U.S. patent application number 11/971112 was filed with the patent office on 2008-10-02 for novel inhibitors of angiogenesis and tumor growth.
This patent application is currently assigned to UNIVERSITY OF SOUTHERN CALIFORNIA. Invention is credited to Parkash S. Gill.
Application Number | 20080242840 11/971112 |
Document ID | / |
Family ID | 22234332 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080242840 |
Kind Code |
A1 |
Gill; Parkash S. |
October 2, 2008 |
NOVEL INHIBITORS OF ANGIOGENESIS AND TUMOR GROWTH
Abstract
This invention provides for polypeptides that have surprising
anti-angiogenic activity. These peptides are derived from Saposin
B, a previously known protein involved in the hydrolysis of
sphingolipids. In addition, methods of treating mammals with these
anti-angiogenic polypeptides are provided, as well as the
pharmaceutical compositions used to treat. Furthermore, the
polypeptides of this invention can be used in fusion proteins,
wherein the fusion proteins also comprise cell targeting or
cytotoxic moieties. Also provided is the receptor to which these
polypeptides bind.
Inventors: |
Gill; Parkash S.; (Agoura,
CA) |
Correspondence
Address: |
HOGAN & HARTSON L.L.P.
1999 AVENUE OF THE STARS, SUITE 1400
LOS ANGELES
CA
90067
US
|
Assignee: |
UNIVERSITY OF SOUTHERN
CALIFORNIA
Los Angeles
CA
|
Family ID: |
22234332 |
Appl. No.: |
11/971112 |
Filed: |
January 8, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09743684 |
Apr 23, 2001 |
7341730 |
|
|
PCT/US99/15772 |
Jul 12, 1999 |
|
|
|
11971112 |
|
|
|
|
60092647 |
Jul 13, 1998 |
|
|
|
Current U.S.
Class: |
530/350 ;
530/387.9 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 43/00 20180101; A61P 9/00 20180101; C07K 2319/00 20130101;
C07K 14/475 20130101; A61P 35/00 20180101 |
Class at
Publication: |
530/350 ;
530/387.9 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C07K 14/00 20060101 C07K014/00 |
Claims
1-23. (canceled)
24. An isolated protein, wherein said protein specifically binds to
Saposin B and is found on the surface of cells selected from the
group consisting of KS Y-1, SLK and HUVEC.
25. The isolated protein of claim 24, wherein said protein is
recombinantly expressed.
26. An antibody that is specifically reactive with the isolated
polypeptide of claim 7.
27. The antibody of claim 26, wherein said monoclonal antibody is a
monoclonal antibody.
28. The antibody of claim 26, wherein said antibody is a single
chain antibody.
29-52. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation in part of U.S.
Provisional Application 60/092,647, filed Jul. 13, 1998, which is
incorporated by reference in its entirety.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Not Applicable.
BACKGROUND OF THE INVENTION
[0003] Angiogenesis is the formation of new blood vessels from
existing blood vessels. To initiate the angiogenic process,
biochemical signals stimulate protease secretion from, among other
cell types, endothelial cells lining the lumen of the vessel. The
secreted proteases degrade the basement membrane and the
endothelial cell layer protrudes through the hole created in the
basement membrane. If the biochemical signals are continuously
present, the migrating endothelial cells undergo mitosis and
divide. The dividing cells form a sprout through the vessel wall.
Again, if the angiogenic stimulus remains, the sprouts merge to
form capillary loops which later mature into new blood vessels.
[0004] Under normal circumstances of wound healing, fetal and
embryonic development and formation of the corpus luteum,
endometrium and placenta, the initial angiogenic signals subside
and other, secondary, signals predominate to turn off the
angiogenic process. However in disease states such as cancer,
angiofibroma, neovascular glaucoma, arteriovenous malformations,
nonunion fractures, arthritis and other connective tissue
disorders, Osler-Weber syndrome, atherosclerotic plaques,
psoriasis, corneal graft neovascularization, pyogenic granuloma,
retrolental fibroplasia, diabetic retinopathy, scleroderma,
hemangioma, trachoma, vascular adhesions and hypertrophic scars,
the local concentration of angiogenic signals never decreases and
new blood vessels continuously form, supplying the diseased tissue
with nutrients. This allows the tumor or diseased tissue to
grow.
[0005] In cancer, undesired angiogenesis provides a steady supply
of nutrients to the tumor. This allows the tumor to grow as well as
metastasize. However, in addition to a general tumor
growth-supporting role, some tumors are highly angiogenic. For
example, Kaposi's Sarcoma (KS) is a tumor characterized by
unregulated growth of blood vessels. It is, in fact, an angiogenic
tumor. Currently, the treatment of Kaposi's Sarcoma, like most
tumors, is based on chemotherapy. However, most chemotherapeutic
agents are universally harmful to all dividing cells; cancerous or
not. Thus, there is a need for compounds that will reduce the
angiogenesis required for many disease states, including cancer and
specifically, Kaposi's Sarcoma. This invention surprising meets
these and other needs.
BRIEF SUMMARY OF THE INVENTION
[0006] A surprising discovery of this invention is that Saposin B,
previously known as a protein involved in the hydrolysis of
sphingolipids has potent anti-angiogenic and antitumoral activity.
In addition, this protein has been found to have anti-proliferative
and anti-migratory activity against endothelial cells. Even more
surprising was the discovery that the activity against tumor and
endothelial cells was conserved in cryptic polypeptides as small as
five amino acids. These small polypeptides can now be used either
in vitro as well as in vivo as anti-angiogenic and anti-tumor
agents.
[0007] One embodiment of this invention is an isolated polypeptide
of about 5 to about 80 amino acids in length and comprising a
contiguous amino acid sequence DX.sub.1CX.sub.2D. X.sub.1 and
X.sub.2 can be any amino acid. In one aspect of this embodiment,
the isolated polypeptide is between 7 and 50 amino acids in length.
In another embodiment, the isolated polypeptide is between 11 and
50 amino acids in length. In yet another embodiment, the isolated
polypeptide is between 5 and 40 amino acids in length. In yet
another embodiment, the isolated polypeptide is between 7 and 40
amino acids in length. In yet another embodiment, the isolated
polypeptide is between 11 and 40 amino acids in length. In yet
another embodiment, the isolated polypeptide is between 5 and 30
amino acids in length. In yet another embodiment, the isolated
polypeptide is between 7 and 30 amino acids in length. In yet
another embodiment, the isolated polypeptide is between 11 and 30
amino acids in length. In yet another embodiment, the isolated
polypeptide is between 5 and 20 amino acids in length. In yet
another embodiment, the isolated polypeptide is between 7 and 20
amino acids in length. In yet another embodiment, the isolated
polypeptide is between 11 and 20 amino acids in length.
[0008] In further embodiments, X.sub.1 is a valine or a
conservatively modified variant thereof or X.sub.2 is a glutamine
or a conservatively modified variant thereof. In a preferred
embodiment, the polypeptide will comprise the contiguous amino acid
sequence DVCQD.
[0009] In yet another embodiment, the isolated polypeptide
specifically binds to an antibody raised against Saposin B. In a
preferred embodiment, the polypeptide comprises an amino acid
sequence substantially identical to that shown in SEQ ID NO:1
beginning at position 7. In a most preferred embodiment, the
polypeptide comprises at least 5 contiguous amino acids, or
conservatively modified variants thereof, said contiguous amino
acids having an amino acid sequence as shown in SEQ ID NO:1,
beginning at position 7.
[0010] In still another embodiment, the isolated polypeptide
comprises R-DVCQD-R'; wherein R is from 0 to about 6 contiguous
amino acids; and wherein R' is from 0 to about 59 contiguous amino
acids. In a preferred embodiment, the polypeptide comprises
R-XDVCQD-R'; wherein R is selected from the group consisting of
Aa.sub.1-Aa.sub.2-Aa.sub.3-Aa.sub.4-Aa.sub.5,
Aa.sub.2-Aa.sub.3-Aa.sub.4-Aa.sub.5, Aa.sub.3-Aa.sub.4-Aa.sub.5,
Aa.sub.4-Aa.sub.5 and Aa.sub.5. Aa.sub.1, Aa.sub.2, Aa.sub.3,
Aa.sub.4 and Aa.sub.5 are selected from the group consisting of
amino acids; X is selected from the group consisting of G, A, S and
T; and wherein R' is from 0 to about 59 contiguous amino acids. In
a more preferred embodiment, Aa.sub.1 is a glutamine or a
conservative substitution thereof, Aa.sub.2 in a proline or a
conservative substitution thereof, Aa.sub.3 in a lysine or a
conservative substitution thereof, Aa.sub.4 in an aspartic acid or
a conservative substitution thereof, or Aa.sub.5 in a asparagine or
a conservative substitution thereof.
[0011] In another embodiment, R' is selected from the group
consisting of Aa.sub.12-Aa.sub.13-Aa.sub.14-Aa.sub.15-Aa.sub.16,
Aa.sub.12-Aa.sub.13-Aa.sub.14-Aa.sub.15,
Aa.sub.12-Aa.sub.13-Aa.sub.14, Aa.sub.12-Aa.sub.13 and Aa.sub.12,
wherein Aa.sub.12, Aa.sub.13, Aa.sub.14, Aa.sub.15 and Aa.sub.16
are selected from the group consisting of amino acids. In a
preferred embodiment, Aa.sub.12 is a cysteine or a conservative
substitution thereof, Aa.sub.13 is an isoleucine or a conservative
substitution thereof, Aa.sub.14 is an glutamine or a conservative
substitution thereof, Aa.sub.15 is a methionine or a conservative
substitution thereof, or Aa.sub.16 is a valine or a conservative
substitution thereof.
[0012] In a most preferred embodiment, the isolated polypeptide has
the amino acid sequence GDVCQDCIQMV.
[0013] In another embodiment of this invention, a receptor is
provided wherein the receptor specifically binds to Saposin B and
is found on the surface of cells selected from the group consisting
of KS Y-1, SLK, HUVEC and murine endothelial cells. In a preferred
embodiment, the receptor is recombinantly expressed.
[0014] In another embodiment, a method of treating a mammal is
provided, wherein said organism has a pathological condition
associated with undesired angiogenesis. The method comprises
administering to the mammal an amount of an isolated polypeptide
comprising a contiguous amino acid sequence DX.sub.1CX.sub.2D,
wherein X.sub.1 and X.sub.2 are selected from the group consisting
of amino acids, wherein the amount of polypeptide effective to
reduce angiogenesis. In a most preferred embodiment, the mammal is
human and the isolated polypeptide is Saposin B.
[0015] In a more preferred embodiment, the pathological condition
to be treated is cancer. In the most preferred embodiment, the
cancer is Kaposi's Sarcoma. Administration of the isolated
polypeptide is selected from the group consisting of subcutaneous,
intramuscular, intravenous, intra-arterial, intrabronchial,
parenteral, transdermal, intraocular, rectal, vaginal, intranasal,
sublingual and intralesional. In the most preferred embodiment, the
administration is selected from the group consisting of
intralesional and transdermal.
[0016] In yet another embodiment, a pharmaceutical composition in
unit dosage form is provided, the composition comprising one or
more pharmaceutically acceptable excipients, and an amount of a
polypeptide comprising a contiguous amino acid sequence
DX.sub.1CX.sub.2D, wherein X.sub.1 and X.sub.2 are selected from
the group consisting of amino acids. The polypeptide is effective
to treat or prevent undesired angiogenesis in an animal or patient
to whom one or more unit doses of said composition are
administered. In this embodiment, it is preferred the unit dosage
form be a solution comprising said polypeptide.
[0017] In still another embodiment, a fusion protein is provided,
wherein the fusion protein comprises a polypeptide of a contiguous
amino acid sequence DX.sub.1CX.sub.2D, wherein X.sub.1 and X.sub.2
are selected from the group consisting of amino acids. The second
moiety of the fusion protein is a cell targeting moiety. The cell
targeting moiety and the polypeptide have functional activity
independent of each other. In a more preferred embodiment, the cell
targeting moiety is a protein. In a most preferred embodiment, the
protein is an antibody. In a further refinement, the antibody is a
monoclonal antibody. In yet another refinement, the antibody is a
single chain Fv antibody.
[0018] In one embodiment of this invention, another fusion protein
is provided, wherein the fusion protein comprises a polypeptide of
a contiguous amino acid sequence DX.sub.1CX.sub.2D, wherein X.sub.1
and X.sub.2 are selected from the group consisting of amino acids.
The fusion protein also comprises a cytotoxic moiety. The cytotoxic
moiety and said polypeptide have functional activity independent of
each other. In a preferred embodiment, the cytotoxic moiety is a
protein. In a more preferred embodiment, the protein is a bacterial
toxin. In a most preferred embodiment, the bacterial toxin is from
Diphtheria, particularly the B chain of Diphtheria toxin.
[0019] In a related embodiment, the bacterial toxin is from
Pseudomonas, in particular Pseudomonas exotoxin. In the most
preferred of this embodiment, the Pseudomonas exotoxin is selected
from the group consisting of recombinant PE38 and PE40.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1: Activity of recombinant Saposin B. Only KS and
endothelial cells showed dose dependent growth inhibition.
[0021] FIG. 1B: Activity of recombinant Saposin B. Only
proliferating and not quiescent endothelial cells (HUVEC) show dose
dependent growth inhibition.
[0022] FIG. 1C: Activity of truncated recombinant Saposin B.
Saposin (Sap) B 1-69 (consisting of the first 69 amino acids), Sap
B 1-42 (consisting of the first 42 amino acids), Sap B 11-81
(consisting of 11-81 amino acids and thus lacking the first 10
amino acids). Notably, Sap B 1-69 and Sap B 1-42, but not Sap B
11-81 showed dose dependent growth inhibition.
[0023] FIG. 1D: Activity of n-terminus decapeptide (DVCQDCIQMV SEQ
ID NO 21). Only endothelial cells show dose dependent growth
inhibition.
[0024] FIG. 1E: Activity of N-terminus pentapeptide (DVCQC, SEQ ID
NO 28). Only endothelial cells show dose dependent growth
inhibition.
[0025] FIG. 2: Saposin B inhibited endothelial cell migration. The
assays were done in double chamber wells separated by
fibrinogen-coated membrane. Chemotaxis was induced by bFGF (25
ng/mL) in the lower chamber. Endothelial cells or KS cells
(5.times.10.sup.4/mL) were placed in the upper chamber in the
presence and absence of test polypeptides. Taxol at 10 ng/mL was
used as a known inhibitor of migration. Cell migration across the
membrane was quantitated after an overnight incubation.
[0026] FIG. 3: Effect of recombinant Saposin B angiogenesis. The
number of blood vessel branches formed in CAMs in response to
angiogenic factor (bFGF) in inhibited by Saposin B in a
dose-dependent fashion.
[0027] FIG. 4: Inhibition of tumor growth in mice. C57BL/6 mice
were implanted with Lewis lung carcinoma, melanoma (B16), and
T-cell lymphoma (EL4). KS Y-1 was implanted in nude mice as a
positive control. One day after implantation 2.5 mg/kg of Saposin B
was injected into the mice subcutaneously.
[0028] FIG. 5: Effect of two daily doses of Saposin B on growth of
an established KS Y-1 tumor. The arrow marks initiation of daily
subcutaneous dosing.
[0029] FIG. 6: Effect of pentapeptide DVCQD (SEQ ID NO 28) on the
growth of established KS Y-1 tumors in mice. Mice were implanted
with the tumor on day one. Treatment with the peptide was started
on the following day at a dose of 50 mg/kg subcutaneously daily.
When compared to the control, the tumor volumes were significantly
smaller. The arrow marks initiation of daily subcutaneous
dosing.
DEFINITIONS
[0030] Unless defined otherwise herein, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY
AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York
(1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF
BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a
general dictionary of many of the terms used in this invention.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, the preferred methods and materials are
described. Numeric ranges are inclusive of the numbers defining the
range. Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation; amino acid sequences are written
left to right in amino to carboxyl orientation, respectively. The
headings provided herein are not limitations of the various aspects
or embodiments of the invention which can be had by reference to
the specification as a whole. Accordingly, the terms defined
immediately below are more fully defined by reference to the
specification as a whole.
[0031] The phrase "administering a therapeutic amount" refers to
the means by which anti-angiogenic polypeptides are used to treat a
mammal. The term "administering" is intended to encompass all
methods which result in the contact between a mammal and the
polypeptides of this invention, including but not limited to,
subcutaneous, intramuscular, intravenous, intra-arterial,
intraocular and intralesional injections; intrabronchial and
intranasal inhalation or instillation; rectal and vaginal
suppositories; sublingual and oral delivery; and absorption across
the dermal and mucosal barriers.
[0032] The term "amino acid" includes reference to an amino acid
that is incorporated into a protein, polypeptide, or peptide
(collectively "polypeptide"). The amino acid can be a naturally
occurring amino acid and, unless otherwise limited, can encompass
known analogs of natural amino acids that can function in a similar
manner as naturally occurring amino acids.
[0033] The amino acids and analogs referred to herein are described
by shorthand designations as follows in Table 1:
TABLE-US-00001 TABLE 1 Amino Acid Nomenclature Name 3-letter
1-letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic
Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q
Glycine Gly G Histidine His H Homoserine Hse -- Isoleucine Ile I
Leucine Leu L Lysine Lys K Methionine Met M Methionine sulfoxide
Met (O) -- Methionine Met (S-Me) -- methylsulfonium Norleucine Nle
-- Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T
Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0034] Unless stated otherwise, "X.sub.n" and "Aa.sub.n" refer to
any amino acid. The amino acid may be a naturally occurring L-amino
acid, a D-amino acid or any synthetic amino acid analog. The phrase
"contiguous amino acid sequence" refers to a linear amino acid
sequence wherein the first amino acid is at the N terminus of the
polypeptide and the last amino acid is at the C terminus. The terms
"R" and "R'" refer to contiguous amino acid sequences. Unless
stated otherwise, "R" is a contiguous amino acid sequence at the N
terminus of a polypeptide and "R'" is a contiguous amino acid
sequence at the C terminus of a polypeptide. R and R' do not
necessarily comprise the same contiguous amino acid sequence.
[0035] As used herein, "antibody" includes reference to an
immunoglobulin molecule immunologically reactive with a particular
antigen, and includes both polyclonal and monoclonal antibodies.
The term also includes genetically engineered forms such as
chimeric antibodies (e.g., humanized murine antibodies),
heteroconjugate antibodies (e.g., bispecific antibodies) and
recombinant single chain Fv fragments (scFv), or disulfide
stabilized (dsFv) Fv fragments (see, U.S. Pat. No. 5,747,654). The
term "antibody" also includes antigen binding forms of antibodies
(e.g., Fab', F(ab').sub.2, Fab, Fv and rIgG. See also, Pierce
Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,
Ill.)).
[0036] An antibody immunologically reactive with a particular
antigen can be generated by recombinant methods such as selection
of libraries of recombinant antibodies in phage or similar vectors.
See, e.g., Huse, et al., Science 246:1275-1281 (1989); Ward, et
al., Nature 341:544-546 (1989); and Vaughan, et al., Nature
Biotech. 14:309-314 (1996).
[0037] Typically, an immunoglobulin has a heavy and light chain.
Each heavy and light chain contains a constant region and a
variable region. Light and heavy chain variable regions contain a
"framework" region interrupted by three hypervariable regions, also
called complementarity-determining regions or CDRs. The extent of
the framework region and CDRs have been defined (see, SEQUENCES OF
PROTEINS OF IMMUNOLOGICAL INTEREST, Kabat, E., et al., U.S.
Department of Health and Human Services, (1987)). The sequences of
the framework regions of different light or heavy chains are
relatively conserved within a species. The framework region of an
antibody, that is the combined framework regions of the constituent
light and heavy chains, serves to position and align the CDRs in
three dimensional space. The CDRs are primarily responsible for
binding to an epitope of an antigen. The CDRs are typically
referred to as CDR1, CDR2, and CDR3, numbered sequentially starting
from the N-terminus.
[0038] The phrase "single chain Fv" or "scFv" refers to an antibody
in which the heavy chain and the light chain of a traditional two
chain antibody have been joined to form one chain. Typically, a
linker peptide is inserted between the two chains to allow for
proper folding and creation of an active binding site. A "linker
peptide" includes, but is not limited to, peptides within an
antibody binding fragment (e.g., Fv fragment) which serve to
indirectly bond the variable heavy chain to the variable light
chain.
[0039] The phrase "aseptic solution" refers to a solution that is
microorganism-free. Making a solution microorganism-free can be
done by removal of or by killing the microorganisms. Methods of
removal of microorganisms consist primarily of filtration using a
membrane with a pore size smaller than the microorganism.
Typically, the pore size is 0.11-0.22 .mu.m. Another, less
preferred method of removing microorganisms is by centrifugation.
Methods of killing microorganisms are well known in the art and
include but are not limited to, pasteurization, treatment with high
pressure and temperature, i.e., autoclaving, contact with
anti-microbial agents, e.g., antibiotics, antivirals, antifungals,
etc. However, one of skill will realize that in some embodiments of
this invention, the solutions are intended to be administered to
mammals in need of treatment. Therefore, the agents used to kill
microorganisms should not have adverse effects on the mammal to be
treated.
[0040] The term "cancer", for purposes of this disclosure, refers
to a pathological condition caused by unregulated in vivo growth of
cells. Thus, for purposes of this disclosure, cancer includes but
is not limited to the following: solid as well as hematopoietic
tumors, malignant and benign tumors, primary and metastatic tumors,
and precancerous conditions. One such cancer is "Kaposi's Sarcoma."
Kaposi's Sarcoma presents in three different classes of
individuals. Classic Kaposi's sarcoma is a rare, indolent, cancer
of mainly elderly men of Jewish or Mediterranean origin
(Lospalleti, M., et al., Dermatology 191(2): 104-8 (1995)). Endemic
Kaposi's Sarcoma (EKS) affects elderly and young Africans,
particularly Bantus. EKS can become particularly aggressive after a
long period of quiescence (Safai, B., Semin Oncol 2 (Suppl 3): 7-12
(1987)). HIV-associated Kaposi's sarcoma is an aggressive cancer
found as an opportunistic disease related to infection with HIV
(Wahman, A., et al., Epidemiol Rev. 13:178-9 (1991)). In all of the
above types of Kaposi's sarcoma, a compromised immune system is
indicated.
[0041] The HIV-related form of Kaposi's sarcoma (AIDS-KS) most
frequently presents with cutaneous lesions. Occasionally, cases
present with lymph node or visceral KS only. Mucosal involvement of
the oral cavity is the second most common site of disease. The
tumor lesions are noted frequently on the palate, gums and can
cause tooth loss, pain and ulceration (Paredes, S., J. Acquir.
Immune Defic. Syndr. Hum. Retroviral 9(2):138-44 (1995)).
[0042] A "conservative substitution," when describing a polypeptide
refers to a change in the amino acid composition of the polypeptide
that does not substantially alter the polypeptide's activity.
"Conservatively modified variations" of a particular amino acid
sequence refers to amino acid substitutions of those amino acids
that are not critical for functional activity or substitution of
amino acids with other amino acids having similar properties (e.g.,
acidic, basic, positively or negatively charged, polar or
non-polar, etc.) such that the substitutions of even critical amino
acids do not substantially alter activity. Conservative
substitution tables providing functionally similar amino acids are
well known in the art.
[0043] A "cell targeting moiety," as used herein, refers generally
to compounds capable of specifically delivering a molecule,
reacting with or otherwise recognizing or binding to a target cell.
Specifically, examples of cell targeting moieties include, but are
not limited to, immunoglobulins or binding fragments thereof,
lymphokines, cytokines, cell surface antigens, solubilized receptor
proteins, hormones, growth factors such as epidermal growth factor
(EOF), and the like which specifically bind desired target cells.
Although the above exemplified cell targeting moieties are
polypeptides, it is not necessary that cell targeting moieties
consist of polypeptides. Cell targeting moieties can also be
carbohydrates, drugs, lipids or any other compound which
selectively binds to a target cell.
[0044] The term "cytotoxic moiety" includes, but is not limited to,
abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT),
botulinum toxin, or modified toxins thereof. For example, PE and DT
are bacterial toxins that typically bring about death through liver
toxicity. PE and DT, however, can be modified into a form for use
in a fusion protein by removing the native targeting component of
the toxin (e.g., domain Ia of PE and the B chain of DT) and
replacing it with a different moiety, such as a polypeptide which
specifically binds to a cell to be killed. "PE38" and "PE40" refer
to a 38 kD and a 40 kD, respectively, cytotoxic moiety derived from
PE. See, for example, U.S. Pat. Nos. 5,082,927 and 5,696,237 as
well as Chaudhary, et al., Nature 339:394 (1989) for descriptions
of and methods of making and using PE40 and Chaudhary, et al.,
Proc. Nat'l Acad. Sci. USA 87:308 (1990) and Benhar, et al,
Bioconjug. Chem. 5:321 (1994) for descriptions of PE 38 as well as
methods for making and using PE38.
[0045] The terms "effective amount" or "amount effective to" or
"therapeutically effective amount" includes reference to a dosage
of a therapeutic agent sufficient to produce a desired result, such
as inhibiting the formation of new blood vessels by at least 25%,
or killing a cell.
[0046] The phrase "functional activity independent of each other",
in the context of this invention, refers to the activity of the two
moieties of the fusion proteins of this invention. For example, the
polypeptides of the fusion proteins have anti-angiogenic activity.
This activity is independent of the cell targeting or cytotoxic
activity of the other moiety of the fusion protein.
[0047] A "fusion protein" refers to a chimeric molecule formed by
the joining of two or more compounds through a bond formed between
one moiety and another moiety. For purposes of this invention, one
moiety is a polypeptide. The bond between the polypeptide and the
other moiety may be covalent or noncovalent. An example of a
covalent bond is the chemical coupling of two polypeptides to form
peptide bond. Examples of non-covalent bond are hydrogen bonds,
electrostatic interactions and van der Waal's forces.
[0048] If the bond is a peptide bond and the other moiety is a
polypeptide as well, the fusion protein may be expressed as a
single polypeptide from a nucleic acid sequence encoding a single
contiguous fusion protein.
[0049] The term "identical" in the context of two polypeptide
sequences refers to the residues in the two sequences that are the
same when aligned for maximum correspondence, as measured using one
of the following "sequence comparison algorithms." The phrase
"substantially identical" in the context of two polypeptides refers
to the residues in the two sequences that have at least 60%
identity when aligned for maximum correspondence over a domain of
the protein. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Si. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by
inspection.
[0050] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments. It can also plot a tree
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method
used is similar to the method described by Higgins & Sharp,
CABIOS 5:151-153 (1989). The program can align up to 300 sequences
of a maximum length of 5,000. The multiple alignment procedure
begins with the pairwise alignment of the two most similar
sequences, producing a cluster of two aligned sequences. This
cluster can then be aligned to the next most related sequence or
cluster of aligned sequences. Two clusters of sequences can be
aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program can also be used
to plot a dendrogram or tree representation of clustering
relationships. The program is run by designating specific sequences
and their amino acid coordinates for regions of sequence
comparison.
[0051] Another example of algorithm that is suitable for
determining sequence similarity is the BLAST algorithm, which is
described in Altschul, et al., J. Mol. Biol. 215:403-410 (1990).
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul, et al, supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Extension of the word
hits in each direction are halted when: the cumulative alignment
score falls off by the quantity X from its maximum achieved value;
the cumulative score goes to zero or below, due to the accumulation
of one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T,
and X determine the sensitivity and speed of the alignment. The
BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M'5, N'-4, and a comparison of both strands.
[0052] The BLAST algorithm performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to an ribonuclease nucleic acid if the smallest sum
probability in a comparison of the test nucleic acid to an
ribonuclease nucleic acid is less than about 0.1, more preferably
less than about 0.01, and most preferably less than about 0.001.
Where the test nucleic acid encodes a ribonuclease polypeptide, it
is considered similar to a specified ribonuclease nucleic acid if
the comparison results in a smallest sum probability of less than
about 0.5, and more preferably less than about 0.2.
[0053] Another indication that two polypeptides are substantially
identical is that the first polypeptide is immunologically
cross-reactive with the second polypeptide. Thus, a polypeptide is
substantially identical to a second polypeptide, for example, where
the two peptides differ only by a conservative substitution.
[0054] The terms "isolated," "purified" or "biologically pure"
refer to material that is substantially or essentially free from
components which normally accompany it as found in its native
state. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography. A
polypeptide that is the predominant species present in a
preparation is substantially purified. The term "purified" denotes
that a polypeptide gives rise to essentially one band in an
electrophoretic gel. Particularly, it means that the polypeptide is
at least 85% pure, more preferably at least 95% pure, and most
preferably at least 99% pure.
[0055] The term "liposome" refers to vesicles comprised of one or
more concentrically ordered lipid bilayers which encapsulate an
aqueous interior. Normally, the encapsulated interior matrix does
not permeate the bilayer. However, if a hole or pore occurs in the
bilayer, if the bilayer is dissolved or degraded, if the bilayer
changes conformation, or if the environmental temperature is
increased to the phase transition temperature, Tc, of the
constituent lipids, the matrix may leak through the liposome.
[0056] The "interior" of a liposome is the aqueous area surrounded
by the lipid bi-layer of the liposome, i.e., encapsulated matrix.
The process of placing a compound within the aqueous matrix is
termed "encapsulating," The "surface" of a liposome is the
hydrophilic portion of the substituent lipids exposed to the
extraliposomal environment. Binding of a polypeptide to the
interior or exterior surface of a liposome can be due to covalent
bonding of the compound to the hydrophilic group, hydrogen bonding,
electrostatic interactions, or hydrophobic/hydrophilic
interactions. In addition to binding to the surface, a compound
with a hydrophobic component can insert into the liposome bilayer
so that the hydrophobic component is within the bilayer and the
hydrophilic portion of the compound extends beyond the surface of
the liposome or into the interior matrix of the liposome.
[0057] "Pharmaceutically acceptable excipients" refers to
ingredients other than the active ingredient in the pharmaceutical
compositions of this invention.
[0058] The phrase "pharmaceutical composition" refers to
compositions of the polypeptides or fusion proteins of this
invention mixed with suitable carriers or excipient(s) in a
therapeutically effective amount, e.g., at doses effective to
reduce angiogenesis.
[0059] As used herein, "polypeptide," "peptide" and "protein" are
used interchangeably and include reference to a polymer of amino
acid residues and/or amino acid analogs. The terms apply to amino
acid polymers in which one or more amino acid residue is an
artificial chemical analog of a corresponding naturally occurring
amino acid, e.g., a peptidomimetic, as well as to naturally
occurring amino acid polymers. The terms also apply to polymers
containing conservative amino acid substitutions such that the
polypeptide remains functional. The term polypeptide also includes
concatemer units of a motif, or a contiguous amino acid sequence
within a larger amino acid sequence, or polypeptides comprising the
motif.
[0060] The phrase "antibody raised against Saposin B" refers to
antibodies that can neutralize the anti-angiogenic activity of
Saposin B or of the active peptides provided herein. The antibodies
can be either polyclonal or monoclonal. These antibodies are
produced or raised by immunogenically exposing Saposin B to the
immune system of an animal able to produce antibodies specific to
Saposin B
[0061] As used herein, "recombinant" includes reference to a
polypeptide produced using cells that do not have, in their native
state, an endogenous copy of the DNA able to express the
polypeptide. The cells produce the recombinant polypeptide because
they have been genetically altered by the introduction of the
appropriate isolated nucleic acid sequence. The term also includes
reference to a cell, or nucleic acid, or vector, that has been
modified by the introduction of a heterologous nucleic acid or the
alteration of a native nucleic acid to a form not native to that
cell, or that the cell is derived from a cell so modified. Thus,
for example, recombinant cells express genes that are not found
within the native (non-recombinant) form of the cell, express
mutants of genes that are found within the native form, or express
native genes that are otherwise abnormally expressed, under
expressed or not expressed at all.
[0062] The term "Saposin B" refers to a polypeptide fragment of
Prosaposin. Prosaposin is a 70 kD glycoprotein which has been
deposited and given GenBank Accession No. 337762. Prosaposin is the
precursor of four small heat-stable sphingolipid-binding
glycoproteins labeled Saposin A, Saposin B, Saposin C and Saposin
D. In addition to binding sphingolipids, Saposin C has been found
to have neurotrophic activity. This peptide and its functional
activity is described in U.S. Pat. No. 5,696,080. Saposin B is
associated with lysosomal hydrolysis of sphingolipids, including
sulfatides, GM1 ganglioside, globotriaosylceramide. In addition to
sphingolipids, Saposin B is involved in the hydrolysis of
glycerolipids (Hiraiwa, et al., Arch. Biochem. Biophys. 303:326
(1993)). In reference to the deposited Prosaposin amino acid
sequence, the Saposin B amino acid sequence resides between
positions 190 and 269, inclusive. Humans with Saposin B deficiency
have an accumulation of cerebroside sulfate and a clinical
presentation of leukodystrophy (Kretz, et al., Proc. Nat'l Acad.
Sci. USA 87:2541 (1990)). See also, Kase, et al., FEBS Lett. 393:74
(1996) and Lamontagne & Potier, J. Biol. Chem. 269:20528
(1994). In addition to its sphingolipid hydrolysis activity,
Saposin B, as disclosed below, surprisingly has anti-angiogenic
activity. Even more surprising, polypeptides of Saposin B, some as
small as five amino acids, have anti-angiogenic activity.
[0063] The phrase "specifically (or selectively) binds" refers to a
binding reaction that is determinative of the presence of a
polypeptide in a heterogeneous population of polypeptides and other
compounds. Thus, under designated binding conditions, the specified
polypeptides bind to a particular compound at least two times the
background and do not substantially bind in a significant amount to
other compounds present in the sample. Specific binding to an
antibody under such conditions may require an antibody that is
selected for its specificity for a particular polypeptide. For
example, antibodies raised to a polypeptide of this invention can
be selected so that a composition of antibodies will comprise only
antibodies that are specifically immunoreactive with the
polypeptides and not with other compounds, except for polymorphic
variants, alleles, and closely related interspecies homologs of the
polypeptides. This selection may be achieved by subtracting out
antibodies that cross-react with other polypeptides, for example,
Saposin C.
[0064] The phrase "surface of cells" refers to the interstitial
aspect of a cellular membrane. In some cases, the surface of a cell
will comprise a compound that is embedded in the membrane and has
an extracellular component that is available for binding to
compounds in the interstitium.
[0065] The term "therapeutic agent" includes any number of
compounds which will be apparent to one of skill upon review of
this disclosure that act as anti-neoplastics, anti-angiogenics or
other agents administered to induce a desired therapeutic effect in
a patient.
[0066] The phrase "treating an mammal" refers to administering a
polypeptide or a fusion protein of this invention to a mammal in
order to obtain a desired result, i.e., decrease undesired
angiogenesis.
[0067] "Undesired angiogenesis" refers to uncontrolled persistent
angiogenesis, or unregulated growth of capillary loops and blood
vessels, occurring in tumor growth, tumor metastasis, and abnormal
endothelial growth among other pathological conditions. The phrase
"reduce angiogenesis" refers to decreasing the undesired formation
of capillary loops and blood vessels. One of skill will realize
that although reduction of undesired angiogenesis is desired,
reduction of desired angiogenesis, i.e., normal growth of capillary
loops and blood vessels, may also be reduced.
[0068] The phrase "unit dosage form" refers essentially to the
final composition form, e.g., the capsules; tablets; suppositories;
solutions; powders, both lyophilized and admixed; impregnated
transdermal patches; vials and the like into which the composition
is ultimately delivered to the marketplace.
SEQUENCE LISTING
[0069] In the Sequence Listing, SEQ ID NO:1 corresponds to the
amino acid sequence of Saposin B. At position 7 of this amino acid
sequence is an aspartic acid residue. SEQ ID NO:2 is the amino acid
sequence of full length Prosaposin. SEQ ID NO:3 and 4 are nucleic
acid primers used to amplify the nucleic acid sequence which
encodes Saposin B. SEQ ID NO:4 and 5 are nucleic acid primers used
to amplify the nucleic acid sequence which encodes Prosaposin. SEQ
ID NO:6 and 7 are nucleic acid primers used to amplify the nucleic
acid sequence which encodes Saposin A. SEQ ID NO: 8 and 9 are
nucleic acid primers used to amplify the nucleic acid sequence
which encodes Saposin C. SEQ ID NO:10 and 11 are nucleic acid
primers used to amplify the nucleic acid sequence which encodes
Saposin D.
[0070] The amino acid sequence of human prosaposin (SEQ ID NO 1).
The sequence of Saposin B mature peptide (SEQ ID NO 2) is indicated
in underlined bold lettering.
TABLE-US-00002 MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQ
TVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKP
NMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNH
QKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQD
CIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSE
IAIQMMMHMQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVE
PIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLP
KSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHV
TQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQ
CDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPS
YWCQNTETAAQCNAVEHCKRHVWN
[0071] Table 2 provides sequences that are anti-angiogenic
polypeptides of this invention.
TABLE-US-00003 TABLE 2 Anti-angiogenic Polypeptides SEQ ID NO Amino
Acid Sequence 13 QPKDNGDVCQDCIQV 14 IQMVTDIQTAVRTNSTF 15
STYQALVEHVKEECDR 16 CDRLGPGMADKNYS 17 YISQYSEIAIQMMMHMQP 18
QMMMHMQPKEICALVG 19 GDVCQDCIQMV 20 GDVSQDSIQMV 21 DVCQDCIQMV 22
GDVCQ 23 DCIQMV 24 DVCQDCIQM 25 DVCQDCIQ 26 DVCQDCI 27 DVCQDC 28
DVCQD 29 VCQDCIQMV 30 CQDCIQMV 31 QDCIQMV 32 GDVSQDCIQMV 33
GDVCQDSIQMV 34 GDVSQD 35 DACQD 36 DICQD 37 DLCQD 38 DVCSD 39 DVCED
40 DVCDD 41 QPKEICALVGFCDEVK 42 CDRLGPGMAKICKNYIS 43
QMVTDIQTQVRTNSTF
DETAILED DESCRIPTION OF THE INVENTION
[0072] This invention is directed towards polypeptides which
possess anti-angiogenic properties as well as anti-tumoral
properties. The polypeptides described herein include cryptic as
well as N-terminal peptides of Saposin B and the full-length
Saposin B, a protein heretofore only known to have activity
associated with the hydrolysis of sphingolipids and
phosphoglycerides. Saposin B is highly conserved and this invention
includes corresponding proteins and peptides from humans as well as
other animal species including mice, rats, chickens, dogs and
primates.
[0073] The polypeptides of this invention have a variety of uses.
For example, they can be used as a therapeutic agent to treat
undesired angiogenesis and tumor growth. In addition, because of
their effect on specific cell types, the polypeptides of this
invention can be used in conjunction with cell cytotoxic moieties
to selectively kill certain cell types. The polypeptides can also
be linked to cell targeting moieties to modulate cells upon which
the polypeptide would not normally be active. In addition to in
vitro uses, the polypeptides of this invention can also be used in
vitro. For example, the polypeptides can be used to generate
antibodies which then can be used to treat diseases caused by an
overproduction of Saposin B or Prosaposin. In another example, the
polypeptides can be used to generate synthetic drugs with similar
activity.
[0074] The diseases and pathological conditions for which the
compounds and methods of this invention include, but are not
limited to, cancer, angiofibroma, neovascular glaucoma,
arteriovenous malformations, nonunion fractures, arthritis and
other connective tissue disorders, Osler-Weber syndrome,
atherosclerotic plaques, psoriasis, corneal graft
neovascularization, pyogenic granuloma, retrolental fibroplasia,
diabetic retinopathy, scleroderma, hemangioma, trachoma, vascular
adhesions and hypertrophic scars. One of skill, upon review of this
disclosure, will appreciate that other diseases states and
pathological conditions are susceptible to treatment with the
compounds and methods of this invention as well.
[0075] The diseases treatable with Saposin B and its derivative
peptides include human as well as veterinary uses such as treating
cats, dogs, horses and cattle.
I. Polypeptides and Fusion Proteins of this Invention
[0076] A. Sources of the Polypeptides and Fusion Proteins
[0077] 1. Natural Sources of the Polypeptides
[0078] The polypeptides of this invention can be obtained from
natural sources. Natural sources in this context comprises mammals
including, but not limited to, humans. In a preferred embodiment,
the polypeptides of this invention are isolated from the body
fluids of humans. In a particularly preferred embodiment, the body
fluid is urine. In this embodiment, the preferred polypeptide is
Saposin B (SEQ ID NO:1).
[0079] One of skill in the art will realize that, because of the
inherent danger of processing body fluids, care should be taken to
avoid contact with the fluid during collection and preparation of
the polypeptides of this invention. During the collection period,
the urine is preferable stored frozen. After a suitable amount has
been collected, the urine is concentrated and fractionated.
[0080] Before concentration, the urine is thawed and centrifuged to
remove solids. Typical centrifuge conditions are 800.times.g for 20
minutes at 4.degree. C. To remove the remaining solids from the
supernatant, the urine is preferably filtered through a 0.43 .mu.m
or another suitable filtration membrane. To concentrate the
clarified urine, it can be precipitated with a compound that
reduces the solubility of proteins in the urine, e.g., ammonium
sulfate or polyethylene glycol. Alternatively, the urine can be
filtered through a membrane in which the molecular weight cut off
is less than the size of the desired polypeptide and therefore is
retained, i.e., 2-5 kD. Examples of these filtration techniques
include, but are not limited to, ultrafiltration and diafiltration,
both of which are well known to those of skill in the art.
[0081] After the initial concentration step, the urine is
fractionated on the basis of size by applying it to a size
exclusion column. Because the polypeptides of this invention are a
small fraction of the total protein found in urine, the absorbance
at 280 nm is not sufficient to determine the presence of the
polypeptides. Therefore, the functional and immunoassays described
below preferably are used to determine which fractions contain
anti-angiogenic polypeptides. If desired, an additional
fractionation step, e.g., ion exchange, affinity and hydrophobic
interaction chromatography, can be performed to further purify
and/or concentrate the polypeptides. Again, these techniques are
well known in the art.
[0082] To produce fusion proteins, another embodiment of this
invention, the polypeptides are linked to a cell targeting or a
cytotoxic moiety ("functional moiety"). The moieties can either be
proteinaceous or another compound that has cell targeting or
cytotoxic activity. The linkage between the polypeptide and the
cell targeting or cytotoxic moiety is produced through chemical
conjugation and is described in greater detail below.
[0083] Chemical modifications before chemical conjugation can be
effected. These modifications include, for example, derivitization
for the purpose of linking the polypeptide to the functional
moiety, either directly or through a linking compound, by methods
that are well known in the art of protein chemistry. In one
preferred chemical conjugation embodiment, the means of linking the
polypeptide and the functional moiety comprises a
heterobifunctional coupling reagent which ultimately contributes to
formation of an intermolecular disulfide bond between the two
moieties. Other types of coupling reagents that are useful in this
capacity for the present invention are described, for example, in
U.S. Pat. No. 4,545,985. Alternatively, an intermolecular disulfide
may conveniently be formed between cysteines in each moiety which
occur naturally or are inserted by genetic engineering (see below).
The means of linking moieties may also use thioether linkages
between heterobifunctional crosslinking reagents or specific low pH
cleavable crosslinkers or specific protease cleavable linkers or
other cleavable or noncleavable chemical linkages. The means of
linking moieties of the fusion proteins may also comprise a
peptidyl bond formed between moieties which are separately
synthesized by standard peptide synthesis chemistry or recombinant
means.
[0084] In the case of chemical conjugation between the polypeptide
and a non-proteinaceous functional moiety, a covalent bond between
the two is preferred. Examples of active sites on the polypeptide
or on the functional moiety for covalent bonds include
sulfhydryl-reactive groups (e.g., methanethiosulfonyl groups,
dithiopyridyl groups, other reactive disulfides, and cystine),
alkylating agents (e.g., .alpha.-halo ketones, .alpha.-diazo
ketones), and acylating agents (e.g., activated esters such as
2,4-dinitrophenyl esters and pentafluorophenyl esters, and certain
anhydrides). Other suitable active sites are known to those of
skill in the art.
[0085] However, covalent bonding of the polypeptide and the
functional moiety of this invention is not required for the
compounds of the present invention. Non-covalent bonding can take
place via suitable electrostatic interactions with, for example,
ammonium ion and carboxylic acid groups present in the polypeptide
or in the functional moiety.
[0086] In one embodiment, the polypeptide and the functional moiety
are linked in a non-continuous manner. For example, a linking group
between the polypeptide and the functional moiety may comprise of
two parts, which are selected to be complimentary binding groups,
for example, two complimentary oligonucleotides or an avidin-biotin
pair. Other complementary binding groups will be apparent to those
of skill upon review of this disclosure.
[0087] In addition to the chemical modifications made to the
polypeptides and the functional moieties prior to linking, chemical
modifications of the polypeptides and the fusion proteins
themselves are envisioned. Such modifications include but are not
limited to, derivitization with polyethylene glycol (PEG) to extend
time of residence in the circulatory system and reduce
immunogenicity, according to well known methods (see for example,
Lisi, et al., Applied Biochem. 4:19 (1982); Beauchamp, et al., Anal
Biochem. 131:25 (1982); and Goodson, et al., Bio/Technology 8:343
(1990)).
[0088] 2. Recombinant Synthesis of Polypeptides and Fusion
Proteins
[0089] In another embodiment, the polypeptides and the fusion
proteins of this invention are synthesized recombinantly.
Recombinant techniques are well known to those of skill and are
described, in brief, below. The nucleic acids which encode the
polypeptides and the functional moieties, whether RNA, cDNA,
genomic DNA, or a hybrid of the various combinations, are isolated
from biological sources or synthesized in vitro.
[0090] The nucleic acids which encode the polypeptides and
functional moieties of this invention can be found in either
genomic or cDNA libraries. For example, nucleic acids which encode
the polypeptides of this invention can be found in human genomic
libraries, nucleic acids which encode cell targeting antibodies can
be found in spleen cells from immunized animals, and nucleic acids
which encode for toxins can be found in the source bacteria, e.g.,
Pseudomonas aeruginosa and Corynebacterium diphtheriae. Methods for
generating these libraries from source organisms, e.g., animals or
bacteria, are known to those of skill and can be found in many
practice guides, including Berger & Kimmel, GUIDE TO MOLECULAR
CLONING TECHNIQUES, METHODS IN ENZYMOLOGY VOL. 152, Academic Press,
Inc., San Diego, Calif. (Berger); Sambrook et al. MOLECULAR
CLONING--A LABORATORY MANUAL (2ND ED.) VOL. 1-3, Cold Springs
Harbor Publishing (1989) (Sambrook); and CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, Ausubel et al. (eds.), Current Protocols, a
joint venture between Greene Publishing Associates, Inc. and John
Wiley & Sons, Inc., (1997 Supplement) (Ausubel). Product
information from manufacturers of biological reagents and
experimental equipment also provide information useful in
established biological methods. Such manufacturers include the
SIGMA chemical company (Saint Louis, Mo.), R&D systems
(Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway,
N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes
Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research,
Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka
Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),
Invitrogen, San Diego, Calif., and Applied Biosystems (Foster City,
Calif.), as well as many other commercial sources known to those of
skill.
[0091] After the libraries have been created, the colonies must be
probed to identify those colonies that contain the DNA of interest.
Nucleic acid probes are nucleotide sequences that specifically
hybridize under stringent conditions to the desired nucleic acid.
Because the amino acid as well as the nucleotide sequence of
Saposin B is known, generating probes to isolate clones with
desired DNA would be considered routine and is not a critical
aspect of this invention. In a preferred embodiment, the probes are
chemically synthesized with a DNA synthesizer, amplified using the
primers as shown in SEQ ID NO:3 and 4, and expanded by cloning into
a bacterial vector. The probes are then labeled by techniques well
known in the art and the library is screened. Screening techniques
with labeled nucleic acid probes is also well known in the art.
[0092] Stringent conditions for hybridization is dependent on the
nucleic acid to be hybridized. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, LABORATORY
TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY--HYBRIDIZATION
WITH NUCLEIC ACID PROBES PARTS I AND II, Elsevier, New York,
(1993). Generally, stringent conditions are selected to be about
5.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength and pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. Highly
stringent conditions are selected to be equal to the Tm point for a
particular probe. Sometimes the term "Td" is used to define the
temperature at which at least half of the probe dissociates from a
perfectly matched target nucleic acid. In any case, a variety of
estimation techniques for estimating the Tm or Td are available,
and generally described in Tijssen, id. Typically, G-C base pairs
in a duplex are estimated to contribute about 3.degree. C. to the
Tm, while A-T base pairs are estimated to contribute about
2.degree. C., up to a theoretical maximum of about 80-100.degree.
C. However, more sophisticated models of TM and Td are available
and appropriate in which G-C stacking interactions, solvent
effects, the desired assay temperature and the like are taken into
account. In one example, PCR primers are designed to have a
dissociation temperature (Td) of approximately 60.degree. C., using
the formula:
Td'(((((3.times.#GC)+(2.times.#AT)).times.37)-562)/#bp)-5, where
#GC, #AT, and #bp are the number of guanine-cytosine base pairs,
the number of adenine-thymine base pairs, and the number of total
base pairs, respectively, involved in the annealing of the primer
to the template DNA.
[0093] In general, a signal to noise ratio of 2.times. (or higher)
than that observed for an unrelated probe in the particular
hybridization assay indicates detection of a specific
hybridization. For highly specific hybridization strategies such as
allele-specific hybridization, an allele-specific probe is usually
hybridized to a marker nucleic acid (e.g., a genomic nucleic acid,
or the like) comprising a polymorphic nucleotide under highly
stringent conditions.
[0094] In addition to using nucleic acid probes for isolating the
class of proteins claimed herein, it is possible to use antibodies
to probe expression libraries for novel forms of active
polypeptides. This is a well known technology (See Young &
Davis, Proc. Nat'l Acad. Sci. USA 80:1194 (1982)). In general, a
cDNA expression library may be prepared from commercially available
kits or using readily available components. Phage vectors are
preferred, but a variety of other vectors are available for the
expression of protein. Such vectors include but are not limited to
yeast, animal cells and Xenopus oocytes. One selects mRNA from a
source that is enriched with the target protein and creates cDNA
which is then ligated into a vector and transformed into the
library host cells for immunoscreening. Screening involves binding
and visualization of antibodies bound to specific proteins on
library host cells or immobilized on a solid support such as
nitrocellulose or nylon membranes. Positive clones are selected for
purification to homogeneity and the isolated cDNA then prepared for
expression in the desired recombinant cells. A general review of
this technology can be found in METHODS OF CELL BIOLOGY, VOL. 37
entitled Antibodies in Cell Biology, Assai (ed.) 1993.
[0095] In addition, one of skill will realize that in some
instances, the nucleic acid encoding a functional moiety does not
have to be generated from a nucleic acid library. For example,
transformed bacteria comprising nucleic acid sequences which encode
bacterial exotoxins are available, as are transformed bacteria and
mammalian cell lines which comprise nucleic acids which encode
monoclonal or single chain antibodies (see Chaudhary, infra).
[0096] After the libraries described above have been screened and
colonies with the appropriate DNA selected, the DNA is cloned
according to techniques described in Sambrook, Ausubel and other
literature available to those in the molecular biology field. To
clone the polypeptides of this invention, the cells of a library
which contain the desired DNA are selected and expanded. The
genomic DNA from the culture is isolated and the inserted DNA of
choice is purified. Typically, as an initial step, the desired
nucleotide sequence is cleaved from the genomic or episomal DNA by
restriction enzymes. After electrophoresis to separate the DNA on
the basis of size, the nucleotide of interest is excised from the
gel and inserted into an expression vector. For expression of the
peptides of this invention, any suitable cell may be used,
including, but not limited to, bacteria, insect, plant and
mammal.
[0097] In vitro amplification techniques suitable for amplifying
sequences for use as molecular probes or generating nucleic acid
fragments for subsequent subcloning are known. Examples of
techniques sufficient to direct persons of skill through such in
vitro amplification methods, including the polymerase chain
reaction (PCR) the ligase chain reaction (LCR), Q.beta.-replicase
amplification and other RNA polymerase mediated techniques (e.g.,
NASBA) are found in Berger, Sambrook, Innis and Ausubel, as well as
U.S. Pat. No. 4,683,202; Arnheim & Levinson, C&EN 36-47
(Oct. 1, 1990); Kwoh, et al., Proc. Nat'l Acad Sci. USA 86:1173
(1989); Guatelli, et al., Proc. Nat'l Acad. Sci. USA 87:1874
(1990); Lomell, et at, J. Clin. Chem. 35:1826 (1989); Landegren, et
al., Science 241:1077-1080 (1988); Van Brunt, Biotechnology
8:291-294 (1990); Wu & Wallace, Gene 4:560 (1989); Barringer,
et al., Gene 89:117 (1990); and Sooknanan & Malek,
Biotechnology 13:563-564 (1995). Improved methods of cloning in
vitro amplified nucleic acids are described in U.S. Pat. No.
5,426,039.
[0098] In a preferred embodiment, the nucleic acid sequences which
encode the polypeptides of this invention are amplified with
primers that correspond to SEQ ID NO:3 through 5. These primers are
specific for Saposin B (SEQ ID NO:3 and 4) and proSaposin (SEQ ID
NO:5 and 6).
[0099] Oligonucleotides for use as probes and primers are typically
synthesized chemically according to the solid phase phosphoramidite
triester method described by Beaucage & Caruthers, Tetrahedron
Letts. 22(20):1859-1862 (1981), e.g., using an automated
synthesizer, e.g., as described in Needham-VanDevanter, et al.,
Nucl Acids Res. 12:6159-6168 (1984). Oligonucleotides can also be
custom made and ordered from a variety of commercial sources known
to persons of skill, for example, Promega (Madison, Wis.).
Purification of oligonucleotides, where necessary, is typically
performed by either native acrylamide gel electrophoresis or by
anion-exchange HPLC as described in Pearson & Regnier, J.
Chrom. 255:137-149 (1983). The sequence of the synthetic
oligonucleotides can be verified using the chemical degradation
method of Maxam & Gilbert, Methods in Enzymology 65:499-560
(1980).
[0100] In some embodiments of this invention, it may be desirable
to change an amino acid within a polypeptide or to truncate the
polypeptide before the naturally occurring carboxyl terminus. There
are many ways of generating alterations in a given nucleic acid
sequence to effect substitutions of amino acid or to insert a stop
codon to truncate the polypeptide. Such well-known methods include
site-directed mutagenesis, PCR amplification using degenerate
oligonucleotides, exposure of cells containing the nucleic acid to
mutagenic agents or radiation, chemical synthesis of a desired
oligonucleotide (e.g., in conjunction with ligation and/or cloning
to generate large nucleic acids) and other well-known techniques.
See, Giliman & Smith, Gene 8:81-97 (1979); Roberts, et al.,
Nature 328:731-734 (1987); Sambrook; Innis, Ausubel, Berger,
Needham VanDevanter and Mullis (all supra).
[0101] Another preferred genetic engineering modifications of the
polypeptides or the fusion proteins of this invention include
combination of the polypeptide and the functional moiety into a
single chain multi-functional protein expressed from a single gene.
(See, for example, PCT published application WO8809344).
Accordingly, the fusion protein will then comprise a polypeptide
beginning at one end with the polypeptide and ending with the
functional moiety. The recombinant linking of the polypeptide and
the functional moiety can take place at either end of either
molecule. For example, the carboxyl terminus of the polypeptide can
be linked to the amino terminal of the functional moiety, or vice
versa. Similarly, if desired, the polypeptide can be inserted into
the interior of the functional moiety amino acid sequence. However,
one must realize that, unless the polypeptide is small, activity of
the functional moiety may be lost.
[0102] Methods of producing recombinant fusion proteins are well
known to those of skill in the art. Thus, for example, Chaudhary,
et al., Nature 339:394 (1989); Batra, et al., J. Biol. Chem.
265:15198 (1990); Batra, et al., Proc. Nat'l Acad. Sci. USA 86:8545
(1989); Chaudhary, et al., Proc. Nat'l Acad. Sci. USA 87:1066
(1990), describe the preparation of various single chain fusion
proteins.
[0103] In general, producing fusion proteins involves separately
preparing the polypeptide nucleic acid sequence and DNA encoding
the functional moiety to be used. The two sequences are combined in
a plasmid or other vector to form a construct encoding the
particular desired fusion protein. A simpler approach involves
inserting the DNA encoding the particular polypeptide into a
construct already encoding the desired functional moiety.
[0104] Thus, for example, DNA encoding a polypeptide-Pseudomonas
exotoxin fusion protein is most easily prepared by inserting the
DNA encoding the polypeptide into constructs already containing DNA
encoding the desired exotoxin using techniques well known to those
of skill in the art.
[0105] Mammalian cells have been used to express and secrete
polypeptides and hybrid molecules such as antibody-cytokines
(Hoogenboom, et al., Biochem. Biophys. Acta 1096:345 (1991);
Hoogenboom, et al., Mol. Immunol. 28:1027 (1991)) and
antibody-enzyme (Casadei, et al., Proc. Nat'l Acad. Sci. USA
87:2047 (1990); Williams, et al., Gene 43:319 (1986)). A drawback
to using recombinant proteins is the potential immunogenicity of
the foreign proteins. Immunogenicity of foreign proteins is
typically due to incorrect glycosylation patterns present on
recombinant proteins. Therefore, because the expressed proteins are
glycosylated, eukaryotic cell lines are preferred over prokaryotic
cells. In particular, human derived cell lines are particularly
preferred in that these cells incorporate a sialic acid as the
terminal glycoside.
[0106] Although human cells are desirable because of decreased
immunogenicity, one of skill will realize that other cells can be
used to express the peptides of this invention. For example,
mammalian cell lines like CHO, COS, 3T3 and L cells can be used,
Other eukaryotic cells that can be used include, but are not
limited to insect cell lines and yeast cells, e.g., Saccharomyces
cerevisiae and Pichia pasteris. In addition, if glycosylation is
not critical, the peptides of this invention can be expressed in
prokaryotic cells, for example, E. coli.
[0107] One of skill will recognize that modifications can be made
to the polypeptides without diminishing their anti-angiogenic
activity. Some modifications may be made to facilitate the cloning,
expression, or incorporation of the anti-angiogenic moiety into a
fusion protein. Such modifications are well known to those of skill
in the art and include, for example, a methionine added at the
amino terminus to provide an initiation site, or additional amino
acids (e.g., poly His) placed on either terminus to create
conveniently located restriction sites or termination codons or
purification sequences.
[0108] Other genetic engineering modifications of the protein
moieties of the fusion proteins of this invention include deletions
of functionally unnecessary domains to reduce the size of the
protein or to modify other parameters which facilitate production
or utility, such as sequence changes to affect the solubility
(e.g., cysteine to serine) or glycosylation sites. One skilled in
the art would appreciate that many additional well known chemical
and genetic modifications of proteins may be advantageously applied
to any protein which, like the present fusion protein, may be
intended for parenteral administration.
[0109] 3. Chemical Synthesis of the Peptides
[0110] The polypeptides of this invention are preferably
synthetically prepared. Polypeptides of relatively short size are
typically synthesized in solution or on a solid support in
accordance with conventional techniques. See, e.g., Merrifield, J.
Am. Chem. Soc. 85:2149 (1963). Various automatic synthesizers and
sequencers are commercially available and can be used in accordance
with known protocols. See, e.g., Stewart & Young, SOLID PHASE
PEPTIDE SYNTHESIS, 2D. ED., Pierce Chemical Co (1984). Solid phase
synthesis in which the carboxyl terminal amino acid of the sequence
is attached to an insoluble support followed by sequential addition
of the remaining amino acids in the sequence is the preferred
method for the chemical synthesis of the polypeptides of this
invention. Techniques for solid phase synthesis are described by
Barany & Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284
in THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY. VOL. 2: SPECIAL
METHODS IN PEPTIDE SYNTHESIS, PART A.; Merrifield, et al., J. Am.
Chem. Soc. 85: 2149-2156 (1963); and Stewart, et al., SOLID PHASE
PEPTIDE SYNTHESIS, 2ND ED. Pierce Chem. Co., Rockford, Ill.
(1984).
[0111] After chemical synthesis or recombinant expression, the
polypeptide(s) may possess a conformation substantially different
than the native conformation of the polypeptides. In this case, it
is helpful to denature and reduce the polypeptide and then cause
the polypeptide to re-fold into the preferred conformation. Methods
of reducing and denaturing polypeptides and inducing re-folding are
well known to those of skill in the art (see, Debinski, et al. J.
Biol. Chem. 268:14065 (1993); Kreitman & Pastan, Bioconjug.
Chem. 4:581 (1993); and Buchner, et al., Anal. Biochem. 205:263
(1992)). Debinski, et al., for example, describe the denaturation
and reduction of inclusion body polypeptides in guanidine-DTE. The
polypeptide is then refolded in a redox buffer containing oxidized
glutathione and L-arginine.
[0112] In addition to polypeptides consisting of a peptide
backbone, peptidomimetics or polypeptide analogs are also provided.
Polypeptide analogs are commonly used in the pharmaceutical
industry as non-peptide drugs with properties analogous to those of
the template polypeptide. These types of non-peptide compound are
termed "peptide mimetics" or "peptidomimetics" (Fauchere, J., Adv.
Drug Res. 15:29 (1986); Veber & Freidinger, TINS p. 392 (1985);
and Evans et al., J. Med. Chem. 30:1229 (1987)). Peptide mimetics
that are structurally similar to the useful polypeptides of this
invention may be used to produce an equivalent or enhanced
anti-angiogenic effect.
[0113] Generally, peptidomimetics are structurally similar to a
paradigm polypeptide, i.e., Saposin B or a polypeptide with
anti-angiogenic activity, but have one or more peptide linkages
optionally replaced by a linkage selected from the group consisting
of: --CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--,
--CH--CH-- (cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--, and
--CH.sub.2SO--, by methods known in the art and further described
in the following references: Spatola, CHEMISTRY AND BIOCHEMISTRY OF
AMINO ACIDS, PEPTIDES, AND PROTEINS, B. Weinstein, (ed.), Marcel
Dekker, New York, p. 267 (1983); Spatola, Vega Data 1(3), Peptide
Backbone Modifications (general review) (March 1983); Morley,
Trends Pharm. Sci. (1980) pp. 463-468 (general review); Hudson, et
al., Int'l J. Pept. Prot. Res. 14:177-185 (1979) (--CH.sub.2NH--,
CH.sub.2CH.sub.2--); Spatola, et al., Life Sci. 38:1243-1249 (1986)
(--CH.sub.2--S); Hann, J. Chem. Soc. Perkin Trans. I 307-314 (1982)
(--CH--CH--, cis and trans); Almquist, et al., J. Med. Chem.
23:1392-1398 (1980) (--COCH.sub.2--); Jennings-White, et al.,
Tetrahedron Lett. 23:2533 (1982) (--COCH.sub.2--); Szelke, et al.,
EP 45665 (1982) (--CH(OH)CH.sub.2--); Holladay, et al., Tetrahedron
Lett. 24:4401-4404 (1983) (--C(OH)CH.sub.2--); and Hruby, Life Sci.
31:189-199 (1982) (--CH.sub.2--S--). Thus, in the instant
invention, an anti-angiogenic peptidomimetic would be structurally
similar to the polypeptides of this invention, i.e., comprises
DX.sub.1CX.sub.2D.
[0114] Peptide mimetics may have significant advantages over
polypeptide embodiments of this invention, including, for example:
more economical production, greater chemical stability, enhanced
pharmacological properties (half-life, absorption, potency,
efficacy, etc.), altered specificity (e.g., a broad-spectrum of
biological activities), reduced antigenicity, and others.
[0115] Chemical conjugation of peptidomimetics to make fusion
proteins usually involves covalent attachment to one or more
binding sites on the functional moieties, directly or through a
spacer (e.g., an amide group), to non-interfering position(s) on
the peptidomimetic that are predicted by quantitative
structure-activity data and/or molecular modeling. Such
non-interfering positions generally are positions that do not form
direct contacts with the surface macromolecules(s) (e.g., Saposin B
receptors on KS cells) to which the peptidomimetic binds to produce
the anti-angiogenic effect. In addition, derivitization (e.g.,
labeling) and conjugation of peptidomimetics should not
substantially interfere with the desired anti-angiogenic activity
of the peptidomimetic.
[0116] In addition to the peptidomimetics, synthetic polypeptides
can comprise systematic substitution of one or more amino acids
with a D-amino acid of the same type (e.g., D-lysine in place of
L-lysine). These substitutions may be used to generate more stable
polypeptides. Also, constrained polypeptides comprising a consensus
sequence or a substantially identical consensus sequence variation
may be generated by methods known in the art (Rizo & Gierasch,
Ann. Rev. Biochem. 61:387 (1992)); for example, by adding internal
cysteine residues capable of forming intramolecular disulfide
bridges which cyclize the peptide.
[0117] In addition to modifications to the peptide backbone,
synthetic or non-naturally occurring amino acids can also be used
to substitute for the amino acids present in the polypeptide or in
the functional moiety of fusion proteins. Synthetic or
non-naturally occurring amino acids refer to amino acids which do
not naturally occur in vivo but which, nevertheless, can be
incorporated into the peptide structures described herein.
Preferred synthetic amino acids are the D-.alpha.-amino acids of
naturally occurring L-.alpha.-amino acid, mentioned above, as well
as non-naturally occurring D- and L-.alpha.-amino acids represented
by the formula H.sub.2NCHR.sup.5COOH where R.sup.5 is 1) a lower
alkyl group, 2) a cycloalkyl group of from 3 to 7 carbon atoms, 3)
a heterocycle of from 3 to 7 carbon atoms and 1 to 2 heteroatoms
selected from the group consisting of oxygen, sulfur, and nitrogen,
4) an aromatic residue of from 6 to 10 carbon atoms optionally
having from 1 to 3 substituents on the aromatic nucleus selected
from the group consisting of hydroxyl, lower alkoxy, amino, and
carboxyl, 5)-alkylene-Y where alkylene is an alkylene group of from
1 to 7 carbon atoms and Y is selected from the group consisting of
(a) hydroxy, (b) amino, (c) cycloalkyl and cycloalkenyl of from 3
to 7 carbon atoms, (d) aryl of from 6 to 10 carbon atoms optionally
having from 1 to 3 substituents on the aromatic nucleus selected
from the group consisting of hydroxyl, lower alkoxy, amino and
carboxyl, (e) heterocyclic of from 3 to 7 carbon atoms and 1 to 2
heteroatoms selected from the group consisting of oxygen, sulfur,
and nitrogen, (f) --C(O)R.sup.2 where R.sup.2 is selected from the
group consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy,
and --NR.sup.3R.sup.4 where R.sup.3 and R.sup.4 are independently
selected from the group consisting of hydrogen and lower alkyl, (g)
--S(O).sub.nR.sup.6 where n is an integer from 1 to 2 and R.sup.6
is lower alkyl and with the proviso that R.sup.5 does not define a
side chain of a naturally occurring amino acid.
[0118] Other preferred synthetic amino acids include amino acids
wherein the amino group is separated from the carboxyl group by
more than one carbon atom such as .beta.-alanine,
.gamma.-aminobutyric acid, and the like.
[0119] Particularly preferred synthetic amino acids include, by way
of example, the D-amino acids of naturally occurring L-amino acids,
L-1-naphthyl-alanine, L-2-naphthylalanine, L-cyclohexylalanine,
L-2-amino isobutyric acid, the sulfoxide and sulfone derivatives of
methionine (i.e.,
HOOC--(H.sub.2NCH)CH.sub.2CH.sub.2--S(O).sub.nR.sup.6 where n and
R.sup.6 are as defined above as well as the lower alkoxy derivative
of methionine (i.e., HOOC--(H.sub.2NCH)CH.sub.2CH.sub.2--OR.sup.6
where R.sup.6 is as defined above).
[0120] B. Characterization of the Peptides and Fusion Proteins
[0121] It is necessary, especially when synthesizing fusion
proteins, to determine whether the polypeptides or fusion proteins
have desired characteristics and thus will be anti-angiogenic.
Characterization can be done either by the structural or chemical
properties of the polypeptides or fusion proteins, or by the
functional properties of the polypeptides or fusion proteins.
[0122] 1. Physical and Chemical Characterization of the
Peptides
[0123] Polypeptides and fusion proteins may be detected or
quantified by a variety of methods. Preferred methods involve the
use of immunological assays utilizing specific antibodies.
[0124] a. Antibodies
[0125] Methods of producing polyclonal and monoclonal antibodies
are known to those of skill in the art. See, e.g., Coligan, CURRENT
PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites, et al.
(eds.) BASIC AND CLINICAL IMMUNOLOGY, (7TH ED.) Lange Medical
Publications, Los Altos, Calif., and references cited therein
("Stites"); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE
(2D ED.) Academic Press, New York, N.Y. (1986); Kohler &
Milstein, Nature 256.495 (1975); and Harlow and Lane. Such
techniques include antibody preparation by selection of antibodies
from libraries of recombinant antibodies in phage or similar
vectors. See, Huse, et al., Science 246:1275 (1989) ("Huse"); and
Ward, et al., Nature 341:544 (1989).
[0126] To produce polyclonal antibodies, in brief, an immunogen,
e.g., a polypeptide of this invention, is mixed with an adjuvant,
and animals are immunized. The animal's immune response to the
immunogen preparation is monitored by taking test bleeds and
determining the titer of reactivity to the immunogen. When
appropriately high titers of antibody to the immunogen are
obtained, blood is collected from the animal and antisera are
prepared. Further fractionation of the antisera to enrich for
antibodies reactive to the protein may be done if desired. (See
Harlow and Lane, supra).
[0127] Antibodies can only be raised against macromolecules.
Therefore, it is likely that an immune response will not be mounted
against the smaller polypeptides of this invention. To generate
antibodies against such small molecules, it is first necessary to
associate them with larger macromolecules which will be recognized
by the animal's immune system. Briefly, the polypeptide is
conjugated to carrier proteins according to the methods described
in the preceding sections. Typical carrier proteins are bovine
serum albumin, keyhole limpet cyanin and ovalbumin. The animals are
immunized with the carrier proteins associated with the polypeptide
and bleeds collected as above. Only the polypeptide (without the
carrier protein) is used to screen the test bleeds for reactivity
to the polypeptide.
[0128] Large amounts of monoclonal antibodies may be obtained by
various techniques familiar to those skilled in the art. Briefly,
spleen cells from an animal immunized with a desired protein
(either a fusion protein or a polypeptide of this invention, or a
polypeptide associated with a carrier protein) are immortalized,
commonly by fusion with a myeloma cell (See, Kohler & Milstein,
Eur. J. Immunol. 6:511 (1976)). Alternative methods of
immortalization include transformation with Epstein Barr Virus,
oncogenes, retroviruses, or other methods well known in the art.
Colonies arising from single immortalized cells are screened for
production of antibodies of the desired specificity and affinity
for the polypeptides or fusion proteins of this invention. The
yield of the monoclonal antibodies produced by such cells may be
enhanced by various techniques, including injection into the
peritoneal cavity of a vertebrate host. Alternatively, one may
isolate DNA sequences which encode a monoclonal antibody or a
binding fragment thereof by screening a DNA library from human B
cells according to the general protocol outlined in Huse.
[0129] 2. Immunological Binding Assays.
[0130] The concentration of the polypeptides and fusion proteins
can be measured by a variety of immunoassay methods. For a review
of immunological and immunoassay procedures in general, see Stites.
Moreover, the immunoassays of the present invention can be
performed in any of several configurations, which are reviewed
extensively in ENZYME IMMUNOASSAY, E. T. Maggio, ed., CRC Press,
Boca Raton, Fla. (1980); Tijssen; and Harlow and Lane.
[0131] In a preferred embodiment, the polypeptides and fusion
proteins of this invention are detected and/or quantified using any
of a number of well recognized immunological binding assays (see,
e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and
4,837,168). For a review of the general immunoassays, see also
METHODS IN CELL BIOLOGY VOL. 37: Antibodies in Cell Biology, Asai,
ed. Academic Press, Inc. New York (1993); and Stites. Immunological
binding assays (or immunoassays) typically utilize a "capture
agent" to specifically bind to and often immobilize the analyte (in
this case polypeptides, fusion proteins and the receptors). The
capture agent is a moiety that specifically binds to the analyte.
In a preferred embodiment, the capture agent is an antibody that
specifically binds to the polypeptides, fusion proteins and
receptors of this invention. The antibody may be produced by any of
a number of means well known to those of skill in the art and as
described above.
[0132] Immunoassays also often utilize a labeling agent to
specifically bind to and label the binding complex formed by the
capture agent and the analyte. The labeling agent may itself be one
of the moieties comprising the antibody/analyte complex. Thus, for
example, the labeling agent may be a labeled polypeptide or a
labeled anti-polypeptide antibody. Alternatively, the labeling
agent may be a third moiety, such as another antibody, that
specifically binds to the antibody/polypeptide complex.
[0133] In a preferred embodiment the labeling agent is a second
polypeptide or receptor antibody bearing a label. Alternatively,
the second antibody may lack a label, but it may, in turn, be bound
by a labeled third antibody specific to antibodies of the species
from which the second antibody is derived. The second can be
modified with a detectable moiety, such as biotin, to which a third
labeled molecule can specifically bind, such as enzyme-labeled
streptavidin.
[0134] Other proteins capable of specifically binding
immunoglobulin constant regions, such as protein A or protein G may
also be used as the label agent. These proteins are normal
constituents of the cell walls of streptococcal bacteria. They
exhibit a strong non-immunogenic reactivity with immunoglobulin
constant regions from a variety of species (see, generally Kronval,
et al., J. Immunol. 111:1401-1406 (1973), and Akerstrom, et al., J.
Immunol. 135:2589-2542 (1985)).
[0135] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, preferably from
about 5 minutes to about 24 hours. However, the incubation time
will depend upon the assay format, analyte, volume of solution,
concentrations, and the like. Usually, the assays will be carried
out at ambient temperature, although they can be conducted over a
range of temperatures, such as 4.degree. C. to 40.degree. C.
[0136] In addition to the EIA based formats described above,
western blot (immunoblot) analysis can be used to detect and
quantify the presence of antifreeze protein in a sample. The
technique generally comprises separating sample proteins by gel
electrophoresis on the basis of molecular weight, transferring the
separated proteins to a suitable solid support, (such as a
nitrocellulose filter, a nylon filter, or derivatized nylon
filter), and incubating the sample with the antibodies that
specifically bind THP. The anti-THP antibodies specifically bind to
THP on the solid support. These antibodies may be directly labeled
or alternatively may be subsequently detected using labeled
antibodies (e.g., labeled sheep anti-mouse antibodies) that
specifically bind to the anti-antifreeze protein.
[0137] Other assay formats include liposome immunoassays (LIA),
which use liposomes designed to bind specific molecules (e.g.,
antibodies) and release encapsulated reagents or markers. The
released chemicals are then detected according to standard
techniques (see, Monroe, et al., Amer. Clin. Prod. Rev. 5:34
(1986)).
[0138] 3. Functional Characterization of the Peptides
[0139] Functional characterization of the polypeptides of this
invention takes advantage of their anti-angiogenic
anti-proliferative properties. In other words, the anti-angiogenic
and/or anti-proliferative potential of the polypeptides is
measured. In a preferred embodiment, the anti-proliferative
activity of the polypeptides of this invention are measured in
cells of endothelial origin, e.g., HUVEC and murine endothelial
cells, and Kaposi's Sarcoma cell lines, e.g., KS, Y-1, KS-SLK and
KS 6-3. However, one of skill will realize that any cell line that
responds to anti-angiogenic or anti-tumoral factors can be used to
functionally characterize the polypeptides of this invention.
[0140] Typically, in an anti-proliferation assay, the suppression
of growth in the presence of the compound to be tested is measured.
For example, the above cells are incubated with the polypeptides of
this invention for a suitable time and the decrease in the growth
rate relative to a negative control, i.e., cells of the same cell
line which have been incubated in media alone, is measured Growth
of the cell culture can be measured by any method acceptable to one
of skill, including but not limited to, 3H-Thymidine uptake, cell
counting and tetrazolium dye uptake.
[0141] Anti-angiogenic assays measure the functional inhibition of
endothelial cells by a compound in the presence of a chemotactic
factor known to activate endothelial cells, e.g., VEGF and bPGF. In
a typical conformation, endothelial cells along with the compound
to be tested are placed in the upper chamber of a two-chamber well,
e.g., Boyden chambers or transwell plates. Media with a chemotactic
factor is placed in the lower chamber. Dividing the two chambers is
a membrane that is permeable to the chemotactic factor but not to
the endothelial cells. The membrane can be covered with a basement
membrane, either natural or artificial, e.g., Matrigel.
Alternatively, the membrane can be coated with fibronectin or
another protein capable of forming a gel susceptible to protease
degradation. The plates with the endothelial cells are incubated
for a sufficient time to allow the cells to traverse the membrane.
To determine the anti-angiogenic activity of the compound, the
number of cells that traversed the membrane in the presence of the
compound is compared to the number of cells that traversed the
membrane without the compound. If the number of cells in the lower
chamber (or on the aspect of the membrane in contact with the
chemotactic factor) is less than the number of cells in the control
chamber, the compound had anti-angiogenic potential.
[0142] In addition to the relatively simple assay described above,
the polypeptides and fusion proteins of this invention may be
functionally characterized in a multi-system angiogenic assay. For
example, the most commonly used multi-system assay utilizes the
allantoic membrane of the developing chick embryo. A window is cut
into the egg shell, exposing the allantoic membrane. The
polypeptide to be tested is added to the membrane and the egg is
incubated long enough for the anti-angiogenic effect of the
compound to be visible, e.g., fewer blood vessels in the allantoic
membrane compared to a negative control.
[0143] In another in vivo assay, tumor cell lines are implanted
into nude mice. The mice are then treated with the polypeptides and
fusion proteins of this invention. After a suitable time, the mice
are observed for signs of tumor regression. If the tumor has
regressed or decreased in size, the compound being tested has
anti-angiogenic or anti-tumor properties. See, Arora, et al.,
Cancer Research 59:183 (1999).
[0144] The fusion proteins of this invention can be further
characterized by the activity of the functional moiety. One of
skill will realize that these proteins can be characterized by the
same assays used to characterize the functional moiety alone. For
example, assays to measure the cytotoxic effect of bacterial toxins
can be used to measure the cytotoxic activity of fusion proteins
comprising the polypeptides of this invention conjugated to
Pseudomonas exotoxin or Diphtheria toxin. One example of a
cytotoxic assay, not intended to be limiting, can be found in
Galloway, J. Immunol. Methods 140:37 (1991).
[0145] C. Receptors of this Invention
[0146] Another embodiment of this invention comprises the isolated
receptors that bind to the polypeptides of this invention. The
isolated receptors can be used to screen for other anti-angiogenic
or anti-tumor drugs. In addition, the isolated receptors may
themselves be used as an angiogenic drug. For example, if
angiogenesis is desired, e.g., wound healing, the isolated
receptors of this invention can be administered to a mammal,
compete for Saposin B binding (or other anti-angiogenic compounds)
with the receptors present on the endothelial cells, and promote
the migration of endothelial cells through the basement membrane of
existing blood vessels.
[0147] Isolation of cell surface receptors is known to those of
skill in the art. Briefly, cells known to comprise the receptors of
this invention are solubilized with a lipophilic agent, typically
an ionic detergent. The cell lysate is then affinity purified by
passing it through a column packed with a resin to which the
polypeptides of this invention have been conjugated. The bound
receptors are eluted from the column, typically in high salt or if
necessary, an ionic detergent. The receptors can be further
purified to homogeneity by further column purification or by
preparative electrophoresis.
[0148] It is anticipated that the numbers of receptors present on
cells is very small and the techniques outlined above will not
generate sufficient receptors to use in drug screening assays or as
angiogenesis promoting pharmaceuticals. Therefore, it may be
necessary to recombinantly express the receptors of this invention.
Again, genomic or cDNA libraries can be probed either with nucleic
acids or with antibodies or polypeptides that specifically bind to
the receptors of this invention. To make degenerate nucleic acid
probes, the naturally occurring receptors described above can be
sequenced according to techniques well known in the art and
described herein. Using the appropriate preferred codons, nucleic
acid probes are synthesized and labeled. Because it is likely that
nucleotide mismatches will occur (because of codon degeneracy), the
hybridization of the probe to the membrane to which the library has
been transferred, should not be as stringent as described above.
Colonies which contain DNA that hybridized to the degenerate probes
are isolated and expanded as described above.
[0149] To screen an expression library, either antibodies raised
against the purified naturally-occurring receptor or the
polypeptides of this invention can be used. The general techniques
of screening the expression library are as described herein.
[0150] The receptors of this invention can be characterized using
the assays described above. However, the desired results will be
the opposite of the results obtained with the polypeptides of this
invention, i.e., increase angiogenesis or proliferation in the
presence of Saposin B or the polypeptides of this invention is
desired.
[0151] D. Saposin B Antibodies
[0152] In addition to characterizing the peptides of this
invention, antibodies directed against Saposin B can be used as a
therapeutic treatment to encourage angiogenesis.
[0153] Saposin B is expressed by cells as an element in the
homeostasis of cell growth. Thus, in situations where angiogenesis
is desired but blocked by the presence of Saposin B, compositions
which inhibit the action of Saposin B will block this protein's
activity. The compositions include, but are not limited to, Saposin
B receptors (see above) and neutralizing antibodies directed
against Saposin B.
[0154] Thus, the present invention provides antibodies targeted to
Saposin B and/or the peptides of this invention. These antibodies
are selectively reactive under immunological conditions to
determinants of Saposin B exposed on the protein surface and
accessible to the antibody from the extracellular milieu.
[0155] The term "selectively reactive" includes reference to the
preferential association of an antibody, in whole or part, with a
Saposin B determinant or peptide and not to proteins, cells or
tissues lacking that target determinant. It is, of course,
recognized that a certain degree of non-specific interaction may
occur between a molecule and a non-target cell or tissue.
Nevertheless, specific binding, may be distinguished as mediated
through specific recognition of the target Saposin B molecule.
Typically specific binding results in a much stronger association
between the delivered antibody and Saposin B than between the bound
molecule and proteins or cells lacking Saposin B. Specific binding
typically results in greater than 2 fold, preferably greater than 5
fold, more preferably greater than 10 fold and most preferably
greater than 100 fold increase in amount of bound ligand (per unit
time) to Saposin B or a cell or tissue bearing Saposin B as
compared to a protein, cell or tissue lacking a Saposin B
determinant. The immunoassay formats listed above are appropriate
for selecting antibodies specifically immunoreactive with a
particular protein.
[0156] In some embodiments of the present invention, the
anti-Saposin B antibody is an antibody binding fragment such as an
scFv or dsFv antibody. Fv fragments are typically about 25 kDa and
contain a complete antigen-binding site. The V.sub.H and V.sub.L
chains of the Fv fragments are held together by noncovalent
interactions. These chains tend to dissociate upon dilution, so
methods have been developed to crosslink the chains through
glutaraldehyde, intermolecular disulfides, or a peptide linker. In
some preferred embodiments, the Fv antibody binding fragment has a
variable heavy chain from an antibody directed against Saposin B or
a conservatively modified variant thereof, and/or a variable light
chain from an antibody directed against Saposin B or conservatively
modified variant thereof. Such conservative variants employed in
dsFv fragments will retain cysteine residues used for disulfide
linkages between the chains. Conservatively modified variants of
the anti-Saposin B V.sub.H and V.sub.L have at least 80% sequence
similarity, preferably at least 85% sequence similarity, more
preferably at least 90% sequence similarity, and most preferably at
least 95% sequence similarity at the amino acid level to a nucleic
acid sequence of the V.sub.H and V.sub.L of a monoclonal antibody
directed against Saposin B.
[0157] Methods of making Fv antibodies have been described. See,
Huse, et al., Science 246:1275-1281 (1989); and Ward, et al.,
Nature 341:544-546 (1989); and Vaughan, et al., Nature
Biotechnology 14:309-314 (1996). In general, suitable monoclonal or
polyclonal antibodies will usually bind with an affinity constant
of at least 10.sup.-6 M, preferably at least 10.sup.-8 M,
preferably at least 10.sup.-9 M, more preferably at least
10.sup.-10 M, most preferably at least 10.sup.-11 M.
[0158] The variable heavy and light chains (V.sub.H and V.sub.L) of
disulfide stabilized Fv fragments are covalently linked via a
disulfide linkage between cysteine residues present in each of the
two chains. The pair of amino acids to be selected are, in order of
decreasing preference:
[0159] V.sub.H 44-V.sub.L 100
[0160] V.sub.H 105-V.sub.L 43,
[0161] V.sub.H 105-V.sub.L 42,
[0162] V.sub.H 106-V.sub.L 43,
[0163] V.sub.H 104-V.sub.L 43,
[0164] V.sub.H 44-V.sub.L 99,
[0165] V.sub.H 45-V.sub.L 98,
[0166] V.sub.H 46-V.sub.L 98,
[0167] V.sub.H 103-V.sub.L 43,
[0168] V.sub.H 103-V.sub.L 44,
[0169] V.sub.H 103-V.sub.L 45.
[0170] Most preferably, substitutions of cysteine are made at the
positions:
[0171] V.sub.H 44-V.sub.L 100; or
[0172] V.sub.H 105-V.sub.L 43.
[0173] The notation V.sub.H 44-V.sub.L 100, for example, refers to
a polypeptide with a V.sub.H having a cysteine at position 44 and a
cysteine in V.sub.L at position 100; the positions being in
accordance with the numbering given in "Sequences of Proteins of
Immunological Interest," E. Kabat, et al., U.S. Government Printing
Office, NIH Publication No. 91-3242 (1991); which is incorporated
herein by reference ("Kabat and Wu"). DsFv fragments comprise at
least one disulfide linkage but may comprise 2, 3, 4, 5 or more
linkages as desired.
[0174] While the two V.sub.H and V.sub.L chains of some antibody
embodiments can be directly joined together, one of skill will
appreciate the molecules may be separated by a peptide linker
consisting of one or more amino acids. Generally the peptide linker
will have no specific biological activity other than to join the
proteins or to preserve some minimum distance or other spatial
relationship between them. However, the constituent amino acids of
the peptide linker may be selected to influence some property of
the molecule such as the folding, net charge, or hydrophobicity.
Single chain Fv (scfv) antibodies optionally include a peptide
linker of no more than 50 amino acids, generally no more than 40
amino acids, preferably no more than 30 amino acids, and more
preferably no more than 20 amino acids in length.
II. Pharmaceutical Compositions of this Invention
[0175] In one embodiment of this invention, the polypeptides of
this invention are formulated into pharmaceutical compositions. In
addition to the polypeptides of this invention, the pharmaceutical
compositions of this invention comprise pharmaceutically acceptable
carriers, including excipients. Throughout this section, the term
polypeptide will be used to indicate the polypeptides, fusion
proteins and receptors of this invention.
[0176] A. Purification of the Polypeptides and Fusion Proteins
[0177] It may be necessary to purify the polypeptides of this
invention prior to formulation into a pharmaceutical composition of
this invention. Protein purification techniques are well known in
the art and can be found in many practice guides, including "Basic
Protein and Peptide Protocols," METHODS IN MOLEC. BIOL. VOL. 32,
Walker, ed., Humana Press (1994).
[0178] After the polypeptides have been chemically synthesized by,
for example, the peptide synthesis techniques described above, it
may be necessary to remove the excess amino acids from the reaction
mixture containing the polypeptides. Purification techniques
suitable for removing amino acids are well known in the art. For
example, the polypeptides may be purified using known
chromatographic procedures such as reverse phase HPLC, gel
permeation, ion exchange, size exclusion, affinity, partition, or
countercurrent distribution (see, generally, R. Scopes, POLYPEPTIDE
PUIRFICATION, Springer-Verlag, N.Y. (1982), Deutscher, METHODS IN
ENZYMOLOGY VOL. 182: Guide to Polypeptide Purification., Academic
Press, Inc. N.Y. (1990)). Once purified, partially or to
homogeneity as desired, the polypeptides may then be used (e.g., as
immunogens for antibody production, as anti-angiogenic moieties in
fusion proteins, or as active compounds in pharmaceutical
compositions).
[0179] B. Pharmaceutically Acceptable Carriers
[0180] Suitable formulations for use in the present invention are
found in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing
Company, Philadelphia, Pa., 17th ed. (1985)). Moreover, for a brief
review of methods for drug delivery, see, Langer, Science
249:1527-1533 (1990). The pharmaceutical compositions described
herein can be manufactured in a manner that is known to those of
skill in the art, i.e., by means of conventional mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or lyophilizing processes. The following
methods and excipients are merely exemplary and are in no way
limiting.
[0181] For injection, the polypeptides of this invention can be
formulated into preparations by dissolving, suspending or
emulsifying them in an aqueous or nonaqueous solvent, such as
vegetable or other similar oils, synthetic aliphatic acid
glycerides, esters of higher aliphatic acids or propylene glycol;
and if desired, with conventional additives such as solubilizers,
isotonic agents, suspending agents, emulsifying agents, stabilizers
and preservatives. Preferably, the compounds of the invention may
be formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks's solution, Ringer's solution, or
physiological saline 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.
[0182] For oral administration, the polypeptides can be formulated
readily by combining with pharmaceutically acceptable carriers that
are well known in the art. Such carriers enable the compounds to be
formulated as tablets, pills, dragees, capsules, emulsions,
lipophilic and hydrophilic suspensions, liquids, gels, syrups,
slurries, suspensions and the like, for oral ingestion by a patient
to be treated. Pharmaceutical preparations for oral use can be
obtained by mixing the compounds with a solid excipient, optionally
grinding a 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, hydroxypropylmethylcellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0183] 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, and/or 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.
[0184] Pharmaceutical preparations 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 can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds 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 such administration.
[0185] For buccal and sublingual administration, the compositions
may take the form of tablets or lozenges formulated in conventional
manner.
[0186] For administration by instillation or inhalation, the
compounds for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebulizer, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas, or
from propellant-free, dry-powder inhalers. 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 an inhaler or insufflator may be
formulated containing a powder mix of the compound and a suitable
powder base such as lactose or starch.
[0187] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0188] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
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 compounds to allow for
the preparation of highly concentrated solutions. Alternatively,
the active ingredient may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0189] The compounds may also be formulated in vaginal or rectal
compositions such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter,
carbowaxes, polyethylene glycols or other glycerides, all of which
melt at body temperature, yet are solidified at room
temperature.
[0190] In addition to the formulations described previously, the
compounds may 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 compounds may be formulated 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.
[0191] Alternatively, other delivery systems for the polypeptides
and fusion proteins of this invention may be employed. Liposomes
and emulsions are well known examples of delivery vehicles or
carriers for drugs. For transdermal delivery, certain organic
solvents such as dimethylsulfoxide also may be employed, although
usually at the cost of greater toxicity. Additionally, the
compounds may be delivered using a sustained-release system, such
as semipermeable matrices of solid hydrophobic polymers containing
the therapeutic polypeptide. Various types of sustained-release
materials have been established and are well known by those skilled
in the art. Sustained-release capsules may, depending on their
chemical nature, release the compounds for a few weeks up to over
100 days.
[0192] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0193] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in a therapeutically effective amount. The amount
administered to the patient will vary depending upon severity of
the undesired angiogenesis, the activity of the specific
polypeptide being administered, the overall health of the patient
and the manner of administration. The pharmaceutical compositions
of this invention are administered to a patient already suffering
undesirable angiogenesis and therefore, the composition is
administered in an amount sufficient to ameliorate undesirable
angiogenesis and its complications. An amount adequate to
accomplish this is defined as "therapeutically effective dose."
Generally, the dose for systemic use, e.g., intravenous,
intrathecal, and intraarterial, will be in the range of about 0.1
mg/kg to about 50 mg/kg per day, preferably about 5 mg/kg to about
20 kg/mg per day, for a 70 kg patient. One of skill will recognize
upon review of this disclosure that the dose will also depend on
the route of administration. For example, for local administration,
e.g., topical, intravaginal, intrarectal, subcutaneous, and
intraocular, the dose will be in the range of about 0.1 mg/cm.sup.2
to about 2.5 mg/cm.sup.2 per dose, preferably about 1 mg/cm.sup.2
per dose.
III. Examples
Example 1
Activity of Recombinant Saposin B and Other Prosaposin Chains
[0194] In order to determine whether recombinant Saposin B has
anti-angiogenic activity, the coding region of each of the
Prosaposin chains was amplified from fibroblast cell lines and
cloned into both bacterial (pGEX-KG) and eukaryotic (pcDNA 3.1His
A) expression vectors.
[0195] A Ti fibroblast cell line (ATCC, Rockville, Md.) was grown
in DMEM media containing 10% FBS. Total RNA was extracted from
4.times.106 cells by RNAzol reagent (Tel-Test, Friendswood, Tex.).
About 5 .mu.g of total RNA was used to synthesize cDNA (Life
technologies, Gaithersburg, Md.) using either oligo dT or random
primers. To amplify, Saposin B cDNA, two primers were synthesized
to correspond to the 5' and 3' ends of the coding region (ATT CGA
ATT CAA GGG GAC GTT TGC CAG GAC TGC (SEQ ID NO:3) and TTC TGT GAT
GAG GTG AAA TAG CTC GAG CTC GAG (SEQ ID NO:4)). The primers were
designed so that an Eco RI restriction site was located at the 5'
end and a Xho I restriction site was located at the 3' end.
Prosaposin, Saposin A, C and D were amplified from the same cDNA
using primers that added a Xba1 restriction site at the 5' end and
a Xho1 restriction site at the 3' end of the amplified DNA. The
primers used in the PCR amplification were: Prosaposin-CTA GAT CTA
GAA ATG TAC GCC CTC TTC CTC CTG GCC (SEQ ID NO:5) and CTC GAG CTC
GAG CTA GTT CCA CAC ATG GCG TTT GCA (SEQ ID NO:6); Saposin A-CTA
GAT CTA GAA TCC CTT CCC TGC GAC ATA TCC (SEQ ID NO:7) and CTC GAG
CTC GAG TCA CTT CTG GAG AGA CTC GCA GAG (SEQ ID NO: 8); Saposin
C-CTA GAT CTA GAA TCT GAT GTT TAC TGT GAG GTG (SEQ ID NO:9) and CTC
GAG CTC GAG TCA TGC CAG AGC AGA GGT GCA GCA (SEQ ID NO:100); and
Saposin D-CTA GAT CTA GAA GAC GGT GGC TTC TGC GAA GTG (SEQ ID
NO:11) and CTC GAG CTC GAG TCA CTT ATG GGC CGA GGG GCA GGC (SEQ ID
NO:12). The amplification reactions included incubation at
94.degree. C. for 1 min to denature double stranded DNA, 55.degree.
C. for 2.5 min to anneal primers and 72.degree. C. for 3 min to
polymerase double stranded DNA. The reaction was repeated for 30
cycles. A final polymerization step was done at 72.degree. C. for
10 min.
[0196] The PCR products were digested with the respective
restriction enzymes and the restriction products were inserted into
the pGEX-KG vector for bacterial expression. pGEX-KG vector
comprises a glutathione S transferase (GST)-tag at the 3' end of
the insertion site so that a GST-fusion protein is expressed. The
GST-tag is used to aid in purification of the recombinant protein.
After elution from a glutathione-Sepharose 4B column, the
GST-tagged proteins were incubated with thrombin (4 .mu.g/ml) for 4
hr at room temperature (22-25.degree. C.) in 50 mM Tris, pH 8.3, 3
mM CaCl.sub.2, 150 mM NaCl to cleave GST from the fusion protein.
Free GST was removed from the preparation by passing the digested
fusion protein through the glutathione-Sepharose 4B column.
[0197] By SDS-PAGE, the bacterial product was smaller than the
eukaryotic protein. Because prokaryotically expressed proteins are
not glycosylated, the difference in molecular weights probably
represents the presence of carbohydrate in the eukaryotic protein.
Recombinant proteins were then tested in KS, endothelial and
control cell lines. Activity was found only in KS cell lines and
endothelial cells.
[0198] Recombinant Saposin C had no activity against KS cell lines
and endothelial cells even at the highest concentration tested (50
.mu.g/mL). Similarly Saposin A and D had no activity (data not
shown). Naturally occurring full-length Prosaposin was also tested
with, again, no activity. These findings demonstrate that only
Saposin B exhibits anti-angiogenic activity. Similar results with
anti-angiogenic proteins have previously been reported. For
example, angiostatin is a cryptic peptide of prothrombin and
endostatin is a carboxy peptide of collagen XVIII. Both angiostatin
and endostatin have been reported to possess anti-angiogenic
activity while their precursor proteins do not.
Example 2
Saposin B Inhibits Endothelial Cell Migration
[0199] Angiogenesis is mediated by complex biochemical processes
including degradation of the basement membrane under existing blood
vessel endothelial cells, followed by proliferation and migration
of endothelial cells, followed by formation of capillary loops, and
recruitment of vascular smooth muscle cells to encase the newly
formed vessels and provide stability. KS and endothelial cell
migration in the presence of Saposin B was studied in transwell
culture plates with 8 .mu.m pore membranes (Costar, Cambridge,
Mass.).
[0200] Briefly, wells were coated with fibronectin (25 .mu.g/mL)
overnight, and endothelial cells or KS cells were plated in the
upper chamber with 100 .mu.L of DMEM/0.4% FCS. 500 .mu.L of
DMEM/0.4% FCS was added to the lower chamber and incubated at
37.degree. C. for one hour. The test polypeptides, at various
concentrations, were added to the upper chamber, and chemotactic
agents (VEGF or bFGF at 20 ng/mL) were added to the lower
cell-culture chambers. The plates were incubated for 5 hr at
37.degree. C. and the cells crossing the fibronectin-coated
membrane were counted after wiping the cells off the membrane in
the upper chamber with a cotton swab.
[0201] The cells that traversed the membrane were fixed with
Diff-Quik stain according to the manufacturer's instruction (Dade
Diagnostics Inc., Aguada, PR). The cells were counted under a
microscope at 320.times. in four randomly selected fields. The
experiments were done in duplicate and repeated at least three
times.
[0202] Recombinant Saposin B was found to be highly active with
compete inhibition of cell migration. Thus, Saposin B is a strong
inhibitor of endothelial and KS cell migration. For comparison,
paclitaxel did not completely inhibit endothelial cell
migration.
Example 3
Saposin B Inhibits Angiogenesis in CAM Assays
[0203] In order to test anti-angiogenic activity, chicken allantoic
membrane (CAM) assays were performed with recombinant Saposin B.
Ten day old fertilized chicken eggs were prepared for assay by
creating a window in the egg shell, and placing filter paper discs
saturated with VEGF or bFGF as positive controls, test compounds,
or carrier buffer (negative controls) on the allantoic membrane.
The membranes were harvested after 48 hours and analyzed using an
Olympus stereomicroscope. The number of branching blood vessels
that infiltrated under the discs were counted and photographed.
Eight CAMs were studied for test group, and the experiments were
repeated at least twice.
[0204] Recombinant Saposin B effectively blocked angiogenesis
induced by VEGF or bFGF. See FIG. 3. Moreover, the inhibition of
angiogenesis induced by both proteins was blocked by adding
anti-Saposin B antibodies to the filter paper. These results
demonstrate that Saposin B has anti-angiogenic activity in the CAM
assay.
Example 4
Saposin B is Cytotoxic to CD34+/Flk-1+ Cells
[0205] A bone marrow derived human CD34+/Flk-1+ progenitor cell can
be mobilized into the blood stream in response to tumor induced
signals such as VEGF and cytokine production. These cells can
target to sites of angiogenesis where they mature into activated
endothelial cells and thus contribute to the generation of new
blood vessels at the tumor site. By blocking the production of
these cells or by targeting these cells (i.e., the CD34+/Flk-1+
progenitors and the activated endothelial cell they can become),
the angiogenesis/vasculogenesis required for tumor growth can be
reduced or halted. CD34+/Flk-1+ selected subpopulations of bone
marrow derived stem cells or peripheral mononuclear blood cells
have been shown to differentiate into vascular endothelial cells
when plated on fibronectin-coated dishes as described in Asahara,
et al. Science 1997, 275, 965-967. The adherent cells take on a
spindle-shaped morphology characteristic of the activated vascular
endothelial cells typical of the angiogenic process. These cells
express endothelial-specific markers including factor VIII, ulex
europaeus agglutinin-1 (UEA-1), endothelial constitutive nitric
oxide synthase (ecNOS), and E selectin. Further, when these cells
(peripheral blood Flk+ progenitors) are infused into mice, they
incorporate into newly formed blood vessels at site of injury.
These observation identify CD34+/Flk-1+ bone marrow derived
progenitors as a putative precursor to vascular endothelial cells.
Preventing these cells from colonizing at distal sites of
angiogenesis in tumors has enormous implication for the treatment
of tumors.
[0206] To determine whether Saposin B was cytotoxic to
CD34.sup.+/Flk-1.sup.+ progenitors, CD34.sup.+/Flk-1.sup.+ cells
from cord blood were isolated and plated on fibronectin coated
dishes in the presence or absence of 50 ng/mL Saposin B. In the
absence of Saposin B, adherent cells were observed to proliferate
and mature into spindle shaped endothelial cells. However in the
presence of Saposin B, CD34.sup.+/Flk-1.sup.+ progenitor grwptj was
inhibited.
[0207] Saposin B was found not to be toxic to most CD34.sup.+ cord
blood derived cells, since the difference in viability of Saposin B
treated CD34.sup.+ cells was only around 20% when compared to
control cells. Thus Saposin B not only targets fully differentiated
and activated endothelial cells, but also a circulating progenitor,
previously shown to target angiogenic sites.
Example 5
Effect of Saposin B on Bone Marrow Progenitor Cells
[0208] Human bone marrow cells and peripheral blood mononuclear
cells were harvested from a subject after the administration of
high doses of cyclophosphamide and G-CSF. Stem cell clonogenic
assays in methylcellulose were performed with IL-3, erytropoietin,
and VEGF (10 ng/mL, and 100 ng/mL). VEGF was used to determine if
there are progenitor cells with self replication capacity but
without adherence to extracellular matrix such as fibronectin. It
was also desirable to determine if Saposin B was toxic to
endothelial cell specific progenitors for hematopoietic (myeloid or
erythroid) lineage cells.
[0209] It was found that VEGF treatment resulted in the formation
of colonies in methylcellulose, thus proving there are progenitor
cells responsive to VEGF. These colonies contained mixture of cells
indicating more than one cell lineage generated in response to
VEGF. The colonies also contained spindle shaped cells which mark
the presence of VEGF receptors.
[0210] Saposin B treatment had no cytotoxic effect on the colonies
developed with either IL-3 or erythropoietin, suggesting that
Saposin B is not toxic to these cell types. However, colonies
generated with VEGF had loss of spindle shaped cells, while round
compact cells remained. This indicated that Saposin B was toxic to
endothelial cells with VEGF receptors; cells likely to engage in
the angiogenic and vasculogenic processes.
Example 6
Saposin B is Active In Vivo
[0211] In order to determine the anti-angiogenic activity of the
recombinant Saposin B, tumor cells were implanted in
immuno-deficient mice and treated with either Saposin B or buffer
alone.
[0212] Nude mice were injected with 2.times.10.sup.6 KS-SLK or KS
Y-1 cells subcutaneously in a total volume of 100 .mu.L. After one
week of tumor development, the mice were injected subcutaneously
daily with either PBS or Saposin B at concentrations of 1, 10 and
20 mg/kg for a total protein concentration of 100 .mu.g/kg body
weight. The tumor size was measured three times a week. The results
represent the median of 4 mice in each group.
[0213] Tumor growth was markedly retarded by Saposin B, and the
effect was dose dependent. Saposin B had no effect on the growth of
these cells. Thus, Saposin B is a potent anti-tumor protein with
activity both in vitro and in vivo.
[0214] To determine whether this anti-tumor effect was reproducible
with different classes of syngeneic tumors, C57BL/6 mice were
implanted with Lewis lung carcinoma, melanoma (B16), and T-cell
lymphoma (EL-4) cell lines. KS Y-1 was implanted into nude mice for
a positive control. The day following tumor implantation, 2.5 mg/kg
of Saposin B was administered intraperitoneally. Tumor size was
measured three times a week for two weeks at which time the mice
were sacrificed for analysis. Saposin B treated mice showed a
profound inhibition of tumor growth in all tumor types tested. See,
FIG. 4
[0215] In further experiments, KS tumors were allowed to grow for
five days before treatment with Saposin B (1 and 5 mg/kg daily at a
distal site from the tumor). In control mice, tumors grew to a
weight of 555.+-.mg. Inhibition of tumor growth was observed in
Saposin B treated mice with weights of excised tumors being
approximately 23% that of controls. See FIG. 5. Tumors excised at
the conclusion of the experiments were also examined for apoptosis,
blood vessel density, and mitotic index. Saposin B treated tumors
showed an increase in apoptosis and decrease in blood vessel
density. Thus, contrary to the results in vitro, Saposin B has an
inhibitory effect against non-KS tumors.
Example 7
Anti-Angiogenic Activity of Saposin B Polypeptides
[0216] To determine if Saposin B polypeptides had anti-angiogenic
activity, a series of overlapping polypeptides were synthesized and
tested in the KS Y-1 cell proliferation assay. The overlapping
polypeptides are SEQ ID NO:13 though 42. The results are tabulated
in Tables 4 through 8. Cell Proliferation assays in either KS cells
or fibroblast. Cells were plated in 48 well plates at equal numbers
in appropriate culture medium. Cells were treated with the test
compounds at various concentrations on day one, and on day 3. MTT
was done on day 5. KS cells were used to represent the activated
endothelial cells, while fibroblast cells represent the control
cells. The results are remarkable for the lack of toxicity to the
fibroblast relative to the activated endothelial cells/KS cells.
Similar results were seen in proliferating endothelial cells. These
results support the findings of antiangiogenic properties of these
test compounds.
TABLE-US-00004 TABLE 4 KS Cell Proliferation in the Presence of
Saposin B Polypeptides.sup.1 Control % of Control OD.sub.490 nm 1
.mu.g/mL 5 .mu.g/mL 10 .mu.g/mL 50 .mu.g/mL 100 .mu.g/mL 500
.mu.g/mL SEQ ID 0.56 51.8 17.9 1.8 1.8 1.8 NO: 13 SEQ ID 0.56 89.3
60.7 28.6 NO: 14 SEQ ID 0.56 101.8 60.7 28.6 NO: 15 SEQ ID 96.3
86.4 46.9 NO: 17 SEQ ID 0.81 88.9 86.4 58.0 NO: 18 SEQ ID 0.56
100.0 96.4 92.9 82.1 NO: 20 SEQ ID 1.15 88.7 68.7 26.1 7.0 NO: 21
SEQ ID 1.15 158.3 77.4 59.1 37.4 NO: 22 SEQ ID 1.15 88.7 83.5 68.7
51.3 NO: 23 .sup.1Cell proliferation assay performed with 7,500 KS
Y-1 cells/well in 48-well plates. Incubation for 5 days at
37.degree. C. MTT added and plates read as described in the
text.
TABLE-US-00005 TABLE 5 Fibroblast Proliferation in the Presence of
Saposin B Polypeptides.sup.2 Control % of Control OD.sub.490-650 nm
1 .mu.g/mL 10 .mu.g/mL 50 .mu.g/mL SEQ ID NO: 19 0.23 91.3 91.3
82.6 SEQ ID NO: 20 0.23 91.3 95.7 95.7 SEQ ID NO: 21 0.23 100.0
91.3 87.0 SEQ ID NO: 22 0.23 95.7 95.7 87.0 .sup.2Cell
proliferation assay performed with 5000 T1 cells/well in 48-well
plates. Incubation for 7 days at 37.degree. C. MTT added and plates
read as described in the text.
TABLE-US-00006 TABLE 6 Effect of SEQ ID NO: 19 Saposin B
polypeptide on Migration of KS Cell Lines.sup.3 SEQ ID NO: 19 %
Cells Migrating Through Membrane (of Control) 0 .mu.g/mL (control)
100 1 .mu.g/mL 78.23 10 .mu.g/mL 40.32 50 .mu.g/mL 20.98 .sup.3Cell
migration assay was performed with 50,000 KS Y-1 cells/well. Wells
were coated with fibronectin prior to use. 25 ng/mL bFGF was added
to thelower chamber to act as a chemotactic agent. cells counted
after overnight incubation with polypeptide.
TABLE-US-00007 TABLE 7 Cell Proliferation in the Presence of
Saposin B Polypeptides.sup.4 Cell Proliferation Activity (% of
control) SEQ ID NO KS Y-1 T1 SEQ ID NO: 19 28 8 SEQ ID NO: 20 93 84
SEQ ID NO: 21 29 67 SEQ ID NO: 22 59 28 SEQ ID NO: 23 68 51 SEQ ID
NO: 24 68 46 96 84 SEQ ID NO: 25 72 39 98 89 SEQ ID NO: 26 72 40 96
81 SEQ ID NO: 27 60 22 88 75 SEQ ID NO: 28 30 10 91 87 SEQ ID NO:
29 65 33 95 86 SEQ ID NO: 30 77 35 92 84 SEQ ID NO: 31 74 87 80
.sup.4Cell proliferation performed with fibroblast T1 cells/well in
48-well plates. Incubation for 5 days at 37.degree. C. MTT added
and plates read as described in the text.
TABLE-US-00008 TABLE 8 Saposin B Peptides Activity Summary Seq IC50
(mM) Core Labe ID Activity Name code/group No. Sequence in KS Y-1
internal peptide scan G1-V11 20PG 19 GDVCQDCIQMV 4.9 Q9-F15 2PG 43
QMVTDIQTQVRTNSTF 14 S23-R39 3PG 15 STFVQALVEHVKEECDR 22 C37-S53 4PG
42 CDRLGPGMAKICKNYIS 9.7 Y51-P68 5PG 17 YISQYSEIAIQMMMHMQP 20
Q67-E80 36PG 41 QPKEICALVGFCDEVK 14 Bisection of 20PG G1-Q5 23PG 22
GDVCQ 49.4 D6-V12 24PG 23 DCIQMV 57.3 C-terminal deletions D2-M10
25PG 24 DVCQDCIQM 16.2 D2-Q9 28PG 25 DVCQDCIQ 16.6 D2-I8 27PG 26
DVCQDCI 20 D2-C7 28PG 27 DVCQDC 19 D2-D5 29PG 31 DVCQD 11.8
N-terminal deletions D2-V11 22PG 21 DVCQDCIQMV 4.4 V3-V11 30PG 29
VCQDCIQMV 11.9 C4-V11 31PG 30 CQDCIQMV 13.2 Q5-V11 32PG 31 QDCIQMV
16.6 C-S mutations G1-(S4,S7)-V11 21PG 20 GDVSQDSIQMV >400
G1-(S4)-V11 33PG 32 GDVSQDCIQMV 18 G1-(S7)-V11 34PG 33 GDVCQDSIQMV
7.8 G1-(S4)-D6 35PG 34 GDVSQD 40 V3 and Q5 mutations D2-(A3)-D6
37PG 35 DACQD 42.9 D2-(I3)-D6 38PG 36 DICQD 34.6 D2-(L3)-D6 39PG 37
DLCQD 39.8 D2-(S5)-D6 40PG 38 DVCSD 25.5 D2-(E5)-D6 41PG 39 DVCED
26.3 D2-(D5)-D6 42PG 40 DVCDD 12.2
[0217] In vivo results for experiments similar to those described
in Example 6 demonstrate that the pentapeptide DVCQD (SEQ ID NO 28)
was active in vivo (see FIG. 6).
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 63 <210> SEQ ID NO 1 <211> LENGTH: 524 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: prosaposin <220> FEATURE:
<221> NAME/KEY: PEPTIDE <222> LOCATION: (195)..(275)
<223> OTHER INFORMATION: Saposin B <400> SEQUENCE: 1
Met Tyr Ala Leu Phe Leu Leu Ala Ser Leu Leu Gly Ala Ala Leu Ala 1 5
10 15 Gly Pro Val Leu Gly Leu Lys Glu Cys Thr Arg Gly Ser Ala Val
Trp 20 25 30 Cys Gln Asn Val Lys Thr Ala Ser Asp Cys Gly Ala Val
Lys His Cys 35 40 45 Leu Gln Thr Val Trp Asn Lys Pro Thr Val Lys
Ser Leu Pro Cys Asp 50 55 60 Ile Cys Lys Asp Val Val Thr Ala Ala
Gly Asp Met Leu Lys Asp Asn 65 70 75 80 Ala Thr Glu Glu Glu Ile Leu
Val Tyr Leu Glu Lys Thr Cys Asp Trp 85 90 95 Leu Pro Lys Pro Asn
Met Ser Ala Ser Cys Lys Glu Ile Val Asp Ser 100 105 110 Tyr Leu Pro
Val Ile Leu Asp Ile Ile Lys Gly Glu Met Ser Arg Pro 115 120 125 Gly
Glu Val Cys Ser Ala Leu Asn Leu Cys Glu Ser Leu Gln Lys His 130 135
140 Leu Ala Glu Leu Asn His Gln Lys Gln Leu Glu Ser Asn Lys Ile Pro
145 150 155 160 Glu Leu Asp Met Thr Glu Val Val Ala Pro Phe Met Ala
Asn Ile Pro 165 170 175 Leu Leu Leu Tyr Pro Gln Asp Gly Pro Arg Ser
Lys Pro Gln Pro Lys 180 185 190 Asp Asn Gly Asp Val Cys Gln Asp Cys
Ile Gln Met Val Thr Asp Ile 195 200 205 Gln Thr Ala Val Arg Thr Asn
Ser Thr Phe Val Gln Ala Leu Val Glu 210 215 220 His Val Lys Glu Glu
Cys Asp Arg Leu Gly Pro Gly Met Ala Asp Ile 225 230 235 240 Cys Lys
Asn Tyr Ile Ser Gln Tyr Ser Glu Ile Ala Ile Gln Met Met 245 250 255
Met His Met Gln Pro Lys Glu Ile Cys Ala Leu Val Gly Phe Cys Asp 260
265 270 Glu Val Lys Glu Met Pro Met Gln Thr Leu Val Pro Ala Lys Val
Ala 275 280 285 Ser Lys Asn Val Ile Pro Ala Leu Glu Leu Val Glu Pro
Ile Lys Lys 290 295 300 His Glu Val Pro Ala Lys Ser Asp Val Tyr Cys
Glu Val Cys Glu Phe 305 310 315 320 Leu Val Lys Glu Val Thr Lys Leu
Ile Asp Asn Asn Lys Thr Glu Lys 325 330 335 Glu Ile Leu Asp Ala Phe
Asp Lys Met Cys Ser Lys Leu Pro Lys Ser 340 345 350 Leu Ser Glu Glu
Cys Gln Glu Val Val Asp Thr Tyr Gly Ser Ser Ile 355 360 365 Leu Ser
Ile Leu Leu Glu Glu Val Ser Pro Glu Leu Val Cys Ser Met 370 375 380
Leu His Leu Cys Ser Gly Thr Arg Leu Pro Ala Leu Thr Val His Val 385
390 395 400 Thr Gln Pro Lys Asp Gly Gly Phe Cys Glu Val Cys Lys Lys
Leu Val 405 410 415 Gly Tyr Leu Asp Arg Asn Leu Glu Lys Asn Ser Thr
Lys Gln Glu Ile 420 425 430 Leu Ala Ala Leu Glu Lys Gly Cys Ser Phe
Leu Pro Asp Pro Tyr Gln 435 440 445 Lys Gln Cys Asp Gln Phe Val Ala
Glu Tyr Glu Pro Val Leu Ile Glu 450 455 460 Ile Leu Val Glu Val Met
Asp Pro Ser Phe Val Cys Leu Lys Ile Gly 465 470 475 480 Ala Cys Pro
Ser Ala His Lys Pro Leu Leu Gly Thr Glu Lys Cys Ile 485 490 495 Trp
Gly Pro Ser Tyr Trp Cys Gln Asn Thr Glu Thr Ala Ala Gln Cys 500 505
510 Asn Ala Val Glu His Cys Lys Arg His Val Trp Asn 515 520
<210> SEQ ID NO 2 <211> LENGTH: 81 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: Saposin B <400> SEQUENCE: 2
Gly Asp Val Cys Gln Asp Cys Ile Gln Met Val Thr Asp Ile Gln Thr 1 5
10 15 Ala Val Arg Thr Asn Ser Thr Phe Val Gln Ala Leu Val Glu His
Val 20 25 30 Lys Glu Glu Cys Asp Arg Leu Gly Pro Gly Met Ala Asp
Ile Cys Lys 35 40 45 Asn Tyr Ile Ser Gln Tyr Ser Glu Ile Ala Ile
Gln Met Met Met His 50 55 60 Met Gln Pro Lys Glu Ile Cys Ala Leu
Val Gly Phe Cys Asp Glu Val 65 70 75 80 Lys <210> SEQ ID NO 3
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: 5' primer for
amplifying Saposin B cDNA <400> SEQUENCE: 3 attcgaattc
aaggggacgt ttgccaggac tgc 33 <210> SEQ ID NO 4 <211>
LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: 3' primer for amplifying
Saposin B cDNA <400> SEQUENCE: 4 ttctgtgatg aggtgaaata
gctcgagctc gag 33 <210> SEQ ID NO 5 <211> LENGTH: 36
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: 5' primer for PCR amplification of Prosaposin
<400> SEQUENCE: 5 ctagatctag aaatgtacgc cctcttcctc ctggcc 36
<210> SEQ ID NO 6 <211> LENGTH: 36 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
3' primer for PCR amplification of Prosaposin <400> SEQUENCE:
6 ctcgagctcg agctagttcc acacatggcg tttgca 36 <210> SEQ ID NO
7 <211> LENGTH: 33 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: 5' primer
for PCR amplification of Saposin A <400> SEQUENCE: 7
ctagatctag aatcccttcc ctgcgacata tcc 33 <210> SEQ ID NO 8
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: 3' primer for PCR
amplification of Saposin A <400> SEQUENCE: 8 ctcgagctcg
agtcacttct ggagagactc gcagag 36 <210> SEQ ID NO 9 <211>
LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: 5' primer for PCR amplification
of Saposin C <400> SEQUENCE: 9 ctagatctag aatctgatgt
ttactgtgag gtg 33 <210> SEQ ID NO 10 <211> LENGTH: 36
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: 3' primer for PCR amplification of Saposin C
<400> SEQUENCE: 10 ctcgagctcg agtcatgcca gagcagaggt gcagca 36
<210> SEQ ID NO 11 <211> LENGTH: 33 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
5' primer for PCR amplification of Saposin D <400> SEQUENCE:
11 ctagatctag aagacggtgg cttctgcgaa gtg 33 <210> SEQ ID NO 12
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: 3' primer for PCR
amplification of Saposin D <400> SEQUENCE: 12 ctcgagctcg
agtcacttat gggccgaggg gcaggc 36 <210> SEQ ID NO 13
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: anti-angiogenic
polypeptide <400> SEQUENCE: 13 Gln Pro Lys Asp Asn Gly Asp
Val Cys Gln Asp Cys Ile Gln Val 1 5 10 15 <210> SEQ ID NO 14
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: anti-angiogenic
polypeptide <400> SEQUENCE: 14 Ile Gln Met Val Thr Asp Ile
Gln Thr Ala Val Arg Thr Asn Ser Thr 1 5 10 15 Phe <210> SEQ
ID NO 15 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: S23-R39
anti-angiogenic polypeptide <400> SEQUENCE: 15 Ser Thr Phe
Val Gln Ala Leu Val Glu His Val Lys Glu Glu Cys Asp 1 5 10 15 Arg
<210> SEQ ID NO 16 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
anti-angiogenic polypeptide <400> SEQUENCE: 16 Cys Asp Arg
Leu Gly Pro Gly Met Ala Asp Lys Asn Tyr Ser 1 5 10 <210> SEQ
ID NO 17 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Y51-P68
anti-angiogenic polypeptide <400> SEQUENCE: 17 Tyr Ile Ser
Gln Tyr Ser Glu Ile Ala Ile Gln Met Met Met His Met 1 5 10 15 Gln
Pro <210> SEQ ID NO 18 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: anti-angiogenic polypeptide <400> SEQUENCE: 18 Gln
Met Met Met His Met Gln Pro Lys Glu Ile Cys Ala Leu Val Gly 1 5 10
15 <210> SEQ ID NO 19 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: G1-V11 anti-angiogenic polypeptide <400> SEQUENCE:
19 Gly Asp Val Cys Gln Asp Cys Ile Gln Met Val 1 5 10 <210>
SEQ ID NO 20 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
G1-(S4, S7)-V11 anti-angiogenic polypeptide <400> SEQUENCE:
20 Gly Asp Val Ser Gln Asp Ser Ile Gln Met Val 1 5 10 <210>
SEQ ID NO 21 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
D2-V11 anti-angiogenic polypeptide <400> SEQUENCE: 21 Asp Val
Cys Gln Asp Cys Ile Gln Met Val 1 5 10 <210> SEQ ID NO 22
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: G1-Q5
anti-angiogenic polypeptide <400> SEQUENCE: 22 Gly Asp Val
Cys Gln 1 5 <210> SEQ ID NO 23 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: D6-V12 anti-angiogenic polypeptide <400>
SEQUENCE: 23 Asp Cys Ile Gln Met Val 1 5 <210> SEQ ID NO 24
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: D2-M10
anti-angiogenic polypeptide <400> SEQUENCE: 24 Asp Val Cys
Gln Asp Cys Ile Gln Met 1 5 <210> SEQ ID NO 25 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: D2-Q9 anti-angiogenic
polypeptide <400> SEQUENCE: 25 Asp Val Cys Gln Asp Cys Ile
Gln 1 5 <210> SEQ ID NO 26 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: D2-I8 anti-angiogenic polypeptide <400> SEQUENCE:
26 Asp Val Cys Gln Asp Cys Ile 1 5 <210> SEQ ID NO 27
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: D2-C7
anti-angiogenic polypeptide <400> SEQUENCE: 27 Asp Val Cys
Gln Asp Cys 1 5 <210> SEQ ID NO 28 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: D2-D6 anti-angiogenic polypeptide <400>
SEQUENCE: 28 Asp Val Cys Gln Asp 1 5 <210> SEQ ID NO 29
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: V3-V11
anti-angiogenic polypeptide <400> SEQUENCE: 29 Val Cys Gln
Asp Cys Ile Gln Met Val 1 5 <210> SEQ ID NO 30 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: C4-V11 anti-angiogenic
polypeptide <400> SEQUENCE: 30 Cys Gln Asp Cys Ile Gln Met
Val 1 5 <210> SEQ ID NO 31 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Q5-V11 anti-angiogenic polypeptide <400> SEQUENCE:
31 Gln Asp Cys Ile Gln Met Val 1 5 <210> SEQ ID NO 32
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: G1-(S4)-V11
anti-angiogenic polypeptide <400> SEQUENCE: 32 Gly Asp Val
Ser Gln Asp Cys Ile Gln Met Val 1 5 10 <210> SEQ ID NO 33
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: G1-(S7)-V11
anti-angiogenic polypeptide <400> SEQUENCE: 33 Gly Asp Val
Cys Gln Asp Ser Ile Gln Met Val 1 5 10 <210> SEQ ID NO 34
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: G1-(S4)-D6
anti-angiogenic polypeptide <400> SEQUENCE: 34 Gly Asp Val
Ser Gln Asp 1 5 <210> SEQ ID NO 35 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: D2-(A3)-D6 anti-angiogenic polypeptide
<400> SEQUENCE: 35 Asp Ala Cys Gln Asp 1 5 <210> SEQ ID
NO 36 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: D2-(I3)-D6
anti-angiogenic polypeptide <400> SEQUENCE: 36 Asp Ile Cys
Gln Asp 1 5 <210> SEQ ID NO 37 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: D2-(L3)-D6 anti-angiogenic polypeptide
<400> SEQUENCE: 37 Asp Leu Cys Gln Asp 1 5 <210> SEQ ID
NO 38 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: D2-(S5)-D6
anti-angiogenic polypeptide <400> SEQUENCE: 38 Asp Val Cys
Ser Asp 1 5 <210> SEQ ID NO 39 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: D2-(E5)-D6 anti-angiogenic polypeptide
<400> SEQUENCE: 39 Asp Val Cys Glu Asp 1 5 <210> SEQ ID
NO 40 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: D2-(D5)-D6
anti-angiogenic polypeptide <400> SEQUENCE: 40 Asp Val Cys
Asp Asp 1 5 <210> SEQ ID NO 41 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Q67-E80 anti-angiogenic polypeptide
<400> SEQUENCE: 41 Gln Pro Lys Glu Ile Cys Ala Leu Val Gly
Phe Cys Asp Glu Val Lys 1 5 10 15 <210> SEQ ID NO 42
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: C37-S53
anti-angiogenic polypeptide <400> SEQUENCE: 42 Cys Asp Arg
Leu Gly Pro Gly Met Ala Lys Ile Cys Lys Asn Tyr Ile 1 5 10 15 Ser
<210> SEQ ID NO 43 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Q9-F15 anti-angiogenic polypeptide <400> SEQUENCE: 43 Gln Met
Val Thr Asp Ile Gln Thr Gln Val Arg Thr Asn Ser Thr Phe 1 5 10 15
<210> SEQ ID NO 44 <211> LENGTH: 70 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
anti-angiogenic polypeptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1)..(6) <223> OTHER
INFORMATION: Xaa = any amino acid, Xaa at positions 1-6 may be
present or absent <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (12)..(70) <223> OTHER
INFORMATION: Xaa = any amino acid, Xaa at positions 12-70 may be
present or absent <400> SEQUENCE: 44 Xaa Xaa Xaa Xaa Xaa Xaa
Asp Val Cys Gln Asp Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50
55 60 Xaa Xaa Xaa Xaa Xaa Xaa 65 70 <210> SEQ ID NO 45
<211> LENGTH: 70 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: anti-angiogenic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1)..(5) <223> OTHER INFORMATION: Xaa =
any amino acid, Xaa at positions 1-5 may range from 1-5 residues
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (6) <223> OTHER INFORMATION: Xaa = Gly, Ala, Ser or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12)..(70) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 12-70 may be present or absent <400>
SEQUENCE: 45 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa
Xaa 65 70 <210> SEQ ID NO 46 <211> LENGTH: 70
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6)
<223> OTHER INFORMATION: Xaa = variable amino acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12)..(70) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 12-70 may be present or absent <400>
SEQUENCE: 46 Gln Pro Lys Asp Asn Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa
Xaa 65 70 <210> SEQ ID NO 47 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(6) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-6 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(16) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 13-16 may be present or absent <400> SEQUENCE:
47 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa Xaa Xaa Xaa
1 5 10 15 <210> SEQ ID NO 48 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(6) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-6 may be present or absent <400> SEQUENCE: 48
Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Cys Ile Gln Met Val 1 5
10 15 <210> SEQ ID NO 49 <211> LENGTH: 70 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: anti-angiogenic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (2)..(5)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6)
<223> OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12)..(70) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 12-70 may be present or absent <400>
SEQUENCE: 49 Gln Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa
Xaa 65 70 <210> SEQ ID NO 50 <211> LENGTH: 70
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
position 1 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (3)..(5)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6)
<223> OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12)..(70) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 12-70 may be present or absent <400>
SEQUENCE: 50 Xaa Pro Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa
Xaa 65 70 <210> SEQ ID NO 51 <211> LENGTH: 70
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(2) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1 and 2 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6)
<223> OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12)..(70) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 12-70 may be present or absent <400>
SEQUENCE: 51 Xaa Xaa Lys Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa
Xaa 65 70 <210> SEQ ID NO 52 <211> LENGTH: 70
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(3) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-3 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)..(70)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
positions 12-70 maybe present or absent <400> SEQUENCE: 52
Xaa Xaa Xaa Asp Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa 65 70
<210> SEQ ID NO 53 <211> LENGTH: 70 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
anti-angiogenic polypeptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1)..(4) <223> OTHER
INFORMATION: Xaa = any amino acid, Xaa at positions 1-4 may be
present or absent <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION:
Xaa = Gly, Ala, Ser or Thr <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (12)..(70) <223>
OTHER INFORMATION: Xaa = any amino acid, Xaa at positions 12-70 may
be present or absent <400> SEQUENCE: 53 Xaa Xaa Xaa Xaa Asn
Xaa Asp Val Cys Gln Asp Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40
45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa 65 70 <210> SEQ ID NO 54
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: anti-angiogenic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1)..(4) <223> OTHER INFORMATION: Xaa =
any amino acid, Xaa at positions 1-4 may be present or absent
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (5) <223> OTHER INFORMATION: Xaa = any amino acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (6) <223> OTHER INFORMATION: Xaa = Gly, Ala, Ser or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12) <223> OTHER INFORMATION: Xaa = any amino acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(16) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 13-16 may be present or absent <400>
SEQUENCE: 54 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 <210> SEQ ID NO 55 <211> LENGTH:
16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(4) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-4 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (13)..(16)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
positions 13-16 may be present or absent <400> SEQUENCE: 55
Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Cys Xaa Xaa Xaa Xaa 1 5
10 15 <210> SEQ ID NO 56 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: anti-angiogenic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(4)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
positions 1-4 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(14)..(16) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 14-16 may be present or absent <400> SEQUENCE:
56 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Ile Xaa Xaa Xaa
1 5 10 15 <210> SEQ ID NO 57 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(4) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-4 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)..(13)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(15)..(16) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 15 and 16 may be present or absent <400>
SEQUENCE: 57 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Gln Xaa Xaa 1 5 10 15 <210> SEQ ID NO 58 <211> LENGTH:
16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(4) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-4 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)..(14)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (16)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
position 16 may be present or absent <400> SEQUENCE: 58 Xaa
Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa Xaa Met Xaa 1 5 10
15 <210> SEQ ID NO 59 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: anti-angiogenic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(4)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
positions 1-4 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)..(15)
<223> OTHER INFORMATION: Xaa = any amino acid <400>
SEQUENCE: 59 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Val 1 5 10 15 <210> SEQ ID NO 60 <211> LENGTH:
5 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Amino acids 1-5
of SEQ ID NO:13 <400> SEQUENCE: 60 Gln Pro Lys Asp Asn 1 5
<210> SEQ ID NO 61 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Amino acids 12-15 of SEQ ID NO:13
<400> SEQUENCE: 61 Cys Ile Gln Val 1 <210> SEQ ID NO 62
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Amino acids 7-11 of SEQ ID NO:19 <400> SEQUENCE:
62 Cys Ile Gln Met Val 1 5 <210> SEQ ID NO 63 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Amino
acids 6-9 of SEQ ID NO:24 <400> SEQUENCE: 63 Cys Ile Gln Met
1
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 63 <210>
SEQ ID NO 1 <211> LENGTH: 524 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <220> FEATURE: <223>
OTHER INFORMATION: prosaposin <220> FEATURE: <221>
NAME/KEY: PEPTIDE <222> LOCATION: (195)..(275) <223>
OTHER INFORMATION: Saposin B <400> SEQUENCE: 1 Met Tyr Ala
Leu Phe Leu Leu Ala Ser Leu Leu Gly Ala Ala Leu Ala 1 5 10 15 Gly
Pro Val Leu Gly Leu Lys Glu Cys Thr Arg Gly Ser Ala Val Trp 20 25
30 Cys Gln Asn Val Lys Thr Ala Ser Asp Cys Gly Ala Val Lys His Cys
35 40 45 Leu Gln Thr Val Trp Asn Lys Pro Thr Val Lys Ser Leu Pro
Cys Asp 50 55 60 Ile Cys Lys Asp Val Val Thr Ala Ala Gly Asp Met
Leu Lys Asp Asn 65 70 75 80 Ala Thr Glu Glu Glu Ile Leu Val Tyr Leu
Glu Lys Thr Cys Asp Trp 85 90 95 Leu Pro Lys Pro Asn Met Ser Ala
Ser Cys Lys Glu Ile Val Asp Ser 100 105 110 Tyr Leu Pro Val Ile Leu
Asp Ile Ile Lys Gly Glu Met Ser Arg Pro 115 120 125 Gly Glu Val Cys
Ser Ala Leu Asn Leu Cys Glu Ser Leu Gln Lys His 130 135 140 Leu Ala
Glu Leu Asn His Gln Lys Gln Leu Glu Ser Asn Lys Ile Pro 145 150 155
160 Glu Leu Asp Met Thr Glu Val Val Ala Pro Phe Met Ala Asn Ile Pro
165 170 175 Leu Leu Leu Tyr Pro Gln Asp Gly Pro Arg Ser Lys Pro Gln
Pro Lys 180 185 190 Asp Asn Gly Asp Val Cys Gln Asp Cys Ile Gln Met
Val Thr Asp Ile 195 200 205 Gln Thr Ala Val Arg Thr Asn Ser Thr Phe
Val Gln Ala Leu Val Glu 210 215 220 His Val Lys Glu Glu Cys Asp Arg
Leu Gly Pro Gly Met Ala Asp Ile 225 230 235 240 Cys Lys Asn Tyr Ile
Ser Gln Tyr Ser Glu Ile Ala Ile Gln Met Met 245 250 255 Met His Met
Gln Pro Lys Glu Ile Cys Ala Leu Val Gly Phe Cys Asp 260 265 270 Glu
Val Lys Glu Met Pro Met Gln Thr Leu Val Pro Ala Lys Val Ala 275 280
285 Ser Lys Asn Val Ile Pro Ala Leu Glu Leu Val Glu Pro Ile Lys Lys
290 295 300 His Glu Val Pro Ala Lys Ser Asp Val Tyr Cys Glu Val Cys
Glu Phe 305 310 315 320 Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn
Asn Lys Thr Glu Lys 325 330 335 Glu Ile Leu Asp Ala Phe Asp Lys Met
Cys Ser Lys Leu Pro Lys Ser 340 345 350 Leu Ser Glu Glu Cys Gln Glu
Val Val Asp Thr Tyr Gly Ser Ser Ile 355 360 365 Leu Ser Ile Leu Leu
Glu Glu Val Ser Pro Glu Leu Val Cys Ser Met 370 375 380 Leu His Leu
Cys Ser Gly Thr Arg Leu Pro Ala Leu Thr Val His Val 385 390 395 400
Thr Gln Pro Lys Asp Gly Gly Phe Cys Glu Val Cys Lys Lys Leu Val 405
410 415 Gly Tyr Leu Asp Arg Asn Leu Glu Lys Asn Ser Thr Lys Gln Glu
Ile 420 425 430 Leu Ala Ala Leu Glu Lys Gly Cys Ser Phe Leu Pro Asp
Pro Tyr Gln 435 440 445 Lys Gln Cys Asp Gln Phe Val Ala Glu Tyr Glu
Pro Val Leu Ile Glu 450 455 460 Ile Leu Val Glu Val Met Asp Pro Ser
Phe Val Cys Leu Lys Ile Gly 465 470 475 480 Ala Cys Pro Ser Ala His
Lys Pro Leu Leu Gly Thr Glu Lys Cys Ile 485 490 495 Trp Gly Pro Ser
Tyr Trp Cys Gln Asn Thr Glu Thr Ala Ala Gln Cys 500 505 510 Asn Ala
Val Glu His Cys Lys Arg His Val Trp Asn 515 520 <210> SEQ ID
NO 2 <211> LENGTH: 81 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: Saposin B <400> SEQUENCE: 2 Gly Asp Val Cys Gln
Asp Cys Ile Gln Met Val Thr Asp Ile Gln Thr 1 5 10 15 Ala Val Arg
Thr Asn Ser Thr Phe Val Gln Ala Leu Val Glu His Val 20 25 30 Lys
Glu Glu Cys Asp Arg Leu Gly Pro Gly Met Ala Asp Ile Cys Lys 35 40
45 Asn Tyr Ile Ser Gln Tyr Ser Glu Ile Ala Ile Gln Met Met Met His
50 55 60 Met Gln Pro Lys Glu Ile Cys Ala Leu Val Gly Phe Cys Asp
Glu Val 65 70 75 80 Lys <210> SEQ ID NO 3 <211> LENGTH:
33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: 5' primer for amplifying Saposin B cDNA
<400> SEQUENCE: 3 attcgaattc aaggggacgt ttgccaggac tgc 33
<210> SEQ ID NO 4 <211> LENGTH: 33 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
3' primer for amplifying Saposin B cDNA <400> SEQUENCE: 4
ttctgtgatg aggtgaaata gctcgagctc gag 33 <210> SEQ ID NO 5
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: 5' primer for PCR
amplification of Prosaposin <400> SEQUENCE: 5 ctagatctag
aaatgtacgc cctcttcctc ctggcc 36 <210> SEQ ID NO 6 <211>
LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: 3' primer for PCR amplification
of Prosaposin <400> SEQUENCE: 6 ctcgagctcg agctagttcc
acacatggcg tttgca 36 <210> SEQ ID NO 7 <211> LENGTH: 33
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: 5' primer for PCR amplification of Saposin A
<400> SEQUENCE: 7 ctagatctag aatcccttcc ctgcgacata tcc 33
<210> SEQ ID NO 8 <211> LENGTH: 36 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
3' primer for PCR amplification of Saposin A <400> SEQUENCE:
8 ctcgagctcg agtcacttct ggagagactc gcagag 36 <210> SEQ ID NO
9 <211> LENGTH: 33 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: 5' primer
for PCR amplification of Saposin C <400> SEQUENCE: 9
ctagatctag aatctgatgt ttactgtgag gtg 33 <210> SEQ ID NO 10
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: 3'
primer for PCR amplification of Saposin C <400> SEQUENCE: 10
ctcgagctcg agtcatgcca gagcagaggt gcagca 36 <210> SEQ ID NO 11
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: 5' primer for PCR
amplification of Saposin D <400> SEQUENCE: 11 ctagatctag
aagacggtgg cttctgcgaa gtg 33 <210> SEQ ID NO 12 <211>
LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: 3' primer for PCR amplification
of Saposin D <400> SEQUENCE: 12 ctcgagctcg agtcacttat
gggccgaggg gcaggc 36 <210> SEQ ID NO 13 <211> LENGTH:
15 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <400>
SEQUENCE: 13 Gln Pro Lys Asp Asn Gly Asp Val Cys Gln Asp Cys Ile
Gln Val 1 5 10 15 <210> SEQ ID NO 14 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <400>
SEQUENCE: 14 Ile Gln Met Val Thr Asp Ile Gln Thr Ala Val Arg Thr
Asn Ser Thr 1 5 10 15 Phe <210> SEQ ID NO 15 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: S23-R39 anti-angiogenic
polypeptide <400> SEQUENCE: 15 Ser Thr Phe Val Gln Ala Leu
Val Glu His Val Lys Glu Glu Cys Asp 1 5 10 15 Arg <210> SEQ
ID NO 16 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence:
anti-angiogenic polypeptide <400> SEQUENCE: 16 Cys Asp Arg
Leu Gly Pro Gly Met Ala Asp Lys Asn Tyr Ser 1 5 10 <210> SEQ
ID NO 17 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Y51-P68
anti-angiogenic polypeptide <400> SEQUENCE: 17 Tyr Ile Ser
Gln Tyr Ser Glu Ile Ala Ile Gln Met Met Met His Met 1 5 10 15 Gln
Pro <210> SEQ ID NO 18 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: anti-angiogenic polypeptide <400> SEQUENCE: 18 Gln
Met Met Met His Met Gln Pro Lys Glu Ile Cys Ala Leu Val Gly 1 5 10
15 <210> SEQ ID NO 19 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: G1-V11 anti-angiogenic polypeptide <400> SEQUENCE:
19 Gly Asp Val Cys Gln Asp Cys Ile Gln Met Val 1 5 10 <210>
SEQ ID NO 20 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
G1-(S4, S7)-V11 anti-angiogenic polypeptide <400> SEQUENCE:
20 Gly Asp Val Ser Gln Asp Ser Ile Gln Met Val 1 5 10 <210>
SEQ ID NO 21 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
D2-V11 anti-angiogenic polypeptide <400> SEQUENCE: 21 Asp Val
Cys Gln Asp Cys Ile Gln Met Val 1 5 10 <210> SEQ ID NO 22
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: G1-Q5
anti-angiogenic polypeptide <400> SEQUENCE: 22 Gly Asp Val
Cys Gln 1 5 <210> SEQ ID NO 23 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: D6-V12 anti-angiogenic polypeptide <400>
SEQUENCE: 23 Asp Cys Ile Gln Met Val 1 5 <210> SEQ ID NO 24
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: D2-M10
anti-angiogenic polypeptide <400> SEQUENCE: 24 Asp Val Cys
Gln Asp Cys Ile Gln Met 1 5 <210> SEQ ID NO 25 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: D2-Q9 anti-angiogenic
polypeptide <400> SEQUENCE: 25 Asp Val Cys Gln Asp Cys Ile
Gln 1 5 <210> SEQ ID NO 26 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: D2-I8 anti-angiogenic polypeptide <400> SEQUENCE:
26 Asp Val Cys Gln Asp Cys Ile 1 5 <210> SEQ ID NO 27
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: D2-C7
anti-angiogenic polypeptide <400> SEQUENCE: 27 Asp Val Cys
Gln Asp Cys
1 5 <210> SEQ ID NO 28 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: D2-D6 anti-angiogenic polypeptide <400> SEQUENCE:
28 Asp Val Cys Gln Asp 1 5 <210> SEQ ID NO 29 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: V3-V11 anti-angiogenic
polypeptide <400> SEQUENCE: 29 Val Cys Gln Asp Cys Ile Gln
Met Val 1 5 <210> SEQ ID NO 30 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: C4-V11 anti-angiogenic polypeptide <400>
SEQUENCE: 30 Cys Gln Asp Cys Ile Gln Met Val 1 5 <210> SEQ ID
NO 31 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Q5-V11
anti-angiogenic polypeptide <400> SEQUENCE: 31 Gln Asp Cys
Ile Gln Met Val 1 5 <210> SEQ ID NO 32 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: G1-(S4)-V11 anti-angiogenic polypeptide
<400> SEQUENCE: 32 Gly Asp Val Ser Gln Asp Cys Ile Gln Met
Val 1 5 10 <210> SEQ ID NO 33 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: G1-(S7)-V11 anti-angiogenic polypeptide
<400> SEQUENCE: 33 Gly Asp Val Cys Gln Asp Ser Ile Gln Met
Val 1 5 10 <210> SEQ ID NO 34 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: G1-(S4)-D6 anti-angiogenic polypeptide
<400> SEQUENCE: 34 Gly Asp Val Ser Gln Asp 1 5 <210>
SEQ ID NO 35 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
D2-(A3)-D6 anti-angiogenic polypeptide <400> SEQUENCE: 35 Asp
Ala Cys Gln Asp 1 5 <210> SEQ ID NO 36 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: D2-(I3)-D6 anti-angiogenic polypeptide
<400> SEQUENCE: 36 Asp Ile Cys Gln Asp 1 5 <210> SEQ ID
NO 37 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: D2-(L3)-D6
anti-angiogenic polypeptide <400> SEQUENCE: 37 Asp Leu Cys
Gln Asp 1 5 <210> SEQ ID NO 38 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: D2-(S5)-D6 anti-angiogenic polypeptide
<400> SEQUENCE: 38 Asp Val Cys Ser Asp 1 5 <210> SEQ ID
NO 39 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: D2-(E5)-D6
anti-angiogenic polypeptide <400> SEQUENCE: 39 Asp Val Cys
Glu Asp 1 5 <210> SEQ ID NO 40 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: D2-(D5)-D6 anti-angiogenic polypeptide
<400> SEQUENCE: 40 Asp Val Cys Asp Asp 1 5 <210> SEQ ID
NO 41 <211> LENGTH: 16 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Q67-E80
anti-angiogenic polypeptide <400> SEQUENCE: 41 Gln Pro Lys
Glu Ile Cys Ala Leu Val Gly Phe Cys Asp Glu Val Lys 1 5 10 15
<210> SEQ ID NO 42 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
C37-S53 anti-angiogenic polypeptide <400> SEQUENCE: 42 Cys
Asp Arg Leu Gly Pro Gly Met Ala Lys Ile Cys Lys Asn Tyr Ile 1 5 10
15 Ser <210> SEQ ID NO 43 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Q9-F15 anti-angiogenic polypeptide <400> SEQUENCE:
43 Gln Met Val Thr Asp Ile Gln Thr Gln Val Arg Thr Asn Ser Thr Phe
1 5 10 15 <210> SEQ ID NO 44 <211> LENGTH: 70
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(6) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-6
may be present or absent <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (12)..(70) <223> OTHER
INFORMATION: Xaa = any amino acid, Xaa at positions 12-70 may be
present or absent <400> SEQUENCE: 44 Xaa Xaa Xaa Xaa Xaa Xaa
Asp Val Cys Gln Asp Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50
55 60 Xaa Xaa Xaa Xaa Xaa Xaa 65 70 <210> SEQ ID NO 45
<211> LENGTH: 70 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: anti-angiogenic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1)..(5) <223> OTHER INFORMATION: Xaa =
any amino acid, Xaa at positions 1-5 may range from 1-5 residues
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (6) <223> OTHER INFORMATION: Xaa = Gly, Ala, Ser or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12)..(70) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 12-70 may be present or absent <400>
SEQUENCE: 45 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa
Xaa 65 70 <210> SEQ ID NO 46 <211> LENGTH: 70
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6)
<223> OTHER INFORMATION: Xaa = variable amino acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12)..(70) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 12-70 may be present or absent <400>
SEQUENCE: 46 Gln Pro Lys Asp Asn Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa
Xaa 65 70 <210> SEQ ID NO 47 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(6) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-6 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(16) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 13-16 may be present or absent <400> SEQUENCE:
47 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa Xaa Xaa Xaa
1 5 10 15 <210> SEQ ID NO 48 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(6) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-6 may be present or absent <400> SEQUENCE: 48
Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Cys Ile Gln Met Val 1 5
10 15 <210> SEQ ID NO 49 <211> LENGTH: 70 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: anti-angiogenic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (2)..(5)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6)
<223> OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12)..(70) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 12-70 may be present or absent <400>
SEQUENCE: 49 Gln Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa
Xaa 65 70 <210> SEQ ID NO 50 <211> LENGTH: 70
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
position 1 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (3)..(5)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6)
<223> OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12)..(70) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 12-70 may be present or absent <400>
SEQUENCE: 50 Xaa Pro Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa
Xaa 65 70 <210> SEQ ID NO 51 <211> LENGTH: 70
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(2) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1 and 2 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: Xaa = any amino acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (6) <223> OTHER INFORMATION: Xaa = Gly, Ala, Ser or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12)..(70) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 12-70 may be present or absent <400>
SEQUENCE: 51 Xaa Xaa Lys Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa
Xaa 65 70 <210> SEQ ID NO 52 <211> LENGTH: 70
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(3) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-3 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)..(70)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
positions 12-70 maybe present or absent <400> SEQUENCE: 52
Xaa Xaa Xaa Asp Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa 65 70
<210> SEQ ID NO 53 <211> LENGTH: 70 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
anti-angiogenic polypeptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1)..(4) <223> OTHER
INFORMATION: Xaa = any amino acid, Xaa at positions 1-4 may be
present or absent <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION:
Xaa = Gly, Ala, Ser or Thr <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (12)..(70) <223>
OTHER INFORMATION: Xaa = any amino acid, Xaa at positions 12-70 may
be present or absent <400> SEQUENCE: 53 Xaa Xaa Xaa Xaa Asn
Xaa Asp Val Cys Gln Asp Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40
45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa 65 70 <210> SEQ ID NO 54
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: anti-angiogenic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1)..(4) <223> OTHER INFORMATION: Xaa =
any amino acid, Xaa at positions 1-4 may be present or absent
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (5) <223> OTHER INFORMATION: Xaa = any amino acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (6) <223> OTHER INFORMATION: Xaa = Gly, Ala, Ser or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12) <223> OTHER INFORMATION: Xaa = any amino acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(16) <223> OTHER INFORMATION: Xaa = any amino
acid, Xaa at positions 13-16 may be present or absent <400>
SEQUENCE: 54 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 <210> SEQ ID NO 55 <211> LENGTH:
16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(4) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-4 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (13)..(16)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
positions 13-16 may be present or absent <400> SEQUENCE: 55
Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Cys Xaa Xaa Xaa Xaa 1 5
10 15 <210> SEQ ID NO 56 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: anti-angiogenic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(4)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
positions 1-4 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(14)..(16) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 14-16 may be present or absent <400> SEQUENCE:
56 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Ile Xaa Xaa Xaa
1 5 10 15 <210> SEQ ID NO 57 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(4) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-4 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)..(13)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (15)..(16)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
positions 15 and 16 may be present or absent <400> SEQUENCE:
57 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa Gln Xaa Xaa
1 5 10 15 <210> SEQ ID NO 58 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: anti-angiogenic polypeptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(4) <223> OTHER INFORMATION: Xaa = any amino acid, Xaa
at positions 1-4 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)..(14)
<223> OTHER INFORMATION: Xaa = any amino acid <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (16)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
position 16 may be present or absent <400> SEQUENCE: 58 Xaa
Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa Xaa Met Xaa 1 5 10
15 <210> SEQ ID NO 59 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: anti-angiogenic polypeptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(4)
<223> OTHER INFORMATION: Xaa = any amino acid, Xaa at
positions 1-4 may be present or absent <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>
OTHER INFORMATION: Xaa = any amino acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>
OTHER INFORMATION: Xaa = Gly, Ala, Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (12)..(15)
<223> OTHER INFORMATION: Xaa = any amino acid <400>
SEQUENCE: 59 Xaa Xaa Xaa Xaa Xaa Xaa Asp Val Cys Gln Asp Xaa Xaa
Xaa Xaa Val 1 5 10 15 <210> SEQ ID NO 60 <211> LENGTH:
5 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Amino acids 1-5
of SEQ ID NO:13 <400> SEQUENCE: 60 Gln Pro Lys Asp Asn 1 5
<210> SEQ ID NO 61 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Amino acids 12-15 of SEQ ID NO:13
<400> SEQUENCE: 61 Cys Ile Gln Val 1 <210> SEQ ID NO 62
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Amino acids 7-11 of SEQ ID NO:19 <400> SEQUENCE:
62 Cys Ile Gln Met Val 1 5 <210> SEQ ID NO 63 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Amino
acids 6-9 of SEQ ID NO:24 <400> SEQUENCE: 63 Cys Ile Gln Met
1
* * * * *
References