LAMININ RECEPTOR BINDING MOLECULE

FIELD OF THE INVENTION

This invention relates to a laminin receptor binding molecule. More particularly, the present invention relates to a laminin receptor binding molecule derived from PSP94 and its use for promoting signal transduction, such as laminin receptor mediated signal transduction, in a mammalian cell and its use to inhibit laminin and its receptor mediated signaling.

BACKGROUND OF THE INVENTION

Laminins are a family of heterotrimeric protein that resides primarily in the basal lamina. They function via binding interactions with neighboring cell receptors, and are important signaling molecules that can strongly influence cellular function.

Laminins are composed of one each of the five known alpha, three known beta, and three known gamma chains. Laminins may be assembled into at least 15 isoforms made from combinations of the various alpha, beta and gamma chains. Laminin-1 , for example, is composed of alpha-1 , beta-1 and gamma-1 isoforms.

Four structurally-defined family groups of laminins have been identified. The first group of five identified laminin molecules all share the beta-1 and gamma-1 chains, and vary by their alpha-chain composition (alpha-1 to alpha-5 chain). The second group of five identified laminin molecules all share the beta-1 and gamma-1 chain, and again vary by their alpha-chain composition. The third group of identified laminin molecules has one identified member, laminin 5, with a chain composition of alpha-3, beta-3 and gamma-2. The fourth group of identified laminin molecules has one identified member, laminin 12, with the gamma-3 chain (alpha-2, beta-1 and gamma-3).

Laminins mediate the attachment of both epithelial and neoplastic cells to the basement membrane, a ubiquitous, specialized type of extracellular matrix. Interaction of cells with this matrix is an important aspect of both normal and neoplastic cellular processes. Normal cells appear to require an extracellular matrix for survival, proliferation, and differentiation, while migratory cells, both normal and neoplastic, must traverse the basement membrane in moving from one tissue to another.

Laminins are therefore important in both maintaining cell/tissue phenotype as well as promoting cell growth and differentiation in tissue repair and development. The laminin molecule integrates various matrix and cell interactive functions into one molecule.

Laminin alpha chain may have specific as well as overlapping functions. Unique phenotypes have linked alpha-2 to a muscular dystrophy subset (Tome F.M., et al., C R Acad Sci III 317:351 , 1994). In fact, the absence of this laminin chain, in 13 patients seems to be associated with Fukuyama congenital muscular dystrophy. Impaired laminin alpha-3 chain expression seems to be involved in epidermolysis bullosa (Baudoin C, et al., J Invest Dermatol 104:597, 1995). The alpha-5 chain seems to contribute to vascular occlusion in sickle cell disease (Lee, SP. et al. Blood, Vol. 92 No. 8, 1998: pp. 2951-2958).

One class of laminin receptors is the integrins, which are cell surface receptors that mediate many cell-matrix and cell-cell interactions. Some integrins have only one or a few known ligands, whereas others appear to be very promiscuous. Integrins, activated through binding to their ligands, transduce signals via kinase activation cascades, such as focal adhesion and mitogen-activated kinases. Several different integrins may bind different laminin isoforms. (Aumailley et al., In The Laminins, Timpl and Ekblom, eds., Harwood Academic Publishers, Amsterdam, pp. 127-158 (1996))

Other laminin receptors have been identified and include dystroglycan, heparan sulfate proteoglycans, sulfatides, HNK-1 (alpha-1 chain) and lutheran (alpha-5 chain).

Laminin receptor, by mediating the attachment of both epithelial and neoplastic cells to the basement membrane, plays a critical role in controlling, for example, the metastatic process. The relationship between laminin and MMP-9 regulation has been the subject of several papers (JBC, 1995, vol 270, p 10365-68; J. Immunology, 2003, vol 171, p 398-406; Cancer Research, 2004, vol 64, p 4810-4816)

PSP94 inhibits the growth of tumor cells (see U.S. Pat. No.: 5,428,011 to Seth et al., the entire content of which is incorporated herein by reference). Tumor growth inhibition by PSP94 fragment such as PCK3145, has also been observed in animal models (see International application No. PCT/CA01/01463 to Garde, S. et al., published under No.: WO02/33090, the entire content of which is incorporated herein by reference). PSP94 also reduces the development of skeletal metastasis (see International application No.: PCT/CA02/01737 to Rabbani. S. et al., published under

No.: WO03/039576, the entire content of which is incorporated herein by reference). This latter characteristic was observed by a reduction in calcium levels and hind limb paralysis following administration of PSP94 to animal modeling prostate cancer. It was also found that PCK3145 is able to reduce matrix metalloproteinases (MMPs) plasma levels. These results were observed by administering the drug to patients characterized as having metastatic adenocarcinoma of the prostate, stage IV prostatic cancer and as having a metastatic hormone resistant prostatic cancer as described in Canadian Patent application no. 2,441,695 published on March 26, 2005, the entire content of which is incorporated herein by reference.

SUMMARY OF THE INVENTION

This invention relates to molecule derived from PSP94 (PSP94 fragment) and its use to promote signal transduction in a mammalian cell, such as laminin receptor signal transduction.

This invention also relates to the use of a molecule derived from PSP94 (PSP94 fragment) to inhibit laminin and its receptor mediated signaling.

This invention also relates to targeting of cell surface laminin binding activities by a PSP94 fragment (PCK3145 (SEQ ID NO.:5, SEQ ID NO.:7 and derivatives).

This invention also relates to regulation of HuR expression by a PSP94 fragment (PCK3145 (SEQ ID NO.:5, SEQ ID NO.:7 and derivatives) and the use of PSP94 fragments in the treatment of diseases affected by (associated with, linked with) HuR expression.

HuR regulation by a PSP94 fragment may be, for example, at the transcriptional level and/or at the level of protein expression.

The present invention also relates to a laminin receptor binding molecule derived from PSP94 (PSP94 fragment) and its use to trigger (e.g., partially) laminin receptor signal transduction and/or laminin receptor signal transduction-mediated event.

The present invention further relates to the use of a PSP94 fragment in the treatment of a condition or disease associated with an inefficient activation of a laminin receptor

or an inefficient (insufficient) activation of a laminin receptor downstream effector (e.g., ERK, etc.).

The present invention also relates to a soluble laminin-like peptidic molecule which may be useful to promote activation of a laminin receptor.

The present invention also relates to the use of a laminin receptor binding molecule in the detection of a laminin receptor or a laminin receptor carrying cell.

The present invention further relates to the use of a PSP94 fragment in the treatment of leukemia and for treating diseases mediated by laminin binding to a laminin receptor (e.g., other cancers such as prostate cancer, breast cancer, etc.).

The present invention provides, in one aspect thereof, a method of promoting laminin receptor signal transduction and/or laminin receptor signal transduction-mediated event in a mammal which may comprise the step of administering to the mammal, a compound (or pharmaceutical composition comprising a compound) selected from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to bind a laminin receptor, and e) combination of any one of a) through d) thereof or any other biologically active PSP94 derived molecule.

The present invention further provides, in another aspect, a method of promoting laminin signal transduction-mediated event in a mammal which may comprise the step of administering to the mammal, a compound (or pharmaceutical composition comprising a compound) selected from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to bind a laminin receptor, and e) combination of any one of a) through d) thereof or any other PSP94 derived molecule.

In an additional aspect the present invention relates to the use of a compound selected, for example, from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a

laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to bind a laminin receptor, and e) combination of any one of a) through d) thereof or any other PSP94 derived molecule for promoting laminin receptor signal transduction and/or laminin receptor signal transduction-mediated event, or for promoting laminin signal transduction-mediated event in a mammal in need thereof.

In another aspect the present invention relates to the use of a compound selected, for example, from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to bind a laminin receptor, and e) combination of any one of a) through d) thereof or any other PSP94 derived molecule in the manufacture of a pharmaceutical composition (or drug) for promoting laminin receptor signal transduction and laminin receptor signal transduction-mediated event, or for promoting laminin signal transduction-mediated event, for inhibiting laminin and laminin-induced laminin receptor mediated events (e.g., signal transduction) in a mammal.

In another aspect, the present invention provides the use of a compound which may be selected from the group consisting of; a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative, c) a SEQ ID NO.:5 biologically active fragment, d) a SEQ ID NO.:5 biologically active analog, and e) combination of any one of a) through d), in the preparation of a pharmaceutical composition (drug) for treating leukemia or other cancers (prostate cancer, breast cancer, etc.). In accordance with the present invention, a suitable biologically active fragment, derivative or analog is one that may be able to inhibit cell growth in an assay as described herein.

In yet another aspect the present invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound selected, for example, from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to

bind a laminin receptor, and e) combination of any one of a) through d) thereof or any other PSP94 derived molecule for promoting laminin receptor signal transduction and/or laminin receptor signal transduction-mediated event, or for promoting laminin signal transduction-mediated event in a mammal, for inhibiting laminin and laminin- induced laminin receptor mediated signal transduction in a mammal and/or for treating cancer (leukemia) in a mammal in need thereof.

The present invention further provides a method for detecting a laminin receptor (in a cell (e.g., on a cell surface), in a tissue, in a mammal, etc.) or a laminin receptor- expressing cell or tissue, the method may comprise contacting a cell or tissue with a laminin receptor binding molecule of the present invention.

In accordance with the present invention biologically active compounds may comprise, for example, at least one amino acid substitution in the SEQ ID NO.:5 sequence.

Also in accordance with the present invention, the compound may be viewed, for example, as a laminin receptor agonist. More particularly, the compound may be viewed as a laminin receptor partial agonist. Also more particularly and further in accordance with the present invention, the compound may be viewed as a laminin receptor partial competitive agonist, laminin (and/or SIKVAV, and analogues thereof) being natural ligands of the laminin receptor.

In accordance with the present invention, the laminin receptor binding molecule may be peptidic. Also in accordance with the present invention, the laminin receptor binding molecule may be a PSP94 derived molecule. In addition, further in accordance with the present invention, the laminin receptor binding molecule may be selected, for example, from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to bind a laminin receptor, and e) combination of any one of a) through d).

Further in accordance with the present invention, the laminin-induced laminin receptor mediated events (e.g., signal transduction) which may be inhibited by the compounds of the present invention, may comprise, for example, MMP (and/or pro-MMP) secretion, expression and/or activity, and metastatis as well as events other than MMP (and/or pro-MMP) secretion, expression and/or activity or metastasis. The compounds

of the present invention may thus be used, for example, in the inhibition of differentiation of endothelial cells or the inhibition of leukemia cells (acute myeloid leukemia).

A "molecule derived from PSP94" is to be understood herein as a polypeptide originating from PSP94. For example, a PSP94 fragment, a PSP94 derivative, a PSP94 analogue, PCK3145 (SEQ ID NO.:5), a PCK3145 fragment, a PCK3145 derivative, a PCK3145 analogue, etc.

Therefore, in accordance with the present invention, a "molecule derived from PSP94" may be selected, for example, from the group consisting of a) a SEQ ID NO.:1 biologically active derivative, b) a SEQ ID NO.:1 biologically active fragment, c) SEQ ID NO.:1 biologically active analogue, d) SEQ ID NO.:5, e) a SEQ ID NO.:5 biologically active derivative, f) a SEQ ID NO.:5 biologically active fragment, g) a SEQ ID NO.:5 biologically active analogue, h) SEQ ID NO.:7, and i) combination of any one of a) through h) thereof.

A "molecule derived from PSP94" may also include, for example, SEQ ID NO.: 3, SEQ ID NO.:4, SEQ ID NO.:6, as well as SEQ ID NO.: 9 to 98.

In accordance with the present invention the SEQ ID NO.:1 fragment may be selected, for example, from the group consisting of SEQ ID NO.:4 and SEQ ID NO.:6.

Also in accordance with the present invention the SEQ ID NO.:1 derivative may be selected, for example, from the group consisting of SEQ ID NO.:2 and SEQ ID NO.:3.

A "fragment" is to be understood herein as a polypeptide originating from a portion of an original or parent sequence. Fragments encompass polypeptides having truncations of one or more amino acids, wherein the truncation may originate from the amino terminus (N-terminus), carboxy terminus (C-terminus), or from the interior of the protein. A fragment may comprise the same sequence as the corresponding portion of the original sequence. For example, SEQ ID NO.: 4, SEQ ID NO.: 5 and SEQ ID NO.: 6 fall into the definition of "a PSP94 fragment"; when considering PSP94 (SEQ ID NO.:1) as an original sequence.

A "derivative" is to be understood herein as a polypeptide originating from an original sequence or from a portion of an original sequence and which may comprise one or more modification; for example, one or more modification in the amino acid sequence (e.g., an amino acid addition, deletion, insertion, substitution etc.), one or more modification in the backbone or side-chain of one or more amino acid, or an addition of a group or another molecule to one or more amino acids (side-chains or backbone). For example, SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 7 fall into the definition of "a PSP94 derivative"; when considering PSP94 (SEQ ID NO.:1) as an original sequence.

It is to be understood herein that SEQ ID NO.: 7 may fall into the definition of "a PCK3145 derivative" or "SEQ ID NO.:5 derivative when considering PCK3145 (SEQ ID NO.:5) as an original sequence. The addition of polyethylene glycol group (i.e., pegylation) to PCK3145 (SEQ ID NO.:5 or SEQ ID NO.: 7) also falls within the definition of "a PCK3145 derivative". It is to be understood that SEQ ID NO.: 5 and SEQ ID NO.: 7 are biologically equivalent as demonstrated herein and may be interchanged.

An "analogue" is to be understood herein as a molecule having a biological activity and chemical structure similar to that of a polypeptide described herein. An "analogue" may have sequence similarity with that of an original sequence or a portion of an original sequence and may also have a modification of its structure as discussed herein. For example, an "analogue" may have at least 90 % sequence similarity with an original sequence or a portion of an original sequence. An "analogue" may also have, for example; at least 70 % or even 50 % sequence similarity (or less, i.e., at least 40%) with an original sequence or a portion of an original sequence. Also, an "analogue" may have, for example, 50 % sequence similarity to an original sequence with a combination of one or more modification in a backbone or side-chain of an amino acid, or an addition of a group or another molecule, etc.

Thus, biologically active polypeptides in the form of the original polypeptides, fragments (modified or not), analogues (modified or not), derivatives (modified or not), homologues, (modified or not) of PSP94 and PCK3145 are encompassed by the present invention.

Therefore, any polypeptide having a modification compared to an original polypeptide (e.g., PSP94, PCK3145) which does not destroy significantly a desired biological

activity is encompassed herein. It is well known in the art, that a number of modifications may be made to the polypeptides of the present invention without deleteriously affecting their biological activity. These modifications may, on the other hand, keep or increase the biological activity of the original polypeptide or may optimize one or more of the particularity (e.g. stability, bioavailability, etc.) of the polypeptides of the present invention which, in some instance might be desirable. Polypeptides of the present invention comprises for example, those containing amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are known in the art. Modifications may occur anywhere in a polypeptide including the polypeptide backbone, the amino acid side-chains and the amino- or carboxy-terminus. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslational natural processes or may be made by synthetic methods. Modifications comprise for example, without limitation, pegylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to fiavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of drug, covalent attachment of a marker (e.g., fluorescent, radioactive, etc.), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination, etc. It is to be understood herein that more than one modification to the polypeptides described herein are encompassed by the present invention to the extent that the biological activity is similar to the original (parent) polypeptide.

As discussed above, polypeptide modification may comprise, for example, amino acid insertion (i.e., addition), deletion and substitution (i.e., replacement), either conservative or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide sequence where such changes do not substantially alter the overall

biological activity of the polypeptide which is, for example, to reduce the level of expression of matrix metalloproteinases or pro-matrix metalloproteinases and/or to reduce their enzymatic activity.

Example of substitutions may be those, which are conservative (i.e., wherein a residue is replaced by another of the same general type or group) or when wanted, non- conservative (i.e., wherein a residue is replaced by an amino acid of another type). In addition, a non-naturally occurring amino acid may substitute for a naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non- naturally occurring non-conservative amino acid substitution).

As is understood, naturally occurring amino acids may be sub-classified as acidic, basic, neutral and polar, or neutral and non-polar. Furthermore, three of the encoded amino acids are aromatic. It may be of use that encoded polypeptides differing from the determined polypeptide of the present invention contain substituted codons for amino acids, which are from the same type or group as that of the amino acid to be replaced. Thus, in some cases, the basic amino acids Lys, Arg and His may be interchangeable; the acidic amino acids Asp and GIu may be interchangeable; the neutral polar amino acids Ser, Thr, Cys, GIn, and Asn may be interchangeable; the non-polar aliphatic amino acids GIy, Ala, VaI, lie, and Leu are interchangeable but because of size GIy and Ala are more closely related and VaI, lie and Leu are more closely related to each other, and the aromatic amino acids Phe, Trp and Tyr may be interchangeable.

It should be further noted that if the polypeptides are made synthetically, substitutions by amino acids, which are not naturally encoded by DNA (non-naturally occurring or unnatural amino acid) may also be made.

A non-naturally occurring amino acid is to be understood herein as an amino acid which is not naturally produced or found in a mammal. A non-naturally occurring amino acid comprises a D-amino acid, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, etc. The inclusion of a non-naturally occurring amino acid in a defined polypeptide sequence will therefore generate a derivative of the original polypeptide. Non-naturally occurring amino acids (residues) include also the omega amino acids of the formula

NH ₂ (CH ₂ ) _n COOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t- butyl alanine, t-butyl glycine, N-methyl isoleucine, norleucine, etc. Phenylglycine may

substitute for Trp, Tyr or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.

It is known in the art that analogues may be generated by substitutional mutagenesis and retain the biological activity of the polypeptides of the present invention. These analogues have at least one amino acid residue in the protein molecule removed and a different residue inserted in its place. For example, one site of interest for substitutional mutagenesis may include but are not restricted to sites identified as the active site(s), or immunological site(s). Other sites of interest may be those, for example, in which particular residues obtained from various species are identical. These positions may be important for biological activity. Examples of substitutions identified as "conservative substitutions" are shown in table 1. If such substitutions result in a change not desired, then other type of substitutions, denominated "exemplary substitutions" in table 1 , or as further described herein in reference to amino acid classes, are introduced and the products screened.

In some cases it may be of interest to modify the biological activity of a polypeptide by amino acid substitution, insertion, or deletion. For example, modification of a polypeptide may result in an increase in the polypeptide's biological activity, may modulate its toxicity, may result in changes in bioavailability or in stability, or may modulate its immunological activity or immunological identity. Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties:

(1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (VaI),

Leucine (Leu), lsoleucine (lie)

(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr)

(3) acidic: Aspartic acid (Asp), Glutamic acid (GIu)

(4) basic: Asparagine (Asn), Glutamine (GIn), Histidine (His), Lysine (Lys), Arginine (Arg)

(5) residues that influence chain orientation: Glycine (GIy), Proline (Pro); and aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe)

Non-conservative substitutions will entail exchanging a member of one of these classes for another.

Example of biologically active analogues of PCK3145 (SEQ ID NO: 5) exemplified by amino acid substitutions is illustrated below.

Position 1 5 10 15 PCK3145 E W Q T D N C E T C T C Y E T (SEQ ID

X ₁ W Q X ₂ D X ₁ C X ₁ X ₂ C X ₂ C X ₃ X ₁ X ₂ NO.:88)

For example, Xi may be glutamic acid (i.e., glutamate) (GIu), aspartic acid (aspartate) (Asp), or asparagine (Asn), X ₂ may be threonine (Thr) or serine (Ser) and X ₃ may be tyrosine (Tyr) or phenylalanine (Phe). Any replacement of an original residue in SEQ ID NO.:5 with a conserved amino acid (i.e. conservative substitution) is encompassed by the present invention.

Another example of a PCK3145 (SEQ ID NO: 5) analogue may include, for example, a polypeptide as exemplified in SEQ ID NO.:88 or any other polypeptide having at least one conservative amino acid substitution (illustrated in bold below) as defined in Table 1 , such as, for example;

GIu Tyr GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr (SEQ ID NO.:92)

GIu Trp Asn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr (SEQ ID NO.:93)

GIu Trp GIn Thr Asp GIn Ser GIu Thr Cys Thr Cys Tyr Asp Thr (SEQ ID NO.:94)

Examples of a PCK3145 (SEQ ID NO: 5) derivative may include, for example, a polypeptide having an addition in one or both of the terminal region (amino-terminal or carboxy-terminal) as illustrated in SEQ IDs No.: 9 to 87, a peptide having a stabilizing group such as exemplified in SEQ ID NO.:7, or a peptide having one or more repeats of SEQ ID No.:5 such as exemplified in SEQ ID NOs.: 89 to 91, a polypeptide having at least one D-amino acid as exemplified in SEQ ID No. 98 and combination thereof.

An example of a PCK3145 (SEQ ID NO: 5) fragment may include, for example, a polypeptide having a truncation in one or both of the terminal regions (amino-terminal or carboxy-terminal) as illustrated below.

Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr (SEQ ID NO.:95)

GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr (SEQ ID NO.:96)

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys (SEQ ID NO.:97)

Polypeptides may be either naturally occurring (that is to say, substantially purified or isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the encoding DNA or made by other synthetic methods such as

chemical synthesis). It is thus apparent that the polypeptides of the invention can be either naturally occurring or recombinant (that is to say prepared from the recombinant DNA techniques) or made by chemical synthesis (e.g., organic synthesis).

As used herein, "pharmaceutical composition" means therapeutically effective amounts of the agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. A "therapeutically effective amount" as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCI., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts). Solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermal^, subcutaneously, intraperitonealy, intraventricularly, intracranially and intratumorally. The formulations include those suitable for oral, rectal, ophthalmic, (including intravitreal or intracameral) nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active

ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into associate the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally coated or scored and may be formulated so as to provide a slow or controlled release of the active ingredient therein.

Formulations suitable for topical administration in the mouth include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier.

Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the ingredient to be administered in a pharmaceutical acceptable carrier. An example of a topical delivery system is a transdermal patch containing the ingredient to be administered.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of 20 to 500 microns which is administered in the manner in which snuff is administered, i.e., by rapid

inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented as pessaries, tamports, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Further, as used herein "pharmaceutically acceptable carrier" or "pharmaceutical carrier" are known in the art and include, but are not limited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8 % saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

It is to be understood herein, that if a "range" or "group" of substances (e.g. amino acids), substituents" or the like is mentioned or if other types of a particular characteristic (e.g. temperature, pressure, chemical structure, time, etc.) is mentioned,

the present invention relates to and explicitly incorporates herein each and every specific member and combination of sub-ranges or sub-groups therein whatsoever. Thus, any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein. Thus, for example,

with respect to a temperature greater than 100° C, this is to be understood as specifically incorporating herein each and every individual temperature state, as well as sub-range, above 100° C, such as for example 101° C, 105° C and up,

110° C and up, 115° C and up, 110 to 135° C, 115° c to 135° C, 102° C to 150° C, up to 210° C, etc.; and similarly with respect to other parameters such as, concentrations, elements, etc...

It is in particular to be understood herein that the polypeptides of the present invention each include each and every individual polypeptide described thereby as well as each and every possible mutant, variant, homolog, analogue or else whether such mutant, variant, homolog, analogue or else is defined as positively including particular polypeptides, as excluding particular polypeptides or a combination thereof; for example an exclusionary definition for a polypeptide analogue (e.g. X _I WQX ₂ DX _I CX ₁ X ₂ CX ₂ CX ₃ XIX ₂ (SEQ ID NO.88)) may read as follows: "provided that when one of Xi is glutamic acid and X ₂ is threonine X ₃ may not be phenylalanine" or else.

It is also to be understood herein that "g" or "gm" is a reference to the gram weight unit; that "C" is a reference to the Celsius temperature unit.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrates exemplary embodiments of the present invention;

Fig. 1A is a picture of a zymography gel showing the effect of the PCK3145 derivative (SEQ ID NO.:7) on MMP-9 levels and activity on collagen type 1- treated MatLyLu cells (first lane:marker; second lane:cells; third lane:cells and collagen; fourth lane:cells, collagen and 500μg/ml of SEQ ID NO.:7; fifth laneicells, collagen and 1mg/ml of SEQ ID NO.:7),

Fig. 1B is a picture of a western blot membrane showing the effect of the PCK3145 derivative (SEQ ID NO.:7) on MMP-9 expression level (fist lane: MMP9 standard; second lane:cells; third lane:cells and collagen; fourth lanexells, collagen and 100μg/ml of SEQ ID NO.:7; fifth lane: cells, collagen and 500μg/ml of SEQ ID N0..7; sixth lane: cells, collagen and 1mg/ml of SEQ ID NO.:7),

Fig. 1C is a picture of a time-course/dose-response experiment of a MMP-9 secretion (upper panel) and a graph (lower panel) expressing these results in a quantitative manner;

Fig. 2 is a picture of a MMP-9 zymography assay performed on cells induced with cytokines in the presence or absence of PCK3145;

Fig. 3A is the result of a sequence homology search performed on the PCK3145 amino acid sequence (SEQ ID NO.:5) with the Gene Bank internet database,

Fig. 3B is a picture of a westernblot illustrating the inhibitory effect of laminin on PCK3145 (PCK) induced ERK phosphorylation,

Fig. 3C is a picture of a further westernblot illustrating the inhibitory effect of laminin on PCK3145 (PCK) induced ERK phosphorylation,

Fig. 4A is a picture of a further westernblot illustrating expression of the laminin receptor in several cell lines,

Fig. 4B represents pictures of western blots of PCK3145-induced ERK phosphorylation in laminin receptor transfected U-87 cells or non-transfected cells (mock) and a graph illustrating the results in a quantitative manner,

Fig. 5A is a picture of a westernblot performed with anti-laminin receptor or beta-actin antibody on various fractions collected from an affinity column coupled with PCK3145 (Affi-PCK) or not (AfR-CtI) ₁

Fig. 5B is a histogram illustrating the results of Fig. 5A in a quantitative manner,

Fig. 6A is a graph illustrating cell binding of Iabeled-PCK3145 at 4 ⁰ C and intracellular uptake of labeled-PCK3145 at 37 ⁰ C and the dilution effect of added unlabelled PCK3145 (cold),

Fig. 6B is a histogram illustrating PCK3145 uptake competition experiments performed with scrambled polypeptides 1 (s1) and 2 (s2), native PSP94 (PSP), unlabelled PCK3145 (PCK) and laminin (LAM), Ctl=control,

Fig. 6C is a histogram illustrating PCK3145 uptake competition experiments performed with green tea catechin (EGCg), green tea catechin lacking gallate moiety (EGC), unlabelled PCK3145 (cold PCK) and laminin (LAM), Ctl=control,

Fig. 7A is dose-response of FITC-labeled PCK3145 binding to the cell surface measured by FACS analysis;

Fig. 7B is a graph obtained from FACS analysis of FITC-labeled PCK3145 binding to the cell surface in the absence (upper panel) and in the presence of laminin (lower panel);

Fig. 8A is a picture of a zymography gel showing the effect of the PCK3145 on MMP-9 levels in the presence or absence of laminin or the laminin-derived SIKVAV peptide;

Fig. 8B is a histogram quantifying the results obtained in Fig. 8A;

Fig. 9A is a picture illustrating the results of a time-course ERK phosphorylation experiment determined by a Western blot performed on lyzates of cells incubated with various concentration of PCK3145 for;

Fig. 9B is a picture illustrating ERK-phophorylation determined by a Western blot performed on lyzates of cells incubated for 1 minute with various concentration of PCK3145 with or without the SIKVAV peptide;

Fig. 10A are pictures of cells transfected with a laminin receptor or not (mock) adhering to PCK3145-coated dishes;

Fig. 10B is a graph of a time-course of cell (transfected with a laminin receptor or not) adhesion experiment performed on PCK3145-coated dish;

Fig. 11 A are pictures of stained PVDF membranes obtained following electrophoresis and transfer of proteins eluted from a PCK3145 coupled affinity matrix;

Fig. 11B represents the sequence of a first PCK3145 binding protein; serum albumin;

Fig. 11C represents the sequence of a first PCK3145 binding protein; enolase;

Fig. 12A is a graph showing inhibition of cell adhesion to PCK3145-coated dishes by enolase or EGCg;

Fig. 12B is a histogram showing results of competition of FITC-labeled PCK3145 binding to cells by enolase by FACS analysis;

Fig. 13A -panel A. represents graphs obtained from FACS analysis showing binding of biotinilated-laminin on cell surface (control; upper left panel) and inhibition of binding by excess unlabelled laminin (upper right panel), by PCK3145 (lower left panel) and by EGCg (lower right panel); panel B. is a histogram illustrating the results of panel A. in a quantitative manner;

Fig. 13B are pictures of PVDF membranes obtained following electrophoresis of cell lyzates or enolase and transfer to membranes; detection was performed with biotinilated-laminin and streptavidin-HRP (horse radish peroxidase) or biotinilated- laminin and an anti-biotin antibody;

Fig. 14 -panel A. is a picture of a gel loaded with RT-PCR-amplified HuR RNA obtained from cells incubated with PCK3145 in the presence or absence of laminin (Lam) or the SIKVAV peptide; panel B. is a picture of a western blot performed on lyzates obtained from cells incubated with PCK3145 in the presence or absence of laminin (Lam) or the SIKVAV peptide where the level of HuR protein expression is detected with an anti-HuR antibody;

Fig. 15 -panel A. represents graphs obtained from FACS analysis of cells incubated or not with PCK3145 and detected with either a labeled anti-CD44 antibody or a labeled anti-MMP-9 antibody; panel B. are histograms illustrating the results of panel A. in a quantitative manner (white bars; without PCK3145, grey bars; with PCK3145);

Fig. 16 is a schematic showing the biological effect of PCK3145;

Fig.17A are graphs illustrating binding (left) of [ ¹⁴ C]labelled-PCK3145 to HT-1080 fibrosarcoma cells at 4 ⁰ C and uptake (right) of [ ¹⁴ C]labelled-PCK3145 by HT-1080 fibrosarcoma cells at 37 ⁰ C;

Fig. 17B is a graph illustrating binding and uptake of increasing concentrations of [ ¹⁴ C]labelled-PCK3145 to HT-1080 fibrosarcoma cells;

Fig. 18A is a histogram illustrating cell surface binding (white bars) or uptake (grey bars) of PCK3145 in the presence or not of laminin, hyaluronic acid (HA), or type-l collage, probability values of less than 0.05 were considered significant, and an asterisk ( ^** ) identifies such significance cell surface binding and uptake experiments;

Fig. 18B are graphs illustrating the relative PCK3145 binding or uptake in the presence or not of type-l collagen, insert represents the representative dose response as monitored for PCK3145 uptake in the presence of low type-l collagen concentrations (data are expressed as the % of control basal PCK3145 uptake and cell surface binding);

Fig. 19-Left panel, represents a picture of a Far-western blot of a 10% SDS-PAGE loaded with lysate from confluent HT-1080 cells and transferred to PVDF membranes incubated (+) or not (-) with biotin-labeled PCK3145 and bands revealed using Streptavidin-HRP coupled to ECL detection; -Right panel, represents a picture of the same PVDF membrane blotted with an anti-67 kDa laminin receptor (67LR) antibody.

Fig. 20-panel A. are pictures showing several cell density (10 ⁴ to 10 ⁶ cells/well); panel B. s Cell lysates were isolated in duplicate from each cell confluence culture condition. SDS-PAGE and immunoblotting with the anti-67LR was performed at 24 hrs.; panel C. is a histogram of results of panel B. expressed in a quantitative manner; panel D. shows a time-course experiment of a laminin receptor expression in cells at a density of 10 ⁵ by Western blot;

Fig. 21 -panel A. is an histogram representing the effect of the 67-kDa laminin receptor expression (dependent of cell confluence) on the inhibition of cell surface PCK3145 binding by EGCg in HT-1080 cells (10 ⁴ cells/well, white bars; 10 ⁶ cells/well, grey bars), control is without EGCg;-panel B. is a picture showing proMMP-9 secretion from low

and high confluent cells monitored by gelatin zymography in serum-starved cells that were incubated or not with 300 μg/ml PCK3145 for 48 hrs, -panel C. is an histogram representing the results of panel B (white bars; without PCK3145, grey bars; with PCK3145);

Fig. 22A is an immunoblot performed on lysates of HT-1080 cells which have been transfected or not with a cDNA encoding the 67LR (laminin receptor of 67 Kda), and;

Fig. 22B is a histogram representing PCK3145 cell surface binding and uptake on HT- 1080 cells which have been transfected or not with a cDNA encoding the 67LR Mock (white bars) and 67LR-transfected (grey bars).

DETAILED DESCRIPTION OF THE INVENTION

Test compound. The wild type amino acid sequence of PCK3145 has been disclosed, for example, in international application No.: PCT/CA01/01463 and is defined herein in SEQ ID NO.: 5. A PCK3145 derivative has been generated by attaching an acetylaminomethyl group to the sulfur atom of each of the three cysteines of PCK3145. These groups stabilize the compound by preventing formation of peptide dinners or polymer by blocking the sulfhydryl group of cysteines. This PCK3145 derivative is defined in SEQ ID NO.: 7. The drug was manufactured by Multiple Peptide Systems (3550) (General Atomics Court, San Diego, Calif.) using standard solid-phase peptide chemistry and lyophilized into a powder. Other type of synthesis or manufacture method may however be performed to make a peptide or polypeptide of the invention. Other PCK3145 derivatives, analogs and fragments (e.g., SEQ IDs NO: 88, 98, etc.) may be generated similarly.

Materials. Sodium dodecylsulfate (SDS) and bovine serum albumin (BSA) were purchased from Sigma (Oakville, ON). FUGENE-6 transfection reagent was from

Roche Diagnostics Canada (Laval, QC). Cell culture media was obtained from Life

Technologies (Burlington, ON). Electrophoresis reagents were purchased from Bio-

Rad (Mississauga, ON). The polyclonal antibody against the 67-kDa laminin receptor

(67LR) was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA), while the mouse β-actin monoclonal antibody was from Sigma. The enhanced chemiluminescence (ECL) reagents were from Amersham Pharmacia Biotech (Baie d'Urfe, QC). Micro bicinchoninic acid protein assay reagents were from Pierce

22

(Rockford, IL). Fluorescein isothiocyanate (FITC)-N-terminal-labeled PCK3145 was synthesized by EZBiolab (Westfield, IN). All other reagents were from Sigma-AIdrich Canada.

Ce// culture and cDNA transfection method : The HT- 1080 cell line was purchased from American Type Culture Collection and maintained in Dulbecco Minimum Essential Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT), 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and were cultured at 37 ⁰ C under a humidified atmosphere containing 5% CO ₂ . The human 67LR full-length cDNA was from OriGene Technologies (Rockville, MD). HT-1080 cells were transiently transfected with cDNA using the non-liposomal formulation FUGENE-6 transfection reagent. Transfection efficiency was confirmed by western blotting. All experiments involving these cells were performed 36 hrs following transfection. Mock transfections of HT-1080 cultures with pcDNA (3.1+) expression vector alone were used as controls.

Membrane protein labeling : Endothelial or cancer cell membrane proteins will be biotin labeled with EZ-Link Sulfo-NHS-Biotin (Pierce). Confluent dishes containing approximately 3*10 ⁶ cells will be rinsed with cold PBS and incubated at 4 ⁰ C with 4 mg of Sulfo-NHS-Biotin. The biotin reagent will be neutralized with Tris-buffer saline. Cells will then be scraped off the dish, pelleted by centrifugation, and lysed with 0.5% Nonidet P-40 in PBS (NP/PBS). Soluble-labeled membrane proteins will be collected after centrifugation at 11,000g at 4°C and used for either BIAcore studies, immunoprecipitations or affinity gel chromatography approaches.

Affinity chromatography will be performed with PCK3145 or scrambled peptide linked to Affi-gel 10 or 15. Four mg of peptide will be coupled overnight to Affi-gel resin in 100 mmol/L sodium carbonate buffer, pH 8.5. Remaining active groups blocked with 100 mmol/L diethanolamine, and the resin equilibrated with NP/PBS. Beads blocked with diethanolamine or to which scrambled peptide or PCK3145 were coupled will be used as control. Biotin-labeled membrane proteins will be passed through the column and allowed to incubate for 30 min. After washing with 50 ml of NP/PBS, bound proteins will be eluted successively with either increasing concentrations of PCK3145, or with successively 5 mmol/L EDTA, 1 mol/L NaCI, and 4 mol/L urea. Fractions will be collected (500 μl) and screened for the presence of biotin-labeled proteins. Biotin- containing fractions will be pooled and dialyzed against water. Aliquots from each

fraction will be electrophoresed on 7-12% SDS-PAGE and transferred to nitrocellulose membranes, and biotin-labeled proteins detected with streptavidin-horseradish peroxidase and enhanced chemiluminescence (ECL). Differentially expressed proteins will then be sent for amino acid sequencing.

Materials. Cell culture media were obtained from Life Technologies (Burlington, Ontario, Canada) and serum was purchased from Hyclone Laboratories (Logan, UT). Electrophoresis reagents were purchased from Bio-Rad (Mississauga, Ontario, Canada). The polyclonal (C-1158) and monoclonal (A3) antibodies, used for precipitation and detection, respectively, of VEGFR-2, and the anti-PDGFR pAb (958) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Antiphosphotyrosine mAb PY99 was also purchased from Santa Cruz Biotechnologies. Anti-phospho-ERK polyclonal antibodies were from Cell Signaling Technology (Beverly, MA). Antimouse and anti-rabbit horseradish peroxidase-linked secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) and enhanced chemiluminescence (ECL) reagents were from Amersham Pharmacia Biotech (Baie d'Urfe, Quebec, Canada). Human recombinant PDGF was obtained from R&D Systems (Minneapolis, MN). Micro bicinchoninic acid protein assay reagents were from Pierce (Rockford, IL). All other reagents were from Sigma-Aldrich Canada.

lmmunoprecipitation and immunoblotting procedures. After treatment with cytokines, cells were washed once with phosphate-buffered saline (PBS) containing 1 mM sodium orthovanadate and were incubated in the same medium for 1 h at 4°C. The cells were solubilized on ice in lysis buffer (150 mM NaCI, 10 mM Tris-HCI, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, 1% Triton X-100) containing 1 mM sodium orthovanadate. The cells were then scraped from the culture dishes and the resulting lysates were clarified by centrifugation at 10,00Og for 10 min. Protein concentrations were determined using the micro bicinchoninic acid method. For immunoprecipitation studies, lysates were clarified by a 1 h incubation at 4 ⁰ C with a mixture of Protein A/Protein G Sepharose beads. After removal of the Sepharose beads by low-speed centrifugation, identical amounts of protein (200 μg) from each sample were transferred to fresh tubes and incubated in lysis buffer overnight at 4°C in the presence of 2 μg/ml of specific antibodies. Immunocomplexes were collected by incubating the mixture with 25 μl (50% suspension) of Protein A- (rabbit primary antibody) or Protein G- (mouse primary antibody) Sepharose beads, for 2 h. Nonspecifically-bound material was removed by washing the beads three times in 1 ml

of lysis buffer containing 1 mM sodium orthovanadate, and bound material was solubilized in 25 μl of two-fold concentrated Laemmli sample buffer, boiled 5 min, and resolved by SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, blocked 1 h at room temperature with Tris-buffered saline/Tween 20 (147 mM NaCI, 20 mM Tris/HCI, pH 7.5, and 0.1% Tween 20) containing 2% bovine serum albumin and incubated overnight at 4 ⁰ C with primary antibody. Immunoreactive bands were revealed after a 1 h incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies, and the signals were visualized by enhanced chemiluminescence (Amersham Biosciences, Baie d'Urfee, QC).

General lmmunoblotting procedures: Proteins from control and treated cells were separated by SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were electrotransferred to polyvinylidene difluoride membranes which were then blocked overnight at 4°C with 5% non-fat dry milk in Tris-buffered saline (150 mM NaCI, 20 mM Tris-HCI, pH 7.5) containing 0.3% Tween-20 (TBST). Membranes were further washed in TBST and incubated with the primary antibodies (1/1 ,000 dilution) in TBST containing 3% bovine serum albumin, followed by a 1 hr incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1/10,000 dilution for 67LR) or anti- mouse IgG (1/5,000 dilution) in TBST containing 5% non-fat dry milk. Immunoreactive material was visualized by enhanced chemiluminescence (Amersham Biosciences, Baie d'Urfee, QC).

Biologically active molecule; Fragments, derivatives and analogues may be prepared by techniques known in the art (recombinant technology, solid phase synthesis, etc.). The biological activity of derivatives, fragments and analogues may be determined by any of the techniques described herein (effect on ERK phosphorylation) or known in the field to be relevant for any of the biological activity described herein.

Statistical data analysis : Data are representative of three or more independent experiments. Statistical significance was assessed using Student's unpaired West and was used to compare the PCK3145 effect to vehicle treated cells. Probability values of less than 0.05 were considered significant, and an asterisk (*) identifies such significance in each figure.

EXAMPLE 1 Effect on MMP-9 extracellular levels

Zymography assays and western blots were performed on cell lines incubated with a PCK3145 derivative (SEQ ID NO.:7).

In the experiment presented in Fig. 1A, 2.5x10 ⁵ MatLyLu tumor cells (American Type Culture Collection No.: JHU-5)) were seeded in T-25 flasks containing RPMI with 10% fetal bovine serum (FBS). After overnight incubation, the cells were washed once with serum free medium and treated with various concentrations of the PCK3145 derivative (500ug/ml and 1 mg/ml) in the presence of 50ug/ml collagen type-l in serum free RPMI for 72 hrs. Control cells received 50ug/ml collagen or only serum free medium.

The media were collected after 72 hours of exposure to the PCK3145 derivative and subjected to gelatin zymography. Zymography for MMP-2 and MMP-9 was performed in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (10%) containing 0.1% gelatin (Invitrogen). Twenty-four microliters of culture media was mixed with non- reducing sample buffer and subjected to electrophoresis without boiling. After electrophoresis, gels were soaked for 30 minutes in 2.5% Triton X-100 solution with 2- 3 washing steps. The gels were then incubated for 18 hours at 37 ⁰ C in buffer containing 50 mM Tris/HCI, pH 7.6, 50 mM NaCI, 1OmM CaCI ₂ and 0.05% Brij-35. After incubation, the gels were stained with 0.2% Coomassie blue and de-stained until clear proteolytic bands appeared. Gels were scanned with Microtek flatbed scanner (Scanmaker 5 software; Microtek lab, Redondo Beach, CA). The band intensities were determined using the Image Quant software (version 5.0) from molecular Dynamics.

The MMP-9 and MMP-2 gelatinase zymography standard were purchased from Chemicon (catalogue no. CC073). One nanogram of purified human pro-MMP-2 and pro-MMP-9 standards were used in every gel run.

Results of this experiment are illustrated in Fig.1A and indicate that PCK3145 derivative treatment of MatLyLu cells resulted in a dose-dependent reduction of MMP- 9 secreted in the cell culture media (a reduction in MMP-9 extracellular levels), as detected by zymography.

Western blot

A separate western blot experiment was performed in which MatLyLu cells were treated with 100ug/ml, 500ug/ml and 1 mg/ml of the PCK3145 derivative for 72 hrs. At the end of the experiment, the media were collected and concentrated 5 times using Amicon centrifugal filter devices (3500 molecular weight cut-off).

Twenty five microliters samples were separated on SDS-PAGE gel under reducing conditions using pre-cast gels of 4-12% Bis-Tris (Invitrogen). Following electrophoresis, the proteins were transferred on nitrocellulose membrane. Non- specific binding sites were blocked using 5 % skimmed milk in 1OmM phosphate buffer saline (PBS) containing 0.05% Tween-20 for 1 hour at room temperature. The membrane was later incubated with a primary antibody (monoclonal, RDI-MMP-9abm- 2A5) at a concentration of 1 ug/ml (in 10 mM PBS, containing 0.5% bovine serum albumin (BSA) and 0.05% Tween-20) for 3 hours at room temperature.

The membranes were washed three times in PBS (5 minutes each wash) to remove non-specific binding and they were incubated with the secondary antibody (Rabbit anti- mouse IgG horseradish peroxidase-conjugated (Dako no. 0260)) at a dilution of 1 :5000 for one hour.

Detection of specific MMP-9 protein was made by incubating the membrane in ECL™ reagent (enhanced-chemoluminescence, Roche) and exposing to the X-ray film.

Results of this experiment are illustrated in Fig.1B and again indicate that treatment of MatLyLu cells PCK3145 derivative resulted in a dose-dependent reduction of MMP-9 extracellular levels.

A time-course/dose-response experiment of a MMP-9 secretion (upper panel) was also performed in the presence or absence (control) of PCK3145 (Fig. 1C).

In order to investigate the effect of PCK3145 on MMP-9 secretion in vitro, we treated serum-starved HT-1080 fibrosarcoma cells with increasing doses of PCK3145 for 24 and 48 hrs. Doses of PCK3145 were found not to be cytotoxic as assessed by the measurement of the pro-apoptotic caspase-3 activity (not shown). MMP-9 extracellular levels were then assessed by western blotting and immunodetection. While MMP-9 extracellular levels continued to increase from 24 to 48 hrs in untreated cells, those from PCK3145-treatments decreased in a dose-dependent manner. Interestingly, a 24

hours treatment with PCK3145 decreased MMP-9 by 20%, while a treatment of 48 hrs was needed to inhibit by approximately 80% the extracellular levels of MMP-9. This result confirms those observed in the phase Ua clinical trial on the efficacy of PCK to decrease MMP-9 levels up to 90%, and suggests that PCK3145 inhibits intracellular signal transduction mechanisms that regulate MMP-9 secretion.

EXAMPLE 2 Effect on cytokine-induced MMP-9 extracellular levels PCK3145 has been shown to inhibit basal MMP-9 secretion from cells including HT- 1080 cells, a human fibrosarcoma cell line provided by American Type Culture Collection (ATCC, Manassas, USA) (data not shown). The inhibitory effect was also observed on phorbol ester (PMA)- and tumor necrosis factor (TNF)-induced MMP-9 secretion (Fig. 2). These two agents trigger specific intracellular signaling that lead either to intracellular Ca ⁺⁺ release and protein kinase C (PKC) activation, or to TNF- induced signaling that collectively result into strong MMP-9 production. PCK3145 can thus be considered as a potential inhibitor of intracellular signal transduction as it antagonizes both PMA- and TNF-induced MMP-9 secretion as observed by gelatin- zymography (Fig 2).

EXAMPLE 3 Sequence homology

Using GeneBank internet data base, a homology search was performed using the 15 amino acid sequence of PCK3145. Homology was found with different chain precursors of laminin (Fig.3A). These included the alpha-2, alpha-5, and beta-l chains of laminin.

The unexpected observation that PCK3145 shares, to some extent, homology with different structural chains of laminin suggests that it may indeed also share the same cell surface receptor.

EXAMPLE 4 Competition assays Competitions experiments using several extra-cellular matrix (ECM) purified proteins (including laminin), on the potential of PCK3145 to induce ERK {Extracellular-signal- Regulated protein Kinases 1 or 2) phosphorylation in U-87 cells were then performed.

Indeed, laminin was able to inhibit PCK3145-induced ERK phosphorylation but not Fibrinogen (FN), hyaluronic acid (HA), or Fibrin (Fib) (Fig. 3B). The specific antagonistic effect of laminin on PCK3145 was further confirmed in Fig. 3C.

EXAMPLE 5 PCK3145 binding/uptake assays

Results of Example 4 strongly suggest that the laminin receptor potentially triggers some of the PCK3145 effects and may potentially be involved in transducing the signaling of PCK3145 from the cell surface. As such, the expression of the 67-kDa laminin receptor (metastasis-associated 67-kDa laminin receptor) in several cell lines was observed using specific immunodetection on cell lysates (20 ug) (Fig. 4A).

In fact, transfection of U-87 cells with the cDNA expressing the recombinant 67-kDa laminin receptor resulted in a significantly sustained PCK-induced ERK phosphorylation between 10 and 20 minutes (Fig. 4B).

Affinity chromatography was performed to analyze PCK3145 binding to laminin receptor. Briefly, native PCK3145 was cross-linked to Sepharose-activated gel beads, followed by the loading of U-87 cell lysate and extensive washing of the gel. Immunodetection was then performed for laminin receptor in gels that have been either coupled or not to PCK3145. This experiment indicate that in fact the laminin receptor possesses affinity to PCK3145 (Fig. 5A) as shown by the increase in laminin receptor immunoreactivity.

Reprobing of that same membrane showed that a house keeping gene such as β-actin remained fairly constant and this was further quantified (Fig. 5B).

In order to shed some light into the kinetics of PCK3145 binding and uptake, PCK3145 was iodinated on its unique tyrosine and was used to perform binding assays at 4 ⁰ C and uptake at 37 ⁰ C. PCK3145 is seen to be rapidly taken up into the U-87 cells within the first 20 minutes of incubation, and this uptake kinetic reached a plateau after about

1 hour of incubation (Fig. 6A). When the same kinetic was performed at 4 ⁰ C, at which condition only binding to the cell surface is thought to happen with minimum intracellular uptake, a much lower saturable time-dependent binding of the radiolabeled PCK3145 was observed, lsotopic dilution is also observed in uptake experiments at 37 ⁰ C when 10-times excess cold PCK3145 is co-incubated with

radiolabeled-PCK3145, and this is reflected by a decreased uptake of PCK3145 to levels close to those found after 1 hr at 4 ⁰ C. Competition experiments were also performed with different substrates and this was measured at 37 ⁰ C after a one hour incubation. While Scrambled peptides 1 (s1) and 2 (s2) (e.g., SEQ ID NO.:99) had barely no effect on radiolabeled PCK3145 uptake, laminin (lam) and cold PCK3145 (PCK) were both effective in decreasing that uptake (Fig. 6B). Intriguingly, the native PSP-94 protein (e.g., SEQ ID NO.:1; PSP) only partially inhibited the PCK3145 uptake. This may potentially be due to incorrect or non-optimal folding of the native protein in solution.

Finally, a second set of competitors was also used to compete with the potential PCK3145 cell surface receptor.

Recent published evidence suggests that the laminin receptor binds the green tea catechin EGCg (Nat Struct MoI Biol. (2004) 11 :380-381). EGCg was therefore used in uptake competition experiments. These uptake experiments were also performed at

37 ⁰ C. Results of this experiment indicate that both laminin and EGCg, significantly antagonized the PCK3145 uptake (Fig. 6C). EGC, which is a catechin found in green tea but that lacks the gallate moiety, failed to compete with PCK3145. As an inhibitory effect of EGCg adhesion of murine melanoma cells to laminin has been observed, a similar effect of PCK3145 is likely to occur (Nat. Struct. MoI. Biol. 2004, 11 :380-381).

Further characterization studies on PCK3145 properties to bind to the cell surface and to subsequently be internalized were initiated. This is basically achieved by either performing the experiments at 4 ⁰ C, a condition known to minimize internalization/uptake mechanisms, or at 37 ⁰ C, a condition that favors both cell surface binding and internalization. The synthesis of [ ¹⁴ C]-radiolabeled PCK3145 (H-EW-[U-

¹⁴ C]Q-TDNC(Acm)ETC(Acm)TC(Acm)YET-OH) was achieved and this allowed us to develop assays with very low background. Assays were performed in 6-well plates with

80% confluent HT-1080 fibrosarcoma cells. Prior to the assay, cells were washed with

HBSS buffer and stabilized at either 4 ⁰ C (binding assay) or 37 ⁰ C (uptake assay) for approximately 15 min. Reactions were initiated by adding 500 μl of Ringer/HEPES containing 0.5% ovalbumin and 0.5-1.0 μCi of [ ¹⁴ C]PCK3145 (30 μg/ml [ ¹⁴ C]labelled- PCK3145). PCK3145 binding/uptake assays were performed for up to 5 minutes. Data illustrated in Fig. 17A are from one representative experiment. Reactions were stopped with rapid aspiration of the media, and cells washed rapidly with 1 ml

Ringer/HEPES containing 0.5% ovalbumin. Radioactivity was then counted in the whole cells after the addition of 500 μl NaOH 1 N. Similarly, increasing concentrations of PCK3145 were used to monitor cell surface binding and uptake (Fig. 17B.)

These observations are consistent with the "hit-and-run" hypothesis that we have previously suggested in PCK3145 intracellular cell signalling and that rapidly led to extracellular signal-regulated kinase (ERK) phosphorylation. When radiolabeled- PCK3145 binding and uptake experiments were performed with increasing PCK3145 concentrations (Fig.1b), PCK3145 cell surface binding was lower than uptake (1087 ± 49 vs 1087 ± 49). Dissociation constants (Kd) were also computed and were not statistically different for both processes (9.0 ± 1.1 μg/ml vs 7.7 ± 0.5 μg/ml for binding and uptake respectively) and one can conclude that the relative affinity for PCK3145, whether it is in binding or internalization processes, is approximately equivalent.

The effect of laminin, hyaluronic acid (HA), or type-l collagen on PCK3145 cell surface binding and internalization processes was monitored. Cells were thus incubated for 15 seconds with 30 μg/ml [ ¹⁴ C]-labelled-PCK3145in combination or not of laminin, hyaluronic acid (HA), or type-l collagen (Fig. 18A). Cell surface binding (white bars) or uptake (grey bars) were monitored as described herein. Data are expressed as the % of untreated controls, and are the mean of three independent experiments. Probability values of less than 0.05 were considered significant, and an asterisk (**) identifies such significance cell surface binding and uptake experiments. The relative PCK3145 binding or uptake was also monitored in cells that were incubated for 15 sec with 30 μg/ml [ ¹⁴ C]-labelled-PCK3145 in the presence or not of type-l collagen (Fig. 18B). Insert represents the representative dose response as monitored for PCK3145 uptake in the presence of low type-l collagen concentrations. Data of Fig. 18B are expressed as the % of control basal PCK3145 uptake and cell surface binding

This set of experiments suggests that potential alternate cell surface receptors may be involved in transducing PCK3145 intracellular effects. Moreover, this also implies that PCK3145 may have differential effects that would be dictated by the composition of the ECM tumor microenvironment.

EXAMPLE 6 PCK3145 binds to the cell surface of HT-1080 cells

Binding (and internalization) of iodinated-PCK3145 to the cell surface of cancer cells has been demonstrated herein. An assay has been designed to monitor such binding to the cell surface with the use of flow cytometry (FACS). Fluoresceinisothiocyanate (FITC) is currently the most commonly-used fluorescent dye for FACS analysis and was conjugated to the N-terminus of PCK3145. This assay enables to visualize cell binding of FITC-PCK3145 through the shift in fluorescence associated with cells that bind to it. 80-90% confluent HT-1080 cells were trypsinized and counted. Labeling was performed on 10 ⁶ cells for 1 hr at 4 ⁰ C. FITC-PCK3145 effectively bound to the cell surface in a dose-dependent manner with a plateau reached around 10 μg/ml (Fig 7A). More importantly, FITC-PCK3145 (10 μg/ml) cell surface binding was substantially inhibited by the incubation of excess 30 μg/ml laminin-1 (Fig 7B). This latter observation supports that a common cell surface receptor or that some laminin- binding activity is shared between PCK3145 and laminin and may trigger the subsequent signaling by PCK3145.

It is to be understood herein that any other markers may be attached to PCK3145 (e.g., biotin, etc.). When a FACS analysis is to be performed it is useful to use a fluorescent marker.

EXAMPLE 7

Laminin and laminin-derived peptide SIKVAV antagonize PCK3145 inhibitory action on MMP-9 secretion

In order to confirm the molecular mechanism involved in PCK3145 inhibitory action on MMP-9 secretion, HT-1080 cells were incubated with either the native laminin-1 protein (Fig 8A, 8B), or with a laminin-1 -derived peptide SIKVAV known for its capacity to induce MMP-9 secretion [Freitas et a/., (2004) Oral Oncology 40:483-489]. Confluent serum-starved cells were treated with these agents in the presence or not of 300 μg/ml PCK3145 for 24 hours. Conditioned media was isolated and gelatin zymography performed to monitor the extent of MMP-9 extracellular levels. Results show that PCK3145 is able to inhibit by 50% MMP-9 extracellular levels, while laminin antagonized PCK3145 inhibitory action. Interestingly, SIKVAV-induced MMP-9 secretion was also antagonized by PCK3145 (Fig 8A, 8B). This result also strongly

support that laminin and laminin-derived peptides interfere with PCK3145 actions and therefore share common cell surface receptor(s).

EXAMPLE 8 PCK3145 triggers rapid intracellular ERK phosphorylation

As illustrated in Fig. 9A and 9B, PCK3145 has the capacity to trigger ERK (Extracellular-signal-Regulated protein Kinases) phosphorylation. A time-course/dose- response experiment was performed. Overnight serum-starved quiescent HT-1080 cells were incubated with vehicle (phosphate-buffered saline (PBS) pH 7.4) or PCK3145 (3-100 μg/ml) for 1 , 2 or 5 minutes at 37 ⁰ C. The cells were scraped from the culture dishes in PBS containing NaF/Na ₃ VO ₄ and incubated in the same medium buffer for 1 h at 4°C. The resulting lysates were further clarified by centrifugation. Western blotting and immunodetection using anti-phosphoERK and anti-ERK antibodies was then performed.

The results show that PCK3145 was able to promote ERK phosphorylation as soon as in 1 minute of incubation and maximally at a concentration of 3 μg/ml (Fig 9A) while total ERK levels remained unaffected. Determination of the capacity of laminin-1- derived peptide SIKVAV to inhibit PCK3145-induced ERK phosphorylation was evaluated. Cells were thus similarly as above treated with either PCK3145, SIKVAV, or an equimolar combination of both and ERK phosphorylation monitored at 1 minute of incubation. While SIKVAV had virtually no effect on ERK phosphorylation, it significantly antagonized that ERK phosphorylation triggered by PCK3145 at 1 and 3 μg/ml (Fig 9B).

These results indicate that PCK3145 mediates cellular events in a manner which is faster than expected and also that the biological effect is observed at a low dose. These results indicate that PCK1345 may be administered in humans at a lower dose than previously demonstrated and still it will be sufficient to cause a desired biological effect. These results have an impact in the administration scheme (dose, time of administration, etc.) of the drug (PCK3145).

EXAMPLE 9

HT-1080 cells adhere to PCK3145-coated dishes through the 67-kDa laminin receptor

As shown above, the overexpression of the recombinant 67-kDa laminin receptor is able to sustain PCK-induced ERK phosphorylation. A cell adhesion assay was designed where increasing concentrations of PCK3145 were coated on dishes and cells subsequently incubated. This coating may be performed for either 1 hour at 37 ⁰ C or overnight at 4 ⁰ C. Blocking with albumin was avoided due to a potential interaction between PCK3145 and albumin (see below). Confluent HT-1080 cells were trypsinised, seeded onto the PCK3145-coated dishes, and adhesion left to proceed for 1 hour at 37 ⁰ C. Results show that cells adhered with more avidity to increasing doses of PCK3145 (Fig 10A). When the 67-kDa laminin receptor cDNA was transiently transfected into HT-1080 cells, adhesion to PCK3145 was then found increased in comparison to mock cells (Fig 10B). These results further evidence laminin binding activities to PCK3145 actions.

The effect of cell confluence on the regulation of the 67-kDa laminin receptor expression and PCK3145 cell surface binding was assessed. HT-1080 cells were seeded in individual wells of a 6-well plate at increasing confluence of 10 ⁴ to 10 ⁶ cells/well and pictures taken 24 hrs later (Fig 20-panel A). Cell lysates were isolated in duplicate from each cell confluence culture condition. SDS-PAGE and immunoblotting with the anti-67LR was performed at 24 hrs (Fig 20-panels B and C). HT-1080 cells were seeded at 10 ⁵ cells/well in a 6-well culture plate and lysates were isolated at 24, 48, and 72 hrs (Fig 20-panel D). It was noticed from the setting of these experimental conditions, that cell confluence regulates the laminin receptor protein expression. Indeed, it was observed that the laminin receptor was expressed at very low levels in low (10 ⁴ cells / well) density, while its expression drastically increased at high (10 ⁶ cells / well). This effect was also observed when cells were harvested at different time of incubation (from 24 to 78 hours).

The relationship between the level of expression of the 67-kDa laminin receptor and EGCg inhibition of cell surface PCK3145 binding was assessed. Low confluent HT- 1080 cells (10 ⁴ cells/well, white bars) were trypsinized and incubated with biotin- labeled PCK3145 at 4 ⁰ C as described herein. PCK3145 cell surface binding was monitored by flow cytometry in cells that were incubated or not (i.e., control) with 100 μM EGCg and binding compared to that of high confluent cells (10 ⁶ cells/well, grey bars) (Fig. 21 -panel A). proMMP-9 secretion from low and high confluent cells was

also monitored by gelatin zymography in serum-starved cells that were incubated or not with 300 μg/ml PCK3145 for 48 hrs (Fig. 21-panel B and panel C).;

This observation correlates and supports the fact that the higher expression of the 67LR at the cell surface may better transduce the effect of PCK3145. This highlights the potential contribution of 67LR as one of the cell surface laminin receptors that may transduce the effects of PCK3145. Noteworthy, an alternative interpretation of these results may also rely on the fact that the increase in 67LR protein expression in high confluent cells represents approximately 5-6 times that expression in low confluent cells. In contrast, an increase of only 25% is observed in PCK3145 cell surface binding. This suggests that a potential cell surface 67LR sub-population may not be as effective in binding PCK3145 or that these may not be in the right conformation. Collectively, we certainly provide more evidence for the involvement of the 67LR as one of the PCK3145 cell surface binding protein as EGCg is being able to efficiently compete with PCK3145 for cell surface binding. Since the 67LR expression is induced in high confluent cells, we may safely infer that the 67LR may indeed transduce, at least, in part, the effects of PCK3145.

EXAMPLE 10

Potential PCK3145-binding proteins revealed by affinity-gel chromatography

In order to identify potential PCK3145-binding proteins, an affinity chromatography assay was designed in which PCK3145 was coupled to activated Sepharose-beads. Cell lysates were then overlaid onto the respective gels and columns washed extensively to minimize non-specific protein binding. Three independent PCK3145 coupling were performed. It was systematically observed that proteins at approximately MW of 58, 47, and 35 kDa were differentially bound to PCK3145- coupled gels (Fig 11A). The corresponding PVDF membranes were cut and sent for protein sequencing. The identity of two of these three PCK-binding candidates was found to be Albumin (Fig 11B) and enolase (Fig 11C). However, it is tempting to suggest that i) Albumin may interact with PCK3145 and act as a carrier protein (bovine serum albumin and human serum albumin share conserved regions) and that H) PCK3145 may interact with a cell surface enolase which is known to possess both laminin and plasminogen binding activities.

EXAMPLE 11 Enolase inhibits ceil adhesion to PCK3145

The identification of enolase as a potential PCK3145-binding protein is supported by its recently published ability to also bind to laminin [Cameiro et al. (2004) Microbes Infect. 6:604-608]. In order to provide evidence of a potential role of enolase in PCK3145 effects, a test of cell adhesion to 10 ug/ml PCK3145-coated dished was performed. Cell adhesion was performed for 2 hours at 37 ⁰ C in the presence or not of 30 μg/ml enolase or with 30 μM EGCg, a green tea catechin that we have been shown herein to both inhibit MMP secretion and PCK3145 internalization. These results indicate that both enolase and EGCg inhibit cell adhesion to PCK3145-coated dishes (Fig 12A). Although, only very little inhibition (-12%) was observed when cell surface binding of FITC-PCK3145 was assessed by flow cytometry (Fig 12B).

EXAMPLE 12

Far-Western ligand binding assay

Far-Western is a proteomic approach aimed at identifying potential protein-protein interactions using electrotransferred proteins onto PVDF membranes. Cell lysates were run on a 7.5% or 12% SDS-gels and transferred to PVDF membranes. Membranes were then blocked with either BSA or powder milk. Binding was then performed with biotinilated-laminin-1 overnight at 4 ⁰ C. Biotinilated-laminin was previously monitored for its ability to bind HT-1080 cell surface and to be functionally inhibited by excess unlabelled laminin, PCK3145, or EGCg (Fig 13A). We show that both laminin and PCK3145 were significantly able to inhibit the binding of biotinilated- Laminin (-20% inhibition), while 30 μM EGCg was extremely potent to inhibit biotinilated-laminin binding. Furthermore, biotinilated-laminin was able to bind to electrotransferred proteins as shown by the detection using either streptavidin-HRP or an anti-biotin antibody method (Fig 13B). Interestingly, when the 47 kDa commercially available enolase was run on the 12% SDS-gel it was also specifically recognized by the biotinilated-laminin.

Alternatively, PCK3145 was biotin labeled according to Pierce and purified by FPLC- chromatography using Akta-explorer with a resource RPC-30mI column (Amersham Bioscience, QC). The biotin-labeled peptide was eluted with a gradient from 20% CH ₃ CN to 80% CH ₃ CN (+0.05%TFA) at a flow rate of 4 ml/min. Products were monitored at different wavelengths (205, 229 and 254 nm) with a UV-900 cell-10.

Confluent HT-1080 cell lysates (-50-100 μg/well) were migrated using 10% SDS-gels and then electrotransferred onto PVDF membranes. Membranes were then incubated with 10 μg/ml of unlabeled PCK3145 or 10 μg/ml biotin-labeled PCK3145 for 1 hr, followed with incubation with HRP-Streptavidin for another hour and ECL detection.

The results illustrated in the left panel of Fig 19 shows proteins from HT-1080 cell lysate that are recognized by biotin-labeled PCK3145 ((+) incubated or (-) not incubated with biotin-labeled PCK3145). The right panel of Fig. 19 represents the same PVDF membrane blotted with an anti-67 kDa laminin receptor (67LR) antibody. Numbers (1 to 11) next to the left panel represent the potential proteins that interact with PCK3145. Numbers (6, 8, and 9) represent the 67LR immunoreactive bands that are similar to those identified in the left panel.

The results to Fig. 19 therefore show that the 67-kDa laminin receptor is among the potential PCK3145-binding proteins.

EXAMPLE 13

The expression of the 67-kDa laminin receptor regulates PCK3145 cell surface binding but not internalization

The impact of the expression of the 67-kDa laminin receptor on the regulation of PCK3145 cell surface binding was assessed. HT-1080 cells were transiently transfected with a cDNA encoding the 67LR. Cell lysates were isolated and immunoblot performed as described herein (mock; white bars and 67LR-transfected cells; grey bars). HT-1080 cells were trypsinized and PCK3145 cell surface binding and uptake monitored as described herein.

Results of Figs. 22A and B indicate that the expression of the 67-kDa laminin receptor regulates PCK3145 cell surface binding but not internalization.

EXAMPLE 14 PCK3145 inhibits HuR gene and protein expression Recent articles indicate that MMP-9 expression to be induced through a stabilizing nuclear factor HuR [Huwiler et al. (2003) J Biol Chem. 278:51748-69], and that a3b1

integrin (an integrin known to bind laminin) regulates MMP-9 mRNA [Lyer et al., (2005) J Cell Science 118:1185-1195]. These published evidence link Hur, an mRNA stabilizing factor that is ubiquitously expressed and that has the ability to bind and stabilize mRNA degradation to AU-rich elements (AREs). These elements are expressed in the 3'-untranslated region of several RNAs encoding, for instance, VEGF and MMP-9. Whether PCK3145 may regulate HuR expression was therefore tested. Serum-starved cells were treated or not with 300 μg/ml PCK3145 for 24 hours in the presence or not of laminin or SIKVAV, and cells harvested for either RNA extraction or cell homogenates. RT-PCR with specific primers revealed that HuR gene expression was downregulated by PCK3145 and that both laminin and laminin-derived peptide SIKVAV antagonized PCK3145's inhibitory effect (Fig 14). This was further confirmed independently with specific anti-HuR antibody and immunodetection. This effect of PCK3145 on HuR may explain partly the inhibition of MMP-9 expression and subsequent diminished extracellular levels.

EXAMPLE 15

PCK3145 triggers CD44 cell surface shedding and inhibits MMP-9 docking at the cell surface Decreased cell migration and adhesion on HA was observed when cells were pretreated with PCK3145 (data not shown). It was shown that this was the results of an increased CD44 cell surface shedding as demonstrated by a strong immunoreactive band observed in the cells which had been pre-treated with PCK3145. This effect was also shown to happen in parallel with MT1-MMP-transfected cells. Interestingly, an increase in MT1-MMP expression in the PCK3145-treated cells was shown which may partially explain how PCK may lead to CD44 shedding. The potential implication of these observations on MMP-9 cell surface binding was investigated, since CD44 is also reported to be the docking receptor for MMP-9. HT- 1080 cells were treated or not with 300 μg/ml PCK3145 for 24 hours, trypsinised and either labeled with anti-CD44 or anti-MMP-9 antibodies. Flow cytometry measurements clearly show a 25% downregulation in CD44 cell surface expression that is correlated to a 50% inhibition of cell surface-associated MMP-9 (Fig 15). Altogether, these observations provide a rational for an additional regulation of MMP-9 functions (inhibition of secretion being the first one) by PCK3145 of diminished cell surface docking of MMP-9 to CD44 which may control both ECM degradation and cell migration by MMP-9.

PCK3145 inhibits extracellular matrix (ECM) degradation by MMP-9; a matrix metalloproteinase involved in prostate cancer progression and several other conditions such as, for example, angiogenesis, wound healing, etc. While PCK3145 did not affect MMP-9 enzymatic activity per se, it however significantly reduces its (type-l collagen-induced) gene expression which, consequently, led to decreased extracellular MMP-9 secreted levels.

Although most published studies have focused on transcriptional control of MMP-9, there is increasing evidence that its expression can also be regulated at the steps of mRNA stability, translation and protein secretion. The ability to modulate MMP-9 expression at multiple steps through distinct signaling pathways may be particularly important during malignant conversion and metastasis, when tumor cells need to induce or maintain MMP-9 levels in response to changing environmental cues.

EXAMPLE 16 PCK3145 inhibits growth of leukemia cell The therapeutic effect of PCK3145 on the inhibition of leukemia cell growth was evaluated. Human leukemia cells RPMI-8226 and SR (available at the National Cancer Institute (http://www.dtp.nci.nih.gov/branches/btb/services.html)) were incubated with PCK3145 and cell growth was monitored. Results are expressed in Table 2A and 2B below.

Table 2A

Log 10 concentration

Time Mean optical densities zero CtI -1.9 -0.9 0.1 1.1 2.1

Leukemia

RPMI-8226 0.367 0.557 0.501 0.522 0.413 0.357 0.279

SR 0.309 0.965 0.956 0.909 0.861 0.526 0.451

Table 2B

Log 10 concentration

Percent growth

-1.9 -0.9 0.1 1.1 2.1 GI50 TGI LC50

Leukemia

RPMI-8226 71 82 24 -3 -24 4.44 E-1 9.80 EO >1.25E2

SR 99 92 84 33 22 5.83 EO >1.25E2 >1.25E2

Interestingly the laminin receptor (67LR) has been found expressed in several acute myeloid leukemia cells (Monturi, N. Clin. Cancer Res. 5:1465-1472, 1999) and is associated with monocytic differentiation.

Cell surface laminin binding activities as primary targets for PCK3145.

The novel mechanism of laminin receptor-mediated MMP-9 gene expression provides solid foundation to suggest that PCK3145 acts through receptor-mediated signaling. Several lines of evidence indeed show that PCK3145 targets laminin binding activities (including MMP-9 regulation);

1. FITC-labeled PCK3145 effectively binds to the cell surface, and that this binding is inhibited by either laminin-1 or a laminin-1 -derived peptide (SIKVAV)

2. EGCg, a green tea catechin that inhibits MMP-9 secretion, is also a laminin receptor ligand that antagonized cell binding to PCK3145

3. Overexpression of the 67-kDa non-integrin laminin receptor in cells promotes binding/recognition to PCK3145

4. PCK3145-affinity chromatography as identified enolase, an enzyme recently thought to also possess laminin-binding properties, and which have been shown herein to inhibit cell binding to PCK3145.

Increased expression of the 67-kDa laminin receptor has been reported in a variety of human carcinomas (colon, breast, stomach, liver, and ovary) and directly correlates with a higher proliferation rate of malignant cells and tendency to metastasize. In

addition, the 67-kDa laminin receptor is detectable in anaplastic large cell lymphomas and in small subsets of high-grade B-cell non-Hodgkin's or Hodgkin's lymphomas. More recently, expression of the 67-kDa laminin receptor has been found to mediate acute myeloid leukemia cell adhesion to laminin and to be frequently associated with monocytic differentiation. In light of these documented expression of the 67-kDa laminin receptor and in light of the results presented herein, it is thus tempting to conclude that cancer (e.g., of lymphoid or monocytic origin, etc.) other than prostate cancer, or specific stage of development such as in monocytic-oriented acute myeloid leukemia may be targeted by PCK3145.

PCK3145 triggers intracellular signaling that regulates HuR expression.

The unexpected observation that PCK3145 shares, to some extent, homology with different structural chains of laminin suggests that it may indeed also share the same cell surface receptor. The results disclosed herein support this suggestion. Indeed, laminin and PCK3145 competed for ERK phosphorylation as well as for the binding/uptake of PCK3145. It was shown herein that intracellular signaling leads to MMP-9 decreased secretion and that HuR, a MMP-9 mRNA stabilizing factor, is targeted by PCK3145. Indeed, both of HuR gene and protein expression were downregulated by PCK3145, and this downregulation was reversed by laminin receptor ligands. PCK3145 is therefore a laminin receptor-mediated signal transduction inhibitor.

As HuR also binds to the AU-rich elements of RNAs encoding genes for cytokines, growth factors, tumor suppressor genes, proto-oncogene, and cell cycle regulators, one can envisioned that downregulation of HuR by PCK3145 inhibits several cellular processes and may therefore be used to treat several diseases associated with HuR expression.

Fig. 16 is a schematic summarizing the effect of PCK on its target and the biological event and signaling event occurring thereafter.

The content of each publication, patent and patent application mentioned in the present application is incorporated herein by reference.

Although the present invention has been described in details herein and illustrated in the accompanying drawings, it is to be understood that the invention is not limited to the embodiments described herein and that various changes and modifications may be effected without departing from the scope or spirit of the present invention.

Examplarv embodiment of sequences

SEQUENCE DESCRIPTION: SEQ ID NO: 1: Ser Cys Tyr Phe lie Pro Asn GIu GIy VaI Pro GIy Asp Ser Thr Arg 1 5 10 15

Lys Cys Met Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp

20 25 30

GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu lie Ser 35 40 45

Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp Asn Cys 50 55 60

GIn Arg lie Phe Lys Lys GIu Asp Cys Lys Tyr lie VaI VaI GIu Lys 65 70 75 80 Lys Asp Pro Lys Lys Thr Cys Ser VaI Ser GIu Trp lie lie

85 90

SEQUENCE DESCRIPTION: SEQ ID NO: 2:

GIu Ala GIu Ala Tyr VaI GIu Phe Ser Cys Tyr Phe lie Pro Asn GIu 1 5 10 15

GIy VaI Pro GIy Asp Ser Thr Arg Lys Cys Met Asp Leu Lys GIy Asn 20 25 30 Lys His Pro lie Asn Ser GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys 35 40 45

Thr Cys Tyr GIu Thr GIu lie Ser Cys Cys Thr Leu VaI Ser Thr Pro 50 55 60

VaI GIy Tyr Asp Lys Asp Asn Cys GIn Arg He Phe Lys Lys GIu Asp 65 70 75 80

Cys Lys Tyr He VaI VaI GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser 85 90 95

VaI Ser GIu Trp He He 100

SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Tyr Thr Cys Ser VaI Ser GIu Pro GIy He i 5 io

SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Asn GIu GIy VaI Pro GIy Asp Ser Thr Arg Lys Cys Met Asp Leu 1 5 10 15

SEQUENCE DESCRIPTION: SEQ ID NO: 5:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 1 5 10 15

SEQUENCE DESCRIPTION: SEQ ID NO : 6 : lie VaI VaI GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser VaI Ser GIu 1 5 10 15

Trp He He

SEQUENCE DESCRIPTION: SEQ ID NO: 7 (an acetylaminomethyl group may be attached to the sulfur atom of cysteine 7, of cysteine 10 and/or of cysteine 12)

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 1 5 10 15

SEQUENCE DESCRIPTION: SEQ ID NO: 8:

TCATGCTATT TCATACCTAA TGAGGGAGTT CCAGGAGATT CAACCAGGAA ATGCATGGAT 60 CTCAAAGGAA ACAAACACCC AΆTAAACTCG GAGTGGCAGA CTGACAACTG TGAGACATGC 120

ACTTGCTACG AAACAGAAAT TTCATGTTGC ACCCTTGTTT CTACACCTGT GGGTTATGAC 180 AAAGACAACT GCCAAΆGAAT CTTCAAGAAG GAGGACTGCA AGTATATCGT GGTGGAGAAG 240

AAGGACCCAA AAAAGACCTG TTCTGTCAGT GAATGGATAA TCTAA 285

SEQUENCE DESCRIPTION: SEQ ID NO: 9:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

SEQUENCE DESCRIPTION: SEQ ID NO : 10 :

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He

SEQUENCE DESCRIPTION : SEQ ID NO : 11 :

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser

SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys

SEQUENCE DESCRIPTION: SEQ ID NO : 13 :

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys 20

SEQUENCE DESCRIPTION: SEQ ID NO: 14:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr 20

SEQUENCE DESCRIPTION: SEQ ID NO: 15:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu 20

SEQUENCE DESCRIPTION: SEQ ID NO: 16:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

lie Ser Cys Cys Thr Leu VaI 20

SEQUENCE DESCRIPTION: SEQ ID NO : 17 :

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser 20

SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr 20 25

SEQUENCE DESCRIPTION: SEQ ID NO: 19:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro 20 25

SEQUENCE DESCRIPTION: SEQ ID NO: 20:

GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI 20 25

SEQUENCE DESCRIPTION: SEQ ID NO: 21:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy

20 25

SEQUENCE DESCRIPTION: SEQ ID NO: 22: GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr 20 25

SEQUENCE DESCRIPTION : SEQ ID NO : 23 :

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp 20 25 30

SEQUENCE DESCRIPTION: SEQ ID NO : 24 : GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys

20 25 30

SEQUENCE DESCRIPTION: SEQ ID NO: 25:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

SEQUENCE DESCRIPTION: SEQ ID NO: 26:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn

SEQUENCE DESCRIPTION: SEQ ID NO: 27:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp

20 25 30

Asn Cys

SEQUENCE DESCRIPTION: SEQ ID NO: 28:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp

20 25 30

Asn Cys Gin

35

SEQUENCE DESCRIPTION: SEQ ID NO: 29:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg 35

SEQUENCE DESCRIPTION: SEQ ID NO: 30:

GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys Gin Arg He 35

SEQUENCE DESCRIPTION: SEQ ID NO: 31:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30 Asn Cys Gin Arg He Phe 35

SEQUENCE DESCRIPTION: SEQ ID NO: 32: GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp

20 25 30

Asn Cys GIn Arg He Phe Lys 35

SEQUENCE DESCRIPTION: SEQ ID NO: 33:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg He Phe Lys Lys 35 40

SEQUENCE DESCRIPTION: SEQ ID NO : 34 :

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg lie Phe Lys Lys GIu 35 40

SEQUENCE DESCRIPTION: SEQ ID NO: 35:

GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg He Phe Lys Lys GIu Asp 35 40

SEQUENCE DESCRIPTION: SEQ ID NO: 36:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys 35 40

SEQUENCE DESCRIPTION: SEQ ID NO: 37:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30 Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys Lys 35 40

SEQUENCE DESCRIPTION: SEQ ID NO: 38: GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys Lys Tyr 35 40 45

SEQUENCE DESCRIPTION: SEQ ID NO : 39 :

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg lie Phe Lys Lys GIu Asp Cys Lys Tyr lie 35 40 45

SEQUENCE DESCRIPTION: SEQ ID NO: 40:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys Gin Arg He Phe Lys Lys GIu Asp Cys Lys Tyr He VaI 35 40 45

SEQUENCE DESCRIPTION: SEQ ID NO: 41:

GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30 Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys Lys Tyr He VaI VaI 35 40 45

SEQUENCE DESCRIPTION: SEQ ID NO: 42: GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys Lys Tyr He VaI VaI 35 40 45

GIu

SEQUENCE DESCRIPTION: SEQ ID NO: 43:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys Lys Tyr He VaI VaI 35 40 45

GIu Lys 50

SEQUENCE DESCRIPTION: SEQ ID NO: 44:

GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys Gin Arg lie Phe Lys Lys GIu Asp Cys Lys Tyr lie VaI VaI 35 40 45 GIu Lys Lys 50

SEQUENCE DESCRIPTION: SEQ ID NO: 45: GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp

20 25 30

Asn Cys GIn Arg lie Phe Lys Lys GIu Asp Cys Lys Tyr lie VaI VaI 35 40 45

GIu Lys Lys Asp so

SEQUENCE DESCRIPTION: SEQ ID NO: 46:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30 Asn Cys GIn Arg lie Phe Lys Lys GIu Asp Cys Lys Tyr lie VaI VaI 35 40 45

GIu Lys Lys Asp Pro

50

SEQUENCE DESCRIPTION: SEQ ID NO: 47:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30 Asn Cys GIn Arg lie Phe Lys Lys GIu Asp Cys Lys Tyr lie VaI VaI 35 40 45

GIu Lys Lys Asp Pro Lys 50

SEQUENCE DESCRIPTION: SEQ ID NO: 48:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys Lys Tyr He VaI VaI 35 40 45

GIu Lys Lys Asp Pro Lys Lys 50 55

SEQUENCE DESCRIPTION: SEQ ID NO: 49:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu

1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys Lys Tyr He VaI VaI

35 40 45 GIu Lys Lys Asp Pro Lys Lys Thr 50 55

SEQUENCE DESCRIPTION: SEQ ID NO: 50: GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys Lys Tyr He VaI VaI 35 40 45

GIu Lys Lys Asp Pro Lys Lys Thr Cys 50 55

SEQUENCE DESCRIPTION: SEQ ID NO: 51: GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp

20 25 30

Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys Lys Tyr He VaI VaI 35 40 45

GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser 50 55

SEQUENCE DESCRIPTION: SEQ ID NO: 52:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys Gin Arg He Phe Lys Lys GIu Asp Cys Lys Tyr He VaI VaI 35 40 45

GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser VaI 50 55

SEQUENCE DESCRIPTION: SEQ ID NO: 53:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys Lys Tyr He VaI VaI 35 40 45 GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser VaI Ser 50 55 60

SEQUENCE DESCRIPTION: SEQ ID NO: 54: GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30

Asn Cys GIn Arg He Phe Lys Lys GIu Asp Cys Lys Tyr He VaI VaI 35 40 45

GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser VaI Ser GIu 50 55 60

SEQUENCE DESCRIPTION: SEQ ID NO: 55: GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

He Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp

20 25 30

Asn Cys GIn Arg lie Phe Lys Lys GIu Asp Cys Lys Tyr lie VaI VaI 35 40 45

GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser VaI Ser GIu Trp 50 55 60

SEQUENCE DESCRIPTION: SEQ ID NO: 56:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 25 30 Asn Cys GIn Arg lie Phe Lys Lys GIu Asp Cys Lys Tyr lie VaI VaI 35 40 45

GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser VaI Ser GIu Trp lie 50 55 60

SEQUENCE DESCRIPTION: SEQ ID NO: 57:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp 20 ^' 25 30

Asn Cys GIn Arg lie Phe Lys Lys GIu Asp Cys Lys Tyr lie VaI VaI 35 40 45

GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser VaI Ser GIu Trp lie lie 50 55 60

SEQUENCE DESCRIPTION: SEQ ID NO: 58:

Ser GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 1 5 10 15

SEQUENCE DESCRIPTION: SEQ ID NO: 59:

Asn Ser GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu 1 5 10 15 Thr

SEQUENCE DESCRIPTION: SEQ ID NO: 60:

He Asn Ser GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr 1 5 10 15

GIu Thr

SEQUENCE DESCRIPTION: SEQ ID NO: 61:

Pro lie Asn Ser GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys 1 5 10 15

Tyr GIu Thr

SEQUENCE DESCRIPTION: SEQ ID NO: 62: His Pro lie Asn Ser GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr 1 5 10 15

Cys Tyr GIu Thr 20

SEQUENCE DESCRIPTION: SEQ ID NO: 63:

Lys His Pro lie Asn Ser GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys 1 5 10 15

Thr Cys Tyr GIu Thr 20

SEQUENCE DESCRIPTION: SEQ ID NO: 64:

Asn Lys His Pro lie Asn Ser GIu Trp GIn Thr Asp Asn Cys GIu Thr 1 5 10 15

Cys Thr Cys Tyr GIu Thr 20

SEQUENCE DESCRIPTION: SEQ ID NO: 65:

GIy Asn Lys His Pro lie Asn Ser GIu Trp Gin Thr Asp Asn Cys GIu 1 5 10 15

Thr Cys Thr Cys Tyr GIu Thr 20

SEQUENCE DESCRIPTION: SEQ ID NO: 66:

Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn Thr Asp Asn Cys 1 5 10 15

GIu Thr Cys Thr Cys Tyr GIu Thr 20

SEQUENCE DESCRIPTION: SEQ ID NO: 67:

Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn Thr Asp Asn 1 5 10 15

Cys GIu Thr Cys Thr Cys Tyr GIu Thr 20 25

SEQUENCE DESCRIPTION: SEQ ID NO: 68:

Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn Thr Asp 1 5 10 15

Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 20 25

SEQUENCE DESCRIPTION: SEQ ID NO: 69:

Met Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn Thr 1 5 10 15

Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 20 25

SEQUENCE DESCRIPTION: SEQ ID NO: 70:

Cys Met Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn

1 5 10 15

Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 20 25

SEQUENCE DESCRIPTION: SEQ ID NO: 71: Lys Cys Met Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp 1 5 10 15

Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 20 25

SEQUENCE DESCRIPTION: SEQ ID NO: 72:

Arg Lys Cys Met Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu 1 5 10 15

Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 20 25 30

SEQUENCE DESCRIPTION: SEQ ID NO: 73:

Thr Arg Lys Cys Met Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser 1 5 10 15

GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 20 25 30

SEQUENCE DESCRIPTION: SEQ ID NO: 74:

Ser Thr Arg Lys Cys Met Asp Leu Lys GIy Asn Lys His Pro lie Asn 1 5 10 15

Ser GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 20 25 30

SEQUENCE DESCRIPTION: SEQ ID NO : 75 :

Asp Ser Thr Arg Lys Cys Met Asp Leu Lys GIy Asn Lys His Pro lie 1 5 10 15

Asn Ser GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu 20 25 30

Thr

SEQUENCE DESCRIPTION: SEQ ID NO: 76:

GIy Asp Ser Thr Arg Lys Cys Met Asp Leu Lys GIy Asn Lys His Pro 1 5 10 15

He Asn Ser GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr 20 25 30

GIu Thr

SEQUENCE DESCRIPTION: SEQ ID NO: 77:

Pro GIy Asp Ser Thr Arg Lys Cys Met Asp Leu Lys GIy Asn Lys His 1 5 10 15 Pro lie Asn Ser GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys 20 25 30

Tyr GIu Thr 35

SEQUENCE DESCRIPTION: SEQ ID NO: 78:

VaI Pro GIy Asp Ser Thr Arg Lys Cys Met Asp Leu Lys GIy Asn Lys 1 5 10 15 His Pro He Asn Ser GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr 20 25 30

Cys Tyr GIu Thr 35

SEQUENCE DESCRIPTION: SEQ ID NO: 79:

GIy VaI Pro GIy Asp Ser Thr Arg Lys Cys Met Asp Leu Lys GIy Asn 1 5 10 15 Lys His Pro He Asn Ser GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys 20 25 30

Thr Cys Tyr GIu Thr 35 SEQUENCE DESCRIPTION: SEQ ID NO: 80:

Glu GIy VaI Pro GIy Asp Ser Thr Arg Lys Cys Met Asp Leu Lys GIy 1 5 10 15

Asn Lys His Pro lie Asn Ser GIu Trp GIn Thr Asp Asn Cys GIu Thr 20 25 30

Cys Thr Cys Tyr GIu Thr 35

SEQUENCE DESCRIPTION: SEQ ID NO : 81 :

Asn GIu GIy VaI Pro GIy Asp Ser Thr Arg Lys Cys Met Asp Leu Lys 1 5 10 15

GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn Thr Asp Asn Cys GIu 20 25 30

Thr Cys Thr Cys Tyr GIu Thr 35

SEQUENCE DESCRIPTION: SEQ ID NO: 82:

Pro Asn GIu GIy VaI Pro GIy Asp Ser Thr Arg Lys Cys Met Asp Leu

1 5 10 15

Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn Thr Asp Asn Cys 20 25 30

GIu Thr Cys Thr Cys Tyr GIu Thr 35 40

SEQUENCE DESCRIPTION: SEQ ID NO: 83; lie Pro Asn GIu GIy VaI Pro GIy Asp Ser Thr Arg Lys Cys Met Asp 1 5 10 15

Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp Gin Thr Asp Asn 20 25 30

Cys GIu Thr Cys Thr Cys Tyr GIu Thr 35 40

SEQUENCE DESCRIPTION: SEQ ID NO: 84:

Phe lie Pro Asn GIu GIy VaI Pro GIy Asp Ser Thr Arg Lys Cys Met 1 5 10 15

Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp Gin Thr Asp 20 25 30

Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 35 40

SEQUENCE DESCRIPTION: SEQ ID NO: 85:

Tyr Phe He Pro Asn GIu GIy VaI Pro GIy Asp Ser Thr Arg Lys Cys 1 5 10 15

Met Asp Leu Lys GIy Asn Lys His Pro He Asn Ser GIu Trp GIn Thr 20 25 30

Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 35 40

SEQUENCE DESCRIPTION: SEQ ID NO: 86:

Cys Tyr Phe lie Pro Asn GIu GIy VaI Pro GIy Asp Ser Thr Arg Lys 1 5 10 15 Cys Met Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn 20 25 30

Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 35 40

SEQUENCE DESCRIPTION: SEQ ID NO: 87:

Ser Cys Tyr Phe lie Pro Asn GIu GIy VaI Pro GIy Asp Ser Thr Arg 1 5 10 15 Lys Cys Met Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp 20 25 30

GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 35 40 45

INFORMATION FOR SEQ ID NO: 88:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 (B) TYPE: AMINO ACID

(C) STRANDEDNESS : SINGLE

(D) TOPOLOGY : LINEAR ( U) MOLECULE TYPE :

(ix) FEATURE : NAME/KEY : Modified site

LOCATION : 1

OTHER INFORMATION : The residue in this position is either glutamic acid, asparagine, or aspartic acid, (ix) FEATURE : NAME/KEY : Modified site

LOCATION : 4

(D)OTHER INFORMATION : The residue in this position is either threonine, or serine, (ix) FEATURE : NAME/KEY : Modified site

LOCATION : 6

(D)OTHER INFORMATION : The residue in this position is either glutamic acid, asparagine, or aspartic acid. (ix) FEATURE :

NAME/KEY : Modified site LOCATION : 8

(D)OTHER INFORMATION : The residue in this position is either glutamic acid, asparagine, or aspartic acid.

(ix) FEATURE :

NAME/KEY : Modified site LOCATION : 9

(D)OTHER INFORMATION : The residue in this position is either threonine, or serine, (ix) FEATURE :

NAME/KEY : Modified site (B)LOCATION : 11

(D)OTHER INFORMATION : The residue in this position is either threonine, or serine, (ix) FEATURE :

(A)NAME/KEY : Modified site (B)LOCATION : 13

(D)OTHER INFORMATION : The residue in this position is either tyrosine, or phenylalanine, (ix) FEATURE :

NAME/KEY : Modified site (B)LOCATION : 14

(D)OTHER INFORMATION : The residue in this position is either glutamic acid, asparagine, or aspartic acid.

(ix) FEATURE : (A)NAME/KEY : Modified site

(B)LOCATION : 15

(D)OTHER INFORMATION : The residue in this position is either threonine, or serine. (Vi)ORIGINAL SOURCE:

(A) ORGANISM:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:

Xaa Trp GIn Xaa Asp Xaa Cys Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 15

SEQUENCE DESCRIPTION: SEQ ID NO: 89:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 20 25 30

SEQUENCE DESCRIPTION: SEQ ID NO: 90:

Glu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15

Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr Glu Trp 20 25 30

GIn Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr 35 40 45

SEQUENCE DESCRIPTION: SEQ ID NO: 91:

Glu Trp GIn Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu 1 5 10 15

Trp GIn Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu Trp 20 25 30

GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu Trp GIn

35 40 45

Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 50 55 60

SEQUENCE DESCRIPTION: SEQ ID NO: 92:

GIu Tyr GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 1 5 10 15

SEQUENCE DESCRIPTION: SEQ ID NO: 93:

GIu Trp Asn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 1 5 10 15

SEQUENCE DESCRIPTION: SEQ ID NO: 94: GIu Trp Gin Thr Asp GIn Ser GIu Thr Cys Thr Cys Tyr Asp Thr 1 5 10 15

SEQUENCE DESCRIPTION: SEQ ID NO: 95:

Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 1 5 10

SEQUENCE DESCRIPTION: SEQ ID NO: 96:

GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr 1 5 10

SEQUENCE DESCRIPTION: SEQ ID NO: 97:

GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys 1 5 10

SEQUENCE DESCRIPTION: SEQ ID NO: 98:

GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys D-Tyr GIu Thr 1 5 10 15

SEQUENCE DESCRIPTION: SEQ ID NO: 99 (scarmbled peptide) :

Thr-Cys (Acm) -Glu-Asn-Cys (Acm) -Thr-Glu-Thr-Gln-Trp-Cys (Acm) -Glu-Thr- Asp-Tyr