Antiangiogenic peptides and methods for inhibiting angiogenesis

Mammalian kringle 5 peptide fragments are disclosed for treating angiogenic diseases Methods and compositions for inhibiting angiogenic diseases are also disclosed.

TECHNICAL FIELD 
The present invention relates to the field of peptide chemistry. More 
particularly, the invention relates to the preparation and use of peptides 
containing amino acid sequences substantially similiar to the 
corresponding sequences of the kringle 5 region of mammalian plasminogen, 
pharmaceutical compositions containing the peptides, antibodies specific 
for the angiostatin receptor, means for angiostatin detection and 
measurement, cytotoxic agents linked to angiostatin proteins and treatment 
of diseases which arise from or are exacerbated by angiogenesis. 
BACKGROUND OF THE INVENTION 
Angiogenesis, the process by which new blood vessels are formed, is 
essential for normal body activities including reproduction, development 
and wound repair. Although the process is not completely understood, it is 
believed to involve a complex interplay of molecules which regulate the 
growth of endothelial cells (the primary cells of capillary blood 
vessels). Under normal conditions, these molecules appear to maintain the 
microvasculature in a quiescent state (i.e. one of no capillary growth) 
for prolonged periods which may last for as long as weeks or, in some 
cases, decades. When necessary (such as during wound repair), these same 
cells can undergo rapid proliferation and turnover within a 5 day period 
(Folkman, J. and Shing, Y., The Journal of Biological Chemistry, 267 (16), 
10931-10934, and Folkman, J. and Klagsbrun, M., Science, 235, 442-447 
(1987). 
Although angiogenesis is a highly regulated process under normal 
conditions, many diseases (characterized as angiogenic diseases) are 
driven by persistent unregulated angiogenesis. Otherwise stated, 
unregulated angiogenesis may either cause a particular disease directly or 
exascerbate an existing pathological condition. For example, ocular 
neovacularization has been implicated as the most common cause of 
blindness and dominates approximately 20 eye diseases. In certain existing 
conditions, such as arthritis, newly formed capillary blood vessels invade 
the joints and destroy cartilage. In diabetes, new capillaries formed in 
the retina invade the vitreous, bleed, and cause blindness. Growth and 
metastasis of solid tumors are also dependent on angiogenesis (Folkman, 
J., Cancer Research, 46, 467-473 (1986), Folkman, J., Journal of the 
National Cancer Institute, 82, 4-6 (1989). It has been shown, for example, 
that tumors which enlarge to greater than 2 mm must obtain their own blood 
supply and do so by inducing the growth of new capillary blood vessels. 
Once these new blood vessels become embedded in the tumor, they provide a 
means for tumor cells to enter the circulation and metastasize to distant 
sites such as liver, lung or bone (Weidner, N., et al., The New England 
Journal of Medicine, 324 (1), 1-8 (1991). 
To date, several naturally occurring angiogenic factors have been described 
and characterized (Fidler, J. I. and Ellis, L. M., Cell, 79, 185-189 
(1994). Recently, O'Reilly, et al. have isolated and purified a 38 
kilodalton (kDa) protein from serum and urine of tumor-bearing mice that 
inhibits endothelial cell proliferation (O'Reilly, M. et al., Cell, 79, 
315-328 (1994) and International Application WO 95/29242, published Nov. 
2, 1995. Microsequence analysis of this endothelial inhibitor showed 98% 
sequence homology to an internal fragment of murine plasminogen. 
Angiostatin, as the murine inhibitory fragment was named, was a peptide 
which included the first four kringle regions of murine plasminogen. A 
peptide fragment from the same region of human plasminogen (i.e. 
containing kringles 1-4) also strongly inhibited proliferation of 
capillary endothelial cells in vitro and in vivo. The intact plasminogen 
from which this peptide fragment was derived did not possess as potent an 
inhibitory effect. 
Several angiogenesis inhibitors are currently under development for use in 
treating angiogenic diseases (Gasparini, G. and Harris, A. L., J. Clin. 
Oncol., 13 (3): 765-782, (1995), but there are disadvantages associated 
with these compounds. Suramin, for example, is a potent angiogenesis 
inhibitor but causes severe systemic toxicity in humans at doses required 
for antitumor activity. Compounds such as retinoids, interferons and 
antiestrogens are safe for human use but have weak antiangiogenic effects. 
Thus, there is a need for compounds useful in treating angiogenic diseases 
in mammals. More specifically, there is a need for angiogenesis inhibitors 
which are safe for therapeutic use and which exhibit selective toxicity 
with respect to the pathological condition such as by selectively 
inhibiting the proliferation of cancer cells while exhibiting no or a low 
degree of toxicity to normal (ie. non-cancerous) cells. Such compounds 
should also be easily and cost-effectively made. 
SUMMARY OF THE INVENTION 
In its principle embodiment, the present invention provides a kringle 5 
peptide fragment represented by the structural formula 
A-B-C-X-Y (I), or a pharmaceutically acceptible salt thereof, wherein 
A is absent or a nitrogen protecting group; 
Y is absent or a carboxylic acid protecting group; 
B is absent, a naturally-occuring amino acid residue or a peptide of 
between 2 and 197 amino acids (inclusive), the .alpha.-N-terminal 
optionally capped with A, the .alpha.-C-terminal optionally capped with Y 
and a substantial sequence homology to the corresponding amino acid 
sequence from Asp.sup.334 to Arg.sup.530 (inclusive) of SEQ ID NO: 1; 
C is absent or R.sup.1 -R.sup.2 -R.sup.3 -R.sup.4 wherein 
R.sup.1 is lysyl, 
R.sup.2 is selected from leucyl and arginyl, 
R.sup.3 is selected from tyrosyl, 3-I-tyrosyl and phenylalanyl and 
R.sup.4 is aspartyl, 
with the proviso that at least one of B or C is present, and 
X is absent, a naturally occuring amino acid residue or a peptide of 
between 2 and 11 amino acids (inclusive), the .alpha.-N-terminal 
optionally capped with A, the .alpha.-C-terminal optionally capped with Y 
and a substantial sequence homology to the corresponding amino acid 
sequence beginning at Tyr.sup.535 and ending at Phe.sup.546 of SEQ ID NO: 
1. 
The present invention also includes a method for treating a patient in need 
of antiangiogenesis therapy comprising adminstering to the patient a 
compound containing a kringle 5 peptide fragment. 
The present invention also includes a composition for treating a patient in 
need of anti-angiogenesis therapy comprising a compound containing a 
kringle 5 peptide fragment, kringle 5 antisera, kringle 5 receptor 
agonists and antagonists and kringle 5 antagonists linked to cytotoxic 
agents either alone or in combination with a pharmaceutically acceptible 
excipient and/or optionally sustained release compounds to form a 
therapeutic composition. 
The present invention also includes a composition for the treatment of a 
disease selected from the group consisting of cancer, arthritis, macular 
degeneration and diabetic retinopathy comprising a compound containing a 
kringle 5 peptide fragment. 
The present invention also includes a composition comprising an isolated 
single or double-stranded polynucleotide sequence that encodes a kringle 5 
peptide fragment or kringle 5 peptide fragment conjugate. Such a 
polynucleotide is preferably a DNA molecule. The present invention also 
includes a vector containing a DNA sequence encoding a kringle 5 peptide 
fragment or kringle 5 peptide fragment conjugate wherein the vector is 
capable of expressing a kringle 5 peptide fragment or kringle 5 peptide 
conjugate when present in a cell and a composition comprising a cell 
containing a vector wherein the vector contains a DNA sequence encoding a 
kringle 5 peptide fragment or kringle 5 peptide conjugate. The present 
invention further encompasses gene therapy methods whereby DNA sequences 
encoding a kringle 5 peptide fragment or kringle 5 peptide fragment 
conjugate are introduced into a patient to modify in vivo kringle 5 
levels. 
The present invention also includes a method of making a kringle 5 peptide 
fragment comprising the steps of: (a) exposing mammalian plasminogen to 
human or porcine elastase at a ratio of about 1:100 to about 1:300 to form 
a mixture of said plasminogen and said elastase; (b) incubating said 
mixture and (c) isolating the kringle 5 peptide fragment from said 
mixture. 
The present invention also includes a method of making a kringle 5 peptide 
fragment comprising the steps of: (a) exposing mammalian plasminogen to 
human or porcine elastase at an elastase:plasminogen ratio of about 1:100 
to about 1:300 to form a mixture of said elastase and said plasminogen; 
(b) incubating said mixture; and (c) isolating a protein conjugate of a 
kringle 5 peptide fragment from said mixture; (d) exposing said protein 
conjugate of the kringle 5 peptide fragment to pepsin at a ratio of about 
1:0.2 to form a mixture of said pepsin and said plasminogen and (d) 
isolating said kringle 5 peptide fragment from said mixture. 
The present invention also includes antibodies specific for the kringle 5 
binding site and methods for the production of antibodies specific for the 
kringle 5 binding site. Antibodies can be monoclonal and polyclonal and 
can be used in diagnostic kits for detection and measurement of kringle 5 
peptide fragment concentrations and for localization of kringle 5 proteins 
in tissues and cells. The antibodies specific for kringle 5 can be used in 
the diagnostic kits to detect the presence and quantity of kringle 5 
peptide fragments which are indicative of diseases caused or exacerbated 
by angiogenesis, to isolate pure kringle 5 peptide fragments from mixtures 
containing kringle 5 peptide fragments and to isolate the kringle 5 
receptor. 
The present invention also includes methods and kits for the detection and 
measurement of kringle 5 peptide fragments in body fluid or tissue. The 
diagnostic kit would be in any configuration well-known to those of 
ordinary skill in the art and would provide instructions and the necessary 
kringle 5 peptide fragments and antisera for the measurement of kringle 5 
peptide fragments in biological fluids and tissue extracts of animals and 
humans with and without tumors. 
The present invention also includes a method for preparing kringle 5 
peptide fragments or prodrugs of kringle 5 peptide fragments synthetically 
by standard methods of solid phase or solution phase chemistry or 
recombinant technology known to those of ordinary skill in the art. 
The present invention also includes kringle 5 peptide fragments which can 
be labeled isotopically or with other molecules or proteins for use in the 
detection and visualization of kringle 5 binding sites with techniques 
including, but not limited to, radioimmunoassays, competitive and 
noncompetitive assays, bioluminescence and enzyme-linked immunoabsorbent 
assays.

DETAILED DESCRIPTION OF THE INVENTION 
As used herein, the term "kringle 5" refers to the region of mammalian 
plasminogen having three disulfide bonds which contribute to the specific 
three-dimensional confirmation defined by the fifth kringle region of the 
mammalian plasminogen molecule. One such disulfide bond links the cysteine 
residues located at amino acid positions 462 and 541, a second links the 
cysteine residues located at amino acid positions 483 and 524 and a third 
links the cysteine residues located at amino acid positions 512 and 536. 
The amino acid sequence of a complete mammalian plasminogen molecule (the 
human plasminogen molecule), including its kringle 5 region, is shown in 
FIG. 1 (SEQ ID NO: 1). 
As used herein, the term "kringle 5 peptide fragment" refers to a peptide 
of between 4 and 104 amino acids (inclusive) with a substantial sequence 
homology to the corresponding peptide fragment of mammalian plasminogen, 
an .alpha.-N-terminus at about amino acid position 443 of intact mammalian 
plasminogen and an .alpha.-C-terminus at about position 546. The total 
length of the a kringle 5 peptide fragment may vary depending upon the 
manner in which the kringle 5 peptide is obtained or may vary somewhat in 
sequence depending upon the species from which it is obtained. For 
example, certain forms of kringle 5 peptide fragments may be produced by 
proteolytic cleavage of glu-plasminogen, lys-plasminogen or 
miniplasminogen using the enzymes human or porcine elastase. When produced 
in this manner, the .alpha.-C-terminal of the peptide resides at about 
amino acid 543 of SEQ ID NO: 1, but the .alpha.-N-terminal amino acid may 
begin at amino acid position 443, 449 or 454. Thus, a kringle 5 peptide 
fragment resulting from human or porcine elastase digestion of 
glu-plasminogen, lys-plasminogen or miniplasminogen may have a total 
length of either 101 (SEQ ID NO: 2), 95 (SEQ ID NO: 3) or 90 (SEQ ID NO: 
4) amino acids. A summary of these kringle 5 peptide fragments is shown in 
Table 1. When produced in the aformentioned manner, a pool of these three 
fragments is obtained wherein about 60% of the fragments have a length of 
95 amino acids, about 35% of the fragments have the length of 101 amino 
acids and about 5% of the fragments have a length of 90 amino acids. If 
desired, these various fragments may be further purified by reverse phase 
HPLC, a technique well-known to those skilled in the art. 
Alternatively, kringle 5 peptide fragments or kringle 5 peptide fragments 
bound to protein conjugates may be obtained by expression of a recombinant 
molecule comprising a polynucleotide having a sequence which encodes 
proteins having kringle 5 peptide fragment with an .alpha.-N terminal at 
amino acid position 443, 449 or 454 (preferably at position 443) and an 
.alpha.-C-terminal at amino acid position 543 and 546 (preferably at 
position 543) and then purifying the peptide product which is expressed 
(see Menhart, N., et al, Biochemistry, 32: 8799-8806 (1993). The DNA 
sequence of human plasminogen has been published (Browne, M. J. et al. 
Fibrinolysis, 5 (4): 257-260 (1991) and is shown in FIGS. 6(a-b) (SEQ ID 
NO: 12). A polynucleotide sequence encoding kringle 5 begins at about 
nucleotide position 1421 of SEQ ID NO: 12 and ends at about nucleotide 
position 1723. This method of making a kringle 5 peptide fragment employs 
conventional techniques of molecular biology, microbiology, recombinant 
DNA and immunology, all of which are within the skill of the art and fully 
explained in the literature. (See for example, "Molecular Cloning: A 
Laboratory Manual" Second Edition by Sambrook et al., Cold Spring Harbor 
Press, 1989. For example, the gene for a kringle 5 peptide fragment may be 
isolated from cells or tissues that express high levels of kringle 5 
peptide fragments by (1) isolating messenger RNA from the tissue or cells, 
(2) using reverse transcriptase to generate the corresponding DNA sequence 
and (3) using the polymerase chain reaction (PCR) with the appropriate 
primers to amplify the DNA sequence coding for the active kringle 5 amino 
acid sequence. Furthermore, a polynucleotide encoding a kringle 5 peptide 
fragment may be cloned into any commercially available expression vector 
(such as pBR322, pUC vectors and the like) or expression/purification 
vectors (such as a GST fusion vector (Pharmacia, Piscataway, N.J.)) and 
then expressed in a suitable procaryotic, viral or eucaryotic host. 
Purification may then be achieved by conventional means or, in the case of 
a commercial expression/purification system, in accordance with 
manufacturer's instructions. 
Kringle 5 peptide fragments may also be synthesized by standard methods of 
solid phase chemistry known to those of ordinary skill in the art For 
example kringle 5 peptide fragments may be synthesized by solid phase 
chemistry techniques following the procedures described by Steward and 
Young (Steward, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd 
Ed., Pierce Chemical Company, Rockford, Ill., (1984) using an Applied 
Biosystem synthesizer. Similarly, multiple fragments may be synthesized 
then linked together to form larger fragments. These synthetic peptide 
fragments can also be made with amino acid substitutions at specific 
locations to test for kringle 5 fragment-like activity in vitro and in 
vivo. For solid phase peptide synthesis, a summary of the many techniques 
may be found in J. M. Stewart and J. D. Young, Solid Phase Peptide 
Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J. Meienhofer, 
Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 
1973. For classical solution synthesis see G. Schroder and K. Lupke, The 
Peptides, Vol. 1, Academic Press (New York). In general, these methods 
comprise the sequential addition of one or more amino acids or suitably 
protected amino acids to a growing peptide chain. Normally, either the 
amino or carboxyl group of the first amino acid is protected by a suitable 
protecting group. The protected or derivatized amino acid is then either 
attached to an inert solid support or utilized in solution by adding the 
next amino acid in the sequence having the complimentary (amino or 
carboxyl) group suitably protected and under conditions suitable for 
forming the amide linkage. The protecting group is then removed from this 
newly added amino acid residue and the next amino acid (suitably 
protected) is added, and so forth. After all the desired amino acids have 
been linked in the proper sequence, any remaining protecting groups (and 
any solid support) are removed sequentially or concurrently to afford the 
final polypeptide. By simple modification of this general procedure, it is 
possible to add more than one amino acid at a time to a growing chain, for 
example, by coupling (under conditions which do not racemize chiral 
centers) a protected tripeptide with a properly protected dipeptide to 
form, after deprotection, a pentapeptide. 
A particularly preferred method of preparing compounds of the present 
invention involves solid phase peptide synthesis wherein the amino acid 
.alpha.-N-terminal is protected by an acid or base sensitive group. Such 
protecting groups should have the properties of being stable to the 
conditions of peptide linkage formation while being readily removable 
without destruction of the growing peptide chain or racemization of any of 
the chiral centers contained therein. Suitable protecting groups are 
9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), 
benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, 
isobornyloxycarbonyl, 
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl, 
o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like. The 
9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularly 
preferred for the synthesis of kringle 5 peptide fragments. Other 
preferred side chain protecting groups are, for side chain amino groups 
like lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), 
nitro, p-toluenesulfonyl, 4-methoxybenzene- sulfonyl, Cbz, Boc, and 
adamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy-carbonyl, 
2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl and 
acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; for 
histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; 
for tryptophan, formyl; for asparticacid and glutamic acid, benzyl and 
t-butyl and for cysteine, triphenylmethyl (trityl). In the solid phase 
peptide synthesis method, the .alpha.-C-terminal amino acid is attached to 
a suitable solid support or resin. Suitable solid supports useful for the 
above synthesis are those materials which are inert to the reagents and 
reaction conditions of the stepwise condensation-deprotection reactions, 
as well as being insoluble in the media used. The preferred solid support 
for synthesis of .alpha.-C-terminal carboxy peptides is 
4-hydroxymethylphenoxymethyl-copoly(styrene-1% divinylbenzene). The 
preferred solid support for .alpha.-C-terminal amide peptides is the 
4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin 
available from Applied Biosystems (Foster City, Calif.). The 
.alpha.-C-terminal amino acid is coupled to the resin by means of 
N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) or 
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate 
(HBTU), with or without 4-dimethylaminopyridine (DMAP), 
1-hydroxybenzotriazole (HOBT), 
benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate 
(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), mediated 
coupling for from about 1 to about 24 hours at a temperature of between 
10.degree. and 50.degree. C. in a solvent such as dichloromethane or DMF. 
When the solid support is 
4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, 
the Fmoc group is cleaved with a secondary amine, preferably piperidine, 
prior to coupling with the .alpha.-C-terminal amino acid as described 
above. The preferred method for coupling to the deprotected 4 
(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is is 
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU, 
1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. The coupling 
of successive protected amino acids can be carried out in an automatic 
polypeptide synthesizer as is well known in the art. In a preferred 
embodiment, the .alpha.-N-terminal in the amino acids of the growing 
peptide chain are protected with Fmoc. The removal of the Fmoc protecting 
group from the .alpha.-N-terminal side of the growing peptide is 
accomplished by treatment with a secondary amine, preferably piperidine. 
Each protected amino acid is then introduced in about 3-fold molar excess, 
and the coupling is preferably carried out in DMF. The coupling agent is 
normally 
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU, 
1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.). At the end of the 
solid phase synthesis, the polypeptide is removed from the resin and 
deprotected, either in successively or in a single operation. Removal of 
the polypeptide and deprotection can be accomplished in a single operation 
by treating the resin-bound polypeptide with a cleavage reagent comprising 
thianisole, water, ethanedithiol and trifluoroacetic acid. In cases 
wherein the .alpha.-C-terminal of the polypeptide is an alkylamide, the 
resin is cleaved by aminolysis with an alkylamine. Alternatively, the 
peptide may be removed by transesterification, e.g. with methanol, 
followed by aminolysis or by direct transamidation. The protected peptide 
may be purified at this point or taken to the next step directly. The 
removal of the side chain protecting groups is accomplished using the 
cleavage cocktail described above. The fully deprotected peptide is 
purified by a sequence of chromatographic steps employing any or all of 
the following types: ion exchange on a weakly basic resin (acetate form); 
hydrophobic adsorption chromatography on underivitized 
polystyrene-divinylbenzene (for example, Amberlite XAD); silica gel 
adsorption chromatography; ion exchange chromatography on 
carboxymethylcellulose; partition chromatography, e.g. on Sephadex G-25, 
LH-20 or countercurrent distribution; high performance liquid 
chromatography (HPLC), especially reverse-phase HPLC on octyl- or 
octadecylsilyl-silica bonded phase column packing. Molecular weights of 
these kringle 5 peptide fragments are determined using Fast Atom 
Bombardment (FAB) Mass Spectroscopy. Solid phase kringle 5 peptide 
fragment synthesis is illustrated in Examples 1 to 12. 
Depending on how they are produced, kringle 5 peptide fragments may exist 
with or without the aformentioned disulfide bonds of the kringle 5 region 
of mammalian plasminogen or may exist with disulfide bonds forming a 
tertiary structure which differs from the tertiary structure found in 
native mammalian plasminogen (for example, disulfide bonds between 
Cys.sup.462 and Cys.sup.483, between Cys.sup.512 and and between 
Cys.sup.524 and Cys.sup.536 and Cys.sup.541. Kringle 5 peptide fragments 
produced by enzymatic cleavage of Glu-, Lys- or miniplasminogen with 
elastase and/or pepsin (enzymes which cleave at sites removed from the 
cysteine linkages) will contain the native tertiary kringle 5 protein 
structure; kringle 5 peptide fragments prepared by solid phase peptide 
synthesis may or may not contain cystyl amino acyl residues and kringle 5 
peptide fragments prepared by expression may contain disulfide bonds at 
different positions than those found in kringle 5 peptide fragments 
produced by enzymatic cleavage. 
As used herein, the term "conjugate of a kringle 5 peptide fragment" means 
a kringle 5 peptide fragment chemically coupled to another protein to form 
a conjugate. Examples of conjugates of kringle 5 peptide fragments include 
a kringle 5 peptide fragment coupled to albumin or to a peptide fragment 
from another kringle region of mammalian plasminogen. Molecular weights of 
conjugates of kringle 5 peptide fragments are between about 1,000 and 
about 25,000 kDa. 
As used herein, the term "substantial sequence homology" means 
approximately 60% amino acid identity, desirably at least approximately 
70% amino acid identity, more desirably approximately 80% amino acid 
identity and most desirably approximately 95% amino acid identity of the 
corresponding peptide sequence of human plasminogen. Because the amino 
acid sequence or the number of amino acids in a kringle 5 peptide fragment 
may vary from species to species or from the method of production, the 
total number of amino acids in a kringle 5 peptide fragment cannot, in 
some instances, be defined exactly. Given that these sequences are 
identical in at least 73% of their amino acids, it is to be understood 
that the amino acid sequence of a kringle 5 peptide fragment is 
substantially similar among species and that methods of production of 
kringle 5 peptide fragments provide kringle 5 peptide fragments with 
substantial sequence homology to the corresponding amino acid sequences of 
human plasminogen. FIG. 2 shows the amino acid sequence of a human kringle 
5 peptide fragment having 95 amino acids (SEQ ID NO: 2) is in comparison 
with the sequences of kringle 5 fragments from murine (SEQ ID NO: 8), 
Rhesus monkey (SEQ ID NO: 9), bovine (SEQ ID NO: 10) and porcine (SEQ ID 
NO: 11) plasminogen. 
Thus, the present invention contemplates amino acid residue sequences that 
have substantial sequence homology to the sequences set forth herein such 
that those sequences demonstrate like biological activity to disclosed 
kringle 5 peptide fragment sequences. It is well known in the art that 
modifications and changes can be made without substantially altering the 
biological function of that peptide. For example, alterations to kringle 5 
peptide fragments may enhance the peptide's potency or stability to 
enzymatic breakdown. Such contemplated sequences include those analogous 
sequences characterized by a change in amino acid residue sequence or type 
wherein the change does not alter the fundamental nature and biological 
activity of the aforementioned kringle 5 peptide fragments. 
A kringle 5 peptide fragment of the present invention may be characterized 
on the basis of potency when tested for its ability to inhibit the growth 
of bovine capillary (BCE) cells in vitro. The data in Table 1 illustrate 
that the kringle 5 peptide fragment SEQ ID NO: 3 has a 100-fold increase 
in activity (i.e. at inhibiting BCE cell proliferation) when compared to 
the kringle 5 peptide fragment SEQ ID NO: 6 and a 400-fold increase in 
activity when compared to kringle 14 peptide fragments. 
As used herein, the term ".alpha.-N-terminal" refers to the free 
alpha-amino group of an amino acid in a peptide, and the term 
".alpha.-C-terminal" refers to the free alpha-carboxylic acid terminus of 
an amino acid in a peptide. 
All peptide sequences are written according to the generally accepted 
convention whereby the .alpha.-N-terminal amino acid residue is on the 
left and the .alpha.-C-terminal is on the right. 
As used herein, the term "N-protecting group" refers to those groups 
intended to protect the .alpha.-N-terminal of an amino acid or peptide or 
to otherwise protect the amino group of an amino acid or peptide against 
undesirable reactions during synthetic procedures. Commonly used 
N-protecting groups are disclosed in Greene, "Protective Groups In Organic 
Synthesis," (John Wiley & Sons, New York (1981)), which is hereby 
incorporated by reference. Additionally, protecting groups can be used as 
prodrugs which are readily cleaved in vivo, for example, by enzymatic 
hydrolysis, to release the biologically active parent. N-protecting groups 
comprise loweralkanoyl groups such as formyl, acetyl ("Ac"), propionyl, 
pivaloyl, t-butylacetyl and the like; other acyl groups include 
2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, 
o-nitrophenoxyacetyl, .alpha.-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 
4-bromobenzoyl, 4-nitrobenzoyl and the like; sulfonyl groups such as 
benzenesulfonyl, p-toluenesulfonyl and the like; carbamate forming groups 
such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, 
p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 
2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 
3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 
2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 
2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 
1-(p-biphenylyl)-1-methylethoxycarbonyl, 
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl, 
benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, 
isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 
2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, 
fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, 
cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; arylalkyl groups 
such as benzyl, triphenylmethyl, benzyloxymethyl, 
9-fluorenylmethyloxycarbonyl (Fmoc) and the like and silyl groups such as 
trimethylsilyl and the like. Preferred N-protecting groups are formyl, 
acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, 
t-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz). For example, lysine 
may be protected at the .alpha.-N-terminal by an acid labile group (e.g. 
Boc) and protected at the .epsilon.-N-terminal by a base labile group 
(e.g. Fmoc) then deprotected selectively during synthesis. 
As used herein, the term "carboxy protecting group" refers to a carboxylic 
acid protecting ester or amide group employed to block or protect the 
carboxylic acid functionality while the reactions involving other 
functional sites of the compound are performed. Carboxy protecting groups 
are disclosed in Greene, "Protective Groups in Organic Synthesis" pp. 
152-186 (1981), which is hereby incorporated by reference. Additionally, a 
carboxy protecting group can be used as a prodrug whereby the carboxy 
protecting group can be readily cleaved in vivo , for example by enzymatic 
hydrolysis, to release the biologically active parent Such carboxy 
protecting groups are well known to those skilled in the art, having been 
extensively used in the protection of carboxyl groups in the penicillin 
and cephalosporin fields as described in U.S. Pat. Nos. 3,840,556 and 
3,719,667, the disclosures of which are hereby incorporated herein by 
reference. Representative carboxy protecting groups are C.sub.1 -C.sub.8 
loweralkyl (e.g., methyl, ethyl or t-butyl and the like); arylalkyl such 
as phenethyl or benzyl and substituted derivatives thereof such as 
alkoxybenzyl or nitrobenzyl groups and the like; arylalkenyl such as 
phenylethenyl and the like; aryl and substituted derivatives thereof such 
as 5-indanyl and the like; dialkylaminoalkyl such as dimethylaminoethyl 
and the like); alkanoyloxyalkyl groups such as acetoxymethyl, 
butyryloxymethyl, valeryloxymethyl, isobutyryloxymethyl, 
isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl, 1-(pivaloyloxyl)-1-ethyl, 
1-methyl-1-(propionyloxy)-1-ethyl, pivaloyloxymethyl, propionyloxymethyl 
and the like; cycloalkanoyloxyalkyl groups such as 
cyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl, 
cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl and the like; 
aroyloxyalkyl such as benzoyloxymethyl, benzoyloxyethyl and the like; 
arylalkylcarbonyloxyalkyl such as benzylcarbonyloxymethyl, 
2-benzylcarbonyloxyethyl and the like; alkoxycarbonylalkyl or 
cycloalkyloxycarbonylalkyl such as methoxycarbonylmethyl, 
cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl and the like; 
alkoxycarbonyloxyalkyl or cycloalkyloxycarbonyloxyalkyl such as 
methoxycarbonyloxymethyl, t-butyloxycarbonyloxymethyl, 
1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl and the 
like; aryloxycarbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl, 
2-(5-indanyloxycarbonyloxy)ethyl and the like; alkoxyalkylcarbonyloxyalkyl 
such as 2-(1-methoxy-2-methylpropan-2-oyloxy)ethyl and like; 
arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl and the 
like; arylalkenyloxycarbonyloxyalkyl such as 
2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl and the like; 
alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and the 
like; alkylaminocarbonylaminoalkyl such as methylaminocarbonylaminomethyl 
and the like; alkanoylaminoalkyl such as acetylaminomethyl and the like; 
heterocycliccarbonyloxyalkyl such as 4-methylpiperazinylcarbonyloxymethyl 
and the like; dialkylaminocarbonylalkyl such as 
dimethylaminocarbonylmethyl, diethylaminocarbonylmethyl and the like; 
(5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as 
(5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like; and 
(5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as 
(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like. 
Representative amide carboxy protecting groups are aminocarbonyl and 
loweralkylaminocarbonyl groups. 
Preferred carboxy-protected compounds of the invention are compounds 
wherein the protected carboxy group is a loweralkyl, cycloalkyl or 
arylalkyl ester, for example, methyl ester, ethyl ester, propyl ester, 
isopropyl ester, butyl ester, sec-butyl ester, isobutyl ester, amyl ester, 
isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl ester and the 
like or an alkanoyloxyalkyl, cycloalkanoyloxyalkyl, aroyloxyalkyl or an 
arylalkylcarbonyloxyalkyl ester. Preferred amide carboxy protecting groups 
are loweralkylaminocarbonyl groups. For example, aspartic acid may be 
protected at the .alpha.-C-terminal by an acid labile group (e.g. t-butyl) 
and protected at the .beta.-C-terminal by a hydrogenation labile group 
(e.g. benzyl) then deprotected selectively during synthesis. 
As used herein, the term "loweralkylaminocarbonyl" means a --C(O)NHR.sup.10 
group which caps the .alpha.-C-terminal of a synthetic, kringle 5 peptide 
fragment wherein R.sup.10 is C.sub.1 -C.sub.4 alkyl. 
As used herein, the term "aminocarbonyl" indicates a --C(O)NH.sub.2 group 
which caps the .alpha.-C-terminal of a synthetic, kringle 5 peptide 
fragment. 
As used herein, the term "prodrug" refers to compounds which are rapidly 
transformed in vivo to yield the parent compound, for example, by 
enzymatic hydrolysis in blood. A thorough discussion is provided in T. 
Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the 
A.C.S. Symposium Series and in Edward B. Roche, ed., Bioreversible 
Carriers in Drug Design, American Pharmaceutical Association and Permagon 
Press, 1987. 
As used herein, the term "pharmaceutically acceptible prodrug" refers to 
(1) those prodrugs of the compounds of the present invention which are, 
within the scope of sound medical judgement, suitable for use in contact 
with the tissues of humans and lower animals without undue toxicity, 
irritation, allergic response and the like, commensurate with a suitable 
benefit-to-risk ratio and effective for their intended use and (2) 
zwitterionic forms, where possible, of the parent compound. 
As used herein, the term "antiangiogenesis activity" refers to the 
capability of a molecule to inhibit the growth of blood vessels. 
As used herein, the term "endothelial inhibiting activity" refers to the 
capability of a molecule to inhibit angiogenesis in general and, for 
example, to inhibit the growth or migration of bovine capillary 
endothelial cells in culture in the presence of fibroblast growth factor 
or other known growth factors. 
As used herein, the term "ED.sub.50 " is an abbreviation for the dose of a 
kringle 5 peptide fragment which is effective to inhibit the growth of 
blood vessels or inhibit the growth of bovine capillary endothelial cells 
in culture in the presence of fibroblast growth factor or other known 
growth factors or inhibit the migration of endeothelial cells by one-half 
of what the growth or migration would be in the absence of the inhibitor. 
As used herein, for the most part, the names of naturally-occuring amino 
acids and aminoacyl residues used herein follow the naming conventions 
suggested by the IU Commission on the Nomenclature of Organic Chemistry 
and the IU-IUB Commission on Biochemical Nomenclature as set out in 
Nomenclature of .alpha.-Amino Acids (Recommendations, 1974), Biochemistry, 
14 (2), (1975). Accordingly, the terms "Ala," "Arg," "Asn," "Asp," "Cys," 
"Gln," "Glu," "Gly," "His," "Ile," "Leu," "Lys," "Met," "Phe," "Pro," 
"Ser," "Thr," "Trp," "Tyr" and "Val" refer to the amino acids alanine, 
arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, 
glycine, histidine, isoleucine, leucine, lysine, methionine, 
phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine 
and their corresponding aminoacyl residues in peptides in their L-, D- or 
D, L- forms. Where no specific configuration is indicated, one skilled in 
the art would understand that the stereochemistry of the .alpha.-carbon of 
the amino acids and aminoacyl residues in peptides described in this 
specification and the appended claims is the naturally occuring or "L" 
configuration with the exception of the achiral molecule glycine and with 
the further exception of any amino acids which are achiral or otherwise 
designated as "D-." 
As used herein, the term "3-I-Tyr" means a L-, D-, or D, L-tyrosyl residue 
wherein a hydrogen radical ortho to the phenolic hydroxyl is replaced by 
an iodide radical. The iodide radical may be radioactive or 
nonradioactive. 
The present invention also contemplates amino acid residues with 
non-naturally occuring side chain residues such as homophenylalanine, 
phenylglycine, norvaline, norleucine, ornithine, thiazoylalanine (2-, 4- 
and 5- substituted) and the like. 
Thus, it is to be understood that the present invention is contemplated to 
encompass any derivatives of kringle 5 peptide fragments which have 
antiangiogenic activity and includes the entire class of kringle 5 peptide 
fragments described herein and derivatives of those kringle 5 peptide 
fragments. Additionally, the invention is not dependent on the manner in 
which the kringle 5 peptide fragment is produced, i.e. by (1) proteolytic 
cleavage of an isolated mammalian plasminogen, (2) by expression of a 
recombinant molecule having a polynucleotide which encodes the amino acid 
sequence of a kringle 5 peptide fragment or a conjugate containing a 
kringle 5 peptide fragment and (3) solid phase synthetic techniques known 
to those of ordinary skill in the art. 
In one embodiment, the present invention provides peptides with the general 
structure B-C-X wherein B is a 88-mer peptide beginning at Val.sup.443 and 
ending at Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and R.sup.4 are 
previously defined, R.sup.2 is leucyl; R.sup.3 is tyrosyl and X is a 9-mer 
peptide beginning at Tyr.sup.535 and ending at Ala.sup.543 of SEQ ID NO: 1 
(SEQ ID NO: 2). 
In another embodiment, the present invention provides peptides with the 
general structure B-C-X wherein B is a 82-mer peptide beginning at 
Val.sup.449 and ending at Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and 
R.sup.4 are previously defined; R.sup.2 is leucyl; R.sup.3 is tyrosyl and 
X is a 9-mer peptide beginning at Tyr.sup.535 and ending at Ala.sup.543 of 
SEQ ID NO: 1 (SEQ ID NO: 3). 
In yet another embodiment, the present invention provides peptides with the 
general structure B-C-X wherein B is a 77-mer peptide beginning at 
Val.sup.454 and ending at Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and 
R.sup.4 are previously defined; R.sup.2 is leucyl; R.sup.3 is tyrosyl and 
X is a 9-mer peptide beginning at Tyr.sup.535 and ending at Ala.sup.543 of 
SEQ ID NO: 1 (SEQ ID NO: 4). 
In yet another embodiment, the present invention provides peptides with the 
general structure B-C-X wherein B is a 88-mer peptide beginning at 
Val.sup.443 and ending at Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and 
R.sup.4 are previously defined; R.sup.2 is leucyl; R.sup.3 is tyrosyl and 
X is a 12-mer peptide beginning at Tyr.sup.535 and ending at Phe.sup.546 
of SEQ ID NO: 1 (SEQ ID NO: 5). 
In yet another embodiment, the present invention provides peptides with the 
general structure structure B-C-X wherein B is a 82-mer peptide beginning 
at Val.sup.449 and ending at Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and 
R.sup.4 are previously defined; R.sup.2 is leucyl; R.sup.3 is tyrosyl and 
X is a 12-mer peptide beginning at Tyr.sup.535 and ending at Phe.sup.546 
of SEQ ID NO: 1 (SEQ ID NO: 6). 
In yet another embodiment, the present invention provides peptides with the 
general structure B-C-X wherein B is a 77-mer peptide beginning at 
Val.sup.454 and ending at Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and 
R.sup.4 are previously defined; R.sup.2 is leucyl, R.sup.3 is tyrosyl and 
X is a 12-mer peptide beginning at Tyr.sup.535 and ending at Phe.sup.546 
of SEQ ID NO: 1 (SEQ ID NO: 7). 
In yet another embodiment, the present invention provides peptides with the 
general structure B-C-X wherein B is a 176-mer peptide beginning at 
Val.sup.355 and ending at Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and 
R.sup.4 are previously defined; R.sup.2 is leucyl, R.sup.3 is tyrosyl and 
X is a 12-mer peptide beginning at Tyr.sup.535 and ending at Ala.sup.543 
of SEQ ID NO: 1. 
In yet another embodiment, the present invention provides peptides with the 
general structure B-C-X wherein B is a 176-mer peptide beginning at 
Val.sup.355 and ending at Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and 
R.sup.4 are previously defined; R.sup.2 is leucyl, R.sup.3 is tyrosyl and 
X is a 12-mer peptide beginning at Tyr.sup.535 and ending at Phe.sup.546 
of SEQ ID NO: 1. 
In yet another embodiment, the present invention provides peptides with the 
general structure A-B-Y wherein A is acetyl, B is a 13-mer peptide 
beginning at amino acid Val.sup.449 and ending at Asp.sup.461 of SEQ ID 
NO: 1 and Y is aminocarbonyl. 
In yet another embodiment, the present invention provides peptides with the 
general structure A-B-Y wherein A is acetyl, B is a 20-mer peptide 
beginning at amino acid Met.sup.463 and ending at Pro.sup.482 of SEQ ID 
NO: 1 and Y is aminocarbonyl. 
In yet another embodiment, the present invention provides peptides with the 
general structure A-B-Y wherein A is acetyl, B is a 28-mer peptide 
beginning at amino acid Gln.sup.484 and ending at amino acid Tyr.sup.511 
of SEQ ID NO: 1 and Y is aminocarbonyl. 
In yet another embodiment, the present invention provides peptides with the 
general structure A-B-Y wherein A is acetyl; B is an 11-mer peptide 
beginning at amino acid position Arg.sup.513 and ending at amino acid 
position Trp.sup.523 of SEQ ID NO: 1 and Y is aminocarbonyl. 
In yet another embodiment, the present invention provides peptides with the 
general structure A-B-C-Y wherein A is acetyl; B is a dipeptide beginning 
at amino acid position Pro.sup.529 and ending at amino acid position 
Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and R.sup.4 are previously 
defined; R.sup.2 is leucyl; R.sup.3 is tyrosyl and Y is aminocarbonyl. 
In yet another embodiment, the present invention provides peptides with the 
general structure A-B-C-Y wherein A is acetyl; B is a dipeptide beginning 
at amino acid position Pro.sup.529 and ending at amino acid position 
Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and R.sup.4 are previously 
defined; R.sup.2 is leucyl; R.sup.3 is tyrosyl and Y is aminocarbonyl. 
In yet another embodiment, the present invention provides peptides with the 
general structure A-B-C-X-Y wherein A is acetyl; B is a hexapeptide 
beginning at amino acid position Tyr.sup.525 and ending at amino acid 
position Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and R.sup.4 are 
previously defined; R.sup.2 is leucyl, R.sup.3 is tyrosyl, X is tyrosyl 
and Y is aminocarbonyl. 
In yet another embodiment, the present invention provides peptides with the 
general structure A-B-C-X-Y wherein A is acetyl; B is arginyl; C, R.sup.1 
and R.sup.4 are previously defined; R.sup.2 is leucyl; R.sup.3 is tyrosyl; 
X is tyrosyl and Y is aminocarbonyl. 
In yet another embodiment, the present invention provides peptides with the 
general structure A-B-C-X-Y wherein A is acetyl, B is a dipeptide 
beginning at amino acid position Pro.sup.529 and ending at amino acid 
position Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and R.sup.4 are 
previously defined; R.sup.2 is leucyl; R.sup.3 is tyrosyl; X is 
3-I-tyrosyl and Y is aminocarbonyl. 
In yet another embodiment, the present invention provides peptides with the 
general structure A-B-C-X-Y wherein A is acetyl, B is a dipeptide 
beginning at amino acid position Pro.sup.529 and ending at amino acid 
position Arg.sup.530 of SEQ ID NO: 1; C, R.sup.1 and R.sup.4 are 
previously defined; R.sup.2 is leucyl; R.sup.3 is 3-I-tyrosyl; X is 
tyrosyl and Y is aminocarbonyl. 
In yet another embodiment, the present invention provides peptides with the 
general structure A-C-Y wherein A is acetyl; C, R.sup.1 and R.sup.4 are 
previously defined; R.sup.2 is leucyl, R.sup.3 is tyrosyl, X is tyrosyl 
and Y is aminocarbonyl. 
Representative compounds of formula (I) include: 
H.sub.2 N-Val.sup.443 -Ala.sup.543 -CO.sub.2 H of SEQ ID NO: 1 (SEQ ID NO: 
2) 
H.sub.2 N-Val.sup.449 -Ala.sup.543 -CO.sub.2 H of SEQ ID NO: 1 (SEQ ID NO: 
3) 
H.sub.2 N-Val.sup.454 -Ala.sup.543 -CO.sub.2 H of SEQ ID NO: 1 (SEQ ID NO: 
4) 
H.sub.2 N-Val.sup.443 -Phe.sup.546 -CO.sub.2 H of SEQ ID NO: 1 (SEQ ID NO: 
5) 
H.sub.2 N-Val.sup.449 -Phe.sup.546 -CO.sub.2 H of SEQ ID NO: 1 (SEQ ID NO: 
6) 
H.sub.2 N-Val.sup.454 -Phe.sup.546 -CO.sub.2 H of SEQ ID NO: 1 (SEQ ID NO: 
7) 
N-Ac-Val.sup.449 -Asp.sup.461 -NH.sub.2 of SEQ ID NO: 1 
N-Ac-Met.sup.463 -Pro.sup.482 -NH.sub.2 of SEQ ID NO: 1 
N-Ac-Gln.sup.484 -Tyr.sup.511 -NH.sub.2 of SEQ ID NO: 1 
N-Ac-Arg.sup.513 -Trp.sup.523 -NH.sub.2 of SEQ ID NO: 1 
N-Ac-Tyr.sup.525 -Tyr.sup.535 -NH.sub.2 of SEQ ID NO: 1 
N-Ac-Pro.sup.529 -Tyr.sup.535 -NH.sub.2 of SEQ ID NO: 1 
N-Ac-Pro.sup.529 -Asp.sup.534 -NH.sub.2 of SEQ ID NO: 1 
N-Ac-Arg.sup.530 -Tyr.sup.535 -NH.sub.2 of SEQ ID NO: 1 
N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I-Tyr-NH.sub.2 (SEQ ID NO: 13) 
N-Ac-Pro-Arg-Lys-Leu-3-I-Tyr-Asp-Tyr-NH.sub.2 (SEQ ID NO: 14) 
N-Ac-H-Lys.sup.531 -Asp.sup.534 -NH.sub.2 of SEQ ID NO: 1 
H.sub.2 N-Val.sup.355 -Ala.sup.543 -CO.sub.2 H of SEQ ID NO: 1 
H.sub.2 N-Val.sup.355 -Phe.sup.546 -CO.sub.2 H of SEQ ID NO: 1 
The compounds of the invention, including but not limited to those 
specified in the examples, possess anti-angiogenic activity. As 
angiogenesis inhibitors, such compounds are useful in the treatment of 
both primary and metastatic solid tumors and carcinomas of the breast; 
colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach; 
pancreas; liver; gallbladder; bile ducts; small intestine; urinary tract 
including kidney, bladder and urothelium; female genital tract including 
cervix, uterus, ovaries, choriocarcinoma and gestational trophoblastic 
disease; male genital tract including prostate, seminal vesicles, testes 
and germ cell tumors; endocrine glands including thyroid, adrenal, and 
pituitary; skin including hemangiomas, melanomas, sarcomas arising from 
bone or soft tissues and Kaposi's sarcoma; tumors of the brain, nerves, 
eyes, and meninges including astrocytomas, gliomas, glioblastomas, 
retinoblastomas, neuromas, neuroblastomas, Schwannomas and meningiomas; 
solid tumors arising from hematopoietic malignancies such as leukemias and 
including chloromas, plasmacytomas, plaques and tumors of mycosis 
fungoides and cutaneous T-cell lymphoma/leukemia; lymphomas including both 
Hodgkin's and non-Hodgkin's lymphomas; prophylaxis of autoimmune diseases 
including rheumatoid, immune and degenerative arthritis; ocular diseases 
including diabetic retinopathy, retinopathy of prematurity, corneal graft 
rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis, 
retinal neovascularization due to macular degeneration and hypoxia; 
abnormal neovascularization conditions of the eye; skin diseases including 
psoriasis; blood vessel diseases including hemagiomas and capillary 
proliferation within atherosclerotic plaques; Osler-Webber Syndrome; 
myocardial angiogenesis; plaque neovascularization; telangiectasia; 
hemophiliac joints; angiofibroma; wound granulation; diseases 
characterized by excessive or abnormal stimulation of endothelial cells 
including intestinal adhesions, Crohn's disease, atherosclerosis, 
scleroderma and hypertrophic scars (i.e. keloids) and diseases which have 
angiogenesis as a pathologic consequence including cat scratch disease 
(Rochele minalia quintosa) and ulcers (Helicobacter pylori). Another use 
is as a birth control agent which inhibits ovulation and establishment of 
the placenta. 
The compounds of the present invention may also be useful for the 
prevention of metastases from the tumors described above either when used 
alone or in combination with radiotherapy and/or other chemotherapeutic 
treatments conventionally administered to patients for treating angiogenic 
diseases. For example, when used in the treatment of solid tumors, 
compounds of the present invention may be administered with 
chemotherapeutic agents such as alpha inteferon, COMP (cyclophosphamide, 
vincristine, methotrexate and prednisone), etoposide, mBACOD 
(methortrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and 
dexamethasone), PROMACE/MOPP (prednisone, methotrexate (w/leucovin 
rescue), doxorubicin, cyclophosphamide, taxol, etoposide/mechlorethamine, 
vincristine, prednisone and procarbazine), vincristine, vinblastine, 
angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4, 
angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG and the 
like. Other chemotherapeutic agents include alkylating agents such as 
nitrogen mustards including mechloethamine, melphan, chlorambucil, 
cyclophosphamide and ifosfamide; nitrosoureas including carmustine, 
lomustine, semustine and streptozocin; alkyl sulfonates including 
busulfan; triazines including dacarbazine; ethyenimines including thiotepa 
and hexamethylmelamine; folic acid analogs including methotrexate; 
pyrimidine analogues including 5-fluorouracil, cytosine arabinoside; 
purine analogs including 6-mercaptopurine and 6-thioguanine; antitumor 
antibiotics including actinomycin D; the anthracyclines including 
doxorubicin, bleomycin, mitomycin C and methramycin; hormones and hormone 
antagonists including tamoxifen and cortiosteroids and miscellaneous 
agents including cisplatin and brequinar. For example, a tumor may be 
treated conventionally with surgery, radiation or chemotherapy and kringle 
5 administration with subsequent kringle 5 adminsteration to extend the 
dormancy of micrometastases and to stabilize and inhibit the growth of any 
residual primary tumor. 
Cytotoxic agents such as ricin may be linked to kringle 5 peptide fragments 
and thereby provide a tool for destruction of cells that bind kringle 5. 
Peptides linked to cytotoxic agents may be infused in a manner designed to 
maximize delivery to the desired location. For example, ricin-linked high 
affinity kringle 5 fragments may be delivered via cannula directly into 
the target or into vessels supplying the target site. Such agents may also 
be delivered in a controlled manner through osmotic pumps coupled to 
infusion cannulae. A combination of kringle 5 antagonists may be 
co-applied with stimulators of angiogenesis to increase vascularization of 
tissue. Therapeutic regimens of this type could provide an effective means 
of destroying metastatic cancer. 
The compounds of the present invention may be used in the form of 
pharmaceutically acceptible salts derived from inorganic or organic acids. 
By "pharmaceutically acceptible salt" is meant those salts which are, 
within the scope of sound medical judgement, suitable for use in contact 
with the tissues of humans and lower animals without undue toxicity, 
irritation, allergic response and the like and are commensurate with a 
reasonable benefit/risk ratio. Pharmaceutically acceptible salts are 
well-known in the art. For example, S. M. Berge, et al. describe 
pharmaceutically acceptible salts in detail in J. Pharmaceutical Sciences, 
1977, 66: 1 et seq., which is hereby incorporated herein by reference. The 
salts may be prepared in situ during the final isolation and purification 
of the compounds of the invention or separately by reacting a free base 
function with a suitable organic acid. Representative acid addition salts 
include, but are not limited to acetate, adipate, alginate, citrate, 
aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, 
camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptanoate, 
hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 
2-hydroxyethansulfonate (isethionate), lactate, maleate, methanesulfonate, 
nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, 
persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, 
tartrate, thiocyanate, phosphate, glutamate, bicarbonate, 
p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing 
groups can be quarternized with such agents as lower alkyl halides such as 
methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl 
sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain 
halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides 
and iodides; arylalkyl halides like benzyl and phenethyl bromides and 
others. Water or oil-soluble or dispersible products are thereby obtained. 
Examples of acids which may be employed to form pharmaceutically 
acceptable acid addition salts include such inorganic acids as 
hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid 
and such organic acids as oxalic acid, maleic acid, succinic acid and 
citric acid. 
Basic addition salts can be prepared in situ during the final isolation and 
purification of kringle 5 peptide fragments by reacting a carboxylic 
acid-containing moiety with a suitable base such as the hydroxide, 
carbonate or bicarbonate of a pharmaceutically acceptible metal cation or 
with ammonia or an organic primary, secondary or tertiary amine. 
Pharmaceutically acceptible salts include, but are not limited to, cations 
based on alkali metals or alkaline earth metals such as lithium, sodium, 
potassium, calcium, magnesium and aluminum salts and the like and nontoxic 
quaternary ammonia and amine cations including ammonium, 
tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, 
trimethylamine, triethylamine, diethylamine, ethylamine and the like. 
Other representative organic anines useful for the formation of base 
addition salts include ethylenediamine, ethanolamine, diethanolamine, 
piperidine, piperazine and the like. Preferred salts of the compounds of 
the invention include phosphate, tris and acetate. 
Kringle 5 peptide fragments, kringle 5 antisera, kringle 5 receptor 
agonists, kringle 5 receptor antagonists or combinations thereof may be 
combined with pharmaceutically acceptible sustained-release matrices, such 
as biodegradable polymers, to form therapeutic compositions. A 
sustained-release matrix, as used herein, is a matrix made of materials, 
usually polymers, which are degradable by enzymatic or acid-base 
hydrolysis or by dissolution. Once inserted into the body, the matrix is 
acted upon by enzymes and body fluids. A sustained-release matrix is 
desirably chosen from biocompatible materials such as liposomes, 
polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), 
polylactide co-glycolide (copolymers of lactic acid and glycolic acid) 
polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, 
collagen, chondroitin sulfate, carboxylic acids, fatty acids, 
phospholipids, polysaccharides, nucleic acids, polyamino acids, amino 
acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, 
polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred 
biodegradable matrix is a matrix of one of either polylactide, 
polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and 
glycolic acid). 
Kringle 5 peptide fragments, kringle 5 receptor agonists, kringle 5 
receptor antagonists or combinations thereof may be combined with 
pharmaceutically acceptable excipients or carriers to form therapeutic 
compositions. A pharmaceutically acceptable carrier or excipient refers to 
a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating 
material or formulation auxiliary of any type. The compositions may be 
administered parenterally, sublingually, intracisternally, intravaginally, 
intraperitoneally, rectally, bucally or topically (as by powder, ointment, 
drops, transdermal patch or iontophoresis device). 
The term "parenteral," as used herein, refers to modes of administration 
which include intravenous, intramuscular, intraperitoneal, intrasternal, 
subcutaneous and intraarticular injection and infusion. Pharmaceutical 
compositions for parenteral injection comprise pharmaceutically acceptable 
sterile aqueous or nonaqueous solutions, dispersions, suspensions or 
emulsions as well as sterile powders for reconstitution into sterile 
injectable solutions or dispersions just prior to use. Examples of 
suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles 
include water, ethanol, polyols (such as glycerol, propylene glycol, 
polyethylene glycol and the like), carboxymethylcellulose and suitable 
mixtures thereof, vegetable oils (such as olive oil) and injectable 
organic esters such as ethyl oleate. Proper fluidity may be maintained, 
for example, by the use of coating materials such as lecithin, by the 
maintenance of the required particle size in the case of dispersions and 
by the use of surfactants. These compositions may also contain adjuvants 
such as preservatives, wetting agents, emulsifying agents and dispersing 
agents. Prevention of the action of microorganisms may be ensured by the 
inclusion of various antibacterial and antifungal agents such as paraben, 
chlorobutanol, phenol sorbic acid and the like. It may also be desirable 
to include isotonic agents such as sugars, sodium chloride and the like. 
Prolonged absorption of the injectable pharmaceutical form may be brought 
about by the inclusion of agents, such as aluminum monostearate and 
gelatin, which delay absorption. Injectable depot forms are made by 
forming microencapsule matrices of the drug in biodegradable polymers such 
as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). 
Depending upon the ratio of drug to polymer and the nature of the 
particular polymer employed, the rate of drug release can be controlled. 
Depot injectable formulations are also prepared by entrapping the drug in 
liposomes or microemulsions which are compatible with body tissues. The 
injectable formulations may be sterilized, for example, by filtration 
through a bacterial-retaining filter or by incorporating sterilizing 
agents in the form of sterile solid compositions which can be dissolved or 
dispersed in sterile water or other sterile injectable media just prior to 
use. 
Topical administration includes administration to the skin, mucosa and 
surfaces of the lung and eye. Compositions for topical administration, 
including those for inhalation, may be prepared as a dry powder which may 
be pressurized or non-pressurized. In non-pressurized powder compositions, 
the active ingredient in finely divided form may be used in admixture with 
a larger-sized pharmaceutically acceptable inert carrier comprising 
particles having a size, for example, of up to 100 micrometers in 
diameter. Suitable inert carriers include sugars such as lactose. 
Desirably, at least 95% by weight of the particles of the active 
ingredient have an effective particle size in the range of 0.01 to 10 
micrometers. For topical administration to the eye, a compound of the 
invention is delivered in a pharmaceutically acceptable ophthalmic vehicle 
such that the compound is maintained in contact with the ocular surface 
for a sufficient time period to allow the compound to penetrate the 
corneal and internal regions of the eye, as, for example, the anterior 
chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, 
cornea, iris/cilary, lens, choroid/retina and sclera. The pharmaceutically 
acceptable ophthalmic vehicle may, for example, be an ointment, vegetable 
oil or an encapsulating material. Alternatively, a compound of the 
invention may be injected directly into the vitrious and aqueous humor. 
The composition may be pressurized and contain a compressed gas such as 
nitrogen or a liquified gas propellant. The liquified propellant medium 
and indeed the total composition is preferably such that the active 
ingredient does not dissolve therein to any substantial extent. The 
pressurized composition may also contain a surface active agent such as a 
liquid or solid non-ionic surface active agent or may be a solid anionic 
surface active agent. It is preferred to use the solid anionic surface 
active agent in the form of a sodium salt. 
Compositions for rectal or vaginal administration are preferably 
suppositories which may be prepared by mixing the compounds of this 
invention with suitable non-irritating excipients or carriers such as 
cocoa butter, polyethylene glycol or a suppository wax which are solids at 
room temperature but liquids at body temperature and therefore melt in the 
rectum or vaginal cavity and release the active compound. 
Compounds of the present invention may also be administered in the form of 
liposomes. As is known in the art, liposomes are generally derived from 
phospholipids or other lipid substances. Liposomes are formed by mono- or 
multi-lamellar hydrated liquid crystals that are dispersed in an aqueous 
medium. Any non-toxic, physiologically acceptable and metabolizable lipid 
capable of forming liposomes can be used. The present compositions in 
liposome form may contain, in addition to a compound of the present 
invention, stabilizers, preservatives, excipients and the like. The 
preferred lipids are the phospholipids and the phosphatidyl cholines 
(lecithins), both natural and synthetic. Methods to form liposomes are 
known in the art. See, for example, Prescott, Ed., Methods in Cell 
Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., 
which is hereby incorporated herein by reference. 
When used in the above or other treatments, a therapeutically effective 
amount of one of the compounds of the present invention may be employed in 
pure form or, where such forms exist, in pharmaceutically acceptable salt 
form and with or without a pharmaceutically acceptible excipient. A 
"therapeutically effective amount" of the compound of the invention means 
a sufficient amount of the compound to treat an angiogenic disease (for 
example, to limit tumor growth or to slow or block tumor metastasis) at a 
reasonable benefit/risk ratio applicable to any medical treatment. It will 
be understood, however, that the total daily usage of the compounds and 
compositions of the present invention will be decided by the attending 
physician within the scope of sound medical judgment. The specific 
therapeutically effective dose level for any particular patient will 
depend upon a variety of factors including the disorder being treated and 
the severity of the disorder; activity of the specific compound employed; 
the specific composition employed; the age, body weight, general health, 
sex and diet of the patient; the time of administration; the route of 
administration; the rate of excretion of the specific compound employed; 
the duration of the treatment; drugs used in combination or coincidential 
with the specific compound employed and like factors well known in the 
medical arts. For example, it is well within the skill of the art to start 
doses of the compound at levels lower than those required to achieve the 
desired therapeutic effect and to gradually increase the dosage until the 
desired effect is achieved. Total daily dose of kringle 5 peptide 
fragments to be administered locally or systemically to a human or other 
mammal host in single or divided doses may be in amounts, for example, 
from 0.0001 to 200 mg/kg body weight daily and more usually 1 to 300 mg/kg 
body weight. If desired, the effective daily dose may be divided into 
multiple doses for purposes of administration. Consequently, single dose 
compositions may contain such amounts or submultiples thereof to make up 
the daily dose. 
It will be understood that agents which can be combined with the compound 
of the present invention for the inhibition, treatment or prophylaxis of 
angiogenic diseases are not limited to those listed above, but include, in 
principle, any agents useful for the treatment or prophylaxis of 
angiogenic diseases. 
The present invention also encompasses gene therapy whereby the gene 
encoding kringle 5 peptide fragments or kringle 5 peptide fragment 
conjugates is regulated in a patient. Various methods of transferring or 
delivering DNA to cells for expression of the gene product protein, 
otherwise referred to as gene therapy, are disclosed in Gene Transfer into 
Mammalian Somatic Cells in vivo, N. Yang, Crit. Rev. Biotechn. 12 (4): 
335-356 (1992), which is hereby incorporated herein by reference. Gene 
therapy encompasses incorporation of DNA sequences into somatic cells or 
germ line cells for use in either ex vivo or in vivo therapy. Gene therapy 
functions to replace genes, to augment normal or abnormal gene function 
and to combat infectious diseases and other pathologies. 
Strategies for treating medical problems with gene therapy include 
therapeutic strategies such as identifying the defective gene and then 
adding a functional gene to either replace the function of the defective 
gene or to augment a slightly functional gene or prophylactic strategies 
such as adding a gene which encodes a protein product that will treat the 
condition or that will make the tissue or organ more susceptible to a 
treatment regimen. As an example of a prophylactic strategy, a gene 
encoding a kringle 5 peptide fragment or a kringle 5 peptide fragment 
conjugate may be placed in a patient and thus prevent occurrence of 
angiogenesis or a gene that makes tumor cells more susceptible to 
radiation could be inserted so that radiation of the tumor would cause 
increased killing of the tumor cells. 
Many protocols for the transfer of kringle 5 peptide fragment DNA or 
kringle 5 peptide fragment regulatory sequences are envisioned in this 
invention. Transfection of promoter sequences, other than ones 
specifically associated with a kringle 5 peptide fragment or other 
sequences which would increase production of kringle 5 peptide fragments, 
are also envisioned as methods of gene therapy. An example of this 
technology is found in Transkaryotic Therapies, Inc., of Cambridge, Mass., 
using homologous recombination to insert a "genetic switch" which turns on 
an erythropoietin gene in cells as disclosed in Genetic Engineering News, 
Apr. 15, 1994, which is hereby incorporated herein by reference. Such 
"genetic switches" could be used to activate a kringle 5 peptide fragment 
(or a kringle 5 receptor) in cells not normally expressing these proteins. 
Gene transfer methods for gene therapy fall into three broad categories: 
(1) physical (e.g., electroporation, direct gene transfer and particle 
bombardment), (2) chemical (e.g. lipid-based carriers and other non-viral 
vectors) and (3) biological (e.g. virus derived vectors). For example, 
non-viral vectors such as liposomes coated with DNA may be directly 
injected intravenously into the patient It is believed that the 
liposome/DNA complexes are concentrated in the liver where they deliver 
the DNA to macrophages and Kupffer cells. Vectors or the "naked" DNA of 
the gene may also be directly injected into the desired organ, tissue or 
tumor for targeted delivery of the therapeutic DNA. 
Gene therapy methodologies can also be described by delivery site. 
Fundamental ways to deliver genes include ex vivo gene transfer, in vivo 
gene transfer and in vitro gene transfer. In ex vivo gene transfer, cells 
are taken from the patient and grown in cell culture. The DNA is 
transfected into the cells, and the transfected cells are expanded in 
number and then reimplanted in the patient. In in vitro gene transfer, the 
transformed cells are cells growing in culture, such as tissue culture 
cells, and not particular cells from a particular patient. These 
"laboratory cells" are transfected, and the transfected cells are selected 
and expanded for either implantation into a patient or for other uses. In 
vivo gene transfer involves introducing the DNA into the cells of the 
patient when the cells are within the patient All three of the broad based 
categories described above may be used to achieve gene transfer in vivo, 
ex vivo and in vitro. 
Mechanical (i.e. physical) methods of DNA delivery can be achieved by 
microinjection of DNA into germ or somatic cells, pneumatically delivered 
DNA-coated particles such as the gold particles used in a "gene gun" and 
inorganic chemical approaches such as calcium phosphate transfection. It 
has been found that physical injection of plasmid DNA into muscle cells 
yields a high percentage of cells which are transfected and have a 
sustained expression of marker genes. The plasmid DNA may or may not 
integrate into the genome of the cells. Non-integration of the transfected 
DNA would allow the transfection and expression of gene product proteins 
in terminally differentiated, non-proliferative tissues for a prolonged 
period of time without fear of mutational insertions, deletions or 
alterations in the cellular or mitochondrial genome. Long-term, but not 
necessarily permanent, transfer of therapeutic genes into specific cells 
may provide treatments for genetic diseases or for prophylactic use. The 
DNA could be reinjected periodically to maintain the gene product level 
without mutations occurring in the genomes of the recipient cells. 
Non-integration of exogenous DNAs may allow for the presence of several 
different exogenous DNA constructs within one cell with all of the 
constructs expressing various gene products. 
Particle-mediated gene transfer may also be employed for injecting DNA into 
cells, tissues and organs. With a particle bombardment device, or "gene 
gun," a motive force is generated to accelerate DNA-coated high density 
particles (such as gold or tungsten) to a high velocity that allows 
penetration of the target organs, tissues or cells. Electroporation for 
gene transfer uses an electrical current to make cells or tissues 
susceptible to electroporation-mediated gene transfer. A brief electric 
impulse with a given field strength is used to increase the permeability 
of a membrane in such a way that DNA molecules can penetrate into the 
cells. The techniques of particle-mediated gene transfer and 
electroporation are well known to those of ordinary skill in the art 
Chemical methods of gene therapy involve carrier-mediated gene transfer 
through the use of fusogenic lipid vesicles such as liposomes or other 
vesicles for membrane fusion. A carrier harboring a DNA of interest can be 
conveniently introduced into body fluids or the bloodstream and then site 
specifically directed to the target organ or tissue in the body. Cell or 
organ-specific DNA-carrying liposomes, for example, can be developed and 
the foreign DNA carried by the liposome absorbed by those specific cells. 
Injection of immunoliposomes that are targeted to a specific receptor on 
certain cells can be used as a convenient method of inserting the DNA into 
the cells bearing that receptor. Another carrier system that has been used 
is the asialoglycoprotein/polylysine conjugate system for carrying DNA to 
hepatocytes for in vivo gene transfer. 
Transfected DNA may also be complexed with other kinds of carriers so that 
the DNA is carried to the recipient cell and then deposited in the 
cytoplasm or in the nucleoplasm. DNA can be coupled to carrier nuclear 
proteins in specifically engineered vesicle complexes and carried directly 
into the nucleus. 
Carrier mediated gene transfer may also involve the use of lipid-based 
compounds which are not liposomes. For example, lipofectins and 
cytofectins are lipid-based positive ions that bind to negatively charged 
DNA and form a complex that can ferry the DNA across a cell membrane. 
Another method of carrier mediated gene transfer involves receptor-based 
endocytosis. In this method, a ligand (specific to a cell surface 
receptor) is made to form a complex with a gene of interest and then 
injected into the bloodstream. Target cells that have the cell surface 
receptor will specifically bind the ligand and transport the ligand-DNA 
complex into the cell. 
Biological gene therapy methodologies employ viral vectors to insert genes 
into cells. The term "vector" as used herein means a carrier which may 
contain or associate with specific polynucleotide sequences and which 
functions to transport the specific polynucleotide sequences into a cell. 
The transfected cells may be cells derived from the patient's normal 
tissue, the patient's diseased tissue or non-patient cells. Examples of 
vectors include plasmids and infective microorganisms such as viruses or 
nonviral vectors such as the ligand-DNA conjugates, liposomes and the 
lipid-DNA complexes discussed above. 
It may be desirable that a recombinant DNA molecule comprising a kringle 5 
peptide fragment DNA sequence is operatively linked to an expression 
control sequence to form an expression vector capable of expressing a 
kringle 5 peptide fragment. Alternatively, gene regulation of kringle 5 
peptide fragments may be accomplished by administering compounds that bind 
to the kringle 5 gene or control regions associated with the kringle 5 
gene or its corresponding RNA transcript to modify the rate of 
transcription or translation. 
Viral vectors that have been used for gene therapy protocols include, but 
are not limited to, retroviruses, other RNA viruses such as poliovirus or 
Sindbis virus, adenovirus, adeno-associated virus, herpes viruses, SV 40, 
vaccinia and other DNA viruses. Replication-defective murine retroviral 
vectors are the most widely utilized gene transfer vectors. Murine 
leukemia retroviruses are composed of a single strand RNA completed with a 
nuclear core protein and polymerase (pol) enzymes encased by a protein 
core (gag) and surrounded by a glycoprotein envelope (env) that determines 
host range. The genomic structure of retroviruses include gag, pol, and 
env genes enclosed at the 5' and 3' long terminal repeats (LTRs). 
Retroviral vector systems exploit the fact that a minimal vector 
containing the 5' and 3' LTRs and the packaging signal are sufficient to 
allow vector packaging and infection and integration into target cells 
providing that the viral structural proteins are supplied in trans in the 
packaging cell line. Fundamental advantages of retroviral vectors for gene 
transfer include efficient infection and gene expression in most cell 
types, precise single copy vector integration into target cell chromosomal 
DNA and ease of manipulation of the retroviral genome. For example, 
altered retrovirus vectors have been used in ex vivo methods to introduce 
genes into peripheral and tumor-infiltrating lymphocytes, hepatocytes, 
epidermal cells, myocytes or other somatic cells (which may then be 
introduced into the patient to provide the gene product from the inserted 
DNA). 
The adenovirus is composed of linear, double stranded DNA complexed with 
core proteins and surrounded with capsid proteins. Advances in molecular 
virology have led to the ability to exploit the biology of these organisms 
to create vectors capable of transducing novel genetic sequences into 
target cells in vivo. Adenoviral-based vectors will express gene product 
peptides at high levels. Adenoviral vectors have high efficiencies of 
infectivity, even with low titers of virus. Additionally, the virus is 
fully infective as a cell-free virion so injection of producer cell lines 
are not necessary. Another potential advantage to adenoviral vectors is 
the ability to achieve long term expression of heterologous genes in vivo. 
Viral vectors have also been used to insert genes into cells using in vivo 
protocols. To direct tissue-specific expression of foreign genes, 
cis-acting regulatory elements or promoters that are known to be 
tissue-specific may be used. Alternatively, this can be achieved using in 
situ delivery of DNA or viral vectors to specific anatomical sites in 
vivo. For example, gene transfer to blood vessels in vivo was achieved by 
implanting in vitro transduced endothelial cells in chosen sites on 
arterial walls. The virus-infected surrounding cells, in turn, also 
expressed the gene product. A viral vector can be delivered directly to 
the in vivo site (by catheter, for example) thus allowing only certain 
areas to be infected by the virus and providing long-term, site-specific 
gene expression. In vivo gene transfer using retrovirus vectors has also 
been demonstrated in mammary tissue and hepatic tissue by injection of the 
altered virus into blood vessels leading to the organs. 
Kringle 5 peptide fragments may also be produced and used in a variety of 
applications. As examples, different peptide fragments of kringle 5 can be 
used (1) as agonists and antagonists active at kringle 5 binding sites, 
(2) as antigens for the development of specific antisera, (3) as peptides 
for use in diagnostic kits and (4) as peptides linked to or used in 
combination with cytotoxic agents for targeted killing of cells that bind 
kringle 5 peptide fragments. The amino acid sequences that comprise these 
peptide fragments may be selected on the basis of their position on the 
exterior regions of the molecule which are accessible for binding to 
antisera or the inhibitory potency of the peptide fragments toward 
processes arising from or exaserbated by angiogenesis. Furthermore, these 
peptide sequences may be compared to known sequences using protein 
sequence databases such as GenBank, Brookhaven Protein, SWISS-PROT, and 
PIR to determine potential sequence homologies. This information 
facilitates elimination of sequences that exhibit a high degree of 
sequence homology to other molecules and thereby enhances the potential 
for high specificity in the development of antisera, agonists and 
antagonists to kringle 5. 
Kringle 5 peptide fragments may also be used as a means to isolate a 
kringle 5 receptor by immobilization of the kringle 5 peptide fragment on 
a solid support in, for example, an affinity column through which cultured 
endothelial cells or membrane extracts are passed. As is known in the art, 
isolation and purification of a kringle 5 receptor may be followed by 
amino acid sequencing to identify and isolate polynucleotides which encode 
the kringle 5 receptor. Such polynucleotides may then be cloned into a 
suitable expression vector and transfected into tumor cells. Expression of 
the receptor by the transfected tumor cells would enhance the 
responsiveness of these cells to endogenous or exogenous kringle 5 peptide 
fragments and thereby decrease the rate of metastatic growth. Furthermore, 
recombinant expression of this receptor would allow greater amounts of 
receptor to be produced, e.g. to produce a sufficient quantity for use in 
high throughput screening assays to identify smaller antagonists which 
mimic the action of kringle 5. 
Systematic substitution of amino acids within these synthesized peptides 
may yield high affinity peptide agonists and antagonists to the kringle 5 
receptor that enhance or diminish kringle 5 peptide fragment binding to 
its receptor. Such agonists may be used to suppress the growth of 
micrometastases and thereby limit the spread of cancer. In cases of 
inadequate vascularization, antagonists to kringle 5 peptide fragments may 
be applied to block the inhibitory effects of kringle 5 peptide fragments 
and promote angiogenesis. For example, this type of treatment may have 
therapeutic effects in promoting wound healing in diabetics. 
Kringle 5 peptide fragments of the present invention can also be used as 
antigens to generate polyclonal or monoclonal antibodies which are 
specific for the kringle 5 inhibitor. One way in which such antibodies 
could be used is in diagnostic methods and kits to detect or quantify 
kringle 5 peptide fragments in a body fluid or tissue. Results from these 
tests could be used to diagnose or determine the prognostic relevance of 
kringle 5 peptide fragments. 
Kringle 5 peptide fragments may be labeled with radioactive isotopes (See 
Example 13) or chemically coupled to proteins to form conjugates. 
Conjugates include enzymes, carrier proteins, cytotoxic agents, 
fluorescent, chemiluminescent and bioluminescent molecules which are used 
to facilitate the testing of the ability of compounds containing kringle 5 
peptide fragments to bind kringle 5 antisera, detect cell types which 
possess a kringle 5 peptide fragment receptor or aid in purification of 
kringle 5 peptide fragments. The coupling technique is generally chosen on 
the basis of the functional groups available on the amino acids of the 
kringle 5 peptide fragment sequence including, but not limited to alkyl, 
amino, sulfhydryl, carboxyl, amide, phenol, indolyl and imidazoyl. Various 
reagents used to effect such couplings include, among others, 
glutaraldehyde, diazotized benzidine, carbodiimides and p-benzoquinone. 
The efficiency of the coupling reaction is determined using different 
techniques appropriate for the specific reaction. For example, 
radiolabeling of a kringle 5 peptide or a biologically active fragment 
thereof with I.sup.125 may be accomplished using chloramine T and 
NaI.sup.125 of high specific activity. The reaction is terminated with 
sodium metabisulfite and the mixture is desalted on disposable columns. 
The labeled peptide is eluted from the column and the fractions are 
collected. Aliquots are removed from each fraction and radioactivity is 
measured in a gamma counter. This procedure provides the radiolabeled 
kringle 5 peptide fragment free from unreacted NaI.sup.125. In another 
example, blood or tissue extracts containing a kringle 5 peptide fragment 
coupled to kringle 4 may be purified on a polylysine resin affinity column 
whereby the kringle 4-kringle 5 peptide fragment binds to the resin 
through the affinity of the kringle 4 peptide fragment for lysine. Elution 
of the bound protein would provide a purified kringle 4-kringle 5 peptide 
fragment 
Another application of peptide conjugation is the production of polyclonal 
antisera. The production of antiserum against kringle 5 peptide fragments, 
kringle 5 peptide fragment analogs and the kringle 5 receptor can be 
performed using established techniques known to those skilled in the art. 
For example, kringle 5 peptide fragments containing lysine residues may be 
linked to purified bovine serum albumin (BSA) using glutaraldehyde. The 
efficiency of this reaction may be determined by measuring the 
incorporation of radiolabeled peptide. Unreacted glutaraldehyde and 
peptide may be separated by dialysis, and the conjugate may be use to 
raise polyclonal antisera in rabbits, sheep, goats or other animals. 
Kringle 5 peptide fragments conjugated to a carrier molecule such as BSA 
may be combined with an adjuvant mixture, emulsified and injected 
subcutaneously at multiple sites on the back, neck, flanks, and sometimes 
in the footpads of a suitable host. Generally, booster injections are then 
given at regular intervals, such as every 2 to 4 weeks. Approximately 7 to 
10 days after each injection, blood samples are obtained by venipuncture 
using, for example, the marginal ear veins after dilation. The blood 
samples are allowed to clot overnight at 4.degree. C. and are centrifuged 
at approximately 2400.times.g at 4.degree. C. for about 30 minutes. The 
serum is removed, aliquoted and stored at 4.degree. C. for immediate use 
or at -20 to -90.degree. C. for subsequent analysis. 
Serum samples from generation of polyclonal antisera or media samples from 
production of monoclonal antisera may be analyzed for determination of 
antibody titer and, in particular, for the determination of high titer 
antisera Subsequently, the highest titer kringle 5 peptide fragment 
antisera may be tested to establish the following: a) optimal antiserum 
dilution for highest specific binding of the antigen and lowest 
non-specific binding, b) ability to bind increasing amounts of kringle 5 
peptide fragments in a standard displacement curve, c) potential 
cross-reactivity with related peptides and proteins including plasminogen 
and kringle 5 peptide fragments of related species and d) ability to 
detect kringle 5 peptide fragments in cell culture media and in extracts 
of plasma, urine and tissues. Titer may be established through several 
means known in the art, such as by dot blot and density analysis and also 
by precipitation of radiolabeled peptide-antibody complexes using protein 
A, secondary antisera, cold ethanol or charcoal-dextran followed by 
activity measurement with a gamma counter. If desired, the highest titer 
antisera may be purified on affinity columns. For example, kringle 5 
peptide fragments may be coupled to a commercially available resin and 
used to form an affinity column. Antiserum samples may then be passed 
through the column so that kringle 5 antibodies bind (via kringle 5 
peptide fragments) to the column. These bound antibodies are subsequently 
eluted, collected and evaluated for determination of titer and 
specificity. 
Kits for measurement of kringle 5 peptide fragments and the kringle 5 
receptor are also contemplated as part of the present invention. Antisera 
that possess the highest titer and specificity and can detect kringle 5 
peptide fragments in extracts of plasma, urine, tissues and cell culture 
media may be used to establish assay kits for rapid, reliable, sensitive 
and specific measurement and localization of kringle 5 peptide fragments. 
These assay kits may employ, but are not limited to, the following 
techniques: competitive and non-competitive assays, radioimmunoassays, 
bioluminescence and chemilumenescence assays, fluorometric assays, 
sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays 
including ELISAs, microtiter plates, immunocytochemistry and 
antibody-coated strips or dipsticks for rapid monitoring of urine or 
blood. For each kit the range, sensitivity, precision, reliability, 
specificity and reproducibility of the assay are established by means well 
known to those skilled in the art. 
One example of an assay kit commonly used in research and in the clinic is 
a radioimmunoassay (RIA) kit. A kringle 5 peptide fragment RIA may be 
established in the following manner: After successful radioiodination and 
purification of a kringle 5 peptide fragment, antiserum possessing the 
highest titer of anti-kringle 5 peptide fragment antibodies is added at 
several dilutions to tubes containing a relatively constant amount of 
radioactivity, such as 10,000 cpm, in a suitable buffer system. (Buffer or 
preimmune serum is added to other tubes to determine non-specific 
binding). After incubation at 4.degree. C. for 24 hours, protein A is 
added to all tubes and the tubes are vortexed, incubated at room 
temperature for 90 minutes and centrifuged at approximately 
2000-2500.times.g at 4.degree. C. to precipitate the complexes of antibody 
bound to labeled antigen. The supernatant is removed by aspiration and 
radioactivity in the pellets counted in a gamma counter. The antiserum 
dilution that binds approximately 10 to 40% of the labeled peptide after 
subtraction of the non-specific binding is selected for further 
characterization. 
Next, a dilution range (approximately 0.1 pg to 10 ng) of the kringle 5 
peptide fragment used for development of the antiserum is evaluated by 
adding known amounts of the peptide to tubes containing radiolabeled 
peptide and antiserum. After an incubation period (24 or 48 hours, for 
example), protein A is added and the tubes are centrifuged, the 
supernatant is removed and the radioactivity in the pellet is counted. The 
displacement of the binding of radiolabeled the kringle 5 peptide fragment 
by the unlabeled kringle 5 peptide fragment (standard) provides a standard 
curve. Additionally, several concentrations of other kringle 5 peptide 
fragments, plasminogens, kringle 5 peptide fragments from different 
species and homologous peptides may be added to the assay tubes to 
characterize the specificity of the kringle 5 peptide fragment antiserum. 
Thereafter, extracts of various tissues including, but not limited to, 
primary and secondary tumors, Lewis lung carcinoma, cultures of kringle 5 
peptide fragment-producing cells, placenta, uterus and other tissues such 
as brain, liver and intestine are prepared using extraction techniques 
that have been successfully employed to extract kringle 5 peptide 
fragments. After workup of the tisssue extracts, assay buffer is added and 
different aliquots are placed into the RIA tubes. Extracts of known 
kringle 5 peptide fragment-producing cells produce displacement curves 
that are parallel to the standard curve whereas extracts of tissues that 
do not produce kringle 5 peptide fragments do not displace radiolabeled 
kringle 5 peptide fragments from the kringle 5 peptide fragment antiserum. 
Such displacement curves indicate the utility of the kringle 5 peptide 
fragment assay to measure kringle 5 peptide fragments in tissues and body 
fluids. 
Tissue extracts that contain kringle 5 peptide fragments may also be 
characterized by subjecting aliquots to reverse phase HPLC. Eluate 
fractions are collected, dried in Speed Vac, reconstituted in RIA buffer 
and analyzed in the kringle 5 RIA. In this case, the maximal amount of 
kringle 5 peptide fragment immunoreactivity is located in the fractions 
corresponding to the elution position of the kringle 5 peptide fragment. 
The above described assay kit would provide instructions, antiserum, a 
kringle 5 peptide fragment and possibly a radiolabeled kringle 5 peptide 
fragment and/or reagents for precipitation of bound kringle 5 peptide 
fragment/kringle 5 antibody complexes. Such a kit would be useful for the 
measurement of kringle 5 peptide fragments in biological fluids and tissue 
extracts of animals and humans with and without tumors. 
Another kit may be used to visualize or localize kringle 5 peptide 
fragments in tissues and cells. For example, immunohistochemistry 
techniques and kits which employ such techniques are well known to those 
of ordinary skill in the art. As is known in the art, an 
immunohistochemistry kit would provide kringle 5 peptide fragment 
antiserum; and possibly blocking serum and secondary antiserum linked to a 
fluorescent molecule such as fluorescein isothiocyanate or to some other 
reagent used to visualize the primary antiserum. Using this methodology, 
biopsied tumors may be examined for sites of kringle 5 peptide fragment 
production or for sites of the kringle 5 peptide fragment receptor. 
Alternatively, a kit may supply radiolabeled nucleic acids for use in in 
situ hybridization to probe for kringle 5 peptide fragment messenger RNA. 
The compounds of the invention may be prepared using processes well known 
to those of ordinary skill in the art. (See for example, Sottrup-Jensen et 
al., Progress in Chemical Fibrinolysis and Thrombolysis, Vol. 3, Davidson, 
J. F., Rowan, R. M., Samama, M. M. and Desnoyers, P. C. editors, Raven 
Press, New York, 1978. One manner of preparing kringle 5 peptide fragments 
is by enzymatic cleavage of the native protein (glu-plasminogen) or a 
variant thereof (meaning a truncated form of the full length protein which 
is amenable to cleavage by enzymatic digestion and which comprises at 
least a kringle 5 sequence as defined above such as lys-plasminogen or 
miniplasminogen). This method first requires isolating the protein from 
human plasma in the absence of plasmin inhibitors and thereby promoting 
the conversion of glu-plasminogen to lys-plasminogen (see Novokhatny, V 
and Kudinov, S. A., J. Mol. Biol. 179: 215-232 (1984). Subsequently, the 
truncated molecule is treated with an proteolytic enzyme at a 
concentration sufficient to cleave kringle 5 peptide fragments from the 
polypeptide and then purified from the remaining fragments by means known 
to those skilled in the art. A preferred proteolytic enzyme is human or 
porcine elastase which cleaves plasminogen and its truncated variants 
between kringle regions 3-4 and 4-5 (and is thereby capable of forming 
peptide fragments containing kringles 1-3 and 1-4 or kringles 4 or 5 
alone). For example, lys-plasminogen or glu-plasminogen may be treated 
with porcine or human neutrophyl elastase at a ratio of about 1:100-1:300 
lys-plasminogen:elastase (preferably at a ratio of 1:150-1:250 and most 
preferably at a ratio of 1:150 in a buffer solution (such as Tris-HCl, 
NaCl, sodium phosphate and the like). Alternatively, the elastase may 
first be immobilized (such as to a resin) to facilitate purification of 
the cleavage products. The glu-plasminogen or lys-plaminogen is generally 
treated with human or porcine elastase at temperatures ranging from about 
10.degree. C. to about 40.degree. C. and for time periods ranging from 
about 4 to about 24 hours depending on the extent of cleavage desired. To 
achieve complete digestion of glu-plasminogen, lys-plasminogen or 
miniplasminogen with human or porcine elastase requires exposure of these 
polypeptides to the enzyme for at least about 12 hours at room 
temperature. Varying the pH and exposure time to the enzyme results in 
less or partial cleavage at one or more of the susceptible cleavage sites. 
The cleavage products are then purified by any means well known in the art 
(such as column chromatography). A preferred purification scheme involves 
applying the cleavage products to a lysine-Sepharose column as described 
in Example 14. 
Solid Phase Synthesis of Kringle 5 Peptide Fragments 
The following examples will serve to further illustrate the preparation of 
the novel compounds of the invention: 
EXAMPLE 1 
N-Ac-Val-Leu-Leu-Pro-Asp-Val-Glu-Thr-Pro-Ser-Glu-Glu-Asp-NH.sub.2 
An amide peptide synthesis column (Applied Biosystems) was placed in the 
peptide synthesis column position of a Perkin Elmer/Applied Biosynthesis 
"Synergy" peptide synthesizer, and the following synthetic sequence was 
used: 
1. Solvating the resin with DMF for about 5 minutes; 
2. Deblocking the Fmoc group from the .alpha.-N-terminal of the resin-bound 
amino acid using 20% piperidine in DMF for about 15 minutes; 
3. Washing the resin with DMF for about 5 minutes; 
4. Activating the .alpha.-C-terminal of amino acid No. 1 
(Fmoc-Asp(.beta.-O.sup.t Bu), 25 .mu.mol) using a 0.2 M solution of HBTU 
(25 .mu.mol) and HOBT (25 .mu.mol) in DMSO-NMP (N-methylpyrrolidone) and a 
0.4 M solution of diisopropylethylamine (25 .mu.mol) in DMSO- NMP and 
coupling the activated amino acid to the resin; 
5. Coupling the activated Fmoc-protected amino acid (prepared in step 5) to 
the resin-bound amino acid (prepared in step 2) in DMF for about 30 
minutes; 
6. Washing with DMF for 5 minutes; 
7. Repeating steps 3 through 6 with the following amino acids: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Glu(.gamma.-O.sup.t Bu) 
3. Fmoc-Glu(.gamma.-O.sup.t Bu) 
4. Fmoc-Ser(.sup.t Bu) 
5. Fmoc-Pro 
6. Fmoc-Thr(.sup.t Bu) 
7. Fmoc-Glu(.gamma.-O.sup.t Bu) 
8. Fmoc-Val 
9. Fmoc-Asp(.beta.-O.sup.t Bu) 
10. Fmoc-Pro 
11. Fmoc-Leu 
12. Fmoc-Leu 
13. Fmoc-Val 
______________________________________ 
8. Coupling acetic acid to the .alpha.-N-terminal of the resin-bound 
peptide via the conditions of steps 4 and 5. 
9. Washing the resin with THF for about 5 minutes to remove DMF and shrink 
the resin, then drying the resin with argon for 10 minutes and nitrogen 
for 10 minutes more to provide clean, resin-bound peptide. 
10. Cleaving of the peptide from the resin with concomitant deprotection of 
amino acid side chains by stirring with cleavage reagent (freshly-prepared 
thioanisole (100 .mu.L), water (50 .mu.L), ethanedithiol (50 .mu.L) and 
trifluoroacetic acid (1.8 mL) mixed in the above order at -5.degree. C. to 
-10.degree. C.) at 0.degree. C. for 10-15 minutes and then at ambient 
temperature for an additional 1.75 hours (plus an additional 0.5 hour for 
each Arg(Pmc), if present). The amount of cleavage reagent used was 
determined by the following formula: 
______________________________________ 
weight of resin with bound peptide (mg) 
amount of cleavage reagent (.mu.L) 
______________________________________ 
0-10 100 
10-25 200 
25-50 400 
50-100 700 
100-200 1200 
______________________________________ 
11. Filtering and rinsing the produdct with neat trifluoroacetic acid, 
adding the filtrate in 0.5 mL portions to a centrifuge tube containing 
about 8 mL of cold diethyl ether, centrifuging and decanting and repeating 
the process until all of the peptide precipitated (if the peptide did not 
precipitate upon addition to ether, the mixture was extracted with aqueous 
30% aqueous acetic acid (3.times.1 mL), and the combined aqueous extracts 
were lyophilized to provide the product). 
12. Using the peptide crude or purifying the peptide by HPLC using a 7 
.mu.m Symmetry Prep C18 column (7.8.times.300 mm) with solvent mixtures 
varying in a gradient from 5% to 100% acetonitrile-(water, 0.1% TFA) over 
a period of 50 minutes followed by lyophilizing to provide 35 mg of 
N-Ac-Val-Leu-Leu-Pro-Asp-Val-Glu-Thr-Pro-Ser-Glu-Glu-Asp-NH.sub.2. 
EXAMPLE 2 
N-Ac-Met-Phe-Gly-Asn-Gly-Lys-Gly-Tyr-Arg-Gly-Lys-Arg-Ala-Thr-Thr-Val-Thr-Gl 
y-Thr-Pro-NH.sub.2 
The title compound was prepared using the synthetic sequence described in 
Example 1 and using Fmoc-Pro as amino acid No. 1. The following amino 
acids were added using the conditions indicated: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Thr(.sup.t Bu) 
3. Fmoc-Gly 
4. Fmoc-Thr(.sup.t Bu) 
5. Fmoc-Val 
6. Fmoc-Thr(.sup.t Bu) 
7. Fmoc-Thr(.sup.t Bu) 
8. Fmoc-Ala 
9. Fmoc-Arg(Pmc) 
10. Fmoc-Lys(Boc) 
11. Fmoc-Gly 
12. Fmoc-Arg(Pmc) 
13. Fmoc-Tyr(.sup.t Bu) 
14. Fmoc-Gly 
15. Fmoc-Lys(Boc) 
16. Fmoc-Gly 
17. Fmoc-Asn(Trt) 
18. Fmoc-Gly 
19. Fmoc-Phe 
20. Fmoc-Met 
______________________________________ 
to provide 35 mg of 
N-Ac-Met-Phe-Gly-Asn-Gly-Lys-Gly-Tyr-Arg-Gly-Lys-Arg-Ala-Thr-Thr-Val-Thr-G 
ly-Thr-Pro-NH.sub.2. 
EXAMPLE 3 
Ac-Gln-Asp-Trp-Ala-Ala-Gln-Glu-Pro-His-Arg-His-Ser-Ile-Phe-Thr-Pro-Glu-Thr- 
Asn-Pro-Arg-Ala-Gly-Leu-Glu-Lys-Asn-Tyr-NH.sub.2 
The title compound was prepared using the synthetic sequence described in 
Example 1 and using Fmoc-Tyr(.sup.t Bu) as amino acid No. 1. The following 
amino acids were added using the conditions indicated: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Asn(Trt) 
3. Fmoc-Lys(Boc) 
4. Fmoc-Glu(.gamma.-O.sup.t Bu) 
5. Fmoc-Leu 
6. Fmoc-Gly 
7. Fmoc-Ala 
8. Fmoc-Arg(Pmc) 
9. Fmoc-Pro 
10. Fmoc-Asn(Trt) 
11. Fmoc-Thr(.sup.t Bu) 
12. Fmoc-Glu(.gamma.-O.sup.t Bu) 
13. Fmoc-Pro 
14. Fmoc-Thr(.sup.t Bu) 
15. Fmoc-Phe 
16. Fmoc-Ile 
17. Fmoc-Ser(.sup.t Bu) 
18. Fmoc-His(Trt) 
19. Fmoc-Arg(Pmc) 
20. Fmoc-His(Trt) 
21. Fmoc-Pro 
22. Fmoc-Glu(.gamma.-O.sup.t Bu) 
23. Fmoc-Gln(Trt) 
24. Fmoc-Ala 
25. Fmoc-Ala 
26. Fmoc-Trp 
27. Fmoc-Asp(.beta.-O.sup.t Bu) 
28. Fmoc-Gln(Trt) 
______________________________________ 
to provide 40 mg of 
N-Ac-Gln-Asp-Trp-Ala-Ala-Gln-Glu-Pro-His-Arg-His-Ser-Ile-Phe-Thr-Pro-Glu-T 
hr-Asn-Pro-Arg-Ala-Gly-Leu-Glu-Lys-Asn-Tyr-NH.sub.2. 
EXAMPLE 4 
N-Ac-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-NH.sub.2 
The title compound was prepared using the synthetic sequence described in 
Example 1 and using Fmoc-Trp as amino acid No. 1. The following amino 
acids were added using the conditions indicated: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Pro 
3. Fmoc-Gly 
4. Fmoc-Gly 
5. Fmoc-Val 
6. Fmoc-Asp(.beta.-O.sup.t -Bu) 
7. Fmoc-Gly 
8. Fmoc-Asp(.beta.-O.sup.t -Bu) 
9. Fmoc-Pro 
10. Fmoc-Asn(Trt) 
11. Fmoc-Arg(Pmt) 
______________________________________ 
to provide 20 mg of 
N-Ac-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-NH.sub.2. 
EXAMPLE 5 
N-Ac-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH.sub.2 
The title compound was prepared using the synthetic sequence described in 
example 1 and using Fmoc-Tyr(.sup.t Bu) as amino acid No. 1. The following 
amino acids were added using the conditions indicated: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Asp(.beta.- O.sup.t Bu) 
3. Fmoc-Tyr(.sup.t Bu) 
4. Fmoc-Leu 
5. Fmoc-Lys(Boc) 
6. Fmoc-Arg(Pmc) 
7. Fmoc-Pro 
8. Fmoc-Asn(Trt) 
9. Fmoc-Thr(.sup.t Bu) 
10. Fmoc-Thr(.sup.t Bu) 
11. Fmoc-Tyr(.sup.t Bu) 
______________________________________ 
to provide 10 mg of 
N-Ac-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH.sub.2. 
EXAMPLE 6 
N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH.sub.2 
The title compound was prepared using the synthetic sequence described in 
Example 1 and using Fmoc-Tyr(.sup.t Bu) as amino acid No. 1. The following 
amino acids were added using the conditions indicated: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Asp(.beta.-O.sup.t Bu) 
3. Fmoc-Tyr(.sup.t Bu) 
4. Fmoc-Leu 
5. Fmoc-Lys(Boc) 
6. Fmoc-Arg(Pmc) 
7. Fmoc-Pro 
______________________________________ 
to provide N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH.sub.2 (4 mg). MS (FAB) m/z 
995 (M+H).sup.+. 
EXAMPLE 7 
N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-NH.sub.2 
The title compound was prepared using the synthetic sequence described in 
Example 1. The following amino acids were added using the conditions 
indicated: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Tyr(.sup.t Bu) 
3. Fmoc-Leu 
4. Fmoc-Lys(Boc) 
5. Fmoc-Arg(Pmc) 
6. Fmoc-Leu 
______________________________________ 
to provide N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-NH.sub.2 (6 mg). MS (ESI) m/z 832 
(M+H).sup.+. 
EXAMPLE 8 
N-Ac-Pro-Glu-Lys-Arg-Tyr-Asp-Tyr-NH.sub.2 
The title compound was prepared using the synthetic sequence described in 
Example 1 and using Fmoc-Tyr(.sup.t Bu) as amino acid No. 1. The following 
amino acids were added using the conditions indicated: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Asp(.beta.-O.sup.t Bu) 
3. Fmoc-Tyr(.sup.t Bu) 
4. Fmoc-Arg(Pmc) 
5. Fmoc-Lys(Boc) 
6. Fmoc-Glu 
7. Fmoc-Pro 
______________________________________ 
to provide N-Ac-Pro-Glu-Lys-Arg-Tyr-Asp-Tyr-NH.sub.2 (6 mg). MS (FAB) m/z 
(1101) (M+H).sup.+. 
EXAMPLE 9 
N-Ac-Arg-Lys-Leu-Tyr-Asp-Tyr-NH.sub.2 
The title compound was prepared using the synthetic sequence described in 
Example 1 and using Fmoc-Tyr(.sup.t Bu) as amino acid No. 1. The following 
amino acids were added using the conditions indicated: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Asp(.beta.-O.sup.t Bu) 
3. Fmoc-Tyr(.sup.t Bu) 
4. Fmoc-Leu 
5. Fmoc-Lys(Boc) 
6. Fmoc-Arg(Pmc) 
______________________________________ 
to provide N-Ac-Arg-Lys-Leu-Tyr-Asp-Tyr-NH.sub.2 (8 mg). MS (ESI) m/z 898 
(M+H).sup.+. 
EXAMPLE 10 
N-Ac-Pro-Arg-Lys-Leu-3-I-Tyr-Asp-Tyr-NH.sub.2 (SEQ ID NO: 13) 
The title compound was prepared using the synthetic sequence described in 
Example 1 and using Fmoc-Tyr(.sup.t Bu) as amino acid No. 1. The following 
amino acids were added using the conditions indicated: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Asp(.beta.-O.sup.t Bu) 
3. Fmoc-3-I-Tyr(.sup.t Bu) 
4. Fmoc-Leu 
5. Fmoc-Lys(Boc) 
6. Fmoc-Arg(Pmc) 
7. Fmoc-Pro 
______________________________________ 
to provide N-Ac-Pro-Arg-Lys-Leu-3-I-Tyr-Asp-Tyr-NH.sub.2 (2 mg). MS (ESI) 
m/z (1121) (M+H).sup.+. 
EXAMPLE 11 
N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I-Tyr-NH.sub.2 (SEQ ID NO: 14) 
The title compound was prepared using the synthetic sequence described in 
Example 1 and using Fmoc-3-I-Tyr(.sup.t Bu) as amino acid No. 1. The 
following amino acids were added using the conditions indicated: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Asp(.beta.-O.sup.t Bu) 
3. Fmoc-Tyr(.sup.t Bu) 
4. Fmoc-Leu 
5. Fmoc-Lys(Boc) 
6. Fmoc-Arg(Pmc) 
7. Fmoc-Pro 
______________________________________ 
to provide N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I-Tyr-NH.sub.2 (2.5 mg). MS (ESI) 
m/z 1121 (M+H).sup.+. 
EXAMPLE 12 
N-Ac-Lys-Leu-Tyr-Asp-NH.sub.2 
The title compound was prepared using the synthetic sequence described in 
Example 1 and using Fmoc-Asp(.beta.-O.sup.t Bu) as amino acid No. 1. The 
following amino acids were added using the conditions indicated: 
______________________________________ 
No. Amino Acid 
______________________________________ 
2. Fmoc-Tyr(.sup.t Bu) 
3. Fmoc-Leu 
4. Fmoc-Lys 
______________________________________ 
to provide 2 mg of N-Ac-Lys-Leu-Tyr-Asp-NH.sub.2 (2 mg). 
EXAMPLE 13 
Preparation and separation of a mixture 
N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I.sup.125 -Tyr.sup.535 -NH.sub.2 and 
N-Ac-Pro-Arg-Lys-Leu-3-I.sup.125 -Tyr.sup.533 -Asp-Tyr-NH.sub.2 (SEQ ID 
NO: 13) and (SEQ NO: ID 14), Respectively 
To a solution of 30 .mu.g of 
N-acetyl-prolyl-arginyl-lysyl-leucyl-tyrosyl-aspartyl-tyrosylamide in 80 
mL of phosphate buffered saline (PBS) was added one iodobead (Pierce, 
Rockford, Ill.) and 100 .mu.Ci of NaI.sup.125. After 10 minutes, the 
excess NaI.sup.125 reagent was removed by applying the reaction mixture to 
a Waters C18-Light SepPack column and eluting with water then 0.1% TFA in 
1:1 CH.sub.3 CN/water and collecting 3.times.200 .mu.L fractions to 
provide a mixture of Tyr.sup.533 - and Tyr.sup.535 - radiolabeled 
peptides. 
The hot peptide mixture was coinjected onto a C18 HPLC column with an 
equimolar solution of cold carriers 
N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I-Tyr-NH.sub.2 and 
N-Ac-Pro-Arg-Lys-Leu-3-I-Tyr-Asp-Tyr-NH.sub.2, the elution times of which 
had been predetermined as 36 and 38 minutes, respectively. Repeated 
elutions with the solvent system in Example 1 and lyophylization of the 
combined, relevant fractions provided the desired compound 
N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I-Tyr-NH.sub.2 with a minimal impurity 
N-Ac-Pro-Arg-Lys-Leu-3-I-Tyr-Asp-Tyr-NH.sub.2. 
General Methodologies 
EXAMPLE 14 
Isolation and Purification of Kringle 5 Peptide Fragments 
The kringle 5 peptide fragments were prepared from the digestion of Lys 
plasminogen (Lys-HPg, Abbott Laboratories, Abbott Park, Ill.) with porcine 
elastase (SIGMA, St. Louis, Mo.) by a modification of the method of Powell 
et al. (Arch Biochem. Biophys. 248 (1): 390-400 (1986), which is hereby 
incorporated herein by reference). 1.5 mg of porcine elastase was 
incubated with 200 mg of Lys-HPg in 50 mM Tris-HCl pH 8.0 and rocked 
overnight at room temperature. The reaction was terminated by the addition 
of DPF (diisopropyl fluorophosphate, SIGMA) to a final concentration of 1 
mM. The mixture was rocked for an additional 30 minutes, dialysed against 
50 mM Tris pH 8.0 overnight and concentrated. The cleaved plasminogen was 
placed over a 2.5 cm.times.15 cm lysine-Sepharose 4B column (Brockway, W. 
J. and Castellino, F. J., Arch. Biochem. Biophys. 151: 194-199 (1972), 
which is hereby incorporated by reference) and equilibrated with 50 mM 
Tris pH 8.0 until an absorbance of 0.05 (at 280 nm) was reached. (This 
step was performed to remove any fragments containing a kringle 1 region 
and/or a kringle 4 region (both of which bind lysine)). The non-absorbed 
kringle 5 peptide fragments were dialysed against 50 mM Na.sub.2 PO.sub.4 
buffer, pH 5.0 then applied to a BioRad Mono-S column equilibrated with 
the same buffer. The cleaved kringle 5 portion, uncut mini-HPg and 
remaining protease domain fraction were eluted with a 0-20%, 20-50% and 
50-70% step gradient of 20 mM Phosphate/1 M KCl pH 5.0. The kringle 5 
peptide fragments eluted at the 50% step as determined by gel 
electrophesis. The collected peak was dialysed overnight against 20 mM 
Tris pH 8.0. 
The separated kringle 5 fragments were determined to be at least 95% pure 
by FPLC chromatography and DodSO4/PAGE with silver staining (Coomasie 
Blue). Sequence analysis of the amino terminal portion of the purified 
fragments revealed the presence of three polypeptides having 
.alpha.-N-terminus sequences of VLLPDVETPS, VAPPPVVLL and VETPSEED whch 
correspond to amino acid positions Val.sup.449 -Ser.sup.458, Val.sup.443 
-Leu.sup.450 and Val.sup.454 -Asp.sup.461 of SEQ ID. NO: 1, respectively. 
EXAMPLE 15 
Endothelial Proliferation Assay 
The in vitro proliferation of endothelial cells was determined as described 
by Lingen, et al., in Laboratory Investigation, 74: 476-483 (1996), which 
is hereby incorporated herein by reference, using the Cell Titer 96 
Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega Corporation, 
Madison, Wis.). Bovine capillary (adrenal) endothelial cells were plated 
at a density of 1000 cells per well in a 96-well plate in Dulbecco's 
Modified Eagle Medium (DMEM) containing 10% donor calf serum and 1% BSA 
(bovine serum albumin, GIBCO BRL, Gaithersburg, Md.). After 8 hours, the 
cells were starved overnight in DMEM containing 0.1% BSA then re-fed with 
media containing specified concentrations of inhibitor and 5 ng/mL bFGF 
(basic fibroblast growth factor). The results of the assay were corrected 
both for unstimulated cells (i.e. no bFGF added) as the baseline and for 
cells stimulated with bFGF alone (i.e. no inhibitor added) as the maximal 
proliferation. When multple experiments were combined, the results were 
represented as the percent change in cell number as compared to bFGF 
alone. 
EXAMPLE 16 
Endothelial Cell Migration Assay 
The endothelial cell migration assay was performed essentially as described 
by Polverini, P. J. et al., Methods Enzymol, 198: 440-450 (1991), which is 
hereby incorporated herein by reference. Briefly, bovine capillary 
(adrenal) endothelial cells (BCE, supplied by Judah Folkman, Harvard 
University Medical School) were starved overnight in DMEM containing 0.1% 
bovine serum albumin (BSA). Cells were then harvested with trypsin and 
resuspended in DMEM with 0.1% BSA at a concentration of 1.5.times.10.sup.6 
cells/mL. Cells were added to the bottom of a 48-well modified Boyden 
chamber (Nucleopore Corporation, Cabin John, Md.). The chamber was 
assembled and inverted, and cells were allowed to attach for 2 hours at 
37.degree. C. to polycarbonate chemotaxis membranes (5 .mu.m pore size) 
that had been soaked in 0.1% gelatin overnight and dried. The chamber was 
then reinverted and test substances were added to the wells of the upper 
chamber (to a total volume of 50 .mu.L); the apparatus was then incubated 
for 4 hours at 37.degree. C. Membranes were recovered, fixed and stained 
(DiffQuick, Fisher Scientific, Pittsburgh, Pa.) and the number of cells 
that had migrated to the upper chamber per 10 high power fields were 
counted. Background migration to DMEM+0.1% BSA was subtracted and the data 
reported as the number of cells migrated per 10 high power fields (400X) 
or when results from multiple experiments were combined, as the percent 
inhibition of migration compared to a positive control. The results are 
shown in Table 1. 
EXAMPLE 17 
Effect of Kringle 5 Peptide Fragments on Endothetial Cell Proliferation in 
vitro 
The effect of kringle 5 peptide fragments on endothelial cell proliferation 
was determined in vitro using the above described endothelial cell 
proliferation assay. For these experiments, kringle 5 peptide fragments 
was prepared as illustrated in Examples 1 through 14 and tested at various 
concentrations ranging from about 100 to 1000 pM with bFGF used as a 
maximum proliferation control. The kringle 5 peptide fragment SEQ ID NO: 3 
was effective at inhibiting BCE cell proliferation in a dose-dependent 
manner. The concentration of kringle 5 peptide fragment SEQ ID NO: 3 
required to reach 50% inhibition (ED.sub.50) was determined at about 300 
pM. In contrast, the ED.sub.50 of kringles 1-4 was shown to be 135 nM. 
A summary of the effect of other kringle peptide fragments on inhibition of 
BCE cell proliferation is shown in Table 1. The kringle 3 peptide fragment 
was least effective at inhibitng BCE cell proliferation (ED.sub.50 =460 
nM), followed by the kringle 1 peptide fragment (ED.sub.50 =320 nM), 
kringle 1-4 peptide fragments (ED.sub.50 =135 nM) and kringles 1-3 peptide 
fragments (ED.sub.50 =75 nM). The kringle 5 peptide fragment was the most 
effective at inhibiting BCE cell proliferation with an ED.sub.50 of 0.3 
nM. 
EXAMPLE 18 
Effect of Kringle 5 Peptide Fragments on Endothelial Cell Migration in vito 
The effect of kringle 5 peptide fragments on endothelial cell migration was 
also determined in vitro using the above described endothelial cell 
migration assay. Kringle 5 peptide fragments inhibited BCE cell migration 
in a dose-dependent fashion with an ED.sub.50 of approximately 300 pM. At 
the concentration of kringle 5 peptide fragments required for maximal 
inhibition of BCE cells, PC-3 cells and MDA 486 cells were also inhibited. 
This result, taken together with the result in Example 2, indicates that 
the inhibition of stimulated proliferation and migration of BCE cells by 
kringle 5 peptide fragments is both potent and specific to endothelial 
cells and not to normal or tumor cells. 
The foregoing are merely illustrative of the invention and are not intended 
to limit the invention to the disclosed compounds. Variations and changes 
which are obvious to one skilled in the art are intended to be within the 
scope and nature of the invention which are defined in the appended 
claims. 
Table 1 shows a summary of ED.sub.50 values obtained from the inhibition of 
various kringle fragments on BCE cell proliferation and cell migration in 
vitro. In the table, kringle peptide fragments are labeled according to 
their corresponding sequence homology to SEQ ID NO: 1. The symbol "*" 
indicates data taken from Marti, D., et al., Eur. J. Biochem., 219: 
455-462 (1994), which is hereby incorporated by reference. 
TABLE 1 
______________________________________ 
Antiproliferative 
Migratory 
Activity of Inhibition of 
Protein Fragment from BCE Cells HMVEC Cells 
SEQ ID NO:1 (ED.sub.50) (ED.sub.50) 
______________________________________ 
kringles 1-4(angiostatin)* 
135 nM 160 nM 
kringle 1 (Tyr.sup.80 -Glu.sup.163)* 320 nM -- 
kringle 2 (Glu.sup.161 -Thr.sup.245)* no activity -- 
kringle 3 (Thr.sup.253 -Ser.sup.335)* 460 nM -- 
kringle 4 (Val.sup.354 -Val.sup.443)* no activity -- 
kringles 1-3 (Tyr.sup.80 -Pro.sup.353)* 75 nM 60 nM 
kringles 2-3 (Glu.sup.161 -Ser.sup.335)* -- -- 
kringle 5 (Val.sup.443 -Ala.sup.543) 250 pM 200 pM 
kringle 5 (Val.sup.449 -Ala.sup.543) -- 240 pM 
kringle 5 (Val.sup.454 -Ala.sup.543) -- 220 pM 
kringle 5 (Val.sup.443 -Phe.sup.546) 60 nM 55 nM 
kringle 5 (Val.sup.449 -Phe.sup.546) -- -- 
kringle 5 (Val.sup.454 -Phe.sup.546) -- -- 
kringles 4-5 (Val.sup.355 -Ala.sup.543) -- 280 pM 
kringles 4-5 (Val.sup.355 -Phe.sup.546) -- -- 
N-Ac-Val.sup.449 -Asp.sup.461 -NH.sub.2 -- &gt;1 mM 
N-Ac-Met.sup.463 -Pro.sup.482 -NH.sub.2 -- &gt;1 mM 
N-Ac-Gln.sup.484 -Tyr.sup.511 -NH.sub.2 -- &gt;100 .mu.M 
N-Ac-Arg.sup.513 -Trp.sup.523 -NH.sub.2 -- 500 pM 
N-Ac-Tyr.sup.525 -Trp.sup.535 -NH.sub.2 -- 200 pM 
N-Ac-Pro.sup.529 -Tyr.sup.535 -NH.sub.2 -- 120 pM 
N-Ac-Pro.sup.529 -Asp.sup.534 -NH.sub.2 -- 123 pM 
N-Ac-Pro.sup.150 -Tyr.sup.156 -NH.sub.2 -- 160 nM 
N-Ac-Arg.sup.530 -Tyr.sup.535 -NH.sub.2 -- 80 nM 
N-Ac-Pro-Arg-Lys-Leu-3-I- -- &gt;100 nM 
Tyr-Asp-Tyr-NH.sub.2 
N-Ac-Pro-Arg-Lys-Leu-Tyr- -- 400 pM 
Asp-3-I-Tyr-NH.sub.2 
N-Ac-Lys.sup.531 -Tyr.sup.534 -NH.sub.2 -- -- 
______________________________________ 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 14 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 791 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: prote - #in 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:1: 
- - Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gl - #y Ala Ser Leu Phe 
Ser 
1 5 - # 10 - # 15 
- - Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Il - #e Glu Glu Cys Ala Ala 
20 - # 25 - # 30 
- - Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Ar - #g Ala Phe Gln Tyr His 
35 - # 40 - # 45 
- - Ser Lys Glu Gln Gln Cys Val Ile Met Ala Gl - #u Asn Arg Lys Ser Ser 
50 - # 55 - # 60 
- - Ile Ile Ile Arg Met Arg Asp Val Val Leu Ph - #e Glu Lys Lys Val Tyr 
65 - # 70 - # 75 - # 80 
- - Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys As - #n Tyr Arg Gly Thr Met 
85 - # 90 - # 95 
- - Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Ly - #s Trp Ser Ser Thr Ser 
100 - # 105 - # 110 
- - Pro His Arg Pro Arg Phe Ser Pro Ala Thr Hi - #s Pro Ser Glu Gly Leu 
115 - # 120 - # 125 
- - Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn As - #p Pro Gln Gly Pro Trp 
130 - # 135 - # 140 
- - Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr As - #p Tyr Cys Asp Ile Leu 
145 1 - #50 1 - #55 1 - 
#60 
- - Glu Cys Glu Glu Glu Cys Met His Cys Ser Gl - #y Glu Asn Tyr Asp 
Gly 
165 - # 170 - # 175 
- - Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cy - #s Gln Ala Trp Asp Ser 
180 - # 185 - # 190 
- - Gln Ser Pro His Ala His Gly Tyr Ile Pro Se - #r Lys Phe Pro Asn Lys 
195 - # 200 - # 205 
- - Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro As - #p Arg Glu Leu Arg Pro 
210 - # 215 - # 220 
- - Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Tr - #p Glu Leu Cys Asp Ile 
225 2 - #30 2 - #35 2 - 
#40 
- - Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gl - #y Pro Thr Tyr Gln 
Cys 
245 - # 250 - # 255 
- - Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly As - #n Val Ala Val Thr Val 
260 - # 265 - # 270 
- - Ser Gly His Thr Cys Gln His Trp Ser Ala Gl - #n Thr Pro His Thr His 
275 - # 280 - # 285 
- - Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys As - #n Leu Asp Glu Asn Tyr 
290 - # 295 - # 300 
- - Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Tr - #p Cys His Thr Thr Asn 
305 3 - #10 3 - #15 3 - 
#20 
- - Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pr - #o Ser Cys Asp Ser 
Ser 
325 - # 330 - # 335 
- - Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Al - #a Pro Pro Glu Leu Thr 
340 - # 345 - # 350 
- - Pro Val Val Gln Asp Cys Tyr His Gly Asp Gl - #y Gln Ser Tyr Arg Gly 
355 - # 360 - # 365 
- - Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cy - #s Gln Ser Trp Ser Ser 
370 - # 375 - # 380 
- - Met Thr Pro His Arg His Gln Lys Thr Pro Gl - #u Asn Tyr Pro Asn Ala 
385 3 - #90 3 - #95 4 - 
#00 
- - Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro As - #p Ala Asp Lys Gly 
Pro 
405 - # 410 - # 415 
- - Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Tr - #p Glu Tyr Cys Asn Leu 
420 - # 425 - # 430 
- - Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Va - #l Ala Pro Pro Pro Val 
435 - # 440 - # 445 
- - Val Leu Leu Pro Asp Val Glu Thr Pro Ser Gl - #u Glu Asp Cys Met Phe 
450 - # 455 - # 460 
- - Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Al - #a Thr Thr Val Thr Gly 
465 4 - #70 4 - #75 4 - 
#80 
- - Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pr - #o His Arg His Ser 
Ile 
485 - # 490 - # 495 
- - Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Le - #u Glu Lys Asn Tyr Cys 
500 - # 505 - # 510 
- - Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Tr - #p Cys Tyr Thr Thr Asn 
515 - # 520 - # 525 
- - Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pr - #o Gln Cys Ala Ala Pro 
530 - # 535 - # 540 
- - Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pr - #o Lys Lys Cys Pro Gly 
545 5 - #50 5 - #55 5 - 
#60 
- - Arg Val Val Gly Gly Cys Val Ala His Pro Hi - #s Ser Trp Pro Trp 
Gln 
565 - # 570 - # 575 
- - Val Ser Leu Arg Thr Arg Phe Gly Met His Ph - #e Cys Gly Gly Thr Leu 
580 - # 585 - # 590 
- - Ile Ser Pro Glu Trp Val Leu Thr Ala Ala Hi - #s Cys Leu Glu Lys Ser 
595 - # 600 - # 605 
- - Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gl - #y Ala His Gln Glu Val 
610 - # 615 - # 620 
- - Asn Leu Glu Pro His Val Gln Glu Ile Glu Va - #l Ser Arg Leu Phe Leu 
625 6 - #30 6 - #35 6 - 
#40 
- - Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Ly - #s Leu Ser Ser Pro 
Ala 
645 - # 650 - # 655 
- - Val Ile Thr Asp Lys Val Ile Pro Ala Cys Le - #u Pro Ser Pro Asn Tyr 
660 - # 665 - # 670 
- - Val Val Ala Asp Arg Thr Glu Cys Phe Ile Th - #r Gly Trp Gly Glu Thr 
675 - # 680 - # 685 
- - Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Gl - #u Ala Gln Leu Pro Val 
690 - # 695 - # 700 
- - Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Ph - #e Leu Asn Gly Arg Val 
705 7 - #10 7 - #15 7 - 
#20 
- - Gln Ser Thr Glu Leu Cys Ala Gly His Leu Al - #a Gly Gly Thr Asp 
Ser 
725 - # 730 - # 735 
- - Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cy - #s Phe Glu Lys Asp Lys 
740 - # 745 - # 750 
- - Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Le - #u Gly Cys Ala Arg Pro 
755 - # 760 - # 765 
- - Asn Lys Pro Gly Val Tyr Val Arg Val Ser Ar - #g Phe Val Thr Trp Ile 
770 - # 775 - # 780 
- - Glu Gly Val Met Arg Asn Asn 
785 7 - #90 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 101 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: prote - #in 
- - (v) FRAGMENT TYPE: internal 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:2: 
- - Val Ala Pro Pro Pro Val Val Leu Leu Pro As - #p Val Glu Thr Pro Ser 
1 5 - # 10 - # 15 
- - Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gl - #y Tyr Arg Gly Lys Arg 
20 - # 25 - # 30 
- - Ala Thr Thr Val Thr Gly Thr Pro Cys Gln As - #p Trp Ala Ala Gln Glu 
35 - # 40 - # 45 
- - Pro His Arg His Ser Ile Phe Thr Pro Glu Th - #r Asn Pro Arg Ala Gly 
50 - # 55 - # 60 
- - Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gl - #y Asp Val Gly Gly Pro 
65 - #70 - #75 - #80 
- - Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Ty - #r Asp Tyr Cys Asp Val 
85 - # 90 - # 95 
- - Pro Gln Cys Ala Ala 
100 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 95 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: protein 
- - (v) FRAGMENT TYPE: internal 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:3: 
- - Val Leu Leu Pro Asp Val Glu Thr Pro Ser Gl - #u Glu Asp Cys Met Phe 
1 5 - # 10 - # 15 
- - Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Al - #a Thr Thr Val Thr Gly 
20 - # 25 - # 30 
- - Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pr - #o His Arg His Ser Ile 
35 - # 40 - # 45 
- - Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Le - #u Glu Lys Asn Tyr Cys 
50 - # 55 - # 60 
- - Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Tr - #p Cys Tyr Thr Thr Asn 
65 - #70 - #75 - #80 
- - Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pr - #o Gln Cys Ala Ala 
85 - # 90 - # 95 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 90 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: prote - #in 
- - (v) FRAGMENT TYPE: internal 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:4: 
- - Val Glu Thr Pro Ser Glu Glu Asp Cys Met Ph - #e Gly Asn Gly Lys Gly 
1 5 - # 10 - # 15 
- - Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gl - #y Thr Pro Cys Gln Asp 
20 - # 25 - # 30 
- - Trp Ala Ala Gln Glu Pro His Arg His Ser Il - #e Phe Thr Pro Glu Thr 
35 - # 40 - # 45 
- - Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cy - #s Arg Asn Pro Asp Gly 
50 - # 55 - # 60 
- - Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr As - #n Pro Arg Lys Leu Tyr 
65 - #70 - #75 - #80 
- - Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala 
85 - # 90 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 104 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: prote - #in 
- - (v) FRAGMENT TYPE: internal 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:5: 
- - Val Ala Pro Pro Pro Val Val Leu Leu Pro As - #p Val Glu Thr Pro Ser 
1 5 - # 10 - # 15 
- - Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gl - #y Tyr Arg Gly Lys Arg 
20 - # 25 - # 30 
- - Ala Thr Thr Val Thr Gly Thr Pro Cys Gln As - #p Trp Ala Ala Gln Glu 
35 - # 40 - # 45 
- - Pro His Arg His Ser Ile Phe Thr Pro Glu Th - #r Asn Pro Arg Ala Gly 
50 - # 55 - # 60 
- - Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gl - #y Asp Val Gly Gly Pro 
65 - #70 - #75 - #80 
- - Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Ty - #r Asp Tyr Cys Asp Val 
85 - # 90 - # 95 
- - Pro Gln Cys Ala Ala Pro Ser Phe 
100 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 98 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: prote - #in 
- - (v) FRAGMENT TYPE: internal 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:6: 
- - Val Leu Leu Pro Asp Val Glu Thr Pro Ser Gl - #u Glu Asp Cys Met Phe 
1 5 - # 10 - # 15 
- - Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Al - #a Thr Thr Val Thr Gly 
20 - # 25 - # 30 
- - Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pr - #o His Arg His Ser Ile 
35 - # 40 - # 45 
- - Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Le - #u Glu Lys Asn Tyr Cys 
50 - # 55 - # 60 
- - Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Tr - #p Cys Tyr Thr Thr Asn 
65 - #70 - #75 - #80 
- - Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pr - #o Gln Cys Ala Ala Pro 
85 - # 90 - # 95 
- - Ser Phe 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 93 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: prote - #in 
- - (v) FRAGMENT TYPE: internal 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:7: 
- - Val Glu Thr Pro Ser Glu Glu Asp Cys Met Ph - #e Gly Asn Gly Lys Gly 
1 5 - # 10 - # 15 
- - Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gl - #y Thr Pro Cys Gln Asp 
20 - # 25 - # 30 
- - Trp Ala Ala Gln Glu Pro His Arg His Ser Il - #e Phe Thr Pro Glu Thr 
35 - # 40 - # 45 
- - Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cy - #s Arg Asn Pro Asp Gly 
50 - # 55 - # 60 
- - Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr As - #n Pro Arg Lys Leu Tyr 
65 - #70 - #75 - #80 
- - Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pr - #o Ser Phe 
85 - # 90 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 102 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: prote - #in 
- - (v) FRAGMENT TYPE: internal 
- - (ix) FEATURE: 
(A) NAME/KEY: mouse - #sequence 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:8: 
- - Val Glu Leu Pro Thr Val Ser Gln Glu Pro Se - #r Gly Pro Ser Asp Ser 
1 5 - # 10 - # 15 
- - Glu Thr Asp Cys Met Tyr Gly Asn Asp Lys As - #p Tyr Arg Thr Lys Thr 
20 - # 25 - # 30 
- - Ala Val Ala Ala Ala Gly Thr Pro Gly Gln Gl - #y Trp Ala Ala Gln Glu 
35 - # 40 - # 45 
- - Pro His Arg His Ser Ile Phe Thr Pro Gln Th - #r Asn Pro Arg Ala Gly 
50 - # 55 - # 60 
- - Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gl - #y Asp Val Asn Gly Pro 
65 - #70 - #75 - #80 
- - Trp Cys Tyr Thr Thr Asn Pro Arg Ser Leu Ty - #r Asp Tyr Cys Asp Ile 
- # 85 - # 90 - # 95 
- - Pro Leu Cys Ala Ser Ala 
100 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 100 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: prote - #in 
- - (v) FRAGMENT TYPE: internal 
- - (ix) FEATURE: 
(A) NAME/KEY: monkey - #sequence 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:9: 
- - Ala Ala Pro Pro Pro Val Ala Gln Leu Pro As - #p Ala Glu Thr Pro Ser 
1 5 - # 10 - # 15 
- - Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gl - #y Tyr Arg Gly Lys Lys 
20 - # 25 - # 30 
- - Ala Thr Thr Val Thr Gly Thr Pro Cys Gln As - #p Trp Ala Ala Gln Glu 
35 - # 40 - # 45 
- - Pro His Ser His Arg Ile Phe Thr Pro Glu Th - #r Asn Pro Arg Ala Gly 
50 - # 55 - # 60 
- - Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gl - #y Asp Val Gly Gly Pro 
65 - #70 - #75 - #80 
- - Trp Cys Tyr Thr Thr Asn Pro Arg Ser Leu Ph - #e Asp Tyr Cys Asp Val 
85 - # 90 - # 95 
- - Pro Gln Cys Ala 
100 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 97 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: prote - #in 
- - (v) FRAGMENT TYPE: internal 
- - (ix) FEATURE: 
(A) NAME/KEY: bovine se - #quence 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:10: 
- - Pro Ala Ala Pro Gln Ala Pro Gly Val Glu As - #n Pro Pro Glu Ala Asp 
1 5 - # 10 - # 15 
- - Cys Met Ile Gly Thr Gly Lys Ser Tyr Arg Gl - #y Lys Lys Ala Thr Thr 
20 - # 25 - # 30 
- - Val Ala Gly Val Pro Cys Gln Glu Trp Ala Al - #a Gln Glu Pro His His 
35 - # 40 - # 45 
- - His Ser Ile Phe Thr Pro Glu Thr Asn Pro Gl - #n Ser Gly Leu Glu Arg 
50 - # 55 - # 60 
- - Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val As - #n Gly Pro Trp Cys Tyr 
65 - #70 - #75 - #80 
- - Thr Met Asn Pro Arg Ser Leu Phe Asp Tyr Cy - #s Asp Val Pro Gln Cys 
85 - # 90 - # 95 
- - Glu 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 101 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: prote - #in 
- - (v) FRAGMENT TYPE: internal 
- - (ix) FEATURE: 
(A) NAME/KEY: porcine s - #equence 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:11: 
- - Thr Asn Phe Pro Ala Ile Ala Gln Val Pro Se - #r Val Glu Asp Leu Ser 
1 5 - # 10 - # 15 
- - Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Ar - #g Tyr Arg Gly Lys Arg 
20 - # 25 - # 30 
- - Ala Thr Thr Val Ala Gly Val Pro Cys Gln Gl - #u Trp Ala Ala Gln Glu 
35 - # 40 - # 45 
- - Pro His Arg His Ser Ile Phe Thr Pro Glu Th - #r Asn Pro Arg Ala Gly 
50 - # 55 - # 60 
- - Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gl - #y Asp Asp Asn Gly Pro 
65 - #70 - #75 - #80 
- - Trp Cys Tyr Thr Thr Asn Pro Gln Lys Leu Ph - #e Asp Tyr Cys Asp Val 
85 - # 90 - # 95 
- - Pro Gln Cys Val Thr 
100 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2497 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:12: 
- - CATCCTGGGA TTGGGACCCA CTTTCTGGGC ACTGCTGGCC AGTCCCAAAA TG - 
#GAACATAA 60 
- - GGAAGTGGTT CTTCTACTTC TTTTATTTCT GAAATCAGGT CAAGGAGAGC CT - 
#CTGGATGA 120 
- - CTATGTGAAT ACCCAGGGGG CTTCACTGTT CAGTGTCACT AAGAAGCAGC TG - 
#GGAGCAGG 180 
- - AAGTATAGAA GAATGTGCAG CAAAATGTGA GGAGGACGAA GAATTCACCT GC - 
#AGGGCATT 240 
- - CCAATATCAC AGTAAAGAGC AACAATGTGT GATAATGGCT GAAAACAGGA AG - 
#TCCTCCAT 300 
- - AATCATTAGG ATGAGAGATG TAGTTTTATT TGAAAAGAAA GTGTATCTCT CA - 
#GAGTGCAA 360 
- - GACTGGGAAT GGAAAGAACT ACAGAGGGAC GATGTCCAAA ACAAAAAATG GC - 
#ATCACCTG 420 
- - TCAAAAATGG AGTTCCACTT CTCCCCACAG ACCTAGATTC TCACCTGCTA CA - 
#CACCCCTC 480 
- - AGAGGGACTG GAGGAGAACT ACTGCAGGAA TCCAGACAAC GATCCGCAGG GG - 
#CCCTGGTG 540 
- - CTATACTACT GATCCAGAAA AGAGATATGA CTACTGCGAC ATTCTTGAGT GT - 
#GAAGAGGA 600 
- - ATGTATGCAT TGCAGTGGAG AAAACTATGA CGGCAAAATT TCCAAGACCA TG - 
#TCTGGACT 660 
- - GGAATGCCAG GCCTGGGACT CTCAGAGCCC ACACGCTCAT GGATACATTC CT - 
#TCCAAATT 720 
- - TCCAAACAAG AACCTGAAGA AGAATTACTG TCGTAACCCC GATAGGGAGC TG - 
#CGGCCTTG 780 
- - GTGTTTCACC ACCGACCCCA ACAAGCGCTG GGAACTTTGT GACATCCCCC GC - 
#TGCACAAC 840 
- - ACCTCCACCA TCTTCTGGTC CCACCTACCA GTGTCTGAAG GGAACAGGTG AA - 
#AACTATCG 900 
- - CGGGAATGTG GCTGTTACCG TGTCCGGGCA CACCTGTCAG CACTGGAGTG CA - 
#CAGACCCC 960 
- - TCACACACAT AACAGGACAC CAGAAAACTT CCCCTGCAAA AATTTGGATG AA - 
#AACTACTG 1020 
- - CCGCAATCCT GACGGAAAAA GGGCCCCATG GTGCCATACA ACCAACAGCC AA - 
#GTGCGGTG 1080 
- - GGAGTACTGT AAGATACCGT CCTGTGACTC CTCCCCAGTA TCCACGGAAC AA - 
#TTGGCTCC 1140 
- - CACAGCACCA CCTGAGCTAA CCCCTGTGGT CCAGGACTGC TACCATGGTG AT - 
#GGACAGAG 1200 
- - CTACCGAGGC ACATCCTCCA CCACCACCAC AGGAAAGAAG TGTCAGTCTT GG - 
#TCATCTAT 1260 
- - GACACCACAC CGGCACCAGA AGACCCCAGA AAACTACCCA AATGCTGGCC TG - 
#ACAATGAA 1320 
- - CTACTGCAGG AATCCAGATG CCGATAAAGG CCCCTGGTGT TTTACCACAG AC - 
#CCCAGCGT 1380 
- - CAGGTGGGAG TACTGCAACC TGAAAAAATG CTCAGGAACA GAAGCGAGTG TT - 
#GTAGCACC 1440 
- - TCCGCCTGTT GTCCTGCTTC CAGATGTAGA GACTCCTTCC GAAGAAGACT GT - 
#ATGTTTGG 1500 
- - GAATGGGAAA GGATACCGAG GCAAGAGGGC GACCACTGTT ACTGGGACGC CA - 
#TGCCAGGA 1560 
- - CTGGGCTGCC CAGGAGCCCC ATAGACACAG CATTTTCACT CCAGAGACAA AT - 
#CCACGGGC 1620 
- - GGGTCTGGAA AAAAATTACT GCCGTAACCC TGATGGTGAT GTAGGTGGTC CC - 
#TGGTGCTA 1680 
- - CACGACAAAT CCAAGAAAAC TTTACGACTA CTGTGATGTC CCTCAGTGTG CG - 
#GCCCCTTC 1740 
- - ATTTGATTGT GGGAAGCCTC AAGTGGAGCC GAAGAAATGT CCTGGAAGGG TT - 
#GTAGGGGG 1800 
- - GTGTGTGGCC CACCCACATT CCTGGCCCTG GCAAGTCAGT CTTAGAACAA GG - 
#TTTGGAAT 1860 
- - GCACTTCTGT GGAGGCACCT TGATATCCCC AGAGTGGGTG TTGACTGCTG CC - 
#CACTGCTT 1920 
- - GGAGAAGTCC CCAAGGCCTT CATCCTACAA GGTCATCCTG GGTGCACACC AA - 
#GAAGTGAA 1980 
- - TCTCGAACCG CATGTTCAGG AAATAGAAGT GTCTAGGCTG TTCTTGGAGC CC - 
#ACACGAAA 2040 
- - AGATATTGCC TTGCTAAAGC TAAGCAGTCC TGCCGTCATC ACTGACAAAG TA - 
#ATCCCAGC 2100 
- - TTGTCTGCCA TCCCCAAATT ATGTGGTCGC TGACCGGACC GAATGTTTCG TC - 
#ACTGGCTG 2160 
- - GGGAGAAACC CAAGGTACTT TTGGAGCTGG CCTTCTCAAG GAAGCCCAGC TC - 
#CCTGTGAT 2220 
- - TGAGAATAAA GTGTGCAATC GCTATGAGTT TCTGAATGGA AGAGTCCAAT CC - 
#ACCGAACT 2280 
- - CTGTGCTGGG CATTTGGCCG GAGGCACTGA CAGTTGCCAG GGTGACAGTG GA - 
#GGTCCTCT 2340 
- - GGTTTGCTTC GAGAAGGACA AATACATTTT ACAAGGAGTC ACTTCTTGGG GT - 
#CTTGGCTG 2400 
- - TGCACGCCCC AATAAGCCTG GTGTCTATGT TCGTGTTTCA AGGTTTGTTA CT - 
#TGGATTGA 2460 
- - GGGAGTGATG AGAAATAATT AATTGGACGG GAGACAG - # 
- # 2497 
- - - - (2) INFORMATION FOR SEQ ID NO:13: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: pepti - #de 
- - (v) FRAGMENT TYPE: internal fragment 
- - (ix) FEATURE: 
(A) OTHER INFORMATION: - #Tyr number 7 is 3-I-Tyr. 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:13: 
- - Pro Arg Lys Leu Tyr Asp Tyr 
1 5 
- - - - (2) INFORMATION FOR SEQ ID NO:14: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: 
(A) DESCRIPTION: pepti - #de 
- - (v) FRAGMENT TYPE: internal fragment 
- - (ix) FEATURE: 
(A) OTHER INFORMATION: - #Tyr number 5 is 3-I-Tyr. 
- - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:14: 
- - Pro Arg Lys Leu Tyr Asp Tyr 
1 5 
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