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1. (WO2018045346) METHODS FOR TREATING CANCER AND DETERMINING EFFICACY OF TREATMENT
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METHODS FOR TREATING CANCER AND DETERMINING EFFICACY OF

TREATMENT

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62/485,188 filed on April 13, 2017, 62/478,817 filed March 30, 2017 and 62/382,773 filed September 1, 2016, the contents of each which are herein incorporated by reference in their entirety.

FIELD OF INVENTION

[0002] The invention relates to medicine and oncology, including treating cancer, determining efficacy of treatment and determining prognosis.

BACKGROUND

[0003] All publications cited herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Pancreatic cancer is a devastating disease. It represents one of the worst solid tumors and has a diagnosis to mortality ratio approaching one. There are approximately 40,000 diagnoses per year in the US and 350,000 diagnoses worldwide. Combinations of radiotherapy (RT), chemotherapy and surgery have been used to treat resectable, locally advanced un-resectable and metastatic cancer patients. However, the doses of treatments (e.g., RT and chemotherapy) are dictated by their associated toxicities. This in turn impacts the clinical efficacy of these treatments. There is an unmet need in the art for cancer therapies and methods for determining efficacies of the cancer therapies.

SUMMARY

[0005] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0006] Provided herein are methods for assessing efficacy of Trabedersen therapy in a subject in need thereof. The methods include determining the levels of cytokines in a sample obtained from the subject, wherein the subject is undergoing or has undergone treatment comprising administration of therapeutically effective amount of Trabedersen; and wherein a spike in the level of cytokines relative to reference value indicates that the treatment is efficacious.

[0007] Further provided herein are methods for prognosing cancer in a subject in need thereof. The methods include determining the levels of cytokines in a sample obtained from the subject, wherein the subject is undergoing or has undergone treatment comprising administration of therapeutically effective amount of Trabedersen; and wherein a spike in the level of cytokines relative to reference value indicates that the treatment is efficacious. A determination that the treatment comprising Trabedersen is efficacious is indicative of good prognosis.

[0008] In some embodiments, the subject has undergone at least one cycle of treatment with Trabedersen. In some embodiments, the subject is undergoing treatment comprising Trabedersen.

[0009] In various embodiments, the sample from the subject is obtained after administration of Trabedersen.

[0010] In some embodiments, the subject has undergone one, two, three, four or more cycles of treatment with Trabedersen.

[0011] In one embodiment, the spike in the cytokine levels after the first and/or second cycle of treatment comprising Trabedersen indicates that the treatment is efficacious.

[0012] In one embodiment, the spike in the cytokine levels after the first and/or second cycle of treatment comprising Trabedersen indicates that the prognosis is good.

[0013] In one embodiment, absence of a spike in the cytokine levels after the first and second cycle of treatment comprising Trabedersen indicates that the treatment is not efficacious.

[0014] In one embodiment, absence of a spike in the cytokine levels after the first and second cycle of treatment comprising Trabedersen is indicative of poor prognosis.

[0015] In one embodiment, treatment comprising Trabedersen comprises administering Trabedersen to the subject for at least one cycle to sensitize the tumor to chemotherapeutic agent. In another embodiments, treatment comprising Trabedersen comprises administering Trabedersen to the subject for at least one cycle to sensitize the tumor to chemotherapeutic agent and subsequently administering the chemotherapeutic agent.

[0016] In exemplary embodiments, the chemotherapeutic agents are paclitaxel, dacarbazine, alkylating agents or checkpoint inhibitors.

[0017] In one embodiment, the cytokine is IL-8. In one embodiment, the cytokine is IL-15. In one embodiment, the cytokine is IL-6. In some embodiments, the cytokine is IL-8, IL-15, IL6 or a combination thereof.

[0018] In an embodiment, the subject has cancer. In exemplary embodiments, the cancer is pancreatic cancer, melanoma or ovarian cancer.

[0019] In some embodiments, the spike in levels of the cytokine is relative to a reference value. In one embodiment, the reference value is the level of the cytokine in the sample obtained from the same subject prior to treatment with Trabedersen. In another embodiment, the reference value is the level of the cytokine in a sample obtained from a subject that has cancer and has not been treated with Trabedersen. In a further embodiment, the reference value is the level of the cytokine in a sample obtained from a subject whose cancer is in remission.

[0020] Also provided herein is a method, comprising: administering a therapeutically effective amount of Trabedersen so a subject diagnosed with cancer, so as to sensitize a tumor in the subject to one or more chemotherapeutic agents; subsequently administering a therapeutically effective amount of one or more chemotherapeutic agents to the subject; and determining the levels of cytokines in a sample obtained from the subject, wherein a spike in the level of cytokines indicates that the treatment is efficacious. A determination that the treatment comprising Trabedersen is efficacious is indicative of good prognosis. In one embodiment, the cytokine is IL-8. In one embodiment, the cytokine is IL-15. In one embodiment, the cytokine is IL-6. In some embodiments, the cytokine is IL-8, IL-15, IL6 or a combination thereof. In an embodiment, the subject has cancer. In exemplary embodiments, the cancer is pancreatic cancer, melanoma or ovarian cancer. In some embodiments, the sample is obtained after administration of Trabedersen.

[0021] Further provided herein is a method, comprising: administering a therapeutically effective amount of Trabedersen so a subject diagnosed with cancer, so as to sensitize a tumor in the subject to one or more chemotherapeutic agents; and determining the levels of cytokines in a sample obtained from the subject, wherein a spike in the level of cytokines indicates that the treatment is efficacious. A determination that the treatment comprising Trabedersen is efficacious is indicative of good prognosis. In one embodiment, the cytokine is IL-8. In one embodiment, the cytokine is IL-15. In one embodiment, the cytokine is IL-6. In some embodiments, the cytokine is IL-8, IL-15, IL6 or a combination thereof. In an embodiment, the subject has cancer. In exemplary embodiments, the cancer is pancreatic cancer, melanoma or ovarian cancer. In some embodiments, the sample is obtained after administration of Trabedersen.

[0022] In some embodiments, the spike in levels of the cytokine is relative to a reference value. In an embodiment, the reference value is the level of the cytokine in the sample obtained from the same subject prior to treatment with Trabedersen.

[0023] Provided herein is a method, comprising: obtaining a sample from a subject that has been treated with Trabedersen and measuring the level of one or more cytokines in the sample. In one embodiment, an increase in the level of the one or more cytokines relative to a reference value indicates that the treatment with Trabedersen is efficacious. In one embodiment, the method further comprises selecting or prescribing if efficacy is determined. In an embodiment, the increase in the level of one or more cytokines is a transient increase in the level of one or more cytokines as described herein. In one embodiment, the subject is undergoing treatment for cancer. In an embodiment, the Trabedersen is administered as a sensitizing agent prior to administration of a chemotherapeutic agent, wherein the Trabedersen sensitizes the tumor to the chemotherapeutic agent.

[0024] Also provided herein is a method for assessing efficacy of Trabedersen therapy in a subject in need thereof, comprising: determining the level of one or more cytokines in a subject that has been treated with Trabedersen and determining that Trabedersen therapy is efficacious if the level of one or more cytokines in the subject is increased relative to a reference value. In an embodiment, the increase in the level of one or more cytokines is a transient increase in the level of one or more cytokines as described herein. In one embodiment, the subject is undergoing treatment for cancer. In an embodiment, the Trabedersen is administered as a sensitizing agent prior to administration of a

chemotherapeutic agent, wherein the Trabedersen sensitizes the tumor to the chemotherapeutic agent.

[0025] In one embodiment, the subject has undergone at least one cycle of Trabedersen treatment. In one embodiment, the subject has been treated with Trabedersen and a chemotherapeutic agent, wherein the Trabedersen is administered to the subject for at least one cycle prior to treatment with the chemotherapeutic agent.

[0026] In one embodiment, the subject has been treated with Trabedersen and any one of paclitaxel, dacarbazine, or a checkpoint inhibitor, wherein the Trabedersen is administered to the subject for at least one cycle prior to treatment with any of paclitaxel, dacarbazine, or a checkpoint inhibitor.

[0027] In one embodiment, the cytokine is IL-8. In another embodiment, the cytokine is IL-15. In a further embodiment, the cytokine is IL-6.

[0028] In one embodiment, the subject has cancer. In exemplary embodiments, the cancer is pancreatic cancer, melanoma or ovarian cancer.

[0029] In some embodiments, the increase in levels of the cytokine is relative to a reference value. In one embodiment, reference value is the level of the cytokine in a sample obtained from the same subject prior to treatment. In another embodiment, the reference value is the level of the cytokine in a sample obtained from a subject that has cancer and has not been treated with Trabedersen. In a further embodiment, the reference value is the level of the cytokine in a sample obtained from a subject whose cancer is in remission.

[0030] In one embodiment, the cancer is melanoma. In one embodiment of the methods described herein, the tumor is a pancreatic tumor.

BRIEF DESCRIPTION OF DRAWINGS

[0031] Fig. 1 depicts in accordance with various embodiments of the invention, the dose dependent increase in overall survival (OS) rate in pancreatic cancer patients when Trabedersen is used as a second line treatment. OS and PFS of patients treated with Trabedersen were compared to reported median OS and PFS from 65 references from 1997-2015. OS at 140 mg/m2/d is in line with literature data and mOS of patients at 167 and 196 mg/m2/d is superior compared to literature. PFS of patients at all doses is in line with

historical data from literature. While PFS was not significantly different, the OS was higher than reported in the literature.

[0032] Fig. 2 depicts in accordance with various embodiments of the invention, that there is an increase in response to chemotherapy following Trabedersen therapy as indicated by an increase in overall survival rate. Sequencing of Trabedersen treatment in 2nd line followed by subsequent therapies has higher OS (9.93 months) versus the reverse- 2nd line chemotherapy followed by Trabedersen (2.80 months) (p=0.0354, log-rank). This suggests that Trabedersen sensitizes the tumor to chemotherapy.

[0033] Fig. 3 depicts in accordance with various embodiments of the invention, that Trabedersen alone does not have a significant effect on the tumor as indicated by a low overall survival rate but when Trabedersen therapy is followed by chemotherapy, there is a significant increase in the overall survival rate. Sequencing and Subsequent Chemotherapy in 2nd Line demonstrated patients receiving subsequent chemotherapies exhibit significantly greater OS (14.73 months) than patients without subsequent chemotherapies (2.93 months) (p=0.0023, log-rank). This suggests that Trabedersen sensitizes the tumor to chemotherapy.

[0034] Fig. 4A-Fig. 4E depicts, in accordance with various embodiments of the invention, Fig. 4A: shows the impact of Trabedersen on 31 cytokine/chemokines in plasma samples from 12 patients. Blood samples were drawn and plasma levels of 31 cyto-/chemokine were tracked over 8 time points during 3 cycles of trabedersen therapy (140 mg/m2/day) for 12 PAC patients. Clustering of correlation coefficients resulted in the identification of three highly correlated subsets of cytokines/chemokines following the trabedersen treatment. (Cluster 1 : EGF, MIP-la, MIP-lb; Cluster 2: FGF-2, IL-1R, MIG, IP-10, IL-15, IFN-a, IL-12A; Cluster 3 : IL-2R, IL-6, IL-8, HGF) identified following Trabedersen therapy (r ranged from 0.34 (p = 0.002) to 0.65 (p < 0.0001)). Of the 31 cytokines and chemokines examined, 19 are shown and the rest were at a level too low to detect. Suppression of TGFb2 signaling by Trabedersen leads to upregulation of IL-8, IL-6 and IL-15. Protein-protein interaction networks were constructed using STRING10 algorithm for co-regulated cytokines/chemokines to identify potential hub proteins connecting TGFBR2 pathways to cytokines/chemokines. IL-8 is linked to TGF betaR2; Fig 4B: IL-8 Expression in 12 pancreatic cancer patients following 3 cycles of trabedersen treatment. Heat map represents standardized expression levels (blue to red depicts increasing expression levels relative to mean and standard deviation of log 10 transformed IL-8 expression level in each patient.

Patients exhibited variable dynamics in IL-8 levels whereby increases were observed during cycles 1, 2 and 3. Patients 1035 and 1047 showed marked increases early in cycle 1 Fig. 4C: Induction of IL-8 by Trabedersen. The graphs show the secretion of IL-8 following a preincubation (1 h) with the test substance (Trabedersen) and a consecutive culture period (48 h), either without or with stimuli (LPS/SEB), in order to allow for the formation and secretion of cytokines and chemokines under the influence of the test drug Biologically relevant effects of Trabedersen were found in the whole-blood cultures of all three healthy volunteers that were tested. ; Fig. 4D: induction of IL-8 by Paclitaxel and Dacarbazine. Performed the search in The Comparative Toxicogenomics Database, 735 gene related to paclitaxel while 25 genes related Dacarbazine. IL-8 is among the 17 genes common to paclitaxel and dacarbazine; Fig. 4E: synergistic anti-tumor activity and survival observed with Trabedersen and DTIC or Paclitaxel against three human tumor xenograft models in athymic nude mice.

[0035] Fig. 5A-Fig. 5B depicts, in accordance with various embodiments of the invention, Fig. 5A: IL-8 spike predicts overall survival (OS) of patients treated with or without chemotherapy following OT-101 (Trabedersen). (Left) LoglO standardized IL-8 levels per patient across 8 time points depicted as a heat map. (Right) Difference of Maximum Day 5 IL-8 expression from Maximum Day 2 expression stratified by OT-101 treatment followed with or without chemotherapy. OT101 followed by Chemotherapy (R2=0.99, P=0.0686). OT-101 treatment only (R2=0.77, P=0.0020) IL-8 as a biomarker in TGF-β immunotherapy; Fig. 5B: Cytokine levels predict OS of patients with or without chemotherapy following OT-101. Cytokine levels of Max Day 2- Max Day 5 plotted against OS and stratified by with (red) or without (blue) subsequent chemotherapy. Intersection of statistically significant linear regression lines at OS=0 observed in IL-8 and IL-15. Survival in months can be predicted based on expression levels of these two cytokines where intersection at OS=0 w/ or w/o chemotherapy provide additional evidence of common mechanism of action in both subset of patients.

[0036] Fig. 6 depicts, in accordance with various embodiments of the invention, effect of Trabedersen on orthotopic L3.6pl paclitaxel xenograft mouse model. The study was to evaluate the effect of trabedersen on the weight of the primary tumor on day 29 and significant suppression of primary tumor with both Gem and trabedersen. However , there was no synergy observed. The inhibition of tumor vascularization by trabedersen was also evaluated for CD31+ vessel area in tumor sections. The results showed significant

suppression of tumor vascularization by both Gem and trabedersen, but no synergy was observed

[0037] Fig. 7A-7B depicts, in accordance with various embodiments of the invention, synergistic benefits of Trabedersen and Paclitaxel are observed when Trabedersen is administered first to sensitize cancer to paclitaxel (Fig. 7B) but not when Trabedersen and Paclitaxel are administered simultaneously (Fig. 7A).

DESCRIPTION OF THE INVENTION

[0038] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (September 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7.

[0039] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. Indeed, the present invention is in no way limited to the methods and materials described. For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0040] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The definitions and terminology used herein are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims.

Definitions

[0041] In the event of any inconsistency with a term defined herein and those set forth in patents, published application and non-patent literature that are incorporated herein by reference, the definitions herein will control.

[0042] Unless stated otherwise, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the application (especially in the context of claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0043] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). Although the open-ended term "comprising," as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as "consisting of or "consisting essentially of."

[0044] As used herein, the term "about" refers to a measurable value such as an amount, a time duration, and the like, and encompasses variations of ±20%, ±10%, ±5%, ±1%, ±0.5% or ±0.1%) from the specified value.

[0045] "Sample" or "biological sample" as used herein refers to tissues or body fluids removed from a mammal, preferably human. Samples can be blood and/or blood fractions, including peripheral blood sample like peripheral blood mononuclear cell (PBMC) sample or blood, bone marrow cell sample. A sample can also include any specific tissues/organ sample of interest, including, without limitation, lymphoid, thymus, pancreas, eye, heart, liver, nerves, intestine, skin, muscle, cartilage, ligament, synovial fluid, and/or joints. The samples can be taken from any individual including a healthy individual or an individual having cells, tissues, and/or an organ afflicted with the unwanted immune response. Methods for obtaining such samples are well known to a person of ordinary skill in the art of immunology and medicine. They include drawing and processing blood and blood components using routine procedures, or obtaining biopsies from the bone marrow or other tissue or organ using standard medical techniques.

[0046] As used herein, the terms "treat," "treatment," "treating," or "amelioration" when used in reference to a disease, disorder or medical condition, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, reverse, alleviate, ameliorate, inhibit, lessen, slow down or stop the progression or severity of a symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease, disorder or medical condition is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Also, "treatment" may mean to pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented.

[0047] "Beneficial results" or "desired results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition, decreasing morbidity and mortality, and prolonging a patient's life or life expectancy. As non-limiting examples, "beneficial results" or "desired results" may be alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of pancreatic cancer, delay or slowing of pancreatic cancer, and amelioration or palliation of symptoms associated with pancreatic cancer.

[0048] The term "statistically significant" or "significantly" refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.

[0049] As used herein, the terms "categorizing", "classifying", "stratifying", "subtyping", and "subgrouping" are interchangeable. As used herein, the terms "category", "class", "strata", "subtype", and "subgroup" are interchangeable.

[0050] The term "sensitization" as used herein refers to making the tumors sensitive to treatment by uncloaking the tumors and thus making the tumors more accessible to the subsequent exogenous therapies and the endogenous immune system. In one embodiment, Trabedersen or a variant, derivative or analog thereof sensitizes tumors to subsequent exogenously administered therapies such as chemotherapy, radiation therapy, hormonal therapy or combination thereof. In an embodiment, Trabedersen or a variant, derivative or analog thereof sensitizes tumors to the patient's own endogenous immune system. In another

embodiment, Trabedersen or a variant, derivative or analog thereof sensitizes tumors to one or more chemotherapeutic agent. In exemplary embodiments, when a tumor is sensitized with Trabedersen or a variant, derivative or analog thereof prior to chemotherapy, administration of one or more chemotherapeutic agents following treatment with Trabedersen or a variant, derivative or analog thereof results in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%), 80%), 90% or 100% improved response to the chemotherapeutic agents compared to treatment with the chemotherapeutic agents without sensitization with Trabedersen or a variant, derivative or analog thereof.

[0051] The term "cycle" as used herein refers to the number of days when the inhibitor of TGF is administered and number of days when the inhibitor of TGF is not administered. In an embodiment, one cycle is defined as administering the inhibitor for 7 days at a specific dosage per day and then not administering the inhibitor for 7 days. This is referred to as "7 days on and 7 days off (7/7) cycle. In another embodiment, one cycle is defined as administering the inhibitor for 4 days at a specific dosage per day and then not administering the inhibitor for 10 days. This is referred to as "4 days on and 10 days off (4/10) cycle. In exemplary embodiments, Trabedersen is administered for 1, 2, 3, 4, 5 or more 4/10 cycles with exogenous treatments (for example, chemotherapy) between each cycle. In further exemplary embodiments, Trabedersen is administered for 1, 2, 3, 4, 5 or more 7/7 cycles with exogenous treatments (for example, chemotherapy) between each cycle

[0052] The term "PFS" as used herein refers to progression free survival and is a measure of the activity of a treatment on a disease. The term "OS" as used herein refers to overall survival and is a measure of the activity of the treatment and subsequent treatments, on a disease. As described herein, in some embodiments, if PFS is not improved upon administration of Trabedersen or a variant, derivative or analog thereof but OS is improved upon administration of Trabedersen or a variant, derivative or analog thereof, it is indicative of sensitization of the tumors by Trabedersen or a variant, derivative or analog thereof to subsequent treatments.

[0053] As used herein, a "spike" refers to a transient increase in the levels of cytokines within 1, 2, 3 or 4 cycles after treatment with Trabedersen. A "spike" or a "transient increase" refers to a temporary increase in the levels of cytokines (i.e., the cytokine levels increase for about 1, 2, 3, 4 or more days after administration of Trabedersen and then decrease to the subject' s normal levels of the cytokines). In an embodiment, the spike in the cytokine levels is during the first cycle of treatment with Trabedersen for example, about one to two days, about two to three days, about three to four days, about four to five days, about one to three days, about two to four days or a combination thereof, after the treatment with Trabedersen. In a further embodiment, the spike in the cytokine levels is during the second cycle of treatment with Trabedersen for example, about one to two days, about two to three days, about three to four days, about four to five days, about one to three days, about two to four days or a combination thereof, after the second cycle of treatment with Trabedersen. In a further embodiment, the spike in the cytokine levels is during the third cycle of treatment with Trabedersen for example, about one to two days, about two to three days, about three to four days, about four to five days, about one to three days, about two to four days or a combination thereof, after the third cycle of treatment with Trabedersen. In a further embodiment, the spike in the cytokine levels is during the fourth or higher cycle of treatment with Trabedersen for example, about one to two days, about two to three days, about three to four days, about four to five days, about one to three days, about two to four days or a combination thereof, after the fourth or higher cycle of treatment with Trabedersen. In some embodiments, one or more exogenous therapies (for example, chemotherapeutic agents) have been administered to the subject after treatment with Trabedersen, including after the first, second, third, fourth and/or more cycles of Trabedersen treatment. In various embodiments, the cytokines are IL-8, IL-6, IL-15 or a combination thereof.

[0054] "Poor Prognosis" means the prospect of survival and recovery in subjects in whom the cytokine (for example, IL-8, IL-6, IL-15 or a combination thereof) levels do not spike after the first, second, third or fourth cycle of Trabedersen treatment are less than the median survival in subjects in whom the cytokine levels spike after the first, second, third or fourth cycle of Trabedersen treatment. In some embodiments, a poor prognosis is indicative of poor clinical outcome, wherein a poor clinical outcome comprises lower survival rates compared to the mean or median reported survival rates. In an exemplary embodiment, absence of a spike in the cytokine levels after 1st and 2nd cycle of Trabedersen treatment indicates that the therapy that includes sensitization with Trabedersen is not efficacious and is indicative of poor prognosis. In another exemplary embodiment, absence of a spike in the cytokine levels after 1st and 2nd and 3rd cycle of Trabedersen treatment indicates that the therapy that includes sensitization with Trabedersen is not efficacious and is indicative of poor prognosis. In an exemplary embodiment, absence of a spike in the cytokine levels after 1st and 2nd and 3rd and 4th cycle of Trabedersen treatment indicates that the therapy that includes sensitization with Trabedersen is not efficacious and is indicative of poor prognosis. In one embodiment, the spike is observed after treatment (sensitization) with Trabedersen. In some embodiments, the spike is observed after sensitization with Trabedersen and treatment with a chemotherapy agent.

[0055] "Good Prognosis" means the prospect of survival and recovery in subjects in whom the cytokine (for example, IL-8, IL-6, IL-15 or a combination thereof) levels spike after the first or second cycle of Trabedersen treatment are greater than or equal to the median survival in subjects in whom the cytokine levels do not spike after the first or second cycle of Trabedersen treatment. In some embodiments, a good prognosis is indicative of good clinical outcome, wherein a good clinical outcome comprises higher survival rates compared to the mean or median reported survival rates. In some embodiments, presence of a spike in the cytokine levels after the 1st cycle of Trabedersen treatment indicates that the therapy that includes sensitization with Trabedersen is efficacious and is indicative of good prognosis. In various embodiments, presence of a spike in the cytokine levels after the 2nd cycle of Trabedersen treatment indicates that the therapy that includes sensitization with Trabedersen is efficacious and is indicative of good prognosis. In various embodiments, presence of a spike in the cytokine levels after 1st or 2nd cycle of Trabedersen treatment indicates that the therapy that includes sensitization with Trabedersen is efficacious and is indicative of good prognosis. In various embodiments, presence of a spike in the cytokine levels after 1st and 2nd cycle of Trabedersen treatment indicates that the therapy that includes sensitization with Trabedersen is efficacious and is indicative of good prognosis. In various embodiments, the spike is observed after sensitization with Trabedersen and treatment with a chemotherapy agent.

[0056] "Diseases", "conditions" and "disease conditions," as used herein may include, but are in no way limited to any form of malignant neoplastic cell proliferative disorders or diseases. Examples of such disorders include but are not limited to cancer and tumor.

[0057] A "cancer" or "tumor" as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems, and/or all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant tumors, as well as dormant tumors or micrometastasis. Cancers

which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. As used herein, the term "invasive" refers to the ability to infiltrate and destroy surrounding tissue. Melanoma is an invasive form of skin tumor. As used herein, the term "carcinoma" refers to a cancer arising from epithelial cells. Examples of cancer include, but are not limited to, nervous system tumor, brain tumor, nerve sheath tumor, breast cancer, colorectal cancer, colon cancer, rectal cancer, bowel cancer, carcinoma, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer. Examples of brain tumor include, but are not limited to, benign brain tumor, malignant brain tumor, primary brain tumor, secondary brain tumor, metastatic brain tumor, glioma, glioblastoma, glioblastoma multiforme (GBM), medulloblastoma, ependymoma, astrocytoma, pilocytic astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma, mixed glioma such as oligoastrocytoma, low-grade glioma, high-grade glioma, supratentorial glioma, infratentorial glioma, pontine glioma, meningioma, pituitary adenoma, and nerve sheath tumor. Nervous system tumor or nervous system neoplasm refers to any tumor affecting the nervous system. A nervous system tumor can be a tumor in the central nervous system (CNS), in the peripheral nervous system (PNS), or in both CNS and PNS. Examples of nervous system tumor include but are not limited to brain tumor, nerve sheath tumor, and optic nerve glioma.

[0058] As used herein, the term "administering," refers to the placement of an agent or a composition as disclosed herein into a subject by a method or route which results in at least partial localization of the agents or composition at a desired site. "Route of administration" may refer to any administration pathway known in the art, including but not limited to oral, topical, aerosol, nasal, via inhalation, anal, intra-anal, peri-anal, transmucosal, transdermal, parenteral, enteral, or local. "Parenteral" refers to a route of administration that is generally associated with injection, including intratumoral, intracranial, intraventricular, intrathecal, epidural, intradural, intraorbital, infusion, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravascular, intravenous, intraarterial, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the agent or

composition may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the enteral route, the agent or composition can be in the form of capsules, gel capsules, tablets, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the topical route, the agent or composition can be in the form of aerosol, lotion, cream, gel, ointment, suspensions, solutions or emulsions. In an embodiment, agent or composition may be provided in a powder form and mixed with a liquid, such as water, to form a beverage. In accordance with the present invention, "administering" can be self-administering. For example, it is considered as "administering" that a subject consumes a composition as disclosed herein.

[0059] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. The terms, "patient", "individual" and "subject" are used interchangeably herein. In an embodiment, the subject is mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In addition, the methods described herein can be used to treat domesticated animals and/or pets.

[0060] "Mammal" as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

[0061] A "subject" can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., pancreatic cancer) or one or more complications related to the condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a

condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for a condition or one or more complications related to the condition or a subject who does not exhibit risk factors. For example, a subject can be one who exhibits one or more symptoms for a condition or one or more complications related to the condition or a subject who does not exhibit symptoms. A "subject in need" of diagnosis or treatment for a particular condition can be a subject suspected of having that condition, diagnosed as having that condition, already treated or being treated for that condition, not treated for that condition, or at risk of developing that condition.

[0062] The term "functional" when used in conjunction with "equivalent", "analog", "derivative" or "variant" or "fragment" refers to an entity or molecule which possess a biological activity that is substantially similar to a biological activity of the entity or molecule of which it is an equivalent, analog, derivative, variant or fragment thereof.

[0063] As used herein, the term "small molecule" refers to a chemical agent including, but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0064] A "nucleic acid", as described herein, can be RNA or DNA, and can be single or double stranded, and can be selected, for example, from a group including: nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA) etc. Such nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.

[0065] An "inhibitor" of TGFP, as the term is used herein, can function in a competitive or non-competitive manner, and can function, in one embodiment, by interfering with the expression of the TGFP protein. In one embodiment, the TGFP protein is TGFP2, having the

sequence set forth in SEQ ID NO: 2 (NM_003238.3). Any of a number of different approaches can be taken to inhibit TGFp (for example, TGFP2) expression or activity. A TGFP (for example, TGFP2) inhibitor includes any chemical or biological entity that, upon treatment of a cell, results in inhibition of the biological activity caused by activation of TGFp (for example, TGFP2) in response to cellular signals. TGFp (for example, TGFP2) inhibitors, include, but are not limited to, small molecules, antibodies or antigen-binding antibody fragments, intrabodies, aptamers, antisense constructs, RNA interference agents, and ribozymes. In an exemplary embodiments, a small molecule inhibitor of TGF β is a small molecule inhibitor of TGFP receptor type I kinase (ALK5; an inhibitor is an ALK5 inhibitor). In another exemplary embodiment, an antibody inhibitor of ΤΟΡβ is a neutralizing anti-TGF-beta-1, -2, -3 antibody or TGF-beta binding fragments thereof or a neutralizing anti-TGF-beta receptor type I,- type II or type III antibody or TGF-beta receptors binding fragments thereof or a TGF trap. In a further exemplary embodiment, an inhibitor of ΤΟΡβ is an antisense oligonucleotide specific for mRNA encoding TGF-beta 1, -2, and -3 isotypes or other components of TGF-beta signaling assembly optionally comprising a modified nucleoside such as 2 -0, 4 -C-methylene linked bicyclic ribonucleotides, known as locked nucleic acids LNA (e.g., oxy-LNA, amino-LNA, thio-LNA), phosphorodiamidate morphoHno oligomers (PMO), phosphorothioate (PS), 2 -0-methyl (2 -Ome), 2 -fiuoro (2 -fluoro (2 -F), or 2 -methoxyethyl (2 -MOE) derivatives. In an additional exemplary embodiment, an inhibitor of ΤίΐΡβ includes an antisense RNA molecule specific for TGF-beta2-mRNA like belagenpumatucei -L and/or TGF-betal -mRNA or TGF-beta3 -mRNA or other components of mRNA encoding TGF-beta signaling assembly. In a further exemplar}' embodiment, an inhibitor of ΤΟΡβ includes a silencing RNA molecule (siRNA) specific for mRNA encoding TGF -beta!, -2, and/or -3 isotypes or other components of TGF-beta signaling assembly. In an additional exemplar}' embodiment, an inhibitor of TGFP includes a short hairpin RNA (shRNA) specific for mRNA encoding TGF-betal, -2, and/or -3 isotypes or other components of TGF-beta signaling assembly. In an additional embodiment, an inhibitor of ΤΟΡβ includes a niiRNA molecule specific for mRNA encoding TGF-betal, -2, and/or -3 isotypes or other components of TGF-beta signaling assembly. In a further exemplary embodiment, an inhibitor of TGF β includes an aptamer and/or spiegelmer molecule specific for TGF-betal, -2, and/or -3 isotypes or other components of the TGF-beta signaling assembly. In a further exemplary embodiment, an inhibitor of Τ6Ρ'β includes a ribozyme molecule specific for mR A encoding TGF-betal, -2, and/or -3 isotypes or other components of the TGF-beta

signaling assembly. Antisense oligonucleotides (ASOs) are single-stranded polynucleotide molecules comprising 13-25 nucleotides, preferably 15-20 nucleotides, more preferred 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides, that hybridize to complementary R A, inhibiting mRNA function, and preventing protein synthesis for example through accelerated mRNA degradation by RNase H or steric blockade. In some embodiments, the TGFp2 inhibitor is an antisense oligonucleotide ASPH_0047 (from Isama Therapeutics as described in WO2014/154835) which is a selective LNA-modified ASO gapnier targeting TGF-p2. In an embodiment, ASPH 0047 (ISTH0047) has the sequence 5 ' -C AAAGTATTTGGTCTCC- 3', as set forth in SEQ ID NO: 3.

[0066] In additional embodiments, TGF signaling inhibitors for use with the methods described herein include but are not limited to antisense oligonucleotides from Isarna Therapeutics as described in WO2014/154835 having the sequence C AAAGTATTTGGTCTCC (ASPH47; SEQ ID NO: 3), ACCTCCTTGGCGTAGTA (ASPH01; SEQ ID NO: 4), ACCTCCTTGGCGTAGTA (ASPH02; SEQ ID NO: 5), CCTCCTTGGCGTAGTA (ASPH03; SEQ ID NO: 6), CCTCCTTGGCGTAGTA (ASPH04; SEQ ID NO: 7), CTCCTTGGCGTAGTA (ASPH05; SEQ ID NO: 8), CTCCTTGGCGTAGTA (ASPH06; SEQ ID NO: 9), CTCCTTGGCGTAGTA (ASPH07; SEQ ID NO: 10), TCCTTGGCGTAGTA (ASPH08; SEQ ID NO: 11), CAGAAGTTGGCAT (ASPH09; SEQ ID NO: 12), CAGAAGTTGGCAT (ASPH10; SEQ ID NO: 13), CTGCCCGCGGAT (ASPH15; SEQ ID NO: 14), TCTGCCCGCGGAT (ASPH17; SEQ ID NO: 15), TCGCGCTCGCAGGC (ASPH22; SEQ ID NO: 16), GGATCTGCCCGCGGA (ASPH26; SEQ ID NO: 17), GGATCTGCCCGCGGA (ASPH27; SEQ ID NO: 18), CGATCCTCTTGCGCAT (ASPH30; SEQ ID NO: 19), GGCGGGATGGCAT (ASPH35; SEQ ID NO: 20), GACCAGATGCAGGA (ASPH36; SEQ ID NO: 21), CTTGCTCAGGATCTGCC (ASPH37; SEQ ID NO: 22), TCTGTAGGAGGGC (ASPH45; SEQ ID NO: 23), CCTTAAGCCATCCATGA (ASPH48; SEQ ID NO: 24), TCTGAACTAGTACCGCC (ASPH65; SEQ ID NO: 25), TACTATTATGGCATCCC (ASPH69; SEQ ID NO: 26), AGCGTAATTGGTCATCA (ASPH71; SEQ ID NO: 27), GCGACCGTGACCAGAT (ASPH80; SEQ ID NO: 28), AACTAGTACCGCCTTT (ASPH82; SEQ ID NO: 29), GCGCGACCGTGACC (ASPH98; SEQ ID NO: 30), ACCACTAGAGCACC (ASPH105; SEQ ID NO: 31), AGCGCGACCGTGA (ASPH111; SEQ ID NO: 32), GGATCGCCTCGAT (ASPH112; SEQ ID NO: 33), CTAGTACCGCCTT (ASPH115; SEQ ID NO: 34), CCGCGGATCGCC (ASPH119; SEQ ID NO: 35),

GACCGTGACCAGAT (ASPH121 ; SEQ ID NO: 36), and/or GACCGTGACCAGAT (ASPH153; SEQ ID NO: 37). In various embodiment, one or more nucleotide(s) of the oligonucleotide set forth herein (for example, in SEQ ID Nos. 1-37) is/are a LNA modified, wherein the modified nucleotide is a LNA, and/or an ENA, polyalkylene oxide-, 2'-fluoro-, 2'- 0-methoxy-, and/or 2'0-methyl-modified nucleotide.

[0067] "Trabedersen" or "Trabedersen" as used herein refers to a transforming growth factor (TGF)-beta2 (TGF 2) specific phosphorothioate antisense oligodeoxynucleotide with the sequence 5'-CGGCATGTCTATTTTGTA-3', as shown in SEQ ID No: 1. In exemplary embodiments, inhibitors of TGF , including inhibitors of TGF 2 (such as Trabedersen) are described in W094/25588, WO95/17507, WO95/02051, WO98/33904, W099/63975, WO01/68146, WO01/68122, WO03/064457, WO2005/014812, WO2004/093945, WO2005/059133, WO2005/084712, WO2006/11740, WO2008/077956A2, WO2010/055148, WO2011/012713A1, WO2011/154542, EP application no. 20100191103, WO2014154835 A2, WO2015/140150A1, the contents of each of which are herein incorporated by reference. In one embodiment, Trabedersen is further LNA modified. A LNA is a modified RNA nucleotide, wherein the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon (2'- 4 'ribonucleoside). The bridge "locks" the ribose in the 3'-endo (North) conformation, which is often found in the A- form duplexes. LNA nucleosides and nucleotides, respectively, comprise for example the forms of thio-LNA, oxy-LNA, or amino-LNA, in alpha-D- or beta-L-configuration, and are mixable and combineable, respectively, with DNA or RNA residues in the oligonucleotide.

Prognostic Methods

[0068] Without wishing to be bound by a particular theory, the inventors found that a spike in the levels of certain cytokines (for example, IL-8, IL-6 and/or IL-15) after treatment with Trabedersen resulted in increased overall survival rate of the subject compared to subjects that did not show an early spike in the cytokine levels. The inventors found that subjects that showed a spike in the cytokine levels during the first cycle of Trabedersen treatment showed an increased overall survival rate. The inventors also found that subjects that showed a spike in the cytokine levels during the second but not the first cycle of Trabedersen treatment showed an increased overall survival rate. Further, the inventors found that subjects that did not show a spike in the cytokine levels during the first or second cycle of Trabedersen treatment showed a decreased overall survival rate.

[0069] Accordingly, provided herein are methods for prognosing cancer in a subject desiring cancer prognosis and methods for determining efficacy of sensitization treatment with Trabedersen. In various embodiments, the methods comprise, consist of or consist essentially of determining the levels of cytokines in a sample from a subject that has undergone or is undergoing Trabedersen treatment. In some embodiments, the cytokines are IL-8, IL-6, IL-15 or a combination thereof. In an embodiment, the sample is obtained after treatment with Trabedersen. In one embodiment, the subject has been diagnosed with cancer and is undergoing a first cycle of Trabedersen treatment to sensitize the tumor to a chemotherapeutic agent, wherein a spike in cytokine levels during the first cycle of Trabedersen treatment relative to a reference value is indicative of good prognosis. In an embodiment, a spike in cytokine levels during the first cycle of treatment comprising Trabedersen relative to a reference value indicates that the treatment is efficacious. In some embodiments, the spike in the cytokine levels is within 1-5 hours, 5-15 hours, 15-25 hours, 1-2 days, 2-3 days, 3-4 days, 4-5 days or 5-6 days after administration of Trabedersen. In an embodiment, the reference value is the level of cytokines (such as IL-8, IL-6, IL-15 or a combination thereof) in the subject prior to administration of Trabedersen.

[0070] Also provided herein are methods for prognosing cancer in a subject desiring cancer prognosis and methods for determining efficacy of sensitization treatment with Trabedersen. In various embodiments, the methods comprise, consist of or consist essentially of determining the levels of cytokines in a sample from a subject that has undergone or is undergoing Trabedersen treatment. In some embodiments, the cytokines are IL-8, IL-6, IL-15 or a combination thereof. In an embodiment, the sample is obtained after treatment with Trabedersen. In an embodiment, the subject has been diagnosed with cancer and is undergoing a first cycle of Trabedersen treatment to sensitize the tumor to a chemotherapeutic agent and has been treated with a chemotherapeutic agent, wherein a spike in the cytokine levels during the first cycle of Trabedersen treatment relative to a reference value is indicative of good prognosis. In an embodiment, a spike in the cytokine levels during the first cycle of treatment comprising Trabedersen relative to a reference value indicates that the treatment is efficacious. In some embodiments, the spike in the cytokine levels is within 1-5 hours, 5-15 hours, 15-25 hours, 1-2 days, 2-3 days, 3-4 days, 4-5 days or 5-6 days after administration of Trabedersen. In an embodiment, the reference value is the level of cytokines (such as IL-8, IL-6, IL-15 or a combination thereof) in the subject prior to administration of Trabedersen.

[0071] Further provided herein are methods for prognosing cancer in a subject desiring cancer prognosis and methods for determining efficacy of sensitization treatment with Trabedersen. In various embodiments, the methods comprise, consist of or consist essentially of determining the levels of cytokines in a sample from a subject that has undergone or is undergoing Trabedersen treatment. In some embodiments, the cytokines are IL-8, IL-6, IL-15 or a combination thereof. In an embodiment, the sample is obtained after treatment with Trabedersen. In one embodiment, the subject has been diagnosed with cancer and is undergoing a second cycle of Trabedersen treatment to sensitize the tumor to a chemotherapeutic agent, wherein a spike in cytokine levels during the second cycle of Trabedersen treatment relative to a reference value is indicative of good prognosis. In an embodiment, a spike in cytokine levels during the second cycle of treatment comprising Trabedersen relative to a reference value indicates that the treatment is efficacious. In one embodiment, the subject showing a spike during the second cycle of Trabedersen treatment did not show a spike in the cytokine levels during the first cycle of Trabedersen treatment. In some embodiments, the spike in the cytokine levels is within 1-5 hours, 5-15 hours, 15-25 hours, 1-2 days, 2-3 days, 3-4 days, 4-5 days or 5-6 days after administration of Trabedersen. In an embodiment, the reference value is the level of cytokines (such as IL-8, IL-6, IL-15 or a combination thereof), in the subject prior to administration of Trabedersen.

[0072] Also provided herein are methods for prognosing cancer in a subject desiring cancer prognosis and methods for determining efficacy of sensitization treatment with Trabedersen. In various embodiments, the methods comprise, consist of or consist essentially of determining the levels of cytokines in a sample from a subject that has undergone or is undergoing Trabedersen treatment. In some embodiments, the cytokines are IL-8, IL-6, IL-15 or a combination thereof. In an embodiment, the sample is obtained after treatment with Trabedersen. In an embodiment, the subject has been diagnosed with cancer and is undergoing a second cycle of Trabedersen treatment to sensitize the tumor to a chemotherapeutic agent and has been treated with a chemotherapeutic agent, wherein a spike in cytokine levels during the second cycle of Trabedersen treatment relative to a reference value is indicative of good prognosis. In an embodiment, a spike in cytokine levels relative to a reference value during the second cycle of treatment comprising Trabedersen indicates that the treatment is efficacious. In one embodiment, the subject showing a spike during the second cycle of Trabedersen treatment did not show a spike in the cytokine levels during the first cycle of Trabedersen treatment. In some embodiments, the spike in the cytokine levels is within 1-5 hours, 5-15 hours, 15-25 hours, 1-2 days, 2-3 days, 3-4 days, 4-5 days or 5-6 days after administration of Trabedersen. In an embodiment, the reference value is the level of cytokines (such as IL-8, IL-6, IL-15 or a combination thereof), in the subject prior to administration of Trabedersen.

[0073] Also provided herein are methods for prognosing cancer in a subject desiring cancer prognosis and methods for determining efficacy of sensitization treatment with Trabedersen. In various embodiments, the methods comprise, consist of or consist essentially of determining the levels of cytokines in a sample from a subject that has undergone or is undergoing Trabedersen treatment. In some embodiments, the cytokines are IL-8, IL-6, IL-15 or a combination thereof. In an embodiment, the sample is obtained after treatment with Trabedersen. In one embodiment, the subject has been diagnosed with cancer and is undergoing a second cycle of Trabedersen treatment to sensitize the tumor to a chemotherapeutic agent, wherein an absence of a spike in the cytokine levels during the second cycle of Trabedersen treatment relative to a reference value indicates that the treatment is not efficacious. In one embodiment, the subject has been diagnosed with cancer and is undergoing a second cycle of Trabedersen treatment to sensitize the tumor to a chemotherapeutic agent, wherein an absence of a spike in the cytokine levels during the second cycle of Trabedersen treatment relative to a reference value is indicative of poor prognosis. In an embodiment, the subject showing absence of a spike in cytokine levels relative to the reference value during the second cycle of treatment comprising Trabedersen also showed an absence of a spike in the cytokine levels during the first cycle of Trabedersen treatment. In an embodiment, absence of a spike in cytokine levels relative to a reference value during the first and second cycle of treatment comprising Trabedersen indicates that the treatment is not efficacious and the prognosis is poor. In some embodiments, the spike in the cytokine levels is within 1-5 hours, 5-15 hours, 15-25 hours, 1-2 days, 2-3 days, 3-4 days, 4-5 days or 5-6 days after administration of Trabedersen. In an embodiment, the reference value is the level of cytokines (such as IL-8, IL-6, IL-15 or a combination thereof), in the subject prior to administration of Trabedersen.

[0074] Further provided herein are methods for prognosing cancer in a subject desiring cancer prognosis and methods for determining efficacy of sensitization treatment with Trabedersen. In various embodiments, the methods comprise, consist of or consist essentially of determining the levels of cytokines in a sample from a subject that has undergone or is undergoing Trabedersen treatment. In some embodiments, the cytokines are IL-8, IL-6, IL-

15 or a combination thereof. In an embodiment, the sample is obtained after treatment with Trabedersen. In one embodiment, the subject has been diagnosed with cancer and is undergoing a second cycle of Trabedersen treatment to sensitize the tumor to a chemotherapeutic agent and has been treated with a chemotherapeutic agent, wherein an absence of a spike in cytokine levels during the second cycle of Trabedersen treatment relative to a reference value indicates that the treatment is not efficacious. In one embodiment, the subject has been diagnosed with cancer and is undergoing a second cycle of Trabedersen treatment to sensitize the tumor to a chemotherapeutic agent and has been treated with a chemotherapeutic agent, wherein an absence of a spike in cytokine levels during the second cycle of Trabedersen treatment relative to a reference value is indicative of poor prognosis. In an embodiment, the subject showing absence of a spike in cytokine levels relative to the reference value during the second cycle of Trabedersen treatment also showed an absence of a spike in the cytokine levels during the first cycle of Trabedersen treatment. In an embodiment, absence of a spike in cytokine levels relative to a reference value during the first and second cycle of treatment comprising Trabedersen indicates that the treatment is not efficacious and the prognosis is poor. In some embodiments, the spike in the cytokine levels is within 1-5 hours, 5-15 hours, 15-25 hours, 1-2 days, 2-3 days, 3-4 days, 4-5 days or 5-6 days after administration of Trabedersen. In an embodiment, absence of a spike in cytokine levels relative to a reference value during the first and second cycle of treatment comprising Trabedersen indicates that the treatment is not efficacious.

[0075] In various embodiments, the prognostic methods and methods for determining efficacy as described herein comprise the steps of obtaining a sample from a subject that is undergoing Trabedersen therapy and subsequently assaying the sample to determine the levels of cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) in a sample.

[0076] In some embodiments of the prognostic and efficacy methods described herein, determining the levels of cytokines in a sample from a subject that has undergone or is undergoing Trabedersen treatment comprises obtaining the results of assays performed on the sample to determine the levels of the cytokines in the sample.

[0077] Further provided herein are methods comprising administering Trabedersen to a subject having cancer so as to sensitize the tumor to one or more exogenous therapies as described herein and subsequently determining prognosis of the cancer and efficacy of the Trabedersen treatment by the methods described herein. In some embodiments, the

exogenous therapy comprises chemotherapy wherein the chemotherapeutic agents are any one or more of Paclitaxel, Dacarbazine or alkylating agents such as Mechlorethamine, Cyclophosphamide, Chlorambucil, Melphalan, and Ifosfamide.

[0078] Also provided herein are methods comprising administering Trabedersen to a subject having cancer so as to sensitize the tumor to one or more one or more exogenous therapies as described herein, subsequently administering one or more exogenous therapies and determining the cancer prognosis and efficacy to Trabedersen treatment by the methods described herein. In some embodiments, the exogenous therapy is chemotherapy and the chemotherapeutic agents are any one or more of Paclitaxel, Dacarbazine or alkylating agents such as Mechlorethamine, Cyclophosphamide, Chlorambucil, Melphalan, and Ifosfamide.

[0079] In one exemplary embodiment, the cancer is pancreatic cancer. In another exemplary embodiment, the cancer is a melanoma.

Biological Samples

[0080] In various embodiments, the sample is cheek swab; mucus; whole blood; blood; serum; plasma; urine; saliva; semen; lymph; fecal extract; sputum; other body fluid or biofluid; cell sample; or tissue sample; or a combination thereof. In various embodiments, the sample comprises a nucleic acid from the individual. In some embodiments, the nucleic acid comprises genomic DNA, or mitochondrial DNA, or both. In various embodiments, the sample is a body fluid. In some embodiments, the body fluid is whole blood, plasma, saliva, mucus, or cheek swab. In various embodiments, the sample is a cell or tissue. In some embodiments, the cell is a blood cell.

Subjects

[0081] In various embodiments, the subject is a human. In some embodiments, the subject is a child. In some embodiments, the subject is a teenager. In other embodiments, the subject is an adult. In various embodiments, the subject has been diagnosed with cancer. In some embodiments, the subject has been diagnosed with cancer and is undergoing sensitization treatment with Trabedersen for at least one, two, three, four or more cycles. In various embodiments, the subject is undergoing sensitization treatment with Trabedersen followed by exogenous therapy such as chemotherapy.

Reference Values

[0082] Various methods described herein may compare a subject's cytokine (for example, IL-8, IL-6, IL-15 or a combination thereof) to a pre-determined reference levels of cytokines.

[0083] In one embodiment, the reference cytokine level is the level of cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) in the subject (from whom the sample is obtained) prior to administration of the 1st, 2nd, 3rd, 4th or higher cycle Trabedersen. In one embodiment, the references cytokine level is the cytokine level in the subject (from whom the sample is obtained) prior to administration of first cycle of Trabedersen. In one embodiment, the references cytokine level is the cytokine level in the subject (from whom the sample is obtained) prior to administration of second cycle of Trabedersen. In one embodiment, the references cytokine level is the cytokine level in the subject (from whom the sample is obtained) prior to administration of third cycle of Trabedersen. In one embodiment, the references cytokine level is the cytokine level in the subject (from whom the sample is obtained) prior to administration of fourth or higher cycle of Trabedersen. Trabedersen.

[0084] In another embodiment, the reference cytokine level is the median or mean cytokine (for example, IL-8, IL-6, IL-15 or a combination thereof) level in the subject being tested wherein the cytokine level is from an earlier time point (for example, 1 day, 2 days, 5-10 days, 10-15 days, 15-30 days or more). In a further embodiment, the reference cytokine level is the median or mean cytokine (for example, IL-8, IL-6, IL-15 or a combination thereof) level in the subject being tested prior (for example, 1-5 hours, 5-10 hours, 10-15 hours, 15-20 hours or 1 day) to starting treatment with Trabedersen.

[0085] Reference values may be obtained by various methods known in the field. For example, one or more samples from one individual may be collected, processed and analyzed to obtain the individual's cytokine levels. The same step is used to obtain cytokine levels in another 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more individuals, that is, "cytokine-n" (n is 1, 2, 3, 4, 5, 6, 7, ...). Then, the median or mean of cytokine levels may be used as the reference cytokine levels, to which the subject's cytokine levels are compared to. In some embodiments, the cytokine are IL-8, IL-6, IL-15 or a combination thereof.

[0086] Various statistical methods, for example, a two-tailed student t-test with unequal variation, may be used to measure the differences between the subject's cytokine levels and a reference cytokine value generated by pooling many individuals of the same ancestry as the subject, as described herein. A significant difference may be achieved where the p value is equal to or less than 0.05.

[0087] In various embodiments, the expression level of cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) in the cancer subject compared to the reference value is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In various embodiments, the expression level of cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) in the cancer subject compared to the reference value is increased by at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold or a combination thereof. As described herein, an increase in the level of cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) relative to the reference value is indicative of good prognosis.

[0088] Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are the selection of constituent modules for the inventive methods, compositions, kits, and systems, and the various conditions, diseases, and disorders that may be diagnosed, prognosed or treated therewith. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

[0089] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0090] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Detection Methods

[0091] In various embodiments of the processes, assays and methods described herein, assaying for the levels of cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) comprises measuring the amount of nucleic acid encoding the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) present in the sample, measuring the amount of cytokine (for example, IL-8, IL-6, IL-15 or a combination thereof) proteins, or a combination thereof.

[0092] In various embodiments of the processes, assays and methods described herein, analyzing the sample includes detecting the level of cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) with an antibody specific to the said cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof). In various embodiments, the antibody is any one or more of a monoclonal antibody or fragment thereof, a polyclonal antibody or a fragment thereof, chimeric antibodies, humanized antibodies, human antibodies, and a single chain antibody. In an embodiment, the antibody is a monoclonal antibody.

[0093] In some embodiments of the processes, assays and methods described herein, analyzing the sample includes measuring the levels mRNA that encode the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof), present in the sample with a polynucleotide capable of hybridizing with mRNA specific for the said cytokines under stringent hybridization conditions.

[0094] Techniques that may be used to assess the amount of nucleic acid encoding the said cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof), present in the sample include but are not limited to in situ hybridization (e.g., Angerer (1987) Meth. Enzymol 152: 649). Preferred hybridization-based assays include, but are not limited to, traditional "direct probe" methods such as Southern blots or in situ hybridization {e.g., FISH and FISH plus

SKY), and "comparative probe" methods such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonucleotide-based CGH. The methods can be used in a wide variety of formats including, but not limited to, substrate (e.g. membrane or glass) bound methods or array-based approaches. Probes that may be used for nucleic acid analysis are typically labeled, e.g., with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. The preferred size range is from about 200 bases to about 1000 bases. Hybridization protocols suitable for use with the methods of the invention are described, e.g., in Albertson (1984) EMBO J. 3 : 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33 : In situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994), Pinkel, et al. (1998) Nature Genetics 20: 207-211, and/or Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992).

[0095] Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Measurement of DNA copy number at microsatellite loci using quantitative PCR anlaysis is described in Ginzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR may also be used in the methods of the invention. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and sybr green.

[0096] Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.

[0097] A two-tailed student t-test with unequal variation may be used to measure the differences between the patient's expression of the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) and a normal blood sample, or the patient's own blood (matched control), or a reference generate by computer algorithm pooling many control samples, as described herein. A significant difference may be achieved where the p value is equal to or less than 0.05. As described herein, the level of cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) after treatment with Trabedersen is be used to determine patient's prognosis where the expression of mRNA encoding the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) is separated into two groups: those with increased cytokine expression after treatment with Trabedersen and those with unchanged level of cytokine expression after treatment with Trabedersen. The groups may be separated by the median cytokine expression and plotted over time with a Kaplan-Meier curve.

[0098] Suitable methods for assaying the expression level of the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) include but are not limited to using DNA sequencing, comparative genomic hybridization (CGH), array CGH (aCGH), SNP analysis, mRNA expression assay, RT-PCR, real-time PCR, or a combination thereof. In various embodiments, the assay to detect the nucleic acid encoding or protein levels of, the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof), is any one or more of Northern blot analysis, Southern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), radio-immuno assay (RIA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis or a combination thereof.

[0099] Antibodies, both polyclonal and monoclonal, can be produced by a skilled artisan either by themselves using well known methods or they can be manufactured by service providers who specialize making antibodies based on known protein sequences. In the present invention, the protein sequences are known and thus production of antibodies against them is a matter of routine.

[0100] For example, production of monoclonal antibodies can be performed using the traditional hybridoma method by first immunizing mice with an antigen which may be an isolated protein of choice or fragment thereof (for example, the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof)) and making hybridoma cell lines that each produce a specific monoclonal antibody. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen using, e.g., ELISA or Antigen Microarray Assay, or immuno-dot blot techniques. The antibodies that are most specific for the detection of the protein of interest can be selected using routine methods and using the antigen used for immunization and other antigens as controls. The antibody that most specifically detects the desired antigen and protein and no other antigens or proteins are selected for the processes, assays and methods described herein.

[0101] The best clones can then be grown indefinitely in a suitable cell culture medium. They can also be injected into mice (in the peritoneal cavity, surrounding the gut) where they produce an antibody-rich ascites fluid from which the antibodies can be isolated and purified. The antibodies can be purified using techniques that are well known to one of ordinary skill in the art.

[0102] In the methods and assays of the invention, the change in the level of the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) after treatment with Trabedersen is determined using antibodies specific for the said cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) and detecting immunospecific binding of each antibody to its respective cognate marker.

[0103] Any suitable immunoassay method may be utilized, including those which are commercially available, to determine the level the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) measured according to the invention. Extensive discussion of the known immunoassay techniques is not required here since these are known to those of skill in the art. Typical suitable immunoassay techniques include sandwich enzyme-linked immunoassays (ELISA), radioimmunoassays (RIA), competitive binding assays, homogeneous assays, heterogeneous assays, etc. Various known immunoassay methods are reviewed, e.g., in Methods in Enzymology, 70, pp. 30-70 and 166-198 (1980).

[0104] In the assays of the invention, "sandwich-type" assay formats can be used. Some examples of such sandwich-type assays are described in by U.S. Pat. No. 4, 168,146 to Grubb, et al. and U.S. Pat. No. 4,366,241 to Tom, et al. An alternative technique is the "competitive-type" assay. In a competitive assay, the labeled probe is generally conjugated with a molecule that is identical to, or an analog of, the analyte. Thus, the labeled probe competes with the analyte of interest for the available receptive material. Competitive assays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are described in U.S. Pat. No. 4,235,601 to Deutsch, et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler, et al.

[0105] The antibodies can be labeled. In some embodiments, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, label with a chemiluminescent compound. For example, the detection antibody can be labeled with catalase and the conversion uses a colorimetric substrate composition comprises potassium iodide, hydrogen peroxide and sodium thiosulphate; the enzyme can be alcohol dehydrogenase and the conversion uses a colorimetric substrate composition comprises an alcohol, a pH indicator and a pH buffer, wherein the pH indicator is neutral red and the pH buffer is glycine-sodium hydroxide; the enzyme can also be hypoxanthine oxidase and the conversion uses a colorimetric substrate composition comprises xanthine, a tetrazolium salt and 4,5-dihydroxy-l,3-benzene disulphonic acid. In one embodiment, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, or label with a chemiluminescent compound.

[0106] Direct and indirect labels can be used in immunoassays. A direct label can be defined as an entity, which in its natural state, is visible either to the naked eye or with the aid of an optical filter and/or applied stimulation, e.g., ultraviolet light, to promote fluorescence. Examples of colored labels which can be used include metallic sol particles, gold sol particles, dye sol particles, dyed latex particles or dyes encapsulated in liposomes. Other direct labels include radionuclides and fluorescent or luminescent moieties. Indirect labels such as enzymes can also be used according to the invention. Various enzymes are known for use as labels such as, for example, alkaline phosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and urease. For a detailed discussion of enzymes in immunoassays see Engvall, Enzyme Immunoassay ELISA and EMIT, Methods of Enzymology, 70, 419-439 (1980).

[0107] The antibody can be attached to a surface. Examples of useful surfaces on which the antibody can be attached for the purposes of detecting the desired antigen include nitrocellulose, PVDF, polystyrene, and nylon. The surface or support may also be a porous support (see, e.g., U.S. Patent No. 7,939,342). The assays can be carried out in various assay device formats including those described in U.S. Pat. Nos. 4,906,439; 5,051,237 and 5, 147,609 to PB Diagnostic Systems, Inc.

[0108] In some embodiments of the processes, assays and methods described herein, detecting the level of antibodies reactive to the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) includes contacting the sample from the cancer patient with an antibody or a fragment thereof that specifically binds to the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof), forming an antibody-protein complex between the antibody and the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) present in the sample, washing the sample to remove the unbound antibody, adding a detection antibody that is labeled and is reactive to the antibody bound to to the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) in the sample, washing to remove the unbound labeled detection antibody and converting the label to a detectable signal, wherein the detectable signal is indicative of the level of to the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) in the sample from the patient. In some embodiments, the effector component is a detectable moiety selected from the group consisting of a fluorescent label, a radioactive compound, an enzyme, a substrate, an epitope tag, electron-dense reagent, biotin, digonigenin, hapten and a combination thereof. In some embodiments, the detection antibody is labeled by covalently linking to an enzyme, labeled with a fluorescent compound or metal, labeled with a chemiluminescent compound. The level of to the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) may be obtained by measuring a light scattering intensity resulting from the formation of an antibody-protein complex formed by a reaction of the cytokines (for example, IL-8, IL-6, IL-15 or a combination thereof) in the sample with the antibody, wherein the light scattering intensity of at least 10% above a control light scattering intensity indicates the likelihood of chemotherapy resistance.

[0109] In various embodiments of the processes, assays and methods of the invention, an increased likelihood of chemotherapy resistance may result in poor prognosis wherein the poor prognosis comprises decreased survival likelihood, shortened life expectancy, or enhanced tumor sternness.

[0110] In various embodiments of the processes, assays and methods of the invention, the process described herein further comprises prescribing a first therapy to the subject if the subject has a good prognosis or prescribing a second therapy, or both the first therapy and the second therapy, to the subject if the subject has a poor prognosis.

Therapies

[0111] In various embodiments, the methods described herein further comprise selecting or prescribing a therapy to the subject wherein it has been determined that the subject has poor prognosis. In various embodiments, the methods described herein further comprise selecting or prescribing a therapy to the subject wherein it has been determined that the subject has good prognosis.

[0112] In some embodiments of the methods described herein, Trabedersen or a variant, derivative or analog thereof is administered intravenously via continuous infusion for one, two, three, four, five or more cycles of 7 days and on 7 days off, prior to administration of a chemotherapeutic agent or radiation therapy. In some embodiments, Trabedersen or a variant, derivative or analog thereof is administered intravenously via continuous infusion for one, two, three, four, five or more cycles of 4 days on and 10 days off, prior to administration of a chemotherapeutic agent or radiation therapy. In one embodiment, Trabedersen or a variant, derivative or analog thereof is administered intravenously via continuous infusion for one cycle of 4 days on and 10 days off, prior to administration of a chemotherapeutic agent or radiation therapy. In one embodiment, Trabedersen or a variant, derivative or analog thereof is administered intravenously via continuous infusion two cycles of 4 days on and 10 days off, prior to administration of a chemotherapeutic agent or radiation therapy. In one embodiment, Trabedersen or a variant, derivative or analog thereof is administered intravenously via continuous infusion three cycles of 4 days on and 10 days off, prior to administration of a chemotherapeutic agent or radiation therapy. In one embodiment, Trabedersen or a variant, derivative or analog thereof is administered intravenously via continuous infusion four cycles of 4 days on and 10 days off, prior to administration of a chemotherapeutic agent or radiation therapy.

[0113] In various embodiments of the methods described herein, the chemotherapeutic agent for use with the methods described herein include but are not limited to Temozolomide, Actinomycin, Alitretinoin, All-trans retinoic acid, Azacitidine, Azathioprine, Bevacizumab, Bexatotene, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cetuximab, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Dacarbazine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, liposome-encapsulated Doxorubicin such as as Doxil (pegylated form), Myocet (nonpegylated form) and Caelyx, Epirubicin, Epothilone, Erlotinib, Etoposide, Fluorouracil, Folinic acid, Gefitinib, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Imatinib, Ipilimumab, Irinotecan, Nanoliposomal Irinotecan (Nal-IRI), Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Ocrelizumab, Ofatumumab, Oxaliplatin, Paclitaxel, Taxol, Abraxane, Genexol, Protein-Bound Paclitaxel, Nab-Paclitaxel, Panitumab, Pemetrexed, Rituximab, Tafluposide, Teniposide, Tioguanine, Topotecan, Tretinoin, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine,

Vorinostat, Romidepsin, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), Cladribine, Clofarabine, Floxuridine, Fludarabine, Pentostatin, Mitomycin, ixabepilone, Estramustine, prednisone, methylprednisolone, dexamethasone or a combination thereof.

[0114] In various embodiments of the methods described herein, exogenous therapies for use with the methods described herein include radiation therapy. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., J. B. Lippencott Company, Philadelphia. The radiation therapy can be administered as external beam radiation or tele-therapy wherein the radiation is directed from a remote source. The radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Also encompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and 2BA-2-DMHA.

[0115] In various embodiments of the methods described herein, exogenous therapies for use with the methods described herein include hormonal therapy. Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

[0116] As described herein, typical dosages of an effective amount of the TGF inhibitor (for example, TGF 2 inhibitor) can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about an order of magnitude in concentration or amount without losing relevant biological activity. The actual dosage can depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of relevant cultured cells or histocultured tissue sample, such as biopsied malignant tumors, or the responses observed in the appropriate animal models. In various embodiments, the compositions of the invention comprising the TGF inhibitor (for example, TGF 2 inhibitor) may be administered once a day (SID/QD), twice a day (BID), three times a day (TID), four times a day (QID), or more, so as to administer an effective amount of the TGF inhibitor (for example, TGF 2 inhibitor) to the subject, where the effective amount is any one or more of the doses described herein.

[0117] In various embodiments, the subject is selected from the group consisting of human, non-human primate, monkey, ape, dog, cat, cow, horse, rabbit, mouse and rat.

[0118] Typical dosages of an effective amount of the TGFP signaling inhibitor (for example, TGF 2 inhibitor or a composition comprising TGF 2 inhibitor) for use with methods of the invention are described herein. These dosages can be in the ranges recommended by the manufacturer where known molecules or compounds are used, and also as indicated to the skilled artisan by the in vitro responses in cells or in vivo responses in animal models. Such dosages typically can be reduced by up to about an order of magnitude in concentration or amount without losing relevant biological activity. The actual dosage can depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of relevant cultured cells or histocultured tissue sample, or the responses observed in the appropriate animal models.

[0119] In various embodiments, the TGFP signaling inhibitor is administered at about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 mg inhibitor/kg body weight, or a combination thereof. In various embodiments, the TGFP signaling inhibitor is administered at about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 mg inhibitor/m2 body surface area, or a combination thereof. Here, "mg inhibitor/kg body weight" refers to mg inhibitor per kg body weight of the subject, and "mg inhibitor/m2 body surface area" refers to mg inhibitor per m2 body surface area of the subject.

[0120] In various embodiments, the effective amount of the TGFP signaling inhibitor is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 μg/kg/day, or a combination thereof. In various embodiments, the effective amount of the TGFP signaling inhibitor is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 μg/m2/day, or a combination thereof. In various embodiments, the effective amount of the TGFP signaling inhibitor is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 mg/kg/day, or a combination thereof. In various embodiments, the effective amount of the TGFP signaling inhibitor is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 mg/m2/day, or a combination thereof. Here, '^g/kg/day" or "mg/kg/day" refers to μg or mg inhibitor per kg body weight of the subject per day, and '^g/m2/day" or "mg/m2/day" refers to μg or mg inhibitor per m2 body surface area of the subject per day.

[0121] In various embodiments, the TGFP signaling inhibitor is Trabedersen (AP12009) or a variant, derivative or analog thereof, and it is administered at about 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 mg/m2/day. In some embodiments, Trabedersen or a variant, derivative or analog thereof is administered at about 140, 150, 160, 170, or 180mg/m2/day. In some embodiments, Trabedersen or a variant, derivative or analog thereof is administered at about 310, 320, 330, 340, or 350mg/m2/day.

[0122] In various embodiments, the TGFP signaling inhibitor may be administered once, twice, three or more times. In various embodiments, the TGFP signaling inhibitor may be administered 1-3 times per day, 1-7 times per week, 1-9 times per month, or 1-12 times per year. In various embodiments, the TGFP signaling inhibitor may be administered for about 1-10 days, 10-20 days, 20-30 days, 30-40 days, 40-50 days, 50-60 days, 60-70 days, 70-80 days, 80-90 days, 90-100 days, 1-6 months, 6-12 months, or 1-5 years. In various embodiments, the TGFP signaling inhibitor may be administered once a day (SID/QD), twice a day (BID), three times a day (TID), four times a day (QID), or more, so as to deliver an effective amount of the TGFP signaling inhibitor to the subject, where the effective amount is any one or more of the doses described herein.

EXAMPLES

[0123] The invention will be further explained by the following Examples, which are intended to be purely exemplary of the invention, and should not be considered as limiting the invention in any way. The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1

Cytokine induction by Trabedersen (OT-101) treatment - results from a phase I/II trial in pancreatic cancer patients

[0124] Without being bound to a particular theory, the inventors hypothesize that cytokines, especially IL-6 and IL-8, play important roles in immune mediated antitumor response. These cytokines should increase with elimination of immunosuppression activity of TGF- β2 via Trabedersen treatment.

[0125] Overexpression of the transforming growth factor-beta 2 (TGF-P2) has been suggested as a pivotal factor for malignant progression of pancreatic carcinogenesis. OT-101 (Trabedersen) is an 18-mer phosphorothioate antisense oligodeoxynucleotide designed to specifically target the human TGF-P2 messenger RNA. In this phase I/II trial of advanced pancreatic cancer patients, we examined the decloaking of the pancreatic tumors by anti-TGF-P2 antisense oligonucleotide.

[0126] A total of 37 patients with advanced pancreatic cancer were treated with OT-101 by intravenous infusion. Pearson pairwise correlation coefficients (r) of 19 cyto/chemokine levels across all time points and 12 patients were utilized to construct a two-way hierarchical correlation cluster figure to identify co-regulated cyto-/chemokines. Dose-dependent effects of OT-101 on the cyto-/chemokine response to LPS/SE-B stimulated and non-stimulated human whole-blood cultures was quantitated using Luminex platform. Xenograft studies were conducted to compare IL-6 and IL-8 inducing Dacarbazine and Paclitaxel monotherapy versus OT-101 combination therapy to demonstrate a synergistic effect.

[0127] MTD for 7/7 regimen (N=l l) was 160 mg/m2/d; whereas MTD was not reached on 4/10 regimen (N=27) even at highest dose of 330 mg/m2/d. There was 11 2nd line patients and 16 3rd line and beyond on 4/10 regimen. The OS of this 2nd line 4/10 cohort treated with subsequent chemotherapies was 14.5 mos and 2.90 mos with and without subsequent chemotherapies, p = 0.0023. Correlation analysis identified 3 distinct positively correlated clusters, one of which consisted of IL-6, IL-8, HGF, IL-2R (r ranged from 0.34 (P = 0.002) to 0.65 (p < 0.0001)). Both Paclitaxel and Dacarbazine were shown to exhibit drug induced IL-8 and IL-6 expression. Trabedersen synergized with these agents when tested in xenograft tumor model.

[0128] These results demonstrate that IL-6 and IL-8 are potentially important prognostic and/or diagnostic biomarkers for Trabedersen and chemo combination therapy, wherein Trabedersen is administered prior to chemotherapy for at least one cycle. Patients treated with immune-checkpoint drug ipilimumab following Trabedersen have a trend to improved OS

Example 2

Impact of Antisense OT-101 (Trabedersen) Therapy on the Cytokine and Chemokine Profile in Patients with Pancreatic Cancer

[0129] Overexpression of the transforming growth factor-beta 2 (TGF-P2) has been suggested as a pivotal factor for malignant progression of pancreatic carcinogenesis. OT-101 (Trabedersen) is an 18-mer phosphorothioate antisense oligodeoxynucleotide designed to specifically target the human TGF-P2 messenger RNA. In a Phase I/II study, OT-101 treatment was characterized by outstanding overall survival in pancreatic cancer (PAC) patients. In this study, we sought to identify (i) co-regulated sets of cytokine/chemokines; (ii) identify potential mechanisms that link TGF R2 receptor inhibition and; (iii) examine patient to patient variability in the response profiles for cyto-/chemokine signatures in OT-101-treated PAC patients.

[0130] Time evolution of a panel of 31 cytokine/chemokine levels in plasma were tracked over a period of 11 days following intravenous OT-101 therapy (140 mg/m2/day) for 12 PAC patients. Samples were acquired before onset of OT-101 therapy and at selected time points (cycle 1 [day 2 and 5], cycle 2 [days 15, 16, and 19], cycle 3 [day 5]) during the therapy. Samples were measured in duplicate and concentrations were expressed in pg/ml.

[0131] Clustering of correlation coefficients resulted in the identification of three highly correlated subsets of cyto-/chemokines (Cluster 1 : EGF, CCL3/MIP-la, CCL4/MIP-1□; Cluster 2: FGF-2, CXCL9/MIG, CXCLlO/IP-10, IL-15, IFNAl/IFN-a, IL-12A/IL-12; Cluster 3 : HGF, IL-6, IL-8). A cluster of 4 out of the 12 patients exhibited a consistent increase in ΜΙΡ-Ια and IL-Ιβ. The mixed ANCOVA model explained a significant proportion of the observed data for the 19 cyto-/chemokines. Significant differences were observed between cytokine levels (P<0.0001), and levels of cytokines across time points (P = 0.0002) with no interaction between cytokine levels and regression across time points (P = 0.398). Examination of the parameter estimates of the model across time points to identify individually affected cyto/chemokines showed that only EGF increased significantly across time points (P = 0.0097). Though there was a trend of increase observed among 13 of the 19 cytokines examined.

[0132] The cluster analysis of standardized expression levels stratified across patient and 31 cytokine/chemokines did not reveal an overt signature for the emergence of a cytokine/chemokine pattern following OT-101 therapy, except for the EGF profile identified from the regression model.

Example 3

[0133] Cytokines- especially IL-6 and IL-8- play important roles in immune mediated antitumor response. These cytokines should increase with elimination of immunosuppression activity of TGF- β2 via Trabedersen treatment. Transforming growth factor-beta (TGF-β) -comprising 3 different, though closely related, isoforms: TGF- βΐ, β2 and β3 - is a crucial factor promoting tumor malignancy in advanced tumors via multiple pathological mechanisms. TGF-β is the most potent immunosuppressor described to date. Overexpression of TGF^2 has been suggested as a pivotal factor for malignant progression of pancreatic carcinogenesis. OT-101 (Trabedersen) is an antisense oligodeoxynucleotide undergoing clinical development by OncoTelic Inc for the treatment of TGF^2 overexpressing tumors. OT-101 is a synthetic 18-mer phosphorothioate oligodeoxynucleotide complementary to the messenger ribonucleic acid (mRNA) of the human TGF^2 gene. In this phase I/II trial of advanced pancreatic cancer patients, we examined the decloaking of the pancreatic tumors by anti-TGF^2 antisense oligonucleotide to allow for deep sustained overall survival (OS) as well as favorable changes in cytokine profile - especially interleukin-6 (IL-6) and IL-8. [0134] A total of 37 patients with advanced pancreatic cancer (PAC) were treated with OT-101 by IV infusion. A panel of 31 cyto-/chemokine levels in plasma were tracked for 1 1 days following OT-101 therapy (140 mg/m2/day) for 12 PAC patients. Pearson pairwise correlation coefficients (r) of 19 cyto-/chemokine levels across all time points and 12 patients were utilized to construct a two-way hierarchical correlation cluster figure to identify co-regulated cyto-/chemokines. Effects of OT-101 on the cytokine/chemokine response to LPS/SE-B stimulated and non-stimulated human whole-blood cultures was quantitated using Luminex platform. Xenograft studies compared IL-6 and IL-8 inducing Dacarbazine and Paclitaxel monotherapy versus OT-101 combination therapy to demonstrate a synergistic effect, when Trabedersen is administered prior to the chemotherapeutic agent.

[0135] MTD for 7/7 regimen (N=l l) was 160 mg/m2/d; whereas MTD was not reached on 4/10 regimen (N=27) even at highest dose of 330 mg/m2/d. There was 1 1 2nd line patients and 16 3rd line and beyond on 4/10 regimen. The OS of this 2nd line 4/10 cohort treated with subsequent chemotherapies was 14.5 and 2.90 mos with and without subsequent chemotherapies, p = 0.0023. (Fig. 4)

Demographics

Patient Characteristic 7-days-on, 7-days-off 4-days-on, 10-days-off Total

N = 11 N = 26 N = 37

Gender [N (%)]

Male 5 (45.6) 12 (46.2) 17 (45.95)

Female 6 (54.6) 14 (53.9) 20 (54.05)

Age

Mean ± SD 61.0 ± 8.6 60.9 ± 9.8 60.9 ± 9.3

Median (Ql, Q3) 61.0 (58.5, 65.5) 63.5 (54.3. 66.8) 64.0 (55.0, 66.0)

Weight

Mean ± SD 65.7 ± 15.6 65.3 ± 11.4 65.4 ± 12.6

Median (Ql, Q3) 64.0 (56.0, 74.0) 63.0 (55.3, 72.0) 63.0 (55.0, 73.0)

Treatment Line [N (%)]

2 4 (36.4) 11 (42.31) 15 (40.54)

3 5 (45.5) 9 (34.62) 14 (37.84)

4 1 (9.1) 4 (15.38) 5 (13.51)

5 1 (9.1) 1 (3.85) 2 (5.41)

6 0 (0.00) 1 (3.85) 1 (2.70)

Extent of Patient Exposure to Trabedersen

No of cycles

Treatment Schedule No of patient t>diiH!

Cohort Total fmin; mas)

7-days-on, 7-days-off

Cohort 1: 40 rag/ray day 2.0 (1; 4)

Cohort 2: 80 rag/iir'/day 5.0 (4; 10)

Cohort 3: 160 mg/mVday 2.5 (1; 4)

Cohort 4: 240 mg m:7day 1,5 U; 2)

4-days-on, 10-days-off

Cohort Ai: 140 mg./m'/da'v 4.0 (1; 10)

Cohort A2: 190 mg/m'/da 4.0 (2; 8)

Cohort A3: 250 tng rn!/da> 4.0 (1: 10)

Cohort A4: 330 mg/nryda' 2.0 (2; 10)

4-days-on, 10-days-off

140 mg/m-/day 4.0 (1; 8)

[0136] Clustering of correlation coefficients resulted in the identification of three highly correlated subsets of cyto/chemokines, one of which consisted of IL-6, IL-8, HGF, IL-2R (r ranged from 0.34 (P = 0.002) to 0.65 (p < 0.0001)). Protein-Protein interaction networks were constructed using STRINGIO algorithm (http://string-db.org/) for co-regulated cyto/chemokines to identify potential hub proteins connecting TGFBR2 pathways to cyto/chemokines. IL-8 is linked to TGF R2 (Fig. 4A-Fig. 4D).

[0137] Both Paclitaxel and Dacarbazine were shown to exhibit drug-IL-8/CXCL8 (16 and 4 associations with Paclitaxel [PTX] and Dacarbazine [DTIC], respectively) and drug-IL6 interactions (2 associations for Paclitaxel and Dacarbazine) identified using text-mining in CTDbase (http://ctdbase.org/). Of 718 genes affected by Paclitaxel and 25 genes affected by Dacarbazine - 17 were affected by both agents including IL-6 and IL-8. Of the 222 genes affected by Temozolomide (TZM) - 52 were affected by both Temozolomide and Paclitaxel-including IL-6. Trabedersen synergized with these agents when tested in three xenograft tumor models with enhanced anti-tumor activity and prolonged survival. (Fig 4E)

[0138] IL-6 and IL-8 are potentially important prognostic and/or diagnostic biomarkers for Trabedersen + chemo combinations. Patients treated with immune-checkpoint drug ipilimumab following Trabedersen have a trend to improved OS. Additional clinical trial will be conducted to further explore cytokine expression and Trabedersen and Ipilimumab combination therapy, wherein Trabedersen is administered prior to the chemotherapeutic agent for at least one cycle.

Example 4

[0139] Transforming growth factor-beta (TGF-β) - comprising 3 different, though closely related, isoforms: TGF- βΐ, β2 and β3 - is a crucial factor promoting tumor malignancy in advanced tumors via multiple pathological mechanisms. TGF-β is the most potent immunosuppressor described to date. Overexpression of TGF^2 has been suggested as a pivotal factor for malignant progression of pancreatic carcinogenesis. OT-101 (Trabedersen) is a novel antisense oligodeoxynucleotide undergoing clinical development by Oncotelic Inc for the treatment of TGF^2 overexpressing tumors. OT-101 is a synthetic 18-mer phosphorothioate oligodeoxynucleotide complementary to the messenger ribonucleic acid (mRNA) of the human TGF^2 gene. In a Phase I/II study, OT-101 treatment was characterized by outstanding overall survival in pancreatic cancer (PAC) patients. In this study, we sought to identify (i) co-regulated sets of cyto-/chemokines; (ii) identify potential mechanisms that link TGFBR2 receptor inhibition and; (iii) identify correlations between cyto-/chemokines with OS to determine potential biomarkers for OT-101 treatment. Increased expression of IL-8 and/or its receptors has been characterized in cancer cells, endothelial cells, infiltrating neutrophils, and tumor-associated macrophages, suggesting that IL-8 may function as a significant regulatory factor within the tumor microenvironment. Herein, we examined the role of IL-8 in OT-101 treatment followed with chemotherapies during our Phase II trial in patients with advanced pancreatic cancer (PAC).

[0140] Time evolution of a panel of 31 cyto-/chemokine levels in plasma were tracked over 3 cycles of OT-101 administration (140 mg/m2/day) for 12 PAC patients. Samples were acquired before onset of OT-101 therapy and at 8 selected time points (Cycle 1 [Day 2 and 5], Cycle 2 [Days 1, 2, and 5], Final Visit, Cycle 3 [Day 5]) during the therapy. Samples were measured in duplicate and concentrations were expressed in pg/mL. Standardized log 10 transformed values calculated from the mean and standard deviation of each cyto-/chemokine in each patient was utilized in an ANCOVA model to investigate the correlation with Overall Survival (OS).

[0141] Clustering of correlation coefficients resulted in the identification of three highly correlated subsets of cyto-/chemokines (Cluster 1 : EGF, MIP-la, MIP-Ιβ; Cluster 2: FGF-2, IL-1RA, MIG, IP-10, IL-15, IFN-a, IL-12A/IL-12; Cluster 3 : HGF, IL-2R, IL-6, IL-8). Protein-Protein Interaction networks constructed using STRING10 algorithm identified a relationship between IL-8 and ΤΟΡβΡ2 inhibition. The ANCOVA model explained a

significant proportion of the observed data for Cycle l [Day 2] measurements of cyto-/chemokines (R2 = 0.3, F59 217 = 1.575, P = 0.0103). Other time points did not exhibit a significant model fit or significant relationships in the interaction term. IL-8 expression showed significant association with OS (positive association, N=12, T-value = 2.92, P = 0.0039) at Cycle l [Day 2] measurements (7% False Discovery Rate). Linear regression of the increase in IL-8 levels during Cycle l [Day 2] treatment with OT-101 was related to an increase in OS outcome (R2 = 0.54). To further define the appropriate chemoagent to combine with OT-101, we evaluated paclitaxel (PTX), gemcitabine (GEM), and dacarbazine (DTIC) in animal model. Synergy was observed with PTX and DTIC, but not with GEM. Both PTX and DTIC induced IL-8 expression whereas, surprisingly, GEM reduced IL-8 expression.

[0142] IL-8 expression during early phase of OT-101 treatment cycle was positively associated with OS across 12 patients. IL-8 is also induced by PTX and DTIC which synergized with OT-101. The data suggests that PAC and DTIC are acting on the same target as our TGF- 2 inhibitor; and potentially, the beneficial effect of these chemoagents is a result of chemoagent-induced immune/IL-8 response. (Fig. 5A- Fig. 5B).

[0143] Cluster analysis and Protein-Protein Interaction networks identified a relationship between IL-8 and TGF-β inhibition. An ANCOVA model developed to investigate the association of cytokine levels with OS identified IL-8 as a significant covariate. Increasing difference Day 2 and Day 5 IL-8 levels showed significant correlation with OS when stratifying by treatment with (R2 = 0.99) or without (R2 = 0.77) chemotherapy following OT-101 treatment. Analysis of all cytokines show positive correlations with IFNa, IL-12, IL-15, IL-6, IP-10, and IL-8. However, accurate prediction is observed only from IL-15 and IL-8 where fit lines intersect when OS = 0.

Example 5

[0144] Transforming growth factor-beta (TGF-β) - comprising 3 different, though closely related, isoforms: TGF- βΐ, β2 and β3 - is a crucial factor promoting tumor malignancy via multiple pathological mechanisms. TGF-β is the most potent immunosuppressor described to date. Overexpression of TGF^2 has been suggested as a pivotal factor for malignant progression by inducing immunosuppression, metastasis, angiogenesis and proliferation OT-101 (Trabedersen) is a novel antisense oligonucleotide under clinical development for the treatment of TGF^2 overexpressing tumors. OT-101 is a synthetic 18-mer phosphorothioate oligodeoxynucleotide complementary to the mRNA of the human TGF^2 gene. In vivo

pharmacology studies were conducted in mice to provide additional preclinical evidence to support ΟΤ-101/chemotherapy combinations. The tumor models were selected based on intended initial clinical indications and readiness of TGF-P2 expression/detection. Herein, we report the synergizing effect of OT-101 with chemotherapy in multiple human tumor xenograft models for further exploration of clinical combinations.

[0145] The in vivo efficacy studies of intraperitoneal (IP, 16 or 50 mg/kg) or subcutaneous (SC, 1-64 mg/kg) repeated administration (qdx3/wk or qdx21) of OT-101 as single agent and in combination with Gemcitabine (GEM, 15 mg/kg, qdx2/wk, IP), Dacarbazine (DTIC, 1-10 mg/kg, qdx4/wk, IP) or Paclitaxel (PTX, 10 mg/kg, qdx5, IV) were evaluated in nude mice (10/subgroup) bearing either (i) orthotopic human L3.6pl pancreatic cancer (PAC), (ii) human metastatic C8161 melanoma, (iii) SC glioblastoma (U87-MG) or (iv) SC ovarian (SKOV-3) tumors. Mice were monitored thrice a week for adverse effects, body weight loss, tumor size and survival outcome. The incidence of lymph node and liver surface and micro-metastases as well as size and weight of the pancreatic tumors were determined. Tumor sections were stained with anti-5-bromo-2'-deoxy-uridine antibody (anti-BrdU) to determine tumor cell proliferation and with anti-CD31/PECAM-l antibody to determine vascularization.

Orthotopic and Subcutaneous Xenograft Tumor Models

[0146] Intraperitoneal (IP, 16 or 50 mg/kg) or subcutaneous (SC, 1-64 mg/kg) repeated administration (qdx3/wk or qdx21) of OT-101 as single agent and in combination with GEM (15 mg/kg, qdx2/wk, IP), DTIC (1-10 mg/kg, qdx4/wk, IP) or PTX (10 mg/kg, qdx5, IV) were evaluated in nude mice (10/subgroup) bearing either orthotopic or subcutaneous tumors.

Orthotopic human L3.6pl pancreatic cancer (PAC) model

[0147] In brief, 1 x 106 human pancreatic cancer cells (L3.6pl) were injected into the pancreatic tail. Eight-week-old male athymic nude mice were randomized into groups (n = 9-10 per group) receiving either vehicle (controls) or OT-101. Treatment with OT-101 by IP injection was started 2 days after tumor implantation with an initial loading dose of 50 mg/ kg. For further treatment, OT-101 was administered at doses of 16 mg/kg three times a week. On day 29 after tumor inoculation, the mice were killed. The incidence of liver and lymph node metastases was determined and pancreatic tumors were measured and weighed.

Human metastatic C8161 melanoma model

[0148] Sixty female athymic nude mice were intradermally inoculated with 0.5 x 106 C8161 human melanoma cells and randomized into six groups of 10 mice. Three groups received monotherapy treatment with either OT-101 (16 mg/kg) (Group 2) or DTIC (1 or 10 mg/kg; Groups 3, 4). Two groups received combination therapy with OT-101/DTIC at 16/1 mg/kg or 16/10 mg/kg (Groups 5 and 6). Vehicle (0.9% saline, Groups 1, 3, and 4) and OT-101 were administered 3 times/week via subcutaneous injection (SC). Vehicle (0.9% saline, Groups 1 and 2) and DTIC (1 or 10 mg/kg) were administered via intraperitoneal injection four times/week beginning day 14. Mice were monitored for adverse effects, body weight and tumor size three times weekly. The tumor, lungs, liver and kidneys were excised from all mice at termination and weighed.

Human U87-MG glioblastoma model

[0149] 3 x 106 U87 glioblastoma cells were injected into the right flank of Balb/c nude mice and tumor-bearing (50-250 mm3) mice were randomized into 10 groups (10 mice/group) and the antitumor activities of OT-101 and PTX as single agents and in combinations were evaluated. OT-101 (16 or 32 mg/kg, QDx21) in saline was given by SC administration. Taxol® was administered IV in 5 doses (QDx5) either on Day 0 or Day 7.

Human SKOV-3 ovarian tumor model

[0150] 5 x 106 SKOV-3 cancer cells were injected into the right flank of female BALB/c nude mice and tumor-bearing (100-300 mm3) mice were randomized into 10 groups (10 mice/group). The antitumor activities of OT-101 and PTX as single agents and in combinations were evaluated: OT-101 (32 or 64 mg/kg, QDx20) in saline was administered SC and Taxol® was administered IV in 5 doses (QDx5) on Day 0 or Day 7. Animals with tumor burdens greater than 2000 mm3, body weight (BW) loss more than 20% or found in a moribund condition were euthanized. Mice were monitored thrice a week for adverse effects, body weight loss, tumor size and survival outcome.

Effects of OT-101 in subcutaneous xenograft mouse models

[0151] In three xenograft models, OT-101 displayed a significant synergistic relationship in vivo with a schedule of OT-101 followed by chemotherapy that enhanced antitumor activity and increased survival in mice.

C8161 melanoma tumor model

[0152] Tumor growth was suppressed vs. control group 1 by 2%, -2%, 78%, 27%, and 92% on day 42 for group 2, 3, 4, 5, and 6, respectively. DTIC inhibition of tumor growth was enhanced by OT-101 (group 5 vs. 3 or group 6 vs. 4). Anti-tumor activity of the combination, group 6, was significantly superior (P = 0.038). OT-101 plus DTIC induced a synergistic effect on tumor inhibition. U87 glioblastoma and SKOV-3 ovarian xenograft models. Taxol® alone (D7-D1 1) significantly inhibited tumor growth following 5 days of treatment (P <0.0001 or 0.006 vs. vehicle, two-way ANOVA). OT-101 alone lacked antitumor activity. Combined treatment of U87 glioblastoma cells or SKOV-3 ovarian cancer cells with Taxol® (D7-D1 1) and OT-101 (D0-D20) resulted in further enhancement of antitumor activity (P = 0.002 or 0.0045 vs. Taxol®, two-way ANOVA). Kaplan-Meir Survival Curves: Melanoma: The median survival of group 1, 2, 3, 4, 5, and 6 were 48, 47, 47, 60, 53 and 81 days, respectively. Maximum survival was apparent with the combination, group 6, vs. corresponding high dose DTIC, group 4 (P = 0.0035, log-rank test). Glioblastoma: The survival time of vehicle-treated mice and Taxol®-treated mice were 24 days and 31 days respectively, whereas in mice treated with Taxol® plus OT-101 (32 mg/kg), survival was significantly increased to 40 days (P = 0.0076, log-rank test). Ovarian: The survival time of vehicle-treated mice and Taxol®-treated mice were 28 days and 39 days respectively, whereas mice treated with Taxol® plus OT-101(32 or 64 mg/kg, D0-D20), survival was significantly increased (49-51 days, log-rank test, P = 0.0026 and P <0.0001) (Fig. 4E).

Orthotopic human L3.6pl pancreatic cancer (PAC) model:

[0153] OT-101 induced inhibition of tumor cell proliferation as well as reduced lymph node and liver metastasis. However, no synergy was observed with Gemcitabine (PAC) (Fig. 6).

Effect of OT-101 on metastasis

[0154] Metastasis to the liver and the lymph node was suppressed by both Gem and OT-101, no synergy was observed. (Fisher' s exact test, P = 0.0230 for OT-101.


[0155] OT-101 significantly reduced tumor growth (p = 0.0084), lymph node metastasis (p = 0.023), and tumor angiogenesis (p <0.0001) in the orthotopic PAC model. Mean tumor vessel density was significantly reduced (p <0.0001) in all groups in comparison to untreated control. OT-101 demonstrated synergy in tumor growth inhibition and increased survival in human malignant melanoma (C8161, p = 0.038, vs. DTIC alone), glioblastoma (U87-MG, p = 0.001 vs. PTX) and ovarian (SKOV-3, p <0.05 vs. PTX) cancer models when combined with either DTIC (C8161) or PTX (U87-MG and SKOV-3). No synergy was observed with GEM (PAC). The combination regimen tested was effective and tolerable. Significant antitumor activity was achieved at human dose equivalent of 80 mg/m2/day which is well below the optimized clinical dose used for IV infusion of patients at 140 mg/m2/day.

[0156] The preclinical data lay the groundwork for further exploration of combination therapy in the clinic as well as demonstrating that TGF-P2 is a druggable target. Of interest is the preferential synergy between OT-101 and PTX or DTIC, but not with GEM.

[0157] Studies in functional in vivo test systems demonstrated that OT-101 has minor antitumor activities on its own. Tumor cell proliferation, metastases and tumor angiogenesis were reduced by OT-101. GEM and OT-101 demonstrated modest antitumor activity in an orthotopic xenograft mouse model of PAC. There was no synergy between OT-101 and GEM. However, OT-101 was able to synergize and increase the antitumor activity of DTIC and PTX. The combination of OT-101 with DTIC and PTX was shown to have a significant synergistic relationship in vivo with a schedule of OT-101 followed by chemotherapy resulting in enhanced antitumor activity as well as increased survival in mice. Significant antitumor activity was achieved at human dose equivalent to 80 mg/m2/day which is well below the optimized clinical dose used for IV infusion of patients at 140 mg/m2/day.

Example 6

[0158] Synergistic benefits of Trabedersen and Paclitaxel (Taxol®) are observed only when Trabedersen is administered sequentially for at least one cycle prior to subsequent administration of Paclitaxel. 3 x 106 U87 glioblastoma cells were injected into the right flank of Balb/c nude mice and tumor-bearing (50-250 mm3) mice were randomized into 10 groups (10 mice/group) and the antitumor activities of Trabedersen (OT-101) and Paclitaxel (Taxol) as single agents and in various combinations were evaluated. Trabedersen (16 or 32 mg/kg, QDx21) in saline was given by subcutaneous administration. Paclitaxel (Taxol®) was administered intravenously in 5 doses (QDx5) either on Day 0 or Day 7.

[0159] Fig. 7A shows the effect of simultaneous combination therapy with Trabedersen and Paclitaxel. Fig. 7B shows the effect of sequential combination therapy, wherein Trabedersen is sequentially administered first and Paclitaxel is subsequently administered on Day 7 (Taxol D7). Paclitaxel significantly inhibited tumor growth following 5 days of treatment (P <0.0001 or 0.006 versus vehicle). Trabedersen alone lacked antitumor activity. Sequential combination therapy (Trabederson administered first for one cycle followed by subsequent administration of Paclitaxel) of U87 glioblastoma cells with Trabedersen and Paclitaxel resulted in further enhancement of antitumor activity (P = 0.002 or 0.0045 vs. Taxol®, two-way ANOVA), i.e. Trabedersen displayed a significant synergistic relationship in vivo with a schedule of Trabedersen followed by Paclitaxel which enhanced antitumor activity and increased survival in mice. The survival time of vehicle-treated mice and Paclitaxel-treated mice were 24 days and 31 days respectively, whereas in mice treated with Paclitaxel plus Trabedersen, sequentially, (32 mg/kg), survival was significantly increased to 40 days (P = 0.0076, log-rank test).

[0160] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[0161] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[0162] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

[0163] Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[0164] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.

[0165] It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

[0166] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is

understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0167] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0168] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention.