COMBINATION THERAPIES FOR THE TREATMENT OF CANCER
20230022045 · 2023-01-26
Assignee
Inventors
Cpc classification
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2039/545
HUMAN NECESSITIES
International classification
A61K39/395
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
Combination therapies for the treatment of cancers are provided. In some embodiments, a flagellin derivative such as CBLB502 is administered in combination with an immune checkpoint therapy (e.g., an anti-PD 1 antibody and an anti-CTLA4 antibody) to treat a cancer in a mammalian subject. In some embodiments, the combination therapy is administered intratumorally or peritumorally.
Claims
1. A method of treating a cancer in a mammalian subject, comprising administering to the subject a therapeutically effective amount of: (i) a TLR5 agonist; and (ii) an immune checkpoint therapy (ICT).
2. The method of claim 1, wherein the TLR5 agonist is flagellin or a flagellin derivative.
3. The method of claim 2, wherein the TLR5 agonist is flagellin, CBLB502, or a CBLB502 derivative.
4. The method of claim 3, wherein the immune checkpoint therapy comprises an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody; an anti-LAG3 antibody, an anti-TIM-3 antibody, an anti-VISTA antibody, an anti-TIGIT antibody, an anti-KIR antibody, an anti-CD47 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an ICOS agonist, an OX40 agonist, and/or an IDO inhibitor.
5. The method of claim 4, wherein the immune checkpoint therapy comprises: (a) an anti-PD1 antibody or an anti-PD-L1 antibody; and (b) an anti-CTLA4 antibody.
6. The method of any one of claims 2-5, wherein the anti-PD1 antibody is Pembrolizumab, Nivolumab, REGN2810, BMS-936558, SHR1210, IBI308, PDR001, BGB-A317, BCD-100, or JS001.
7. The method of any one of claims 2-6, wherein the anti-PDL1 antibody is Avelumab, Atezolizumab, Durvalumab, KN035, MPDL3280A, MEDI4736, or BMS-936559.
8. The method of any claims 2-7, wherein the anti-CTLA4 antibody is Ipilimumab or Tremelimumab.
9. The method of any one of claims 3-8, wherein the CBLB502 is administered to the subject.
10. The method of claim 9, wherein about 5 μg/mL to 150 μg/mL of CBLB502 is administered to the subject.
11. The method of any one of claims 2-10, wherein the administration is intratumoral, peritumoral, intravenous, parenteral, subcutaneous, or intrathecal.
12. The method of claim 11, wherein the administration is intratumoral or peritumoral.
13. The method of claim 3, wherein CBLB502, an anti-PD1 antibody, and an anti-CTLA4 antibody are administered to the subject.
14. The method of claim 13, wherein the administration is intratumoral, peritumoral, intravenous, parenteral, subcutaneous, or intrathecal.
15. The method of claim 14, wherein the administration is intratumoral or peritumoral.
16. The method of any of claims 2-15, wherein the cancer is an ICT-refractory cancer or an ICT-refractory solid tumor.
17. The method of any of claims 2-16, wherein the cancer is a melanoma, a breast cancer, a lung cancer, a prostate cancer, a pancreatic cancer, a head and neck cancer, a liver cancer, an ovarian cancer, a nonpalpable cancer, or a lymphoma.
18. The method of claim 17, wherein the cancer is a melanoma or a breast cancer.
19. The method of claim 18, wherein the breast cancer is triple negative breast cancer.
20. The method of any one of claims 2-18, wherein the subject is a human, dog, cat, horse, or cow.
21. The method of claim 20, wherein the subject is a human.
22. A pharmaceutical composition comprising CBLB502, and an immune checkpoint therapy (ICT), wherein the pharmaceutical composition is formulated for injection, intratumoral administration, or peritumoral administration.
23. The composition of claim 22, wherein the immune checkpoint therapy is an anti-PD1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody.
24. The composition of claim 23, wherein the pharmaceutical composition comprises both an anti-PD1 antibody and an anti-CTLA4 antibody.
25. The composition of claim 23, wherein the pharmaceutical composition comprises both an anti-PD-L1 antibody and an anti-CTLA4 antibody.
26. The composition of claim 23, wherein the pharmaceutical composition comprises an anti-PD1 antibody, an anti-PD1 antibody, and an anti-CTLA4 antibody.
27. The composition of any one of claims 22-26, wherein the anti-PD1 antibody is Pembrolizumab, Nivolumab, REGN2810, BMS-936558, SHR1210, IBI308, PDR001, BGB-A317, BCD-100, or JS001.
28. The composition of any one of claims 22-27, wherein the anti-PDL1 antibody is Avelumab, Atezolizumab, Durvalumab, KN035, MPDL3280A, MEDI4736, or BMS-936559.
29. The composition of any claims 22-28, wherein the anti-CTLA4 antibody is Ipilimumab or Tremelimumab.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings(s) will be provided by the Office upon request and payment of the necessary fee.
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DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0029] The present invention, in some aspects, provides combination therapies for the treatment of cancers. In some aspects, a flagellin (e.g., CBLB502) is administered in combination with an ICT (e.g., antibody inhibitors of CTLA-4, PD-L1, and/or PD-1) to treat a cancer (e.g., a triple negative breast cancer). As shown in the below examples, these combinations may result in the synergistic treatment of cancers that do not respond to the ICT alone. In some embodiments, the ICT (e.g., antibody inhibitors of CTLA-4, PD-L1, and/or PD-1) and flagellin (e.g., CBLB502) are administered intratumorally.
I. Flagellin and Flagellin Derivatives
[0030] Recent evidence indicates that therapies that harness innate immunity show promising antitumor potential (Goldberg and Sondel, 2015). In a number of studies, Salmonella typhimurium, a flagellated facultative intracellular bacteria, induce tumor regression in pre-clinical models (Frahm et al., 2015; Pawelek et al., 2003; Pawelek et al., 1997; Yu et al., 2012; Flentie et al., 2012; Ganai et al., 2009; Forbes, 2010; al-Ramadi et al., 2009). Building on this concept, therapeutic trials with Salmonella species are underway (multiple myeloma trial of orally administered Salmonella based Survivin vaccine. ClinicalTrials.gov Identifier: NCT03762291). Without wishing to be bound by any theory, the therapeutic effects of Salmonella are likely driven by bacteria antigenicity and activation of host immunity-mediated recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) (Flentie et al., 2012; Kawasaki and Kawai, 2014; Rakoff-Nahoum and Medzhitov, 2009; Zheng et al., 2017). Many TLRs agonist have been shown to elicit antitumor activity (Garaude et al., 2012; Lu, 2014; Nguyen et al., 2013; Simone et al., 2009). In particular, treatment with bacterial flagellin, a TLR5 agonist (Hayashi et al., 2001), results in potent antitumor responses in various xenograft models for colon, breast, and prostate cancer as well as a number of mouse spontaneous tumor models (Rhee et al., 2008; Cai et al., 2011; Galli et al., 2010; Sfondrini et al., 2006). Interestingly, higher TLR5 expression levels correlate with enhanced survival in breast, lung, and ovarian cancer patients (Flentie et al., 2018). Although the precise mechanisms of TLR5-mediated antitumor effects remain to be elucidated, it is known that TLR5 mediates innate immune responses against bacterial flagellin (Hayashi et al., 2001), likely through activation of pro-inflammatory pathways, including NF-κB (Rhee et al., 2008; Flentie et al., 2018; Menendez et al., 2011). Thus, it is possible that the antitumor responses are a collateral effect of host immune response to flagellin. Bacterial flagellin has been viewed as a virulence factor that can contribute adhesion and invasion of host cells, but this protein may also function as an immune activator (Hajam et al., 2017). TLR5-mediated immunogenic response has led to the exploration of flagellin-derived reagents suitable for clinical application.
[0031] A. CBLB502
[0032] CBLB502 (Entolimod), is a recombinant flagellin protein fragment derived from Salmonella enterica that can act as a TLR5 agonist and can activate the NF-κB inflammatory response (Burdelya et al., 2008; Zhou et al., 2012). In pre-clinical studies, treatment with CBLB502 showed antitumor and anti-metastatic effects through activation of components of the innate immune system (Leigh et al., 2014; Hossain et al., 2014; Brackett et al., 2016; Yang et al., 2016; Burdelya et al., 2013). Safe systemic administration of CBLB502 has been demonstrated in rodents, non-human primates, and humans (Burdelya et al., 2013; ClinicalTrials.gov Identifier: NCT01527136). As shown in the below examples, in some embodiments, CBLB502 administered in combination with an immune checkpoint therapy (ICT) can be used to treat a cancer and may synergistically interact. The 4T1 breast cancer solid tumor model, a highly aggressive cancer refractory to standard therapies (Lechner et al., 2013; Song et al., 2018), was used in the studies in the examples and provides in vivo evidence that such combinations may be particularly useful for the treatment of cancers that are refractory to other therapies. In some embodiments, CBLB502 and the ICT are administered intratumorally.
[0033] CBLB502 (also referred to as Entolimod) is a toll-like receptor 5 (TLR5) agonist derived from Salmonella flagellin. CBLB502 has displayed some anti-inflammatory effects towards gut mucosal tissues (Xu et al., 2016). CBLB502 is further described in U.S. Pat. No. 10,265,390 and U.S. Pub. No. 2012/0208871, which are incorporated by reference herein in their entirety.
[0034] A variety of dosages of CBLB502 may be administered to a subject. It is anticipated that the therapeutically effective dosage of CBLB502 to produce an anti-cancer effect may be significantly reduced when administered in combination with an ICT. For example, CBLB502 can be administered to a subject, such as a human patient, in combination with an ICT to treat a cancer in the subject, wherein the CBLB502 is in a range of from about 0.001 mg/kg to about 200 mg/kg per day, from about 1 mg/kg to about 100 mg/kg per day, or about 1-50 mg/kg. The dosage may be at any dosage such as about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 275 mg/kg, 300 mg/kg, 325 mg/kg, 350 mg/kg, 375 mg/kg, 400 mg/kg, 425 mg/kg, 450 mg/kg, 475 mg/kg, 500 mg/kg, 525 mg/kg, 550 mg/kg, 575 mg/kg, 600 mg/kg, 625 mg/kg, 650 mg/kg, 675 mg/kg, 700 mg/kg, 725 mg/kg, 750 mg/kg, 775 mg/kg, 800 mg/kg, 825 mg/kg, 850 mg/kg, 875 mg/kg, 900 mg/kg, 925 mg/kg, 950 mg/kg, 975 mg/kg or 1 g/kg. In some preferred embodiments, the CBLB502 is administered intratumorally or peritumorally.
[0035] The therapeutically effective amount required for use in therapy varies with the nature of the condition being treated, the length of time desired to activate TLR activity, and the age/condition of the patient. The desired dose may be conveniently administered (e.g., intratumorally or peritumorally) in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day.
II. IMMUNE CHECKPOINT THERAPIES (ICT)
[0036] Immune checkpoint therapies (ICT), also referred to as immune checkpoint inhibitors (ICIs) or immune checkpoint blockade (ICB), have provided significant improvements in the treatment of cancers. Some cancers can evade immunosurveillance by activation of immune checkpoint pathways that suppress antitumor immune responses. ICT can in many instances promote antitumor immune responses by interrupting co-inhibitory signaling pathways and promote immune-mediated elimination of tumor cells. In some preferred embodiments, flagellin or a flagellin derivative, such as CBLB502, is administered to a mammalian subject to treat a cancer in combination with both (i) an anti-PD1 antibody or an anti-PD-L1 antibody and (ii) an anti-CTLA4 antibody.
[0037] A variety of ICT can be used in various embodiments of the present invention. For example, the ICT may be an anti-CTLA4 antibody, such as for example ipilimumab (Schachter et al., 2017). Ipilimumab has been observed to reduce or prevent T-cell inhibition and promote the activation and proliferation of effector T cells.
[0038] In some embodiments, the ICT is an antibody that targets or selectively binds programmed death-1 (PD-1) or programmed death-ligand 1 (PD-L1). Examples of anti-PD-1 antibodies that can be used in various embodiments include pembrolizumab, nivolumab, and cemiplimab (Larkin, et al. 2015). Immune checkpoints, including PD-1 and CTLA-4, expressed on activated T cells can lead to inhibition of T-cell activation upon binding to their ligands on tumor cells/antigen-presenting cells. These interactions can be blocked using monoclonal antibodies, leading to the activation of T cells targeting tumor cells through the release of effector cytokines and cytotoxic granules (Liakou, et al., 2008) Anti-PD-L1 antibodies that may be used include, e.g., MPDL3280A, MEDI4736, and BMS-936559.
[0039] In some embodiments, the ICT comprises or consists of an anti-PD1 antibody or an anti-PDL1 antibody, such as, e.g., pembrolizumab, nivolumab, cemiplimab, avelumab, atezolizumab, durvalumab, nivolumab, REGN2810, MPDL3280A, MEDI4736, BMS-936558, SHR1210, KN035, IBI308, PDR001, BGB-A317, BCD-100, or JS001. The anti-CTLA-4 antibody may be ipilimumab or tremelimumab. In some preferred embodiments, the ICT comprises administering both: (i) an anti-PD1 antibody or an anti-PDL1 antibody, and (ii) an anti-CTLA-4 antibody to a mammalian subject to treat a cancer. In some embodiments, the ICT may comprise or consist of an anti-LAG3, anti-TIM-3, anti-VISTA, anti-TIGIT, anti-KIR, anti-CD47, anti-B7-H3, or anti-B7-H4 antibodies, ICOS and OX40 agonists; and IDO inhibitors.
[0040] Specific treatment regimens of an ICT that may be administered in combination with CBLB502 include, e.g., treatment doses ranging from 5 μg/day to 150 μg/day daily or every other day to a mammalian subject to treat a cancer, such as for example NSCLC, small cell lung cancer, head and neck squamous cell carcinoma, glioblastoma and other brain tumors, renal cell carcinoma, gastric adenocarcinoma, nasopharyngeal neoplasms, urothelial carcinoma, colorectal cancer, pleural mesothelioma, breast cancer, TNBC, esophageal neoplasms, multiple myeloma, gastric and gastroesophageal junction cancer, gastric adenocarcinoma, melanoma, Hodgkin lymphoma, hepatocellular carcinoma, lung cancer, mesothelioma, non-Hodgkin lymphoma, ovarian cancer, fallopian tube cancer, peritoneal neoplasms, or a solid malignancy. The ICT may be an OX40 inhibitor or an IDO inhibitors, a B7H3 inhibitor, or a B7H4 inhibitor.
III. PHARMACEUTICAL PREPARATIONS
[0041] Pharmaceutical compositions of the present invention comprise an effective amount of a flagellin (e.g., CBLB5020) and/or an ICT, or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. In some embodiments, the CBLB502 and ICT are comprised in separate pharmaceutical preparations. Nonetheless, in some embodiments, it is anticipated that a flagellin (e.g., CBLB5020) and an ICT can be formulated in the same pharmaceutical preparation. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one of CBLB502 and/or an ICT will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 2.sup.nd Ed., Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should typically meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
[0042] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
[0043] The flagellin (e.g., CBLB5020) and/or ICT may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The composition of the present disclosure can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
[0044] The flagellin (e.g., CBLB5020) and/or ICT may be provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein, its use in administrable composition for use in practicing the methods of the present disclosure is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
[0045] The composition can be combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
[0046] In a specific embodiment, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
[0047] In further embodiments, a pharmaceutical lipid vehicle can comprise the flagellin (e.g., CBLB5020) and/or ICT, one or more lipids, and an aqueous solvent. As used herein, the term “lipid” will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term “lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present disclosure.
[0048] The actual dosage amount of a composition of the present disclosure administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
[0049] In some embodiments, a flagellin (e.g., CBLB5020) and/or an ICT are formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may, e.g., be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablet.
[0050] A flagellin (e.g., CBLB5020) and/or ICT can be included a liquid formulations such as aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The agents may also be formulated as a dry product for constitution with water or other suitable vehicle before use. Liquid preparations may contain additives such as suspending agents, emulsifying agents, nonaqueous vehicles and preservatives. Suspending agent may be sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents that can be used include lecithin, sorbitan monooleate, and acacia. Nonaqueous vehicles that can be used include edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives such as methyl or propyl p-hydroxybenzoate and sorbic acid can be included in the formulations.
[0051] Agents provided herein may also be formulated for parenteral administration such as by injection, intratumor injection, peritumoral injection, or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents. The agent may also be provided in a powder form for reconstitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water.
[0052] As used herein, the term “parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intratumorally, peritumorally, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).
[0053] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, intratumoral, peritumoral, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Ed., pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
[0054] The flagellin (e.g., CBLB5020) and/or ICT can be formulated as a depot preparation, which may be administered by implantation or by intratumoral or peritumoral injection. The agents may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).
[0055] In some embodiments, the flagellin (e.g., CBLB5020) and/or ICT may be formulated for administration via various miscellaneous routes, for example, topical or transdermal administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
IV. CANCERS
[0056] The therapies as described herein (e.g., CBLB502 in combination with an ICT, such as an anti-PD1 antibody and an anti-CTLA4 antibody) can be used to treat a variety of cancers. For example, in some embodiments, the cancer is NSCLC, small cell lung cancer, head and neck squamous cell carcinoma, glioblastoma, brain tumors, renal cell carcinoma, gastric adenocarcinoma, nasopharyngeal neoplasms, urothelial carcinoma, colorectal cancer, pleural mesothelioma, breast cancer, TNBC, esophageal neoplasms, multiple myeloma, gastric and gastroesophageal junction cancer, gastric adenocarcinoma, melanoma, Hodgkin lymphoma, hepatocellular carcinoma, lung cancer, mesothelioma, non-Hodgkin lymphoma, ovarian cancer, fallopian tube cancer, peritoneal neoplasms, or a solid malignancy.
V. EXAMPLES
[0057] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
TLR5 Agonists Enhance Anti-Tumor Immunity and Overcome Therapy Resistance to Checkpoint Blockade
In Vitro Characterization of 4T1 Tumor Cell Line
[0058] In Vitro NF-κB Activation of 4T1 Cells
[0059] To evaluate flagella- and CBLB502-mediated NF-κB activation of 4T1 mammary carcinoma cells, 4T1 cells we stably transfected with a κB5:IκBa-FLuc transcriptional reporter comprised of a concatenated κB5 promoter region, followed by the bioluminescent IκBa-FLuc fusion reporter gene (Gross and Piwnica-Worms, 2005; Moss et al., 2008). This reporter provides a readout of endogenous ligand-induced IκB degradation and production of new IκB
-FLuc fusion protein (Moss et al., 2008). In the cytoplasm, IκB
sequesters and inactivates NF-κB dimers. The binding of flagellin (or CBLB502) to TLR5 on the cell surface initiates TKK-mediated kinase activity, and the subsequent phosphorylation, ubiquination and targeting for proteasomal degradation of endogenous IκB
as well as the reporter fusion protein (Flentie et al., 2018; Moss et al., 2008). This resulted in a reduction of bioluminescent activity during the first 100 minutes in flagellin-treated cultures (
[0060] In Vitro Cytokine Profile of 4T1 Cells
[0061] CBLB502 activation of NF-κB pro-inflammatory signaling is mediated through TLR5 (Burdelya et al., 2008), a known activator of the innate immune system (Hayashi et al., 2001). Given that CLB502 is a potent activator of the NF-κB signaling in 4T1 carcinoma cells (
TABLE-US-00001 TABLE 1 In vitro Cytokine Profile Cytokine Vehicle (PBS)* CBLB502 (1 μg/mL)* Fold Difference Axl 254 258 1.0 BCL 158 62 0.4 CD30 Ligand 78 67 0.9 CD30 40 35 0.9 CD40 80 65 0.8 CFG-2 116 214 1.8 CTACK 899 1,000 1.1 CXCL16 466 1,330 2.8 Eotaxin-1 30 239 8.1 Eotaxin-2 33 82 2.5 Fas Ligand 134 164 1.2 Fractalkine 2,270 2,440 1.1 G-CSF 1,670 4,260 2.6 GM-CSF 247 228 0.9 IFN-γ 78 74 1.0 IGFBP-3 983 1,190 1.2 IGFBP-5 110 396 3.6 IGFBP-6 194 489 2.5 IL-1α 880 1,150 1.3 IL-1β 136 279 2.1 IL-2 106 283 2.7 IL-3 28 308 11 IL-3 RB 37 319 8.7 IL-4 905 728 0.8 IL-5 126 124 1.0 IL-6 144 83 0.6 IL-9 227 209 0.9 IL-10 41 120 2.9 IL-12 p40/p70 57 324 5.7 IL-12 p70 746 1230 1.7 IL-13 178 610 3.4 IL-17A 255 530 2.1 CXCL1 4,100 10,600 2.6 Leptin R 154 692 4.5 Leptin 102 870 8.6 CXCL5 3,080 8,880 2.9 L-Selectin 9 370 40 XCL1 168 328 2.0 CCL2 975 6,390 6.6 CCL12 325 429 1.3 M-CSF 752 1,139 1.5 CXCL9 88 229 2.6 CCL3 52 244 4.6 CCL9 93 726 7.8 CXCL2 831 1,820 2.2 CCL19 200 608 3.0 CCL20 252 1,910 7.6 CXCL4 611 1020 1.7 P-Selectin 508 703 1.4 CCL5 1,550 3,130 2.0 SCF 1 176 180 CXCL12 141 74 0.5 CCL17 237 251 1.1 I-309 963 1,020 1.1 CCL25 170 233 1.4 TIMP-1 804 949 1.2 TNFα 93 255 2.7 TNF RI 191 582 3.0 TNF RII 237 620 2.6 TPO 76 270 3.5 VCAM-1 382 1,100 2.9 VEGF-A 12 73 6.3 *Normalized densitometry
[0062] Treatment of ICT Refractory 4T1 Carcinomas
[0063] Next, the inventors investigated whether administration of flagellin or CBLB502 could elicit antitumor responses in a syngeneic triple negative breast cancer 4T1 tumor model in vivo. Mammary cell carcinomas were generated in BALB/c mice (5-6 weeks old) by orthotopic injection of 4T1 FUGW-FL tumor cells into the right fourth mammary fat pad. Tumor progression of each mouse was assessed weekly using bioluminescence imaging and caliper measurements of tumor volume (
TABLE-US-00002 TABLE 2 Treatment Doses and Delivery Routes Initial dose and Subsequent dose and Treatment delivery site delivery site Flagellin 10 μg i.t. or i.p. 2 μg (8x) i.t. or i.p. CBLB502 High Dose 10 μg i.t. or i.p. 2 μg (8x) i.t. or i.p. CBLB502 Low Dose 1 μg i.t. or i.p. 0.5 μg (8x) i.t. or i.p. anti-CTLA-4 (9D9) 200 μg i.p. 100 μg i.p. (3x) anti-PD-1 (RPM1-1) 200 μg i.p. 100 μg i.p. (3x)
TABLE-US-00003 TABLE 3 Murine 4T1 Carcinoma Experiments. 4T1 Murine Carcinoma Experiments Innate immune activating treatment ICT (i.p.) Experiment 1 Flagellin (i.t.) With or without Experiment 2 Flagellin (i.t.) CBLB502 With or High Dose (i.t.) without Experiment 3 Flagellin (i.t.) CBLB502 CBLB502 With or High Dose (i.t.) Low Dose (i.t.) without Experiment 4 Flagellin (i.t.) CBLB502 CBLB502 With or High Dose (i.t.) Low Dose (i.t.) without Experiment 5 CBLB502 CBLB502 With or Low Dose (i.t.) Low Dose (i.p.) without Experiment 6 CBLB502 CBLB502 With or Low Dose (i.t.) Low Dose (i.p.) without Experiment 7 No Treatment No Treatment No Treatment No Treatment No (Re-challenge Treatment experiment)
Intratumoral Injection (i.t.); Intraperitoneal Injection (i.p.).
[0064] Flagellin Treatment
[0065] The overall survival combined from four independent experiments is shown in
[0066] CBLB502 Treatment
[0067] Given that CBLB502 showed greater potency and efficacy for activation of NF-κB signaling than flagellin (
[0068] A higher intratumoral CBLB502 dose (CBLB502 high dose) was tested, which is comparable to the dose administered to mice in the flagellin treatment cohort (Table 2). In three independent experiments (Table 3), treatment with CBLB502 (high dose) resulted in one tumor free mouse in the CBLB502 (high dose) only treatment (n=15) with no survivors in any of the other treatments (vehicle (n=19), ICT (n=17), CBLB502 (high dose)+ICT (n=15)) (
[0069] Whether systemic delivery of CBLB502 (low dose) via intraperitoneal (i.p.) injections could elicit a similar response to intra-tumoral delivery of CBLB502 (low dose) was tested. It was observed in two independent experiments (Table 3) that i.p. delivered CBLB502 (low dose)+ICT treatment resulted in 10% long-term survivors (n=20) (
Tumor Re-Challenge Experiments and Immune Memory
[0070] Mice that showed complete tumor regression were re-challenged by orthotopic injections of 4T1 FUGW-FL cells into the opposite (left) fourth mammary fat pad without any additional therapy.
TABLE-US-00004 TABLE 4 Re-challenge Experiment Time Alive Post-tumor Outcome Implantation (Post-re- Original cohort Mouse I.D. (weeks) challenge) Tumor-Naïve FIG. 6B-Naïve Mouse 1 51* Dead W6 Tumor-Naïve FIG. 6B-Naïve Mouse 2 51* Dead W6 Tumor-Naïve FIG. 6B-Naïve Mouse 3 51* Dead W6 Tumor-Naïve FIG. 6B-Naïve Mouse 4 51* Dead W5 Tumor-Naïve FIG. 6B-Naïve Mouse 5 51* Dead W5 Tumor-Naïve FIG. 6B-Naïve Mouse 6 22* Dead W6 Tumor-Naïve FIG. 6B-Naïve Mouse 7 22* Dead W6 Tumor-Naïve FIG. 6B-Naïve Mouse 8 22* Dead W4 Tumor-Naïve FIG. 6B-Naïve Mouse 9 22* Dead W6 Tumor-Naïve FIG. 6B-Naïve 22* Dead W6 Mouse 10 Tumor-Naïve FIG. 6B-Naïve 18* Dead W6 Mouse 11 Tumor-Naïve FIG. 6B-Naïve 18* Dead W6 Mouse 12 Tumor-Naïve FIG. 6B-Naïve 18* Dead W6 Mouse 13 Tumor-Naïve FIG. 6B-Naïve 18* Dead W6 Mouse 14 Vehicle FIG. 5A-Mouse 25 18 Alive Flagellin FIG. 3C-Mouse 13 40 Alive CBLB502 high dose FIG. 10C-Mouse 5 51 Dead W7 ICT FIG. 5B-Mouse 23 22 Dead W6 ICT FIG. 12B-Mouse 3 18 Alive Flagellin (i.t.) + ICT FIG. 3D-Mouse 2 54 Alive Flagellin (i.t.) + ICT FIG. 3D-Mouse 6 51 Alive Flagellin (i.t.) + ICT FIG. 3D-Mouse 7 51 Alive CBLB502 low dose FIG. 5D-Mouse 1 40 Dead W6 (i.t.) + ICT CBLB502 low dose FIG. 5D-Mouse 4 40 Alive (i.t.) + ICT CBLB502 low dose FIG. 5D-Mouse 10 38 Alive (i.t.) + ICT CBLB502 low dose FIG. 5D-Mouse 19 22 Alive (i.t.) + ICT CBLB502 low dose FIG. 5D-Mouse 22 18 Alive (i.t.) + ICT CBLB502 low dose FIG. 5D-Mouse 26 18 Alive (i.t.) + ICT CBLB502 low dose FIG. 12D-Mouse 1 22 Alive (i.t.) + ICT CBLB502 low dose FIG. 12D-Mouse 5 22 Alive (i.t.) + ICT *Tumor-naïve mice are age-matched mice that were part of the original cohort of mice, but were not implanted with tumor cells during the original experiments, W = week.
In Vivo Cytokine Profile
[0071] Characterization of Peripheral Blood Cytokine Profile
[0072] To begin to explore the mechanisms of response and characterize changes elicited by ICT and CBLB502 therapies alone or in combination, the inventors assayed 32 peripheral blood-borne cytokines from aged matched tumor-free mice (healthy mice) and 4T1 FUGW-FL tumor-bearing mice under the treatment cohorts: tumor-bearing, vehicle control; tumor-bearing treatment failure; and tumor-bearing, treatment responders, during weeks 5 to 7 (
TABLE-US-00005 TABLE 5 In vivo cytokine profile: treatment response. Naïve Tumor-free Vehicle Fold Failed Fold Long-term Fold mice Control Change Treatment Change survivors change Cytokine (pg/mL) (pg/mL) Veh. (pg/mL) Failed (pg/mL) Survivors G-CSF 524 7,750 15 12,100 23 161 0.3 GM-CSF 53 Eotaxin 805 308 0.4 407 0.5 607 0.8 IL-13 23 217 9 IL-1α 1,900 215 0.1 3,720 5.0 3,420 2 IL-1β 504 35 0.1 IL-2 75 IL-3 14 IL-4 252 12 0.05 IL-5 239 11 0.05 11 0.05 IL-6 601 8 0.01 43 0.1 178 0.3 IL-7 928 148 IL-9 247 142 0.6 223 0.9 786 3 IFN-γ 24 IL-10 1,450 134 0.1 23 0.02 IL-12 p40 3,700 IL-12 p70 2,340 114 0.05 55 0.02 LIF 67 61 CXCL5 13,600 9,300 0.7 11,100 0.8 10,200 0.7 IL-15 148 3,790 26 2,230 15 IL-17 58 5 0.1 CXCL10 194 370 2 417 2 186 1 CXCL1 64 7 0.1 28 0.4 103 2 CCL2 412 12 0.03 52 0.1 60 0.1 CCL3 39 189 5 CCL4 31 84 3 56 2 58 2 M-CSF 6 3,020 536 3,170 561 CXCL2 157 103 0.7 2,700 17 2,890 18 CXCL9 145 472 3 427 3 269 2 CCL5 103 11 0.1 23 0.2 20 0.2 VEGF 12 TNF-α 642 9 0.01 Cytokines highlighted in Bold showed higher levels in responsive mice compared to mice that failed therapies.
[0073] Characterization of Peripheral Blood Cytokine Profile of Re-Challenged Mice
[0074] Tumor-re-challenged and tumor-naïve mice were assayed three weeks post 4T1 FUGW-FL implantation. Mice that were tumor-free for at least 60 weeks post re-challenge (tumor survivor, re-challenge survivor) revealed a distinctive cytokine profile from those mice that were re-challenged, but developed tumors (tumor survivor, re-challenge failure) (
TABLE-US-00006 TABLE 6 In vivo cytokine profile: re-challenge experiment. Tumor- Failed Fold-change Long-term Fold-change naïve survivors Failed survivors Long-term Cytokine (pg/mL) (pg/mL) survivors (pg/mL) survivors G-CSF 10,100 10,000 1.0 540 0.1 GM-CSF 490 400 0.8 1,100 2 Eotaxin 44 130 3 1,200 27 IL-13 16 22 1 848 53 IL-1α 870 3,400 4 19,000 22 IL-1β 58 170 3 360 6 IL-2 7 46 7 1,800 260 IL-3 20 7 0.4 14 0.7 IL-4 2 41 18 81 35 IL-5 98 30 0.3 50 0.5 IL-6 65 44 0.7 120 2 IL-7 3 310 120 11,000 4,400 IL-9 460 1,900 4 3,400 7 IFN-γ 110 130 1 930 8 IL-10 38 2,100 55 8,000 213 IL-12 p40 451 195 0.4 1,000 2 IL-12 p70 5 51 10 1,200 250 LIF 38 180 5 16,000 420 CXCL5 11,000 11,000 1.0 8,900 0.8 IL-15 34 5,800 170 650,000 19,000 IL-17 34 52 2 32 0.9 CXCL10 450 140 0.3 210 0.5 CXCL1 31 77 3 3,800 120 CCL2 120 240 2 260 2 CCL3 92 240 3 1,800 20 CCL4 38 94 3 2,200 57 M-CSF 47 1,900 40 2,900 61 CXCL2 260 2,200 8 4,500 17 CXCL9 220 250 1 2,600 12 CCL5 38 45 1 120 3 VEGF 1 3 3 620 590 TNF-α 35 16 0.5 62 2 Cytokines highlighted in Bold showed higher levels in long-term survivor mice mice that failed therapies.
[0075] Treatment of ICT Refractory B16-F10 Melanoma Tumor
[0076] We further investigated whether combination treatment of CBLB502 and ICT could also elicit antitumor responses in a poorly immunogenic tumor such as B16-F10 melanoma tumor model. Melanoma tumors were generated in C57BL/6J (6-9 weeks old) by subcutaneous injection of B16-F10 tumor cells into the right dorsal flank. Three days after tumor implantation, mice were randomized into four different treatment controls: vehicle control, ICT (anti-PD-1 and anti-CTLA-4), CBLB502 treatment, and CBLB502 in combination with ICT treatment at the indicated dose and delivery method (Table 7). Tumor progression of each mouse was assessed bi-weekly using caliper measurements of tumor volume (
TABLE-US-00007 TABLE 7 B16-F10 Melanoma Experiments B16-F10 Vehicle Innate immune Melanoma Control activating Experiments (i.t. and i.p) treatment control ICT (i.p.) Experiment 10 PBS CBLB502 (i.t) Only with ICT Experiment 13 PBS CBLB502 (i.t) With or without Experiment 15 PBS CBLB502 (i.t.) With or without Experiment 19 PBS CBLB502 (i.t.) With or without
[0077] Intratumoral Injection (i.t.); Intraperitoneal Injection (i.p.).
[0078] The overall survival combined from four independent experiments is shown in
[0079] 4T1 mammary carcinoma is a robust murine model to study human triple negative breast cancer, which is highly invasive, metastatic and resistant to immune check point therapies (Dexter et al., 1978; Aslakson and Miller, 1992). Herein it was observed that: 1) the successful treatment of established ICT-refractory murine 4T1 mammary carcinoma through the combination of standard ICT treatment plus potent innate immune activating TLR5 agonists, 2) immune-related treatments elicited immune memory against tumor antigens in most long-term survivors, 3) systemic cytokine profiles implicated engagement of both innate and adaptive immunity in response to treatment, and 4) the data supports the idea that G-CSF may function as a bio-marker for positive response to treatment.
[0080] These support the approach of using a combination of these therapeutics to harnesses both innate and adaptive components of the immune system to elicit a lasting antitumor response. On the one hand, bacterial derived-flagellin has been shown to elicit a targeted antitumor response by binding to TLR5 on the tumor surface, initiating a cascade of signals that produce a pro-inflammatory response via activation of the transcription factor NF-kB (Flentie et al., 2018). The results provided herein show that in vitro CBLB502, a potent activator of the NF-kB signaling pathways, was sufficient to elicit a TLR5-mediated immunogenic cytokine response in tumor cells. Given that deficiencies in antigen presentation underlie many mechanisms of resistance against immune checkpoint therapies, it is possible to hypothesize that a potent activator of innate immunity may modulate tumor homeostasis, shifting the tumor microenvironment state from immune suppression to immune activation. Several lines of evidence support this model. First, only treatment with either flagella or CBLB502 in combination with ICT increased survival in mice bearing highly ICT-refractory 4T1 tumors, whereas monotherapies of flagellin, CBLB502, or ICT did not show significant curative effects. Second, the peripheral blood cytokine profiles of mice that responded to treatment and showed complete tumor regression reflected a concerted antitumor response, with IL-13, IL-9, CCL3 and CXCL1 showing higher levels in mice that responded to treatment. Third, nearly all survivor mice that were re-challenged with the same tumor rejected it, implying an adaptive response against tumor cells. Fourth, the peripheral cytokine profiles of those mice that were re-challenged and rejected the tumor aligned with a strong adaptive immune-activating response. While it has been shown that mice bearing tumor cells lacking TLR5 fail to respond to treatment with flagellin (Rhee et al., 2008), another study suggests that antitumor effects are mediated by TLR5 agonists acting on immune cells (Geng et al., 2015). It remains to be studied whether flagella and CBLB502 act on the tumor cells or on immune cells to elicit a curative immune response in the context of combination treatment with ICT.
[0081] Finally, the remarkable increase of blood-borne G-CSF protein levels in tumor-bearing mice that either failed treatment or served as tumor-bearing untreated controls suggested that G-CSF levels may be explored as a potential biomarker. Conversely, low serum level of G-CSF in tumor-bearing mice that responded to treatment or developed long-term tumor immunity suggested utility as a predictive marker for treatment response. Moreover, these results further raise caution against the use of G-CSF to prevent neutropenia in cancer patients. Although a recent meta-analysis study showed some benefit of supportive G-CSF therapy in overall survival of patients receiving chemotherapy, data also show an increased risk of developing secondary malignancies (Lyman et al., 2018). G-CSF therapy in the context of ICT remain to be explored.
[0082] Additional studies demonstrated that CBLB502 in combination with an anti-PDL1 therapy improved responses in an immunotherapy-resistant model of triple negative breast cancer. Experiments were performed as follows: 10,000 4T1 EF1a luc FUGW-FL were implanted via Orthotopic mammary fat pad injection into 6 weeks old female Balb/c mice. After two weeks of tumor growth mice were treated for two weeks with: 1. Vehicle (n=3), 2. α-PD-L1 (n=6), 3. α-PD-L1+CBLB502 (n=10), 4. α-PD-L1+α-CTLA-4+CBLB502 (n=6). Dosing for each treatment was as follows: CBLB502—Initial (i) 1 μg/mouse follow by 200 ng/mouse every other day; Immune Checkpoint Therapy—Initial dose (i) of 200 μg/mouse per Ab (α-PD-L1 alone, or PD-L1 and α-CTLA-4) followed by 100 μg/mouse on days 17, 19 and 22 post initial dose. Antibodies InVivoMAb anti-mouse PD-L1 (B7-H1) (Cat. #BE0101, Clone: 10F.9G2), InVivoPlus anti-mouse CTLA-4 (CD152) (Cat. #BP0164, Clone: 9D9), and InVivoPlus anti-mouse PD-1 (CD279) (Cat. #BP0146, Clone: RMP1-14), all from BioXCell (Lebanon, N.H.) were used. The timing of the experiment, including tumor implantation and subsequent administration of therapeutic agents or vehicle (control group) are shown in
[0083] As shown in
[0084] In conclusion, these success of immune checkpoint therapy in eliciting long lasting curative responses against various types of cancers in subsets of patients make worthwhile efforts to expand the number of patients that respond to this type of treatment. The results provided herein support innate immune activators of TLR5, such as flagellin and CBLB502, in combination with immune checkpoint therapies may be used to treat cancers in vivo, and may beneficial for the treatment of previously unresponsive patients.
Example 2
Materials and Methods
[0085] Reagents
[0086] Salmonella typhimurium flagellin (FLA-ST) was purchased from Invivogen. CBLB502 was a gift from Cleveland Biolabs, Inc. Monoclonal antibodies 9D9 (anti-CTLA-4) and RPM1-14 (anti-PD-1) were purchased from BioX Cell and maintained in 6.5 mg/mL and 6.7 mg/mL stocks, respectively, and stored at 4° C. before use. d-luciferin (d-Luc) (BioGold), the substrate for firefly luciferase, was maintained in a 30 mg/mL solution of phosphate-buffered saline (PBS). Matrigel was obtain from Corning and maintain at −20° C.
[0087] Creation of 4T1 κB.sub.5:IκBα-FLuc-Expressing Cell Line for In Vitro Study
[0088] 4T1 mammary carcinoma cells (ATCC) at 95% confluency were co-transfected with 10 μg of pκB.sub.5:IκBα-FLuc (Moss et al., 2012) and 3 μg of pIRES-puro plasmid DNA using Fugene 6 (Roche) in 10 cm dishes (BD Bioscience). After 24 hours, the media was replaced with fresh RPMI supplemented with 10% heat-inactivated FBS media. 24 hours later, cells were split at multiple dilutions into media containing 0.5 μg/ml puromycin to select for stable transformants. After two weeks, isolated cell colonies were imaged to confirm reporter gene expression and bioluminescent colonies were harvested and expanded. Reporter cells were continuously cultured in the presence of 0.5 μg/ml puromycin to maintain expression of the reporter plasmid.
[0089] In Vivo Study Cell Lines
[0090] 4T1 mammary carcinoma cells were stably transfected with the EF1α:FLuc plasmid producing a constitutive florescent and bioluminescent dual imaging reporter cell line (4T1 FUGW-FL) (Luker et al. 2004). Cells were cultured according to ATCC protocols and kept under selection with 0.5 μg/ml puromycin. B16-F10 parental cells were cultured according to ATCC protocols (Fidler I J, 1975).
[0091] In Cellulo Analysis of NF-κB Signaling
[0092] 4T1 κB.sub.5:IκBα-FLuc reporter cells (7,000 cells) were added to a 96-well plate and incubated overnight at 37° C. One hour prior to imaging, cell media were aspirated and replaced with RPMI with L-Glutamate (4T1 cells) supplemented with 10% heat-inactivated FBS and 150 μg/ml d-luciferin (BioGold). Cells were imaged in an IVIS 100 imaging system, with images being acquired every 5 minutes for 4 hours, unless otherwise indicated. Cells were maintained in the imaging chamber by a heated stage (37° C.) and 5% CO.sub.2 air flow. Acquisition parameters were: acquisition time, 60 sec; binning, 4-8; filter, open; f stop, 1; FOV, 12-23 cm. Stimuli included: TNFα (20 ng/ml) (R & D systems); flagellin (various concentrations ranging from 1 μg/mL to 0.1 ng/mL); CBLB502 (various concentrations ranging from 1 μg/mL to 0.1 ng/mL); and nuclease-free water (vector only control) added to triplicate wells. Bioluminescence photon flux data (photons/sec) represent the mean of triplicate wells for the indicated number of independent experiments, and were analyzed by region of interest (ROI) measurements with Living Image 3.2 (Caliper Life Sciences). Data were imported into Excel (Microsoft Corp.), averaged, and normalized to both initial (t=0) values (fold-initial) and vehicle-treated controls (fold-vehicle) for presentation in dynamic plots (Gross and Piwnica-Worms, 2005). The normalized results from repeated experiments were averaged for each time point, and the results graphed as normalized photon flux versus time, with the y-axis on a log 2 scale. Positive error bars present standard error of the mean for repeated experiments.
[0093] Mice
[0094] All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas M.D. Anderson Cancer Center, protocol 00001179-RN01. Female BALB/c mice (4 weeks old) were purchased from The Jackson Laboratory. Animals were allowed at least one week to acclimate to the animal facility before start of experiments. Age-matched control groups were used where indicated.
[0095] Mouse Endpoint Protocol
[0096] Mice that reached end point (moribund condition or having one tumor measurement in the sagittal or axial plane greater than 1.5 cm) were euthanized according to University of Texas M.D. Anderson Cancer Center IACUC euthanasia protocols.
[0097] Allograft Model of Human Breast Cancer
[0098] A mammary cell carcinoma allograft was established in each BALB/c mouse (5-6 week old mice) by orthotopic injection into the fourth mammary fat pad of approximately 10,000 4T1 FUGW-FL cells mixed with Matrigel at a 2:1 ratio. The total volume injected into each mouse was 30 μL.
[0099] Administration of Flagellin, CBLB502, and Immune Checkpoint Therapy for Allograph for Breast Cancer Carcinoma Model
[0100] Flagellin, CBLB502, 9D9 (anti-CTLA-4), and RPM1-14 (anti-PD-1) were suspended in filtered PBS; filtered PBS was used as a vehicle control. Two weeks post orthotopic injection of 4T1 FUGW-FL cells into the mammary fat pad, each mouse was randomly sorted into groups receiving vehicle control (n=40, total), or treatment with flagellin only (n=22), CBLB502 only (n=30), ICT only (9D9 plus RPM1-1) (n=37), flagellin combined with ICT or CBLB502 combined with ICT (n=52). Flagellin or CBLB502 was administered every two days for two weeks. On the first day of treatment, 10 μg of flagellin solution in 50 μL (PBS), or 10 μg (high dose) or 1 μg (low dose) of CBLB502 solution in 50 μL (PBS) were administered as an intra-tumoral or intraperitoneal injection into designated animals. For each subsequent treatment, 2 μg of flagellin solution in 50 μL (PBS) or 2 μg (high dose) or 0.5 μg (low dose) of CBLB502 solution in 50 μL (PBS) was used. ICT was administered on days 1, 3, 5, and 8 of treatment. On the first day, mice receiving ICT treatment were injected intraperitoneally with 200 μg in 100 μL of both 9D9 and RPM1-14 (200 μL total per mouse). On subsequent days, each mouse was injected with 100 μg in 100 μL of each antibody. On days when both flagellin and ICT were administered, vehicle control mice were given two intraperitoneal injections of 100 μL PBS and one intra-tumoral injection of 50 μL PBS. On days when only flagellin was administered, vehicle mice received only 50 μL PBS intra-tumoral injection, and on the day that only ICT was administered, they received only two 100 μL intraperitoneal injections of PBS.
[0101] In Vivo Bioluminescence Imaging
[0102] The mice were imaged using the PerkinElmer IVIS Spectrum Imaging System weekly beginning one week after orthotopic injection of 4T1 FUGW-FL cells into the mammary fat pad. The mice were weighed at the beginning of each imaging session, and 165 μg d-luciferin (prepared at 30 mg/mL in PBS) was injected intraperitoneally per gram of mouse. Mice were imaged ten minutes after injection with d-luciferin (Gross and Piwnica-Worms, 2005).
[0103] Allograft Model of Melanoma Tumor
[0104] A melanoma tumor was established in each C57BL/6J mouse (Jackson Laboratory, 6-9 week old mice) by subcutaneous injection into the right dorsal flank of approximately 12,000 B16-F10 cells. The total volume injected into each mouse was 50 μL of cells resuspend in RPMI 1640 with L-glutamine media (Millipore Sigma).
[0105] Administration of CBLB502 and Immune Checkpoint Therapy Treatments for Melanoma Tumor Model
[0106] Three days post subcutaneous injection of B16-F10 cells into the posterior right flank, each mouse was randomly sorted into a group receiving treatment with vehicle control, CBLB502 only, ICT only (9D9 plus RPM1-1), or CBLB502 combined with ICT. CBLB502, 9D9, and RPM1-14 were suspended in filtered PBS, and filtered PBS was used as a vehicle control. CBLB502 was administered every two days for two weeks. On the first day of treatment, 1 μg in 50 μL of CBLB502 was administered as an intra-tumoral injection into designated animals. For each subsequent treatment, 500 ng in 50 μL of CBLB502 was used. ICT was administered on days 1, 3, 5, and 8 of treatment. On the first day, mice receiving ICT treatment were injected intraperitoneally with 200 μg in 100 μL of both 9D9 and RPM1-14 (200 μL total per mouse). On subsequent treatment days, each mouse was injected with 100 μg in 100 μL of each antibody. On days when both CBLB502 and ICT were administered, vehicle control mice were given an intraperitoneal injections of 200 μL PBS and one intra-tumoral injection of 50 μL PBS. On days when only CBLB502 was administered, vehicle mice received only 50 μL PBS intra-tumoral injection, and on the day that only ICT was administered, they received only a 200 μL intraperitoneal injections of PBS.
[0107] Caliper Measurement of Tumor Volume
[0108] Tumor volume was determined by measuring length (1, longest measurement) and width (w) for each tumor at least once a week by caliper, using the standard triangular prism formula for volume: V=(l×w.sup.2)/2.
[0109] In Vitro Cytokine Profile
[0110] 4T1 FUGW-FL florescent and bioluminescent dual reporter cells were plated in 100 mm tissue culture plates (BD) (750,000 cells per plate) and incubated overnight at 37° C. with RPMI supplemented with 10% heat-inactivated FBS. On day two, cell media were aspirated and replaced with RPMI without 10% heat-inactivated FBS. On day three, cultures were treated with either CBLB502 at 1 μg/ml or PBS as vector control in triplicates. On day four, media was collected into 15 ml tubes, centrifuge at 2,000 rpm at 4° C. for 10 minutes. The supernatant was assayed using Mouse Cytokine Antibody Array C series 1000 (RayBiotech).
[0111] Serum Collection
[0112] Serum from mice was obtained by mandibular bleeding once a week for the duration of the experiment. Samples were allowed to clot at room temperature for 1 hour, centrifuge at 2,000 g for 10 minutes at room temperature and sera (upper phase) was collected into Eppendorf tubes. All serum samples were stored at −80° C.
[0113] Luminex Multiplex Quantitative Analysis
[0114] Serum samples were analyzed at the Antibody-Based Proteomics Core at Baylor College of Medicine, Houston, Tex. The core used the Milliplex Mouse 32-Plex Cytokine Panel (Millipore), which included the following cytokines: G-CSF, GM-CSF, IFN-7, IL-1α, IL-10, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, CXCL10, CXCL-1-like, LIF, CXCL5, CCL2, M-CSF, CXCL9, CCL3, CCL4, CXCL2, CCL5, TNF-α, VEGF and appropriate controls and calibration standards.
[0115] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
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