WO2012068412A2 - Method and compositions for treatment of stat3-responsive cancers and/or renal cancer - Google Patents

Method and compositions for treatment of stat3-responsive cancers and/or renal cancer Download PDF

Info

Publication number
WO2012068412A2
WO2012068412A2 PCT/US2011/061260 US2011061260W WO2012068412A2 WO 2012068412 A2 WO2012068412 A2 WO 2012068412A2 US 2011061260 W US2011061260 W US 2011061260W WO 2012068412 A2 WO2012068412 A2 WO 2012068412A2
Authority
WO
WIPO (PCT)
Prior art keywords
stat3
scv
cells
treatment
subject
Prior art date
Application number
PCT/US2011/061260
Other languages
French (fr)
Other versions
WO2012068412A3 (en
Inventor
Cynthia Tuthill
Israel Rios
Original Assignee
Sciclone Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sciclone Pharmaceuticals, Inc. filed Critical Sciclone Pharmaceuticals, Inc.
Publication of WO2012068412A2 publication Critical patent/WO2012068412A2/en
Publication of WO2012068412A3 publication Critical patent/WO2012068412A3/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention is based in part on the discovery that neoplasia associated with inducible expression of STAT3 can be treated with compounds of the invention, e.g., compounds of Formula A. Accordingly, the present invention provides methods for treating subjects with inducibly expressed STAT3 as well as methods for determining treatment regimen and/or predicting treatment efficacy for a subject with neoplasia.
  • the present invention provides methods for treating a subject with neoplasia comprising administering to a subject in need of such treatment an effective amount of a therapeutic entity, wherein the subject has inducible expression of STAT3 and wherein the therapeutic entity comprises a compound of Formula A
  • n 1 or 2
  • R is hydrogen, acyl, alkyl or a peptidyl
  • X is an aromatic or heterocyclic amino acid or a derivative thereof.
  • the present invention provides methods for treating a subject with neoplasia comprising determining the presence of inducible expression of STAT3 in a biological sample of the subject and administering a therapeutic entity to the subject upon determination of the presence of inducible expression of STAT3 in the subject, wherein the therapeutic entity comprises a compound of Formula A
  • the present invention provides methods for determining the treatment regimen for a subject with neoplasia comprising selecting a treatment regimen comprising a therapeutic entity for the subject based on the presence of inducible expression of STAT3, wherein the therapeutic entity comprises a compound of Formula A
  • the method further comprises determining the presence of inducible expression of STAT3 in a biological sample from the subject.
  • the present invention provides methods for predicting the treatment efficacy of a therapeutic entity for the treatment of neoplasia comprising detecting inducible expression of STAT3 in a biological sample of a subject, wherein the presence of inducible expression of STAT3 is indicative of the treatment efficacy of the therapeutic entity for the subject, and wherein said therapeutic entity comprises a compound of Formula A
  • n 1 or 2
  • R is hydrogen, acyl, alkyl or a peptidyl
  • X is an aromatic or heterocyclic amino acid or a derivative thereof.
  • the present invention provides methods for determining the treatment efficacy of a therapeutic entity for the treatment of neoplasia comprising detecting in a biological sample of a subject treated with the therapeutic entity the presence of one or more markers selected from the group consisting of markers for inhibition of signal transduction through STAT3, markers for inhibition of phosphorylation of STAT3, markers for inhibition of nuclear translocation of STAT3, markers for inhibition of IL-6 mediated STAT3 activation, markers for inhibition of IL-10 mediated STAT3 activation, markers for inhibition of IFN-a mediated STAT3 activation and markers for inhibition of IL- 4 mediated STAT3 activation,
  • said therapeutic entity comprises a compound of Formula A
  • the present invention provides methods of providing useful information for determining the treatment regimen for a subject with neoplasia comprising detecting the presence or absence of inducible expression of STAT3 in a biological sample of a subject and providing the result of the detection to an entity that determines the treatment regimen based on the presence or absence of inducible expression of STAT3,
  • said therapeutic entity comprises a compound of Formula A
  • n 1 or 2
  • R is hydrogen, acyl, alkyl or a peptidyl
  • X is an aromatic or heterocyclic amino acid or a derivative thereof.
  • Figure 1 is a cartoon showing the STAT3 pathway.
  • Figure 2 is a cartoon showing a proposed mechanism of action for SCV-07.
  • Figure 3 is a graph showing % activity relative to the log of the peptide concentration.
  • the protein is human STAT3 Protein (amino acids 127-722); the ligand is pYLPQTV-NH 2 (SEQ ID NO:2) labeled with 5-carboxyfluorescein.
  • pY indicates pYLPQTV (SEQ ID NO:2);
  • Ac indicates Ac-YLPQTV (SEQ ID NO:3).
  • Figure 4 is a series of photographs showing staining results for STAT3.
  • Figure 8A no treatment
  • Figure 8B IL-6
  • Figure 8C IL-6 + SCV-07 0.04 ⁇ (0.01 ⁇ g/mL)
  • Figure 8D IL-6 + SCV-07 0.4 ⁇ (0.1 ⁇ g/mL)
  • Figure 8E IL-6 + SCV-07 10 ⁇ (2.48 ⁇ g/mL)
  • Figure 8F IL-6 + CP 0.04 ⁇
  • Figure 8G Figure 8G (IL-6 + CP0.4 ⁇
  • Figure 8H IL-6 + CP 10 ⁇ ).
  • Figure 5 is a graph showing the effect of SCV-07 on B 16F0 tumor growth in C57BL-6 mice.
  • Figure 6 is a diagram comparing the means of pSTAT3 levels in tumors of SCV- 07-treated and untreated mice.
  • Figure 7 is diagram comparing the means of NK cell accumulation in tumors of SCV-07-treated and untreated mice.
  • Figure 8 is a graph showing tumor growth curves for the renal carcinoma study of Example 3.
  • Figure 9 is a bar graph showing tumor weights for each indicated treatment group on Day 17.
  • Figure 10 is a graph showing animal growth over the course of treatment for the indicated treatment groups.
  • Figure 11 is a graph showing mean weight change in mice receiving the indicated treatment with SCV-07, with no accompanying radiation treatment. Error bars indicate the SEM.
  • Figure 12 is a graph showing mean weight change in mice receiving the indicated treatment with SCV-07, with accompanying radiation treatment. Error bars indicate the SEM.
  • Figure 13 is a graph presenting the AUC for mean weight change in mice receiving the indicated treatment with SCV-07, with no accompanying radiation treatment.
  • Figure 14 is a graph presenting the AUC for mean weight change in mice receiving the indicated treatment with SCV-07, with accompanying radiation treatment.
  • Figure 15 is a graph indicating changes in mean tumor volume in mice receiving the indicated treatment with SCV-07, with no accompanying radiation treatment.
  • Figure 16 is a graph indicating changes in mean tumor volume in mice receiving the indicated treatment with SCV-07, with accompanying radiation treatment.
  • Figure 17 is a graph showing data relating to mean tumor volume AUC for mice receiving the indicated treatment with SCV-07, with no accompanying radiation treatment.
  • Figure 18 is a graph showing data relating to mean tumor volume AUC for mice receiving the indicated treatment with SCV-07, with accompanying radiation treatment.
  • Figures 19A, 19B and 19C describe inhibitory screening of the compound by the STAT3/luciferase reporter assay.
  • Figures 20A, 20B and 20C describe STAT3 phosphorylation inhibition assay in Jurkat cells.
  • Figures 21A, 21B and 21C describe STAT3 phosphorylation inhibition assay in THP-1 cells.
  • Figures 22A, 22B and 22C describe the effect of SCV-07 on STAT3
  • Figures 23 A, 23B and 23C describe the effect of SCV-07 on STAT3
  • Figures 24A and 24B describe effects of SCV-07 on constitutive STAT3 phosphorylation on a set of tumor cell lines.
  • Figures 25 A, 25B, 25C and 25D describe the effect of SCV-07 on STAT3 phosphorylation in Jurkat cells treated with pervanadate.
  • Figures 26A and 26B describe the effect of SCV-07 on STAT3 phosphorylation in Kasumi-1 cells treated with pervanadate.
  • Figures 27A and 27B describe effects of SCV-07 on various kinase
  • Figures 28A, 28B, 28C, 28D, 28E and 28F describes quantitation data for the human phospho-kinase array tests.
  • Figure 29 shows normalized quantitation data for the human phospho-kinase array tests.
  • Figure 30 shows normalized quantitation data for the human phospho-kinase array tests.
  • Figures 31 A and 3 IB describe the effect of SCV-07 on STAT3 phosphorylation in differentiated THP-1 cells.
  • Figures 32A and 32B describe the effect of SCV-07 on STAT3 phosphorylation in NK-92 cells.
  • Figure 33 describes the effect of SCV-07 on STAT3 phosphorylation in various tumor cell lines.
  • Figure 34 describes the effect of SCV-07 on STAT3 phosphorylation in Jurkat cells treated with pervanadate.
  • Figures 35A and 35B describe that SCV-07 does not inhibit constitutive tyrosine phosphorylation of STAT3 in a variety of tumor cell lines.
  • Figures 36A and 36B describe the effect of SCV-07 on STAT3 phosphorylation in CCRF-CEM cell lines.
  • Figure 42A, 42B, 42C and 42D describe SCV-07 inhibition of cytokine-induced STAT3 tyrosine phosphorylation (PY) in MOLT-4, used for the xenograft studies.
  • Figure 43 describes that SCV-07 inhibits IFNa-induced STAT3 PY in Kasumi-1 cells and IL-4-induced STAT3 PY in CCRF-CEM cells. Western blots with antibodies against STAT3-Tyr705 or Total STAT3.
  • Figure 44 describes that SCV-07 inhibits IFNa-induced STAT3 PY in Jurkat T cells. Western blots with antibodies against STAT3-Tyr705 or Total STAT3.
  • Figure 45 describes that SCV-07 does not inhibit constitutive STAT3 PY in various cell lines.
  • Figure 46 describes that SCV-07 also leads to decreased STAT5a/b tyrosine phosphorylation in Jurkat T cells.
  • Figure 47 describes that SCV-07 treatment leads to inhibition of IL-6 induced STAT3 -regulated gene expression.
  • Figure 48 describes that a tyrosine phosphatase is required for SCV-07 effects on STAT3 PY in Jurkat T cells.
  • SCV-07 inhibition of STAT3 PY is blocked by addition of pervanadate, a tyrosine phosphatase inhibitor.
  • Cells were pretreated with 50 ⁇ pervanadate for 4 h.
  • the pervanadate-treated and untreated control cells (5 x 10 6 cells/sample) were further treated with 1 and 10 ⁇ g/ml SCV-07 for 2 h, and then stimulated with 25 ng/ml IFNa for 0, 15, 30, 60 and 120 minutes.
  • Jurkat cells were then lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti- pSTAT3xyr705 antibody ⁇ upper panels of A and B). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A and B). The graph indicates the relative quantitation of pSTAT3 band intensities (B and C). A and C: pervanadate-treated cells. B and D: untreated control cells.
  • Figure 49 describes that CD45 is not the phosphatase required for SCV-07 effects on STAT3 PY. SCV-07 can still decrease STAT3 PY in cells lacking transmembrane
  • CD45 Western blots with antibodies against STAT3-Tyr705 or Total STAT3.
  • Cells (5 x 10 6 cells/sample) were pretreated with 1 and 10 ⁇ g/ml SCV-07 for 2 h. Cells were then stimulated with 25 ng/ml IFNa, 200 ng/ml IL-6 or 100 ng/ml IL-10 for 0, 10 and 30 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3x yr 705 antibody (upper panels of A). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A). The graph indicates the relative quantitation of pSTAT3 band intensities (B).
  • Figure 50 describes that SHP-2 tyrosine phosphorylation increased by different concentrations of SCV-07 in both untreated and IFNa-treated Jurkat cells.
  • Figure 51 shows survival data. The percent survival was calculated for each group on each day of the study. No significant changes in survival were seen.
  • Figure 52 shows the mean percent weight change. The percentage daily weight change for each animal and the means for each treatment group were calculated. Error bars represent the SEM.
  • Figure 53 shows the Mean Weight Change AUC.
  • the area under the curve (AUC) was calculated for the percent weight change exhibited by each animal in the study. This calculation was made using the trapezoidal rule transformation. Group means were calculated and are shown with error bars representing SEM for each group. P values indicate statistically significant differences between that group and the vehicle control group.
  • Figure 54 shows the Mean Tumor Volumes. Mean Tumor Volumes were calculated from the length and width measurements. Group means were calculated and are shown with error bars representing SEM for each group.
  • Figure 55 shows the Tumor Volume Change AUC.
  • Figure 56A, 56B, and 56C show IFNa stimulation time points for induction of pSHP-2 and pSHP-1 in Jurkat cells.
  • Cells (5 x 10 6 cells/sample) were stimulated with 25 ng/ml IFNa for 0, 5, 10, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3x yr 705 ⁇ upper panel of A), anti-pSHP-l T y r 536 ⁇ upper panel ofB), anti-pSHP- 2 ⁇ ⁇ ⁇ 542 (first panel of C) and anti-pSHP-2 T y r 58o (second panel of C) antibodies.
  • the membranes were probed with anti-pSTAT3x yr 705 ⁇ upper panel of A), anti-pSHP-l T y r 536 ⁇ upper panel ofB), anti-pSHP- 2 ⁇ ⁇ ⁇ 542 (first panel of C) and anti-
  • Figure 57A, 57B, 57C, 57D, 57E and 57F show the effect of SCV-07 on pSTAT3, pSHP-1 and pSHP-2 in IFNa-stimulated Jurkat cells (Experiment 1).
  • Cells (5 x 10 6 cells/sample) were pretreated with 1 and 10 ⁇ g/ml SCV-07 for 2 h and then stimulated with 25 ng/ml IFNa for 0, 10 and 30 minutes.
  • Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with anti- pSTAT3xy r 705 (upper panel of A), anti-pSHP-lx yr 536 (upper panel ofB), antipSHP- 2x yr 54 2 (first panel of C) and anti-pSHP-2 Tyr58 o (third panel of C) antibodies.
  • the membranes were then stripped and reprobed with anti-STAT3 (lower panel of A), anti-SHP-1 (lower panel of B) and anti-SHP-2 (second and fourth panels of C) antibodies.
  • the graphs indicate the relative quantitation of pSTAT3 (D), pSHP-2 T y r5 4 2 (E) and pSHP-2 Tyr5 8o (F) band intensities.
  • Figure 58A, 58B, 58C, 58D, 58E and 58F show the effect of SCV-07 on pSTAT3, pSHP-1 and pSHP-2 in IFNa-stimulated Jurkat cells (Experiment 2).
  • Cells (5 x 10 6 cells/sample) were pretreated with 1 and 10 ⁇ g/ml SCV-07 for 2 h and then stimulated with 25 ng/ml IFNa for 0, 10 and 30 minutes.
  • Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with anti- pSTAT3xy r 705 (upper panel of A), anti-pSHP-lx yr 536 (upper panel ofB), antipSHP-2x yr 54 2 (first panel of C) and anti-pSHP-2 Tyr58 o (third panel of C) antibodies.
  • the membranes were then stripped and reprobed with anti-STAT3 (lower panel of A), anti-SHP-1 (lower panel of B) and anti-SHP-2 (second and fourth panels of C) antibodies.
  • the graphs indicate the relative quantitation of pSTAT3 (D), pSHP-2 Tyr5 4 2 (E) and pSHP-2 Tyr5 8o (F) band intensities.
  • Figure 59A, 59B and 59C show selection of cytokines used for induction of pSTAT3 in primary mouse macrophages.
  • Mouse peritoneal macrophages (3-5 x 10 6 cells/sample) were stimulated with 25 ng/ml IFNa (A), 200 ng/ml IL-6 (B) or 100 ng/ml IL- 10 (C) for 0, 5, 10, 30 and 60 minutes.
  • Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with anti- pSTAT3xyr705 antibody (upper panels). The membranes were then stripped and reprobed with anti-STAT3 (lower panels) antibody.
  • Figure 60A, 60B and 60C show selection of cytokines used for induction of pSHP-2 in primary mouse macrophages.
  • Mouse peritoneal macrophages (3-5 x 10 6 cells/sample) were stimulated with 25 ng/ml IFNa (A), 200 ng/ml IL-6 (B) or 100 ng/ml IL- 10 (C) for 0, 5, 10, 30 and 60 minutes.
  • Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with antipSHP-2 T y r 542 (upper panels)or pSHP-2 Tyr5 8o (middle panels) antibody. The membranes were then stripped and reprobed with anti-SHP-2 antibody (lower panels).
  • FIG. 61A, 61B and 61C show selection of cytokines used for induction of pSHP-1 in primary mouse macrophages.
  • Mouse peritoneal macrophages (3-5 x 10 6 cells/sample) were stimulated with 25 ng/ml IFNa (A), 200 ng/ml IL-6 (B) or 100 ng/ml IL- 10 (C) for 0, 5, 10, 30 and 60 minutes.
  • Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with antipSHP-l T y r 536 antibody (upper panels). The membranes were then stripped and reprobed with anti-SHP-1 antibody (lower panels).
  • Figure 62A, 62B, 62C, 62D, 62E and 62F show the effect of SCV-07 on pSTAT3, pSHP-1 and pSHP-2 in IL-6-stimulated primary mouse macrophages (Experiment 1).
  • Mouse peritoneal macrophages (3-5 x 10 6 cells/sample) were pretreated with 1 and 10 ⁇ g/ml SCV- 07 for 2 h and then stimulated with 200 ng/ml IL-6 for 0, 10 and 30 minutes.
  • Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with anti-pSTAT3 Tyr705 (upper panel of A), anti-pSHP-l Tyr5 3 6
  • Figure 63 A, 63B, 63C, 63D, 63E, and 63F show the effect of SCV-07 on pSTAT3, pSHP-1 and pSHP-2 in IL-6-stimulated primary mouse macrophages (Experiment 2).
  • Mouse peritoneal macrophages (3-5 x 10 6 cells/sample) were pretreated with 1 and 10 ⁇ g/ml SCV-07 for 2 h and then stimulated with 200 ng/ml IL-6 for 0, 10, 20 and 30 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice.
  • the lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with anti-pSTAT3x yr 705 (upper panel of A), anti- pSHP-l T y r 536 (upper panel ofB), anti-pSHP-2 T y r 542 (first panel of C) and anti-pSHP-2 T y r 58o (third panel of C) antibodies.
  • the membranes were then stripped and reprobed with anti- STAT3 (lower panel of A), anti-SHP-1 (lower panel of B) and anti-SHP-2 (second and fourth panels of C) antibodies.
  • the graphs indicate the relative quantitation of pSTAT3 (D), pSHP- 2 ⁇ ⁇ ⁇ 542 (E) and pSHP-2 Tyr5 8o (F) band intensities.
  • Figure 64A and 64B show the effect of SCV-07 on MCP-1 and IL-12p40 induction in primary mouse macrophages that were stimulated with IL-6.
  • Mouse peritoneal macrophages (3-5 x 10 6 cells/sample) were pretreated with 1 and 10 ⁇ g/ml SCV-07 for 2 h and then stimulated with 200 ng/ml IL-6 for 18 h.
  • Cell culture supernatants were harvested and analyzed by ELISA.
  • Induction levels of MCP-1 (A) and IL-12p40 (B) were then analyzed.
  • the present invention is based in part on the discovery that neoplasia associated with inducible expression of STAT3 can be treated with compounds of the invention, e.g., compounds of Formula A. Accordingly, the present invention provides methods for treating subjects with inducible expression of STAT3 as well as methods for determining treatment regimen and/or predicting treatment efficacy for a subject with neoplasia. [0077] According to one aspect of the invention, it provides methods for treating a subject, e.g., mammal including humans with neoplasia associated with an inducible expression of STAT3 by administering to the subject an effective amount of a therapeutic entity comprising a compound of the present invention.
  • a subject e.g., mammal including humans with neoplasia associated with an inducible expression of STAT3 by administering to the subject an effective amount of a therapeutic entity comprising a compound of the present invention.
  • neoplasia includes any abnormal growth or cell proliferation, e.g. , uncoordinated with the proliferation of the tissues (normal) around it.
  • neoplasia includes any cancer or tumor growth in a subject.
  • neoplasia includes any pre-cancerous or pre-tumor growth in a subject.
  • neoplasia includes any solid or circulating cancer or tumor growth in a subject.
  • neoplasia includes any abnormal growth that is capable of metastasizing or spreading to other locations of the subject.
  • neoplasia examples include, but are not limited to carcinoma, sarcoma, blastoma, lymphoma, leukemia, and germ cell tumors.
  • neoplasia includes head and neck, skin, colon, oral, glioblastoma, breast, laryngeal, esophageal, endothelial, endometrial, ovarian, lung, urogenital, rectal, prostate, kidney, melanoma, renal, and papilloma virus-induced cancer.
  • neoplasia associated with inducible expression of STAT3 includes any neoplasia accompanied by or having detectable inducible expression of STAT3.
  • neoplasia associated with inducible expression of STAT3 includes neoplasia with inducible expression of STAT3 within the site or cell or tissue of neoplasia.
  • neoplasia associated with inducible expression of STAT3 includes neoplasia with inducible expression of STAT3 outside of the original site or cell or tissue of neoplasia, but within cells or tissues directly associated with cells or tissues with neoplasia.
  • neoplasia associated with inducible expression of STAT3 includes neoplasia with inducible expression of STAT3 in cells or tissues in direct contact with or in the proximity of cells or tissues with neoplasia.
  • inducible expression of STAT3 includes any STAT3 expression or activation, e.g., phosphorylation that is induced, activated or triggered by one or more elements in a subject, e.g., human.
  • inducible expression of STAT3 includes any STAT3 expression or activation that is induced by a cytokine, e.g., IFN-a, IL-4, IL-6, IL-10, and IL-27, etc.
  • inducible expression of STAT3 includes any STAT3 expression or activation that is induced by one or more factors/elements within the pathway of a cytokine that induces expression or activation of STAT3, e.g., IFN- ⁇ , IL-4, IL-6, IL-10, and IL-27, etc.
  • inducible expression of STAT3 includes any STAT3 expression or activation that is induced by an exogenous agent or an endogenous agent that is itself inducibly expressed or present in a temporal or spatial pattern.
  • inducible expression of STAT3 includes any increased expression or activation of STAT3, e.g., comparing to a
  • inducible expression of STAT3 includes any expression or activation of STAT3 in one or more cells in response to the cells being exposed to an agent, e.g., cytokine in vivo or in vitro.
  • inducible expression of STAT3 includes any STAT3 expression or activation that is not present constantly, but only present with a temporal or spatial pattern. In still another embodiment, inducible expression of STAT3 includes any STAT3 expression or activation that is not associated with a genetic
  • inducible expression of STAT3 includes expression or activation of any STAT3 that is not a constitutively phosphorylated form of STAT3 (including forms with mutated phosphorylation sites), inappropriately truncated forms of STAT3 that are constantly active, as well as other forms of STAT3 that are unable to be inactivated.
  • STAT3 Signal transducer and activator of transcription 3
  • STAT3 is of great importance in the development of neoplasia.
  • STAT3 is a point of convergence for numerous oncogenic signaling pathways. It is involved in numerous signal transduction pathways, and can be constitutively activated in both tumor cells and immune cells in the tumor microenvironment.
  • Constitutively activated STAT3 inhibits the expression of a variety of cytokines, including IL-12, IFN- ⁇ , and type I interferons as well as up-regulates co-stimulatory molecules such as B7-1 and 2, which are necessary for immune activation against tumor cells.
  • cytokines including IL-12, IFN- ⁇ , and type I interferons
  • co-stimulatory molecules such as B7-1 and 2
  • STAT3 activity can promote the production of immunosuppressive factors such as IL-6, IL- 10, TGF- ⁇ and VEGF, which in turn activate STAT3 in diverse immune cell subsets and alter gene expression profiles.
  • STAT3 is present in the cytoplasm in the inactive monomeric form.
  • Molecules that activate STAT3 include, among others, IL-6, IL-10, VEGF, oncostatin M and a number of growth factor receptors including epidermal growth factor receptors EGFR and HER2, fibroblast growth factor receptor (FGFR), insulin- like growth factor receptor (IGFR), hepatocyte growth factor receptor (HGFR), platelet- derived growth factor receptor (PDGFR) and transforming growth factor beta receptors (TGFBR).
  • FGFR fibroblast growth factor receptor
  • IGFR insulin- like growth factor receptor
  • HGFR hepatocyte growth factor receptor
  • PDGFR platelet- derived growth factor receptor
  • TGFBR transforming growth factor beta receptors
  • Detection of inducible expression of STAT3 can be carried out by any suitable means in the field.
  • detection of inducible expression of STAT3 includes detecting the level of proteins, nucleic acids or gene expression. Methods for detecting the levels of nucleic acids and proteins are well known in the art and any standard methods for detection of nucleic acid or protein levels can be employed with the methods of the present invention and used for detecting inducible expression of STAT3.
  • Methods for detecting the levels of nucleic acids, such as RNA or DNA have been well described and are well known to those of skill in the art.
  • Methods for detecting RNA can include but are not limited to RT-PCR, northern blot analyses, gene expression analyses, microarray analyses, gene expression chip analyses, hybridization techniques (including FISH), expression beadchip arrays, and chromatography as well as any other techniques known in the art.
  • Methods for detecting DNA can include but are not limited to PCR, realtime PCR, digital PCR, hybridization (including FISH), microarray analyses, and
  • Methods for detecting proteins and polypeptides can include but are not limited to spectrophotometric determination of protein concentration, quantitative amino acid analysis, protein concentration assays, chromatography assays, western blot analyses, gel
  • Protein detection can also include detecting the levels of stable or active proteins and methods such as kinetic assays, kinase assays, phosphatase assays, enzyme assays and post-translation modification assays (for example, assays for determining phosphorylation and glycosylation state) can also be employed.
  • Methods for quantitating nucleic acid and protein levels have also been well described. Methods can include traditional methods, such as western blot quantization as well as computer based methods, such as microarray assay or genechip assay analyses, for analyzing nucleic acid or protein levels. These standard methods known in the art can be employed to determine whether the level of STAT3 is increased or induced in a sample. In some embodiments, determination of an increased expression or induced expression of STAT3 can be based on a comparison of the level of STAT3 in a sample with a
  • the determination of an increased expression or induced expression of STAT3 can be based on a comparison of the level of STAT3 in a sample before treatment with an agent and to the level after treatment with an agent, e.g., a cytokine.
  • Predetermined standard levels of STAT3 can be defined using a variety of methods known to those of skill in the art. Generally, standard levels are determined by determining the level of STAT3 in a sufficiently large number of samples obtained from normal, healthy control subjects. Further, standard level information can be obtained from publically available databases, as well as other sources. (See, e.g., Bunk, D.M., "Reference Materials and Reference Measurement Procedures: An Overview from a National Metrology Institute," Clin. Biochem. Rev., 28(4): 131-137 (2007); Suraj Peril , et al, "Development of Human Protein Reference Database as an Initial Platform for Approaching Systems Biology in Humans" Genome Res. 13: 2363-2371 (2003); Remington: The Science and Practice of Pharmacy, Twenty First Edition (2005).)
  • inducible expression of STAT3 in a subject can be detected by exposing a cell population of the subject to an agent, e.g., cytokine and determining the expression or activation, e.g., phosphorylation of STAT3 in the cell population in response to the agent.
  • agent e.g., cytokine
  • inducible expression of STAT3 in a subject can be determined by detecting an increased level of STAT3 expression or phosphorylation in response to a cytokine.
  • the therapeutic entity used in the methods of the present invention includes any composition comprising a compound of Formula A or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof.
  • Formula A is
  • n 1 or 2
  • R is hydrogen, acyl, alkyl or a peptidyl
  • X is an aromatic or heterocyclic amino acid or a derivative thereof.
  • X is L-tryptophan or D-tryptophan.
  • Derivatives of the aromatic or heterocyclic amino acids for "X” can include amides, mono-or di-(Cr C6) alklyl substituted amides, arylamides, and (Cr C6) alkyl or aryl esters.
  • Acyl or alkyl moieties for "R” can include branched or unbranched alkyl groups of 1 to about 6 carbons, acyl groups from 2 to about 10 carbon atoms, and blocking groups such as carbobenzyloxy and t-butyloxycarbonyl.
  • the carbon of the CH group shown in Formula A has a stereoconfiguration, when n is 2, that is different from the
  • the compound of Formula A includes ⁇ -D-glutamyl-L- tryptophan, ⁇ -L-glutamyl-L-tryptophan, ⁇ -L-glutamyl-N m -formyl-L-tryptophan, N-methyl- ⁇ - L-glutamyl-L-tryptophan, N-acetyl-y-D-glutamyl-L-tryptophan, ⁇ -L-glutamyl-D-tryptophan, ⁇ -L-aspartyl-L-tryptophan, and ⁇ -D-aspartyl-L-tryptophan.
  • the compound of Formula A is ⁇ -D-glutamyl-L-tryptophan (SCV-07).
  • compositions can be administered as a pharmaceutical composition or formulation.
  • these pharmaceutical compositions can also contain pharmaceutically acceptable carriers or excipients.
  • pharmaceutically acceptable carriers or excipients As used herein, the term
  • pharmaceutically acceptable carrier or “excipient” and variants thereof include but are not limited to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and others that are physiologically compatible, as well as pharmaceutically acceptable salts.
  • Pharmaceutically acceptable carriers can include but are not limited to sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions. The preparation of compositions containing nVmrmflPfflitirflll arrive substances is well known in the art and any well known methods can be employed with the methods of the present invention. (See, e.g., Remington: The Science and Practice of Pharmacy, Twenty First Edition (2005); US Patent No. 5,916,878.)
  • compositions of the present invention can be formulated for parenteral, intravenous, intraperitoneal, intramuscular, intradermal, sublingual or oral administration.
  • the pharmaceutical compositions can be formulated to contain the therapeutic entity and one or more chemotherapeutics, radiations therapeutics or chemoradiation therapeutics as a single composition for administration.
  • chemotherapeutics, radiations therapeutics or chemoradiation therapeutics as a single composition for administration.
  • the pharmaceutical compositions can be formulated to contain the therapeutic entity as one composition and one or more chemotherapeutics, radiations therapeutics and chemoradiation therapeutics as a separate composition for administration.
  • the separate compositions can be administered together or separately, at the same site or different sites, and can be administered sequentially or concurrently.
  • Administration can include a variety of methods and routes for administration.
  • the therapeutic entity of the present invention can be administered with other therapeutics. Other therapeutics included but are not limited to chemotherapeutics, targeted therapeutics, radiation therapeutics, or chemoradiation therapeutics.
  • the therapeutic entity is administered with one or more one or more chemotherapeutics, radiation therapeutics, or chemoradiation therapeutics.
  • the therapeutic entity is co-administered with one or more chemotherapeutics, targeted therapeutics, radiation therapeutics, or chemoradiation therapeutics.
  • the therapeutic entity is administered after administration of one or more one or more chemotherapeutics, targeted therapeutics, radiation therapeutics, or chemoradiation therapeutics.
  • the therapeutic entity is administered in the absence of any other therapeutics.
  • Co-administration can include administration at the same site or at different body sites in a subject. Co -administration can further include sequential or concurrent
  • Sequential administration can include administration of the therapeutic entity either before or after another therapeutic.
  • Concurrent administration can include
  • the therapeutic entity can be administered at the same site as another therapeutic. In other embodiments, the therapeutic entity can be administered at a different site than another therapeutic. In yet other embodiments, the therapeutic entity can be administered sequentially or concurrently with another therapeutic.
  • an effective amount of a therapeutic entity is an amount that causes a therapeutic effect or benefit for the subject treated.
  • the therapeutic entity of the present invention can be administrated at a variety of effective dosages.
  • the effective dosage is in the range of about 0.01 to 100 milligrams per kilogram (mg/kg) subject body weight.
  • the effective dosage is in the range of about 0.1-10 milligrams per kilogram subject body weight.
  • the effective dosage is in the range of about 0.1-5.0 milligrams per kilogram subject body weight.
  • the effective dosage is in the range of about 1.0-5.0 milligrams per kilogram subject body weight.
  • the effective dosage is in the range of about 1.0, 2.0, 3.0, 4.0 or 5.0 milligrams per kilogram subject body weight. In yet other embodiments, the effective dosage is in the range of 5.0 milligrams per kilogram. In yet other embodiments, the effective dosage is in the range of 1.0 milligrams per kilogram. In yet other embodiments, the effective dosage is in the range of 0.1 milligrams per kilogram. In further embodiments the effective dosage is 0.02 mg/kg subject body weight.
  • neoplasia provides methods for treating neoplasia by first determining the presence of inducible expression of STAT3 in a subject and then administering to the subject a therapeutic entity comprising a compound of Formula A upon determination that the subjection is positive for inducible expression of STAT3, e.g., the neoplasia is associated with inducible expression of STAT3.
  • determining the presence of inducible expression of STAT3 in a subject can be carried out by any suitable means, e.g., by clinician asking another entity and/or person to test the presence of inducible expression of STAT3 in a biological sample of a subject or by clinician studying the test results provided by another entity or person regarding STAT3 expression or activation.
  • a biological sample of a subject can be any suitable sample for detecting inducible expression of STAT3, e.g., liquid, solid, cell, or tissue samples, etc.
  • biological samples of the present invention can be obtained by any methods known in the art.
  • biological samples suitable for the present invention include but are not limited to serum, blood, plasma, whole blood and derivatives thereof, skin, hair, hair follicles, saliva, oral mucous, vaginal mucous, sweat, tears, epithelial tissues, urine, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), excreta, ascites, cerebrospinal fluid, lymph, and tissue extract sample or biopsy.
  • the invention provides methods for determining the proper treatment and/or regimen for neoplasia by selecting a treatment regimen using a therapeutic entity comprising a compound of Formula A based on the determination that the subject is positive for inducible expression of STAT3, e.g., detection of inducible expression of STAT3 (associated with the neoplasia) in the subject.
  • a subject is determined to be suitable for a treatment using a therapeutic entity comprising a compound of Formula A if such subject is determined to have inducible expression of STAT3.
  • the subject is consequently treated using a therapeutic entity comprising a compound of Formula A.
  • a therapeutic entity comprising a compound of Formula A by detecting the presence of inducible expression of STAT3 in a biological sample of the subject and predicting treatment efficacy of the therapeutic entity if the subject is positive for inducible expression of STAT3.
  • a treatment using a therapeutic entity comprising a compound of Formula A is likely to be efficacious for a subject if the subject is determined to have inducible expression of STAT3.
  • a therapeutic entity e.g., a therapeutic entity comprising a compound of Formula A.
  • These methods comprise detecting in a subject treated with the therapeutic entity the presence of one or more markers for inhibition of signal transduction through STAT3, inhibition of phosphorylation of STAT3, inhibition of nuclear translocation of STAT3, inhibition of IL-6 mediated STAT3 activation, inhibition of IFN-a mediated STAT3 activation, inhibition of IL-4 mediated STAT3 activation, inhibition of IL-10 mediated STAT3 activation, or increase of IFN-a or IL-6 mediated SHP-2 activation, e.g., increase of phosphorylation of SHP-2 TYR542 .
  • the presence of one or more markers is indicative of the therapeutic efficacy of the therapeutic entity.
  • the presence of one or more markers for inhibition of signal transduction through STAT3 can include one or more markers for inhibition of signal transduction through STAT3 via an increase in SHP-2 or SHP-2 activity, e.g., phosphorylation of SHP-2.
  • one or more markers for inhibition of signal transduction through STAT3 can include one or more markers for inhibition of signal transduction through STAT3 via an increase in SHP-2 TYR542 .
  • one or more markers for inhibition of phosphorylation of STAT3 can include one or more markers for inhibition of phosphorylation of pSTAT3x YR 705.
  • SHP-2 such as PSHP-2 TYR542 can be used as a marker for determining or monitoring the treatment efficacy of the therapeutic entity of the present invention .
  • one or more markers for the inhibition of signal transduction through STAT3 can include one or more markers for the inhibition of signal transduction through STAT3 via an increase and/or decrease in kinase phosphorylation, including phosphorylation of those kinases listed in Figures 27-30.
  • STAT4 e.g., phosphorylation levels of these kinases are increased in response to treatment with the therapeutic entity of the present invention in the presence of inducible expression of STAT3.
  • phosphorylation levels of these kinases are decreased in response to treatment with the therapeutic entity of the present invention in the presence of inducible expression of STAT3.
  • the subject has inducible expression of STAT3 and wherein the one or more markers include without any limitation p38a (T180/Y182), ERKl/2
  • a decrease in phosphorylation levels of GSK-3a/p (S21/S9), ⁇ -Catenin, Paxillin (Y118), Lyn (Y397), p27 (T157), STATl (Y701), STAT3 (Y705), STAT5a (Y699), STAT5b (Y699), STAT5a (Y699), PLCy-l (Y783), c-Jun (S63), Pyk2 (Y402) and eNOS (SI 177) is indicative of treatment efficacy of the therapeutic entity of the present invention.
  • the presence of these markers can be determined by standard assays known in the art. For example, inhibition of signal transduction through STAT3 can be determined by examining phosphorylation of STAT3 as well as activation of any members of the STAT3 pathway. Inhibition of phosphorylation of STAT3 can be examined by standard
  • Inhibition of nuclear translocation of STAT3 can be examined by a variety of assays including cellular fraction, immunohistochemistry, or other well known assays.
  • Inhibition of IL-6 mediated STAT3 activation can be examined using standard IL-6 inhibitors and IL-6 detection assays (which are commercially available, from companies such as for example Thermo-Scientific, USA).
  • Inhibition of IFN-a mediated STAT3 activation can be examined using standard IFN-a inhibitor and IFN-a detection assays (also, commercially available, from companies such as for example Thermo- Scientific, USA).
  • Inhibition of IL-4 mediated STAT3 activation can be examined using standard IL-4 inhibitor and IL-4 detection assays (also, commercially available, from companies such as for example Thermo-Scientific, USA). Increases and decreases in phosphorylation levels can be readily measured using a variety of well known techniques in the art and any techniques can be employed. For example, phosphorylation of PSHP-2 TYR542 can be measured using phospho-specific antibodies for detecting the presence of pSHP- 2 TYR542 . Phosphorylation of STAT3 can be measured using phospho-specific antibodies for detecting the nresence of pSTAT3x YR705 .
  • the present invention provides methods for providing useful information for predicting or determining the treatment regimen for a subject with neoplasia. These methods comprise detecting inducible expression of STAT3 in a biological sample of a subject and providing the results to an entity and/or clinician to predict or determine the treatment regimen based on the results provided, e.g., based on the presence or absence of inducible expression of STAT3.
  • Example 1 Effect of SCV-07 on STAT3 driven gene expression, SCV-07 Binding to STAT3 and Nuclear Translocation of STAT3.
  • SCV-07 reduces progression of melanoma in an animal model, and since STAT3 inhibitors are effective in inducing anti-tumor immunity in an antigen nonspecific manner as seen with SCV-07, the following experiments were conducted to determine whether SCV-07 could inhibit STAT3 -dependent gene expression.
  • HEK293 cells were transfected with the luciferase gene driven by STAT3 responsive elements. These cells were treated with either medium (control) or with 4 ⁇ (1 ⁇ g/mL) SCV-07.
  • the luciferase reporter assay to determine the effect of SCV-07 on STAT3 driven gene products was performed as previously described in Turkson et ah, Mol. Cell. Biol. 18:2545-2552, 1998, the disclosures of which are hereby incorporated by reference.
  • the pGL2 vector containing luciferase driven by SV40 promoter and 2X copies of STAT responsive element was transiently transfected into either HEK (Human embryonic kidney cells) or B16F0 melanoma cells. Briefly, the cell lines were seeded in a six-well tissue culture plate, allowed to reach -50-60% confluency and then transfected with the reporter carrying the STAT3 response element. The transfected cells were allowed to recover for 24 hours and were then treated with PBS as negative control, or with SCV-07. The cells were incubated for another 24 hours and harvested for analysis.
  • the cells were lysed in the presence of protease inhibitors, protein concentration estimated, and the luciferase activity in the cell lysate was measured using PromegaTM luciferase kit and a TecanTM luminometer. Based on luciferase expression, when compared with luciferase expression in cells incubated in medium alone (control), incubation with SCV-07 inhibited STAT3 expression 40%. [00110] To confirm that this phenomenon is seen in B16F0 melanoma cells also, B16F0 melanoma cells were transfected with the pGL2 vector containing STAT3 responsive elements driving luciferase.
  • the fluorescence polarization assay is a screen for small molecules that bind to the STAT3 SH2 domain and thereby inhibit or antagonize STAT3 activity.
  • the basis of this assay is the binding of a fluorescein-labeled phosphotyrosine-peptide derived from the interleukin-6 receptor subunit gpl30 to unphosphorylated STAT3.
  • the assay was performed in 96 well plates.
  • STAT3 protein amino acids 127-721 was added to each well at a final concentration of 1 ⁇ g in buffer (50 mM NaCl, 10 mM HEPES, 1 mM EDTA, 0.1% NP-40).
  • the controls included were STAT3 alone, STAT3 plus labeled ligand, labeled ligand alone and buffer alone. The reaction mixture was incubated for 1 hour at 37°C.
  • the carboxyfluorescein-labeled STAT3 ligand then was added to a final concentration of 5 nM/well and the reaction mixture incubated for 15 minutes at 37°C. After incubation, polarization readings were taken using a TECANTM fluorescent polarizer instrument. The extent of inhibition by the test peptides was calculated using an equation relating the difference in polarization between protein + probe and protein + probe + compound, to give the extent of inhibition due to compound addition. See Figure 3. The reported IC50 is 0.3-0.5 ⁇ ; the IC50 obtained here was 0.3 ⁇ .
  • SCV-07 Since SCV-07 was capable of inhibiting STAT3 driven gene expression, we sought to determine if this was due to direct binding to STAT3. The STAT3 binding ability of SCV-07 therefore was assessed at various concentrations. As seen in Table I, the PY peptide (positive control) binds strongly to the SH2 domain of STAT3, indicated by 98% inhibition of depolarization. Neither SCV-07 nor CP showed any significant ability to bind STAT3 directly (Table I, below).
  • B16F0 cells were grown on poly-L-lysine-coated cover slips in 6-well plates (50,000 cells per well) and incubated overnight. The next day, cells were treated with varying concentrations of SCV-07 or control peptide in the presence or absence of rIL-6. rIL-6 was added to inducibly express STAT3. Following a 30-minute incubation with IL-6, cells were incubated for 48 hours and then were fixed with 3% formaldehyde for 10 minutes and washed with PBS. The cells were permeabilized with absolute methanol, blocked with 1% BSA and incubated overnight at 4°C with anti-STAT3 monoclonal antibody
  • STAT3 constitutive expression of STAT3 in tumor tissue leads to upregulation of STAT3 -dependent genes such as c-Myc, JunB and Mcl-1. Activation of these genes induces proliferation of cancerous cells. Inhibition of STAT3 leads to inhibition of these genes resulting in an anti-proliferative effect on cancerous cells, thereby reducing tumor growth.
  • SCV-07 multiple cell lines originating from various forms of cancers were incubated with SCV-07 and their proliferation measured after 96 hours.
  • XTT 2,3-bis(2- methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide
  • the XTT assay measures metabolically active, proliferating cells that are capable of reducing XTT to water-soluble formazan. Dying cells (metabolically compromised cells) are incapable of this reaction.
  • VX-680 a small molecule with known ability to inhibit tumor growth, inhibited proliferation to the extent of 70-90% in a variety of cell lines (A-375, B16F0, A-549, HT-29 and MDA-MB-453) as measured by the XTT assay.
  • VX- 680 had only a marginal effect on the prostate carcinoma cell line (PC-3) and on MDA- MB-231.
  • CP and SCV-07 at several different concentrations did not show an appreciable effect on inhibition of cell proliferation, suggesting that SCV-07 does not mediate its antitumor action by directly inhibiting proliferation of tumor cells.
  • Values shown in Table III shown are % growth inhibition as measured in the XTT assay.
  • B16F0 cells were assayed for the presence of these cytokines after treatment with SCV-07 or CP.
  • B16F0 cells were seeded in 6-well plates (1 x 10 5 cells/well in 2 mL of DMEM, in duplicate) and incubated overnight. The cells then were treated with SCV-07 and CP in combination with LPS (2.0 ⁇ g/mL) and incubated for 8 hours. Cells alone and LPS alone served as negative controls. Following incubation, cell supernatants were collected and analyzed for TNFa and IL-12 by ELISA.
  • mice were conducted in C57BL-6 mice to study the effect of 5 mg/kg SCV07 administered subcutaneously on progression of melanomas.
  • B16F0 tumors were established in C57BL-6 mice.
  • Ten mice per experimental group were administered SCV-07 subcutaneously once daily.
  • the mice were followed over the course of 14 days for clinical signs including evaluation of skin and fur; eyes, mucous membranes; respiratory, circulatory, autonomic and central nervous systems, behavioral pattern, signs of tremors, convulsions, lethargy, excessive salivation and diarrhea, as well as for general morbidity.
  • mice were weighed and tumor size measured daily. Mice did not show any significant clinical signs or altered behavior.
  • Table IV shows that body weight reduction in the SCV-07-treated mice was not significantly different from the control group.
  • the tumors were dissociated. Single cell suspensions were prepared and stained with STAT3 antibody (pTyrosine 705, Cell SignalingTM) followed by goat, anti- rabbit antibodies conjugated to Alexa Fluor 488 (InvitrogenTM). The percentage of cells staining for phosphorylated STAT3 was measured in vehicle- and SCV-07-treated groups. In addition, dissociated tumor cells were stained with anti-NKl .l-FITC (BioligandTM) to quantitate NK cells. As shown in Table V below, the starting volume of tumors in both groups was comparable (day 0). Mice receiving subcutaneous SCV-07 (5 mg/kg) showed 33% tumor growth inhibition, which was statistically significant (P ⁇ 0.001). See Table V and Figure 5. A second comparable study revealed a 40%> reduction in tumor volume.
  • a reduction in pSTAT3 expression is accompanied by an increase in natural killer cell accumulation at the tumor site.
  • Tumor tissue from mice treated as described above was dissociated and stained for NK cells and analyzed by flow cytometry.
  • the accumulation of NK cells in the SCV-07 treated mice was 33% greater than the accumulation seen at the tumor site in vehicle treated mice.
  • mice were implanted subcutaneously with murine renal carcinoma cells (Renca cells, 8 x 10 5 in a volume of 0.1 mL normal saline), followed by treatment with SCV-07, cyclophosphamide (CTX) or both for 14 consecutive days.
  • murine renal carcinoma cells Renca cells, 8 x 10 5 in a volume of 0.1 mL normal saline
  • mice Thirty-five male and thirty-five female healthy, naive, Balb/C mice were involved in the study. The animals were four to six weeks old, weighing between 16 and 20 grams at the start of the study. The animals were group-housed in autoclaved shoe box cages with autoclaved wood chips as the bedding materials. The temperature of the animal room was maintained at 22 to 25°C, and the relative humidity was maintained at 40 to 60%. A 12-hour light/12-hour dark cycle was maintained except when interrupted by study-related events. Animals were fed ad libitum with sterile water and Beijing KeAoXieLi Rodent Diet
  • Murine renal carcinoma cells were adapted in Balb/C mice before use as follows. Using aseptic tissue culture procedures, one vial of murine renal carcinoma (Renca) cells was thawed and centrifuged with a TD5A-WS centrifuge at 1000 rpm, 20-25°C for 5 minutes. The cell pellets were suspended in 0.1-0.5 mL DMEM with 10% PBS, then
  • T25 flask When grown to confluency, they were digested and passaged to T75 and later further split into three T75 flasks. At the time of cell implantation, the cells were collected from the flasks, washed three times with normal saline (NS), and subcutaneously injected into the right axilla of 3 mice (approximately 1 xlO 6 or 8xl0 5 cells/mouse). The day of tumor inoculation was defined as Day 0. When the tumor diameter was approximately 1 cm (measured by vernier caliper), the animals were euthanized with C0 2 asphyxiation and the tumors excised. Tumor cells were dispersed and suspended in normal saline as
  • CTX was administered by intraperitoneal injection every other day using the same dose volume.
  • the vehicle (control) was also administered once daily for 14 consecutive days subcutaneously (0.1 mL/20 g body weight).
  • Treatment regimens for all groups are outlined in Table VIII.
  • CTX other day [001 31 ] SCV-07 was administered daily via subcutaneous injection; CTX was administered every other day via intraperitoneal injection.
  • the mice were divided into 7 groups: Group 1 (vehicle), Group 2 (CTX 20 mg/kg), Group 3 (CTX 40 mg/kg), Group 4 (SCV-07 5 mg/kg), Group 5 (SCV-07 10 mg/kg), Group 6 (SCV-07 5 mg/kg plus CTX 20 mg/kg), and Group 7 (SCV-07 10 mg/kg plus CTX 20 mg/kg).
  • Tumor volumes and body weights were measured every three days, and tumor weights were measured on Day 17 (necropsy day) at the end of the study.
  • CTX was aliquoted to 10 mg/vial.
  • Tumor size was measured using calipers and recorded along with the animal's body weight once every three days throughout the course of the study. Based on the tumor size, the tumor volume (TV) was calculated with the following formula.
  • IR (TV) (TVvehicle TVdrug treated)/ TV ve hicle 100
  • TW tumor weight
  • IR (TW) (TWveh!de— TW dmg seated)/ TW vehl cle X 100
  • SCV-07 showed no evidence of toxicity in this study based on observations of survival and weight change.
  • Daily administration of SCV-07 (5 mg/kg or 10 mg/kg) for 14 days inhibited tumor growth as reflected by the lower tumor weights in these groups than that of the vehicle control group (less than 40%).
  • the combination of SCV-07 (5 or 10 mg/kg) with CTX (20 mg/kg) resulted in higher inhibition in comparison to SCV-07 treatment alone, however, there were no statistically significant differences between any of the combination treatment groups and CTX (20 mg/kg) treatment alone group.
  • Tumor measurement data showed that the mean tumor sizes of Group 3 and Group 7 were statistically significantly smaller than that of Group 1 (vehicle control) on Day 6. On Day 9 and Day 12, the mean tumor sizes in all groups except Group 4 and Group 5 were statistically significantly smaller in comparison to that of Group 1. On Day 15, the mean tumor sizes of Group 2, Group 3 and Group 6 were statistically significantly smaller. On Day 17, the mean tumor weights of all treatment groups were statistically significantly lower than that of Group 1.
  • the inhibition calculated based on tumor weight were 55.97% (p ⁇ 0.01), 90.12% (p ⁇ 0.01), 30.02% (p ⁇ 0.01), 28.33% (p ⁇ 0.01), 59.65% (p ⁇ 0.01) and 47.78%) (p ⁇ 0.01), for Group 2, Group 3, Group 4, Group 5, Group 6, and Group 7, respectively.
  • the body weights showed no statistically significant differences between the vehicle control group and any of the treatment groups (i.e., Groups 2-7).
  • Table X Mean Tumor Volume (cm ) on Day 6.
  • HI 46 human lung cancer cells were obtained from ATCC. The cells were grown in DMEM supplemented with 10% fetal calf serum (FCS), 1% penicillin and streptomycin, and 2 mM L-glutamine. Cells were sub-cultured by removing the medium, rinsing twice with sterile calcium- and magnesium-free phosphate buffered saline (PBS) and adding 1 to 2 mL of 0.25% trypsin/ 0.03% EDTA solution. The flask was incubated at 37°C until cells detached. Cells then were sub-cultured at a ratio of 1 :3.
  • FCS fetal calf serum
  • PBS sterile calcium- and magnesium-free phosphate buffered saline
  • mice Female nude mice, homozygous for the nu gene (nu+/nu+), aged 5 to 6 weeks, with a mean pre-treatment body weight of 24 grams were used in the study. Animals were individually numbered using an ear punch, housed in groups of 6 animals per cage, and acclimatized prior to study commencement. During the acclimatization period of at least 2 days, the animals were observed daily in order to reject animals that presented in poor condition. [00145] The study was performed in animal rooms provided with filtered air at a temperature of 70°F+/-5 0 F and 50% +/-20% relative humidity. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour.
  • Sterilized Bed-O- Cobs ® bedding was used. Bedding was changed a minimum of once per week. Cages, tops, bottles, etc. were washed with a commercial detergent and allowed to air dry. Prior to use, these items were wrapped and autoclaved. A commercial disinfectant was used to disinfect surfaces and materials introduced into the hood. Floors were swept daily and mopped a minimum of twice weekly with a commercial detergent. Walls and cage racks were sponged a minimum of once per month with a dilute bleach solution. A cage card or label with the appropriate information necessary to identify the study, dose, animal number and treatment group marked all cages. The temperature and relative humidity were recorded during the study, and the records retained. Animals were fed with sterile Labdiet ® 5053 (pre-sterilized) rodent chow and sterile water was provided ad libitum.
  • TGI tumor growth index
  • mice in groups 1 and 4 received vehicle by subcutaneous (sc) injection for 20 days.
  • Mice in groups 2-4 and 6-8 received SCV-07 in vehicle once a day by subcutaneous injection on days 1 through 20, and mice in groups 6-8 received radiation (2 doses of 4 Gy/dose on days 0 and 2).
  • Radiation was done by anesthetizing the mice in these groups with ketamine (120 mg/kg) and xylazine (6 mg/kg), and placing them under a lead shield such that the region of the flank with tumor was exposed to the radiation.
  • Radiotherapy was delivered using a Philips 160 kV source at a focal distance of approximately 40 cm, and a dose rate of approximately 1.0 Gy/min. Tumors were measured on alternating days throughout the duration of the study. Mice in groups 1-8 were sacrificed on day 21 and remaining tumors were excised, measured, weighed, photographed and fixed in formalin for later analysis.
  • the AUC data is shown in Figures 13 (no radiation) and 14 (plus radiation).
  • the AUC was calculated for the percent weight change exhibited by each animal in the study. This calculation was made usign the trapezoidal rule
  • Tumor volumes were calculated from the length and width measurements taken on alternating days by calculating the mean radius (r), which was the sum of length and width divided by 4, and using the formula 4/3 ⁇ 3 to calculate the volume.
  • the mean tumor volume data is shown in Figures 15 (no radiation) and 16 (plus radiation). Tumors from animals treated with 100 ⁇ g/mL grew at rates faster than vehicle control animals. Among the non- irradiated animals, mice treated with 10 mg/kg of SCV-07 showed the best improvement in tumor growth inhibition.
  • mice treated with 10 mg/kg of SCV-07 showed the best improvement in tumor growth inhibition.
  • the mean tumor volume at the end of the study period for vehicle-treated animals was 1618.5 mm 2 , 1322.3 mm 2 for 100 ⁇ g/kg SCV- 07-treated animals, 1923.9 mm 2 for 1 mg/kg SCV-07-treated animals, and 962.8 mm 2 for 10 mg/kg SCV-07-treated animals.
  • TGI tumor growth inhibition
  • Vc is the mean volume of the tumors in the contol group
  • Vt is the mean volume of the tumors in the test group.
  • Table XXII shows the tumor growth inhibition for animals treated with 100 ⁇ g/kg, 1 mg/kg, 10 mg/kg SCV-07 alone or in combination with radiation. When compared to unirradiated controls, animals treated with 1 mg/kg SCV-07 alone had a tumor growth inhibition of 9.1%, and animals treated with 10 mg/kg SCV-07 alone had a tumor growth inhibition of 35.9%.
  • Table XXII ** indicates that mean tumor volumes in groups 2 and 7 exceeded the vehicle control animals (9.8% and 15.87%), respectively).
  • TGI Tumor Growth Inhibition
  • Example 5 Exemplary Treatment for Cancer and Pre-malignant Conditions (Precancer).
  • a cancer patient is administered a STAT3 antagonist agent at a dose of 1000-3000 mg/day for up to 48 weeks.
  • a cancer patient is administered SCV-07 at a dose of 0.1-5.0 mg/kg/day, three times per week, for up to 48 weeks.
  • a patient suffering from a pre-malignant condition is administered a STAT3 antagonist agent at a dose of 1000- 3000 mg/day for up to 48 weeks.
  • a patient suffering from a pre-malignant condition is administered SCV-07 at a dose of 0.1-5.0 mg/kg/day, three times per week, for up to 48 weeks.
  • Example 6 Screening Utilizing Tumor Cells with Inducibly Expressed STAT3.
  • Target Compounds used in the study include the test compound SCV-07 (8-D-Glutamyl-L-Tryptophan-Na; [Ci 6 Hi 8 N 3 0 5 Na], MW 355.3) and the control compound (L)Glu(L)Leu.Na + ([CiiHi 9 N 2 0 5 Na], MW 282).
  • STAT3 inhibitor test by STAT3/luciferase reporter assay A cell-based assay based on the STAT3/luciferase reporter system (SABiosciences, Cat. #CCS-9028L) is used to test the target compound-mediated inhibitory effect on STAT3 upon IL-6 stimulation in HEK 293T cells.
  • STAT3 inhibitor test by STAT3/luciferase reporter assay test procedures.
  • Cells were plated in 24-well plates at 2.5 x 10 5 cells per well for 16 h. Cells were transiently transfected with STAT3/Luc reporter and incubated for 24 h. Cells were pretreated with the compounds for lh followed by stimulation with 20 ng/ml IL-6 for 6 h. Cells were then lysed with the Passive Lysis Buffer (Promega, Cat. #E1941) and the lysates were collected by centrifugation. Luciferase activity of each sample was measured using the Dual-Luciferase Reporter Assay Substrate System (Promega, Cat. #E1910).
  • STAT3 inhibitor test by ST AT3/lucif erase reporter assay. The data were analyzed with Excel as shown in Figure 19.
  • Figure 19 describes inhibitory screening of the compound by the STAT3/luciferase reporter assay.
  • HEK 293T cells were plated in a 24-well plate at 2.5 10 5 cells/well for 16 h. Cells were transiently transfected with STAT3/LUC reporter for 24 h. Cells were pretreated with 0.01 , 0.1 , 1 , 10 and 50 ⁇ of test (SCV-07) or control (L- Glu-L-Leu) compound for 1 h. Cells were then stimulated with 20 ng/ml IL-6 for 6 h in order to promote inducible expression of the construct.
  • V vehicle control
  • Cells were lysed and luciferase activity of each lysate sample was measured. Results are shown as the mean ⁇ SD, and determinations were made in triplicate. Data consist of the assay sets 1 (A) and 2 (B) as well as a positive control (C).
  • curcumin (Imgenex, Cat. #IMG ⁇ '2010), which is a known inhibitor for IL-6 inducible STAT3 phosphorylation, was used as a positive STAT3 inhibitor control (C).
  • the vehicle control (V) for curcumin was DMSO.
  • SCV-07 inhibited IL-6-mediated STAT3 activation in HEK 293T cells, yielding around 50% inhibition at 10 ⁇ g/ml when compared with vehicle control in ST AT3 -based luciferase reporter assay.
  • the control compound L-Glu-L-Leu
  • the control compound also exhibited similar inhibitory effect on STAT3 activity (see, Figure 19).
  • STAT3 inhibitor test by Western blot analysis Western blot analysis was performed to test the target compound-mediated inhibitory effect on STAT3 phosphorylation upon IFN-a stimulation at various time points in Jurkat and THP-1 cells. Western blotting was performed using anti-phospho-STAT3 antibody as well as anti-STAT3 antibody according to the Western standard protocols. The Western data were further qunatitated using TotalLab Quant software (Gentel Biosciences) as shown in Figures 20C and 21C.
  • Cells were plated in 60 x 15-mm tissue culture dishes at 5 x 10 6 cells per dish. Cells were pretreated with 0.01 , 0.1 , 1 or 10 ⁇ g/ml SCV-07 for 1 h and then stimulated with 25 ng/ml IFN-a for 0, 15, 30, 60 and 120 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels (Invitrogen, Cat.
  • Figure 20 describes the STAT3 phosphorylation inhibition assay in Jurkat cells.
  • Cells were plated in 60 x 15-mm tissue culture dishes at 5 x 10 6 cells/dish. Cells were pretreated with 0.01, 0.1, 1 or 10 ⁇ g/ml SCV-07 for 1 h, and then stimulated with 25 ng/ml IFN-a for 0, 15, 30, 60 and 120 min in order to inducibly express STAT3.
  • PBS was used as vehicle control.
  • Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with anti-pSTAT3 antibody ⁇ upper panels of A and B). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A and B). The graph indicates the relative quantitation of pSTAT3 band intensities (C). Note that Piceatannol (EMD Biosciences, Cat. #527948), which selectively inhibits the tyrosine phosphorylation of STAT3, was used as a positive control (B).
  • Figure 21 describes the STAT3 phosphorylation inhibition assay in THP-1 cells.
  • Cells were plated in 60 x 15-mm tissue culture dishes at 5 x 10 6 cells/dish. Cells were pretreated with 0.01, 0.1, 1 or 10 ⁇ g/ml SCV-07 for 1 h, and then stimulated with 25 ng/ml IFN-a for 0, 15, 30, 60 and 120 min. PBS was used as vehicle control. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3 antibody (upper panels of A and B).
  • the membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A and B).
  • the graph indicates the relative quantitation of pSTAT3 band intensities (C). Note that Piceatannol, which selectively inhibits the tyrosine phosphorylation of STAT3, was used as a positive control (B).
  • Figure 36 describes the effect of SCV-07 on STAT3 phosphorylation in the human lymphoblastic leukemia, CCRF-CEM, cell line.
  • Cells (5 x 10 6 cells/sample) were pretreated with 1 and 10 ⁇ g/ml SCV-07 for 2 hours and then stimulated with 100 ng/ml IL-4 for 0, 10 and 30 minutes in order to inducibly express STAT3.
  • Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to a nitrocellulose membrane.
  • the membrane was probed with anti-pSTAT3 Tyr 705 antibody ( Figure 36A, upper panel).
  • the membrane was then stripped and reprobed with anti-STAT3 antibody ( Figure 36A, lower panel).
  • the graph indicates the relative quantitation of pSTAT3x yr 705 band intensities ( Figure 36B).
  • SCV-07 significantly reduced IFN-a-mediated STAT3 phosphorylation in Jurkat cells whereas SCV-07 showed no significant inhibitory effect on STAT3 phosphorylation in THP-1 cell ( Figures 20 and 21). SCV-07 also significantly reduced IL-4 mediated STAT3 phosphorylation in CCRF-CEM cells ( Figure 36).
  • Example 7 Screening Utilizing Tumor Cells with Constitutively Active STAT3 or Inducibly Expressed STAT3.
  • the target compound used in this study included the test compound SCV-07 (8-D-Glutamyl-L-Tryptophan-Na, [Ci 6 Hi 8 N 3 0 5 Na], MW 355.3).
  • the compound stock preparation was SCV-07 was dissolved in 1 x phosphate-buffered saline (HyClone, Cat. #SH30264.01) at 5 mg/ml, divided into useable aliquots, and stored at -20°C.
  • the condition for PMA treatment was previously determined by a pilot experiment.
  • the differentiated THP-1 or NK-92 cells (5 x 10 6 cells per each treatment) were pretreated with vehicle (PBS), SCV-07 (0.01, 0.1, 1 and 10 ⁇ g/ml) and 10 ⁇ g/ml Piceatannol (positive control) for 2 h. Cells were then stimulated with 25 ng/ml IFN-a for 15, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels (Invitrogen, Cat.
  • Figure 22 describes the effect of SCV-07 on STAT3 phosphorylation in differentiated THP-1 cells.
  • Cells were differentiated by treatment of 25 ng/ml PMA for 20 h.
  • the differentiated cells (5 X 10 6 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 ⁇ g/ml SCV-07 for 2 h.
  • Cells were then stimulated with 25 ng/ml IFN-a for 0, 15, 30 and 60 minutes.
  • Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with anti-pSTAT3x yr 705 antibody ⁇ upper panels of A and B). The membranes were then stripped and reprobed with anti- STAT3 antibody (lower panels of A and B). The graph indicates the relative quantitation of pSTAT3 band intensities (C).
  • Figure 31 also describes the effects of SCV-07 on STAT3 phosphorylation in differentiated THP-1 cells.
  • Cells were differentiated by treatment of 25 ng/ml PMA for 20 h.
  • the differentiated cells (5 x 10 6 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 ⁇ g/ml SCV-07 for 2 h.
  • Cells were then stimulated with 25 ng/ml IFN-a for 0, 15, 30 and 60 minutes.
  • Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with anti-pSTAT3x yr 705 antibody (upper panels of A and B). The membranes were then stripped and reprobed with anti- STAT3 antibody (lower panels of A and B). (See, also Figures 22A and 22B.)
  • Figure 23 describes the effect of SCV-07 on STAT3 phosphorylation in NK-92 cells.
  • Cells (5 x 10 6 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 ⁇ g/ml SCV-07 for 2 h. Cells were then stimulated with 25 ng/ml IFN-a for 0, 15, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose
  • Figure 32 also describes the effect of SCV-07 on STAT3 phosphorylation in NK- 92 cells.
  • Cells (5 10 6 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 ⁇ SCV-07 for 2 h. Cells were then stimulated with 25 ng/ml IFN-a for 0, 15, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose
  • SCV-07 did not specifically reduce IFN-a-induced STAT3 phosphorylation in PMA-differentiated THP-1 cells ( Figures 22 and 31). SCV-07 showed no significant inhibitory effect on STAT3 phosphorylation in NK-92 cells; instead, SCV-07 somewhat enhanced IFN-a-induced STAT3 phosphorylation in NK-92 cells ( Figures 23 and 32).
  • Table XXIII The five tumor cell lines used in the study.
  • Figure 24 describes the effect of SCV-07 on STAT3 phosphorylation in various tumor cell lines.
  • Cells (5 x 10 6 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 ⁇ g/ml SCV-07 for 2 h. Cells were then lysed with RIPA lysis buffer containing
  • Figure 33 also describes the effect of SCV-07 on STAT3 phosphorylation in various tumor cell lines.
  • Cells (5 x 10 6 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 ⁇ g/ml SCV-07 for 2 h. Cells were then lysed with RIPA lysis buffer containing
  • Figure 35 shows that SCV-07 does not inhibit constitutive tyrosine
  • Piceatannol EMD Biosciences, Cat. #527948
  • P piceatannol
  • the pervanadate-treated cells as well as untreated control cells were further incubated with 0.01, 0.1, 1 and 10 ⁇ SCV-07 for 2 h.
  • Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice.
  • the lysates were run on 4- 20% Tris-Glycine gels (Invitrogen, Cat. #EC60255) and transferred to nitrocellulose membranes.
  • the membranes were probed with anti-phospho-STAT3 Tyr 705 antibody (Cell Signaling Technology, Cat. #9138).
  • the membranes were stripped and reprobed with anti-STAT3 antibody (Cell Signaling Technology, Cat. #9139) to analyze the cellular level of STAT3 from each sample.
  • Figure 25 also describes the effect of SCV-07 on STAT3 phosphorylation in
  • the pervanadate-treated and untreated control cells (5 x 10 6 cells/sample) were further treated with 1 and 10 ⁇ g/ml SCV-07 for 2 h, and then stimulated with 25 ng/ml IFN-a for 0, 15, 30,
  • Figure 26 describes the effect of SCV-07 on STAT3 phosphorylation in Kasumi-1 cells treated with pervanadate.
  • Cells were pretreated with 50 ⁇ pervanadate for 4 h.
  • the pervanadate-treated and untreated control cells (5 x 10 6 cells/sample) were further treated with 0.01, 0.1, 1 and 10 ⁇ g/ml SCV-07 for 2 h.
  • Cells were then lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice.
  • the lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with anti-pSTAT3 Tyr 705 antibody (upper panels of A).
  • Figure 34 also describes the effect of SCV-07 on STAT3 phosphorylation in Jurkat cells treated with pervanadate.
  • Cells were pretreated with 50 ⁇ pervanadate for 4 h.
  • the pervanadate treated cells (5 x 10 6 cells/sample) were further treated with 1 and 10 ⁇ g/ml SCV-07 for 2 h, and then stimulated with 25 ng/ml IFN-a for 0, 15, 30, 60 and 120 minutes.
  • Cells were then lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice.
  • the lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with anti-pSTAT3xy r 705 antibody (upper panels).
  • the membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels). (See Figure 25 A.)
  • SCV-07 somewhat reversed the pervanadate activity that enhanced both IFN-a- induced and constitutive STAT3 phosphorylation, respectively, in Jurkat and Kasumi-1 cells; suggesting that tyrosine phosphatases are involved in SCV-07-mediated inhibition of STAT3 activation (see, Figures 25, 26 and 34).
  • Effects of SCV-07 on kinase phosphorylation profiles test description. The phosphorylation profiles of kinases in Jurkat cells when stimulated with IFN-a in the presence or absence of SCV-07 were analyzed using the Human Phospho-kinase Array Kit (R & D Systems, Cat. #ARY003).
  • Test procedures Jurkat cells were plated in 60 x 15 -mm tissue culture dishes at 1 x 10 7 cells per dish. Cells were pretreated with vehicle (PBS) and SCV-07 (1 and 10 ⁇ g/ml) for 2 h. Cells were then stimulated with 25 ng/ml IFN-a for 30 minutes in order to inducibly express STAT3. (The time point of 30 min was previously determined by Western analysis.) Two cell samples, which were treated only with vehicle and 10 ⁇ g/ml SCV-07 for 30 minutes, were also prepared. Cells were lysed with the Array Lysis Buffer (R & D Systems).
  • Figure 27 describes effects of SCV-07 on various kinase phosphorylation in Jurkat cells.
  • Cells were plated in 60 x 15 -mm tissue culture dishes at 1 ⁇ 10 7 cells/dish. Cells were pretreated with vehicle and SCV-07 (1 and 10 ⁇ g/ml) for 2 h, and then stimulated with 25 ng/ml IFN-a or vehicle control for 30 min. IFN-a was added in order to inducibly express STAT3. Two cell samples treated only with vehicle and 10 ⁇ g/ml SCV-07 for 30 min were also prepared (as indicated with asterisks in panel A).
  • Figure 28 describes quantitation data for the human phospho-kinase array tests. Array signals from scanned X-ray film images (as shown in Figure 29A) were analyzed using the TotalLab Quant software (Gentel Biosciences).
  • Figure 29 shows normalized quantitation data for the human phospho-kinase array tests ( Figure 27A, Non-activated Jurkat cells).
  • the quantitation values for the SCV-07 (1 ⁇ / ⁇ 1)/- ⁇ - ⁇ and SCV-07 (10 ⁇ / ⁇ 1)/- ⁇ - ⁇ samples were normalized by the Vehicle/- IFN-a sample values. Note that value 1 is a basal level (no change). >1, fold increase; ⁇ 1, fold decrease.
  • Figure 30 shows normalized quantitation data for the human phospho-kinase array tests (Fig. 27A, IFN-a-activated Jurkat cells).
  • the quantitation values for the SCV-07 (1 ⁇ g/ml)/+IFN- and SCV-07 (10 ⁇ g/ml)/+IFN- samples were normalized by the
  • Vehicle/+IFN-a sample values. Note that value 1 is a basal level (no change). >1, fold increase; ⁇ 1, fold decrease.
  • Example 8 The peptide immunomodulator SCV-07 requires phosphatase activity for inhibition of STAT3 signaling.
  • the novel immunomodulatory peptide, gamma-D-glutamyl-L- tryptophan (SCV-07) is being evaluated as an intervention for oral mucositis in radiation treatment of head and neck cancer (Adkins 2010), and has also been shown in various xenograft cancer models to decrease tumor growth and improve survival (Tuthill 2009).
  • Preliminary evaluation of the mechanism of action of SCV-07 demonstrated inhibition of IL- 6 dependent STAT3 signaling in B16 melanoma cells (Nagabhushanam 2008, Tuthill 2009).
  • the objective of the current mechanistic studies was to understand further details of the involvement of SCV-07 in the STAT3 signaling pathway.
  • the lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes and probed with anti-pSTAT3 Tyr 705 antibody. The membranes were then stripped and reprobed with anti-STAT3 antibody to analyze the cellular level of STAT3 from each sample.
  • the phosphorylation profile of kinases in Jurkat cells stimulated with IFN-a in order to inducibly express STAT3 in the presence or absence of SCV-07 were analyzed using the Human Phosphokinase Array Kit, using 400 ⁇ g total protein from cells lysed with the Array Lysis Buffer.
  • the human phosphokinase assay revealed that SCV-07 not only suppresses pSTAT3 Tyr 705 but also significantly inhibits pSTAT5( a & b)Tyr699 and pSTATl Tyr 7oi , while it somewhat enhances pSTAT4 Tyr6 93.
  • Example 9 The Effect of the Immunomodulatory Peptide Gamma-D-Glutamyl-L- Tryptophan in Leukemia, Lymphoma, and Head and Neck Cancer Xenograft Models.
  • SCV-07 ( ⁇ -D-glutamyl-L-tryptophan) is a synthetic dipeptide which has been shown to stimulate the immune system. SCV-07 is effective in various preclinical models in which immune stimulation would be expected to be helpful, including vaccine enhancement (Tuthill, et al., Cold Spring Harbor, Harnessing Immunity to Prevent and Treat Disease (2009)), asthma (Regal, et al., Society of Toxicology abstracts, #1502 (2009)), and infectious disease (Mossel, et al., (2005) Microbes in a Changing World IUMS meeting, San Francisco, CA (2005); and Rose, et al., International Journal of Antimicrobial Agents 32: 262-266 (2008)).
  • SCV-07 demonstrated efficacy in attenuating acute and fractionated radiation-induced mucosal injury in the clinically predictive hamster model (Watkins, et al., Oral Diseases, 16: 655-60 (2010)), an effect which was noted to be dose and schedule dependent. SCV-07 has demonstrated a broad spectrum of immune stimulation, enhancing the Thl-type immune response and increasing antigen-specific T cell responses (Simbirtsev, et al., Russian Journal of Immunology 8: 11-22 (2003)).
  • the efficacy of SCV-07 in preventing radiation-induced oral mucositis is expected to arise from the drug's ability to stimulate the Thl type immune response and to block signal transducers and activator of transcription 3 (STAT3) mediated signaling (Tuthill, et al, AACR-NCI-EORTC Molecular Targets and Cancer Therapeutics (2009); and Papkoff, et al., AACR Tumor Immunology: Basic and Clinical Advances, Miami Beach, FL (2010)).
  • STAT3 signal transducers and activator of transcription 3
  • SCV-07 was further investigated in a phase 2a clinical trial, in which the compound demonstrated a trend toward delay of onset of ulcerative (World Health
  • mice Female mice aged 5 - 6 weeks were individually numbered using an ear punch and housed in groups of 10 animals per cage. Animals were acclimatized for at least 3 days and only those in good condition were used for the study.
  • Tumor cell lines from ATCC were grown in medium supplemented with 10% Fetal Calf Serum (FCS), 1% penicillin and streptomycin, and 2 mM L-Glutamine and sub- cultured by dilution at a ratio of 1 :3.
  • FCS Fetal Calf Serum
  • penicillin and streptomycin 1% penicillin and streptomycin
  • 2 mM L-Glutamine sub- cultured by dilution at a ratio of 1 :3.
  • mice were inoculated with tumor cells subcutaneously in the left flank, and when tumors were established, mice were randomly and prospectively divided into treatment groups prior to the initiation of daily treatment with either vehicle or SCV-07 of various doses.
  • Tumors were measured once every two days with micro-calipers, and tumor volume was calculated as (length x width x width)/2.
  • the tumor growth index (TGI) was calculated using the formula 100-(Vc* 100/Vt), where Vc is the mean volume of the tumors in the control group and Vt is the mean volume of the tumors in the test group.
  • MOLT-4 cells were plated in 60 x 15 -mm tissue culture dishes at 5 x 10 6 cells per dish. Cells were pretreated with vehicle (PBS) or SCV-07 (1 and 10 ⁇ g/mL) for 2 hours and then stimulated with 25 ng/mL IFNa, 200 ng/mL IL-6 or 100 ng/mL IL-10 for 0, 10, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice, run on 4-20% Tris-Glycine gels (Invitrogen, Cat. #EC60255), and transferred to nitrocellulose membranes.
  • vehicle PBS
  • SCV-07 1 and 10 ⁇ g/mL
  • the membranes were probed with anti-pSTAT3Tyr705 (Cell Signaling Technology, Cat. #9138). After analysis of pSTAT3, the membranes were stripped and reprobed with anti-STAT3 antibody (Cell Signaling Technology, Cat. #9139).
  • SCV-07 did not inhibit m vz ' vo proliferation of any tumor cell lines tested (NCI60; data on file). SCV-07 was shown to inhibit cytokine-induced STAT3 tyrosine
  • the immune modulating peptide SCV-07 is able to inhibit growth of a variety of tumor cell line types, including a human head and neck tumor line in nude mice. These results suggest that the use of SCV-07 as an intervention for mucositis should not interfere with therapy, but may be useful in enhancing the tumor response to conventional therapy. SCV-07 may also be useful alone for the treatment of tumors with inducible STAT3.
  • SCV-07 is currently under clinical investigation for attenuation of oral mucositis in FiNSCC patients receiving chemoradiation therapy. Based on its
  • SCV-07 could also have antitumor activity.
  • SCV-07 decreased tumor growth of a variety of murine and human cancer cell lines.
  • SCV-07 decreased tumor growth of a variety of murine and human cancer cell lines.
  • STAT3 inhibition and changes in cytokine production could shift macrophage and T cell subsets to promote an immune regulatory environment that inhibits both mucositis and tumor progression.
  • SHP-2 phosphatase SHP-2 and altered phosphorylation of Src family kinases and STAT5a/b, known substrates for SHP-2.
  • SCV-07 also decreased IFNa stimulated phosphorylation of SHP-2 associated immunoreceptors Siglec3 (CD33) and SIRPbetal (CD172B).
  • SHP-2 also increases Src-family kinase activity via dephosphoryation of the regulatory tyrosine.
  • SCV-07 leads to increased phosphorylation of various Src family kinases (potentially autophosphoryation).
  • Summary of Results SCV-07 has anti-tumor efficacy with human and murine tumor cell line xenograft models (see Example 9). Inhibition of cytokine -induced, but not constitutive, STAT3 protein tyrosine 705 phosphorylation (STAT3 PY) in some monocyte and T cell tumor lines. While not a direct inhibitor of STAT3, SCV-07 does lead to dose- dependent inhibition of IL-6-induced STAT3 reporter gene activity in transfected HEK-293T cells.
  • SCV-07 activates a pathway that leads to SHP-2 activation and STAT3 and STAT5 de-phosphorylation. No in vitro inhibition of proliferation was observed for any of the tumor cell lines treated with SCV-07.
  • SCV-07 binds directly with high affinity to mouse macrophages and thymocytes.
  • In vitro effects of SCV-07 on STAT3 PY are observed in cell lines of T cell and monocyte origin. Efficacy observed for multiple tumor cell types was observed in both syngeneic and nude mice.
  • SCV-07 may mediate anti-tumor activity by creating an environment that inhibits tumor progression through reducing chemotaxis of tumor-growth promoting macrophages, secretion of tumor growth-promoting cytokines in the tumor microenvironment, promoting the shift from M2 to Ml profile in macrophage cells, and/or promoting the shift from Th2 to Thl profile of local T cells. Further experimentation will be required to define the "SCV-07 receptor", other signaling molecules in SCV-07 pathway, and to explore additional cell biological consequences of SCV-07 action.
  • Example 11 Study of Tumor Growth in MOLT-4 Lymphoblastic Leukemia Model.
  • mice Eighty (80) nude mice (nu/nu) were randomly and prospectively divided into four groups of ten mice and two groups of twenty mice. Mice were inoculated s.c. in the left flank with MOLT-4 tumor cells, at an inoculum of 5 x 10 6 cells per mouse. Animals were treated with vehicle or SCV-07 as shown in Table XXVII. Each animal's weights and condition were recorded daily and tumors were measured three times per week.
  • mice per group in groups 1 and 6 were sacrificed on Day 33 for cytokine analysis
  • mice Female nude mice (Taconic Labs), aged 5 to 6 weeks, with a mean pre- treatment body weight 30.5 grams were used. Animals were individually numbered using an ear punch and housed in groups of 10 animals per cage. Animals were acclimatized prior to tumor implantation. During this period of at least 3 days, the animals were observed daily in order to reject animals that presented in poor condition.
  • mice were randomly and prospectively divided into four (4) groups prior to the initiation of treatment. Each animal was identified by ear punching corresponding to an individual number. A cage card was used to identify each cage and marked with the study number, treatment group number and animal numbers.
  • Tumors were measured once every two days with micro-calipers, and tumor volume was calculated as (length x width x width)/2.
  • the tumor growth index (TGI) was calculated using the formula 100-(Vc* 100/Vt), where Vc is the mean volume of the tumors in the control group and Vt is the mean volume of the tumors in the test group.
  • MOLT-4 (ATCC # CRL-1852) adult lymphoblastic leukemia (ATL) cells were obtained from ATCC. These cells were grown in RPMI-1640 medium supplemented with 10% Fetal Calf Serum (FCS), 1% penicillin and streptomycin, and 2mM L-Glutamine. Cells were sub-cultured by dilution at a ratio of 1 :3.
  • SCV-07 dosing solutions were prepared freshly each week. All groups were treated with a volume of 0.1 mL, and the concentration for dosing solutions were based on mean group weights on the day that drug dosing solutions are prepared for each week. For example, for mice with a mean weight of 25 g and dosed at 40 mg/kg, a 10 mg/mL dosing solution was prepared by dissolving 80 mg of SCV-07 in 8 mL of PBS. For the 20 mg/kg group, dosing solution was prepared by diluting the 10 mg/mL solution 1 :2 in PBS (5 mg/mL).
  • dosing solution was prepared by diluting the 5 mg/mL solution 1 :2 in PBS (2.5 mg/mL).
  • dosing solution was prepared by diluting the 10 mg/mL solution 1 :2 in PBS (1.25 mg/mL).
  • dosing solution was prepared by diluting the 5 mg/mL solution 1 :5 in PBS (0.25 mg/mL). All dosing solutions were made freshly at the beginning of each week of dosing, and stored at 4°C protected from light.
  • mice receiving vehicle had a mean gain of 4.3% of their starting weight at the end of the study.
  • Mice treated with SCV-07 at 1.0 mg/kg had a mean gain of 7.5% of their starting weight at the end of the study.
  • Mice treated with SCV-07 at 5.0 mg/kg had a mean gain of 9.9% of their starting weight at the end of the study.
  • Mice treated with SCV-07 at 10.0 mg/kg had a mean gain of 2.7% of their starting weight at the end of the study.
  • Mice treated with SCV-07 at 20.0 mg/kg had a mean gain of 7.9% of their starting weight at the end of the study.
  • Mice treated with SCV-07 at 40.0 mg/kg had a mean gain of 8.4% of their starting weight at the end of the study.
  • Tumor Volumes ( Figures 54 and 55). Tumor volume was analyzed to Day 36, when the first animal to be euthanized for exceeding maximum allowable tumor volume was euthanized. These data are shown in Figure 54.
  • the mean tumor volume for the vehicle control group increased from 108 mm 3 on Day 1 to 961 mm 3 on Day 36.
  • the group treated with SCV-07 at 1.0 mg/kg had a mean tumor volume of 114 mm 3 on Day 1, increasing to 763 mm 3 on Day 36.
  • the group treated with SCV-07 at 5.0 mg/kg had a mean tumor volume of 104 mm 3 on Day 1, increasing to 551 mm 3 on Day 36.
  • the group treated with SCV-07 at 10.0 mg/kg had a mean tumor volume of 111 mm 3 on Day 1, increasing to 405 mm 3 on Day 36.
  • the group treated with SCV-07 at 20.0 mg/kg had a mean tumor volume of 117 mm 3 on Day 1, which had increased to 431 mm 3 on Day 36.
  • the mean tumor volume was 107 mm 3 on Day 1 which had increased to 227 mm 3 on Day 36.
  • test compound was SCV-07 (gamma-D- glutamyl-L-tryptophan).
  • SCV-07 was dissolved in 1 x phosphate-buffered saline (HyClone, Cat. #SH30264.01) at 5 mg/ml, divided into useable aliquots, and stored at -20°C prior to use.
  • Jurkat cells were plated in 60 x 15-mm tissue culture dishes at 5 x 10 6 cells per dish. Cells were then pretreated with vehicle (PBS) or SCV-07 (1 and 10 ⁇ g/mL) for 2h and then stimulated with 25 ng/mL IFNafor 10 and 30 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4- 20%) Tris-Glycine gels (Invitrogen, Cat. #EC60255) and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3 Tyr 705 (Cell Signaling
  • the graphs indicate the relative quantitation of pSTAT3 (Fig. 57D & 58D), pSHP-2 Tyr54 2 (Fig. 57E & 58E) and pSHP-2 Tyr58 o (Fig. 57F & 58F) band intensities.
  • mouse peritoneal macrophages (3-5 x 10 6 cells/sample) were stimulated with 25 ng/mL IFNa ( Fig. 59 A), 200 ng/mL IL-6 (Fig. 59B) or 100 ng/mL IL-10 (Fig. 59 for 0, 5, 10, 30 and 60 minutes.
  • Cells were lysed with RIPA lysis buffer containing
  • protease/phosphatase inhibitor cocktail on ice.
  • the lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with antipSTAT3x yr 705 antibody (upper panels Fig. 59).
  • the membranes were then stripped and reprobed with anti-STAT3 (lower panels Fig. 59) antibody.
  • mouse peritoneal macrophages (3-5 x 10 6 cells/sample) were stimulated with 25 ng/mL IFNa (Fig. 60 A), 200 ng/mL IL-6 (Fig. 60B) or 100 ng/mL IL-10 (Fig. 60 for 0, 5, 10, 30 and 60 minutes.
  • Cells were lysed with RIPA lysis buffer containing
  • protease/phosphatase inhibitor cocktail on ice.
  • the lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes.
  • the membranes were probed with anti-p- SHP-2 T y r54 2 (upper panels)ox pSHP-2 Tyr5 8o (middle panels Fig. 60) antibody.
  • the membranes were then stripped and reprobed with anti-SHP-2 antibody (lower panels Fig. 60).
  • mouse peritoneal macrophages (3-5 x 10 6 cells/sample) were stimulated with 25 ng/mL IFNa (Fig. 61 A), 200 ng/mL IL-6 (Fig. 6 IB) or 100 ng/mLIL-10 (Fig. 61 for 0, 5, 10, 30 and 60 minutes.
  • Cells were lysed with RIPA lysis buffer containing
  • mice On day of harvest, euthanized mice were injected with cold lx PBS (8-10 mL) to wash the peritoneal cavity and lavage was collected using the syringe (up to 5-6 mL per mouse). Lavage (Peritoneal wash) containing cells was pooled and centrifuged for 10 min. The pellets were washed once with culture media. Cells were re-suspended, counted and plated in 6-well plates to get adherent macrophages at 3-5 x 10 6 cells per well. Cells were then incubated at 37°C and 16h later, non-adherent cells were removed and adherent cells (macrophages) were washed with fresh media.
  • Lavage Peritoneal wash
  • peritoneal Macrophages were isolated from Thioglycollate-stimulated mice and were then cultured in complete DMEM media in a 6-well plate. Sixteen hours later, non-adherent cells were removed and adherent cells (macrophages) were harvested and stained with PE- conjugated F4/80, a peritoneal macrophage marker, or with PE-conjugated Rat IgG2a isotype control at ⁇ g/10 6 cells for 30 min on ice. Cells were washed twice with flow buffer and resuspended in flow buffer. Cells were then analyzed using CellQuest-based FACSCalibur (BD Biosciences). The cell population was then confirmed as peritoneal macrophages.
  • Mouse peritoneal macrophages plated in 6-well plates were pretreated with vehicle (PBS) or SCV-07 (1 and 10 ⁇ g/mL) for 2 h and then stimulated with 200 ng/mL IL-6 for 10 minutes and 30 minutes.
  • Cells were then lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice.
  • the lysates were run on 4-20% Tris-Glycine gels (Invitrogen, Cat. #EC60255) and transferred to nitrocellulose membranes.
  • the membranes were probed with anti-pSTAT3 Tyr 705 antibody (Cell Signaling Technology, Cat.
  • the graphs indicate the relative quantitation of pSTAT3 ⁇ Fig. 62D & 63E), pSHP-2 Tyr5 42 ⁇ Fig. 62E & 63E) and pSHP-2 Tyr5 8o (Fig. 62F & 63F) band intensities.
  • SCV-07 enhanced activation of pSHP-2 Tyr54 2 in IL-6- stimulated primary mouse macrophages while pSHP-2 Tyr5 8o was not significantly affected by SCV-07 ( Figures 62 & 63).
  • pSHP-l Tyr5 3 6 was not induced by IL-6 stimulation in primary mouse macrophages ( Figures 62 & 63).
  • SCV-07 suppressed both constitutive and IL-6- mediated levels of MCP-1 in primary mouse macrophages while IL-12p40 was unlikely induced by IL-6 in primary mouse macrophages ( Figure 64).
  • SCV-07 likely targets upstream of STAT3, potentially directly catalyzing SHP-2 phosphorylation or synergizing with (or activating) an upstream regulator for SHP-2.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Organic Chemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention relates to the use of compounds of Formula A for the treatment of neoplasia with inducibly expressed STAT3. Further, the present invention relates to determining treatment regimens and therapeutic efficacy with respect to treatment of neoplasia with inducibly expressed STAT3.

Description

METHOD AND COMPOSITIONS FOR TREATMENT
OF STAT3-RESPONSIVE CANCERS AND/OR RENAL CANCER
RELATED APPLICATIONS
[001] This application claims priority to U.S. Provisional Application No: 61/414,844, filed November 17, 2010; U.S. Provisional Application No: 61/418,828 , filed December 01, 2010; and U.S. Provisional Application No: 61/487,191, filed May 17, 2011, each of which are hereby incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
[002] Personalized medicine has become an ever more popular and growing field. Given the vast number of therapies available for the treatment of any given disease indication, the ability to tailor therapies to individual patients is becoming increasingly important to those being treated as well as to physicians deciding which therapeutic compound should be used for a given patient, e.g. , at a given time point of the treatment cycle. Physicians, patients, and third-party payers all seek therapies tailored to the individual needs of the patient.
[003] As such, while correct diagnosis of an indication is important for successful treatment, equally important is determining and/or predicting the suitability, responsiveness, and/or efficacy of treatment with a particular therapeutic agent. This is especially relevant in cases when treatment is prolonged or cycled as is the case in many anti-cancer regimens. A clinician's ability to recognize at early phases of treatment which patients are responding to therapy can provide a platform for accurate decision making for continued and future treatment. As such, methods for determining which patients are likely to respond to a given treatment will provide physicians with information necessary to specifically tailor treatments to individual patients.
[004] There is a need in the field to develop therapies for the treatment of neoplasia, especially in terms of developing therapies in response to certain characteristics of the neoplastic condition, e.g., a neoplastic growth with certain pathways activated. SUMMARY OF THE INVENTION
[005] The present invention is based in part on the discovery that neoplasia associated with inducible expression of STAT3 can be treated with compounds of the invention, e.g., compounds of Formula A. Accordingly, the present invention provides methods for treating subjects with inducibly expressed STAT3 as well as methods for determining treatment regimen and/or predicting treatment efficacy for a subject with neoplasia.
[006] In one embodiment, the present invention provides methods for treating a subject with neoplasia comprising administering to a subject in need of such treatment an effective amount of a therapeutic entity, wherein the subject has inducible expression of STAT3 and wherein the therapeutic entity comprises a compound of Formula A
R— NH— CH (CH2)„— C— X
COOH O
(A)
or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof.
[007] In another embodiment, the present invention provides methods for treating a subject with neoplasia comprising determining the presence of inducible expression of STAT3 in a biological sample of the subject and administering a therapeutic entity to the subject upon determination of the presence of inducible expression of STAT3 in the subject, wherein the therapeutic entity comprises a compound of Formula A
R— NH— CH (CH2)„— C— X
COOH O
(A)
or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof. [008] In yet another embodiment, the present invention provides methods for determining the treatment regimen for a subject with neoplasia comprising selecting a treatment regimen comprising a therapeutic entity for the subject based on the presence of inducible expression of STAT3, wherein the therapeutic entity comprises a compound of Formula A
R— NH— CH (CH2)W— C— X
COOH O
(A)
or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof. In a further embodiment, the method further comprises determining the presence of inducible expression of STAT3 in a biological sample from the subject.
[009] In still another embodiment, the present invention provides methods for predicting the treatment efficacy of a therapeutic entity for the treatment of neoplasia comprising detecting inducible expression of STAT3 in a biological sample of a subject, wherein the presence of inducible expression of STAT3 is indicative of the treatment efficacy of the therapeutic entity for the subject, and wherein said therapeutic entity comprises a compound of Formula A
Figure imgf000004_0001
(A)
or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof.
[0010] In still yet another embodiment, the present invention provides methods for determining the treatment efficacy of a therapeutic entity for the treatment of neoplasia comprising detecting in a biological sample of a subject treated with the therapeutic entity the presence of one or more markers selected from the group consisting of markers for inhibition of signal transduction through STAT3, markers for inhibition of phosphorylation of STAT3, markers for inhibition of nuclear translocation of STAT3, markers for inhibition of IL-6 mediated STAT3 activation, markers for inhibition of IL-10 mediated STAT3 activation, markers for inhibition of IFN-a mediated STAT3 activation and markers for inhibition of IL- 4 mediated STAT3 activation,
wherein the presence of one or more markers is indicative of the therapeutic efficacy of the therapeutic entity,
wherein said therapeutic entity comprises a compound of Formula A
Figure imgf000005_0001
(A) or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof.
[0011 ] In still yet another embodiment, the present invention provides methods of providing useful information for determining the treatment regimen for a subject with neoplasia comprising detecting the presence or absence of inducible expression of STAT3 in a biological sample of a subject and providing the result of the detection to an entity that determines the treatment regimen based on the presence or absence of inducible expression of STAT3,
wherein said therapeutic entity comprises a compound of Formula A
Figure imgf000005_0002
(A)
or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 is a cartoon showing the STAT3 pathway.
[0013] Figure 2 is a cartoon showing a proposed mechanism of action for SCV-07.
[0014] Figure 3 is a graph showing % activity relative to the log of the peptide concentration. The protein is human STAT3 Protein (amino acids 127-722); the ligand is pYLPQTV-NH2 (SEQ ID NO:2) labeled with 5-carboxyfluorescein. pY indicates pYLPQTV (SEQ ID NO:2); Ac indicates Ac-YLPQTV (SEQ ID NO:3).
[0015] Figure 4 is a series of photographs showing staining results for STAT3. Figure 8A (no treatment); Figure 8B (IL-6); Figure 8C (IL-6 + SCV-07 0.04 μΜ (0.01 μg/mL)); Figure 8D (IL-6 + SCV-07 0.4 μΜ (0.1 μg/mL)); Figure 8E (IL-6 + SCV-07 10 μΜ (2.48 μg/mL)); Figure 8F (IL-6 + CP 0.04 μΜ); Figure 8G (IL-6 + CP0.4 μΜ); Figure 8H (IL-6 + CP 10 μΜ).
[0016] Figure 5 is a graph showing the effect of SCV-07 on B 16F0 tumor growth in C57BL-6 mice.
[0017] Figure 6 is a diagram comparing the means of pSTAT3 levels in tumors of SCV- 07-treated and untreated mice.
[0018] Figure 7 is diagram comparing the means of NK cell accumulation in tumors of SCV-07-treated and untreated mice.
[0019] Figure 8 is a graph showing tumor growth curves for the renal carcinoma study of Example 3.
[0020] Figure 9 is a bar graph showing tumor weights for each indicated treatment group on Day 17.
[0021] Figure 10 is a graph showing animal growth over the course of treatment for the indicated treatment groups.
[0022] Figure 11 is a graph showing mean weight change in mice receiving the indicated treatment with SCV-07, with no accompanying radiation treatment. Error bars indicate the SEM.
[0023] Figure 12 is a graph showing mean weight change in mice receiving the indicated treatment with SCV-07, with accompanying radiation treatment. Error bars indicate the SEM.
[0024] Figure 13 is a graph presenting the AUC for mean weight change in mice receiving the indicated treatment with SCV-07, with no accompanying radiation treatment. [0025] Figure 14 is a graph presenting the AUC for mean weight change in mice receiving the indicated treatment with SCV-07, with accompanying radiation treatment.
[0026] Figure 15 is a graph indicating changes in mean tumor volume in mice receiving the indicated treatment with SCV-07, with no accompanying radiation treatment.
[0027] Figure 16 is a graph indicating changes in mean tumor volume in mice receiving the indicated treatment with SCV-07, with accompanying radiation treatment.
[0028] Figure 17 is a graph showing data relating to mean tumor volume AUC for mice receiving the indicated treatment with SCV-07, with no accompanying radiation treatment.
[0029] Figure 18 is a graph showing data relating to mean tumor volume AUC for mice receiving the indicated treatment with SCV-07, with accompanying radiation treatment.
[0030] Figures 19A, 19B and 19C describe inhibitory screening of the compound by the STAT3/luciferase reporter assay.
[0031] Figures 20A, 20B and 20C describe STAT3 phosphorylation inhibition assay in Jurkat cells.
[0032] Figures 21A, 21B and 21C describe STAT3 phosphorylation inhibition assay in THP-1 cells.
[0033] Figures 22A, 22B and 22C describe the effect of SCV-07 on STAT3
phosphorylation in differentiated THP-1 cells.
[0034] Figures 23 A, 23B and 23C describe the effect of SCV-07 on STAT3
phosphorylation in NK-92 cells.
[0035] Figures 24A and 24B describe effects of SCV-07 on constitutive STAT3 phosphorylation on a set of tumor cell lines.
[0036] Figures 25 A, 25B, 25C and 25D describe the effect of SCV-07 on STAT3 phosphorylation in Jurkat cells treated with pervanadate.
[0037] Figures 26A and 26B describe the effect of SCV-07 on STAT3 phosphorylation in Kasumi-1 cells treated with pervanadate.
[0038] Figures 27A and 27B describe effects of SCV-07 on various kinase
phosphorylation in Jurkat cells.
[0039] Figures 28A, 28B, 28C, 28D, 28E and 28F describes quantitation data for the human phospho-kinase array tests.
[0040] Figure 29 shows normalized quantitation data for the human phospho-kinase array tests. [0041] Figure 30 shows normalized quantitation data for the human phospho-kinase array tests.
[0042] Figures 31 A and 3 IB describe the effect of SCV-07 on STAT3 phosphorylation in differentiated THP-1 cells.
[0043] Figures 32A and 32B describe the effect of SCV-07 on STAT3 phosphorylation in NK-92 cells.
[0044] Figure 33 describes the effect of SCV-07 on STAT3 phosphorylation in various tumor cell lines.
[0045] Figure 34 describes the effect of SCV-07 on STAT3 phosphorylation in Jurkat cells treated with pervanadate.
[0046] Figures 35A and 35B describe that SCV-07 does not inhibit constitutive tyrosine phosphorylation of STAT3 in a variety of tumor cell lines.
[0047] Figures 36A and 36B describe the effect of SCV-07 on STAT3 phosphorylation in CCRF-CEM cell lines.
[0048] Figure 37 describes the effect of SCV-07 on MOLT -4 tumors in mice. Tumors in SCV-07-treated animals grew more slowly than those in control animals; the reduction was generally dose-dependent and statistically significant at higher doses. For MOLT-4: 10 mg/kg (p=0.003), 20 mg/kg (p=0.002), 40 mg/kg (p<0.001).
[0049] Figure 38 describes the effect of SCV-07 on HL-60 tumors in mice. Tumors in SCV-07-treated animals grew more slowly than those in control animals; the reduction was generally dose-dependent and statistically significant at higher doses. For HL-60: 20 mg/kg (p=0.025), 40 mg/kg (p=0.005).
[0050] Figure 39 describes the effect of SCV-07 on EL-4 tumors in mice. Tumors in SCV-07-treated animals grew more slowly than those in control animals; the reduction was generally dose-dependent and statistically significant at higher doses. For EL-4: 20 mg/kg (p=0.001), 40 mg/kg (p<0.001).
[0051 ] Figure 40 describes the effect of SCV-07 on FaDu tumors in mice. Tumors in SCV-07-treated animals grew more slowly than those in control animals; the reduction was generally dose-dependent and statistically significant at higher doses. For FaDu: 20 mg/kg (p=0.004), 40 mg/kg (p=0.004).
[0052] Figure 41 describes the effect of SCV-07 on SK-MEL tumors in mice. Tumors in SCV-07-treated animals grew more slowly than those in control animals; the reduction was generally dose-dependent and statistically significant at higher doses. For SK-MEL: 20 mg/kg (p=0.02) .
[0053] Figure 42A, 42B, 42C and 42D describe SCV-07 inhibition of cytokine-induced STAT3 tyrosine phosphorylation (PY) in MOLT-4, used for the xenograft studies.
[0054] Figure 43 describes that SCV-07 inhibits IFNa-induced STAT3 PY in Kasumi-1 cells and IL-4-induced STAT3 PY in CCRF-CEM cells. Western blots with antibodies against STAT3-Tyr705 or Total STAT3.
[0055] Figure 44 describes that SCV-07 inhibits IFNa-induced STAT3 PY in Jurkat T cells. Western blots with antibodies against STAT3-Tyr705 or Total STAT3.
[0056] Figure 45 describes that SCV-07 does not inhibit constitutive STAT3 PY in various cell lines.
[0057] Figure 46 describes that SCV-07 also leads to decreased STAT5a/b tyrosine phosphorylation in Jurkat T cells. Western blots with antibodies against STAT3-Tyr694, STAT1-Tyr701 or Total STAT5 and STAT1 or Total STAT3.
[0058] Figure 47 describes that SCV-07 treatment leads to inhibition of IL-6 induced STAT3 -regulated gene expression.
[0059] Figure 48 describes that a tyrosine phosphatase is required for SCV-07 effects on STAT3 PY in Jurkat T cells. SCV-07 inhibition of STAT3 PY is blocked by addition of pervanadate, a tyrosine phosphatase inhibitor. Western blots with antibodies against STAT3- Tyr705 or Total STAT3. Cells were pretreated with 50 μΜ pervanadate for 4 h. The pervanadate-treated and untreated control cells (5 x 106 cells/sample) were further treated with 1 and 10 μg/ml SCV-07 for 2 h, and then stimulated with 25 ng/ml IFNa for 0, 15, 30, 60 and 120 minutes. Jurkat cells were then lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti- pSTAT3xyr705 antibody {upper panels of A and B). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A and B). The graph indicates the relative quantitation of pSTAT3 band intensities (B and C). A and C: pervanadate-treated cells. B and D: untreated control cells.
[0060] Figure 49 describes that CD45 is not the phosphatase required for SCV-07 effects on STAT3 PY. SCV-07 can still decrease STAT3 PY in cells lacking transmembrane
CD45. Western blots with antibodies against STAT3-Tyr705 or Total STAT3. Cells (5 x 106 cells/sample) were pretreated with 1 and 10 μg/ml SCV-07 for 2 h. Cells were then stimulated with 25 ng/ml IFNa, 200 ng/ml IL-6 or 100 ng/ml IL-10 for 0, 10 and 30 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3xyr705 antibody (upper panels of A). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A). The graph indicates the relative quantitation of pSTAT3 band intensities (B).
[0061] Figure 50 describes that SHP-2 tyrosine phosphorylation increased by different concentrations of SCV-07 in both untreated and IFNa-treated Jurkat cells. Example of data from R&D Systems phosphoprotein detection array with INFa treated Jurkat cells.
[0062] Figure 51 shows survival data. The percent survival was calculated for each group on each day of the study. No significant changes in survival were seen.
[0063] Figure 52 shows the mean percent weight change. The percentage daily weight change for each animal and the means for each treatment group were calculated. Error bars represent the SEM.
[0064] Figure 53 shows the Mean Weight Change AUC. The area under the curve (AUC) was calculated for the percent weight change exhibited by each animal in the study. This calculation was made using the trapezoidal rule transformation. Group means were calculated and are shown with error bars representing SEM for each group. P values indicate statistically significant differences between that group and the vehicle control group.
[0065] Figure 54 shows the Mean Tumor Volumes. Mean Tumor Volumes were calculated from the length and width measurements. Group means were calculated and are shown with error bars representing SEM for each group.
[0066] Figure 55 shows the Tumor Volume Change AUC. The area under the curve (AUC) was calculated for the tumor volume measured on each animal in the study. This calculation was made using the trapezoidal rule transformation. Group means were calculated and are shown with error bars representing SEM for each group. Groups were compared using an ANOVA on ranks test, and statistically significant differences were seen between the vehicle control group and the groups treated with SCV-7 at 10 mg/kg (p=0.002), 20 mg/kg (p=0.002) and 40 mg/kg (p<0.001). [0067] Figure 56A, 56B, and 56C show IFNa stimulation time points for induction of pSHP-2 and pSHP-1 in Jurkat cells. Cells (5 x 106 cells/sample) were stimulated with 25 ng/ml IFNa for 0, 5, 10, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3xyr705 {upper panel of A), anti-pSHP-lTyr536 {upper panel ofB), anti-pSHP- 2τΥι542 (first panel of C) and anti-pSHP-2Tyr58o (second panel of C) antibodies. The
membranes were then stripped and reprobed with anti-STAT3 (lower panel of A) ), anti-SHP- 1 (lower panel of B) and anti-SHP-2 (third panel of C) antibodies.
[0068] Figure 57A, 57B, 57C, 57D, 57E and 57F show the effect of SCV-07 on pSTAT3, pSHP-1 and pSHP-2 in IFNa-stimulated Jurkat cells (Experiment 1). Cells (5 x 106 cells/sample) were pretreated with 1 and 10 μg/ml SCV-07 for 2 h and then stimulated with 25 ng/ml IFNa for 0, 10 and 30 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with anti- pSTAT3xyr705 (upper panel of A), anti-pSHP-lxyr536 (upper panel ofB), antipSHP- 2xyr542 (first panel of C) and anti-pSHP-2Tyr58o (third panel of C) antibodies. The membranes were then stripped and reprobed with anti-STAT3 (lower panel of A), anti-SHP-1 (lower panel of B) and anti-SHP-2 (second and fourth panels of C) antibodies. The graphs indicate the relative quantitation of pSTAT3 (D), pSHP-2Tyr542 (E) and pSHP-2Tyr58o (F) band intensities.
[0069] Figure 58A, 58B, 58C, 58D, 58E and 58F show the effect of SCV-07 on pSTAT3, pSHP-1 and pSHP-2 in IFNa-stimulated Jurkat cells (Experiment 2). Cells (5 x 106 cells/sample) were pretreated with 1 and 10 μg/ml SCV-07 for 2 h and then stimulated with 25 ng/ml IFNa for 0, 10 and 30 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with anti- pSTAT3xyr705 (upper panel of A), anti-pSHP-lxyr536 (upper panel ofB), antipSHP-2xyr542 (first panel of C) and anti-pSHP-2Tyr58o (third panel of C) antibodies. The membranes were then stripped and reprobed with anti-STAT3 (lower panel of A), anti-SHP-1 (lower panel of B) and anti-SHP-2 (second and fourth panels of C) antibodies. The graphs indicate the relative quantitation of pSTAT3 (D), pSHP-2Tyr542 (E) and pSHP-2Tyr58o (F) band intensities. [0070] Figure 59A, 59B and 59C show selection of cytokines used for induction of pSTAT3 in primary mouse macrophages. Mouse peritoneal macrophages (3-5 x 106 cells/sample) were stimulated with 25 ng/ml IFNa (A), 200 ng/ml IL-6 (B) or 100 ng/ml IL- 10 (C) for 0, 5, 10, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with anti- pSTAT3xyr705 antibody (upper panels). The membranes were then stripped and reprobed with anti-STAT3 (lower panels) antibody.
[0071] Figure 60A, 60B and 60C show selection of cytokines used for induction of pSHP-2 in primary mouse macrophages. Mouse peritoneal macrophages (3-5 x 106 cells/sample) were stimulated with 25 ng/ml IFNa (A), 200 ng/ml IL-6 (B) or 100 ng/ml IL- 10 (C) for 0, 5, 10, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with antipSHP-2Tyr542 (upper panels)or pSHP-2Tyr58o (middle panels) antibody. The membranes were then stripped and reprobed with anti-SHP-2 antibody (lower panels).
[0072] Figure 61A, 61B and 61C show selection of cytokines used for induction of pSHP-1 in primary mouse macrophages. Mouse peritoneal macrophages (3-5 x 106 cells/sample) were stimulated with 25 ng/ml IFNa (A), 200 ng/ml IL-6 (B) or 100 ng/ml IL- 10 (C) for 0, 5, 10, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with antipSHP-lTyr536 antibody (upper panels). The membranes were then stripped and reprobed with anti-SHP-1 antibody (lower panels).
[0073] Figure 62A, 62B, 62C, 62D, 62E and 62F show the effect of SCV-07 on pSTAT3, pSHP-1 and pSHP-2 in IL-6-stimulated primary mouse macrophages (Experiment 1). Mouse peritoneal macrophages (3-5 x 106 cells/sample) were pretreated with 1 and 10 μg/ml SCV- 07 for 2 h and then stimulated with 200 ng/ml IL-6 for 0, 10 and 30 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3Tyr705 (upper panel of A), anti-pSHP-lTyr536
(upper panel of B), anti-pSHP-2xyr542 (first panel of C) and anti-pSHP-2xyr58o (third panel of C) antibodies. The membranes were then stripped and reprobed with anti-STAT3 (lower panel of A) ), anti-SHP-1 (lower panel ofB) and anti-SHP-2 (second and fourth panels of C) antibodies. The graphs indicate the relative quantitation of pSTAT3 (D), pSHP-2Tyr542 (E) and pSHP-2Tyr58o (F) band intensities.
[0074] Figure 63 A, 63B, 63C, 63D, 63E, and 63F show the effect of SCV-07 on pSTAT3, pSHP-1 and pSHP-2 in IL-6-stimulated primary mouse macrophages (Experiment 2). Mouse peritoneal macrophages (3-5 x 106 cells/sample) were pretreated with 1 and 10 μg/ml SCV-07 for 2 h and then stimulated with 200 ng/ml IL-6 for 0, 10, 20 and 30 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3xyr705 (upper panel of A), anti- pSHP-lTyr536 (upper panel ofB), anti-pSHP-2Tyr542 (first panel of C) and anti-pSHP-2Tyr58o (third panel of C) antibodies. The membranes were then stripped and reprobed with anti- STAT3 (lower panel of A), anti-SHP-1 (lower panel of B) and anti-SHP-2 (second and fourth panels of C) antibodies. The graphs indicate the relative quantitation of pSTAT3 (D), pSHP- 2τΥι542 (E) and pSHP-2Tyr58o (F) band intensities.
[0075] Figure 64A and 64B show the effect of SCV-07 on MCP-1 and IL-12p40 induction in primary mouse macrophages that were stimulated with IL-6. Mouse peritoneal macrophages (3-5 x 106 cells/sample) were pretreated with 1 and 10 μg/ml SCV-07 for 2 h and then stimulated with 200 ng/ml IL-6 for 18 h. Cell culture supernatants were harvested and analyzed by ELISA. Induction levels of MCP-1 (A) and IL-12p40 (B) were then analyzed. *p = 0.0495 versus vehicle control 1 (VC-1; without SCV-07 and IL-6); **p = 0.0495 versus vehicle control 2 (VC-2; IL-6 only) (Mann- Whitney U test).
DETAILED DESCRIPTION OF THE INVENTION
[0076] The present invention is based in part on the discovery that neoplasia associated with inducible expression of STAT3 can be treated with compounds of the invention, e.g., compounds of Formula A. Accordingly, the present invention provides methods for treating subjects with inducible expression of STAT3 as well as methods for determining treatment regimen and/or predicting treatment efficacy for a subject with neoplasia. [0077] According to one aspect of the invention, it provides methods for treating a subject, e.g., mammal including humans with neoplasia associated with an inducible expression of STAT3 by administering to the subject an effective amount of a therapeutic entity comprising a compound of the present invention.
[0078] According to the present invention, neoplasia includes any abnormal growth or cell proliferation, e.g. , uncoordinated with the proliferation of the tissues (normal) around it. In one embodiment, neoplasia includes any cancer or tumor growth in a subject. In another embodiment, neoplasia includes any pre-cancerous or pre-tumor growth in a subject. In yet another embodiment, neoplasia includes any solid or circulating cancer or tumor growth in a subject. In still another embodiment, neoplasia includes any abnormal growth that is capable of metastasizing or spreading to other locations of the subject. Examples of neoplasia include, but are not limited to carcinoma, sarcoma, blastoma, lymphoma, leukemia, and germ cell tumors. In some embodiments, neoplasia includes head and neck, skin, colon, oral, glioblastoma, breast, laryngeal, esophageal, endothelial, endometrial, ovarian, lung, urogenital, rectal, prostate, kidney, melanoma, renal, and papilloma virus-induced cancer.
[0079] According to the present invention, neoplasia associated with inducible expression of STAT3 includes any neoplasia accompanied by or having detectable inducible expression of STAT3. In one embodiment, neoplasia associated with inducible expression of STAT3 includes neoplasia with inducible expression of STAT3 within the site or cell or tissue of neoplasia. In another embodiment, neoplasia associated with inducible expression of STAT3 includes neoplasia with inducible expression of STAT3 outside of the original site or cell or tissue of neoplasia, but within cells or tissues directly associated with cells or tissues with neoplasia. In yet another embodiment, neoplasia associated with inducible expression of STAT3 includes neoplasia with inducible expression of STAT3 in cells or tissues in direct contact with or in the proximity of cells or tissues with neoplasia.
[0080] According to the present invention, inducible expression of STAT3 includes any STAT3 expression or activation, e.g., phosphorylation that is induced, activated or triggered by one or more elements in a subject, e.g., human. In one embodiment, inducible expression of STAT3 includes any STAT3 expression or activation that is induced by a cytokine, e.g., IFN-a, IL-4, IL-6, IL-10, and IL-27, etc. In another embodiment, inducible expression of STAT3 includes any STAT3 expression or activation that is induced by one or more factors/elements within the pathway of a cytokine that induces expression or activation of STAT3, e.g., IFN-α, IL-4, IL-6, IL-10, and IL-27, etc. In yet another embodiment, inducible expression of STAT3 includes any STAT3 expression or activation that is induced by an exogenous agent or an endogenous agent that is itself inducibly expressed or present in a temporal or spatial pattern. In still another embodiment, inducible expression of STAT3 includes any increased expression or activation of STAT3, e.g., comparing to a
predetermined base level or constitutive level of STAT3 expression or activation. In still yet another embodiment, inducible expression of STAT3 includes any expression or activation of STAT3 in one or more cells in response to the cells being exposed to an agent, e.g., cytokine in vivo or in vitro.
[0081] In still yet another embodiment, inducible expression of STAT3 includes any STAT3 expression or activation that is not present constantly, but only present with a temporal or spatial pattern. In still another embodiment, inducible expression of STAT3 includes any STAT3 expression or activation that is not associated with a genetic
modification which causes constant expression or activation of STAT3. In still another embodiment, inducible expression of STAT3 includes expression or activation of any STAT3 that is not a constitutively phosphorylated form of STAT3 (including forms with mutated phosphorylation sites), inappropriately truncated forms of STAT3 that are constantly active, as well as other forms of STAT3 that are unable to be inactivated.
[0082] Signal transducer and activator of transcription 3 (STAT3) is of great importance in the development of neoplasia. STAT3 is a point of convergence for numerous oncogenic signaling pathways. It is involved in numerous signal transduction pathways, and can be constitutively activated in both tumor cells and immune cells in the tumor microenvironment. Constitutively activated STAT3 inhibits the expression of a variety of cytokines, including IL-12, IFN-γ, and type I interferons as well as up-regulates co-stimulatory molecules such as B7-1 and 2, which are necessary for immune activation against tumor cells. Additionally, STAT3 activity can promote the production of immunosuppressive factors such as IL-6, IL- 10, TGF-β and VEGF, which in turn activate STAT3 in diverse immune cell subsets and alter gene expression profiles.
[0083] The STAT3 pathway is shown in Figure 1. STAT3 is present in the cytoplasm in the inactive monomeric form. Molecules that activate STAT3 include, among others, IL-6, IL-10, VEGF, oncostatin M and a number of growth factor receptors including epidermal growth factor receptors EGFR and HER2, fibroblast growth factor receptor (FGFR), insulin- like growth factor receptor (IGFR), hepatocyte growth factor receptor (HGFR), platelet- derived growth factor receptor (PDGFR) and transforming growth factor beta receptors (TGFBR). These molecules or receptors induce phosphorylation of STAT3 monomers. The phosphorylated STAT3 monomers then dimerize and translocate to the nucleus where they bind STAT3 responsive elements (RE) and initiate transcription of ST AT3 -dependent genes.
[0084] Detection of inducible expression of STAT3 can be carried out by any suitable means in the field. For example, detection of inducible expression of STAT3 includes detecting the level of proteins, nucleic acids or gene expression. Methods for detecting the levels of nucleic acids and proteins are well known in the art and any standard methods for detection of nucleic acid or protein levels can be employed with the methods of the present invention and used for detecting inducible expression of STAT3.
[0085] Methods for detecting the levels of nucleic acids, such as RNA or DNA have been well described and are well known to those of skill in the art. Methods for detecting RNA can include but are not limited to RT-PCR, northern blot analyses, gene expression analyses, microarray analyses, gene expression chip analyses, hybridization techniques (including FISH), expression beadchip arrays, and chromatography as well as any other techniques known in the art. Methods for detecting DNA can include but are not limited to PCR, realtime PCR, digital PCR, hybridization (including FISH), microarray analyses, and
chromatography as well as any other techniques known in the art.
[0086] Methods for detecting proteins and polypeptides can include but are not limited to spectrophotometric determination of protein concentration, quantitative amino acid analysis, protein concentration assays, chromatography assays, western blot analyses, gel
electrophoresis, (followed by staining procedures including but not limited to Coomassie Blue, Silver stain, Syber Green, Syber Gold), hybridization, multiplex cytokine assays, ELISA, bicinchoninic acid (BCA) protein assays, Bradford protein assays, and Lowry protein assays as well as any other techniques known in the art. Protein detection can also include detecting the levels of stable or active proteins and methods such as kinetic assays, kinase assays, phosphatase assays, enzyme assays and post-translation modification assays (for example, assays for determining phosphorylation and glycosylation state) can also be employed.
[0087] Methods for quantitating nucleic acid and protein levels have also been well described. Methods can include traditional methods, such as western blot quantization as well as computer based methods, such as microarray assay or genechip assay analyses, for analyzing nucleic acid or protein levels. These standard methods known in the art can be employed to determine whether the level of STAT3 is increased or induced in a sample. In some embodiments, determination of an increased expression or induced expression of STAT3 can be based on a comparison of the level of STAT3 in a sample with a
predetermined standard, e.g., base or constant level of STAT3, e.g., corresponding to the cell or tissue type used in the sample. In other embodiments, the determination of an increased expression or induced expression of STAT3 can be based on a comparison of the level of STAT3 in a sample before treatment with an agent and to the level after treatment with an agent, e.g., a cytokine.
[0088] Predetermined standard levels of STAT3 can be defined using a variety of methods known to those of skill in the art. Generally, standard levels are determined by determining the level of STAT3 in a sufficiently large number of samples obtained from normal, healthy control subjects. Further, standard level information can be obtained from publically available databases, as well as other sources. (See, e.g., Bunk, D.M., "Reference Materials and Reference Measurement Procedures: An Overview from a National Metrology Institute," Clin. Biochem. Rev., 28(4): 131-137 (2007); Suraj Peril , et al, "Development of Human Protein Reference Database as an Initial Platform for Approaching Systems Biology in Humans" Genome Res. 13: 2363-2371 (2003); Remington: The Science and Practice of Pharmacy, Twenty First Edition (2005).)
[0089] In one embodiment, inducible expression of STAT3 in a subject can be detected by exposing a cell population of the subject to an agent, e.g., cytokine and determining the expression or activation, e.g., phosphorylation of STAT3 in the cell population in response to the agent. For example, inducible expression of STAT3 in a subject can be determined by detecting an increased level of STAT3 expression or phosphorylation in response to a cytokine.
[0090] According to the present invention, the therapeutic entity used in the methods of the present invention includes any composition comprising a compound of Formula A or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof. Formula A is
Figure imgf000018_0001
(A) wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof. In some embodiments, X is L-tryptophan or D-tryptophan.
[0091] Derivatives of the aromatic or heterocyclic amino acids for "X" can include amides, mono-or di-(Cr C6) alklyl substituted amides, arylamides, and (Cr C6) alkyl or aryl esters. Acyl or alkyl moieties for "R" can include branched or unbranched alkyl groups of 1 to about 6 carbons, acyl groups from 2 to about 10 carbon atoms, and blocking groups such as carbobenzyloxy and t-butyloxycarbonyl. Preferably the carbon of the CH group shown in Formula A has a stereoconfiguration, when n is 2, that is different from the
stereoconfiguration of X.
[0092] In some embodiments, the compound of Formula A includes γ-D-glutamyl-L- tryptophan, γ -L-glutamyl-L-tryptophan, γ-L-glutamyl-Nm-formyl-L-tryptophan, N-methyl-γ- L-glutamyl-L-tryptophan, N-acetyl-y-D-glutamyl-L-tryptophan, γ-L-glutamyl-D-tryptophan, β-L-aspartyl-L-tryptophan, and β -D-aspartyl-L-tryptophan. In some other embodiments, the compound of Formula A is γ-D-glutamyl-L-tryptophan (SCV-07).
[0093] The therapeutics and compounds contemplated for use with the methods of the present invention can be administered as a pharmaceutical composition or formulation. In addition to the therapeutic entity, these pharmaceutical compositions can also contain pharmaceutically acceptable carriers or excipients. As used herein, the term
"pharmaceutically acceptable carrier" or "excipient" and variants thereof include but are not limited to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and others that are physiologically compatible, as well as pharmaceutically acceptable salts. Pharmaceutically acceptable carriers can include but are not limited to sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions. The preparation of compositions containing nVmrmflPfflitirflll arrive substances is well known in the art and any well known methods can be employed with the methods of the present invention. (See, e.g., Remington: The Science and Practice of Pharmacy, Twenty First Edition (2005); US Patent No. 5,916,878.)
[0094] The pharmaceutical compositions of the present invention can be formulated for parenteral, intravenous, intraperitoneal, intramuscular, intradermal, sublingual or oral administration. In some embodiments, the pharmaceutical compositions can be formulated to contain the therapeutic entity and one or more chemotherapeutics, radiations therapeutics or chemoradiation therapeutics as a single composition for administration. In other
embodiments, the pharmaceutical compositions can be formulated to contain the therapeutic entity as one composition and one or more chemotherapeutics, radiations therapeutics and chemoradiation therapeutics as a separate composition for administration. The separate compositions can be administered together or separately, at the same site or different sites, and can be administered sequentially or concurrently.
[0095] Administration can include a variety of methods and routes for administration. The therapeutic entity of the present invention can be administered with other therapeutics. Other therapeutics included but are not limited to chemotherapeutics, targeted therapeutics, radiation therapeutics, or chemoradiation therapeutics. In some embodiments of the methods of the present invention, the therapeutic entity is administered with one or more one or more chemotherapeutics, radiation therapeutics, or chemoradiation therapeutics. In some embodiments, the therapeutic entity is co-administered with one or more chemotherapeutics, targeted therapeutics, radiation therapeutics, or chemoradiation therapeutics. In other embodiments, the therapeutic entity is administered after administration of one or more one or more chemotherapeutics, targeted therapeutics, radiation therapeutics, or chemoradiation therapeutics. In other embodiments, the therapeutic entity is administered in the absence of any other therapeutics.
[0096] Co-administration can include administration at the same site or at different body sites in a subject. Co -administration can further include sequential or concurrent
administration. Sequential administration can include administration of the therapeutic entity either before or after another therapeutic. Concurrent administration can include
administration of the therapeutic entity at the same site as another therapeutic, as well as administration of the therapeutic entity at a different site than another therapeutic but at the same time. In some embodiments, the therapeutic entity can be administered at the same site as another therapeutic. In other embodiments, the therapeutic entity can be administered at a different site than another therapeutic. In yet other embodiments, the therapeutic entity can be administered sequentially or concurrently with another therapeutic.
[0097] According to the present invention, an effective amount of a therapeutic entity is an amount that causes a therapeutic effect or benefit for the subject treated. The therapeutic entity of the present invention can be administrated at a variety of effective dosages. In some embodiments, the effective dosage is in the range of about 0.01 to 100 milligrams per kilogram (mg/kg) subject body weight. In other embodiments, the effective dosage is in the range of about 0.1-10 milligrams per kilogram subject body weight. In other embodiments, the effective dosage is in the range of about 0.1-5.0 milligrams per kilogram subject body weight. In other embodiments, the effective dosage is in the range of about 1.0-5.0 milligrams per kilogram subject body weight. In other embodiments, the effective dosage is in the range of about 1.0, 2.0, 3.0, 4.0 or 5.0 milligrams per kilogram subject body weight. In yet other embodiments, the effective dosage is in the range of 5.0 milligrams per kilogram. In yet other embodiments, the effective dosage is in the range of 1.0 milligrams per kilogram. In yet other embodiments, the effective dosage is in the range of 0.1 milligrams per kilogram. In further embodiments the effective dosage is 0.02 mg/kg subject body weight.
[0098] According to another aspect of the invention, it provides methods for treating neoplasia by first determining the presence of inducible expression of STAT3 in a subject and then administering to the subject a therapeutic entity comprising a compound of Formula A upon determination that the subjection is positive for inducible expression of STAT3, e.g., the neoplasia is associated with inducible expression of STAT3. According to the present invention, determining the presence of inducible expression of STAT3 in a subject can be carried out by any suitable means, e.g., by clinician asking another entity and/or person to test the presence of inducible expression of STAT3 in a biological sample of a subject or by clinician studying the test results provided by another entity or person regarding STAT3 expression or activation.
[0099] According to the present invention, a biological sample of a subject can be any suitable sample for detecting inducible expression of STAT3, e.g., liquid, solid, cell, or tissue samples, etc. In addition, biological samples of the present invention can be obtained by any methods known in the art. Examples of biological samples suitable for the present invention include but are not limited to serum, blood, plasma, whole blood and derivatives thereof, skin, hair, hair follicles, saliva, oral mucous, vaginal mucous, sweat, tears, epithelial tissues, urine, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), excreta, ascites, cerebrospinal fluid, lymph, and tissue extract sample or biopsy.
[00100] According to yet another aspect of the invention, it provides methods for determining the proper treatment and/or regimen for neoplasia by selecting a treatment regimen using a therapeutic entity comprising a compound of Formula A based on the determination that the subject is positive for inducible expression of STAT3, e.g., detection of inducible expression of STAT3 (associated with the neoplasia) in the subject. For example, a subject is determined to be suitable for a treatment using a therapeutic entity comprising a compound of Formula A if such subject is determined to have inducible expression of STAT3. The subject is consequently treated using a therapeutic entity comprising a compound of Formula A.
[00101 ] According to still another aspect of the invention, it provides methods for predicting treatment efficacy of a therapeutic entity comprising a compound of Formula A by detecting the presence of inducible expression of STAT3 in a biological sample of the subject and predicting treatment efficacy of the therapeutic entity if the subject is positive for inducible expression of STAT3. For example, a treatment using a therapeutic entity comprising a compound of Formula A is likely to be efficacious for a subject if the subject is determined to have inducible expression of STAT3.
[00102] According to still another aspect of the invention, it provides methods for determining the treatment efficacy or monitoring the treatment effect of a therapeutic entity, e.g., a therapeutic entity comprising a compound of Formula A. These methods comprise detecting in a subject treated with the therapeutic entity the presence of one or more markers for inhibition of signal transduction through STAT3, inhibition of phosphorylation of STAT3, inhibition of nuclear translocation of STAT3, inhibition of IL-6 mediated STAT3 activation, inhibition of IFN-a mediated STAT3 activation, inhibition of IL-4 mediated STAT3 activation, inhibition of IL-10 mediated STAT3 activation, or increase of IFN-a or IL-6 mediated SHP-2 activation, e.g., increase of phosphorylation of SHP-2TYR542. In this embodiment, the presence of one or more markers is indicative of the therapeutic efficacy of the therapeutic entity. In some embodiments, the presence of one or more markers for inhibition of signal transduction through STAT3 can include one or more markers for inhibition of signal transduction through STAT3 via an increase in SHP-2 or SHP-2 activity, e.g., phosphorylation of SHP-2. In some embodiments, one or more markers for inhibition of signal transduction through STAT3 can include one or more markers for inhibition of signal transduction through STAT3 via an increase in SHP-2TYR542. In some embodiments, one or more markers for inhibition of phosphorylation of STAT3 can include one or more markers for inhibition of phosphorylation of pSTAT3xYR705. In further embodiments, where the subject has inducible expression of STAT3, e.g., SHP-2 such as PSHP-2TYR542 can be used as a marker for determining or monitoring the treatment efficacy of the therapeutic entity of the present invention .
[00103] In still some other embodiments, one or more markers for the inhibition of signal transduction through STAT3 can include one or more markers for the inhibition of signal transduction through STAT3 via an increase and/or decrease in kinase phosphorylation, including phosphorylation of those kinases listed in Figures 27-30. In some embodiments, one or more markers for the inhibition of signal transduction through STAT3 via an increase of p38a (T180/Y182), ERKl/2 (T202/Y204, T185/Y187), INK pan (T183/Y185,
T221/Y223), p53 (S392), p53 (S46), p53 (S I 5), MEK1/2 (S218/S222, S222/S226), MSK1/2
(S376/S360), AMPKal (T174), Akt (S473), Akt (T308), TOR (S2448), HSP27 (S78/S82),
Src (Y419), Lck (Y394), p70 S6 Kinase (T421/S424), p70 S6 Kinase (T229), RSK1/2/3
(S380), RSK1/2 (S221), Fyn (Y420), Yes (Y426), Fgr (Y412), Chk-2 (T68), FAK (Y394),
STAT4 (Y693), e.g., phosphorylation levels of these kinases are increased in response to treatment with the therapeutic entity of the present invention in the presence of inducible expression of STAT3. In some embodiments, one or more markers for the inhibition of signal transduction through STAT3 via a decrease of GSK-3a/p (S21/S9), β-Catenin, Paxillin
(Y118), Lyn (Y397), p27 (T157), STATl (Y701), STAT3 (Y705), STAT5a (Y699), STAT5b
(Y699), STAT5a/b (Y699), PLCy-l (Y783), c-Jun (S63), Pyk2 (Y402) and eNOS (S I 177), e.g., phosphorylation levels of these kinases are decreased in response to treatment with the therapeutic entity of the present invention in the presence of inducible expression of STAT3.
[00104] In further embodiments, the subject has inducible expression of STAT3 and wherein the one or more markers include without any limitation p38a (T180/Y182), ERKl/2
(T202/Y204, T185/Y187), JNK pan (T183/Y185, T221/Y223), p53 (S392), p53 (S46), p53
(SI 5), MEK1/2 (S218/S222, S222/S226), MSK1/2 (S376/S360), AMPKal (T174), Akt
(S473), Akt (T308), TOR (S2448), HSP27 (S78/S82), Src (Y419), Lck (Y394), p70 S6
Kinase (T421/S424), p70 S6 Kinase (T229), RSK1/2/3 (S380), RSK1/2 (S221), Fyn (Y420),
Yes ΓΥ4261 Fer ΓΥ412), Chk-2 (T68), FAK (Y394), STAT4 (Y693), GSK-3a/p (S21/S9), β-Catenin, Paxillin (Y118), Lyn (Y397), p27 (T157), STATl (Y701), STAT3 (Y705), STAT5a (Y699), STAT5b (Y699), STAT5a/b (Y699), PLCy-l (Y783), c-Jun (S63), Pyk2 (Y402) and eNOS (SI 177). For example, an increase in phosphorylation levels of p38a (T180/Y182), ERK1/2 (T202/Y204, T185/Y187), JNK pan (T183/Y185, T221/Y223), p53 (S392), p53 (S46), p53 (SI 5), MEK1/2 (S218/S222, S222/S226), MSK1/2 (S376/S360), AMPKal (T174), Akt (S473), Akt (T308), TOR (S2448), HSP27 (S78/S82), Src (Y419), Lck (Y394), p70 S6 Kinase (T421/S424), p70 S6 Kinase (T229), RSK1/2/3 (S380), RSK1/2 (S221), Fyn (Y420), Yes (Y426), Fgr (Y412), Chk-2 (T68), FAK (Y394), STAT4 (Y693) is indicative of treatment efficacy of the therapeutic entity of the present invention. In another example, a decrease in phosphorylation levels of GSK-3a/p (S21/S9), β-Catenin, Paxillin (Y118), Lyn (Y397), p27 (T157), STATl (Y701), STAT3 (Y705), STAT5a (Y699), STAT5b (Y699), STAT5a (Y699), PLCy-l (Y783), c-Jun (S63), Pyk2 (Y402) and eNOS (SI 177) is indicative of treatment efficacy of the therapeutic entity of the present invention.
[00105] The presence of these markers can be determined by standard assays known in the art. For example, inhibition of signal transduction through STAT3 can be determined by examining phosphorylation of STAT3 as well as activation of any members of the STAT3 pathway. Inhibition of phosphorylation of STAT3 can be examined by standard
phosphorylation assays known in the art. Inhibition of nuclear translocation of STAT3 can be examined by a variety of assays including cellular fraction, immunohistochemistry, or other well known assays. Inhibition of IL-6 mediated STAT3 activation can be examined using standard IL-6 inhibitors and IL-6 detection assays (which are commercially available, from companies such as for example Thermo-Scientific, USA). Inhibition of IFN-a mediated STAT3 activation can be examined using standard IFN-a inhibitor and IFN-a detection assays (also, commercially available, from companies such as for example Thermo- Scientific, USA). Inhibition of IL-4 mediated STAT3 activation can be examined using standard IL-4 inhibitor and IL-4 detection assays (also, commercially available, from companies such as for example Thermo-Scientific, USA). Increases and decreases in phosphorylation levels can be readily measured using a variety of well known techniques in the art and any techniques can be employed. For example, phosphorylation of PSHP-2TYR542 can be measured using phospho-specific antibodies for detecting the presence of pSHP- 2TYR542. Phosphorylation of STAT3 can be measured using phospho-specific antibodies for detecting the nresence of pSTAT3xYR705. [00106] According to yet another aspect of the present invention, it provides methods for providing useful information for predicting or determining the treatment regimen for a subject with neoplasia. These methods comprise detecting inducible expression of STAT3 in a biological sample of a subject and providing the results to an entity and/or clinician to predict or determine the treatment regimen based on the results provided, e.g., based on the presence or absence of inducible expression of STAT3.
EXAMPLES
Example 1. Effect of SCV-07 on STAT3 driven gene expression, SCV-07 Binding to STAT3 and Nuclear Translocation of STAT3.
[00107] Since SCV-07 reduces progression of melanoma in an animal model, and since STAT3 inhibitors are effective in inducing anti-tumor immunity in an antigen nonspecific manner as seen with SCV-07, the following experiments were conducted to determine whether SCV-07 could inhibit STAT3 -dependent gene expression.
[00108] HEK293 cells were transfected with the luciferase gene driven by STAT3 responsive elements. These cells were treated with either medium (control) or with 4 μΜ (1 μg/mL) SCV-07. The luciferase reporter assay to determine the effect of SCV-07 on STAT3 driven gene products was performed as previously described in Turkson et ah, Mol. Cell. Biol. 18:2545-2552, 1998, the disclosures of which are hereby incorporated by reference.
[00109] The pGL2 vector containing luciferase driven by SV40 promoter and 2X copies of STAT responsive element was transiently transfected into either HEK (Human embryonic kidney cells) or B16F0 melanoma cells. Briefly, the cell lines were seeded in a six-well tissue culture plate, allowed to reach -50-60% confluency and then transfected with the reporter carrying the STAT3 response element. The transfected cells were allowed to recover for 24 hours and were then treated with PBS as negative control, or with SCV-07. The cells were incubated for another 24 hours and harvested for analysis. The cells were lysed in the presence of protease inhibitors, protein concentration estimated, and the luciferase activity in the cell lysate was measured using Promega™ luciferase kit and a Tecan™ luminometer. Based on luciferase expression, when compared with luciferase expression in cells incubated in medium alone (control), incubation with SCV-07 inhibited STAT3 expression 40%. [00110] To confirm that this phenomenon is seen in B16F0 melanoma cells also, B16F0 melanoma cells were transfected with the pGL2 vector containing STAT3 responsive elements driving luciferase. While the transfection efficiency was lower in B16F0 cells, the data showed the same inhibition of STAT3 activity. SCV-07 at concentrations ranging from 0.01 to 1 μg/mL was capable of significantly (-70%) inhibiting luciferase expression driven by STAT3. This suggests that SCV-07 mediates its antitumor effects by inhibiting STAT3 activity. The control peptide (CP) showed little or no inhibitory activity. This phenomenon was repeatable in subsequent experiments. Percent inhibition with CP was 0 under all conditions.
[00111] To investigate direct interaction of SCV-07 with STAT3, a fluorescence polarization assay was used. This assay was performed as described in Schust et ah, Anal. Biochem. 330: 114-118, 2004, the disclosures of which are hereby incorporated by reference. In brief, the fluorescence polarization assay is a screen for small molecules that bind to the STAT3 SH2 domain and thereby inhibit or antagonize STAT3 activity. The basis of this assay is the binding of a fluorescein-labeled phosphotyrosine-peptide derived from the interleukin-6 receptor subunit gpl30 to unphosphorylated STAT3.
[00112] The assay was performed in 96 well plates. STAT3 protein amino acids 127-721 was added to each well at a final concentration of 1 μg in buffer (50 mM NaCl, 10 mM HEPES, 1 mM EDTA, 0.1% NP-40). SCV-07 and control peptide (AAK ; SEQ ID NO: l), referred to as CP, were added at different concentrations in a constant volume such that the total reaction mixture volume was 100
Figure imgf000025_0001
The controls included were STAT3 alone, STAT3 plus labeled ligand, labeled ligand alone and buffer alone. The reaction mixture was incubated for 1 hour at 37°C. The carboxyfluorescein-labeled STAT3 ligand then was added to a final concentration of 5 nM/well and the reaction mixture incubated for 15 minutes at 37°C. After incubation, polarization readings were taken using a TECAN™ fluorescent polarizer instrument. The extent of inhibition by the test peptides was calculated using an equation relating the difference in polarization between protein + probe and protein + probe + compound, to give the extent of inhibition due to compound addition. See Figure 3. The reported IC50 is 0.3-0.5 μΜ; the IC50 obtained here was 0.3 μΜ.
[00113] Since SCV-07 was capable of inhibiting STAT3 driven gene expression, we sought to determine if this was due to direct binding to STAT3. The STAT3 binding ability of SCV-07 therefore was assessed at various concentrations. As seen in Table I, the PY peptide (positive control) binds strongly to the SH2 domain of STAT3, indicated by 98% inhibition of depolarization. Neither SCV-07 nor CP showed any significant ability to bind STAT3 directly (Table I, below).
Table I. Direct Binding of SCV-07 to STAT3.
Figure imgf000026_0001
[00114] Having determined that SCV-07 inhibited STAT3 driven gene expression, but was not capable of directly binding STAT3, experiments then were conducted to determine whether SCV-07 prevented STAT3 from entering the nucleus. As previously mentioned, STAT3 inhibitors interfere with STAT3 -dependent responses by preventing STAT3 dimerization (competitive inhibitors) or by preventing translocation to the nucleus.
Immunofluorescence staining of phosphorylated STAT3 was conducted to localize STAT3 in B16F0 cells treated with SCV-07 or CP in the presence or absence of IL-6, which activates STAT3. See Figure 4.
[00115] B16F0 cells were grown on poly-L-lysine-coated cover slips in 6-well plates (50,000 cells per well) and incubated overnight. The next day, cells were treated with varying concentrations of SCV-07 or control peptide in the presence or absence of rIL-6. rIL-6 was added to inducibly express STAT3. Following a 30-minute incubation with IL-6, cells were incubated for 48 hours and then were fixed with 3% formaldehyde for 10 minutes and washed with PBS. The cells were permeabilized with absolute methanol, blocked with 1% BSA and incubated overnight at 4°C with anti-STAT3 monoclonal antibody
(Invitrogen™) at 1 :500 dilution. The cells were washed the next day with PBS and incubated for 3 hours at room temperature with goat anti-mouse antibody conjugated to Alexa Fluor488 (Invitrogen™) at 1 : 1000 dilution. Cells were washed, stained with propidium iodide, mounted on glass slides and observed under a NIKON™ fluorescence microscope.
[00116] As shown in Figures 4 A and 4B, treatment with IL-6 results in nuclear translocation of STAT3 (indicated by punctate staining). As shown in Figures 4F, 4G and 4H, CP at varying concentrations has no effect on nuclear translocation of STAT3. SCV-07, however, has a clear inhibitory effect on nuclear translocation of STAT3, as shown in Figures 4C, 4D and 4E (staining for STAT3 is chiefly cytoplasmic).
[00117] Constitutive expression of STAT3 in tumor tissue leads to upregulation of STAT3 -dependent genes such as c-Myc, JunB and Mcl-1. Activation of these genes induces proliferation of cancerous cells. Inhibition of STAT3 leads to inhibition of these genes resulting in an anti-proliferative effect on cancerous cells, thereby reducing tumor growth. To show this an anti-proliferative effect of SCV-07, multiple cell lines originating from various forms of cancers were incubated with SCV-07 and their proliferation measured after 96 hours.
[00118] The cell lines listed in Table II, below, were seeded at 2 x 103/well in triplicate in 100 μΐ of the corresponding medium supplemented with 2% FBS in 96-well plates. The plates were incubated overnight at 37°C and 5% C02. The following day, SCV-07 and CP were added at various concentrations. PBS was added as a vehicle control and cells in media alone as a control for maximal proliferation. Cells were incubated at 37°C and 5% C02 for 96 hours. After the incubation, 50 μΐ of the modified tetrazolium salt, XTT (2,3-bis(2- methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide ), was added to all wells and colorimetric measurements made at 465 nm at one and two hours post addition. The XTT assay measures metabolically active, proliferating cells that are capable of reducing XTT to water-soluble formazan. Dying cells (metabolically compromised cells) are incapable of this reaction.
Table II. Cell lines studied. Cell Line Organ of Origin Culture Medium
A-375 human melanoma DMEM
HT-29 colon McCoy's
A549 lung Ham's F12K
B16F0 mouse melanoma DMEM
PC-3 prostate Ham's F12K
MDA-MB-231 breast DMEM
MDA-MB-453 breast DMEM
[00119] As shown in Table III, the positive control, VX-680, a small molecule with known ability to inhibit tumor growth, inhibited proliferation to the extent of 70-90% in a variety of cell lines (A-375, B16F0, A-549, HT-29 and MDA-MB-453) as measured by the XTT assay. VX- 680 had only a marginal effect on the prostate carcinoma cell line (PC-3) and on MDA- MB-231. CP and SCV-07 at several different concentrations did not show an appreciable effect on inhibition of cell proliferation, suggesting that SCV-07 does not mediate its antitumor action by directly inhibiting proliferation of tumor cells. Values shown in Table III shown are % growth inhibition as measured in the XTT assay.
Table III. Effect of SCV-07 on Inhibition of Proliferation of Cancer Cell Lines.
Figure imgf000028_0001
CP
5.7 2.6 15.7 12.7 0.04 μΜ
[00120] Since STAT3 inhibitors can relieve immunosuppression by inducing cytokines such as TNFa and/or IL-12, supernatants from B16F0 cells were assayed for the presence of these cytokines after treatment with SCV-07 or CP. B16F0 cells were seeded in 6-well plates (1 x 105 cells/well in 2 mL of DMEM, in duplicate) and incubated overnight. The cells then were treated with SCV-07 and CP in combination with LPS (2.0 μg/mL) and incubated for 8 hours. Cells alone and LPS alone served as negative controls. Following incubation, cell supernatants were collected and analyzed for TNFa and IL-12 by ELISA.
[00121 ] No significant production of either cytokine was seen with medium alone or with LPS. However, SCV-07 (4 μΜ) did induce significant levels of TNFa and IL-12 in B16F0 cells when compared with CP. The levels of the cytokines were variable across 3 assays performed, but induction of TNFa by SCV-07 was consistent across all assays. The control peptide showed little or no effect in the induction of these cytokines. Therefore, these data suggest that induction of proinflammatory cytokines is at least one of several mechanisms by which STAT3 inhibition by SCV-07 relieves immunosuppression.
Example 2. Efficacy of subcutaneously administered SCV-07 on progression of
melanoma in C57B1/6 mice.
[00122] These studies were conducted in C57BL-6 mice to study the effect of 5 mg/kg SCV07 administered subcutaneously on progression of melanomas. B16F0 tumors were established in C57BL-6 mice. Ten mice per experimental group were administered SCV-07 subcutaneously once daily. The mice were followed over the course of 14 days for clinical signs including evaluation of skin and fur; eyes, mucous membranes; respiratory, circulatory, autonomic and central nervous systems, behavioral pattern, signs of tremors, convulsions, lethargy, excessive salivation and diarrhea, as well as for general morbidity. In addition, mice were weighed and tumor size measured daily. Mice did not show any significant clinical signs or altered behavior. As shown in Table IV, below, body weight reduction in the SCV-07-treated mice was not significantly different from the control group.
Table IV. Body Weight Reduction in SCV-07 Treated Mice, versus Placebo. Body Weight Reduction
Group Day 0 Day 14 Difference % reduction in body weight n= 10
Vehicle 23.77 25.11 -1.34 0.00
Control
SCV-7 (5 23.45 25.50 2.05 0.00
mg/kg)
[00123] For analysis, the tumors were dissociated. Single cell suspensions were prepared and stained with STAT3 antibody (pTyrosine 705, Cell Signaling™) followed by goat, anti- rabbit antibodies conjugated to Alexa Fluor 488 (Invitrogen™). The percentage of cells staining for phosphorylated STAT3 was measured in vehicle- and SCV-07-treated groups. In addition, dissociated tumor cells were stained with anti-NKl .l-FITC (Bioligand™) to quantitate NK cells. As shown in Table V below, the starting volume of tumors in both groups was comparable (day 0). Mice receiving subcutaneous SCV-07 (5 mg/kg) showed 33% tumor growth inhibition, which was statistically significant (P<0.001). See Table V and Figure 5. A second comparable study revealed a 40%> reduction in tumor volume.
Table V. Effect of SCV-07 on Tumor Volume.
Figure imgf000030_0001
[00124] Since SCV-07 inhibits STAT3-driven responses in vitro and decreased pSTAT3 positive cells in in vivo tumor models, repeat studies were performed to demonstrate that treatment of animals with SCV-07 had an effect on STAT3 expression in tumor tissue.
Tumor tissue from treated and control mice was dissociated and the expression levels of pSTAT3 measured in the tumors by flow cytometry. Treatment with SCV-07 resulted in reduced expression of pSTAT3; comparison of the means of treated and untreated groups showed a 23% reduction in STAT3. See Table VI and Figure 6.
[00125] Although the individual mice varied in their expression of pSTAT3 levels following SCV-07 treatment, the data show a significant reduction (p=0.005) in pSTAT3 levels when compared with vehicle treated mice. Overall, the group of mice treated with SCV-07 showed a 24% reduction in pSTAT3 when compared with vehicle treated mice.
Table VI. Reduction in pSTAT3 Following Treatment with SCV-07.
Figure imgf000031_0001
[00126] A reduction in pSTAT3 expression is accompanied by an increase in natural killer cell accumulation at the tumor site. Tumor tissue from mice treated as described above was dissociated and stained for NK cells and analyzed by flow cytometry. As shown in Table VII and Figure 7, the NK cell accumulation in SCV-07 treated mice was significantly greater (p=0.001) than that in vehicle-treated mice. The accumulation of NK cells in the SCV-07 treated mice was 33% greater than the accumulation seen at the tumor site in vehicle treated mice.
Table VII. Increase in NK Cell Accumulation in Tumors Following SCV-07 Treatment. Mouse No. Mouse No. (SCV-
(Vehicle Alone % NK Cells 07, 5 mg/kg % NK Cells
Group) Group)
1 23.82 11 23.96
2 23.63 12 25.95
3 8.98 13 24.06
4 14.94 14 23.16
5 18.16 15 20.37
6 17.34 16 26.30
7 11.74 17 20.13
8 23.13 18 26.13
9 11.54 19 29.13
10 8.29 20 21.12
Example 3. In Vivo Efficacy of SCV-07 in Mice Bearing Renal Carcinoma Tumors.
[00127] The inhibitory effect of SCV-07 on growth of murine cancer in Balb/C mice was evaluated. The study was designed to evaluate the anti-tumor effect of SCV-07 in a well- known murine renal cancer model (Balb/C) compared to the positive control drug, cyclophosphamide (CTX). The effects of the combination of SCV-07 and CTX also was tested. The negative control was PBS. Throughout the course of the study, no animal died as a result of treatment. In summary, the tumor model used in this study was validated because the positive control drug CTX effectively reduced the tumor growth. A total of 70 mice were implanted subcutaneously with murine renal carcinoma cells (Renca cells, 8 x 105 in a volume of 0.1 mL normal saline), followed by treatment with SCV-07, cyclophosphamide (CTX) or both for 14 consecutive days.
[00128] Thirty-five male and thirty-five female healthy, naive, Balb/C mice were involved in the study. The animals were four to six weeks old, weighing between 16 and 20 grams at the start of the study. The animals were group-housed in autoclaved shoe box cages with autoclaved wood chips as the bedding materials. The temperature of the animal room was maintained at 22 to 25°C, and the relative humidity was maintained at 40 to 60%. A 12-hour light/12-hour dark cycle was maintained except when interrupted by study-related events. Animals were fed ad libitum with sterile water and Beijing KeAoXieLi Rodent Diet
(certified). All animals were acclimated for 3 days prior to tumor inoculation. [00129] Murine renal carcinoma cells (Renca) were adapted in Balb/C mice before use as follows. Using aseptic tissue culture procedures, one vial of murine renal carcinoma (Renca) cells was thawed and centrifuged with a TD5A-WS centrifuge at 1000 rpm, 20-25°C for 5 minutes. The cell pellets were suspended in 0.1-0.5 mL DMEM with 10% PBS, then
transferred to T25 flask. When grown to confluency, they were digested and passaged to T75 and later further split into three T75 flasks. At the time of cell implantation, the cells were collected from the flasks, washed three times with normal saline (NS), and subcutaneously injected into the right axilla of 3 mice (approximately 1 xlO6 or 8xl05 cells/mouse). The day of tumor inoculation was defined as Day 0. When the tumor diameter was approximately 1 cm (measured by vernier caliper), the animals were euthanized with C02 asphyxiation and the tumors excised. Tumor cells were dispersed and suspended in normal saline as
previously described and the cell adaptation cycle was repeated once.
[00130] On Day 1 , the animals were randomized into seven groups based on their body weights so that the mean body weights were not statistically significantly different among groups. Dosing was initiated on Day 1. SCV-07 was administered once daily for 14
consecutive days subcutaneously in a volume of 0.1 mL/20 g body weight, and CTX was administered by intraperitoneal injection every other day using the same dose volume. The vehicle (control) was also administered once daily for 14 consecutive days subcutaneously (0.1 mL/20 g body weight). Treatment regimens for all groups are outlined in Table VIII.
Table VIII. Study Design.
Number
Group Dosing Necropsy
Treatment of Dose/Schedule
Number Days Day
Animals
0
1 Vehicle 10 Days 1-14 Day 17
20 mg/kg, ip, every other day
2 CTX 10 Days 1-14 Day 17
40 mg/kg, ip, every other day
3 CTX 10 Days 1-14 Day 17
5 mg/kg, sc
4 SCV-07 10 Days 1-14 Day 17
10 mg/kg, sc
5 SCV-07 10 Days 1-14 Day 17
SCV-07 + 5 mg/kg, sc + 20 mg/kg, ip, every
6 10 Days 1-14 Day 17
CTX other day
SCV-07 + 10 mg/kg, sc + 20 mg/kg, ip, every
7 10 Days 1-14 Day 17
CTX other day [001 31 ] SCV-07 was administered daily via subcutaneous injection; CTX was administered every other day via intraperitoneal injection. The mice were divided into 7 groups: Group 1 (vehicle), Group 2 (CTX 20 mg/kg), Group 3 (CTX 40 mg/kg), Group 4 (SCV-07 5 mg/kg), Group 5 (SCV-07 10 mg/kg), Group 6 (SCV-07 5 mg/kg plus CTX 20 mg/kg), and Group 7 (SCV-07 10 mg/kg plus CTX 20 mg/kg). Tumor volumes and body weights were measured every three days, and tumor weights were measured on Day 17 (necropsy day) at the end of the study. CTX was aliquoted to 10 mg/vial. PBS was added to achieve the proper dose level as indicated in the study design table (Table VIII). The formulation was kept on ice, protected from light, and used immediately. SCV-07 was dissolved in PBS to achieve the proper dose levels as indicated on the study design table; kept on ice, protected from light, and used within one week.
[001 32] Tumor size was measured using calipers and recorded along with the animal's body weight once every three days throughout the course of the study. Based on the tumor size, the tumor volume (TV) was calculated with the following formula.
TV = (Length x Width x Width) 12
[001 33] Tumor inhibition as reflected by the inhibitory ratio (IR) of TV was calculated according to the equation below.
IR (TV) = (TVvehicle TVdrug treated)/ TVvehicle 100
[001 34] The calculation was performed using an Excel™ spreadsheet. The anti-tumor effect of the drugs also was evaluated by tumor weight (TW). TW for every mouse was measured at the end of the study. Tumor inhibition, as reflected by the inhibitory ratio of TW, was calculated according to the equation below.
IR (TW) = (TWveh!de— TWdmg seated)/ TWvehlcle X 100
[001 35] Mean and standard deviations were calculated using Excel and the student's t test was used for statistical analysis. [00136] SCV-07 showed no evidence of toxicity in this study based on observations of survival and weight change. Daily administration of SCV-07 (5 mg/kg or 10 mg/kg) for 14 days inhibited tumor growth as reflected by the lower tumor weights in these groups than that of the vehicle control group (less than 40%). The combination of SCV-07 (5 or 10 mg/kg) with CTX (20 mg/kg) resulted in higher inhibition in comparison to SCV-07 treatment alone, however, there were no statistically significant differences between any of the combination treatment groups and CTX (20 mg/kg) treatment alone group.
[00137] Tumor measurement data showed that the mean tumor sizes of Group 3 and Group 7 were statistically significantly smaller than that of Group 1 (vehicle control) on Day 6. On Day 9 and Day 12, the mean tumor sizes in all groups except Group 4 and Group 5 were statistically significantly smaller in comparison to that of Group 1. On Day 15, the mean tumor sizes of Group 2, Group 3 and Group 6 were statistically significantly smaller. On Day 17, the mean tumor weights of all treatment groups were statistically significantly lower than that of Group 1. The inhibition calculated based on tumor weight were 55.97% (p<0.01), 90.12% (p<0.01), 30.02% (p<0.01), 28.33% (p<0.01), 59.65% (p<0.01) and 47.78%) (p<0.01), for Group 2, Group 3, Group 4, Group 5, Group 6, and Group 7, respectively. The body weights showed no statistically significant differences between the vehicle control group and any of the treatment groups (i.e., Groups 2-7).
[00138] The calculated tumor inhibition and statistical comparison results of each treatment group versus the vehicle group are listed in Tables IX-XIII. Tumors were not measurable on Day 3. On Day 6, tumors were measurable in all groups except Group 3 (CTX 40 mg/kg) and Group 7 (SCV-07 10 mg/kg + CTX 20 mg/kg). From Day 9 onwards, tumors were measurable in all groups. Based on the calculated tumor volume, all treatment groups except Group 4 (SCV-07 5 mg/kg) and Group 5 (SCV-07 10 mg/kg) showed inhibition (p < 0.05) in comparison to Group 1 (vehicle control group) on Day 9 and Day 12. On Day 15, the mean tumor sizes of Group 2 (CTX 20 mg/kg), Group 3 (CTX 40 mg/kg) and Group 6 (SCV-07 5 mg/kg + CTX 20 mg/kg) were statistically significantly smaller than Group 1. The tumor growth curves are shown in Figure 8.
Table IX. Mean Tumor Volume (cm ) on Day 3.
Figure imgf000036_0001
Table X. Mean Tumor Volume (cm ) on Day 6.
Figure imgf000036_0002
Table XI. Mean Tumor Volume (cm ) on Day 9.
Figure imgf000037_0001
Figure imgf000038_0001
Table XIII. Mean Tumor Volume (cm ) on Day 15.
Figure imgf000038_0002
[00139] Mean tumor weights of all test groups were illustrated in Figure 9. The results of statistical comparison between each of treatment groups and vehicle control group were tabulated in Table XIV. As shown in Table XIV and Figure 9, the mean tumor weights measured on Day 17 of all treatment groups were lower than that of the vehicle control group. The tumor inhibition of Group 2, Group 3, Group 4, Group 5, Group 6, and Group 7 were 55.97% (p<0.01), 90.12% (p<0.01), 30.02% (p<0.01), 28.33% (p<0.01), 59.65% (p<0.01), and 47.78%) (p<0.01), respectively. Although the combination treatment groups (Group 6 and Group 7) showed increased inhibition in comparison to SCV-07 treatment alone, there was no statistically significant difference between any of these combination treatment groups and the group receiving CTX treatment alone, indicating no additive antitumor effect resulting from the combination treatment. Table XIV. Mean Tumor Weight on Day
Figure imgf000039_0001
[00140] Animal growth curves were illustrated in Figure 10. The results of statistical comparison of each treatment group versus the vehicle control group were tabulated in the Tables XV-XX. As shown in these Tables, there were no statistically significant differences between each of the treatment groups and the vehicle control group throughout the course of the study, in terms of body weight.
Table XV. Mean Body Weight on Day 0.
Figure imgf000039_0002
Figure imgf000040_0001
Table XVI. Mean Body Weight on Day 3.
Figure imgf000040_0002
Table XVII. Mean Body Weight on Day 6.
Number
Group of Body Weight
Group Name Treatment IR (BW) P value Number Animals (mean ± SD)
Surviving
Vehicle
1 PBS 10 19.10tl .65
Control
Figure imgf000041_0001
Table XVIII. Mean Body Weight on Day 9.
Figure imgf000041_0002
Table XIX. Mean Body Weight on Day 12.
Figure imgf000042_0001
Table XX. Mean Body Weight on Day 15.
Figure imgf000042_0002
Figure imgf000043_0001
[00141] In conclusion, daily administration of the SCV-07 at 5 mg/kg or 10 mg/kg for 14 days inhibited tumor growth, as reflected by the lower tumor weights measured on Day 17 in comparison to that of the vehicle control group. Inhibition calculated from the tumor weight data were 30.02% and 28.33% for the animals receiving SCV-07 treatment at 5 mg/kg and 10 mg/kg, respectively. The combined use of 5 or 10 mg/kg SCV-07 and 20 mg/kg CTX collectively produced 59.65%> or 47.78%> inhibition of tumor growth, which was not different from the inhibition achieved with 20 mg/kg CTX treatment alone (55.97%).
Example 4. In Vivo Treatment of Lung Cancer Tumors.
[00142] These studies were undertaken to demonstrate the efficacy of SCV-07 in inhibiting tumor growth in a well-established lung cancer model, NCI HI 46 small cell lung cancer in mice. Both monotherapy with SCV-07 and combined chemotherapy with SCV-07 and radiation treatment were investigated.
[00143] HI 46 human lung cancer cells were obtained from ATCC. The cells were grown in DMEM supplemented with 10% fetal calf serum (FCS), 1% penicillin and streptomycin, and 2 mM L-glutamine. Cells were sub-cultured by removing the medium, rinsing twice with sterile calcium- and magnesium-free phosphate buffered saline (PBS) and adding 1 to 2 mL of 0.25% trypsin/ 0.03% EDTA solution. The flask was incubated at 37°C until cells detached. Cells then were sub-cultured at a ratio of 1 :3.
[00144] Female nude mice, homozygous for the nu gene (nu+/nu+), aged 5 to 6 weeks, with a mean pre-treatment body weight of 24 grams were used in the study. Animals were individually numbered using an ear punch, housed in groups of 6 animals per cage, and acclimatized prior to study commencement. During the acclimatization period of at least 2 days, the animals were observed daily in order to reject animals that presented in poor condition. [00145] The study was performed in animal rooms provided with filtered air at a temperature of 70°F+/-5 0 F and 50% +/-20% relative humidity. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour. The room was on an automatic timer for a light/dark cycle of 12 hours on and 12 hours off with no twilight. Sterilized Bed-O- Cobs® bedding was used. Bedding was changed a minimum of once per week. Cages, tops, bottles, etc. were washed with a commercial detergent and allowed to air dry. Prior to use, these items were wrapped and autoclaved. A commercial disinfectant was used to disinfect surfaces and materials introduced into the hood. Floors were swept daily and mopped a minimum of twice weekly with a commercial detergent. Walls and cage racks were sponged a minimum of once per month with a dilute bleach solution. A cage card or label with the appropriate information necessary to identify the study, dose, animal number and treatment group marked all cages. The temperature and relative humidity were recorded during the study, and the records retained. Animals were fed with sterile Labdiet® 5053 (pre-sterilized) rodent chow and sterile water was provided ad libitum.
[00146] Ninety-Six (96) female nude mice (nu/nu) were randomly assigned into 8 treatment groups prior to the initiation of treatment. The animals were identified by an ear punch which corresponded to an individual number. Each mouse was inoculated into their lower left flank with lxl 05 NCI-H146 (HI 46) lung cancer cells in a volume of 0.1 mL with Matrigel™. Treatment began once tumors reached a volume of 75-125 mm3. Tumors were measured once every two days with micro-calipers, and tumor volume was calculated as 4/3πΓ3, where r is equal to the sum of the length and the width divided by 4. The tumor growth index (TGI) was calculated using the formula 100-(Vc* 100/Vt), where Vc is the mean volume of the tumors in the contol group and Vt is the mean volume of the tumors in the test group. Statistical differences between treatment groups were determined using Student's t-test, Mann- Whitney U test and chi-square analysis with a critical value of 0.05.
[00147] To assess possible toxicity, animals were weighed every day and their survival recorded. Any animals exhibiting a loss of >20% of starting weight during the course of the study were euthanized. Any animals in which tumors grew to over 4000 mm3 were euthanized. No animal deaths occurred as a direct result of treatment during the course of this study.
[00148] The groups were treated with vehicle, radiation, SCV-07 or radiation and SCV-07 as detailed in Table XXI. Initiation of drug treatment was designated as Day 1. Mice in groups 1 and 4 received vehicle by subcutaneous (sc) injection for 20 days. Mice in groups 2-4 and 6-8 received SCV-07 in vehicle once a day by subcutaneous injection on days 1 through 20, and mice in groups 6-8 received radiation (2 doses of 4 Gy/dose on days 0 and 2). Radiation was done by anesthetizing the mice in these groups with ketamine (120 mg/kg) and xylazine (6 mg/kg), and placing them under a lead shield such that the region of the flank with tumor was exposed to the radiation. Radiation was delivered using a Philips 160 kV source at a focal distance of approximately 40 cm, and a dose rate of approximately 1.0 Gy/min. Tumors were measured on alternating days throughout the duration of the study. Mice in groups 1-8 were sacrificed on day 21 and remaining tumors were excised, measured, weighed, photographed and fixed in formalin for later analysis.
Table XXI. Lung Cancer In Vivo Treatment Study Design.
Figure imgf000045_0001
[00149] There were no significant differences in mean daily weight changes between vehicle-treated groups and animals who received SCV-07 as a monotherapy (p=0.7) or in animals that received radiation only or SCV-07 in conjunction with radiotherapy (p=0.68). The mean daily percentage weight change for each treatment group is shown in Figures 11 (no radiation) and 12 (plus radiation). The mice receiving vehicle only gained an average of 13.2% of their starting weight by Day 21. Mice treated with either 100 μg/kg, 1.0 mg/kg or 10 mg/kg SCV-07 gained between 10.2% and 12.3% of their starting weight by Day 21. Mice treated with vehicle and exposed to radiation gained an average on 3.2% of their starting weight by Day 21. Mice treated with either 100 μg/kg, 1.0 mg/kg or 10 mg/kg SCV- 07 and were exposed to radiation gained between 2.8% and 3.6%> of their starting weight by Day 21. The significance of these differences was evaluated by calculating the mean area under the curve (AUC) for the percentage weight change for each animal and comparing the groups using a One- Way ANOVA test. The AUC data is shown in Figures 13 (no radiation) and 14 (plus radiation). The AUC was calculated for the percent weight change exhibited by each animal in the study. This calculation was made usign the trapezoidal rule
transformation. Group means were calculated and are shown with error bars representing SEM for each group. Groups were compared using the One- Way ANOVA method; no statistically significant differences in weight change were seen between SCV-07-treated and control groups.
[00150] Tumor volumes were calculated from the length and width measurements taken on alternating days by calculating the mean radius (r), which was the sum of length and width divided by 4, and using the formula 4/3πΓ3 to calculate the volume. The mean tumor volume data is shown in Figures 15 (no radiation) and 16 (plus radiation). Tumors from animals treated with 100 μg/mL grew at rates faster than vehicle control animals. Among the non- irradiated animals, mice treated with 10 mg/kg of SCV-07 showed the best improvement in tumor growth inhibition. The mean tumor volume at the end of the study period for vehicle treated animals was 4436.6 mm2, 4923 mm2 for 100μg/kg SCV-07-treated animals, 4033.4 mm2 for 1 mg/kg SCV-07-treated animals, and 2842.4 mm2 for 10 mg/kg SCV-07-treated animals. Among the irradiated animals, mice treated with 10 mg/kg of SCV-07 showed the best improvement in tumor growth inhibition. The mean tumor volume at the end of the study period for vehicle-treated animals was 1618.5 mm2, 1322.3 mm2 for 100μg/kg SCV- 07-treated animals, 1923.9 mm2 for 1 mg/kg SCV-07-treated animals, and 962.8 mm2 for 10 mg/kg SCV-07-treated animals.
[00151 ] Further analysis of the data was performed by calculating the mean area under the curve (AUC) for the tumor volume for each animal (made using the trapezoidal rule transformation) and comparing the groups using a One-Way ANOVA test. See Figures 17 (no radiation) and 18 (plus radiation). Group means are shown with error bars representing SEM for each group. This analysis did not reveal significant differences between any of the treated groups and the saline control group (p=0.13 for the non-irradiated animals, and p= 0.14 for irradiated animals). However, a direct comparison of treatment with vehicle and lOmg/kg SCV-07 with a Mann- Whitney Rank Sum analysis was significantly different (p = 0.026). In Figure 17, * shows data directly compared to vehicle control animals using a Mann- Whitney rank sum analysis (p=0.026).
[00152] The tumor growth inhibition (TGI) was calculated from the last tumor
measurement using the formula 100-(Vc* 100/Vt), where Vc is the mean volume of the tumors in the contol group and Vt is the mean volume of the tumors in the test group. Table XXII shows the tumor growth inhibition for animals treated with 100 μg/kg, 1 mg/kg, 10 mg/kg SCV-07 alone or in combination with radiation. When compared to unirradiated controls, animals treated with 1 mg/kg SCV-07 alone had a tumor growth inhibition of 9.1%, and animals treated with 10 mg/kg SCV-07 alone had a tumor growth inhibition of 35.9%. Animals treated with radiation alone had a TGI of 63.5% when compared to unirradiated controls, while animals treated with SCV-07 plus radiation had TGI values of 70.2% (100 μg/kg), 50.3%( lmg/kg) and 78.3% (10 mg/kg). When compared to the group receiving radiation plus vehicle the groups treated with radiation plus SCV-07 had a TGI of 18.3 % at 100 μg/kg and 40.5% at 10 mg/kg. See Table XXII. ** indicates that mean tumor volumes in groups 2 and 7 exceeded the vehicle control animals (9.8% and 15.87%), respectively).
Table XXII. Tumor Growth Inhibition (TGI) in Lung Cancer In Vivo Model by SCV-07.
Figure imgf000047_0001
[00153] SCV-07 showed no evidence of toxicity in this study based on observations of survival and weight change. Animals treated with 10 mg/kg SCV-07 alone showed significant reduction in tumor growth inhibition (TGI=68%) compared to vehicle control animals (P=0.026). Animals treated with 100 μg/kg SCV-07 alone or with 1 mg/kg SCV-07 alone showed reductions in tumor growth compared to animals who received vehicle only, but these improvements were not statistically significant. Irradiated animals treated with SCV-07 at 100 μg/kg or 10 mg/kg showed reductions in tumor growth relative to irradiated vehicle control animals, but these differences were not statistically significant.
Example 5. Exemplary Treatment for Cancer and Pre-malignant Conditions (Precancer).
[00154] Assay Description. A cancer patient is administered a STAT3 antagonist agent at a dose of 1000-3000 mg/day for up to 48 weeks. A cancer patient is administered SCV-07 at a dose of 0.1-5.0 mg/kg/day, three times per week, for up to 48 weeks. A patient suffering from a pre-malignant condition is administered a STAT3 antagonist agent at a dose of 1000- 3000 mg/day for up to 48 weeks. A patient suffering from a pre-malignant condition is administered SCV-07 at a dose of 0.1-5.0 mg/kg/day, three times per week, for up to 48 weeks.
Example 6. Screening Utilizing Tumor Cells with Inducibly Expressed STAT3.
[00155] Assay Description. Target Compounds used in the study include the test compound SCV-07 (8-D-Glutamyl-L-Tryptophan-Na; [Ci6Hi8N305Na], MW 355.3) and the control compound (L)Glu(L)Leu.Na+ ([CiiHi9N205Na], MW 282).
[00156] STAT3 inhibitor test by STAT3/luciferase reporter assay. A cell-based assay based on the STAT3/luciferase reporter system (SABiosciences, Cat. #CCS-9028L) is used to test the target compound-mediated inhibitory effect on STAT3 upon IL-6 stimulation in HEK 293T cells.
[00157] STAT3 inhibitor test by STAT3/luciferase reporter assay test procedures. Cells were plated in 24-well plates at 2.5 x 105 cells per well for 16 h. Cells were transiently transfected with STAT3/Luc reporter and incubated for 24 h. Cells were pretreated with the compounds for lh followed by stimulation with 20 ng/ml IL-6 for 6 h. Cells were then lysed with the Passive Lysis Buffer (Promega, Cat. #E1941) and the lysates were collected by centrifugation. Luciferase activity of each sample was measured using the Dual-Luciferase Reporter Assay Substrate System (Promega, Cat. #E1910).
[00158] STAT3 inhibitor test by ST AT3/lucif erase reporter assay. The data were analyzed with Excel as shown in Figure 19. Figure 19 describes inhibitory screening of the compound by the STAT3/luciferase reporter assay. HEK 293T cells were plated in a 24-well plate at 2.5 105 cells/well for 16 h. Cells were transiently transfected with STAT3/LUC reporter for 24 h. Cells were pretreated with 0.01 , 0.1 , 1 , 10 and 50 μ^πιΐ of test (SCV-07) or control (L- Glu-L-Leu) compound for 1 h. Cells were then stimulated with 20 ng/ml IL-6 for 6 h in order to promote inducible expression of the construct. PBS was used as vehicle control (V). Cells were lysed and luciferase activity of each lysate sample was measured. Results are shown as the mean ± SD, and determinations were made in triplicate. Data consist of the assay sets 1 (A) and 2 (B) as well as a positive control (C). Note that curcumin (Imgenex, Cat. #IMG~'2010), which is a known inhibitor for IL-6 inducible STAT3 phosphorylation, was used as a positive STAT3 inhibitor control (C). The vehicle control (V) for curcumin was DMSO.
[00159] SCV-07 inhibited IL-6-mediated STAT3 activation in HEK 293T cells, yielding around 50% inhibition at 10 μg/ml when compared with vehicle control in ST AT3 -based luciferase reporter assay. However, the control compound (L-Glu-L-Leu) also exhibited similar inhibitory effect on STAT3 activity (see, Figure 19).
[00160] STAT3 inhibitor test by Western blot analysis. Western blot analysis was performed to test the target compound-mediated inhibitory effect on STAT3 phosphorylation upon IFN-a stimulation at various time points in Jurkat and THP-1 cells. Western blotting was performed using anti-phospho-STAT3 antibody as well as anti-STAT3 antibody according to the Western standard protocols. The Western data were further qunatitated using TotalLab Quant software (Gentel Biosciences) as shown in Figures 20C and 21C.
[00161 ] STAT3 inhibitor test by Western blot analysis test procedure. Cells were plated in 60 x 15-mm tissue culture dishes at 5 x 106 cells per dish. Cells were pretreated with 0.01 , 0.1 , 1 or 10 μg/ml SCV-07 for 1 h and then stimulated with 25 ng/ml IFN-a for 0, 15, 30, 60 and 120 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels (Invitrogen, Cat.
#EC60255) and transferred to nitrocellulose membranes. The membranes were probed with anti-phospo-STAT3 antibody (Cell Signaling Technology, Cat. #9138). After analysis of phospho-STAT3 (pSTAT3), the membranes were stripped and reprobed with anti-STAT3 antibody (Imgenex, Cat. #IMG-3095) to analyze the cellular level of STAT3 from each sample.
[00162] Figure 20 describes the STAT3 phosphorylation inhibition assay in Jurkat cells. Cells were plated in 60 x 15-mm tissue culture dishes at 5 x 106 cells/dish. Cells were pretreated with 0.01, 0.1, 1 or 10 μg/ml SCV-07 for 1 h, and then stimulated with 25 ng/ml IFN-a for 0, 15, 30, 60 and 120 min in order to inducibly express STAT3. PBS was used as vehicle control. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3 antibody {upper panels of A and B). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A and B). The graph indicates the relative quantitation of pSTAT3 band intensities (C). Note that Piceatannol (EMD Biosciences, Cat. #527948), which selectively inhibits the tyrosine phosphorylation of STAT3, was used as a positive control (B).
[00163] Figure 21 describes the STAT3 phosphorylation inhibition assay in THP-1 cells. Cells were plated in 60 x 15-mm tissue culture dishes at 5 x 106 cells/dish. Cells were pretreated with 0.01, 0.1, 1 or 10 μg/ml SCV-07 for 1 h, and then stimulated with 25 ng/ml IFN-a for 0, 15, 30, 60 and 120 min. PBS was used as vehicle control. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3 antibody (upper panels of A and B). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A and B). The graph indicates the relative quantitation of pSTAT3 band intensities (C). Note that Piceatannol, which selectively inhibits the tyrosine phosphorylation of STAT3, was used as a positive control (B).
[00164] Figure 36 describes the effect of SCV-07 on STAT3 phosphorylation in the human lymphoblastic leukemia, CCRF-CEM, cell line. Cells (5 x 106 cells/sample) were pretreated with 1 and 10 μg/ml SCV-07 for 2 hours and then stimulated with 100 ng/ml IL-4 for 0, 10 and 30 minutes in order to inducibly express STAT3. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to a nitrocellulose membrane. The membrane was probed with anti-pSTAT3Tyr705 antibody (Figure 36A, upper panel). The membrane was then stripped and reprobed with anti-STAT3 antibody (Figure 36A, lower panel). The graph indicates the relative quantitation of pSTAT3xyr705 band intensities (Figure 36B).
[00165] SCV-07 significantly reduced IFN-a-mediated STAT3 phosphorylation in Jurkat cells whereas SCV-07 showed no significant inhibitory effect on STAT3 phosphorylation in THP-1 cell (Figures 20 and 21). SCV-07 also significantly reduced IL-4 mediated STAT3 phosphorylation in CCRF-CEM cells (Figure 36).
Example 7. Screening Utilizing Tumor Cells with Constitutively Active STAT3 or Inducibly Expressed STAT3.
[00166] Assay description. The target compound used in this study included the test compound SCV-07 (8-D-Glutamyl-L-Tryptophan-Na, [Ci6Hi8N305Na], MW 355.3). The compound stock preparation was SCV-07 was dissolved in 1 x phosphate-buffered saline (HyClone, Cat. #SH30264.01) at 5 mg/ml, divided into useable aliquots, and stored at -20°C.
[00167] Effects of SCV-07 on STAT3 phosphorylation in cell lines test procedure. Western blot analysis was performed to test the SCV-07-mediated inhibitory effect on STAT3 phosphorylation upon IFN-a stimulation at various time points in the phorbol 12- myristate 13 -acetate (PMA)-differentiated THP-1 cells as well as NK-92 cells. For THP-1 differentiation, cells were treated with 25 ng/ml PMA for 20 h, and then replaced with fresh medium. (The condition for PMA treatment was previously determined by a pilot experiment.) The differentiated THP-1 or NK-92 cells (5 x 106 cells per each treatment) were pretreated with vehicle (PBS), SCV-07 (0.01, 0.1, 1 and 10 μg/ml) and 10 μg/ml Piceatannol (positive control) for 2 h. Cells were then stimulated with 25 ng/ml IFN-a for 15, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels (Invitrogen, Cat.
#EC60255) and transferred to nitrocellulose membranes. The membranes were probed with anti-phospho-STAT3Tyr705 antibody (Cell Signaling Technology, Cat. #9138). After analysis of phospho-STAT3 (pSTAT3), the membranes were stripped and reprobed with anti-STAT3 antibody (Cell Signaling Technology, Cat. #9139) to analyze the cellular level of STAT3 from each sample. [00168] Effects of SCV-07 on STAT3 phosphorylation in cell lines. Western blotting was performed using anti-phospho-STAT3xyr705 antibody as well as anti-STAT3 antibody according to the Western standard protocols. The Western data were further quantitated using TotalLab Quant software (Gentel Biosciences).
[00169] Figure 22 describes the effect of SCV-07 on STAT3 phosphorylation in differentiated THP-1 cells. Cells were differentiated by treatment of 25 ng/ml PMA for 20 h. The differentiated cells (5 X 106 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 μg/ml SCV-07 for 2 h. Cells were then stimulated with 25 ng/ml IFN-a for 0, 15, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3xyr705 antibody {upper panels of A and B). The membranes were then stripped and reprobed with anti- STAT3 antibody (lower panels of A and B). The graph indicates the relative quantitation of pSTAT3 band intensities (C).
Note that Piceatannol (EMD Biosciences, Cat. #527948), which selectively inhibits the tyrosine phosphorylation of STAT3, was used as a positive control at 10 μg/ml (B).
[00170] Figure 31 also describes the effects of SCV-07 on STAT3 phosphorylation in differentiated THP-1 cells. Cells were differentiated by treatment of 25 ng/ml PMA for 20 h. The differentiated cells (5 x 106 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 μg/ml SCV-07 for 2 h. Cells were then stimulated with 25 ng/ml IFN-a for 0, 15, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3xyr705 antibody (upper panels of A and B). The membranes were then stripped and reprobed with anti- STAT3 antibody (lower panels of A and B). (See, also Figures 22A and 22B.)
[00171 ] Figure 23 describes the effect of SCV-07 on STAT3 phosphorylation in NK-92 cells. Cells (5 x 106 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 μg/ml SCV-07 for 2 h. Cells were then stimulated with 25 ng/ml IFN-a for 0, 15, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose
membranes. The membranes were probed with anti-pSTAT3Tyr705 antibody (upper panels of A and B). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A and B). The graph indicates the relative quantitation of pSTAT3 band intensities (C). Note that Piceatannol (EMD Biosciences, Cat. #527948), which selectively inhibits the tyrosine phosphorylation of STAT3, was used as a positive control at 10 μg/ml (B).
[00172] Figure 32 also describes the effect of SCV-07 on STAT3 phosphorylation in NK- 92 cells. Cells (5 106 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 μ^πιΐ SCV-07 for 2 h. Cells were then stimulated with 25 ng/ml IFN-a for 0, 15, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose
membranes. The membranes were probed with anti-pSTAT3Tyr705 antibody (upper panels of A and B). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A and B). (See, also Figures 23 A and 23B.)
[00173] SCV-07 did not specifically reduce IFN-a-induced STAT3 phosphorylation in PMA-differentiated THP-1 cells (Figures 22 and 31). SCV-07 showed no significant inhibitory effect on STAT3 phosphorylation in NK-92 cells; instead, SCV-07 somewhat enhanced IFN-a-induced STAT3 phosphorylation in NK-92 cells (Figures 23 and 32).
[00174] Effects of SCV-07 on constitutive STAT3 phosphorylation on a set of tumor cell lines test description. Western blot analysis was performed to test the SCV-07- mediated inhibitory effect on constitutive STAT3 phosphorylation in various tumor cell lines. The five cell lines were obtained from ATCC as described in Table XXIII. Western blotting was performed using anti-phospho-STAT3Tyr705 antibody as well as anti-STAT3 antibody according to the Western standard protocols (Figure 24). The Western data were further quantitated using TotalLab Quant software (Gentel Biosciences).
Table XXIII. The five tumor cell lines used in the study.
Figure imgf000053_0001
[00175] Test procedures. Cells were plated in 60 x 15-mm tissue culture dishes at 5 x 106 cells per dish. Cells were pretreated with vehicle (PBS), SCV-07 (0.01, 0.1, 1 and 10 μg/ml) and 10 ug/ml Piceatannol (positive control) for 2 h. Cells were then lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4- 20% Tris-Glycine gels (Invitrogen, Cat. #EC60255) and transferred to nitrocellulose membranes. The membranes were probed with anti-phospho-STAT3xyr705 antibody (Cell Signaling Technology, Cat. #9138). After analysis of pSTAT3, the membranes were stripped and reprobed with anti-STAT3 antibody (Cell Signaling Technology, Cat. #9139) to analyze the cellular level of STAT3 from each sample.
[00176] Figure 24 describes the effect of SCV-07 on STAT3 phosphorylation in various tumor cell lines. Cells (5 x 106 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 μg/ml SCV-07 for 2 h. Cells were then lysed with RIPA lysis buffer containing
protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti- pSTAT3xyr705 antibody {upper panels of A). The membranes were then stripped and reprobed with anti-STAT3 antibody {lower panels of A). The graph indicates the relative quantitation of pSTAT3 band intensities (B). Note that Piceatannol (EMD Biosciences, Cat. #527948), which selectively inhibits the tyrosine phosphorylation of STAT3, was used as a positive control at 10 μg/ml (A). V, PBS vehicle; P, piceatannol.
[00177] Figure 33 also describes the effect of SCV-07 on STAT3 phosphorylation in various tumor cell lines. Cells (5 x 106 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 μg/ml SCV-07 for 2 h. Cells were then lysed with RIPA lysis buffer containing
protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti- pSTAT3xyr705 antibody {upper panels). The membranes were then stripped and reprobed with anti-STAT3 antibody {lower panels). The graph indicates the relative quantitation of pSTAT3 band intensities (B). V, PBS vehicle; P, piceatannol. (See, also Figure 24A.)
[00178] Figure 35 shows that SCV-07 does not inhibit constitutive tyrosine
phosphorylation of STAT3 in a variety of tumor cell lines. Cells (5 x 106 cells/sample) were pretreated with 0.01, 0.1, 1 and 10 μ^ιηΐ SCV-07 for 2 hours. Cells were then lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3xyr705 antibody (Figure 35A, upper panels). The membranes were then stripped and reprobed with anti-STAT3 antibody (Figure 35A, lower panels). The graph indicates the relative quantitation of pSTAT3 band intensities (Figure 35B). Note that Piceatannol (EMD Biosciences, Cat. #527948), which selectively inhibits the tyrosine phosphorylation of STAT3, was used as a positive control at 10 μ§/ηι1. V, PBS vehicle; P, piceatannol.
[00179] When tested with five tumor cell lines, SCV-07 only slightly suppressed the constitutive phosphorylation of STAT3 in Kasumi-1, U266 and HepG2 cell lines (See, Figures 24, 33 and 35).
[00180] Effects of SCV-07 on pervanadate-treated cells test description. Western blot analysis was performed with pervanadate-treated Jurkat and Kasumi-1 cells to determine whether SCV-07 effects on STAT3 phosphorylation are due to tyrosine phosphatase activity.
[00181] Test procedures. Cells were plated in 60 x 15-mm tissue culture dishes at 5 x 106 cells per dish. Cells were pretreated with 50 μΜ pervanadate for 4 h. After treatment of pervanadate, cells were retained without replacing media until the end of SCV-07 treatment and IFN-a stimulation. For Jurkat, the pervanadate-treated cells as well as untreated control cells were further incubated with 1 and 10 μg/ml SCV-07 for 2 h, and then stimulated with 25 ng/ml IFN-a for 0, 15, 30, 60 and 120 minutes in order to inducibly express STAT3. For Kasumi-1, the pervanadate-treated cells as well as untreated control cells were further incubated with 0.01, 0.1, 1 and 10 μ^πιΐ SCV-07 for 2 h. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4- 20% Tris-Glycine gels (Invitrogen, Cat. #EC60255) and transferred to nitrocellulose membranes. The membranes were probed with anti-phospho-STAT3Tyr705 antibody (Cell Signaling Technology, Cat. #9138). After analysis of pSTAT3, the membranes were stripped and reprobed with anti-STAT3 antibody (Cell Signaling Technology, Cat. #9139) to analyze the cellular level of STAT3 from each sample.
[00182] Effects of SCV-07 on pervanadate treated cells. Western blotting was performed using anti-phospho-STAT3xyr705 antibody as well as anti-STAT3 antibody according to the Western standard protocols (Figures 25 and 26). The Western data were further quantitated using TotalLab Quant software (Gentel Biosciences).
[00183] Figure 25 also describes the effect of SCV-07 on STAT3 phosphorylation in
Jurkat cells treated with pervanadate. Cells were pretreated with 50 μΜ pervanadate for 4 h.
The pervanadate-treated and untreated control cells (5 x 106 cells/sample) were further treated with 1 and 10 μg/ml SCV-07 for 2 h, and then stimulated with 25 ng/ml IFN-a for 0, 15, 30,
60 and 120 minutes in order to inducibly express STAT3. Cells were then lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3xyr705 antibody {upper panels of A and B). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A and B). The graph indicates the relative quantitation of pSTAT3 band intensities (B and C). A and C: pervanadate -treated cells; B and D: untreated control cells.
[00184] Figure 26 describes the effect of SCV-07 on STAT3 phosphorylation in Kasumi-1 cells treated with pervanadate. Cells were pretreated with 50 μΜ pervanadate for 4 h. The pervanadate-treated and untreated control cells (5 x 106 cells/sample) were further treated with 0.01, 0.1, 1 and 10 μg/ml SCV-07 for 2 h. Cells were then lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3Tyr705 antibody (upper panels of A). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels of A). The graph indicates the relative quantitation of pSTAT3 band intensities (B). Note that Piceatannol (EMD Biosciences, Cat. #527948), which selectively inhibits the tyrosine phosphorylation of STAT3, was used as a positive control at 10 μg/ml (A). Pic, piceatannol. Note that total protein concentration of each sample was measured by Bio-Rad Protein Assay (Bio-Rad) to confirm that equal amounts of total proteins were analyzed.
[00185] Figure 34 also describes the effect of SCV-07 on STAT3 phosphorylation in Jurkat cells treated with pervanadate. Cells were pretreated with 50 μΜ pervanadate for 4 h. The pervanadate treated cells (5 x 106 cells/sample) were further treated with 1 and 10 μg/ml SCV-07 for 2 h, and then stimulated with 25 ng/ml IFN-a for 0, 15, 30, 60 and 120 minutes. Cells were then lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3xyr705 antibody (upper panels). The membranes were then stripped and reprobed with anti-STAT3 antibody (lower panels). (See Figure 25 A.)
[00186] SCV-07 somewhat reversed the pervanadate activity that enhanced both IFN-a- induced and constitutive STAT3 phosphorylation, respectively, in Jurkat and Kasumi-1 cells; suggesting that tyrosine phosphatases are involved in SCV-07-mediated inhibition of STAT3 activation (see, Figures 25, 26 and 34). [00187] Effects of SCV-07 on kinase phosphorylation profiles test description. The phosphorylation profiles of kinases in Jurkat cells when stimulated with IFN-a in the presence or absence of SCV-07 were analyzed using the Human Phospho-kinase Array Kit (R & D Systems, Cat. #ARY003).
[00188] Test procedures. Jurkat cells were plated in 60 x 15 -mm tissue culture dishes at 1 x 107 cells per dish. Cells were pretreated with vehicle (PBS) and SCV-07 (1 and 10 μg/ml) for 2 h. Cells were then stimulated with 25 ng/ml IFN-a for 30 minutes in order to inducibly express STAT3. (The time point of 30 min was previously determined by Western analysis.) Two cell samples, which were treated only with vehicle and 10 μg/ml SCV-07 for 30 minutes, were also prepared. Cells were lysed with the Array Lysis Buffer (R & D Systems). Each lysate sample of 400 μg total proteins was incubated with each array membrane for 16 h at 4°C, and further processed according to the manufacturer's instructions (R & D Systems). The positive signals detected on the developed X-ray film were analyzed using TotalLab Quant software (Gentel Biosciences).
[00189] Effects of SCV-07 on kinase phosphorylation profiles. Kinase array test was performed using the Human Phospho-kinase Array Kit (R & D Systems) according to the manufacturer's instructions (Figures 55, 56, 57 and 58, Table XXIV). The array data were further analyzed using TotalLab Quant software (Gentel Biosciences).
[00190] Figure 27 describes effects of SCV-07 on various kinase phosphorylation in Jurkat cells. Cells were plated in 60 x 15 -mm tissue culture dishes at 1 χ 107 cells/dish. Cells were pretreated with vehicle and SCV-07 (1 and 10 μg/ml) for 2 h, and then stimulated with 25 ng/ml IFN-a or vehicle control for 30 min. IFN-a was added in order to inducibly express STAT3. Two cell samples treated only with vehicle and 10 μg/ml SCV-07 for 30 min were also prepared (as indicated with asterisks in panel A). Cells were lysed with the Aray Lysis Buffer (R & D Systems), and each lysate sample of 400 μg total proteins was used for array tests. Array images are shown in panel A. The coordinated kinase position information is described in panel B.
[00191] Figure 28 describes quantitation data for the human phospho-kinase array tests. Array signals from scanned X-ray film images (as shown in Figure 29A) were analyzed using the TotalLab Quant software (Gentel Biosciences).
[00192] Figure 29 shows normalized quantitation data for the human phospho-kinase array tests (Figure 27A, Non-activated Jurkat cells). The quantitation values for the SCV-07 (1 μ /ηι1)/-ΙΡΝ-α and SCV-07 (10 μ /ηι1)/-ΙΡΝ-α samples were normalized by the Vehicle/- IFN-a sample values. Note that value 1 is a basal level (no change). >1, fold increase; <1, fold decrease.
[00193] Figure 30 shows normalized quantitation data for the human phospho-kinase array tests (Fig. 27A, IFN-a-activated Jurkat cells). The quantitation values for the SCV-07 (1 μg/ml)/+IFN- and SCV-07 (10 μg/ml)/+IFN- samples were normalized by the
Vehicle/+IFN-a sample values. Note that value 1 is a basal level (no change). >1, fold increase; <1, fold decrease.
Table XXIV. Phosphorylation patterns of different human phospho-kinases affected by
SCV-07.
Figure imgf000058_0001
[00194] The human phospho-kinase assay revealed that SCV-07 not only suppresses pSTAT3Tyr705 but also significantly inhibits pSTAT5(a & b)Tyr699 and pSTATlTyr7oi, while it somewhat enhances pSTAT4Tyr693. Interestingly, SCV-07 constantly enhanced IFN-a- induced activation of p53seris, p53ser46 and p53ser392- See Table XXIV for the regulation patterns of all other kinases. (See, Figures 27, 28, 29, 30 and Table XXIV.)
Example 8. The peptide immunomodulator SCV-07 requires phosphatase activity for inhibition of STAT3 signaling. [00195] Introduction. The novel immunomodulatory peptide, gamma-D-glutamyl-L- tryptophan (SCV-07) is being evaluated as an intervention for oral mucositis in radiation treatment of head and neck cancer (Adkins 2010), and has also been shown in various xenograft cancer models to decrease tumor growth and improve survival (Tuthill 2009). Preliminary evaluation of the mechanism of action of SCV-07 demonstrated inhibition of IL- 6 dependent STAT3 signaling in B16 melanoma cells (Nagabhushanam 2008, Tuthill 2009). The objective of the current mechanistic studies was to understand further details of the involvement of SCV-07 in the STAT3 signaling pathway.
[00196] Experimental procedures. Western blot analysis was performed to test the target compound-mediated inhibitory effect on STAT3 phosphorylation after IFN-a stimulation at various time points in Jurkat, THP- 1 , and NK-92 cells. Cells were treated with 0.01, 0.1, 1.0 or 10 μg/ml SCV-07 for 1 or 2 hr, and then stimulated with 25 ng/ml IFN-a for 0, 15, 30, 60 or 120 min. Jurkat cells were also tested after a 4-hr pretreatment with the tyrosine phosphatase inhibitor pervanadate at 50 uM. PBS was used as vehicle control. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glysine gels and transferred to nitrocellulose membranes and probed with anti-pSTAT3Tyr705 antibody. The membranes were then stripped and reprobed with anti-STAT3 antibody to analyze the cellular level of STAT3 from each sample. The phosphorylation profile of kinases in Jurkat cells stimulated with IFN-a in order to inducibly express STAT3 in the presence or absence of SCV-07 were analyzed using the Human Phosphokinase Array Kit, using 400 μg total protein from cells lysed with the Array Lysis Buffer.
[00197] Summary of Results. SCV-07 significantly reduced IFN-a-mediated ST AT3 phosphorylation in Jurkat T cells, whereas SCV-07 showed no significant inhibitory effect on STAT3 phosphorylation in THP-1 cells, THP-1 cells differentiated by treatment with 25 ng/ml PMA for 20 h, or NK-92 cells. The addition of pervanadate blocked the ability of SCV-07 to reduce STAT3 phosphorylation in Jurkat cells. The human phosphokinase assay revealed that SCV-07 not only suppresses pSTAT3Tyr705 but also significantly inhibits pSTAT5(a & b)Tyr699 and pSTATlTyr7oi , while it somewhat enhances pSTAT4Tyr693.
Interestingly, SCV-07 enhances IFN-a-induced activation of p53seris, p53ser46 and p53ser392- [00198] Conclusions. The inhibition of IFN-a induced STAT3 signaling pathway with SCV-07, seen in a T cell line but not a macrophage or NK cell line, requires the presence of phosphatase activity.
Example 9. The Effect of the Immunomodulatory Peptide Gamma-D-Glutamyl-L- Tryptophan in Leukemia, Lymphoma, and Head and Neck Cancer Xenograft Models.
[00199] Introduction. SCV-07 (γ-D-glutamyl-L-tryptophan) is a synthetic dipeptide which has been shown to stimulate the immune system. SCV-07 is effective in various preclinical models in which immune stimulation would be expected to be helpful, including vaccine enhancement (Tuthill, et al., Cold Spring Harbor, Harnessing Immunity to Prevent and Treat Disease (2009)), asthma (Regal, et al., Society of Toxicology abstracts, #1502 (2009)), and infectious disease (Mossel, et al., (2005) Microbes in a Changing World IUMS meeting, San Francisco, CA (2005); and Rose, et al., International Journal of Antimicrobial Agents 32: 262-266 (2008)).
[00200] Recently SCV-07 demonstrated efficacy in attenuating acute and fractionated radiation-induced mucosal injury in the clinically predictive hamster model (Watkins, et al., Oral Diseases, 16: 655-60 (2010)), an effect which was noted to be dose and schedule dependent. SCV-07 has demonstrated a broad spectrum of immune stimulation, enhancing the Thl-type immune response and increasing antigen-specific T cell responses (Simbirtsev, et al., Russian Journal of Immunology 8: 11-22 (2003)). The efficacy of SCV-07 in preventing radiation-induced oral mucositis is expected to arise from the drug's ability to stimulate the Thl type immune response and to block signal transducers and activator of transcription 3 (STAT3) mediated signaling (Tuthill, et al, AACR-NCI-EORTC Molecular Targets and Cancer Therapeutics (2009); and Papkoff, et al., AACR Tumor Immunology: Basic and Clinical Advances, Miami Beach, FL (2010)).
[00201 ] SCV-07 was further investigated in a phase 2a clinical trial, in which the compound demonstrated a trend toward delay of onset of ulcerative (World Health
Organization [WHO] Grade > 2) and severe (WHO Grade > 3) OM in subjects being treated with chemoradiation (ChemoRT) therapy for HNC (Adkins, et al., (2010) Journal of Clinical
Oncology 28: suppl abstr el 9693 (2010); and Adkins, et al., International Society for
Bioloeical Therapv of Cancer 25th Annual Meeting, Washington, DC (2010)). A phase 2b study in this indication has recently begun and is currently enrolling patients (ClinicalTrials.gov identifier: NCT01247246).
[00202] A challenge for agents which target regimen-related toxicities is to provide clinical benefit without detracting from the anti-tumor effects of the cancer therapy. Results of animal studies have shown that SCV-07 decreased tumor growth in various xenograft models in melanoma (Tuthill, et al. , Cold Spring Harbor, Harnessing Immunity to Prevent and Treat Disease (2009)) and lung cancer (Zou, et al., Journal of Clinical Oncology 26: May 20 suppl; abstract 14012 (2008)) and did not alter the tumoricidal effect of radiation (data on file). In this study, we further describe SCV-07's anti-tumor efficacy.
[00203] Methods. Female mice aged 5 - 6 weeks were individually numbered using an ear punch and housed in groups of 10 animals per cage. Animals were acclimatized for at least 3 days and only those in good condition were used for the study.
[00204] Tumor cell lines from ATCC were grown in medium supplemented with 10% Fetal Calf Serum (FCS), 1% penicillin and streptomycin, and 2 mM L-Glutamine and sub- cultured by dilution at a ratio of 1 :3.
[00205] Mice were inoculated with tumor cells subcutaneously in the left flank, and when tumors were established, mice were randomly and prospectively divided into treatment groups prior to the initiation of daily treatment with either vehicle or SCV-07 of various doses.
[00206] All groups were treated with a volume of 0.1 mL, and the concentrations for dosing solutions were based on mean group weights on the day that drug solutions were prepared for each week. All SCV-07 solutions were made fresh at the beginning of each week, and stored at 4°C protected from light.
[00207] Tumors were measured once every two days with micro-calipers, and tumor volume was calculated as (length x width x width)/2. The tumor growth index (TGI) was calculated using the formula 100-(Vc* 100/Vt), where Vc is the mean volume of the tumors in the control group and Vt is the mean volume of the tumors in the test group.
[00208] All animals were weighed daily in order to assess differences between groups as an indication of possible toxicity resulting from the treatments. Any animals exhibiting a loss of >20% of starting weight during the course of the study or whose tumor grew to over 1,500 mm3 were euthanized and survival was recorded daily. [00209] Statistical differences in tumor growth between treatment groups were determined using Mann- Whitney Rank Sum or ANOVA tests with a critical value of 0.05.
Table XXV. Experimental information for cell lines used in Example 9.
Figure imgf000062_0001
[00210] For analysis of effects in culture, MOLT-4 cells were plated in 60 x 15 -mm tissue culture dishes at 5 x 106 cells per dish. Cells were pretreated with vehicle (PBS) or SCV-07 (1 and 10 μg/mL) for 2 hours and then stimulated with 25 ng/mL IFNa, 200 ng/mL IL-6 or 100 ng/mL IL-10 for 0, 10, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice, run on 4-20% Tris-Glycine gels (Invitrogen, Cat. #EC60255), and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3Tyr705 (Cell Signaling Technology, Cat. #9138). After analysis of pSTAT3, the membranes were stripped and reprobed with anti-STAT3 antibody (Cell Signaling Technology, Cat. #9139).
[00211] Results. SCV-07 was safe and well tolerated: no animal deaths occurred as a direct result of SCV-07 treatment and no statistically significant differences in weight gain were seen between the SCV-07-treated and control groups.
[00212] Tumors in SCV-07-treated animals grew more slowly than those in control animals; the reduction was generally dose-dependent and statistically significant at higher doses: MOLT-4: 10 mg/kg (p=0.003), 20 mg/kg (p=0.002), 40 mg/kg (p<0.001) (Figure 37)
HL-60: 20 mg/kg (p=0.025), 40 mg/kg (p=0.005) (Figure 38)
EL-4: 20 mg/kg (p=0.001), 40 mg/kg (p<0.001) (Figure 39)
FaDu: 20 mg/kg (p=0.004), 40 mg/kg (p=0.004) (Figure 40)
SK-MEL: 20 mg/kg (p=0.02) (Figure 41)
[00213] SCV-07 did not inhibit m vz'vo proliferation of any tumor cell lines tested (NCI60; data on file). SCV-07 was shown to inhibit cytokine-induced STAT3 tyrosine
phosphorylation (PY) in some human tumor cell lines in culture, all of myeloid or T cell lineage. MOLT-4, used for the xenograft studies, showed SCV-07 inhibition of STAT3 phosphorylation (Figure 42).
[00214] Conclusions. The immune modulating peptide SCV-07 is able to inhibit growth of a variety of tumor cell line types, including a human head and neck tumor line in nude mice. These results suggest that the use of SCV-07 as an intervention for mucositis should not interfere with therapy, but may be useful in enhancing the tumor response to conventional therapy. SCV-07 may also be useful alone for the treatment of tumors with inducible STAT3.
Example 10. Identification of Signaling Pathways Involved in the Mechanism of Action of the Immunomodulatory Peptide Gamma-D-Glutamyl-L-Tryptophan.
[00215] Introduction. Gamma-D-glutamyl-L-tryptophan (SCV-07) is an
immunomodulatory peptide with well documented stimulatory activity in vaccine and anti- infective settings. SCV-07 is currently under clinical investigation for attenuation of oral mucositis in FiNSCC patients receiving chemoradiation therapy. Based on its
immunomodulatory properties, we wanted to evaluate whether SCV-07 could also have antitumor activity. In xenograft efficacy experiments, SCV-07 decreased tumor growth of a variety of murine and human cancer cell lines. In this study we explored further the molecular and cell biological mechanisms by which SCV-07 interferes with mucositis pathobiology and concurrently exhibits anti-tumor activity. These studies have focused on validating monocyte and T cell types as key target cells and broadly explored changes in phosphorylation of signaling proteins, particularly including STAT3. Our data suggest a model where SCV-07, via SHP-2 activation, STAT3 inhibition and changes in cytokine production could shift macrophage and T cell subsets to promote an immune regulatory environment that inhibits both mucositis and tumor progression.
[00216] Effects of SCV-07 on tumor growth. Reproducible and statistically significant anti-tumor efficacy of SCV-07 demonstrated in B16F0 and other xenograft models. SCV-07 does not inhibit proliferation of B16F0 or other cell lines in culture.
[00217] Inhibition of Cytokine-Induced STAT3 PY in Cells of Macrophage and T Cell Lineage. SCV-07 significantly inhibited IL-10 and IFNa induced STAT3 PY in Jurkat cells which was blockerd by pervanadate treatment, implicating tyrosine phosphatase activity. Phosphatase CD45 was not required since STAT3 PY inhibition was observed in CD45 mutant Jurkat cells. However, SCV-07 induced activating phosphorylation of the
phosphatase SHP-2 and altered phosphorylation of Src family kinases and STAT5a/b, known substrates for SHP-2. SCV-07 also decreased IFNa stimulated phosphorylation of SHP-2 associated immunoreceptors Siglec3 (CD33) and SIRPbetal (CD172B).
Table XXVI. Summary of SCV-07 effects on cytokine-induced STA T3 PY in cell culture (no inhibition of proliferation)
Figure imgf000065_0001
* No cells tested by NCI60 showed significant growth inhibition
** SCV-07 inhibits growth in xenograft studies
[00218] Potential role for SHP-2 in SCV-07 signal transduction. SCV-07 treatment leads to increased activation and tyrosine phosphorylation of SHP-2. SHP-2
dephosphorylates STAT3 and STAT5. As such, SCV-07 leads to decreased phosphorylation of STAT3 and STAT5.
[00219] SHP-2 also increases Src-family kinase activity via dephosphoryation of the regulatory tyrosine. SCV-07 leads to increased phosphorylation of various Src family kinases (potentially autophosphoryation). [00220] Summary of Results. SCV-07 has anti-tumor efficacy with human and murine tumor cell line xenograft models (see Example 9). Inhibition of cytokine -induced, but not constitutive, STAT3 protein tyrosine 705 phosphorylation (STAT3 PY) in some monocyte and T cell tumor lines. While not a direct inhibitor of STAT3, SCV-07 does lead to dose- dependent inhibition of IL-6-induced STAT3 reporter gene activity in transfected HEK-293T cells.
[00221] We also obtained additional mechanism of action (MO A) information using in Jurkat cells. Inhibition of STAT3 PY requires tyrosine phosphatase activity. These data implicate SHP-2 but not CD45 as having a role in SCV-07 mechanism of action. SCV-07 also inhibits STAT5a/b PY and may possibly inhibit other STATs. SCV-07 may also lead to some inhibition of Jakl and Tyk2 phosphorylation. Changes in phosphorylation of other cytoplasmic and membrane proteins may also be induced by SCV-07.
[00222] Conclusions. SCV-07 activates a pathway that leads to SHP-2 activation and STAT3 and STAT5 de-phosphorylation. No in vitro inhibition of proliferation was observed for any of the tumor cell lines treated with SCV-07.
[00223] Macrophages and T cells are likely target cells for SCV-07 efficacy. SCV-07 binds directly with high affinity to mouse macrophages and thymocytes. In vitro effects of SCV-07 on STAT3 PY are observed in cell lines of T cell and monocyte origin. Efficacy observed for multiple tumor cell types was observed in both syngeneic and nude mice.
Effects of SCV-07 on tumor growth were seen even in cell lines showing no anti-proliferative effects or inhibition of STAT3 PY in culture (in vitro).
[00224] SCV-07 may mediate anti-tumor activity by creating an environment that inhibits tumor progression through reducing chemotaxis of tumor-growth promoting macrophages, secretion of tumor growth-promoting cytokines in the tumor microenvironment, promoting the shift from M2 to Ml profile in macrophage cells, and/or promoting the shift from Th2 to Thl profile of local T cells. Further experimentation will be required to define the "SCV-07 receptor", other signaling molecules in SCV-07 pathway, and to explore additional cell biological consequences of SCV-07 action. Example 11. Study of Tumor Growth in MOLT-4 Lymphoblastic Leukemia Model.
[00225] Introduction. In this study, the impact of SCV-07 on tumor growth was tested using the Molt-4 adult lymphoblastic leukemia xenograft model in nude mice. Tumor bearing mice were treated with saline or SCV-07 once daily for forty-three days. SCV-07 showed no evidence of toxicity in this study based on observations of survival and weight change. SCV-07 reduced Molt-4 tumor growth in a dose dependant manner, with statistically significant reductions seen in groups treated with SCV-07 at 10 mg/kg (p=0.002), 20 mg/kg daily (p=0.002) and 40 mg/kg daily (p<0.001) at the point that the first animal was sacrificed on Day 25. In addition, animals treated with SCV-07 daily at 1, 10, 20 and 40 mg/kg showed increased survival relative to the vehicle control group (p=0.029).
[00226] Methods & Results. The objective of this study was to evaluate the effect of SCV-07 on the growth of Molt-4 tumors in immune-deficient mice.
[00227] Eighty (80) nude mice (nu/nu) were randomly and prospectively divided into four groups of ten mice and two groups of twenty mice. Mice were inoculated s.c. in the left flank with MOLT-4 tumor cells, at an inoculum of 5 x 106 cells per mouse. Animals were treated with vehicle or SCV-07 as shown in Table XXVII. Each animal's weights and condition were recorded daily and tumors were measured three times per week.
Table XXVII: Study Groups - Main Study
Figure imgf000067_0001
* Ten mice per group in groups 1 and 6 were sacrificed on Day 33 for cytokine analysis
[00228] Animals. Female nude mice (Taconic Labs), aged 5 to 6 weeks, with a mean pre- treatment body weight 30.5 grams were used. Animals were individually numbered using an ear punch and housed in groups of 10 animals per cage. Animals were acclimatized prior to tumor implantation. During this period of at least 3 days, the animals were observed daily in order to reject animals that presented in poor condition.
[00229] Housing. The study was performed in animal rooms provided with filtered air at a temperature of 70°F+/-5° F and 50% +/-20% relative humidity. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour. The room was on an automatic timer for a light/dark cycle of 12 hours on and 12 hours off with no twilight.
[00230] Sterilized Bed-O-Cobs® bedding was used. Bedding was changed a minimum of once per week.
[00231] Cages, tops, bottles, etc. were washed with a commercial detergent and allowed to air dry. Prior to use, these items were wrapped and autoclaved. A commercial disinfectant was used to disinfect surfaces and materials introduced into the hood. Floors were swept daily and mopped a minimum of twice weekly with a commercial detergent. Walls and cage racks were sponged a minimum of once per month with a dilute bleach solution. A cage card or label with the appropriate information necessary to identify the study, dose, animal number and treatment group marked all cages. The temperature and relative humidity were recorded during the study, and the records retained.
[00232] Diet. Animals were fed with sterile Labdiet® 5053 (pre-sterilized) rodent chow and sterile water was provided ad libitum.
[00233] Animal Randomization and Allocations. Mice were randomly and prospectively divided into four (4) groups prior to the initiation of treatment. Each animal was identified by ear punching corresponding to an individual number. A cage card was used to identify each cage and marked with the study number, treatment group number and animal numbers.
[00234] Assessment of Results. Statistical differences between treatment groups were determined using Mann- Whitney Rank Sum or ANOVA tests with a critical value of 0.05.
[00235] Experimental Procedures. Tumors were measured once every two days with micro-calipers, and tumor volume was calculated as (length x width x width)/2. The tumor growth index (TGI) was calculated using the formula 100-(Vc* 100/Vt), where Vc is the mean volume of the tumors in the control group and Vt is the mean volume of the tumors in the test group.
[00236] Tissue Culture. MOLT-4 (ATCC # CRL-1852) adult lymphoblastic leukemia (ATL) cells were obtained from ATCC. These cells were grown in RPMI-1640 medium supplemented with 10% Fetal Calf Serum (FCS), 1% penicillin and streptomycin, and 2mM L-Glutamine. Cells were sub-cultured by dilution at a ratio of 1 :3.
[00237] Weights and Survival. All animals were weighed every day in order to assess possible differences in animal weight among treatment groups as an indication of possible toxicity resulting from the treatments. Any animals exhibiting a loss of >20% of starting weight during the course of the study were euthanized. Any animals whose tumor grew to over 1500 mm3 were also euthanized. Survival was recorded daily.
[00238] Test Article Preparation. SCV-07 dosing solutions were prepared freshly each week. All groups were treated with a volume of 0.1 mL, and the concentration for dosing solutions were based on mean group weights on the day that drug dosing solutions are prepared for each week. For example, for mice with a mean weight of 25 g and dosed at 40 mg/kg, a 10 mg/mL dosing solution was prepared by dissolving 80 mg of SCV-07 in 8 mL of PBS. For the 20 mg/kg group, dosing solution was prepared by diluting the 10 mg/mL solution 1 :2 in PBS (5 mg/mL). For the 10 mg/kg group, dosing solution was prepared by diluting the 5 mg/mL solution 1 :2 in PBS (2.5 mg/mL). For the 5 mg/kg group, dosing solution was prepared by diluting the 10 mg/mL solution 1 :2 in PBS (1.25 mg/mL). For the 1 mg/kg group, dosing solution was prepared by diluting the 5 mg/mL solution 1 :5 in PBS (0.25 mg/mL). All dosing solutions were made freshly at the beginning of each week of dosing, and stored at 4°C protected from light.
[00239] Survival was recorded (see, Figure 51). No animal deaths occurred as a direct result of treatment during the course of this study. Five of the animals on this study were euthanized because the tumor reached maximal acceptable volume; the first of these on Day 36. The remaining animals reached the final scheduled timepoint (Day 43). The survival data are shown in Figure 51. The significance of the differences observed was evaluated using a Kaplan-Meier log rank test, and statistically significant differences were seen between the vehicle control group and the groups treated with SCV-07 at 1, 10, 20 and 40 mg/kg (p=0.029 for all comparisons). The difference between the vehicle control group and the group treated with SCV-07 at 5 mg/kg was not statistically significant.
[00240] Animal Weight was recorded (see, Figures 52 and 53). The mean daily percentage weight changes for each treatment group are shown in Figure 52. The mice receiving vehicle had a mean gain of 4.3% of their starting weight at the end of the study. Mice treated with SCV-07 at 1.0 mg/kg had a mean gain of 7.5% of their starting weight at the end of the study. Mice treated with SCV-07 at 5.0 mg/kg had a mean gain of 9.9% of their starting weight at the end of the study. Mice treated with SCV-07 at 10.0 mg/kg had a mean gain of 2.7% of their starting weight at the end of the study. Mice treated with SCV-07 at 20.0 mg/kg had a mean gain of 7.9% of their starting weight at the end of the study. Mice treated with SCV-07 at 40.0 mg/kg had a mean gain of 8.4% of their starting weight at the end of the study.
[00241] The significance of these differences was evaluated by calculating the mean area under the curve (AUC) for the percentage weight change for each animal and comparing the groups using a one-way ANOVA test. A statistically significant difference in weight gain was seen between the vehicle-treated group and the group that received SCV-07 at 40 mg/kg (p=0.004). The differences between the vehicle control group and the other treatment groups were not statistically significant.
[00242] Tumor Volumes (Figures 54 and 55). Tumor volume was analyzed to Day 36, when the first animal to be euthanized for exceeding maximum allowable tumor volume was euthanized. These data are shown in Figure 54. The mean tumor volume for the vehicle control group increased from 108 mm3 on Day 1 to 961 mm3 on Day 36. The group treated with SCV-07 at 1.0 mg/kg had a mean tumor volume of 114 mm3 on Day 1, increasing to 763 mm3 on Day 36. The group treated with SCV-07 at 5.0 mg/kg had a mean tumor volume of 104 mm3 on Day 1, increasing to 551 mm3 on Day 36. The group treated with SCV-07 at 10.0 mg/kg had a mean tumor volume of 111 mm3 on Day 1, increasing to 405 mm3 on Day 36. The group treated with SCV-07 at 20.0 mg/kg had a mean tumor volume of 117 mm3 on Day 1, which had increased to 431 mm3 on Day 36. In the Group dosed with SCV-07 at 40.0 mg/kg, the mean tumor volume was 107 mm3 on Day 1 which had increased to 227 mm3 on Day 36.
[00243] Further analysis of the data was performed by calculating the mean area under the curve (AUC) for the tumor volume for each animal and comparing the groups using an ANOVA on ranks test with a Tukey post test. This analysis indicated that there were statistically significant differences the vehicle control group and the groups treated with SCV-07 at 10 mg/kg (p=0.002), 20 mg/kg (p=0.002) and 40 mg/kg (pO.001).
[00244] Conclusions. SCV-07 showed no evidence of toxicity in this study based on observations of survival and weight change. Molt-4 Tumors in animals treated with SCV-07 grew more slowly than tumors in animals treated with vehicle and this reduction in tumor growth was dose dependant, and statistically significant in the groups treated with SCV-07 at 10 mg/kg (p=0.002), 20 mg/kg daily (p=0.002) and 40 mg/kg daily (p<0.001). Animals treated with SCV-07 daily at 1, 10, 20 and 40 mg/kg showed increased survival relative to the vehicle control group (p=0.029 for all groups).
Example 12. Analysis of effects of SCV-07 on SHP-1 and SHP-2 activation.
[00245] Introduction. In this study, the effects of SCV-07 on activation of SHP-1 and SHP-2 molecules were examined.
[00246] Methods & Results. For the study, the test compound was SCV-07 (gamma-D- glutamyl-L-tryptophan). SCV-07 was dissolved in 1 x phosphate-buffered saline (HyClone, Cat. #SH30264.01) at 5 mg/ml, divided into useable aliquots, and stored at -20°C prior to use.
[00247] The specificity test of antibodies to used in the study tested. Jurkat cells (5 x 106 cells/sample) were stimulated with 50 μΜ pervanadate for 10 min. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. Various amounts of lysates (1-10 μΐ/lane) were run on 4-20% Tris-Glycine gels and transferred to
nitrocellulose membranes. The membranes were probed with anti-pSHP-lxyr536, anti-pSHP- 2τΥι542 and anti-pSHP-2Tyr58o antibodies. The membranes were then stripped and reprobed with anti-SHP-1 and anti-SHP-2 antibodies. (PV=pervanadate.)
[00248] For examining the effects of SCV-07 on activation of SHP-2 PY and SHP- 1 PY in Jurkat cells, western blot assays were used. Specifically, western blot analysis was performed to test SCV-07 effects on activation of pSHP-2Tyr542, pSHP-2Tyr58o, and pSHP- 1τγι536 in Jurkat cells when stimulated with IFNa. The effect of SCV-07 on pSTAT3Tyr705 was used as a control. [00249] For examining IFNa stimulation time points for induction of pSHP-2 and pSHP-1 in Jurkat cells, cells (5 xlO6 cells/sample) were stimulated with 25 ng/mL IFNa for 0, 5, 10, 30 and 60 minutes. Cells were lysed with PJPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with antipSTAT3Tyr705 {upper panel of 56 A), anti-pSHP-lTyr536 {upper panel of Fig. 56B), anti-pSHP-2Tyr542 {first panel of Fig. 56C) and anti-pSHP-2Tyr58o {second panel of Fig. 56C) antibodies. The membranes were then stripped and reprobed with anti-STAT3 {lower panel of Fig. 56 A) ), anti-SHP-1 {lower panel of Fig. 56B) and anti-SHP-2 {third panel of Fig. 56C) antibodies.
[00250] Jurkat cells were plated in 60 x 15-mm tissue culture dishes at 5 x 106 cells per dish. Cells were then pretreated with vehicle (PBS) or SCV-07 (1 and 10 μg/mL) for 2h and then stimulated with 25 ng/mL IFNafor 10 and 30 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4- 20%) Tris-Glycine gels (Invitrogen, Cat. #EC60255) and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3Tyr705 (Cell Signaling
Technology, Cat. #9138), pSHP-2Tyr542, pSHP-2Tyr58o, and pSHP-lTyr536 antibodies (Assay Biotech, Cat. #A0027, A0028 and A0026). After analysis of phospho-proteins, the membranes were stripped and reprobed with anti-STAT3, anti-SHP-2 and anti-SHP-1 antibodies (Cell Signaling Technology, Cat. #9139, 3752 and 3759) to analyze the total cellular levels of STAT3, SHP-2 and SHP-1 from each sample.
[00251 ] For examining the effects of SCV-07 on activation of SHP-2 PY and SHP- 1 PY in Jurkat cells, western blotting was performed using anti-pSTAT3Tyr705, anti-pSHP-2Tyr542, antipSHP-2Tyr58o, and anti-pSHP-lTyr536 antibodies according to the Western standard protocols. After the lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3xyr705 {upper panel of Fig. 57 A & 58A), anti-pSHP-lTyr536 {upper panel of57B & 57 B), antipSHP- 2Tyr542 (first panel of Fig. 57C and 58C) and anti pSHP-2Tyr58o {third panel of Fig. 57C & 58C) antibodies. The membranes were then stripped and reprobed with anti-STAT3 {lower panel of Fig. 57 A & 58 A), anti-SHP-1 {lower panel of Fig. 57 B & 58B) and anti-SHP-2 {second and fourth panels of Fig. 57 C & 58C) antibodies. The Western data were further quantitated using TotalLab Quant software (Gentel Biosciences). The graphs indicate the relative quantitation of pSTAT3 (Fig. 57D & 58D), pSHP-2Tyr542 (Fig. 57E & 58E) and pSHP-2Tyr58o (Fig. 57F & 58F) band intensities.
[00252] For examining effects of SCV-07 on cytokine -induced STAT3 phosphorylation as well as activation of SHP-2 PY and SHP-1 PY in primary mouse macrophages, western blot analysis was again employed. Western blot analysis was performed to test SCV-07 effects on phosphorylation of STAT3 as well as activation of pSHP-2Tyr542, pSHP-2Tyr58o, and pSHP- 1τγι536 in primary mouse macrophages when stimulated with a selected cytokine.
[00253] For selection of cytokines used for induction of pSTAT3 in primary mouse macrophages, mouse peritoneal macrophages (3-5 x 106 cells/sample) were stimulated with 25 ng/mL IFNa ( Fig. 59 A), 200 ng/mL IL-6 (Fig. 59B) or 100 ng/mL IL-10 (Fig. 59 for 0, 5, 10, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing
protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with antipSTAT3xyr705 antibody (upper panels Fig. 59). The membranes were then stripped and reprobed with anti-STAT3 (lower panels Fig. 59) antibody.
[00254] For selection of cytokines used for induction of pSHP-2 in primary mouse macrophages, mouse peritoneal macrophages (3-5 x 106 cells/sample) were stimulated with 25 ng/mL IFNa (Fig. 60 A), 200 ng/mL IL-6 (Fig. 60B) or 100 ng/mL IL-10 (Fig. 60 for 0, 5, 10, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing
protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-p- SHP-2Tyr542 (upper panels)ox pSHP-2Tyr58o (middle panels Fig. 60) antibody. The membranes were then stripped and reprobed with anti-SHP-2 antibody (lower panels Fig. 60).
[00255] For selection of cytokines used for induction of pSHP-1 in primary mouse macrophages, mouse peritoneal macrophages (3-5 x 106 cells/sample) were stimulated with 25 ng/mL IFNa (Fig. 61 A), 200 ng/mL IL-6 (Fig. 6 IB) or 100 ng/mLIL-10 (Fig. 61 for 0, 5, 10, 30 and 60 minutes. Cells were lysed with RIPA lysis buffer containing
protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-p- SHP-lTyr536 antibody (upper panels Fig. 61). The membranes were then stripped and reprobed with anti-SHP-1 antibody (lower panels Fig. 61). [00256] For preparation of primary mouse macrophage (Peritoneal macrophages), the following procedure was employed. C57BL/6 female mice (10 weeks old) were injected with 1.5 mL of 4% Thiogly collate into the peritoneal cavity. Mice were then left for 4 days until macrophages were accumulated into the peritoneal cavity. On day of harvest, euthanized mice were injected with cold lx PBS (8-10 mL) to wash the peritoneal cavity and lavage was collected using the syringe (up to 5-6 mL per mouse). Lavage (Peritoneal wash) containing cells was pooled and centrifuged for 10 min. The pellets were washed once with culture media. Cells were re-suspended, counted and plated in 6-well plates to get adherent macrophages at 3-5 x 106 cells per well. Cells were then incubated at 37°C and 16h later, non-adherent cells were removed and adherent cells (macrophages) were washed with fresh media. Western blotting was performed using anti-pSTAT3xyr705, anti-pSHP-2Tyr542, anti-p- SHP-2 Tyr58o, and anti-pSHP-lxyr536 antibodies according to the Western standard protocols. The Western data were further quantitated using TotalLab Quant software (Gentel
Biosciences).
[00257] For phenotypic analysis of purified peritoneal macrophages by flow cytometry, peritoneal Macrophages were isolated from Thioglycollate-stimulated mice and were then cultured in complete DMEM media in a 6-well plate. Sixteen hours later, non-adherent cells were removed and adherent cells (macrophages) were harvested and stained with PE- conjugated F4/80, a peritoneal macrophage marker, or with PE-conjugated Rat IgG2a isotype control at ^g/106 cells for 30 min on ice. Cells were washed twice with flow buffer and resuspended in flow buffer. Cells were then analyzed using CellQuest-based FACSCalibur (BD Biosciences). The cell population was then confirmed as peritoneal macrophages.
[00258] Mouse peritoneal macrophages plated in 6-well plates (3-5 x 106 cells/well) were pretreated with vehicle (PBS) or SCV-07 (1 and 10 μg/mL) for 2 h and then stimulated with 200 ng/mL IL-6 for 10 minutes and 30 minutes. Cells were then lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels (Invitrogen, Cat. #EC60255) and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3Tyr705 antibody (Cell Signaling Technology, Cat. #9138), pSHP-2 Tyr542, pSHP-2xyr58o, and pSHP-lxyr536 antibodies (Assay Biotech, Cat. #A0027, A0028 and A0026). After analysis of phospho-proteins, the membranes were stripped and reprobed with anti-STAT3, anti-SHP-2 and anti-SHP-1 antibodies (Cell Signaling Technology, Cat. #9139, 3752 and 3759) to analyze the cellular levels of STAT3, SHP-2 and SHP-1 from each sample.
[00259] For examining the effect of SCV-07 on pSTAT3, pSHP-1 and pSHP-2 in Unstimulated primary mouse macrophages. Mouse peritoneal macrophages (3-5 x 106 cells/sample) were pretreated with 1 and 10 μg/mL SCV-07 for 2 h and then stimulated with 200 ng/mL IL-6 for 0, 10 and 30 minutes. Cells were lysed with RIPA lysis buffer containing protease/phosphatase inhibitor cocktail on ice. The lysates were run on 4-20% Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were probed with anti-pSTAT3xyr705 (upper panel of Fig. 62 A & 63 A), anti-pSHP-lTyr536 (upper panel of Fig. 62B & 63B), anti-pSHP-2Tyr542 (first panel of Fig. 62C & 63C) and anti-pSHP-2Tyr58o {third panel of Fig. 62C & 63C) antibodies. (Figures 62 and 63). The membranes were then stripped and reprobed with anti-STAT3 {lower panel of Fig. 62 A & 63 A ), anti-SHP-1 {lower panel of Fig. 62B & 63B) and anti-SHP-2 {second and fourth panels of C) antibodies. The graphs indicate the relative quantitation of pSTAT3 {Fig. 62D & 63E), pSHP-2Tyr542 {Fig. 62E & 63E) and pSHP-2Tyr58o (Fig. 62F & 63F) band intensities.
[00260] The effects of SCV-07 on cytokine-mediated MCP-1 and IL-12p40 production in primary mouse macrophages were then examined. Mouse peritoneal macrophages were plated in 6-well plates (3-5 x 106 cells/well) were pretreated with vehicle (PBS) or SCV-07 (1 and 10 μg/mL) for 2 h. Cells were then stimulated with 200 ng/mL IL-6 for 18 h. Cell culture supernatants were harvested. To determine the effects of SCV-07 on cytokine- mediated MCP-1 and IL-12p40 production in primary mouse macrophages that were stimulated with IL-6 in the present or absence of SCV-07, ELISA was performed on the harvested cell culture supernatants. The levels of MCP-1 and IL 12p40 induction in mouse peritoneal macrophages when stimulated with IL-6 in the presence or absence of SCV-07 were measured. Induction levels of MCP-1 {Fig. 98 A) and IL 12p40 {Fig. 98B) were then analyzed. *p = 0.0495 versus vehicle control 1 (VC-1; without SCV-07 and IL-6); **p = 0.0495 versus vehicle control 2 (VC-2; IL-6 only) (Mann- Whitney U test).
[00261] Conclusions. SCV-07 enhanced activation of pSHP-2Tyr542 in IFNa-stimulated Jurkat cells while pSHP-2Tyr58o was not significantly affected by SCV-07 (Figures 57 & 58). pSHP-lTyr536 was not induced by IFNa stimulation in Jurkat cells (Figures 57 & 58). SCV-07 inhibited STAT3 phosphorylation induced by IL-6 stimulation in primary mouse
macrophages (Figures 96 & 97). SCV-07 enhanced activation of pSHP-2Tyr542 in IL-6- stimulated primary mouse macrophages while pSHP-2Tyr58o was not significantly affected by SCV-07 (Figures 62 & 63). pSHP-lTyr536 was not induced by IL-6 stimulation in primary mouse macrophages (Figures 62 & 63). SCV-07 suppressed both constitutive and IL-6- mediated levels of MCP-1 in primary mouse macrophages while IL-12p40 was unlikely induced by IL-6 in primary mouse macrophages (Figure 64).
[00262] The results of the study indicated that SHP-2 functions as a negative regulator of STAT3 phosphorylation in Jurkat and primary mouse macrophages, and suggest that SHP-2 is an upstream phosphatase for STAT3. The inhibitory effect of SCV-07 on STAT3 phsphorylation is reflected from the SCV-07-mediated upregulation of pSHP-2Tyr542.
Therefore, SCV-07 likely targets upstream of STAT3, potentially directly catalyzing SHP-2 phosphorylation or synergizing with (or activating) an upstream regulator for SHP-2.
[00263] All publications discussed and cited herein are incorporated herein by reference in their entireties. It is understood that the disclosed invention is not limited to the particular methodology, protocols and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
[00264] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.

Claims

CLAIMS:
1. A method for treating a subject with inducible expression of STAT3 and neoplasia comprising administering to the subject in need of such treatment an effective amount of a therapeutic entity, wherein the therapeutic entity comprises a compound of Formula A
Figure imgf000077_0001
(A)
or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof.
2. A method for treating a subject with neoplasia comprising determining the presence of inducible expression of STAT3 in a biological sample of the subject and administering a therapeutic entity to the subject upon determination of the presence of inducible expression of STAT3 in the subject,
wherein the therapeutic entity comprises a compound of Formula A
Figure imgf000077_0002
(A)
or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof.
3. A method for determining the treatment regimen for a subject with neoplasia
comprising selecting a treatment regimen for the subject based on the presence of inducible expression of STAT3, wherein the treatment regimen includes a therapeutic entity comprising a compound of Formula A R— NH— CH (CH2)„— C— X
COOH O
(A)
or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof.
4. The method of claim 3 wherein the method further comprises determining the
presence of inducible expression of STAT3 in a biological sample from the subject.
5. A method for predicting the treatment efficacy of a therapeutic entity for the treatment of neoplasia comprising detecting inducible expression of STAT3 in a biological sample of a subject, wherein the presence of inducible expression of STAT3 is indicative of the treatment efficacy of the therapeutic entity for the subject, and
wherein said therapeutic entity comprises a compound of Formula A
R— NH— CH (CH2)„— C— X
COOH O
(A)
or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof.
6. A method for determining the treatment efficacy of a therapeutic entity for the
treatment of neoplasia in a subject comprising detecting in a biological sample of a subject treated with the therapeutic entity the presence of one or more markers selected from the group consisting of a marker for inhibition of signal transduction through STAT3, a marker for inhibition of phosphorylation of STAT3, a marker for inhibition of nuclear translocation of STAT3, a marker for inhibition of IL-6 mediated STAT3 activation, a marker for inhibition of IL-10 mediated STAT3 activation, a marker for inhibition of IFN-a mediated STAT3 activation and a marker for inhibition >f TT _4 moated STAT3 activation, wherein the presence of one or more markers is indicative of the therapeutic efficacy of the therapeutic entity,
wherein said therapeutic entity comprises a compound of Formula A
Figure imgf000079_0001
(A) or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof.
7. The method of claim 6, wherein the subject has inducible expression of STAT3.
8. A method of providing useful information for determining the treatment regimen for a subject with neoplasia comprising detecting the presence or absence of inducible expression of STAT3 in a biological sample of a subject and providing the result of the detection to an entity that determines the treatment regimen based on the presence or absence of inducible expression of STAT3,
wherein said therapeutic entity comprises a compound of Formula A
Figure imgf000079_0002
(A)
or a pharmaceutically acceptable salt thereof, wherein, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptidyl, and X is an aromatic or heterocyclic amino acid or a derivative thereof.
9. The method of any of the claims above, wherein X is L-tryptophan or D-tryptophan.
10. The method of any of the claims above, wherein said therapeutic entity is γ-D- glutamyl-L-tryptophan.
11. The method of claim 2, 4, 5, 6, or 7, wherein said biological sample is selected from the group consisting of serum, blood, plasma, whole blood and derivatives thereof, skin, hair, hair follicles, saliva, oral mucous, vaginal mucous, sweat, tears, epithelial tissues, urine, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), excreta, ascites, cerebrospinal fluid, lymph, and tissue extract sample and biopsy.
12. The method of claim 11, wherein said biological sample is tumor or neoplastic tissue.
13. The method of any of the claims above, wherein said neoplasia is selected from the group consisting of carcinoma, sarcoma, blastoma, lymphoma, leukemia, and germ cell tumors.
14. The method of any of the claims above, wherein said neoplasia is selected from the group consisting of head and neck, skin, colon, oral, glioblastoma, breast, laryngeal, esophageal, endothelial, endometrial, ovarian, lung, urogenital, rectal, prostate, kidney, melanoma, renal, and papilloma virus-induced cancer.
15. The method of any of the claims above, wherein the presence of inducible STAT3 is determined by detecting STAT3 expression or activation.
16. The method of claim 15, wherein detection of inducible expression of STAT3
includes detecting the level of STAT3 phosphorylation, STAT3 protein, STAT3 nucleic acid or STAT3 gene expression.
PCT/US2011/061260 2010-11-17 2011-11-17 Method and compositions for treatment of stat3-responsive cancers and/or renal cancer WO2012068412A2 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US41484410P 2010-11-17 2010-11-17
US61/414,844 2010-11-17
US41882810P 2010-12-01 2010-12-01
US61/418,828 2010-12-01
US201161487191P 2011-05-17 2011-05-17
US61/487,191 2011-05-17

Publications (2)

Publication Number Publication Date
WO2012068412A2 true WO2012068412A2 (en) 2012-05-24
WO2012068412A3 WO2012068412A3 (en) 2013-01-17

Family

ID=46084659

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/061260 WO2012068412A2 (en) 2010-11-17 2011-11-17 Method and compositions for treatment of stat3-responsive cancers and/or renal cancer

Country Status (1)

Country Link
WO (1) WO2012068412A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3548095A4 (en) * 2016-11-29 2020-07-29 Crown Bioscience, Inc. (Taicang) Methods of mouse clinical trial

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010017178A1 (en) * 2008-08-06 2010-02-11 Sciclone Pharmaceuticals, Inc. Treatment or prevention of hepatitis c with immunomodulator compounds
WO2012040656A2 (en) * 2010-09-24 2012-03-29 Sciclone Pharmaceuticals, Inc. Biomarkers to track response to scv-07

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010017178A1 (en) * 2008-08-06 2010-02-11 Sciclone Pharmaceuticals, Inc. Treatment or prevention of hepatitis c with immunomodulator compounds
WO2012040656A2 (en) * 2010-09-24 2012-03-29 Sciclone Pharmaceuticals, Inc. Biomarkers to track response to scv-07

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NAGABHUSHANAM, V. ET AL.: 'Inhibition of STAT3 driven gene expression in melanoma cells by SCV-07' 2008 AMERICAN SOCIETY CLINICAL ONCOLOGY ANNUAL MEETING *
WATKINS, B. ET AL.: 'Attenuation of radiation- and chemoradiation-induced mucositis using gamma-d-glutamyl-1-tryptophan (SCV-07)' ORAL DIS. vol. 16, no. 7, October 2010, pages 655 - 660 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3548095A4 (en) * 2016-11-29 2020-07-29 Crown Bioscience, Inc. (Taicang) Methods of mouse clinical trial

Also Published As

Publication number Publication date
WO2012068412A3 (en) 2013-01-17

Similar Documents

Publication Publication Date Title
ES2863996T3 (en) Combination therapy for cancer treatment
Maines et al. Efficacy of a novel sphingosine kinase inhibitor in experimental Crohn’s disease
US20230148436A2 (en) Novel small molecule immune agonists and immune targeting compounds and application thereof
Sylvester et al. Effect of Crohn's disease on bone metabolism in vitro: a role for interleukin‐6
Lu et al. Celastrol, a novel HSP90 inhibitor, depletes Bcr–Abl and induces apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation
US9259399B2 (en) Targeting CDK4 and CDK6 in cancer therapy
Zheng et al. Novel phosphatidylinositol 3-kinase inhibitor NVP-BKM120 induces apoptosis in myeloma cells and shows synergistic anti-myeloma activity with dexamethasone
KR20170001962A (en) An anticancer agent composition
US10758522B2 (en) Small molecule analogs of the nemo binding peptide
Ku et al. Transglutaminase 2 inhibitor abrogates renal cell carcinoma in xenograft models
Zhang et al. Fluvastatin Inhibits HMG-CoA Reductase and Prevents Non–Small Cell Lung Carcinogenesis
Shangguan et al. Niclosamide inhibits ovarian carcinoma growth by interrupting cellular bioenergetics
Liu et al. Fenofibrate increases radiosensitivity in head and neck squamous cell carcinoma via inducing G2/M arrest and apoptosis
Liuzzi et al. Interplay between autophagy and zinc
CN107683139A (en) Match degree for treating B cell malignant tumour replaces Buddhist nun
Chai et al. Antihelminthic niclosamide induces autophagy and delayed apoptosis in human non-small lung cancer cells in vitro and in vivo
US8754094B2 (en) Methods for heat shock protein dependent cancer treatment
Ciesielski et al. KX2-361: A novel orally bioavailable small molecule dual Src/tubulin inhibitor that provides long term survival in a murine model of glioblastoma
Haegebarth et al. Induction of Protein Tyrosine Kinase 6 in Mouse Intestinal Crypt Epithelial Cells Promotes DNA Damage–Induced Apoptosis
Chadha et al. Focus on the multimodal role of autophagy in rheumatoid arthritis
US20140121278A1 (en) Anti-tumor agent otx-008 targets human galectin-1
JP2023519424A (en) Deuterated oxophenylarsine compounds and uses thereof
WO2012068412A2 (en) Method and compositions for treatment of stat3-responsive cancers and/or renal cancer
US9873705B2 (en) Vinylogous thioester compounds and methods of use
KR20200135743A (en) Novel use of pyrolo-pyridine derivatives for preventing or treating cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11841773

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11841773

Country of ref document: EP

Kind code of ref document: A2