeric proteins of the
invention are produced in plant cells.
[0146] In order to express the polypeptide, an isolated
polynucleotide comprising a nucleic acid sequence encoding the
chimeric polypeptide as described herein is ligated into a "plant
nucleic acid expression construct".
[0147] As used herein the term "plant nucleic acid expression
construct" refers to a nucleic acid construct which includes the
nucleic acid of some embodiments of the invention and at least one
promoter for directing transcription of nucleic acid in a host
plant cell. Further details of suitable transformation approaches
are provided hereinbelow.
[0148] According to some embodiments of the invention, there is
provided a nucleic acid expression construct comprising the nucleic
acid sequence of the invention, and a promoter for directing
transcription of the nucleic acid sequence in a plant host
cell.
[0149] As used herein the term "nucleic acid sequence" refers to a
single or double stranded nucleic acid sequence 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).
[0150] As used herein the phrase "complementary polynucleotide
sequence" refers to a sequence, which results from reverse
transcription of messenger RNA using a reverse transcriptase or any
other RNA dependent DNA polymerase. Such a sequence can be
subsequently amplified in vivo or in vitro using a DNA dependent
DNA polymerase.
[0151] As used herein the phrase "genomic polynucleotide sequence"
refers to a sequence derived (isolated) from a chromosome and thus
it represents a contiguous portion of a chromosome.
[0152] As used herein the phrase "composite polynucleotide
sequence" refers to a sequence, which is at least partially
complementary and at least partially genomic. A composite sequence
can include some exonal sequences required to encode the
polypeptide of the present invention, as well as some intronic
sequences interposing therebetween. The intronic sequences can be
of any source, including of other genes, and typically will include
conserved splicing signal sequences. Such intronic sequences may
further include cis acting expression regulatory elements.
[0153] According to some embodiments of the present invention, the
nucleic acid sequences encoding the polypeptides of the present
invention are optimized for expression in plants. Examples of such
sequence modifications include, but are not limited to, an altered
G/C content to more closely approach that typically found in the
plant species of interest, and the removal of codons atypically
found in the plant species commonly referred to as codon
optimization. In one embodiment, the codon usage of the nucleic
acid sequence encoding the chimeric polypeptide is optimized for
Nicotiana tabacuum or Nicotiana benthamiana.
[0154] The phrase "codon optimization" refers to the selection of
appropriate DNA nucleotides for use within a structural gene or
fragment thereof that approaches codon usage within the plant of
interest. Therefore, an optimized gene or nucleic acid sequence
refers to a gene in which the nucleotide sequence of a native or
naturally occurring gene has been modified in order to utilize
statistically-preferred or statistically-favored codons within the
plant. The nucleotide sequence typically is examined at the DNA
level and the coding region optimized for expression in the plant
species determined using any suitable procedure, for example as
described in Sardana et al. (1996, Plant Cell Reports 15:677-681).
In this method, the standard deviation of codon usage, a measure of
codon usage bias, may be calculated by first finding the squared
proportional deviation of usage of each codon of the native gene
relative to that of highly expressed plant genes, followed by a
calculation of the average squared deviation. The formula used is:
1 SDCU=n=1 N [(Xn-Yn)/Yn]2/N, where Xn refers to the frequency of
usage of codon n in highly expressed plant genes, where Yn to the
frequency of usage of codon n in the gene of interest and N refers
to the total number of codons in the gene of interest. A table of
codon usage from highly expressed genes of dicotyledonous plants
has been compiled using the data of Murray et al. (1989, Nuc Acids
Res. 17:477-498).
[0155] One method of optimizing the nucleic acid sequence in
accordance with the preferred codon usage for a particular plant
cell type is based on the direct use, without performing any extra
statistical calculations, of codon optimization tables such as
those provided on-line at the Codon Usage Database through the NIAS
(National Institute of Agrobiological Sciences) DNA bank in Japan
(Hypertext Transfer Protocol://World Wide Web (dot) kazusa (dot) or
(dot) jp/codon/). The Codon Usage Database contains codon usage
tables for a number of different species, with each codon usage
table having been statistically determined based on the data
present in Genbank.
[0156] By using such codon optimization tables to determine the
most preferred or most favored codons for each amino acid in a
particular species (for example, rice), a naturally-occurring
nucleotide sequence encoding a protein of interest can be codon
optimized for that particular plant species. This is effected by
replacing codons that may have a low statistical incidence in the
particular species genome with corresponding codons, in regard to
an amino acid, that are statistically more favored. However, one or
more less-favored codons may be selected to delete existing
restriction sites, to create new ones at potentially useful
junctions (5' and 3' ends to add signal peptide or termination
cassettes, internal sites that might be used to cut and splice
segments together to produce a correct full-length sequence), or to
eliminate nucleotide sequences that may negatively affect mRNA
stability or expression.
[0157] The desired encoding nucleotide sequence may already, in
advance of any modification, contain a number of codons that
correspond to a statistically-favored codon in a particular plant
species. Therefore, codon optimization of the native nucleotide
sequence may comprise determining which codons, within the desired
nucleotide sequence, are not statistically-favored with regards to
a particular plant, and modifying these codons in accordance with a
codon usage table of the particular plant to produce a codon
optimized derivative. A modified nucleotide sequence may be fully
or partially optimized for plant codon usage provided that the
protein encoded by the modified nucleotide sequence is produced at
a level higher than the protein encoded by the corresponding
naturally occurring or native gene. Construction of synthetic genes
by altering the codon usage is described in for example PCT Patent
Application 93/07278.
[0158] Thus according to a specific embodiment, there is provided a
Nicotinia tobaccum optimized sequence as set forth in SEQ ID NO:
5.
[0159] According to some embodiments of the invention, the nucleic
acid sequence coding for the cimeric polypeptide is operably linked
to a cis-acting regulatory sequence active in plant cells, such as
a plant promoter sequence.
[0160] A coding nucleic acid sequence is "operably linked" to a
regulatory sequence (e.g., promoter) if the regulatory sequence is
capable of exerting a regulatory effect on (e.g. effect on the
expression of) the coding sequence linked thereto.
[0161] Any suitable promoter sequence can be used by the nucleic
acid construct of the present invention. Preferably the promoter is
a constitutive promoter, a tissue-specific, or an inducible
promoter.
[0162] As used herein the phrase "plant-expressible" refers to a
promoter sequence, including any additional regulatory elements
added thereto or contained therein, is at least capable of
inducing, conferring, activating or enhancing expression in a plant
cell, tissue or organ, preferably a monocotyledonous or
dicotyledonous plant cell, tissue, or organ. Such a promoter can be
constitutive, i.e., capable of directing high level of gene
expression in a plurality of tissues, tissue specific, i.e.,
capable of directing gene expression in a particular tissue or
tissues, inducible, i.e., capable of directing gene expression
under a stimulus, or chimeric, i.e., formed of portions of at least
two different promoters.
[0163] Examples of preferred promoters useful for the methods of
some embodiments of the invention are presented in Table I, II, III
and IV.
TABLE-US-00001 TABLE I Exemplary constitutive promoters for use in
the performance of some embodiments of the invention Gene Source
Expression Pattern Reference Actin constitutive McElroy et al,
Plant Cell, 2: 163-171, 1990 CAMV 35S constitutive Odell et al,
Nature, 313: 810-812, 1985 CaMV 19S constitutive Nilsson et al.,
Physiol. Plant 100: 456-462, 1997 GOS2 constitutive de Pater et al,
Plant J Nov; 2(6): 837-44, 1992 ubiquitin constitutive Christensen
et al, Plant Mol. Biol. 18: 675-689, 1992 Rice cyclophilin
constitutive Bucholz et al, Plant Mol Biol. 25(5): 837-43, 1994
Maize H3 histone constitutive Lepetit et al, Mol. Gen. Genet. 231:
276-285, 1992 Actin 2 constitutive An et al, Plant J. 10(1);
107-121, 1996
TABLE-US-00002 TABLE II Exemplary seed-preferred promoters for use
in the performance of some embodiments of the invention Expression
Gene Source Pattern Reference Seed specific genes seed Simon, et
al., Plant Mol. Biol. 5. 191, 1985; Scofield, etal., J. Biol. Chem.
262: 12202, 1987.; Baszczynski, et al., Plant Mol. Biol. 14: 633,
1990. Brazil Nut albumin seed Pearson' et al., Plant Mol. Biol. 18:
235-245, 1992. legumin seed Ellis, et al. Plant Mol. Biol. 10:
203-214, 1988 Glutelin (rice) seed Takaiwa, et al., Mol. Gen.
Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47,
1987 Zein seed Matzke et al Plant Mol Biol, 143).323-32 1990 napA
seed Stalberg, et al, Planta 199: 515-519, 1996 wheat LMW and
endosperm Mol Gen Genet 216: 81-90, HMW, glutenin-1 1989; NAR 17:
461-2, Wheat SPA seed Albanietal, Plant Cell, 9: 171-184, 1997
wheat a, b and g endosperm EMBO3: 1409-15, 1984 gliadins Barley
ltrl promoter endosperm barley B1, C, D endosperm Theor Appl Gen
98: 1253-62, hordein 1999; Plant J 4: 343-55, 1993; Mol Gen Genet
250: 750-60, 1996 Barley DOF endosperm Mena et al, The Plant
Journal, 116(1): 53-62, 1998 Biz2 endosperm EP99106056.7 Synthetic
promoter endosperm Vicente-Carbajosa et al., Plant J. 13: 629-640,
1998 rice prolamin endosperm Wu et al, Plant Cell Physiology NRP33
39(8) 885-889, 1998 rice-globulin Glb-1 endosperm Wu et al, Plant
Cell Physiology 398) 885-889, 1998 rice OSH1 embryo Sato et al,
Proc. Nati. Acad. Sci. USA, 93: 8117-8122 rice alpha-globulin
endosperm Nakase et al. Plant Mol. Biol. REB/OHP-1 33: 513-S22,
1997 rice ADP-glucose endosperm Trans Res 6: 157-68, 1997 PP maize
ESR gene endosperm Plant J 12: 235-46, 1997 family sorghum gamma-
endosperm PMB 32: 1029-35, 1996 kafirin KNOX embryo Postma-Haarsma
ef al, Plant Mol. Biol. 39: 257-71, 1999 rice oleosin Embryo Wu et
at, J. Biochem., 123: 386, and aleuton 1998 sunflower oleosin Seed
(embryo Cummins, etal., Plant Mol. and dry seed) Biol. 19: 873-876,
1992
TABLE-US-00003 TABLE III Exemplary flower-specific promoters for
use in the performance of the invention Expression Gene Source
Pattern Reference AtPRP4 flowers
www(dot)salus(dot)medium(dot)edu/mmg/ tierney/html chalene flowers
Van der Meer, et al., Plant Mol. Biol. synthase 15, 95-109, 1990.
(chsA) LAT52 anther Twell et al Mol. Gen Genet. 217: 240-245 (1989)
apetala-3 flowers
TABLE-US-00004 TABLE IV Alternative rice promoters for use in the
performance of the invention PRO # gene expression PR00001
Metallothionein Mte transfer layer of embryo + calli PR00005
putative beta-amylase transfer layer of embryo PR00009 Putative
cellulose synthase Weak in roots PR00012 lipase (putative) PR00014
Transferase (putative) PR00016 peptidyl prolyl cis-trans isomerase
(putative) PR00019 unknown PR00020 prp protein (putative) PR00029
noduline (putative) PR00058 Proteinase inhibitor Rgpi9 seed PR00061
beta expansine EXPB9 Weak in young flowers PR00063 Structural
protein young tissues + calli + embryo PR00069 xylosidase
(putative) PR00075 Prolamine 10 Kda strong in endosperm PR00076
allergen RA2 strong in endosperm PR00077 prolamine RP7 strong in
endosperm PR00078 CBP80 PR00079 starch branching enzyme 1 PR00080
Metallothioneine-like ML2 transfer layer of embryo + calli PR00081
putative caffeoyl-CoA 3-0 shoot methyltransferase PR00087 prolamine
RM9 strong in endosperm PR00090 prolamine RP6 strong in endosperm
PR00091 prolamine RP5 strong in endosperm PR00092 allergen RA5
PR00095 Putative methionine embryo aminopeptidase PR00098
ras-related GTP binding protein PR00104 beta expansine EXPB1
PR00105 Glycine rich protein PR00108 metallothionein like protein
(putative) PR00110 RCc3 strong root PR00111 uclacyanin 3-like
protein weak discrimination center/ shoot meristem PR00116 26S
proteasome regulatory very weak meristem particle non-ATPase
subunit 11 specific PR00117 putative 40S ribosomal protein weak in
endosperm PR00122 chlorophyll a/lo-binding protein very weak in
shoot precursor (Cab27) PR00123 putative protochlorophyllide Strong
leaves reductase PR00126 metallothionein RiCMT strong
discrimination center shoot meristem PR00129 GOS2 Strong
constitutive PR00131 GOS9 PR00133 chitinase Cht-3 very weak
meristem specific PR00135 alpha-globulin Strong in endosperm
PR00136 alanine aminotransferase Weak in endosperm PR00138 Cyclin
A2 PR00139 Cyclin D2 PR00140 Cyclin D3 PR00141 Cyclophyllin 2 Shoot
and seed PR00146 sucrose synthase SS1 (barley) medium constitutive
PR00147 trypsin inhibitor ITR1 (barley) weak in endosperm PR00149
ubiquitine 2 with intron strong constitutive PR00151 WSI18 Embryo
and stress PR00156 HVA22 homologue (putative) PR00157 EL2 PR00169
aquaporine medium constitutive in young plants PR00170 High
mobility group protein Strong constitutive PR00171 reversibly
glycosylated protein weak constitutive RGP1 PR00173 cytosolic MDH
shoot PR00175 RAB21 Embryo and stress PR00176 CDPK7 PR00177 Cdc2-1
very weak in meristem PR00197 sucrose synthase 3 PRO0198 OsVP1
PRO0200 OSH1 very weak in young plant meristem PRO0208 putative
chlorophyllase PRO0210 OsNRT1 PRO0211 EXP3 PRO0216 phosphate
transporter OjPT1 PRO0218 oleosin 18 kd aleurone + embryo PRO0219
ubiquitine 2 without intron PRO0220 RFL PRO0221 maize UBI delta
intron not detected PRO0223 glutelin-1 PRO0224 fragment of prolamin
RP6 promoter PRO0225 4xABRE PRO0226 glutelin OSGLUA3 PRO0227
BLZ-2_short (barley) PR00228 BLZ-2_long (barley)
[0164] The nucleic acid construct of some embodiments of the
invention can further include an appropriate selectable marker
and/or an origin of replication. According to some embodiments of
the invention, 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 with propagation in cells. 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.
[0165] The nucleic acid construct of some embodiments of the
invention can be utilized to stably or transiently transform plant
cells. In stable transformation, the nucleic acid is integrated
into the plant genome and as such it represents a stable and
inherited trait. In transient transformation, the exogenous
polynucleotide is expressed by the cell transformed but it is not
integrated into the genome and as such it represents a transient
trait.
[0166] Thus, according to some aspects of the present invention,
there is provided an isolated cell comprising the nucleic acid
construct of the invention.
[0167] As used herein, the term "isolated cell" refers to a cell at
least partially separated from the natural environment e.g., from a
plant. In some embodiments, the isolated cell is a plant cell of a
whole plant. In some embodiments, the isolated cell is a plant
cell, for example, a plant cell in culture.
[0168] The term "plant" as used herein encompasses whole plants,
ancestors and progeny of the plants and plant parts, including
seeds, shoots, stems, roots (including tubers), and plant cells,
tissues and organs. The plant may be in any form including
suspension cultures, embryos, meristematic regions, callus tissue,
leaves, gametophytes, sporophytes, pollen, and microspores. Plants
that are particularly useful in the methods of the invention
include all plants which belong to the superfamily Viridiplantae,
in particular monocotyledonous and dicotyledonous plants including
a fodder or forage legume, ornamental plant, food crop, tree, or
shrub selected from the list comprising Acacia spp., Acer spp.,
Actinidia spp., Aesculus spp., Agathis australis, Albizia amara,
Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu,
Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula
spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea
frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna
indica, Capsicum spp., Cassia spp., Centroema pubescens,
Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum
mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp.,
Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga,
Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia
oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp.,
Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos
spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp.,
Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalypfus
spp., Euclea schimperi, Eulalia vi/losa, Pagopyrum spp., Feijoa
sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli,
Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia
spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma,
Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum
vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia
dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,
Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia
simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus
spp., Manihot esculenta, Medicago saliva, Metasequoia
glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp.,
Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum
spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix
canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus
spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii,
Pogonaffhria squarrosa, Populus spp., Prosopis cineraria,
Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis,
Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus
natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia,
Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum,
Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron
giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus,
Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp,
Taxodium distichum, Themeda triandra, Trifolium spp., Triticum
spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis
vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays,
amaranth, artichoke, asparagus, broccoli, Brussels sprouts,
cabbage, canola, carrot, cauliflower, celery, collard greens, flax,
kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,
straw, sugar beet, sugar cane, sunflower, tomato, squash tea,
maize, wheat, barley, rye, oat, peanut, pea, lentil and alfalfa,
cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant,
eucalyptus, a tree, an ornamental plant, a perennial grass and a
forage crop. Alternatively algae and other non-Viridiplantae can be
used for the methods of the present invention.
[0169] According to some embodiments of the invention, the plant or
plant cell is a duckweed plant, cell or nodule. Duckweed (members
of the monocotyledonous family Lemnaceae, or Lemna) plant or
duckweed nodule cultures can be efficiently transformed with an
expression cassette containing a nucleotide sequence of interest by
any one of a number of methods including Agrobacterium-mediated
gene transfer, ballistic bombardment, or electroporation. Methods
for molecular engineering of duckweed cells and detailed
description of duckweed expression systems useful for commercial
production of polypeptides are known in the art (see, for example,
U.S. Pat. Nos. 6,040,498 and 6,815,184 to Stomp, et al, and U.S.
Pat. No. 8,022,270 to Dickey et al, all of which are incorporated
fully by reference herein).
[0170] According to some embodiments of the invention, the plant or
plant cell used by the method of the invention is a crop plant or
cell of a crop plant such as rice, maize, wheat, barley, peanut,
potato, sesame, olive tree, palm oil, banana, soybean, sunflower,
canola, sugarcane, alfalfa, millet, leguminosae (bean, pea), flax,
lupinus, rapeseed, tobacco, poplar and cotton.
[0171] According to further embodiments the plant cells includes
tobacco cells, Agrobacterium rhizogenes transformed root cell,
celery cell, ginger cell, horseradish cell and carrot cells. In one
embodiment the tobacco cells are from a tobacco cell line, such as,
but not limited to Nicotiana tabacum L. cv Bright Yellow (BY-2)
cells. The plant cells may be grown according to any type of
suitable culturing method, including but not limited to, culture on
a solid surface (such as a plastic culturing vessel or plate for
example) or in suspension. It will be noted that some cells, such
as the BY-2 and carrot cells can be cultured and grown in
suspension. Suitable devices and methods for culturing plant cells
in suspension are known in the art, for example, as described in
International Patent Application PCT IL2008/000614. In yet another
embodiment the cells are cells of whole tobacco plants or plant
tissues, including, but not limited to Nicotiana benthamiana.
[0172] There are various methods of introducing foreign genes into
both monocotyledonous and dicotyledonous plants (Potrykus, I.,
Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225;
Shimamoto et al., Nature (1989) 338:274-276).
[0173] The principle methods of causing stable integration of
exogenous DNA into plant genomic DNA include two main
approaches:
[0174] (i) Agrobacterium-mediated gene transfer: Klee et al. (1987)
Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell
Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular
Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K.,
Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in
Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth
Publishers, Boston, Mass. (1989) p. 93-112.
[0175] (ii) Direct DNA uptake: Paszkowski et al., in Cell Culture
and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of
Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic
Publishers, San Diego, Calif. (1989) p. 52-68; including methods
for direct uptake of DNA into protoplasts, Toriyama, K. et al.
(1988) Bio/Technology 6:1072-1074. DNA uptake induced by brief
electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988)
7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection
into plant cells or tissues by particle bombardment, Klein et al.
Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology
(1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by
the use of micropipette systems: Neuhaus et al., Theor. Appl.
Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.
(1990) 79:213-217; glass fibers or silicon carbide whisker
transformation of cell cultures, embryos or callus tissue, U.S.
Pat. No. 5,464,765 or by the direct incubation of DNA with
germinating pollen, DeWet et al. in Experimental Manipulation of
Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels,
W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad.
Sci. USA (1986) 83:715-719.
[0176] The Agrobacterium system includes the use of plasmid vectors
that contain defined DNA segments that integrate into the plant
genomic DNA. Methods of inoculation of the plant tissue vary
depending upon the plant species and the Agrobacterium delivery
system. A widely used approach is the leaf disc procedure which can
be performed with any tissue explant that provides a good source
for initiation of whole plant differentiation. See, e.g., Horsch et
al. in Plant Molecular Biology Manual A5, Kluwer Academic
Publishers, Dordrecht (1988) p. 1-9. A supplementary approach
employs the Agrobacterium delivery system in combination with
vacuum infiltration. The Agrobacterium system is especially viable
in the creation of transgenic dicotyledonous plants.
[0177] There are various methods of direct DNA transfer into plant
cells. In electroporation, the protoplasts are briefly exposed to a
strong electric field. In microinjection, the DNA is mechanically
injected directly into the cells using very small micropipettes. In
microparticle bombardment, the DNA is adsorbed on microprojectiles
such as magnesium sulfate crystals or tungsten particles, and the
microprojectiles are physically accelerated into cells or plant
tissues.
[0178] Following stable transformation plant propagation is
exercised. The most common method of plant propagation is by seed.
Regeneration by seed propagation, however, has the deficiency that
due to heterozygosity there is a lack of uniformity in the crop,
since seeds are produced by plants according to the genetic
variances governed by Mendelian rules. Basically, each seed is
genetically different and each will grow with its own specific
traits. Therefore, it is preferred that the transformed plant be
produced such that the regenerated plant has the identical traits
and characteristics of the parent transgenic plant. Therefore, it
is preferred that the transformed plant be regenerated by
micropropagation which provides a rapid, consistent reproduction of
the transformed plants.
[0179] Micropropagation is a process of growing new generation
plants from a single piece of tissue that has been excised from a
selected parent plant or cultivar. This process permits the mass
reproduction of plants having the preferred tissue expressing the
fusion protein. The new generation plants which are produced are
genetically identical to, and have all of the characteristics of,
the original plant. Micropropagation allows mass production of
quality plant material in a short period of time and offers a rapid
multiplication of selected cultivars in the preservation of the
characteristics of the original transgenic or transformed plant.
The advantages of cloning plants are the speed of plant
multiplication and the quality and uniformity of plants
produced.
[0180] Micropropagation is a multi-stage procedure that requires
alteration of culture medium or growth conditions between stages.
Thus, the micropropagation process involves four basic stages:
Stage one, initial tissue culturing; stage two, tissue culture
multiplication; stage three, differentiation and plant formation;
and stage four, greenhouse culturing and hardening. During stage
one, initial tissue culturing, the tissue culture is established
and certified contaminant-free. During stage two, the initial
tissue culture is multiplied until a sufficient number of tissue
samples are produced to meet production goals. During stage three,
the tissue samples grown in stage two are divided and grown into
individual plantlets. At stage four, the transformed plantlets are
transferred to a greenhouse for hardening where the plants'
tolerance to light is gradually increased so that it can be grown
in the natural environment.
[0181] According to some embodiments of the invention, the
transgenic plants are generated by transient transformation of leaf
cells, meristematic cells or the whole plant.
[0182] Transient transformation can be effected by any of the
direct DNA transfer methods described above or by viral infection
using modified plant viruses.
[0183] Viruses that have been shown to be useful for the
transformation of plant hosts include CaMV, Tobacco mosaic virus
(TMV), brome mosaic virus (BMV) and Bean Common Mosaic Virus (BV or
BCMV). Transformation of plants using plant viruses is described in
U.S. Pat. No. 4,855,237 (bean golden mosaic virus; BGV), EP-A
67,553 (TMV), Japanese Published Application No. 63-14693 (TMV),
EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al.,
Communications in Molecular Biology: Viral Vectors, Cold Spring
Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus
particles for use in expressing foreign DNA in many hosts,
including plants are described in WO 87/06261.
[0184] According to some embodiments of the invention, the virus
used for transient transformations is avirulent and thus is
incapable of causing severe symptoms such as reduced growth rate,
mosaic, ring spots, leaf roll, yellowing, streaking, pox formation,
tumor formation and pitting. A suitable avirulent virus may be a
naturally occurring avirulent virus or an artificially attenuated
virus. Virus attenuation may be effected by using methods well
known in the art including, but not limited to, sub-lethal heating,
chemical treatment or by directed mutagenesis techniques such as
described, for example, by Kurihara and Watanabe (Molecular Plant
Pathology 4:259-269, 2003), Gal-on et al. (1992), Atreya et al.
(1992) and Huet et al. (1994).
[0185] Suitable virus strains can be obtained from available
sources such as, for example, the American Type culture Collection
(ATCC) or by isolation from infected plants. Isolation of viruses
from infected plant tissues can be effected by techniques well
known in the art such as described, for example by Foster and
Tatlor, Eds. "Plant Virology Protocols: From Virus Isolation to
Transgenic Resistance (Methods in Molecular Biology (Humana Pr),
Vol 81)", Humana Press, 1998. Briefly, tissues of an infected plant
believed to contain a high concentration of a suitable virus,
preferably young leaves and flower petals, are ground in a buffer
solution (e.g., phosphate buffer solution) to produce a virus
infected sap which can be used in subsequent inoculations.
[0186] Construction of plant RNA viruses for the introduction and
expression of non-viral nucleic acid sequences in plants is
demonstrated by the above references as well as by Dawson, W. O. et
al., Virology (1989) 172:285-292; Takamatsu et al. EMBO J. (1987)
6:307-311; French et al. Science (1986) 231:1294-1297; Takamatsu et
al. FEBS Letters (1990) 269:73-76; and U.S. Pat. No. 5,316,931.
[0187] When the virus is a DNA virus, suitable modifications can be
made to the virus itself. Alternatively, the virus can first be
cloned into a bacterial plasmid for ease of constructing the
desired viral vector with the foreign DNA. The virus can then be
excised from the plasmid. If the virus is a DNA virus, a bacterial
origin of replication can be attached to the viral DNA, which is
then replicated by the bacteria. Transcription and translation of
this DNA will produce the coat protein which will encapsidate the
viral DNA. If the virus is an RNA virus, the virus is generally
cloned as a cDNA and inserted into a plasmid. The plasmid is then
used to make all of the constructions. The RNA virus is then
produced by transcribing the viral sequence of the plasmid and
translation of the viral genes to produce the coat protein(s) which
encapsidate the viral RNA.
[0188] In one embodiment, a plant viral polynucleotide is provided
in which the native coat protein coding sequence has been deleted
from a viral polynucleotide, a non-native plant viral coat protein
coding sequence and a non-native promoter, preferably the
subgenomic promoter of the non-native coat protein coding sequence,
capable of expression in the plant host, packaging of the
recombinant plant viral polynucleotide, and ensuring a systemic
infection of the host by the recombinant plant viral
polynucleotide, has been inserted. Alternatively, the coat protein
gene may be inactivated by insertion of the non-native
polynucleotide sequence within it, such that a protein is produced.
The recombinant plant viral polynucleotide may contain one or more
additional non-native subgenomic promoters. Each non-native
subgenomic promoter is capable of transcribing or expressing
adjacent genes or polynucleotide sequences in the plant host and
incapable of recombination with each other and with native
subgenomic promoters. Non-native (foreign) polynucleotide sequences
may be inserted adjacent the native plant viral subgenomic promoter
or the native and a non-native plant viral subgenomic promoters if
more than one polynucleotide sequence is included. The non-native
polynucleotide sequences are transcribed or expressed in the host
plant under control of the subgenomic promoter to produce the
desired products.
[0189] In a second embodiment, a recombinant plant viral
polynucleotide is provided as in the first embodiment except that
the native coat protein coding sequence is placed adjacent one of
the non-native coat protein subgenomic promoters instead of a
non-native coat protein coding sequence.
[0190] In a third embodiment, a recombinant plant viral
polynucleotide is provided in which the native coat protein gene is
adjacent its subgenomic promoter and one or more non-native
subgenomic promoters have been inserted into the viral
polynucleotide. The inserted non-native subgenomic promoters are
capable of transcribing or expressing adjacent genes in a plant
host and are incapable of recombination with each other and with
native subgenomic promoters. Non-native polynucleotide sequences
may be inserted adjacent the non-native subgenomic plant viral
promoters such that the sequences are transcribed or expressed in
the host plant under control of the subgenomic promoters to produce
the desired product.
[0191] In a fourth embodiment, a recombinant plant viral
polynucleotide is provided as in the third embodiment except that
the native coat protein coding sequence is replaced by a non-native
coat protein coding sequence.
[0192] The viral vectors are encapsidated by the coat proteins
encoded by the recombinant plant viral polynucleotide to produce a
recombinant plant virus. The recombinant plant viral polynucleotide
or recombinant plant virus is used to infect appropriate host
plants. The recombinant plant viral polynucleotide is capable of
replication in the host, systemic spread in the host, and
transcription or expression of foreign gene(s) (exogenous
polynucleotide) in the host to produce the desired protein.
[0193] Techniques for inoculation of viruses to plants may be found
in Foster and Taylor, eds. "Plant Virology Protocols: From Virus
Isolation to Transgenic Resistance (Methods in Molecular Biology
(Humana Pr), Vol 81)", Humana Press, 1998; Maramorosh and
Koprowski, eds. "Methods in Virology" 7 vols, Academic Press, New
York 1967-1984; Hill, S. A. "Methods in Plant Virology", Blackwell,
Oxford, 1984; Walkey, D. G. A. "Applied Plant Virology", Wiley, New
York, 1985; and Kado and Agrawa, eds. "Principles and Techniques in
Plant Virology", Van Nostrand-Reinhold, New York.
[0194] In addition to the above, the polynucleotide of the present
invention can also be introduced into a chloroplast genome thereby
enabling chloroplast expression.
[0195] A technique for introducing exogenous nucleic acid sequences
to the genome of the chloroplasts is known. This technique involves
the following procedures. First, plant cells are chemically treated
so as to reduce the number of chloroplasts per cell to about one.
Then, the exogenous polynucleotide is introduced via particle
bombardment into the cells with the aim of introducing at least one
exogenous polynucleotide molecule into the chloroplasts. The
exogenous polynucleotides selected such that it is integratable
into the chloroplast's genome via homologous recombination which is
readily effected by enzymes inherent to the chloroplast. To this
end, the nucleic acid sequence includes, in addition to a gene of
interest, at least one polynucleotide stretch which is derived from
the chloroplast's genome. In addition, the exogenous polynucleotide
includes a selectable marker, which serves by sequential selection
procedures to ascertain that all or substantially all of the copies
of the chloroplast genomes following such selection will include
the exogenous polynucleotide. Further details relating to this
technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507
which are incorporated herein by reference. A polypeptide can thus
be produced by the protein expression system of the chloroplast and
become integrated into the chloroplast's inner membrane.
[0196] According to some embodiments of the invention, the method
further comprises growing the plant cell expressing the nucleic
acid. The plant cells can be any plant cells desired. The plant
cells can be cultured cells, cells in cultured tissue or cultured
organs, or cells in a plant. In some embodiments, the plant cells
are cultured cells, or cells in cultured tissue or cultured organs.
In yet further embodiments, the plant cells are any type of plant
that is used in gene transference. The plant cell can be grown as
part of a whole plant, or, alternatively, in plant cell
culture.
[0197] According to some aspects of the invention, the plant cells
are grown in a plant cell suspension culture. As used herein, the
term "suspension culture" refers to the growth of cells separate
from the organism. Suspension culture can be facilitated via use of
a liquid medium (a "suspension medium"). Suspension culture can
refer to the growth of cells in liquid nutrient media. Methods and
devices suitable for growing plant cells of the invention in plant
cell suspension culture are described in detail in, for example,
PCT WO2008/135991, U.S. Pat. No. 6,391,683, U.S. patent application
Ser. No. 10/784,295; International Patent Publications PCT
Nos.WO2004/091475, WO2005/080544 and WO 2006/040761, all of which
are hereby incorporated by reference as if fully set forth
herein.
[0198] Thus, the invention encompasses plants or plant cultures
expressing the nucleic acid sequences, so as to produce the
recombinant chimeric polypeptide of the invention. Once expressed
within the plant cell or the entire plant, the level of the
chimeric polypeptide encoded by the nucleic acid sequence can be
determined by methods well known in the art such as, activity
assays, Western blots using antibodies capable of specifically
binding the chimeric polypeptide (anti TNFR2, and anti Fc, See
Examples section which follows), Enzyme-Linked Immuno Sorbent Assay
(ELISA), radio-immuno-assays (RIA), immunohistochemistry,
immunocytochemistry, immunofluorescence and the like.
[0199] Methods of determining the level in the plant of the RNA
transcribed from the nucleic acid sequence are well known in the
art and include, for example, Northern blot analysis, reverse
transcription polymerase chain reaction (RT-PCR) analysis
(including quantitative, semi-quantitative or real-time RT-PCR) and
RNA-in situ hybridization.
[0200] According to some embodiments of the invention, the
expressed recombinant chimeric polypeptide of the present invention
is glycosylated in the plant cell, resulting in a chimeric
polypeptide having one, or two or three or more glycan structures
having plant specific glycan residues. Thus, according to some
embodiments of the invention, the cells expressing the expression
vector of the invention produce a chimeric polypeptide having
various amounts of glycan structures arranged in one, two, three or
more antennae. All structures may contain a core structure of two
GlcNAcs and one mannose, and variations of different amounts of
mannose, in addition to core alpha (1,3) fucose, beta (1,2) xylose,
and/or GlcNAc residues. Structures can be of the high mannose type,
having at least one, optionally at least two, optionally at least
three or optionally at least four or more mannose residues in
addition to the core structure; or complex type having both mannose
and other glycan types on each glycan, or of the hybrid type having
both high mannose and complex antennae.
[0201] In other embodiments the cells expressing the expression
vector of the invention produce a chimeric polypeptide having at
least one, optionally at least two, optionally at least three or
optionally at least four or more core xylose residues. In yet other
embodiments the cells expressing the expression vector of the
invention produce a chimeric polypeptide having at least one,
optionally at least two, optionally at least three or optionally at
least four or more core .alpha.-(1,3) fucose residues. In one
embodiment the cells expressing the expression vector of the
invention produce a chimeric polypeptide protein having at least
one exposed mannose residue, at least one core xylose residue and
at least one .alpha.-(1,3) fucose residue. In yet further
embodiments, the cells expressing the expression vector of the
invention produce a chimeric polypeptide having at least one, at
least two, at least 3 or more terminal N-acetyl glucosamine
substitutions on the outer mannose sugars.
[0202] According to a specific embodiment the chimeric polypeptide
lacks sialic acid residues. Yet further according to a specific
embodiment, the chimeric polypeptide comprises at least 40%, 45%,
50%, 55%, 60%, 65%, 70% or more complex glycans. According to a
specific embodiment, the chimeric polypeptide comprises 40-70%
complex glycans.
[0203] Purification of the secreted plant cell-expressed human
chimeric polypeptide from the cell yields a highly purified
composition comprising the prhTNFR2:Fc (also referred to herein as
TNFR2:Fc or PRX-106). Thus, in some embodiments the chimeric
polypeptide protein is purified to a homogeneity of at least 98%.
Thus the purified preparation is characterized by a purity of at
least 85%, at least 87%, at least 90%, at least 91%, at least
91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.1%,
at least 93.2%, at least 93.3%, at least 93.4%, at least 93.5%, at
least 93.6%, at least 93.7%, at least 93.8%, at least 93.9%, at
least 94%, at least 94.5%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at
least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at
least 99.7%, at least 99.8%, at least 99.9%, in a range of at least
95.0-99.8% or 100% purity. In some embodiments, purity of the
chimeric polypeptide is measured by HPLC.
[0204] In some embodiments the plant-expressed chimeric polypeptide
preparation comprises impurities derived from the plant host cell,
such as, but not limited to nucleic acids and polynucleotides,
amino acids, oligopeptides and polypeptides, glycans and other
carbohydrates, lipids and the like. In some embodiments the
host-cell derived impurities comprise biologically active
molecules, such as enzymes. In other embodiments, the
plant-expressed chimeric polypeptide composition comprises plant
beta-N-acetylhexosaminidase. Where the host cell is a tobacco cell,
or tobacco cell line cell, the plant beta-N-acetylhexosaminidase is
a tobacco beta-N-acetylhexosaminidase.
[0205] In further embodiments the plant beta-N-acetylhexosaminidase
is inactivated plant beta-N-acetylhexosaminidase. Inactivation of
plant beta-N-acetylhexosaminidase can be effected by physical
means, chemical means or biochemical means. Physical inactivation
can be performed by heating, freezing, desiccation, etc. Chemical
inactivation can be performed by extremes of pH, chemical
denaturation, addition or removal of side chains, glycans, amino
acids, etc. Biochemical inactivation includes, but is not limited
to inhibition by reversible or irreversible inhibitors. Exemplary
beta-N-acetylhexosaminidase inhibitors include end-product
inhibitors such as N-acetyl-D-glucosamine and beta-methyl-N-acetyl
glucosamine, and selective inhibitors such as the compounds
disclosed in US Patent Applications US2010016386, US20110237631,
US20100087477 and US20120046337. It will be appreciated that
preferred methods for inhibition and/or inactivation of the plant
beta-N-acetylhexosaminidase are those which also effectively
preserve the structural and functional integrity of the
plant-expressed chimeric polypeptide.
[0206] In some embodiments the plant beta-N-acetylhexosaminidase is
inactivated by heating the composition comprising the chimeric
polypeptide. Suitable temperatures for plant
beta-N-acetylhexosaminidase inhibition and/or activation include
heating within a range of 37-60.degree. C. for a period of 2 to 5,
10, 20, 30, 40, 50, 60 or more minutes. It will be appreciated that
effective inhibition and/or inactivation of the plant
beta-N-acetylhexosaminidase is achieved more rapidly at higher
temperatures and more slowly at lower temperatures of the range. In
some embodiments, the plant-expressed chimeric polypeptide
composition is heated in the range of 45-55.degree. C. for 2-10
minutes. In some embodiments, the inhibition/inactivation results
in 20, 30, 40, 50, 60, 70, 80% or greater inactivation of the plant
beta-N-acetylhexosaminidase.
[0207] The chimeric polypeptide of the invention is utilized for
the treatment of TNF.alpha.-associated medical conditions.
[0208] The term "treating" refers to inhibiting, preventing or
arresting the development of a pathology (disease, disorder or
condition) and/or causing the reduction, remission, or regression
of a pathology. Those of skill in the art will understand that
various methodologies and assays can be used to assess the
development of a pathology, and similarly, various methodologies
and assays may be used to assess the reduction, remission or
regression of a pathology.
[0209] As used herein, the term "preventing" refers to keeping a
disease, disorder or condition from occurring in a subject who may
be at risk for the disease, but has not yet been diagnosed as
having the disease.
[0210] As used herein "a TNF.alpha.-associated medical condition"
refers to a medical condition in which TNF.alpha. activity is
associated with onset, progression of the medical conditions and/or
related symptoms in a subject.
[0211] Thus, TNF.alpha.-associated medical condition disease, in a
cell, tissue, organ, animal, or subject in need thereof including,
but not limited to, at least one of obesity, an immune related
disease, a cardiovascular disease, an infectious disease, a
malignant disease or a neurologic disease (see WO0212502).
[0212] Specific examples of a TNF.alpha.-associated medical
condition include, but are not limited to, rheumatoid arthritis,
psoriatic arthritis, ankylosing spondylitis, Wegener's disease
(granulomatosis), Crohn's disease, inflammatory bowel disease,
short bowel syndrome, cholitis, ulcerative cholitis, chronic
obstructive pulmonary disease (COPD), Hepatitis C, endometriosis,
asthma, cachexia, psoriasis, and atopic dermatitis. Additional
diseases or disorders that can be treated with the chimeric
polypeptide of the invention include those described in WO
00/62790, WO 01/62272 and U.S. Patent Application No. 2001/0021380,
U.S. Pat. No. 7,648,702, the relevant portions of which are
incorporated herein by reference.
[0213] Other examples of TNF.alpha.-associated medical conditions
include, but are not limited to those disclosed in Kuek et al.
Postgrad. Med. J. 2007; 83; 251-260, which is herein incorporated
by reference in its entirety.
[0214] Thus, exemplary indications include, Sjorgen's syndrome,
polymyositis, dermatomyositis, Wegener's vasculitis, Bechet's,
giant cell arteritis (GCA), Polymyalgia rheumatic, Takayasu's
arteritis, Polyarteritis nodosa, Sarcoidosis, adult onset Still's
disease, Kawasaki disease, Cryoglobulinemic vasculitis, relapsing
polychondritis, Hidradenitis suppurativa, Coeliac disease,
myelodysplastic syndromes, Pyoderma gangrenosum, Erythema nodosum,
SAPHO syndrome, graft versus host disease, chronic hepatitis B/C,
thrombic/idiopathic thrombocytopenic purpura, refractory asthma,
lupus, amyloidosis, Multicentric reticulohistiocytosis, pemphigus,
Grave's disease, antiphospholipid syndrome, idiopathic membranous
glomerulonephritis, Hep C associated glomerulonephritis, myasthenia
gravis and multiple sclerosis.
[0215] According to a specific embodiment, TNF.alpha.-associated
medical condition is an inflammatory bowel disease. According to a
specific embodiment, the polypeptide is administered enterally,
e.g., orally, such as comprised in the plant cells.
[0216] According to a specific embodiment, the inflammatory bowel
disease is ulcerative colitis or Crohn's disease. According to a
specific embodiment, the polypeptide is administered enterally,
e.g., orally, such as comprised in the plant cells.
[0217] Additional examples of TNF.alpha.-associated medical
condition include, but are not limited to, immune related disease,
such as rheumatoid arthritis, juvenile rheumatoid arthritis,
systemic onset juvenile rheumatoid arthritis, psoriatic arthritis,
ankylosing spondilitis, gastric ulcer, seronegative arthropathies,
osteoarthritis, inflammatory bowel disease, short bowel syndrome,
ulcerative colitis, systemic lupus erythematosis, antiphospholipid
syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic
pulmonary fibrosis, systemic vasculitis/wegener's granulomatosis,
sarcoidosis, orchitis/vasectomy reversal procedures,
allergic/atopic diseases, asthma, allergic rhinitis, eczema,
allergic contact dermatitis, allergic conjunctivitis,
hypersensitivity pneumonitis, transplants, organ transplant
rejection, graft-versus-host disease, systemic inflammatory
response syndrome, sepsis syndrome, gram positive sepsis, gram
negative sepsis, culture negative sepsis, fungal sepsis,
neutropenic fever, urosepsis, meningococcemia, trauma/hemorrhage,
burns, ionizing radiation exposure, acute pancreatitis, adult
respiratory distress syndrome, rheumatoid arthritis,
alcohol-induced hepatitis, chronic inflammatory pathologies,
sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes,
nephrosis, atopic diseases, hypersensitity reactions, allergic
rhinitis, hay fever, perennial rhinitis, conjunctivitis,
endometriosis, asthma, urticaria, systemic anaphalaxis, dermatitis,
pernicious anemia, hemolytic disease, thrombocytopenia, graft
rejection of any organ or tissue, kidney translplant rejection,
heart transplant rejection, liver transplant rejection, pancreas
transplant rejection, lung transplant rejection, bone marrow
transplant (BMT) rejection, skin allograft rejection, cartilage
transplant rejection, bone graft rejection, small bowel transplant
rejection, fetal thymus implant rejection, parathyroid transplant
rejection, xenograft rejection of any organ or tissue, allograft
rejection, anti-receptor hypersensitivity reactions-, Graves
disease, Raynoud's disease, type B insulin-resistant diabetes,
asthma, myasthenia gravis, antibody-meditated cytotoxicity, type
III hypersensitivity reactions, systemic lupus erythematosus, POEMS
syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal
gammopathy, and skin changes syndrome), polyneuropathy,
organomegaly, endocrinopathy, monoclonal gammopathy, skin changes
syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed
connective tissue disease, idiopathic Addison's disease, diabetes
mellitus, chronic active hepatitis, primary billiary cirrhosis,
vitiligo, vasculitis, post-MI cardiotomy syndrome, type IV
hypersensitivity, contact dermatitis, hypersensitivity pneumonitis,
allograft rejection, granulomas due to intracellular organisms,
drug sensitivity, metabolic/idiopathic, Wilson's disease,
hemachromatosis, alpha-1-antitrypsin deficiency, diabetic
retinopathy, hashimoto's thyroiditis, osteoporosis,
hypothalamic-pituitary-adrenal axis evaluation, primary biliary
cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic
fibrosis, neonatal chronic lung disease, chronic obstructive
pulmonary disease (COPD), familial hematophagocytic
lymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,
nephrotic syndrome, nephritis, glomerular nephritis, acute renal
failure, hemodialysis, uremia, toxicity, preeclampsia, okt3
therapy, anti-cd3 therapy, cytokine therapy, chemotherapy,
radiation therapy (e.g, including but not limited toasthenia,
anemia, cachexia, and the like), chronic salicylate intoxication,
and the like. See, e.g, the Merck Manual, 12th-17th Editions, Merck
& Company, Rahway, N.J. (1972, 1977, 1982, 1987, 1992, 1999),
Pharmacotherapy Handbook, Wells et al, eds. Second Edition,
Appleton and Lange, Stamford, Conn. (1998, 2000), each entirely
incorporated by reference. The present invention also provides a
method for treating at least one cardiovascular disease in a cell,
tissue, organ, animal, or patient, including, but not limited to,
at least one of cardiac stun syndrome, myocardial infarction,
congestive heart failure, stroke, ischemic stroke, hemorrhage,
arteriosclerosis, atherosclerosis, restenosis, diabetic
ateriosclerotic disease, hypertension, arterial hypertension,
renovascular hypertension, syncope, shock, syphilis of the
cardiovascular system, heart failure, cor pulmonale, primary
pulmonary hypertension, cardiac arrhythmias, atrial ectopic beats,
atrial flutter, atrial fibrillation (sustained or paroxysmal), post
perfusion syndrome, cardiopulmonary bypass inflammation response,
chaotic or multifocal atrial tachycardia, regular narrow QRS
tachycardia, specific arrythmias, ventricular fibrillation, His
bundle arrythmias, atrioventricular block, bundle branch block,
myocardial ischemic disorders, coronary artery disease, angina
pectoris, myocardial infarction, cardiomyopathy, dilated congestive
cardiomyopathy, restrictive cardiomyopathy, valvular heart
diseases, endocarditis, pericardial disease, cardiac tumors, aordic
and peripheral aneuryisms, aortic dissection, inflammation of the
aorta, occulsion of the abdominal aorta and its branches,
peripheral vascular disorders, occulsive arterial disorders,
peripheral atherlosclerotic disease, thromboangitis obliterans,
functional peripheral arterial disorders, Raynaud's phenomenon and
disease, acrocyanosis, erythromelalgia, venous diseases, venous
thrombosis, varicose veins, arteriovenous fistula, lymphederma,
lipedema, unstable angina, reperfusion injury, post pump syndrome,
ischemia-reperfusion injury, and the like.
[0218] Additional examples of a TNF.alpha.-associated medical
condition include, but are not limited to, infectious diseases,
such as acute or chronic bacterial infection, acute and chronic
parasitic or infectious processes, including bacterial, viral and
fungal infections, HIV infection/HIV neuropathy, meningitis,
hepatitis (A, B or C, or the like), septic arthritis, peritonitis,
pneumonia, epiglottitis, e. coli 0157:h7, hemolytic uremic
syndrome/thrombolytic thrombocytopenic purpura, malaria, dengue
hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome,
streptococcal myositis, gas gangrene, mycobacterium tuberculosis,
mycobacterium avium intracellulare, pneumocystis carinii pneumonia,
pelvic inflammatory disease, orchitis/epidydimitis, legionella,
lyme disease, influenza a, epstein-barr virus, vital-associated
hemaphagocytic syndrome, vital encephalitis/aseptic meningitis, and
the like.
[0219] Additional examples of a TNF.alpha.-associated medical
condition include, but are not limited to, malignant diseases such
as leukemia, acute leukemia, acute lymphoblastic leukemia (ALL),
B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic
myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL),
hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma,
Hodgkin's disease, a malignant lymphoma, non-hodgkin's lymphoma,
Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal
carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma,
malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of
malignancy, solid tumors, adenocarcinomas, sarcomas, malignant
melanoma, hemangioma, metastatic disease, cancer related bone
resorption, cancer related bone pain, and the like.
[0220] Additional examples of a TNF.alpha.-associated medical
condition include, but are not limited to, neurologic diseases,
such as neurodegenerative diseases, multiple sclerosis, migraine
headache, AIDS dementia complex, demyelinating diseases, such as
multiple sclerosis and acute transverse myelitis; extrapyramidal
and cerebellar disorders' such as lesions of the corticospinal
system; disorders of the basal ganglia or cerebellar disorders;
hyperkinetic movement disorders such as Huntington's Chorea and
senile chorea; drug-induced movement disorders, such as those
induced by drugs which block CNS dopamine receptors; hypokinetic
movement disorders, such as Parkinson's disease; Progressive
supranucleo Palsy; structural lesions of the cerebellum;
spinocerebellar degenerations, such as spinal ataxia, Friedreich's
ataxia, cerebellar cortical degenerations, multiple systems
degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and
Machado-Joseph); systemic disorders (Refsum's disease,
abetalipoprotemia, ataxia, telangiectasia, and mitochondrial
multisystem disorder); demyelinating core disorders, such as
multiple sclerosis, acute transverse myelitis; and disorders of the
motor unit, such as neurogenic muscular atrophies (anterior horn
cell degeneration, such as amyofrophic lateral sclerosis, infantile
spinal muscular atrophy and juvenile spinal muscular atrophy);
Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy
body disease; Senile Dementia of Lewy body type; Wernicke-Korsakoff
syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute
sclerosing panencephalitis, Hallerrorden-Spatz disease; and
Dementia pugilistica, and the like.
[0221] As used herein, the term "subject" includes mammals, e.g.,
human beings at any age which suffer from the pathology. According
to a specific embodiment, this term encompasses individuals who are
at risk to develop the pathology.
[0222] It has been shown that plant cells expressing biologically
active human recombinant polypeptides can be used as an effective
systemic delivery system, when provided for enteral administration
to the subject (see WO2007/010533). Thus, in some embodiments, the
chimeric polypeptide can be formulated in a pharmaceutical
composition for oral or enteral delivery comprising transformed
plant cell expressing the chimeric polypeptide and a
pharmaceutically acceptable carrier. In some embodiments, the
transformed plant cells of the pharmaceutical composition are
lyophilized plant cells.
[0223] As used herein the phrase "enteral administration" refers to
administration through any part of the gastro-intestinal tract,
such as rectal administration, colonic administration, intestinal
administration (proximal or distal) and gastric administration. In
some embodiments, enteral administration refers to oral
administration.
[0224] It will be appreciated that the present teachings are also
directed at mucosal administration of plant cells expressing the
chimeric polypeptide of the invention.
[0225] The cells may be formulated as a solid, formulated as a
liquid or formulated as a powder. In some embodiments, the cells
are resuspended, lyophilized cells.
[0226] Thus, in some embodiments, the chimeric polypeptide can be
formulated in a pharmaceutical composition for oral or enteral
delivery comprising transformed plant cell expressing the chimeric
polypeptide and a pharmaceutically acceptable carrier. In some
embodiments, the transformed plant cells of the pharmaceutical
composition are lyophilized plant cells, although the use of fresh
(non-lyophilized cells), plant tissues, plant parts or whole plants
is also contemplated herein.
[0227] Prior to lyophilization the cells may be washed to remove
any cell debris that may be present in the growth medium.
[0228] As the cells are being prepared for lyophilization, it is
sometimes desirable to incubate the cells in a maintenance medium
to reduce the metabolic processes of the cells.
[0229] Pretreatment (although not necessary) can be performed at
room temperature or at temperatures in which the plant cells are
typically cultured. Pretreatment is performed at about room
temperature (20.degree. C.) for ease of handling and as most plant
cells are fairly stable at room temperature. Stabilizers can be
added directly to the medium and replenished as necessary during
the pretreatment process.
[0230] Pretreatments may also involve incubating cells in the
presence of one or more osmotic agents. Examples of useful osmotic
agents include sugars such as saccharides and saccharide
derivatives, amino or imino acids such as proline and proline
derivatives, or combinations of these agents. Some of the more
useful sugars and sugar derivatives are fructose, glucose, maltose,
mannitol, sorbitol, sucrose and trehalose. Osmotic agents are
utilized at a concentration that prepares cells for subsequent
lyophilization.
[0231] Lyophilization is directed at reducing the water content of
the cells by vacuum evaporation. Vacuum evaporation involves
placing the cells in an environment with reduced air pressure.
Depending on the rate of water removal desired, the reduced ambient
pressure operating at temperatures of between about -30.degree. C.
to -50.degree. C. may be at 100 torr, 1 torr, 0.01 torr or less.
According to a specific embodiment, the cells are lyophilized by
freezing to -40.degree. C. and then applying a vacuum to a pressure
of 0.1 mbar. The cells are then heated to -10.degree. C. so all the
ice content will be sublimated and evaporated. Under conditions of
reduced pressure, the rate of water evaporation is increased such
that up to 60-95% of the water in a cell can be removed.
[0232] According to a specific embodiment, lyophilization removes
over 60%, 70%, 80% or specifically over 90%, 91%, 92%, 93%, 94%,
95% or 98% of the water from the cells. According to a specific
embodiment, the final water content is about 5-10%, 5-8% or
6-7%.
[0233] Thus, the oral dosage form may be provided as an oral
nutritional form (e.g., as long as the protein is not exposed to
denaturing conditions which include heating above 37.degree. C. and
compression), as a complete meal, as a powder for dissolution, e.g.
health drinks, as a solution, as a ready-made drink, optionally low
calorie, such as a soft drink, including juices, milk-shake,
yoghurt drink, smoothie or soy-based drink, in a bar, or dispersed
in foods of any sort, such as baked products, cereal bars, dairy
bars, snack-foods, breakfast cereals, muesli, candies, tabs,
cookies, biscuits, crackers (such as a rice crackers), chocolate,
and dairy products.
[0234] Of note is the use of plant cells expressing the chimeric
polypeptide in the treatment of inflammatory bowel disease. The
plant's cell wall is expected to protect the chimeric polypeptide
while moving through the stomach and small intestine. In the colon,
where the polysaccharides are digested, the plant cell is expected
to release its content and hence prh TNRF:Fc is available to bind
its cytokine ligand. Moreover, prh TNRF2:Fc is a chimeric protein
carries an Fc segment of human IgG1. In the epithelial monolayer
lining the mucosal barrier, the FcRn receptor transcytoses IgG
molecules across by binding to their Fc. Therefore, prh TNRF:Fc can
also cross the epithelial barrier to bind its cytokine ligand on
the serosal side of the epithelia.
[0235] It will be appreciated that the present teachings exclude
the use of plant cells expressing the chimeric polypeptide by
enteral administration for the treatment of medical conditions
directly associated with obesity, metabolic syndrome, diabetes and
a liver disease or disorder.
[0236] The metabolic syndrome is a constellation of interrelated
risk factors of metabolic origin--metabolic risk factors--that
appear to directly promote the development of atherosclerotic
cardiovascular disease (ASCVD). Patients with the metabolic
syndrome also are at increased risk for developing type 2 diabetes
mellitus. The multiple components and criteria that define the
metabolic syndrome have varied somewhat in specific elements, but
in general they include a combination of both underlying and
metabolic risk factors. The most widely recognized of the metabolic
risk factors are atherogenic dyslipidemia, elevated blood pressure,
and elevated plasma glucose. Individuals with these characteristics
commonly manifest a prothrombotic state and a proinflammatory state
as well. Atherogenic dyslipidemia consists of an aggregation of
lipoprotein abnormalities including elevated serum triglyceride and
apolipoprotein B (apoB), increased small LDL particles, and a
reduced level of HDL cholesterol (HDL-C). Available data suggest
that it truly is a syndrome, i.e., a grouping of ASCVD risk
factors, but one that probably has more than one cause.
[0237] According to a specific embodiment, the liver disease or
disorder is selected from the group consisting of hepatitis, liver
cirrhosis, liver cancer, hepatotoxicity, chronic liver disease,
fatty liver disease and non-alcoholic steatohepatitis (NASH).
[0238] According to a specific embodiment, when the TNFR2:Fc is
administered enterally (e.g., orally) in plant cells, the medical
condition is not an obesity, metabolic syndrome, diabetes and a
liver disease or disorder.
[0239] According to a specific embodiment, the hepatotoxicity is
induced by a chemical agent selected from the group consisting of
acetaminophen, NSAIDS, glucocorticoid, isoniazid, arsenic,
chemotherapy, carbon tetrachloride and vinyl chloride.
[0240] According to a specific embodiment, the diabetes is selected
from the group consisting of type I diabetes, type II diabetes and
LADA disease.
[0241] Alternatively or additionally, the chimeric polypeptide of
some embodiments of the invention can be administered to an
organism per se, or in a pharmaceutical composition where it is
mixed with suitable carriers or excipients.
[0242] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0243] Herein the term "active ingredient" refers to the chimeric
polypeptide or cells expressing same accountable for the biological
effect.
[0244] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0245] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0246] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0247] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intracardiac, e.g., into the right or left
ventricular cavity, into the common coronary artery, intravenous,
intraperitoneal, intranasal, or intraocular injections.
[0248] The pharmaceutical compositions of this invention are
particularly useful for parenteral administration, i.e.,
subcutaneously, intramuscularly, intravenously, intraperitoneal,
intracerebrospinal, intra-articular, intrasynovial, and/or
intrathecal. Parenteral administration can be by bolus injection or
continuous infusion. Pharmaceutical compositions for injection may
be presented in unit dosage form, e.g., in ampoules or in
multi-dose containers, with an added preservative. In addition, a
number of recent drug delivery approaches have been developed and
the pharmaceutical compositions of the present invention are
suitable for administration using these new methods, e.g.,
Inject-Ease.TM., Genject.TM., injector pens such as GenPen.TM., and
needleless devices such as MediJector.TM. and BioJector.TM..
[0249] The pharmaceutical composition can also be formulated as a
depot preparation. Such long acting formulations may be
administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the formulations may be modified with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0250] Pharmaceutical compositions of some embodiments of the
invention may be manufactured by processes well known in the art,
e.g., by means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping
or lyophilizing processes.
[0251] Pharmaceutical compositions for use in accordance with some
embodiments of the invention thus may be formulated in conventional
manner using one or more physiologically acceptable carriers
comprising excipients and auxiliaries, which facilitate processing
of the active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0252] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0253] For oral administration, the pharmaceutical composition can
be formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0254] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0255] Pharmaceutical compositions which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0256] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0257] For administration by nasal inhalation, the active
ingredients for use according to some embodiments of the invention
are conveniently delivered in the form of an aerosol spray
presentation from a pressurized pack or a nebulizer with the use of
a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichloro-tetrafluoroethane or carbon
dioxide. In the case of a pressurized aerosol, the dosage unit may
be determined by providing a valve to deliver a metered amount.
Capsules and cartridges of, e.g., gelatin for use in a dispenser
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0258] The pharmaceutical composition described herein may be
formulated for parenteral administration, e.g., by bolus injection
or continuous infusion. Formulations for injection may be presented
in unit dosage form, e.g., in ampoules or in multidose containers
with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0259] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0260] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0261] The pharmaceutical composition of some embodiments of the
invention may also be formulated in rectal compositions such as
suppositories or retention enemas, using, e.g., conventional
suppository bases such as cocoa butter or other glycerides.
[0262] Pharmaceutical compositions suitable for use in context of
some embodiments of the invention include compositions wherein the
active ingredients are contained in an amount effective to achieve
the intended purpose. More specifically, a therapeutically
effective amount means an amount of active ingredients (chimeric
polypeptide) effective to prevent, alleviate or ameliorate symptoms
of a disorder or prolong the survival of the subject being
treated.
[0263] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0264] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. For example, a
dose can be formulated in animal models to achieve a desired
concentration or titer. Such information can be used to more
accurately determine useful doses in humans.
[0265] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl, et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1).
[0266] Dosage amount and interval may be adjusted individually to
provide the chimeric polypeptide (the target tissue) levels of the
active ingredient are sufficient to induce or suppress the
biological effect (minimal effective concentration, MEC). The MEC
will vary for each preparation, but can be estimated from in vitro
data. Dosages necessary to achieve the MEC will depend on
individual characteristics and route of administration. Detection
assays can be used to determine plasma concentrations.
[0267] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0268] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0269] In one embodiment, the effective chimeric polypeptide amount
per adult dose ranges from about 1-500 mg/m.sup.2, or from about
1-200 mg/m.sup.2, or from about 1-40 mg/m.sup.2 or about 5-25
mg/m.sup.2. Alternatively, a flat dose may be administered, whose
amount may range from 2-500 mg/dose, 2-100 mg/dose or from about
10-80 mg/dose.
[0270] In one embodiment, the effective chimeric polypeptide amount
per adult dose is about 1-500 mg/m.sup.2, or about 1-200
mg/m.sup.2, or about 1-40 mg/m.sup.2 or about 5-25 mg/m.sup.2.
Alternatively, a flat dose may be administered, whose amount may
range about 2-500 mg/dose, 2-100 mg/dose or from about 10-80
mg/dose.
[0271] In another embodiment the effective chimeric polypeptide
amount per adult dose range is about 0.0002 mg/kg to 2 mg/kg, about
0.002-2 mg/kg, about 0.02-2 mg/kg, about 0.2-2 mg/kg, about
0.002-0.2 mg/kg, about 0.0002-1 mg/kg, about 0.002-0.1 mg/kg, about
0.002-0.02 mg/kg, about 0.002-0.01 mg/kg, about 0.002-0.008 mg/kg,
about 0.02-0.1 mg/kg, about 0.001-0.05 mg/kg, about 0.001-0.01
mg/kg, about 0.01-1 mg/kg, about 0.01-15 mg/kg, about 0.005-1
mg/kg, about 0.01-5 mg/kg, about 0.005-0.01 mg/kg or about 0.05-0.1
mg/kg. According to a further specific embodiment, the effective
chimeric polypeptide amount per adult dose range is about 0.02-0.3
mg/kg, 0.02-0.7 mg/kg, 0.02-0.247 mg/kg, 0.02-0.12 mg/kg, 0.12-0.24
mg/kg, 0.02-0.1 mg/kg or 0.15-0.247 mg/kg.
[0272] According to a specific embodiment, these dose ranges are
used for oral administration such as of plant cells expressing the
chimeric protein.
[0273] According to a specific embodiment, the effective chimeric
polypeptide amount per adult dose ranges about 0.002-0.2 mg/kg or
0.02-0.3 mg/kg. According to a specific embodiment the effective
chimeric polypeptide amount per adult dose ranges about 0.02-0.3
mg/kg, 0.02-0.27 mg/kg or 0.02-0.247 mg/kg. According to a specific
embodiment, this dose range is used for oral administration such as
of plant cells expressing the chimeric protein.
[0274] Alternatively, a flat dose may be administered, whose amount
may range about 2-500 mg/dose, 2-100 mg/dose or from 10-80 mg/dose.
According to a specific embodiment, this dose range is used for
oral administration such as of plant cells expressing the chimeric
protein.
[0275] According to a specific embodiment, a flat dose of 0.01-100
mg, 0.1-100 mg, 0.1-50 mg, 1-20 mg, 1-10 mg, 10-20 mg, 15-20 mg,
0.1-20 mg, 0.1-10 mg, 0.1-5 mg is administered. According to a
specific embodiment, this dose range is used for oral
administration such as of plant cells expressing the chimeric
protein.
[0276] Any of these doses can be packed in a unit dosage form which
comprises plant cells expressing (per batch and not per cell) the
polypeptide in an amount of 1-20 mg, 1-10 mg, 10-20 mg or 15-20
mg.
[0277] According to a specific embodiment the flat dose is about
0.1-10 mg. According to a specific embodiment, this dose range is
used for oral administration such as of plant cells expressing the
chimeric protein.
[0278] According to a specific embodiment, the oral dose is
administered daily. The dose may be divided for a number of
administrations during the day (say 2-4 times a day). The dose can
also be administered every two days, two times a week, three times
a week, biweekly, weekly doses, or separated by several weeks (for
example 2 to 8).
[0279] According to a specific embodiment, if the dose is to be
administered more than one time per week, an exemplary dose range
is the same as the foregoing described dose ranges or lower and
administered two or more times per week (e.g., 25-100 mg/dose). In
another embodiment, an acceptable dose for administration by
injection contains 80-100 mg/dose, or alternatively, containing 80
mg per dose.
[0280] The dose may be modified for children and infants.
[0281] The dose can be administered at biweekly, weekly doses, or
separated by several weeks (for example 2 to 8). According to a
specific embodiment the chimeric polypeptide is generally
administered at 25 mg by a single subcutaneous (SC) injection.
[0282] In many instances, an improvement in a patient's condition
will be obtained by a dose of up to about 100 mg of the
pharmaceutical composition one to three times per week over a
period of at least three weeks, though treatment for longer periods
may be necessary to induce the desired degree of improvement. For
incurable chronic conditions the regimen may be continued
indefinitely. For pediatric patients (ages 4-17), a suitable
regimen involves a dose of 0.4 mg/kg to 5 mg/kg of the chimeric
polypeptides of the invention by injection, administered one or
more times per week.
[0283] In another embodiment, it is contemplated that the
pharmaceutical formulation of the invention is prepared in a bulk
formulation and as such, the components of the pharmaceutical
composition are adjusted so that it is higher than would be
required for administration and diluted appropriately prior to
administration.
[0284] Compositions of some embodiments of the invention may, if
desired, be presented in a pack or dispenser device, such as an FDA
approved kit, which may contain one or more unit dosage forms
containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be accommodated by a
notice associated with the container in a form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the compositions or human or veterinary
administration. Such notice, for example, may be of labeling
approved by the U.S. Food and Drug Administration for prescription
drugs or of an approved product insert. Compositions comprising a
preparation of the invention formulated in a compatible
pharmaceutical carrier may also be prepared, placed in an
appropriate container, and labeled for treatment of an indicated
condition, as is further detailed above. The concentration of the
polypeptide in the aqueous pharmaceutical composition can vary over
a wide range, but is generally within the range of from about 0.05
to about 20,000 micrograms per milliliter .mu./ml) of aqueous
formulation.
[0285] Of note dosage forms which comprise the plant cells may
include additives such as one or more of calcium, magnesium, iron,
zinc, phosphorus, vitamin D and vitamin K. A suitable daily amount
is 0.1 mg to 3.6 g calcium, preferably 320 to 530 mg. In general,
the daily dosage of vitamins and minerals in the nutritional
formulation or medicament of the invention is 25-100% by weight of
the dosages recommended by the health authorities. Dietary fiber
may also be a component of the compositions of the invention.
Further components of the supplement may include any bioactive
compounds or extracts which are known to have health benefits,
especially for improving physical performance.
[0286] Generally the unit dosage form may further comprise an
antioxidant (exemplary embodiments are provided above. In another
embodiment, the antioxidant is a pharmaceutically acceptable
antioxidant. In another embodiment, the antioxidant is selected
from the group consisting of vitamin E, superoxide dismutase (SOD),
omega-3, and beta-carotene.
[0287] In another embodiment, the unit dosage form further
comprises an enhancer of the biologically active protein or
peptide. In another embodiment, the unit dosage form further
comprises a cofactor of the biologically active protein or
peptide.
[0288] In another embodiment, a unit dosage form of the present
invention further comprises pharmaceutical-grade surfactant.
Surfactants are well known in the art, and are described, inter
alia, in the Handbook of Pharmaceutical Excipients (eds. Raymond C
Rowe, Paul J Sheskey, and Sian C Owen, copyright Pharmaceutical
Press, 2005). In another embodiment, the surfactant is any other
surfactant known in the art.
[0289] In another embodiment, a unit dosage form of the present
invention further comprises pharmaceutical-grade emulsifier or
emulgator (emollient). Emulsifiers and emulgators are well known in
the art, and are described, inter alia, in the Handbook of
Pharmaceutical Excipients (ibid). Non-limiting examples of
emulsifiers and emulgators are eumulgin, Eumulgin B1 PH, Eumulgin
B2 PH, hydrogenated castor oil cetostearyl alcohol, and cetyl
alcohol. In another embodiment, the emulsifier or emulgator is any
other emulsifier or emulgator known in the art.
[0290] In another embodiment, a unit dosage form of the present
invention further comprises pharmaceutical-grade stabilizer.
Stabilizers are well known in the art, and are described, inter
alia, in the Handbook of Pharmaceutical Excipients (ibid). In
another embodiment, the stabilizer is any other stabilizer known in
the art.
[0291] In another embodiment, a unit dosage form of the present
invention further comprises an amino acid selected from the group
consisting of arginine, lysine, aspartate, glutamate, and
histidine. In another embodiment, analogues and modified versions
of arginine, lysine, aspartate, glutamate and histidine are
included in the terms "arginine," "lysine," "aspartate",
"glutamate" and "histidine," respectively. In another embodiment,
the amino acid provides additional protection of ribonuclease or
other active molecules. In another embodiment, the amino acid
promotes interaction of biologically active protein or peptide with
a target cell. In another embodiment, the amino acid is contained
in an oil component of the unit dosage form.
[0292] In another embodiment, a unit dosage form of the present
invention further comprises one or more pharmaceutically acceptable
excipients, into which the matrix carrier unit dosage form is
mixed. In another embodiment, the excipients include one or more
additional polysaccharides. In another embodiment, the excipients
include one or more waxes. In another embodiment, the excipients
provide a desired taste to the unit dosage form. In another
embodiment, the excipients influence the drug consistency, and the
final dosage form such as a gel capsule or a hard gelatin
capsule.
[0293] Non limiting examples of excipients include: Antifoaming
agents (dimethicone, simethicone); Antimicrobial preservatives
(benzalkonium chloride, benzelthonium chloride, butylparaben,
cetylpyridinium chloride, chlorobutanol, chlorocresol, cresol,
ethylparaben, methylparaben, methylparaben sodium, phenol,
phenylethyl alcohol, phenylmercuric acetate, phenylmercuric
nitrate, potassium benzoate, potassium sorbate, propylparaben,
propylparaben sodium, sodium benzoate, sodium dehydroacetate,
sodium propionate, sorbic acid, thimerosal, thymol); Chelating
agents (edetate disodium, ethylenediaminetetraacetic acid and
salts, edetic acid); Coating agents (sodium
carboxymethyl-cellulose, cellulose acetate, cellulose acetate
phthalate, ethylcellulose, gelatin, pharmaceutical glaze,
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
hydroxypropyl methylcellulose phthalate, methacrylic acid
copolymer, methylcellulose, polyethylene glycol, polyvinyl acetate
phthalate, shellac, sucrose, titanium dioxide, carnauba wax,
microcrystalline wax, zein); Colorants (caramel, red, yellow, black
or blends, ferric oxide); Complexing agents
(ethylenediaminetetraacetic acid and salts (EDTA), edetic acid,
gentisic acid ethanolmaide, oxyquinoline sulfate); Desiccants
(calcium chloride, calcium sulfate, silicon dioxide); Emulsifying
and/or solubilizing agents (acacia, cholesterol, diethanolamine
(adjunct), glyceryl monostearate, lanolin alcohols, lecithin, mono-
and di-glycerides, monoethanolamine (adjunct), oleic acid
(adjunct), oleyl alcohol (stabilizer), poloxamer, polyoxyethylene
50 stearate, polyoxyl 35 caster oil, polyoxyl 40 hydrogenated
castor oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether,
polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate
60, polysorbate 80, propylene glycol diacetate, propylene glycol
monostearate, sodium lauryl sulfate, sodium stearate, sorbitan
monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan
monostearate, stearic acid, trolamine, emulsifying wax); Flavors
and perfumes (anethole, benzaldehyde, ethyl vanillin, menthol,
methyl salicylate, monosodium glutamate, orange flower oil,
peppermint, peppermint oil, peppermint spirit, rose oil, stronger
rose water, thymol, tolu balsam tincture, vanilla, vanilla
tincture, vanillin); Humectants (glycerin, hexylene glycol,
propylene glycol, sorbitol); Polymers (e.g., cellulose acetate,
alkyl celluloses, hydroxyalkylcelluloses, acrylic polymers and
copolymers); Suspending and/or viscosity-increasing agents (acacia,
agar, alginic acid, aluminum monostearate, bentonite, purified
bentonite, magma bentonite, carbomer 934p, carboxymethylcellulose
calcium, carboxymethylcellulose sodium, carboxymethycellulose
sodium 12, carrageenan, microcrystalline and carboxymethylcellulose
sodium cellulose, dextrin, gelatin, guar gum, hydroxyethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
magnesium aluminum silicate, methylcellulose, pectin, polyethylene
oxide, polyvinyl alcohol, povidone, propylene glycol alginate,
silicon dioxide, colloidal silicon dioxide, sodium alginate,
tragacanth, xanthan gum); Sweetening agents (aspartame, dextrates,
dextrose, excipient dextrose, fructose, mannitol, saccharin,
calcium saccharin, sodium saccharin, sorbitol, solution sorbitol,
sucrose, compressible sugar, confectioner's sugar, syrup). This
list is not meant to be exclusive, but instead merely
representative of the classes of excipients and the particular
excipients which may be used in oral dosage unit dosage forms of
the present invention.
[0294] Conventional additives may be included in the compositions
of the invention, including any of those selected from
preservatives, chelating agents, effervescing agents, natural or
artificial sweeteners, flavoring agents, coloring agents, taste
masking agents, acidulants, emulsifiers, thickening agents,
suspending agents, dispersing or wetting agents, antioxidants, and
the like. Flavoring agents can be added to the compositions of the
invention to aid in compliance with a dosing regimen. Typical
flavoring agents include, but are not limited to natural or
synthetic essences, oils and/or extracts of pineapple, orange,
lemon, mint, berry, chocolate, vanilla and melon.
[0295] As used herein the term "about" refers to .+-.10%.
[0296] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0297] The term "consisting of" means "including and limited
to".
[0298] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0299] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0300] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0301] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0302] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0303] It is appreciated that certain features of the invention,
which are, for clarity, described in the context 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 subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0304] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0305] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
[0306] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
Materials and Experimental Procedures
[0307] Expression Constructs and Expression
[0308] cDNA encoding prh TNFR2:Fc was optimized and synthesized by
GENEART AG (Regensburg, Germany). The codon usage was adapted to
the codon bias of Nicotiana tabacum genes. The IgG1 portion was
cloned from Fc IgG1 heavy chain constant region [Homo sapiens]
ACCESSION AEV43323.
[0309] During the optimization process the following cis-acting
sequence motifs were avoided: Internal TATA-boxes, chi-sites and
ribosomal entry sites, AT-rich or GC-rich sequence stretches, RNA
instability elements ("Killer motifs"), Repeat sequences and RNA
secondary structures, splice donor (cryptic) and acceptor sites,
branch points. In addition, regions of very high (>80%) or very
low (<30%) GC content were avoided. The resultant DNA sequence
is as set forth in SEQ ID NO: 1. The encoded polypeptide is as set
forth in SEQ ID NO: 2. To the native cDNA sequence, a signal
peptide (e.g. endoplasmic reticulum target signal peptide) from N.
plumbaginifolia Calreticulin protein was added to the N' terminus
of the gene, allowing efficient targeting of prh TNFR2:Fc to the
secretory pathway and is then cleaved from the polypeptide, by
signal peptidase, once the protein has been translocated into the
endoplasmic reticulum (SEQ ID NO: 3, SEQ ID NO: 4, representing the
DNA and peptide sequences of the ER signal peptide, respectively).
Additionally, an ER retention signal SEKDEL was added to the C'
terminus of the gene. This signal allows protein retrieval from the
Golgi apparatus to the ER, and localization in the ER. The entire
coding sequence (signal peptide-prh TNFR2:Fc-SEKDEL) is as set
forth in SEQ ID NO: 5 and the encoded polypeptide is as set forth
in SEQ ID NO: 6. The resultant protein following cleavage is as set
forth in SEQ ID NO: 7, 204 or 205 (prh TNFR2:Fc-SEKDEL).
[0310] Stable Expression in N. tabacum BY2 Cells
[0311] Agrobacterium mediated transformation is widely used to
introduce foreign genes into a plant cell genome. Using this
approach, a T-DNA molecule consisting of a foreign gene and its
regulatory elements is randomly introduced into the plant genome.
Since the site of integration, as well as the copy number of the
gene insertions cannot be controlled, the transformation process
results in a highly heterogeneous transgenic `pool` composed of
cells with various levels of transgene expression. The transgenic
`pool` is subsequently used for clone isolation. The transformation
process, results in establishment of numerous single cell lines,
each representing an individual transformation event, from which
the clone with the highest expression level of the foreign gene is
selected. For prh TNFR2:Fc, the transformation was conducted with a
plasmid carrying the prh TNFR2:Fc cassette (FIG. 1 SEQ ID NOs: 7
and 8). As a result, the recombinant protein is targeted to the
Endoplasmic reticulum (ER) of the cells. The transformations of the
BY2 cells with the prh TNFR2:FC-ER expression vector were performed
by the Agrobacterium tumefaciens mediated plant transformation
procedure as follow: BY2 (Bright Yellow 2) suspension culture was
co-cultivated, for 48 hours, with the Agrobacterium tumefactiens
strain carrying the vector harboring the prhTNFR2:FC-gene and the
neomycin phosphotransferase (NPTII) selection gene. Subsequently,
cells were kept in media supplemented with 50 mg/L of Kanamaycin
and 250 mg/L Cefotaxime. The NPTII gene confers resistance to
Kanamycin, thus only NPTII positive BY2 cells survive in this
selection media. The Cefotaxime was used to selectively kill the
agrobacterium, the plant cells being resistant to this
antibiotic.
[0312] Screening for the Optimal Expressing Clone
[0313] In order to select individual cell lines, aliquots of highly
diluted cell suspension were spread on solid BY-2 medium (Toshiyuki
Nagata & Fumi Kumagai Methods in Cell Science 21: 123-127,
1999). The cells were then grown until small calli developed. Each
callus was then re-suspended in liquid culture. Cells were then
sampled and evaluated for prh TNFR2:FC. About 500 cell lines were
screened by Western blot under denaturing conditions (FIG. 4). The
lines with high expression levels were further re-analyzed by the
same method to select the highest expressing clone of prh TNFR2:FC
producing clone.
[0314] Gel Electrophoresis:
[0315] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE) separates proteins on an electrical field according to
their size. Proteins in the presence of the detergent SDS migrate
as a linear function of the logarithm of their molecular weight.
Migration pattern and identification of prh TNFR2:FC on SDS-PAGE
was compared to commercial molecular weight standard proteins (New
England BioLabs; cat No. P7708S) and to the commercially available,
mammalian-cell derived Enbrel expressed in CHO cells (Entanercept;
Wyeth). prh TNFR2:FC was extracted from cells either by reducing
sample buffer containing .beta.-mercaptoethanol or by native
extraction buffer. The native extraction supernatant was mixed with
non-reducing sample buffer prior to analysis. Electrophoresis was
performed using Criterion.TM. cell vertical electrophoresis
apparatus (Bio-Rad Lab.) with premixed electrophoresis
Tris-Glycine-SDS running buffer (Bio-Rad Laboratories). Following
electrophoresis, the proteins were transferred from the
Polyacrylamide gel to a protein binding nitrocellulose membrane
(iBlot.TM.). Membranes were blocked for 1 hr at RT with 5% milk
buffer containing 0.1% Tween 20. For identification of the Fc
portion of the molecule, Goat anti human IgG conjugated to HRP (cat
#109-035-098, Jackson.) was used. For TNFR2 detection, a Rabbit
Anti-TNFRII (ID: ab109853, Abcam) followed by Goat anti Rabbit HRP
(cat #111-035-003, Jackson) were employed. Detection was carried
out with ECL detection kit (Pierce). The immunoreactivity of prh
TNFR2:FC was compared to that of commercial Enbrel (Entanercept;
Wyeth). Bands were detected using the Molecular Imager Gel Doc XR
System (Bio-Rad Laboratories).
[0316] Amino Acid Sequencing by Mass-Spectrometry
[0317] prhTNFR2:FC is sent for sequencing analysis at the Smoler
Proteomics Center at the Technion--Israel Institute of Technology
(Haifa, Israel). The protein is extracted from the gel, reduced
with 2.8 mM DTT (60.degree. C. for 30 min), modified with 8.8 mM
iodoacetamide in 100 mM ammonium bicarbonate (in the dark, room
temperature for 30 min) and digested in 10% ACN and 10 mM ammonium
bicarbonate with modified Trypsin (Promega) or with ChymoTrypsin
overnight at 37.degree. C. in a 1:50 enzyme-to-substrate ratio. 3%
of the resulting peptides are resolved by reverse-phase
chromatography on 0.075.times.200-mm fused silica capillaries
(J&W) packed with Reprosil reversed phase material (Dr Maisch
GmbH, Germany). The peptides are eluted with linear 60 minutes
gradients of 5 to 45% and 15 minutes at 95% acetonitrile with 0.1%
formic acid in water at flow rates of 0.25 .mu.min. On line mass
spectrometry is performed by an ion-trap mass spectrometer
(Orbitrap, Thermo) in a positive mode using repetitively full MS
scan followed by collision induces dissociation (CID) of the 7 most
dominant ion selected from the first MS scan.
[0318] The mass spectrometry data is analyzed using the Sequest
3.31 software (J. Eng and J. Yates, University of Washington and
Finnigan, San Jose) vs a specific sequence.
[0319] Glycosylation Analysis
[0320] The major difference between glycoproteins produced in
Chinese Hamster Ovary (CHO) cell and plant cell systems is the
glycosylation profile and glycan structure. Preliminary analysis
has been performed to characterize the various N-linked glycan
structures attached to the protein. These results are compared to
results of the N-glycosylation profile found in commercial Enbrel.
The presence of O-linked glycans, and glycan site analysis is
determined.
[0321] Samples of prh TNFR2:FC and commercial Enbrel are reduced,
alkylated and separated on SDS-PAGE. The protein bands at .about.75
KDa (a total of about 200 .mu.g protein) are taken for glycan
analysis using Trypsin digestion followed by either PNGase A or
PNGase F digestion (.about.80% and .about.20% of the total protein,
respectively) for prh TNFR2:FC and PNGase F digestion only for
commercial Enbrel. Digestion with Trypsin, followed by PNGase A
releases all the N-linked glycans and digestion with PNGase F
releases all glycans except those containing alpha 1-3 core fucose
(found in plants). The released glycans are extracted, cleaned and
then labeled with the fluorescent reagent anthranilamide
(2-aminobenzamide, 2AB) followed by removal of excess 2AB. The
analytical method includes separation of the glycans on a Waters
HPLC system with a normal phase amide-based column (Tosoh TSK
Amide-80 column), coupled with a fluorescence detector (330 nm
excitation, 420 nm emission). Sequencing of the labeled glycan pool
is achieved by sequential digestion with various exoglycosidases
followed by additional HPLC analysis. Using sequential digestion
with various exoglycosidases provides additional information on the
profile of the glycans structures and their relative amounts. The
exoglycosidase digestions that are carried out for the glycans
released from prh TNFR2:FC are with JBH (Jack bean
beta-N-Acetylhexosaminidase) that removes beta 1-2, 3, 4 and 6
N-acetylglucosamine (GlcNAc), with JBM (Jack bean mannosidase) that
removes mannose alpha 1-2, 6>3 mannose and with BKF (Bovine
testis fucosidase) that removes alpha 1-6 and alpha 1-3 core
fucose. The fluorescence labeling enables a semi-quantitative
analysis of the distribution of the various glycan structures in
the total digested glycan pool. The glycans are then separated
according to unique glycan linkages and in order of increasing size
using a gradient solvent flow consisting of ammonium formate and
acetonitrile. Retention time of individual glycans is compared to
the retention times of a standard mix of partially hydrolysed
dextran fragments, giving a ladder of glucose units (GU). The
glycans are assigned to peaks according to their GU values, based
on standards and a comparison to an external data base
(www(dot)glycobase(dot)nibrt(dot)ie:8080/database/show_glycobase(dot)acti-
on). The final assignment and relative peak areas are calculated
from the chromatogram of the PNGase A digestion.
[0322] Enzyme-Linked Immunosorbent Assay (ELISA)
[0323] Binding ELISA:
[0324] TNF.alpha. binding ELISA is a combination of a commercial
TNF.alpha. detection ELISA kit (Human TNF-.alpha.; Hycult Biotech
Inc. #HK307) and a commercial anti human IgG antibody (Goat anti
human IgG FC specific HRP; Sigma). The assay is a quantitative non
radioactive assay for prhTNFR2:FC binding activity. This binding
ELISA enables to detect functional (capable of binding TNF.alpha.)
molecules comprising both the TNFR and IgG domains.
[0325] An ELISA plate pre-coated with antibodies against TNF.alpha.
was incubated with TNF.alpha. (60 ng/ml, Sigma) for 1 hour at room
temperature. Between each ELISA step the plate was washed three
times with commercial wash buffer. Commercial Enbrel and
supernatant from BY2 cells expressing prh TNFR2:FC (serial
dilutions) were incubated on ELISA plate for 2 hr at RT. Goat anti
human IgG Fc HRP was diluted 1:10,000 and incubated on plate for 1
hr at RT. TMB was used as substrate for HRP. The colorimetric
reaction was stopped with 10% HCL and absorbance determined at 450
nm.
[0326] Prevention of TNF .alpha. Induced Apoptosis in A375
Cells
[0327] A375 cells (human melanoma cells) were grown in suspension
in culture medium (ATCC, #30-2002, supplemented with 10% FBS).
10.sup.4/well cells were plated in 96-well assay plates and
incubated overnight in assay medium (ATCC, #30-2002, supplemented
with 5% FBS). Recombinant TNF.alpha. (2 ng/ml, ProSpec, Rehovot,
Israel) was incubated for 2 hr at 37.degree. C. in the presence of
different concentrations (1.562-100 ng/ml) of prhTNFR2:FC or
commercial Enbrel (Entanercept; Wyeth). Following incubation, the
mixed solution was added to A375 cells in the presence of
actinomycin-D (0.8 .mu.g/ml), incubated for further 24 hr at
37.degree. C., 5% CO.sub.2 in a humidified incubator and
quantification of apoptosis was determined by MTT assay (Sigma Cat.
No. M5655). The plate was read at 570-650 nm and the inhibition of
TNF-.alpha. induced cytotoxicity (%) was calculated.
Example 2
Protein Analysis
[0328] prhTNFR2:FC was analyzed under reducing (FIG. 2A) and
non-reducing conditions (native extraction in the FIG. 2B).
prhTNFR2:FC (Lane 1) and commercial Enbrel (lane 2) were detected
using anti Fc antibody (upper panel) and anti TNFR2 antibody (lower
panel). The two proteins demonstrate a slight difference in
migration characteristics, presumably due to differences in
glycosylation patterns between the plant and mammalian
cell-expressed enzymes.
[0329] TNF.alpha. binding by both commercial Enbrel and prh
TNFR2:FC was examined by comparing serial dilutions of lysates of
BY2 cells expressing prh TNFR2:Fc (PRX-106) to commercial Enbrel.
prh TNFR2:FC serial dilutions demonstrate a dose response binding
pattern similar to the commercial protein (see FIG. 3). The
selection of transgenic cell lines according to protein expression
was done by Western blotting. Thus, to allow for the selection of
individual cell lines, aliquots of highly diluted cell suspension
were spread on solid BY-2 medium. The cells were then grown until
small calli developed. Each callus was then re-suspended in liquid
culture. Cells were then sampled and evaluated for prh TNFR2:Fc
expression levels by extraction under reducing conditions followed
by Western Blot identification (anti FC antibody) of the produced
target protein (FIG. 4). The functionality of the expressed protein
was established by its ability to prevent TNF.alpha. induced
apoptosis. Specifically, TNF.alpha. activity can be measured by its
ability to induce cell death of certain cell lines in the presence
of the transcriptional inhibitor, actinomycin D. Pre-incubation
with a neutralizing protein of TNF.alpha. prevents binding to the
receptors (TNF-R1 and TNF-R2), thereby inhibiting the cytokine
effect and preventing TNF.alpha. induced cell death. Quantification
of cell viability by MTT assay provides an in-cell activity assay
for TNF.alpha. cytotoxicity. The results are shown in FIGS. 5A-5G
on melanoma cells A375 and in FIGS. 6A-6G on L929 fibroblasts.
Example 3
prh TNFR2:FC Suppresses Inflammatory Bowel Disease (IBD)
[0330] Inflammatory bowel disease (IBD) is a chronic intestinal
inflammatory condition that is medicated by genetic, immune, and
environmental factors. About 0.2-0.3% of the population are
diagnosed with IBD annually. IBD is characterized by a tendency for
chronic or relapsing immune activation and inflammation within the
Gastro-intestinal Tract (GIT). It has two presentations: Crohn's
disease (CD), a chronic inflammation potentially involving any
location of the GIT from mouth to anus, and Ulcerative colitis
(UC), an inflammatory disorder that affects the rectum and extends
proximally to affect variable extent of the colon. CD is regulated
more by the TH1 immune cell response, overproducing the cytokines
IL-12, IFN-gamma and TNFalpha among others. UC, on the other hand,
is mainly regulated by the TH2 immune cell response.
[0331] PRX106 is a soluble receptor for a cytokine overproduced in
inflammation involving among others, the TH1 immune cell response.
PRX106 was shown to be very effective when injected IV in other
models of inflammation (Rheumatoid Arthritis). PRX106 is
overexpressed in Protalix's ProCellEx.TM. system, in BY2 plant
cells. Being a plant cell, BY2 has a cell wall that can help
protect PRX106 while moving through the stomach and small
intestine. In the colon, where the polysaccharides are digested,
the plant cell releases its content and hence PRX106 is free to
bind its cytokine ligand. Moreover, PRX106 is a chimeric protein
carrying an Fc segment of human IgG1. In the epithelial monolayer
lining the mucosal barrier, the FcRn receptor transcytoses IgG
molecules across by binding to their Fc. Therefore, PRX106 can also
cross the epithelial barrier to bind its cytokine ligand on the
serosal side of the epithelia.
[0332] IBD models are classified into five major groups: chemically
induced model, cell-transfer model, spontaneous model, congenital
(spontaneous gene mutation) model, and genetically engineered
model. In the most widely used chemically induced models, colitis
is induced by intrarectal administration of the covalently reactive
reagents TNBS/oxazolone, which are believed to induce a
T-cell-mediated response against hapten-modified autologous
proteins/luminal antigens. In the DSS model, mice are subjected
several days to drinking water supplemented with DSS (dextran
sodium sulfate), which seems to be directly toxic to colonic
epithelial cells of the basal crypts. The disease severity is
evaluated by scoring 3 major clinical signs (weight loss, diarrhea,
and rectal bleeding). The mouse models TNBS (Example 3A) and DSS
(Example 3B) are used to determine the therapeutic efficacy of the
plant cells expressing the chimeric polypeptide in vivo.
Example 3A
PRX106-Expressing Cells are Effective in Alleviating Symptoms of
IBD as Evidenced in an In Vivo Trinitrobenzene-Sulphonic Acid
(TNBS) Model
[0333] Materials and Methods
[0334] Ethics Statement--
[0335] All procedures were strictly performed in accordance with
the Guide for the Care and Use of Laboratory Animals.
[0336] Animals
[0337] Male Balb/c mice, 8-9 weeks old were used in all
experiments. Each experimental group included 5 to 10 mice. The
mice were purchased from Harlan Laboratories, Israel. All mice were
moved to SPF-free room (natural bacterial flora) several days
before starting the experiment.
[0338] TNBS Induction
[0339] TNBS was induced in mice by rectal installation of TNBS [M.
F. Neurath, I. Fuss et al: j exp. Med 182' 1281-1290 (1995)]. Mice
were sensitized by painting 100 .mu.L of 1% TNBS in ethanol onto
the shaved skin of their abdomens 7 days before challenge. On the
day of challenge, the mice were given 120 .mu.L of 1% TNBS (Sigma
Aldrich) slowly injected into the lumen of the colon via a
catheter. Following TNBS treatment, mice were treated per os (PO)
daily from day 0 to day 4 with BY-2 cells expressing PRX-106,
equivalent to 5 .mu.g protein (Dose I) and 30 .mu.g protein (Dose
II); BY-2(-) control cells in the same orally administered volumes
of the PRX-106 expressing cells; and saline. TNBS control mice
received PBS alone. The animals were monitored once daily for
weight. Weight loss was calculated by subtracting the weight on
each day from the weight on day 0. After the experiment, the
animals were sacrificed and dissected. On day 5, blood samples were
collected by cardiac puncture, and were left to clot and then
centrifuged to obtain serum for determination of serum cytokine
levels. (Cury et al. Cell Immunol. 2013, 282 (1); 66-70.
Experiments were performed on 5-15 mice per group in three separate
experiments; results followed the same pattern in all
experiments.
[0340] Oral Administration of Recombinant Plant Cells
[0341] Oral administration of plant cells expressing recombinant
TNFR2:Fc was initiated 6 hours after administration of TNBS. Mice
received plant cells expressing recombinant TNFR2:Fc, resuspended
in 350-500 .mu.L. Negative controls received the same orally
administered volumes of host Mock plant cells, instead of the plant
cells expressing recombinant TNFR2:Fc. Oral administration was
performed by gavage. Two more controls were untreated mice and TNBS
treated mice that received saline.
[0342] Analysis of Cytokine Profiles
[0343] Serum levels of cytokines TNF-.alpha., and IL-10 were
determined using ELISA kits following the manufacturer's
instructions (R&D Systems, Minneapolis, Minn., USA).
[0344] Antibody Array
[0345] Serum qualitative measurement of cytokine content was
performed using the Mouse Cytokine Antibody Array (R&D Systems,
Minneapolis, Minn., USA), according to the manufacturer's
manual.
[0346] Immunohistochemistry
[0347] Paraffin-embedded colonic tissue sections (5 .mu.m) were
deparaffinized, rehydrated, washed and incubated in 3%
H.sub.2O.sub.2 and blocked (Bar Sela et al 2006). Slides were
incubated with In-alpha pSer32/Ser36 antibodies (Abcam) Color was
developed using the DAB substrate kit (Thermo Scientific) or Zymed
AEC substrate kit (Zymed Laboratories), followed by counterstaining
with Mayer's hematoxylin. Controls without addition of primary
antibody showed low or no background staining in all cases.
Blocking was performed according to Bar-Sela et al. Histopathology.
2006; 49:188-193.
[0348] Flow Cytometry
[0349] Spleens were harvested from mice in RPMI 1640 medium. Cell
suspensions were prepared by dicing spleens with a razor blade,
followed by passage through a 40 .mu.M Nylon filter (BD Falcon).
Splenocytes were incubated with anti-mouse CD4 (R&D Systems,
Minneapolis, Minn., USA) and anti-mouse CD25 (R&D Systems,
Minneapolis, Minn., USA). Cells were then fixed and permeabilized
for 20 min at 4.degree. C. and then incubated with anti-mouse Foxp3
(Mouse Regulatory T cell 3-Color Flow kit, R&D Systems,
Minneapolis, Minn., USA) diluted in permeabilization buffer for 30
min. Ten thousand CD4.sup.+ cells were analyzed by FACS.
[0350] Macroscopic Colon Damage
[0351] Macroscopic appearance of the colon was assessed using the
Wallace macroscopic scoring system [W. Vermeulen, J. G. de Man, S.
Nullens, P. A. Pelckmans, B. Y. de Winter, and T. G. Moreels, "The
use of colonoscopy to follow the inflammatory time course of TNBS
colitis in rats," Acta Gastro-Enterologica Belgica, vol. 74, no. 2,
pp. 304-311, 2011]. In this scoring system, the inflammation is
assessed on the following scale from 0 to 10 based on ulceration,
inflammation, and extent of disease: 0=normal aspect of the mucosa,
1=localized hyperemia without ulcerations, 2=ulceration,
3=ulceration with thickening of bowel wall at one site, 4=two or
more sites of ulceration and thickening of the bowel wall, 5=major
sites of damage extending <2 cm along the length of the colon,
and 6-10=damage extending >2 cm (with the score increasing by 1
for each centimeter of damaged tissue).
[0352] Results
[0353] Oral administration of plant cells expressing recombinant
TNFR2:Fc improved TNBS-Induced body weight loss as monitored 4 days
after initiation of TNBS administration (FIGS. 7A-7B).
[0354] Colon lengths were measured as morphological indicators of
colon inflammation in TNBS-treated mice; short colon indicating an
inflammatory state. As shown in FIG. 8, the colon length of mice
treated with TNBS was significantly shortened compared to the
control mice. The length of colon of the treated group (oral
administration of cells expressing prTNFR2:Fc) was significantly
longer than that in the TNBS-treated group.
[0355] Oral administration of plant cells expressing recombinant
TNFR2:Fc also improved the macroscopic features of TNBS-induced
colitis. Macroscopic examination of colons, showed reduced colon
damage severity compared with the non treated colons (FIG. 9).
[0356] Oral administration of plant cells expressing recombinant
TNFR2:Fc reduced the expression of proinflammatory cytokines in
mice with TNBS-induced colitis (FIGS. 10A-10C). The effect of the
treatment on the serum levels of proinflammatory cytokines linked
to TNBS colitis and anti-inflammatory cytokines was evaluated. Note
that in most of the TNFR2:Fc treated groups the expression levels
of proinflammatory cytokines IL-6 and TNF-.alpha. were reduced, and
the expression levels of anti-inflammatory cytokine IL-10 were
elevated.
[0357] FIGS. 11A-11B showed that treatment with oral administration
of plant cells expressing recombinant TNFR2:Fc reduced level of
inflammatory mediators like granulocyte colony-stimulating factor
G-CSF, macrophage colony-stimulating factor (M-CSF), potentially
indicating reduced systemic inflammation by lowering systemic
recruitment of bone marrow derived cells from the bloodstream.
[0358] Recently, imbalance of the development and function of
IL-17-producing Th17 cells and CD4.sup.+CD25.sup.+FOXP3.sup.+ Treg
cells has been demonstrated to play an important role in autoimmune
diseases, including IBD. Treg cells, also known as
CD4.sup.+CD25.sup.+, FOXP3.sup.+, are involved in the maintenance
of peripheral tolerance and in controlling the immune response by
initiating suppressive effects on activated immune cells. The
present analysis shows that oral administration of plant cells
expressing TNFR2:Fc expands population of functional regulatory T
(T reg) cells in the spleen (FIG. 12).
[0359] To conclude the above-results demonstrate that oral
administration of plant cells expressing recombinant TNFR2:Fc is an
anti-inflammatory agent that ameliorates TNBS-induced colitis.
Example 3B
PRX106-Expressing Cells are Effective in Alleviating Symptoms of
IBD as Evidenced in an In Vivo Dextran Sulfate Sodium-Induced
(DSS-Induced) Model
[0360] The Dextran Sulfate Sodium-induced (DSS-induced) mouse model
of IBD is used for compounds for efficacy in Inflammatory Bowel
Disease. This is an experimental acute Ulcerative Colitis model
with symptoms similar to those observed in human UC, such as
diarrhea, bloody feces, body weight loss, mucosal ulceration and
shortening of the large intestine.
[0361] Ethics Statement
[0362] All procedures were strictly performed in accordance with
the Guide for the Care and Use of Laboratory Animals.
[0363] Animals
[0364] Male C67/Bl mice, 8-9 weeks old were used in all
experiments. Each experimental group included 10 mice. The mice
were purchased from Harlan Laboratories, Israel. All mice were
moved to SPF-free room (natural bacterial flora) several days
before starting the experiment.
[0365] Induction and Evaluation of Colitis in Mice Treated with DSS
and Following Oral Administration of Plant Cells Expressing
Recombinant TNFR2:Fc
[0366] Colitis was induced by administration of 1.5% (wt/vol) DSS
(reagent-grade DSS salt; molecular mass=36-50 kD; MP Biomedicals)
in normal drinking water for 5 days, followed by 5 days of normal
water consumption. Daily treatment with orally administered plant
cells expressing recombinant TNFR2:Fc; Mock cells comprising vector
alone or control treatment (saline) began 24 hours following DSS
induction, for a period of 7 days. Colonic inflammation was
assessed 5 days after DSS treatment by punch biopsies and
histological score. The animals were monitored once daily for
weight, weight loss was calculated by subtracting the daily weight
from the weight on day 0. After the experiment, the animals were
sacrificed and dissected; colon shortening was assessed by colon
length measurements in comparison to untreated colon; Blood samples
were collected by cardiac puncture, and were left to clot and then
centrifuged to obtain serum for determination of serum cytokine
levels.
[0367] Oral Administration of Recombinant Plant Cells:
[0368] Oral administration of plant cells expressing recombinant
TNFR2:Fc was initiated 24 hours following DSS administration. Mice
received plant cells expressing recombinant TNFR2:Fc (comprising 30
.mu.g of protein), suspended in 500 .mu.l saline. Negative controls
received the equivalent volumes of host Mock plant cells, to the
plant cells expressing recombinant TNFR:Fc. Two more control groups
were DSS treated mice administered with saline and untreated mice.
Oral administration was performed by gavage.
[0369] Analysis of Colon Inflammation.
[0370] Paraffin-embedded colon tissue sections were stained with
hematoxylin and eosin for light microscopic examination to assess
colon injury and inflammation. Samples from entire colon were
analyzed pathologically by a pathologist blinded to treatment
conditions. A scoring system including degree of inflammation,
crypt damage, percentage of area involved by inflammation and depth
of inflammation was used.
[0371] Punch Biopsies.
[0372] Mouse colons were flushed 3 times with PBS containing
antibiotics and opened along a longitudinal axis. Thereafter,
4-mm.sup.2 punch biopsies were obtained and incubated for 24 hours
in RPMI-1640 medium supplemented with antibiotics. Supernatants
were collected and kept in -20.degree. C. until assessed for
cytokine expression. Qualitative measurement of cytokine content in
medium conditioned by colonic explants was performed using the
Magnetic Luminex Screening Assay according to the manufacturer's
manual R&D Systems, Minneapolis, Minn., USA).
[0373] Analysis of Cytokine Profiles
[0374] Serum levels of cytokines TNF-.alpha., IL-6 and IL-10 were
determined using Magnetic Luminex Screening Assay following the
manufacturer's instructions (R&D Systems, Minneapolis, Minn.,
USA).
[0375] Results
[0376] 1. Oral Administration of Plant Cells Expressing Recombinant
TNFR2:Fc Improved DSS-Induced Body Weight Loss
[0377] Body weight was monitored each day following DSS
administration (FIGS. 13A-13B). As can be seen, treatment of mice
orally with plant cells expressing recombinant TNFR2:Fc attenuated
the weight loss induced by DSS.
[0378] 2. Oral Administration of Plant Cells Expressing Recombinant
TNFR2:Fc Suppressed DSS-Induced Colitis in Mice
[0379] Colon lengths were measured as it is well established that a
short colon can be used as a morphological indicator of colon
inflammation in DSS-treated mice. As shown in FIGS. 14A-14B, the
colon length of mice treated with DSS was significantly shortened
compared with the control mice. The length of colon in the oral
administration of plant cells expressing recombinant TNFR2:Fc group
was significantly longer than that in the DSS-treated group.
[0380] 3. Effect of Oral Administration of Plant Cells Expressing
Recombinant TNFR2:Fc on Gut Inflammatory Cytokines Following DSS
Colitis
[0381] FIGS. 15A-15C show a statistically significant decrease in
gut proinflammatory cytokines following oral treatment with plant
cells expressing recombinant TNFR2:Fc.
[0382] 4. Oral Administration of Plant Cells Expressing Recombinant
TNFR2:Fc Reduced the Expression of Proinflammatory Cytokines in
Mice with DSS-Induced Colitis
[0383] The effect of Oral administration of plant cells expressing
recombinant TNFR2:Fc on the production of proinflammatory cytokines
linked to DSS colitis was evaluated. As shown in FIGS. 16A-16C, DSS
induced protein expression of proinflammatory cytokines, such as
IL-6 and TNF-.alpha., in sera, whereas Oral administration of plant
cells expressing recombinant TNFR2:Fc suppressed the host protein
secretion of proinflammatory cytokines. These results pointed out
that oral administration of plant cells expressing recombinant
TNFR2:Fc inhibited the production of proinflammatory cytokine in
the DSS-induced colitis model.
[0384] 5. Histopathological Examination as an Indication for Colon
Inflammation
[0385] The severity of colon inflammation was further evaluated by
histological examinations (FIGS. 17A-17B). Following DSS
administration, colons exhibited transmural inflammation and
intense infiltration of inflammatory cells. This cell influx
associated with ulceration, loss of goblet cells and marked
disruption in the crypts throughout the colon. On note, oral
treatment with plant cells expressing TNFR2:Fc markedly improved
the hisological features of DSS-induced colitis. Histological
examination of colons, showed reduced colon damage severity in
colons of orally administered plant cells expressing recombinant
TNFR2:Fc treated mice, compared with the DSS and Mock treated
colons.
[0386] In conclusion, the present study supports a role for orally
administered plant cells expressing recombinant TNFR2:Fc as an
anti-inflammatory agent with the capacity to ameliorate IBD.
Example 4
Evaluating the Recombinant TNFR2:Fc Protein Pharmacokinetic Profile
in Rat Plasma at Various Time Points Post Feeding of Plant Cells
Expressing Recombinant TNFR2:Fc
[0387] Materials and Methods
[0388] Animals
[0389] Rats (SD, females/9-10 weeks/n=6) were subject to a 20 hours
fast and then fed (free feeding) with cells expressing recombinant
TNFR2:Fc (PRX-106) and host BY2(-). Following two hours from
feeding, food consumption was measured. Young suckling rats (SD,
males and females/16 days/n=6) were 3 hours fasted and fed (by
gavage) with cells expressing recombinant TNFR2: Fc (PRX-106) and
host BY2(-) cells.
[0390] Analysis of TNFR2 Profiles
[0391] Blood samples were collected in the each time point (e.g.,
0, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h), left to clot and then
centrifuged to obtain serum. Serum levels of human TNFRII were
determined using ELISA kits following the manufacturer's
instructions (R&D Systems, Minneapolis, Minn., USA).
[0392] Results
[0393] TNFR2:Fc level in serum is shown in FIG. 18. Results
demonstrate the elevation of TNFR2:Fc level in plasma following
oral administration of plant cells expressing the protein. TNFR2:Fc
level in serum was detected at 8 h, and was still detectable at 24
hours. TNFR2:Fc level in rat's serum fed with host BY2(-) was not
detectable.
[0394] The experiment was then followed by analysis of recombinant
TNFR2:Fc protein pharmacokinetic profile in the suckling rat plasma
various time points post feeding of plant cells expressing
TNFR2:Fc. TNFR2:Fc level in serum is shown in FIG. 19. Results
demonstrate the significant elevation of TNFR2:Fc level in plasma
following oral administration of TNFR2:Fc. TNFR2:Fc level in plasma
peaked at 4 h. Probably, increased level of TNFR2:Fc in serum of
suckling rats relative to adult rat was due to expression of FcNR
in intestine of suckling rats. TNFR2:Fc level in rat's serum fed
with host BY2(-) was not detectable.
Example 5
Toxicology Studies in Mice
[0395] Methods
[0396] Animals
[0397] Male and female SD Rats (Harlan Laboratories, Israel) 8
weeks at study initiation were housed under standard laboratory
conditions. Mean weight at study initiation was approximately 6.8
gr for males and 6.3 gr for females. Animals were fed with
commercial rodent diet (Teklad Certified Global 18% Protein Diet
cat #: 2018SC) and had free access to autoclaved and acidified
drinking water (pH between 2.5 and 3.5).
[0398] Study Design
[0399] Four groups, 3 dosing groups comprising 12 rats per group (6
males and 6 females) and a control group comprising 6 rats per
group (3 males and 3 females), were assigned. In each gender, the
control group received dilution buffer (0.2 M mannitol) and three
treated groups received cells expressing TNFR2:Fc at dose levels of
0.1, 0.5 and 1 mg TNFR2:Fc/Kg body weight. Cells were aliquoted in
accordance with requested expressed protein amount. Each aliquot
was mixed with 30 grams powder of commercial rodent diet and
dilution buffer, to create a pellet. The control pellet was made
with dilution buffer and commercial rodent diet powder alone. All
animals were daily orally fed with the pellets for 14 days. During
the study, mortality and general clinical observation were
performed, bodyweight was monitored daily. At study termination
(Day 15) after light anesthesia with carbon dioxide inhalation,
three blood samples were drawn from all animals from the retro
orbital sinus gross, after which, animals were sacrificed,
pathology was executed and selected organs were harvested.
[0400] Results
[0401] No adverse clinical symptoms were recorded throughout the
14-day safety study. All blood parameters were within the normal
range with no significant deviations. Body weight gain was
persistent and normal with no significant difference between the
groups (treated or Control). Cells expressing were found to be safe
and well tolerated with no adverse effects. No effect on
biochemical parameters or clinical symptoms was found. Gross
necropsy observation did not reveal pathological findings. No
animal was found in a moribund state or under severe distress
conditions. There were no observations of animals presenting severe
pain or decreased body weight.
Example 6
Sequencing of PRX-106
[0402] N Terminus Sequencing by Edman Degradation
[0403] Analysis was performed at Alphalyse (Denmark) uainf, an ABI
Procise 494 sequencer. The procedure determines the N-terminal
amino acid sequence of proteins and peptides by the Edman
degradation chemistry. The Edman degradation is a cyclic procedure
where amino acid residues are cleaved off one at a time and
identified by chromatography. Here are 3 steps in the cyclic
procedure. In step 1, the PITC reagent is coupled to the N-terminal
amino group under alkaline conditions. In step 2, the N-terminal
residue is cleaved in acidic media. In step 3, the PITC coupled
residue is transferred to a flask, converted to a PTH-residue and
identified by HPLC chromatography. The next cycle is then started
for identification of the next N-terminal residue.
[0404] Results:
[0405] The sequence was determined to be LPAQV (SEQ ID NO: 18).
[0406] Amino Acid Sequence Verification by Reverse Phase HPLC
Coupled to a Mass Spectrometry Detector.
[0407] Sequencing was performed at the Smoler Proteomics Center
(Technion--Israel Institute of Technology, Haifa, Israel). Analyses
were carried out using reverse-phase HPLC coupled to a mass
spectrometry detector.
[0408] Method
[0409] Proteolysis
[0410] The analyzed samples were resuspended in 8 M Urea, 100 mM
ammonium bicabonate (ABC) followed by reduction with 2.8 mM DTT
(60.degree. C. for 30 min) and modified with 8.8 mM iodoacetamide
in 100 mM ABC in the dark, at ambient temperature for an additional
30 min. The proteins were digested overnight at 37.degree. C. using
modified trypsin (Promega) at a 1:50 enzyme-to-substrate ratio in 2
M Urea, 25 mM ABC.
[0411] Mass Spectrometry Analysis
[0412] The tryptic or chymotryptic peptides were desalted using
stage tips (home-made C18), the residual buffer was evaporated and
the pellet was resuspended in 0.1% (v/v) formic acid. Twenty
nanogram of the resulting peptides were resolved by reversed-phase
liquid chromatography on a 0.075.times.200-mm fused silica
capillaries (J and W) packed with Reprosil reversed phase material
(Dr Maisch GmbH, Germany). Peptides were eluted with a linear 60
minutes gradient of 5 to 45% followed by 15 minutes at 95%
acetonitrile with 0.1% formic acid in water at flow rates of 0.25
.mu.L/min. On-line mass spectrometry was performed on an ion-trap
mass spectrometer (Orbitrap, Thermo) in a positive mode using
repetitively full MS scan followed by collision induced
dissociation (CID) of the 7 most dominant ions selected from the
first MS scan. The mass spectrometry data was analyzed using the
Discoverer software version 1.3 software using a specific protein
derived database.
[0413] Results
[0414] The sequence was compared to the peptide sequence of the
Etanercept sequence. The identified sequences are presented in
Table V, below. Presented is 84.8% coverage of the reference
sequence (see green color, FIG. 20).
TABLE-US-00005 TABLE V Peptides Identified Following Digestion with
Trypsin (SEQ ID NO: 19-203, ordered) WQQGnVFScSVMHEALHnHYTQK
WQQGNVFScSVMHEALHNHYTqK GFYPSDIAVEWESNGqPENnYKT
qYNSTYRVVSVLTVLHqDWLNGK WQqGNVFScSVMHEALHNHYTqKS
VVSVLTVLHQDWLNGKEYKc VVSVLTVLHqDWLnGKEYK
SqHTqPTPEPSTAPSTSFLLPmGPSPPAEGSTGDEPK WQQGnVFScSVMHEALHNHY
ScDKTHTcPPcPAPELLGGPSVFLFPPKPKD GQPREPqVYTLPPSREEMTK
GFYPSDIAVEWESNGQPEnNYKT LPAqVAFTPYAPEPGSTcR EALHnHYTqK
qNRIcTcRPGWYcALSKQEGcR WQQGNVFScSVmHEALHnHYTQK
SqHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPK GQPREPqVYTLPPSREEmTK
GFYPSDIAVEWESnGQPENNYK SqHTQPTPEPSTAPSTSFLLPmGPSPPAEGSTGDEPK
VVSVLTVLHQDWLnGK TYTqLWNWVPEcLScGSRcSSDqVETQAcTR
WQQGNVFScSVMHEALHNHYTQK GFYPSDIAVEWESnGQPEnnYKT VVVDVSHEDPEVK
PSTSFLLPMGPSPPAEGSTGDEPK LPAQVAFTPYAPEPGSTcR TTPPVLDSDGSFFL LSLSPGK
EPQVYTLPPSREEMTKN SmAPGAVHLPQ TTPPVLDSDGSFFLYSK
WQQGNVFScSVmHEALHNHYTQK SMAPGAVH SVmHEALHNHYTQK VVSVLTVLH
SQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPK GQPREPQVY AQVAFTPYAPEPGSTcR
cAPLRK EPQVYTLPPSREEmTKnQVSLTcLVK SmAPGAVH VVSVLTVLHQD LFPPKPK
GSFFLYSK IcTcRPGWY SQHTQPTPEPS SVLTVLHQDWLnGKEYK QVETQAcTR SLSLSPGK
SDGSFFLYSK KALPAPIEK ALPAPIEK AVcTSTSPTR SQHTQPTPEPSTAPSTSF
QVSLTcLVK LREYYDQTAqmccSKcSPGQHAK WQQGNVFScSVMHEALH DTLmISR
PmGPSPPAEGSTGDEPK THTcPPcPAPELLGGPSVF DTLMISR SDQVETQAcTR
KcRPGFGVAR WYVDGVEVHNAK YVDGVEVHNAK TTPPVLDSDGSFF
THTcPPcPAPELLGGPSVFLFPPKPK PSPPAEGSTGDEPK SLSLSPGKSEKD
MAPGAVHLPQPVSTR VDGVEVHNAK ScDKTHTcPPcPAPELLGGPSVF VVSVLTVLHQDWLNGK
SLSLSPGKSEK PPcPAPELLGGPSVFLFPPKPK SFFLYSK FNWYVDGVEVHNAK
FLLPMGPSPPAEGSTGDEPK DAVcTSTSPTR NQVSLTcLVK NqVSLTcLVKG SLSPGKSEK
TPEVTcVVVDVSHEDPEVK LREYYDQTAQM GFYPSDIAVEWESNGQPENNYK FNWYVDGVEVHN
VVSVLTVLHQDWLN SQHTQPTPEPSTAPST RTPEVTcVVVDVSHEDPEVK SLSLSPGKS
LSPGKSEKDEL LPQPVSTR TTPPVLDSDGSFFLY TSDTVcDScEDSTYTQLWN
ALPAQVAFTPYAPEPGSTcR EEQYNSTYR ScDKTHTcPPcPAPELLGGPSVFLFPPKPK
cSPGQHAKVFcTK TPEVTcVVVDVSHED SMAPGAVHLPQPV TcRPGWYcALSK
TcPPcPAPELLGGPSVFLFPPKPK TSDTVcDScEDSTYTQLWNWVPEcLScGSR LcAPLRK
SPPAEGSTGDEPK WVPEcLScGSR GPSPPAEGSTGDEPK SSDQVETQAcTR EEQYnSTYR
VAFTPYAPEPGSTcR PGWYcALSK cRPGFGVAR ScSVmHEALHnHYTqK
VVSVLTVLHQDWLNGKEYK LcAPLR EPQVYTLPPSREEMTKnQVSLTcLVK
LLPMGPSPPAEGSTGDEPK SQHTQPTPEPSTAPSTSFLLPmGPSPPAEGSTGDEPK
SLSLSPGKSE EEMTKNqV SVMHEALHNHYTQK
SQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKScDK EEmTKnQVSLTcLVKG
LREYYDQTAQmccSK cSSDqVETQAcTR EPQVYTLPPSREEMTK NQVSLTcLVKG
cSSDQVETQAcTR nQVSLTcLVK TKPREEQYNSTYR PAQVAFTPYAPEPGSTcR
SLSLSPGKSEKDEL AFTPYAPEPGSTcR APGAVHLPQPVSTR SDGSFFLYSKLTVDK
THTcPPcPAPELLG VVSVLTVLHQDWLn EPQVYTLPPSR SmAPGAVHLPQPVSTR
GQPREPQVYTLPPSREEmTK TPYAPEPGSTcR EVTcVVVDVSHEDPEVK TKPREEQYnSTYR
VSnKALPAPIEK LREYYDQTAQMccSK FTPYAPEPGSTcR SMAPGAVHLPQPVSTR
GPSVFLFPPKPK VVSVLTVLHQDWLnGKEYK SQHTQPTPEPSTAPS SMAPGAVHLPQPVS
AVHLPQPVSTR GQPREPQVYTLPPSR PGAVHLPQPVSTR TLMISR
KNqVSLTcLVKGFYPSDIAVEWESNGqPENnYK LREYYDQTAQMc SmAPGAVHLPQPV
LPAPIEK EYYDQTAQMccSK NWVPEcLScGSR SLSPGKSEKDEL IcTcRPGWYcALSK
SMAPGAVHLPQPVST EYYDQTAQmccSK ASMDAVcTSTSPTR SQHTQPTPEPSTAPSTS
TLPPSREEMTK SQHTQPTPEPSTAPSTSFL TLmISR EPQVYTLPPSREEmTK
GQPREPQVYTLPPSREEMTK TPEVTcVVVDVSHEDPEVKFN ScDKTHTcPPcPAPELLG
GFYPSDIAVEWESNGqPENnYK AKGQPREPQVYTLPPSR LREYYDQTAQMcc
LPmGPSPPAEGSTGDEPK ScSVMHEALHNHYTQK FNWYVDGVEVHnAK
PMGPSPPAEGSTGDEPK SMAPGAVHLPqPVSTR SMAPGAVHLPQ
LPMGPSPPAEGSTGDEPK
Example 7
Evaluation of Orally Administered PRX-106 Plant Cells Expressing
Recombinant TNFR2:Fc in the Clinical Context
[0415] In order to assess safety of administration of orally
administered plant cells expressing recombinant TNFR2:Fc, also
referred to herein as OPRX-106 and to evaluate pharmacokinetic
parameters of the OPRX-106, OPRX-106 is administered to healthy
subjects as further described herein below.
[0416] Primary Objective
[0417] The primary objective of this trial is to evaluate the
safety of OPRX-106, in healthy male volunteers.
[0418] Secondary Objective
[0419] The secondary objective of this clinical study is to
evaluate the pharmacokinetics of TNFR2-Fc following administration
of OPRX-106 in healthy male volunteers.
[0420] Overall Study Design and Plan--Description
[0421] This is a Phase 1, randomized, open-label study to evaluate
the safety of OPRX-106 and pharmacokinetics of TNFR-Fc following
oral administration of OPRX-106 in healthy volunteers. Up to 18
healthy male subjects (age 18 years and older) are randomly
allocated to one of three dose cohorts (up to 6 and not less than 4
subjects per cohort) receiving OPRX-106 doses equivalent to 2 mg, 8
mg or 16 mg TNFR-Fc. Subjects receive daily oral administrations of
OPRX-106 for 5 consecutive days. Subjects remain at the clinical
center for 12 hours after the first administration of OPRX-106 and
then return to the site as needed for additional administrations
and study procedures. The cells are provided as a cell
suspension.
[0422] At screening and on Days 1 (0 and 24 hours), 5 and 10, vital
signs are measured, a physical examination is effected and blood
samples are retrieved for laboratory tests. Adverse events (AEs)
and concomitant medications are evaluated at all study visits.
[0423] Blood samples for Plasma TNFR-Fc levels are drawn three
times (in 2 hour intervals) at screening, on Days 1 and 4:
pre-administration, 2, 4, 6, 8, 10, 12, .+-.15 minutes at each time
point, and 24.+-.2 hours post administration.
[0424] A control (placebo) group is not necessary, as all safety
endpoints are assessed as change from baseline. Subjects are
allocated to a dose cohort to limit bias but the study is
open-label in order to identify any dose-dependent adverse effects
of OPRX-106.
[0425] Selection of Study Population
[0426] Inclusion Criteria
[0427] To be eligible for the study, subjects must meet the
inclusion criteria listed below:
1. Healthy male age 18 or older; 2. BMI 20-25 kg/m.sup.2; 3. Male
subjects or their partners must use an adequate method of
contraception at all times during the study; 4. Negative laboratory
tests for HIV, HBcAb and anti HCV at the screening visit; 5. Naive
to any previous recombinant protein therapy; 6. Provide written
informed consent; and 7. Have the ability to understand the
requirements of the study and to comply with the study protocol and
dosing regimen.
[0428] Exclusion Criteria
[0429] To be eligible for the study, subjects must not meet any of
the exclusion criteria listed below:
1. Clinical evidence of any active significant disease that could
potentially compromise the ability of the Investigator to evaluate
or interpret the effects of the study treatment on safety
assessment, thus increasing the risk to the subject to unacceptable
levels; 2. Presence of any acute or chronic diseases; 3. History of
any allergies or protein-drug hypersensitivity; 4. Suspected
Tuberculosis (previous, active or latent); 5. Exposure to long-term
steroid treatment within the last 12 months prior to the study; 6.
Subject had a major operation in last 6 months; 7. Subject has
received immunosuppressive treatment; 8. Chronic use of any
medication including vitamins; 9. Participation in another clinical
trial during the previous 3 months (subject report); 10. Reported
history of alcohol or drug abuse; 11. Subjects with short bowel
(more than 1 m removed of small bowel);
[0430] Removal of Subjects from Therapy or Assessment
[0431] Reasons for permanent discontinuation include the
following:
[0432] Clinically significant adverse events (AEs);
[0433] Serious protocol violation;
[0434] Sponsor elects to terminate the study;
[0435] Subject requires the use of prohibited treatment or
medication;
[0436] Intolerance to treatment;
[0437] The subject experiences progressive or severe
hypersensitivity--to be treated appropriately and withdrawn from
the study;
[0438] The subject requests to discontinue treatment or withdraws
consent;
[0439] The Investigator feels that it is not in the best interest
of the subject to continue treatment and/or if the Investigator
believes that the subject can no longer be compliant with the
requirements of the study;
[0440] Replacement Policy
[0441] Withdrawn subjects may be replaced upon Medical Director
decision.
[0442] Primary Endpoint
[0443] The safety of each of the three doses of OPRX-106 as
assessed by AEs, physical examinations, vital signs, concomitant
medications and laboratory test results.
[0444] Vital signs are measured at screening and on Days 1 (0
pre-dose and 24 hours), 5 and 10. Vital signs comprising systolic
and diastolic arterial blood pressure, pulse and body temperature
are assessed at all study visits. Systolic and diastolic blood
pressure is measured after the subject has been in supine position
for at least 1 minute. All recordings are performed using standard
equipment. Clinically significant abnormal findings are reported as
AEs.
[0445] Clinical Laboratory Tests--Blood samples for clinical
laboratory safety tests are collected after 8 hours fasting, at
screening and pre-dose administration on Days 1, 2, and 5, and on
Day 10. Clinical laboratory tests include total protein, albumin,
alanine transaminase (ALT), aspartate aminotransferase (AST),
alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGTP),
Lactate dehydrogenase (LDH), cholesterol, uric acid, creatinine,
blood urea nitrogen (BUN), sodium (Na), potassium (K), fasting
glucose, total bilirubin, and complete blood cell count (CBC) with
differential. Clinically significant abnormal findings are reported
as AEs.
[0446] Secondary Endpoint
[0447] The PK profile of TNFR-Fc following administration of each
of the three doses of OPRX-106 on the first (Day 1) and last (Day
4) of administration as assessed by AUC.sub.0-t, C.sub.max,
t.sub.max and additional PK parameters based on observed
results.
PK Parameters
[0448] Blood samples for Plasma TNFR-Fc levels are drawn three
times (in 2 hour intervals) at screening, on Day 1 and 4:
pre-administration, 2, 4, 6, 8, 10, 12, .+-.15 minutes at each time
point, and 24.+-.2 hours post administration.
[0449] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0450] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
Sequence CWU 1
1
2051705DNAArtificial sequenceDNA encoding a TNFR2 derived sequence
1cttccagctc aggttgcttt tactccatat gctccggagc caggatctac ttgtaggctt
60agggaatact acgatcagac tgctcaaatg tgctgctcta agtgctctcc aggacagcac
120gctaaggttt tctgcactaa gacttcagat actgtttgcg attcttgcga
ggattctact 180tacactcagc tttggaattg ggttccagag tgtctttctt
gtggatctag gtgctcttct 240gatcaggttg agactcaggc ttgtactagg
gagcagaata ggatttgtac ttgcaggcca 300ggatggtatt gtgctctttc
taagcaagag ggatgtaggc tttgtgctcc acttagaaag 360tgcaggcctg
gttttggagt tgctagacca ggaactgaga cttctgacgt tgtttgcaag
420ccatgtgctc caggaacttt ctctaatact acttcttcta ctgatatttg
caggccacat 480caaatttgca atgttgttgc tattccaggt aatgcttcta
tggatgctgt ttgcacttct 540acttctccaa ctaggtctat ggctccagga
gctgttcatc ttccacaacc agtttctact 600aggtcacaac atactcagcc
aactccagaa ccatctactg ctccatctac ttcattcctt 660ttgccaatgg
gaccatctcc accagctgaa ggatctactg gagat 7052235PRTArtificial
sequenceAmino acid sequence encoding TNFR2 derived sequence 2Leu
Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1 5 10
15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys
20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr
Lys Thr 35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr
Tyr Thr Gln Leu 50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys
Gly Ser Arg Cys Ser Ser 65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys
Thr Arg Glu Gln Asn Arg Ile Cys 85 90 95 Thr Cys Arg Pro Gly Trp
Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys 100 105 110 Arg Leu Cys Ala
Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 115 120 125 Arg Pro
Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro 130 135 140
Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145
150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met
Asp Ala 165 170 175 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala
Pro Gly Ala Val 180 185 190 His Leu Pro Gln Pro Val Ser Thr Arg Ser
Gln His Thr Gln Pro Thr 195 200 205 Pro Glu Pro Ser Thr Ala Pro Ser
Thr Ser Phe Leu Leu Pro Met Gly 210 215 220 Pro Ser Pro Pro Ala Glu
Gly Ser Thr Gly Asp 225 230 235 381DNAArtificial sequenceN.
plumbaginifolia Calreticulin protein derived signal peptide
encoding sequence 3atggctactc aaaggagggc taatccatct tctcttcatc
ttattactgt tttctctctt 60cttgttgctg ttgtttctgc a 81427PRTArtificial
sequenceAmino acid sequence encoding signal peptide from N.
plumbaginifolia Calreticulin protein 4Met Ala Thr Gln Arg Arg Ala
Asn Pro Ser Ser Leu His Leu Ile Thr 1 5 10 15 Val Phe Ser Leu Leu
Val Ala Val Val Ser Ala 20 25 51506DNAArtificial sequenceNucleic
acid sequence encoding prh TNFR2Fc (PRX106) 5atggctactc aaaggagggc
taatccatct tctcttcatc ttattactgt tttctctctt 60cttgttgctg ttgtttctgc
acttccagct caggttgctt ttactccata tgctccggag 120ccaggatcta
cttgtaggct tagggaatac tacgatcaga ctgctcaaat gtgctgctct
180aagtgctctc caggacagca cgctaaggtt ttctgcacta agacttcaga
tactgtttgc 240gattcttgcg aggattctac ttacactcag ctttggaatt
gggttccaga gtgtctttct 300tgtggatcta ggtgctcttc tgatcaggtt
gagactcagg cttgtactag ggagcagaat 360aggatttgta cttgcaggcc
aggatggtat tgtgctcttt ctaagcaaga gggatgtagg 420ctttgtgctc
cacttagaaa gtgcaggcct ggttttggag ttgctagacc aggaactgag
480acttctgacg ttgtttgcaa gccatgtgct ccaggaactt tctctaatac
tacttcttct 540actgatattt gcaggccaca tcaaatttgc aatgttgttg
ctattccagg taatgcttct 600atggatgctg tttgcacttc tacttctcca
actaggtcta tggctccagg agctgttcat 660cttccacaac cagtttctac
taggtcacaa catactcagc caactccaga accatctact 720gctccatcta
cttcattcct tttgccaatg ggaccatctc caccagctga aggatctact
780ggagatgagc caaagtcttg cgataagact catacttgtc caccatgtcc
agctccagaa 840cttcttggag gaccatctgt tttccttttc ccaccaaagc
caaaggatac tcttatgatt 900tctaggactc cagaggttac ttgcgttgtt
gttgatgttt cacatgaaga tccagaggtg 960aagttcaatt ggtacgttga
tggagttgag gttcataatg ctaagactaa gccaagggag 1020gagcaataca
attcaacata cagggttgtt tctgttctta ctgttcttca tcaagattgg
1080cttaatggaa aggaatacaa gtgcaaggtt tctaataagg ctttgccagc
accaattgaa 1140aagactattt ctaaggctaa gggacaacca agagagccac
aagtttacac tcttccacca 1200tctagggagg agatgactaa gaatcaagtt
tctcttactt gccttgttaa gggattctac 1260ccatctgata ttgctgttga
gtgggagtct aacggacagc ctgagaataa ttacaagact 1320actccaccag
ttcttgattc tgatggatct ttcttccttt actctaagtt gactgttgat
1380aagtctaggt ggcaacaggg aaatgttttc tcttgctctg ttatgcatga
ggctcttcat 1440aatcattaca ctcagaaatc actttctctt tctccaggta
agagtgagaa ggacgagctc 1500tgatga 15066500PRTArtificial sequenceprh
TNFR2Fc (PRX106) polypeptide 6Met Ala Thr Gln Arg Arg Ala Asn Pro
Ser Ser Leu His Leu Ile Thr 1 5 10 15 Val Phe Ser Leu Leu Val Ala
Val Val Ser Ala Leu Pro Ala Gln Val 20 25 30 Ala Phe Thr Pro Tyr
Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg 35 40 45 Glu Tyr Tyr
Asp Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro 50 55 60 Gly
Gln His Ala Lys Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys 65 70
75 80 Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val
Pro 85 90 95 Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln
Val Glu Thr 100 105 110 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys
Thr Cys Arg Pro Gly 115 120 125 Trp Tyr Cys Ala Leu Ser Lys Gln Glu
Gly Cys Arg Leu Cys Ala Pro 130 135 140 Leu Arg Lys Cys Arg Pro Gly
Phe Gly Val Ala Arg Pro Gly Thr Glu 145 150 155 160 Thr Ser Asp Val
Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn 165 170 175 Thr Thr
Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val 180 185 190
Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr 195
200 205 Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Leu Pro Gln
Pro 210 215 220 Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro Glu
Pro Ser Thr 225 230 235 240 Ala Pro Ser Thr Ser Phe Leu Leu Pro Met
Gly Pro Ser Pro Pro Ala 245 250 255 Glu Gly Ser Thr Gly Asp Glu Pro
Lys Ser Cys Asp Lys Thr His Thr 260 265 270 Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 275 280 285 Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 290 295 300 Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 305 310 315
320 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
325 330 335 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val 340 345 350 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys 355 360 365 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser 370 375 380 Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro 385 390 395 400 Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 405 410 415 Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 420 425 430 Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 435 440
445 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
450 455 460 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His 465 470 475 480 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys Ser Glu 485 490 495 Lys Asp Glu Leu 500
7473PRTArtificial sequenceprh TNFR2Fc (PRX106) mature polypeptide
7Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1
5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys
Cys 20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys
Thr Lys Thr 35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser
Thr Tyr Thr Gln Leu 50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser
Cys Gly Ser Arg Cys Ser Ser 65 70 75 80 Asp Gln Val Glu Thr Gln Ala
Cys Thr Arg Glu Gln Asn Arg Ile Cys 85 90 95 Thr Cys Arg Pro Gly
Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys 100 105 110 Arg Leu Cys
Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 115 120 125 Arg
Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro 130 135
140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His
145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser
Met Asp Ala 165 170 175 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met
Ala Pro Gly Ala Val 180 185 190 His Leu Pro Gln Pro Val Ser Thr Arg
Ser Gln His Thr Gln Pro Thr 195 200 205 Pro Glu Pro Ser Thr Ala Pro
Ser Thr Ser Phe Leu Leu Pro Met Gly 210 215 220 Pro Ser Pro Pro Ala
Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260
265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385
390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys Ser Glu Lys
Asp Glu Leu 465 470 8696DNAArtificial sequenceIgG1-FC encoding
sequence 8gagccaaagt cttgcgataa gactcatact tgtccaccat gtccagctcc
agaacttctt 60ggaggaccat ctgttttcct tttcccacca aagccaaagg atactcttat
gatttctagg 120actccagagg ttacttgcgt tgttgttgat gtttcacatg
aagatccaga ggtgaagttc 180aattggtacg ttgatggagt tgaggttcat
aatgctaaga ctaagccaag ggaggagcaa 240tacaattcaa catacagggt
tgtttctgtt cttactgttc ttcatcaaga ttggcttaat 300ggaaaggaat
acaagtgcaa ggtttctaat aaggctttgc cagcaccaat tgaaaagact
360atttctaagg ctaagggaca accaagagag ccacaagttt acactcttcc
accatctagg 420gaggagatga ctaagaatca agtttctctt acttgccttg
ttaagggatt ctacccatct 480gatattgctg ttgagtggga gtctaacgga
cagcctgaga ataattacaa gactactcca 540ccagttcttg attctgatgg
atctttcttc ctttactcta agttgactgt tgataagtct 600aggtggcaac
agggaaatgt tttctcttgc tctgttatgc atgaggctct tcataatcat
660tacactcaga aatcactttc tctttctcca ggtaag 6969232PRTArtificial
sequenceIgG1-FC 9Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100
105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys 225 230 10467PRTArtificial
sequenceEtanercept polypeptide 10Leu Pro Ala Gln Val Ala Phe Thr
Pro Tyr Ala Pro Glu Pro Gly Ser 1 5 10 15 Thr Cys Arg Leu Arg Glu
Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 20 25 30 Ser Lys Cys Ser
Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 35 40 45 Ser Asp
Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55 60
Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65
70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg
Ile Cys 85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys
Gln Glu Gly Cys 100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg
Pro Gly Phe Gly Val Ala 115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp
Val Val Cys Lys Pro Cys Ala Pro 130 135 140 Gly Thr Phe Ser Asn Thr
Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys
Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 165 170 175 Val
Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 180 185
190 His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr
195 200 205 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro
Met Gly 210 215 220 Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu
Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe
Pro Pro
Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325
330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450
455 460 Pro Gly Lys 465 114PRTArtificial sequenceAmino acid
sequence encoding TNFR2 derived sequence 11Leu Cys Ala Pro 1
124PRTArtificial sequenceAmino acid sequence encoding TNFR2 derived
sequence 12Val Phe Cys Thr 1 1318PRTArtificial sequenceAmino acid
sequence encoding TNFR2 derived sequencemisc_feature(8)..(8)Xaa can
be any naturally occurring amino acid 13Leu Pro Ala Gln Val Ala Phe
Xaa Pro Tyr Ala Pro Glu Pro Gly Ser 1 5 10 15 Thr Cys
144PRTArtificial sequenceER retention signal peptide 14His Asp Glu
Leu 1 154PRTArtificial sequenceER retention signal peptide 15Lys
Asp Glu Leu 1 166PRTArtificial sequenceER retention signal peptide
16Ser Glu Lys Asp Glu Leu 1 5 1718PRTArtificial SequenceAmino acid
sequence encoding TNFR2 derived sequence 17Leu Pro Ala Gln Val Ala
Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1 5 10 15 Thr Cys
185PRTArtificial SequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 18Leu Pro Ala Gln Val 1 5 1923PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 19Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 1 5 10 15 His Asn His Tyr Thr Gln Lys 20 2023PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 20Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 1 5 10 15 His Asn His Tyr Thr Gln Lys 20 2123PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 21Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln 1 5 10 15 Pro Glu Asn Asn Tyr Lys Thr 20 2223PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 22Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His 1 5 10 15 Gln Asp Trp Leu Asn Gly Lys 20 2324PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 23Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 1 5 10 15 His Asn His Tyr Thr Gln Lys Ser 20
2420PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 24Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys 1 5 10 15 Glu Tyr Lys Cys 20 2519PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 25Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys 1 5 10 15 Glu Tyr Lys 2637PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 26Ser Gln His
Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr 1 5 10 15 Ser
Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr 20 25
30 Gly Asp Glu Pro Lys 35 2720PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 27Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 1 5 10 15 His
Asn His Tyr 20 2831PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 28Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 20 25 30
2920PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 29Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu 1 5 10 15 Glu Met Thr Lys 20 3023PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 30Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln 1 5 10 15 Pro Glu Asn Asn Tyr Lys Thr 20 3119PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 31Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro
Gly Ser 1 5 10 15 Thr Cys Arg 3210PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 32Glu Ala Leu
His Asn His Tyr Thr Gln Lys 1 5 10 3322PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 33Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala
Leu Ser 1 5 10 15 Lys Gln Glu Gly Cys Arg 20 3423PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 34Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 1 5 10 15 His Asn His Tyr Thr Gln Lys 20 3537PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 35Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro
Ser Thr 1 5 10 15 Ser Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala
Glu Gly Ser Thr 20 25 30 Gly Asp Glu Pro Lys 35 3620PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 36Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu 1 5 10 15 Glu Met Thr Lys 20 3722PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 37Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln 1 5 10 15 Pro Glu Asn Asn Tyr Lys 20 3837PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 38Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro
Ser Thr 1 5 10 15 Ser Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala
Glu Gly Ser Thr 20 25 30 Gly Asp Glu Pro Lys 35 3916PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 39Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys 1 5 10 15 4031PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 40Thr Tyr Thr Gln Leu Trp
Asn Trp Val Pro Glu Cys Leu Ser Cys Gly 1 5 10 15 Ser Arg Cys Ser
Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg 20 25 30
4123PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 41Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 1 5 10 15 His Asn His Tyr Thr Gln Lys 20
4223PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 42Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln 1 5 10 15 Pro Glu Asn Asn Tyr Lys Thr 20
4313PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 43Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys 1 5 10 4424PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 44Pro Ser Thr Ser Phe Leu
Leu Pro Met Gly Pro Ser Pro Pro Ala Glu 1 5 10 15 Gly Ser Thr Gly
Asp Glu Pro Lys 20 4519PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 45Leu Pro Ala Gln Val Ala
Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1 5 10 15 Thr Cys Arg
4614PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 46Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu 1 5 10 477PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 47Leu Ser Leu Ser Pro Gly
Lys 1 5 4817PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 48Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys 1 5 10 15 Asn 4911PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 49Ser Met Ala Pro Gly Ala Val His Leu Pro Gln 1 5 10
5017PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 50Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 1 5 10 15 Lys 5123PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 51Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 1 5 10 15 His
Asn His Tyr Thr Gln Lys 20 528PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 52Ser Met Ala
Pro Gly Ala Val His 1 5 5314PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 53Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 1 5 10 549PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 54Val Val Ser Val Leu Thr Val Leu His 1 5
5537PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 55Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser
Thr Ala Pro Ser Thr 1 5 10 15 Ser Phe Leu Leu Pro Met Gly Pro Ser
Pro Pro Ala Glu Gly Ser Thr 20 25 30 Gly Asp Glu Pro Lys 35
569PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 56Gly Gln Pro Arg Glu Pro Gln Val Tyr 1 5
5717PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 57Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu
Pro Gly Ser Thr Cys 1 5 10 15 Arg 586PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 58Cys Ala Pro
Leu Arg Lys 1 5 5926PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 59Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 1 5 10 15 Asn Gln Val Ser
Leu Thr Cys Leu Val Lys 20 25 608PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 60Ser Met Ala
Pro Gly Ala Val His 1 5 6111PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 61Val Val Ser
Val Leu Thr Val Leu His Gln Asp 1 5 10 627PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 62Leu Phe Pro Pro Lys Pro Lys 1 5 638PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 63Gly Ser Phe Phe Leu Tyr Ser Lys 1 5 649PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 64Ile Cys Thr Cys Arg Pro Gly Trp Tyr 1 5
6511PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 65Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser
1 5 10 6617PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 66Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr 1 5 10 15 Lys 679PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 67Gln Val Glu Thr Gln Ala Cys Thr Arg 1 5 688PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 68Ser Leu Ser Leu Ser Pro Gly Lys 1 5 6910PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 69Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 1 5 10
709PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 70Lys Ala Leu Pro Ala Pro Ile Glu Lys 1 5
718PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 71Ala Leu Pro Ala Pro Ile Glu Lys 1 5
7210PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 72Ala Val Cys Thr Ser Thr Ser Pro Thr Arg 1 5
10 7318PRTArtificial sequencePeptide degradation product derived
from prh TNFR2Fc (PRX106) 73Ser Gln His Thr Gln Pro Thr Pro Glu Pro
Ser Thr Ala Pro Ser Thr 1 5 10 15 Ser Phe 749PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 74Gln Val Ser Leu Thr Cys Leu Val Lys 1 5
7523PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 75Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met
Cys Cys Ser Lys Cys 1 5 10 15 Ser Pro Gly Gln His Ala Lys 20
7617PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 76Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 1 5 10 15 His 777PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 77Asp Thr Leu
Met Ile Ser Arg 1 5 7817PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 78Pro Met Gly Pro Ser Pro
Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro 1 5 10 15 Lys
7919PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 79Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro 1 5 10 15 Ser Val Phe 807PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 80Asp Thr Leu Met Ile Ser Arg 1 5 8111PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 81Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg 1 5 10
8210PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 82Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 1 5
10 8312PRTArtificial sequencePeptide degradation product derived
from prh TNFR2Fc (PRX106) 83Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 1 5 10 8411PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 84Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys 1 5 10 8513PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 85Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 1 5
10 8626PRTArtificial sequencePeptide degradation product derived
from prh TNFR2Fc (PRX106) 86Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro 1 5 10 15 Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 20 25 8714PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 87Pro Ser Pro Pro Ala Glu Gly Ser
Thr Gly Asp Glu Pro Lys 1 5 10 8812PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 88Ser Leu Ser
Leu Ser Pro Gly Lys Ser Glu Lys Asp 1 5 10 8915PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 89Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr
Arg 1 5 10 15 9010PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 90Val Asp Gly Val Glu Val His Asn
Ala Lys 1 5 10 9123PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 91Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly Gly Pro
Ser Val Phe 20 9216PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 92Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys 1 5 10 15 9311PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 93Ser Leu Ser Leu Ser Pro Gly Lys Ser Glu Lys 1 5 10
9422PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 94Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu 1 5 10 15 Phe Pro Pro Lys Pro Lys 20
957PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 95Ser Phe Phe Leu Tyr Ser Lys 1 5
9614PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 96Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys 1 5 10 9720PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 97Phe Leu Leu Pro Met Gly
Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly 1 5 10 15 Asp Glu Pro Lys
20 9811PRTArtificial sequencePeptide degradation product derived
from prh TNFR2Fc (PRX106) 98Asp Ala Val Cys Thr Ser Thr Ser Pro Thr
Arg 1 5 10 9910PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 99Asn Gln Val Ser Leu Thr Cys Leu
Val Lys 1 5 10 10011PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 100Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly 1 5 10 1019PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 101Ser Leu
Ser Pro Gly Lys Ser Glu Lys 1 5 10219PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 102Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 1 5 10 15
Glu Val Lys 10311PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 103Leu Arg Glu Tyr Tyr Asp Gln
Thr Ala Gln Met 1 5 10 10422PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 104Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 1 5 10 15
Pro Glu Asn Asn Tyr Lys 20 10512PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 105Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn 1 5 10 10614PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 106Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
1 5 10 10716PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 107Ser Gln His Thr Gln Pro Thr
Pro Glu Pro Ser Thr Ala Pro Ser Thr 1 5 10 15 10820PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 108Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 1 5 10 15 Pro Glu Val Lys 20 1099PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 109Ser Leu Ser Leu Ser Pro Gly Lys Ser 1 5
11011PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 110Leu Ser Pro Gly Lys Ser Glu Lys Asp Glu Leu
1 5 10 1118PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 111Leu Pro Gln Pro Val Ser Thr
Arg 1 5 11215PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 112Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr 1 5 10 15 11319PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 113Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr
Thr Gln 1 5 10 15 Leu Trp Asn 11420PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 114Ala Leu
Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly 1 5 10 15
Ser Thr Cys Arg 20 1159PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 115Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg 1 5 11630PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 116Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30
11713PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 117Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys
Thr Lys 1 5 10 11815PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 118Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp 1 5 10 15
11913PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 119Ser Met Ala Pro Gly Ala Val His Leu Pro Gln
Pro Val 1 5 10 12012PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 120Thr Cys Arg Pro Gly
Trp Tyr Cys Ala Leu Ser Lys 1 5 10 12124PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 121Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val 1 5 10 15 Phe Leu Phe Pro Pro Lys Pro Lys 20
12230PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 122Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp
Ser Thr Tyr Thr Gln 1 5 10 15 Leu Trp Asn Trp Val Pro Glu Cys Leu
Ser Cys Gly Ser Arg 20 25 30 1237PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 123Leu Cys
Ala Pro Leu Arg Lys 1 5 12413PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 124Ser Pro
Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys 1 5 10
12511PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 125Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg
1 5 10 12615PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 126Gly Pro Ser Pro Pro Ala Glu
Gly Ser Thr Gly Asp Glu Pro Lys 1 5 10 15 12712PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 127Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg 1 5 10
1289PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 128Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 1 5
12915PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 129Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly
Ser Thr Cys Arg 1 5 10 15 1309PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 130Pro Gly
Trp Tyr Cys Ala Leu Ser Lys 1 5 1319PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 131Cys Arg
Pro Gly Phe Gly Val Ala Arg 1 5 13216PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 132Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 1 5 10 15
13319PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 133Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys 1 5 10 15 Glu Tyr Lys 1346PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 134Leu Cys Ala Pro Leu Arg 1 5 13526PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 135Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys 1 5 10 15 Asn Gln Val Ser Leu Thr Cys Leu Val Lys 20 25
13619PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 136Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu
Gly Ser Thr Gly Asp 1 5 10 15 Glu Pro Lys 13737PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 137Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro
Ser Thr 1 5 10 15 Ser Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala
Glu Gly Ser Thr 20 25 30 Gly Asp Glu Pro Lys 35 13810PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 138Ser Leu Ser Leu Ser Pro Gly Lys Ser Glu 1 5 10
1398PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 139Glu Glu Met Thr Lys Asn Gln Val 1 5
14014PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 140Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys 1 5 10 14141PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 141Ser Gln His Thr Gln
Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr 1 5 10 15 Ser Phe Leu
Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr 20 25 30 Gly
Asp Glu Pro Lys Ser Cys Asp Lys 35 40 14216PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 142Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly 1 5 10 15 14315PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 143Leu Arg Glu Tyr Tyr
Asp Gln Thr Ala Gln Met Cys Cys Ser Lys 1 5 10 15
14413PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 144Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys
Thr Arg 1 5 10 14516PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 145Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 1 5 10 15
14611PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 146Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
1 5 10 14713PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 147Cys Ser Ser Asp Gln Val Glu
Thr Gln Ala Cys Thr Arg 1 5 10 14810PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 148Asn Gln
Val Ser Leu Thr Cys Leu Val Lys 1 5 10 14913PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 149Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 1 5
10 15018PRTArtificial sequencePeptide degradation product derived
from prh TNFR2Fc (PRX106) 150Pro Ala Gln Val Ala Phe Thr Pro Tyr
Ala Pro Glu Pro Gly Ser Thr 1 5 10 15 Cys Arg 15114PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 151Ser Leu Ser Leu Ser Pro Gly Lys Ser Glu Lys Asp Glu Leu
1 5 10 15214PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 152Ala Phe Thr Pro Tyr Ala Pro
Glu Pro Gly Ser Thr Cys Arg 1 5 10 15314PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 153Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg
1 5 10 15415PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 154Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys 1 5 10 15 15514PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 155Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15614PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 156Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn 1 5 10 15711PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 157Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 1 5 10
15816PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 158Ser Met Ala Pro Gly Ala Val His Leu Pro Gln
Pro Val Ser Thr Arg 1 5 10 15 15920PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 159Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 1 5 10 15
Glu Met Thr Lys 20 16012PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 160Thr Pro Tyr Ala Pro
Glu Pro Gly Ser Thr Cys Arg 1 5 10 16117PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 161Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val 1 5 10 15 Lys 16213PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 162Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 1 5 10
16312PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 163Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 1 5 10 16415PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 164Leu Arg Glu Tyr Tyr Asp Gln
Thr Ala Gln Met Cys Cys Ser Lys 1 5 10 15 16513PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 165Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg 1 5
10 16616PRTArtificial sequencePeptide degradation product derived
from prh TNFR2Fc (PRX106) 166Ser Met Ala Pro Gly Ala Val His Leu
Pro Gln Pro
Val Ser Thr Arg 1 5 10 15 16712PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 167Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 16819PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 168Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys 1 5 10 15 Glu Tyr Lys 16915PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 169Ser Gln
His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser 1 5 10 15
17014PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 170Ser Met Ala Pro Gly Ala Val His Leu Pro Gln
Pro Val Ser 1 5 10 17111PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 171Ala Val His Leu Pro
Gln Pro Val Ser Thr Arg 1 5 10 17215PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 172Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 1 5 10 15
17313PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 173Pro Gly Ala Val His Leu Pro Gln Pro Val Ser
Thr Arg 1 5 10 1746PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 174Thr Leu Met Ile Ser
Arg 1 5 17533PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 175Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser 1 5 10 15 Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 20 25 30 Lys
17612PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 176Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met
Cys 1 5 10 17713PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 177Ser Met Ala Pro Gly Ala Val
His Leu Pro Gln Pro Val 1 5 10 1787PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 178Leu Pro
Ala Pro Ile Glu Lys 1 5 17913PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 179Glu Tyr
Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser Lys 1 5 10
18012PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 180Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser
Arg 1 5 10 18112PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 181Ser Leu Ser Pro Gly Lys Ser
Glu Lys Asp Glu Leu 1 5 10 18214PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 182Ile Cys
Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys 1 5 10
18315PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 183Ser Met Ala Pro Gly Ala Val His Leu Pro Gln
Pro Val Ser Thr 1 5 10 15 18413PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 184Glu Tyr
Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser Lys 1 5 10
18514PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 185Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser
Pro Thr Arg 1 5 10 18617PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 186Ser Gln His Thr Gln
Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr 1 5 10 15 Ser
18711PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 187Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
1 5 10 18819PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 188Ser Gln His Thr Gln Pro Thr
Pro Glu Pro Ser Thr Ala Pro Ser Thr 1 5 10 15 Ser Phe Leu
1896PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 189Thr Leu Met Ile Ser Arg 1 5
19016PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 190Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys 1 5 10 15 19120PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 191Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 1 5 10 15
Glu Met Thr Lys 20 19221PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 192Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 1 5 10 15 Glu Val Lys
Phe Asn 20 19318PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 193Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly
19422PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 194Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln 1 5 10 15 Pro Glu Asn Asn Tyr Lys 20
19517PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 195Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser 1 5 10 15 Arg 19613PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 196Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 1 5
10 19718PRTArtificial sequencePeptide degradation product derived
from prh TNFR2Fc (PRX106) 197Leu Pro Met Gly Pro Ser Pro Pro Ala
Glu Gly Ser Thr Gly Asp Glu 1 5 10 15 Pro Lys 19816PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 198Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys 1 5 10 15 19914PRTArtificial sequencePeptide degradation
product derived from prh TNFR2Fc (PRX106) 199Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 1 5 10 20017PRTArtificial
sequencePeptide degradation product derived from prh TNFR2Fc
(PRX106) 200Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp
Glu Pro 1 5 10 15 Lys 20116PRTArtificial sequencePeptide
degradation product derived from prh TNFR2Fc (PRX106) 201Ser Met
Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg 1 5 10 15
20211PRTArtificial sequencePeptide degradation product derived from
prh TNFR2Fc (PRX106) 202Ser Met Ala Pro Gly Ala Val His Leu Pro Gln
1 5 10 20318PRTArtificial sequencePeptide degradation product
derived from prh TNFR2Fc (PRX106) 203Leu Pro Met Gly Pro Ser Pro
Pro Ala Glu Gly Ser Thr Gly Asp Glu 1 5 10 15 Pro Lys
204474PRTArtificial SequencePRX106 204Ala Leu Pro Ala Gln Val Ala
Phe Thr Pro Tyr Ala Pro Glu Pro Gly 1 5 10 15 Ser Thr Cys Arg Leu
Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys 20 25 30 Cys Ser Lys
Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys 35 40 45 Thr
Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln 50 55
60 Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser
65 70 75 80 Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn
Arg Ile 85 90 95 Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser
Lys Gln Glu Gly 100 105 110 Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys
Arg Pro Gly Phe Gly Val 115 120 125 Ala Arg Pro Gly Thr Glu Thr Ser
Asp Val Val Cys Lys Pro Cys Ala 130 135 140 Pro Gly Thr Phe Ser Asn
Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro 145 150 155 160 His Gln Ile
Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp 165 170 175 Ala
Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala 180 185
190 Val His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro
195 200 205 Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu
Pro Met 210 215 220 Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp
Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310
315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435
440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu 450 455 460 Ser Pro Gly Lys Ser Glu Lys Asp Glu Leu 465 470
205472PRTArtificial SequencePRX106 205Pro Ala Gln Val Ala Phe Thr
Pro Tyr Ala Pro Glu Pro Gly Ser Thr 1 5 10 15 Cys Arg Leu Arg Glu
Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser 20 25 30 Lys Cys Ser
Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr Ser 35 40 45 Asp
Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp 50 55
60 Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp
65 70 75 80 Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile
Cys Thr 85 90 95 Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln
Glu Gly Cys Arg 100 105 110 Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro
Gly Phe Gly Val Ala Arg 115 120 125 Pro Gly Thr Glu Thr Ser Asp Val
Val Cys Lys Pro Cys Ala Pro Gly 130 135 140 Thr Phe Ser Asn Thr Thr
Ser Ser Thr Asp Ile Cys Arg Pro His Gln 145 150 155 160 Ile Cys Asn
Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val 165 170 175 Cys
Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His 180 185
190 Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro
195 200 205 Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met
Gly Pro 210 215 220 Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro
Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310
315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435
440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 450 455 460 Gly Lys Ser Glu Lys Asp Glu Leu 465 470
* * * * *