WO2015127227A1 - Uses of flagellin for improved chemotherapy - Google Patents

Uses of flagellin for improved chemotherapy Download PDF

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Publication number
WO2015127227A1
WO2015127227A1 PCT/US2015/016850 US2015016850W WO2015127227A1 WO 2015127227 A1 WO2015127227 A1 WO 2015127227A1 US 2015016850 W US2015016850 W US 2015016850W WO 2015127227 A1 WO2015127227 A1 WO 2015127227A1
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cblb502
cancer
mice
irinotecan
effective amount
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PCT/US2015/016850
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French (fr)
Inventor
Andrei Gudkov
Lyudmila G. BURDELYA
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Cleveland Biolabs, Inc.
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Priority to EA201592012A priority Critical patent/EA201592012A8/en
Publication of WO2015127227A1 publication Critical patent/WO2015127227A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to methods and compositions that are useful for the treatment of various diseases, including cancer.
  • the invention relates to, in part, combination therapeutic strategies to increase the effectiveness of chemotherapies.
  • Chemotherapy is a cancer treatment that uses drugs to destroy cancer cells.
  • chemotherapy agents may destroy not only cancer cells but also normal, healthy cells. This damage to healthy cells causes side effects of chemotherapies. These side effects can have the effect of dramatically reducing a patient's quality of life. Additionally, such side effects can limit the ability of a patient to receive treatment and therefore lower the likelihood of recovery.
  • the present invention provides for methods for treating cancer in patients undergoing treatment with a chemotherapeutic agent by administering an agent that increases the effectiveness and/or reduces toxic side effects of chemotherapies, specifically, TLR agonist, including a flagellin-related composition, such as CBLB502.
  • the invention provides a method for treating cancer, comprising administering an effective amount of a TLR agonist, including a flagellin-related composition, such as CBLB502, to a patient undergoing treatment with fluorouracil (5-FU).
  • a method for treating cancer comprising administering an effective amount of a TLR agonist, including a flagellin-related composition, such as CBLB502, after administering an effective amount of 5-FU to a cancer patient in need thereof.
  • the invention provides a method for treating cancer, comprising administering an effective amount of a TLR agonist, including a flagellin-related composition, such as CBLB502, to a patient undergoing treatment with a topoisomerase inhibitor.
  • the topoisomerase inhibitor may be selected from irinotecan and doxorubicin.
  • a method for treating cancer comprising administering an effective amount of a TLR agonist, including a flagellin-related composition, such as CBLB502, sequentially or simultaneously with administering an effective amount of a topoisomerase inhibitor, optionally selected from irinotecan and doxorubicin, to a cancer patient in need thereof
  • a TLR agonist including a flagellin-related composition, such as CBLB502 mitigates the side effects of certain chemotherapies, including 5-FU, irinotecan, and/or doxorubicin, and may expand the therapeutic window of these treatments.
  • administering a TLR agonist, including a flagellin-related composition, such as CBLB502 increases the ability of a patient to receive an adequate dose, and/or an increased or complete regimen of a therapy and/or increased or complete maintenance regimen of a therapy.
  • some patients including those having preexistent impaired hepatic function or a deficiency of dipyrimidine dehydrogenase (DPD) activity, typically cannot sustain full 5-FU treatment.
  • DPD dipyrimidine dehydrogenase
  • TLR agonist including a flagellin-related composition, such as CBLB502
  • a flagellin-related composition such as CBLB502
  • cancer patients classified as *28 variants typically cannot sustain full irinotecan treatment.
  • Administration of a TLR agonist, including a flagellin-related composition, such as CBLB502 increases the likelihood that these patients can receive this treatment.
  • Fig. 1 shows the effect of CBLB502 on 5-FU-induced mortality in mice.
  • Panel A shows a body weight (percentage of starting weight) growth curve of BALB/c mice treated with 5-FU (200mg/kg or 400 mg/kg) alone or with subsequent injection of CBLB502 (1 ⁇ g/mouse) 24 and 48 h after the last 5-FU dose.
  • Panel B shows a percent survival growth curve of BALB/c mice treated with 5-FU (200 mg/kg (second from bottom curve) or 400 mg/kg (bottom curve)) alone or with subsequent injection of CBLB502 (1 ⁇ g/mouse) 24 and 48 h after the last 5- FU dose: with 200 mg/kg 5-FU (top curve) or 400 mg/kg 5-FU (second from top curve).
  • Panel C shows a percent survival growth curve of C57BL/6 mice injected with a single dose of 400 mg/kg 5-FU (second from bottom curve) or 3 daily doses of 100 mg/kg 5-FU (bottom curve) with or without CBLB502 (1 ⁇ g/mouse) injected 24 and 48 h after the last 5-FU dose: 400 mg/kg 5-FU (top curve) or 3 daily doses of 100 mg/kg 5-FU (second from top curve).
  • Fig. 2 demonstrates the mitigation of body weight loss and increase of survival of BALB/c mice after 5-FU injections (200 mg/kg) by CBLB502 treatment.
  • Panel A shows a body weight (percentage of starting weight) growth curve after 5-FU (200 mg/kg) treatment (bottom curve) with and without CBLB502 injections given in different regimens: 24 h post-5-FU (second from bottom curve), 1 and 24 h post-5-FU (third from top curve), and 1 , 48 and 96h post-5-FU (fourth from top curve).
  • CBLB502 (top curve) and vehicle (second from top curve) injected mice without 5-FU were weighed as controls.
  • Panel B shows a survival growth curve of BALB/c mice after 5-FU (200 mg/kg) treatment (bottom curve) with and without CBLB502 injections given in different regimens: 24 h post-5-FU (second from top curve), 1 and 24 h post-5-FU (second from bottom curve), and 1 , 48 and 96h post-5-FU (top curve).
  • FIG. 3 shows that the CBLB502-mediated protection against 5-FU toxicity is TLR5-dependent.
  • Panel A shows a body weight (percentage of starting weight) growth curve in wild type C57BL/C mice injected with 5-FU 100 mg/kg x 3 times with 24 h interval with CBLB502 injected 24 and 48 h after the last 5-FU treatment.
  • Panel B shows a body weight (percentage of starting weight) growth curve in wild type C57BL/C mice injected with 5-FU 200 mg/kg x 2 times with 6 h interval with and without CBLB502 injected 24 and 48 h post-5-FU.
  • Panel C shows a percent survival growth curve of wild type C57BL/6 and TLR5-KO mice injected with 5-FU - 200 mg/kg x 2 times with 6 h interval with (top curve and second from top curve, respectively) and without (second from bottom curve and bottom curve, respectively) CBLB502 injected 24 and 48 h post-5-FU.
  • Fig. 4 demonstrates the protection and restoration of hematopoiesis.
  • Panel A shows a blood cell analysis growth chart of white blood cells (WBC) and neutrophils for the indicated days after 5-FU injection performed using blood samples from mice treated with vehicle, 5-FU alone or 5-FU+CBLB502.
  • 5-FU was injected i.p. at a dose of 200 mg/kg followed by three s.c. CBLB502 (1 ⁇ g/mouse) injections 1 , 48 and 96 h post- 5-FU.
  • Panel B shows a blood cell analysis growth chart of white blood cells (WBC), neutrophils, lymphocytes and platelets for the indicated days after 5-FU injection performed using blood samples from mice treated with vehicle, 5-FU alone or 5-FU+CBLB502.
  • 5-FU was injected i.p. at a dose of 100 mg/kg followed by three s.c. CBLB502 (1 ⁇ g/mouse) injections 1 , 48 and 96 h post-5-FU.
  • Fig. 5 shows the restoration of blood cell count in BALB/c mice following 5-FU treatment alone and in combination with CBLB502. Specifically, Fig. 5 shows growth charts of white blood cells, neutrophils, lymphocytes and platelets from BALB/c mice treated with 5-FU injected i.p. (150 mg/kg) followed by CBLB502 24 h and 48 h post 5-FU. Two out of 5 mice in the 5-FU injected group died on day 8, all 5 mice in the 5- FU+CBLB502-treated group survived. Vehicle injected mice were used as controls.
  • FIG. 6 shows the effect of CBLB502 on 5-FU-induced changes in bone marrow morphology. Specifically, Fig. 6 shows representative pictures of H&E-stained bone marrow sections (10x objective magnification) prepared 3 and 7 days after 5-FU injection. BALB/c mice were treated with 200 mg/kg 5-FU with or without injection of CBLB502 24 and 48 h post-5-FU. The lower row shows selected areas outlined in white at two fold higher magnification.
  • FIG. 7 demonstrates the effect of CBLB502 on 5-FU-induced changes in small intestine morphology.
  • Panel A shows representative H&E-stained transverse sections (250x objective magnification) of small intestines from vehicle injected BALB/c mice (intact) and treated with 5-FU (200 mg/kg) alone or in combination with CBLB502 24 and 48 h post-5-FU. Sections were prepared 3 and 7 days after 5-FU treatment. Enterocytes lining the villi are indicated by arrows, lamina intestinal by asterisks, and crypts by arrowheads. The lower row shows selected crypt areas outlined in white at two fold higher magnification.
  • Panel B shows representative H&E- stained transverse sections (250x objective magnification) of small intestines from vehicle injected BALB/c mice (intact) and treated with 5-FU (400 mg/kg) alone or in combination with CBLB502 24 and 48 h post-5-FU. Sections were prepared 3 and 7 days after 5-FU treatment. Enterocytes lining the villi are indicated by arrows, lamina intestinal by asterisks, and crypts by arrowheads. The lower row shows selected crypt areas outlined in white at two fold higher magnification.
  • Panel C shows a bar chart of the average injury score of small intestine sections described in (Panels A-B).
  • the degree of pathomorphological changes in the surface epithelium, villi, crypts, lamina intestinal, stroma, transitory and lymphoid elements and submucosa was scored as: 4 - Severe; 3 - Markedly abnormal; 2 - Moderately abnormal; 1 - Mild; and 0 - Normal, including non-integer scores.
  • the average morphological score for 5 mice/group is shown.
  • Panel D shows a bar chart of the mitotic index in mice treated as described in (Panels A-B) calculated in crypts of 4 transverse sections of small intestine per mouse as the number of mitoses per crypt.
  • the mitotic index of vehicle treated animals is plotted as "No-5-FU" control or mean.
  • each set of four bars from left to right represent vehicle injected BALB/c mice (intact) and treated with 5-FU (200 mg/kg or 400 mg/kg) alone (collected on day 3 (first bar), collected on day 7 (third bar)) or in combination with CBLB502 24 and 48 h post-5-FU (collected on day 3 (second bar), collected on day 7 (fourth bar)).
  • FIG. 8 shows the effect of CBLB502 on 5-FU-induced changes in colon morphology.
  • Panel A shows representative H&E-stained transverse sections (250x objective magnification) of colon from untreated "intact" BALB/c mice and those treated with 5-FU injections (200 mg/kg) alone or in combination with CBLB502 treatment 24 and 48 h post-5-FU. Sections were prepared 3 or 7 days after 5-FU treatment. Morphology of crypt areas outlined in white is shown at two fold higher magnification in the lower rows. Atrophic and degenerative changes in the surface epithelium are indicated by arrows and in the crypt cells and mucin globules by arrowheads.
  • Panel B shows representative H&E-stained transverse sections (250x objective magnification) of colon from untreated "intact" BALB/c mice and those treated with 5-FU injections (400 mg/kg) alone or in combination with CBLB502 treatment 24 and 48 h post-5-FU. Sections were prepared 3 or 7 days after 5-FU treatment. Morphology of crypt areas outlined in white is shown at two fold higher magnification in the lower rows. Atrophic and degenerative changes in the surface epithelium are indicated by arrows and in the crypt cells and mucin globules by arrowheads.
  • Panel C shows a bar chart of the average injury score determined by pathomorphological changes in the surface epithelium, number of surface enterocytes and goblet cells, size and shape of crypts, lymphoid elements and state of the submucosa in colon sections from mice described in (Panels A-B).
  • each set of four bars from left to right represent vehicle injected BALB/c mice (intact) and treated with 5-FU (200 mg/kg or 400 mg/kg) alone (collected on day 3 (first bar), collected on day 7 (third bar)) or in combination with CBLB502 24 and 48 h post-5-FU (collected on day 3 (second bar), collected on day 7 (fourth bar)).
  • 5-FU 200 mg/kg or 400 mg/kg
  • Fig. 9 demonstrates that CBLB502 induces IL-6 production in 5-FU-treated mice.
  • Fig. 9 shows a growth chart of IL-6 concentration in plasma samples from BALB/c mice analyzed by MILLIPLEX kit. Plasma was collected 3 and 7 days after injection (labeled from left to right) of vehicle (DMSO) (first bar), CBLB502 alone (second bar), 200 mg/kg 5-FU alone (third bar), or 5-FU+CBLB502 (fourth bar).
  • DMSO vehicle
  • FIG. 10 shows the effect of CBLB502 on 5-FU toxicity in IL-6 knockout mice.
  • Panel A shows a survival of WT BALB/c and IL-6-KO mice growth curve after treatment with 200 mg/kg 5-FU (bottom curve and second from bottom curve, respectively), with or without CBLB502 treatment (1 ⁇ g/mouse) 24 and 48 h after 5- FU (top curve and second from top curve, respectively).
  • Panel B shows a body weight growth curve in IL-6-KO mice treated with 5-FU (400 mg/kg), with (top curve) or without (bottom curve) CBLB502 injected 24 and 48 h after the last 5-FU dose.
  • Panel C shows a survival of IL-6-KO and wild type (WT) mice growth curve after treatment with 5-FU (400 mg/kg) (second from bottom curve and bottom curve, respectively) or 5-FU+CBLB502 (second from top curve and top curve, respectively).
  • 5-FU 400 mg/kg
  • 5-FU+CBLB502 second from top curve and top curve, respectively.
  • Panel D shows H&E-stained small intestine sections with crypts from IL-6- KO mice euthanized on Day 3 after 5-FU (400 mg/kg) with or without CBLB502. Enterocytes lining the villi are indicated by arrows, lamina intestinal by asterisks and crypts by arrowheads.
  • Panel E shows a growth chart (labeled in sets of three from left to right) of concentrations of white blood cells (WBC) in blood samples from IL-6-KO mice treated with CBLB502 alone (first bar), 5-FU alone (second bar) or 5-FU+CBLB502 (third bar). 5-FU (100 mg/kg) was followed by CBLB502 24 and 48 h later.
  • WBC white blood cells
  • Fig. 11 shows the mitotic index in IL-6-KO mice after 5-FU treatment with and without CBLB502. Specifically, Fig. 11 shows a bar chart (labeled from left to right) of the mitotic index calculated in crypts of 4 transverse sections of small intestine per mouse as the number of mitoses per crypt in 3 mice/ group. The samples were obtained 3 days after 5-FU (400 mg/kg) injections with (third bar) and without (second bar) CBLB502 24 and 48 h post-5-FU. The first bar corresponds to vehicle injected BALB/c mice (intact).
  • Fig. 12 demonstrates that CBLB502 induces expression of soluble IL-1 receptor, SOD2, and IL-1 receptor antagonist (IL1 -RN).
  • Panel A shows a bar chart (labeled in pairs from left to right) of the concentration of soluble IL-1 receptor in plasma prepared from BALB/c mice 72 h after 5-FU injections (200 and 400 mg/kg) with (second bar) and without (first bar) CBLB502 injected 24 and 48 h after 5- FU that was determined using a MILLIPLEX kit.
  • Panel B shows a Western blot for SOD-2 expression in small intestine samples from N IH-Swiss mice at the indicated time-points after CBLB502 injection (without 5-FU).
  • Panel C shows the expression of IL-1 receptor antagonist (IL-1 RN) detected by RT-PCR using total RNA prepared from small intestine isolated from a BALB/c mouse treated with CBLB502 (1 mg/ mouse x2 injections 24h apart) 24 h after the second injection.
  • Small intestine RNA from a PBS-injected mouse was used as an "intact" control.
  • GADPH expression was used a housekeeping gene for loading control.
  • Fig. 13 shows a representative experiment showing the absence of any effect of CBLB502 on s.c. growing CT26 tumors in syngeneic BALB/c mice. Specifically, Fig.
  • FIG. 13 shows a tumor volume growth curve of the treatment of CT-26 tumor-bearing BALB/c mice with CBLB502 initiated when tumors reached about 5 mm in diameter.
  • CBLB502 (1 mg/mouse) was injected s.c. 24 h apart on days 1 , 2 and 3.
  • Fig. 14 demonstrates the protection of mice, but not tumors, from 5-FU toxicity by CBLB502 in CT26 tumor-bearing mice.
  • Panel A shows a CT26 tumor growth curve in mice treated with vehicle ("untreated") (top curve), 5-FU alone (100 (second from top curve) or 200 mg/kg (bottom curve)), or 5-FU (100 (third from top curve) or 200 mg/kg (second from bottom curve)) + CBLB502 injected 1 , 48, and 96 h post-5-FU.
  • Panel B shows a body weight change (percentage of starting weight) growth curve of mice described in Panel A.
  • mice treated with vehicle (“untreated”) second from bottom curve
  • 5-FU alone 100 (top curve) or 200 mg/kg (bottom curve)
  • 5-FU 100 (second from top curve) or 200 mg/kg (third from top curve)
  • Panel C shows a percent survival growth curve of mice described in Panel A.
  • mice treated with vehicle (“untreated") bottom curve
  • 5-FU alone 100 (second from top curve) or 200 mg/kg (second from bottom curve)
  • 5-FU 100 (third from top curve) or 200 mg/kg (top curve) + CBLB502 injected 1 , 48, and 96 h post-5-FU.
  • mice in this group died due to 5-FU toxicity with small or no tumors.
  • Fig. 15 shows the toxicity of irinotecan in Fischer rats bearing Ward colorectal carcinoma with or without CBLB502 (administered before or concurrently).
  • FIG. 16 shows the effect of CBLB502 treatment with and without 5-FU against 4T1 tumor growth.
  • Panel B shows mouse survival after i.v. injection of 4T1 cells and treatment with 5-FU (100 mg/kg, on day 4), CBLB502 (1 ⁇ g or PBS vehicle on days 5 and 6) or a combination of both.
  • the difference from vehicle control and (**) from any other group is statistically significant (pO.05).
  • FIG. 17 shows the effect of CBLB502 on myeloid derived suppressor cells (MDSC) in 4T1 tumor- bearing mice.
  • Mice with orthotopically (mammary fat pad) growing 4T1 tumors were injected with 5-FU (50 and 100 mg/kg, i.p.) and vehicle control on day 14, as indicated by the arrow.
  • the amount of neutrophils in the blood was determined by complete blood count (CBC) on days 10, 12, 14, 17 and 20 (panel A).
  • MDSC myeloid derived suppressor cells
  • the present invention is based, in part, on the discovery that the TLR agonists, including flagellin- related compositions, such as CBLB502, mitigate the side effects of certain chemotherapies, including 5-FU, irinotecan, and doxorubicin, and therefore expand the therapeutic window of these treatments. Accordingly, the present invention provides improved methods of treating cancers with genotoxic chemotherapy agents.
  • the term "about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication.
  • the language “about 50” covers the range of 45 to 55.
  • an "effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.
  • compositional percentages are by weight of the total composition, unless otherwise specified.
  • the word "include,” and its variants is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology.
  • the terms “can” and “may” and their variants are intended to be non- limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
  • the words "preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.
  • compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose.
  • therapeutic agents e.g. flagellin related compositions (and/or additional agents, including 5-FU, irinotecan, and doxorubicin) described herein
  • the therapeutic agents are given at a pharmacologically effective dose.
  • a “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease.
  • An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease.
  • administration of therapeutic agents to a patient suffering from cancer provides a therapeutic benefit not only when the underlying condition is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the symptoms associated with the disease, e.g., a decrease in tumor burden, a decrease in circulating tumor cells, an increase in progression free survival.
  • Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model.
  • Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder or reduction in toxicity, regardless of whether improvement is realized. [0039] In certain embodiments, a pharmacologically effective amount that will treat cancer will modulate the symptoms typically by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.
  • such modulations will result in, for example, statistically significant and quantifiable changes in the numbers of cancerous cells or indicia of toxicity as described herein (e.g. number of certain cells in a blood test, number of liver enzymes in a blood test, etc.).
  • the present invention provides methods of treating cancer with a TLR agonist, including a flagellin-related composition, such as CBLB502, in a patient undergoing treatment with another therapy, including 5-FU and/or irinotecan and/or doxorubicin.
  • a TLR agonist including a flagellin-related composition, such as CBLB502
  • the present invention provides for the use of a TLR agonist, including a flagellin-related composition, such as CBLB502, for the treatment of cancer in a patient undergoing treatment with another therapy, including 5-FU and/or irinotecan and/or doxorubicin.
  • the TLR agonist may be a flagellin related composition, including a flagellin-related polypeptide.
  • the flagellin related compositions may be from various sources, including a variety of Gram-positive and Gram- negative bacterial species.
  • the flagellin related compositions may have an amino acid sequence that is derived from any of the flagellins from bacterial species that are depicted in FIG. 7 of U.S. Patent Publication No. 2003/0044429, the contents of which are incorporated herein by reference in their entirety.
  • the flagellin related compositions may have nucleotide sequences related to those encoding the flagellin polypeptides listed in FIG. 7 of U.S. 2003/0044429, which are publicly available at sources including the NCBI Genbank database.
  • the flagellin related compositions may be the major component of bacterial flagellum.
  • the flagellin related compositions may be composed of one, or two, or three, or four, or five, or six, or seven domains or fragments thereof (see, e.g. FIG. 10 of US Patent 8,324,163, the contents of which are incorporated herein by reference in their entirety).
  • the domains may be selected from NDO, ND1 , ND2, D3, CD2, CD1 , and CDO.
  • Domains 0 (DO), 1 (D1 ), and 2 (D2) may be discontinuous and may be formed when residues in the amino terminus and carboxy terminus are juxtaposed by the formation of a hairpin structure.
  • the amino and carboxy terminus comprising the D1 and D2 domains may be most conserved, whereas the middle hypervariable domain (D3) may be highly variable.
  • the non-conserved D3 domain may be on the surface of the flagellar filament and may contain the major antigenic epitopes.
  • the potent proinflammatory activity of flagellin may reside in the highly conserved N and CD1 and D2 regions.
  • the flagellin related compositions may be from a species of Salmonella, representative examples of which are S. typhimurium and S. dublin (encoded by GenBank Accession Number M84972).
  • the flagellin related-polypeptide may be a fragment, variant, analog, homolog, or derivative of wild type flagellin (SEQ ID NO: 1 ), or combination thereof:
  • Lys Thr Met Phe lie Asp Lys Thr Ala Ser Gly Val Ser Thr Leu lie Asn Glu Asp Ala Ala Ala
  • a fragment, variant, analog, homolog, or derivative of flagellin may be obtained by rational-based design based on the domain structure of flagellin and the conserved structure recognized by TLR5.
  • the flagellin related compositions may be related to a flagellin polypeptide from any Gram-positive or Gram-negative bacterial species including, but not limited to, the flagellin polypeptides disclosed in U.S. Pat. Pub. 2003/000044429, the contents of which are incorporated herein, and the flagellin peptides corresponding to the Accession numbers listed in the BLAST results shown in FIG. 7 (7AA-7F) of U.S. Patent Pub. 2003/000044429, or variants thereof.
  • the flagellin-related polypeptide comprises a CBLB502 molecule.
  • CBLB502 (aka “Entolimod") as used herein comprises the sequence of SEQ ID NO: 2.
  • Gin Asp Asn Gin Met Lys lie Gin Val Gly Ala Asn Asp Gly Glu Thr He Thr He Asp Leu Gin
  • the flagellin-related polypeptide of the present invention comprises a truncation in one or more domains of SEQ ID NO: 1 or 2 or variants thereof.
  • the flagellin-related composition comprises a deletion in a N-terminal domain of SEQ ID NO: 1 or 2 or variants thereof.
  • the flagellin-related composition comprises a deletion in the NDO domain of SEQ ID NO: 1 or 2 or variants thereof.
  • the flagellin-related composition comprises a deletion of the entire NDO domain of SEQ ID NO: 1 or 2 or variants thereof.
  • the flagellin- related composition comprises a deletion in a C-terminal domain of SEQ ID NO: 1 or 2 or variants thereof. In yet another embodiment, the flagellin-related composition comprises a deletion in the CDO domain of SEQ ID NO: 1 or 2 or variants thereof. In yet another embodiment, the flagellin-related composition retains amino acids 470-485 of the CDO domain of SEQ ID NO: 1 or 2 or variants thereof.
  • the flagellin related compositions may be at least 30-99% identical to amino acids 1-174 and 418- 505 of SEQ ID NO: 1.
  • the flagellin related compositions may be a species of the genera disclosed in US Patent No. 8,932,609, the contents of which are hereby incorporated by reference.
  • the flagellin related compositions may stimulate Toll-like receptor activity (e.g. TLR1 , and/or TLR2, and/or TLR3, and/or TLR4, and/or TLR5, and/or TLR6, and/or TLR7, and/or TLR8, and/or TLR9, and/or TLR10, and/or TLR11 , and/or TLR12, and/or TLR13).
  • TLR1 Toll-like receptor activity
  • TLR2 and/or TLR3, and/or TLR4, and/or TLR5, and/or TLR6, and/or TLR7, and/or TLR8, and/or TLR9, and/or TLR10, and/or TLR11 , and/or TLR12, and/or TLR13 The TLR family is composed of at least 10 members and is essential for innate immune defense against pathogens. The innate immune system recognizes conserved pathogen-associated molecular patterns (PAMPs).
  • PAMPs pathogen-associated molecular patterns
  • TLR may recognize a conserved structure that is particular to bacterial flagellin which may be composed of a large group of residues that are somewhat permissive to variation in amino acid content.
  • the flagellin-related composition activates TLR5 signaling.
  • the flagellin-related composition activates TLR5 at the same levels, or levels similar to, CBLB502. Activation of TLR5 induces expression of the nuclear factor NF- ⁇ , which in turn activates numerous inflammatory-related cytokines.
  • the flagellin related compositions induce expression of proinflammatory cytokines. In further embodiments, the flagellin related compositions induce expression of antiinflammatory molecules. In another embodiment, the flagellin related compositions induce expression of anti- apoptotic molecules. In yet a further embodiment, the flagellin related compositions induce expression of antibacterial molecules.
  • the targets of NF- ⁇ include, but are not limited to, IL- ⁇ , TNF-a, IL-6, IL-8, IL-18, G-CSF, TNFSF13B, keratinocyte chemoattractant (KC), BLIMP1 /PRDM1 , CCL5, CCL15, CCL17, CCL19, CCL20, CCL22, CCL23, CXCL1 ,CCL28, CXCL1 1 , CXCL10, CXCL3, CXCL1 , GRO-beta, GRO-gamma, CXCL1 , ICOS, IFNG, IL-1A, IL-1 B, IL1 RN, IL-2, IL-9, IL-10, IL-11 , IL-12, IL-12B, IL-12A, IL-13, IL-15, IL-17, IL-23A, IL-27, EBI3, IFNB1 , CXCL5, KC, liGpl ,
  • the effective amount of flagellin related composition is about 0.001 mg to about 1000 mg per day, about 1 mg to about 600 mg per day, or about 5 mg to about 30 mg per day.
  • the present invention provides for methods of treating cancer, in patients undergoing treatment with a chemotherapy, including 5-FU and/or irinotecan and/or doxorubicin, by administering an effective amount of a flagellin related composition, such as CBLB502.
  • a chemotherapy including 5-FU and/or irinotecan and/or doxorubicin
  • the present invention provides for methods of treating cancer, involving administering an effective amount of a flagellin related composition, such as CBLB502 after administering an effective amount of 5-FU to a cancer patient in need thereof.
  • the present invention provides for provides for methods of treating cancer, involving administering an effective amount of CBLB502 sequentially or simultaneously (including coadministration and/or co-formulation) with administering an effective amount of irinotecan to a cancer patient in need thereof.
  • the present invention provides for provides for methods of treating cancer, involving administering an effective amount of CBLB502 sequentially or simultaneously (including coadministration and/or co-formulation) with administering an effective amount of doxorubicin to a cancer patient in need thereof.
  • 5-FU and/or “irinotecan” and/or “doxorubicin” may refer to any forms of these agents, including various formulations as described herein (including, but not limited to iv, topical, oral, nanoformulations, slow-release, gels, etc.). Further “5-FU” and/or “irinotecan” and/or “doxorubicin” may refer to prodrugs of these agents.
  • “5-FU” may encompass one or more of, for example, 5'-deoxy-5- fluorouhdine, capecitabine (e.g. Xeloda), BOF-A2, ftorafur, UFT, and S-1.
  • 5-FU and/or “irinotecan” and/or “doxorubicin” may refer to in vivo active metabolites of these agents.
  • “irinotecan” may refer to one or more of CPT1 1 , SN-38, SN-38-G, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1 - piperidino]carbonyloxycamptothecin (APC), and 7-ethyl-10-(4-amino-1 -piperidino) carbonyloxycamptothecin (NPC).
  • doxorubicin also known as hydroxydaunorubicin
  • hydroxydaunomycin may refer to one or more of Adriamycin PFS, Adriamycin RDF, or Rubex, Doxil, Myocet, and Caelyx.
  • administering a flagellin related composition, such as CBLB502 after 5-FU encompasses after as single dose of 5-FU, irinotecan, and/or doxorubicin, or after a partial or full cycle of 5-FU, irinotecan, and/or doxorubicin therapy (e.g. at an intermediate step or at the completion of any of the regimens described herein), or after the onset of toxicity from 5-FU, irinotecan, and/or doxorubicin treatment.
  • the administration of a flagellin related composition protects and/or reduces and/or mitigates one or more side effects of a patient's cancer therapy.
  • the patient's cancer therapy is 5-FU and the side effect is one or more of stomatitis, esophagopharyngitis, leukopenia and/or reduced white blood count, risk of infection, vomiting, diarrhea, nausea, poor appetite, damage to the Gl tract, including gastrointestinal ulceration and/or bleeding, thrombocytopenia, hand-foot syndrome, alopecia, mucositis, impaired hepatic function, and hemorrhage.
  • the administration of a flagellin related composition may permit a patient to receive a specific mode of administration of 5-FU by reducing side effects.
  • myelotoxicity is the major toxic effect in patients receiving bolus doses of 5-FU.
  • a flagellin related composition, including CBLB502 offsets this side effect and therefore permits bolus doses.
  • hand- foot syndrome palmar-plantar erythrodysesthesia
  • stomatitis neuro- and cardiotoxicity are associated with continuous infusions of 5-FU.
  • a flagellin related composition, including CBLB502 offsets this side effect and therefore permits continuous infusions.
  • the side effect of 5-FU treatment is hematological damage.
  • the hematological damage is one or more of leukopenia and/or reduced white blood count and reduction of other types of blood cells, including neutrophils, lymphocytes and platelets.
  • hematological damage is assessed by a blood test.
  • a patient is administered 5-FU and is subsequently evaluated for a hematological damage by, for example, a blood test that involves a cell count, and if presenting with lowered cell counts, is administered an effective amount of a flagellin related composition, including CBLB502.
  • a patient is administered 5-FU and is subsequently evaluated for a hematological damage by, for example, a blood test that involves a cell count, and if presenting with normal counts, is not administered an effective amount of a flagellin related composition, including CBLB502.
  • the side effect of 5-FU treatment is impaired hepatic function.
  • impaired hepatic function is assessed by testing for levels of various liver enzymes.
  • Exemplary indicia of impaired hepatic function include, by way of non-limiting example, elevated aminotransferase levels, elevated plasma ammonia levels, changes in one or more of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, 5' nucleotidase, a gamma-glutamyl transpeptidase (GGT), and bilirubin.
  • a patient is administered 5-FU and is subsequently evaluated for impaired hepatic function by, for example, a blood test and/or biopsy that involves a screen for markers of liver function (e.g. liver enzymes) and, if presenting with abnormal levels, is administered an effective amount of a flagellin related composition, including CBLB502.
  • a patient is administered 5-FU and is subsequently evaluated for an impaired hepatic function by, for example, a blood test and/or biopsy that involves a screen for markers of liver function (e.g. liver enzymes) and, if presenting with normal levels, is not administered an effective amount of a flagellin related composition, including CBLB502.
  • hepatic imaging may be used to test for impaired hepatic function.
  • the hepatic imaging may be one or more of ultrasounds, CT, MR, and PET, PET/CT and may involve various imaging agents (e.g. gadolinium chelates).
  • imaging agents e.g. gadolinium chelates.
  • the side effect of 5-FU treatment is damage to the Gl tract.
  • the damage to the Gl tract is assessed by measurement one or more of (a) the height and width of villi of the small intestines and/or number of surface enterocytes and goblet cells in the small and large intestines; (b) status of crypts (one or more of depth, size and shape, presence of apoptotic bodies, number of and granules in Paneth's cells, luminal migration of epithelial nuclei, loss of goblet cells, presence of atrophy and distortion); and (c) state of the lamina limbal, one or more of presence of transitory cells, lymphoid accumulations, edema, blood vessel congestion and hemorrhage) in, for example, a biopsy.
  • the side effect of 5-FU treatment is diarrhea.
  • administering an effective amount of CBLB502 reduces a dose and/or frequency of an anti-diarrheal agent (e.g. one or more of loperamide, diphenoxylate, and atropine) administered to the cancer patient.
  • an anti-diarrheal agent e.g. one or more of loperamide, diphenoxylate, and atropine
  • the side effect of 5-FU treatment is vomiting.
  • administering an effective amount of CBLB502 reduces a dose and/or frequency of an antiemetic, including, for example, 5-HT3 blockers with or without dexamethasone, administered to the cancer patient.,
  • the patient's cancer therapy is 5-FU and the side effect is predicted by a genetic test, including, for example, genetic tests for one or more of genes DPYD and TYMS.
  • a patient is tested for the likelihood of a side effect of 5-FU treatment before beginning a 5-FU regimen and, if there is a high likelihood of side effects, is administered a flagellin related composition, including CBLB502, to mitigate the side effects.
  • a patient is tested for the likelihood of a side effect of 5-FU treatment before beginning a 5-FU regimen and, if there is a low likelihood of side effects, is not administered a flagellin related composition, including CBLB502, to mitigate the side effects.
  • the patient's cancer therapy is 5-FU and the side effect is measured by monitoring a patient's temperature.
  • temperature in excess of about 37.5°C (99.5° F) or about 38°C (100.4°F) and beyond indicates 5-FU side effects and directs administration of an effective amount of a flagellin related composition, including CBLB502.
  • the present invention provides for administration of a flagellin related composition, including CBLB502, to a cancer patient receiving 5-FU and also having a preexistent impaired hepatic function including, for example, hepatitis.
  • a flagellin related composition, including CBLB502 serves to protect the patient's compromised liver from further damage.
  • the present invention provides for administration of a flagellin related composition, including CBLB502 to a cancer patient receiving 5-FU and also having a deficiency of dipyrimidine dehydrogenase (DPD) activity.
  • DPD deficient patients tend to experience exacerbated 5-FU side effects (e.g. severe toxicity (e.g., stomatitis, diarrhea, neutropenia and neurotoxicity) and a flagellin related composition, including CBLB502, serves to mitigate these side effects.
  • a flagellin related composition including CBLB502
  • a cancer patients with risk factors for 5-FU including for example, a history of high-dose pelvic irradiation or previous use of alkylating agents, who have a widespread involvement of bone marrow by metastatic tumors or those with impaired hepatic or renal function, allows for administration of 5- FU.
  • the present invention provides for administration of a flagellin related composition, including CBLB502 to a female cancer patient receiving 5-FU. Women tend to have more severe stomatitis and leukopenia than men when receiving 5 consecutive days of 5-FU at doses of 370 to 450 mg/m 2 (see, e.g. Sloan, et al. JCO 20(6): 1491 -1498, the contents of which are hereby incorporated by reference). Administration of a flagellin related composition, including CBLB502, to such patients allows the full course and/or dose of treatment to occur with suspension of treatment because of, for example, stomatitis and/or leukopenia.
  • the patient's cancer therapy is a chemotherapeutic agent classified as a topoisomerase inhibitor.
  • Topoisomerase inhibitors are agents that interfere with the action of topoisomerase enzymes which regulate DNA structure by catalyzing the breaking and rejoining of the phosphodiester backbone of DNA strands during the cell cycle. Topoisomerase inhibitors can be classified into topoisomerase I and topoisomerase II inhibitors. Topoisomerase I inhibitors, such as irinotecan, topotecan, camptothecin and lamellarin D, target the topoisomerase I enzymes which catalyze the breaking of one strand of the DNA double helix.
  • Topoisomerase II inhibitors such as doxorubicin, etoposide, teniposide, daunorubicin, mitoxantrone, amsacrine, and ellipticines, target the topoisomerase II enzymes which can break both strands of the DNA double helix.
  • the patient's cancer therapy is a chemotherapeutic agent classified as a topoisomerase inhibitor I or a topoisomerase inhibitor II.
  • the patient's cancer therapy is the topoisomerase I inhibitor irinotecan.
  • the patient's cancer therapy is the topoisomerase II inhibitor, doxorubicin.
  • the patient's cancer therapy is irinotecan and the side effect is one or more of diarrhea, dehydration, suppression of the immune system, hematological damage, lowered white blood cell counts, neutropenia, cholinergic syndrome, blood clots, infection, and abdominal pain.
  • hematological damage is one or more of leukopenia and/or reduced white blood count and reduction of other types of blood cells, including neutrophils, lymphocytes and platelets.
  • hematological damage is assessed by a blood test as described herein.
  • hematological damage in a patient undergoing irinotecan treatment directs administration of a flagellin related composition, including CBLB502, to mitigate this side effect.
  • the patient's cancer therapy is irinotecan and the side effect is diarrhea.
  • administering an effective amount of CBLB502 reduces a dose and/or frequency of an anti- diarrheal agent (e.g. one or more of loperamide, diphenoxylate, and atropine) administered to the cancer patient.
  • an anti- diarrheal agent e.g. one or more of loperamide, diphenoxylate, and atropine
  • the side effect of irinotecan treatment is vomiting.
  • administering an effective amount of CBLB502 reduces a dose and/or frequency of an antiemetic, including, for example, 5-HT3 blockers with or without dexamethasone, administered to the cancer patient.
  • the patient's cancer therapy is irinotecan and the side effect is measured by monitoring a patient's temperature.
  • temperature in excess of about 37.5°C (99.5° F) or about 38°C (100.4°F) and beyond indicates irinotecan side effects and directs administration of an effective amount of a flagellin related composition, including CBLB502.
  • a patient is administered irinotecan and is subsequently evaluated for impaired hepatic function by, for example, a blood test and/or biopsy and/or hepatic imaging.
  • this directs administration of a flagellin related composition, including CBLB502, to mitigate this side effect.
  • the present invention provides for administration of a flagellin related composition, including CBLB502, to a cancer patient receiving irinotecan and also classified as a *28 variant.
  • a flagellin related composition including CBLB502
  • Patients with variants of the UGT1A1 called TA 7 also known as the "*28 variant” express fewer UGT1A1 enzymes in their liver and often suffer from Gilbert's syndrome.
  • these patients may effectively receive a larger than expected dose because their bodies are not able to clear irinotecan as fast as others. This often corresponds with higher incidences of severe neutropenia and diarrhea.
  • irinotecan recipients with a homozygous polymorphism in UGT1A1 gene may receive standard doses if also receiving a flagellin related composition, including CBLB502.
  • the patient's cancer therapy is doxorubicin and the side effect is one or more of vomiting, nausea, diarrhea, dehydration, suppression of the immune system, hematological damage, lowered white blood cell counts, infection, cardiomyopathy, congestive heart failure, typhlitis, hand-foot syndrome.
  • hematological damage is one or more of leukopenia and/or reduced white blood count and reduction of other types of blood cells, including neutrophils, lymphocytes and platelets.
  • hematological damage is assessed by a blood test as described herein.
  • hematological damage in a patient undergoing doxorubicin treatment directs administration of a flagellin related composition, including CBLB502, to mitigate this side effect.
  • the patient's cancer therapy is doxorubicin and the side effect is diarrhea.
  • administering an effective amount of CBLB502 reduces a dose and/or frequency of an anti- diarrheal agent (e.g. one or more of loperamide, diphenoxylate, and atropine) administered to the cancer patient.
  • an anti- diarrheal agent e.g. one or more of loperamide, diphenoxylate, and atropine
  • the side effect of doxorubicin treatment is vomiting.
  • administering an effective amount of CBLB502 reduces a dose and/or frequency of an antiemetic, including, for example, 5-HT3 blockers with or without dexamethasone, administered to the cancer patient.
  • the patient's cancer therapy is doxorubicin and the side effect is measured by monitoring a patient's temperature.
  • temperature in excess of about 37.5°C (99.5 ) or about 38°C (100.4°F) and beyond indicates doxorubicin side effects and directs administration of an effective amount of a flagellin related composition, including CBLB502.
  • the present invention provides for uses of the flagellin related polypeptides, including CBLB502, to increase a therapeutic window of a therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin.
  • the increased therapeutic window of a therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin comprises one or more of increasing a cancer patient's likelihood receiving therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin maintenance therapy; increasing a cancer patient's likelihood of receiving a complete regime of a therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin; increasing a cancer patient's likelihood of receiving more than a complete regimen of a therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin; and increasing the dose or length of a therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin treatment.
  • uses of the flagellin related polypeptides, including CBLB502 allow for a cancer patient to receive dose-dense chemotherapy. In various embodiments, uses of the flagellin related polypeptides, including CBLB502, allow for a cancer patient to avoid dose delay.
  • the standard 5-FU treatment regime comprises administering about 12 mg/kg once daily for four successive days and optionally about 6 mg/kg on the sixth, eighth, tenth and twelfth days unless toxicity occurs.
  • administration of a flagellin related polypeptide, including CBLB502 may allow a patient to be administered a full regimen without having to cease treatment because of, for example, side effects.
  • administration of a flagellin related polypeptide, including CBLB502 allows a patient to be administered increased dosages (e.g.
  • administration of a flagellin related polypeptide may allow a patient to be administered a dose of 5-FU that exceeds about 200 mg daily, about 300 mg daily, about 400 mg daily, about 500 mg daily, about 600 mg daily, about 700 mg daily, or about 800 mg daily, or about 900 mg daily, or about 1000 mg daily.
  • the standard 5-FU maintenance therapy comprises dosage of about 12 mg/kg every 30 days after the last day of a previous course of treatment or about 10 to 15 mg/kg/week as a single dose.
  • administration of a flagellin related polypeptide, including CBLB502 may allow a patient to be administered this 5-FU maintenance therapy.
  • administration of a flagellin related polypeptide, including CBLB502 may allow a patient to be administered a greater 5-FU maintenance therapy (e.g. more frequently and/or at a larger dose).
  • administration of a flagellin related polypeptide may allow a patient to be administered a dose of irinotecan that is greater than about 125 mg/m 2 , about 150 mg/m 2 , about 175 mg/m 2 , about 200 mg/m 2 , about 225 mg/m 2 , about 250 mg/m 2 , about 275 mg/m 2 , about 300 mg/m 2 , about 325 mg/m 2 , about 350 mg/m 2 , about 400 mg/m 2 , or about 500 mg/m 2 , or about 1000 mg/m 2 .
  • the standard monotherapy regimen of irinotecan is (a) weekly: 125 mg/m 2 IV infusion over 90 minutes on days 1 , 8, 15, 22, then 2 weeks off, then repeat or (b) once every 3 weeks: 350 mg/m 2 IV infusion over 30-90 minutes every 3 weeks.
  • administration of a flagellin related polypeptide, including CBLB502 may allow a patient to be administered a complete regimen of irinotecan without having to cease treatment because of, for example, side effects.
  • administration of a flagellin related polypeptide, including CBLB502 may allow a patient to be administered an increased dose or frequency of irinotecan than the standard regimen (e.g.
  • administration of a flagellin related polypeptide may allow a patient to be administered a dose of doxorubicin that is greater than about 20 mg/m 2 , about 25 mg/m 2 , about 30 mg/m 2 , about 35 mg/m 2 , about 40 mg/m 2 , about 45 mg/m 2 , about 50 mg/m 2 , about 55 mg/m 2 , about 60 mg/m 2 , about 65 mg/m 2 , about 70 mg/m 2 , about 75 mg/m 2 , about 80 mg/m 2 , about 85 mg/m 2 , about 90 mg/m 2 , or about 95 mg/m 2 , or about 100 mg/m 2 .
  • the standard monotherapy regimen of doxorubicin is a 60-75 mg/m 2 IV infusion every 3-4 weeks.
  • administration of a flagellin related polypeptide, including CBLB502 may allow a patient to be administered a complete regimen of doxorubicin without having to cease treatment because of, for example, side effects.
  • administration of a flagellin related polypeptide, including CBLB502 may allow a patient to be administered an increased dose or frequency of doxorubicin than the standard regimen (e.g.
  • the present invention provides for uses of the flagellin related polypeptides, including CBLB502, to increase an ability of a cancer patient to receive a combination therapy with irinotecan and/or 5-FU and or doxorubicin.
  • administering an effective amount of a flagellin related polypeptide, including CBLB502 increases the ability of a cancer patient to receive a combination therapy with 5-FU.
  • the combination therapy is selected from leucovorin, oxaliplatin, folinic acid, and irinotecan.
  • administering an effective amount of a flagellin related polypeptide increases the ability of a cancer patient to receive a combination therapy with irinotecan or doxorubicin.
  • the combination therapy is selected from oxaliplatin, 5-fluorouracil, folinic acid, leucovorin, cetuximab, temozolomide, sorafenib, capecitabine, bevacizumab, vincristine, vinblastine, dacarbazine, prednisone, rapamycin, cyclophosphamide, and bleomycin,.
  • administering an effective amount of a flagellin related polypeptide, including CBLB502 increases the ability of a cancer patient to receive a complete regimen of a combination therapy with 5-FU and/or irinotecan and/or doxorubicin and another agent and not have to cease treatment because of, for example, side effects.
  • administering an effective amount of a flagellin related polypeptide, including CBLB502 increases the ability of a cancer patient to receive a greater dose or longer duration of combination therapy with 5-FU and/or irinotecan and/or doxorubicin and another agent.
  • a combination therapy regimen for irinotecan with 5-fluorouracil/ leucovorin may be (a) 6 week cycle with infusional 5-fluorouracil/leucovorin: 180 mg/m 2 IV infusion over 30-90 minutes once on days 1 , 15, and 29 IV (infuse over 30-90 min), followed by infusion with leucovorin and 5-fluorouracil; next cycle begins on day 43 or (b) 6 week cycle with bolus 5-fluorouracil/ leucovorin: 125 mg/m 2 on days 1 , 8, 15, and 22 (infuse over 90 min), followed by bolus doses of leucovorin and 5-fluorouracil.
  • administering an effective amount of a flagellin related polypeptide, including CBLB502 increases the dose and/or frequency of this regimen.
  • this regimen can be extended beyond 6 weeks to about 45, or about 50, or about 55, or about 60, or about 65, or about 70, or about 75, or about 100 days (including about 7, or about 8, or about 9, or about 10, or about 1 1 , or about 12 weeks).
  • this regimen can allow for doses of irinotecan that exceed about 125 mg/m 2 , or about 150 mg/m 2 , or about 200 mg/m 2 , or about 250 mg/m 2 , or about 300 mg/m 2 , or about 500 mg/m 2 .
  • administering an effective amount of a flagellin related polypeptide increases the ability of a cancer patient to receive a complete regimen of any one of the FOLFOX, FOLFIRI, IFL, FL ( Mayo), QUASAR, Machover schedule, CAF, CMF, ECF, and FEC regimens and not have to cease treatment because of, for example, side effects.
  • administering an effective amount of a flagellin related polypeptide, including CBLB502 increases the ability of a cancer patient to receive a greater dose or longer duration of therapy in the FOLFOX, FOLFIRI, IFL, FL ( Mayo), QUASAR, Machover schedule, CAF, CMF, ECF, and FEC regimens.
  • combination agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (e.g., alkylating agents such as
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, ADRIAMYCIN doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxy doxorubicin), epirub
  • the methods of treatment can further include the following: oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-a, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the methods of treatment can further include the
  • the present invention provides a method for improving treatment with the flagellin related compositions in the context of oncology.
  • the flagellin related compositions may be paired with one or more agents as described herein (e.g. 5-FU) to enhance CBLB502-mediated antitumor immune response.
  • agents as described herein e.g. 5-FU
  • such a combination may reduce tumor-associated immunosuppression and/or increase tumor cell immunogenicity.
  • such a combination may reduce the activity or activation of one or more cells, or reduce or eliminate the infiltration to a tumor site and/or TME of one or more cells, including, but not limited to: myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs); tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs).
  • MDSCs myeloid-derived suppressor cells
  • Tregs tumor associated neutrophils
  • TANs tumor associated neutrophils
  • TAMs tumor associated macrophages
  • the flagellin related compositions (and/or additional agents) described herein include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition.
  • derivatives include composition that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of turicamycin, etc. Additionally, the derivative can contain one or more non- classical amino acids.
  • the flagellin related compositions (and/or additional agents) described herein may be modified post-translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
  • effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
  • the present invention pertains to cancers and/or tumors; for example, the treatment or prevention of cancers and/or tumors.
  • a cancer to be treated in the context of 5- FU is one or more of breast cancer, head and neck cancer, anal cancer, stomach cancer, colon cancer, and skin cancer.
  • a cancer to be treated in the context of irinotecan is one or more of lung cancer, ovarian cancer, non-small cell lung cancer, leukemia, lymphoma, pancreatic tumor, metastatic cancer, breast tumor, rectal tumor, colonic neoplasms, stomach tumor, metastatic cancer colon or rectum and glioma.
  • a cancer to be treated in the context of doxorubicin is one or more of leukemia, lymphoma (e.g., Hodgkin's lymphoma), bladder cancer, breast cancer, head and neck cancer, stomach cancer, liver cancer, lung cancer, mesothelioma, ovarian cancer, thyroid cancer, pancreatic cancer, prostate cancer, sarcomas (e.g., soft tissue sarcoma), neuroblastoma, uterine cancer, testis cancer, and multiple myeloma.
  • lymphoma e.g., Hodgkin's lymphoma
  • bladder cancer e.g., breast cancer, head and neck cancer
  • stomach cancer e.g., liver cancer, lung cancer, mesothelioma, ovarian cancer, thyroid cancer, pancreatic cancer, prostate cancer
  • sarcomas e.g., soft tissue sarcoma
  • neuroblastoma uterine cancer
  • testis cancer e
  • cancer refers to an uncontrolled growth of cells and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of the bodily organs and systems. Included are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Also, included are cells having abnormal proliferation that is not impeded by the immune system (e.g. virus infected cells).
  • a subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. 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.
  • Hematopoietic cancers such as leukemia, are able to out-compete the normal hematopoietic compartments in a subject, thereby leading to hematopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.
  • the cancer may be a primary cancer or a metastatic cancer.
  • the primary cancer may be an area of cancer cells at an originating site that becomes clinically detectable, and may be a primary tumor.
  • the metastatic cancer may be the spread of a disease from one organ or part to another non-adjacent organ or part.
  • the metastatic cancer may be caused by a cancer cell that acquires the ability to penetrate and infiltrate surrounding normal tissues in a local area, forming a new tumor, which may be a local metastasis.
  • “Metastasis” refers to the spread of cancer from a primary site to other places in the body.
  • Metastasis can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life -threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant. Metastases may be detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.
  • MRI magnetic resonance imaging
  • CT computed tomography
  • the cancer may also be caused by a cancer cell that acquires the ability to penetrate the walls of lymphatic and/or blood vessels, after which the cancer cell is able to circulate through the bloodstream (thereby being a circulating tumor cell) to other sites and tissues in the body.
  • the cancer may be due to a process such as lymphatic or hematogeneous spread.
  • the cancer may also be caused by a tumor cell that comes to rest at another site, re-penetrates through the vessel or walls, continues to multiply, and eventually forms another clinically detectable tumor.
  • the cancer may be this new tumor, which may be a metastatic (or secondary) tumor.
  • the cancer may be caused by tumor cells that have metastasized, which may be a secondary or metastatic tumor.
  • the cells of the tumor may be like those in the original tumor.
  • the secondary tumor while present in the liver, is made up of abnormal breast or colon cells, not of abnormal liver cells.
  • the tumor in the liver may thus be a metastatic breast cancer or a metastatic colon cancer, not liver cancer.
  • the cancer may have an origin from any tissue.
  • the cancer may originate from melanoma, colon, breast, or prostate, and thus may be made up of cells that were originally skin, colon, breast, or prostate, respectively.
  • the cancer may also be a hematological malignancy, which may be lymphoma.
  • Representative cancers and/or tumors of the present invention include, but are not limited to, a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small- cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx);
  • the flagellin related compositions (and/or additional agents) described herein can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt.
  • a pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art.
  • Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.
  • Pharmaceutically acceptable salts include, by way of non-limiting example, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,
  • Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such as mono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert- butylamine, or tri
  • compositions described herein are in the form of a pharmaceutically acceptable salt.
  • any flagellin related compositions (and/or additional agents) described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle.
  • Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.
  • Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like.
  • auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used.
  • the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions.
  • suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the present invention includes the described flagellin related compositions (and/or additional agents) in various formulations.
  • Any flagellin related composition (and/or additional agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • the composition is in the form of a capsule (see, e.g., U.S. Patent No. 5,698,155).
  • suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.
  • the flagellin related compositions can also include a solubilizing agent.
  • the agents can be delivered with a suitable vehicle or delivery device as known in the art.
  • Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device.
  • Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.
  • the formulations comprising the flagellin related compositions (and/or additional agents) of the present invention may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art)
  • a carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed
  • any flagellin related composition (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.
  • Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
  • the administering is effected orally or by parenteral injection.
  • the mode of administration can be left to the discretion of the practitioner, and depends in-part upon the site of the medical condition. In most instances, administration results in the release of any agent described herein into the bloodstream.
  • flagellin related composition (and/or additional agents) described herein can be administered orally.
  • flagellin related compositions (and/or additional agents) can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local.
  • Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer.
  • any flagellin related composition (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for oral administration to humans.
  • Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
  • Orally administered compositions can comprise one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
  • compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time.
  • Selectively permeable membranes surrounding an osmotically active driving any flagellin related composition (and/or additional agents) described herein are also suitable for orally administered compositions.
  • fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture.
  • delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations.
  • a time-delay material such as glycerol monostearate or glycerol stearate can also be useful.
  • Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate.
  • the excipients are of pharmaceutical grade.
  • Suspensions in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, etc., and mixtures thereof.
  • Dosage forms suitable for parenteral administration include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
  • any flagellin related composition (and/or additional agents) described herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject's general health, and the administering physician's discretion.
  • Any agent described herein can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional therapeutic agent, to a subject in need thereof.
  • any agent described herein is administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart.
  • any flagellin related composition (and/or additional agents) described herein that is admixed with the carrier materials to produce a single dosage can vary depending upon the subject being treated and the particular mode of administration. In vitro or in vivo assays can be employed to help identify optimal dosage ranges.
  • doses that are useful are known to those in the art.
  • doses may be determined with reference Physicians' Desk Reference, 66th Edition, PDR Network; 2012 Edition (December 27, 2011 ), the contents of which are incorporated by reference in its entirety.
  • the present invention allows a patient to receive doses that exceed those determined with reference Physicians' Desk Reference.
  • any flagellin related composition (and/or additional agents) described herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.
  • the dosage of any flagellin related composition (and/or additional agents) described herein may be 0.001 mg/kg/day to 100 mg/kg/day, 0.01 mg/kg/day to 50 mg/kg/day, or 0.1 mg/kg/day to 10 mg/kg/day.
  • the dosage of any agent described herein is normally 0.001 mg to 1000 mg per day, 1 mg to 600 mg per day, or 5 mg to 30 mg per day.
  • the dosage is normally 0.1 mg to 250 mg per day, 1 mg to 20 mg per day, or 3 mg to 5 mg per day. Injections may be given up to four times daily.
  • the dosage of any agent described herein is normally 0.1 mg to 1500 mg per day, or 0.5 mg to 10 mg per day, or 0.5 mg to 5 mg per day. A dosage of up to 3000 mg per day can be administered.
  • delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et a/., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).
  • a liposome see Langer, 1990, Science 249:1527-1533; Treat et a/., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).
  • Any flagellin related composition (and/or additional agents) described herein can be administered by controlled-release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety.
  • Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions.
  • Suitable controlled- or sustained-release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the agents described herein.
  • the invention thus provides single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled- or sustained-release.
  • Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability : Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem.
  • a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • Other controlled-release systems discussed in the review by Langer, 1990, Science 249:1527-1533 may be used.
  • Administration of any flagellin related composition (and/or additional agents) described herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the subject. Chronic, long-term administration will be indicated in many cases.
  • the dosage may be administered as a single dose or divided into multiple doses. In general, the desired dosage should be administered at set intervals for a prolonged period, usually at least over several weeks or months, although longer periods of administration of several months or years or more may be needed.
  • the dosage regimen utilizing any flagellin related composition (and/or additional agents) described herein can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the specific compound of the invention employed.
  • Any flagellin related composition (and/or additional agents) described herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily.
  • any flagellin related composition (and/or additional agents) described herein can be administered continuously rather than intermittently throughout the dosage regimen.
  • the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon.
  • the subject and/or animal is a non-mammal, such, for example, a zebrafish.
  • the subject and/or animal may comprise fluorescently-tagged cells (with e.g. GFP).
  • the subject and/or animal is a transgenic animal comprising a fluorescent cell.
  • the subject and/or animal is a human.
  • the human is a pediatric human.
  • the human is an adult human.
  • the human is a geriatric human.
  • the human may be referred to as a patient.
  • the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
  • the subject is a non-human animal, and therefore the invention pertains to veterinary use.
  • the non-human animal is a household pet.
  • the non-human animal is a livestock animal.
  • kits that can simplify the administration of any agent described herein.
  • An exemplary kit of the invention comprises any composition described herein in unit dosage form.
  • the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle.
  • the kit can further comprise a label or printed instructions instructing the use of any agent described herein.
  • the kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location.
  • the kit can also further comprise one or more additional agent described herein.
  • the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those described herein.
  • mice BALB/c and C57BL/6 female mice, 10-14 weeks old (Jackson Laboratory, Bar Harbor, ME) were used in the study.
  • IL-6 deficient mice C.129S2-IL6 tm1/kopf /J (BALB/c background) were originally purchased from Jackson Laboratory; TLR5 knockout mice B6.129P2-Tlr5 tm1Aki (C57BL/6 background) were a generous gift of Dr. Shizuo Akira (University of Tokyo, Japan); mice of both strains were bred at Roswell Park Cancer Institute (RPCI). All animal experiments followed protocols approved by the RPCI IACUC.
  • CBLB502 was obtained from Cleveland BioLabs, Inc. (Buffalo, NY). 5-Fluorouracil (5-FU) was purchased from Sigma (St. Louis, MO).
  • Tumor cells Murine colon undifferentiated carcinoma CT26 cells (ATCC) were cultured in RPMI media with 10% FBS, standard supplements (2 mmol/L L-glutamine, ⁇ ⁇ /L nonessential amino acids) and 1 % penicillin-streptomycin (Invitrogen) at 37°C in a 5% CO2 incubator.
  • CT26 tumor cells (5x10 5 /100 ⁇ PBS) were injected s.c. into syngeneic BALB/c mice (2 flanks/mouse). When tumors reached ⁇ 5 mm in diameter, mice were randomly divided into 5 treatment groups. 5-FU (100 or 200 mg/kg) was injected i.p. with CBLB502 (1 g/ 100 ⁇ ) or PBS injected s.c. 1 , 48 and 96 h after 5-FU. A group of mice injected with vehicle solutions was used as an "untreated" control. Tumors were measured by two diameters with a digital caliper every second day.
  • the mitotic index was determined by counting the mitotic figures in crypt cells of the small intestine using light microscopy at 100 ⁇ magnification. The mitotic index is calculated in longitudinally sectioned crypts as an average value per crypt in each treatment-group (4 samples per mouse, 3 mice/group).
  • Cytokine analysis Concentrations of IL-6 and soluble IL-1 receptor in mouse plasma were determined using MILLIPLEX kit (EMD Millipore, St. Charles, MO). Plasma was prepared from blood collected from BALB/c mice by heart puncture upon euthanasia 3 or 7 days after 5-FU injections (200 mg/kg) with or without CBLB502 (1 ⁇ g/ mouse) injected 24 and 48 h after 5-FU. Plasma from vehicle-injected mice was used as an untreated control. There were 5 mice per treatment group and plasma from 2-3 mice was pooled together for analysis (data is shown as the average of 2 pools for each treatment group).
  • RT-PCR Expression of IL-1 receptor antagonist (IL-1 RN) was detected by RT-PCR using total RNA prepared from the small intestine isolated from a mouse treated with CBLB502 (1 ⁇ g/ mouse x2 injections 24 h apart) 24 h after the second injection. The small intestine from a PBS-injected mouse was used as an "intact" control. Total RNA was extracted using TRIzol reagent according to manufacturer's instructions (Invitrogen, Carlsbad, CA). cDNAs were synthesized using iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) according to manufacturer's protocol from 250 ng RNA/sample.
  • mice Two mouse strains, BALB/c and C57BL/6, which differ in their sensitivity to 5-FU were used to determine whether CBLB502 is capable of protecting normal tissues from the toxicity of 5-FU. Mice were administered 5-FU alone or in combination with subsequent CBLB502 treatment using different doses and schedules and toxicity was assessed by monitoring body weight loss and mortality.
  • Fig. 1 shows body weight (A) and survival (B) of BALB/c mice treated with 5-FU (200 mg/kg or 400 mg/kg given in two equal fractions 6 h apart) alone or with subsequent injection of CBLB502 (1 ⁇ g/mouse) 24 and 48 h after the last 5-FU dose (10 animals/group).
  • Body weight in Fig. 1 panel A is shown as a percentage of starting weight; mean ⁇ SEM.
  • the kinetics of body weight loss showed an initial drop in mean body weight during the first 3-5 days after injection, partial improvement by day 7-8, and then a second drop with no recovery.
  • a higher dose of 5-FU 400 mg/kg caused more rapid single-phasic body weight loss and death of 100% of animals within 7 days.
  • administration of CBLB502 two s.c. injections given 24 and 48 h after 5-FU
  • mean body weight loss was minimal ( ⁇ 10%) and all mice survived to the end of the 30 day observation period.
  • CBLB502 and vehicle injected mice without 5-FU were weighed as controls. Mean body weight (as a percentage of starting weight) +/- SEM is shown.
  • Body weight beyond this time-point was based on 3 surviving mice in the group treated with CBLB502 1 and 24 h post-5-FU.
  • C57BL/6 mice were more resistant to 5-FU toxicity than BALB/c mice.
  • Administration of 200 mg/kg 5- FU to C57BL/6 mice was not lethal, but caused transient body weight loss (only about 10-15%) with complete recovery by Day 10.
  • 400 mg/kg 5-FU which caused early death of BALB/c mice by day 7, was lethal for majority of C57BL/6 mice by day 15 (Fig. 1 , panel C).
  • CBLB502 was evaluated to determine whether it specifically reduces HP damage caused by 5-FU.
  • treatment of BALB/c mice with 200 or 100 mg/kg 5-FU resulted in rapid elimination of practically all types of blood cells, including neutrophils, lymphocytes and platelets, correlating with lethality observed within 1 1-14 days after 5-FU administration.
  • CBLB502 treatment after 200 mg/kg 5-FU did not ameliorate 5-FU-induced depletion of blood cell populations as observed on Days 7 and 1 1 post-5-FU (Fig. 4 panel A).
  • mice treated with CBLB502 after 200 mg/kg 5-FU administration showed gradual recovery of hematopoiesis leading to complete restoration of normal WBC and neutrophil levels by Day 14 and improved mouse survival.
  • mice treated with CBLB502 after 5-FU consistently displayed more rapid recovery of peripheral blood cell populations than mice given 5-FU alone.
  • Fig. 5 shows the restoration of blood cell count in BALB/c mice following 5-FU treatment alone and in combination with CBLB502. Specifically, 5-FU was injected i.p.
  • surviving mice displayed temporary overcompensation for the loss of HP cells: numbers of lymphocytes, neutrophils and platelets were higher in treated mice than in intact controls at the day 14-15 time-points (Fig. 4 panel B (see above text), but normalized by day 21.
  • BM bone marrow
  • Fig. 6 shows the effect of CBLB502 on 5-FU-induced changes in bone marrow morphology.
  • Representative pictures of H&E-stained bone marrow sections (10x objective magnification) were prepared 3 and 7 days after 5-FU injection.
  • BALB/c mice were treated with 200 mg/kg 5-FU with or without injection of CBLB502 24 and 48 h post-5-FU; 5 mice/group. The lower row shows selected areas outlined in white at two fold higher magnification.
  • mice In both 5-FU-treated mice and those given 5-FU in combination with CBLB502, the BM was drastically depopulated in contrast to BM samples from mice treated with CBLB502 alone or intact mice which displayed normal morphology with a meshwork of bony spicules, open areas with active hematopoiesis and sinusoids filled with erythrocytes.
  • the BM of mice treated with 200 mg/kg 5-FU alone contained only single HP and stromal cells that were rounded and detached from each other (indicative of necrosis and necrobiosis). In addition, hemorrhage and an abundant fat component were observed in these BM samples.
  • the BM of mice injected with CBLB502 after 200 mg/kg 5-FU showed signs of HP recovery including islets of preserved HP cells with prevalent hyperplastic megakaryocytes suggesting that CBLB502 promotes recovery of hematopoiesis in the BM and restoration of peripheral HP cell populations after 5-FU-induced damage.
  • Fig. 7 shows the effect of CBLB502 on 5-FU- induced changes in small intestine morphology.
  • Fig. 7A-B correspond to: representative H&E-stained transverse sections (250x objective magnification) of small intestines from vehicle injected BALB/c mice (intact) and treated with 5-FU (200 mg/kg (Fig. 7 panel A) or 400 mg/kg (Fig.
  • FIG. 7 panel B shows the average injury score of the small intestine sections described in (Fig. 7 panels A-B).
  • FIG. 8 Similar analysis of the morphology of the large intestine (colon) in BALB/c mice after 200 mg/kg or 400 mg/kg 5-FU treatment showed dose-dependent injury of the colonic mucosa on day 3 and 7 post-5-FU, with atrophic and degenerative changes observed mainly in the crypt cells (e.g., loss of mucin globules, loss of distinct boundaries between adjacent cells, disappearance of the apical cytoplasm, and extended crypt lumens) (Fig. 8).
  • Fig. 8 panel A or 400 mg/kg (Fig. 8 panel B)) alone or in combination with CBLB502 treatment 24 and 48 h post-5-FU. Sections were prepared 3 or 7 days after 5-FU treatment. Morphology of crypt areas outlined in white is shown at two fold higher magnification in the lower rows. Atrophic and degenerative changes in the surface epithelium are indicated by arrows and in the crypt cells and mucin globules by arrowheads.
  • Fig. 8 panel C shows the average injury score determined by pathomorphological changes in the surface epithelium, number of surface enterocytes and goblet cells, size and shape of crypts, lymphoid elements and state of the submucosa in colon sections from mice described in (Fig.
  • Fig. 9 shows IL-6 concentration in plasma samples from BALB/c mice that were analyzed by a MILLIPLEX kit (average of 2 pools of 3 mouse samples each at each data-point). Plasma was collected 3 and 7 days after injection of vehicle (DMSO), CBLB502 alone, 200 mg/kg 5-FU alone, or 5-FU+CBLB502.
  • FIG. 10 panel A shows survival of WT BALB/c and IL-6- KO mice after treatment with 200 mg/kg 5-FU, with or without CBLB502 treatment (1 ⁇ g/mouse) 24 and 48 h after 5-FU.
  • Fig. 10 panel B shows the kinetics of body weight changes in IL-6-KO mice treated with 5-FU (400 mg/kg) and CBLB502 injected 24 and 48 h after the last 5-FU dose (mean ⁇ SEM).
  • Fig. 10 panel C shows survival of IL-6-KO and wild type (WT) mice treated with 5-FU (400 mg/kg) or 5-FU+CBLB502. The combined results of two independent experiments are presented in (Fig. 10 panel B) and Fig. 10 panel C).
  • FIG. 10 panel D shows H&E-stained small intestine sections showing crypts from IL-6-KO mice euthanized on Day 3 after 5-FU (400 mg/kg) with or without CBLB502. Enterocytes lining the villi are indicated by arrows, lamina intestinal by asterisks and crypts by arrowheads. Normal morphology of crypts in an intact mouse is shown as a control. Fig.
  • 5-FU 100 mg/kg was followed by CBLB502 24 and 48 h later; m ⁇ SEM.
  • Differences between the 5-FU and 5-FU+CBLB502 groups were p ⁇ 0.05.
  • CBLB502 treatment did provide some survival benefit to IL-6-KO mice, increasing mean survival time from 8 to 10 days (PO.001 ) (Fig. Fig. 10 panel C (see above text)).
  • Fig. 10 panel B shows the mitotic index in IL-6-KO mice after 5-FU treatment with and without CBLB502. The mitotic index was calculated in crypts of 4 transverse sections of small intestine per mouse as the number of mitoses per crypt in 3 mice/ group.
  • Fig. 12 panel A shows (Fig. 12 panel A): the concentration of soluble IL-1 receptor in plasma prepared from BALB/c mice 72 h after 5-FU injections (200 and 400 mg/kg) with and without CBLB502 injected 24 and 48 h after 5- FU was determined using MILLIPLEX kit (average of 2 pools of 3 mouse samples at each data-point).
  • CBLB502 to improve anticancer chemotherapy by reducing its adverse side effects requires that the protective effects of the drug are strictly limited to normal (non-tumor) cells and tissues. Since 5- FU is commonly used as a single agent or in combination with other chemotherapeutic drugs to treat colorectal cancer, the mouse CT26 colorectal carcinoma model was employed to test the effect of CBLB502 on tumor sensitivity to 5-FU. CT26 cells do not express TLR5 and their growth as s.c. tumors is not suppressed by CBLB502 treatment (Fig. 13). Fig. 13 shows the treatment of CT-26 tumor-bearing BALB/c mice with CBLB502 initiated when tumors reached about 5 mm in diameter.
  • CT26 tumors were, however, sensitive to 5- FU treatment, showing 5-FU dose-dependent reduction of tumor growth (Fig. 14 panel A).
  • Fig. 14 panel A kinetics of s.c. CT26 tumor growth in mice treated with vehicle ("untreated"), 5-FU alone (100 or 200 mg/kg), or 5-FU + CBLB502 injected 1 , 48, and 96 h post-5-FU (10 mice, 20 tumors per group in 5-FU treated groups, and 5 mice, 10 tumors, in "untreated” group, mean ⁇ SEM); Fig.
  • FIG. 14 panel B kinetics of body weight changes (percentage of starting weight); and Fig. 14 panel C: survival of mice described in Fig. 14 panel A.
  • (*) Body weight and tumor volume are shown for only 4/10 mice injected with 200 mg/kg 5-FU alone that were surviving on Day 18 after CT26 cell inoculation; mice in this group died due to 5-FU toxicity with small or no tumors. In the other groups, mice were euthanized due to large size or ulceration of tumors; 2/10 mice in the group treated with 200 mg/kg 5-FU + CBLB502 remained tumor-free for the entire period of observation (60 days).
  • CBLB502 treatment also did not change survival of tumor-bearing mice treated with 100 mg/kg 5-FU; 100% of mice in these two groups were euthanized by Day 29 after tumor cell inoculation due to tumors reaching the size endpoint or developing ulcerations (Fig. 14 panel B (see above text)).
  • 100 % mice injected with 200 mg/kg 5-FU without CBLB502 died due to 5-FU toxicity by Day 27 post tumor cell implantation (Day 23 post 5-FU injection), while those treated with the combination of 200 mg/kg 5-FU and CBLB502 experienced significantly less toxicity.
  • Tumor-bearing mice given CBLB502 after 200 mg/kg 5-FU displayed less weight loss (Fig. 14 panel B (see above text)) and prolonged survival (Fig.
  • Rats (Ward colorectal cancer Rat model), weighing 125-150gm, were transplanted s.c. with a 50 mg viable tumor fragment on day 1 and then the tumors were allowed to grow to approximately 1.0 g by day 7.
  • CBLB502 and/or irinotecan was administered via intraperitoneal injection utilizing the following schedules: 1 ) 0.2 mg/kg CBLB502 d5-15 (10 doses) following implant of tumors (7 rats/group)- 4 rats kept up to 52 days (length of study) and 3 rats euthanized 2h after last 502 injection for histology tumor/gastrointestinal organs and liver evaluation; 2) 0.2 mg/kg CBLB502 d5-9 (5 doses) following implant of tumors (10 rats/group)- 4 rats kept up to 52 days and 3 rats euthanized 2h after last 502 injection for histology tumor/gastrointestinal organs and liver evaluation; 3) 0.2 mg/kg CBLB502 d7-9 (3 doses) following implant of tumors (10 rats/group)- 4 rats kept up to 52 days, 3 rats euthanized 2h after last 502 injection for histology tumor/gastrointestinal organs and liver evaluation; 4) Irinotecan alone (200 mg/kg iv for 3 days) (7 rats)
  • Fig. 15 shows the toxicity of irinotecan in Fischer rats bearing Ward colorectal carcinoma with or without CBLB502 (administered before or concurrently).
  • Irinotecan an agent active in the treatment of colorectal cancer, the dose of 200 mg/kg/dx3 compared with the maximum tolerated dose (100 mg/kg/dx3), was highly detrimental with 100% of rats having the side effect diarrhea and lethality (Fig. 15). All three schedules of CBLB502 offered protection/mitigation against the high levels of diarrhea and lethality induced by irinotecan (Fig. 15).
  • immunosuppression prevents the generation of a durable antitumor immune response and may interfere with effective cancer treatment.
  • Immunosuppression is defined as the expansion of T regulatory cells (Tregs) and myeloid derived suppressor cells (MDSC), which are predominately immature neutrophils called granulocytic MDSC (gMDSC), that directly prevent activation of antitumor T cell-dependent immunity.
  • Tregs and MDSC represent a potential strategy to improve anticancer immunotherapy.
  • chemotherapeutic agents such as 5-FU may enhance CBLB502 mediated antitumor immune response by reducing tumor-associated immunosuppression and increasing tumor cell immunogenicity.
  • a highly tumorigenic and invasive mouse 4T1 mammary carcinoma model was utilized to study whether chemotherapeutic drugs such as 5-FU can reduce immunosuppression and thus allow improved efficacy of CBLB502.
  • the 4T1 model shares many of the characteristics of human breast cancer, particularly its ability to spontaneously metastasize to the lungs and increase the numbers of cells with immunosuppressive phenotypes through production of TGF and generation of MDSCs.
  • chemotherapeutic treatment can be combined with TLR5 agonistic agents (e.g., CBLB502) for effective cancer treatment.
  • chemotherapeutic agents promote anticancer immunotherapy by reducing immunosuppressive cells and enhancing the antitumor effect of CBLB502 when applied in a neoadjuvant setting in respect to immunotherapy.
  • Toll-like receptor 5 agonist protects mice from dermatitis and oral mucositis caused by local radiation: implications for head-and-neck cancer radiotherapy. International journal of radiation oncology, biology, physics. 2012; 83(1 ):228-234.
  • Pritchard DM Potten CS and Hickman JA. The relationships between p53-dependent apoptosis, inhibition of proliferation, and 5-fluorouracil-induced histopathology in murine intestinal epithelia. Cancer research. 1998; 58(23):5453-5465.

Abstract

This invention relates to methods and compositions that are useful for the treatment of various diseases, including cancer. The invention relates to, in part, combination therapeutic strategies to increase the effectiveness of chemotherapies.

Description

USES OF FLAGELLIN FOR IMPROVED CHEMOTHERAPY
PRIORITY
[0001] The present application claims priority to U.S. Provisional Application No. 61/942,927, filed February 21 , 2014, the contents of which are incorporated by reference herein
FIELD OF THE INVENTION
[0002] This invention relates to methods and compositions that are useful for the treatment of various diseases, including cancer. The invention relates to, in part, combination therapeutic strategies to increase the effectiveness of chemotherapies.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0003] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: CLE013PC_SequenceListing.txt; date recorded: February 19, 2015; file size: 8 KB).
BACKGROUND
[0004] Chemotherapy is a cancer treatment that uses drugs to destroy cancer cells. Unfortunately, chemotherapy agents may destroy not only cancer cells but also normal, healthy cells. This damage to healthy cells causes side effects of chemotherapies. These side effects can have the effect of dramatically reducing a patient's quality of life. Additionally, such side effects can limit the ability of a patient to receive treatment and therefore lower the likelihood of recovery.
[0005] There remains a need for agents that can increase the effectiveness of chemotherapies.
SUMMARY OF THE INVENTION
[0006] Accordingly, the present invention provides for methods for treating cancer in patients undergoing treatment with a chemotherapeutic agent by administering an agent that increases the effectiveness and/or reduces toxic side effects of chemotherapies, specifically, TLR agonist, including a flagellin-related composition, such as CBLB502.
[0007] In one aspect, the invention provides a method for treating cancer, comprising administering an effective amount of a TLR agonist, including a flagellin-related composition, such as CBLB502, to a patient undergoing treatment with fluorouracil (5-FU). In another aspect, there is provided a method for treating cancer, comprising administering an effective amount of a TLR agonist, including a flagellin-related composition, such as CBLB502, after administering an effective amount of 5-FU to a cancer patient in need thereof. [0008] In another aspect, the invention provides a method for treating cancer, comprising administering an effective amount of a TLR agonist, including a flagellin-related composition, such as CBLB502, to a patient undergoing treatment with a topoisomerase inhibitor. The topoisomerase inhibitor may be selected from irinotecan and doxorubicin. In another aspect, there is provided a method for treating cancer comprising administering an effective amount of a TLR agonist, including a flagellin-related composition, such as CBLB502, sequentially or simultaneously with administering an effective amount of a topoisomerase inhibitor, optionally selected from irinotecan and doxorubicin, to a cancer patient in need thereof
[0009] In various embodiments, a TLR agonist, including a flagellin-related composition, such as CBLB502, mitigates the side effects of certain chemotherapies, including 5-FU, irinotecan, and/or doxorubicin, and may expand the therapeutic window of these treatments. For example, administering a TLR agonist, including a flagellin-related composition, such as CBLB502, increases the ability of a patient to receive an adequate dose, and/or an increased or complete regimen of a therapy and/or increased or complete maintenance regimen of a therapy. Further, some patients, including those having preexistent impaired hepatic function or a deficiency of dipyrimidine dehydrogenase (DPD) activity, typically cannot sustain full 5-FU treatment. Administration of a TLR agonist, including a flagellin-related composition, such as CBLB502, increases the likelihood that these patients can receive this treatment. Also, for example, cancer patients classified as *28 variants typically cannot sustain full irinotecan treatment. Administration of a TLR agonist, including a flagellin-related composition, such as CBLB502, increases the likelihood that these patients can receive this treatment.
BRIEF DESCRIPTION OF THE FIGURES
[0010] Fig. 1 shows the effect of CBLB502 on 5-FU-induced mortality in mice. Panel A shows a body weight (percentage of starting weight) growth curve of BALB/c mice treated with 5-FU (200mg/kg or 400 mg/kg) alone or with subsequent injection of CBLB502 (1 μg/mouse) 24 and 48 h after the last 5-FU dose. Panel B shows a percent survival growth curve of BALB/c mice treated with 5-FU (200 mg/kg (second from bottom curve) or 400 mg/kg (bottom curve)) alone or with subsequent injection of CBLB502 (1 μg/mouse) 24 and 48 h after the last 5- FU dose: with 200 mg/kg 5-FU (top curve) or 400 mg/kg 5-FU (second from top curve). Panel C shows a percent survival growth curve of C57BL/6 mice injected with a single dose of 400 mg/kg 5-FU (second from bottom curve) or 3 daily doses of 100 mg/kg 5-FU (bottom curve) with or without CBLB502 (1 μg/mouse) injected 24 and 48 h after the last 5-FU dose: 400 mg/kg 5-FU (top curve) or 3 daily doses of 100 mg/kg 5-FU (second from top curve).
[0011] Fig. 2 demonstrates the mitigation of body weight loss and increase of survival of BALB/c mice after 5-FU injections (200 mg/kg) by CBLB502 treatment. Panel A shows a body weight (percentage of starting weight) growth curve after 5-FU (200 mg/kg) treatment (bottom curve) with and without CBLB502 injections given in different regimens: 24 h post-5-FU (second from bottom curve), 1 and 24 h post-5-FU (third from top curve), and 1 , 48 and 96h post-5-FU (fourth from top curve). CBLB502 (top curve) and vehicle (second from top curve) injected mice without 5-FU were weighed as controls. (*) Body weight beyond this time-point is based on 3 surviving mice in the group treated with CBLB502 1 and 24 h post-5-FU. Panel B shows a survival growth curve of BALB/c mice after 5-FU (200 mg/kg) treatment (bottom curve) with and without CBLB502 injections given in different regimens: 24 h post-5-FU (second from top curve), 1 and 24 h post-5-FU (second from bottom curve), and 1 , 48 and 96h post-5-FU (top curve).
[0012] Fig. 3 shows that the CBLB502-mediated protection against 5-FU toxicity is TLR5-dependent. Panel A shows a body weight (percentage of starting weight) growth curve in wild type C57BL/C mice injected with 5-FU 100 mg/kg x 3 times with 24 h interval with CBLB502 injected 24 and 48 h after the last 5-FU treatment. (*) The results of the group treated with 5-FU and CBLB502 at this point and later are based on 3 surviving mice. Panel B shows a body weight (percentage of starting weight) growth curve in wild type C57BL/C mice injected with 5-FU 200 mg/kg x 2 times with 6 h interval with and without CBLB502 injected 24 and 48 h post-5-FU. Panel C shows a percent survival growth curve of wild type C57BL/6 and TLR5-KO mice injected with 5-FU - 200 mg/kg x 2 times with 6 h interval with (top curve and second from top curve, respectively) and without (second from bottom curve and bottom curve, respectively) CBLB502 injected 24 and 48 h post-5-FU.
[0013] Fig. 4 demonstrates the protection and restoration of hematopoiesis. Panel A shows a blood cell analysis growth chart of white blood cells (WBC) and neutrophils for the indicated days after 5-FU injection performed using blood samples from mice treated with vehicle, 5-FU alone or 5-FU+CBLB502. 5-FU was injected i.p. at a dose of 200 mg/kg followed by three s.c. CBLB502 (1 μg/mouse) injections 1 , 48 and 96 h post- 5-FU. Panel B shows a blood cell analysis growth chart of white blood cells (WBC), neutrophils, lymphocytes and platelets for the indicated days after 5-FU injection performed using blood samples from mice treated with vehicle, 5-FU alone or 5-FU+CBLB502. 5-FU was injected i.p. at a dose of 100 mg/kg followed by three s.c. CBLB502 (1 μg/mouse) injections 1 , 48 and 96 h post-5-FU.
[0014] Fig. 5 shows the restoration of blood cell count in BALB/c mice following 5-FU treatment alone and in combination with CBLB502. Specifically, Fig. 5 shows growth charts of white blood cells, neutrophils, lymphocytes and platelets from BALB/c mice treated with 5-FU injected i.p. (150 mg/kg) followed by CBLB502 24 h and 48 h post 5-FU. Two out of 5 mice in the 5-FU injected group died on day 8, all 5 mice in the 5- FU+CBLB502-treated group survived. Vehicle injected mice were used as controls.
[0015] Fig. 6 shows the effect of CBLB502 on 5-FU-induced changes in bone marrow morphology. Specifically, Fig. 6 shows representative pictures of H&E-stained bone marrow sections (10x objective magnification) prepared 3 and 7 days after 5-FU injection. BALB/c mice were treated with 200 mg/kg 5-FU with or without injection of CBLB502 24 and 48 h post-5-FU. The lower row shows selected areas outlined in white at two fold higher magnification.
[0016] Fig. 7 demonstrates the effect of CBLB502 on 5-FU-induced changes in small intestine morphology. Panel A shows representative H&E-stained transverse sections (250x objective magnification) of small intestines from vehicle injected BALB/c mice (intact) and treated with 5-FU (200 mg/kg) alone or in combination with CBLB502 24 and 48 h post-5-FU. Sections were prepared 3 and 7 days after 5-FU treatment. Enterocytes lining the villi are indicated by arrows, lamina propria by asterisks, and crypts by arrowheads. The lower row shows selected crypt areas outlined in white at two fold higher magnification. Panel B shows representative H&E- stained transverse sections (250x objective magnification) of small intestines from vehicle injected BALB/c mice (intact) and treated with 5-FU (400 mg/kg) alone or in combination with CBLB502 24 and 48 h post-5-FU. Sections were prepared 3 and 7 days after 5-FU treatment. Enterocytes lining the villi are indicated by arrows, lamina propria by asterisks, and crypts by arrowheads. The lower row shows selected crypt areas outlined in white at two fold higher magnification. Panel C shows a bar chart of the average injury score of small intestine sections described in (Panels A-B). The degree of pathomorphological changes in the surface epithelium, villi, crypts, lamina propria, stroma, transitory and lymphoid elements and submucosa was scored as: 4 - Severe; 3 - Markedly abnormal; 2 - Moderately abnormal; 1 - Mild; and 0 - Normal, including non-integer scores. The average morphological score for 5 mice/group is shown. Panel D shows a bar chart of the mitotic index in mice treated as described in (Panels A-B) calculated in crypts of 4 transverse sections of small intestine per mouse as the number of mitoses per crypt. The mitotic index of vehicle treated animals is plotted as "No-5-FU" control or mean. For both panels C and D, each set of four bars from left to right represent vehicle injected BALB/c mice (intact) and treated with 5-FU (200 mg/kg or 400 mg/kg) alone (collected on day 3 (first bar), collected on day 7 (third bar)) or in combination with CBLB502 24 and 48 h post-5-FU (collected on day 3 (second bar), collected on day 7 (fourth bar)).
[0017] Fig. 8 shows the effect of CBLB502 on 5-FU-induced changes in colon morphology. Panel A shows representative H&E-stained transverse sections (250x objective magnification) of colon from untreated "intact" BALB/c mice and those treated with 5-FU injections (200 mg/kg) alone or in combination with CBLB502 treatment 24 and 48 h post-5-FU. Sections were prepared 3 or 7 days after 5-FU treatment. Morphology of crypt areas outlined in white is shown at two fold higher magnification in the lower rows. Atrophic and degenerative changes in the surface epithelium are indicated by arrows and in the crypt cells and mucin globules by arrowheads. Panel B shows representative H&E-stained transverse sections (250x objective magnification) of colon from untreated "intact" BALB/c mice and those treated with 5-FU injections (400 mg/kg) alone or in combination with CBLB502 treatment 24 and 48 h post-5-FU. Sections were prepared 3 or 7 days after 5-FU treatment. Morphology of crypt areas outlined in white is shown at two fold higher magnification in the lower rows. Atrophic and degenerative changes in the surface epithelium are indicated by arrows and in the crypt cells and mucin globules by arrowheads. Panel C shows a bar chart of the average injury score determined by pathomorphological changes in the surface epithelium, number of surface enterocytes and goblet cells, size and shape of crypts, lymphoid elements and state of the submucosa in colon sections from mice described in (Panels A-B). For panel C, each set of four bars from left to right represent vehicle injected BALB/c mice (intact) and treated with 5-FU (200 mg/kg or 400 mg/kg) alone (collected on day 3 (first bar), collected on day 7 (third bar)) or in combination with CBLB502 24 and 48 h post-5-FU (collected on day 3 (second bar), collected on day 7 (fourth bar)). [0018] Fig. 9 demonstrates that CBLB502 induces IL-6 production in 5-FU-treated mice. Specifically, Fig. 9 shows a growth chart of IL-6 concentration in plasma samples from BALB/c mice analyzed by MILLIPLEX kit. Plasma was collected 3 and 7 days after injection (labeled from left to right) of vehicle (DMSO) (first bar), CBLB502 alone (second bar), 200 mg/kg 5-FU alone (third bar), or 5-FU+CBLB502 (fourth bar).
[0019] Fig. 10 shows the effect of CBLB502 on 5-FU toxicity in IL-6 knockout mice. Panel A shows a survival of WT BALB/c and IL-6-KO mice growth curve after treatment with 200 mg/kg 5-FU (bottom curve and second from bottom curve, respectively), with or without CBLB502 treatment (1 μg/mouse) 24 and 48 h after 5- FU (top curve and second from top curve, respectively). Panel B shows a body weight growth curve in IL-6-KO mice treated with 5-FU (400 mg/kg), with (top curve) or without (bottom curve) CBLB502 injected 24 and 48 h after the last 5-FU dose. Panel C shows a survival of IL-6-KO and wild type (WT) mice growth curve after treatment with 5-FU (400 mg/kg) (second from bottom curve and bottom curve, respectively) or 5-FU+CBLB502 (second from top curve and top curve, respectively). The combined results of two independent experiments are presented in (Panel B) and (Panel C). Panel D shows H&E-stained small intestine sections with crypts from IL-6- KO mice euthanized on Day 3 after 5-FU (400 mg/kg) with or without CBLB502. Enterocytes lining the villi are indicated by arrows, lamina propria by asterisks and crypts by arrowheads. Normal morphology of crypts in an intact mouse is shown as a control. Panel E shows a growth chart (labeled in sets of three from left to right) of concentrations of white blood cells (WBC) in blood samples from IL-6-KO mice treated with CBLB502 alone (first bar), 5-FU alone (second bar) or 5-FU+CBLB502 (third bar). 5-FU (100 mg/kg) was followed by CBLB502 24 and 48 h later.
[0020] Fig. 11 shows the mitotic index in IL-6-KO mice after 5-FU treatment with and without CBLB502. Specifically, Fig. 11 shows a bar chart (labeled from left to right) of the mitotic index calculated in crypts of 4 transverse sections of small intestine per mouse as the number of mitoses per crypt in 3 mice/ group. The samples were obtained 3 days after 5-FU (400 mg/kg) injections with (third bar) and without (second bar) CBLB502 24 and 48 h post-5-FU. The first bar corresponds to vehicle injected BALB/c mice (intact).
[0021] Fig. 12 demonstrates that CBLB502 induces expression of soluble IL-1 receptor, SOD2, and IL-1 receptor antagonist (IL1 -RN). Panel A shows a bar chart (labeled in pairs from left to right) of the concentration of soluble IL-1 receptor in plasma prepared from BALB/c mice 72 h after 5-FU injections (200 and 400 mg/kg) with (second bar) and without (first bar) CBLB502 injected 24 and 48 h after 5- FU that was determined using a MILLIPLEX kit. Panel B shows a Western blot for SOD-2 expression in small intestine samples from N IH-Swiss mice at the indicated time-points after CBLB502 injection (without 5-FU). Actin was used as a loading control. Panel C shows the expression of IL-1 receptor antagonist (IL-1 RN) detected by RT-PCR using total RNA prepared from small intestine isolated from a BALB/c mouse treated with CBLB502 (1 mg/ mouse x2 injections 24h apart) 24 h after the second injection. Small intestine RNA from a PBS-injected mouse was used as an "intact" control. GADPH expression was used a housekeeping gene for loading control. [0022] Fig. 13 shows a representative experiment showing the absence of any effect of CBLB502 on s.c. growing CT26 tumors in syngeneic BALB/c mice. Specifically, Fig. 13 shows a tumor volume growth curve of the treatment of CT-26 tumor-bearing BALB/c mice with CBLB502 initiated when tumors reached about 5 mm in diameter. CBLB502 (1 mg/mouse) was injected s.c. 24 h apart on days 1 , 2 and 3.
[0023] Fig. 14 demonstrates the protection of mice, but not tumors, from 5-FU toxicity by CBLB502 in CT26 tumor-bearing mice. Panel A shows a CT26 tumor growth curve in mice treated with vehicle ("untreated") (top curve), 5-FU alone (100 (second from top curve) or 200 mg/kg (bottom curve)), or 5-FU (100 (third from top curve) or 200 mg/kg (second from bottom curve)) + CBLB502 injected 1 , 48, and 96 h post-5-FU. Panel B shows a body weight change (percentage of starting weight) growth curve of mice described in Panel A. Specifically, mice treated with vehicle ("untreated") (second from bottom curve), 5-FU alone (100 (top curve) or 200 mg/kg (bottom curve)), or 5-FU (100 (second from top curve) or 200 mg/kg (third from top curve)) + CBLB502 injected 1 , 48, and 96 h post-5-FU. Panel C shows a percent survival growth curve of mice described in Panel A. Specifically, mice treated with vehicle ("untreated") (bottom curve), 5-FU alone (100 (second from top curve) or 200 mg/kg (second from bottom curve)), or 5-FU (100 (third from top curve) or 200 mg/kg (top curve)) + CBLB502 injected 1 , 48, and 96 h post-5-FU. (*, Panels A-B) - Body weight and tumor volume are shown for only 4/10 mice injected with 200 mg/kg 5-FU alone that were surviving on Day 18 after CT26 cell inoculation; mice in this group died due to 5-FU toxicity with small or no tumors. In the other groups, mice were euthanized due to large size or ulceration of tumors; 2/10 mice in the group treated with 200 mg/kg 5-FU + CBLB502 remained tumor-free for the entire period of observation (60 days).
[0024] Fig. 15 shows the toxicity of irinotecan in Fischer rats bearing Ward colorectal carcinoma with or without CBLB502 (administered before or concurrently).
[0025] Fig. 16 shows the effect of CBLB502 treatment with and without 5-FU against 4T1 tumor growth. Panel A shows orthotopic (mammary fat pad) 4T1 tumor growth as measured after treatment with 5-FU (100 mg/kg, i.p.), CBLB502 (1 μg, s.c.) or a combination of both; n=10 (5 mice, 2 tumors/ mouse). Mice received either CBLB502 or PBS at 24 and 48 hours post-5-FU treatment. Panel B shows mouse survival after i.v. injection of 4T1 cells and treatment with 5-FU (100 mg/kg, on day 4), CBLB502 (1 μg or PBS vehicle on days 5 and 6) or a combination of both. (*) The difference from vehicle control and (**) from any other group is statistically significant (pO.05).
[0026] Fig. 17 shows the effect of CBLB502 on myeloid derived suppressor cells (MDSC) in 4T1 tumor- bearing mice. Mice with orthotopically (mammary fat pad) growing 4T1 tumors were injected with 5-FU (50 and 100 mg/kg, i.p.) and vehicle control on day 14, as indicated by the arrow. The amount of neutrophils in the blood was determined by complete blood count (CBC) on days 10, 12, 14, 17 and 20 (panel A). Spleen weight (panel B) and the percent of granulocytic MDSC within hematopoietic cell population (panel C) were analyzed on day 20 post 4T1 cell s.c. implantation that is day 7 post 5-FU injection (n=2-3). One intact, tumor-free, mouse was used as a control. [0027] Fig. 18 shows the effect of doxorubicin on myeloid derived suppressor cells (MDSC) in 4T1 tumor- bearing mice. Percentage of neutrophils and lymphocytes among total white blood cells in the blood of mice with s.c. growing 4T1 tumors was determined by complete blood cell count on the indicated days. Doxorubicin (2.5 mg/kg) was injected i.v. on day 7 after tumor cell implantation as shown by green arrows (n=3).
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention is based, in part, on the discovery that the TLR agonists, including flagellin- related compositions, such as CBLB502, mitigate the side effects of certain chemotherapies, including 5-FU, irinotecan, and doxorubicin, and therefore expand the therapeutic window of these treatments. Accordingly, the present invention provides improved methods of treating cancers with genotoxic chemotherapy agents.
Definitions
[0029] The following definitions are used in connection with the invention disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs.
[0030] As used herein, "a," "an," or "the" can mean one or more than one.
[0031] Further, the term "about" when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language "about 50" covers the range of 45 to 55.
[0032] An "effective amount," when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.
[0033] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word "include," and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms "can" and "may" and their variants are intended to be non- limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
[0034] 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."
[0035] As used herein, the words "preferred" and "preferably" refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.
[0036] The amount of compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents (e.g. flagellin related compositions (and/or additional agents, including 5-FU, irinotecan, and doxorubicin) described herein) for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A "pharmacologically effective amount," "pharmacologically effective dose," "therapeutically effective amount," or "effective amount" refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. For example, administration of therapeutic agents to a patient suffering from cancer provides a therapeutic benefit not only when the underlying condition is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the symptoms associated with the disease, e.g., a decrease in tumor burden, a decrease in circulating tumor cells, an increase in progression free survival. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
[0037] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. In some embodiments, compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
[0038] In certain embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder or reduction in toxicity, regardless of whether improvement is realized. [0039] In certain embodiments, a pharmacologically effective amount that will treat cancer will modulate the symptoms typically by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In exemplary embodiments, such modulations will result in, for example, statistically significant and quantifiable changes in the numbers of cancerous cells or indicia of toxicity as described herein (e.g. number of certain cells in a blood test, number of liver enzymes in a blood test, etc.).
[0040] In some aspects, the present invention provides methods of treating cancer with a TLR agonist, including a flagellin-related composition, such as CBLB502, in a patient undergoing treatment with another therapy, including 5-FU and/or irinotecan and/or doxorubicin. In some aspects, the present invention provides for the use of a TLR agonist, including a flagellin-related composition, such as CBLB502, for the treatment of cancer in a patient undergoing treatment with another therapy, including 5-FU and/or irinotecan and/or doxorubicin.
[0041] The TLR agonist may be a flagellin related composition, including a flagellin-related polypeptide. The flagellin related compositions may be from various sources, including a variety of Gram-positive and Gram- negative bacterial species. In some embodiments, the flagellin related compositions may have an amino acid sequence that is derived from any of the flagellins from bacterial species that are depicted in FIG. 7 of U.S. Patent Publication No. 2003/0044429, the contents of which are incorporated herein by reference in their entirety. The flagellin related compositions may have nucleotide sequences related to those encoding the flagellin polypeptides listed in FIG. 7 of U.S. 2003/0044429, which are publicly available at sources including the NCBI Genbank database.
[0042] The flagellin related compositions may be the major component of bacterial flagellum. The flagellin related compositions may be composed of one, or two, or three, or four, or five, or six, or seven domains or fragments thereof (see, e.g. FIG. 10 of US Patent 8,324,163, the contents of which are incorporated herein by reference in their entirety). The domains may be selected from NDO, ND1 , ND2, D3, CD2, CD1 , and CDO. Domains 0 (DO), 1 (D1 ), and 2 (D2) may be discontinuous and may be formed when residues in the amino terminus and carboxy terminus are juxtaposed by the formation of a hairpin structure. The amino and carboxy terminus comprising the D1 and D2 domains may be most conserved, whereas the middle hypervariable domain (D3) may be highly variable. The non-conserved D3 domain may be on the surface of the flagellar filament and may contain the major antigenic epitopes. The potent proinflammatory activity of flagellin may reside in the highly conserved N and CD1 and D2 regions.
[0043] The flagellin related compositions may be from a species of Salmonella, representative examples of which are S. typhimurium and S. dublin (encoded by GenBank Accession Number M84972). The flagellin related-polypeptide may be a fragment, variant, analog, homolog, or derivative of wild type flagellin (SEQ ID NO: 1 ), or combination thereof:
SEQ ID NO: 1
Met Ala Gin Val He Asn Thr Asn Ser Leu Ser Leu Leu Thr Gin Asn Asn Leu Asn Lys Ser
Gin Ser Ser Leu Ser Ser Ala He Glu Arg Leu Ser Ser Gly Leu Arg He Asn Ser Ala Lys Asp Asp Ala Ala Gly Gin Ala lie Ala Asn Arg Phe Thr Ser Asn lie Lys Gly Leu Thr Gin Ala Ser
Arg Asn Ala Asn Asp Gly He Ser He Ala Gin Thr Thr Glu Gly Ala Leu Asn Glu He Asn Asn
Asn Leu Gin Arg Val Arg Glu Leu Ser Val Gin Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp
Leu Lys Ser He Gin Asp Glu He Gin Gin Arg Leu Glu Glu He Asp Arg Val Ser Asn Gin Thr
Gin Phe Asn Gly Val Lys Val Leu Ser Gin Asp Asn Gin Met Lys He Gin Val Gly Ala Asn
Asp Gly Glu Thr He Thr He Asp Leu Gin Lys He Asp Val Lys Ser Leu Gly Leu Asp Gly Phe
Asn Val Asn Gly Pro Lys Glu Ala Thr Val Gly Asp Leu Lys Ser Ser Phe Lys Asn Val Thr
Gly Tyr Asp Thr Tyr Ala Ala Gly Ala Asp Lys Tyr Arg Val Asp He Asn Ser Gly Ala Val Val
Thr Asp Ala Ala Ala Pro Asp Lys Val Tyr Val Asn Ala Ala Asn Gly Gin Leu Thr Thr Asp
Asp Ala Glu Asn Asn Thr Ala Val Asp Leu Phe Lys Thr Thr Lys Ser Thr Ala Gly Thr Ala
Glu Ala Lys Ala He Ala Gly Ala lie Lys Gly Gly Lys Glu Gly Asp Thr Phe Asp Tyr Lys Gly
Val Thr Phe Thr He Asp Thr Lys Thr Gly Asp Asp Gly Asn Gly Lys Val Ser Thr Thr He Asn
Gly Glu Lys Val Thr Leu Thr Val Ala Asp lie Ala Thr Gly Ala Ala Asp Val Asn Ala Ala Thr
Leu Gin Ser Ser Lys Asn Val Tyr Thr Ser Val Val Asn Gly Gin Phe Thr Phe Asp Asp Lys
Thr Lys Asn Glu Ser Ala Lys Leu Ser Asp Leu Glu Ala Asn Asn Ala Val Lys Gly Glu Ser
Lys He Thr Val Asn Gly Ala Glu Tyr Thr Ala Asn Ala Thr Gly Asp Lys He Thr Leu Ala Gly
Lys Thr Met Phe lie Asp Lys Thr Ala Ser Gly Val Ser Thr Leu lie Asn Glu Asp Ala Ala Ala
Ala Lys Lys Ser Thr Ala Asn Pro Leu Ala Ser He Asp Ser Ala Leu Ser Lys Val Asp Ala Val
Arg Ser Ser Leu Gly Ala He Gin Asn Arg Phe Asp Ser Ala He Thr Asn Leu Gly Asn Thr Val
Thr Asn Leu Asn Ser Ala Arg Ser Arg lie Glu Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn
Met Ser Lys Ala Gin He Leu Gin Gin Ala Gly Thr Ser Val Leu Ala Gin Ala Asn Gin Val Pro
Gin Asn Val Leu Ser Leu Leu Arg.
[0044] A fragment, variant, analog, homolog, or derivative of flagellin may be obtained by rational-based design based on the domain structure of flagellin and the conserved structure recognized by TLR5.
[0045] The flagellin related compositions may be related to a flagellin polypeptide from any Gram-positive or Gram-negative bacterial species including, but not limited to, the flagellin polypeptides disclosed in U.S. Pat. Pub. 2003/000044429, the contents of which are incorporated herein, and the flagellin peptides corresponding to the Accession numbers listed in the BLAST results shown in FIG. 7 (7AA-7F) of U.S. Patent Pub. 2003/000044429, or variants thereof.
[0046] In some embodiments, the flagellin-related polypeptide comprises a CBLB502 molecule. "CBLB502" (aka "Entolimod") as used herein comprises the sequence of SEQ ID NO: 2.
SEQ ID NO: 2.
Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly Gly Gin Gin Met Gly
Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp Pro Met Ala Gin Val He Asn Thr Asn Ser Leu
Ser Leu Leu Thr Gin Asn Asn Leu Asn Lys Ser Gin Ser Ser Leu Ser Ser Ala He Glu Arg Leu Ser Ser Gly Leu Arg He Asn Ser Ala Lys Asp Asp Ala Ala Gly Gin Ala He Ala Asn Arg
Phe Thr Ser Asn lie Lys Gly Leu Thr Gin Ala Ser Arg Asn Ala Asn Asp Gly He Ser lie Ala
Gin Thr Thr Glu Gly Ala Leu Asn Glu He Asn Asn Asn Leu Gin Arg Val Arg Glu Leu Ser
Val Gin Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser He Gin Asp Glu He Gin Gin
Arg Leu Glu Glu lie Asp Arg Val Ser Asn Gin Thr Gin Phe Asn Gly Val Lys Val Leu Ser
Gin Asp Asn Gin Met Lys lie Gin Val Gly Ala Asn Asp Gly Glu Thr He Thr He Asp Leu Gin
Lys He Asp Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val Asn Ser Pro Gly He Ser Gly Gly
Gly Gly Gly He Leu Asp Ser Met Gly Thr Leu He Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser
Thr Ala Asn Pro Leu Ala Ser He Asp Ser Ala Leu Ser Lys Val Asp Ala Val Arg Ser Ser Leu
Gly Ala He Gin Asn Arg Phe Asp Ser Ala He Thr Asn Leu Gly Asn Thr Val Thr Asn Leu
Asn Ser Ala Arg Ser Arg He Glu Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala
Gin He Leu Gin Gin Ala Gly Thr Ser Val Leu Ala Gin Ala Asn Gin Val Pro Gin Asn Val Leu
Ser Leu Leu Arg.
[0047] In some embodiments, the flagellin-related polypeptide of the present invention comprises a truncation in one or more domains of SEQ ID NO: 1 or 2 or variants thereof. In a further embodiment, the flagellin-related composition comprises a deletion in a N-terminal domain of SEQ ID NO: 1 or 2 or variants thereof. In yet a further embodiment, the flagellin-related composition comprises a deletion in the NDO domain of SEQ ID NO: 1 or 2 or variants thereof. In yet a further embodiment, the flagellin-related composition comprises a deletion of the entire NDO domain of SEQ ID NO: 1 or 2 or variants thereof. In a further embodiment, the flagellin- related composition comprises a deletion in a C-terminal domain of SEQ ID NO: 1 or 2 or variants thereof. In yet another embodiment, the flagellin-related composition comprises a deletion in the CDO domain of SEQ ID NO: 1 or 2 or variants thereof. In yet another embodiment, the flagellin-related composition retains amino acids 470-485 of the CDO domain of SEQ ID NO: 1 or 2 or variants thereof.
[0048] The flagellin related compositions may be at least 30-99% identical to amino acids 1-174 and 418- 505 of SEQ ID NO: 1. In some embodiments, the flagellin related compositions may be a species of the genera disclosed in US Patent No. 8,932,609, the contents of which are hereby incorporated by reference.
[0049] In some embodiments, the flagellin related compositions may stimulate Toll-like receptor activity (e.g. TLR1 , and/or TLR2, and/or TLR3, and/or TLR4, and/or TLR5, and/or TLR6, and/or TLR7, and/or TLR8, and/or TLR9, and/or TLR10, and/or TLR11 , and/or TLR12, and/or TLR13). The TLR family is composed of at least 10 members and is essential for innate immune defense against pathogens. The innate immune system recognizes conserved pathogen-associated molecular patterns (PAMPs). TLR may recognize a conserved structure that is particular to bacterial flagellin which may be composed of a large group of residues that are somewhat permissive to variation in amino acid content. Smith et ai, Nat. Immunol. 4:1247-53 (2003), the contents of which are hereby incorporated by reference, have identified 13 conserved amino acids in flagellin that are part of the conserved structure recognized by TLR5. [0050] In some embodiments, the flagellin-related composition activates TLR5 signaling. In some embodiments, the flagellin-related composition activates TLR5 at the same levels, or levels similar to, CBLB502. Activation of TLR5 induces expression of the nuclear factor NF-κΒ, which in turn activates numerous inflammatory-related cytokines. In further embodiments, the flagellin related compositions induce expression of proinflammatory cytokines. In further embodiments, the flagellin related compositions induce expression of antiinflammatory molecules. In another embodiment, the flagellin related compositions induce expression of anti- apoptotic molecules. In yet a further embodiment, the flagellin related compositions induce expression of antibacterial molecules. The targets of NF-κΒ, include, but are not limited to, IL-β, TNF-a, IL-6, IL-8, IL-18, G-CSF, TNFSF13B, keratinocyte chemoattractant (KC), BLIMP1 /PRDM1 , CCL5, CCL15, CCL17, CCL19, CCL20, CCL22, CCL23, CXCL1 ,CCL28, CXCL1 1 , CXCL10, CXCL3, CXCL1 , GRO-beta, GRO-gamma, CXCL1 , ICOS, IFNG, IL-1A, IL-1 B, IL1 RN, IL-2, IL-9, IL-10, IL-11 , IL-12, IL-12B, IL-12A, IL-13, IL-15, IL-17, IL-23A, IL-27, EBI3, IFNB1 , CXCL5, KC, liGpl , CXCL5, CXCL6, LTA, LTB, CCL2, CXCL9, MCP-1/JE, CCL3, CCL4, CXCL3, CCL20, CXCL10, CXCL5, CCL5, CCL1 , TNFbeta, TNFSF10, TFF3, TNFSF15, CD86, complement component 8a, CCL27, defensin- 3, MIG, MIP-2, and/or NOD2/CARD15.
[0051] In various embodiments, the effective amount of flagellin related composition, including CBLB502, is about 0.001 mg to about 1000 mg per day, about 1 mg to about 600 mg per day, or about 5 mg to about 30 mg per day.
[0052] In some aspects, the present invention provides for methods of treating cancer, in patients undergoing treatment with a chemotherapy, including 5-FU and/or irinotecan and/or doxorubicin, by administering an effective amount of a flagellin related composition, such as CBLB502. In some aspects, the present invention provides for methods of treating cancer, involving administering an effective amount of a flagellin related composition, such as CBLB502 after administering an effective amount of 5-FU to a cancer patient in need thereof. In still other aspects, the present invention provides for provides for methods of treating cancer, involving administering an effective amount of CBLB502 sequentially or simultaneously (including coadministration and/or co-formulation) with administering an effective amount of irinotecan to a cancer patient in need thereof. In further aspects, the present invention provides for provides for methods of treating cancer, involving administering an effective amount of CBLB502 sequentially or simultaneously (including coadministration and/or co-formulation) with administering an effective amount of doxorubicin to a cancer patient in need thereof.
[0053] As used herein "5-FU" and/or "irinotecan" and/or "doxorubicin" may refer to any forms of these agents, including various formulations as described herein (including, but not limited to iv, topical, oral, nanoformulations, slow-release, gels, etc.). Further "5-FU" and/or "irinotecan" and/or "doxorubicin" may refer to prodrugs of these agents. For example, "5-FU" may encompass one or more of, for example, 5'-deoxy-5- fluorouhdine, capecitabine (e.g. Xeloda), BOF-A2, ftorafur, UFT, and S-1. Further, "5-FU" and/or "irinotecan" and/or "doxorubicin" may refer to in vivo active metabolites of these agents. For example, "irinotecan" may refer to one or more of CPT1 1 , SN-38, SN-38-G, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1 - piperidino]carbonyloxycamptothecin (APC), and 7-ethyl-10-(4-amino-1 -piperidino) carbonyloxycamptothecin (NPC). In other examples, "doxorubicin" also known as hydroxydaunorubicin, hydroxydaunomycin may refer to one or more of Adriamycin PFS, Adriamycin RDF, or Rubex, Doxil, Myocet, and Caelyx.
[0054] In certain embodiments, administering a flagellin related composition, such as CBLB502 after 5-FU encompasses after as single dose of 5-FU, irinotecan, and/or doxorubicin, or after a partial or full cycle of 5-FU, irinotecan, and/or doxorubicin therapy (e.g. at an intermediate step or at the completion of any of the regimens described herein), or after the onset of toxicity from 5-FU, irinotecan, and/or doxorubicin treatment.
[0055] In some embodiments, the administration of a flagellin related composition, including CBLB502, protects and/or reduces and/or mitigates one or more side effects of a patient's cancer therapy.
[0056] In some embodiments the patient's cancer therapy is 5-FU and the side effect is one or more of stomatitis, esophagopharyngitis, leukopenia and/or reduced white blood count, risk of infection, vomiting, diarrhea, nausea, poor appetite, damage to the Gl tract, including gastrointestinal ulceration and/or bleeding, thrombocytopenia, hand-foot syndrome, alopecia, mucositis, impaired hepatic function, and hemorrhage.
[0057] In some embodiments, the administration of a flagellin related composition, including CBLB502, may permit a patient to receive a specific mode of administration of 5-FU by reducing side effects. For example, myelotoxicity is the major toxic effect in patients receiving bolus doses of 5-FU. In some embodiments, a flagellin related composition, including CBLB502 offsets this side effect and therefore permits bolus doses. Also, hand- foot syndrome (palmar-plantar erythrodysesthesia), stomatitis, neuro- and cardiotoxicity are associated with continuous infusions of 5-FU. In some embodiments, a flagellin related composition, including CBLB502 offsets this side effect and therefore permits continuous infusions.
[0058] In another embodiment, the side effect of 5-FU treatment is hematological damage. In some embodiments, the hematological damage is one or more of leukopenia and/or reduced white blood count and reduction of other types of blood cells, including neutrophils, lymphocytes and platelets. In some embodiments, hematological damage is assessed by a blood test.
[0059] In some embodiments, a patient is administered 5-FU and is subsequently evaluated for a hematological damage by, for example, a blood test that involves a cell count, and if presenting with lowered cell counts, is administered an effective amount of a flagellin related composition, including CBLB502. In some embodiments, a patient is administered 5-FU and is subsequently evaluated for a hematological damage by, for example, a blood test that involves a cell count, and if presenting with normal counts, is not administered an effective amount of a flagellin related composition, including CBLB502.
[0060] In another embodiment, the side effect of 5-FU treatment is impaired hepatic function. In various embodiments, impaired hepatic function is assessed by testing for levels of various liver enzymes. Exemplary indicia of impaired hepatic function include, by way of non-limiting example, elevated aminotransferase levels, elevated plasma ammonia levels, changes in one or more of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, 5' nucleotidase, a gamma-glutamyl transpeptidase (GGT), and bilirubin.
[0061] In some embodiments, a patient is administered 5-FU and is subsequently evaluated for impaired hepatic function by, for example, a blood test and/or biopsy that involves a screen for markers of liver function (e.g. liver enzymes) and, if presenting with abnormal levels, is administered an effective amount of a flagellin related composition, including CBLB502. In some embodiments, a patient is administered 5-FU and is subsequently evaluated for an impaired hepatic function by, for example, a blood test and/or biopsy that involves a screen for markers of liver function (e.g. liver enzymes) and, if presenting with normal levels, is not administered an effective amount of a flagellin related composition, including CBLB502.
[0062] Alternatively, in some embodiments, hepatic imaging may be used to test for impaired hepatic function. In some embodiments, the hepatic imaging may be one or more of ultrasounds, CT, MR, and PET, PET/CT and may involve various imaging agents (e.g. gadolinium chelates). Techniques for imaging may be found in Ros and Mortele, Hepatic Imaging. An overview Clin Liver Dis. 2002 Feb;6(1 ):1 -16, the contents of which are hereby incorporated by reference in their entirety.
[0063] In a specific embodiment, the side effect of 5-FU treatment is damage to the Gl tract. In some embodiments, the damage to the Gl tract is assessed by measurement one or more of (a) the height and width of villi of the small intestines and/or number of surface enterocytes and goblet cells in the small and large intestines; (b) status of crypts (one or more of depth, size and shape, presence of apoptotic bodies, number of and granules in Paneth's cells, luminal migration of epithelial nuclei, loss of goblet cells, presence of atrophy and distortion); and (c) state of the lamina propria (one or more of presence of transitory cells, lymphoid accumulations, edema, blood vessel congestion and hemorrhage) in, for example, a biopsy.
[0064] In another embodiment, the side effect of 5-FU treatment is diarrhea. In those embodiments, administering an effective amount of CBLB502 reduces a dose and/or frequency of an anti-diarrheal agent (e.g. one or more of loperamide, diphenoxylate, and atropine) administered to the cancer patient.
[0065] In another embodiment, the side effect of 5-FU treatment is vomiting. In those embodiments, administering an effective amount of CBLB502 reduces a dose and/or frequency of an antiemetic, including, for example, 5-HT3 blockers with or without dexamethasone, administered to the cancer patient.,
[0066] In one embodiment, the patient's cancer therapy is 5-FU and the side effect is predicted by a genetic test, including, for example, genetic tests for one or more of genes DPYD and TYMS. In some embodiments, a patient is tested for the likelihood of a side effect of 5-FU treatment before beginning a 5-FU regimen and, if there is a high likelihood of side effects, is administered a flagellin related composition, including CBLB502, to mitigate the side effects. In some embodiments, a patient is tested for the likelihood of a side effect of 5-FU treatment before beginning a 5-FU regimen and, if there is a low likelihood of side effects, is not administered a flagellin related composition, including CBLB502, to mitigate the side effects. [0067] In one embodiment, the patient's cancer therapy is 5-FU and the side effect is measured by monitoring a patient's temperature. For example, temperature in excess of about 37.5°C (99.5° F) or about 38°C (100.4°F) and beyond indicates 5-FU side effects and directs administration of an effective amount of a flagellin related composition, including CBLB502.
[0068] In various embodiments, the present invention provides for administration of a flagellin related composition, including CBLB502, to a cancer patient receiving 5-FU and also having a preexistent impaired hepatic function including, for example, hepatitis. In these embodiments, a flagellin related composition, including CBLB502, serves to protect the patient's compromised liver from further damage.
[0069] In various embodiments, the present invention provides for administration of a flagellin related composition, including CBLB502 to a cancer patient receiving 5-FU and also having a deficiency of dipyrimidine dehydrogenase (DPD) activity. DPD deficient patients tend to experience exacerbated 5-FU side effects (e.g. severe toxicity (e.g., stomatitis, diarrhea, neutropenia and neurotoxicity) and a flagellin related composition, including CBLB502, serves to mitigate these side effects. In some embodiments, administration of a flagellin related composition, including CBLB502, to a cancer patients with risk factors for 5-FU, including for example, a history of high-dose pelvic irradiation or previous use of alkylating agents, who have a widespread involvement of bone marrow by metastatic tumors or those with impaired hepatic or renal function, allows for administration of 5- FU.
[0070] In various embodiments, the present invention provides for administration of a flagellin related composition, including CBLB502 to a female cancer patient receiving 5-FU. Women tend to have more severe stomatitis and leukopenia than men when receiving 5 consecutive days of 5-FU at doses of 370 to 450 mg/m2 (see, e.g. Sloan, et al. JCO 20(6): 1491 -1498, the contents of which are hereby incorporated by reference). Administration of a flagellin related composition, including CBLB502, to such patients allows the full course and/or dose of treatment to occur with suspension of treatment because of, for example, stomatitis and/or leukopenia.
[0071] In some embodiments, the patient's cancer therapy is a chemotherapeutic agent classified as a topoisomerase inhibitor. Topoisomerase inhibitors are agents that interfere with the action of topoisomerase enzymes which regulate DNA structure by catalyzing the breaking and rejoining of the phosphodiester backbone of DNA strands during the cell cycle. Topoisomerase inhibitors can be classified into topoisomerase I and topoisomerase II inhibitors. Topoisomerase I inhibitors, such as irinotecan, topotecan, camptothecin and lamellarin D, target the topoisomerase I enzymes which catalyze the breaking of one strand of the DNA double helix. Topoisomerase II inhibitors, such as doxorubicin, etoposide, teniposide, daunorubicin, mitoxantrone, amsacrine, and ellipticines, target the topoisomerase II enzymes which can break both strands of the DNA double helix. In some embodiments, the patient's cancer therapy is a chemotherapeutic agent classified as a topoisomerase inhibitor I or a topoisomerase inhibitor II. In an embodiment, the patient's cancer therapy is the topoisomerase I inhibitor irinotecan. In another embodiment, the patient's cancer therapy is the topoisomerase II inhibitor, doxorubicin.
[0072] In some embodiments the patient's cancer therapy is irinotecan and the side effect is one or more of diarrhea, dehydration, suppression of the immune system, hematological damage, lowered white blood cell counts, neutropenia, cholinergic syndrome, blood clots, infection, and abdominal pain. In some embodiments, hematological damage is one or more of leukopenia and/or reduced white blood count and reduction of other types of blood cells, including neutrophils, lymphocytes and platelets. In some embodiments, hematological damage is assessed by a blood test as described herein. In various embodiments, hematological damage in a patient undergoing irinotecan treatment directs administration of a flagellin related composition, including CBLB502, to mitigate this side effect.
[0073] In one embodiment, the patient's cancer therapy is irinotecan and the side effect is diarrhea. In those embodiments, administering an effective amount of CBLB502 reduces a dose and/or frequency of an anti- diarrheal agent (e.g. one or more of loperamide, diphenoxylate, and atropine) administered to the cancer patient.
[0074] In another embodiment, the side effect of irinotecan treatment is vomiting. In those embodiments, administering an effective amount of CBLB502 reduces a dose and/or frequency of an antiemetic, including, for example, 5-HT3 blockers with or without dexamethasone, administered to the cancer patient.
[0075] In one embodiment, the patient's cancer therapy is irinotecan and the side effect is measured by monitoring a patient's temperature. For example, temperature in excess of about 37.5°C (99.5° F) or about 38°C (100.4°F) and beyond indicates irinotecan side effects and directs administration of an effective amount of a flagellin related composition, including CBLB502.
[0076] In some embodiments, a patient is administered irinotecan and is subsequently evaluated for impaired hepatic function by, for example, a blood test and/or biopsy and/or hepatic imaging. In some embodiments, if a patient presents with impaired hepatic function, as described above, this directs administration of a flagellin related composition, including CBLB502, to mitigate this side effect.
[0077] In various embodiments, the present invention provides for administration of a flagellin related composition, including CBLB502, to a cancer patient receiving irinotecan and also classified as a *28 variant. Patients with variants of the UGT1A1 called TA7, also known as the "*28 variant" express fewer UGT1A1 enzymes in their liver and often suffer from Gilbert's syndrome. During chemotherapy, these patients may effectively receive a larger than expected dose because their bodies are not able to clear irinotecan as fast as others. This often corresponds with higher incidences of severe neutropenia and diarrhea. In some embodiments, irinotecan recipients with a homozygous polymorphism in UGT1A1 gene, may receive standard doses if also receiving a flagellin related composition, including CBLB502.
[0078] In some embodiments the patient's cancer therapy is doxorubicin and the side effect is one or more of vomiting, nausea, diarrhea, dehydration, suppression of the immune system, hematological damage, lowered white blood cell counts, infection, cardiomyopathy, congestive heart failure, typhlitis, hand-foot syndrome. In some embodiments, hematological damage is one or more of leukopenia and/or reduced white blood count and reduction of other types of blood cells, including neutrophils, lymphocytes and platelets. In some embodiments, hematological damage is assessed by a blood test as described herein. In various embodiments, hematological damage in a patient undergoing doxorubicin treatment directs administration of a flagellin related composition, including CBLB502, to mitigate this side effect.
[0079] In one embodiment, the patient's cancer therapy is doxorubicin and the side effect is diarrhea. In those embodiments, administering an effective amount of CBLB502 reduces a dose and/or frequency of an anti- diarrheal agent (e.g. one or more of loperamide, diphenoxylate, and atropine) administered to the cancer patient.
[0080] In another embodiment, the side effect of doxorubicin treatment is vomiting. In those embodiments, administering an effective amount of CBLB502 reduces a dose and/or frequency of an antiemetic, including, for example, 5-HT3 blockers with or without dexamethasone, administered to the cancer patient.
[0081] In one embodiment, the patient's cancer therapy is doxorubicin and the side effect is measured by monitoring a patient's temperature. For example, temperature in excess of about 37.5°C (99.5 ) or about 38°C (100.4°F) and beyond indicates doxorubicin side effects and directs administration of an effective amount of a flagellin related composition, including CBLB502.
[0082] In some embodiments, the present invention provides for uses of the flagellin related polypeptides, including CBLB502, to increase a therapeutic window of a therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin. In various embodiments, the increased therapeutic window of a therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin comprises one or more of increasing a cancer patient's likelihood receiving therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin maintenance therapy; increasing a cancer patient's likelihood of receiving a complete regime of a therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin; increasing a cancer patient's likelihood of receiving more than a complete regimen of a therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin; and increasing the dose or length of a therapeutic agent, including 5-FU and/or irinotecan and/or doxorubicin treatment.
[0083] In some embodiments, uses of the flagellin related polypeptides, including CBLB502, allow for a cancer patient to receive dose-dense chemotherapy. In various embodiments, uses of the flagellin related polypeptides, including CBLB502, allow for a cancer patient to avoid dose delay.
[0084] In some embodiments, the standard 5-FU treatment regime comprises administering about 12 mg/kg once daily for four successive days and optionally about 6 mg/kg on the sixth, eighth, tenth and twelfth days unless toxicity occurs. In various embodiments, administration of a flagellin related polypeptide, including CBLB502, may allow a patient to be administered a full regimen without having to cease treatment because of, for example, side effects. In some embodiments, administration of a flagellin related polypeptide, including CBLB502, allows a patient to be administered increased dosages (e.g. greater than about 20 mg/kg once daily, or about 18 mg/kg once daily, or about 16 mg/kg once daily, or about 14 mg/kg once daily, or about 12 mg/kg once daily) on greater than four (e.g. about 10, about 9, about 8, about 7, about 6, or about 5) successive days; greater than about 15 mg/kg once daily, or about 12 mg/kg once daily, or about 10 mg/kg once daily, or about 8 mg/kg once daily, or about 6 mg/kg once daily on the sixth, eighth, tenth and twelfth day; and/or length of administration (e.g. beyond 12 days, including, for example, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 4 weeks, or about one month).
[0085] In some embodiments, administration of a flagellin related polypeptide, including CBLB502, may allow a patient to be administered a dose of 5-FU that exceeds about 200 mg daily, about 300 mg daily, about 400 mg daily, about 500 mg daily, about 600 mg daily, about 700 mg daily, or about 800 mg daily, or about 900 mg daily, or about 1000 mg daily.
[0086] In some embodiments, the standard 5-FU maintenance therapy comprises dosage of about 12 mg/kg every 30 days after the last day of a previous course of treatment or about 10 to 15 mg/kg/week as a single dose. In some embodiments, administration of a flagellin related polypeptide, including CBLB502, may allow a patient to be administered this 5-FU maintenance therapy. In some embodiments, administration of a flagellin related polypeptide, including CBLB502, may allow a patient to be administered a greater 5-FU maintenance therapy (e.g. more frequently and/or at a larger dose).
[0087] In some embodiments, administration of a flagellin related polypeptide, including CBLB502, may allow a patient to be administered a dose of irinotecan that is greater than about 125 mg/m2, about 150 mg/m2, about 175 mg/m2, about 200 mg/m2, about 225 mg/m2, about 250 mg/m2, about 275 mg/m2, about 300 mg/m2, about 325 mg/m2, about 350 mg/m2, about 400 mg/m2, or about 500 mg/m2, or about 1000 mg/m2. In some embodiments, the standard monotherapy regimen of irinotecan is (a) weekly: 125 mg/m2 IV infusion over 90 minutes on days 1 , 8, 15, 22, then 2 weeks off, then repeat or (b) once every 3 weeks: 350 mg/m2 IV infusion over 30-90 minutes every 3 weeks. In some embodiments, administration of a flagellin related polypeptide, including CBLB502, may allow a patient to be administered a complete regimen of irinotecan without having to cease treatment because of, for example, side effects. In some embodiments, administration of a flagellin related polypeptide, including CBLB502, may allow a patient to be administered an increased dose or frequency of irinotecan than the standard regimen (e.g. greater than about 125 mg/m2 weekly, or greater than about 150 mg/m2 weekly, or greater than about 175 mg/m2 weekly, or greater than about 200 mg/m2 weekly, or greater than about 300 mg/m2 weekly, or greater than about 500 mg/m2 weekly, or greater than about 1000 mg/m2 weekly or greater than about 350 mg/ m2 every three weeks, or greater than about 400 mg/ m2 every three weeks, or greater than about 450 mg/ m2 every three weeks, or greater than about 500 mg/ m2 every three weeks, or greater than about 600 mg/ m2 every three weeks, or greater than about 700 mg/ m2 every three weeks, or greater than about 800 mg/ m2 every three weeks, or greater than about 1000 mg/ m2 every three weeks or about 125 mg/m2 about every 5 days, or about every 3 days, or about every 2 days, or about daily, or about 350 mg/m2 about every 18 days, or about 350 mg/m2 about every 15 days, or about 350 mg/m2 about every 2 weeks, or about 350 mg/m2 about every 10 days, or about 350 mg/m2 about every week, or about 350 mg/m2 about every day). [0088] In some embodiments, administration of a flagellin related polypeptide, including CBLB502, may allow a patient to be administered a dose of doxorubicin that is greater than about 20 mg/m2, about 25 mg/m2, about 30 mg/m2, about 35 mg/m2, about 40 mg/m2, about 45 mg/m2, about 50 mg/m2, about 55 mg/m2, about 60 mg/m2, about 65 mg/m2, about 70 mg/m2, about 75 mg/m2, about 80 mg/m2, about 85 mg/m2, about 90 mg/m2, or about 95 mg/m2, or about 100 mg/m2. In some embodiments, the standard monotherapy regimen of doxorubicin is a 60-75 mg/m2 IV infusion every 3-4 weeks. In some embodiments, administration of a flagellin related polypeptide, including CBLB502, may allow a patient to be administered a complete regimen of doxorubicin without having to cease treatment because of, for example, side effects. In some embodiments, administration of a flagellin related polypeptide, including CBLB502, may allow a patient to be administered an increased dose or frequency of doxorubicin than the standard regimen (e.g. greater than about 60 mg/m2 every 3-4 weeks, about 65 mg/m2 every 3-4 weeks, about 70 mg/m2 every 3-4 weeks, about 75 mg/m2 every 3-4 weeks, about 80 mg/m2 every 3-4 weeks, about 85 mg/m2 every 3-4 weeks, about 90 mg/m2 every 3-4 weeks, or about 95 mg/m2 every 3- 4 weeks, or about 100 mg/m2 every 3-4 weeks).
[0089] In some embodiments, the present invention provides for uses of the flagellin related polypeptides, including CBLB502, to increase an ability of a cancer patient to receive a combination therapy with irinotecan and/or 5-FU and or doxorubicin.
[0090] In one embodiment, administering an effective amount of a flagellin related polypeptide, including CBLB502, increases the ability of a cancer patient to receive a combination therapy with 5-FU. In specific embodiments the combination therapy is selected from leucovorin, oxaliplatin, folinic acid, and irinotecan.
[0091] In one embodiment, administering an effective amount of a flagellin related polypeptide, including CBLB502, increases the ability of a cancer patient to receive a combination therapy with irinotecan or doxorubicin. In specific embodiments the combination therapy is selected from oxaliplatin, 5-fluorouracil, folinic acid, leucovorin, cetuximab, temozolomide, sorafenib, capecitabine, bevacizumab, vincristine, vinblastine, dacarbazine, prednisone, rapamycin, cyclophosphamide, and bleomycin,.
[0092] In some embodiments, administering an effective amount of a flagellin related polypeptide, including CBLB502, increases the ability of a cancer patient to receive a complete regimen of a combination therapy with 5-FU and/or irinotecan and/or doxorubicin and another agent and not have to cease treatment because of, for example, side effects. In some embodiments, administering an effective amount of a flagellin related polypeptide, including CBLB502, increases the ability of a cancer patient to receive a greater dose or longer duration of combination therapy with 5-FU and/or irinotecan and/or doxorubicin and another agent. For example, a combination therapy regimen for irinotecan with 5-fluorouracil/ leucovorin may be (a) 6 week cycle with infusional 5-fluorouracil/leucovorin: 180 mg/m2 IV infusion over 30-90 minutes once on days 1 , 15, and 29 IV (infuse over 30-90 min), followed by infusion with leucovorin and 5-fluorouracil; next cycle begins on day 43 or (b) 6 week cycle with bolus 5-fluorouracil/ leucovorin: 125 mg/m2 on days 1 , 8, 15, and 22 (infuse over 90 min), followed by bolus doses of leucovorin and 5-fluorouracil. In various embodiments, administering an effective amount of a flagellin related polypeptide, including CBLB502, increases the dose and/or frequency of this regimen. For example, this regimen can be extended beyond 6 weeks to about 45, or about 50, or about 55, or about 60, or about 65, or about 70, or about 75, or about 100 days (including about 7, or about 8, or about 9, or about 10, or about 1 1 , or about 12 weeks). By way of further example, this regimen can allow for doses of irinotecan that exceed about 125 mg/m2, or about 150 mg/m2, or about 200 mg/m2, or about 250 mg/m2, or about 300 mg/m2, or about 500 mg/m2.
[0093] In some embodiments, administering an effective amount of a flagellin related polypeptide, including CBLB502, increases the ability of a cancer patient to receive a complete regimen of any one of the FOLFOX, FOLFIRI, IFL, FL (Mayo), QUASAR, Machover schedule, CAF, CMF, ECF, and FEC regimens and not have to cease treatment because of, for example, side effects. In some embodiments, administering an effective amount of a flagellin related polypeptide, including CBLB502, increases the ability of a cancer patient to receive a greater dose or longer duration of therapy in the FOLFOX, FOLFIRI, IFL, FL (Mayo), QUASAR, Machover schedule, CAF, CMF, ECF, and FEC regimens.
[0094] Further examples of combination agents are also provided and include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1 -TM1 ); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, ADRIAMYCIN doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as minoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; def of amine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 11 1.), and TAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11 ) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-a, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the methods of treatment can further include the use of radiation. In addition, the methods of treatment can further include the use of photodynamic therapy.
[0095] In various embodiments, the present invention provides a method for improving treatment with the flagellin related compositions in the context of oncology. For instance, the flagellin related compositions may be paired with one or more agents as described herein (e.g. 5-FU) to enhance CBLB502-mediated antitumor immune response. In some embodiments, such a combination may reduce tumor-associated immunosuppression and/or increase tumor cell immunogenicity. In some embodiments, such a combination may reduce the activity or activation of one or more cells, or reduce or eliminate the infiltration to a tumor site and/or TME of one or more cells, including, but not limited to: myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs); tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs). In some embodiments, such a combination may eliminate Tregs and/or MDSC.
[0096] In some embodiments, the flagellin related compositions (and/or additional agents) described herein, include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition. For example, but not by way of limitation, derivatives include composition that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of turicamycin, etc. Additionally, the derivative can contain one or more non- classical amino acids.
[0097] In still other embodiments, the flagellin related compositions (and/or additional agents) described herein may be modified post-translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
[0098] In various embodiments, the present invention pertains to cancers and/or tumors; for example, the treatment or prevention of cancers and/or tumors. In one embodiment, a cancer to be treated in the context of 5- FU is one or more of breast cancer, head and neck cancer, anal cancer, stomach cancer, colon cancer, and skin cancer. In one embodiment, a cancer to be treated in the context of irinotecan is one or more of lung cancer, ovarian cancer, non-small cell lung cancer, leukemia, lymphoma, pancreatic tumor, metastatic cancer, breast tumor, rectal tumor, colonic neoplasms, stomach tumor, metastatic cancer colon or rectum and glioma. In one embodiment, a cancer to be treated in the context of doxorubicin is one or more of leukemia, lymphoma (e.g., Hodgkin's lymphoma), bladder cancer, breast cancer, head and neck cancer, stomach cancer, liver cancer, lung cancer, mesothelioma, ovarian cancer, thyroid cancer, pancreatic cancer, prostate cancer, sarcomas (e.g., soft tissue sarcoma), neuroblastoma, uterine cancer, testis cancer, and multiple myeloma.
[0099] As used herein, "cancer" or "tumor" refers to an uncontrolled growth of cells and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of the bodily organs and systems. Included are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Also, included are cells having abnormal proliferation that is not impeded by the immune system (e.g. virus infected cells). A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. 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. Hematopoietic cancers, such as leukemia, are able to out-compete the normal hematopoietic compartments in a subject, thereby leading to hematopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.
[00100] The cancer may be a primary cancer or a metastatic cancer. The primary cancer may be an area of cancer cells at an originating site that becomes clinically detectable, and may be a primary tumor. In contrast, the metastatic cancer may be the spread of a disease from one organ or part to another non-adjacent organ or part. The metastatic cancer may be caused by a cancer cell that acquires the ability to penetrate and infiltrate surrounding normal tissues in a local area, forming a new tumor, which may be a local metastasis. "Metastasis" refers to the spread of cancer from a primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life -threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant. Metastases may be detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.
[00101] The cancer may also be caused by a cancer cell that acquires the ability to penetrate the walls of lymphatic and/or blood vessels, after which the cancer cell is able to circulate through the bloodstream (thereby being a circulating tumor cell) to other sites and tissues in the body. The cancer may be due to a process such as lymphatic or hematogeneous spread. The cancer may also be caused by a tumor cell that comes to rest at another site, re-penetrates through the vessel or walls, continues to multiply, and eventually forms another clinically detectable tumor. The cancer may be this new tumor, which may be a metastatic (or secondary) tumor.
[00102] The cancer may be caused by tumor cells that have metastasized, which may be a secondary or metastatic tumor. The cells of the tumor may be like those in the original tumor. As an example, if a breast cancer or colon cancer metastasizes to the liver, the secondary tumor, while present in the liver, is made up of abnormal breast or colon cells, not of abnormal liver cells. The tumor in the liver may thus be a metastatic breast cancer or a metastatic colon cancer, not liver cancer.
[00103] The cancer may have an origin from any tissue. The cancer may originate from melanoma, colon, breast, or prostate, and thus may be made up of cells that were originally skin, colon, breast, or prostate, respectively. The cancer may also be a hematological malignancy, which may be lymphoma.
[00104] Representative cancers and/or tumors of the present invention include, but are not limited to, a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small- cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
[00105] The flagellin related compositions (and/or additional agents) described herein can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. A pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art. Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.
[00106] Pharmaceutically acceptable salts include, by way of non-limiting example, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, a-hydroxybutyrate, butyne-1 ,4- dicarboxylate, hexyne-1 ,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1 -sulfonate, naphthalene-2-sulfonate, naphthalene-1 ,5-sulfonate, xylenesulfonate, and tartarate salts.
[00107] The term "pharmaceutically acceptable salt" also refers to a salt of the compositions of the present invention having an acidic functional group, such as a carboxylic acid functional group, and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such as mono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert- butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.
[00108] In some embodiments, the compositions described herein are in the form of a pharmaceutically acceptable salt.
[00109] Further, any flagellin related compositions (and/or additional agents) described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.
[00110] Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.
[00111] The present invention includes the described flagellin related compositions (and/or additional agents) in various formulations. Any flagellin related composition (and/or additional agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the composition is in the form of a capsule (see, e.g., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.
[00112] Where necessary, the flagellin related compositions (and/or additional agents) can also include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device. Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.
[00113] The formulations comprising the flagellin related compositions (and/or additional agents) of the present invention may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art)
[00114] In one embodiment, any flagellin related composition (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.
[00115] Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. In some embodiments, the administering is effected orally or by parenteral injection. The mode of administration can be left to the discretion of the practitioner, and depends in-part upon the site of the medical condition. In most instances, administration results in the release of any agent described herein into the bloodstream.
[00116] Any flagellin related composition (and/or additional agents) described herein can be administered orally. Such flagellin related compositions (and/or additional agents) can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer.
[00117] In specific embodiments, it may be desirable to administer locally to the area in need of treatment.
[00118] In one embodiment, any flagellin related composition (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for oral administration to humans. Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can comprise one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving any flagellin related composition (and/or additional agents) described herein are also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time-delay material such as glycerol monostearate or glycerol stearate can also be useful. Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipients are of pharmaceutical grade. Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, etc., and mixtures thereof.
[00119] Dosage forms suitable for parenteral administration (e.g. intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
[00120] The dosage of any flagellin related composition (and/or additional agents) described herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject's general health, and the administering physician's discretion. Any agent described herein, can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional therapeutic agent, to a subject in need thereof. In various embodiments any agent described herein is administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart.
[00121] The amount of any flagellin related composition (and/or additional agents) described herein that is admixed with the carrier materials to produce a single dosage can vary depending upon the subject being treated and the particular mode of administration. In vitro or in vivo assays can be employed to help identify optimal dosage ranges.
[00122] In general, the doses that are useful are known to those in the art. For example, doses may be determined with reference Physicians' Desk Reference, 66th Edition, PDR Network; 2012 Edition (December 27, 2011 ), the contents of which are incorporated by reference in its entirety. In some embodiment, the present invention allows a patient to receive doses that exceed those determined with reference Physicians' Desk Reference.
[00123] The dosage of any flagellin related composition (and/or additional agents) described herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.
[00124] Generally, when orally administered to a mammal, the dosage of any flagellin related composition (and/or additional agents) described herein may be 0.001 mg/kg/day to 100 mg/kg/day, 0.01 mg/kg/day to 50 mg/kg/day, or 0.1 mg/kg/day to 10 mg/kg/day. When orally administered to a human, the dosage of any agent described herein is normally 0.001 mg to 1000 mg per day, 1 mg to 600 mg per day, or 5 mg to 30 mg per day.
[00125] For administration of any flagellin related composition (and/or additional agents) described herein by parenteral injection, the dosage is normally 0.1 mg to 250 mg per day, 1 mg to 20 mg per day, or 3 mg to 5 mg per day. Injections may be given up to four times daily. Generally, when orally or parenterally administered, the dosage of any agent described herein is normally 0.1 mg to 1500 mg per day, or 0.5 mg to 10 mg per day, or 0.5 mg to 5 mg per day. A dosage of up to 3000 mg per day can be administered.
[00126] In another embodiment, delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et a/., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).
[00127] Any flagellin related composition (and/or additional agents) described herein can be administered by controlled-release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled- or sustained-release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the agents described herein. The invention thus provides single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled- or sustained-release.
[00128] Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds. [00129] In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability: Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 ; see also Levy et al, 1985, Science 228:190; During et al, 1989, Ann. Neurol. 25:351 ; Howard et al., 1989, J. Neurosurg. 71 :105).
[00130] In another embodiment, a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer, 1990, Science 249:1527-1533) may be used.
[00131] Administration of any flagellin related composition (and/or additional agents) described herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the subject. Chronic, long-term administration will be indicated in many cases. The dosage may be administered as a single dose or divided into multiple doses. In general, the desired dosage should be administered at set intervals for a prolonged period, usually at least over several weeks or months, although longer periods of administration of several months or years or more may be needed.
[00132] The dosage regimen utilizing any flagellin related composition (and/or additional agents) described herein can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the specific compound of the invention employed. Any flagellin related composition (and/or additional agents) described herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily. Furthermore, any flagellin related composition (and/or additional agents) described herein can be administered continuously rather than intermittently throughout the dosage regimen.
[00133] In some embodiments, the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In other embodiments, the subject and/or animal is a non-mammal, such, for example, a zebrafish. In some embodiments, the subject and/or animal may comprise fluorescently-tagged cells (with e.g. GFP). In some embodiments, the subject and/or animal is a transgenic animal comprising a fluorescent cell.
[00134] In some embodiments, the subject and/or animal is a human. In some embodiments, the human is a pediatric human. In other embodiments, the human is an adult human. In other embodiments, the human is a geriatric human. In other embodiments, the human may be referred to as a patient. [00135] In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
[00136] In other embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet. In another specific embodiment, the non-human animal is a livestock animal.
[00137] The invention provides kits that can simplify the administration of any agent described herein. An exemplary kit of the invention comprises any composition described herein in unit dosage form. In one embodiment, the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit can further comprise a label or printed instructions instructing the use of any agent described herein. The kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location. The kit can also further comprise one or more additional agent described herein. In one embodiment, the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those described herein.
[00138] This invention is further illustrated by the following non-limiting examples.
EXAMPLES
Materials and Methods
[00139] Mice: BALB/c and C57BL/6 female mice, 10-14 weeks old (Jackson Laboratory, Bar Harbor, ME) were used in the study. IL-6 deficient mice C.129S2-IL6tm1/kopf/J (BALB/c background) were originally purchased from Jackson Laboratory; TLR5 knockout mice B6.129P2-Tlr5tm1Aki (C57BL/6 background) were a generous gift of Dr. Shizuo Akira (University of Tokyo, Japan); mice of both strains were bred at Roswell Park Cancer Institute (RPCI). All animal experiments followed protocols approved by the RPCI IACUC.
[00140] Reagents: CBLB502was obtained from Cleveland BioLabs, Inc. (Buffalo, NY). 5-Fluorouracil (5-FU) was purchased from Sigma (St. Louis, MO). [00141] Tumor cells: Murine colon undifferentiated carcinoma CT26 cells (ATCC) were cultured in RPMI media with 10% FBS, standard supplements (2 mmol/L L-glutamine, Ι ΟΟμιτιοΙ/L nonessential amino acids) and 1 % penicillin-streptomycin (Invitrogen) at 37°C in a 5% CO2 incubator.
[00142] In vivo model of 5-FU toxicity: Mice were weighed and randomly divided into treatment groups. 5-FU was diluted in DMSO (40%) and injected i.p. (100 or 200 mg/kg / 200 μΙ/ injection). High dose 5-FU (400 mg/kg) was given as two i.p. injections of 200 mg/kg 6 h apart. CBLB502 (1 g/ 100 μΙ) was injected s.c. at the indicated times after the first 5-FU injection. When mice were given three i.p. injections of 5-FU (100 mg/kg/day, once/day), CBLB502 was injected 24 and 48 h after the last 5-FU injection. DMSO (40%) and PBS were used as vehicle controls for 5-FU and CBLB502, respectively. Mice were monitored daily or at least 3 times a week for survival and signs of morbidity, including changes in body weight.
[00143] In vivo model of 5-FU antitumor therapy: CT26 tumor cells (5x105 /100μΙ PBS) were injected s.c. into syngeneic BALB/c mice (2 flanks/mouse). When tumors reached ~5 mm in diameter, mice were randomly divided into 5 treatment groups. 5-FU (100 or 200 mg/kg) was injected i.p. with CBLB502 (1 g/ 100 μΙ) or PBS injected s.c. 1 , 48 and 96 h after 5-FU. A group of mice injected with vehicle solutions was used as an "untreated" control. Tumors were measured by two diameters with a digital caliper every second day. Tumor volume (V) was calculated as: V=TT/6 X di2 x cfe, where di< cfe. Mice were euthanized according to IACUC protocol when signs of morbidity were observed or tumors reached about 13 mm in diameter or developed ulceration.
[00144] Complete blood cell analysis: 25 μί. of whole blood from the orbital sinus were analyzed to determine complete and differential blood cell counts using a Hemavet 950 Hematology System (Drew Scientific, Dallas, TX).
[00145] Histological analysis: Immediately after euthanasia on day 3 or 7 after 5-FU injection, 1.0 cm samples of duodenum, jejunum, ileum, colon and femora with bone marrow (BM) were harvested from mice processed for hematoxylin-eosin (H&E) staining and analyzed in a blinded fashion by a trained pathologist using ImagePro software (at 10x magnification unless otherwise stated). Pathomorphological changes in intestinal sections were scored as: 4 - Severe; 3 - Markedly abnormal; 2 - Moderate; 1 - Mild I; or 0 - Normal, with intermediate non- integer scores assigned based on the pathologist's judgment. The mitotic index was determined by counting the mitotic figures in crypt cells of the small intestine using light microscopy at 100χ magnification. The mitotic index is calculated in longitudinally sectioned crypts as an average value per crypt in each treatment-group (4 samples per mouse, 3 mice/group).
[00146] Statistical analysis: Differences in body weight loss (expressed as percent initial body weight) between groups were analyzed by two-way (time and treatment) repeated measures ANOVA; differences in histomorphological scores of tissue damage and mitotic index were analyzed by Student's t-test (two-tailed, unequal variances); differences in survival kinetics (mean survival time) were analyzed by log-rank test using GraphPad Prism software; differences in discrete blood cell populations between treatment groups were determined using two-tailed unpaired Student's t-test. Significance level was set at P O.05.
[00147] Cytokine analysis: Concentrations of IL-6 and soluble IL-1 receptor in mouse plasma were determined using MILLIPLEX kit (EMD Millipore, St. Charles, MO). Plasma was prepared from blood collected from BALB/c mice by heart puncture upon euthanasia 3 or 7 days after 5-FU injections (200 mg/kg) with or without CBLB502 (1 μg/ mouse) injected 24 and 48 h after 5-FU. Plasma from vehicle-injected mice was used as an untreated control. There were 5 mice per treatment group and plasma from 2-3 mice was pooled together for analysis (data is shown as the average of 2 pools for each treatment group).
[00148] Western blotting: The levels of SOD-2 expression in mouse small intestine samples collected 2, 4, 6 or 24 h after CBLB502 (1 μg/ mouse) s.c. injections were determined by Western blot analysis using rabbit polyclonal anti-SOD-2 antibody followed by secondary goat anti-rabbit HRP-conjugated antibody (Santa Cruz Biotechnology, Inc.). The small intestine of a PBS-injected mouse was used as an "intact" control, β-actin was analyzed as a loading control.
[00149] RT-PCR: Expression of IL-1 receptor antagonist (IL-1 RN) was detected by RT-PCR using total RNA prepared from the small intestine isolated from a mouse treated with CBLB502 (1 μg/ mouse x2 injections 24 h apart) 24 h after the second injection. The small intestine from a PBS-injected mouse was used as an "intact" control. Total RNA was extracted using TRIzol reagent according to manufacturer's instructions (Invitrogen, Carlsbad, CA). cDNAs were synthesized using iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) according to manufacturer's protocol from 250 ng RNA/sample. 1 μί cDNA in 12.5 μί TAQ PCR Master Mix, 2X (Affymetrix, Santa Clara, CA) and 9.5 μί DNase, RNase free water was amplified by PCR using forward and reverse primers for IL-1 RN and GAPDH (house-keeping gene for loading control). The RT-PCR conditions were 95°C for 2 min, followed by 32 cycles for IL-1 RN and 25 cycles for GAPDH of 95°C for 30 sec, 55.2°C (IL-1 RN) or 52.1 °C (GAPDH) for 30 sec, and 72°C for 1 min, and 1 cycle of 72°C for 3 min (for final extension). The PCR products were visualized on 1.2% agarose gels stained with ethidium bromide.
Example 1: Effect of CBLB502 on 5-FU-induced Mortality in Mice
[00150] Two mouse strains, BALB/c and C57BL/6, which differ in their sensitivity to 5-FU were used to determine whether CBLB502 is capable of protecting normal tissues from the toxicity of 5-FU. Mice were administered 5-FU alone or in combination with subsequent CBLB502 treatment using different doses and schedules and toxicity was assessed by monitoring body weight loss and mortality.
[00151] In BALB/c mice, injection of 100 mg/kg 5-FU induced only transient body weight loss without mortality, but a single injection of 200 mg/kg 5-FU caused severe weight loss and 80-100% mortality within 2 weeks after 5-FU injection (Fig. 1 (panels A and B)). Fig. 1 shows body weight (A) and survival (B) of BALB/c mice treated with 5-FU (200 mg/kg or 400 mg/kg given in two equal fractions 6 h apart) alone or with subsequent injection of CBLB502 (1 μg/mouse) 24 and 48 h after the last 5-FU dose (10 animals/group). Body weight in Fig. 1 , panel A is shown as a percentage of starting weight; mean ± SEM. The kinetics of body weight loss showed an initial drop in mean body weight during the first 3-5 days after injection, partial improvement by day 7-8, and then a second drop with no recovery. A higher dose of 5-FU (400 mg/kg) caused more rapid single-phasic body weight loss and death of 100% of animals within 7 days. At both tested doses of 5-FU, administration of CBLB502 (two s.c. injections given 24 and 48 h after 5-FU) reduced body weight loss and prevented mouse mortality. With 200 mg/kg 5-FU + CBLB502, mean body weight loss was minimal (<10%) and all mice survived to the end of the 30 day observation period. With 400 mg/kg 5-FU + CBLB502, the initial 5-FU-induced drop in mean body weight was observed, but this was reversed at Day 5, and on Day 30, mean body weight was restored to normal and 80% of animals were alive. Improved resistance of BALB/c mice to 5-FU-induced toxicity was also observed when CBLB502 was applied 24 h (single injection), or 1 , 48 and 96 h (three injections) after 200 mg/kg 5-FU as evidenced by substantially reduced weight loss and an increase in 30-day survival to 90% (versus 0% in the group given 5-FU alone) (Fig. 2).
[00152] Fig. 2 shows body weight (panel A) and (panel B) survival of BALB/c mice after 5-FU (200 mg/kg) with and without CBLB502 injections given in different regimens: 24 h post-5-FU, 1 and 24 h post-5-FU, and 1 , 48 and 96h post-5-FU; n=10 mice/group. CBLB502 and vehicle injected mice without 5-FU were weighed as controls. Mean body weight (as a percentage of starting weight) +/- SEM is shown. (*) Body weight beyond this time-point was based on 3 surviving mice in the group treated with CBLB502 1 and 24 h post-5-FU. (**) No difference from 5-FU alone (p>0.05). Under the same conditions, two injections of CBLB502 given 1 h and 24 h post-5FU prevented weight loss and mortality in only 3/10 mice a statistical insignificant effect.
[00153] C57BL/6 mice were more resistant to 5-FU toxicity than BALB/c mice. Administration of 200 mg/kg 5- FU to C57BL/6 mice was not lethal, but caused transient body weight loss (only about 10-15%) with complete recovery by Day 10. However, 400 mg/kg 5-FU, which caused early death of BALB/c mice by day 7, was lethal for majority of C57BL/6 mice by day 15 (Fig. 1 , panel C). Fig. 1 , panel C shows the survival of C57BL/6 mice injected with a single dose of 400 mg/kg 5-FU or 3 daily doses of 100 mg/kg 5-FU with or without CBLB502 (1 μg/mouse) injected 24 and 48 h after the last 5-FU dose; n=10 mice/group. The differences in mortality kinetics between corresponding 5-FU and 5-FU+CBLB502 groups were: (*) p<0.001 ; (**) p<0.03 by Log-rank test for 30- day survival. Toxicity with similar kinetics was observed after three injections of 100 mg/kg 5-FU given 24 h apart, with severe weight loss and death occurring about 2 weeks post-treatment (Fig. 1 panel C (see above text), Fig. 3 panels A-B). Fig. 3 panel A and panel B correspond to body weight changes in wild type C57BL/C mice injected with 5-FU: (Fig. 3 panel A) 100 mg/kg x 3 times with 24 h interval with CBLB502 injected 24 and 48 h after the last 5-FU treatment, and (Fig. 3 panel B) 200 mg/kg x 2 times with 6 h interval with and without CBLB502 injected 24 and 48 h post-5-FU (Fig. 3 panel B); n=10; (*) The results of the group treated with 5-FU and CBLB502 at this point and later were based on 3 surviving mice. With both toxic regimens of 5-FU treatment, injection of CBLB502 (1 μg/mouse) 24 and 48 h after injection of the last fraction of 5-FU resulted in reduction of body weight loss and improved survival. With addition of CBLB502 to 400 mg/kg 5-FU treatment, 30-day survival was increased from 10% to 70% (p=0.0079, Fig. 1 panel C (see above text)). Similarly, when CBLB502 was given to mice treated with 3x100 mg/kg 5-FU, mortality was delayed. Mean survival time was increased from 11.5 days to 14 days (p= 0.0002) and 30-day survival was increased from 0% to 30%. The ability of CBLB502 to reduce 5-FU-associated mortality was confirmed to be TLR5-specific since the effect was not observed in similarly treated TLR5 knockout mice (Fig. 3 panel C). Fig. 3 panel C shows the survival of wild type C57BL/6 and TLR5-KO mice injected with 5-FU - 200 mg/kg x 2 times with 6 h interval with and without CBLB502 injected 24 and 48 h post-5-FU. (**) p=0.0022 for comparison of WT mice treated with 5-FU+CBLB502 to WT mice treated with 5-FU only.
Example 2 Effect of CBLB502 on 5-FU-induced Hematopoietic Damage
[00154] CBLB502 was evaluated to determine whether it specifically reduces HP damage caused by 5-FU. As shown in Fig. 4, treatment of BALB/c mice with 200 or 100 mg/kg 5-FU resulted in rapid elimination of practically all types of blood cells, including neutrophils, lymphocytes and platelets, correlating with lethality observed within 1 1-14 days after 5-FU administration. CBLB502 treatment after 200 mg/kg 5-FU did not ameliorate 5-FU-induced depletion of blood cell populations as observed on Days 7 and 1 1 post-5-FU (Fig. 4 panel A). Fig. 4 shows complete blood cell analysis performed using blood samples from mice treated with vehicle (n=5), 5-FU alone (n=10) or 5-FU+CBLB502 (n=10). 5-FU was injected i.p. at a dose of 200 mg/kg (Fig. 4 panel A) or 100 mg/kg (Fig. 4 panel B) followed by three s.c. CBLB502 (1 μg/mouse) injections 1 , 48 and 96 h post-5-FU. Mean concentrations (Κ/μΙ peripheral blood) of white blood cells (WBC), neutrophils, lymphocytes and platelets ± SEM are shown for the indicated days after 5-FU injection. The differences between 5-FU and 5- FU+CBLB502 groups were: (*) p<0.001 ; (**) p<0.05 by two-tailed unpaired t-test. However, in contrast to mice treated with 200 mg/kg 5-FU alone, mice treated with CBLB502 after 200 mg/kg 5-FU administration showed gradual recovery of hematopoiesis leading to complete restoration of normal WBC and neutrophil levels by Day 14 and improved mouse survival. With sub-lethal doses of 100 mg/kg and 150 mg/kg 5-FU (Fig. 4 panel B (see above text) and Fig. 5, respectively), mice treated with CBLB502 after 5-FU consistently displayed more rapid recovery of peripheral blood cell populations than mice given 5-FU alone. Fig. 5 shows the restoration of blood cell count in BALB/c mice following 5-FU treatment alone and in combination with CBLB502. Specifically, 5-FU was injected i.p. (150 mg/kg) followed by CBLB502 24 h and 48 h post 5-FU (n=5 mice/group). Two out of 5 mice in the 5-FU injected group died on day 8, all 5 mice in the 5-FU+CBLB502-treated group survived. Vehicle injected mice were used as controls (n=3-5). In some cases in response to 5-FU with or without CBLB502, surviving mice displayed temporary overcompensation for the loss of HP cells: numbers of lymphocytes, neutrophils and platelets were higher in treated mice than in intact controls at the day 14-15 time-points (Fig. 4 panel B (see above text), but normalized by day 21.
[00155] The effect of CBLB502 treatment on 5-FU-induced HP damage in BALB/c mice was also assessed through morphological analysis of H&E-stained bone marrow (BM) sections (Fig. 6). Fig. 6 shows the effect of CBLB502 on 5-FU-induced changes in bone marrow morphology. Representative pictures of H&E-stained bone marrow sections (10x objective magnification) were prepared 3 and 7 days after 5-FU injection. BALB/c mice were treated with 200 mg/kg 5-FU with or without injection of CBLB502 24 and 48 h post-5-FU; 5 mice/group. The lower row shows selected areas outlined in white at two fold higher magnification. This result showed that the application of 200 mg/kg 5-FU resulted in severe aplasia of the BM, with near-complete absence of HP cells, remnants of stromal cells, expanded sinusoids and hemorrhage observed in BM sections prepared on Day 3 (72 h) after 5-FU administration. Treatment with CBLB502 (1 μg/mouse) 24 and 48 h after 200 mg/kg 5-FU had no or very little beneficial effect on BM morphology at this time point. In both 5-FU-treated mice and those given 5-FU in combination with CBLB502, the BM was drastically depopulated in contrast to BM samples from mice treated with CBLB502 alone or intact mice which displayed normal morphology with a meshwork of bony spicules, open areas with active hematopoiesis and sinusoids filled with erythrocytes.
[00156] At Day 7, the BM of mice treated with 200 mg/kg 5-FU alone contained only single HP and stromal cells that were rounded and detached from each other (indicative of necrosis and necrobiosis). In addition, hemorrhage and an abundant fat component were observed in these BM samples. In contrast, the BM of mice injected with CBLB502 after 200 mg/kg 5-FU showed signs of HP recovery including islets of preserved HP cells with prevalent hyperplastic megakaryocytes suggesting that CBLB502 promotes recovery of hematopoiesis in the BM and restoration of peripheral HP cell populations after 5-FU-induced damage.
[00157] Taken together, without wishing to be bound by theory, these data demonstrate that 5-FU causes severe myelosuppression, neutropenia and thrombocytopenia which likely contribute to the lethality of the treatment in BALB/c mice. Furthermore, CBLB502 promotes recovery of hematopoiesis in the BM and restoration of peripheral HP cell populations after 5-FU-induced damage.
Example 3 Effect of CBLB502 on 5-FU-induced Gastrointestinal Damage
[00158] Tissue morphology in H&E-stained transverse sections of small and large intestines from untreated "intact" mice and mice treated with different regimens of 5-FU with or without CBLB502 was compared to assess the effect of CBLB502 treatment on the Gl toxicity of 5-FU. Specific morphological features were evaluated, including (a) the height and width of villi of the small intestines and number of surface enterocytes and goblet cells in the small and large intestines; (b) status of crypts (depth, size and shape, presence of apoptotic bodies, number of and granules in Paneth's cells, luminal migration of epithelial nuclei, loss of goblet cells, presence of atrophy and distortion); and (c) state of the lamina propria (presence of transitory cells, lymphoid accumulations, edema, blood vessel congestion and hemorrhage). The extent of damage related to these features was scored according to the following semi-quantitative scale: 4 - Severe, 3 - Markedly abnormal, 2 - Moderate, 1 - Mild and 0 - No damage.
[00159] Administration of 5-FU (200 mg/kg or 400 mg/kg) into BALB/c mice caused dose-dependent damage in the small and large intestines. The small intestine showed atrophy and necrosis in the crypts and surface epithelium at 3 and 7 days after 5-FU administration (Fig. 7). Fig. 7 shows the effect of CBLB502 on 5-FU- induced changes in small intestine morphology. Fig. 7A-B correspond to: representative H&E-stained transverse sections (250x objective magnification) of small intestines from vehicle injected BALB/c mice (intact) and treated with 5-FU (200 mg/kg (Fig. 7 panel A) or 400 mg/kg (Fig. 7 panel B)) alone or in combination with CBLB502 24 and 48 h post-5-FU. Sections were prepared 3 and 7 days after 5-FU treatment. Enterocytes lining the villi are indicated by arrows, lamina propria by asterisks, and crypts by arrowheads. The lower row shows selected crypt areas outlined in white at two fold higher magnification. Fig. 7 panel C shows the average injury score of the small intestine sections described in (Fig. 7 panels A-B). The degree of pathimorphological changes in the surface epithelium, villi, crypts, lamina propria, stroma, transitory and lymphoid elements and submucosa were scored as: 4 - Severe; 3 - Markedly abnormal; 2 - Moderately abnormal; 1 - Mild; and 0 - Normal, including non-integer scores. The average morphological score for 5 mice/group is shown ± SEM. Differences between corresponding 5-FU and 5-FU+CBLB502 groups were: (*) pO.05; (**) p<0.001. Fig. 7 panel D shows the mitotic index in mice treated as described in (Fig. 7 panels A-B) was calculated in crypts of 4 transverse sections of small intestine per mouse as the number of mitoses per crypt (12 samples/ group). The mitotic index of vehicle treated animals was plotted as "No-5-FU" control; mean ± SEM; (*) Differences between corresponding 5-FU and 5-FU+CBLB502 groups were p<0.05. 5-FU-induced injury was most pronounced in the crypt layer, with smaller and denser crypts, scarce and degranulated Paneth's cells, abnormal differentiation and maturation of the crypt epithelium, absence of goblet cells, and enlarged and vesicular nuclei. The observed toxicity was dose- and time-dependent, with greater damage observed after treatment with 400 mg/kg 5-FU versus 200 mg/kg 5-FU and at day 3 versus day 7 indicating Gl recovery at the latter time point. However, after 400 mg/kg 5-FU, crypt damage remained even at day 7. In addition to the above described changes in the crypts, severe 5-FU-induced injury was observed in the surface epithelium of the villi, in the lamina propria and in the submucosa. For both tested 5-FU doses, CBLB502 injection at 24 and 48 h post-5-FU led to mitigation of 5-FU-induced damage to the small intestine mucosa and morphology closer to normal (Fig. 7 panels A-C (see above text)). The most dramatic evidence of this was the preservation of secretory Paneth's cells in the crypts of all mice treated with CBLB502 in addition to 5-FU. The beneficial effect of CBLB502 was clearly illustrated by the near-complete restoration of normal small intestine morphology on day 7 after 5-FU injection in CBLB502-treated mice, while those given 5- FU alone showed severe damage with no signs of recovery at the same time point. In addition, the average overall Gl injury scores obtained by semi-quantitative scoring of the stained tissue sections confirmed efficacy of CBLB502 in reducing small intestine toxicity of 200 or 400 mg/kg doses of 5-FU at both day 3 and day 7 (Fig. 7 panel C (see above text)).
[00160] Similar analysis of the morphology of the large intestine (colon) in BALB/c mice after 200 mg/kg or 400 mg/kg 5-FU treatment showed dose-dependent injury of the colonic mucosa on day 3 and 7 post-5-FU, with atrophic and degenerative changes observed mainly in the crypt cells (e.g., loss of mucin globules, loss of distinct boundaries between adjacent cells, disappearance of the apical cytoplasm, and extended crypt lumens) (Fig. 8). Fig. 8 panels A-C: representative H&E-stained transverse sections (250x objective magnification) of colon from untreated "intact" BALB/c mice and those treated with 5-FU injections (200 mg/kg (Fig. 8 panel A) or 400 mg/kg (Fig. 8 panel B)) alone or in combination with CBLB502 treatment 24 and 48 h post-5-FU. Sections were prepared 3 or 7 days after 5-FU treatment. Morphology of crypt areas outlined in white is shown at two fold higher magnification in the lower rows. Atrophic and degenerative changes in the surface epithelium are indicated by arrows and in the crypt cells and mucin globules by arrowheads. Fig. 8 panel C shows the average injury score determined by pathomorphological changes in the surface epithelium, number of surface enterocytes and goblet cells, size and shape of crypts, lymphoid elements and state of the submucosa in colon sections from mice described in (Fig. 8 panel A-B); 5 mice/group; mean ± SEM. Differences between corresponding 5-FU and 5-FU+CBLB502 groups were (*) p<0.05. Treatment of 5-FU-injected mice with CBLB502 led to improved preservation of crypt cells and mucin globules and overall closer-to-normal morphology in the large intestine. The beneficial effect of CBLB502 on 5-FU-induced large intestine injury at day 3 and day 7 post-5-FU was found for groups treated with high dose (400 mg/kg) of 5-FU (Fig. 8 panel C (see above text)). The Gl toxicity of 5-FU and potential mitigative effect of CBLB502 were examined further by determining the number of mitoses per crypt ("mitotic index") in transverse small intestine sections as an indicator of the proliferative capacity/health of the crypts (Fig. 7 panel D (see above text)). 5-FU administration (200 or 400 mg/kg) was found to cause a dose- dependent reduction in mitotic index on day 3 post-injection. At both 5-FU dose levels, CBLB502 treatment ameliorated the drop in mitotic index on day 3 (although only significantly so with 400 mg/kg 5-FU). By day 7 after 200 mg/kg 5-FU injection, the mitotic index was restored to normal in both mice treated with 5-FU alone and those treated with 5-FU+CBLB502; however, on day 7 after 400 mg/kg 5-FU injection, efficacy of CBLB502 in promoting restoration of the small intestine mitotic index was evident. The mitotic index on day 7 in mice treated with CBLB502 after 400 mg/kg 5-FU was the same as in intact mice, while that in mice treated with 5-FU alone remained -50% lower than normal (Fig. 7 panel D (see above text)).
[00161] Taken together, the results of these direct analyses of the Gl tract demonstrate that CBLB502 counteracts the Gl toxicity of 5-FU, primarily by stimulating regeneration of damaged Gl tissue.
Example 4 Role of IL-6 in the Mitigating Effects of CBLB502 Against 5-FU Toxicity
[00162] Induction of the NF-KB-regulated cytokine, IL-6, in 5-FU-treated mice was a result of CBLB502 injection. A strong CBLB502-dependent spike in plasma levels of IL-6 (58-fold over the level observed with vehicle treatment) was observed in BALB/c mice at 7 days after 200 mg/kg 3-FU treatment (Fig. 9). Fig. 9 shows IL-6 concentration in plasma samples from BALB/c mice that were analyzed by a MILLIPLEX kit (average of 2 pools of 3 mouse samples each at each data-point). Plasma was collected 3 and 7 days after injection of vehicle (DMSO), CBLB502 alone, 200 mg/kg 5-FU alone, or 5-FU+CBLB502. In order to determine whether CBLB502- induced IL-6 plays a role in the drug's ability to protect mice against 5-FU toxicity, the effects of CBLB502 treatment on 5-FU-associated body weight loss, mortality and morphological Gl damage in IL-6-KO mice versus wild type (WT) BALB/c mice were compared. IL-6-KO and WT mice displayed similar sensitivity to 3 different doses of 5-FU as determined by body weight loss and survival (100, 150 and 200 mg/kg). However, while CBLB502 was able to rescue WT mice from death induced by 200 mg/kg 5-FU, it did not reduce the lethality of the same dose of 5-FU in IL-6-KO mice (Fig. 10 panel A). Fig. 10 panel A shows survival of WT BALB/c and IL-6- KO mice after treatment with 200 mg/kg 5-FU, with or without CBLB502 treatment (1 μg/mouse) 24 and 48 h after 5-FU. Fig. 10 panel B shows the kinetics of body weight changes in IL-6-KO mice treated with 5-FU (400 mg/kg) and CBLB502 injected 24 and 48 h after the last 5-FU dose (mean ± SEM). Fig. 10 panel C shows survival of IL-6-KO and wild type (WT) mice treated with 5-FU (400 mg/kg) or 5-FU+CBLB502. The combined results of two independent experiments are presented in (Fig. 10 panel B) and Fig. 10 panel C). (*) Differences between corresponding 5-FU and 5-FU+CBLB502 groups were p<0.001. Fig. 10 panel D shows H&E-stained small intestine sections showing crypts from IL-6-KO mice euthanized on Day 3 after 5-FU (400 mg/kg) with or without CBLB502. Enterocytes lining the villi are indicated by arrows, lamina propria by asterisks and crypts by arrowheads. Normal morphology of crypts in an intact mouse is shown as a control. Fig. 10 panel E shows concentrations of white blood cells (WBC) in blood samples from IL-6-KO mice treated with CBLB502 alone (n=3), 5-FU alone (n=5) or 5-FU+CBLB502 (n=5). 5-FU (100 mg/kg) was followed by CBLB502 24 and 48 h later; m ± SEM. (**) Differences between the 5-FU and 5-FU+CBLB502 groups were p<0.05. In contrast, with a higher dose of 5-FU (400 mg/kg), CBLB502 treatment did provide some survival benefit to IL-6-KO mice, increasing mean survival time from 8 to 10 days (PO.001 ) (Fig. Fig. 10 panel C (see above text)). This improvement in survival of IL-6-KO mice dosed with 400 mg/kg 5-FU was associated with CBLB502-mediated recovery of body weight after initial 5-FU-induced weight loss (Fig. 10 panel B (see above text)), consistent with mitigation of Gl damage in KO animals with preservation of crypt cells in small intestine and higher mitotic index (Fig. 10 panel D (see above text) and Fig. 11 ) as in WT animals (Fig. 7 panel B and panel D (see above text)). Fig. 11 shows the mitotic index in IL-6-KO mice after 5-FU treatment with and without CBLB502. The mitotic index was calculated in crypts of 4 transverse sections of small intestine per mouse as the number of mitoses per crypt in 3 mice/ group. The samples were obtained 3 days after 5-FU (400 mg/kg) injections with and without CBLB502 24 and 48 h post-5-FU (12 samples/ group). Mean ± SEM is shown. (*) The difference in mitotic index between 5-FU and 5-FU+CBLB502 groups was p<0.05. CBLB502 did not, however, prevent mouse lethality and all KO mice treated with 400 mg/kg 5-FU + CBLB502 died by day 13 post-5-FU (Fig. 10 panel C (see above text)). Using a sub-lethal dose of 5-FU (100 mg/kg), CBLB502-mediated acceleration of HP recovery was confirmed to be absent in IL-6-KO mice (Fig. 10 panel E (see above text)) in contrast to WT mice (Fig. 4 panel B (see above text)). These data demonstrate, without wishing to be bound by theory, that IL-6 production is an essential mechanism of CBLB502-mediated restoration of hematopoiesis after 5-FU treatment, but is not required for mitigation of Gl toxicity under the same conditions.
[00163] In addition to its direct cytotoxic effects, the plasma concentration of soluble IL-1 receptor was elevated in mice treated with CBLB502 alone or following 5-FU as compared to untreated "intact" mice (Fig. 12 panel A). Fig. 12 shows (Fig. 12 panel A): the concentration of soluble IL-1 receptor in plasma prepared from BALB/c mice 72 h after 5-FU injections (200 and 400 mg/kg) with and without CBLB502 injected 24 and 48 h after 5- FU was determined using MILLIPLEX kit (average of 2 pools of 3 mouse samples at each data-point). (12B): western blotting for SOD-2 expression in small intestine samples from NIH-Swiss mice at the indicated time-points after CBLB502 injection (without 5-FU). Actin was used as a loading control (Fig. 12 panel C): expression of IL-1 receptor antagonist (IL-1 RN) was detected by RT-PCR using total RNA prepared from small intestine isolated from a BALB/c mouse treated with CBLB502 (1 mg/ mouse x2 injections 24h apart) 24 h after the second injection. Small intestine RNA from a PBS-injected mouse was used as an "intact" control. GADPH expression was used a housekeeping gene for loading control. In addition, upregulation of IL-1 RN expression in the small intestine was detected following CBLB502 treatment of mice (Fig. 12 panel B (see above text)). These data suggest, without wishing to be bound by theory, that neutralization of IL-1 activity as a possible mechanism underlying CBLB502-mediated mitigation of 5-FU toxicity in Gl tissues. Additionally, production of the antioxidant enzyme SOD2 in the small intestine was increased following CBLB502 treatment of mice and presents, without wishing to be bound by theory, neutralization of ROS in Gl tissues as another possible mechanism involved in the protective effects of CBLB502 against 5-FU-induced tissue damage (Fig. 12 panel C (see above text)).
Example 5 CBLB502 does not Protect Tumors from 5-FU
[00164] The use of CBLB502 to improve anticancer chemotherapy by reducing its adverse side effects requires that the protective effects of the drug are strictly limited to normal (non-tumor) cells and tissues. Since 5- FU is commonly used as a single agent or in combination with other chemotherapeutic drugs to treat colorectal cancer, the mouse CT26 colorectal carcinoma model was employed to test the effect of CBLB502 on tumor sensitivity to 5-FU. CT26 cells do not express TLR5 and their growth as s.c. tumors is not suppressed by CBLB502 treatment (Fig. 13). Fig. 13 shows the treatment of CT-26 tumor-bearing BALB/c mice with CBLB502 initiated when tumors reached about 5 mm in diameter. CBLB502 (1 mg/mouse) was injected s.c. 24 h apart on days 1 , 2 and 3; mean ±SEM, 5 mice x2 tumors per group (n=10). CT26 tumors were, however, sensitive to 5- FU treatment, showing 5-FU dose-dependent reduction of tumor growth (Fig. 14 panel A). Fig. 14 panel A: kinetics of s.c. CT26 tumor growth in mice treated with vehicle ("untreated"), 5-FU alone (100 or 200 mg/kg), or 5-FU + CBLB502 injected 1 , 48, and 96 h post-5-FU (10 mice, 20 tumors per group in 5-FU treated groups, and 5 mice, 10 tumors, in "untreated" group, mean ± SEM); Fig. 14 panel B: kinetics of body weight changes (percentage of starting weight); and Fig. 14 panel C: survival of mice described in Fig. 14 panel A. (*) - Body weight and tumor volume are shown for only 4/10 mice injected with 200 mg/kg 5-FU alone that were surviving on Day 18 after CT26 cell inoculation; mice in this group died due to 5-FU toxicity with small or no tumors. In the other groups, mice were euthanized due to large size or ulceration of tumors; 2/10 mice in the group treated with 200 mg/kg 5-FU + CBLB502 remained tumor-free for the entire period of observation (60 days). (**) Differences in mouse survival between 5-FU (200 mg/kg) + CBLB502 group and any other group were p<0.05 by Log-rank test for mean survival. Inclusion of CBLB502 in the treatment regimen with either 100 mg/kg or 200 mg/kg 5-FU did not have any effect on 5-FU-induced tumor suppression (Fig. 14 panel A (see above text)).
[00165] CBLB502 treatment also did not change survival of tumor-bearing mice treated with 100 mg/kg 5-FU; 100% of mice in these two groups were euthanized by Day 29 after tumor cell inoculation due to tumors reaching the size endpoint or developing ulcerations (Fig. 14 panel B (see above text)). In contrast, 100 % mice injected with 200 mg/kg 5-FU without CBLB502 died due to 5-FU toxicity by Day 27 post tumor cell implantation (Day 23 post 5-FU injection), while those treated with the combination of 200 mg/kg 5-FU and CBLB502 experienced significantly less toxicity. Tumor-bearing mice given CBLB502 after 200 mg/kg 5-FU displayed less weight loss (Fig. 14 panel B (see above text)) and prolonged survival (Fig. 14 panel C (see above text)) compared to those treated with 200 mg/kg 5-FU alone. Moreover, 2 of the 10 mice treated with 200 mg/kg 5-FU+CBLB502 survived tumor-free for the entire period of observation (>60 days). These results illustrate that treatment of CT26 tumor- bearing mice with CBLB502 in combination with 5-FU reduced the systemic toxicity of 5-FU without affecting its antitumor efficacy.
Example 6 Effects CBLB502 Against Irinotecan Toxicity
[00166] Rats (Ward colorectal cancer Rat model), weighing 125-150gm, were transplanted s.c. with a 50 mg viable tumor fragment on day 1 and then the tumors were allowed to grow to approximately 1.0 g by day 7. CBLB502 and/or irinotecan was administered via intraperitoneal injection utilizing the following schedules: 1 ) 0.2 mg/kg CBLB502 d5-15 (10 doses) following implant of tumors (7 rats/group)- 4 rats kept up to 52 days (length of study) and 3 rats euthanized 2h after last 502 injection for histology tumor/gastrointestinal organs and liver evaluation; 2) 0.2 mg/kg CBLB502 d5-9 (5 doses) following implant of tumors (10 rats/group)- 4 rats kept up to 52 days and 3 rats euthanized 2h after last 502 injection for histology tumor/gastrointestinal organs and liver evaluation; 3) 0.2 mg/kg CBLB502 d7-9 (3 doses) following implant of tumors (10 rats/group)- 4 rats kept up to 52 days, 3 rats euthanized 2h after last 502 injection for histology tumor/gastrointestinal organs and liver evaluation; 4) Irinotecan alone (200 mg/kg iv for 3 days) (7 rats)- 4 rats kept up to 52 days (length of study), 3 rats used for histology samples, tumor, liver and organs of the gastrointestinal tract collected Day 4 following first irinotecan injection; 5) 0.2 mg/kg CBLB502 d5-15 (10 doses) following implant of tumors and irinotecan administered starting on 6th day of CBLB502 treatment (200 mg/kg for 3 days) (4 rats)- 4 rats kept up to 52 days (length of study); 6) 0.2 mg/kg CBLB502 d5-9 (5 doses) following implant of tumors and irinotecan (200 mg/kg iv for 3 days) administered starting the 3rd day of CBLB502 administration (7 rats/group)- 4 rats kept up to 52 days (length of study); 3 rats used for histology samples, tumor, liver and organs of the gastrointestinal tract t collected 4th day following first irinotecan injection; and 7) 0.2 mg/kg CBLB502 d7-9 (iv, 3 doses) and irinotecan (200 mg/kg, iv for 3 days) following implant of tumors followed by (7 rats/group)- 4 rats kept up to 52 days (length of study), b. 3 rats used for histology samples, tumor, liver and organs of the gastrointestinal tract collected 4th day following first irinotecan injection.
[00167] Fig. 15 shows the toxicity of irinotecan in Fischer rats bearing Ward colorectal carcinoma with or without CBLB502 (administered before or concurrently). Irinotecan, an agent active in the treatment of colorectal cancer, the dose of 200 mg/kg/dx3 compared with the maximum tolerated dose (100 mg/kg/dx3), was highly detrimental with 100% of rats having the side effect diarrhea and lethality (Fig. 15). All three schedules of CBLB502 offered protection/mitigation against the high levels of diarrhea and lethality induced by irinotecan (Fig. 15).
Example 7 Effects of 5-FU on CBLB502 Mediated Antitumor Immune Response
[00168] Without wishing to be bound by theory, immunosuppression prevents the generation of a durable antitumor immune response and may interfere with effective cancer treatment. Immunosuppression is defined as the expansion of T regulatory cells (Tregs) and myeloid derived suppressor cells (MDSC), which are predominately immature neutrophils called granulocytic MDSC (gMDSC), that directly prevent activation of antitumor T cell-dependent immunity. Thus, elimination of Tregs and MDSC represent a potential strategy to improve anticancer immunotherapy.
[00169] Studies are carried out to evaluate the effectiveness of using chemotherapeutic drugs to enhance TLR5 mediated antitumor immune response. Without wishing to be bound by theory, it is believed that chemotherapeutic agents such as 5-FU may enhance CBLB502 mediated antitumor immune response by reducing tumor-associated immunosuppression and increasing tumor cell immunogenicity.
[00170] A highly tumorigenic and invasive mouse 4T1 mammary carcinoma model was utilized to study whether chemotherapeutic drugs such as 5-FU can reduce immunosuppression and thus allow improved efficacy of CBLB502. The 4T1 model shares many of the characteristics of human breast cancer, particularly its ability to spontaneously metastasize to the lungs and increase the numbers of cells with immunosuppressive phenotypes through production of TGF and generation of MDSCs.
[00171] In mice bearing 4T1 tumors (orthotopic in the mammary fat pad or established in the lungs following i.v. injection), combined treatment with CBLB502 and 5-FU had a much stronger antitumor effect than treatment with either drug alone (Fig. 16, panels A and B). Complete blood count (CBC) analysis showed strong expansion of gMDSC in 4T1 tumor-bearing mice (Fig. 17, panels A and C). Treatment with two different doses of 5-FU (50 mg/kg and 100 mg/kg) reduced gMDSC numbers in the blood of 4T1 tumor-bearing mice. Spleen size reflected gMDSC levels and was significantly smaller in 5-FU-treated 4T1 tumor-bearing mice than in vehicle-treated controls (Figure 17, panel B). Without wishing to be bound by theory, it is believed that the enhanced antitumor efficacy observed upon combination of CBLB502 and 5-FU treatments may be due, at least in part, to 5-FU- mediated reduction of immunosuppressive cells.
Example 8 Effects of Doxorubicin on CBLB502 Mediated Antitumor Immune Response
[00172] Studies were carried out to evaluate the antitumor effects of a combination treatment using CBLB502 and doxorubicin. Doxorubicin is able to eliminate MDSC in breast cancer models (Alizadeh et al 2014) and enhance dendritic cell and CD8 T-cell mediated tumor cell apoptosis by increasing tumor cell immunogenicity. Results showed that treatment of 4T1 tumor-bearing mice with a sub-clinical dose of doxorubicin (2.5 mg/kg) effectively prevented expansion of neutrophils (i.e., gMDSC) while maintaining the constant ratio of neutrophils to lymphocytes for about 7 days following the treatment thereby providing favorable conditions for immunotherapeutic intervention using CBLB502 (Fig. 18).
[00173] Altogether, the results using 5-FU and doxorubicin demonstrate that chemotherapeutic treatment can be combined with TLR5 agonistic agents (e.g., CBLB502) for effective cancer treatment. In particular, chemotherapeutic agents promote anticancer immunotherapy by reducing immunosuppressive cells and enhancing the antitumor effect of CBLB502 when applied in a neoadjuvant setting in respect to immunotherapy. EQUIVALENTS
[00174] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.
[00175] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
INCORPORATION BY REFERENCE
[00176] All patents and publications referenced herein are hereby incorporated by reference in their entireties.
[00177] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
[00178] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.
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The following references are incorporated by reference in their entireties.
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Claims

CLAIMS What is claimed is:
1. A method for treating cancer, comprising administering an effective amount of CBLB502 to a patient undergoing treatment with fluorouracil (5-FU).
2. A method for treating cancer, comprising administering an effective amount of CBLB502 after administering an effective amount of 5-FU to a cancer patient in need thereof.
3. The method of any of the above claims, wherein administering an effective amount of CBLB502 reduces and/or mitigates one or more side effects of 5-FU therapy.
4. The method of claim 3, wherein the side effects of 5-FU therapy are one or more of stomatitis, esophagopharyngitis, leukopenia and/or reduced white blood count, reduced neutrophils, reduced lymphocytes, reduced platelets, increased risk of infection, vomiting, diarrhea, nausea, poor appetite, gastrointestinal ulceration and/or bleeding, thrombocytopenia, hand-foot syndrome, alopecia, mucositis, impaired hepatic function, impaired hepatic function, and hemorrhage.
5. The method of claims 3 or 4, wherein the side effects of 5-FU therapy are predicted and/or assessed by one or more of increased or decreased blood cell counts, elevated aminotransferase levels, elevated plasma ammonia levels, changes in one or more of ALT, AST, alkaline phosphatase and bilirubin, hepatic imaging, genetic tests for one or more the genes DPYD and TYMS, and biopsies.
6. The method of any of the above claims, wherein administering an effective amount of CBLB502 increases a therapeutic window of 5-FU.
7. The method of claim 6, wherein the increased therapeutic window of 5-FU comprises one or more of increasing a cancer patient's likelihood receiving 5-FU maintenance therapy; increasing a cancer patient's likelihood of receiving a complete regime of 5-FU; increasing a cancer patient's likelihood of receiving more than a complete regime of 5-FU; and increasing the dose or length of 5-FU treatment.
8. The method of claim 7, wherein the standard 5-FU treatment regime comprises administering about 12 mg/kg once daily for four successive days and optionally about 6 mg/kg on the sixth, eighth, tenth and twelfth days unless toxicity occurs.
9. The method of any of the above claims, wherein the dose of 5-FU exceeds about 200 mg daily, about 300 mg daily, about 400 mg daily, about 500 mg daily, about 600 mg daily, about 700 mg daily, or about 800 mg daily.
10. The method of any of the above claims, wherein the maintenance therapy comprises dosage of about 12 mg/kg every 30 days after the last day of a previous course of treatment or about 10 to 15 mg/kg/week as a single dose.
11. The method of any of the above claims, wherein the cancer patient has impaired hepatic function.
12. The method of claim 1 1 , wherein the impaired hepatic function is hepatitis.
13. The method of any of the above claims, wherein the cancer patient has a deficiency of dipyrimidine dehydrogenase (DPD) activity.
14. The method of any of the above claims, wherein administering an effective amount of CBLB502 increases the ability of a cancer patient to receive a combination therapy with 5-FU.
15. The method of claim 14, wherein the combination therapy is selected from leucovorin, oxaliplatin, and irinotecan.
16. The method of any of the above claims, wherein the cancer is breast cancer, head and neck cancer, anal cancer, stomach cancer, colon cancer, and skin cancer.
17. The method of any of the above claims, wherein the effective amount of CBLB502 is 0.001 mg to 1000 mg per day, 1 mg to 600 mg per day, or 5 mg to 30 mg per day.
18. A method for treating cancer, comprising administering an effective amount of CBLB502 to a patient undergoing treatment with a topoisomerase inhibitor, optionally selected from irinotecan and doxorubicin.
19. A method for treating cancer, comprising administering an effective amount of CBLB502 sequentially or simultaneously with administering an effective amount of a topoisomerase inhibitor, optionally selected from irinotecan and doxorubicin, to a cancer patient in need thereof.
20. The method of any one of claims 18 or 19, wherein administering an effective amount of CBLB502 protects and/or reduces and/or mitigates one or more side effects of irinotecan therapy.
21. The method of claim 19, wherein the side effect of irinotecan therapy is one or more of diarrhea, dehydration, suppression of the immune system, lowered white blood cell counts, neutropenia, cholinergic syndrome, blood clots, infection, and abdominal pain.
22. The method of claims 20 or 21 , wherein the side effects of irinotecan therapy are predicted and/or assessed by one or more of increased or decreased blood cell counts, elevated plasma ammonia levels, changes in one or more of ALT, AST, alkaline phosphatase and bilirubin, hepatic imaging, and biopsies.
23. The method of any one of claims 18-22, wherein administering an effective amount of CBLB502 increases a therapeutic window of irinotecan.
24. The method of claim 23, wherein the increased therapeutic window of irinotecan comprises one of more of increasing a cancer patient's likelihood receiving irinotecan maintenance therapy; increasing a cancer patient's likelihood of receiving a complete regime of irinotecan; increasing a cancer patient's likelihood of receiving more than a complete regime of irinotecan; and increasing the dose of length of irinotecan treatment.
25. The method of any one of claims 18-24, wherein the dose of irinotecan is greater than about 125 mg/m2.
26. The method of any one of claims 18-24, wherein the dose of irinotecan is greater than about 350 mg/m2.
27. The method of any one of claims 18-26, wherein the cancer patient is classified as a *28 variant.
28. The method of any one of claims 18-27, wherein administering an effective amount of CBLB502 reduces a dose and/or frequency of an anti-diarrheal agent administered to the cancer patient.
29. The method of claim 28, wherein the anti-diarrheal agent is one or more of loperamide, diphenoxylate, and atropine.
30. The method of any one of claims 18-29, wherein administering an effective amount of CBLB502 increases an ability of a cancer patient to receive a combination therapy with irinotecan.
31. The method of claim 30, wherein the combination therapy is one or more of oxaliplatin, 5-fluorouracil, leucovorin, cetuximab, temozolomide, sorafenib, capecitabine, bevacizumab, and vincristine.
32. The method of any one of claims 18-31 , wherein the effective amount of CBLB502 is 0.001 mg to 1000 mg per day, 1 mg to 600 mg per day, or 5 mg to 30 mg per day.
33. The method of any one of claims 18-32, wherein the cancer is one or more of lung cancer, ovarian cancer, non-small cell lung cancer, leukemia, lymphoma, pancreatic tumor, metastatic cancer, breast tumor, rectal tumor, colonic neoplasms, stomach tumor, metastatic cancer colon or rectum and glioma.
PCT/US2015/016850 2014-02-21 2015-02-20 Uses of flagellin for improved chemotherapy WO2015127227A1 (en)

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