Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
  • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
  • A61K49/0043—Fluorescein, used in vivo
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
  • A61K49/0056—Peptides, proteins, polyamino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
  • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01T—MEASUREMENT OF NUCLEAR OR X-RADIATION
  • G01T1/00—Measuring X-radiation, gamma radiation, corpuscular radiation, or cosmic radiation
  • G01T1/16—Measuring radiation intensity
  • G01T1/24—Measuring radiation intensity with semiconductor detectors
  • G01T1/249—Measuring radiation intensity with semiconductor detectors specially adapted for use in SPECT or PET
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

• the present invention relates to novel peptides and their use in compositions and methods for identifying cells expressing RHAMM, and in compositions and methods of treatment of disorders or conditions that result from the formation of RHAMM/ligand complex.
• Hyaluronan (HA, an anionic polymer of repeating units of glucuronic acid and N- acetylglucosamine) is one extracellular matrix (ECM) component of stroma, that is associated with cancer progression: increased accumulation of tumor HA is prognostic of poor outcome in cancer patients [1 , 2, 3].
• ECM extracellular matrix
• HA stimulates cancer cell motility in vitro, suggesting its importance in cancer cell invasion in vivo.
• Two HA cellular receptors that have been implicated in cancer progression are CD44 and RHAMM (receptor for HA-mediated motility).
• CD44+/CD24-/ESA+ Progenitor or "tumour initiating cells” are now generally accepted to disproportionately contribute to the tumour forming potential of leukemias.
• a similar phenomenon has more recently been described in breast cancer (BC) and other solid tumours and the phenotype of these tumour cell subsets has been partially characterized as CD44+/CD24-/ESA+.
• CD44+ cells sorted by FACS from primary BC express a gene signature that predicts poor clinical outcome RHAMM mRNA and protein hyper-expression also occur in primary human breast tumour cell subsets and the presence of these RHAMM positive subsets is predictive of poor clinical outcome and increased risk of peripheral metastasis in BC.
• CD44 is expressed in many normal tissues, RHAMM expression is not detected in these tissues but appears following wound repair and during pathological processes such as cancer progression. Imaging of CD44- positive and RHAMM-positive tumour cell subsets would therefore be relevant in identifying patients at risk for poor outcome. However, as to the date of this document these and/or other tumour progenitor cells have not yet been directly imaged. It would, therefore, be advantageous to find a molecular imaging probe capable of targeting RHAMM to provide a means of imaging progenitor cell status non-invasively. This targeting method could also provide a method of selectively targeting progenitor cells with a therapeutic drug agent, either directly through the ligand-receptor interaction, or indirectly by delivering a therapeutic payload to the targeted cells.
• a chemotherapeutic eg. Cisplatin.
• the present invention provides for novel peptide ligands for targeting RHAMM, which has not been previously reported.
• the present invention relates to the discovery of novel peptides that are capable of binding to RHAMM.
• the present invention provides for an isolated peptide comprising at least one amino acid sequence selected from the group comprising at least the following amino acid sequences: SEQ ID NO: 1 to SEQ ID NO: 17.
• the present invention provides for an isolated DNA, said DNA comprising a nucleic sequence encoding peptides according to SEQ. ID No 1 to SEQ ID NO: 17.
• the present invention also relates to the discovery of peptides binding with specificity and affinity to a RHAMM, wherein said peptides are derived from tubulin.
• the RHAMM-binding peptides of the present invention are derived from the carboxy terminal tails of tubulin.
• the present invention provides for a RHAMM- binding peptide, wherein said RHAMM-binding peptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO. 17.
• the present invention provides for a RHAMM- binding peptide characterized in that said RHAMM-binding peptide comprises a sequence having a formula EEXEEZ (SEQ ID NO: 18), wherein X is selected from alanine or glycine, and Z is selected from tyrosine or glutamic acid.
• the present invention provides for a pharmaceutical composition characterized in that said pharmaceutical composition comprises an effective amount of one or more of the RHAMM-binding peptides of the present invention and a pharmaceutically acceptable carrier.
• the present invention provides for a probe comprising a RHAMM-binding peptide of the present invention and a detectable label capable of being detected by a detection technique.
• the present invention provides for a method of imaging RHAMM expression in tissues or organs of a subject, characterized in that the method comprises: (a) administering a probe comprising a RHAMM-binding peptide of the present invention and a detectable label capable of being detected by a detection technique; and (b) applying the imaging technique for detecting the label in the tissues or organs of the subject.
• a method of determining the presence of RHAMM in cells comprises contacting cells with a probe comprising a RHAMM-binding peptide of the present invention and a detectable label capable of being detected by a detection technique, and applying the imaging technique for detecting the label in the cells, wherein a detection of labels in the cells indicates the presence of RHAMM in the cells.
• the present invention provides for a method of imaging tumor progenitor cells in a subject characterized in that said method comprises administering a probe comprising a RHAMM-binding peptide of the present invention and a detectable label capable of being detected by a detection technique to the subject and applying the imaging technique for detecting the label in the subject, wherein a detection of labels in the subject indicates the presence of tumor progenitor cells in the subject.
• the present invention is a method of imaging tumor progenitor cells in animal cells, tissues or organs characterized in that said method comprises contacting the cells, tissues or organs with a probe comprising a RHAMM-binding peptide of the present invention and a detectable label capable of being detected by a detection technique, and applying the imaging technique for detecting the label in the cells, tissues or organs, wherein a detection of labels in the cells, tissues or organs indicates the presence of tumor progenitor cells in the cells, tissues or organs.
• the present invention provides for a method for determining a prognosis for a cancer patient characterized in that said method comprises: (a) obtaining a tumor tissue sample from the patient; (b) contacting said sample with a probe comprising a RHAMM-binding peptide of the present invention and a detectable label capable of being detected by a detection technique; (c) applying the imaging technique for detecting the label in the sample; and (d) determining the prognosis of the patient, wherein the prognosis predicts a probability of aggressiveness or metastasis of the cancer in the patient, and wherein the detection of RHAMM expression in the sample indicates a poor prognosis.
• the present invention provides for a method for determining a course of treatment for a cancer patient characterized in that the method comprises: (a) obtaining a tumor tissue sample from the patient; (b) contacting said sample with a probe comprising a RHAMM-binding peptide of the present invention and a detectable label capable of being detected by a detection technique; (c) applying the imaging technique for detecting the label in the sample; and (d) determining the prognosis of the patient, wherein the prognosis predicts a probability of aggressiveness of the cancer in the patient, and wherein the detection of RHAMM expression in the sample indicates a poor prognosis; and prescribing a course of treatment for the patient based on the prognosis.
• the present invention provides for a method for diagnosing a patient of a disorder or condition associated with RHAMM expression in cells characterized in that said method comprises: (a) obtaining a tissue sample from the patient; (b) contacting said sample with a probe comprising a RHAMM- binding peptide of the present invention and a detectable label capable of being detected by a detection technique; and (c) applying the imagining technique for detecting the label in the sample; wherein detection of RHAMM expression in the sample indicates a positive diagnosis of the disorder or condition.
• the present invention provides for a method for treating a subject suffering from a disorder or condition associated with RHAMM expression in cells characterized in that said method comprises administering to the subject an effective amount of a composition comprising an effective amount of one or more RHAMM-binding peptides or nucleic acids molecules encoding said one or more RHAMM-binding peptides and a pharmaceutically acceptable carrier, wherein said one or more RHAMM-binding peptides comprise a sequence having a formula EEXEEZ (SEQ ID NO: 18), wherein X is selected from alanine or glycine, and Z is selected from tyrosine or glutamic acid.
• SEQ ID NO: 18 formula EEXEEZ
• the present invention provides for a method for treating a subject suffering from a disorder or condition associated with RHAMM expression in cells characterized in that said method comprises administering to the subject an effective amount of a composition comprising an effective amount of one or more RHAMM-binding peptides or nucleic acids molecules encoding said one or more RHAMM-binding peptides and a pharmaceutically acceptable carrier, wherein said one or more RHAMM-binding peptides is or are selected from the group consisting of SEQ ID NOs. 1-17.
• the present invention provides for a method of inhibiting proliferation of cells expressing RHAMM characterized in that said method comprises contacting the cells with an effective amount of one or more RHAMM- binding peptides or nucleic acids molecules encoding said one or more RHAMM- binding peptides, wherein said one or more RHAMM-binding peptides comprise a sequence having a formula EEXEEZ (SEQ ID NO: 18), wherein X is selected from alanine or glycine, and Z is selected from tyrosine or glutamic acid.
• SEQ ID NO: 18 formula EEXEEZ
• the present invention provides for a method of inhibiting proliferation of cells expressing RHAMM characterized in that said method comprises contacting cells with an effective amount one or more RHAMM-binding peptides or nucleic acids molecules encoding said one or more RHAMM-binding peptides, wherein said one or more RHAMM-binding peptides is or are selected from the group consisting of SEQ ID NOs. 1-17.
• the present invention provides for a method of inhibiting the motility of cells expressing RHAMM characterized in that said method comprises contacting the cells with an effective of one or more RHAMM-binding peptides or nucleic acids molecules encoding said one or more RHAMM-binding peptides, wherein said one or more RHAMM-binding peptides comprise a sequence having a formula EEXEEZ (SEQ ID NO: 18), wherein X is selected from alanine or glycine, and Z is selected from tyrosine or glutamic acid.
• SEQ ID NO: 18 formula EEXEEZ
• the present invention provides for a method of inhibiting the motility of cells expressing RHAMM characterized in that said method comprises contacting the cells with an effective amount of one or more RHAMM- binding peptides or nucleic acids molecules encoding said one or more RHAMM- binding peptides, wherein said one or more RHAMM-binding peptides is or are selected from the group consisting of SEQ ID NOs. 1-17.
• said cells expressing RHAMM are cancer cells.
• the present invention provides for a method of preventing metastasis in a patient having cancer characterized in that said method comprises administering to the patient an effective amount of a composition comprising one or more RHAMM-binding peptides or nucleic acids molecules encoding said one or more RHAMM-binding peptides, wherein said one or more RHAMM-binding peptides comprise a sequence having a formula EEXEEZ (SEQ ID NO: 18), wherein X is selected from alanine or glycine, and Z is selected from tyrosine or glutamic acid.
• SEQ ID NO: 18 formula EEXEEZ
• the present invention provides for a method of preventing metastasis in a patient having cancer characterized in that said method comprises administering to the patient an effective amount of a composition comprising one or more RHAMM-binding peptides or nucleic acids molecules encoding said one or more RHAMM-binding peptides, wherein said one or more RHAMM-binding peptides is or are selected from the group consisting of SEQ ID NOs. 1-17.
• the present invention provides for a use of a RHAMM-binding peptide in the treatment, amelioration or prevention of a disorder or condition associated with RHAMM expression characterized in that said RHAMM-binding peptide comprises a sequence having a formula EEXEEZ (SEQ ID NO: 18), wherein X is selected from alanine or glycine, and Z is selected from tyrosine or glutamic acid.
• RHAMM-binding peptide comprises a sequence having a formula EEXEEZ (SEQ ID NO: 18), wherein X is selected from alanine or glycine, and Z is selected from tyrosine or glutamic acid.
• the present invention provides for a use of a RHAMM- binding peptide or nucleic acid coding for said RHAMM-binding peptide in the treatment, amelioration or prevention of a disorder or condition associated with RHAMM expression, characterized in that said RHAM -binding peptide is selected from the group consisting of SEQ ID NOs: 1-17.
• the disorder or condition associated with RHAMM expression is tissue scarring. In aspects of the invention the disorder or condition associated with RHAMM expression is cancer.
• the peptides of the invention may have added; cysteines to one or both ends of the peptide to cyclize by means of disulfide bond formation, phosphorous groups and acetyl groups.
• a multimer according to the invention can either be a homomer, consisting of a multitude of the same peptide, or a heteromer consisting of different peptides.
• the characteristic amino acid sequences of the peptides according to the invention can be flanked by random amino acid sequences. Preferred are flanking sequences that have a stabilizing effect on the peptides, thus increasing their biological availability. Flanking sequences may also be used to improve pharmacokinetic behaviour.
• fusion peptides and pegylated peptides that can improve metabolic stability, increase bioavailability, lower susceptibility to proteolysis.
• peptides characterized by at least one amino acid being replaced by another amino acid with similar chemical properties; at least one amino acid being replaced by an unnatural amino acid, such as an N- methylated amino acid, D-amino acid, or other unnatural amino acid construct; at least one additional amino acid being present at the N-or/and C-terminus; at least one amino acid being deleted; and at least one amino acid being chemically modified.
• an unnatural amino acid such as an N- methylated amino acid, D-amino acid, or other unnatural amino acid construct
• at least one additional amino acid being present at the N-or/and C-terminus
• at least one amino acid being deleted and at least one amino acid being chemically modified.
• FIG 1 A illustrates a diagram of 72 kDa RHAMM protein containing the HA-binding domain (amino acid residues 718 - 750, SEQ ID NO: 19) necessary for RHAMM/ hyaluronan (HA) interaction.
• FIG 1 B illustrates a flow chart of molecular imaging probe design and optimization.
• FIG 2 A illustrate the general structure of (a) unmodified tubulin-derived peptides, (b) tubulin-derived peptides conjugated to N-acetyl cysteine, and (c) tubulin- derived peptides conjugated to fluorescein isothiocyanate.
• FIG 2 B is a table of 17 synthesized tubulin-derived peptides, including peptides derivatized with fluorescein or N-acetyi cysteine, tubulin fragment type, calculated and observed mass spectrometry m/z values (m is representative of molecular or atomic mass and z is representative of the number of elementary charges carried by the ion), percent purity and binding constant Kd.
• Figure 2 B discloses SEQ ID NOs: 1 , 24-25, 2-3, 26-27, 4-9, 28-29, 10-1 1 , 30-31 , 12, 32-33, 13-14, 34-35 and 15-1 , respectively, in order of appearance.
• FIG 3 B illustrates surface plasmon resonance (SPR) - based screening of purified tubulin-derived peptides (SEQ ID NOs: 1-17) against RHAMM to determine high affinity ligands. Screening-resulted in six high affinity ligands (SEQ ID NOs: 1 , 3, 9, 1 1 , 12 and 14) for RHAMM.
• SPR surface plasmon resonance
• FIG 3 C is an ELISA binding assay to verify the affinity of six peptides (SEQ ID NOs: 1 , 3, 9, 1 1 , 12 and 14) after SPR-screening illustrating the binding of fluoresce] n-labelled peptides of the present invention to RHAMM. Negative control (no immobilized RHAMM) was subtracted for each of the measurements.
• FIG 4 A illustrates seven sets of sensograms showing global fits to each specific peptide-RHAMM interaction, with negative control and reference sensogram plot.
• Anti-RHAMM mAb was used as a positive control.
• Each set of sensogram corresponds to the responses of three RHAMM concentrations (1000 nM, 750 nM and 500 nM) interacting with immobilized peptide.
• FIG 4 B is a table illustrating the kinetic profile of selected tubulin-derived peptides (SEQ ID NOs: 1 , 3, 9, 1 1 , 12 and 14) and RHAMM monoclonal antibody (mAb).
• the table shows calculated association rates (KON) > dissociation rates (KOFF) and binding constants (KD). Errors are standard deviation of the mean KD-
• FIG 5 A is a graph which shows competitive displacement of six selected fluorescein-labelled RHAMM-binding peptides SEQ ID NOs: 1 , 3, 9, 1 1 , 12 and 14 (corresponding to SEQ ID NOs: 25, 27, 29, 31 , 33 and 35 respectively) of the present invention by HA (0, 1 ,25, 2,5, 5, and 10 pg/mL) to immobilized RHAMM- CT. Data shows mean of three measurements in two independent experiments.
• FIG 5 B is a graph which shows competitive displacement of dye-labelled HA by six selected non-labelled RHAMM-binding peptides (SEQ ID NOs: 1 , 3, 9, 1 1 , 12 and 14) of the present invention by HA to immobilized RHAMM-CT. Data shows mean of three measurements in two independent experiments.
• FIG 5 C is a graph which shows ELISA binding assay of six fluorescein- conjugated RHAMM-binding peptides SEQ ID NOs: 1 , 3, 9, 11 , 12 and 14 (corresponding to SEQ ID NOs: 25, 27, 29, 31 , 33 and 35 respectively) of the present invention and purified CD44 or RHAMM-CT. Data shows mean of three measurements in two independent experiments.
• FIG 6 A is a microphotograph which illustrates visualization of uptake of fluorescein-conjugated SEQ ID NO: 14 (SEQ ID NO: 35) in breast tumour cells (MDA-MB-231 ) using fluorescence imaging.
• DAPI channel denotes nucleus while FITC channel illustrates localization of the fluorescein-labelled peptide.
• FIG 6 B is a graph which illustrates quantification of uptake of fluorescein- conjugated SEQ ID NOs: 1 , 9 and 14 (SEQ ID NOs: 25, 29, 35) in breast tumour cells (MDA-MB-231 ). Each bar represents the mean of 1048 measurements from each of the three experiments. All treatment groups were significantly higher (p ⁇ 0.001 ) compared to groups which received antt-RHAMM treatment. Data were analyzed using ANOVA and errors are standard deviation of the mean
• FIG 7 is a microphotograph which illustrates visualization of uptake of FITC- conjugated SEQ ID NO: 1 and SEQ ID NO: 9 (SEQ ID NOs:. 25 and 29) in breast tumour cells (MDA-MB-231) using fluorescence imaging.
• DAPI channel denotes nucleus while FITC channel illustrates localization of the fluorescein-labelled peptide.
• FIG 8 A is an amino acid sequence of HA-binding domain (aa 718-750; SEQ ID 5 NO: 19) of RHA M Basic amino acid residues required for HA binding are underlined.
• FIG 8 B is an amino acid sequence alignment of four RHAMM-binding peptides (SEQ ID NOs: 1 , 3, 9 and 1 1). Identical sequences are highlighted in grey while semi-conserved residues are boxed in white. Acidic amino acid residues which I 0 may interact with the basic amino acid residues on RHAMM via ionic bonding are underlined.
• FIG 9 illustrates serum stability study of SEQ ID NOs: 1 , 3, 9, 10, 12 and 14. Graphs show percent intact peptides (as detected by RP-HPLC) at 30 minutes increments for 11 hours. Data were fitted to a first order decay curve.
• FIG 10 A is a graph which illustrates the affinity of alanine-substituted fluorescein- conjugated peptides of SEQ ID NO: 1 (SEQ ID NO: 25) to RHAMM (SEQ ID NOs: 1 and 36-47, respectively, in columns from left to right).
• Asterisks show significant decrease (p ⁇ 0.01 ) in observed fluorescence due to alanine substitutions of residues important for RHAMM binding (no immobilized RHAMM was subtracted
• FIG 10 B illustrates the competitive displacement of twelve alanine-substituted fluorescein-conjugated peptides of SEQ ID NO: 1 (SEQ ID NO: 25) by HA to immobilized RHAMM (SEQ ID NOs: 1 and 36-47, respectively, from left to right). 25 Negative control (no immobilized RHAMM was subtracted from each measurement and all data shows mean of three measurements in two independent experiments.)
• FIG 11 is a graph illustrating a proliferation assay of human epithelial ovarian cancer (EOC) cells in the presence of different concentrations (1 ⁇ and 10 ⁇ ) 30 of RHAMM-binding peptide SEQ ID NO: 1. In this assay, cells were treated on day 1 and proliferation was followed for six days.
• FIG 12 is a graph illustrating reduced viability of ascites-derived human EOC cells by Rhamm-binding peptide SEQ ID NO: 3 of the present invention (p ⁇ 0.05).
• FIG 13 is a photomicrograph illustrating uptake fluorescein-conjugated peptide SEQ ID NO: 1 (SEQ ID NO: 25) in ovarian tumor cells using fluorescence microscopy.
• DAPl channel denotes nucleus while FITC channel illustrates localization of the fluorescein-labelled peptide.
• FIG 14 A shows (a) uptake of the fluorescein-conjugated SEQ ID NO: 9 (SEQ ID NO: 29) of the present invention by PC3mLN4 human prostate cancer cells using fluorescence microscopy, (b) uptake of fluorescein-SEQ ID NO: 9 is not blocked by a specific anti-CD44 mAb, however, (c) uptake of fluorescein-conjugated SEQ I D NO: 9 is blocked by a specific anti-RHAM mAb.
• FIG 14 B shows quantification of SEQ ID NO: 9 of the present invention by PC3mLN4 human prostate cancer cells. Data shows no statistical difference in cells which receive no antibody treatment (a) and cells blocked with anti-CD44 (b). Uptake of the probe drastically decreased in cells treated with anti-RHAMM antibody (c).
• FIG 15 A illustrates the synthesis of Ga-DOTA-conjugated SEQ ID NO: 1 peptide.
• FIG 15 B is a RP-HPLC chromatogram of purified Ga-DOTA-conjugated SEQ ID NO: 1 peptide at 9.73 minutes.
• FIG 15 C is an ESI mass spectrum of purified Ga-DOTA-conjugated SEQ ID NO: 1 peptide.
• FIG 16 A illustrates the synthesis of Re(CO) 3 + - coordinated SEQ ID NO: 1 peptide.
• FIG 16 B is a RP-HPLC chromatogram of purified Re(CO) 3 * - coordinated SEQ ID NO: 1 peptide at 1 1.06 minutes.
• FIG 16 C is an ESI mass spectrum of purified Re(CO) 3 + - coordinated SEQ ID NO: 1 peptide.
• FIG 17 A illustrates the affinity of alanine-substituted fluorescein-conjugated peptides Of SEQ ID NO: 14 (SEQ ID NO: 35) to RHAMM (SEQ ID NOs: 48-55, 14 and 56-58, respectively, in columns from left to right).
• Asterisks show significant decrease (p ⁇ 0.01 ) in observed fluorescence due to alanine substitutions of residues important for RHA M binding (no immobilized RHAMM was subtracted from each measurement and all data shows mean of three measurements in two independent experiments.)
• FIG 17 6 illustrates the competitive displacement of eleven alanine-substituted fluorescein-conjugated peptides of SEQ ID NO: 14 (SEQ ID NO: 35) by HA to immobilized RHAMM (SEQ ID NOs: 48-55, 14 and 56-58, respectively, from left to right). Negative control (no immobilized RHAMM was subtracted from each measurement and all data shows mean of three measurements in two independent experiments.)
• A Ala-alanine
• R Arg- Arginine
• N Asn-Asparagine
• D Asp-Aspartic acid
• C Cys-Cysteine
• Q Gln- Glutamine
• E Glu-Glutamic acid
• G Gly--Glycine
• H His— Histidine
• I lie— Isoleucine
• L Leu-Leucine
• K Lys-Lysine
• M Met-Methionine
• F Phe- Phenyalanine
• P Pro-Proline
• S Ser-Serine
• T Thr-Threonine
• W Trp- Tryptophan
• Y Tyr-Tyrosine
• V Val— Valine.
• Analog includes any peptide having an amino acid residue sequence substantially identical to the sequence of the RHAMM-binding peptides of the present invention in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the ability to mimic a HA binding peptide:
• “Derivative” refers to a peptide having one or more residues chemically derivatized by reaction of a functional side group.
• Such derivatized molecules may include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
• Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
• Free hydroxy! groups may be derivatized to form O-acyl or O-alkyl derivatives.
• the imidazole nitrogen of histidine may be derivatized to form N-im-benzy!histidine.
• derivatives include those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine, homoserine may be substituted for serine; and ornithine may be substituted for lysine.
• fragment refers to any subject peptide having an amino acid residue sequence shorter than that of a peptide whose amino acid residue sequence is shown herein.
• isolated peptide or isolated DNA
• isolated DNA may be defined as a peptide or DNA molecule, as the case may be, which is substantially separated from other cellular components which may naturally accompany the peptide and DNA molecules.
• isolated peptide or isolated DNA
• isolated DNA includes, without limitation, recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
• peptide as used herein is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term “peptide” is mutually inclusive of the terms “peptides” and “proteins”.
• RHAMM refers to a Receptor for Hyaluronic Acid Mediated Motility, also known as CD 168.
• RHAMM is a non-integral cell surface (CD 168) and intracellular hyaluronan binding protein that promotes cell motility in vitro and whose expression is strongly upregulated in aggressive tumors [WO 2008/140587].
• RHAMM-binding-peptide(s) as used herein means peptides which are capable of binding RHAMM.
• Subject or “patient” refers to an animal, including humans, in need of treatment for a condition, disorder or disease.
• the present invention relates to peptides and nucleic acid sequences coding for said peptides, which may be capable of binding RHAMM.
• the present invention further relates to methods of using said RHAMM-binding peptides and nucleic acid sequences coding for said RHAMM-binding peptides in the treatment of diseases, conditions or disorders that may be associated with RHAMM expression in cells.
• the RHAMM-binding peptides of the present invention may be used in probes which may be capable of identifying cells expressing RHAMM.
• the present invention further relates to methods of using the RHAMM-binding peptides for the identification of samples which may include cells expressing RHAMM.
• Advantages of the present invention include: (a) Novel small peptides have been discovered that bind RHAMM with specificity; (b) To the inventors' knowledge tumor progenitor cells have not been directly imaged. The novel peptides may be used as molecular imaging probes to image progenitor ceil status non-invasively; (c) The capacity of the novel peptides to specifically bind RHAMM may be exploited to selectively targeting progenitor cells with a therapeutic drug agent, either directly through the ligand-receptor interaction, or indirectly by delivering a therapeutic payload to the targeted cells; (d) Binding of the peptides of the present invention to RHAMM increased epitope exposure and anti-RHAMM Mab binding, which may allow for imaging enhancement as well as to therapeutically target RHAMM positive cells.
• the present inventors have isolated, sequenced and characterized novel peptides of about 6 to 14 amino acid residues.
• the present invention provides for an isolated DNA coding for a peptide, said peptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 17.
• the present invention provides for an isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 17.
• novel peptides of the present invention may be capable of binding RHAMM, In aspects of the invention the novel RHAMM-binding peptides of the present invention may be capable of specifically binding RHAMM. In aspects of the invention the novel RHA M-binding peptides of the present invention may be capable of binding RHAMM with substantially high affinity. In aspects of the invention the novel RHAMM-binding peptides of the present invention may be capable of specifically binding RHAMM with substantially high affinity.
• novel RHAMM-binding peptides of the present invention may comprise a sequence of the formula (1):
• X is selected from A or G, and Z is selected from Y or E.
• novel RHAMM-binding peptides of the present invention may comprise a sequence selected from SEQ ID NO: 1 to SEQ ID NO:
• the present invention provides for a RHAMM-binding peptide, wherein said peptide is selected from the group consisting of SEQ ID NOs: 1, 3, 9, 10, 11 , 12 and 14.
• the present invention provides for a RHAMM-binding peptide, wherein said peptide is selected from the group consisting of SEQ ID NOs: 1, 9 and 14.
• the peptides of the present invention may be derived from the carboxy terminal tails (CTT) region of tubulin.
• CTT carboxy terminal tails
• RHAMM- binding peptides may be derived from for sequences directly adjacent to the tubulin's CTT region.
• RHAMM shares structural and functional similarities with proteins which may be essential to the maintenance of mitotic spindles.
• RHAMM's HA binding domain may show sequence homology to the tubulin binding domain of many kinesins and microtubule associated proteins (MAPs).
• MAPs microtubule associated proteins
• human breast cancer cell line MDA-MB-231 which is known to express RHAMM, showed intra-cellular fluorescence when incubated with dye- conjugated peptides of the present invention, as illustrated in FIG. 6A and FIG. 7.
• Cells blocked with anti-RHAMM showed about 65% to about 85% reduction in intra-cellular fluorescence as illustrated in FIG. 6 B.
• Other RHAMM expressing cancer cells such as PC3mLN4 and patient derived ovarian tumor cells also show intra-cellular fluorescence when incubated with RHAMM peptides of the present invention, as illustrated in FIG. 13 and FIG. 14.
• the binding of the RHAMM-binding peptides of the present invention to RHAMM may increase epitope exposure and anti- RHAMM monoclonal antibody (mAb) binding.
• This capacity of the RHAMM- binding peptides of the present invention to increase epitope exposure to anti- RHAMM mabs may be used therapeutically as well as for image enhancement.
• the peptides of the present invention may be used, for example, to "activate" available RHAMM, for example in tumors (but not limited to this disease).
• a therapeutic or imaging (Fab fragment for example) anti-RHAMM mAb may be administered and may be more effective in targeting the (tumor) cell expressing RHAMM.
• the present invention demonstrates a set of RHAMM-binding peptides which in aspects may be derived from the CTT of tubulin sequences and in other aspects may be based on artificial peptide sequences.
• the peptides of the present invention may be modified by the addition cysteine residues to one or both ends of the peptides to cyclize the peptides by the formation of disulfide bond formation.
• the peptides of the present invention may be modified by the addition of phosphorus and acetyl groups. Phosphorylation is one of the most common protein modifications that occur in animal cells [4]. It occurs most commonly on threonine, serine and tyrosine residues and plays critical roles in the regulation of many cellular processes including: cell cycle, growth, apoptosis and differentiation [4].
• peptides of the present invention contain tyrosine and because an acyl group may be added to N-terminal amino acid [5].
• aromatic amino acids phenylalanine, tryptophan and tyrosine in the peptides of the invention may be substituted with other aromatic amino acids to evaluate their effects on peptide activities.
• aliphatic amino acids such as glycine, alanine, valine, isoleucine and leucine in the peptides of the invention may be substituted with other aliphatic amino acids to evaluate their effects on peptide activities.
• the peptides of the invention may be of about 6 to about 14 amino acids in length and may include any ranges of length therein (i.e 6-13, 6-12, 6-11 , 6-10, 6-9, 6-8, 6-7, 7-13, 7-12, 7-1 1 , 7-10, 7-9, 7-8, 8-13, 8-12, 8-1 1 , 8-10, 8-9, 9-14, 9-13, 9-12, 9-11 , 9-10, 10-1 , 10-12, 10-13 and 10-14) as is understood by one of skill in the art.
• Peptides of over about 14 amino acids in length may also be encompassed by the present invention. The length of peptide being only restricted by its ability to bind RHAMM.
• the peptides of the invention may also include dimers and trimers of the peptides as well as additional stabilizing flanking sequences as is understood by those of skill in the art and described for example in U.S. Pat. No. 5,824,315 and U.S. Pat. No. 6, 184,204 (the disclosures of which are incorporated herein by reference in their entirety).
• a multimer according to the invention can either be a homomer, consisting of a multitude of the same peptide, or a heteromer consisting of different peptides.
• the amino acid sequences of the peptides according to the invention may be flanked by random amino acid sequences. Preferred may be flanking sequences which have a stabilizing effect on the peptides, thus increasing their biological availability.
• peptides of the invention may also encompass peptides which may have been modified by, for example, phosphorylation, glycosylation, pegylation or lipidation.
• the peptides of the present invention may also encompass functionally equivalent variants or analogues of the peptides of the present invention. As such, this may include but not be limited to peptides with partial sequence homology, peptides having one or more specific conservative and/or non-conservative amino acid changes and peptide conjugates which do not alter the biological or structural properties of the peptide (i.e. the ability to bind to RHAMM).
• Functional analogues may be generated by conservative or non-conservative amino acid substitutions.
• Amino acid substitutions may be generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size and the like.
• conservative amino acid changes means an amino acid change at a particular position which may be of the same type as originally present; i.e. a hydrophobic amino acid exchanged for a hydrophobic amino acid, a basic amino acid for a basic amino acid, etc.
• conservative substitutions may include, without limitation, the substitution of non-polar (hydrophobic) residues such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another, the substitution of a branched chain amino acid, such as isoleucine, leucine, or valine for another, the substitution of one aromatic amino acid, such as phenylalanine, tyrosine or tryptophan for another.
• Such amino acid changes may result in functional analogues in that they may not significantly alter the overall charge and/or configuration of the peptide. Examples of such conservative changes are well-known to the skilled artisan and are within the scope of the present invention. Conservative substitution may also include the use of a chemically derivatized residue in place of a non-derivatized residue provided that the resulting peptide is a biologically functional equivalent to the peptides of the invention. Therefore, the peptides of the present invention may encompass a peptide having an amino acid sequence that differs from SEQ ID NOs: 1-17.
• the peptides of the invention may also encompass a peptide having an amino acid sequence that may differ from SEQ ID NOs: 1-17 by a single mutation, where the single mutation represents a single amino acid deletion, insertion or substitution.
• FIG. 10 A and FIG. 10 B illustrate that in the case of RHAMM- binding peptide SEQ ID NO: 1, alanine substitution at positions 1 , 3, 5, 6 and 11 do not appear to affect proper binding of the substituted RHAMM-binding peptide to RHA M.
• FIG. 17 A and FIG. 17 B illustrate that in the case of RHAMM- binding peptide SEQ ID NO: 14, alanine substitution at positions 1 , 4, 7, 8, 11 and 12 do not appear to affect proper binding of the substituted RHAMM-binding peptide to RHAMM.
• the peptides of the present invention may be made by methods known to those of skill in the art most notably and preferably by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis [6, 7, 8, which are hereby incorporated by reference] or synthesis in homogenous solution [9, which is hereby incorporated by reference] to generate synthetic peptides.
• the peptides of the invention may be made by the use of recombinant DNA techniques known to one skilled in the art.
• the invention encompasses vectors which may include nucleic acids coding for at least one of the peptides of the present invention.
• RHAMM-binding peptides may be isolated by assaying a sample for peptides that bind to RHAMM. Any assay system or testing method that detects protein-protein interactions may be used including, without limitation, co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns. Biological samples and commercially available libraries may be tested for RHAMM-binding peptides. For example, labelled RHAMM may be used to probe phage display libraries as is described in greater detail in Example 1. In addition, antibodies prepared to the peptides of the invention may be used to isolate other peptides with RHAMM binding affinity. For example, labelled antibodies may be used to probe phage display libraries or biological samples. Additionally, a DNA sequence encoding a RHAMM protein may be used to probe biological samples or libraries for nucleic acids that encode HA-binding proteins.
• the present invention provides for compositions which may comprise one or more RHAMM-binding peptides of this invention for administration to subjects in a biologically compatible form suitable for administration in vivo.
• the present invention provides for compositions which may comprise one or more RHAMM-binding peptides for use to study samples in vitro. Samples which may be used include, without limitation, cells, tissues and organs.
• biologically compatible form suitable for administration in vivo is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects.
• the substances may be administered to any animal, preferably, humans.
• Administration of a therapeutically active amount of the pharmaceutical compositions of the present invention, or an "effective amount" may be defined as an amount effective at dosages and for periods of time, necessary to achieve the desired result of eliciting an immune response in a human.
• Suitable administration routes may be intramuscular injections, subcutaneous injections, intravenous injections or intraperitoneal injections, oral and intranasal administration.
• Acceptable carriers are well known to those skilled in the art and include, for example, sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil and deionised water.
• composition according to the invention may comprise one or more stabilizers such as, for example, carbohydrates including sorbitol, mannitol, starch, sucrose, dextrin and glucose, proteins such as albumin or casein, and buffers like alkaline phosphates.
• stabilizers such as, for example, carbohydrates including sorbitol, mannitol, starch, sucrose, dextrin and glucose, proteins such as albumin or casein, and buffers like alkaline phosphates.
• the composition of the present invention may comprise one or more adjuvants which may enhance the anti cell proliferative properties of the peptides of the invention.
• compositions for injection may include, albeit not exclusively, the peptides or nucleic acids of the present invention in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
• Any pharmaceutically suitable diluent may be used in the composition for injections: distilled water, physiological or a salt solution, and/or a buffer solution.
• the composition for injections may be prepared by conventional volume-weight procedures. A certain amount of the peptide may be diluted to the necessary volume with a diluent or solvent. The solution may then filtered through sterilized filters, bottled or ampouled. The resultant solution may be a stable transparent liquid, and may not contain any chemical or other impurities.
• Detection of RHAMM expression in samples may be carried out by a suitable probe.
• the probe may include at least a RHAMM-binding peptide of the present invention and a detectable label.
• the detectable label may be capable of being detected by a detection means.
• the detection means may be any detection technique which may be capable of providing a measurable response to the detectable label. In aspects of the invention, the measurable response may be provided in the form of an image.
• the detectable label may allow detection of the location of the RHAMM positive cells with a suitable detection means.
• the probe of the present invention may allow following movement and development of RHAMM positive cells. As such, in one embodiment, the probes of the present invention may be used, without limitation, to study RHAMM positive cells, in diagnosis and prognosis methods.
• Preferred labels may be those which are suitable for use in in vivo imaging.
• the anti-RHAMM probes may be detectably labelled prior to detection.
• a detectable label which may bind to the hybridization product may be used.
• detectable labels may include, without limitation, any material having a detectable physical or chemical property and have been well-developed in the field of immunoassays.
• a label for use in the present invention may be any composition detectable by detection means such as, without limitation, spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
• Labels which may be used in the present invention include biotin-based label, magnetic label (e.g. DYNABEADSTM), radioactive label (e.g.
• the RHAMM-binding peptides of the invention may be provided with a carrier such as for example coupled to bovine serum albumin (BSA) or keyhole limpet haemocyanin.
• BSA bovine serum albumin
• the RHAMM-binding peptides may be covalently or non-covalently coupled to a solid carrier such as a microsphere of gold or polystyrene, a slide, chip or to a wall of a microtitre plate.
• the RHAMM-binding peptides may be labelled directly or indirectly with a label selected from but not limited to biotin, fluorescein and an enzyme such as horseradish peroxidase.
• the imaging component may be a radionuclide (e.g. 18 F, 11 C, 13 N, 64 Cu, 68 Ga, 123 1, 111 In, 99m Tc, etc.) due to the ease of using such techniques as SPECT, CT and PET imaging for in vivo detection of RHAMM/RHAMM-binding peptide complexes and tumor progenitor cells.
• SPECT single photonuclide
• CT computed tomography
• PET imaging component for agents used in SPECT or PET imaging may also be determined by whether the radionuclide is generated by generator or cyclotron or is an chelator or organic/halide.
• FIGS. 15 and 16 illustrate the synthesis and mass spectrometry characterization of Ga-DOTA- conjugated peptide SEQ ID NO: 1 , and of Re(CO) 3 + - coordinated peptide SEQ ID NO: 1.
• a direct labelled probe may be a probe to which a detectable label is attached. Because the direct label is already attached to the probe, no subsequent steps may be required to associate the probe with the detectable label.
• an indirect labeled probe may be one which bears a moiety to which a detectable label is subsequently bound, typically after the RHAMM- binding peptide is hybridized with the target RHAMM.
• monoclonal antibodies which may recognize any of the RHAMM-binding peptides of the invention may also be made and used to detect the presence of the RHAMM-binding peptides in a sample.
• Mab may provide a rapid and simple method of testing the compositions of the invention for their quality.
• methods for the preparation of antibodies are well known.
• methods to produce mAb which specifically recognize the RHAMM- binding peptides of the invention are well known to those of skill in the art.
• peptides are injected in Freund's adjuvant into mice.
• mice spleens After being injected 9 times over a three week period, the mice spleens are removed and re- suspended in phosphate buffered saline (PBS),
• PBS phosphate buffered saline
• the spleen cells may serve as a source of lymphocytes, some of which may be producing antibody of the appropriate specificity. These may then fused with a permanently growing myeloma partner cell, and the products of the fusion may be plated into a number of tissue culture wells in the presence of a selective agent such as HAT.
• the wells may then be screened to identify those containing cells making useful antibody by ELISA. These may then be freshly plated. After a period of growth, these wells may again be screened to identify antibody-producing cells.
• the present invention may encompass detection, imaging, diagnostic and therapeutic strategies which may involve, without limitation, targeting RHAMM with the RHAMM-binding peptides of the present invention, which may disrupt the formation of RHAMM/ligand complexes which may play a role in stimulating the ligand-RHAMM mediated cell motility response. These methods may be used in combination with other known therapies for treating conditions related to a pathogenic condition, inflammation and pathogenic infections. 1. Detection of RHAMM Positive Cells
• the RHAMM-binding peptides of the present invention have been shown to have moderate stability in a biological environment (about 110 to about 250 minutes half-life), which may be long enough to facilitate in vitro or in vivo imaging.
• the RHAMM-binding peptides may be used in methods to identify RHAMM positive cells.
• the method for identifying RHAMM positive cells may include contacting cells with a probe of the present invention and applying a suitable detection technique for detecting the detectable label in the cells, identification of the RHAMM positive cells may be determined based on detection of the detectable label with the detection technique.
• a method of detecting RHAMM expression in tissues or organs of a subject may comprise: (a) administering a probe according to the present invention to the subject; (b) applying the detection technique for detecting the label in the tissues or organs of the subject.
• the RHAMM-binding peptides of the present invention may be used in diagnostic methods to determine whether samples such as tumor biopsies may include tumorigenic progenitor cells, which may put the cancer patient at risk for developing metastases.
• One such diagnostic method may comprise using one or more of RHAMM-binding peptides to detect for the presence of RHAMM in a sample. RHAMM positive samples may be indicative that the sample contains cancer progenitor cells.
• a method for diagnosing a patient of cancer may comprise: (a) obtaining a tissue sample from the patient, (b) contacting said sample with a probe of the present invention; (c) applying the detection technique for detecting the probe's detectable label in the sample Detection of RHAMM expression in the sample may be indicative of a cancer diagnosis.
• a method for diagnosing a patient of cancer according to another embodiment of the present invention may comprise: (a) administering a probe according to the present invention to the subject; and (b) applying the detection technique for detecting the probe's label in the tissues or organs of the subject. Detection of RHAMM in the subject may be indicative of a cancer diagnosis.
• a method for determining a prognosis for a cancer patient may comprise: (a) obtaining a tumor tissue sample from the patient; (b) contacting said sample with a probe of the present invention; (c) applying the detection technique for detecting the probe's label in the sample; and (d) determining the prognosis of the patient.
• the prognosis may predict a probability of aggressiveness or metastasis of the cancer in the patient. Detection of RHAMM expression in the sample may be indicative of a poor prognosis.
• the present invention provides for a method for determining a course of treatment for a cancer patient.
• the method may comprise: (a) obtaining a tumor tissue sample from the patient; (b) contacting said sample with a probe of the present invention; (c) applying the detection technique for detecting the probe's label in the sample; and (d) determining the prognosis of the patient.
• the prognosis may predict a probability of aggressiveness or metastasis of the cancer in the patient.
• detection of a measurable RHAMM expression in the sample may indicate a poor prognosis; and prescribing a course of treatment for the patient based on the prognosis.
• RHAMM expression in a sample may be compared to a known negative control or a known standard. Detection of a measurable RHAMM expression in the sample relative to the negative control or the known standard may be indicative of: presence of RHAMM in the sample, a diagnosis of cancer, or a poor prognosis, as the case may be.
• FIGs. 6 and 7 illustrate the visualization of human breast cancer cells using fluorescein-conjugated RHAMM-binding peptides SEQ ID NO: 14 (FIG. 6A; SEQ ID NO. 35) and SEQ ID NOs: 1 and 9 (FIG. 7; SEQ ID NOs: 25 and 29).
• FIG, 13 illustrates the visualization of human ovarian cancer cells using fluorescein-conjugated RHAMM-binding peptides SEQ ID NO: 1 (SEQ ID NO: 25), while FIG 14 demonstrates the visualization of human prostate cancer cells using fluorescein-conjugated RHAMM-binding peptides SEQ ID NO: 9 (SEQ ID NO: 29).
• PC3mLN4 human prostate cancer lines which are aggressively invasive and metastatic, are strongly positive for fluorescent staining indicating rapid uptake of the HA peptide mimetic SEQ ID NO: 9.
• RHAMM-binding peptides of the present invention may be 5 blocked by a specific anti-RHAMM mAb but not by a specific anti-CD44 mAb, which may however block binding of HA to CD44 ⁇ see FIGs 6, 7, 13 and 14). These results may indicate that the HA peptide mimetics of the present invention may associate with and may be taken up by prostate, breast and ovarian cancer cells by a RHAM dependent mechanism.
• the RHAMM-binding peptides of the present invention may have substantial stability in a biological environment (about 110 to about 250 minutes half-life), which may be long enough to facilitate their use in therapeutic methods.
• the RHAMM-binding peptides of the invention may be used in the prophylaxis or treatment of pathological conditions involving cell locomotion such as cancer, inflammatory and autoimmune disorders, and fibrotic disorders associated with tissue trauma and its recovery in a mammal.
• RHAMM-binding peptides of the present invention may be used in compositions0 and methods of treatment of disorders or conditions which may result from the formation of RHAMM/ligand complex by disrupting the formation of said RHAMM/ligand complex.
• the present invention may provide for a method for treating a subject who may be suffering from a disorder or condition associated with5 RHAMM expression in cells.
• the method may include at least administering to the subject an effective amount of a composition comprising an effective amount of one or more RHAMM-binding peptides or nucleic acids molecules encoding said one or more RHAMM-binding peptides and a pharmaceutically acceptable carrier.
• the present invention provides a method of preventing or0 reducing tissue scarring.
• the method may include at least administering an effective amount of one or more RHAMM binding peptide or nucleic acid molecules encoding said one or more RHAMM binding peptides of the invention to a subject in need thereof.
• the ability of the RHAMM-binding peptides to inhibit the proliferation of cancer cells may implicate their effectiveness in preventing tumour metastasis and their utility as cancer chemotherapeutic agents. Accordingly, the present invention provides a method of preventing or reducing tumour metastasis comprising administering an effective amount of a RHAMM-binding peptide or a nucleic acid molecule encoding a RHAMM-binding peptide of the invention to a subject in need thereof.
• the RHAMM-binding peptides of the invention may be useful in treating cancers which may be associated with RH AMM expression including, without limitation, cancer of the lung, gastrointestinal, breast, bladder, skin cancer (melanoma and non-melanoma), brain, cervix, and leukemia.
• the present invention may provide a method of preventing or treating cancer in a subject. This method may include at least administering an effective amount of one or more RHAMM binding peptides or nucleic acid molecules encoding said one or more RHAMM binding peptides of this invention to a subject in need thereof
• the RHAMM-binding peptides of the present invention may be used in combination with existing treatments to reduce morbidity and mortality in patients with a disorder or condition associated with RHAMM expression, such as cancer.
• a method for treating a subject for a disorder or condition associated with RHAMM expression may include at least administering to the subject a RHAMM-binding peptide of the present invention in combination with at least one other therapy for said RHAMM-related disorder.
• the combination of the RHAMM- binding peptide and at lease one other therapy may increase the efficacy of the disease therapy.
• the subject may also be administered a therapy which may be capable of substantially reducing angiogenesis, such as small-molecule drugs, siRNAs, antisense therapy or any combinations thereof which may target one or more growth factors which may act as angiogenesis switches as well as providing proliferative signals for trophoblast and endothelial cells.
• growth factors may be needed for the growth and survival of various cancers and may be essential for cancer progression. Therefore the anticancer methods of the invention using RHAMM-binding molecules may be combined with therapies targeted to one or more growth factors, including VEGF, FGR and hCG.
• the RHAMM-binding peptides of the present invention may also be combined with radiotherapy or chemotherapy. Such combination therapy contributes to a synergistic effect to treat cancer.
• RHAMM-binding peptides of the present invention may be capable of inhibiting proliferation of human epithelial ovarian cancer (EOC) cells (see FIG. 11).
• EOC epithelial ovarian cancer
• the inventors have also shown that RHAMM-binding peptides of the present invention may be capable of reducing the viability of human EOC cells (see FIG 12).
• the detection methods of the present invention may allow for the development of novel therapeutics to selectively kill or force terminal differentiation of these progenitor cells.
• the technology of the present invention may be used for in vivo imaging of tumors which may contain highly tumorigenic progenitor cell subsets, which may allow for selective treatment of patients at risk for metastasis vs. those not at risk.
• the technology of the present invention may be used to deliver anti-cancer drugs to the highly tumorigenic progenitor subsets present in primary cancers, such as breast cancers.
• the peptide may be conjugated to a cytotoxic agent in order to enable the targeted delivery of the cytotoxic to the cells expressing RHAMM.
• the delivery of other therapeutic entities may be possible, including but not limited to, alkylating agents, anti-angiogenic agents, antimitotics, hormonal therapeutics, nucleoside analogues, or prodrugs thereof.
• radiotherapeutics may be envisioned whereby a RHAMM-binding peptide of the present invention may be linked to a particle emitting radionuclide thereby providing a mechanism of delivering a cell destructive entity selectively to the RHAMM positive cells.
• a therapeutic radionuclide can include a beta-emitting radionuclide such as 90 Y, or 186 Re; a beta/gamma emitting radionuclide such as 47 Sc, 15 Sm, 177 Lu, or 188 Re; alpha-emitting radionuclides such as 213 Bi, Z23 Ra, or 225 Ac; or Auger emitting radionuclides such as 67 Ga or 1 ln,
• the probes and/or the peptides of the present invention may be administered directly to a mammalian subject using any route known in the art, including without limitation e.g., by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, or intradermal), inhalation, transdermal application, rectal administration, intranasal or oral administration.
• the detecting probes and/or peptides of the present invention may be administered subcutaneously.
• the detecting probes and/or peptides of the present invention may be administered intravenously.
• an effective amount of the probe or RHAMM-binding peptide may be administered via non-systemic, local administration, such as by peripheral administration which includes peripheral intramuscular, intraglandular, and subcutaneous administration routes, and allowing the probe and/or peptide several hours in vivo to be carried to sites having cells expressing RHAMM and tumorigenic cell populations.
• Another embodiment of the present invention may be administering the RHAMM- binding peptides via an implantable device.
• the implantable device may be capable of controlled release of the RHAMM-binding peptides.
• the RHAMM-binding peptides of the present invention may be provided systemically as described herein above. As understood by one of skill in the art, the RHAMM-binding peptides of the invention may be used within conventional liposomes, specialized liposomes, lipid formulations and immunoliposomes.
• the liposomes may be unilamellar or multilamellar and may be formed of constituents selected from phosphatidylcholine, dipalmitoylphosphatidylcholine, cholesterol, phosphatidy!ethanolamine, phosphatidylserine, demyristoylphosphatidylcholine and combinations thereof.
• the multilamellar liposomes may include multilamellar vesicles of similar composition to unilamellar vesicles, but may be prepared so as to result in a plurality of compartments in which the silver component in solution or emulsion is entrapped. Additionally, other adjuvants and modifiers may be included in the liposomal formulation such as polyethyleneglycol, or other materials.
• liposome may include dipalmitoyl- phosphatidylcholine:cholesterol (1 :1) it is understood by those skilled in the art that any number of liposome bilayer compositions may be used in the composition of the present invention.
• Liposomes may be prepared by a variety of known methods such as those disclosed in U.S. Pat. No. 4,235,871 and in RRC, Liposomes: A Practical Approach. IRL Press, Oxford, 1990, pages 33-101, which are hereby incorporated by reference.
• the liposomes containing the RHAMM-binding peptides may have modifications such as having non-polymer molecules bound to the exterior of the liposome such as haptens, enzymes, antibodies or antibody fragments, cytokines and hormones and other small proteins, polypeptides or non-protein molecules which confer a desired enzymatic or surface recognition feature to the liposome.
• Surface molecules which preferentially target the liposome to specific organs or cell types include for example antibodies which target the liposomes to cells bearing specific antigens. Techniques for coupling such molecules are well known to those skilled in the art (see for example U.S. Pat. No. 4,762,915 the disclosure of which is incorporated herein by reference).
• any number of lipids bearing a positive or negative net charge may be used to alter the surface charge or surface charge density of the liposome membrane.
• the liposomes may also incorporate thermal sensitive or pH sensitive lipids as a component of the lipid bilayer to provide controlled degradation of the lipid vesicle membrane.
• the dose administered to a patient may be a suitable dose to effectively image sites of RHAMM/RHAMM-binding ligand complexes and tumorigenic cell populations with sufficient specificity such that a surgeon may perform a biopsy or other procedure to remove tumorigenic cell populations detected by imaging RHAMM/RHAMM-binding peptide complexes.
• the dose may be determined by the efficacy of the particular vector ⁇ e.g. peptide or nucleic acid) employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
• the size of the dose also may be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a probe in a particular patient.
• detecting probes of the present invention may be administered at a rate determined by the LD-50 of the polypeptide or nucleic acid, and the side- effects of the polypeptide or nucleic acid at various concentrations, as applied to the mass and overall health of the patient.
• Administration may be accomplished via single or divided doses, e.g., doses administered on a regular basis (e.g., daily) for a period of time (e.g., 2, 3, 4, 5, 6, days or 1-3 weeks or more).
• about 5 to about 2000 LD 50 units of the labeled probe may be administered to said subject using recognized clinical standards and practices.
• FIG. 1 B illustrates a flow chart of molecular imaging probe design and optimization.
• the HA-binding domain was used as the query sequence in BLAST to determine homologous protein sequences.
• putative binding partners ligands
• Library of peptides were synthesized based on truncation of ligand sequences and screened using surface plasmon resonance (SPR) binding assay to determine high affinity iigands.
• SPR surface plasmon resonance
• Pair-wise comparisons between RHAMM and microtubule binding domain of MAPs and kinesins revealed moderate sequence homology (about 17%-24% as calculated using ClustalX2 [12]) to the HA binding domain of RHAMM; however, MAPs and RHAMM share similar physiochemical properties regarding the amino acid sequence of their tubulin binding site. In other words, both RHAMM and MAPs have a stretch of basic residues which are postulated to bind to tubulins. 5 Furthermore, the secondary structures of the tubulin binding sites Of MAPs and hyaluronan binding site of RHAMM has the same degree of helicity and both could be classified as a basic-zipper domain [13].
• RHAMM has been reported to use a common binding site to associate with HA in the extracellular surface and microtubules in the cytosol [14]. Since microtubule-
• RHAMM could show direct interaction with synthetic peptides representing the CTTs of different tubulin subtypes. Since HA interacts with RHAMM mainly through ionic interactions between its negatively charged groups and the positively charged groups on RHAMM, molecules possessing an
• tubulin CTTs may mimic the carboxylates on HA.
• the binding of synthetic CTTs to the HA binding domain of RHAMM might utilize the same charged side chains thought to be important for HA binding.
• N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride EDAC
• N-hydroxysulfosuccinimide sodium salt sulfo-NHS
• fluorescein isothiocyanate FITC
• FBS fetal bovine serum0
• NHS-Biotin was obtained from Nova BioChem.
• Antibodies such as anti-RHAMM (Santa Cruz Biotechnology, USA), anti-CD44 (Pharmigen) and IgG ab (Santa Cruz Biotechnology USA) were obtained commercially.
• Peptide Synthesis Seventeen (17) peptides having 12 amino acid residues corresponding to the CTT and H 2 region of different tubulin subtypes were synthesized using solid-phase peptide synthesis following Fmoc protocols.
• the filtrate was collected, precipitated using cold tert-butyl methyl ether and pelleted via centrifugatlon at 3000 rpm in -5°C for 10 minutes. Pellets were then dissolved in distilied-deionized water and lyophilized yielding solid powders,
• Recombinant protein RHAMM-CT (a truncated RHAMM corresponding only to the carboxy terminus which contains the HA binding domain, amino acids 706-766, sequence: RDSYAQLLGH QNLKQKIKHV VKLKDENSQL KSEVSKLRSQ
• ProteON XPR36 system was used for the selection and ranking of different tubulin derived peptides against RHAMM.
• the ProteON GLC sensor chip surfaces were activated by amine coupling using 100 mM EDAC and 24 mM sulfo-NHS.
• RHAMM (30 g/mL in sodium bicarbonate buffer, pH 9.7) was injected at a flow a rate of 30 ⁇ /min.
• a buffer sample was injected on a different sensor plate for use as a reference.
• Ethanolamine HCI (1 M, pH 8.5) was then injected to deactivate any remaining surface groups.
• Peptides (10 ⁇ in PBS-T, 2% DMSO) were injected to a RHAMM functionaiized surface at 50 iUm ⁇ n for 3 minutes followed by a 10 minute dissociation (i.e. injection of PBS-T buffer) period.
• the surfaces were regenerated using two injections of 30 [it of 1 M NaCI prior to the injection of the next peptide.
• reference subtraction was performed using data obtained from reference plate (No RHAMM) and RHAMM functionaiized plate.
• CompetMve ELISA experiments using fluorescein-labelled peptides ELISA was carried out to test the ability of FITC-labelled tubultn-derived peptides compete with HA for binding site with RHAMM.
• ELISA was carried out to test the ability of non-labeled tubulin-derived peptides to compete with dye (Alexa Fluor 6 7)-conjugated HA for RHAMM.
• the sensor chip surface was regenerated for the next peptide sample injection, via treatment with two 10 min pulse injections of regenerating buffer (2 M NaCI in HBS-EP, pH 7.4) at 100 mL/min. Baseline returned to the initial value after the regeneration step, confirming the removal of all bound analytes. Data were analyzed and the corresponding dissociation constants (Ko) were obtained via non-linear regression fitting to a Langmuir binding model [17]. In all experiments, reference subtraction was performed using data obtained from reference plate (no peptide) and peptide functionalized plate.
• Peptides were eluted from the cartridge using 3 mL ethanol (70% v/v) with 0.1 % TFA, lyophilized, and analyzed using analytical RP-HPLC (Grace Vydac Protein/Peptide RP-C18 column 4.6 x 250 mm, 5 pm).
• analytical RP-HPLC Grace Vydac Protein/Peptide RP-C18 column 4.6 x 250 mm, 5 pm.
• Employed mobile phase of this system were 0.1 % TFA in water (eluent A) and 0.1% TFA in CH 3 CN (eluent B). Linear gradient of 10-95% of eluent 8 at a flow rate of 1.5 ml/min over 20 minutes was used for each peptide sample.
• Percent intact peptides were detected using Waters 2998 Photodiode array detector set at 220 and 254 nm, and identified using ESI-MS. Haif-life of peptides were determined using GraphPad
• Candidate peptides showing high affinity for RHAMM-CT were conjugated to a fluorescent dye (FITC) and their specificities for RHA M expressing cancer cells (MDA-MB-231) were evaluated using a cellular fluorescence imaging assay.
• FITC fluorescent dye
• DA-MB-231 cells cultured in DM EM media and 10% FBS up to 90% cofluency. Then the cells were seeded on a glass cover slip (12 x 12 mm, coated with 50 ug/ml fibronectin) in a 2 x 24-well tissue culture place (confluency of 20000/well) one day after seeding. Starvation step carried out with DMEM + 0.1 % FCS overnight at 37°C. Then culture medium was aspirated, and was rinsed with DMEM + 0.1 % FCS overnight at 37°C. Cells were blocked with 3% BSA in DMEM + 0.1 % FCS for 1 hour at room temperature.
• antibodies (dilution 1 : 100, mouse IgG ab, goat, anti-RHAMM mAb, or mouse anti-CD44 mAb in DMEM + 0.1 % FCS media) were added and incubated at 37°C for 1 hour. Cells blocked with anti-mouse IgG serve as positive control since the antibody shows no binding affinity to RHAMM.
• ROI Regions of interest
• FIG. 2 A shows the general structure of unmodified tubulin-derived peptides (FIG.
• FIG. 2 A a is a table which describes all 17 peptides prepared for this study. Sequences of 17 synthesized peptides encompass the CTTs of different tubulin subtypes as well as sequences directly flanking a1 a- (SEQ ID NOs: 6, 7 and 8) and pilla-CTT (SEQ ID NOs: 13 and 14).
• FIG. 2 B also includes derivatized peptides.
• Derivatized peptides were prepared with either fluorescein (peptides 1 c, 3c, 9c, 1 1 c, 12c, 14c of FIG. 2B; SEQ ID NOs: 25, 27, 29, 31 , 33 and 35) or N-acetyl cysteine modified N-terminus (peptides 1 b, 3b, 9b, 1 1 b, 12b, 14b of FIG. 2B; SEQ ID NOs: 25, 27, 29, 31 , 33 and 35) or N-acetyl cysteine modified N-terminus (peptides 1 b, 3b, 9b, 1 1 b, 12b, 14b of FIG. 2B; SEQ ID NOs: 25, 27, 29, 31 , 33 and 35) or N-acetyl cysteine modified N-terminus (peptides 1 b, 3b, 9b, 1 1 b, 12b, 14b of FIG. 2B; SEQ ID NOs: 25, 27, 29, 31 , 33 and 35) or N-acetyl cyste
• FIG. 2 B is a table illustrating the analysis of the 17 synthesized tubulin-derived peptides using ESI-MS and RP HPLC. The calculated and observed m/z values are based on the prominent observed signals as determined by ESI. Percent purity determined by RP HPLC with detection at 220 nm.
• a SPR (surface plasmon resonance)-based screening method was utilized for rapid and accurate determination of potential peptide candidates that are able to recognize the HA-binding domain of RHAMM. Seventeen tubulin-derived peptides were screened, and the resulting sensograms were used to deduce peptides which show affinity to RHAMM. Prior to screening, the optimal condition for ligand immobilization, in this instance RHAMM, to the sensor plate was determined. The optimal pH for the immobilization buffer balances the electrostatic attraction of the protein to the sensor plate while minimizing protein deactivation. In this study the optimal pH for immobilization was determined to be the pH that yielded the highest ligand density.
• RHAMM was coupled to the sensor plate using sodium bicarbonate buffer of varying pH, at a flow rate of 30 ⁇ / ⁇ - ⁇ and with fixed concentration of EDAC and sulfo-NHS.
• the RHAMM ligand density for each pH immobilization condition was determined from the average SPR response of six measurements and the maximum ligand immobilization occurred at pH 9.7 (FIG. 3 A).
• Ligand immobilization was slightly lower than pH 10.1 due in part to loss of net charge on RHAMM at its Isoelectric point.
• FIG. 3 B shows the experimental sensograms obtained at a concentration of 10 ⁇ .
• control sensograms no immobilized RHAMM and buffer injection
• FIG. 4 A depicts the resulting sensograms. Again, each sensograms has been corrected by subtracting each curve from a reference sensorgram (data acquired from the sensorgram without immobilized peptide) in order to account for refractive index differences between running buffer and sample solutions.
• the relative binding affinities of each of the 6 peptides were also measured using ELISA.
• peptides were labelled with fluorescein, with the dye situated away from the peptide by the addition of an aminohexanoic acid linker.
• ELISA results also indicated that peptides SEQ ID NOs: 1 , 9 and 14 had the highest relative binding affinity in all concentrations. It may be possible that the fluorophore modification to the peptide could have an effect on the ligand- HAMM interaction or on non-specific binding, thus SPR and ELISA results might not exactly match.
• a competitive ELISA was done to determine the selectivity of the peptides to the HA binding domain of RHAMM. In this assay, fluorescein-labelled peptides were used and the effectiveness of unlabe!led HA to block the binding of peptides to RHAMM was evaluated. Fluorescein-labelled peptides SEQ ID NOs: 1 , 3, 9, 11 , 12 and 14, corresponding to peptides 1c, 3c, 9c, 11c, 12c and 14c in FIG.
• 5 A shows competitive displacement of peptide binding to RHAMM by HA, and a concentration dependent decrease in fluorescence is observed for all six ligands as the HA competitor concentration increases, with the most effective relative displacement occurring for fluorescein-labelled peptides SEQ ID NOs: 1 , 12 and 14 (SEQ ID NOs: 25, 33 and 35), although peptide of SEQ ID NO: 9 (SEQ ID NO: 29) was also displaced.
• Serum Stability To test their potential as targeting entities or therapeutics, the stability of the RHAMM-binding peptides in biological environments needs to be investigated.
• the peptides are subjected to fetal bovine serum at 37°C, and aliquots were taken at 30 minutes intervals. At each time point, the reaction was stopped by precipitating serum protein with trifluoroacetic acid (TFA), and centrifugation was used to obtain a crude solution containing the peptides.
• TFA trifluoroacetic acid
• MDA-MB-231 Uptake Studies To determine the specificity of tubulin derived peptides of the present invention to RHAMM expressing cells and their potential as a targeting entity, a cellular fluorescence assay was performed using MDA-MB-231 cells treated with anti-RHAMM antibody. MDA-MB-231 breast tumour cell line shows high expression of RHAMM receptors, thus this cell line was an ideal choice for evaluation of the targeting and specificity of synthetic tubulin peptides. MDA-MB-231 cells were incubated with fluorescein-conjugated peptides SEQ ID NOs. 1 , 9 and 14 (SEQ ID NOs: 25, 29 and 35) and cellular uptake of the probe was measured.
• IgG antibody non-specific antibody
• cells blocked with anti-CD44 showed no reduction of signal.
• cells blocked with anti-RHAMM mAb showed significant decrease in cellular fluorescence confirming the specificity of peptides SEQ ID NOs: 1 , 9 and 14 to RHAMM (p ⁇ 0.001 ).
• FIG. 6A for SEQ ID NO: 35
• FIG. 7 for SEQ ID NOs: 25 and 29
• RHAMM is an oncogene that is over-expressed in a variety of cancers.
• increased accumulation of HA, the extracellular ligand of RHAMM, within RHAMM- expressing cancer cells is a prognostic factor for poor clinical outcome.
• low molecular weight ligands targeting RHAMM that are able to compete with HA for binding may be useful for diagnostic and therapeutic purposes.
• the inventors sought to identify ligands that could target the HA binding domain of RHAMM.
• RHAMM which contains the HA binding domain, mediates the localization at the microtubule network within the cell
• database searches and pairwise comparison between RHAMM and microtubule binding domain of known tubulin associated proteins e.g. MAPs
• these proteins show direct binding to CTTs of tubulin
• the inventors hypothesize that RHAMM may show direct interaction with synthetic peptides representing the CTTs of different tubulin subtypes. Due to moderate homology between the tubulin binding domain of MAPs and HA-binding domain of RHAMM, further investigation was performed to determine which peptide fragments of tubulin interacts with RHAMM.
• This method was utilized because it allows for high throughput screening using a single RHAMM-charged sensor plate and the SPR-bound RHAMM could reflect its native membrane bound state as a cellular receptor.
• the screening afforded six high-affinity ligands that are able to compete with HA for the HA binding region of RHAMM as determined by ELISA. Further evaluation determined that these peptides showed a higher specificity to RHAMM compared to CD44, a known hyaladerin that also binds HA.
• Peptides showed moderate stability (approximately 110-250 minutes half-life) in a biological environment (bovine serum), which may be long enough to facilitate in vitro, ex vivo or in vivo imaging.
• the specificity of the three HA peptide mimics (SEQ ID NOs:. 1 , 9 and 14) showing the higher affinity for RHAMM was analyzed by quantifying the relative uptake of the ligands in RHAMM-expressing cancer cells.
• peptides of SEQ ID NOs:. 1 , 9 and 14 were conjugated with fluorescein (SEQ ID NOs: 25, 29 and 35) and incubated with MDA-MB-231 ceils, a cell line which over-expresses RHAMM.
• This study discloses the discovery of at least six novel ligands that interact with the HA binding domain of RHAMM. These six peptides showed strong affinity to RHAMM and were able to be displaced by endogenous HA, thus demonstrating the specific targeting to the HA-binding domain. In addition, the greater propensity of these ligands to bind to RHAMM as compared to another hyaladerin CD , both in non-cell based (i.e. ELISA) and cell based (i.e. cellular fluorescence) assays, may demonstrate their potential as a targeting entity. The over-expression of RHAMM and its resulting interaction with HA has been implicated in the pathology of cancers. Therefore, these peptides may serve as antagonists that could bloc the HA M-HA interaction, thus limiting the transforming potential of RHA M.
• Solvents were used without further purification, and purchased from VWR, Fisher Scientific, or Sigma Aldrich, Fluorenylmethyloxycarbonyl (Fmoc)- Rink amide MBHA (100-200 mesh) resin, Fmoc amino acids, Fmoc-protected aminohexanoic acid (Fmoc-Ahx) and HBTU (2-(1 H-benzotriazole 1-yl)-1 , 1 ,3,3- tetramethyluronium hexafluorophosphate) coupling reagent for peptide synthesis were obtained from Peptides International,
• the ensuing resin was treated with cleavage cocktail consisting of 88% TFA and 12% scavengers (5% water, 5% triisopropyisilane, and 2% phenol). The resulting resin was shaken for 3 hours at 700 rpm and purified via RP-HPLC.
• FIG. 10 A illustrates the affinity of twelve aianine-substituted (alanine scan) fluorescein-conjugated peptide SEQ ID NO: 1 (SEQ ID NO: 25) to RHAMM. Data show that residues 2, 4, 7-10 and 12 of SEQ ID NO: 1 may be important for proper binding to RHAMM.
• FIG. 10 B illustrates competitive displacement of the 12 aianine-substituted fluorescein-labeiled peptides of SEQ ID NO: 1 (SEQ ID NO: 25) by HA to immobilized Rhamm-CT. Data shows that alanine substitution of SEQ ID NO: 1 at positions 2, 4, 7-10 and 12 abolishes the peptide's ability to compete with HA. Negative control (no immobilized Rhamm) was subtracted for each measurement and all data shows mean of three measurements in two independent experiments.
• FIG. 17 A illustrates the affinity of 11 aianine-substituted fluorescein-conjugated peptide SEQ ID NO: 14 (SEQ ID NO: 35) to the HA-binding region of RHAMM. Data show that residue 2, 3, 5, 6, 9 and 10 of SEQ ID NO 14 may be important for proper binding to RHAMM.
• FIG. 17 B illustrates competitive displacement of 11 aianine-substituted fluorescein-labeiled SEQ ID NO: 14 (SEQ ID NO: 35) by HA to immobilized RHAMM-CT. Data shows that alanine substitution of SEQ ID NO: 14 at positions 2, 3, 5, 6, 9 and 10 abolishes the peptide's ability to compete with HA. Negative control (no immobilized RHAMM) was subtracted for each measurement and all data shows mean of three measurements in two independent experiments.
• Buffer solutions and media were purchased from Invitrogen, Sigma, and VWR, AlamarBlue assay kits were purchased from Invitrogen. Dimehylsulfoxide (DMSO) was purchased from Aldrich. Epithelial ovarian cancer (EOC) cells were obtained from London Regional Cancer Center following patient consents.
• RHAMM-binding peptide SEQ ID NO: 1
• SEQ ID NO: 3 RHAMM-binding peptide
• epithelial ovarian cancer cells derived from ascites of patients
• Peptides (1 uM and 10 uM) were added to cells on day one and proliferation was measured for 6 days.
• ROI Region of interest
• FIG. 11 illustrates a proliferation assay of peptide SEQ ID NO: 1 in human epithelial ovarian cancer (EOC) cells.
• EOC cells are samples from the ascites of patients with epithelial ovarian cancer.
• the alamarBlue assay was used to measure the level of proliferation.
• Peptides of SEQ ID NO: 1 were added once on day 1 and the effect over time was measured. Data showed inhibition of proliferation in 3 of 4 patient OC cell samples
• Figure 13 illustrates the visualization of uptake of fluorescein-conjugated peptide SEQ ID NO: 1 (SEQ ID NO: 25) in ovarian tumor cells using fluorescence microscopy.
• uptake of the fluorescein-labelled SEQ ID NO: 1 (SEQ ID NO: 25) decreased when EOC cells were incubated with anti-RHAMM and uptake of the probe was unaffected with cells were treated with anti-lgG or anti- CD44.
• FIG. 12 is a graph illustrating reduced viability of ascites-derived human EOC cells by a RHAMM-binding peptide (p ⁇ 0.05).
• Primary cells from patient sample EOC were seeded to a 24-well culture dish and 24 hours later treatment was initiated with the RHAMM-binding peptide SEQ ID NO. 3 (7 and 0.7 uM concentrations) or DMSO vehicle control. Re-dosing occurred everyday for up to 6 days and cell viability was assessed by fluorometry using alamarBlueTM reagent (measured in fluorescence units, FU).
• the two hyaluronan receptors expressed by invasive/metastatic prostate tumor cells are CD44 and RHAMM.
• the ability of peptides of the present invention to selectively target prostate cancer cells was evaluated.
• PC3mLN4 human prostate cancer cells were cultured in DMEM media + 10% FBS up to 90% confluency. Then cells were seeded on glass cover slips (12 x 12 mm, coated with 50 ug/ml fibronectin) in 2 x 24-well tissue culture plates (confluency of 20,000 cells/well). Then cells were blocked with 3% BSA in DMEM + 0.1% FCS for 1 hour at room temperature.
• Figure 14 A shows the uptake of the fiuorescein-conjugated peptide SEQ ID NO: 9 mimetic of HA (SEQ ID NO. 29) by PC3mLN4 human prostate cancer lines, which are aggressively invasive and metastatic.
• FITC signal decreases when RHAMM proteins are blocked with anti-RHAMM mAb (FIG. 14A (c)). This indicates that probe uptake is blocked by a specific anti- RHAMM monoclonal antibody.
• Solvents were used purchased either from WVR, Fisher Scientific, or Sigma Aldrich.
• Rink amide MBHA resin 100-200 mesh, 0.56 mmol/g
• standard Fmoc amino acids and HBTU coupling reagents were obtained from Peptides International.
• 1 -Bromohexanocic acid, glycine, Bromo ethylacetate and gallium (III) nitrate were purchased from Sigma Aldrich. Cyclen (1 ,4,7, 10- tetraazacyclododecane) and Re(CO)sBr were purchased from Strem Chemicals
• Peptide Synthesis Peptides were synthesized on rink amide MBHA resins (0.1 mmol) using standard solid phase peptide synthesis involving Fmoc deprotection and amino acid coupling cycles. Repeated Fmoc deprotection throughout the synthesis (15 and 20 minutes periods) was carried out using 20% piperidine solution in ⁇ /, ⁇ -dimethylformide (D F). All amino acid couplings were carried out using 0.05 or higher concentration of Fmoc-protected amino acid and HBTU, 5 equivalent of ⁇ /,/V-diisopropy!ethylamine (DIPEA) in DMF at 30 and 90 minutes intervals.
• DIPEA ⁇ /,/V-diisopropy!ethylamine
• Analytical HPLC was performed using a Grace Vydac Protein/Peptide RP-C 8 column (4.6 mm x 250 pm, 5 Mm), and preparative HPLC was performed using a Grace Vydac protein/peptide RP- C 8 column (22.0 mm x 250 mm, 10 pm), Absorbance was detected at wavelengths of 220 nm and 254 nm using a Waters 2998 Photodiode Array detector. During purification, fractions were collected, lyophilized, and analyzed by ESI-MS (Waters Micromass Quattro MicroTM API).
• Ga ⁇ 69/71 Labelling Gallium coordination was performed using 69 71 Ga(N0 3 ) 3 in acetate buffer at pH 5.5 for 15 minutes. Products were isolated using C18 reverse- phase (C18 Sep-Pak ⁇ ) cartridge. Products was eluted from the cartridge using 30% acetonitrile in de-ionized water with 1 % trifluoroacetic acid. Resulting product was characterized using mass spectrometry, and purity was analyzed using RP- HPLC. Synthesis and Characterization of Ga-DOTA-conjugated peptide
• FIG 15 A describes the gallium labelling of DOTA- conjugated peptide.
• FIG. 15 B illustrates RP-HPLC chromatogram of purified gallium coordinated peptide at 9.73 minutes.
• FIG. 15 C illustrates an ESI mass spectrum of the compound of FIG. 15 A (observed: 936.7 [M+2H] Z ⁇ calculated: 934.87 [M+2H] 2+ ).
• Solvents were used purchased either from VWR. Fisher Scientific, or Sigma Aldrich.
• Rink amide BHA resin 100-200 mesh, 0.56 mmol/g
• standard Fmoc amino acids and HBTU coupling reagents were obtained from Peptides International.
• Glycine, sodium cyanoborohydride and 3-Pyridinecarboxaldehyde were purchased from Sigma Aldrich.
• Re(CO) s Br was purchased from Strem Chemicals
• FIG 16 A describes the synthesis of Re(CO)3* - coordinated peptide SEQ ID NO: 1 and FIG. 16 B illustrates RP-HPLC chromatogram of purified rhenium coordinated peptide at 1 1.06 minutes.
• FIG. 16 C illustrates an ESI mass spectrum of the rhenium coordinated peptide (observed: 939.1 [M+2H] 2+ , calculated: 940.2 [M+2H] Z+ ).
• RHAMM is a centrosomal protein that interacts with dynein and maintains spindle pole stability. Mol. Biol. Cell., 14, 2262-2276.

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