# Patent

## Title
Peptide modulators of angiogenesis and use thereof

## Abstract

Compositions and methods that are useful for modulating blood vessel formation, as well as methods that provide for the systematic and efficient identification of angiogenesis modulators are described. As discussed in more detail below, a systematic computational methodology based on bioinformatics was used to identify novel peptide modulators of angiogenesis that have been characterized in vitro and/or in vivo.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of the following U.S. Provisional Application No. 60/878,579, filed on Jan. 3, 2007, the entire contents of which are incorporated herein by reference.


STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
This work was supported by the following grant from the National Institutes of Health, Grant No.: HL079653 and CA103175. The government may have certain rights in the invention.


BACKGROUND OF THE INVENTION
Angiogenesis, the process of developing a novel vascular network from a pre-existing one, is tightly controlled by various endogenous regulators. These regulatory elements include both pro- and anti-angiogenic proteins that finely modulate the neovascular morphological and functional characteristics. Where the regulation of such processes is disrupted a variety of pathological conditions can ensue, including neoplasia, hematologic malignancies, rheumatoid arthritis, diabetic retinopathy, age-related macular degeneration, atherosclerosis, endometriosis, pathologic obesity, and ischemic heart and limb disease. An urgent need exists for angiogenesis modulators that can be used as therapeutics for these and other numerous angiogenesis related diseases and conditions. While some promising angiogenesis modulators have been identified, to date, the quest for the experimental identification of such agents has been an empirical time-consuming process. Improved angiogenesis modulators and methods for systematically identifying and assessing the biological activity of such agents are urgently required.
SUMMARY OF THE INVENTION
As described below, the present invention generally features angiogenesis modulators, related prophylactic and therapeutic methods, as well as screening methods for the identification of such agents.
The invention generally provides peptides that reduce blood vessel formation in a cell, tissue, or organ. Accordingly, in one aspect the invention features an isolated peptide or analog thereof containing one of the following amino acid sequences:










TSP Motif:
W-X(2)-C-X(3)-C-X(2)-G,


 



CXC Motif:
G-X(3)-C-L


 



Collagen Motif:
C-N-X(3)-V-C


 



Collagen Motif:
P-F-X(2)-C


 



Somatotropin Motif:
L-X(3)-L-L-X(3)-S-X-L


 



Serpin Motif:
L-X(2)-E-E-X-P;






where X denotes a variable amino acid and the number in parentheses denotes the number of variable amino acids; W denotes tryptophan; C denotes cysteine, G denotes glycine, V denotes valine; L denotes leucine, P is proline, and where the peptide reduces blood vessel formation in a cell, tissue or organ. In one embodiment, the peptide contains an amino acid sequence shown in Table 1-6, 8 and 9. In yet another embodiment, the peptide further contains at least 5, 10, 15, or 20 amino acids flanking the naturally occurring sequence.

In another aspect, the invention features an isolated peptide or analog thereof having at least 85%, 90%, 95%, or 100% identity to an amino acid sequence shown in Table 1-10 or otherwise disclosed herein. In one embodiment, the peptide contains an amino acid sequence shown in Table 1-10. In another embodiment, the peptide consists essentially of an amino acid sequence shown in Table 1-10. In yet another embodiment, the peptide further contains at least 5, 10, 15, or 20 amino acids flanking the naturally occurring sequence.
In yet another aspect, the invention features an isolated peptide or analog thereof containing or consisting essentially of a sequence having at least 85% 90%, 95%, or 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of:










Placental Lactogen
LLRISLLLIESWLE


 



hGH-V
LLRISLLLTQSWLE


 



GH2
LLHISLLLIQSWLE


 



Chorionic somatomammotropin
LLRLLLLIESWLE


 



Chorionic somatomammotropin
LLHISLLLIESRLE



hormone-like 1



 



Transmembrane protein 45A
LLRSSLILLQGSWF


 



IL-17 receptor C
RLRLLTLQSWLL


 



Neuropeptide FF receptor 2
LLIVALLFILSWL


 



Brush border myosin-I
LMRKSQILISSWF






where the peptide reduces blood vessel formation in a cell, tissue or organ.

In yet another aspect, the invention features an isolated peptide or analog thereof containing or consisting essentially of a sequence having at least 85%, 90%, 95%, or 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of:










DEAH box polypeptide 8
EIELVEEEPPF


 



Caspase 10
AEDLLSEEDPF


 



CKIP-1
TLDLIQEEDPS






where the peptide reduces blood vessel formation in a cell, tissue or organ.

In yet another aspect, the invention features an isolated peptide or analog thereof containing or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:










Collagen type IV, alpha6
LPRFSTMPFIYCNINEVCHY



fibril







where the peptide reduces blood vessel formation in a cell, tissue or organ.

In another aspect, the invention features a pharmaceutical composition containing an effective amount of an isolated peptide containing an amino acid sequence shown in Table 1-10 or a peptide analog thereof in a pharmacologically acceptable excipient. In one embodiment, the composition contains at least one peptide that is a TSP, CXC, Collagen IV, Somatotropin, or Serpin derived peptide. In another embodiment, the composition contains at least two, three, four, or five peptides selected from the group consisting of TSP, CXC, Collagen IV, Somatotropin, and Serpin derived peptides. In one embodiment, the composition contains at least a CXC derived peptide and a TSP1 derived peptide. In another embodiment, the CXC derived peptide contains the amino acid sequence NGRKACLNPASPIVKKIIEKMLNS. In yet another embodiment, the TSP1 repeat-containing protein contains the amino acid sequence GPWEPCSVTCSKGTRTRRR.
In a related aspect, the invention features an isolated nucleic acid molecule encoding the peptide of any previous aspect.
In another related aspect, the invention features an expression vector containing the nucleic acid molecule of the previous aspect, where the nucleic acid molecule is positioned for expression. In one embodiment, the vector includes a promoter suitable for expressing the nucleic acid molecule in a mammalian cell.
In yet another related aspect, the invention features a host cell containing the peptide of any previous aspect or a nucleic acid molecule encoding the peptide. In one embodiment, the cell is a prokaryotic or eukaryotic cell (e.g., mammalian, human). In another embodiment, the cell is in vitro or in vivo.
In another aspect, the invention features a method of reducing blood vessel formation in a tissue or organ, the method involving contacting an endothelial cell, or a tissue or organ containing an endothelial cell with an effective amount of a peptide of any previous aspect, thereby reducing blood vessel formation in the tissue or organ.
In yet another aspect, the invention features a method of reducing endothelial cell proliferation, migration, survival, or stability in a tissue or organ, the method involving contacting tissue or organ containing an endothelial cell with an effective amount of a peptide of any previous aspect.
In still another aspect, the invention features a method of increasing endothelial cell death in a tissue or organ, the method involving contacting a tissue or organ containing an endothelial cell with an effective amount of a peptide of any previous aspect, thereby increasing endothelial cell death in the tissue or organ.
In another aspect, the invention features a method of reducing blood vessel formation in a tissue or organ the method involving contacting the tissue, or organ with a vector encoding a peptide of any previous aspect; and expressing the peptide in a cell of the tissue or organ, thereby reducing blood vessel formation in the tissue or organ.
In another aspect, the invention features a method of modulating angiogenesis in a cell, tissue, or organ, the method involving contacting the cell, tissue, or organ with an effective amount of an agent that binds CD36, CD47 or CXCR3.
In another aspect, the invention features a method for treating a neoplasia in a subject in need thereof, the method involving administering an effective amount of a peptide of any previous aspect. In one embodiment, at least one peptide binds CD36, CD47 or CXCR3. In another embodiment, the method involves administering two peptides, one that binds CD36 or CD47 and one that binds CXCR3. In yet another embodiment, the method reduces angiogenesis in a neoplastic tissue. In yet another embodiment, the neoplasia is lung carcinoma.
In another aspect, the invention features a kit containing an effective amount of a peptide of any previous aspect, and directions for using the peptide to treat a disease characterized by undesirable or excess angiogenesis.
In various embodiments of any of the above aspects, the peptide contains a motif delineated herein or an amino acid sequence delineated herein. In various embodiments of the above aspects, the peptide contains an alteration in one amino acid relative to a reference sequence shown in Tables 1-10. In various embodiments of the above aspects, the peptide contains at least one modification (e.g., a sequence alteration or post-translational modification that increases protease resistance, biodistribution, or therapeutic efficacy). In various embodiments of the above aspects, the peptide is cyclized or pegylated. In other embodiments delineated herein, the sequence alteration replaces a cysteine with aminobutyric acid (Abu), serine or alanine, replaces methionine with isoleucine, or replaces lysine with arginine. In various embodiments of the above aspects, the peptide contains at least 10, 20, 30, 40, or 50 amino acids of a naturally occurring amino acid sequence described by an NCBI reference number listed in Table 1-10. In various embodiments of the above aspects, the tissue or organ is in vitro or in vivo. In other embodiments, the cell is a human cell, tissue, or organ. In yet other embodiments, the cell is a neoplastic cell (lung carcinoma cell). In another embodiment, the method treats a neoplasia (e.g., lung carcinoma). In another embodiment, the method treats corneal or choroidal neovascularization. In another embodiment, the number or volume of blood vessels in the tissue or organ (e.g., mammalian tissue or organ) are reduced by at least 10%, 25%, 30%, 50%, 75% or more relative to a control condition. In another embodiment, the peptide acts on an endothelial cell. In various embodiments of the above aspects, the method involves contacting the cell tissue or organ with two agents, one that binds CD36 or CD47 and one that binds CXCR3 (e.g., a CXC derived peptide or a TSP1 derived peptide). In another embodiment, the method involves administering at least two peptides, such as a CXC derived peptide and a TSP1 derived peptide; a peptide that binds CD36 or CD47 and a peptide that binds BetaI or BetaIII integrin; a TSP derived peptide and a collagen IV derived peptide. In various embodiments of the above aspects, the method involves administering a combination of two, three, four, or more peptides shown in Table 1-10.
DEFINITIONS
By “analog” is meant a chemical compounds having a structure that is different from the general structure of a reference agent, but that functions in a manner similar to the reference agent. For example, a peptide analog having a variation in sequence or having a modified amino acid.
By “thrombospondin (TSP) derived peptide” is meant a peptide comprising a TSP motif: W-X(2)-C-X(3)-C-X(2)-G. Exemplary TSP derived peptides are shown in Tables 1 and 2. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence of the peptide. TSP1 derived peptides include, for example, those derived from proteins WISP-1 (SPWSPCSTSCGLGVSTR1), NOVH (TEWTACSKSCGMGFSTRV) and UNC5C (TEWSVCNSRCGRGYQKRTR).
By “CXC derived peptide” is meant a peptide comprising a CXC Motif: G-X(3)-C-L. Exemplary CXC derived peptides are shown in Table 3. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence. CXC derived peptides include, for example, those derived from proteins GRO-α/CXCL1 (NGRKACLNPASPIVKKIIEKMLNS), GRO-γ/MIP-2β/CXCL3 (NGKKACLNPASPMVQKIIEKIL), and ENA-78/CXCL5 (NGKEICLDPEAPFLKKVIQKILD).
By “Collagen IV derived peptide” is meant a peptide comprising a C-N-X(3)-V-C or P-F-X(2)-C collagen motif. Exemplary collagen IV derived peptides are shown in Table 5. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence. Type IV collagen derived peptides include, for example, LRRFSTMPFMFCNINNVCNF and FCNINNVCNFASRNDYSYWL, and LPRFSTMPFIYCNINEVCHY.
By “Somatotropin derived peptide” is meant a peptide comprising a Somatotropin Motif: L-X(3)-L-L-X(3)-S-X-L. Exemplary somatotropin derived peptides are shown in Table 8. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence. Somatotropin derived peptides include, for example, those shown in FIG. 10A.
By “Serpin derived peptide” is meant a peptide comprising a Serpin Motif: L-X(2)-E-E-X-P. Exemplary serpin derived peptides are shown in Table 9. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence. Serpin derived peptides include, for example, those shown in FIG. 10B.
By “Beta 1 integrin” is meant a polypeptide that binds a collagen IV derived peptide or that has at least about 85% identity to NP—596867 or a fragment thereof.
By “Beta 3 integrin” is meant a polypeptide that binds a collagen IV derived peptide or that has at least about 85% identity to P05106 or a fragment thereof.
By “CD36” is meant a CD36 glycoprotein that binds to a thrombospondin-derived peptide or that has at least about 85% identity to NP—001001548 or a fragment thereof. CD36 is described, for example, by Oquendo et al., “CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes,” Cell 58: 95-101, 1989.
By “CD47” is meant a CD47 glycoprotein that binds to a thrombospondin-derived peptides or that has at least about 85% identity to NP—000315 or a fragment thereof. CD47 is described, for example, by Han et al., “CD47, a ligand for the macrophage fusion receptor, participates in macrophage multinucleation.” J. Biol. Chem. 275: 37984-37992, 2000.
By “CXCR3” is meant a G protein coupled receptor or fragment thereof having at least about 85% identity to NP—001495. CXCR3 is described, for example, by Trentin et al., “The chemokine receptor CXCR3 is expressed on malignant B cells and mediates chemotaxis.” J. Clin. Invest. 104: 115-121, 1999.
By “blood vessel formation” is meant the dynamic process that includes one or more steps of blood vessel development and/or maturation, such as angiogenesis, vasculogenesis, formation of an immature blood vessel network, blood vessel remodeling, blood vessel stabilization, blood vessel maturation, blood vessel differentiation, or establishment of a functional blood vessel network.
By “angiogenesis” is meant the growth of new blood vessels originating from existing blood vessels. Angiogenesis can be assayed by measuring the total length of blood vessel segments per unit area, the functional vascular density (total length of perfused blood vessel per unit area), or the vessel volume density (total of blood vessel volume per unit volume of tissue).
By “vasculogenesis” is meant the development of new blood vessels originating from stem cells, angioblasts, or other precursor cells.
By “blood vessel stability” is meant the maintenance of a blood vessel network.
By “alteration” is meant a change in the sequence or in a modification (e.g., a post-translational modification) of a gene or polypeptide relative to an endogeneous wild-type reference sequence.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
By “antibody” is meant any immunoglobulin polypeptide, or fragment thereof, having immunogen binding ability.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
A “cancer” in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers. In some circumstances, cancer cells will be in the form of a tumor; such cells may exist locally within an animal, or circulate in the blood stream as independent cells, for example, leukemic cells.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
By “an effective amount” is meant the amount required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of an angiogenesis-associated disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
By “isolated nucleic acid molecule” is meant a nucleic acid (e.g., a DNA) that is free of the genes, which, in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
By “neoplasia” is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Solid tumors, hematological disorders, and cancers are examples of neoplasias.
By “operably linked” is meant that a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide.
By “peptide” is meant any fragment of a polypeptide. Typically peptide lengths vary between 5 and 1000 amino acids (e.g., 5, 10, 15, 20, 25, 50, 100, 200, 250, 500, 750, and 1000).
By “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification.
By “promoter” is meant a polynucleotide sufficient to direct transcription.
By “reduce” is meant a decrease in a parameter (e.g., blood vessel formation) as detected by standard art known methods, such as those described herein. As used herein, reduce includes a 10% change, preferably a 25% change, more preferably a 40% change, and even more preferably a 50% or greater change.
By “reference” is meant a standard or control condition.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and even more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
“Sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window, and can take into consideration additions, deletions and substitutions. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (for example, charge or hydrophobicity) and therefore do not deleteriously change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have sequence similarity. Approaches for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17, 1988, for example, as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
“Percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The term “substantial identity” or “homologous” in their various grammatical forms in the context of polynucleotides means that a polynucleotide comprises a sequence that has a desired identity, for example, at least 60% identity, preferably at least 70% sequence identity, more preferably at least 80%, still more preferably at least 90% and even more preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 85%, 90%, and even more preferably at least 95%.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, although such cross-reactivity is not required for two polypeptides to be deemed substantially identical.
An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.
A “recombinant host” may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
The term “operably linked” is used to describe the connection between regulatory elements and a gene or its coding region. That is, gene expression is typically placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 5, 10, or 15 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, about 100 amino acids, or about 150 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides about 300 nucleotides or about 450 nucleotides or any integer thereabout or therebetween.
Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2: 482, 1981; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48: 443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 8: 2444, 1988; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 7 Science Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp, Gene, 73: 237-244, 1988; Corpet, et al., Nucleic Acids Research, 16:881-90, 1988; Huang, et al., Computer Applications in the Biosciences, 8:1-6, 1992; and Pearson, et al., Methods in Molecular Biology, 24:7-331, 1994. The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York, 1995. New versions of the above programs or new programs altogether will undoubtedly become available in the future, and can be used with the present invention.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs, or their successors, using default parameters (Altschul et al., Nucleic Acids Res, 2:3389-3402, 1997). It is to be understood that default settings of these parameters can be readily changed as needed in the future.
As those ordinary skilled in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163, 1993) and XNU (Clayerie and States, Comput. Chem., 17:191-1, 1993) low-complexity filters can be employed alone or in combination.
As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
A “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a set of amino acid sequences that include a shaded 4-letter motif common in all the experimentally tested TSP-1 containing proteins. At the bottom of the figure is the consensus sequence.
FIGS. 2A and 2B show a set of amino acid sequences that included shaded common motifs of the TSP-1 containing peptides using a threshold of 60% (FIG. 2A) and 45% (FIG. 2B).
FIG. 3 shows a set of amino acid sequences that include a shaded 4-letter motif common in all the theoretically predicted TSP-1 containing proteins. In the red insert the predicted motif is identified within TSP-2 domains as well.
FIG. 4 shows a set of amino acid sequences that include a shaded 6-letter motif common in all the experimentally tested C-X-C containing proteins.
FIG. 5 shows a set of amino acid sequences that include a shaded common motif in all the theoretically predicted anti-angiogenic C-X-C containing proteins.
FIGS. 6A-6C show a set of amino acid sequences that include in shading the most abundant motif in the theoretically predicted anti-angiogenic type IV collagen derived peptide fragments. Novel motifs occur when the abundant 7-mer is shifted downstream (FIG. 6B) or upstream (FIG. 6C).
FIG. 7 shows a set of amino acid sequences that include in shading a less common motif within the sequences of type IV collagen derived peptide fragments.
FIG. 8 shows a set of amino acid sequences that include in shading a motif identified within the subset of the of type IV collagen derived short anti-angiogenic peptides.
FIG. 9 shows a set of amino acid sequences that include in shading a common motif that occurs in all the predicted anti-angiogenic fragments derived from TIMPs.
FIGS. 10A and 10B show the amino acid sequences of eleven novel anti-angiogenic peptides. Sequences in FIG. 10A are from the somatotropin family and those in FIG. 10B are from the serpin family.
FIG. 11 shows a set of amino acid sequences that include in shading a motif identified within the similarity hits of the Growth Hormone derived anti-angiogenic peptide.
FIG. 12 shows a set of amino acid sequences that include in shading a motif identified within the similarity hits of the PEDF derived short anti-angiogenic peptide.
FIG. 13 shows the amino acid sequence of a novel peptide derived from the alpha4 fibril of type IV collagen and its similarities with known peptides. Common amino acids are shaded.
FIG. 14 shows exemplary amino acid sequence modifications.
FIGS. 15A-15C includes a set of graphs showing that likely receptors for peptides identified herein were identified in the HUVEC proliferation assay after neutralization of various receptors associated with anti-angiogenic activity. FIG. 15A shows the effect of β1- and αvβ3 integrin-neutralizing antibodies on the activity of three collagen IV-derived peptides (red). The collagen derived peptides used in the experiment are derived from the alpha5 fibrils of type IV collagen (LRRFSTMPFMFCNINNVCNF and FCNINNVCNFASRNDYSYWL), and from alpha6 fibrils of type IV collagen (LPRFSTMPFIYCNINEVCHY). FIG. 15B shows the effect of two different concentrations of the CXCR3 receptor-neutralizing antibody on the activity of three CXC chemokine-derived peptides (green). The CXC derived peptides used in this experiment are derived from proteins GRO-α/CXCL1 (NGRKACLNPASPIVKKIIEKMLNS), GRO-γ/MIP-2β/CXCL3 (NGKKACLNPASPMVQKIIEKIL), and ENA-78/CXCL5 (NGKEICLDPEAPFLKKVIQKILD). FIG. 15C shows the effect of CD36 and CD47 receptor-neutralizing antibodies on the activity of three thrombospondin-derived peptides (blue). The TSP1 repeat-containing protein derived peptides used in the experiment are derived from proteins WISP-1 (SPWSPCSTSCGLGVSTR1), NOVH (TEWTACSKSCGMGFSTRV) and UNC5C (TEWSVCNSRCGRGYQKRTR).
FIG. 16 includes a number of graphs showing the evaluation of peptide combinations from different protein families. Two peptides from each of three different protein families were combined serially in the proliferation assay, and the efficiency of the peptide combinations was evaluated after calculating the isobolograms and Combination Index for each of the combinations.
FIG. 17 shows a quantitative description of the peptide combinations. The combinations that induce strong synergism are marked with thicker red lines whereas the combinations that induce antagonism are shown with dotted blue lines.
FIGS. 18A-18C are graphs. FIG. 18A shows the results of the administration of collagen IV, TSP1 and CXC derived peptides, as well as the combination of the TSP1 derived peptide and the CXC derived peptide. Each of the peptides was administered at 20 mg/kg/day i.p. (n=3 per condition). For the combination, the peptides were administered alternately every other day. PBS was administered as a positive control. FIG. 18B shows the effect of the administration of the collagen IV derived peptide on tumor volume. The peptide was administered in an i.p. injection at 10 mg/kg/day for 12 days. Control (n=6); peptide application (green, n=6; red, n=5). These results for n=5 do not include one animal in which the tumor started growing after day 9. FIG. 18C shows the tumor growth rate (% volume change per day) on day 14 after inoculation (day 0 at panel C). Once the tumors reached a volume of approximately 800 mm3 treatment with a TSP1 derived and CXC derived peptides was started. The peptides were administered alternately every other day at a dose of 10 mg/kg. The tumor growth rate dropped to zero after 3 injections.



DETAILED DESCRIPTION OF THE INVENTION
The invention features compositions and methods that are useful for modulating angiogenesis. The invention is based, at least in part, on the discovery of general peptide motifs that are associated with anti-angiogenic properties of peptides.
Angiogenesis
Angiogenesis, which involves the growth or sprouting of new microvessels from pre-existing vasculature, and vasculogenesis, which involves de novo vascular growth, is essential to many physiological and pathological conditions, including embryogenesis, cancer, rheumatoid arthritis, diabetic retinopathy, obesity, atherosclerosis, ischemic heart and limb disease, and wound healing. Over 70 diseases have been identified as angiogenesis dependent (Carmeliet, Nature, 438:932-6, 2005). Under physiological conditions, the growth of new microvessels is tightly regulated and orchestrated by maintaining a balance between endogenous pro- and anti-angiogenic factors. Tipping the balance of this regulation may lead to either excessive neovascularization, as in cancer, age-related macular degeneration, and rheumatoid arthritis, or insufficient vascularization, as in ischemic heart and limb disease, ischemic brain, and neural degeneration.
Angiogenesis is a complex multistep process that involves interactions between endothelial cells (EC), pericytes, vascular smooth muscle cells, and stromal cells (e.g., stem cells and parenchymal cells). These interactions occur through secreted factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF or FGF-2) and angiopoietins, as well as through cell-cell and cell-extracellular matrix (ECM) interactions. Endothelial cell-ECM interactions regulate numerous processes that are critical for angiogenesis, including endothelial cell migration, proliferation, differentiation and apoptosis. Angiogenic processes include network stabilization and remodeling that may involve the recruitment of stromal cells, as well as the pruning of some vessels. In many cases, angiogenesis occurs as a response to hypoxia. A transcription factor called hypoxia-inducible factor, HIF1α, has been demonstrated to act as an oxygen sensor whose activity leads to upregulation of VEGF in parenchymal and stromal cells (Semenza, Physiology (Bethesda), 19:176-82, 2004). VEGF is secreted as a homodimer in the form of several heparin-binding and non-heparin-binding splice-variant isoforms; it diffuses through the interstitial space and can bind to the endothelial cell receptors VEGFR1 and VEGFR2, as well as co-receptors such as Neuropilin-1, thus initiating a signal transduction cascade that leads to endothelial cell proliferation and migration. The production of endothelial cell matrix metalloproteinases, MMPs, increases as a result of endothelial cell activation; MMPs are necessary for selectively clipping the capillary basement membrane and the ECM, which constitute physical barriers to endothelial cell migration and capillary sprouting. MMPs and their associated molecules also play a crucial role in uncovering cryptic sites of the ECM proteins, a number of which have been identified as anti-angiogenic (Davis et al., Anat Rec, 268:252-75, 2002; Folkman, Annu Rev Med, 57:1-18, 2006; Rundhaug, J Cell Mol Med, 9:267-85, 2005; Schenk and Quaranta, Trends Cell Biol, 13:366-75, 2003), and in processing cell-surface receptors (Mott and Werb, Curr Opin Cell Biol, 16:558-64, 2004).
Diseases Associated with Undesirable Angiogenesis
Where the processes regulating angiogenesis are disrupted, pathology may result. Such pathology affects a wide variety of tissues and organ systems. Diseases characterized by excess or undesirable angiogenesis are susceptible to treatment with therapeutic agents described herein.
Excess angiogenesis in numerous organs is associated with cancer and metastasis, including neoplasia and hematologic malignancies.
Angiogenesis-related diseases and disorders are commonly observed in the eye where they may result in blindness. Such disease include, but are not limited to, age-related macular degeneration, choroidal neovascularization, persistent hyperplastic vitreous syndrome, diabetic retinopathy, and retinopathy of prematurity (ROP).
A number of angiogenesis-related diseases are associated with the blood and lymph vessels including transplant arteriopathy and atherosclerosis, where plaques containing blood and lymph vessels form, vascular malformations, DiGeorge syndrome, hereditary hemorrhagic telangiectasia, cavernous hemangioma, cutaneous hemangioma, and lymphatic malformations.
Other angiogenesis diseases and disorders affect the bones, joints, and/or cartilage include, but are not limited to, arthritis, synovitis, osteomyelitis, osteophyte formation, and HIV-induced bone marrow angiogenesis.
The gastro-intestinal tract is also susceptible to angiogenesis diseases and disorders. These include, but are not limited to, inflammatory bowel disease, ascites, peritoneal adhesions, and liver cirrhosis.
Angiogenesis diseases and disorders affecting the kidney include, but are not limited to, diabetic nephropathy (early stage: enlarged glomerular vascular tufts).
Excess angiogenesis in the reproductive system is associated with endometriosis, uterine bleeding, ovarian cysts, ovarian hyperstimulation.
In the lung, excess angiogenesis is associated with primary pulmonary hypertension, asthma, nasal polyps, rhinitis, chronic airway inflammation, cystic fibrosis.
Diseases and disorders characterized by excessive or undesirable angiogenesis in the skin include psoriasis, warts, allergic dermatitis, scar keloids, pyogenic granulomas, blistering disease, Kaposi's sarcoma in AIDS patients, systemic sclerosis.
Obesity is also associated with excess angiogenesis (e.g., angiogenesis induced by fatty diet). Adipose tissue may be reduced by the administration of angiogenesis inhibitors.
Excess angiogenesis is associated with a variety of auto-immune disorders, such as systemic sclerosis, multiple sclerosis, Sjögren's disease (in part by activation of mast cells and leukocytes). Undesirable angiogenesis is also associated with a number of infectious diseases, including those associated with pathogens that express (lymph)-angiogenic genes, that induce a (lymph)-angiogenic program or that transform endothelial cells. Such infectious disease include those bacterial infections that increase HIF-1 levels, HIV-Tat levels, antimicrobial peptides, levels, or those associated with tissue remodeling.
Infectious diseases, such as viral infections, can cause excessive angiogenesis which is susceptible to treatment with agents of the invention. Examples of viruses that have been found in humans include, but are not limited to, Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1 (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phieboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astroviruses).
The present invention provides methods of treating diseases and/or disorders or symptoms thereof associated with excess or undesired angiogenesis, which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formulae herein to a subject (e.g., a mammal, such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to an angiogenesis-related disease or disorder or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the disease or disorder or symptom thereof (e.g., to prevent or reduce angiogenesis) under conditions such that the disease or disorder is treated.
The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein (e.g., a peptide described herein, or mimetic, or analog thereof), or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which angiogenesis may be implicated.
In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with angiogenesis, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.
Treatment of Neoplasia
The methods of the invention are particularly well suited for the treatment of neoplasias. By “neoplasia” is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancer is an example of a proliferative disease. Examples of cancers include, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Lymphoproliferative disorders are also considered to be proliferative diseases.
Peptides of the Invention
The present invention utilizes powerful computational and bioinformatic approaches to identify therapeutic agents (e.g., polypeptides, peptides, analogs, and fragments thereof) having anti-angiogenic activity. The amino acid sequences of such agents are provided herein. The Tables and Figures provide sequences of peptides of the invention, GenBank Accession Nos., and the amino acid positions of the sequences. Amino acids are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission; they can also be referred to by their commonly known three letter symbols.
Angiogenesis Assays
The biological activity of therapeutic agents of the invention is characterized using any method for assaying angiogenic activity known in the art. In vitro angiogenesis assays have been described in detail in recent reviews (Akhtar et al., Angiogenesis, 5:75-80, 2002; Auerbach et al., Cancer Metastasis Rev, 19:167-72, 2000; Auerbach et al., Clin Chem, 49:32-40, 2003; Staton et al., Int J Exp Pathol, 85:233-48, 2004; Vailhe et al., Lab Invest, 81:439-52, 2001). There are a number of different endothelial cell lineages that have been used in angiogenesis assays: bovine aortic, bovine retinal, rat and mouse microvascular, human aortic, human bladder microvascular, human cardiac microvascular, human dermal microvascular, human lung microvascular and human umbilical vein endothelial cells. All of these endothelial cells are capable of differentiating in vitro and forming capillary-like structures. This process occurs when the cells are cultured in a monolayer of extracellular matrix components, such as the Matrigel (extracellular matrix material similar to basement membrane), type I collagen, fibronectin or laminin. An endothelial cell lineage that is commonly used for testing the angiogenic response is the human umbilical vein endothelial cells (HUVECs). The National Cancer Institute (NCI) has issued guidelines for testing the anti-angiogenic efficacy of novel agents; they include proliferation, migration and tube formation assays using HUVECs.
Initially the anti-angiogenic effect of selected standard agents is assessed as a positive control by adding them into the wells containing cultured endothelial cells. Such standard anti-angiogenic agents include the fumigillin analog TNP-470 that is available by request from NCI. The standard cell culture medium is usually used as a negative control. The experiments described below are repeated several times as required to obtain statistically significant and reproducible results. Once the platform is calibrated and tested for the known agents, the novel inhibitors are tested.
Cell Proliferation Assay
In these assays anti-angiogenic agents are tested for their ability to alter endothelial cell proliferation. A reduction in endothelial cell proliferation identifies an agent that inhibits angiogenesis. The viability and metabolic activity of the cells is measured in the presence of the anti-angiogenic peptides at different concentrations and various time steps. In one example, a cell proliferation reagent, MTT, is used in a substrate/assay that measures the metabolic activity of viable cells. The assay is based on the reduction of the yellow tetrazolium salt, MTT, by viable, metabolically active cells to form the insoluble purple formazan crystals, which are solubilized by the addition of a detergent. MTT is a colorimetric, non-radioactive assay that can be performed in a microplate. It is suitable for measuring cell proliferation, cell viability or cytotoxicity. The procedure involves three steps. First, the cells are cultured in a multi-well plate and then incubated with the yellow MTT for approximately 2 to 4 hours. During this incubation period, viable cells convert, in their mitochondria, the yellow MTT to the purple formazan crystals. The second step involves the solubilization of the crystals. A detergent solution is added to lyse the cells and solubilize the colored crystals. The final step of the assay involves quantifying changes in proliferation by measuring the changes in the color after lysing the cells. The samples are read using an ELISA plate reader at a wavelength of 570 nm. The amount of color produced is directly proportional to the number of viable cells present in a particular well. Other proliferation assays include WST-1, XTT, Trypan Blue, Alamar Blue and BrdU. In contrast to the MTT assay, in the WST-1 assay the formazan crystals do not need to be solubilized by the addition of a detergent; they are soluble to the cell medium.
In another example, cell proliferation is assayed by quantitating bromodeoxyuridine (BrdU) incorporation into the newly synthesized DNA of replicating cells. The assay is a cellular immunoassay that uses a mouse monoclonal antibody directed against BrdU. The procedure involves four steps. First, the cells are cultured in a microtiterplate and pulse-labeled with BrdU. Only proliferating cells incorporate BrdU into their DNA. The cells are then fixed in a denaturing solution. The genomic DNA is denatured, exposing the incorporated BrdU to immunodetection. The BrdU label is located in the DNA with a peroxidase-conjugated anti-BrdU antibody. The antibody is quantitated using a peroxidase substrate. To test anti-proliferative effects of the selected peptides, the endothelial cells are incubated in the presence of varying amounts of the peptides for different time intervals. After labeling of the cells with BrdU the cell proliferation reagent WST-1 is added, and the cells are reincubated. The formazan product is quantified at 450 nm with an absorbance reader. Subsequently, BrdU incorporation is determined using the colorimetric cell proliferation ELISA, BrdU. The results of this assay indicate the effects of the anti-angiogenic peptides either on DNA synthesis (anti-proliferative) or the metabolic activity (pro-apoptotic) of the cell. Kits for implementing these techniques are commercially available.
Preferably, an agent of the invention reduces cell proliferation by at least about 5%, 10%, 20% or 25%. More preferably, cell proliferation is reduced by at least 50%, 75%, or even by 100%.
Cell Apoptosis and Cell Cycle Assay
Agents having anti-angiogenic activity can also be identified in assay that measures the effect of a candidate agent on cell proliferation and survival using a mitogenic assay (incorporation of thymidine, or 5-bromodeoxyuridine) that measures alterations in cell number (direct counts or indirect colorimetric evaluation). Agents that reduce cell proliferation, cell survival, or that increase cell death are identified as having anti-angiogenic activity. Cell death by apoptosis can be measured using a TUNEL assay or by analyzing the expression of apoptosis markers, such as the caspases and annexin V (Fennell et al., J Biomol Screen, 11:296-302, 2006; Loo and Rillema, Methods Cell Biol, 57:251-64, 1998; Otsuki et al., Prog Histochem Cytochem, 38:275-339, 2003).
A number of methods have been developed to study apoptosis in cell populations. Apoptosis is a form of cell death that is characterized by cleavage of the genomic DNA into discrete fragments prior to membrane disintegration. Because DNA cleavage is a hallmark for apoptosis, assays that measure prelytic DNA fragmentation are especially attractive for the determination of apoptotic cell death. DNA fragments obtained from cell populations are assayed on agarose gels to identify the presence of absence of “DNA ladders” or bands of 180 bp multiples, which form the rungs of the ladders, or by quantifying the presence of histone complexed DNA fragments by ELISA.
Other indicators of apoptosis include assaying for the presence caspases that are involved in the early stages of apoptosis. The appearance of caspases sets off a cascade of events that disable a multitude of cell functions. Caspase activation can be analyzed in vitro by utilizing an enzymatic assay. Activity of a specific caspase, for instance caspase 3, can be determined in cellular lysates by capturing of the caspase and measuring proteolytic cleavage of a suitable substrate that is sensitive to the specific protease (Fennell et al., J Biomol Screen, 11:296-302, 2006; Loo and Rillema, Methods Cell Biol, 57:251-64, 1998; Otsuki et al., Prog Histochem Cytochem, 38:275-339, 2003). Agents that increase caspase activity or DNA fragmentation in endothelial cells are identified as useful in the methods of the invention.
In addition to in vitro techniques, apoptosis can be measured using flow cytometry. One of the simplest methods is to use propidium iodide (PI) to stain the DNA and look for sub-diploid cells (Fennell et al., J Biomol Screen, 11:296-302, 2006; Loo and Rillema, Methods Cell Biol, 57:251-64, 1998; Otsuki et al., Prog Histochem Cytochem, 38:275-339, 2003).
The most commonly used dye for DNA content/cell cycle analysis is propidium iodide (PI). PI intercalates into the major groove of double-stranded DNA and produces a highly fluorescent adduct that can be excited at 488 nm with a broad emission centered around 600 nm. Since PI can also bind to double-stranded RNA, it is necessary to treat the cells with RNase for optimal DNA resolution. Other flow cytometric-based methods include the TUNEL assay, which measures DNA strand breaks and Annexin V binding, which detects relocation of membrane phosphatidyl serine from the intracellular surface to the extracellular surface.
Cell Migration and Invasion Assay
Another anti-angiogenic activity is the ability to reduce endothelial cell migration towards an attractant that is present in a chemotactic gradient, such as a growth factor gradient. Endothelial cell motility or migration can be assessed using the Boyden chamber technique (Auerbach et al., Cancer Metastasis Rev, 19:167-72, 2000; Auerbach et al., Clin Chem, 49:32-40, 2003; Taraboletti and Giavazzi, Eur J Cancer, 40:881-9, 2004). In one example, a Boyden chamber assay is used to test endothelial cell migration from one side of the chamber in the presence of an activator. In brief, the lower compartment of the Boyden chamber is separated from the upper (containing the endothelial cells) by a matrix-coated polycarbonate filter with pores small enough to allow only the active passage of the cells (3-8 μm pore size). The matrix may include, for example, extracellular matrix proteins, such as collagen, laminin and fibronectin. Activators include but are not limited to growth factors, such as vascular endothelial growth factor and fibroblast growth factor-2 or conditioned medium (e.g. from tumor cells or NIH-3T3 fibroblasts). Migration typically occurs rapidly typically within 4-20 hours cells have migrated through the filter. The number of migrating cells is quantified using a cell-permeable fluorescent dye in the presence or absence of an inhibitor; it can also be quantified by any means of cell counting. A fluorescence plate reader is used to quantify the migrating cells by measuring the amount of fluorescence and directly correlating it to cell number. A decrease in cell migration identifies a peptide that inhibits angiogenesis. Preferably, cell migration or motility is reduced by at least about 5%, 10%, 20% or 25%. More preferably, cell migration or motility is reduced by at least about 50%, 75%, or even by 100%.
In other embodiments, anti-angiogenic agents of the invention alter the invasiveness of an endothelial cell, for example, by reducing the ability of an endothelial cell to degrade an extracellular matrix component. In one example, an anti-angiogenic inhibitor acts by reducing the proteolytic activity of a matrix metalloproteinase. Methods for assaying protease activity are known in the art. Quantification of the matrix metalloproteinase activity can be accomplished using a zymographic or gelatinase activity assay (Frederiks and Mook, J Histochem Cytochem. 52:711-22, 2004). Preferably, protease activity is reduced by at least about 5%, 10%, 20% or 25%. More preferably, protease activity is reduced by at least about 50%, 75%, or even by 100%.
In another example, the invasive activity of an endothelial cell is measured using a Boyden chamber invasion assay or by measuring phagokinetic tracks. The invasion assay is essentially as described above for the Boyden motility assay, except that the filter is coated with a layer of a matrix several microns thick, usually Matrigel or other basement membrane extracts, which the cells must degrade before migrating through the filter (Auerbach et al., Cancer Metastasis Rev, 19:167-72, 2000; Auerbach et al., Clin Chem, 49:32-40, 2003; Taraboletti and Giavazzi, Eur J Cancer, 40:881-9, 2004). Compounds that reduce extracellular matrix degradation or endothelial cell invasiveness are identified as useful in the methods of the invention.
Tube Formation Assay
Another method of identifying an agent having anti-angiogenic activity involves measuring the agent's ability to reduce or disrupt capillary tube formation. Various types of endothelial cells (e.g., HUVECs, HMVECs (human microvascular endothelial cells)) form tubes when cultured in wells uniformly coated with Matrigel, an extracellular matrix protein, or other substrates. Therefore the assay characterizes endothelial cell differentiation. The endothelial cells are cultured in the presence or the absence of a candidate agent. The agent may be added to the culture media or may be present or applied to the gel. Typically, the effect on tube formation is measured by incubating the cells for a period of time (e.g., one to four days) at 37° C. in 5% CO2 atmosphere. Kits for implementing these techniques are commercially available.
The output of the experiments are images of capillary networks formed. A common metric used for the morphological characteristics of a capillary network is the angiogenic index. This index is calculated as the ratio of the total length of the connected tubes over the total monitored surface of the well. The change of the angiogenic index as a function of the concentration of the anti-angiogenic peptide will be the determinant for the effectiveness of the tested novel angiogenesis inhibitors.
Aortic Ring Assay
The aortic ring assay integrates the advantages of both in vivo and in vitro systems. It is a useful assay to test angiogenic factors or inhibitors in a controlled environment. More importantly, it recapitulates all of the necessary steps involved in angiogenesis (Staton et al., Int J Exp Pathol, 85:233-48, 2004).
In this quantitative method of studying angiogenesis, ring segments of aortas from various animals such as rats and mice are embedded in a three-dimensional matrix composed of fibrin or collagen, and cultured in a defined medium devoid of serum and growth factors. Microvessels sprout spontaneously from the surface of the aortic rings. This angiogenic process is mediated by endogenous growth factors produced from the aorta or can be assisted by applying exogenously specific concentrations of growth factors. The embedded aortas are incubated for 10-12 days and after the incubation period the newly formed vessels are quantified. Microvessels can be counted manually or quantified using computer-assisted image analysis. Test agents can be added to the culture medium to assay for angiogenic or anti-angiogenic activity. Also aortas from animals with different genetic background (e.g., knockout mice) can be used in order to assess specific mechanisms of the effect of the anti-angiogenic peptides on the neovessel formation process.
In Vivo Angiogenesis Assays
A recent review identified over 70 disease conditions that involve angiogenesis, about half of those characterized by abnormal or excessive angiogenesis or lymphangiogenesis (Carmeliet, Nature, 438:932-6, 2005). Agents identified as having anti-angiogenic activity are optionally tested in in vivo assays using animal models that exhibit abnormal or excessive angiogenesis or lymphangiogenesis.
Matrigel Plug Assay
In one in vivo approach, a candidate agent of the invention is tested for anti-angiogenic activity by implanting a polymer matrix subcutaneously in an animal and assaying the matrix for signs of neovascularization. In one embodiment, a Matrigel plug or a similar substrate containing tumor cells and an anti-angiogenic factor is used to study in vivo angiogenesis (Auerbach et al., Cancer Metastasis Rev, 19:167-72, 2000; Staton et al., Int J Exp Pathol, 85:233-48, 2004). Matrigel is a liquid at 4° C., but forms a solid gel at 37° C. A candidate agent is suspended together with an attractant, such as a growth factor, in the gel. The Matrigel is then injected subcutaneously where it forms a solid plug allowing for the prolonged local release of pro- or anti-angiogenic agents present in the gel. The plug is subsequently removed and neovascularization is assessed by any standard methods, including but not limited to, identifying the presence of endothelial cells or endothelial cell tubules in the plug using microscopy. In some embodiments, this approach is combined with an immuno-histological identification of endothelium specific proteins (e.g., CD-31/34 or integrins) on the newly formed vessels.
The Matrigel plug assay can be applied for testing the efficacy of the novel anti-angiogenic peptides identified herein. In one example, Matrigel is mixed with heparin (usually 20 U/ml) and a vascular endothelial growth factor at about 50 ng/ml in the presence or absence of a candidate peptide, which is supplied at a variety of concentrations (e.g., at the IC50). A control animal receives the gel without the anti-angiogenic fragment. The Matrigel is injected into the mice subcutaneously and after one week the animals are sacrificed. The Matrigel plugs are then removed and fixed with 4% paraformaldehyde. The plugs are then embedded in paraffin, sectioned and stained with hematoxylin and eosin. The number of blood vessels as well as any other angiogenic indexes are estimated.
Directed In Vivo Angiogenesis Assay (DIVAA)
Directed in vivo angiogenesis assay (DIVAA) is a reproducible and quantitative in vivo method of assaying angiogenesis. It involves the preparation of silicon cylinders that are closed on one side filled with some type of extracellular matrix (for example Matrigel) with or without premixed angiogenic factors (Guedez et al., Am J Pathol, 162:1431-9, 2003) to form an angioreactor. The angioreactors are then implanted subcutaneously in mice. Vascular endothelial cells migrate into the extracellular matrix and form vessels in the angioreactor. As early as nine days post-implantation, there are enough cells present in the angioreactor to assay the effect of an angiogenic modulating factors. A candidate agent may be included in the matrix together with the angiogenic factors. The design of the angioreactor provides a standardized platform for reproducible and quantifiable in vivo angiogenesis assays.
Advantageously, the angioreactor prevents assay errors due to absorption of the basement membrane extract or the diffusion of the anti-angiogenic agent into the surrounding tissue; may be carried out using only a fraction of the materials required in the plug assay described above; and up to four angioreactors may be implanted in a single animal (e.g., mouse), providing more data for analysis. Vascularization response can be measured by intravenous injection of fluorescein isothiocyanate (FITC)-dextran before the recovery of the angioreactor, followed by spectrofluorimetry. Alternatively, to obtain a quantitative assessment of the angiogenic invasion, the content of the angioreactors, can be removed and the endothelial cells stained using FITC-Lectin. Fluorescence of the FITC-Lectin solution can be quantitated by measuring the fluorescence at 485 nm excitation and 510 nm emission using a fluorescence plate reader e.g., Victor 3V (Perkin Elmer). The intensity of the signal is directly proportional to the number of endothelial cells that are present in the angioreactors. The technique allows dose response analysis and identification of effective doses of angiogenesis-modulating factors in vivo.
Chorioallantoic Membrane Assay
The chorioallantoic membrane assay (CAM) is widely used as an angiogenesis assay Auerbach et al., Cancer Metastasis Rev 19:167-172, 2000; Staton et al., Int J Exp Pathol 85: 233-248, 2004; D'Amato, In: Voest, E. E., and D'Amore, P. A. (eds). Tumor Angiogenesis and Microcirculation, 2001, Marcel Dekker, New York-Basel). In one embodiment, the chorioallantoic membrane of a 7-9 day old chick embryos is exposed by making a window in the egg shell. A candidate agent is provided in a formulation that provides for its extended release (e.g., in a slow-release polymer pellets, absorbed on a gelatin sponge, or air-dried onto a plastic disc). The candidate agent formulation is implanted onto the chorioallantoic membrane through a window in the shell. The window is sealed and the egg is re-incubated. The lack of mature immune system in the 7 day old chick embryos allows the study of angiogenesis without any immunological interference. In the modified version of the in ovo assay, the entire egg content is transferred to a plastic culture dish. After 3-6 days of incubation the testing agents are applied and angiogenesis is monitored using various angiogenesis indexes.
In the case of testing the angiostatic peptides, polymer pellets can be loaded both with the growth factors and the anti-angiogenic fragments and be implanted in the chorioallantoic membrane. The modified version of the assay allows the application of a candidate agent using different strategies to identify effective therapeutic regimens. For example, a candidate agent is applied in a single bolus at a particular concentration; at different time points at lower concentrations; or in different formulations that provide for the extended release of an agent. This provides for the temporal control of candidate agent release and the delineation of temporal variations in drug administration on the angiostatic activity of the candidate agents.
Ocular Angiogenesis Models
Corneal Micropocket:
The cornea is an avascular site and presumably any vessels penetrating from the limbus into the cornea stroma can be identified as newly formed. In this assay a pocket is created in the cornea stroma of the animal. An angiogenic response is usually initiated by implantation of a slow release pellet or polymer containing growth factors (Auerbach et al., Cancer Metastasis Rev, 19:167-72, 2000; Auerbach et al., Clin Chem, 49:32-40, 2003; D'Amato, Tumor Angiogenesis and Microcirculation, 103-110, 2001; Staton et al., Int J Exp Pathol, 85:233-48, 2004).
In order to test an angiogenesis inhibitor, the effect of a candidate agent on an angiogenic response in the cornea is assayed after the implantation of a pellet comprising an angiogenic agent in combination with a candidate inhibitor in the cornea pockets. Also the efficacy of an anti-angiogenic agent can be evaluated using the mouse model of ocular ischemic retinopathy to quantitatively assess anti-angiogenic effects on retinal neovascularization. In addition, a mouse model of laser induced choroidal neovascularization can be used in order to quantitatively assess the anti-angiogenic effects of candidate agents on choroidal neovascularization. The tested peptides can be administered with a bolus injection or any other scheduled administration.
Mouse Model of Choroidal Neovascularization (CNV):
Laser photocoagulation is used on normal mice to rupture Bruch's membrane at three locations in each eye (e.g., To be et al., Am J Pathol 153:1641-1646, 1998); this procedure leads to neovascularization arising from the choroidal circulation. On the day of laser treatment, the mice are injected intravitreously with the peptide being evaluated. The injections are repeated a week later. One eye is injected with peptide, the contralateral eye receives the vehicle or scrambled peptide as control. Two weeks following laser treatment the mice are sacrificed and quantitative assessment of choroidal neovascularization is performed. The eyes are removed and fixed overnight in phosphate-buffered formalin. The cornea and lens are removed and the entire retina is dissected from the eyecup. Radial cuts are made from the edge to the equator and the eyecup is flat mounted with the sclera facing down. Flat mounts are examined by fluorescence microscopy. The area of the CNV lesions in the peptide injected eyes are compared to the area of neovascularization of CNV in the paired vehicle injected eyes.
Mouse Model of Ischemic Retinopathy:
Seven-day-old (P7) mice and their mothers are placed in an airtight incubator and exposed to an atmosphere of 75% oxygen for 5 day (Smith et al., Invest Opthalmol V is Sci. 35:101-111, 1994). The incubator temperature is maintained at 23° C., and oxygen is continuously monitored with an oxygen controller. At P12 the litters are returned to room air. One day following removal from oxygen and return to room air intravitreous injection of peptide into the right eye of each pup and vehicle into the left is carried out. On P17 pups are euthanized, and the eyes are rapidly removed, positioned and frozen in an embedding compound. Ocular sections are then stained with Griffonia Simplicifolia lectin that labels vascular endothelial cells. Histopathological sections demonstrating the presence, extent and location of normal and abnormal blood vessels are then analyzed following preparation of a standardized series of sections in each eye. The area of retinal neovascularization in the peptide injected eye is compared to the area of retinal neovascularization in the vehicle injected eye.
Chamber Assays
Other methods for studying the effect of a candidate agent in vivo on chronic angiogenesis involve the use of an implanted transparent chamber. The chamber is implanted in an accessible site (e.g., the rabbit ear, the dorsal skinfold and the cranial window chamber (Auerbach et al., Clin Chem, 49:32-40, 2003; Staton et al., Int J Exp Pathol, 85:233-48, 2004). In each of these systems a piece of skin (the ear or skinfold chamber) or part of the skull (cranial chamber) is removed from an anesthetized animal. Tumor cells or a pellet containing an angiogenesis stimulus is then placed on the exposed surface and covered by a glass. The animals are allowed to recover, and angiogenesis is subsequently monitored. The models allow for the continuous measurement of various angiogenesis as well as tissue parameters, such as pH or blood flow. Similarly to the corneal pocket assay, the angiostatic agents are administered orally, locally, or systemically using a predefined drug administration schedule. Agents that reduce angiogenesis in a chamber assay are identified as useful in the methods of the invention.
Tumor Models
Many different in vivo models have been developed to test the activity of potential anti-angiogenic or anti-cancer treatments, specifically on tumor vasculature. Tumors are implanted and can be grown syngeneically; i.e., subcutaneously, orthotopically in a tissue of origin, or as xenografts in immunodeficient mice (Auerbach et al., Clin Chem, 49:32-40, 2003; Staton et al., Int J Exp Pathol, 85:233-48, 2004). Any number of histological analyses may be used to examine the effect of a candidate agent on a blood vessel supplying the tumor. In one embodiment, the blood vessel density of a newly formed vasculature in the tumor is monitored; in another embodiment, the vascular architecture is monitored, for example, by counting the number of vascular branches per vessel unit length. In another embodiment, blood flow through the vasculature is measured.
The tumor models provide a variety of different conditions that can be analyzed to assay the efficacy of a candidate anti-angiogenic agent. For example, the effects of a candidate agent on the stability of a well vascularized vs. a poorly vascularized tumor can be assayed; the effect of a candidate agent on tumors of different origin, for example prostate and breast cancer, renal cell carcinoma, and including those of vascular origin such as the chemically induced hemangiosarcomas and Kaposi's sarcomas, can be analyzed. The study of in vivo tumor models provide the closest approximation of human tumor angiogenesis. Moreover, such models provide the opportunity to study the pharmacokinetics of the candidate drug as well as its efficacy simultaneously in a large scale model and under different administration carriers and strategies.
Anti-Angiogenic Peptides and Analogs
The invention is not limited to conventional therapeutic peptides having anti-angiogenic activity, but comprises a variety of modified peptides having properties that enhance their biodistribution, selectivity, or half-life. In particular, the invention provides peptides that are modified in ways that enhance their ability to inhibit angiogenesis in a cell, tissue, or organ in a subject in need thereof.
The invention provides methods for optimizing a transcription factor or protein transduction domain amino acid sequence or nucleic acid sequence by producing an alteration in the sequence. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. The invention further includes analogs of any naturally-occurring polypeptide of the invention. Analogs can differ from a naturally-occurring polypeptide of the invention by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally-occurring amino, acid sequence of the invention. The length of sequence comparison is at least about 5, 10, 15 or 20 amino acid residues, at least about 25, 50, or 75 amino acid residues, or at least about 100 amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the invention by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine, phosphothreonine. “Amino acid analogs” refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, for example, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (for example, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acids and analogs are well known in the art. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” apply to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or similar amino acid sequences and include degenerate sequences. For example, the codons GCA, GCC, GCG and GCU all encode alanine. Thus, at every amino acid position where an alanine is specified, any of these codons can be used interchangeably in constructing a corresponding nucleotide sequence. The resulting nucleic acid variants are conservatively modified variants, since they encode the same protein (assuming that is the only alternation in the sequence). One skilled in the art recognizes that each codon in a nucleic acid, except for AUG (sole codon for methionine) and UGG (tryptophan), can be modified conservatively to yield a functionally-identical peptide or protein molecule. As to amino acid sequences, one skilled in the art will recognize that substitutions, deletions, or additions to a polypeptide or protein sequence which alter, add or delete a single amino acid or a small number (typically less than about ten) of amino acids is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitutions are well known in the art and include, for example, the changes of alanine to serine; arginine to lysine; asparigine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparigine; glutamate to aspartate; glycine to proline; histidine to asparigine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine. Other conservative and semi-conservative substitutions are known in the art and can be employed in practice of the present invention.
The terms “protein”, “peptide” and “polypeptide” are used herein to describe any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation). Thus, the terms can be used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid. Thus, the term “polypeptide” includes full-length, naturally occurring proteins as well as recombinantly or synthetically produced polypeptides that correspond to a full-length naturally occurring protein or to particular domains or portions of a naturally occurring protein. The term also encompasses mature proteins which have an added amino-terminal methionine to facilitate expression in prokaryotic cells.
The polypeptides and peptides of the invention can be chemically synthesized or synthesized by recombinant DNA methods; or, they can be purified from tissues in which they are naturally expressed, according to standard biochemical methods of purification. Also included in the invention are “functional polypeptides,” which possess one or more of the biological functions or activities of a protein or polypeptide of the invention. These functions or activities include the ability to inhibit angiogenesis (e.g., by reducing endothelial cell proliferation, migration, survival, or tube formation). The functional polypeptides may contain a primary amino acid sequence that has been modified from that considered to be the standard sequence of a peptide described herein. Preferably these modifications are conservative amino acid substitutions, as described herein.
In addition to full-length polypeptides, the invention also includes fragments of any one of the polypeptides of the invention. As used herein, the term “a fragment” means at least 5, 10, 13, or 15 amino acids. In other embodiments a fragment is at least 20 contiguous amino acids, at least 21, 22, 23, 24, or 25 contiguous amino acids, or at least 30, 35, 40, or 50 contiguous amino acids, and in other embodiments at least 60 to 80 or more contiguous amino acids. Fragments of the invention can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).
Non-protein transcription factor/protein transduction domain fusion analogs have a chemical structure designed to mimic the fusion proteins functional activity. Such analogs are administered according to methods of the invention. Fusion protein analogs may exceed the physiological activity of the original fusion polypeptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs increase the reprogramming or regenerative activity of a reference transcription factor/protein transduction domain fusion polypeptide. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference fusion polypeptide. Preferably, the fusion protein analogs are relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.
Peptide-Design Approaches
Iterative design approaches (DeFreest et al., J Pept Res, 63:409-19, 2004) offer unique opportunities to optimize the structure and function of the candidate anti-angiogenic peptides. During iterative design an initial set of amino acids is substituted and the effect of the resulting agent on angiogenesis is assayed. The exploration of the structure-function relationships, but most importantly the conservation of the biophysical and biochemical characteristics of the peptides, during the iterative design and synthesis, is expected to contribute to the optimization of the anti-angiogenic activity. To determine which residues are essential to the bioactivity of the predicted peptide a series of analogs is prepared and evaluated.
In order to assess the types of substitutions within the amino acid sequence of the candidate peptide one can initially use computational methods. The most straightforward method for deciphering the importance of each amino acid is to investigate the conservation of these amino acids at multiple orthologues (same locus in different organisms). Amino acids that are conserved among different organisms are identified as functionally significant. From a biophysical point of view electrostatic interactions and hydrophobic partitioning act in concert to promote the interactions of the peptides with their receptors. In this sense, any point substitution should comply with the conservation of the net charge and hydrophobicity of the agent (DeFreest et al., J Pept Res, 63:409-19, 2004). Phage display technology can also be used for performing random substitutions at expressed peptides of 20-25 amino acids length (Scott and Smith, Science, 249:386-90, 1990). In each of the cases the resultant peptide is tested for its effect on angiogenesis using any of the assays described herein.
Design optimization of the activity of the predicted peptides can also be performed by altering specific structural characteristics of the agents. For example, it has been shown (DeFreest et al., J Pept Res, 63:409-19, 2004) that head-to-tail cyclization of the molecules confers an active dose range broader than the linear form of the molecules, and the peptide stability and shelf life are not compromised. The head-to-tail conjunction can occur either by a disulfide bond or by a peptide bond formation. The use of a peptide bond may be advantageous for purposes of shelf life, and elimination of dimers, trimers, and higher-order aggregates formation that can sometimes develop when peptides are stored or used in conditions where the redox state cannot be fully controlled. The cyclization approaches are discussed in the following section.
Cyclization of Linear Peptides
Cyclization of peptides has been shown to be a useful approach to developing diagnostically and therapeutically useful peptidic and peptidomimetic agents. Cyclization of peptides reduces the conformational freedom of these flexible, linear molecules, and often results in higher receptor binding affinities by reducing unfavorable entropic effects. Because of the more constrained structural framework, these agents are more selective in their affinity to specific receptor cavities. By the same reasoning, structurally constrained cyclic peptides confer greater stability against the action of proteolytic enzymes.
Methods for cyclization can be classified into the so called “backbone to backbone” cyclization by the formation of the amide bond between the N-terminal and the C-terminal amino acid residues, and cyclizations involving the side chains of individual amino acids (Li and Roller, Curr Top Med Chem, 2:325-41, 2002). Although many novel approaches have been developed to accomplish the head-to-tail cyclization of linear peptides and peptidomimetics, the most commonly used method is still the solution phase macro-cyclization using peptide coupling reagents. The results of the peptide cyclization are mainly influenced by the conformation of the linear peptide precursors in solution. Synthesis design is affected by the strategy of the ring disconnection, and the rational selection of peptide coupling reagents. A reasonable ring disconnection will significantly facilitate the peptide macro-cyclization reaction, while a poor selection of cyclization site may result in slow reaction speed and low yield accompanied by various side reactions such as racemization, dimerization, and oligomerization.
Cyclization involving the side chains of individual amino acids includes the formation of disulfide bridges between omega-thio amino acid residues (cysteine, homocysteine), the formation of lactam bridges between glutamic/aspartic acid and lysine residues, the formation of lactone or thiolactone bridges between amino acid residues containing carboxyl, hydroxyl or mercapto functional groups, and the formation of thio-ether or ether bridges between the amino acids containing hydroxyl or mercapto functional groups.
Recombinant Polypeptide Expression
The invention provides therapeutic peptides that are most commonly generated by routine methods for peptide synthesis. Such methods are known in the art and are described herein. If an alternative approach is desired, the peptides are expressed recombinantly, either alone, or as part of a larger fusion protein that includes an anti-angiogenic peptide operably linked to a polypeptide that facilitates expression. If desired, the peptide can subsequently be cleaved (e.g., enzymatically) from the fusion protein. Where the fusion protein does not interfere with the anti-angiogenic activity of the peptide such cleavage may not be necessary or even desirable. When the therapeutic peptide or fusion protein comprising the peptide contacts an endothelial cell, tissue, or organ comprising such a cell it reduces angiogenesis. Recombinant polypeptides of the invention are produced using virtually any method known to the skilled artisan. Typically, recombinant polypeptides are produced by transformation of a suitable host cell with all or part of a polypeptide-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle.
Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant protein. The precise host cell used is not critical to the invention. A polypeptide of the invention may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or preferably COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., Current Protocol in Molecular Biology, New York: John Wiley and Sons, 1997). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).
A variety of expression systems exist for the production of the polypeptides of the invention. Expression vectors useful for producing such polypeptides include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof.
One particular bacterial expression system for polypeptide production is the E. coli pET expression system (e.g., pET-28) (Novagen, Inc., Madison, Wis.). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signals, expression of the polypeptide is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using host strains that express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant polypeptide is then isolated according to standard methods known in the art, for example, those described herein.
Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system employs a GST gene fusion system that is designed for high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins expressed in pGEX-2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa.
Alternatively, recombinant polypeptides of the invention are expressed in Pichia pastoris, a methylotrophic yeast. Pichia is capable of metabolizing methanol as the sole carbon source. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde by the enzyme, alcohol oxidase. Expression of this enzyme, which is coded for by the AOX1 gene is induced by methanol. The AOX1 promoter can be used for inducible polypeptide expression or the GAP promoter for constitutive expression of a gene of interest.
Once the recombinant polypeptide of the invention is expressed, it is isolated, for example, using affinity chromatography. In one example, an antibody (e.g., produced as described herein) raised against a polypeptide of the invention may be attached to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra). Alternatively, the polypeptide is isolated using a sequence tag, such as a hexahistidine tag, that binds to nickel column.
Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980). Polypeptides of the invention, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs (described herein).
Combinatorial Peptide Libraries
In addition to the synthetic solid state production of small peptides, the amino acid sequences of predicted fragments can be expressed and produced recombinantly using a variety of genetically modified organisms following insertion of the relevant DNA into their genome. One such widely used organism is Escherichia coli. Combinatorial biology depends on the ability to link peptides to their encoding DNA and create large libraries of encoded peptides. The methods for generating DNA-encoded peptide libraries can be divided into two groups. In vitro methods use libraries in which the peptides are accessible to exogenous ligands or cells. These libraries can be used in direct in vitro binding selections with cell cultures to enrich for peptides that induce particular phenotypes. In contrast, in vivo methods use peptide libraries that are expressed inside living cells. An interaction between a particular library member and the target protein is detected by virtue of an effect on the host cell, such as a selective growth advantage, or changes to a physical property of the host cell (Pelletier and Sidhu, Curr Opin Biotechnol, 12:340-7, 2001).
To optimize a set of peptides, such as those peptides identified herein, in vitro methods for creating and testing peptide libraries are suitable. In one embodiment, oligonucleotide directed mutagenesis of initial sequence is used. In another embodiment, a phage is used to display libraries of peptides.
Oligonucleotide Directed Mutagenesis
Oligonucleotide directed mutagenesis can be used in order to modify a single or multiple amino acids that compose the maternal sequence of the predicted anti-angiogenic fragments (Ryu and Nam, Biotechnol Prog, 16:2-16, 2000). Directed mutagenesis is based on the concept that an oligonucleotide encoding a desired mutation is annealed to one strand of a DNA of interest and serves as a primer for initiation of DNA synthesis. In this manner, the mutagenic oligonucleotide is incorporated into the newly synthesized strand. Mutagenic oligonucleotides incorporate at least one base change but can be designed to generate multiple substitutions, insertions or deletions.
Oligonucleotides can also encode a library of mutations by randomizing the base composition at sites during chemical synthesis resulting in degenerate oligonucleotides. The ability to localize and specify mutations is greatly enhanced by the use of synthetic oligonucleotides hybridized to the DNA insert-containing plasmid vector. The general format for site-directed mutagenesis includes several steps. Plasmid DNA containing the template of interest (cDNA) is denatured to produce single-stranded regions. A synthetic mutant oligonucleotide is annealed to the target strand. DNA polymerase is used to synthesize a new complementary strand, and finally DNA ligase is used to seal the resulting nick between the end of the new strand and the oligonucleotide. The resulting heteroduplex is propagated by transformation in E. coli. 
Phage-Displayed Peptide Library Screening
Phage display is one method for in vitro combinatorial biology. The method stems from the observation that peptides fused to certain bacteriophage coat proteins are displayed on the surfaces of phage particles that also contain the cognate DNA (Landon et al., Curr Drug Discov Technol, 1:113-32, 2004).
Phage display describes a selection technique in which a library of variants of an initial peptide (e.g., a peptide described herein), is expressed on the outside of a phage virion, while the genetic material encoding each variant resides on the inside. This creates a physical linkage between each variant protein sequence and the DNA encoding it, which allows rapid partitioning based on binding affinity to a given target molecule by an in vitro selection process called panning. In its simplest form, panning is carried out by incubating a library of phage-displayed peptides with a plate containing a culture of cells, such as endothelial cells, washing away the unbound phage, and eluting the specifically bound phage. The eluted phage is then amplified and taken through additional binding/amplification cycles to enrich the pool in favor of specific phenotypes, such as suppression of proliferation, of the cells that are cultured. After 3-4 rounds, individual clones are characterized by DNA sequencing and ELISA.
Libraries of “fusion phages” are rapidly sorted to obtain clones with desired properties and phages can be readily amplified by passage through a bacterial host. Phage display was first demonstrated with the Escherichia-coli-specific M13 bacteriophage and this remains the most popular platform. Several other E. coli phages have also been adapted for phage display and eukaryotic systems have also been developed.
Screening Assays
Polypeptides and fragments of the invention are useful as targets for the identification of agents that modulate angiogenesis. In particular, the peptides identified herein are typically polypeptide fragments that are hidden within hydrophobic regions of a larger polypeptide. While the entire polypeptide may be pro-angiogenic, the peptides of the invention are typically anti-angiogenic. As such, the activity of these peptides, when exposed to the cellular or extracellular milleau, may reduce the pro-angiogenic function of the larger polypeptide. Where this antagonistic function is undesirable, agents that bind and/or inhibit the biological activity of these peptides are sought. Once identified, such agents are used to enhance angiogenesis. In another approach, anti-angiogenic agents are identified by screening for agents that bind to and enhance the activity of a peptide of the invention. Once identified, such agents are used to reduce angiogenesis.
Alternatively, or in addition, candidate agents may be identified that specifically bind to and inhibit a peptide of the invention. The efficacy of such a candidate compound is dependent upon its ability to interact with the peptide. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays (e.g., those described in Ausubel et al., supra). For example, a candidate compound may be tested in vitro for interaction and binding with a polypeptide of the invention and its ability to modulate angiogenesis may be assayed by any standard assays (e.g., those described herein).
Potential antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acid ligands, aptamers, and antibodies that bind to a peptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also include small molecules that bind to and occupy the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented.
In one particular example, a candidate compound that binds to a pathogenicity polypeptide may be identified using a chromatography-based technique. For example, a recombinant polypeptide of the invention may be purified by standard techniques from cells engineered to express the polypeptide, or may be chemically synthesized, once purified the peptide is immobilized on a column. A solution of candidate compounds is then passed through the column, and a compound specific for the peptide is identified on the basis of its ability to bind to the peptide and be immobilized on the column. To isolate the compound, the column is washed to remove non-specifically bound molecules, and the compound of interest is then released from the column and collected. Compounds isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography). In addition, these candidate compounds may be tested for their ability to modulate angiogenesis (e.g., as described herein). Compounds isolated by this approach may also be used, for example, as therapeutics to treat or prevent the onset of a disease or disorder characterized by excess or undesirable angiogenesis. Compounds that are identified as binding to peptides with an affinity constant less than or equal to 1 nM, 5 nM, 10 nM, 100 nM, 1 mM or 10 mM are considered particularly useful in the invention.
Methods of the invention are useful for the high-throughput low-cost screening of polypeptides, biologically active fragments or analogs thereof that can be used to modulate angiogenesis. One skilled in the art appreciates that the effects of a candidate peptide on a cell (e.g., an endothelial cell) are typically compared to a corresponding control cell not contacted with the candidate peptide. Thus, the screening methods include comparing the expression profile, phenotype, or biological activity of a cell modulated by a candidate peptide to a reference value of an untreated control cell.
In one example, candidate peptides are added at varying concentrations to the culture medium of an endothelial cell. The survival, tube formation, apoptosis, proliferation, migration of the cell are assayed as indicators of angiogenesis. Peptides that reduce the survival, tube formation, proliferation, or migration of an endothelial cell are identified as useful anti-angiogenic agents. Alternatively, peptides that enhance the survival, tube formation, proliferation, or migration of an endothelial cell are identified as useful angiogenic agents. In another embodiment, the expression of a nucleic acid molecule or polypeptide characteristic of the vasculature is monitored. Typical cell surface markers include the fibronectin extra-domain B, large tenascin-C isoforms, various integrins, VEGF receptors, prostate specific membrane antigen, endoglin and CD44 isoforms and tumor endothelium marker (TEM). Peptides or other agents that alter the expression of such markers are identified as useful modulators of angiogenesis. An agent that reduces the expression of a characteristic polypeptide expressed in the vasculature is considered useful in the invention; such an agent may be used, for example, as a therapeutic to prevent, delay, ameliorate, stabilize, or treat an injury, disease or disorder characterized by an undesirable increase in neovascularization. In other embodiments, agents that increase the expression or activity of a marker characteristically expressed in an endothelial cell are used to prevent, delay, ameliorate, stabilize, or treat an injury, disease or disorder characterized by a reduction in angiogenesis. Agents identified according to the methods described herein maybe administered to a patient in need of angiogenesis modulation. Where such agents are peptides, such as those described herein, one skilled in the art appreciates that the invention further provides nucleic acid sequences encoding such peptides (e.g., a peptide shown in Tables 1-10).
Test Compounds and Extracts
In general, peptides are identified from large libraries of natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries, according to methods known in the art. Such candidate polypeptides or the nucleic acid molecules encoding them may be modified to enhance biodistribution, protease resistance, or specificity. The modified peptides are then screened for a desired activity (e.g., angiogenesis modulating activity). Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Agents used in screens may include known compounds (for example, known polypeptide therapeutics used for other diseases or disorders). Alternatively, virtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as the modification of existing polypeptides.
Libraries of natural polypeptides in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Such polypeptides can be modified to include a protein transduction domain using methods known in the art and described herein. In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA 91:11422, 1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho et al., Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al., J. Med. Chem. 37:1233, 1994. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.
Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of polypeptides, chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, chemical compounds to be used as candidate compounds can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra.).
In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.
When a crude extract is found to have angiogenesis modulating activity further fractionation of the positive lead extract is necessary to isolate molecular constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that alters angiogenesis (increases or decreases). Methods of fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds shown to be useful as therapeutics are chemically modified according to methods known in the art.
Therapeutic Methods
Therapeutic polypeptides, peptides, or analogs or fragments thereof, as well as the nucleic acid molecules encoding such molecules are useful for preventing or ameliorating a disease or injury associated with an undesirable increase or decrease in angiogenesis. Diseases and disorders characterized by excess angiogenesis may be treated using the methods and compositions of the invention. Such diseases and disorders include, but are not limited to, neoplasia, hematologic malignancies, rheumatoid arthritis, diabetic retinopathy, age-related macular degeneration, atherosclerosis, and pathologic obesity. In one embodiment, a peptide of the invention is delivered to one or more endothelial cells at a site of angiogenesis-associated disease or injury.
In other embodiments, a nucleic acid molecule encoding a peptide of the invention is administered to a cell, tissue, or organ in need of a reduction in angiogenesis. If desired, the peptide is expressed as a fusion with a longer polypeptide. The peptide may then be cleaved from the polypeptide to achieve its desired therapeutic effect. Such cleavage is not required where the fusion protein does not interfere with the peptide's biological activity.
Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, a full length gene sialidase gene, or a portion thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest (e.g. endothelial cell). Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77 S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). Most preferably, a viral vector is used to administer the gene of interest systemically or to a cell at the site of neovascularization.
Non-viral approaches can also be employed for the introduction of therapeutic to a cell of a patient having an angiogenesis related disease. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofectin (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.
Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a patient can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue at the site of disease or injury.
cDNA expression for use in such methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types, such as an intestinal epithelial cell, can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
Another therapeutic approach included in the invention involves administration of a recombinant therapeutic, such as a sialidase polypeptide, biologically active fragment, or variant thereof, either directly to the site of a potential or actual disease-affected tissue (for example, by administration to the intestine) or systemically (for example, by any conventional recombinant protein administration technique). The dosage of the administered protein depends on a number of factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Generally, between 0.1 mg and 100 mg, is administered per day to an adult in any pharmaceutically acceptable formulation.
Pharmaceutical Therapeutics
The invention provides a simple means for identifying compositions (including nucleic acids, peptides, small molecule inhibitors, and mimetics) capable of acting as therapeutics for the treatment of a disease associated with altered levels of angiogenesis. Accordingly, a chemical entity discovered to have medicinal value using the methods described herein is useful as a drug or as information for structural modification of existing compounds, e.g., by rational drug design. Such methods are useful for screening compounds having an effect on a variety of conditions characterized by undesired angiogenesis.
For therapeutic uses, the compositions or agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of a therapeutic agent described herein in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the disease or disorder. Generally, amounts will be in the range of those used for other agents used in the treatment of other diseases associated with alterations in angiogenesis, although in certain instances lower amounts will be needed because of the increased specificity of the compound. A compound is administered at a dosage that controls the clinical or physiological symptoms associated with angiogenesis as determined by a diagnostic method known to one skilled in the art.
It would be advantageous to administer therapeutic peptides in a formulation that would slow their elimination from the circulation through renal filtration, enzymatic degradation, uptake by the reticulo-endothelial system (RES), and accumulation in non-targeted organs and tissues. In addition, methods for administering agents that limits their widespread distribution in non-targeted organs and tissues allows lower concentrations of the agent to be administered reducing adverse side-effects and providing economic benefits. A variety of methods are available to slow the elimination of agents of the invention. In one embodiment, an implantable device is used to provide for the controlled release of an agent described herein. Such devices are known in the art and include, but are not limited to, polymeric gels and micro-fabricated chips. Some of these devices are already used in the clinic or are being tested in clinical trials (Moses et al., Cancer Cell, 4:337-41, 2003). Various delivery methods for anti-angiogenic agents are tissue specific, e.g., intraocular and periocular injection or gene transfer in the eye (Akiyama et al., J Cell Physiol, 2006; Saishin et al., Hum Gene Ther, 16:473-8, 2005). Numerous reviews on the subject of anti-angiogenic drug delivery are available.
Enhanced Permeability and Retention Effect
For the treatment of neoplasia or sites of neovascularization, the “enhanced permeability and retention effect” (EPR) constitutes a natural mechanism through which high molecular weight (40 kDa or higher) macromolecules with long circulation half-lives, including peptides and proteins conjugated with water-soluble polymers, accumulate (Shukla and Krag, Expert Opin Biol Ther, 6:39-54, 2006; Torchilin and Lukyanov, Drug Discov Today, 8:259-66, 2003). This effect occurs because of certain characteristics of those tissues. The first is that tumor or newly formed vasculature, unlike the vasculature of healthy tissues, is permeable to macromolecules with a MW up to 50 kDa or even higher. This allows macromolecules to enter into the interstitial space. Another characteristic is that in the case of many tumors the lymphatic system, which is responsible for the drainage of macromolecules from normal tissues, is impaired. Because of this, macromolecules that have entered a neo-vascularized tissue are retained there for a prolonged time. To enhance the retention of a low MW peptide described herein, the peptide may be conjugated to a suitable polymer or delivered using a micro-reservoir system.
Peptide and Protein Polymer Conjugation
Mechanisms that increase the MW of a peptide, such as conjugation with polymer chains or concentration of the drug in micro-reservoir systems tend to increase the retention time of the peptide in the tissue (Duncan, Nat Rev Drug Discov, 2:347-60, 2003). Moreover, renal filtration and excretion are mainly responsible for the rapid clearance from the systemic circulation of proteins with molecular weights (MW) of 40 kDa or lower. Rapid clearance and increased retention of a peptide of interest can be achieved by conjugating the peptides with water-soluble polymers. Preferably, the peptide-polymer conjugate has a molecular weight of at least about 30 kDA, 35 kDa, 40 kDa, 50 kDa, 75 kDa, or 100 kDa. Additional benefits of peptide and protein-polymer conjugation include increased resistance to enzymatic degradation and reduced immunogenicity.
Even endogenous proteins can be susceptible to protease degradation in the bloodstream and interstitial space or induce an immune response. Enzymatic degradation and an immune response against a protein result in its rapid elimination from the systemic circulation. In addition, the development of an immune response is potentially dangerous because of the possibility of allergic reactions and anaphylactic shock upon repetitive administrations. The mechanism of protein protection by polymer attachment is similar in both cases. Polymer molecules attached to the protein create steric hindrances, which interfere with binding to the active sites of proteases, and antigen-processing cells. Examples of peptide/protein-polymer conjugation include conjugates with poly(ethylene glycol) and conjugates with poly(styrene-co-maleic acid anhydride).
Conjugates with poly(ethylene glycol)
Several polymers have been used for protein stabilization with varying degrees of success. Poly(ethylene glycol) (PEG) is one widely used polymer for the modification of proteins with therapeutic potential (Thanou and Duncan, Curr Opin Investig Drugs, 4:701-9, 2003; Vicent and Duncan, Trends Biotechnol, 24:39-47, 2006). This polymer is inexpensive, has low toxicity and has been approved for internal applications by drug regulatory agencies. PEG is commercially available in a variety of molecular weights and in chemically activated, ready-for-use forms for covalent attachment to proteins.
Conjugates with poly(styrene-co-maleic acid anhydride)
In some cases, the circulation time of drugs can be increased by conjugating with polymers that are not large enough to prevent renal clearance themselves, but which can attach themselves, with their conjugated drug, to natural long-circulating blood plasma components, such as serum albumin or lipoproteins (Thanou and Duncan, Curr Opin Investig Drugs, 4:701-9, 2003; Vicent and Duncan, Trends Biotechnol, 24:39-47, 2006).
Because of the small size and low molecular weight of the identified anti-angiogenic peptides and the high probability that the conjugated polymers, which are orders of magnitude larger than the peptides, may sterically hinder the activity of the fragments the method of protein conjugation may not be the most efficient method for increasing the retention of the agent in the neo-vascular site. A more attractive scenario is the administration of the peptide in a micro-reservoir delivery system.
Formulation of Pharmaceutical Compositions
The administration of a compound for the treatment of a disease or disorder associated with altered levels of angiogenesis may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing a disease or disorder associated with altered levels of angiogenesis (e.g., an amount sufficient to reduce neovascularization). The compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
Pharmaceutical compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in the central nervous system or cerebrospinal fluid; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that allow for convenient dosing for metronomic therapy that would require taking small doses of the drug several times a week; (vii) formulations that target a disease or disorder associated with altered levels of angiogenesis by using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., endothelial cell) whose function is perturbed in a disease or disorder associated with altered levels of angiogenesis.
For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.
Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
Micro-Reservoir Delivery Systems
For some applications, micro-reservoir or micro-particulate carriers are used to deliver a peptide of the invention. Such systems include, but are not limited to, liposomes, micelles, polymer micro-particles, and cell ghosts. The use of such carriers results in a much higher ratio of active agent over carrier compared with direct molecular conjugates. They also provide a higher degree of protection against enzymatic degradation and other destructive factors upon parenteral administration because the carrier wall completely isolates drug molecules from the environment. An additional advantage of these carriers is that a single carrier can deliver multiple drug species so that they can be used in combination therapies. All micro-particulates are too large to be lost by renal filtration (Thanou and Duncan, Curr Opin Investig Drugs, 4:701-9, 2003). Exemplary micro-particulate delivery systems include, but are not limited to, liposomes and micelles.
Liposomes
Among particulate drug carriers, liposomes are the most extensively studied and possess suitable characteristics for peptide and protein encapsulation. Liposomes are vesicles formed by concentric spherical phospholipid bilayers encapsulating an aqueous space (Moses et al., Cancer Cell, 4:337-41, 2003). These particles are biocompatible, biologically inert and cause little toxic or antigenic reactions. Their inner aqueous compartment can be used for encapsulation of peptides and proteins. Many techniques for liposome preparation require only manipulations that are compatible with peptide and protein integrity (Allen and Cullis, Science, 303:1818-22, 2004). As with other micro-particulate delivery systems, cells of the RES rapidly eliminate conventional liposomes.
In one embodiment, surface-modified long-circulating liposomes grafted with a flexible hydrophilic polymer, such as PEG, are used. This approach prevents plasma protein adsorption to the liposome surface and the subsequent recognition and uptake of liposomes by the RES. Liposomes, in common with protein conjugated macromolecules, can accumulate in tumors of various origins via the EPR effect. Currently, liposomal forms of at least two conventional anticancer drugs, daunorubicin and doxorubicin, are used in the clinic (Torchilin and Lukyanov, Drug Discov Today, 8:259-66, 2003).
Micelles
In another approach, micelles or polymeric micelles, including those prepared from amphiphilic PEG-phospholipid conjugates, may be used to deliver an agent of the invention. Such formulations are of special interest because of their stability (Torchilin and Lukyanov, Drug Discov Today, 8:259-66, 2003). These particles are smaller than liposomes and lack the internal aqueous space. To load micelles, peptides can be attached to the surface of these particles or incorporated into them via a chemically attached hydrophobic anchor. An example of a biodegradable micelle developed for delivery of pharmacological agents are the poly{[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium iodide]ethyl phosphate} (PCEP) micelles (Wen, Mao et al., J Pharm Sci. 93:2142-57, 2004). Carrying a positive charge in its backbone and a lipophilic cholesterol structure in the side chain, PCEP self-assembles into micelles in aqueous buffer at room temperature with an average size of 60-100 nm. PCEP is an excellent platform for delivering ant-angiogenic agents as by itself shows lower cytotoxicity for endothelial cells than for poly-L-lysine and Lipofectamine.
Nanoparticles
An increasing number of agents are delivered with colloidal nanoparticles. Such formulations overcome non-cellular and cellular based mechanisms of resistance and increase the selectivity of agents to target cells while reducing their toxicity in normal tissues. Nanoparticles are typically submicron (<1 μm) colloidal systems. In some embodiments, nanoparticles are made of polymers (biodegradable or not). According to the process used for the preparation of the nanoparticles, nanospheres or nanocapsules can be obtained. Unlike nanospheres (matrix systems in which the drug is dispersed throughout the particles), nanocapsules are vesicular systems in which an agent is confined to an aqueous or oily cavity surrounded by a single polymeric membrane. Nanocapsules are one form of ‘reservoir’ system.
In some embodiments, nanoparticles are generated using hydrophilic polymers, (poly(ethylene glycol) (PEG), poloxamines, poloxamers, polysaccharides) to efficiently coat a nanoparticle surface. These coatings provide a dynamic ‘cloud’ of hydrophilic and neutral chains at the particle surface that repels plasma proteins. Hydrophilic polymers are introduced at the surface in two ways, either by adsorption of surfactants or by use of block or branched copolymers.
Parenteral Compositions
The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.
Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active therapeutic(s), the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable active angiogenic modulating therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
Controlled Release Parenteral Compositions
Controlled release parenteral compositions may be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions. Alternatively, the active drug may be incorporated in biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.
Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutam-nine) and, poly(lactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof).
Solid Dosage Forms for Oral Use
Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. Such formulations are known to the skilled artisan. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.
The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active drug in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). Furthermore, a time delay material such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.
The solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, (e.g., chemical degradation prior to the release of the active angiogenic modulating therapeutic). The coating may be applied on the solid dosage form in a similar manner as that described in Encyclopedia of Pharmaceutical Technology, supra.
At least two active angiogenic modulating therapeutics may be mixed together in the tablet, or may be partitioned. In one example, the first active in angiogenic modulating therapeutic is contained on the inside of the tablet, and the second active angiogenic modulating therapeutic is on the outside, such that a substantial portion of the second angiogenic modulating therapeutic is released prior to the release of the first angiogenic modulating therapeutic.
Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.
Controlled Release Oral Dosage Forms
Controlled release compositions for oral use are constructed to release the active angiogenic modulating therapeutic by controlling the dissolution and/or the diffusion of the active substance. Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, d1-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.
A controlled release composition containing one or more therapeutic compounds may also be in the form of a buoyant tablet or capsule (i.e., a tablet or capsule that, upon oral administration, floats on top of the gastric content for a certain period of time). A buoyant tablet formulation of the compound(s) can be prepared by granulating a mixture of the compound(s) with excipients and 20-75% w/w of hydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, or hydroxypropylmethylcellulose. The obtained granules can then be compressed into tablets. On contact with the gastric juice, the tablet forms a substantially water-impermeable gel barrier around its surface. This gel barrier takes part in maintaining a density of less than one, thereby allowing the tablet to remain buoyant in the gastric juice.
Polymeric Controlled-Release Implants
In another embodiment, an agent of the invention is delivered by implanting a biodegradable polymeric controlled-release device that stores the pharmaceutical agent and allows its delivery via diffusion into the surrounding tissue. Controlled release devices include Norplant and Gliadel, which are used clinically for the prevention of pregnancy and for brain tumor therapy, respectively. Local delivery of pro- or anti-angiogenic factors can be accomplished by encapsulating the agent within a biocompatible polymer matrix. The controlled-release polymer system is then implanted at the desired tissue site, where it releases the soluble factor directly into the interstitial space of the tissue. The diffusible agent can influence the survival or function of damaged cells within the local tissue, or provide a signal that elicits cell proliferation and migration or apoptosis and suppression of migration within the tissue region.
Controlled release implants are typically composed of inert, biocompatible polymers, such as poly(ethylene-co-vinyl acetate) (EVAc), or biodegradable polymers, such as poly(lactide-co-glycolide) (PLGA) (Torchilin and Lukyanov, Drug Discov Today, 8:259-66, 2003). EVAc-matrix systems have been used to release protein hormones, growth factors, antibodies, antigens and DNA. EVAc matrices allow a high degree of control over agent release, versatility in allowing the release of a wide range of agents, and good retention of biological activity. Biodegradable polymers have also been used to release growth factors, protein hormones, antibodies, antigens and DNA. Biodegradable materials disappear from the implant site after protein release. Polymer gels might also be useful for topical or localized protein delivery. Systems that release multiple protein factors are also possible (Saltzman and Olbricht, Nat Rev Drug Discov, 1:177-86, 2002; Torchilin and Lukyanov, Drug Discov Today, 8:259-66, 2003).
Biodegradable polymers include non-water-soluble polymers that are degraded by surface or bulk erosion in addition to water-soluble gels that dissolve and are cleared from the body without undergoing a decrease in molecular weight. There are many different types of biodegradable polymers that can potentially be used in the preparation of peptide delivery systems. They include both naturally derived and synthetic materials.
Biocompatibility of Polymeric Systems
Polymers used as drug delivery systems for protein pharmaceuticals need to exhibit biocompatible characteristics in terms of both the polymer's effect on the organism receiving the drug delivery system and the polymer's effect on the protein to be delivered. Several aspects of a polymeric delivery system ultimately contribute to its overall biocompatibility, or lack thereof. The polymer itself, which consists of a repeating monomeric species, may potentially be antigenic, carcinogenic, or toxic or have some inherent incompatibility with organisms. The shape of an implanted material has been implicated in its biocompatibility as well, smooth surfaces being less irritating and more biocompatible than rough surfaces (Saltzman and Olbricht, Nat Rev Drug Discov, 1:177-86, 2002).
Pharmaceutical Stability
Interactions between proteins and polymeric materials appear to be protein and polymer specific. At issue are the protein molecular weight, which is an important parameter with regard to diffusion characteristics and the iso-electric point of the protein (and polymer as well in some cases), which governs charge-charge interactions (protein-polymer and protein-protein). Moreover the presence of cysteines on the protein may facilitate the formation of intermolecular (i.e., protein-polymer) disulfide bonds. Furthermore, the primary amino acid sequence of the protein may be rendered susceptible to chemical modification in association with a polymeric material. The presence or absence of carbohydrates on the protein may enhance or prevent interaction with polymeric materials and affect the protein's hydrodynamic volume. The relative hydrophobicity of a protein could interact with hydrophobic sites on a polymer. Finally the heterogeneity of protein pharmaceuticals often exists for proteins produced by recombinant methods (Bilati et al., Eur J Pharm Biopharm, 59:375-88, 2005; Gombotz and Pettit, Bioconjug Chem, 6:332-51, 1995; Saltzman and Olbricht, Nat Rev Drug Discov, 1:177-86, 2002).
Bulk Erosion Polymers
Poly(lactic-co-glycolic acid)
Poly(lactic-co-glycolic acid) (PLGA) has been used successfully for several decades in biodegradable structures and more recently as drug delivery micro-carriers, and as a result of the extended use, much is known about their biocompatibility and physicochemical characteristics. PLGA copolymers are well suited for use in delivery systems since they can be fabricated into a variety of morphologies including films, rods, spheres by solvent casting, compression molding and solvent evaporation techniques. Examples of peptide drug delivery systems made from PLGA copolymers, have successfully met FDA approval and they are available as marketed products are Lupron Depot, Zoladex and Decapeptyl (Frokjaer and Otzen, Nat Rev Drug Discov, 4:298-306, 2005).
Block Copolymers of PEG and PLA
Copolymers of PEG and PLA have been synthesized for use in delivery systems. The net result is a biodegradable polymer with a reduced amount of hydrophobicity that is an inherent property of PLA systems. These copolymer systems can be composed of random blocks of the two polymers, two blocks in which case the molecules are amphiphilic, or triblocks in which hydrophilic microphases are present. Peptides that are incorporated into devices made from these copolymers are less likely to adsorb to the delivery system through hydrophobic interactions. The polymers were shown to swell very rapidly due to microphase separation, and degradation occurred over 2-3 weeks (Bilati et al., Eur J Pharm Biopharm, 59:375-88, 2005; Gombotz and Pettit, Bioconjug Chem, 6:332-51, 1995).
Poly(cyanoacrylates)
Poly(cyanoacrylates) have received attention as delivery systems for proteins and peptides. They undergo spontaneous polymerization at room temperature in the presence of water, and their erosion has been shown to be controlled by the length of the monomer chain and the pH. Once formed, the polymer is slowly hydrolyzed, leading to a chain scission and liberation of formaldehyde. While the polymers are not toxic, the formaldehyde released as the degradation byproduct does create a toxicity concern. A characteristic example of their use are delivery systems for insulin prepared by the interfacial emulsion polymerization of alkyl cyanoacrylate (Gombotz and Pettit, Bioconjug Chem, 6:332-51, 1995).
Surface Erosion Polymers
Poly(anhydrides)
Poly(anhydrides) represent a class of surface eroding polymers. Hydrolysis of the anhydride bond is suppressed by acid, which results in an inhibition of bulk erosion by the acidity of the carboxylic acid products of the polymer hydrolysis process. By varying the ratio of their hydrophobic components, one can control degradation rates ranging from days to years. Several proteins have been successfully incorporated into, and released, from poly-(anhydride) delivery systems. The incorporation of insulin and myoglobin has successfully been achieved in poly(anhydride) microspheres using both a hot-melt microencapsulation technique or microencapsulation by solvent removal (Gombotz and Pettit, Bioconjug Chem, 6:332-51, 1995).
Poly(ortho esters)
Poly(ortho esters) are another example of surface-eroding polymers that have been developed for drug delivery systems. Several proteins and peptides have been incorporated into poly(ortho-ester) delivery systems including the LHRH analog nafarelin, insulin and lysozyme.
Hydrogels
The use of biodegradable hydrogels as delivery systems for proteins is of particular interest due to their biocompatibility and their relative inertness toward protein drugs (Gombotz and Pettit, Bioconjug Chem, 6:332-51, 1995). Hydrogels are the only class of polymer that can enable a protein to permeate through the continuum of the carrier. The initial release rate of proteins from biodegradable hydrogels is therefore generally diffusion controlled through the aqueous channels of the gel and is inversely proportional to the molecular weight of the protein. Once polymer degradation occurs, and if protein still remains in the hydrogel, erosion-controlled release may contribute to the system. Several disadvantages must be considered when using a biodegradable hydrogel system for the release of proteins. Their ability to rapidly swell with water can lead to very fast release rates and polymer degradation rates. In addition, hydrogels can rapidly decrease in mechanical strength upon swelling with water. Examples of hydrogels include, pluronic polyols, poly(vinyl alcohol), poly(vinylpyrrolidone), malein anhydride, callulose, hyaluronic acid derivatives, alginate, collagens, gelatin, starches and dextrans.
Selective Drug Delivery
Selective delivery of therapeutic agents includes any methodology by which the functional concentration of drug is higher at the target site than in normal tissue. A wide variety of methods may fall under the category of “selective delivery,” including interventions as simple and mechanical as selective vascular administration in which the drug is physically isolated in a neovascularized area. An example of that type of mechanical selectivity is also the EPR effect.
Most strategies, however, are pharmaceutical. In these approaches, the differences in the biochemical and physiological nature of normal and the targeted cells and their microenvironment are exploited for selective delivery. In one embodiment, a carrier is used to deliver a peptide of the invention that because of its physical properties, accumulates preferentially at a target site. In another embodiment, a ligand is conjugated to a peptide of the invention that binds to a tissue-associated antigen. In another embodiment, an agent of the invention is maintained in an inactive form that can be activated preferentially at the tissue site. In yet another embodiment, external energy irradiation is used to release a peptide at the delivery site.
A variety of technologies using combinations of different approaches are constantly being developed for selective delivery of therapeutics. These delivery systems employ different targets such as cancer cell and neovascular antigens, hypoxia, or high osmotic pressure; targeting agents such as monoclonal antibodies (mAbs), single chain variable fragments (scFvs), peptides and oligonucleotides; effectors like chemical or biological toxins, radioisotopes, genes, enzymes, immunomodulators, oligonucleotides, imaging and diagnostic agents; vehicles the already mentioned colloidal systems, including liposomes, emulsions, micelles, nanoparticles, polymer conjugates or implants; and drug-releasing switches such as systems that utilize thermal, radiation, ultrasound or magnetic fields (Allen and Cullis, Science, 303:1818-22, 2004; Gombotz and Pettit, Bioconjug Chem, 6:332-51, 1995; Moses et al., Cancer Cell, 4:337-41, 2003; Neri and Bicknell, Nat Rev Cancer, 5:436-46, 2005; Saltzman and Olbricht, Nat Rev Drug Discov, 1:177-86, 2002).
Tumor Marker Targeting
The advent of aptamer and antibody technology has facilitated the use of cancer-specific monoclonal antibodies and aptamers to deliver peptides of the invention to a selected target tissue. Of special interest are antibodies and aptamers that target, in vivo, tumor endothelium. Those targets include, but are not limited to, the fibronectin extra-domain B, large tenascin-C isoforms, various integrins, VEGF receptors, prostate specific membrane antigen, endoglin and CD44 isoforms (Shukla and Krag, Expert Opin Biol Ther, 6:39-54, 2006). Alternatively, the tumor itself may be targeted, exemplary tumor markers include CA-125, gangliosides G(D2), G(M2) and G(D3), CD20, CD52, CD33, Ep-CAM, CEA, bombesin-like peptides, PSA, HER2/neu, epidermal growth factor receptor, erbB2, erbB3, erbB4, CD44v6, Ki-67, cancer-associated mucin, VEGF, VEGFRs (e.g., VEGFR3), estrogen receptors, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, IL-2R and CO17-1A. Aptamers and antibodies of the invention can recognize tumors derived from a wide variety of tissue types, including, but not limited to, breast, prostate, colon, lung, pharynx, thyroid, lymphoid, lymphatic, larynx, esophagus, oral mucosa, bladder, stomach, intestine, liver, pancreas, ovary, uterus, cervix, testes, dermis, bone, blood and brain. In the context of the present invention, a tumor cell is a neoplastic (e.g., cancer) cell or a mass of cancer cells, which can also encompass cells that support the growth and/or propagation of a cancer cell, such as vasculature and/or stroma, but not necessarily macrophages. For instance, therefore, the present invention envisages compositions and methods for reducing growth of a tumor cell in a subject, wherein antibodies or aptamers bind with specificity to cell surface epitopes (or epitopes of receptor-binding molecules) of a cancer cell or a cell that is involved in the growth and/or propagation of a cancer cell such as a cell comprising the vasculature of a tumor or blood vessels that supply tumors and/or stromal cells. Methods of this invention are particularly suitable for administration to humans with neoplastic diseases.
Antibodies
Antibodies are well known to those of ordinary skill in the science of immunology. Particularly useful in the methods of the invention are antibodies that specifically bind a polypeptide that is expressed in a tumor or endothelial cell. As used herein, the term “antibody” means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Accordingly, as used herein, the term “antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab′)2, and Fab. F(ab′)2, and Fab fragments which lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325, 1983). The antibodies of the invention comprise whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab′, single chain V region fragments (scFv) and fusion polypeptides.
In one embodiment, an antibody that binds polypeptide is monoclonal. Alternatively, the antibody is a polyclonal antibody. The preparation and use of polyclonal antibodies are also known to the skilled artisan. The invention also encompasses hybrid antibodies, in which one pair of heavy and light chains is obtained from a first antibody, while the other pair of heavy and light chains is obtained from a different second antibody. Such hybrids may also be formed using humanized heavy and light chains. Such antibodies are often referred to as “chimeric” antibodies.
In general, intact antibodies are said to contain “Fc” and “Fab” regions. The Fc regions are involved in complement activation and are not involved in antigen binding. An antibody from which the Fc′ region has been enzymatically cleaved, or which has been produced without the Fc′ region, designated an “F(ab′)2” fragment, retains both of the antigen binding sites of the intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an “Fab′” fragment, retains one of the antigen binding sites of the intact antibody. Fab′ fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain, denoted “Fd.” The Fd fragments are the major determinants of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to specifically bind to immunogenic epitopes.
Antibodies can be made by any of the methods known in the art utilizing a peptide of the invention (e.g., a peptide shown in Tables 1-10), or immunogenic fragments thereof, as an immunogen. One method of obtaining antibodies is to immunize suitable host animals with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production. The immunogen will facilitate presentation of the immunogen on the cell surface. Immunization of a suitable host can be carried out in a number of ways. Nucleic acid sequences encoding a polypeptide described herein, or immunogenic fragments thereof, can be provided to the host in a delivery vehicle that is taken up by immune cells of the host. The cells will in turn express the receptor on the cell surface generating an immunogenic response in the host. Alternatively, nucleic acid sequences encoding a peptide of the invention (e.g., a peptide shown in Tables 1-10), or immunogenic fragments thereof, can be expressed in cells in vitro, followed by isolation of the polypeptide and administration of the receptor to a suitable host in which antibodies are raised.
Using either approach, antibodies can then be purified from the host. Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column preferably run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin.
Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods of making hybridomas are well known in the art. The hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition; e.g., Pristane.
Monoclonal antibodies (Mabs) produced by methods of the invention can be “humanized” by methods known in the art. “Humanized” antibodies are antibodies in which at least part of the sequence has been altered from its initial form to render it more like human immunoglobulins. Techniques to humanize antibodies are particularly useful when non-human animal (e.g., murine) antibodies are generated. Examples of methods for humanizing a murine antibody are provided in U.S. Pat. Nos. 4,816,567, 5,530,101, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.
Aptamers
Nucleic acid aptamers are single-stranded nucleic acid (DNA or RNA) ligands that function by folding into a specific globular structure that dictates binding to target proteins or other molecules with high affinity and specificity, as described by Osborne et al., Curr. Opin. Chem. Biol. 1:5-9, 1997; and Cerchia et al., FEBS Letters 528:12-16, 2002. By “aptamer” is meant a single-stranded polynucleotide that binds to a protein. Desirably, the aptamers are small, approximately  ̃15 KD. The aptamers are isolated from libraries consisting of some 1014-1015 random oligonucleotide sequences by a procedure termed SELEX (systematic evolution of ligands by exponential enrichment). See Tuerk et al., Science, 249:505-510, 1990; Green et al., Methods Enzymology. 75-86, 1991; Gold et al., Annu. Rev. Biochem., 64: 763-797, 1995; Uphoff et al., Curr. Opin. Struct. Biol., 6: 281-288, 1996. Methods of generating aptamers are known in the art and are described, for example, in U.S. Pat. Nos. 6,344,318, 6,331,398, 6,110,900, 5,817,785, 5,756,291, 5,696,249, 5,670,637, 5,637,461, 5,595,877, 5,527,894, 5,496,938, 5,475,096, 5,270,163, and in U.S. Patent Application Publication Nos. 20040241731, 20030198989, 20030157487, and 20020172962.
An aptamer of the invention is capable of binding with specificity to a polypeptide expressed by a cell of interest (e.g., a tumor cell or an endothelial cell supplying a tumor). “Binding with specificity” means that non-tumor polypeptides are either not specifically bound by the aptamer or are only poorly bound by the aptamer. In general, aptamers typically have binding constants in the picomolar range. Particularly useful in the methods of the invention are aptamers having apparent dissociation constants of 1, 10, 15, 25, 50, 75, or 100 nM.
In one embodiment, an antigen expressed on a blood vessel supplying a tumor is the molecular target of the aptamer. Because aptamers can act as direct antagonists of the biological function of proteins, aptamers that target such polypeptide can be used to modulate angiogenesis, vasculogenesis, blood vessel stabilization or remodeling. The therapeutic benefit of such aptamers derives primarily from the biological antagonism caused by aptamer binding.
The invention encompasses stabilized aptamers having modifications that protect against 3′ and 5′ exonucleases as well as endonucleases. Such modifications desirably maintain target affinity while increasing aptamer stability in vivo. In various embodiments, aptamers of the invention include chemical substitutions at the ribose and/or phosphate and/or base positions of a given nucleobase sequence. For example, aptamers of the invention include chemical modifications at the 2′ position of the ribose moiety, circularization of the aptamer, 3′ capping and ‘spiegelmer’ technology. Aptamers having A and G nucleotides sequentially replaced with their 2′-OCH3 modified counterparts are particularly useful in the methods of the invention. Such modifications are typically well tolerated in terms of retaining aptamer affinity and specificity. In various embodiments, aptamers include at least 10%, 25%, 50%, or 75% modified nucleotides. In other embodiments, as many as 80-90% of the aptamer's nucleotides contain stabilizing substitutions. In other embodiments, T-OMe aptamers are synthesized. Such aptamers are desirable because they are inexpensive to synthesize and natural polymerases do not accept T-OMe nucleotide triphosphates as substrates so that 2′-OMe nucleotides cannot be recycled into host DNA. A fully 2′-O-methyl aptamer, named ARC245, was reported to be so stable that degradation could not be detected after 96 hours in plasma at 37° C. or after autoclaving at 125° C. Using methods described herein, aptamers will be selected for reduced size and increased stability. In one embodiment, aptamers having 2′-F and 2′-OCH3 modifications are used to generate nuclease resistant aptamers. Other modifications that stabilize aptamers are known in the art and are described, for example, in U.S. Pat. No. 5,580,737; and in U.S. Patent Application Publication Nos. 20050037394, 20040253679, 20040197804, and 20040180360.
Using standard methods tumor markers or endothelial call-specific aptamers can be selected that bind virtually any tumor marker or endothelial cell-expressed polypeptide known in the art.
The Fibronectin Extra-Domain B (EDB)
Fibronectin is a large glycoprotein that is present in large amounts in the plasma and tissues. EDB is a 91-amino-acid type III homology domain that becomes inserted into the fibronectin molecule under tissue-remodeling conditions by a mechanism of alternative splicing at the level of the primary transcript. EDB is essentially undetectable in healthy adult individuals. EDB-containing fibronectin is abundant in many aggressive solid tumors and in neo-vascularized endothelium, and displays either predominantly vascular or diffuse stromal patterns of expression, depending on the tissue.
Large Tenascin-C Isoforms
Tenascins are a family of four extracellular matrix glycoproteins that are found in vertebrates. They are typically present in many different connective tissues. Tenascins contribute to matrix structure and influence the behavior of cells that are in contact with the extracellular matrix. Several isoforms of tenascin-C can be generated as a result of different patterns of alternative splicing in the region between domains A1 and D. It has been known for some time that spliced isoforms containing extra domains are tumor-associated antigens, which show a more restricted pattern of expression in normal tissues compared with the “small” tenascin isoforms. The C domain of tenascin-C is the extra domain that shows the most restricted pattern of expression. In normal adult tissue it is undetectable by immunohistochemistry and northern-blot analysis, but it is strongly expressed in aggressive brain tumors and some lung tumors, with a prominent perivascular pattern of staining.
Integrins
During vascular remodeling and angiogenesis, endothelial cells show increased expression of several cell-surface molecules that potentiate cell invasion and proliferation. One such molecule is the integrin αv-β3, which has a key role in endothelial cell survival during angiogenesis in vivo and which might serve as a target for therapeutic molecules, particularly those that require internalization in endothelial cells. Monoclonal antibodies to the αv-β3 have been shown to display anti-angiogenic activities and to preferentially stain tumor blood vessels.
VEGFs and Their Receptors
VEGFs represent a class of proteins that promote angiogenesis, increase vascular permeability and contribute to endothelial-cell survival in blood and lymphatic vessels. The contribution of VEGFA to cancer progression has been highlighted by the recent approval of the humanized anti-VEGF monoclonal antibody bevacizumab (Avastin; Genentech) for first-line cancer treatment. The overexpression of VEGFs and VEGF receptors in tumors is well documented. The selective tumor localization of monoclonal antibodies to VEGFA, VEGF receptor 2 and the VEGFA-VEGF receptor 2 complex can be used as an excellent selectivity mechanism for targeting the angiogenic vasculature.
Prostate-Specific Membrane Antigen
Prostate-specific membrane antigen (PSMA) is a membrane glycoprotein with proteolytic activity. PSMA is predominantly expressed in the prostate and serum concentrations are often increased in patients with prostate cancer. Several studies have reported overexpression of PSMA in the neo-vasculature of different solid tumors, whereas expression in normal vasculature is limited to some vessels of the breast, duodenum, kidney and prostate.
Endoglin
Endoglin (CD105) is a transforming growth factor-beta (TGF) co-receptor that is overexpressed in tumor neo-vasculature and is used as a marker for the tumor endothelium.
CD44 Isoforms
CD44 is a cell-surface receptor of great molecular heterogeneity, which is due to both alternative splicing and extensive post-translational modification. The radio-labeled monoclonal antibody TES-23, which is specific to an isoform of CD44, has shown impressive performance in tumor-targeting experiments in animal models. TES-23 recognizes a widely distributed form of CD44 that lacks variant exons, known as CD44H.
Tumor Endothelial Markers (TEMs)
TEMs is a family of genes encoding proteins that serve as tumor endothelial markers (Carson-Walter, Watkins, et al, Cancer Res. 61:6649-55, 2001). These genes display elevated expression during tumor angiogenesis. From both biological and clinical points of view, TEMs associated with the cell surface membrane are of particular interest. Accordingly, four such genes are characterized, TEM1, TEM5, TEM7, and TEM8, all of which contain putative transmembrane domains. TEM5 appears to be a seven-pass transmembrane receptor, whereas TEM1, TEM7, and TEM8 span the membrane once. Three of these TEMs (TEM1, TEM5, and TEM8) are abundantly expressed in tumor vessels in mouse tumors, embryos, and adult tissues as well as in the vasculature of the developing mouse embryo. The expression of these TEMs in normal adult mice tissues is undetectable.
Selective Delivery Through Pro-Drug Activation
Selective delivery of agents of the invention can be achieved by administering a pro-drug form that is converted into an active drug at the target site. Most pro-drugs are designed to have a “trigger,” “linker” and “effector.” The “trigger,” following the tissue-specific metabolism, modifies the “linker,” resulting in an activation of the “effector.” There are several mechanisms potentially exploitable for selective activation. Some utilize unique aspects of the tissue physiology, such as selective enzyme expression or hypoxia in the case of tumors, whereas others are based on tissue antigen-specific delivery techniques.
Targeting Secreted Enzymes From Cells
The approach uses pro-drugs that are “hidden” from the cells until cleaved by an enzyme produced and secreted preferentially by the cells. A typical example of an enzyme used for pro-drug activation is MMP-9.
Targeting Tumor Hypoxia
Advances in the chemistry of bio-reductive drug activation have led to the design of hypoxia-selective drug delivery systems. These pro-drugs initially undergo one-electron reduction by reductases to give the radical anion, which in normal cells are re-oxidized to the parent compound, but in hypoxic tumor cells they are further reduced to more hydrophilic species and trapped inside. These drugs can be selectively delivered to tumors with defined hypoxic fractions rich in the required activating enzymes.
Antibody-Directed Enzyme Pro-Drug Therapy
Antibody-directed pro-drug therapy (ADEPT) is a 2-step approach in which first the antibody-enzyme construct is administered intravenously. This is composed of an antibody against a tissue-specific target linked to an enzyme that activates a pro-drug. In the second step, after the unbound antibody-enzyme conjugate construct is cleared from the circulation, a pro-drug is administered intravenously. The pro-drug is an agent that has been rendered less active by chemical addition of enzyme-cleavable moieties. The pro-drug is converted to an active form by the tumor-bound antibody-enzyme, which results in local accumulation of the fully active form of the agent.
External Energy-Controlled Delivery
Some selective delivery strategies involve focusing external energy for concentrating or delivering therapeutics at the tissue site. A variety of delivery systems in this category are in the experimental stage, although some have been used in clinical trials as well. Those strategies include selective delivery through photodynamic therapy, magnetically targeted delivery, selective delivery through X-ray exposure, radiation-induced selective delivery and ultrasound-guided delivery.
Methods of Ocular Delivery
The compositions of the invention (e.g., a peptide of the invention shown in Tables 1-10) are also particularly suitable for treating ocular diseases, such as age-related macular degeneration, choroidal neovascularization, persistent hyperplastic vitreous syndrome, diabetic retinopathy, and retinopathy of prematurity that are characterized by excess angiogenesis.
In one approach, the compositions of the invention are administered through an ocular device suitable for direct implantation into the vitreous of the eye. The compositions of the invention may be provided in sustained release compositions, such as those described in, for example, U.S. Pat. Nos. 5,672,659 and 5,595,760. Such devices are found to provide sustained controlled release of various compositions to treat the eye without risk of detrimental local and systemic side effects. An object of the present ocular method of delivery is to maximize the amount of drug contained in an intraocular device or implant while minimizing its size in order to prolong the duration of the implant. See, e.g., U.S. Pat. Nos. 5,378,475; 6,375,972, and 6,756,058 and U.S. Publications 20050096290 and 200501269448. Such implants may be biodegradable and/or biocompatible implants, or may be non-biodegradable implants. Biodegradable ocular implants are described, for example, in U.S. Patent Publication No. 20050048099. The implants may be permeable or impermeable to the active agent, and may be inserted into a chamber of the eye, such as the anterior or posterior chambers or may be implanted in the schlera, transchoroidal space, or an avascularized region exterior to the vitreous. Alternatively, a contact lens that acts as a depot for compositions of the invention may also be used for drug delivery.
In a preferred embodiment, the implant may be positioned over an avascular region, such as on the sclera, so as to allow for transcleral diffusion of the drug to the desired site of treatment, e.g. the intraocular space and macula of the eye. Furthermore, the site of transcleral diffusion is preferably in proximity to the macula. Examples of implants for delivery of a composition include, but are not limited to, the devices described in U.S. Pat. Nos. 3,416,530; 3,828,777; 4,014,335; 4,300,557; 4,327,725; 4,853,224; 4,946,450; 4,997,652; 5,147,647; 5,164,188; 5,178,635; 5,300,114; 5,322,691; 5,403,901; 5,443,505; 5,466,466; 5,476,511; 5,516,522; 5,632,984; 5,679,666; 5,710,165; 5,725,493; 5,743,274; 5,766,242; 5,766,619; 5,770,592; 5,773,019; 5,824,072; 5,824,073; 5,830,173; 5,836,935; 5,869,079, 5,902,598; 5,904,144; 5,916,584; 6,001,386; 6,074,661; 6,110,485; 6,126,687; 6,146,366; 6,251,090; and 6,299,895, and in WO 01/30323 and WO 01/28474, all of which are incorporated herein by reference.
Examples include, but are not limited to the following: a sustained release drug delivery system comprising an inner reservoir comprising an effective amount of an agent effective in obtaining a desired local or systemic physiological or pharmacological effect, an inner tube impermeable to the passage of the agent, the inner tube having first and second ends and covering at least a portion of the inner reservoir, the inner tube sized and formed of a material so that the inner tube is capable of supporting its own weight, an impermeable member positioned at the inner tube first end, the impermeable member preventing passage of the agent out of the reservoir through the inner tube first end, and a permeable member positioned at the inner tube second end, the permeable member allowing diffusion of the agent out of the reservoir through the inner tube second end; a method for administering a compound of the invention to a segment of an eye, the method comprising the step of implanting a sustained release device to deliver the compound of the invention to the vitreous of the eye or an implantable, sustained release device for administering a compound of the invention to a segment of an eye; a sustained release drug delivery device comprising: a) a drug core comprising a therapeutically effective amount of at least one first agent effective in obtaining a diagnostic effect or effective in obtaining a desired local or systemic physiological or pharmacological effect; b) at least one unitary cup essentially impermeable to the passage of the agent that surrounds and defines an internal compartment to accept the drug core, the unitary cup comprising an open top end with at least one recessed groove around at least some portion of the open top end of the unitary cup; c) a permeable plug which is permeable to the passage of the agent, the permeable plug is positioned at the open top end of the unitary cup wherein the groove interacts with the permeable plug holding it in position and closing the open top end, the permeable plug allowing passage of the agent out of the drug core, through the permeable plug, and out the open top end of the unitary cup; and d) at least one second agent effective in obtaining a diagnostic effect or effective in obtaining a desired local or systemic physiological or pharmacological effect; or a sustained release drug delivery device comprising: an inner core comprising an effective amount of an agent having a desired solubility and a polymer coating layer, the polymer layer being permeable to the agent, wherein the polymer coating layer completely covers the inner core.
Other approaches for ocular delivery include the use of liposomes to target a compound of the present invention to the eye, and preferably to retinal pigment epithelial cells and/or Bruch's membrane. For example, the compound may be complexed with liposomes in the manner described above, and this compound/liposome complex injected into patients with an ocular disease, using intravenous injection to direct the compound to the desired ocular tissue or cell. Directly injecting the liposome complex into the proximity of the retinal pigment epithelial cells or Bruch's membrane can also provide for targeting of the complex with some forms of ocular disease. In a specific embodiment, the compound is administered via intra-ocular sustained delivery (such as VITRASERT or ENVISION). In a specific embodiment, the compound is delivered by posterior subtenons injection. In another specific embodiment, microemulsion particles containing the compositions of the invention are delivered to ocular tissue to take up lipid from Bruch's membrane, retinal pigment epithelial cells, or both.
For optical applications, nanoparticles are a colloidal carrier system that has been shown to improve the efficacy of the encapsulated drug by prolonging the serum half-life. Polyalkylcyanoacrylates (PACAs) nanoparticles are a polymer colloidal drug delivery system that is in clinical development, as described by Stella et al., J. Pharm. Sci., 2000. 89: p. 1452-1464; Brigger et al., Int. J. Pharm., 2001. 214: p. 37-42; Calvo et al., Pharm. Res., 2001. 18: p. 1157-1166; and Li et al., Biol. Pharm. Bull., 2001. 24: p. 662-665. Biodegradable poly (hydroxyl acids), such as the copolymers of poly (lactic acid) (PLA) and poly (lactic-co-glycolide) (PLGA) are being extensively used in biomedical applications and have received FDA approval for certain clinical applications. In addition, PEG-PLGA nanoparticles have many desirable carrier features including (i) that the agent to be encapsulated comprises a reasonably high weight fraction (loading) of the total carrier system; (ii) that the amount of agent used in the first step of the encapsulation process is incorporated into the final carrier (entrapment efficiency) at a reasonably high level; (iii) that the carrier have the ability to be freeze-dried and reconstituted in solution without aggregation; (iv) that the carrier be biodegradable; (v) that the carrier system be of small size; and (vi) that the carrier enhance the particles persistence.
Nanoparticles are synthesized using virtually any biodegradable shell known in the art. In one embodiment, a polymer, such as poly (lactic-acid) (PLA) or poly (lactic-co-glycolic acid) (PLGA) is used. Such polymers are biocompatible and biodegradable, and are subject to modifications that desirably increase the photochemical efficacy and circulation lifetime of the nanoparticle. In one embodiment, the polymer is modified with a terminal carboxylic acid group (COOH) that increases the negative charge of the particle and thus limits the interaction with negatively charge nucleic acid aptamers. Nanoparticles are also modified with polyethylene glycol (PEG), which also increases the half-life and stability of the particles in circulation. Alternatively, the COOH group is converted to an N-hydroxysuccinimide (NHS) ester for covalent conjugation to amine-modified aptamers.
Biocompatible polymers useful in the composition and methods of the invention include, but are not limited to, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetage phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinyl chloride polystyrene, polyvinylpryrrolidone, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecl acrylate) and combinations of any of these. In one embodiment, the nanoparticles of the invention include PEG-PLGA polymers.
Compositions of the invention may also be delivered topically. For topical delivery, the compositions are provided in any pharmaceutically acceptable excipient that is approved for ocular delivery. Preferably, the composition is delivered in drop form to the surface of the eye. For some application, the delivery of the composition relies on the diffusion of the compounds through the cornea to the interior of the eye.
Those of skill in the art will recognize that the best treatment regimens for using compounds of the present invention to treat an ocular disease can be straightforwardly determined. This is not a question of experimentation, but rather one of optimization, which is routinely conducted in the medical arts. In vivo studies in nude mice often provide a starting point from which to begin to optimize the dosage and delivery regimes. The frequency of injection will initially be once a week, as has been done in some mice studies. However, this frequency might be optimally adjusted from one day to every two weeks to monthly, depending upon the results obtained from the initial clinical trials and the needs of a particular patient.
Human dosage amounts can initially be determined by extrapolating from the amount of compound used in mice, as a skilled artisan recognizes it is routine in the art to modify the dosage for humans compared to animal models. In certain embodiments it is envisioned that the dosage may vary from between about 1 mg compound/Kg body weight to about 5000 mg compound/Kg body weight; or from about 5 mg/Kg body weight to about 4000 mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg body weight; or from about 100 mg/Kg body weight to about 1000 mg/Kg body weight; or from about 150 mg/Kg body weight to about 500 mg/Kg body weight. In other embodiments this dose may be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000 mg/Kg body weight. In other embodiments, it is envisaged that higher doses may be used, such doses may be in the range of about 5 mg compound/Kg body to about 20 mg compound/Kg body. In other embodiments the doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient.
Combination Therapies
Optionally, an angiogenic modulating therapeutic as described herein may be administered in combination with any other standard active angiogenic modulating therapeutics; such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences by E. W. Martin. For example, an anti-angiogenic peptide of the invention may be administered in combination with any other anti-angiogenic peptide, or with known anti-angiogenic agent. Such agents are listed below (Folkman, Annu Rev Med. 57:1-18, 2006).














Agent
Clinical Trials









 1. Alphastatin




 2. Angiostatin



 3. Arresten



 4. Anti-thrombin III (truncated)



 5. Canstatin



 6. Endostatin
Phase II



 7. Fibulin-5



 8. Fragment of histidine-rich glycoprotein



 9. Interferon-β
Phase III



10. Maspin



11. 2-methoxyestradiol
Phase II



12. PEX



13. Pigment epithelial-derived factor (PEDF)



14. Platelet factor 4 (PF4)



15. Semaphorin 3F



16. sFlt-1



17. Tetrahydrocortisol
Phase III



18. Thrombospondin-1 (and -2)
Phase II



19. TEMP-2



20. Troponin I



21. Tumstatin



22. Vasostatin










For the treatment of a neoplasia, a peptide of the invention is administered in combination with any conventional treatment (e.g., chemotherapy, radiotherapy, hormonal therapy, surgery, cryosurgery). A pharmaceutical composition of the invention may, if desired, include one or more chemotherapeutics typically used in the treatment of a neoplasm, such as abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMS184476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-t-butylamide, cachectin, cemadotin, chlorambucil, cyclophosphamide, 3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine (BCNU), cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin, dolastatin, doxorubicin (adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, 5-fluorouracil, nilutamide, onapristone, paclitaxel, prednimustine, procarbazine, RPR109881, stramustine phosphate, tamoxifen, tasonermin, taxol, thalidomide, tretinoin, vinblastine, vincristine, vindesine sulfate, and vinflunine. Other examples of chemotherapeutic agents can be found in Cancer Principles and Practice of Oncology by V. T. Devita and S. Hellman (editors), 6th edition (Feb. 15, 2001), Lippincott Williams & Wilkins Publishers.
Kits
The invention provides kits for the treatment or prevention of diseases or disorders characterized by excess or undesirable angiogenesis. In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of one or more peptides described herein in unit dosage form. In some embodiments, the kit comprises a sterile container that contains a therapeutic or prophylactic vaccine; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired a peptide of the invention is provided together with instructions for administering it to a subject having or at risk of developing excess or undesired angiogenesis. The instructions will generally include information about the use of the composition for the treatment or prevention of ischemia or for enhancing angiogenesis to a tissue in need thereof. In other embodiments, the instructions include at least one of the following: description of the expression vector; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
Methods of the Invention
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
EXAMPLES
Example 1
Analysis of Peptide Motifs
Using bioinformatic analysis 156 peptides with anti-angiogenic properties were identified based on their sequence similarity with known anti-angiogenic peptides. A number of these peptides were screened for anti-angiogenic activity using an endothelial cell proliferation assay to identify peptide motifs associated with anti-angiogenic activity. Multiple sequence alignments were used to identify peptides having conserved motifs that are common in a variety of sequences. Multiple sequence alignment was performed using the ClustalW algorithm to align sequences of peptides that belong to different protein families including type I thrombospondin repeat-containing proteins, C-X-C chemokines, collagen type IV, somatotropins and serpins. In order to perform the alignment a critical number of peptide sequences were required. The motifs were represented using the single letter abbreviations of the amino acids that are common and the letter “X” to denote a non-common amino acid that intervenes the common letters. If there is more than one non-common amino acid in between, the letter “X” followed by the number of the non-common amino acids was used. For example if there are three non-common amino acids between two conserved letters, we notify it as “a-X3-b”, where a and b is the conserved motif. This notation is commonly used to represent motifs.
Initially multiple sequence alignments to the experimentally tested peptides were performed. The calculation was generalized to all the theoretically predicted fragments. To determine whether the motifs calculated for the experimentally tested fragments were conserved and reproduced in all of the anti-angiogenic predictions. The results obtained were organized by protein family. As described in more detail below, general peptide motifs associated with anti-angiogenic activity were identified for three families of human proteins: Type I thrombospondin (TSP) domain containing proteins, CXC chemokines, and collagens. Using these motifs, 2286 peptides each containing one of the identified motifs were identified in 1977 different proteins present in the human proteome (166 peptides from 54 different proteins listed in Table 2; 1337 peptides from 1170 proteins listed in Table 4; 24 peptides from 24 proteins listed in Table 5; 306 peptides from 288 proteins listed in Table 6; 139 peptides from 139 proteins listed in Table 8; and 314 peptides from 302 different proteins listed in Table 9.
In addition, 12 novel peptide sequences from the Somatotropin, Serpin, and Type IV Collagen families obtained based on the similarity criteria with known anti-angiogenic peptides are listed in Tables 7A, 7B, and 10, respectively.
Example 2
Thrombospondin-1 (TSP-1) Repeat-Containing Proteins Derived Peptides
From the 31 predicted and experimentally tested TSP-1 containing short peptides 29 share a global 4 letter common motif which is the X2-W-X2-C-X3-C-X2-G-X7, or W-X2-C-X3-C-X2-G after removing the uncommon edges, resulting in the generic TSP-1 containing 20-mer (FIG. 1). The first amino acid that succeeds the first cysteine of the motif, or the seventh amino acid of the sequence can alternate between T, S and N. Thus a more generic description of this motif is X2-W-X2-C-(T/S/N)-X2-C-X2-G-X7 with threonine or serine the most abundant alteration for the seventh amino acid position.
By altering the threshold of the conserved amino acids that are common among the sequences of the predicted peptides we can create subsets of peptide families with individual common motifs of greater length than the global 4-letter motif. The threshold here is defined as the percentage of the peptides that share a common motif. Such a subgroup of peptides is one that consists of 18 TSP-1 containing predictions (threshold 60%) that share a seven amino acid long common motif. The motif is the X2-W-X2-C-S-X2-C-G-X1-G-X3-R-X3. A common alteration occurs in the 19th amino acid, which can be either an arginine or a valine with arginine the most abundant amino acid. In that case the motif is written X2-W-X2-C-S-X2-C-G-X1-G-X3-R-X1-(R/V)-X1. Similarly the ninth amino acid can be altered by either arginine, serine or threonine. In that case the motif can be represented as X2-W-X2-C-S-X1-(S/R/T)-C-G-X1-G-X3-R-X1-(R/V)-X1 with threonine the most abundant amino acid (FIG. 2A). Similarly another motif with 45% threshold, common in 13 sequences, is the 5 letter motif X1-P-W-X2-C-X3-C-X2-G-X7. The common alterations of this motif can be described as (S/G/Q)-P-W-X2-C-(T/S)-X2-C-(G/S)-X1-G-X3-(R/S)-X3 (FIG. 2B).
In addition to calculating the motifs that are present within the sequences of the predicted fragments one can analyze all the possible amino acids that are present within the 29 peptide sequences from which the motifs were calculated. This 20-mer with all the possible substitutions is presented in Table 1 along with the frequencies that each amino acid is present in the 29 sequences.







TABLE 1







The TSP-1 containing 20-mer with all the possible amino acid substitutions


























AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA


#1
#2
#3
#4
#5
#6
#7
#8
#9
#10
#11
#12
#13
#14
#15
#16
#17
#18
#19
#20





S(9)
P(13)
W(29)
S(14)
P(9)
C(29)
S(26)
V(7)
T(15)
C(29)
G(26)
G(10)
G(29)
V(8)
Q(11)
T(10)
R(26)
S(5)
R(15)
R(1)


T(9)
E(5)

T(5)
A(5)

N(2)
A(6)
S(10)

S(2)
K(4)

I(4)
S(7)
F(4)
S(2)
T(5)
V(1)



G(6)
S(3)

G(5)
Q(4)

T(1)
R(5)
R(3)

N(1)
R(4)

M(3)
R(6)
K(3)
Q(1)
V(5)




O(2)
A(2)

E(2)
D(3)


K(4)
N(1)


M(4)

T(3)
K(2)
Q(3)

R(3)




A(1)
Q(1)

D(1)
E(3)


G(2)



T(2)

H(2)
Y(2)
S(3)

H(3)





K(1)

R(1)
K(1)


S(2)



L(2)

A(1)
A(1)
L(2)

E(2)







A(1)
R(1)


T(2)



D(1)

E(1)

E(1)

Q(2)








V(1)


E(1)



S(1)

F(1)

M(1)

A(1)















P(1)

K(1)

N(1)

I(1)

















R(1)

V(1)



















S(1)





















Q(1)





















W(1)





















Y(1)









The above motifs, for both the TSP-1 containing proteins were identified from the sequences of the peptide fragments that have already been experimentally tested in proliferation assay. The specific approach for identification of motifs within groups of sequences can be generalized for the case of all the theoretically predicted anti-angiogenic fragments. For the TSP-1 repeat-containing protein derived fragments the multiple sequence alignment calculations are repeated, but now all of the theoretically predicted fragments are included. The same approach is also utilized for the case of collagens where only the experimentally tested pool of sequences is not sufficient to yield statistically significant results. In that case after including all the theoretically predicted fragments we are able to identify common motifs.
For the cases of all the theoretically predicted TSP-1 containing proteins, multiple sequence alignment yields a common motif within 97% of all the tested sequences. This motif is the already identified W-X2-C-X3-C-X2-G (FIG. 3) and a generic 20-mer can be expressed as X2-W-X2-C-X3-C-X2-G-X7. It is interesting that this motif is not a characteristic of only the TSP-1 domains, in other words in not a signature for TSP-1. When its presence was tested for all the TSP-1 containing proteins it was identified only within a subset of this family. Moreover, it is present within the type-2 thrombospondin containing proteins (TSP-2), which have already been shown to be associated with anti-angiogenic activity. In other words we claim that the motif W-X2-C-X3-C-X2-G, although present within a large portion of the TSP-1 containing proteins, is not a signature for a generic TSP-1 containing protein but only for those proteins with putative anti-angiogenic activity that may or may not belong to the specific protein family. Moreover, as observed within the sequences of the experimentally tested fragments and is also reproduced in the case of all the theoretically predicted fragments, the amino acid following the first cysteine of the motif can alternate between T, S and N. Thus a more specific description of the motif is the W-X2-C-(T/S/N)-X2-C-X2-G with serine and threonine being the predominant amino acids in the position following the first cysteine.
A common alteration occurs in the 19th amino acid of the 20-mer which can be either an arginine or a valine with arginine the most abundant amino acid. In that case the motif is written X2-W-X2-C-(T/S/N)-X2-C-X2-G-X5-(R/V)-X.
The most generic 4-common letter motif identified within the peptide sequences is W-X2-C-X3-C-X2-G. The ScanProsite tool can be used to search the human proteome Prosite database at the Swiss Institute of Bioinformatics. Using the aforementioned motif as a query identified this motif in 166 locations of 54 different proteins listed in Table 2 (SEQ ID Nos. 1-166).







TABLE 2







TSPs


Motif: W-X(2)-C-X(3)-C-X(2)-G


Number of Locations: 166


Number of Different Proteins: 54












Accession Number|Protein

Last



#
Name
First Amino acid
Amino acid
Sequence














1
O00622|CYR61_HUMAN
236
246
WsqCsktCgtG


2
O14514|BAI1_HUMAN
270
280
WgeCtrdCggG


3
O14514|BAI1_HUMAN
363
373
WsvCsstCgeG


4
O14514|BAI1_HUMAN
418
428
WslCsstCgrG


5
O14514|BAI1_HUMAN
476
486
WsaCsasCsqG


6
O14514|BAI1_HUMAN
531
541
WgsCsvtCgaG


7
O15072|ATS3_HUMAN
975
985
WseCsvtCgeG


8
O60241|BAI2_HUMAN
306
316
WsvCsltCgqG


9
O60241|BAI2_HUMAN
361
371
WslCsrsCgrG


10
O60241|BAI2_HUMAN
416
426
WgpCstsCanG


11
O60241|BAI2_HUMAN
472
482
WslCsktCdtG


12
O60242|BAI3_HUMAN
300
310
WstCsvtCgqG


13
O60242|BAI3_HUMAN
354
364
WslCsftCgrG


14
O60242|BAI3_HUMAN
409
419
WsqCsvtCsnG


15
O60242|BAI3_HUMAN
464
474
WsgCsksCdgG


16
O75173|ATS4_HUMAN
529
539
WgdCsrtCggG


17
O76076|WISP2_HUMAN
201
211
WgpCsttCglG


18
O95185|UNC5C_HUMAN
269
279
WsvCnsrCgrG


19
O95388|WISP1_HUMAN
223
233
WspCstsCglG


20
O95389|WISP3_HUMAN
216
226
WtpCsrtCgmG


21
O95450|ATS2_HUMAN
863
873
WspCskpCggG


22
O95450|ATS2_HUMAN
984
994
WsqCsvtCgnG


23
P07996|TSP1_HUMAN
388
398
WtsCstsCgnG


24
P07996|TSP1_HUMAN
444
454
WssCsvtCgdG


25
P07996|TSP1_HUMAN
501
511
WdiCsvtCggG


26
P13671|CO6_HUMAN
32
42
WtsCsktCnsG


27
P13671|CO6_HUMAN
75
85
WqrCpinCllG


28
P14222|PERF_HUMAN
374
384
WrdCsrpCppG


29
P27918|PROP_HUMAN
86
96
WapCsvtCseG


30
P27918|PROP_HUMAN
145
155
WepCsvtCskG


31
P27918|PROP_HUMAN
202
212
WtpCsasChgG


32
P29279|CTGF_HUMAN
206
216
WsaCsktCgmG


33
P35442|TSP2_HUMAN
390
400
WtqCsvtCgsG


34
P35442|TSP2_HUMAN
446
456
WssCsvtCgvG


35
P35442|TSP2_HUMAN
503
513
WsaCtvtCagG


36
P48745|NOV_HUMAN
213
223
WtaCsksCgmG


37
P49327|FAS_HUMAN
627
637
WeeCkqrCppG


38
P58397|ATS12_HUMAN
551
561
WshCsrtCgaG


39
P58397|ATS12_HUMAN
832
842
WteCsvtCgtG


40
P58397|ATS12_HUMAN
952
962
WseCsvsCggG


41
P58397|ATS12_HUMAN
1321
1331
WseCsttCglG


42
P58397|ATS12_HUMAN
1372
1382
WskCsrnCsgG


43
P58397|ATS12_HUMAN
1431
1441
WsqCsrsCggG


44
P58397|ATS12_HUMAN
1479
1489
WdlCstsCggG


45
P59510|ATS20_HUMAN
976
986
WsqCsrsCggG


46
P59510|ATS20_HUMAN
1031
1041
WseClvtCgkG


47
P59510|ATS20_HUMAN
1086
1096
WgpCtttCghG


48
P59510|ATS20_HUMAN
1162
1172
WtpCsvsCgrG


49
P59510|ATS20_HUMAN
1217
1227
WspCsasCghG


50
P59510|ATS20_HUMAN
1314
1324
WgsCsssCsgG


51
P59510|ATS20_HUMAN
1368
1378
WgeCsqtCggG


52
P59510|ATS20_HUMAN
1427
1437
WtsCsasCgkG


53
P59510|ATS20_HUMAN
1483
1493
WneCsvtCgsG


54
P59510|ATS20_HUMAN
1664
1674
WskCsvtCgiG


55
P82987|ATL3_HUMAN
84
94
WsdCsrtCggG


56
P82987|ATL3_HUMAN
427
437
WtaCsvsCggG


57
P82987|ATL3_HUMAN
487
497
WsqCtvtCgrG


58
P82987|ATL3_HUMAN
573
583
WsaCsttCgpG


59
P82987|ATL3_HUMAN
712
722
WgpCsatCgvG


60
P82987|ATL3_HUMAN
768
778
WqqCsrtCggG


61
P82987|ATL3_HUMAN
828
838
WskCsvsCgvG


62
P82987|ATL3_HUMAN
1492
1502
WsqCsvsCgeG


63
P82987|ATL3_HUMAN
1606
1616
WkpCtaaCgrG


64
Q13591|SEM5A_HUMAN
604
614
WspCsttCgiG


65
Q13591|SEM5A_HUMAN
662
672
WerCtaqCggG


66
Q13591|SEM5A_HUMAN
793
803
WsqCsrdCsrG


67
Q13591|SEM5A_HUMAN
850
860
WtkCsatCggG


68
Q496M8|CI094_HUMAN
259
269
WsaCtrsCggG


69
Q6S8J7|POTE8_HUMAN
27
37
WccCcfpCcrG


70
Q6UXZ4|UNC5D_HUMAN
261
271
WsaCnvrCgrG


71
Q6UY14|ATL4_HUMAN
53
63
WasCsqpCgvG


72
Q6UY14|ATL4_HUMAN
732
742
WtsCsrsCgpG


73
Q6UY14|ATL4_HUMAN
792
802
WsqCsvrCgrG


74
Q6UY14|ATL4_HUMAN
919
929
WgeCsseCgsG


75
Q6UY14|ATL4_HUMAN
979
989
WspCsrsCqgG


76
Q6ZMM2|ATL5_HUMAN
44
54
WtrCsssCgrG


77
Q76LX8|ATS13_HUMAN
1081
1091
WmeCsvsCgdG


78
Q86TH1|ATL2_HUMAN
56
66
WtaCsrsCggG


79
Q86TH1|ATL2_HUMAN
631
641
WseCsrtCgeG


80
Q86TH1|ATL2_HUMAN
746
756
WgpCsgsCgqG


81
Q86TH1|ATL2_HUMAN
803
813
WerCnttCgrG


82
Q86TH1|ATL2_HUMAN
862
872
WseCtktCgvG


83
Q8IUL8|CILP2_HUMAN
155
165
WgpCsgsCgpG


84
Q8IZJ1|UNC5B_HUMAN
255
265
WspCsnrCgrG


85
Q8N6G6|ATL1_HUMAN
42
52
WseCsrtCggG


86
Q8N6G6|ATL1_HUMAN
385
395
WtaCsssCggG


87
Q8N6G6|ATL1_HUMAN
445
455
WspCtvtCgqG


88
Q8TE56|ATS17_HUMAN
552
562
WsmCsrtCgtG


89
Q8TE56|ATS17_HUMAN
809
819
WegCsvqCggG


90
Q8TE56|ATS17_HUMAN
870
880
WspCsatCekG


91
Q8TE56|ATS17_HUMAN
930
940
WsqCsasCgkG


92
Q8TE56|ATS17_HUMAN
981
991
WstCsstCgkG


93
Q8TE57|ATS16_HUMAN
595
605
WspCsrtCggG


94
Q8TE57|ATS16_HUMAN
936
946
WsaCsrtCggG


95
Q8TE57|ATS16_HUMAN
995
1005
WaeCshtCgkG


96
Q8TE57|ATS16_HUMAN
1060
1070
WsqCsvtCerG


97
Q8TE57|ATS16_HUMAN
1135
1145
WsqCtasCggG


98
Q8TE58|ATS15_HUMAN
848
858
WgpCsasCgsG


99
Q8TE58|ATS15_HUMAN
902
912
WspCsksCgrG


100
Q8TE59|ATS19_HUMAN
642
652
WspCsrtCsaG


101
Q8TE59|ATS19_HUMAN
924
934
WedCdatCggG


102
Q8TE59|ATS19_HUMAN
985
995
WtpCsrtCgkG


103
Q8TE59|ATS19_HUMAN
1096
1106
WskCsitCgkG


104
Q8TE60|ATS18_HUMAN
598
608
WseCsrtCggG


105
Q8TE60|ATS18_HUMAN
940
950
WstCskaCagG


106
Q8TE60|ATS18_HUMAN
1000
1010
WsqCsktCgrG


107
Q8TE60|ATS18_HUMAN
1061
1071
WseCsatCglG


108
Q8TE60|ATS18_HUMAN
1132
1142
WqqCtvtCggG


109
Q8WXS8|ATS14_HUMAN
856
866
WapCskaCggG


110
Q8WXS8|ATS14_HUMAN
977
987
WsqCsatCgeG


111
Q92947|GCDH_HUMAN
225
235
WarCedgCirG


112
Q96RW7|HMCN1_HUMAN
4538
4548
WraCsvtCgkG


113
Q96RW7|HMCN1_HUMAN
4595
4605
WeeCtrsCgrG


114
Q96RW7|HMCN1_HUMAN
4652
4662
WgtCsesCgkG


115
Q96RW7|HMCN1_HUMAN
4709
4719
WsaCsvsCggG


116
Q96RW7|HMCN1_HUMAN
4766
4776
WgtCsrtCngG


117
Q96RW7|HMCN1_HUMAN
4823
4833
WsqCsasCggG


118
Q99732|LITAF_HUMAN
116
126
WlsCgslCllG


119
Q9C0I4|THS7B_HUMAN
49
59
WgrCtgdCgpG


120
Q9C0I4|THS7B_HUMAN
345
355
WspCsktCrsG


121
Q9C0I4|THS7B_HUMAN
746
756
WtpCprmCqaG


122
Q9C0I4|THS7B_HUMAN
1009
1019
WgsCsssCgiG


123
Q9C0I4|THS7B_HUMAN
1258
1268
WteCsqtCghG


124
Q9C0I4|THS7B_HUMAN
1381
1391
WstCeltCidG


125
Q9H324|ATS10_HUMAN
530
540
WgdCsrtCggG


126
Q9H324|ATS10_HUMAN
808
818
WtkCsaqCagG


127
Q9H324|ATS10_HUMAN
867
877
WslCsrsCdaG


128
Q9H324|ATS10_HUMAN
927
937
WseCtpsCgpG


129
Q9H324|ATS10_HUMAN
986
996
WgeCsaqCgvG


130
Q9HCB6|SPON1_HUMAN
510
520
WspCsisCgmG


131
Q9HCB6|SPON1_HUMAN
567
577
WdeCsatCgmG


132
Q9HCB6|SPON1_HUMAN
623
633
WsdCsvtCgkG


133
Q9HCB6|SPON1_HUMAN
677
687
WseCnksCgkG


134
Q9HCB6|SPON1_HUMAN
763
773
WseCtklCggG


135
Q9NS62|THSD1_HUMAN
349
359
WsqCsatCgdG


136
Q9P283|SEM5B_HUMAN
615
625
WalCstsCgiG


137
Q9P283|SEM5B_HUMAN
673
683
WskCssnCggG


138
Q9P283|SEM5B_HUMAN
804
814
WssCsrdCelG


139
Q9P283|SEM5B_HUMAN
861
871
WspCsasCggG


140
Q9P2N4|ATS9_HUMAN
1006
1016
WteCsksCdgG


141
Q9P2N4|ATS9_HUMAN
1061
1071
WseClvtCgkG


142
Q9P2N4|ATS9_HUMAN
1116
1126
WvqCsvtCgqG


143
Q9P2N4|ATS9_HUMAN
1191
1201
WtpCsatCgkG


144
Q9P2N4|ATS9_HUMAN
1247
1257
WssCsvtCgqG


145
Q9P2N4|ATS9_HUMAN
1337
1347
WgaCsstCagG


146
Q9P2N4|ATS9_HUMAN
1391
1401
WgeCtklCggG


147
Q9P2N4|ATS9_HUMAN
1450
1460
WssCsvsCgrG


148
Q9P2N4|ATS9_HUMAN
1506
1516
WsqCsvsCgrG


149
Q9P2N4|ATS9_HUMAN
1564
1574
WqeCtktCgeG


150
Q9P2N4|ATS9_HUMAN
1621
1631
WseCsvtCgkG


151
Q9P2N4|ATS9_HUMAN
1686
1696
WgsCsvsCgvG


152
Q9UHI8|ATS1_HUMAN
568
578
WgdCsrtCggG


153
Q9UHI8|ATS1_HUMAN
863
873
WgeCsksCelG


154
Q9UHI8|ATS1_HUMAN
917
927
WssCsktCgkG


155
Q9UKP4|ATS7_HUMAN
547
557
WsiCsrsCgmG


156
Q9UKP4|ATS7_HUMAN
924
934
WtkCtvtCgrG


157
Q9UKP5|ATS6_HUMAN
519
529
WgeCsrtCggG


158
Q9UKP5|ATS6_HUMAN
801
811
WseCsatCagG


159
Q9UNA0|ATS5_HUMAN
576
586
WgqCsrsCggG


160
Q9UNA0|ATS5_HUMAN
884
894
WlaCsrtCdtG


161
Q9UP79|ATS8_HUMAN
536
546
WgeCsrtCggG


162
Q9UP79|ATS8_HUMAN
842
852
WseCsstCgaG


163
Q9UPZ6|THS7A_HUMAN
203
213
WseCsktCgsG


164
Q9UPZ6|THS7A_HUMAN
780
790
WtsCpssCkeG


165
Q9UPZ6|THS7A_HUMAN
1044
1054
WsrCsksCgsG


166
Q9UPZ6|THS7A_HUMAN
1423
1433
WslCqltCvnG









These peptides are likely to have anti-angiogenic activity. Methods for testing for such activity are described herein.
Example 3
Peptides Derived from C-X-C Chemokines
For the six predicted and experimentally tested C-X-C chemokines, all of them contain a six amino acid common motif. Following the thus far used notation this motif can be described as X-G-X3-C-L-X-P-X10-K-X-L (FIG. 4). There are few common alterations that occur within the sequences of the predicted fragments. For all those cases the motif can be re-written as (N/D)-G-(R/K)-X2-C-L-(N/D)-P-X2-(P/N)-X2-(K/Q)-(K/Q)-(I/V)-(I/V)-(E/Q)-K-X-L.







TABLE 3





The C-X-C chemokine 22-mer with all the possible amino acid substitutions

























AA#1
AA#2
AA#3
AA#4
AA#5
AA#6
AA#7
AA#8
AA#9
AA#10
AA#11





N(4)
G(6)
R(3)
K(3)
A(2)
C(6)
L(6)
D(4)
P(6)
A(2)
A(3)


D(2)

K(3)
E(2)
I(2)


N(2)

E(2)
S(2)





Q(1)
L(1)




D(1)
E(1)






V(1)




K(1)





AA #12
AA #13
AA #14
AA #15
AA #16
AA #17
AA #18
AA #19
AA #20
AA #21
AA #22





P(6)
F(2)
V(3)
K(4)
K(5)
I(3)
I(4)
E(3)
K(6)
I(3)
L(6)



I(1)
L(2)
Q(2)
R(1)
V(3)
V(2)
Q(3)

F(1)




M(1)
I(1)






K(1)




R(1)







M(1)




W(1)









The generic 22-mer of the predicted C-X-C chemokines including all the possible substitutions is presented in Table 3.
The case of the motif calculation for the theoretically predicted C-X-C chemokines is extremely interesting as in this calculation both short and long fragments are included. If the motifs that were identified within the experimentally tested short fragments are present in the longer ones as well, this might help localize possible anti-angiogenic activity within the longer fragments.
When repeating the calculations with all the theoretically predicted C-X-C chemokines this reproduced the X-G-X3-C-L-X-P-X10-K-X-L motif as predicted when the motifs were calculated in the experimentally tested short fragments, but with minimal alterations (FIG. 5).
For the case of all the theoretically predicted C-X-C chemokines a more generic 22-mer can be described as (N/D/K)-G-X3-C-L-(D/N)-(P/L)-X5-(K/Q)-(K/R/N)-(I/V/L)-(I/V/L)-X6. From the above analysis it also becomes obvious that we can localize the activity of the longer predicted fragments at the sites where the predominant motif from the experimentally tested peptides resides.
Similarly to the type I thrombospondin containing proteins one can consider the most generic 3-common letter motif that is identified within the peptide sequences: G-X3-C-L, and search for its existence within the proteome and identify novel peptides that may contain it. Using as a query the aforementioned motif we utilize the ScanProsite tool to search the Prosite database at the Swiss Institute of Bioinformatics in order to identify protein location that may contain it. The G-X3-C-L motif is identified in 1337 locations of 1170 proteins listed in Table 4 (SEQ ID Nos. 167-1503).







TABLE 4







CXCs


Motif: G-X(3)-C-L


Number of Locations: 1337


Number of Different Proteins: 1170













First
Last




Accession
Amino
Amino


#
Number|Protein Name
acid
acid
Sequence














167
O00142|KITM_HUMAN
62
67
GkttCL


168
O00167|EYA2_HUMAN
361
366
GanlCL


169
O00220|TR10A_HUMAN
332
337
GeaqCL


170
O00291|HIP1_HUMAN
699
704
GattCL


171
O00409|FOXN3_HUMAN
465
470
GirsCL


172
O00444|PLK4_HUMAN
775
780
GhriCL


173
O00462|MANBA_HUMAN
744
749
GeavCL


174
O00468|AGRIN_HUMAN
1549
1554
GdhpCL


175
O00468|AGRIN_HUMAN
2012
2017
GfvgCL


176
O00476|NPT4_HUMAN
144
149
GcvcCL


177
O00488|ZN593_HUMAN
41
46
GlhrCL


178
O00501|CLD5_HUMAN
10
15
GlvlCL


179
O00624|NPT3_HUMAN
220
225
GcvcCL


180
O14514|BAI1_HUMAN
243
248
GpenCL


181
O14522|PTPRT_HUMAN
736
741
GtplCL


182
O14548|COX7R_HUMAN
97
102
GtiyCL


183
O14617|AP3D1_HUMAN
1113
1118
GhhvCL


184
O14628|ZN195_HUMAN
51
56
GlitCL


185
O14772|FPGT_HUMAN
515
520
GnktCL


186
O14773|TPP1_HUMAN
2
7
GlqaCL


187
O14792|OST1_HUMAN
261
266
GrdrCL


188
O14817|TSN4_HUMAN
68
73
GfvgCL


189
O14841|OPLA_HUMAN
1240
1245
GdvfCL


190
O14842|FFAR1_HUMAN
166
171
GspvCL


191
O14894|T4S5_HUMAN
100
105
GaiyCL


192
O14981|BTAF1_HUMAN
608
613
GawlCL


193
O15021|MAST4_HUMAN
1534
1539
GsheCL


194
O15031|PLXB2_HUMAN
308
313
GaglCL


195
O15056|SYNJ2_HUMAN
27
32
GrddCL


196
O15060|ZBT39_HUMAN
272
277
GtnsCL


197
O15063|K0355_HUMAN
244
249
GcdgCL


198
O15067|PUR4_HUMAN
914
919
GlvtCL


199
O15067|PUR4_HUMAN
1040
1045
GpsyCL


200
O15084|ANR28_HUMAN
449
454
GnleCL


201
O15084|ANR28_HUMAN
549
554
GhrlCL


202
O15084|ANR28_HUMAN
661
666
GhseCL


203
O15105|SMAD7_HUMAN
293
298
GngfCL


204
O15146|MUSK_HUMAN
648
653
GkpmCL


205
O15229|KMO_HUMAN
320
325
GfedCL


206
O15230|LAMA5_HUMAN
1933
1938
GrtqCL


207
O15296|LX15B_HUMAN
157
162
GwphCL


208
O15305|PMM2_HUMAN
5
10
GpalCL


209
O15354|GPR37_HUMAN
448
453
GcyfCL


210
O15379|HDAC3_HUMAN
214
219
GryyCL


211
O15397|IPO8_HUMAN
148
153
GsllCL


212
O15554|KCNN4_HUMAN
263
268
GkivCL


213
O43156|K0406_HUMAN
642
647
GkdfCL


214
O43175|SERA_HUMAN
111
116
GmimCL


215
O43175|SERA_HUMAN
416
421
GfgeCL


216
O43184|ADA12_HUMAN
407
412
GmgvCL


217
O43283|M3K13_HUMAN
133
138
GlfgCL


218
O43396|TXNL1_HUMAN
32
37
GcgpCL


219
O43396|TXNL1_HUMAN
144
149
GfdnCL


220
O43405|COCH_HUMAN
10
15
GlgvCL


221
O43541|SMAD6_HUMAN
363
368
GsgfCL


222
O43609|SPY1_HUMAN
219
224
GtcmCL


223
O43638|FREA_HUMAN
315
320
GltpCL


224
O43747|AP1G1_HUMAN
65
70
GqleCL


225
O43820|HYAL3_HUMAN
12
17
GvalCL


226
O43837|IDH3B_HUMAN
181
186
GvieCL


227
O43889|CREB3_HUMAN
330
335
GntsCL


228
O60244|CRSP2_HUMAN
447
452
GnseCL


229
O60266|ADCY3_HUMAN
44
49
GsclCL


230
O60266|ADCY3_HUMAN
944
949
GgieCL


231
O60292|SI1L3_HUMAN
658
663
GekvCL


232
O60423|AT8B3_HUMAN
238
243
GdvvCL


233
O60504|VINEX_HUMAN
478
483
GehiCL


234
O60508|PRP17_HUMAN
320
325
GerrCL


235
O60613|SEP15_HUMAN
4
9
GpsgCL


236
O60656|UD19_HUMAN
510
515
GyrkCL


237
O60662|KBTBA_HUMAN
447
452
GmiyCL


238
O60669|MOT2_HUMAN
93
98
GllcCL


239
O60673|DPOLZ_HUMAN
47
52
GqktCL


240
O60704|TPST2_HUMAN
229
234
GkekCL


241
O60706|ABCC9_HUMAN
1046
1051
GiflCL


242
O60883|ETBR2_HUMAN
315
320
GcyfCL


243
O75037|KI21B_HUMAN
1454
1459
GpvmCL


244
O75037|KI21B_HUMAN
1617
1622
GltpCL


245
O75052|CAPON_HUMAN
420
425
GrrdCL


246
O75077|ADA23_HUMAN
487
492
GggaCL


247
O75078|ADA11_HUMAN
429
434
GggsCL


248
O75094|SLIT3_HUMAN
1428
1433
GepyCL


249
O75095|MEGF6_HUMAN
695
700
GaclCL


250
O75173|ATS4_HUMAN
19
24
GaqpCL


251
O75173|ATS4_HUMAN
419
424
GyghCL


252
O75311|GLRA3_HUMAN
387
392
GmgpCL


253
O75326|SEM7A_HUMAN
499
504
GchgCL


254
O75342|LX12B_HUMAN
299
304
GegtCL


255
O75342|LX12B_HUMAN
552
557
GfprCL


256
O75346|ZN253_HUMAN
131
136
GlnqCL


257
O75426|FBX24_HUMAN
119
124
GrrrCL


258
O75436|VP26A_HUMAN
169
174
GiedCL


259
O75443|TECTA_HUMAN
1687
1692
GdgyCL


260
O75445|USH2A_HUMAN
1668
1673
GfvgCL


261
O75445|USH2A_HUMAN
4401
4406
GqglCL


262
O75446|SAP30_HUMAN
64
69
GqlcCL


263
O75508|CLD11_HUMAN
164
169
GavlCL


264
O75569|PRKRA_HUMAN
268
273
GqyqCL


265
O75592|MYCB2_HUMAN
1087
1092
GfgvCL


266
O75636|FCN3_HUMAN
16
21
GgpaCL


267
O75678|RFPL2_HUMAN
117
122
GcavCL


268
O75679|RFPL3_HUMAN
56
61
GctvCL


269
O75689|CENA1_HUMAN
37
42
GvfiCL


270
O75691|UTP20_HUMAN
2132
2137
GalqCL


271
O75694|NU155_HUMAN
230
235
GkdgCL


272
O75843|AP1G2_HUMAN
67
72
GqmeCL


273
O75886|STAM2_HUMAN
42
47
GakdCL


274
O75911|DHRS3_HUMAN
168
173
GhivCL


275
O75916|RGS9_HUMAN
642
647
GsgtCL


276
O75923|DYSF_HUMAN
378
383
GahfCL


277
O75923|DYSF_HUMAN
1574
1579
GpqeCL


278
O75925|PIAS1_HUMAN
431
436
GvdgCL


279
O75954|TSN9_HUMAN
4
9
GclcCL


280
O75954|TSN9_HUMAN
68
73
GflgCL


281
O76000|OR2B3_HUMAN
108
113
GateCL


282
O76013|K1H6_HUMAN
58
63
GlgsCL


283
O76064|RNF8_HUMAN
15
20
GrswCL


284
O76075|DFFB_HUMAN
43
48
GsrlCL


285
O94759|TRPM2_HUMAN
272
277
GnltCL


286
O94759|TRPM2_HUMAN
713
718
GkttCL


287
O94761|RECQ4_HUMAN
543
548
GlppCL


288
O94779|CNTN5_HUMAN
169
174
GhyqCL


289
O94779|CNTN5_HUMAN
265
270
GsyiCL


290
O94779|CNTN5_HUMAN
454
459
GmyqCL


291
O94829|IPO13_HUMAN
159
164
GqgrCL


292
O94856|NFASC_HUMAN
312
317
GeyfCL


293
O94887|FARP2_HUMAN
192
197
GqqhCL


294
O94900|TOX_HUMAN
22
27
GpspCL


295
O94907|DKK1_HUMAN
107
112
GvqiCL


296
O94919|ENDD1_HUMAN
371
376
GiesCL


297
O94933|SLIK3_HUMAN
898
903
GfvdCL


298
O94955|RHBT3_HUMAN
386
391
GkinCL


299
O94956|SO2B1_HUMAN
449
454
GmllCL


300
O95071|EDD1_HUMAN
531
536
GtqvCL


301
O95153|RIMB1_HUMAN
79
84
GaeaCL


302
O95153|RIMB1_HUMAN
1485
1490
GlasCL


303
O95163|IKAP_HUMAN
472
477
GfkvCL


304
O95202|LETM1_HUMAN
43
48
GlrnCL


305
O95210|GET1_HUMAN
285
290
GdheCL


306
O95239|KIF4A_HUMAN
27
32
GcqmCL


307
O95248|MTMR5_HUMAN
159
164
GlnvCL


308
O95248|MTMR5_HUMAN
381
386
GyrwCL


309
O95255|MRP6_HUMAN
845
850
GalvCL


310
O95255|MRP6_HUMAN
943
948
GtplCL


311
O95255|MRP6_HUMAN
992
997
GllgCL


312
O95256|I18RA_HUMAN
447
452
GyslCL


313
O95279|KCNK5_HUMAN
122
127
GvplCL


314
O95294|RASL1_HUMAN
130
135
GqgrCL


315
O95342|ABCBB_HUMAN
327
332
GfvwCL


316
O95373|IPO7_HUMAN
147
152
GillCL


317
O95396|MOCS3_HUMAN
250
255
GvlgCL


318
O95405|ZFYV9_HUMAN
137
142
GnlaCL


319
O95477|ABCA1_HUMAN
2120
2125
GrfrCL


320
O95500|CLD14_HUMAN
178
183
GtllCL


321
O95551|TTRAP_HUMAN
217
222
GnelCL


322
O95602|RPA1_HUMAN
1556
1561
GitrCL


323
O95620|DUS4L_HUMAN
125
130
GygaCL


324
O95633|FSTL3_HUMAN
88
93
GlvhCL


325
O95671|ASML_HUMAN
588
593
GeyqCL


326
O95714|HERC2_HUMAN
717
722
GsthCL


327
O95714|HERC2_HUMAN
3265
3270
GalhCL


328
O95714|HERC2_HUMAN
4047
4052
GgkhCL


329
O95715|SCYBE_HUMAN
68
73
GqehCL


330
O95780|ZN682_HUMAN
132
137
GlnqCL


331
O95803|NDST3_HUMAN
815
820
GktkCL


332
O95858|TSN15_HUMAN
285
290
GtgcCL


333
O95873|CF047_HUMAN
171
176
GpeeCL


334
O95886|DLGP3_HUMAN
284
289
GgpfCL


335
O95967|FBLN4_HUMAN
76
81
GgylCL


336
O95977|EDG6_HUMAN
333
338
GpgdCL


337
O96006|ZBED1_HUMAN
221
226
GapnCL


338
O96008|TOM40_HUMAN
72
77
GacgCL


339
O96009|NAPSA_HUMAN
350
355
GvrlCL


340
P00505|AATM_HUMAN
268
273
GinvCL


341
P00750|TPA_HUMAN
515
520
GplvCL


342
P00751|CFAB_HUMAN
288
293
GakkCL


343
P01130|LDLR_HUMAN
314
319
GtneCL


344
P01133|EGF_HUMAN
741
746
GadpCL


345
P01266|THYG_HUMAN
2020
2025
GevtCL


346
P01375|TNFA_HUMAN
26
31
GsrrCL


347
P01730|CD4_HUMAN
366
371
GmwqCL


348
P01833|PIGR_HUMAN
437
442
GfywCL


349
P02775|SCYB7_HUMAN
101
106
GrkiCL


350
P02776|PLF4_HUMAN
37
42
GdlqCL


351
P02776|PLF4_HUMAN
79
84
GrkiCL


352
P02778|SCYBA_HUMAN
70
75
GekrCL


353
P02787|TRFE_HUMAN
209
214
GafkCL


354
P02787|TRFE_HUMAN
538
543
GafrCL


355
P02788|TRFL_HUMAN
213
218
GafkCL


356
P02788|TRFL_HUMAN
549
554
GafrCL


357
P03986|TCC_HUMAN
28
33
GtylCL


358
P04350|TBB4_HUMAN
235
240
GvttCL


359
P04920|B3A2_HUMAN
751
756
GvvfCL


360
P05108|CP11A_HUMAN
458
463
GvrqCL


361
P05141|ADT2_HUMAN
155
160
GlgdCL


362
P05549|AP2A_HUMAN
371
376
GiqsCL


363
P06401|PRGR_HUMAN
484
489
GasgCL


364
P06756|ITAV_HUMAN
905
910
GvaqCL


365
P07202|PERT_HUMAN
819
824
GgfqCL


366
P07339|CATD_HUMAN
362
367
GktlCL


367
P07357|CO8A_HUMAN
117
122
GdqdCL


368
P07437|TBB5_HUMAN
235
240
GvttCL


369
P07686|HEXB_HUMAN
483
488
GgeaCL


370
P07814|SYEP_HUMAN
261
266
GhscCL


371
P07942|LAMB1_HUMAN
1052
1057
GqclCL


372
P07988|PSPB_HUMAN
244
249
GicqCL


373
P08151|GLI1_HUMAN
14
19
GepcCL


374
P08151|GLI1_HUMAN
828
833
GlapCL


375
P08243|ASNS_HUMAN
8
13
GsddCL


376
P08319|ADH4_HUMAN
241
246
GatdCL


377
P08582|TRFM_HUMAN
212
217
GafrCL


378
P08582|TRFM_HUMAN
558
563
GafrCL


379
P08686|CP21A_HUMAN
424
429
GarvCL


380
P08697|A2AP_HUMAN
139
144
GsgpCL


381
P08709|FA7_HUMAN
14
19
GlqgCL


382
P08922|ROS_HUMAN
2248
2253
GdviCL


383
P09001|RM03_HUMAN
291
296
GhknCL


384
P09326|CD48_HUMAN
5
10
GwdsCL


385
P09341|GROA_HUMAN
81
86
GrkaCL


386
P09848|LPH_HUMAN
1846
1851
GphaCL


387
P10071|GLI3_HUMAN
1359
1364
GpesCL


388
P10109|ADX_HUMAN
151
156
GcqiCL


389
P10145|IL8_HUMAN
73
78
GrelCL


390
P10635|CP2D6_HUMAN
439
444
GrraCL


391
P10646|TFPI1_HUMAN
213
218
GpswCL


392
P10720|PF4V_HUMAN
40
45
GdlqCL


393
P10720|PF4V_HUMAN
82
87
GrkiCL


394
P10745|IRBP_HUMAN
328
333
GvvhCL


395
P11047|LAMC1_HUMAN
903
908
GqceCL


396
P11362|FGFR1_HUMAN
337
342
GeytCL


397
P11717|MPRI_HUMAN
231
236
GtaaCL


398
P12236|ADT3_HUMAN
155
160
GlgdCL


399
P13473|LAMP2_HUMAN
228
233
GndtCL


400
P13498|CY24A_HUMAN
45
50
GvfvCL


401
P13569|CFTR_HUMAN
124
129
GiglCL


402
P13686|PPA5_HUMAN
215
220
GpthCL


403
P13804|ETFA_HUMAN
49
54
GevsCL


404
P13807|GYS1_HUMAN
185
190
GvglCL


405
P13861|KAP2_HUMAN
354
359
GdvkCL


406
P14222|PERF_HUMAN
530
535
GggtCL


407
P14543|NID1_HUMAN
24
29
GpvgCL


408
P14867|GBRA1_HUMAN
6
11
GlsdCL


409
P15151|PVR_HUMAN
119
124
GnytCL


410
P15538|C11B1_HUMAN
446
451
GmrqCL


411
P15692|VEGFA_HUMAN
168
173
GarcCL


412
P16109|LYAM3_HUMAN
271
276
GnmiCL


413
P16112|PGCA_HUMAN
2183
2188
GhviCL


414
P16581|LYAM2_HUMAN
376
381
GymnCL


415
P17038|ZNF43_HUMAN
127
132
GfnqCL


416
P17040|ZNF31_HUMAN
184
189
GnsvCL


417
P17936|IBP3_HUMAN
66
71
GcgcCL


418
P18510|IL1RA_HUMAN
87
92
GgkmCL


419
P18564|ITB6_HUMAN
674
679
GeneCL


420
P18577|RHCE_HUMAN
306
311
GgakCL


421
P19099|C11B2_HUMAN
446
451
GmrqCL


422
P19224|UD16_HUMAN
512
517
GyrkCL


423
P19367|HXK1_HUMAN
713
718
GdngCL


424
P19835|CEL_HUMAN
96
101
GdedCL


425
P19875|MIP2A_HUMAN
81
86
GqkaCL


426
P19876|MIP2B_HUMAN
81
86
GkkaCL


427
P19883|FST_HUMAN
252
257
GgkkCL


428
P20062|TCO2_HUMAN
79
84
GyqqCL


429
P20273|CD22_HUMAN
691
696
GlgsCL


430
P20648|ATP4A_HUMAN
108
113
GglqCL


431
P20701|ITAL_HUMAN
76
81
GtghCL


432
P20701|ITAL_HUMAN
1150
1155
GdpgCL


433
P20813|CP2B6_HUMAN
432
437
GkriCL


434
P20916|MAG_HUMAN
301
306
GvyaCL


435
P20929|NEBU_HUMAN
4517
4522
GvvhCL


436
P21554|CNR1_HUMAN
427
432
GdsdCL


437
P21580|TNAP3_HUMAN
99
104
GdgnCL


438
P21802|FGFR2_HUMAN
5
10
GrfiCL


439
P21802|FGFR2_HUMAN
338
343
GeytCL


440
P21817|RYR1_HUMAN
840
845
GpsrCL


441
P21860|ERBB3_HUMAN
513
518
GpgqCL


442
P21964|COMT_HUMAN
30
35
GwglCL


443
P22064|LTB1S_HUMAN
938
943
GsfrCL


444
P22064|LTB1S_HUMAN
1359
1364
GsykCL


445
P22105|TENX_HUMAN
565
570
GrgqCL


446
P22309|UD11_HUMAN
276
281
GginCL


447
P22309|UD11_HUMAN
513
518
GyrkCL


448
P22310|UD14_HUMAN
514
519
GyrkCL


449
P22314|UBE1_HUMAN
230
235
GvvtCL


450
P22455|FGFR4_HUMAN
97
102
GrylCL


451
P22455|FGFR4_HUMAN
220
225
GtytCL


452
P22455|FGFR4_HUMAN
329
334
GeytCL


453
P22607|FGFR3_HUMAN
335
340
GeytCL


454
P22680|CP7A1_HUMAN
330
335
GnpiCL


455
P22732|GTR5_HUMAN
348
353
GfsiCL


456
P23142|FBLN1_HUMAN
269
274
GihnCL


457
P23142|FBLN1_HUMAN
547
552
GgfrCL


458
P23416|GLRA2_HUMAN
376
381
GmghCL


459
P23759|PAX7_HUMAN
466
471
GqseCL


460
P24386|RAE1_HUMAN
395
400
GgiyCL


461
P24557|THAS_HUMAN
475
480
GprsCL


462
P24592|IBP6_HUMAN
100
105
GrgrCL


463
P24593|IBP5_HUMAN
96
101
GrgvCL


464
P24821|TENA_HUMAN
143
148
GagcCL


465
P24903|CP2F1_HUMAN
432
437
GrrlCL


466
P25205|MCM3_HUMAN
239
244
GtyrCL


467
P25874|UCP1_HUMAN
21
26
GiaaCL


468
P25940|C05A3_HUMAN
1581
1586
GgetCL


469
P26374|RAE2_HUMAN
397
402
GgiyCL


470
P26951|IL3RA_HUMAN
363
368
GleeCL


471
P27487|DPP4_HUMAN
335
340
GrwnCL


472
P27540|ARNT_HUMAN
332
337
GskfCL


473
P27987|IP3KB_HUMAN
284
289
GtrsCL


474
P28332|ADH6_HUMAN
237
242
GateCL


475
P28340|DPOD1_HUMAN
709
714
GklpCL


476
P29274|AA2AR_HUMAN
162
167
GqvaCL


477
P29353|SHC1_HUMAN
570
575
GselCL


478
P29459|IL12A_HUMAN
33
38
GmfpCL


479
P30040|ERP29_HUMAN
153
158
GmpgCL


480
P30530|UFO_HUMAN
106
111
GqyqCL


481
P30532|ACHA5_HUMAN
279
284
GekiCL


482
P30566|PUR8_HUMAN
169
174
GkrcCL


483
P31323|KAP3_HUMAN
368
373
GtvkCL


484
P32004|L1CAM_HUMAN
308
313
GeyrCL


485
P32004|L1CAM_HUMAN
493
498
GryfCL


486
P32314|FOXN2_HUMAN
319
324
GirtCL


487
P32418|NAC1_HUMAN
414
419
GtyqCL


488
P32929|CGL_HUMAN
80
85
GakyCL


489
P32970|TNFL7_HUMAN
29
34
GlviCL


490
P33402|GCYA2_HUMAN
284
289
GncsCL


491
P34913|HYES_HUMAN
258
263
GpavCL


492
P34981|TRFR_HUMAN
94
99
GyvgCL


493
P34998|CRFR1_HUMAN
83
88
GyreCL


494
P35227|PCGF2_HUMAN
316
321
GslnCL


495
P35251|RFC1_HUMAN
402
407
GaenCL


496
P35270|SPRE_HUMAN
6
11
GravCL


497
P35367|HRH1_HUMAN
96
101
GrplCL


498
P35452|HXD12_HUMAN
176
181
GvasCL


499
P35498|SCN1A_HUMAN
964
969
GqamCL


500
P35499|SCN4A_HUMAN
774
779
GqamCL


501
P35503|UD13_HUMAN
514
519
GyrkCL


502
P35504|UD15_HUMAN
514
519
GyrkCL


503
P35555|FBN1_HUMAN
1259
1264
GeyrCL


504
P35555|FBN1_HUMAN
1385
1390
GsyrCL


505
P35555|FBN1_HUMAN
1416
1421
GngqCL


506
P35555|FBN1_HUMAN
1870
1875
GsfyCL


507
P35555|FBN1_HUMAN
2034
2039
GsfkCL


508
P35556|FBN2_HUMAN
1303
1308
GeyrCL


509
P35556|FBN2_HUMAN
1952
1957
GsynCL


510
P35556|FBN2_HUMAN
1994
1999
GsfkCL


511
P35556|FBN2_HUMAN
2076
2081
GgfqCL


512
P35590|TIE1_HUMAN
280
285
GltfCL


513
P35916|VGFR3_HUMAN
4
9
GaalCL


514
P35968|VGFR2_HUMAN
638
643
GdyvCL


515
P36509|UD12_HUMAN
510
515
GyrkCL


516
P36888|FLT3_HUMAN
99
104
GnisCL


517
P37058|DHB3_HUMAN
13
18
GllvCL


518
P38398|BRCA1_HUMAN
949
954
GsrfCL


519
P38571|LICH_HUMAN
7
12
GlvvCL


520
P38571|LICH_HUMAN
58
63
GyilCL


521
P38606|VATA1_HUMAN
390
395
GrvkCL


522
P38607|VATA2_HUMAN
388
393
GrvkCL


523
P39059|COFA1_HUMAN
8
13
GqcwCL


524
P40205|NCYM_HUMAN
100
105
GrppCL


525
P40939|ECHA_HUMAN
709
714
GfppCL


526
P41217|OX2G_HUMAN
117
122
GcymCL


527
P42331|RHG25_HUMAN
4
9
GqsaCL


528
P42345|FRAP_HUMAN
1479
1484
GrmrCL


529
P42785|PCP_HUMAN
339
344
GqvkCL


530
P42830|SCYB5_HUMAN
87
92
GkeiCL


531
P42892|ECE1_HUMAN
79
84
GlvaCL


532
P43378|PTN9_HUMAN
334
339
GdvpCL


533
P43403|ZAP70_HUMAN
113
118
GvfdCL


534
P43403|ZAP70_HUMAN
245
250
GliyCL


535
P46379|BAT3_HUMAN
872
877
GlfeCL


536
P46531|NOTC1_HUMAN
1354
1359
GslrCL


537
P47775|GPR12_HUMAN
166
171
GtsiCL


538
P47804|RGR_HUMAN
275
280
GiwqCL


539
P48048|IRK1_HUMAN
204
209
GgklCL


540
P48052|CBPA2_HUMAN
12
17
GhiyCL


541
P48059|PINC_HUMAN
176
181
GelyCL


542
P48067|SC6A9_HUMAN
457
462
GtqfCL


543
P48230|T4S4_HUMAN
5
10
GcarCL


544
P48745|NOV_HUMAN
60
65
GcscCL


545
P49247|RPIA_HUMAN
100
105
GgggCL


546
P49327|FAS_HUMAN
1455
1460
GlvnCL


547
P49588|SYAC_HUMAN
897
902
GkitCL


548
P49640|EVX1_HUMAN
345
350
GpcsCL


549
P49641|MA2A2_HUMAN
862
867
GwrgCL


550
P49646|YYY1_HUMAN
393
398
GetpCL


551
P49753|ACOT2_HUMAN
296
301
GgelCL


552
P49903|SPS1_HUMAN
323
328
GlliCL


553
P49910|ZN165_HUMAN
32
37
GqdtCL


554
P50851|LRBA_HUMAN
2736
2741
GpenCL


555
P51151|RAB9A_HUMAN
79
84
GsdcCL


556
P51168|SCNNB_HUMAN
532
537
GsvlCL


557
P51589|CP2J2_HUMAN
444
449
GkraCL


558
P51606|RENBP_HUMAN
37
42
GfftCL


559
P51674|GPM6A_HUMAN
170
175
GanlCL


560
P51685|CCR8_HUMAN
150
155
GttlCL


561
P51790|CLCN3_HUMAN
520
525
GaaaCL


562
P51790|CLCN3_HUMAN
723
728
GlrqCL


563
P51793|CLCN4_HUMAN
520
525
GaaaCL


564
P51793|CLCN4_HUMAN
721
726
GlrqCL


565
P51795|CLCN5_HUMAN
506
511
GaaaCL


566
P51795|CLCN5_HUMAN
707
712
GlrqCL


567
P51800|CLCKA_HUMAN
613
618
GhqqCL


568
P51801|CLCKB_HUMAN
613
618
GhqqCL


569
P51957|NEK4_HUMAN
322
327
GegkCL


570
P52306|GDS1_HUMAN
25
30
GcldCL


571
P52306|GDS1_HUMAN
265
270
GlveCL


572
P52429|DGKE_HUMAN
411
416
GtkdCL


573
P52744|ZN138_HUMAN
48
53
GlnqCL


574
P52789|HXK2_HUMAN
713
718
GdngCL


575
P52803|EFNA5_HUMAN
147
152
GrrsCL


576
P52823|STC1_HUMAN
55
60
GafaCL


577
P52848|NDST1_HUMAN
824
829
GktkCL


578
P52849|NDST2_HUMAN
302
307
GkrlCL


579
P52849|NDST2_HUMAN
823
828
GktrCL


580
P52961|NAR1_HUMAN
220
225
GiwtCL


581
P53355|DAPK1_HUMAN
1326
1331
GkdwCL


582
P54132|BLM_HUMAN
891
896
GiiyCL


583
P54277|PMS1_HUMAN
837
842
GmanCL


584
P54750|PDE1A_HUMAN
32
37
GilrCL


585
P54753|EPHB3_HUMAN
297
302
GegpCL


586
P54826|GAS1_HUMAN
19
24
GawlCL


587
P55160|NCKPL_HUMAN
938
943
GpieCL


588
P55268|LAMB2_HUMAN
501
506
GcdrCL


589
P55268|LAMB2_HUMAN
1063
1068
GqcpCL


590
P56192|SYMC_HUMAN
8
13
GvpgCL


591
P56749|CLD12_HUMAN
63
68
GssdCL


592
P57077|TAK1L_HUMAN
68
73
GflkCL


593
P57679|EVC_HUMAN
683
688
GssqCL


594
P58215|LOXL3_HUMAN
13
18
GlllCL


595
P58397|ATS12_HUMAN
447
452
GwgfCL


596
P58418|USH3A_HUMAN
69
74
GscgCL


597
P58512|CU067_HUMAN
166
171
GfpaCL


598
P59047|NALP5_HUMAN
64
69
GlqwCL


599
P59510|ATS20_HUMAN
458
463
GygeCL


600
P60370|KR105_HUMAN
32
37
GtapCL


601
P60371|KR106_HUMAN
16
21
GsrvCL


602
P60409|KR107_HUMAN
16
21
GsrvCL


603
P60413|KR10C_HUMAN
11
16
GsrvCL


604
P60602|CT052_HUMAN
38
43
GtfsCL


605
P61011|SRP54_HUMAN
129
134
GwktCL


606
P61550|ENT1_HUMAN
343
348
GnasCL


607
P61619|S61A1_HUMAN
143
148
GagiCL


608
P62072|TIM10_HUMAN
46
51
GesvCL


609
P62312|LSM6_HUMAN
32
37
GvlaCL


610
P62714|PP2AB_HUMAN
161
166
GqifCL


611
P67775|PP2AA_HUMAN
161
166
GqifCL


612
P68371|TBB2C_HUMAN
235
240
GvttCL


613
P69849|NOMO3_HUMAN
507
512
GkvsCL


614
P78310|CXAR_HUMAN
219
224
GsdqCL


615
P78324|SHPS1_HUMAN
12
17
GpllCL


616
P78325|ADAM8_HUMAN
101
106
GqdhCL


617
P78346|RPP30_HUMAN
253
258
GdedCL


618
P78357|CNTP1_HUMAN
1205
1210
GfsgCL


619
P78423|X3CL1_HUMAN
350
355
GllfCL


620
P78504|JAG1_HUMAN
898
903
GprpCL


621
P78509|RELN_HUMAN
2862
2867
GhgdCL


622
P78524|ST5_HUMAN
127
132
GvaaCL


623
P78549|NTHL1_HUMAN
286
291
GqqtCL


624
P78559|MAP1A_HUMAN
2433
2438
GpqgCX


625
P80162|SCYB6_HUMAN
87
92
GkqvCL


626
P82279|CRUM1_HUMAN
1092
1097
GlqgCL


627
P83105|HTRA4_HUMAN
10
15
GlgrCL


628
P98088|MUC5A_HUMAN
853
858
GcprCL


629
P98095|FBLN2_HUMAN
1047
1052
GsfrCL


630
P98153|IDD_HUMAN
289
294
GddpCL


631
P98160|PGBM_HUMAN
3181
3186
GtyvCL


632
P98161|PKD1_HUMAN
649
654
GaniCL


633
P98164|LRP2_HUMAN
1252
1257
GhpdCL


634
P98164|LRP2_HUMAN
3819
3824
GsadCL


635
P98173|FAM3A_HUMAN
83
88
GpkiCL


636
P98194|AT2C1_HUMAN
158
163
GdtvCL


637
Q00872|MYPC1_HUMAN
447
452
GkeiCL


638
Q00973|B4GN1_HUMAN
408
413
GlgnCL


639
Q01064|PDE1B_HUMAN
243
248
GmvhCL


640
Q01433|AMPD2_HUMAN
103
108
GpapCL


641
Q02246|CNTN2_HUMAN
107
112
GvyqCL


642
Q02246|CNTN2_HUMAN
203
208
GnysCL


643
Q02318|CP27A_HUMAN
472
477
GvraCL


644
Q02985|FHR3_HUMAN
188
193
GsitCL


645
Q03923|ZNF85_HUMAN
133
138
GlnqCL


646
Q03923|ZNF85_HUMAN
184
189
GmisCL


647
Q03924|ZN117_HUMAN
103
108
GlnqCL


648
Q03936|ZNF92_HUMAN
132
137
GlnqCL


649
Q03938|ZNF90_HUMAN
132
137
GlnqCL


650
Q04721|NOTC2_HUMAN
476
481
GgftCL


651
Q05469|LIPS_HUMAN
716
721
GeriCL


652
Q06730|ZN33A_HUMAN
530
535
GktfCL


653
Q06732|ZN11B_HUMAN
531
536
GktfCL


654
Q07325|SCYB9_HUMAN
70
75
GvqtCL


655
Q07617|SPAG1_HUMAN
133
138
GsnsCL


656
Q07954|LRP1_HUMAN
875
880
GdndCL


657
Q07954|LRP1_HUMAN
3001
3006
GsykCL


658
Q08629|TICN1_HUMAN
178
183
GpcpCL


659
Q09428|ABCC8_HUMAN
1073
1078
GivlCL


660
Q10471|GALT2_HUMAN
535
540
GsnlCL


661
Q12796|PNRC1_HUMAN
63
68
GdgpCL


662
Q12805|FBLN3_HUMAN
66
71
GgylCL


663
Q12809|KCNH2_HUMAN
719
724
GfpeCL


664
Q12841|FSTL1_HUMAN
48
53
GeptCL


665
Q12852|M3K12_HUMAN
90
95
GlfgCL


666
Q12860|CNTN1_HUMAN
110
115
GiyyCL


667
Q12882|DPYD_HUMAN
988
993
GctlCL


668
Q12933|TRAF2_HUMAN
387
392
GykmCL


669
Q12986|NFX1_HUMAN
537
542
GdfsCL


670
Q13077|TRAF1_HUMAN
302
307
GyklCL


671
Q13129|RLF_HUMAN
48
53
GlrpCL


672
Q13200|PSMD2_HUMAN
135
140
GereCL


673
Q13224|NMDE2_HUMAN
584
589
GynrCL


674
Q13224|NMDE2_HUMAN
1392
1397
GddqCL


675
Q13255|MGR1_HUMAN
136
141
GinrCL


676
Q13275|SEM3F_HUMAN
305
310
GghcCL


677
Q13308|PTK7_HUMAN
429
434
GyldCL


678
Q13309|SKP2_HUMAN
107
112
GifsCL


679
Q13322|GRB10_HUMAN
219
224
GlerCL


680
Q13370|PDE3B_HUMAN
253
258
GgagCL


681
Q13371|PHLP_HUMAN
200
205
GcmiCL


682
Q13387|JIP2_HUMAN
594
599
GlfsCL


683
Q13410|BT1A1_HUMAN
8
13
GlprCL


684
Q13444|ADA15_HUMAN
405
410
GmgsCL


685
Q13470|TNK1_HUMAN
105
110
GglkCL


686
Q13485|SMAD4_HUMAN
359
364
GdrfCL


687
Q13554|KCC2B_HUMAN
472
477
GpppCL


688
Q13591|SEM5A_HUMAN
819
824
GgmpCL


689
Q13591|SEM5A_HUMAN
876
881
GgdiCL


690
Q13639|5HT4R_HUMAN
89
94
GevfCL


691
Q13642|FHL1_HUMAN
23
28
GhhcCL


692
Q13686|ALKB1_HUMAN
300
305
GlphCL


693
Q13698|CAC1S_HUMAN
1210
1215
GglyCL


694
Q13751|LAMB3_HUMAN
449
454
GrclCL


695
Q13772|NCOA4_HUMAN
97
102
GqfnCL


696
Q13772|NCOA4_HUMAN
364
369
GnlkCL


697
Q13795|ARFRP_HUMAN
159
164
GrrdCL


698
Q13822|ENPP2_HUMAN
21
26
GvniCL


699
Q13885|TBB2A_HUMAN
235
240
GvttCL


700
Q14008|CKAP5_HUMAN
109
114
GieiCL


701
Q14008|CKAP5_HUMAN
1237
1242
GvigCL


702
Q14114|LRP8_HUMAN
175
180
GnrsCL


703
Q14114|LRP8_HUMAN
336
341
GlneCL


704
Q14159|K0146_HUMAN
513
518
GtraCL


705
Q14264|ENR1_HUMAN
358
363
GeltCL


706
Q14315|FLNC_HUMAN
1649
1654
GlgaCL


707
Q14344|GNA13_HUMAN
314
319
GdphCL


708
Q14392|LRC32_HUMAN
360
365
GslpCL


709
Q14393|GAS6_HUMAN
138
143
GnffCL


710
Q14393|GAS6_HUMAN
217
222
GsysCL


711
Q14435|GALT3_HUMAN
93
98
GerpCL


712
Q14435|GALT3_HUMAN
513
518
GqplCL


713
Q14451|GRB7_HUMAN
517
522
GilpCL


714
Q14520|HABP2_HUMAN
121
126
GrgqCL


715
Q14524|SCN5A_HUMAN
911
916
GqslCL


716
Q14566|MCM6_HUMAN
154
159
GtflCL


717
Q14593|ZN273_HUMAN
100
105
GlnqCL


718
Q14656|ITBA1_HUMAN
197
202
GvlsCL


719
Q14669|TRIPC_HUMAN
562
567
GladCL


720
Q14669|TRIPC_HUMAN
1136
1141
GgaeCL


721
Q14703|MBTP1_HUMAN
845
850
GdsnCL


722
Q14714|SSPN_HUMAN
91
96
GiivCL


723
Q14766|LTB1L_HUMAN
1139
1144
GsfrCL


724
Q14766|LTB1L_HUMAN
1560
1565
GsykCL


725
Q14767|LTBP2_HUMAN
990
995
GsytCL


726
Q14767|LTBP2_HUMAN
1156
1161
GsyqCL


727
Q14767|LTBP2_HUMAN
1197
1202
GsffCL


728
Q14767|LTBP2_HUMAN
1238
1243
GsfnCL


729
Q14767|LTBP2_HUMAN
1324
1329
GsfrCL


730
Q14767|LTBP2_HUMAN
1366
1371
GsflCL


731
Q14774|HLX1_HUMAN
483
488
GalgCL


732
Q14916|NPT1_HUMAN
110
115
GfalCL


733
Q14916|NPT1_HUMAN
207
212
GcavCL


734
Q14940|SL9A5_HUMAN
576
581
GsgaCL


735
Q14957|NMDE3_HUMAN
941
946
GpspCL


736
Q15021|CND1_HUMAN
730
735
GtiqCL


737
Q15034|HERC3_HUMAN
145
150
GnwhCL


738
Q15048|LRC14_HUMAN
281
286
GrftCL


739
Q15058|KIF14_HUMAN
438
443
GfntCL


740
Q15061|WDR43_HUMAN
103
108
GtctCL


741
Q15147|PLCB4_HUMAN
987
992
GgsnCL


742
Q15155|NOMO1_HUMAN
507
512
GkvsCL


743
Q15274|NADC_HUMAN
92
97
GpahCL


744
Q15303|ERBB4_HUMAN
516
521
GpdqCL


745
Q15334|L2GL1_HUMAN
722
727
GvvrCL


746
Q15399|TLR1_HUMAN
663
668
GmqiCL


747
Q15413|RYR3_HUMAN
229
234
GhdeCL


748
Q15413|RYR3_HUMAN
1656
1661
GlrtCL


749
Q15418|KS6A1_HUMAN
548
553
GnpeCL


750
Q15546|PAQRB_HUMAN
185
190
GliyCL


751
Q15633|TRBP2_HUMAN
321
326
GlcqCL


752
Q15650|TRIP4_HUMAN
196
201
GsgpCL


753
Q15652|JHD2C_HUMAN
1864
1869
GfvvCL


754
Q15735|PI5PA_HUMAN
379
384
GpgrCL


755
Q15746|MYLK_HUMAN
229
234
GvytCL


756
Q15746|MYLK_HUMAN
579
584
GtytCL


757
Q15858|SCN9A_HUMAN
940
945
GqamCL


758
Q15911|ATBF1_HUMAN
3527
3532
GsyhCL


759
Q16342|PDCD2_HUMAN
121
126
GesvCL


760
Q16363|LAMA4_HUMAN
1001
1006
GfvgCL


761
Q16549|PCSK7_HUMAN
16
21
GlptCL


762
Q16617|NKG7_HUMAN
15
20
GlmfCL


763
Q16647|PTGIS_HUMAN
437
442
GhnhCL


764
Q16787|LAMA3_HUMAN
1526
1531
GvssCL


765
Q30KQ9|DB111_HUMAN
60
65
GthcCL


766
Q32MQ0|ZN750_HUMAN
121
126
GthrCL


767
Q3KNT7|NSN5B_HUMAN
134
139
GaehCL


768
Q3LI83|KR241_HUMAN
153
158
GqlnCL


769
Q3SYG4|PTHB1_HUMAN
822
827
GgrlCL


770
Q3T8J9|GON4L_HUMAN
1740
1745
GcadCL


771
Q495M9|USH1G_HUMAN
76
81
GhlhCL


772
Q496M8|CI094_HUMAN
170
175
GefsCL


773
Q499Z4|ZN672_HUMAN
40
45
GrfrCL


774
Q4G0F5|VP26B_HUMAN
167
172
GiedCL


775
Q4KMG0|CDON_HUMAN
93
98
GyyqCL


776
Q53G59|KLH12_HUMAN
426
431
GviyCL


777
Q53H47|SETMR_HUMAN
72
77
GtcsCL


778
Q53R12|T4S20_HUMAN
213
218
GflgCL


779
Q58EX2|SDK2_HUMAN
469
474
GtytCL


780
Q5HYK3|COQ5_HUMAN
240
245
GrflCL


781
Q5IJ48|CRUM2_HUMAN
243
248
GsfrCL


782
Q5JPE7|NOMO2_HUMAN
507
512
GkvsCL


783
Q5JQC9|AKAP4_HUMAN
242
247
GkskCL


784
Q5JVG8|ZN506_HUMAN
132
137
GlkqCL


785
Q5JWF2|GNAS1_HUMAN
2
7
GvrnCL


786
Q5JWF2|GNAS1_HUMAN
584
589
GtsgCL


787
Q5JWF8|CT134_HUMAN
111
116
GccvCL


788
Q5MJ68|SPDYC_HUMAN
138
143
GkdwCL


789
Q5NUL3|GP120_HUMAN
72
77
GataCL


790
Q5SRN2|CF010_HUMAN
117
122
GsikCL


791
Q5T2D3|OTUD3_HUMAN
72
77
GdgnCL


792
Q5T5C0|STXB5_HUMAN
322
327
GrrpCL


793
Q5T751|LCE1C_HUMAN
72
77
GggcCL


794
Q5T752|LCE1D_HUMAN
68
73
GggcCL


795
Q5T753|LCE1E_HUMAN
72
77
GggcCL


796
Q5T754|LCE1F_HUMAN
72
77
GggcCL


797
Q5T7P2|LCE1A_HUMAN
64
69
GggcCL


798
Q5T7P3|LCE1B_HUMAN
72
77
GggcCL


799
Q5TA78|LCE4A_HUMAN
55
60
GggcCL


800
Q5TA79|LCE2A_HUMAN
64
69
GggcCL


801
Q5TA82|LCE2D_HUMAN
68
73
GggcCL


802
Q5TCM9|LCE5A_HUMAN
64
69
GggcCL


803
Q5TEA3|CT194_HUMAN
465
470
GgngCL


804
Q5TEJ8|ICB1_HUMAN
39
44
GnecCL


805
Q5THJ4|VP13D_HUMAN
1215
1220
GslgCL


806
Q5VST9|OBSCN_HUMAN
3315
3320
GdryCL


807
Q5VST9|OBSCN_HUMAN
4189
4194
GvqwCL


808
Q5VST9|OBSCN_HUMAN
5195
5200
GvyrCL


809
Q5VST9|OBSCN_HUMAN
6425
6430
GvytCL


810
Q5VT25|MRCKA_HUMAN
1325
1330
GaltCL


811
Q5VUA4|ZN318_HUMAN
1984
1989
GpspCL


812
Q5VZ18|SHE_HUMAN
8
13
GasaCL


813
Q5VZM2|RRAGB_HUMAN
366
371
GpkqCL


814
Q5W111|CLLD6_HUMAN
50
55
GtggCL


815
Q5XUX1|FBXW9_HUMAN
184
189
GgslCL


816
Q5ZPR3|CD276_HUMAN
216
221
GtysCL


817
Q5ZPR3|CD276_HUMAN
434
439
GtysCL


818
Q5ZPR3|CD276_HUMAN
472
477
GlsvCL


819
Q63ZY6|NSN5C_HUMAN
216
221
GaehCL


820
Q63ZY6|NSN5C_HUMAN
293
298
GkgrCL


821
Q68CP9|ARID2_HUMAN
566
571
GfykCL


822
Q6BDS2|URFB1_HUMAN
549
554
GnlfCL


823
Q6GQQ9|OTU7B_HUMAN
190
195
GdgnCL


824
Q6GTX8|LAIR1_HUMAN
10
15
GlvlCL


825
Q6IS24|GLTL3_HUMAN
564
569
GtgrCL


826
Q6ISS4|LAIR2_HUMAN
10
15
GlvlCL


827
Q6ISS4|LAIR2_HUMAN
97
102
GlyrCL


828
Q6N022|TEN4_HUMAN
139
144
GrssCL


829
Q6NUM9|RETST_HUMAN
366
371
GnarCL


830
Q6P1M0|S27A4_HUMAN
297
302
GigqCL


831
Q6P1R4|DUS1L_HUMAN
209
214
GniqCL


832
Q6P587|FAHD1_HUMAN
96
101
GyalCL


833
Q6P656|CO026_HUMAN
144
149
GqdfCL


834
Q6PCB7|S27A1_HUMAN
300
305
GvgqCL


835
Q6PCT2|FXL19_HUMAN
222
227
GgdaCL


836
Q6Q0C0|TRAF7_HUMAN
397
402
GpvwCL


837
Q6Q4G3|LAEVR_HUMAN
794
799
GledCL


838
Q6TGC4|PADI6_HUMAN
22
27
GteiCL


839
Q6UB99|ANR11_HUMAN
498
503
GssgCL


840
Q6UWJ8|C16L2_HUMAN
15
20
GgccCL


841
Q6UWN5|LYPD5_HUMAN
15
20
GaalCL


842
Q6UX01|LMBRL_HUMAN
394
399
GncvCL


843
Q6UX53|MET7B_HUMAN
199
204
GdgcCL


844
Q6UX65|TMM77_HUMAN
99
104
GilsCL


845
Q6UXV0|GFRAL_HUMAN
127
132
GmwsCL


846
Q6UY09|CEA20_HUMAN
226
231
GlyrCL


847
Q6V0L0|CP26C_HUMAN
455
460
GarsCL


848
Q6V0L0|CP26C_HUMAN
517
522
GnglCL


849
Q6VVB1|NHLC1_HUMAN
47
52
GhvvCL


850
Q6VVX0|CP2R1_HUMAN
444
449
GrrhCL


851
Q6W4X9|MUC6_HUMAN
1095
1100
GdceCL


852
Q6WN34|CRDL2_HUMAN
54
59
GlmyCL


853
Q6ZN16|M3K15_HUMAN
82
87
GarqCL


854
Q6ZN17|LN28B_HUMAN
103
108
GgspCL


855
Q6ZRI6|CO039_HUMAN
141
146
GlstCL


856
Q6ZRQ5|CF167_HUMAN
1116
1121
GilkCL


857
Q6ZSY5|PPR3F_HUMAN
647
652
GaevCL


858
Q6ZV89|SH2D5_HUMAN
195
200
GghsCL


859
Q6ZVD8|PHLPL_HUMAN
5
10
GsrnCL


860
Q6ZW76|ANKS3_HUMAN
632
637
GqalCL


861
Q75N90|FBN3_HUMAN
551
556
GsfsCL


862
Q75N90|FBN3_HUMAN
1217
1222
GghrCL


863
Q75N90|FBN3_HUMAN
1826
1831
GsymCL


864
Q75N90|FBN3_HUMAN
1866
1871
GsynCL


865
Q75N90|FBN3_HUMAN
1908
1913
GsfhCL


866
Q75N90|FBN3_HUMAN
1990
1995
GsfqCL


867
Q7L099|RUFY3_HUMAN
37
42
GewlCL


868
Q7L0J3|SV2A_HUMAN
230
235
GrrqCL


869
Q7L3T8|SYPM_HUMAN
149
154
GkeyCL


870
Q7L622|K1333_HUMAN
310
315
GitdCL


871
Q7LBC6|JHD2B_HUMAN
1049
1054
GfgvCL


872
Q7LBC6|JHD2B_HUMAN
1388
1393
GrllCL


873
Q7RTN6|STRAD_HUMAN
294
299
GtvpCL


874
Q7RTP0|NIPA1_HUMAN
122
127
GklgCL


875
Q7RTU9|STRC_HUMAN
1077
1082
GacsCL


876
Q7RTX0|TS1R3_HUMAN
20
25
GaplCL


877
Q7Z2W7|TRPM8_HUMAN
652
657
GgsnCL


878
Q7Z333|SETX_HUMAN
1106
1111
GekkCL


879
Q7Z3K3|POGZ_HUMAN
749
754
GrqtCL


880
Q7Z3T1|OR2W3_HUMAN
108
113
GgveCL


881
Q7Z401|MYCPP_HUMAN
948
953
GsadCL


882
Q7Z460|CLAP1_HUMAN
146
151
GiclCL


883
Q7Z4S6|KI21A_HUMAN
1493
1498
GpvmCL


884
Q7Z5G4|GOGA7_HUMAN
68
73
GclaCL


885
Q7Z5K2|WAPL_HUMAN
850
855
GaerCL


886
Q7Z713|ANR37_HUMAN
75
80
GsleCL


887
Q7Z7E8|UB2Q1_HUMAN
36
41
GpgpCL


888
Q7Z7M0|MEGF8_HUMAN
403
408
GcgwCL


889
Q7Z7M1|GP144_HUMAN
343
348
GselCL


890
Q86SG6|NEK8_HUMAN
418
423
GsngCL


891
Q86SQ6|GP123_HUMAN
1058
1063
GraaCL


892
Q86SQ6|GP123_HUMAN
1091
1096
GhasCL


893
Q86T20|CF001_HUMAN
75
80
GvldCL


894
Q86T65|DAAM2_HUMAN
570
575
GappCL


895
Q86TX2|ACOT1_HUMAN
234
239
GgelCL


896
Q86U44|MTA70_HUMAN
479
484
GkehCL


897
Q86UE6|LRTM1_HUMAN
19
24
GvvlCL


898
Q86UK0|ABCAC_HUMAN
1251
1256
GwlcCL


899
Q86UK5|LBN_HUMAN
26
31
GgrgCL


900
Q86UQ4|ABCAD_HUMAN
4056
4061
GppfCL


901
Q86UQ4|ABCAD_HUMAN
4932
4937
GsfkCL


902
Q86UU1|PHLB1_HUMAN
119
124
GcmlCL


903
Q86UU1|PHLB1_HUMAN
1245
1250
GvdtCL


904
Q86UV5|UBP48_HUMAN
50
55
GnpnCL


905
Q86UW9|DTX2_HUMAN
347
352
GlpvCL


906
Q86V24|ADR2_HUMAN
190
195
GailCL


907
Q86V71|ZN429_HUMAN
132
137
GlnqCL


908
Q86VH4|LRTM4_HUMAN
271
276
GtfkCL


909
Q86WB7|UN93A_HUMAN
178
183
GasdCL


910
Q86WG5|MTMRD_HUMAN
369
374
GyrsCL


911
Q86WK7|AMGO3_HUMAN
348
353
GlfvCL


912
Q86WR7|CJ047_HUMAN
84
89
GgvcCL


913
Q86X76|NIT1_HUMAN
288
293
GpglCL


914
Q86XN8|RKHD1_HUMAN
192
197
GtdvCL


915
Q86Y01|DTX1_HUMAN
345
350
GlpvCL


916
Q86Y56|HEAT2_HUMAN
271
276
GwllCL


917
Q86YC3|LRC33_HUMAN
396
401
GlasCL


918
Q8IU80|TMPS6_HUMAN
503
508
GqpdCL


919
Q8IUK8|CBLN2_HUMAN
27
32
GcgsCL


920
Q8IUL8|CILP2_HUMAN
464
469
GcqkCL


921
Q8IVF6|ANR18_HUMAN
706
711
GykkCL


922
Q8IVH4|MMAA_HUMAN
96
101
GqraCL


923
Q8IWB7|WDFY1_HUMAN
200
205
GsvaCL


924
Q8IWN6|CX052_HUMAN
89
94
GskrCL


925
Q8IWV2|CNTN4_HUMAN
380
385
GmyqCL


926
Q8IWY4|SCUB1_HUMAN
342
347
GsfqCL


927
Q8IX30|SCUB3_HUMAN
337
342
GsfqCL


928
Q8IXI1|MIRO2_HUMAN
515
520
GqtpCL


929
Q8IXW0|CK035_HUMAN
268
273
GslpCL


930
Q8IY26|PPAC2_HUMAN
149
154
GtlyCL


931
Q8IY49|PAQRA_HUMAN
216
221
GvfyCL


932
Q8IYB9|ZN595_HUMAN
132
137
GvyqCL


933
Q8IYG6|LRC56_HUMAN
194
199
GnlvCL


934
Q8IZ96|CKLF1_HUMAN
112
117
GgslCL


935
QBIZD0|SAM14_HUMAN
95
100
GgsfCL


936
Q8IZE3|PACE1_HUMAN
322
327
GetpCL


937
Q8IZF4|GP114_HUMAN
521
526
GkllCL


938
Q8IZJ1|UNC5B_HUMAN
547
552
GtfgCL


939
Q8IZL8|PELP1_HUMAN
317
322
GlarCL


940
Q8IZY2|ABCA7_HUMAN
2001
2006
GrfrCL


941
Q8N122|RPTOR_HUMAN
549
554
GqeaCL


942
Q8N122|RPTOR_HUMAN
1302
1307
GaisCL


943
Q8N1F7|NUP93_HUMAN
518
523
GdppCL


944
Q8N1G0|ZN687_HUMAN
1133
1138
GaqqCL


945
Q8N283|ANR35_HUMAN
65
70
GlteCL


946
Q8N283|ANR35_HUMAN
703
708
GlwdCL


947
Q8N357|CB018_HUMAN
57
62
GefsCL


948
Q8N3C7|RSNL2_HUMAN
201
206
GavkCL


949
Q8N3V7|SYNPO_HUMAN
28
33
GsyrCL


950
Q8N441|FGRL1_HUMAN
334
339
GmyiCL


951
Q8N442|GUF1_HUMAN
334
339
GdtlCL


952
Q8N4B4|FBX39_HUMAN
114
119
GllsCL


953
Q8N5D0|WDTC1_HUMAN
48
53
GcvnCL


954
Q8N5D6|GBGT1_HUMAN
9
14
GlgfCL


955
Q8N655|CJ012_HUMAN
468
473
GdvkCL


956
Q8N6F8|WBS27_HUMAN
160
165
GglvCL


957
Q8N6T3|ARFG1_HUMAN
38
43
GiwiCL


958
Q8N6V9|TEX9_HUMAN
3
8
GrslCL


959
Q8N6Y1|PCD20_HUMAN
27
32
GpfsCL


960
Q8N6Y1|PCD20_HUMAN
881
886
GiyiCL


961
Q8N726|CD2A2_HUMAN
160
165
GrarCL


962
Q8N813|CC056_HUMAN
42
47
GsctCL


963
Q8N895|ZN366_HUMAN
695
700
GrdeCL


964
Q8N8A2|ANR44_HUMAN
543
548
GhrqCL


965
Q8N8A2|ANR44_HUMAN
645
650
GhtlCL


966
Q8N8Q9|NIPA2_HUMAN
112
117
GkigCL


967
Q8N8R3|MCATL_HUMAN
133
138
GsldCL


968
Q8N9B4|ANR42_HUMAN
142
147
GrlgCL


969
Q8N9B4|ANR42_HUMAN
281
286
GhieCL


970
Q8N9L9|ACOT4_HUMAN
234
239
GadiCL


971
Q8NB46|ANR52_HUMAN
434
439
GnveCL


972
Q8NB46|ANR52_HUMAN
732
737
GcedCL


973
Q8NB46|ANR52_HUMAN
802
807
GhedCL


974
Q8NB49|AT11C_HUMAN
110
115
GyedCL


975
Q8NBJ9|SIDT2_HUMAN
296
301
GmlfCL


976
Q8NBV4|PPAC3_HUMAN
128
133
GtilCL


977
Q8NCL4|GALT6_HUMAN
505
510
GtnqCL


978
Q8NCL4|GALT6_HUMAN
593
598
GsgtCL


979
Q8NCN4|RN169_HUMAN
67
72
GcagCL


980
Q8NDX1|PSD4_HUMAN
183
188
GlkcCL


981
Q8NDX1|PSD4_HUMAN
821
826
GedhCL


982
Q8NEN9|PDZD8_HUMAN
724
729
GgliCL


983
Q8NFP4|MDGA1_HUMAN
622
627
GsaaCL


984
Q8NFP9|NBEA_HUMAN
2819
2824
GpenCL


985
Q8NFU7|CXXC6_HUMAN
1660
1665
GvtaCL


986
Q8NG94|O11H1_HUMAN
112
117
GtseCL


987
Q8NG99|OR7G2_HUMAN
109
114
GlenCL


988
Q8NGC9|O11H4_HUMAN
118
123
GtteCL


989
Q8NGH6|O52L2_HUMAN
96
101
GytvCL


990
Q8NGH7|O52L1_HUMAN
96
101
GyivCL


991
Q8NGI2|O52N4_HUMAN
95
100
GfdeCL


992
Q8NGJ0|OR5A1_HUMAN
111
116
GlseCL


993
Q8NGK5|O52M1_HUMAN
95
100
GldaCL


994
Q8NGR9|OR1N2_HUMAN
112
117
GldnCL


995
Q8NGS6|O13C3_HUMAN
108
113
GsteCL


996
Q8NGT2|O13J1_HUMAN
108
113
GsteCL


997
Q8NGT5|OR9A2_HUMAN
247
252
GygsCL


998
Q8NGT9|O2A42_HUMAN
107
112
GhseCL


999
Q8NGU2|OR9A4_HUMAN
251
256
GygsCL


1000
Q8NGZ9|O2T10_HUMAN
109
114
GaecCL


1001
Q8NH09|OR8S1_HUMAN
109
114
GteaCL


1002
Q8NH19|O10AG_HUMAN
99
104
GgteCL


1003
Q8NH40|OR6S1_HUMAN
66
71
GnlsCL


1004
Q8NHA8|OR1FC_HUMAN
50
55
GsdhCL


1005
Q8NHU2|CT026_HUMAN
158
163
GnipCL


1006
Q8NHU2|CT026_HUMAN
582
587
GfksCL


1007
Q8NHW6|OTOSP_HUMAN
8
13
GlalCL


1008
Q8NHX4|SPTA3_HUMAN
175
180
GsrsCL


1009
Q8NHY2|RFWD2_HUMAN
628
633
GkpyCL


1010
Q8NHY3|GA2L2_HUMAN
463
468
GpaeCL


1011
Q8TB24|RIN3_HUMAN
31
36
GmrlCL


1012
Q8TB24|RIN3_HUMAN
971
976
GsppCL


1013
Q8TCB7|METL6_HUMAN
89
94
GvgnCL


1014
Q8TCN5|ZN507_HUMAN
142
147
GmyrCL


1015
Q8TCT7|PSL1_HUMAN
262
267
GlysCL


1016
Q8TCT7|PSL1_HUMAN
329
334
GiafCL


1017
Q8TCT8|PSL2_HUMAN
321
326
GiafCL


1018
Q8TD26|CHD6_HUMAN
1627
1632
GnlcCL


1019
Q8TD43|TRPM4_HUMAN
238
243
GthgCL


1020
Q8TD43|TRPM4_HUMAN
306
311
GaadCL


1021
Q8TD43|TRPM4_HUMAN
650
655
GdatCL


1022
Q8TD43|TRPM4_HUMAN
764
769
GgrrCL


1023
Q8TDJ6|DMXL2_HUMAN
188
193
GkddCL


1024
Q8TDM6|DLG5_HUMAN
1672
1677
GvkdCL


1025
Q8TDN4|CABL1_HUMAN
135
140
GsgpCL


1026
Q8TDU6|GPBAR_HUMAN
81
86
GywsCL


1027
Q8TDU9|RL3R2_HUMAN
187
192
GvrlCL


1028
Q8TDV0|GP151_HUMAN
183
188
GvemCL


1029
Q8TDX9|PK1L1_HUMAN
317
322
GealCL


1030
Q8TDY2|RBCC1_HUMAN
897
902
GelvCL


1031
Q8TDZ2|MICA1_HUMAN
743
748
GhfyCL


1032
Q8TE49|OTU7A_HUMAN
206
211
GdgnCL


1033
Q8TE58|ATS15_HUMAN
418
423
GhgdCL


1034
Q8TE85|GRHL3_HUMAN
429
434
GvkgCL


1035
Q8TEM1|PO210_HUMAN
1489
1494
GdvlCL


1036
Q8TF62|AT8B4_HUMAN
282
287
GfliCL


1037
Q8TF76|HASP_HUMAN
190
195
GtsaCL


1038
Q8WTV0|SCRB1_HUMAN
319
324
GfcpCL


1039
Q8WUB8|PHF10_HUMAN
320
325
GhpsCL


1040
Q8WUM0|NU133_HUMAN
112
117
GgwaCL


1041
Q8WWQ8|STAB2_HUMAN
1358
1363
GngiCL


1042
Q8WWQ8|STAB2_HUMAN
2026
2031
GsgqCL


1043
Q8WWX0|ASB5_HUMAN
179
184
GhheCL


1044
Q8WWZ1|IL1FA_HUMAN
63
68
GgsrCL


1045
Q8WXI2|CNKR2_HUMAN
22
27
GlddCL


1046
Q8WXI7|MUC16_HUMAN
22110
22115
GlitCL


1047
Q8WXK4|ASB12_HUMAN
75
80
GhlsCL


1048
Q8WXS8|ATS14_HUMAN
489
494
GyqtCL


1049
Q8WXS8|ATS14_HUMAN
587
592
GgrpCL


1050
Q8WYB5|MYST4_HUMAN
244
249
GhpsCL


1051
Q8WYP5|AHTF1_HUMAN
112
117
GsvlCL


1052
Q8WYP5|AHTF1_HUMAN
318
323
GnrkCL


1053
Q8WYP5|AHTF1_HUMAN
526
531
GynrCL


1054
Q8WZ42|TITIN_HUMAN
4919
4924
GkytCL


1055
Q8WZ42|TITIN_HUMAN
5147
5152
GsavCL


1056
Q8WZ42|TITIN_HUMAN
7829
7834
GdysCL


1057
Q8WZ42|TITIN_HUMAN
16742
16747
GaqdCL


1058
Q8WZ42|TITIN_HUMAN
20237
20242
GtnvCL


1059
Q8WZ73|RFFL_HUMAN
81
86
GprlCL


1060
Q8WZ74|CTTB2_HUMAN
924
929
GfknCL


1061
Q92481|AP2B_HUMAN
379
384
GiqsCL


1062
Q92496|FHR4_HUMAN
130
135
GsitCL


1063
Q92520|FAM3C_HUMAN
82
87
GpkiCL


1064
Q92527|ANKR7_HUMAN
148
153
GeppCL


1065
Q92529|SHC3_HUMAN
581
586
GselCL


1066
Q92546|K0258_HUMAN
248
253
GtvaCL


1067
Q92583|CCL17_HUMAN
30
35
GrecCL


1068
Q92621|NU205_HUMAN
950
955
GfveCL


1069
Q92636|FAN_HUMAN
824
829
GtdgCL


1070
Q92673|SORL_HUMAN
1415
1420
GpstCL


1071
Q92750|TAF4B_HUMAN
410
415
GaaiCL


1072
Q92752|TENR_HUMAN
293
298
GqrqCL


1073
Q92782|DPF1_HUMAN
256
261
GhpsCL


1074
Q92783|STAM1_HUMAN
41
46
GpkdCL


1075
Q92785|REQU_HUMAN
302
307
GhpsCL


1076
Q92794|MYST3_HUMAN
237
242
GhpsCL


1077
Q92832|NELL1_HUMAN
618
623
GgfdCL


1078
Q92854|SEM4D_HUMAN
620
625
GvyqCL


1079
Q92900|RENT1_HUMAN
370
375
GdeiCL


1080
Q92932|PTPR2_HUMAN
35
40
GrlgCL


1081
Q92932|PTPR2_HUMAN
634
639
GliyCL


1082
Q92947|GCDH_HUMAN
285
290
GpfgCL


1083
Q92947|GCDH_HUMAN
346
351
GlhaCL


1084
Q92952|KCNN1_HUMAN
361
366
GkgvCL


1085
Q92956|TNR14_HUMAN
89
94
GlskCL


1086
Q92968|PEX13_HUMAN
216
221
GtvaCL


1087
Q93038|TNR25_HUMAN
66
71
GnstCL


1088
Q969L2|MAL2_HUMAN
37
42
GafvCL


1089
Q969P0|IGSF8_HUMAN
402
407
GtyrCL


1090
Q96A54|ADR1_HUMAN
179
184
GavlCL


1091
Q96AP0|ACD_HUMAN
269
274
GalvCL


1092
Q96AQ2|TM125_HUMAN
71
76
GtvlCL


1093
Q96B26|EXOS8_HUMAN
230
235
GklcCL


1094
Q96B86|RGMA_HUMAN
311
316
GlylCL


1095
Q96BD0|SO4A1_HUMAN
698
703
GletCL


1096
Q96CE8|T4S18_HUMAN
8
13
GclsCL


1097
Q96CW5|GCP3_HUMAN
190
195
GvgdCL


1098
Q96D59|RN183_HUMAN
95
100
GhqlCL


1099
Q96DN5|WDR67_HUMAN
52
57
GtgdCL


1100
Q96DZ5|CLR59_HUMAN
212
217
GaakCL


1101
Q96EP1|CHFR_HUMAN
528
533
GcygCL


1102
Q96EY5|F125A_HUMAN
51
56
GyflCL


1103
Q96EZ4|MYEOV_HUMAN
232
237
GrraCL


1104
Q96F46|I17RA_HUMAN
628
633
GsqaCL


1105
Q96GC6|ZN274_HUMAN
256
261
GttcCL


1106
Q96H40|ZN486_HUMAN
132
137
GlnqCL


1107
Q96H96|COQ2_HUMAN
172
177
GvllCL


1108
Q96I82|KAZD1_HUMAN
249
254
GtyrCL


1109
Q96IV0|NGLY1_HUMAN
70
75
GaveCL


1110
Q96IW7|SC22A_HUMAN
234
239
GtaaCL


1111
Q96J02|ITCH_HUMAN
160
165
GvslCL


1112
Q96J94|PIWL1_HUMAN
674
679
GlkvCL


1113
Q96JH7|VCIP1_HUMAN
215
220
GdghCL


1114
Q96JK2|WDR22_HUMAN
178
183
GepfCL


1115
Q96JT2|S45A3_HUMAN
27
32
GlevCL


1116
Q96JT2|S45A3_HUMAN
485
490
GrgiCL


1117
Q96K31|CH076_HUMAN
98
103
GqarCL


1118
Q96KC8|DNJC1_HUMAN
228
233
GiwfCL


1119
Q96KM6|K1196_HUMAN
782
787
GkyrCL


1120
Q96LC7|SIG10_HUMAN
373
378
GqslCL


1121
Q96LD4|TRI47_HUMAN
25
30
GhnfCL


1122
Q96LQ0|CN050_HUMAN
366
371
GeprCL


1123
Q96ME1|FXL18_HUMAN
352
357
GcvhCL


1124
Q96ME7|ZN512_HUMAN
320
325
GqpeCL


1125
Q96ME7|ZN512_HUMAN
438
443
GkykCL


1126
Q96MU7|YTDC1_HUMAN
485
490
GtqlCL


1127
Q96MU8|KREM1_HUMAN
53
58
GgkpCL


1128
Q96NL3|ZN599_HUMAN
373
378
GktfCL


1129
Q96NX9|DACH2_HUMAN
585
590
GnyyCL


1130
Q96P11|NSUN5_HUMAN
400
405
GaehCL


1131
Q96PH1|NOX5_HUMAN
272
277
GcgqCL


1132
Q96PL5|ERMAP_HUMAN
122
127
GsyrCL


1133
Q96PP9|GBP4_HUMAN
321
326
GavpCL


1134
Q96Q04|LMTK3_HUMAN
676
681
GacsCL


1135
Q96Q15|SMG1_HUMAN
2809
2814
GnvtCL


1136
Q96Q27|ASB2_HUMAN
101
106
GqvgCL


1137
Q96Q27|ASB2_HUMAN
135
140
GhldCL


1138
Q96Q91|B3A4_HUMAN
455
460
GaafCL


1139
Q96QG7|MTMR9_HUMAN
85
90
GmeeCL


1140
Q96QS1|TSN32_HUMAN
258
263
GpthCL


1141
Q96QU8|XPO6_HUMAN
413
418
GyfsCL


1142
Q96R30|OR2V2_HUMAN
103
108
GlfvCL


1143
Q96RV3|PCX1_HUMAN
696
701
GtvaCL


1144
Q96RW7|HMCN1_HUMAN
677
682
GiygCL


1145
Q96RW7|HMCN1_HUMAN
2546
2551
GrytCL


1146
Q96RW7|HMCN1_HUMAN
3595
3600
GrytCL


1147
Q96SM3|CPXM1_HUMAN
262
267
GgapCL


1148
Q96SQ9|CP2S1_HUMAN
436
441
GkrvCL


1149
Q96SU4|OSBL9_HUMAN
542
547
GcvsCL


1150
Q99250|SCN2A_HUMAN
955
960
GqtmCL


1151
Q99466|NOTC4_HUMAN
216
221
GsfqCL


1152
Q99466|NOTC4_HUMAN
375
380
GsfsCL


1153
Q99466|NOTC4_HUMAN
414
419
GstlCL


1154
Q99466|NOTC4_HUMAN
457
462
GsfnCL


1155
Q99466|NOTC4_HUMAN
609
614
GaffCL


1156
Q99466|NOTC4_HUMAN
787
792
GtfsCL


1157
Q99466|NOTC4_HUMAN
1121
1126
GgpdCL


1158
Q99466|NOTC4_HUMAN
1872
1877
GggaCL


1159
Q99558|M3K14_HUMAN
536
541
GhavCL


1160
Q99611|SPS2_HUMAN
373
378
GlliCL


1161
Q99678|GPR20_HUMAN
115
120
GargCL


1162
Q99741|CDC6_HUMAN
207
212
GktaCL


1163
Q99758|ABCA3_HUMAN
1590
1595
GqfkCL


1164
Q99797|PMIP_HUMAN
277
282
GqlkCL


1165
Q99848|EBP2_HUMAN
52
57
GlkqCL


1166
Q99867|TBB4Q_HUMAN
235
240
GvttCL


1167
Q99884|SC6A7_HUMAN
543
548
GllsCL


1168
Q99973|TEP1_HUMAN
1464
1469
GpfaCL


1169
Q99973|TEP1_HUMAN
1486
1491
GarlCL


1170
Q99973|TEP1_HUMAN
1720
1725
GisaCL


1171
Q99973|TEP1_HUMAN
2595
2600
GsusCL


1172
Q99996|AKAP9_HUMAN
3063
3068
GllnCL


1173
Q9BQ08|RSNB_HUMAN
2
7
GpssCL


1174
Q9BQG2|NUD12_HUMAN
348
353
GmftCL


1175
Q9BQR3|PRS27_HUMAN
231
236
GplvCL


1176
Q9BQS2|SYT15_HUMAN
23
28
GascCL


1177
Q9BRB3|PIGQ_HUMAN
373
378
GlsaCL


1178
Q9BRP4|WDR71_HUMAN
206
211
GrsaCL


1179
Q9BRZ2|TRI56_HUMAN
343
348
GpapCL


1180
Q9BS86|ZPBP1_HUMAN
346
351
GaktCL


1181
Q9BT40|SKIP_HUMAN
131
136
GvniCL


1182
Q9BT51|CU122_HUMAN
6
11
GfshCL


1183
Q9BTF0|THUM2_HUMAN
407
412
GikkCL


1184
Q9BTX1|NDC1_HUMAN
310
315
GsdeCL


1185
Q9BUY5|ZN426_HUMAN
14
19
GdpvCL


1186
Q9BUY5|ZN426_HUMAN
430
435
GypsCL


1187
Q9BV38|WDR18_HUMAN
81
86
GpvtCL


1188
Q9BV38|WDR18_HUMAN
139
144
GgkdCL


1189
Q9BV73|CP250_HUMAN
806
811
GevrCL


1190
Q9BV99|LRC61_HUMAN
113
118
GqlqCL


1191
Q9BVA1|TBB2B_HUMAN
235
240
GvttCL


1192
Q9BVH7|SIA7E_HUMAN
8
13
GlavCL


1193
Q9BVK2|ALG8_HUMAN
361
366
GflrCL


1194
Q9BWT7|CAR10_HUMAN
916
921
GkkhCL


1195
Q9BWU0|NADAP_HUMAN
185
190
GtsyCL


1196
Q9BWU0|NADAP_HUMAN
196
201
GcdvCL


1197
Q9BWV1|BOC_HUMAN
1053
1058
GppcCL


1198
Q9BXC9|BBS2_HUMAN
26
31
GthpCL


1199
Q9BXL6|CAR14_HUMAN
850
855
GfkkCL


1200
Q9BXM7|PINK1_HUMAN
408
413
GgngCL


1201
Q9BXR0|TGT_HUMAN
50
55
GcriCL


1202
Q9BXS4|TMM59_HUMAN
229
234
GflrCL


1203
Q9BXT5|TEX15_HUMAN
1099
1104
GekkCL


1204
Q9BXU8|FHL17_HUMAN
78
83
GghiCL


1205
Q9BY15|EMR3_HUMAN
562
567
GctwCL


1206
Q9BY41|HDAC8_HUMAN
283
288
GigkCL


1207
Q9BYB4|GNB1L_HUMAN
163
168
GmpmCL


1208
Q9BYE0|HES7_HUMAN
95
100
GfreCL


1209
Q9BYJ1|LOXE3_HUMAN
309
314
GqdtCL


1210
Q9BYK8|PR285_HUMAN
1908
1913
GfslCL


1211
Q9BYT1|CT059_HUMAN
398
403
GswtCL


1212
Q9BYX4|IFIH1_HUMAN
265
270
GsusCL


1213
Q9BZ11|ADA33_HUMAN
400
405
GggaCL


1214
Q9BZ76|CNTP3_HUMAN
509
514
GfqgCL


1215
Q9BZ76|CNTP3_HUMAN
1163
1168
GftgCL


1216
Q9BZC7|ABCA2_HUMAN
2262
2267
GrlrCL


1217
Q9BZF3|OSBL6_HUMAN
554
559
GrraCL


1218
Q9BZF9|UACA_HUMAN
79
84
GnleCL


1219
Q9BZF9|UACA_HUMAN
112
117
GhalCL


1220
Q9BZH6|BRWD2_HUMAN
79
84
GspyCL


1221
Q9BZS1|FOXP3_HUMAN
228
233
GraqCL


1222
Q9BZY9|TRI31_HUMAN
32
37
GhnfCL


1223
Q9BZZ2|SN_HUMAN
1507
1512
GmyhCL


1224
Q9C004|SPY4_HUMAN
197
202
GtcmCL


1225
Q9C0A0|CNTP4_HUMAN
1163
1168
GftgCL


1226
Q9C0C6|K1737_HUMAN
47
52
GsseCL


1227
Q9GZK3|OR2B2_HUMAN
108
113
GsteCL


1228
Q9GZR3|CFC1_HUMAN
144
149
GalhCL


1229
Q9GZY1|PBOV1_HUMAN
118
123
GlecCL


1230
Q9H013|ADA19_HUMAN
400
405
GggmCL


1231
Q9H093|NUAK2_HUMAN
587
592
GpgsCL


1232
Q9H0A0|NAT10_HUMAN
654
659
GrfpCL


1233
Q9H0B3|K1683_HUMAN
578
583
GkirCL


1234
Q9H0J9|PAR12_HUMAN
272
277
GdqiCL


1235
Q9H0M4|ZCPW1_HUMAN
249
254
GfgqCL


1236
Q9H172|ABCG4_HUMAN
588
593
GdltCL


1237
Q9H195|MUC3B_HUMAN
545
550
GqcaCL


1238
Q9H1B7|CN004_HUMAN
294
299
GgpaCL


1239
Q9H1D0|TRPV6_HUMAN
10
15
GlilCL


1240
Q9H1K4|GHC2_HUMAN
47
52
GmidCL


1241
Q9H1M3|DB129_HUMAN
23
28
GlrrCL


1242
Q9H1M4|DB127_HUMAN
50
55
GrycCL


1243
Q9H1P6|CT085_HUMAN
107
112
GlnkCL


1244
Q9H1R3|MYLK2_HUMAN
240
245
GqalCL


1245
Q9H1V8|S6A17_HUMAN
421
426
GldpCL


1246
Q9H221|ABCG8_HUMAN
421
426
GaeaCL


1247
Q9H228|EDG8_HUMAN
347
352
GlrrCL


1248
Q9H252|KCNH6_HUMAN
571
576
GfpeCL


1249
Q9H2D1|MFTC_HUMAN
64
69
GilhCL


1250
Q9H2G2|SLK_HUMAN
1208
1213
GeseCL


1251
Q9H2M9|RBGPR_HUMAN
387
392
GesiCL


1252
Q9H2S1|KCNN2_HUMAN
371
376
GkgvCL


1253
Q9H2X9|S12A5_HUMAN
602
607
GmslCL


1254
Q9H2Y7|ZF106_HUMAN
975
980
GegnCL


1255
Q9H324|ATS10_HUMAN
422
427
GlglCL


1256
Q9H324|ATS10_HUMAN
556
561
GgkyCL


1257
Q9H3D4|P73L_HUMAN
557
562
GcssCL


1258
Q9H3R1|NDST4_HUMAN
814
819
GktkCL


1259
Q9H4F1|SIA7D_HUMAN
29
34
GlplCL


1260
Q9H5U8|CX045_HUMAN
403
408
GfdsCL


1261
Q9H5V8|CDCP1_HUMAN
373
378
GcfvCL


1262
Q9H6E5|TUT1_HUMAN
15
20
GfrcCL


1263
Q9H6R4|NOL6_HUMAN
391
396
GislCL


1264
Q9H792|SG269_HUMAN
1661
1666
GilqCL


1265
Q9H7F0|AT133_HUMAN
109
114
GhavCL


1266
Q9H7M9|GI24_HUMAN
142
147
GlycCL


1267
Q9H808|TLE6_HUMAN
315
320
GpdaCL


1268
Q9H8X2|IPPK_HUMAN
110
115
GyamCL


1269
Q9H9S3|S61A2_HUMAN
143
148
GagiCL


1270
Q9HAF5|CO028_HUMAN
120
125
GvrmCL


1271
Q9HAS0|NJMU_HUMAN
123
128
GcyyCL


1272
Q9HAT1|LMA1L_HUMAN
8
13
GplfCL


1273
Q9HAV4|XPO5_HUMAN
266
271
GaaeCL


1274
Q9HAW7|UD17_HUMAN
510
515
GyrkCL


1275
Q9HAW8|UD110_HUMAN
510
515
GyrkCL


1276
Q9HAW9|UD18_HUMAN
510
515
GyrkCL


1277
Q9HBX8|LGR6_HUMAN
550
555
GvlgCL


1278
Q9HBZ2|ARNT2_HUMAN
295
300
GskyCL


1279
Q9HC07|TM165_HUMAN
138
143
GlmtCL


1280
Q9HC84|MUC5B_HUMAN
780
785
GklsCL


1281
Q9HC84|MUC5B_HUMAN
1281
1286
GlgaCL


1282
Q9HCC6|HES4_HUMAN
113
118
GfheCL


1283
Q9HCC9|ZFY28_HUMAN
555
560
GatnCL


1284
Q9HCE9|TM16H_HUMAN
541
546
GgrrCL


1285
Q9HCM2|PLXA4_HUMAN
990
995
GkqpCL


1286
Q9HCM4|E41L5_HUMAN
111
116
GspyCL


1287
Q9HCU4|CELR2_HUMAN
1308
1313
GgytCL


1288
Q9HCU4|CELR2_HUMAN
1757
1762
GfrgCL


1289
Q9HCU4|CELR2_HUMAN
1917
1922
GsptCL


1290
Q9NNW5|WDR6_HUMAN
460
465
GvvaCL


1291
Q9NP73|GT281_HUMAN
82
87
GagsCL


1292
Q9NP90|RAB9B_HUMAN
79
84
GadcCL


1293
Q9NPA1|KCMB3_HUMAN
121
126
GkypCL


1294
Q9NPA3|M1IP1_HUMAN
58
63
GsggCL


1295
Q9NPD7|NRN1_HUMAN
37
42
GfsdCL


1296
Q9NPF8|CENA2_HUMAN
41
46
GifiCL


1297
Q9NPG4|PCD12_HUMAN
807
812
GwdpCL


1298
Q9NPH5|NOX4_HUMAN
51
56
GlglCL


1299
Q9NQ25|SLAF7_HUMAN
3
8
GsptCL


1300
Q9NQ30|ESM1_HUMAN
125
130
GtgkCL


1301
Q9NQ75|CT032_HUMAN
50
55
GwwkCL


1302
Q9NQB0|TF7L2_HUMAN
492
497
GegsCL


1303
Q9NQQ7|S35C2_HUMAN
302
307
GfalCL


1304
Q9NQS5|GPR84_HUMAN
195
200
GifyCL


1305
Q9NQU5|PAK6_HUMAN
662
667
GlpeCL


1306
Q9NR09|BIRC6_HUMAN
511
516
GanpCL


1307
Q9NR61|DLL4_HUMAN
204
209
GnlsCL


1308
Q9NR63|CP26B_HUMAN
437
442
GvrtCL


1309
Q9NR81|ARHG3_HUMAN
203
208
GwlpCL


1310
Q9NR99|MXRA5_HUMAN
2414
2419
GnytCL


1311
Q9NRI5|DISC1_HUMAN
23
28
GsrdCL


1312
Q9NRX5|SERC1_HUMAN
19
24
GsapCL


1313
Q9NS15|LTBP3_HUMAN
846
851
GsyrCL


1314
Q9NS40|KCNH7_HUMAN
722
727
GfpeCL


1315
Q9NS62|THSD1_HUMAN
419
424
GislCL


1316
Q9NSD7|RL3R1_HUMAN
243
248
GeelCL


1317
Q9NSI6|BRWD1_HUMAN
204
209
GsddCL


1318
Q9NSN8|SNTG1_HUMAN
242
247
GiiqCL


1319
Q9NST1|ADPN_HUMAN
24
29
GatrCL


1320
Q9NST1|ADPN_HUMAN
97
102
GlckCL


1321
Q9NT68|TEN2_HUMAN
858
863
GlvdCL


1322
Q9NU22|MDN1_HUMAN
427
432
GrgdCL


1323
Q9NUB4|CT141_HUMAN
156
161
GlafCL


1324
Q9NUP1|CNO_HUMAN
67
72
GyaaCL


1325
Q9NVE7|PANK4_HUMAN
304
309
GqlaCL


1326
Q9NVG8|TBC13_HUMAN
38
43
GglrCL


1327
Q9NVX2|NLE1_HUMAN
474
479
GkdkCL


1328
Q9NW08|RPC2_HUMAN
765
770
GfgrCL


1329
Q9NWT1|PK1IP_HUMAN
83
88
GtitCL


1330
Q9NWU5|RM22_HUMAN
142
147
GrgqCL


1331
Q9NWZ3|IRAK4_HUMAN
255
260
GddlCL


1332
Q9NX02|NALP2_HUMAN
139
144
GnviCL


1333
Q9NXJ0|M4A12_HUMAN
106
111
GivlCL


1334
Q9NXR5|ANR10_HUMAN
69
74
GkleCL


1335
Q9NXR5|ANR10_HUMAN
103
108
GhpqCL


1336
Q9NXS3|BTBD5_HUMAN
293
298
GlfaCL


1337
Q9NXW9|ALKB4_HUMAN
19
24
GirtCL


1338
Q9NY15|STAB1_HUMAN
122
127
GhgtCL


1339
Q9NY15|STAB1_HUMAN
177
182
GdgsCL


1340
Q9NY15|STAB1_HUMAN
752
757
GngaCL


1341
Q9NY15|STAB1_HUMAN
1256
1261
GssrCL


1342
Q9NY15|STAB1_HUMAN
1991
1996
GsgqCL


1343
Q9NY15|STAB1_HUMAN
2250
2255
GfhlCL


1344
Q9NY33|DPP3_HUMAN
515
520
GlylCL


1345
Q9NY35|CLDND_HUMAN
213
218
GwsfCL


1346
Q9NY46|SCN3A_HUMAN
956
961
GqtmCL


1347
Q9NY91|SC5A4_HUMAN
507
512
GtgsCL


1348
Q9NY99|SNTG2_HUMAN
14
19
GrqgCL


1349
Q9NYJ7|DLL3_HUMAN
235
240
GecrCL


1350
Q9NYQ6|CELR1_HUMAN
168
173
GrpiCL


1351
Q9NYQ7|CELR3_HUMAN
2070
2075
GsdsCL


1352
Q9NYQ8|FAT2_HUMAN
3908
3913
GfegCL


1353
Q9NYQ8|FAT2_HUMAN
4285
4290
GggpCL


1354
Q9NYW6|TA2R3_HUMAN
104
109
GvlyCL


1355
Q9NZ56|FMN2_HUMAN
1694
1699
GkeqCL


1356
Q9NZ71|RTEL1_HUMAN
47
52
GktlCL


1357
Q9NZ94|NLGN3_HUMAN
19
24
GrslCL


1358
Q9NZH0|GPC5B_HUMAN
164
169
GlalCL


1359
Q9NZH7|IL1F8_HUMAN
68
73
GkdlCL


1360
Q9NZL3|ZN224_HUMAN
550
555
GwasCL


1361
Q9NZR2|LRP1B_HUMAN
866
871
GdddCL


1362
Q9NZR2|LRP1B_HUMAN
2987
2992
GtykCL


1363
Q9NZV5|SEPN1_HUMAN
273
278
GavaCL


1364
Q9P0K1|ADA22_HUMAN
429
434
GggaCL


1365
Q9P0K7|RAI14_HUMAN
64
69
GhveCL


1366
Q9P0L1|ZN167_HUMAN
617
622
GlskCL


1367
Q9P0M9|RM27_HUMAN
84
89
GknkCL


1368
Q9P0U3|SENP1_HUMAN
531
536
GvhwCL


1369
Q9P0X4|CAC1I_HUMAN
290
295
GrecCL


1370
Q9P203|BTBD7_HUMAN
265
270
GnqnCL


1371
Q9P255|ZN492_HUMAN
143
148
GlnqCL


1372
Q9P273|TEN3_HUMAN
142
147
GrssCL


1373
Q9P273|TEN3_HUMAN
1590
1595
GtngCL


1374
Q9P275|UBP36_HUMAN
824
829
GsetCL


1375
Q9P283|SEM5B_HUMAN
589
594
GgldCL


1376
Q9P283|SEM5B_HUMAN
887
892
GediCL


1377
Q9P298|HIG1B_HUMAN
34
39
GlggCL


1378
Q9P2B2|FPRP_HUMAN
844
849
GllsCL


1379
Q9P2C4|TM181_HUMAN
406
411
GerkCL


1380
Q9P2E3|ZNFX1_HUMAN
1162
1167
GqlfCL


1381
Q9P2I0|CPSF2_HUMAN
759
764
GlegCL


1382
Q9P2J9|PDP2_HUMAN
125
130
GvasCL


1383
Q9P2J9|PDP2_HUMAN
298
303
GmwsCL


1384
Q9P2N4|ATS9_HUMAN
490
495
GygeCL


1385
Q9P2P6|STAR9_HUMAN
715
720
GeadCL


1386
Q9P2R3|ANFY1_HUMAN
720
725
GpggCL


1387
Q9P2R7|SUCB1_HUMAN
316
321
GnigCL


1388
Q9P2S2|NRX2A_HUMAN
1061
1066
GfqgCL


1389
Q9UBD9|CLCF1_HUMAN
10
15
GmlaCL


1390
Q9UBE0|ULE1A_HUMAN
338
343
GiveCL


1391
Q9UBG0|MRC2_HUMAN
50
55
GlqgCL


1392
Q9UBG0|MRC2_HUMAN
89
94
GtmqCL


1393
Q9UBG0|MRC2_HUMAN
938
943
GdqrCL


1394
Q9UBG7|RBPSL_HUMAN
56
61
GvrrCL


1395
Q9UBG7|RBPSL_HUMAN
326
331
GtylCL


1396
Q9UBH0|IL1F5_HUMAN
63
68
GgsqCL


1397
Q9UBM4|OPT_HUMAN
124
129
GlptCL


1398
Q9UBP5|HEY2_HUMAN
125
130
GfreCL


1399
Q9UBS8|RNF14_HUMAN
258
263
GqvqCL


1400
Q9UBY5|EDG7_HUMAN
37
42
GtffCL


1401
Q9UBY8|CLN8_HUMAN
145
150
GflgCL


1402
Q9UDX3|S14L4_HUMAN
250
255
GnpkCL


1403
Q9UDX3|S14L4_HUMAN
351
356
GsltCL


1404
Q9UDX4|S14L3_HUMAN
250
255
GnpkCL


1405
Q9UGF7|O12D3_HUMAN
62
67
GnlsCL


1406
Q9UGI6|KCNN3_HUMAN
525
530
GkgvCL


1407
Q9UGU5|HM2L1_HUMAN
567
572
GplaCL


1408
Q9UHA7|IL1F6_HUMAN
69
74
GlnlCL


1409
Q9UHC6|CNTP2_HUMAN
1174
1179
GftgCL


1410
Q9UHD0|IL19_HUMAN
24
29
GlrrCL


1411
Q9UHI8|ATS1_HUMAN
458
463
GhgeCL


1412
Q9UHW9|S12A6_HUMAN
687
692
GmsiCL


1413
Q9UHX3|EMR2_HUMAN
742
747
GctwCL


1414
Q9UIA9|XPO7_HUMAN
933
938
GccsCL


1415
Q9UIE0|N230_HUMAN
286
291
GksfCL


1416
Q9UIF8|BAZ2B_HUMAN
627
632
GmqwCL


1417
Q9UIF9|BAZ2A_HUMAN
1006
1011
GpeeCL


1418
Q9UIH9|KLF15_HUMAN
117
122
GehfCL


1419
Q9UIR0|BTNL2_HUMAN
337
342
GqyrCL


1420
Q9UK10|ZN225_HUMAN
466
471
GwasCL


1421
Q9UK11|ZN223_HUMAN
294
299
GksfCL


1422
Q9UK12|ZN222_HUMAN
263
268
GksfCL


1423
Q9UK13|ZN221_HUMAN
488
493
GwasCL


1424
Q9UK13|ZN221_HUMAN
572
577
GwasCL


1425
Q9UK99|FBX3_HUMAN
189
194
GlkyCL


1426
Q9UKB1|FBW1B_HUMAN
281
286
GsvlCL


1427
Q9UKP4|ATS7_HUMAN
443
448
GwglCL


1428
Q9UKP5|ATS6_HUMAN
545
550
GgkyCL


1429
Q9UKQ2|ADA28_HUMAN
500
505
GkghCL


1430
Q9UKU0|ACSL6_HUMAN
104
109
GngpCL


1431
Q9UL25|RAB21_HUMAN
121
126
GneiCL


1432
Q9ULB1|NRX1A_HUMAN
1048
1053
GfqgCL


1433
Q9ULL4|PLXB3_HUMAN
1191
1196
GrgeCL


1434
Q9ULV0|MYO5B_HUMAN
1496
1501
GtvpCL


1435
Q9UM47|NOTC3_HUMAN
1228
1233
GgfrCL


1436
Q9UM82|SPAT2_HUMAN
37
42
GsdeCL


1437
Q9UMF0|ICAM5_HUMAN
879
884
GeavCL


1438
Q9UMW8|UBP18_HUMAN
61
66
GqtcCL


1439
Q9UNA0|ATS5_HUMAN
467
472
GhgnCL


1440
Q9UNA0|ATS5_HUMAN
525
530
GqmvCL


1441
Q9UNI1|ELA1_HUMAN
208
213
GplhCL


1442
Q9UP79|ATS8_HUMAN
421
426
GhgdCL


1443
Q9UP79|ATS8_HUMAN
562
567
GgryCL


1444
Q9UP95|S12A4_HUMAN
622
627
GmslCL


1445
Q9UPA5|BSN_HUMAN
1765
1770
GspvCL


1446
Q9UPZ6|THS7A_HUMAN
881
886
GiheCL


1447
Q9UQ05|KCNH4_HUMAN
213
218
GgsrCL


1448
Q9UQ49|NEUR3_HUMAN
380
385
GlfgCL


1449
Q9UQ52|CNTN6_HUMAN
96
101
GmyqCL


1450
Q9UQD0|SCN8A_HUMAN
949
954
GqamCL


1451
Q9Y219|JAG2_HUMAN
907
912
GwkpCL


1452
Q9Y236|OSGI2_HUMAN
480
485
GvtrCL


1453
Q9Y263|PLAP_HUMAN
721
726
GkaqCL


1454
Q9Y278|OST2_HUMAN
51
56
GaprCL


1455
Q9Y297|FBW1A_HUMAN
344
349
GsvlCL


1456
Q9Y2H6|FNDC3_HUMAN
790
795
GivtCL


1457
Q9Y2L6|FRM4B_HUMAN
871
876
GsqrCL


1458
Q9Y2P5|S27A5_HUMAN
345
350
GilgCL


1459
Q9Y2P5|S27A5_HUMAN
452
457
GkmsCL


1460
Q9Y2Q1|ZN257_HUMAN
132
137
GlnqCL


1461
Q9Y2T5|GPR52_HUMAN
205
210
GfivCL


1462
Q9Y385|UB2J1_HUMAN
87
92
GkkiCL


1463
Q9Y3B6|CN122_HUMAN
38
43
GeclCL


1464
Q9Y3C8|UFC1_HUMAN
112
117
GgkiCL


1465
Q9Y3I1|FBX7_HUMAN
71
76
GdliCL


1466
Q9Y3N9|OR2W1_HUMAN
108
113
GsveCL


1467
Q9Y3R4|NEUR2_HUMAN
160
165
GpghCL


1468
Q9Y3S2|ZN330_HUMAN
182
187
GqhsCL


1469
Q9Y485|DMXL1_HUMAN
187
192
GkddCL


1470
Q9Y485|DMXL1_HUMAN
2862
2867
XrnvCL


1471
Q9Y493|ZAN_HUMAN
1152
1157
GtatCL


1472
Q9Y4C0|NRX3A_HUMAN
1014
1019
GfqgCL


1473
Q9Y4F1|FARP1_HUMAN
820
825
GvphCL


1474
Q9Y4K1|AIM1_HUMAN
1473
1478
GhypCL


1475
Q9Y4W6|AFG32_HUMAN
31
36
GeqpCL


1476
Q9Y535|RPC8_HUMAN
43
48
GlciCL


1477
Q9Y561|LRP12_HUMAN
241
246
GnidCL


1478
Q9Y574|ASB4_HUMAN
86
91
GhveCL


1479
Q9Y575|ASB3_HUMAN
291
296
GhedCL


1480
Q9Y5F7|PCDGL_HUMAN
729
734
GtcaCL


1481
Q9Y5J3|HEY1_HUMAN
126
131
GfreCL


1482
Q9Y5N5|HEMK2_HUMAN
45
50
GveiCL


1483
Q9Y5Q5|CORIN_HUMAN
424
429
GdqrCL


1484
Q9Y5R5|DMRT2_HUMAN
130
135
GvvsCL


1485
Q9Y5R6|DMRT1_HUMAN
153
158
GsnpCL


1486
Q9Y5S2|MRCKB_HUMAN
1374
1379
GsvqCL


1487
Q9Y5W8|SNX13_HUMAN
73
78
GvpkCL


1488
Q9Y616|IRAK3_HUMAN
395
400
GldsCL


1489
Q9Y644|RFNG_HUMAN
203
208
GagfCL


1490
Q9Y662|OST3B_HUMAN
7
12
GgrsCL


1491
Q9Y666|S12A7_HUMAN
622
627
GmslCL


1492
Q9Y6H5|SNCAP_HUMAN
361
366
GhaeCL


1493
Q9Y6I4|UBP3_HUMAN
449
454
GpesCL


1494
Q9Y6N6|LAMC3_HUMAN
885
890
GqcsCL


1495
Q9Y6R1|S4A4_HUMAN
512
517
GaifCL


1496
Q9Y6R7|FCGBP_HUMAN
1661
1666
GqgvCL


1497
Q9Y6R7|FCGBP_HUMAN
2388
2393
GqcgCL


1498
Q9Y6R7|FCGBP_HUMAN
2862
2867
GqgvCL


1499
Q9Y6R7|FCGBP_HUMAN
3589
3594
GqcgCL


1500
Q9Y6R7|FCGBP_HUMAN
4063
4068
GqgvCL


1501
Q9Y6R7|FCGBP_HUMAN
4790
4795
GqcgCL


1502
Q9Y6R7|FCGBP_HUMAN
4852
4857
GcgrCL


1503
Q9Y6R7|FCGBP_HUMAN
5032
5037
GcpvCL









These peptides are likely to have anti-angiogenic activity. Methods for testing for such activity are described herein.
Example 4
Collagen Derived Peptides
The same procedure as used for the C-X-C chemokines can be repeated for the case of the collagen related fragments. Because the number of the experimentally tested peptides is small in the calculation, all the theoretically predicted fragments are considered. Both the short and long predicted fragments are introduced. Two predominant motifs were calculated. One of them is the most abundant and is characterized by a conserved 4-amino acid repeat. It can be described by the following generic sequence: C-N-X3-V-C (FIG. 6A). This motif can be localized either upstream or downstream of the peptide sequence. If the peptides are separated according to the location of the C-N-X3-V-C motif, it can be either near the amino or carboxy terminal of the peptide. This provides for the identification of a set of two more definitive motifs (FIGS. 6B and 6C). The 4-letter motif appears upstream is the C-N-X3-V-C-X2-A-X-R-N-D-X-S-Y-W-L (FIG. 6B), whereas the motif that appears downstream is the L-X2-F-S-T-X-P-F-X2-C-N-X3-V-C (FIG. 6C).
Apart from the aforementioned 7-mer there is another motif that is present in a smaller subset of collagen derived peptides. Those peptides do not include the C-N-X3-V-C. This motif is described by the generic sequence X2-P-F-X-E-C-X-G-X8-A-N. Common modifications can be described by the sequence X2-P-F-(I/L)-E-C-X-G-X-(R/G)-X-(Y/F)-(Y/F)-A-N (FIG. 7).
If only the short identified anti-angiogenic fragments are considered then the multiple alignment algorithm may be used to identify motifs present only within this subset of the peptides. The alignment is shown in FIG. 8. These motifs are similar to those identified herein. A more generic 3-common letter motif, the P-F-X2-C motif can be distinguished.
In the case of collagens two generic motifs were identified. The first one is the C-N-X3-V-C. Using this motif as a query and scanning the Prosite database 24 hits in 24 different proteins were identified. These candidate anti-angiogenic peptides are listed in Table 5 (SEQ ID NO: 1504-1527).







TABLE 5







Collagens


Motif: C-N-X(3)-V-C


Number of Locations: 24


Number of Different Proteins: 24













First
Last




Accession
Amino
Amino


#
Number|Protein Name
acid
acid
Sequence














1504
O14514|BAI1_HUMAN
400
406
CNnsaVC


1505
O75093|SLIT1_HUMAN
507
513
CNsdvVC


1506
O75534|CSDE1_HUMAN
733
739
CNvwrVC


1507
P02462|CO4A1_HUMAN
1505
1511
CNinnVC


1508
P08572|CO4A2_HUMAN
1549
1555
CNpgdVC


1509
P09758|TACD2_HUMAN
119
125
CNqtsVC


1510
P25391|LAMA1_HUMAN
751
757
CNvhgVC


1511
P29400|CO4A5_HUMAN
1521
1527
CNinnVC


1512
P53420|CO4A4_HUMAN
1525
1531
CNihqVC


1513
P83110|HTRA3_HUMAN
48
54
CNcclVC


1514
Q01955|CO4A3_HUMAN
1505
1511
CNvndVC


1515
Q13625|ASPP2_HUMAN
1002
1008
CNnvqVC


1516
Q13751|LAMB3_HUMAN
572
578
CNrypVC


1517
Q14031|CO4A6_HUMAN
1527
1533
CNineVC


1518
Q8WWQ8|STAB2_HUMAN
1970
1976
CNnrgVC


1519
Q96GX1|TECT2_HUMAN
642
648
CNrneVC


1520
Q99965|ADAM2_HUMAN
621
627
CNdrgVC


1521
Q9BX93|PG12B_HUMAN
112
118
CNqldVC


1522
Q9BYD5|CNFN_HUMAN
32
38
CNdmpVC


1523
Q9H013|ADA19_HUMAN
659
665
CNghgVC


1524
Q9HBG6|IF122_HUMAN
436
442
CNllvVC


1525
Q9P2R7|SUCB1_HUMAN
152
158
CNqvlVC


1526
Q9UBX1|CATF_HUMAN
89
95
CNdpmVC


1527
Q9UKF2|ADA30_HUMAN
638
644
CNtrgVC









The second motif is the P-F-X2-C. Again using this motif as a query at the Prosite 306 locations that contain the specific amino acid sequence were identified in 288 different proteins. The hits included peptides shown in Table 6 (SEQ ID Nos: 1528-1833).







TABLE 6







Collagens


Motif: P-F-X2-C


Number of Locations: 306


Number of Different Proteins: 288













First





Accession
Amino
Last Amino


#
Number|Protein Name
acid
acid
Sequence














1528
O00116|ADAS_HUMAN
561
565
PFstC


1529
O00182|LEG9_HUMAN
98
102
PFdlC


1530
O00206|TLR4_HUMAN
702
706
PFqlC


1531
O00270|GPR31_HUMAN
2
6
PFpnC


1532
O00398|P2Y10_HUMAN
288
292
PFclC


1533
O00507|USP9Y_HUMAN
259
263
PFgqC


1534
O14646|CHD1_HUMAN
450
454
PFkdC


1535
O14843|FFAR3_HUMAN
84
88
PFilC


1536
O14978|ZN263_HUMAN
547
551
PFseC


1537
O15015|ZN646_HUMAN
880
884
PFlcC


1538
O15031|PLXB2_HUMAN
611
615
PFydC


1539
O15037|K0323_HUMAN
423
427
PFtlC


1540
O15453|NBR2_HUMAN
9
13
PFlpC


1541
O15529|GPR42_HUMAN
84
88
PFilC


1542
O43556|SGCE_HUMAN
207
211
PFssC


1543
O60299|K0552_HUMAN
308
312
PFaaC


1544
O60343|TBCD4_HUMAN
89
93
PFlrC


1545
O60431|OR1I1_HUMAN
93
97
PFvgC


1546
O60449|LY75_HUMAN
1250
1254
PFqnC


1547
O60481|ZIC3_HUMAN
331
335
PFpgC


1548
O60486|PLXC1_HUMAN
618
622
PFtaC


1549
O60494|CUBN_HUMAN
3302
3306
PFsiC


1550
O60603|TLR2_HUMAN
669
673
PFklC


1551
O60656|UD19_HUMAN
149
153
PFdnC


1552
O60706|ABCC9_HUMAN
627
631
PFesC


1553
O75152|ZC11A_HUMAN
23
27
PFrhC


1554
O75197|LRP5_HUMAN
317
321
PFytC


1555
O75419|CC45L_HUMAN
444
448
PFlyC


1556
O75473|LGR5_HUMAN
547
551
PFkpC


1557
O75478|TAD2L_HUMAN
38
42
PFflC


1558
O75581|LRP6_HUMAN
304
308
PFyqC


1559
O75794|CD123_HUMAN
147
151
PFihC


1560
O75882|ATRN_HUMAN
969
973
PFgqC


1561
O76031|CLPX_HUMAN
313
317
PFaiC


1562
O95006|OR2F2_HUMAN
93
97
PFqsC


1563
O95007|OR6B1_HUMAN
285
289
PFiyC


1564
O95149|SPN1_HUMAN
195
199
PFydC


1565
O95202|LETM1_HUMAN
51
55
PFgcC


1566
O95409|ZIC2_HUMAN
336
340
PFpgC


1567
O95450|ATS2_HUMAN
569
573
PFgsC


1568
O95759|TBCD8_HUMAN
67
71
PFsrC


1569
O95841|ANGL1_HUMAN
276
280
PFkdC


1570
O95886|DLGP3_HUMAN
98
102
PFdtC


1571
P02461|CO3A1_HUMAN
80
84
PFgeC


1572
P02462|CO4A1_HUMAN
1501
1505
PFlfC


1573
P02462|CO4A1_HUMAN
1612
1616
PFieC


1574
P08151|GLI1_HUMAN
173
177
PFptC


1575
P08572|CO4A2_HUMAN
1545
1549
PFlyC


1576
P08572|CO4A2_HUMAN
1654
1658
PFieC


1577
P08581|MET_HUMAN
534
538
PFvqC


1578
P09172|DOPO_HUMAN
136
140
PFgtC


1579
P0C0L4|CO4A_HUMAN
731
735
PFlsC


1580
P0C0L5|CO4B_HUMAN
731
735
PFlsC


1581
P15309|PPAP_HUMAN
157
161
PFrnC


1582
P17021|ZNF17_HUMAN
350
354
PFycC


1583
P18084|ITB5_HUMAN
546
550
PFceC


1584
P20645|MPRD_HUMAN
3
7
PFysC


1585
P20851|C4BB_HUMAN
130
134
PFpiC


1586
P20933|ASPG_HUMAN
13
17
PFllC


1587
P21673|SAT1_HUMAN
50
54
PFyhC


1588
P21854|CD72_HUMAN
222
226
PFftC


1589
P22309|UD11_HUMAN
152
156
PFlpC


1590
P22362|CCL1_HUMAN
29
33
PFsrC


1591
P22681|CBL_HUMAN
417
421
PFcrC


1592
P23942|RDS_HUMAN
210
214
PFscC


1593
P24043|LAMA2_HUMAN
2679
2683
PFegC


1594
P24043|LAMA2_HUMAN
3083
3087
PFrgC


1595
P24903|CP2F1_HUMAN
483
487
PFqlC


1596
P25098|ARBK1_HUMAN
252
256
PFivC


1597
P25490|TYY1_HUMAN
386
390
PFdgC


1598
P25929|NPY1R_HUMAN
117
121
PFvqC


1599
P26718|NKG2D_HUMAN
52
56
PFffC


1600
P26927|HGFL_HUMAN
439
443
PFdyC


1601
P27987|IP3KB_HUMAN
869
873
PFfkC


1602
P29400|CO4A5_HUMAN
1517
1521
PFmfC


1603
P29400|CO4A5_HUMAN
1628
1632
PFieC


1604
P34896|GLYC_HUMAN
244
248
PFehC


1605
P35504|UD15_HUMAN
153
157
PFhlC


1606
P35523|CLCN1_HUMAN
26
30
PFehC


1607
P35626|ARBK2_HUMAN
252
256
PFivC


1608
P36383|CXA7_HUMAN
205
209
PFyvC


1609
P36508|ZNF76_HUMAN
258
262
PFegC


1610
P36509|UD12_HUMAN
149
153
PFdnC


1611
P36894|BMR1A_HUMAN
57
61
PFlkC


1612
P41180|CASR_HUMAN
538
542
PFsnC


1613
P42338|PK3CB_HUMAN
650
654
PFldC


1614
P42575|CASP2_HUMAN
141
145
PFpvC


1615
P45974|UBP5_HUMAN
528
532
PFssC


1616
P46531|NOTC1_HUMAN
1411
1415
PFyrC


1617
P48637|GSHB_HUMAN
405
409
PFenC


1618
P49257|LMAN1_HUMAN
471
475
PFpsC


1619
P49888|ST1E1_HUMAN
79
83
PFleC


1620
P50052|AGTR2_HUMAN
315
319
PFlyC


1621
P50876|UB7I4_HUMAN
273
277
PFvlC


1622
P51606|RENBP_HUMAN
376
380
PFkgC


1623
P51617|IRAK1_HUMAN
195
199
PFpfC


1624
P51689|ARSD_HUMAN
581
585
PFcsC


1625
P51690|ARSE_HUMAN
576
580
PFplC


1626
P52740|ZN132_HUMAN
369
373
PFecC


1627
P52747|ZN143_HUMAN
318
322
PFegC


1628
P53420|CO4A4_HUMAN
1521
1525
PFayC


1629
P53420|CO4A4_HUMAN
1630
1634
PFleC


1630
P53621|COPA|HUMAN
1165
1169
PFdiC


1631
P54198|HIRA_HUMAN
215
219
PFdeC


1632
P54793|ARSF_HUMAN
570
574
PFclC


1633
P54802|ANAG_HUMAN
401
405
PFiwC


1634
P55157|MTP_HUMAN
823
827
PFlvC


1635
P62079|TSN5_HUMAN
183
187
PFscC


1636
P78357|CNTP1_HUMAN
926
930
PFvgC


1637
P78527|PRKDC_HUMAN
2853
2857
PFvsC


1638
P81133|SIM1_HUMAN
200
204
PFdgC


1639
P98088|MUC5A_HUMAN
290
294
PFkmC


1640
Q01955|CO4A3_HUMAN
1501
1505
PFlfC


1641
Q01955|CO4A3_HUMAN
1612
1616
PFleC


1642
Q02817|MUC2_HUMAN
597
601
PFgrC


1643
Q02817|MUC2_HUMAN
1375
1379
PFglC


1644
Q02817|MUC2_HUMAN
4916
4920
PFywC


1645
Q03395|ROM1_HUMAN
213
217
PFscC


1646
Q07912|ACK1_HUMAN
293
297
PFawC


1647
Q12830|BPTF_HUMAN
2873
2877
PFyqC


1648
Q12836|ZP4_HUMAN
238
242
PFtsC


1649
Q12866|MERTK_HUMAN
313
317
PFrnC


1650
Q12950|FOXD4_HUMAN
291
295
PFpcC


1651
Q12968|NFAC3_HUMAN
327
331
PFqyC


1652
Q13191|CBLB_HUMAN
409
413
PFcrC


1653
Q13258|PD2R_HUMAN
4
8
PFyrC


1654
Q13356|PPIL2_HUMAN
38
42
PFdhC


1655
Q13607|OR2F1_HUMAN
93
97
PFqsC


1656
Q13753|LAMC2_HUMAN
409
413
PFgtC


1657
Q13936|CAC1C_HUMAN
2179
2183
PFvnC


1658
Q14031|CO4A6_HUMAN
1523
1527
PFiyC


1659
Q14031|CO4A6_HUMAN
1632
1636
PFieC


1660
Q14137|BOP1_HUMAN
400
404
PFptC


1661
Q14330|GPR18_HUMAN
247
251
PFhiC


1662
Q14643|ITPR1_HUMAN
526
530
PFtdC


1663
Q15042|RB3GP_HUMAN
267
271
PFgaC


1664
Q15389|ANGP1_HUMAN
282
286
PFrdC


1665
Q15583|TGIF_HUMAN
269
273
PFhsC


1666
Q15583|TGIF_HUMAN
314
318
PFslC


1667
Q15761|NPY5R_HUMAN
128
132
PFlqC


1668
Q15915|ZIC1_HUMAN
305
309
PFpgC


1669
Q16363|LAMA4_HUMAN
1788
1792
PFtgC


1670
Q16572|VACHT_HUMAN
517
521
PFdeC


1671
Q16586|SGCA_HUMAN
205
209
PFstC


1672
Q16773|KAT1_HUMAN
123
127
PFfdC


1673
Q16878|CDO1_HUMAN
160
164
PFdtC


1674
Q2TBC4|CF049_HUMAN
298
302
PFstC


1675
Q49AM1|MTER3_HUMAN
28
32
PFlaC


1676
Q53FE4|CD017_HUMAN
77
81
PFanC


1677
Q53G59|KLH12_HUMAN
240
244
PFirC


1678
Q53T03|RBP22_HUMAN
517
521
PFpvC


1679
Q5IJ48|CRUM2_HUMAN
762
766
PFrgC


1680
Q5T442|CXA12_HUMAN
241
245
PFfpC


1681
Q5VYX0|RENAL_HUMAN
310
314
PFlaC


1682
Q5W0N0|CI057_HUMAN
89
93
PFhgC


1683
Q6NSW7|NANP8_HUMAN
239
243
PFynC


1684
Q6P2Q9|PRP8_HUMAN
1892
1896
PFqaC


1685
Q6PRD1|GP179_HUMAN
232
236
PFleC


1686
Q6TCH4|PAQR6_HUMAN
95
99
PFasC


1687
Q6UB98|ANR12_HUMAN
1949
1953
PFsaC


1688
Q6UB99|ANR11_HUMAN
2552
2556
PFsaC


1689
Q6UXZ4|UNC5D_HUMAN
766
770
PFtaC


1690
Q7Z434|MAVS_HUMAN
431
435
PFsgC


1691
Q7Z6J6|FRMD5_HUMAN
87
91
PFtmC


1692
Q7Z7G8|VP13B_HUMAN
441
445
PFfdC


1693
Q7Z7G8|VP13B_HUMAN
1423
1427
PFrnC


1694
Q7Z7M1|GP144_HUMAN
352
356
PFlcC


1695
Q86SJ6|DSG4_HUMAN
523
527
PFtfC


1696
Q86SQ6|GP123_HUMAN
863
867
PFiiC


1697
Q86T65|DAAM2_HUMAN
548
552
PFacC


1698
Q86V97|KBTB6_HUMAN
355
359
PFlcC


1699
Q86XI2|CNDG2_HUMAN
1043
1047
PFsrC


1700
Q86YT6|MIB1_HUMAN
909
913
PFimC


1701
Q8IUH2|CREG2_HUMAN
152
156
PFgnC


1702
Q8IWU5|SULF2_HUMAN
745
749
PFcaC


1703
Q8IWV8|UBR2_HUMAN
1514
1518
PFlkC


1704
Q8IWX5|SGPP2_HUMAN
257
261
PFflC


1705
Q8IX07|FOG1_HUMAN
293
297
PFpqC


1706
Q8IX29|FBX16_HUMAN
287
291
PFplC


1707
Q8IXT2|DMRTD_HUMAN
224
228
PFttC


1708
Q8IZF5|GP113_HUMAN
62
66
PFpaC


1709
Q8IZQ8|MYCD_HUMAN
403
407
PFqdC


1710
Q8IZW8|TENS4_HUMAN
423
427
PFttC


1711
Q8N0W3|FUK_HUMAN
100
104
PFddC


1712
Q8N122|RPTOR_HUMAN
1033
1037
PFtpC


1713
Q8N1G1|REXO1_HUMAN
278
282
PFgsC


1714
Q8N1G2|K0082_HUMAN
790
794
PFhiC


1715
Q8N201|INT1_HUMAN
1573
1577
PFpaC


1716
Q8N475|FSTL5_HUMAN
61
65
PFgsC


1717
Q8N567|ZCHC9_HUMAN
182
186
PFakC


1718
Q8N7R0|NANG2_HUMAN
166
170
PFynC


1719
Q8N8U9|BMPER_HUMAN
234
238
PFgsC


1720
Q8N9L1|ZIC4_HUMAN
207
211
PFpgC


1721
Q8NB16|MLKL_HUMAN
411
415
PFqgC


1722
Q8NG11|TSN14_HUMAN
183
187
PFscC


1723
Q8NGC3|O10G2_HUMAN
98
102
PFggC


1724
Q8NGC4|O10G3_HUMAN
94
98
PFggC


1725
Q8NGJ1|OR4D6_HUMAN
165
169
PFpfC


1726
Q8NH69|OR5W2_HUMAN
93
97
PFygC


1727
Q8NH85|OR5R1_HUMAN
93
97
PFhaC


1728
Q8NHU2|CT026_HUMAN
442
446
PFntC


1729
Q8NHY3|GA2L2_HUMAN
359
363
PFlrC


1730
Q8N151|BORIS_HUMAN
369
373
PFqcC


1731
Q8TCB0|IFI44_HUMAN
246
250
PFilC


1732
Q8TCE9|PPL13_HUMAN
88
92
PFelC


1733
Q8TCT7|PSL1_HUMAN
275
279
PFgkC


1734
Q8TD94|KLF14_HUMAN
198
202
PFpgC


1735
Q8TF76|HASP_HUMAN
474
478
PFshC


1736
Q8WW14|CJ082_HUMAN
22
26
PFlsC


1737
Q8WW38|FOG2_HUMAN
299
303
PFpqC


1738
Q8WWG1|NRG4_HUMAN
32
36
PFcrC


1739
Q8WWZ7|ABCA5_HUMAN
361
365
PFchC


1740
Q8WXT5|FX4L4_HUMAN
295
299
PFpcC


1741
Q8WYR1|PI3R5_HUMAN
814
818
PFavC


1742
Q8WZ42|TITIN_HUMAN
31091
31095
PFpiC


1743
Q8WZ60|KLHL6_HUMAN
432
436
PFhnC


1744
Q92485|ASM3B_HUMAN
41
45
PFqvC


1745
Q92793|CBP_HUMAN
1279
1283
PFvdC


1746
Q92838|EDA_HUMAN
328
332
PFlqC


1747
Q92995|UBP13_HUMAN
540
544
PFsaC


1748
Q93008|USP9X_HUMAN
251
255
PFgqC


1749
Q96F10|SAT2_HUMAN
50
54
PFyhC


1750
Q96FV3|TSN17_HUMAN
185
189
PFscC


1751
Q96IK0|TM101_HUMAN
27
31
PFwgC


1752
Q96L50|LLR1_HUMAN
344
348
PFhlC


1753
Q96L73|NSD1_HUMAN
456
460
PFedC


1754
Q96P88|GNRR2_HUMAN
184
188
PFtqC


1755
Q96PZ7|CSMD1_HUMAN
2139
2143
PFprC


1756
Q96R06|SPAG5_HUMAN
378
382
PFstC


1757
Q96RG2|PASK_HUMAN
542
546
PFasC


1758
Q96RJ0|TAAR1_HUMAN
266
270
PFfiC


1759
Q96RQ9|OXLA_HUMAN
32
36
PFekC


1760
Q96SE7|ZN347_HUMAN
798
802
PFsiC


1761
Q96T25|ZIC5_HUMAN
470
474
PFpgC


1762
Q99666|RGPD8_HUMAN
517
521
PFpvC


1763
Q99698|LYST_HUMAN
254
258
PFdlC


1764
Q99726|ZNT3_HUMAN
51
55
PFhhC


1765
Q9BSE5|SPEB_HUMAN
204
208
PFrrC


1766
Q9BWQ6|YIPF2_HUMAN
124
128
PFwiC


1767
Q9BXC9|BBS2_HUMAN
530
534
PFqvC


1768
Q9BXJ4|C1QT3_HUMAN
18
22
PFclC


1769
Q9BXK1|KLF16_HUMAN
130
134
PFpdC


1770
Q9BZE2|PUS3_HUMAN
261
265
PFqlC


1771
Q9C0C4|SEM4C_HUMAN
719
723
PFrpC


1772
Q9C0E2|XPO4_HUMAN
50
54
PFavC


1773
Q9C0I4|THS7B_HUMAN
1482
1486
PFsyC


1774
Q9GZN6|S6A16_HUMAN
271
275
PFflC


1775
Q9GZU2|PEG3_HUMAN
1330
1334
PFyeC


1776
Q9GZZ0|HXD1_HUMAN
162
166
PFpaC


1777
Q9H0A6|RNF32_HUMAN
344
348
PFhaC


1778
Q9H0B3|K1683_HUMAN
326
330
PFqiC


1779
Q9H267|VP33B_HUMAN
189
193
PFpnC


1780
Q9H2J1|CI037_HUMAN
102
106
PFekC


1781
Q9H3H5|GPT_HUMAN
77
81
PFlnC


1782
Q9H8V3|ECT2_HUMAN
239
243
PFqdC


1783
Q9H9S0|NANOG_HUMAN
239
243
PFynC


1784
Q9H9V4|RN122_HUMAN
3
7
PFqwC


1785
Q9HAQ2|KIF9_HUMAN
291
295
PFrqC


1786
Q9HAW7|UD17_HUMAN
149
153
PFdaC


1787
Q9HAW8|UD110_HUMAN
149
153
PFdtC


1788
Q9HAW9|UD18_HUMAN
149
153
PFdaC


1789
Q9HBX8|LGR6_HUMAN
412
416
PFkpC


1790
Q9NQW8|CNGB3_HUMAN
309
313
PFdiC


1791
Q9NRZ9|HELLS_HUMAN
273
277
PFlvC


1792
Q9NTG7|SIRT3_HUMAN
30
34
PFqaC


1793
Q9NWZ5|UCKL1_HUMAN
370
374
PFqdC


1794
Q9NY30|BTG4_HUMAN
98
102
PFevC


1795
Q9NYM4|GPR83_HUMAN
342
346
PFiyC


1796
Q9NYV6|RRN3_HUMAN
561
565
PFdpC


1797
Q9NYW1|TA2R9_HUMAN
190
194
PFilC


1798
Q9NYW3|TA2R7_HUMAN
193
197
PFcvC


1799
Q9NZ56|FMN2_HUMAN
716
720
PFsdC


1800
Q9NZ71|RTEL1_HUMAN
495
499
PFpvC


1801
Q9NZD2|GLTP_HUMAN
31
35
PFfdC


1802
Q9P2N4|ATS9_HUMAN
596
600
PFgtC


1803
Q9UBR1|BUP1_HUMAN
124
128
PFafC


1804
Q9UBS0|KS6B2_HUMAN
344
348
PFrpC


1805
Q9UET6|RRMJ1_HUMAN
234
238
PFvtC


1806
Q9UHD4|CIDEB_HUMAN
37
41
PFrvC


1807
Q9UKA4|AKA11_HUMAN
917
921
PFshC


1808
Q9ULC3|RAB23_HUMAN
230
234
PFssC


1809
Q9ULJ3|ZN295_HUMAN
125
129
PFptC


1810
Q9ULK4|CRSP3_HUMAN
1086
1090
PFpnC


1811
Q9ULL4|PLXB3_HUMAN
24
28
PFglC


1812
Q9ULV8|CBLC_HUMAN
387
391
PFcrC


1813
Q9UM47|NOTC3_HUMAN
1357
1361
PFfrC


1814
Q9UNQ2|DIMT1_HUMAN
146
150
PFfrC


1815
Q9Y3D5|RT18C_HUMAN
86
90
PFtgC


1816
Q9Y3F1|TA6P_HUMAN
25
29
PFpsC


1817
Q9Y3R5|CU005_HUMAN
255
259
PFytC


1818
Q9Y450|HBS1L_HUMAN
487
491
PFrlC


1819
Q9Y493|ZAN_HUMAN
1364
1368
PFetC


1820
Q9Y493|ZAN_HUMAN
1751
1755
PFsqC


1821
Q9Y493|ZAN_HUMAN
2556
2560
PFaaC


1822
Q9Y548|YIPF1_HUMAN
123
127
PFwiC


1823
Q9Y5L3|ENP2_HUMAN
324
328
PFsrC


1824
Q9Y5P8|2ACC_HUMAN
272
276
PFqdC


1825
Q9Y664|KPTN_HUMAN
143
147
PFqlC


1826
Q9Y678|COPG_HUMAN
226
230
PFayC


1827
Q9Y6E0|STK24_HUMAN
371
375
PFsqC


1828
Q9Y6R7|FCGBP_HUMAN
683
687
PFavC


1829
Q9Y6R7|FCGBP_HUMAN
1074
1078
PFreC


1830
Q9Y6R7|FCGBP_HUMAN
1888
1892
PFttC


1831
Q9Y6R7|FCGBP_HUMAN
3089
3093
PFttC


1832
Q9Y6R7|FCGBP_HUMAN
4290
4294
PFttC


1833
Q9Y6R7|FCGBP_HUMAN
5059
5063
PFatC









Finally the motifs that are found within the predicted peptides that are derived from tissue inhibitors of metalloproteinases were calculated. Because of the small number of peptides present in the peptide pool the loop-6 fragment of TIMP-2 was also included in the calculation. This loop is known to have anti-angiogenic activity. For this case the common motif among the peptide sequences is the E-C-L-W-X-D-X8-G-X-Y-X5-C as shown in the FIG. 9.
Example 5
Novel Peptides from the Somatotropin and Serpin Protein Families
Growth Hormone (GH) and prolactin proteins contain a somatotropin conserved domain. Pigment epithelium derived factor (PEDF) contains a serpin conserved domain. There are a number of short peptides, smaller than 25 amino acids, from these two protein families that can be used to identify sequences having similarity to these peptides within the human proteome. Such peptides include the recently identified short fragments of GH and prolactin (Nguyen et al., (2006) Proc Natl Acad Sci USA 103, 14319-14324), and short fragments of PEDF (Filleur et al., (2005) Cancer Res 65, 5144-5152).
After searching within the human proteome for similar sequences to those of the short peptides and filtering the results for only the statistically significant similarities using a Monte Carlo algorithm eleven novel similar peptides were identified, eight similar to the short fragment derived from growth hormone (FIG. 10A) and three from the short fragment of PEDF (FIG. 10B). These sequences are also included in Tables 7A and 7B.







TABLE 7A







Table of the amino acid sequences of the peptides


predicted similar to Growth Hormone









Protein Name
Peptide Location
Peptide sequence





Placental Lactogen
AAA98621(101-114)
LLRISLLLIESWLE


 


hGH-V
AAB59548(101-114)
LLRISLLLTQSWLE


 


GH2
CAG46722(101-114)
LLHISLLLIQSWLE


 


Chorionic somatomammotropin
AAA52116(101-113)
LLRLLLLIESWLE


 


Chorionic somatomammotropin hormone-like 1
AAI19748(12-25)
LLHISLLLIESRLE


 


Transmembrane protein 45A
NP_060474(181-194)
LLRSSLILLQGSWF


 


IL-17 receptor C
Q8NAC3(376-387)
RLRLLTLQSWLL


 


Neuropeptide FF receptor 2
Q9Y5X5(378-390)
LLIVALLFILSWL


 


Brush border myosin-I
AAC27437(719-731)
LMRKSQILISSWF
















TABLE 7B







Table of the amino acid sequences of the


peptides predicted similar to PEDF.











Peptide


Protein Name
Peptide Location
sequence





DEAH box polypeptide
AAH47327(438-448)
EIELVEEEPPF


8




 


Caspase 10
CAD32371(67-77)
AEDLLSEEDPF


 


CKIP-1
CAI14263(66-76)
TLDLIQEEDPS









Example 7
Identification of Motifs within the Somatotropin Derived Peptides
By performing multiple sequence alignment to the sequences of the predicted peptides we can investigate the conservation of specific motifs that are common in most of the sequences. Multiple sequence alignment is performed using the ClustalW algorithm. In order to identify a more robust motif within the peptide sequences, in the case of the somatotropin derived peptides, the lowest similarity hits can be excluded to identify the common amino acids. This process identifies the somatotropin common motif: L-X(3)-L-L-X(3)-S-X-L (FIG. 11).
In order to identify the existence of this motif in other protein sequences in the human proteome, the ScanProsite tool was used to search the Prosite database at the Swiss Institute of Bioinformatics. Using the aforementioned motif as a query this motif was identified in 139 locations of 139 different proteins listed in Table 8 (SEQ ID Nos: 1834-1972).







TABLE 8







Amino acid sequences of peptides that contain the somatotropin motif.


Somatotropins


Motif: L-X(3)-L-L-X(3)-S-X-L


Number of Locations: 139


Number of Different Proteins: 139













First






Ami-
Last



Accession
no
Amino


#
Number|Protein Name
acid
acid
Sequence














1834
O14569|C56D2_HUMAN
164
175
LvgyLLgsaSlL


1835
O15287|FANCG_HUMAN
416
427
LceeLLsrtSsL


1836
O15482|TEX28_HUMAN
338
349
LatvLLvfvStL


1837
O43914|TYOBP_HUMAN
11
22
LllpLLlavSgL


1838
O60609|GFRA3_HUMAN
15
26
LmllLLlppSpL


1839
O75844|FACE1_HUMAN
279
290
LfdtLLeeySvL


1840
O95747|OXSR1_HUMAN
90
101
LvmkLLsggSvL


1841
P01241|SOMA_HUMAN
102
113
LrisLLliqSwL


1842
P01242|SOM2_HUMAN
102
113
LrisLLliqSwL


1843
P01243|CSH_HUMAN
102
113
LrisLLlieSwL


1844
P02750|A2GL_HUMAN
83
94
LpanLLqgaSkL


1845
P03891|NU2M_HUMAN
149
160
LnvsLLltlSiL


1846
P04201|MAS_HUMAN
151
162
LvcaLLwalScL


1847
P05783|K1C18_HUMAN
338
349
LngiLLhleSeL


1848
P07359|GP1BA_HUMAN
3
14
LlllLLllpSpL


1849
P09848|LPH_HUMAN
35
46
LtndLLhnlSgL


1850
P11168|GTR2_HUMAN
136
147
LvgaLLmgfSkL


1851
P12034|FGF5_HUMAN
3
14
LsflLLlffShL


1852
P13489|RINI_HUMAN
247
258
LcpgLLhpsSrL


1853
P14902|I23O_HUMAN
196
207
LlkaLLeiaScL


1854
P16278|BGAL_HUMAN
135
146
LpawLLekeSiL


1855
P19838|NFKB1_HUMAN
558
569
LvrdLLevtSgL


1856
P22079|PERL_HUMAN
512
523
LvrgLLakkSkL


1857
P23276|KELL_HUMAN
53
64
LilgLLlcfSvL


1858
P24394|IL4RA_HUMAN
4
15
LcsgLLfpvScL


1859
P29320|EPHA3_HUMAN
5
16
LsilLLlscSvL


1860
P31512|FMO4_HUMAN
524
535
LaslLLickSsL


1861
P35270|SPRE_HUMAN
26
37
LlasLLspgSvL


1862
P41250|SYG_HUMAN
20
31
LpprLLarpSlL


1863
P42575|CASP2_HUMAN
114
125
LedmLLttlSgL


1864
P46721|SO1A2_HUMAN
396
407
LleyLLyflSfL


1865
P51665|PSD7_HUMAN
201
212
LnskLLdirSyL


1866
P59531|T2R12_HUMAN
188
199
LisfLLsliSlL


1867
P69849|NOMO3_HUMAN
1180
1191
LiplLLqltSrL


1868
P98161|PKD1_HUMAN
82
93
LdvgLLanlSaL


1869
P98171|RHG04_HUMAN
153
164
LqdeLLevvSeL


1870
P98196|AT11A_HUMAN
1077
1088
LaivLLvtiSlL


1871
Q08431|MFGM_HUMAN
10
21
LcgaLLcapSlL


1872
Q08AF3|SLFN5_HUMAN
533
544
LvivLLgfkSfL


1873
Q12952|FOXL1_HUMAN
293
304
LgasLLaasSsL


1874
Q13275|SEM3F_HUMAN
2
13
LvagLLlwaSlL


1875
Q13394|MB211_HUMAN
300
311
LngiLLqliScL


1876
Q13609|DNSL3_HUMAN
8
19
LlllLLsihSaL


1877
Q13619|CUL4A_HUMAN
213
224
LlrsLLgmlSdL


1878
Q13620|CUL4B_HUMAN
349
360
LlrsLLsmlSdL


1879
Q14406|CSHL_HUMAN
84
95
LhisLLlieSrL


1880
Q14667|K0100_HUMAN
8
19
LlvlLLvalSaL


1881
Q15155|NOMO1_HUMAN
1180
1191
LiplLLqltSrL


1882
Q15760|GPR19_HUMAN
279
290
LilnLLfllSwL


1883
Q53RE8|ANR39_HUMAN
166
177
LacdLLpcnSdL


1884
Q5FWE3|PRRT3_HUMAN
586
597
LatdLLstwSvL


1885
Q5GH73|XKR6_HUMAN
630
641
LlyeLLqyeSsL


1886
Q5GH77|XKR3_HUMAN
194
205
LnraLLmtfSlL


1887
Q5JPE7|NOMO2_HUMAN
1180
1191
LiplLLqltSrL


1888
Q5JWR5|DOP1_HUMAN
506
517
LpqlLLrmiSaL


1889
Q5UIP0|RIF1_HUMAN
2413
2424
LsknLLaqiSaL


1890
Q5VTE6|ANGE2_HUMAN
175
186
LsqdLLednShL


1891
Q5VU43|MYOME_HUMAN
1932
1943
LreaLLssrShL


1892
Q5VYK3|ECM29_HUMAN
1296
1307
LipaLLeslSvL


1893
Q68D06|SLN13_HUMAN
554
565
LvivLLgfrSlL


1894
Q6GYQ0|GRIPE_HUMAN
641
652
LwddLLsvlSsL


1895
Q6NTF9|RHBD2_HUMAN
166
177
LvpwLLlgaSwL


1896
Q6ZMH5|S39A5_HUMAN
217
228
LavlLLslpSpL


1897
Q6ZMZ3|SYNE3_HUMAN
532
543
LhnsLLqrkSkL


1898
Q6ZVD8|PHLPL_HUMAN
313
324
LfpiLLceiStL


1899
Q6ZVE7|GOT1A_HUMAN
23
34
LfgtLLyfdSvL


1900
Q70J99|UN13D_HUMAN
927
938
LrveLLsasSlL


1901
Q7Z3Z4|PIWL4_HUMAN
139
150
LriaLLyshSeL


1902
Q7Z6Z7|HUWE1_HUMAN
841
852
LqegLLqldSiL


1903
Q7Z7L1|SLN11_HUMAN
554
565
LvivLLgfrSlL


1904
Q86SM5|MRGRG_HUMAN
223
234
LlnfLLpvfSpL


1905
Q86U44|MTA70_HUMAN
78
89
LekkLLhhlSdL


1906
Q86UQ4|ABCAD_HUMAN
3182
3193
LlnsLLdivSsL


1907
Q86WI3|NLRC5_HUMAN
1485
1496
LlqsLLlslSeL


1908
Q86YC3|LRC33_HUMAN
263
274
LffpLLpqySkL


1909
Q8IYK4|GT252_HUMAN
9
20
LawsLLllsSaL


1910
Q8IYS0|GRM1C_HUMAN
485
496
LesdLLieeSvL


1911
Q8IZL8|PELP1_HUMAN
33
44
LrllLLesvSgL


1912
Q8IZY2|ABCA7_HUMAN
1746
1757
LftlLLqhrSqL


1913
Q8N0X7|SPG20_HUMAN
322
333
LfedLLrqmSdL


1914
Q8N6M3|CT142_HUMAN
33
44
LagsLLkelSpL


1915
Q8N816|TMM99_HUMAN
96
107
LlpcLLgvgSwL


1916
Q8NBM4|PDHL1_HUMAN
15
26
LsksLLlvpSaL


1917
Q8NCG7|DGLB_HUMAN
555
566
LtqpLLgeqSlL


1918
Q8NFR9|I17RE_HUMAN
80
91
LcqhLLsggSgL


1919
Q8NGE3|O10P1_HUMAN
9
20
LpefLLlgfSdL


1920
Q8TCV5|WFDC5_HUMAN
8
19
LlgaLLavgSqL


1921
Q8TDL5|LPLC1_HUMAN
165
176
LriqLLhklSfL


1922
Q8TE82|S3TC1_HUMAN
1025
1036
LegqLLetiSqL


1923
Q8TEQ8|PIGO_HUMAN
857
868
LvflLLflqSfL


1924
Q8TEZ7|MPRB_HUMAN
127
138
LlahLLqskSeL


1925
Q8WWN8|CEND3_HUMAN
1481
1492
LeeqLLqelSsL


1926
Q8WZ84|OR8D1_HUMAN
43
54
LgmiLLiavSpL


1927
Q92535|PIGC_HUMAN
253
264
LfalLLmsiScL


1928
Q92538|GBF1_HUMAN
1224
1235
LrilLLmkpSvL


1929
Q92743|HTRA1_HUMAN
262
273
LpvlLLgrsSeL


1930
Q92935|EXTL1_HUMAN
19
30
LllvLLggfSlL


1931
Q93074|MED12_HUMAN
401
412
LqtiLLccpSaL


1932
Q96DN6|MBD6_HUMAN
740
751
LgasLLgdlSsL


1933
Q96GR4|ZDH12_HUMAN
48
59
LfflLLvlgSlL


1934
Q96HP8|T176A_HUMAN
29
40
LaklLLtccSaL


1935
Q96K12|FACR2_HUMAN
380
391
LmnrLLrtvSmL


1936
Q96KP1|EXOC2_HUMAN
339
350
LldkLLetpStL


1937
Q96MX0|CKLF3_HUMAN
40
51
LkgrLLlaeSgL


1938
Q96Q45|AL2S4_HUMAN
387
398
LvvaLLvglSwL


1939
Q96QZ0|PANX3_HUMAN
136
147
LssdLLfiiSeL


1940
Q96RQ9|OXLA_HUMAN
269
280
LpraLLsslSgL


1941
Q9BY08|EBPL_HUMAN
178
189
LipgLLlwqSwL


1942
Q9BZ97|TTY13_HUMAN
30
41
LclmLLlagScL


1943
Q9H1Y0|ATG5_HUMAN
85
96
LlfdLLassSaL


1944
Q9H254|SPTN4_HUMAN
1422
1433
LdkkLLhmeSqL


1945
Q9H330|CI005_HUMAN
430
441
LgkfLLkvdSkL


1946
Q9H4I8|SEHL2_HUMAN
175
186
LlqrLLksnShL


1947
Q9HCN3|TMEM8_HUMAN
200
211
LpqtLLshpSyL


1948
Q9NQ34|TMM9B_HUMAN
4
15
LwggLLrlgSlL


1949
Q9NR09|BIRC6_HUMAN
1400
1411
LlkaLLdnmSfL


1950
Q9NRA0|SPHK2_HUMAN
296
307
LgldLLlncSlL


1951
Q9NRU3|CNNM1_HUMAN
156
167
LgalLLlalSaL


1952
Q9NTT1|U2D3L_HUMAN
99
110
LskvLLsicSlL


1953
Q9NVH2|INT7_HUMAN
623
634
LridLLqafSqL


1954
Q9NVM9|CL011_HUMAN
350
361
LtnfLLngrSvL


1955
Q9NZD1|GPC5D_HUMAN
60
71
LptqLLfllSvL


1956
Q9P2E9|RRBP1_HUMAN
1226
1237
LrqlLLesqSqL


1957
Q9P2G4|K1383_HUMAN
397
408
LlnaLLvelSlL


1958
Q9P2V4|LRIT1_HUMAN
541
552
LpltLLvccSaL


1959
Q9UDY8|MALT1_HUMAN
33
44
LrepLLrrlSeL


1960
Q9UEW8|STK39_HUMAN
138
149
LvmkLLsggSmL


1961
Q9UGN4|CM35H_HUMAN
188
199
LlllLLvgaSlL


1962
Q9UHD4|CIDEB_HUMAN
189
200
LghmLLgisStL


1963
Q9UIG8|SO3A1_HUMAN
270
281
LcgaLLffsSlL


1964
Q9UPA5|BSN_HUMAN
353
364
LgasLLtqaStL


1965
Q9UPX8|SHAN2_HUMAN
609
620
LtgrLLdpsSpL


1966
Q9Y239|NOD1_HUMAN
318
329
LsgkLLkgaSkL


1967
Q9Y2I2|NTNG1_HUMAN
526
537
LlttLLgtaSpL


1968
Q9Y2U2|KCNK7_HUMAN
92
103
LpsaLLfaaSiL


1969
Q9Y2Y8|PRG3_HUMAN
7
18
LpflLLgtvSaL


1970
Q9Y586|MB212_HUMAN
300
311
LngiLLqliScL


1971
Q9Y5X0|SNX10_HUMAN
106
117
LqnaLLlsdSsL


1972
Q9Y5X5|NPFF2_HUMAN
379
390
LivaLLfilSwL









Example 8
Identification of Motifs within the Serpin Derived Peptides
The L-X(2)-E-E-X-P motif of serpin derived peptides identified the sequences of peptides shown in FIG. 12. Using the ScanProsite tool 314 hits in 302 different proteins were identified. The hits are shown in Table 9 (SEQ ID Nos: 1973-2286).







TABLE 9







Table of the amino acid sequences of the peptides identified to contain


the serpin motif.


Serpins


Motif: L-X(2)-E-E-X-P


Number of Locations: 314


Number of Different Proteins: 302













First
Last




Accession
Amino
Amino


#
Number|Protein Name
acid
acid
Sequence














1973
O00160|MYO1F_HUMAN
744
751
LglEErPe


1974
O00507|USP9Y_HUMAN
2474
2481
LcpEEePd


1975
O00625|PIR_HUMAN
134
141
LksEEiPk


1976
O14641|DVL2_HUMAN
20
27
LdeEEtPy


1977
O14686|MLL2_HUMAN
2819
2826
LgpEErPp


1978
O14709|ZN197_HUMAN
193
200
LsqEEnPr


1979
O14795|UN13B_HUMAN
1499
1506
LgnEEgPe


1980
O15013|ARHGA_HUMAN
199
206
LssEEpPt


1981
O15055|PER2_HUMAN
994
1001
LqlEEaPe


1982
O15528|CP27B_HUMAN
297
304
LfrEElPa


1983
O15534|PER1_HUMAN
987
994
LqlEElPr


1984
O43390|HNRPR_HUMAN
12
19
LkeEEePm


1985
O60216|RAD21_HUMAN
504
511
LppEEpPn


1986
O60237|MYPT2_HUMAN
339
346
LyeEEtPk


1987
O60346|PHLPP_HUMAN
483
490
LeaEEkPI


1988
O60779|S19A2_HUMAN
259
266
LnmEEpPv


1989
O60885|BRD4_HUMAN
913
920
LedEEpPa


1990
O75128|COBL_HUMAN
1064
1071
LerEEkPs


1991
O75420|PERQ1_HUMAN
334
341
LeeEEePs


1992
O75787|RENR_HUMAN
116
123
LfsEEtPv


1993
O75914|PAK3_HUMAN
5
12
LdnEEkPp


1994
O94933|SLIK3_HUMAN
227
234
LqlEEnPw


1995
O94966|UBP19_HUMAN
1251
1258
LeaEEePv


1996
O94986|CE152_HUMAN
847
854
LknEEvPv


1997
O94991|SLIK5_HUMAN
230
237
LqlEEnPw


1998
O95153|RIMB1_HUMAN
915
922
LngEEcPp


1999
O95279|KCNK5_HUMAN
443
450
LagEEsPq


2000
O95712|PA24B_HUMAN
772
779
LkiEEpPs


2001
O95881|TXD12_HUMAN
94
101
LedEEePk


2002
O96018|APBA3_HUMAN
116
123
LhcEEcPp


2003
O96024|B3GT4_HUMAN
217
224
LhsEEvPl


2004
P04275|VWF_HUMAN
1012
1019
LqvEEdPv


2005
P05160|F13B_HUMAN
18
25
LyaEEkPc


2006
P06858|LIPL_HUMAN
279
286
LlnEEnPs


2007
P07237|PDIA1_HUMAN
307
314
LkkEEcPa


2008
P07949|RET_HUMAN
1033
1040
LseEEtPl


2009
P08519|APOA_HUMAN
3880
3887
LpsEEaPt


2010
P09769|FGR_HUMAN
497
504
LdpEErPt


2011
P10745|IRBP_HUMAN
708
715
LvvEEaPp


2012
P11532|DMD_HUMAN
2255
2262
LlvEElPl


2013
P14317|HCLS1_HUMAN
352
359
LqvEEePv


2014
P16150|LEUK_HUMAN
369
376
LkgEEePl


2015
P17025|ZN182_HUMAN
79
86
LevEEcPa


2016
P17600|SYN1_HUMAN
239
246
LgtEEfPl


2017
P18583|SON_HUMAN
1149
1156
LppEEpPt


2018
P18583|SON_HUMAN
1160
1167
LppEEpPm


2019
P18583|SON_HUMAN
1171
1178
LppEEpPe


2020
P19484|TFEB_HUMAN
350
357
LpsEEgPg


2021
P21333|FLNA_HUMAN
1034
1041
LprEEgPy


2022
P21802|FGFR2_HUMAN
33
40
LepEEpPt


2023
P22001|KCNA3_HUMAN
152
159
LreEErPl


2024
P31629|ZEP2_FTUMAN
772
779
LvsEEsPs


2025
P34925|RYK_HUMAN
578
585
LdpEErPk


2026
P36955|PEDF_HUMAN
39
46
LveEEdPf


2027
P40189|IL6RB_HUMAN
787
794
LdsEErPe


2028
P42898|MTHR_HUMAN
598
605
LyeEEsPs


2029
P48729|KC1A_HUMAN
266
273
LrfEEaPd


2030
P51512|MMP16_HUMAN
165
172
LtfEEvPy


2031
P52746|ZN142_HUMAN
750
757
LgaEEnPl


2032
P53370|NUDT6_HUMAN
284
291
LtvEElPa


2033
P53801|PTTG_HUMAN
167
174
LfkEEnPy


2034
P53804|TTC3_HUMAN
2001
2008
LltEEsPs


2035
P55285|CADH6_HUMAN
116
123
LdrEEkPv


2036
P55289|CAD12_HUMAN
117
124
LdrEEkPf


2037
P56645|PER3_HUMAN
929
936
LlqEEmPr


2038
P59797|SELV_HUMAN
163
170
LlpEEdPe


2039
Q01826|SATB1_HUMAN
409
416
LrkEEdPk


2040
Q04725|TLE2_HUMAN
200
207
LveEErPs


2041
Q06330|SUH_HUMAN
7
14
LpaEEpPa


2042
Q06889|EGR3_HUMAN
24
31
LypEEiPs


2043
Q07157|ZO1_HUMAN
1155
1162
LrhEEqPa


2044
Q13072|BAGE1_HUMAN
19
26
LmkEEsPv


2045
Q13087|PDIA2_HUMAN
497
504
LptEEpPe


2046
Q13255|GRM1_HUMAN
995
1002
LtaEEtPl


2047
Q13315|ATM_HUMAN
954
961
LpgEEyPl


2048
Q13439|GOGA4_HUMAN
2092
2099
LeqEEnPg


2049
Q13596|SNX1_HUMAN
265
272
LekEElPr


2050
Q13634|CAD18_HUMAN
446
453
LdrEEtPw


2051
Q14028|CNGB1_HUMAN
137
144
LmaEEnPp


2052
Q14126|DSG2_HUMAN
117
124
LdrEEtPf


2053
Q14204|DYHC_HUMAN
3973
3980
LwsEEtPa


2054
Q14315|FLNC_HUMAN
1738
1745
LphEEePs


2055
Q14524|SCN5A_HUMAN
46
53
LpeEEaPr


2056
Q14554|PDIA5_HUMAN
166
173
LkkEEkPl


2057
Q14562|DHX8_HUMAN
411
418
LskEEfPd


2058
Q14562|DHX8_HUMAN
441
448
LveEEpPf


2059
Q14573|ITPR3_HUMAN
315
322
LaaEEnPs


2060
Q14674|ESPL1_HUMAN
613
620
LspEEtPa


2061
Q14676|MDC1_HUMAN
145
152
LtvEEtPr


2062
Q14684|RRP1B_HUMAN
244
251
LsaEEiPe


2063
Q15021|CND1_HUMAN
1179
1186
LgvEEePf


2064
Q15735|PI5PA_HUMAN
189
196
LasEEqPp


2065
Q15788|NCOA1_HUMAN
982
989
LimEErPn


2066
Q15878|CAC1E_HUMAN
797
804
LnrEEaPt


2067
Q2TAL6|VWC2_HUMAN
179
186
LctEEgPl


2068
Q32MZ4|LRRF1_HUMAN
82
89
LrvEErPe


2069
Q32P28|P3H1_HUMAN
215
222
LysEEqPq


2070
Q3KNS1|PTHD3_HUMAN
96
103
LpeEEtPe


2071
Q3ZCX4|ZN568_HUMAN
100
107
LeqEEePw


2072
Q495W5|FUT11_HUMAN
144
151
LlhEEsPl


2073
Q52LD8|RFTN2_HUMAN
123
130
LviEEcPl


2074
Q53GL0|PKHO1_HUMAN
189
196
LiqEEdPs


2075
Q53GL0|PKHO1_HUMAN
289
296
LraEEpPt


2076
Q53GL7|PAR10_HUMAN
693
700
LeaEEpPd


2077
Q53H47|SETMR_HUMAN
499
506
LdqEEaPk


2078
Q567U6|CCD93_HUMAN
300
307
LsaEEsPe


2079
Q580R0|CB027_HUMAN
41
48
LelEEaPe


2080
Q587I9|SFT2C_HUMAN
136
143
LrcEEaPs


2081
Q5H9T9|CN155_HUMAN
427
434
LlpEEaPr


2082
Q5H9T9|CN155_HUMAN
697
704
LpaEEtPi


2083
Q5H9T9|CN155_HUMAN
736
743
LltEEfPi


2084
Q5JUK9|GGED1_HUMAN
38
45
LqqEEpPi


2085
Q5JXB2|UE2NL_HUMAN
58
65
LlaEEyPm


2086
Q5MCW4|ZN569_HUMAN
60
67
LeqEEePw


2087
Q5SYB0|FRPD1_HUMAN
553
560
LikEEqPp


2088
Q5THJ4|VP13D_HUMAN
2943
2950
LtgEEiPf


2089
Q5VYS4|CM033_HUMAN
293
300
LesEEtPn


2090
Q5VZP5|DUS27_HUMAN
942
949
LrtEEkPp


2091
Q5VZY2|PPC1A_HUMAN
247
254
LkkEErPt


2092
Q63HR2|TENC1_HUMAN
564
571
LddEEqPt


2093
Q66K74|MAP1S_HUMAN
777
784
LgaEEtPp


2094
Q68CZ1|FTM_HUMAN
1181
1188
LpaEEtPv


2095
Q68DD2|PA24F_HUMAN
470
477
LyqEEnPa


2096
Q6BDS2|URFB1_HUMAN
1304
1311
LedEEiPv


2097
Q6DCA0|AMERL_HUMAN
183
190
LtrEElPk


2098
Q6DN90|IQEC1_HUMAN
263
270
LhtEEaPa


2099
Q6DT37|MRCKG_HUMAN
1264
1271
LvpEElPp


2100
Q6HA08|ASTL_HUMAN
62
69
LilEEtPe


2101
Q6IFS5|HSN2_HUMAN
298
305
LnqEElPp


2102
Q6NUN7|CK063_HUMAN
74
81
LdeEEsPr


2103
Q6P2Q9|PRP8_HUMAN
1852
1859
LpvEEqPk


2104
Q6P5W5|S39A4_HUMAN
473
480
LvaEEsPe


2105
Q6P6B1|CH047_HUMAN
249
256
LgkEEqPq


2106
Q6PD74|P34_HUMAN
141
148
LspEElPe


2107
Q6PI48|SYDM_HUMAN
488
495
LpkEEnPr


2108
Q6PJ61|FBX46_HUMAN
246
253
LrkEErPg


2109
Q6S8J7|POTE8_HUMAN
307
314
LtsEEePq


2110
Q6SZW1|SARM1_HUMAN
396
403
LlgEEvPr


2111
Q6UX39|AMTN_HUMAN
114
121
LssEElPq


2112
Q6ZMY3|SPOC1_HUMAN
184
191
LskEEpPg


2113
Q6ZN11|ZN793_HUMAN
60
67
LeqEEaPw


2114
Q6ZNL6|FGD5_HUMAN
382
389
LraEEnPm


2115
Q6ZV29|PLPL7_HUMAN
854
861
LhrEEgPa


2116
Q70CQ4|UBP31_HUMAN
527
534
LpqEEqPl


2117
Q70SY1|CR3L2_HUMAN
153
160
LekEEpPl


2118
Q7L8C5|SYT13_HUMAN
229
236
LaeEElPt


2119
Q7Z3E5|ARMC9_HUMAN
570
577
LnsEElPd


2120
Q7Z410|TMPS9_HUMAN
691
698
LacEEaPg


2121
Q86SP6|GP149_HUMAN
217
224
LcsEEpPr


2122
Q86V87|RAI16_HUMAN
496
503
LdlEEdPy


2123
Q86VQ0|CF152_HUMAN
428
435
LerEEkPe


2124
Q86W50|MET10_HUMAN
454
461
LsqEEnPe


2125
Q86Y13|DZIP3_HUMAN
1192
1199
LlpEEfPg


2126
Q86Y27|BAGE5_HUMAN
19
26
LmkEEsPv


2127
Q86Y28|BAGE4_HUMAN
19
26
LmkEEsPv


2128
Q86Y29|BAGE3_HUMAN
19
26
LmkEEsPv


2129
Q86Y30|BAGE2_HUMAN
19
26
LmkEEsPv


2130
Q8IU99|FA26C_HUMAN
315
322
LgqEEpPl


2131
Q8IUA0|WFDC8_HUMAN
217
224
LqdEEcPl


2132
Q8IV63|VRK3_HUMAN
438
445
LtyEEkPp


2133
Q8IWY9|CDAN1_HUMAN
948
955
LlpEEtPa


2134
Q8IXI1|MIRO2_HUMAN
24
31
LvgEEfPe


2135
Q8IXI2|MIRO1_HUMAN
24
31
LvsEEfPe


2136
Q8IYS5|OSCAR_HUMAN
122
129
LvtEElPr


2137
Q8IZ26|ZNF34_HUMAN
251
258
LhtEEkPy


2138
Q8IZH2|XRN1_HUMAN
1143
1150
LfdEEfPg


2139
Q8IZP0|ABI1_HUMAN
7
14
LleEEiPs


2140
Q8N201|INT1_HUMAN
1587
1594
LlqEEePl


2141
Q8N309|LRC43_HUMAN
373
380
LlvEEsPe


2142
Q8N3C0|HELC1_HUMAN
451
458
LsfEEkPv


2143
Q8N3C0|HELC1_HUMAN
1579
1586
LatEEdPk


2144
Q8N475|FSTL5_HUMAN
786
793
LkaEEwPw


2145
Q8N4L2|TM55A_HUMAN
132
139
LisEEqPa


2146
Q8N752|KC1AL_HUMAN
266
273
LrfEEvPd


2147
Q8NC74|CT151_HUMAN
178
185
LrgEEkPa


2148
Q8NE71|ABCF1_HUMAN
701
708
LrmEEtPt


2149
Q8NEG5|ZSWM2_HUMAN
43
50
LlrEEePe


2150
Q8NEM7|FA48A_HUMAN
115
122
LdaEElPp


2151
Q8NEZ4|MLL3_HUMAN
3046
3053
LllEEqPl


2152
Q8NEZ4|MLL3_HUMAN
4023
4030
LvkEEpPe


2153
Q8NFM7|I17RD_HUMAN
702
709
LgeEEpPa


2154
Q8NFP4|MDGA1_HUMAN
489
496
LplEEtPd


2155
Q8NHJ6|LIRB4_HUMAN
60
67
LdkEEsPa


2156
Q8NI51|BORIS_HUMAN
120
127
LwlEEgPr


2157
Q8TBH0|ARRD2_HUMAN
387
394
LysEEdPn


2158
Q8TDX9|PK1L1_HUMAN
1101
1108
LsaEEsPg


2159
Q8TE68|ES8L1_HUMAN
408
415
LspEEgPp


2160
Q8TER0|SNED1_HUMAN
1083
1090
LrgEEhPt


2161
Q8WU49|CG033_HUMAN
8
15
LslEEcPw


2162
Q8WUA2|PPIL4_HUMAN
16
23
LytEErPr


2163
Q8WUI4|HDAC7_HUMAN
943
950
LveEEePm


2164
Q8WWN8|CEND3_HUMAN
1456
1463
LgqEErPp


2165
Q8WZ42|TITIN_HUMAN
12132
12139
LvvEElPv


2166
Q8WZ42|TITIN_HUMAN
13832
13839
LfvEEiPv


2167
Q92538|GBF1_HUMAN
1062
1069
LqrEEtPs


2168
Q92738|US6NL_HUMAN
51
58
LheEElPd


2169
Q92765|SFRP3_HUMAN
134
141
LacEElPv


2170
Q92851|CASPA_HUMAN
70
77
LlsEEdPf


2171
Q92888|ARHG1_HUMAN
390
397
LepEEpPg


2172
Q93008|USP9X_HUMAN
2466
2473
LcpEEePd


2173
Q969V6|MKL1_HUMAN
497
504
LvkEEgPr


2174
Q96B01|R51A1_HUMAN
55
62
LrkEEiPv


2175
Q96D15|RCN3_HUMAN
192
199
LhpEEfPh


2176
Q96DC7|TMCO6_HUMAN
219
226
LqaEEaPe


2177
Q96FT7|ACCN4_HUMAN
90
97
LslEEqPl


2178
Q96G97|BSCL2_HUMAN
326
333
LseEEkPd


2179
Q96GW7|PGCB_HUMAN
880
887
LhpEEdPe


2180
Q96H72|S39AD_HUMAN
340
347
LleEEdPw


2181
Q96H78|S2544_HUMAN
265
272
LmaEEgPw


2182
Q96J42|TXD15_HUMAN
42
49
LwsEEqPa


2183
Q96JI7|SPTCS_HUMAN
1940
1947
LleEEaPd


2184
Q96JL9|ZN333_HUMAN
80
87
LkpEElPs


2185
Q96JQ0|PCD16_HUMAN
3106
3113
LyrEEgPp


2186
Q96MZ0|GD1L1_HUMAN
195
202
LdhEEePq


2187
Q96NZ9|PRAP1_HUMAN
71
78
LttEEkPr


2188
Q96PQ6|ZN317_HUMAN
109
116
LeqEEePr


2189
Q96RE7|BTB14_HUMAN
133
140
LhaEEaPs


2190
Q96RG2|PASK_HUMAN
1196
1203
LvfEEnPf


2191
Q96RL1|UIMC1_HUMAN
388
395
LllEEePt


2192
Q96SB3|NEB2_HUMAN
435
442
LseEEdPa


2193
Q96SJ8|TSN18_HUMAN
167
174
LdsEEvPe


2194
Q99102|MUC4_HUMAN
1306
1313
LhrEErPn


2195
Q99543|DNJC2_HUMAN
68
75
LqlEEfPm


2196
Q9BQS2|SYT15_HUMAN
36
43
LtyEElPg


2197
Q9BVI0|PHF20_HUMAN
483
490
LepEEsPg


2198
Q9BY44|EIF2A_HUMAN
461
468
LheEEpPq


2199
Q9BY78|RNF26_HUMAN
356
363
LneEEpPg


2200
Q9BYD3|RM04_HUMAN
221
228
LthEEmPq


2201
Q9BZA7|PC11X_HUMAN
315
322
LdrEEtPn


2202
Q9BZA8|PC11Y_HUMAN
347
354
LdrEEtPn


2203
Q9C009|FOXQ1_HUMAN
227
234
LrpEEaPg


2204
Q9H095|IQCG_HUMAN
122
129
LitEEgPn


2205
Q9H0D2|ZN541_HUMAN
149
156
LggEEpPg


2206
Q9H2C0|GAN_HUMAN
36
43
LdgEEiPv


2207
Q9H2X9|S12A5_HUMAN
681
688
LrlEEgPp


2208
Q9H334|FOXP1_HUMAN
291
298
LshEEhPh


2209
Q9H3T3|SEM6B_HUMAN
26
33
LfpEEpPp


2210
Q9H579|CT132_HUMAN
138
145
LvqEErPh


2211
Q9H5V8|CDCP1_HUMAN
788
795
LatEEpPp


2212
Q9H6F5|CCD86_HUMAN
227
234
LnkEElPv


2213
Q9H6Z4|RANB3_HUMAN
4
11
LanEEkPa


2214
Q9H7E9|CH033_HUMAN
94
101
LapEEvPl


2215
Q9H8Y1|CN115_HUMAN
137
144
LcsEEsPe


2216
Q9H9E1|ANRA2_HUMAN
13
20
LivEEcPs


2217
Q9H9F9|ARP5_HUMAN
415
422
LfsEEtPg


2218
Q9HAV4|XPO5_HUMAN
521
528
LnrEEiPv


2219
Q9HCE7|SMUF1_HUMAN
364
371
LedEElPa


2220
Q9NPR2|SEM4B_HUMAN
47
54
LgsEErPf


2221
Q9NR50|EI2BG_HUMAN
333
340
LcpEEpPv


2222
Q9NRJ7|PCDBG_HUMAN
200
207
LdrEEePq


2223
Q9NTN9|SEM4G_HUMAN
203
210
LrtEEtPm


2224
Q9NUR3|CT046_HUMAN
104
111
LhsEEgPa


2225
Q9NVR7|TBCC1_HUMAN
138
145
LigEEwPs


2226
Q9NX46|ARHL2_HUMAN
235
242
LgmEErPy


2227
Q9NYB9|ABI2_HUMAN
7
14
LleEEiPg


2228
Q9P1Y5|K1543_HUMAN
827
834
LlaEEtPp


2229
Q9P1Y5|K1543_HUMAN
938
945
LaqEEaPg


2230
Q9P2E7|PCD10_HUMAN
316
323
LdyEEsPv


2231
Q9P2K9|PTHD2_HUMAN
673
680
LevEEePv


2232
Q9UBB4|ATX10_HUMAN
289
296
LasEEpPd


2233
Q9UBN6|TR10D_HUMAN
78
85
LkeEEcPa


2234
Q9UBT6|POLK_HUMAN
251
258
LlfEEsPs


2235
Q9UGF5|OR5U1_HUMAN
303
310
LskEElPq


2236
Q9UGL1|JAD1B_HUMAN
879
886
LlsEEtPs


2237
Q9UHW9|S12A6_HUMAN
743
750
LrlEEgPp


2238
Q9UIF9|BAZ2A_HUMAN
609
616
LsaEEiPs


2239
Q9UIG0|BAZ1B_HUMAN
75
82
LlkEEfPa


2240
Q9ULD6|PDZD6_HUMAN
390
397
LpaEEvPl


2241
Q9ULG1|INOC1_HUMAN
235
242
LssEEsPr


2242
Q9ULI4|KI26A_HUMAN
1396
1403
LrgEEePr


2243
Q9ULQ1|TPC1_HUMAN
29
36
LgqEElPs


2244
Q9UMS0|NFU1_HUMAN
93
100
LvtEEtPs


2245
Q9UN72|PCDA7_HUMAN
200
207
LdrEEtPe


2246
Q9UN73|PCDA6_HUMAN
200
207
LdrEEaPa


2247
Q9UN74|PCDA4_HUMAN
200
207
LdrEEaPe


2248
Q9UNA0|ATS5_HUMAN
481
488
LgpEElPg


2249
Q9UP95|S12A4_HUMAN
678
685
LrlEEgPp


2250
Q9UPQ7|PZRN3_HUMAN
385
392
LlpEEhPs


2251
Q9UPV0|CE164_HUMAN
488
495
LatEEePp


2252
Q9UPW6|SATB2_HUMAN
398
405
LrkEEdPr


2253
Q9UPW8|UN13A_HUMAN
332
339
LeeEElPe


2254
Q9UPX6|K1024_HUMAN
371
378
LntEEvPd


2255
Q9UQ05|KCNH4_HUMAN
761
768
LlgEElPp


2256
Q9UQ26|RIMS2_HUMAN
201
208
LrnEEaPq


2257
Q9UQ26|RIMS2_HUMAN
1327
1334
LsfEEsPq


2258
Q9Y250|LZTS1_HUMAN
293
300
LayEErPr


2259
Q9Y2I6|NLP_HUMAN
759
766
LelEEpPq


2260
Q9Y2K7|JHD1A_HUMAN
661
668
LlnEElPn


2261
Q9Y2L6|FRM4B_HUMAN
438
445
LpsEEdPa


2262
Q9Y2V3|RX_HUMAN
126
133
LseEEqPk


2263
Q9Y343|SNX24_HUMAN
87
94
LenEElPk


2264
Q9Y3I0|CV028_HUMAN
466
473
LvmEEaPe


2265
Q9Y3L3|3BP1_HUMAN
130
137
LseEElPa


2266
Q9Y3L3|3BP1_HUMAN
494
501
LasEElPs


2267
Q9Y3R5|DOP2_HUMAN
1084
1091
LseEElPy


2268
Q9Y426|CU025_HUMAN
98
105
LsfEEdPr


2269
Q9Y566|SHAN1_HUMAN
1838
1845
LpwEEgPg


2270
Q9Y572|RIPK3_HUMAN
352
359
LnlEEpPs


2271
Q9Y5E2|PCDB7_HUMAN
200
207
LdrEEiPe


2272
Q9Y5E3|PCDB6_HUMAN
199
206
LdrEEqPq


2273
Q9Y5E4|PCDB5_HUMAN
200
207
LdrEErPe


2274
Q9Y5E5|PCDB4_HUMAN
199
206
LdrEEqPe


2275
Q9Y5E6|PCDB3_HUMAN
200
207
LdrEEqPe


2276
Q9Y5E7|PCDB2_HUMAN
202
209
LdrEEqPe


2277
Q9Y5F1|PCDBC_HUMAN
200
207
LdyEErPe


2278
Q9Y5F2|PCDBB_HUMAN
200
207
LdyEElPe


2279
Q9Y5F3|PCDB1_HUMAN
200
207
LdrEEqPe


2280
Q9Y5G1|PCDGF_HUMAN
200
207
LdrEEqPh


2281
Q9Y5G2|PCDGE_HUMAN
410
417
LdrEEiPe


2282
Q9Y5H5|PCDA9_HUMAN
200
207
LdrEEtPe


2283
Q9Y5I2|PCDAA_HUMAN
199
206
LdrEEnPq


2284
Q9Y5I3|PCDA1_HUMAN
200
207
LdrEEtPe


2285
Q9Y5Q9|TF3C3_HUMAN
42
49
LsaEEnPd


2286
Q9Y5R2|MMP24_HUMAN
201
208
LtfEEvPy









Example 9
A Novel Peptide Derived from the Alpha6 Fibril of Type 4 Collagen
A peptide similar to the short Tumstatin T3 peptide derived from the alpha3 fibril of type IV collagen was identified. This peptide was derived from the alpha6 fibril of type 4 collagen. Its amino acid sequence is LPRFSTMPFIYCNINEVCHY as shown in FIG. 13.







TABLE 10







Table containing the amino acid sequence of


the peptide predicted similar to Tumstatin/Tum4









Protein Name
Peptide Location
Peptide sequence





Collagen type IV, alpha6 fibril
CAI40758.1:1630-1648
LPRFSTMPFIYCNINEVCHY









Example 10
Peptide Modifications
One skilled in the art will appreciate that peptides disclosed herein may be modified to increase peptide stability for in vivo administration. To demonstrate the desirability of introducing such modifications, three exemplary peptides were selected where in vivo administration in lung carcinoma xenografts of the naked (unmodified) peptides has shown significant efficacy in suppressing the tumor volume increase.
The three exemplary peptides include a peptide derived from the alpha 5 fibril of type IV collagen, a peptide derived from a TSP1 repeat containing protein properdin, and a peptide derived from a CXC chemokine CXCL1 (FIG. 14). The amino acid sequences of mouse and human peptides are shown in FIG. 14. There are minor differences in the amino acid sequences of the mouse and human sequences for TSP1 derived and CXC derived peptide. These differences do not affect the suggested modifications, as the amino acids that may be associated with peptide instability are common in both the mouse and human sequences. The amino acid sequences of the collagen derived peptides are common in both species.
Amino Acid Modifications Controlling Disulfide Bond Formation
Under oxidizing conditions, the sulfide groups from two cysteines may cross react to form a disulfide bond. If the two cysteines exist in the same molecule, this bond can be formed intra-molecularly producing a hairpin-like tertiary structure in a peptide molecule. If those two cysteines exist either in the same molecule or in two different molecules (one cysteine in the amino acid sequence of the peptide) the disulfide bond formation can cause dimerization or multimerization of the molecules. This can induce possible peptide aggregation, thereby reducing therapeutic efficacy. In addition, albumin contains a free cysteine that can react with the peptides' free cysteines again forming disulfide bonds. These bonds can cause the peptide to non-specifically bind on the albumin's surface. The peptide binding on the albumin's surface can reduce the effective concentration of the circulating peptide.
To promote therapeutic efficacy and reduce the formation of disulfide bonds, cysteines are substituted, for example, by an aminobutyric acid (Abu), serine or alanine. These amino acids have similar physicochemical properties as cysteines, i.e., they include a polar in side chain polarity, neutral in side chain acidity and are largely hydrophobic. However, they are devoid of sulfide groups, which cause them to be non-reactive under oxidizing conditions. Serine and alanine have somewhat different molecular dimensions than cysteine (serine is longer and alanine is shorter). Substitution with these amino acids can cause secondary modifications in the structure of the original peptide. Aminobutyric acid is a favorable modification as it conserves the physicochemical and structural characteristics of the cysteine without the reactive sulfide group.
When two or more cysteines exist per peptide there are two strategies that can be used in order to prevent disulfide bond formation. If the hairpin tertiary structure of the peptide is significant for its activity, the intramolecular disulfide bond formation can be preformed during the solid state synthesis of the molecule if the synthesis is performed under oxidizing conditions. The purification step of the peptide, based on its molecular weight, will eventually obliterate any multimers formed under the oxidizing conditions and can yield a high purity peptide with a hairpin-like tertiary structure. If this structure is not significant or reduces the peptide's activity, then the same strategy as in the case of a single cysteine per molecule can be followed. Both of the cysteines can be substituted by aminobutyric acids, serines or alanines.
Amino Acid Modifications Controlling Pegylation Stability
Pegylation involves the conjugation of polyethylene glycol (PEG) to proteins and peptides. Attaching a PEG increases the molecular weight of a molecule, and yield several significant pharmacological advantages over the unmodified form, which include: improved solubility; reduced dosage frequency without diminished efficacy and potentially reduced toxicity; extended circulating life; and enhanced protection from proteolytic degradation.
The presence of methionines in the amino acid sequence of a peptide may induce a low level oxidation reaction at the sulfur containing chain. This can cause the peptide to be unstable in solution or subject to non-specific interactions. The most important potential problem arising from the presence of methionines is the non-specific interactions of these amino acids with PEG chains. These interactions cause binding of the PEG to the methionines, which may present difficulties in purifying Pegylated peptides (i.e., purifying them to greater than 97% which is required by the U.S. Food and Drug Administration for human administration). The most appropriate strategy for minimizing the effect of the methionines on the Pegylation yield is the substitution of the methionines with isoleucines. Isoleucines have many of the same characteristics as methionines, but no cross-reactivity with the PEG chains.
Another amino acid that may interact non-specifically with PEG chains is lysine. This can reduce the yield of the Pegylation reaction. One strategy to minimize nonspecific interactions with lysine is protecting lysine during chemical synthesis. This extra step may increase the cost of Pegylation. A common modification that can be used in order to avoid lysine protection during Pegylation, is substituting arginine for lysine. Arginine has similar characteristics with lysines and does not affect the Pegylation yield.
Example 11
Receptor Identification and Peptide Combinations
There is growing evidence that anti-angiogenic peptides exert their effects by binding to receptors on endothelial cells. Tumstatin has two binding sites for avβ3 integrins (Maeshima et al., (2001) J Biol Chem 276, 31959-31968), although its anti-angiogenic activity has been connected to the site that is located in the amino-terminal of the fragment. Tumstatin has also been shown to interact with a6β1 integrins (Maeshima et al., (2000) J Biol Chem 275, 23745-23750). The major receptor that has been identified for the anti-angiogenic CXC chemokines is CXCR3 (Strieter et al., (2006) Eur J Cancer 42, 768-778). CXCR3 exists in three alternative splice isoforms, CXCR3A, CXCR3B, and CXCR3-alt. The CXC chemokine ligands of CXCR3 inhibit the proliferation and migration of human microvascular endothelial cells in response to a variety of angiogenic factors. Extensive studies on the mechanistic details of the anti-angiogenic activity of thrombospondin 1, the prototype type 1 thrombospondin repeat-containing protein, have implicated CD36, a 88-kDa transmembrane glycoprotein, as the cell-surface receptor that mediates its effects on endothelial cells (Dawson et al., (1997) J Cell Biol 138, 707-717). CD47 and various integrins have also been mechanistically implicated in the effects of thrombospondin 1 on endothelial cells (Gao et al., (1996) J Biol Chem 271, 21-24).
In order to determine whether peptides identified herein share binding partners with previously identified anti-angiogenic peptides, neutralization studies against these receptors were performed. Endothelial cells were pre-incubated with a range of concentrations of neutralizing monoclonal antibodies that target single receptors, and the activity of the peptides in the angiogenesis assay was then compared to that observed in the absence of neutralizing antibody. The results for neutralization studies of the CXC chemokine-derived peptides, the collagen IV-derived peptides, and the TSP1 repeat-containing peptides are presented herein (FIGS. 15A-15C). In each case, a control where the cells were incubated only in the presence of the antibody solutions and without any peptides was carried out. No effect of the antibody alone on the endothelial cells was observed at any concentration.
In order to determine whether CXCR3 is responsible for the binding of the CXC chemokine derived anti-angiogenic peptides, the proliferation experiments were repeated in the presence of different concentrations of a CXCR3-neutralizing antibody. Two concentrations of the antibody were tested, 1 and 10 μg/ml, one below and one above the designated ED50. In most cases, the activity of the peptide was abrogated in the presence of an increasing concentration of the neutralizing antibody against the CXCR3 receptor. Interestingly, in the cases in which the peptide exhibited a biphasic dose response, the monoclonal antibody did not entirely neutralize the activity of the peptide. This suggests that more than one receptor or more than one mechanism is responsible for the activity of these peptides. By performing similar neutralization studies using monoclonal antibodies against all the known CXC receptors, including CXCR1, CXCR2, and CXCR4, none of these receptors appeared to mediate the anti-angiogenic activity of the peptides.
Noting that the effects of tumstatins are primarily attributed to peptides binding to β1 and β3 integrins (Maeshima et al., (2001) J Biol Chem 276, 31959-31968; Maeshima et al., (2001) J Biol Chem 276, 15240-15248), for collagen-derived peptides monoclonal antibodies directed against the β1 and β3 integrins were tested at two antibody concentrations, 1 and 10 ng/ml (FIG. 14). The activity of the highly potent collagen derived peptides was completely abrogated after pre-incubation with either anti-integrin antibody. In the case of the TSP1 repeat-derived peptides, neutralizing CD36, which is the main TSP1 repeat receptor, abolished the peptides' activity. With increasing antibody concentration, increased endothelial cell proliferation was observed relative to the control. It is noteworthy that at these two antibody concentrations for which no direct effect on endothelial cells was observed, the antibodies were potent enough to neutralize the peptide activity. In contrast, blocking CD47, the integrin-associated receptor, only partially neutralized the peptide activity.
Based on the information obtained from the neutralization experiments, a systematic method to create and test the effectiveness of combinations of individual peptides as potent angiogenesis inhibitors was developed. By using combinations of peptides that bind to different receptors, different pathways were targeted to assess whether there was any modulation of the combined activity in our functional assays. In order to evaluate combinations, a sensitive proliferation assay was selected to analyse changes in peptide activity. The use of multiple peptides targeting multiple targets, with different mechanisms or modes of action, creates the possibility for multiple favorable outcomes, including an increased efficacy of the therapeutic effect, the ability to employ a decreased dosage to obtain an analogous or increased level of efficacy (as a strategy to avoid toxicity), as well as a minimization of, or delay in, the development of resistance (Dorrell et al., (2007) Proceedings of the National Academy of Sciences of the United States of America 104, 967-972).
Combinations of two peptides were tested from each of the three major protein families, the type IV collagen fibrils, CXC chemokines, and TSP1 repeat-containing proteins: The peptides used in the combination experiments are derived from the alpha5 fibril of type IV collagen (LRRFSTMPFMFCNINNVCNF), from alpha4 fibril of type IV collagen (YCNIHQVCHYAQRNDRSYWL), from a CXC protein GRO-α/CXCL1 (NGRKACLNPASPIVKKIIEKMLNS), from a CXC protein ENA-78/CXCL5 (NGKEICLDPEAPFLKKVIQKILD), from a TSP1 repeat-containing protein properdin (GPWEPCSVTCSKGTRTRRR), and from a TSP1 repeat-containing protein THSD6 (WTRCSSSCGRGVSVRSR). One peptide from each family was combined at four different concentrations (0.1, 1, 10, and 30 μg/ml), and the efficacy of these combined peptides was evaluated in a proliferation assay. The peptides were applied in series in order to avoid possible interactions between them, and the viability of the cells was then evaluated. Using the information from the dose-response curves, the data was fit to sigmoidal Hill curves (Chou et al., (2006) Pharmacol Rev 58, 621-681). Based on the estimated Hill curves, isobolograms were calculated to obtain the state space of peptide concentrations with equipotent sums of doses. This data was used to generate graphs of equally effective dose pairs (isoboles) with the same level of effectiveness observed for a single peptide application. In addition to the isobolograms the Combination Indexes (C.I.) for different peptide combinations was also calculated (Chou et al., (1984) Adv Enzyme Regul 22, 27-55) to compare the relative efficacy of the various combinations (FIG. 16).
These analyses indicated a significant synergism between CXC chemokines and TSP1 repeat-containing protein-derived peptides. Thus, it is likely that using specific peptide combinations, provides activity levels similar to those obtained when each of the peptides is used alone, but at significantly lower dosages. In the case of combining a CXC derived peptide with a TSP1 derived peptide, dosage was reduced by one order of magnitude while the same level of efficacy was maintained. Furthermore, when applied at higher concentrations, these two peptides in combination yielded a much higher activity than when either one was applied alone. In the case of the combination of collagen IV-derived peptides with either CXC- or TSP1-derived peptides, a synergism was observed only at lower collagen peptide concentrations. At higher concentrations, the collagen-derived peptides were antagonized by the CXC and TSP1 repeat-derived peptides.
These studies indicated that the peptides bind to receptors on the endothelial cell surface. Based on the information from the receptor binding, combinatorial strategies were designed targeting multiple receptors. This analysis supports the conclusion that targeting CD36 or CD47, the primary thrombospondin receptors, and CXCR3, the receptor responsible for the anti-angiogenic activity of CXC chemokine-derived peptides, provided for the synergistic amplification of the peptides' potency.
Example 12
Anti-Angiogenic Peptides Arrest Tumor Growth
To characterize the functional effects of anti-angiogenic peptides in mouse models, tumor xenografts were generated in female nude mice using the NCI H82 lung carcinoma cell line. This cell line was chosen because its aggressiveness results in rapid tumor growth. Three peptides, a collagen derived, a CXC chemokine derived and a TSP1 containing protein derived peptide were administered once a day, intraperitoneally (i.p.), at doses 10 and 20 mg/kg/day, in a 200 microliter solution injection as individual agents and as a combination. The CXC protein GRO-α/CXCL1 derived peptide (human sequence: NGRKACLNPASPIVKKIIEKMLNS; mouse sequence: NGREACLDPEAPLVQKIVQKMLKG), the TSP1 repeat-containing protein WISP-1 derived peptide (human sequence: GPWEPCSVTCSKGTRTRRR; mouse sequence: GPWGPCSVTCSKGTQIRQR), and the type IV collagen alpha5 fibril derived peptide (human sequence: LRRFSTMPFMFCNINNVCNF is the same as mouse sequence: LRRFSTMPFMFCNINNVCNF). An equivalent volume of PBS was injected as control. The injections were repeated for 12 days. At 10 mg/kg/day (FIG. 18A) and 20 mg/kg/day (FIG. 18B) the peptides suppressed the development of tumors as a monotherapy. Injections of the combination of a TSP1 containing protein derived peptide and a CXC chemokine derived peptide in a rapidly developing tumor (Day 14 after inoculation) completely arrested tumor growth within 3 days (FIG. 18C).
The results described in Example 12 were carried out using the following materials and methods.
Cell Culture
Primary human umbilical vein endothelial cells (HUVECs) from a single donor were purchased from Cambrex (Walkersville, Md.). The cells were propagated in EGM-2 medium, consisting of a basal cell medium with 2% FBS, growth factors (hbFGF and VEGF) and antibiotics (gentamicin/amphotericin B). The cells were subcultured according to the supplier's instructions: Once the cells had reached sub-confluence, they were washed with HEPES buffer solution and trypsinized. The trypsin was then neutralized with trypsin neutralizing solution (TNS; Cambrex, Walkersville, Md.), and the cells were collected and centrifuged at 1500 rpm for 5 minutes. The supernatant was aspirated, and the cells were resuspended in fresh medium. All the cells used were from passage 3 to passage 6.
In Vitro Cell Viability Assay
To assess the effects of peptides on the proliferation of endothelial cells the viability and metabolic activity of the cells was monitored in the presence of the agent at different concentrations after various periods of time. The colorimetric cell proliferation reagent WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate) (Roche, Indianapolis, Ind.) was used as the substrate in an assay that measures the metabolic activity of viable cells (Ishiyama et al., (1996) Biol Pharm Bull 19, 1518-1520). The assay is based on the reduction of the red tetrazolium salt WST-1 by viable, metabolically active cells to form yellow formazan crystals that are soluble in the cell culture medium.
The cells were cultured as described above and then trypsinized and resuspended in EGM-2 once they had reached 80% confluence. Cell counts were determined using a hemocytometer.
The proliferation assay involved two steps: during the first step, the cells ( ̃2×103/well in a 96-well microplate) were seeded without any extracellular matrix substrate onto the microwells overnight (8 hours). The initial cell culture medium was then removed, and the candidate peptides, dissolved in cell culture medium with growth factors and serum, were added to the wells. The viability of the cells was determined after a 3-day exposure to the peptide solution. Each peptide was tested at seven different concentrations: 0.01, 0.1, 1 and 10 μg/ml and 20, 30 and 40 μg/ml. Each of the concentrations was tested simultaneously in quadruplicate, and each of the experiments was repeated two times. As a positive control (i.e., decreasing viability) 100 ng/ml (0.22 μM) TNP-470 (O-(chloro-acetyl-carbamoyl) fumagillol, a synthetic analogue of fumagillin was applied; 0.46 kDa, provided by NCI) along with the full medium. As a negative control (equivalent to normal viability) the cells were cultured without any test agent in full medium, containing growth factors and serum. The cells were then incubated with the WST-1 reagent for approximately 3 hours. During the incubation period, viable cells convert, in their mitochondria, the red WST-1 to yellow formazan crystals that dissolve in the medium. The second step of the assay involved the quantification of the changes in proliferation by measuring the changes in the color of the metabolized substrate. The samples were read at a wavelength of 570 nm in an ELISA plate reader Victor 3V (Perkin Elmer). The amount of color produced was directly proportional to the number of viable cells.
Monoclonal Antibody Neutralization Assay
In the monoclonal antibody neutralization experiments the endothelial cell proliferation assay was repeated in the presence of varying concentrations of monoclonal antibodies against specific receptors. The endothelial cells were seeded overnight in 96 well plates in full growth factor and serum medium. The medium was removed and replaced with medium containing different monoclonal antibody solutions for beta1 integrins (R&D Systems, MAB17781) alphavbeta3 integrins (R&D Systems, MAB3050), CXCR3 (R&D Systems, MAB1685), CD36 (BD Pharmingen, CB38 (NL07)) and CD47 (BD Pharmingen, B6H12). The cells were incubated for two hours with the antibody solutions. After the two hours the peptide solutions at different concentrations were added in the wells. As a control a set of cells was incubated only in the presence of the monoclonal antibody solutions and without any peptides. The cells were incubated for three days and a cell viability estimation was performed similarly to the proliferation assay.
Isobologram and Combination Index Calculation
The proliferation experiments described above were carried out with peptide combinations. In the combination experiments, the cells were seeded in 96-well microplates using the same cell density as described above, i.e., approximately 2000 cells per well. The cells were allowed to attach overnight (6-8 hours) in full growth factor and serum medium. The full medium was withdrawn and a solution of a single peptide was applied in dose response concentrations of 0.1, 1, 10 and 30 μg/ml. These solutions were prepared and applied in growth factor and serum free medium. After two hours the solutions of the first peptide were withdrawn and the solutions of the second peptide were applied in a growth factor and serum free medium. The concentrations at which the second peptide was applied were the same as the concentrations of the first, i.e. in the case that the first peptide was applied at 10 μg/ml, the second was also applied at 10 μg/ml. In addition to the combinations each of the peptides was applied alone for reference. After twenty-four hours the WST-1 dye was applied and the number of live cells was estimated by the optical signal. Dose response sigmoidal curves for a condition “i” were estimated by fitting the data to sigmoidal Hill curves of the type:




  
    
      
        
          E
          i
        
        =
        
          
            
              
                E
                i
                max
              
              ·
              
                
                  D
                  i
                  
                    n
                    i
                  
                
                
                  
                    D
                    
                      50
                      ,
                      i
                    
                    
                      n
                      i
                    
                  
                  +
                  
                    D
                    i
                    
                      n
                      i
                    
                  
                
              
            
            →
            
              D
              i
            
          
          =
          
            
              D
              
                50
                ,
                i
              
            
            ·
            
              
                
                  E
                  i
                
                
                  
                    E
                    i
                    max
                  
                  +
                  
                    E
                    i
                  
                
              
              
                n
                i
              
            
          
        
      
    
    
      
        (
        1
        )
      
    
  




where E is the effect of the condition “i”, in this case the fraction of dead cells, Emax is the maximum observed effect, D is the corresponding dose that yields effectiveness E, D50 is the dose at which half of the maximum effectiveness Emax is observed, and n is the Hill coefficient.

Combining a peptide x with a peptide y and Dxcombo is the applied peptide x concentration in the combination experiment and Dycombo is the applied peptide y concentration in combination then due to the set up of the experiment, at each experimental condition Dxcombo=Dycombo=Dcombo. In order to construct an isobologram, a graph of equally effective dose pairs (isoboles) for a single peptide effect level (Chou et al., (2006) Pharmacol Rev 58, 621-681):




  
    
      
        
          
            
              D
              x
              combo
            
            
              D
              x
            
          
          +
          
            
              D
              y
              combo
            
            
              D
              y
            
          
        
        =
        1
      
    
    
      
        (
        2
        )
      
    
  




In the denominator Dx is the dose for Dxcombo alone that inhibits the proliferation by effectiveness E and Dy is the dose for Dycombo alone that inhibits the proliferation by the same effectiveness E. Also Dxcombo=Dycombo=Dcombo. Solving equation 2 for a single dose:





  
    
      
        
          D
          x
        
        =
        
          
            
              D
              y
            
            ·
            
              D
              combo
            
          
          
            
              D
              y
            
            -
            
              D
              combo
            
          
        
      
    
    
      
        (
        3
        )
      
    
  




After substituting the dose response of the combination Dcombo with the corresponding sigmoidal equation 1 as fitted by the experimental data, equation 3 becomes:





  
    
      
        
          D
          x
        
        =
        
          
            
              D
              y
            
            ·
            
              D
              50
              combo
            
            ·
            
              
                
                  E
                  combo
                
                
                  
                    E
                    combo
                    max
                  
                  +
                  
                    E
                    combo
                  
                
              
              
                n
                combo
              
            
          
          
            
              D
              y
            
            -
            
              
                D
                50
                combo
              
              ·
              
                
                  
                    E
                    combo
                  
                  
                    
                      E
                      combo
                      max
                    
                    +
                    
                      E
                      combo
                    
                  
                
                
                  n
                  combo
                
              
            
          
        
      
    
    
      
        (
        4
        )
      
    
  




The isobologram is the plot of these concentrations that the effectiveness of an agent alone is the same as the effectiveness of the same agent in combination, Ecombo=Ey, thus equation 4 becomes:





  
    
      
        
          D
          x
        
        =
        
          
            
              D
              y
            
            ·
            
              D
              50
              combo
            
            ·
            
              
                
                  E
                  y
                
                
                  
                    E
                    combo
                    max
                  
                  +
                  
                    E
                    y
                  
                
              
              
                n
                combo
              
            
          
          
            
              D
              y
            
            -
            
              
                D
                50
                combo
              
              ·
              
                
                  
                    E
                    y
                  
                  
                    
                      E
                      combo
                      max
                    
                    +
                    
                      E
                      y
                    
                  
                
                
                  n
                  combo
                
              
            
          
        
      
    
    
      
        (
        5
        )
      
    
  




But the effectiveness for y alone is defined according to the Hill equation as:





  
    
      
        
          E
          y
        
        =
        
          
            E
            y
            max
          
          ·
          
            
              D
              y
              
                n
                y
              
            
            
              
                D
                
                  50
                  ,
                  y
                
                
                  n
                  y
                
              
              +
              
                D
                y
                
                  n
                  y
                
              
            
          
        
      
    
    
      
        (
        6
        )
      
    
  




Thus after substituting equation 6 into 5:





  
    
      
        
          D
          x
        
        =
        
          
            D
            y
          
          ·
          
            D
            50
            combo
          
          ·
          
            
              
                
                  
                    E
                    y
                    max
                  
                  ·
                  
                    
                      D
                      y
                      
                        n
                        y
                      
                    
                    
                      
                        D
                        
                          50
                          ,
                          y
                        
                        
                          n
                          y
                        
                      
                      +
                      
                        D
                        y
                        
                          n
                          y
                        
                      
                    
                  
                
                
                  
                    E
                    combo
                    max
                  
                  +
                  
                    
                      E
                      y
                      max
                    
                    ·
                    
                      
                        D
                        y
                        
                          n
                          y
                        
                      
                      
                        
                          D
                          
                            50
                            ,
                            y
                          
                          
                            n
                            y
                          
                        
                        +
                        
                          D
                          y
                          
                            n
                            y
                          
                        
                      
                    
                  
                
              
              
                n
                combo
              
            
            
              
                D
                y
              
              -
              
                
                  D
                  50
                  combo
                
                ·
                
                  
                    
                      
                        E
                        y
                        max
                      
                      ·
                      
                        
                          D
                          y
                          
                            n
                            y
                          
                        
                        
                          
                            D
                            
                              50
                              ,
                              y
                            
                            
                              n
                              y
                            
                          
                          +
                          
                            D
                            y
                            
                              n
                              y
                            
                          
                        
                      
                    
                    
                      
                        E
                        combo
                        max
                      
                      +
                      
                        
                          E
                          y
                          max
                        
                        ·
                        
                          
                            D
                            y
                            
                              n
                              y
                            
                          
                          
                            
                              D
                              
                                50
                                ,
                                y
                              
                              
                                n
                                y
                              
                            
                            +
                            
                              D
                              y
                              
                                n
                                y
                              
                            
                          
                        
                      
                    
                  
                  
                    n
                    combo
                  
                
              
            
          
        
      
    
    
      
        (
        7
        )
      
    
  




In order to graph the isobolograms we calculate for each Dy the corresponding Dx and plot the Dx vs. Dy pairs.

The isobolograms are a special case for the combination index equation as introduced by Chou and Talalay (Chou et al., (1984) Adv Enzyme Regul 22, 27-55). The generic equation for the combination index calculation is expressed:




  
    
      
        CI
        =
        
          
            
              D
              x
              combo
            
            
              D
              x
            
          
          +
          
            
              D
              y
              combo
            
            
              D
              y
            
          
        
      
    
    
      
        (
        8
        )
      
    
  




If CI<1 the drug combination effect is synergistic; if CI=1 the drug combination effect is additive; whereas if CI>1 the drug combination effect is antagonistic.

In vivo Tumor Xenograft Models

A population of 106 cells were washed twice in PBS and gently resuspended to generate a single cell suspension. The cells were mixed with Matrigel (BD Biosciences) in a final 60% cell solution. Subsequently, the cells were injected into into the flank area of immunosupressed nude mice in a total volume of 100 μl. Following growth incubation of 5 to 6 days, the tumor size volume was calculated by measurements of tumor dimensions with calipers. Tumor growth was monitored to an initial average size of 100 mm3, which developed within 6 days after inoculation. Peptides were administered once a day, intraperitoneally (i.p.), in doses of 10 mg/kg and 20 mg/kg. In the case of testing a combination each peptide was injected in a two day cycle of a different peptide per day. Equivalent volume of PBS solution was injected as control. The injections were continued for up to 14 days. A total of six animals per group were used for the experiments per peptide per concentration.
Other Embodiments
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
The following International Patent Application No. PCT/US2006/035580, entitled COMPOSITIONS HAVING ANTIANGIOGENIC ACTIVITY AND USES THEREOF, which was filed on Sep. 12, 2006 may include related subject matter, and is hereby incorporated by reference in its entirety.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. In particular, the sequence of each of the individual NCBI reference numbers listed in Tables 1-10 is hereby incorporated by reference in its entirety.

## Claims

1. An isolated peptide or analog thereof consisting of a sequence having at least 85% amino acid sequence identity to Transmembrane protein 45A LLRSSLILLQGSWF (SEQ ID NO: 2296) wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

2. The isolated peptide of claim 1, wherein the peptide comprises at least one modification.

3. The isolated peptide of claim 2, wherein the modification is a sequence alteration or post-translational modification that increases protease resistance, biodistribution, or therapeutic efficacy.

4. A pharmaceutical composition comprising an effective amount of the isolated peptide of claim 1 thereof in a pharmacologically acceptable excipient.

5. A method of reducing blood vessel formation in a tissue or organ, the method comprising contacting an endothelial cell, or a tissue or organ comprising an endothelial cell with an effective amount of the peptide of claim 1, thereby reducing blood vessel formation in the tissue or organ.

6. The method of claim 5, wherein the method reduces endothelial cell proliferation, migration, survival, or stability in a tissue or organ or increases cell death.

7. A method of reducing blood vessel formation in a tissue or organ the method comprising:
(a) contacting the tissue, or organ with a vector encoding the peptide of claim 1; and
(b) expressing the peptide in a cell of the tissue or organ, thereby reducing blood vessel formation in the tissue or organ.

8. A method of modulating angiogenesis in a cell, tissue, or organ, the method comprising contacting the cell, tissue, or organ with an effective amount of the peptide of claim 1.

9. A kit comprising an effective amount of the peptide of claim 1, and directions for using the peptide to treat a disease characterized by undesirable or excess angiogenesis.