Compositions comprising chrna7 agonists and immune checkpoint inhibitors and uses thereof

Abstract

The present disclosure provides a composition, or a pharmaceutically acceptable salt thereof, comprising a a7 nicotinic acetylcholine receptor agonist and an immune checkpoint inhibitor, their pharmaceutical compositions, and methods of use for treating breast cancer with synergistic effect in reducing tumor burden and increasing survival.

Description

[0001] COMPOSITIONS COMPRISING CHRNA7 AGONISTS AND

[0002] IMMUNE CHECKPOINT INHIBITORS AND USES THEREOF

[0003] PRIORITY

[0004] This application claims the benefit of the filing date of U.S. provisional application No. 63 / 727,068, filed December 2, 2024, the disclosure of which is incorporated by reference herein.

[0005] STATEMENT OF GOVERNMENT SUPPORT

[0006] This invention was made with government support under grant number R01 CA170140 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] FIEED OF INVENTION

[0008] The present disclosure pertains to the field of cancer immunotherapy, specifically to compositions and methods involving al nicotinic acetylcholine receptor agonists and immune checkpoint inhibitors for the treatment of breast cancer, including triple negative breast cancer.

[0009] BACKGROUND

[0010] The field of cancer immunotherapy produces therapeutic strategies that aim to utilize the body’s immune defenses to identify and eliminate malignant cells. Over the past decade, approaches that engage T lymphocytes and reinvigorate antitumor responses have significantly influenced clinical practice for various cancer types. Despite notable progress, the full capabilities of the immune system remain underutilized, partly because effective antitumor immunity requires coordinated action by multiple immune cell types within the tumor microenvironment.

[0011] SUMMARY

[0012] In one embodiment, a pharmaceutical composition comprises an al nicotinic acetylcholine receptor agonist together with an immune checkpoint inhibitor, or a pharmaceutically acceptable salt thereof. The al agonist may be selected from acetylcholine; choline; anabasine; various tetrahydropyridine and spirocyclic derivatives; AR-R17779 (Formula I); nicotine; and related bicyclic and heterocyclic compounds or salts or combinations thereof. The immune checkpoint inhibitor inhibits PD-1, PD-L1, CTLA-4, or any combination thereof, and may comprise anti-PD-Ll including, but not limited to, clinically approved antibodies such as pembrolizumab, nivolumab, ipilimumab, atezolizumab, durvalumab, avelumab, cemiplimab, penpulimab, retifanlimab, tiragolumab, or relatlimab. The composition can further include a pharmaceutically acceptable carrier.

[0013] In another embodiment, a method of treating cancer in a subject in need thereof comprises administering to the subject the foregoing composition. The cancer may be selected from melanoma, non-small cell lung cancer, cervical cancer, colorectal, head and neck, Hodgkin lymphoma, liver, gastric, renal, bladder, urothelial, Merkel cell carcinoma, nasopharyngeal carcinoma, triplenegative breast cancer, and other tumors including those with DNA repair deficiencies. Treatment with the combination composition yields improved survival and reduced tumor burden compared to administration of either the al agonist or the immune checkpoint inhibitor alone. The composition activates tumor-associated myeloid immune cells and may be administered by oral, topical, subcutaneous, intramuscular, intraperitoneal, intrathecal, transdermal, or intravenous routes.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed herein.

[0016] FIGS. 1A-1D. Overall survival of mice bearing breast tumors is decreased in the absence of CHRNA7, while treatment with a CHRNA7 agonist increased survival. (A) CHRNA7 KO and WT sibling matched control mice were injected under the 4th mammary fat pad (MFP) with E0771 mouse breast cancer cells (l x 106), an infdtrative mammary adenocarcinoma syngeneic with the C57B1 / 6 background of CHRNA7 KO mice. (Kaplan-Meier, log-rank test, P<0.05, n=6, each arm). (B) Tumor size was monitored for 21 days after injection. *P <0.05 by one-way ANOVA. (C) Tumor wet weight was measured at Day 28, and (D) representative tumors imaged. *P <0.05.

[0017] FIGS. 2A-2E. CHRNA7 expression in myeloid cell types. (A and B) Analysis of CHRNA7 gene expression data in a tissue survey curated by the Immunological Genome Project (immgen.org). (C) CHRNA7 expression is high in bone marrow and spleen (Inset), in splenic CD8- dendritic cells, in BM long term-hematopoietic stem cells and blood monocytes. (D) PCR of CHRNA7. (E) CD45+ cells. FIGS. 3A-3D. CHRNA7 expression in tumor-associated immune cells (A and B) The fluorescently- conjugated a-bungarotoxin (a -bgtx) was used to identify CHRNA7+ CD45+ cells in spleen, (C) blood of WT mice (Top, green arrow) and absent in CHRNA7 KO mice. (D) In tumors, a -bgtx binding was greatest on CD1 IcHi DCs WT mice (arrow) compared to CD1 Iclnt and CDl lcLo cells (Bottom) compared to CHRNA7KO negative controls.

[0018] FIGS 4A-4H. Tumor-bearing CHRNA7KO mice promote changes in tumor-associated myeloid cells (A and B) Primary E0771 breast tumors resected from the mammary fat pads (MFP) at 28 days post-injection, were dissociated and subjected to flow cytometry analysis of tumor-infiltrating leukocytes. A reduction in the mean fluorescent intensity (MFI) of MHC II staining was observed on CD1 lb+ cells in CHRNA7 KO vs WT mice, (C) along with a CHRNA7-mediated reduction in the MFI of CD83+, (D and E) but no significant changes in the MFI of CD80+ subset of CD1 lb+ cells. No changes were observed in the overall number of CD1 Ib+CDl lc+ DCs (not shown). To determine whether the loss of CHRNA7 in APCs was a driver of changes in tumor immune responses, changes in the number of tumor-associated lymphocytes were examined (Fig. 4F, G). Decreases in intratumoral CD8+cytotoxic cell and Tregs in CHRNA7KOmice were observed compared to WT mice, but no changes in spleen (Fig. 4H), supporting a role for CHRNA7 action in adaptive immune responses in tumors.

[0019] FIGS. 5A-5F. CHRNA7 is preferentially expressed in monocytes, splenic DC and BM LT-HSC subsets, with CHRNA7 KO BMDCs have a reduced capacity to stimulate T cell proliferation. (A) CHRNA7 KO and WT mice bearing primary E0771 mammary adenocarcinoma cells were subjected to CD1 lc+ immunostaining: tumor margins (Ma), stromal cells (St) tumor (Tu). Images are representative of 3 independent experiments. Bar = 100 pm. (B) CD45+ tumor infiltrating leukocytes were isolated using magnetic beads and RNA analyzed for expression of myeloid differentiation markers using nCounter (Nanostring). Changes in gene expression >2 fold, with a P <0.05 based on multiple biological replicates (n=3, all replicates and statistical rankings, not shown). (C) Mixed leukocyte reactions (MLR) were performed using BMDCs differentiated from CHRNA7 KO or WT mice and co-cultured with CFSE- labeled CD4+ T cells from WT allogenic donors (D) serial dilution compares the division ratios of T cells and DCs. * P<0.05. (E) A representative overlay of CHRNA7 KO or WT BMDCs co-incubated with CFSE-labeled WT CD8+ T cells, and (F) quantification of the changes in CD8+ cell numbers.

[0020] FIGS. 6A-6F. Administration of the CHRNA7 agonist AR-R17779 increases survival in mouse models of metastatic and triple negative breast cancer. (A and B) Mice bearing E0771 tumor cells were administered AR- R17779 (1 mg / kg, every 2 days) and overall survival and primary weights measured and compared to vehicle control. (C) BALB / c mice injected with 4T1 mammary carcinoma cells were incubated for 10 days until a tumor was palpable in the MFP, randomized, subjected to intraperitoneal injections of AR-R17779 (1 mg / kg, every 2 days) or vehicle control, and overall survival analyzed. P<0.05, Log-rank (Mantel-Cox) test, n=8 in each arm. (D) The effect of AR- R17779 treatment on the growth kinetics of 4T1 tumors measured with calipers and compared to saline control (n=6 in each arm, P<0.05). (E) The effect of AR- R17779 compared to vehicle control was determined in the MMTV-PyMT spontaneous breast cancer and overall survival determined. (F) In the 4T1 tumor model, the effect of AR-R17779 alone, anti-PD-Ll alone, or the combination of AR-R17779 and anti-PD-Ll compared to vehicle control on overall survival were determined (n=6 in each arm, P<0.05).

[0021] DETAILED DESCRIPTION

[0022] Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0023] Cancer immunotherapy has emerged as a promising field for leveraging the body’s immune system to combat malignant cells. However, despite advancements in checkpoint inhibitors targeting T-cell surface interactions, their efficacy remains limited in certain cancers, such as triple negative breast cancer (TNBC). This limitation is largely attributed to the immunosuppressive tumor microenvironment, where antigen-presenting cells (APCs) fail to adequately process and present tumor antigens, thereby hindering the activation of adaptive immune responses. Conventional approaches often focus on reinvigorating T- cell activity but overlook the significant role of APCs in initiating and sustaining effective antitumor immunity. Furthermore, many tumors employ immune evasion strategies, such as downregulating costimulatory molecules and secreting inhibitory cytokines, which further dampen immune responses and reduce the effectiveness of checkpoint inhibitors.

[0024] The present disclosure addresses these limitations by introducing a novel therapeutic strategy that combines al nicotinic acetylcholine receptor (CHRNA7) agonists with immune checkpoint inhibitors. CHRNA7, a receptor highly expressed on myeloid-derived immune cells, has been identified as a modulator of APC activation. By pharmacologically targeting CHRNA7 with a selective agonist, such as AR-R17779, the approach reprograms tumor- associated APCs into a pro-immunogenic state, enhancing their ability to stimulate CD4+ and CD8+ T cells. This activation converts immunologically “cold” tumors into “hot” tumors, characterized by increased lymphocyte infiltration and improved antigen presentation. When combined with immune checkpoint inhibitors, such as anti-PD-Ll antibodies, the therapeutic strategy demonstrates a synergistic effect, significantly increasing survival rates and reducing tumor burden in TNBC models.

[0025] This inventive approach not only overcomes the limitations of conventional monotherapies but also provides a comprehensive solution to address the immunosuppressive tumor microenvironment. By simultaneously activating APCs and blocking inhibitory pathways on T cells, the combination therapy enhances adaptive immune responses, offering a transformative advancement in the treatment of TNBC and potentially other cancers, such as those with similar immune evasion mechanisms.

[0026] Definitions

[0027] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 14th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

[0028] References in the specification to "one embodiment," "an embodiment," etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0029] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0030] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is di -substituted.

[0031] As used herein, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating a listing of items, “and / or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of’.

[0032] As used herein, the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof, are intended to be inclusive similar to the term “comprising.”

[0033] The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. The term about can also modify the endpoints of a recited range as discuss above in this paragraph.

[0034] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

[0035] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more," and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

[0036] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group.

[0037] Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosed subject matter, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0039] It is appreciated that certain features of the disclosed subject matter, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed subject matter, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the disclosed subject matter and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0040] As used herein, a “subject in need thereof’ is a patient, animal, mammal, or human, who will benefit from the method of this invention.

[0041] The terms "treating," "treat" and "treatment" include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and / or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical, therapeutic, and / or prophylactic administration, as appropriate. As used herein, the phrase “treating cancer” refers to inhibition of cancer cell replication, apoptosis, inhibition of cancer spread (metastasis), inhibition of tumor growth, reduction of cancer cell number or tumor growth, decrease in the malignant grade of a cancer (e.g., increased differentiation), or improved cancer- related symptoms.

[0042] “Administering” when used in conjunction with a therapeutic means to administer a therapeutic to a patient whereby the therapeutic positively impacts the tissue to which it is targeted. The compounds described herein can be administered either alone or in combination (concurrently or serially) with other pharmaceuticals. For example, the compounds can be administered in combination with other anti-cancer or anti-neoplastic agents, or in combination with other cancer therapies other than chemotherapy, such as, for example, surgery or radiotherapy. Indeed, administration of an effective amount of a CHRNA7 agonists and checkpoint inhibitor to a patient in need of such treatment can result in reduced doses of another anticancer agent having clinically significant efficacy. Such efficacy of the reduced dose of the other anticancer agent may not be observed absent administration with a CHRNA7 agonist(s) and checkpoint inhibitor(s). Accordingly, the present invention provides methods for treating a tumor or cancer comprising administering a reduced dose of one or more other anticancer agents (or radiation.

[0043] In some embodiments, the anticancer agents may be tamoxifen, toremifen, raloxifene, droloxifene, iodoxyfene, megestrol acetate, anasfrozole, letrazole, borazole, exemestane, flutamide, nilutamide, bicalutamide, cyproterone acetate, goserelin acetate, luprolide, finasteride, herceptin, methotrexate, 5 -fluorouracil, cytosine arabinoside, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, cisplatin, carboplatin, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide, nitrosoureas, thiotephan, vincristine, taxol, taxotere, etoposide, teniposide, amsacrine, Irinotecan, topotecan, an epothilone, gefitinib, erlotinib, sorafenib, angiogenesis inhibitors, EGF inhibitors, VEGF inhibitors, CDK inhibitors, cytokines, Herl and Her2 inhibitors, and monoclonal antibodies.

[0044] A “therapeutically effective amount” or “effective amount” of a composition is a predetermined amount calculated to achieve the desired effect, i.e., to ameliorate, prevent or improve an unwanted condition, disease or symptom of a patient. The activity contemplated by the present methods may include both therapeutic and / or prophylactic treatment, as appropriate. The specific dose of the compounds administered according to this invention to obtain therapeutic and / or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compounds administered, the route of administration, and the condition being treated. The effective amount administered may be determined by a physician in the light of the relevant circumstances including the condition to be treated, the choice of compounds to be administered, and the chosen route of administration. A therapeutically effective amount of the compound of this invention is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the target tissue.

[0045] As used herein, the term “therapeutic” means an agent utilized to discourage, combat, ameliorate, prevent or improve an unwanted condition, disease or symptom of a patient.

[0046] By “pharmaceutically acceptable”, it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0047] As used herein, the phrase “in need thereof’ means that the patient has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the animal or mammal can be in need thereof. In some embodiments, the animal or mammal is in an environment or will be traveling to an environment in which a particular disease, disorder, or condition is prevalent. Embodiments

[0048] Provided herein is the first evidence for CHRNA7 as a biochemical target in activating adaptive immune responses that reduce breast cancer burden and improve overall survival. It is believed that activation of CHRNA7 drives downstream signaling mediated by transcription factors to re-program antigen- presenting cells to stimulate adaptive immune responses that convert immunologically ‘cold’ tumors to ‘hot’ tumors. It is shown that loss of CHRNA7 in KO mice increased tumor burden and reduced animal survival that was associated with decreases in the numbers of antigen-presenting cells. Moreover, BMDCs isolated from these CHRNA7KOmice had a reduced capacity to stimulate proliferation of CD4+and cytotoxic CD8+T cells. Supported by data demonstrating that among tumor-associated immune cells, CHRNA7 is most highly expressed in CD1 lc+DCs, CHRNA7 is a novel biochemical target for activating tumor-associated DCs that promote adaptive immune responses.

[0049] Based on genetic approaches establishing specificity for CHRNA7 action in select cell types in the host, the effect of pharmacological agonists of CHRNA7 validated in the fields of inflammation and neurobehavior was tested. The reduction in tumor burden and increased survival observed using the CHRNA7 agonist AR-R17779, and in combination with immune checkpoint inhibitors, shows that tumor-associated CHRNA7+immune cells are novel therapeutic targets.

[0050] The findings build on studies of CHRNA7KOmice in models of infection and injury where the loss of CHRNA7 leads to unattenuated inflammation repsonses3,4,16that have been primarily attributed to macrophages. A n unexpected role for CHRNA7 in cancer in the regulation of the profile of tumor- associated immune cells and activation of adaptive immunity was identify. In models of injury and endotoxemia, the loss of CHRNA7 increases inflammation responses by uncoupling the protective effects of innervation by the parasympathetic nervous system (PNS) such as by the vagus nerve16. The attenuation of inflammation responses mediated by such PNS inputs is termed the reflex control of immunity27and is dependent on CHRNA7 action in myeloid- derived bone marrow progenitors rather than cell autonomous effects in T cells28. Interestingly, however, T cells are known to synthesize acetylcholine which stimulates CHRNA7 to complete a vagus nerve circuit that controls innate immune responses in endotoxemia29. While reports that the stimulation of the vagus nerve decreased breast cancer metastasis, the effects on the immune system remain unknown30. Provided herein is a model in which CHRNA7 promotes the activation of myeloid-derived DCs that induce lymphocyte- mediated tumor clearance to reduce tumor burden, increase survival, and as a therapeutic target.

[0051] There is a range of inflammatory diseases, including rheumatoid arthritis12,13, sepsis14, experimental autoimmune encephalitis7,15and SIRS (systemic inflammatory response syndrome)4where a protective potential of CHRNA7 agonists have been demonstrated. In these models, CHRNA7 regulates the expression of pro-inflammatory markers of macrophage and dendritic cell activation, which in turn skews the lymphocyte profile towards an anti-inflammatory signaling profile6,7. These findings, largely in disciplines outside of tumor biology4'11led to the consideration of the possibility that CHRNA7 expression in myeloid cells and its capacity to regulate immune responses may be linked with its role in tumor progression, and translational relevance as a combination therapeutic with an immune checkpoint inhibitor. Based on the supporting data showing that CHRNA7 is expressed in tumor-associated APCs and regulates the expression of cytokines in the tumor microenvironment, it is proposed that CHRNA7 is a novel biochemical target in tumor-infdtrating immune cells that modulates the activation of APCs and mediates outcomes in animal models of breast cancer. Therefore, the potential of AR- R 17779 in combination with an immune checkpoint inhibitor was tested and a substantial and significant increase in overall survival in a mouse model of triple negative breast cancer (TNBC) was observed. a7 Nicotinic Acetylcholine Receptor Agonists

[0052] AR-R 1779 is a small molecule that is a selective agonist for al subtype of neural nicotinic acetylcholine receptors. AR-R1779 has been assigned Chemical Abstracts Service (CAS) number 178419-42-6. In various embodiments, the present disclosure provides a al nicotinic acetylcholine receptor agonist (CHRNA7), called AR-R17779, or pharmaceutically acceptable salts thereof, that are of Formula (I):

[0053] AR-R17779 may activate otherwise tumor-suppressed / inactive myeloid immune cells to stimulate immune responses and attack tumor cells. Combining AR-R17779 with one or more immune checkpoint inhibitors can provide a more potent anti -tumor therapy. This approach can address a major limitation to date of therapies based on immune checkpoint inhibitors alone which do not work in TNBC.

[0054] Other al nicotinic acetylcholine receptor agonist (CHRNA7) for use the invention include, but are not limited to, acetylcholine, choline, anabasine (2-(3- pyridyl)-3,4,5,6-tetrahydropyridine), GTS-21 (DMXB-A; 3- (2,4-dimethoxybenzylidene) anabaseine), 4-OH-DMXB-A-primary human metabolite of GTS-21, AZD0328 - furopyridine ((2'R)-spiro[l- azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine]; SMILES notation: C1CN2CCC1[C@@]3(C2)CC4=C(O3)N=CC=C4), TC-1698 - 2-(3-pyridyl)-l-azabicyclo[3.2.2]nonane, EVP-6124 (encenicline; (R)-7-chloro- N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide), BMS-933043 ((2R)-N- (6-(lH-imidazol-l-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'- oxazol]-2'-amine), RG3487 (MEM3454; N-[(3S)-l-azabicyclo[2.2.2]oct-3-yl]- lH-indazole-3-carboxamide; Smiles notation: O=C(C1=NNC2=C1C=CC=C2)N[C@@H]3CN4CCC3CC4), ABT-126 (2- ((3r,4s,5s,7s)-l-azaadamantan-4-yloxy)-5-phenyl-l,3,4-thiadiazole; Smiles notation: C1C2CC3CN(C2)CC1C3OC4=NN=C(S4)C5=CC=CC=C5), TC-5619 (also known as Bradanicline, is N-[(2S,3R)-2-(pyridin-3-ylmethyl)-l- azabicyclo[2.2.2]octan-3-yl]-l-benzofuran-2 -carboxamide; Smiles notation: C1CN2CCC1C@HNC(=O)C4=CC5=CC=CC=C5O4), PNU-282987 (N-[(3R)- 1 -azabicyclo [2.2.2] octan-3 -yl] -4-chlorobenzamide hydrochloride Smiles notation: C1CN2CCC1C@HNC(=O)C3=CC=C(C=C3)C1.C1), PHA-543613 (N- (3R)-l-Azabicyclo[2.2.2]oct-3-yl-furo[2,3-c]pyridine-5-carboxamide hydrochloride; Smiles notation: C1CN2CCC1C@HNC(=O)C3=NC=COC3), Tropisetron ((lR,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl lH-indole-3- carboxylate hydrochloride; Smiles notation: C1.CN1[C@H]2CC[C@@H]1CC@@HOC(=O)C1=CNC2=CC=CC=C12), 4BP-TQS (4-(4-Bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline- 8-sulfonamide; Smiles Notation BrCl=CC=C(C2NC(C=CC(S(=O)(N)=O)=C4)=C4C3C2CC=C3)C=Cl), Nicotine, Epibatidine ((lR,2R,4S)-2-(6-chloropyridin-3-yl)-7- azabicyclo[2.2.1]heptane; Smiles notation: C1C1=CN=C(C=C1)C2C3CCN2C3), Lobeline (2-[(2R,6S)-6-[(2S)-2-hydroxy-2-phenylethyl]-l-methylpiperidin-2- yl]-l -phenylethanone), or variations, analogs, derivatives or a combination thereof.

[0055] Immune Checkpoint Inhibitors

[0056] Immune checkpoints are regulatory pathways in the immune system that help maintain self-tolerance and modulate the strength and duration of immune responses to prevent excessive tissue damage. These checkpoints function as molecular "brakes" to inhibit or turn off immune cell activity, when necessary, thus protecting the body from autoimmune reactions and controlling inflammation.

[0057] Immune checkpoints engage when proteins on the surface of T cells recognize and bind to partner proteins on other cells, such as some tumor cells. These proteins are called immune checkpoint proteins. When the checkpoint and partner proteins bind together, they send an “off’ signal to the T cells which can prevent the immune system from destroying the cancer.

[0058] They primarily involve receptor-ligand interactions between immune cells, such as T cells, and other cells like antigen-presenting cells or tumor cells. For example, inhibitory checkpoints include proteins like CTLA-4 (Cytotoxic T Lymphocyte Antigen-4) and PD-1 (Programmed cell death protein 1) on T cells, which bind to their ligands (e.g., B7 molecules for CTLA-4 and PD-L1 / PD-L2 for PD-1) to suppress T cell activation and limit immune attack.

[0059] While needed for immune homeostasis, many cancers exploit immune checkpoints by overexpressing checkpoint ligands, thereby evading immune surveillance and destruction. Understanding these pathways has led to the development of immune checkpoint inhibitors as cancer therapies.

[0060] Any molecule capable of inhibiting one or more immune checkpoint proteins can be used in the methods disclosed herein. Such molecules are called checkpoint inhibitors. These include, without limitation, antibodies or functional fragments thereof, inhibitory polypeptides, small molecule chemical compounds, and / or inhibitory nucleic acids (such as, but not limited to, antisense oligonucleotides, small inhibitory RNAs (siRNAs), small hairpin RNAs (shRNAs), and / or catalytic nucleic acids such as ribozymes). Immune checkpoint proteins suitable for targeting by checkpoint inhibitors for use in any of the methods disclosed herein include, without limitation, one or more of the adenosine A2A receptor (A2AR), B7-H3 (a.k.a. CD276; e.g., MGA271), cytotoxic T-lymphocyte-associated protein 4 (CTLA4; a.k.a. CD 152; e.g., ipilimumab; AGEN-1884 (Agenus)), programmed cell death ligand 1 (PD-L1; a.k.a. CD274; e.g., MDX-1105 (Bristol Myers Squibb), WBP-3155 (C-stone), LY3300054 (Eli Lilly)), programmed cell death protein 1 (PD-1; a.k.a. CD279; e.g., pembrolizumab, SHR-1210 (Incyte), STI-A1110 (Sorrento), REGN2810 (Regeneron), CT-011 (pidilizumab; Curetech), PDR-001 (Novartis), BGB-A317 (BeiGene), TSR-042 (Tesaro), ENUMC-8 (Enumeral), MGD-013 (Macrogenics; bispecific antibody for PD1 and Lag3), B7-H4 (a.k.a. VTCN1), T-cell immunoglobulin and mucin-domain containing-3 (TIM3; a.k.a. HAVCR2), B and T Lymphocyte Attenuator (BTLA; a.k.a. CD272), indoleamine-pyrrole 2,3- dioxygenase (IDO), killer-cell immunoglobulin-like receptors (KIRs; e.g., lirilumab), lymphocyte-activation gene 3 (LAG-3; e.g., BMS-986016), T cell immunoreceptor with Ig and ITIM domains (TIGIT; a.k.a. WUCAM and Vstm3), ILT-3, ILT-4, and / or V-domain Ig suppressor of T cell activation (VISTA).

[0061] Further examples of immune checkpoints include: CTLA-4 (Cytotoxic T Lymphocyte Antigen-4): interacts with B7-1 (CD80) and B7-2 (CD86) to negatively regulate T cell activation; PD-1 (Programmed cell death protein 1): binds PD-L1 (CD274) and PD-L2 (CD273) to inhibit immune responses; LAG-3 (Lymphocyte activation gene 3): binds MHC class II, plays a negative regulatory role; TIM-3 (T cell immunoglobulin mucin 3): interacts with galectin-9, an inhibitory checkpoint; TIGIT (T cell immunoreceptor with Ig and ITIM domains): binds poliovirus receptor (CD155), functions as an inhibitory checkpoint; KIR (Killer-cell immunoglobulin-like receptor): expressed on NK cells, binds MHC class I molecules to regulate NK function negatively.

[0062] In some embodiments, the checkpoint inhibitor is an antagonistic antibody, such as, but not limited to, one or more of ipilimumab (Bristol-Myers Squibb), nivolumab (Bristol-Myers Squibb), Pembrolizumab (Merck) durvalumab (Medimmune), atezolizumab (Genentech / Roche), tremelimumab (Medimmune), and / or avelumab (Pfizer).

[0063] Immunotherapy drugs called immune checkpoint inhibitors act by blocking checkpoint proteins from binding with their partner proteins. This prevents the “off’ signal from being sent, allowing the T cells to kill cancer cells. The immune checkpoint inhibitor anti-PD-Ll or anti -PD-1 blocks binding of PD-L1 to PD1 One such drug acts against a checkpoint protein called PD-1 or its partner protein PD-L1. Some tumors turn down the T cell response by producing lots of PD-L1. By blocking these inhibitory pathways, ICIs enhance the immune system's ability to recognize and destroy cancer cells.

[0064] Mechanistically, tumors evade the immune response by engaging checkpoints that inhibit T cell activation, enabling cancer cells to escape immune destruction. ICIs target key checkpoints such as CTLA-4, PD-1, and PD-L1. For example, anti-CTLA-4 antibodies like ipilimumab prevent CTLA-4 from downregulating the co-stimulatory receptor CD28 on T cells, thereby promoting sustained T cell activation. Anti-PD-1 antibodies like nivolumab block PD-1 receptors on T cells, preventing them from receiving inhibitory signals from PD- L1 expressed on tumor cells. This releases the brakes on the immune system, allowing T cells to proliferate and attack tumors effectively.

[0065] Immune checkpoint inhibitors have transformed cancer treatment by improving long-term survival and quality of life in multiple cancers, although they can also cause immune -related adverse effects due to increased immune activation.

[0066] In various embodiments, the immune checkpoint inhibitor may comprise antibodies or other agents that block the action of pathways that keep T cells from killing cancer cells. For example, when Programmed Death Ligand (PD- Ll) is expressed on tumor cells and its receptor PD-1 expressed on T cells, the administration of antibodies that block the binding of PD-L1 to PD-1 allows T cells to kill tumor cells. In another example, administration of antibodies that interfere with the binding of B7-1 / B7-2 expressed on antigen presenting cells with its receptor CTLA-4 on T cells allows T cells to kill tumor cells (Bagchi S, Yuan R, Engleman EG. Immune Checkpoint Inhibitors for the Treatment of Cancer: Clinical Impact and Mechanisms of Response and Resistance. Anna Rev Pathol. 2021 Jan 24; 16:223-249). With recent clinical data showing that the administration of immune checkpoint inhibitors increases overall survival in subsets of patients with melanoma (Carlino MS, Larkin J, Long GV. Immune checkpoint inhibitors in melanoma. Lancet. 2021 Sep 11 ;398(10304): 1002- 1014).

[0067] Immune checkpoint inhibitors include, but are not limited to, Pembrolizumab (Keytruda; anti -PD-1 antibody used for multiple cancers including melanoma, NSCLC, and gastric cancer); Nivolumab (Opdivo; Anti- PD-1 antibody approved for melanoma, NSCLC, colorectal cancer, and more); Ipilimumab (Y ervoy; anti-CTLA-4 antibody approved primarily for melanoma and combination therapies); Atezolizumab (Tecentriq; anti-PD-Ll antibody used in urothelial carcinoma, NSCLC, and triple -negative breast cancer); Durvalumab (Imfinzi; Anti-PD-Ll antibody approved for lung and bladder cancers);

[0068] Avelumab (Bavencio; Anti-PD-Ll antibody approved for Merkel cell carcinoma and urothelial carcinoma); Cemiplimab (Libtayo; anti-PD-1 antibody for cutaneous squamous cell carcinoma and NSCLC); Penpulimab (anti-PD-1 antibody approved for nasopharyngeal carcinoma); Retifanlimab (PD-1 inhibitor approved for anal squamous cell carcinoma); others in clinical use or development include tiragolumab (anti-TIGIT), relatlimab (anti-LAG-3), and several combination therapies with anti-CTLA-4 and anti-PD-l / PD-Ll agents. These inhibitors work primarily by blocking immune checkpoint proteins such as PD-1, PD-L1, and CTLA-4 to enhance the immune system's ability to attack cancer cells.

[0069] Provided herein are embodiments for co-administration of an immune checkpoint inhibitor (i.e. anti-PD-Ll, PD1, or CTLA-4 antibodies) with an al nicotinic acetylcholine receptor agonist (CHRNA7), such as AR-R17779, as a combination therapy, which can be more effective than as a monotherapy.

[0070] Pharmaceutical Composition

[0071] The disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compositions (an immune checkpoint inhibitor (i.e. anti-PD-Ll, PD1, or CTLA-4 antibodies) with an al nicotinic acetylcholine receptor agonist (CHRNA7)), as described herein, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further contains, in accordance with accepted practices of pharmaceutical compounding, one or more additional therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers, preservatives, colorants, buffers, flavor imparting agents.

[0072] The pharmaceutical composition of the present disclosure is formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular subject being treated, the clinical condition of the subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0073] The “therapeutically effective amount” of the composition or a pharmaceutically acceptable salt, stereoisomer, and / or tautomer thereof that is administered is governed by such considerations, and it is the minimum amount necessary for supporting CHRNA7 to activate antigen presenting cells, reduce tumor burden, and increase time of survival of animals with cancer, such as breast cancer including triple negative breast cancer. Such amount may be below the amount that is toxic to normal cells, or the subject as a whole. Generally, the initial therapeutically effective amount of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure that is administered is in the range of about 0.01 to about 200 mg / kg or about 0.1 to about 20 mg / kg of patient body weight per day, with the typical initial range being about 0.3 to about 15 mg / kg / day. Oral unit dosage forms, such as tablets and capsules, may contain from about 0.1 mg to about 1000 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In another embodiment, such dosage forms contain from about 50 mg to about 500 mg of the composition (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In yet another embodiment, such dosage forms contain from about 25 mg to about 200 mg of the composition (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In still another embodiment, such dosage forms contain from about 10 mg to about 100 mg of the composition (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In a further embodiment, such dosage forms contain from about 5 mg to about 50 mg of the composition (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In any of the foregoing embodiments the dosage form can be administered once a day or twice per day.

[0074] The compositions of the present disclosure can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal injection or infusion techniques.

[0075] Suitable oral compositions as described herein include without limitation tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, syrups or elixirs.

[0076] In another aspect, also encompassed are pharmaceutical compositions suitable for single unit dosages that comprise a compound of the disclosure or its pharmaceutically acceptable stereoisomer, salt, or tautomer and a pharmaceutically acceptable carrier.

[0077] The compositions of the present disclosure that are suitable for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions. For instance, liquid formulations of the compositions of the present disclosure can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically palatable preparations of the compositions of the present disclosure.

[0078] For tablet compositions, the compositions of the present disclosure in admixture with non-toxic pharmaceutically acceptable excipients is used for the manufacture of tablets. Examples of such excipients include without limitation inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated, or they may be coated by known coating techniques to delay disintegration and absorption in the gastrointestinal tract and thereby to provide a sustained therapeutic action over a desired time period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

[0079] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. For aqueous suspensions, the compositions of the present disclosure may be admixed with excipients suitable for maintaining a stable suspension. Examples of such excipients include without limitation are sodium carboxymethylcellulose, methylcellulose, hydroxpropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.

[0080] Oral suspensions can also contain dispersing or wetting agents, such as naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0081] Oily suspensions may be formulated by suspending the composition of the present disclosure in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.

[0082] Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0083] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide compositions of the present disclosure in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

[0084] Pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally occurring gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides, for example soybean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation reaction products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavoring agents.

[0085] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents.

[0086] The pharmaceutical compositions may be in the form of a sterile injectable, an aqueous suspension or an oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectable s.

[0087] The compositions as described herein may also be administered in the form of suppositories for rectal administration of the drug. The compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

[0088] Compositions for parenteral administrations are administered in a sterile medium. Depending on the vehicle used and concentration the concentration of the drug in the formulation, the parenteral formulation can either be a suspension or a solution containing dissolved drug. Adjuvants such as local anesthetics, preservatives and buffering agents can also be added to parenteral compositions. Methods of Use

[0089] The compositions described herein can be administered to treat subjects, such as animals in need of such treatment, or who may develop a need for such treatment. For example, the compositions can be used to treat cancer, including, but not limited to, skin cancer, including melanoma, lung cancer, including, NonSmall Cell Lung Cancer (NSCLC), cervical cancer, colon cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, gastric cancer, breast cancer (including triple negative breast cancer), colorectal cancer, renal cancer, bladder cancer, Merkel cell carcinoma, urothelial carcinoma, stomach cancer, rectal cancer, cutaneous squamous cell carcinoma, nasopharyngeal carcinoma and other cancers, such as those with DNA repair deficiencies. For example, the compositions can reduce the severity of breast cancer, such as triple negative breast cancer.

[0090] In some embodiments disclosed herein are methods for inhibiting tumor growth in an individual by administering one or more al nicotinic acetylcholine receptor agonists and one or more molecules (such as an antibody, e.g. a monoclonal antibody) that inhibits an immune checkpoint protein. The combination of al nicotinic acetylcholine receptor agonist added to an immune checkpoint inhibitor is as effective or are more effective in inhibiting tumor growth as compared to a combination of two or more antibody-based immune checkpoint inhibitory therapies administered without a combination of al nicotinic acetylcholine receptor agonist.

[0091] In some embodiments, the methods involve administering an al nicotinic acetylcholine receptor agonist in combination with molecules that inhibit one or more of the following: PD-1, PD-L1, and CTLA-4. In some embodiments, the al nicotinic acetylcholine receptor agonist of Formula I is administered in combination with molecules that inhibit one or more of the following: PD-1, PD- Ll, and CTLA-4. In some embodiments, the molecules that inhibit PD-1, PD-L1, and CTLA-4 are antibodies.

[0092] In some embodiments, the method includes administering an al nicotinic acetylcholine receptor agonist in combination with anti-PD-1 antibodies. In some embodiments, the method includes administering an al nicotinic acetylcholine receptor agonist in combination with anti-PD-Ll antibodies. In some embodiments, the method includes administering an al nicotinic acetylcholine receptor agonist in combination with anti -CTLA-4 antibodies. In some embodiments, the method includes administering an al nicotinic acetylcholine receptor agonist in combination with anti-PD-1 and anti-CTLA-4 antibodies. In some embodiments, the method includes administering an al nicotinic acetylcholine receptor agonist in combination with anti-PD-Ll and anti-CTLA-4 antibodies.

[0093] In some embodiments, al nicotinic acetylcholine receptor agonist is administered to the individual in any of the following ranges: about 0.5 to about 1 mg / kg, about 0.5 to about 2 mg / kg, about 0.5 to about 3 mg / kg, about 0.5 to about 4 mg / kg, about 0.5 to about 5 mg / kg, about 0.5 to about 10 mg / kg, about 0.5 to about 20 mg / kg, about 0.5 to about 50 mg / kg, about 1 to about 10 mg / kg, about 1 to about 50 mg / kg, about 10 to about 100 mg / kg, about 10 to about 150 mg / kg, about 50 to about 175 mg / kg, about 175 to about 200 mg / kg, about 200 to about 225 mg / kg, about 225 to about 250 mg / kg, about 250 to about 300 mg / kg, about 300 to about 350 mg / kg, about 350 to about 400 mg / kg, about 400 to about 450 mg / kg, or about 450 to about 500 mg / kg. The dose administered may be every day, every 2 days, every 3 days, every 4 days, every 5 days, every week, every 2 weeks, every 3 weeks, every 4 weeks, and so on until there is remission.

[0094] In some embodiments, the checkpoint inhibitors are administered at dosages: 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.3 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 33.3 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, or 50 mg / kg.

[0095] In an embodiment, antibodies against PD-1, PD-L1 and CTLA-4 are administered at a dose of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, or 30 mg / kg. The dose administered may be every day, every 2 days, every 3 days, every 4 days, every 5 days, every week, every 2 weeks, every 3 weeks, every 4 weeks, and so on until there is remission. In another embodiment, al nicotinic acetylcholine receptor agonist in combination with molecules that inhibit one or more immune checkpoint proteins administered according to any of the methods disclosed herein provide at least about a 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 33.3%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% tumor inhibitory effect when compared to tumors that are not treated with al nicotinic acetylcholine receptor agonist and checkpoint inhibitors.

[0096] The compounds and molecules disclosed herein can be administered in the conventional manner by any route where they are active. Administration can be systemic, parenteral, topical, or oral. For example, administration can be, but is not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, or ocular routes, or intravaginally, by inhalation, by depot injections, or by implants. Thus, modes of administration for the molecules of the present disclosure (either alone or in combination with other pharmaceuticals) can be, but are not limited to, sublingual, injectable (including short-acting, depot, implant and pellet forms injected subcutaneously or intramuscularly), or by use of vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams. In some embodiments, al nicotinic acetylcholine receptor agonist(s) and checkpoint inhibitors can be administered directly to the tumor site.

[0097] In some embodiments, al nicotinic acetylcholine receptor agonists and checkpoint inhibitors can be administered directly to the tumor site. In some embodiments, al nicotinic acetylcholine receptor agonist is administered at the tumor site and checkpoint inhibitors are administered intravenously. In some embodiments, al nicotinic acetylcholine receptor agonist is administered at the tumor site, followed by oral administration of al nicotinic acetylcholine receptor agonist and intravenous administration of checkpoint inhibitors.

[0098] In some embodiments, al nicotinic acetylcholine receptor agonists and checkpoint inhibitors are administered intravenously. In some embodiments, al nicotinic acetylcholine receptor agonist is administered orally, and checkpoint inhibitors are administered intravenously. In some embodiments, al nicotinic acetylcholine receptor agonist and checkpoint inhibitors are administered orally. In some embodiments, al nicotinic acetylcholine receptor agonists and checkpoint inhibitors are administered simultaneously or sequentially. For example, al nicotinic acetylcholine receptor agonist can be administered first followed by administering antibodies to PD-1, PD-L1, and CTLA-4. In some embodiments, antibodies to PD-1, PD-L1, and CTLA-4 can be administered first, followed by administration of al nicotinic acetylcholine receptor agonist.

[0099] Specific modes of administration will depend on the indication. The selection of the specific route of administration and the dose regimen is to be adjusted or titrated by the clinician according to methods known to the clinician in order to obtain the optimal clinical response. The amount of compounds to be administered is that amount which is therapeutically effective. The dosage to be administered will depend on the characteristics of the subject being treated, e.g., the particular animal or human being treated, age, weight, health, types of concurrent treatment, if any, and frequency of treatments, and can be easily determined by one of skill in the art (e.g., by the clinician).

[0100] In another embodiment, a method of killing cancer cells may comprise contacting the cancer cells with a composition comprising a al nicotinic acetylcholine receptor agonist in combination with a molecule that inhibits at least one immune checkpoint protein. In some embodiments, the method may be in vitro or in vivo. In some embodiments, the molecule that inhibit checkpoint proteins may be antibodies against PD-1, PD-L1, and CTLA-4.

[0101] The invention can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting.

[0102] EXAMPLES

[0103] Introduction

[0104] Using genetic and pharmacological approaches in mouse models of breast cancer it was shown that the al nicotinic acetylcholine receptor (CHRNA7) expressed in myeloid immune cells is a novel modifier of the activation of APCs. Loss of CHRNA7 leads to decreased overall survival, increased tumor burden and a reduced capacity to activate APCs in vivo and in vitro. In contrast, administration of a pharmacological agonist of CHRNA7 increased overall survival and reduced tumor burden. Based on the evidence supporting CHRNA7 activating APCs, a combination treatment of a CHRNA7 agonist with an immune checkpoint inhibitor was tested and a synergistic effect was demonstrated with this therapy in increasing survival of a triple negative mouse breast tumor model. These findings support the translation of CHRNA7 as a novel therapeutic target in breast cancer.

[0105] The evasion of immunosurveillance is a hallmark of cancer, characterized by compromised immune cell signaling1. For this reason, therapeutics that harness the adaptive immune system have shown tremendous promise in reducing tumor burden2, demonstrating the importance of lymphocytes for antitumor immunity. Although lymphocyte function is dependent on priming and antigen presentation, there are limited demonstrations of approaches to modulate the function of antigen presenting cells (APCs) in tumors. The studies herein are focused on the identification of a novel modifier of APC activation, the al nicotinic acetylcholine receptor (CHRNA7), a neurotransmitter receptor that is highly expressed on myeloid immune cells in humans and mice, and a mediator of inflammation responses using CHRNA7 knockout mice in models of tissue injury and endotoxemia3'5. For example, loss of CHRNA7 skewed macrophages towards an anti-inflammatory signaling profile6,7through transcriptional reprogramming of immune cells and increased the expression of pro-inflammatory cytokines such as TNFa and / or p, IL-6, and IL-ip3,8. These CHRNA7-mediated effects on cytokine expression appeared to be specific to bone marrow (BM)- derived immune cells and independent of lymphocytes and other stromal cell types9. Consistent with similar observations in mouse models of atherosclerosis and arthritis, the loss of CHRNA7 led to a pro-inflammatory macrophage phenotypes associated with elevated C- reactive protein and MCP-1, respectively10,11. In addition to the insights from genetic approaches, agonists of CHRNA7 have been tested in a range of mouse models of inflammatory disease and shown to have a protective effect upon outcomes in rheumatoid arthritis12,13, sepsis14, experimental autoimmune encephalitis7,15and systemic inflammatory response syndrome4. This field of study has led to the concept that canonical receptors for neurotransmitters associated with the parasympathetic nervous system are expressed and functional in the immune system, and mediate protective effects in the resolution of injury that have not been examined in cancer models16.

[0106] These observations, along with evidence for a role of the sympathetic nervous system inputs in cancer progression has led to the testing of the importance of the CHRNA7 receptor as a functional mediator of myeloid cell subsets relevant in tumor progression, using a combination of genetic and pharmacological approaches. In studies of mouse models of immunocompetent breast cancer, it was shown, using a genetic approach, that the loss of CHRNA7 leads to increased tumor progression, reduces overall survival, decreases the activation of tumor-associated APCs in vivo, and reduces the capacity of BM- derived CHRNA7 knockout (CHRNA7KO) APCs to stimulate lymphocytes. Using a pharmacological approach, a specific agonist of CHRNA7, AR-R17779, was used to treat tumor-bearing mice and show increased survival in two independent models.

[0107] Example 1: Loss of CHRNA7 decreases survival and increases tumor progression in KO mice

[0108] Based on studies of CHRNA7KOmice in models of tissue injury that have established the functional requirement of CHRNA7 in myeloid immune cells, it was examined whether the loss of CHRNA7 expression in the host compartment affected tumor progression in immune- competent breast tumors . CHRNAKOvs . WT mice were implanted in the mammary fat pad (MFP) with E0771 , a triple negative breast adenocarcinoma compatible with the C57BL / 6 background of CHRNAKOmice17'22, and a Kaplan-Meier survival analysis was performed. Statistically significant reductions in survival in CHRNAKOmice compared to WT mice where none of the CHRNAKOmice survived past 50 days vs. 100% survival of WT mice (Fig. 1A). This decreased survival CHRNAKOmice was associated with an increase in tumor growth in CHRNAKOmice compared to WT mice (Fig. IB), and yielded tumors that were four times the wet weight of WT mice upon harvest (Figs. 1C and ID). These findings are the first to report a CHRNA7- mediated change using CHRNAKOmice to assess tumor progression and overall survival and suggests that CHRNA7 has a function in the host compartment in mediating tumor progression. Example 2: CHRNA7 mRNA is expressed on BM, spleen and CD45+immune cells. Consistent with previous studies showing CHRNA7 expression in myeloid immune cells (i.e. monocytes, macrophages and DCs), it was shown that CHRNA7 gene expression is high in BM and spleen (Fig. 2A, black bars), and in particular hematopoietic stem cell (HSC) subsets of BM, DCs from spleen and monocytes from blood (Fig. 2B). Analysis of WT vs. CHRNA7KOBM and spleen demonstrates the specificity of the qPCR reactions (Fig. 2C and D), and the higher levels of CHRNA7 in CD45-enriched BM and spleen cells (Fig. 2E) relative to levels in unfractionated tissues (Fig. 2C; note the difference in units on the y-axis between Fig. 2C and 2E).

[0109] Example 3: CHRNA7 protein is expressed on CD45+peripheral blood and CDllc+tumor- associated immune cells. To identify CHRNA7+cells in dissociated tumor samples using flow cytometry, an assay was developed that identified higher surface CHRNA7 protein on CD 1 lb+myeloid splenocytes (Fig. 3A), compared to CD3+lymphoid splenocytes (Fig. 3B). High CHRNA7 was also observed on CD45+peripheral blood cells (Fig. 3C). This approach has greater specificity than antibodies since it uses a fluorescent analog of a- bungarotoxin (a -Bgtx), a krait snake venom peptide that is a competitive antagonist, highly specific for CHRNA723.

[0110] Example 4: CDllc+tumor APCs express the highest levels of CHRNA7.

[0111] Based on the validation of CHRNA7 levels identified in splenocytes and blood (Fig. 3), the highest levels of CHRNA7 were detected on CD1 lcnion CD1 lb+tumor-associated immune cells (Fig. 3D, Top Right). This subset of immune cells is most commonly associated with APCs and when compared to CD 1 lcIntor CDl lcLocells, (Fig. 3D, Bottom) cells which are commonly identified as tumor macrophages and other myeloid subsets, there were low to no detectable levels of CHRNA7.

[0112] Example 5: Loss of CHRNA7 reduces APCs in vivo. With CHRNA7 expression high in myeloid cells of the spleen and blood (Figs. 2 and 3), and the highest levels of CHRNA7 observed in intratumoral CD1 lc+cells (Fig. 3D), tumor- associated immune cells were characterized for changes in surface markers of APC activation. It was observed that the loss of CHRNA7 led to decreases in surface levels of MHCII, CD 11c, and CD83, while no overall changes in the numbers of CD1 lb+or F4 / 80+tumor macrophages were observed (Fig. 4). These data supported a model that the loss of CHRNA7 reduced levels of tumor- associated APCs in vivo.

[0113] Example 6: Loss of CHRNA7 in APCs is associated with reductions in Tregs and CD8+lymphocytes. To determine whether the loss of CHRNA7 in APCs was a driver of changes in tumor immune responses, changes in the number of tumor-associated lymphocytes were examined (Fig. 4F, G). Decreases in intratumoral CD8+cytotoxic cell and Tregs in CHRNA7KOmice were observed compared to WT mice, but no changes in spleen (Fig. 4H), supporting a role for CHRNA7 action in adaptive immune responses in tumors.

[0114] Example 7: CHRNA7 is a mediator of myeloid gene expression signature in tumor-associated CD45+immune cells. Based on evidence that CHRNA7 activation stimulates the transcription factor JAK2 / STAT3, and regulates cytokine expression, we analyzed CHRNA7- dependent changes in cytokine expression in the tumor microenvironment. Nanostring analysis was used to measure CHRNA7-mediated gene expression with a focus on genes regulating myeloid differentiation (Fig. 5).

[0115] Example 8: DCs isolated from CHRNA7KOmice have reduced capacity to stimulate CD8+ T cells. Based on the in vivo data showing CHRNA7-dependent changes in APCs observed in tumors, in vitro assays were used with CHRNA7KOand WT mice as bone marrow donors from which BM DCs were differentiated and analyzed for CHRNA7-mediated changes in DC activity. It was observed that the loss of CHRNA7 reduced the capacity of DCs to stimulate proliferation ofCD8+T cells. (Fig 5).

[0116] Example 9: The CHRNA7 agonist AR-R17779 increases survival in a murine model of breast cancer. AR-R17779, a conformationally restricted analog of acetylcholine with specificity for CHRNA724, functions as a CHRNA7 agonist. The efficacy of AR-R17779 to reduce inflammation in animal models of arthritis and gut injury has been demonstrated25,26. Since loss of CHRNA7 increased tumor burden, it was hypothesized that activation of CHRNA7 would decrease tumor burden. Using the CHRNA7 agonist, AR-R17779, mice bearing the E0771 breast tumor cells were subjected to a systemic treatment with AR- R17779 and an increase in survival and decrease in primary tumor burden compared to vehicle control was observed (Fig, 6A and B). Put another way, survival of the animal treated with the composition is longer relative to survival of the animal treated with the al nicotinic acetylcholine receptor agonist or the immune checkpoint inhibitor alone and tumor burden of the animal treated with the composition is decreased relative to an animal treated with the al nicotinic acetylcholine receptor agonist or the immune checkpoint inhibitor alone. Next, the effect of the AR-R17779 was tested on a triple negative breast tumor model such as mice bearing 4T1 breast tumor cells in BALB / c mice. It was observed that the AR-R17779-treated group had an increase in overall survival (Fig. 6C) and decreased lung metastases compared to vehicle (Fig. 6D). In a test of a spontaneous breast tumor onset, the effect of AR-R17779 on overall survival was tested and a significant increase in survival in AR-R17779-treated mice compared mice was observed (Fig. 6E). Based on the protective effects of AR- R17779 on several models ofinvasive breast cancer such as the E0771 PyMT, and 4T1 model, further testing of AR-R17779 was focused on one of the most widely studied mouse models of TNBC using 4T1 cells and further tested as a combination with an immune checkpoint inhibitor. AR-R17779 alone, anti-PD- L1 alone, and the combination of AR-R17779 and anti-PD-Ll were tested in comparison to vehicle control in the 4T1 tumor model. Administration of AR- R17779 or anti-PD-Ll alone provided limited but significant increases in overall survival, however, the combination of AR-R17779 with anti-PD-Ll in twice a week (n=8 each arm) dosing led to a substantial and significant increase in overall survival (Fig. 6F). Together these findings support the potential of combination therapy of immune checkpoint inhibition with an activator of tumor-associated APCs such as AR-R17779 as a novel therapeutic in TNBC.

[0117] Example 10: Methods

[0118] Animals and tumor studies. CHRNA7KO(B6.129S1 -Chrna7tmlBay / J) and sibling-matched WTmice were originally generated by Dr. A. Beaudet31, backcrossed approximately eight generations (“N8F8”), prior to being made commercially available through Jackson Labs (#003232). Once obtained, mice were bred as heterozygotes in the UCSD Animal Care Program and subjected to genotyping (Transnetyx) to identify appropriate genotypes. Unless otherwise specified, wild type mice were matched siblings or matched WT purchase from Jackson Labs. All studies were approved by the UCSD Animal Care and Use Committee. For tumor studies, female 8-12-week-old mice were subjected to injections of the 4thmammary fat pad with le6E0771 mammary adenocarcinoma cells cultured in RPMI 1640 in 10% fetal calf serum as previously described17. Tumor survival and burden was assessed every 3-5 days and the kinetics of tumor growth determined with calipers where tumor volume =1 / 2 (length x width2)32. Wet tumor weights were assessed upon euthanasia, with tumors analyzed by immunohistochemistry, flow cytometry and cell separation as described below. Both male and female mice were used as cell donors for in vitro studies.

[0119] Immunohistochemistry: Tumor tissues resected upon harvest were embedded in formalin, 10 pm sections, subjected to antigen retrieval with Signal Stain Citrate Unmasking solution (Cell Signaling Technology, CST #14746), incubated with anti-CDl lc antibody (CST, #97585S), and detected with a horseradish peroxidase detection system, Signal Stain Boost, per the manufacturer’s protocol (CST, #8112). Slides were imaged with an FSX100 light microscope (Olympus America).

[0120] Flow Cytometry: Tumors and spleen were processed by enzymatic digestion to obtain single cells using the Tumor Dissociation Kit (Miltenyi Biotech, #130- 096-730) and the Spleen Dissociation Kit (Miltenyi Biotech, MB #130-095-926) per manufacturer’s recommendations. Viable cells were identified by staining with propidium iodide (MB, #130-093-233) and incubated with the following antibodies from Miltenyi-Biotec: CD1 lb clone REA592, CD11c clone REA754, MHC II clone M5 / 114.15.2, CD80 clone 16-10A1, CD83 clone REA304, Grl clone RB6-8C5, F4 / 80 clone REA126. The fluorescently labeled D-bgtx-AF647 was obtained from Thermo-Fisher. The specificity of cell staining was determined using fluorescence minus one control, and all cell staining data acquired with a MACSQuantlO flow cytometer obtained using compensation settings determined weekly, as recommended by the manufacturer. Data was analyzed using MACSQuant V2. 11 to gate cell populations and measure percentages, and FlowJo V10 (Becton-Dickinson) to determine MFI and generate histogram overlays and analyses.

[0121] Cell Separation and Gene Expression: Tumor-infiltrating CD45+cells were isolated from primary tumors according to manufacturer’s procedures (MB, #130- 110-618). Cells were lysed in TRIZOL (Thermo-Fisher, #15596018), and RNA prepared using Direct-zol (Zymo, #R2070). Quantitative RT-PCR reactions were used to measure CHRNA7 gene expression using cDNA prepared with iScript (Bio-Rad, #1708891), SsoAdvance SYBR green (Bio-Rad, #172-5271).

[0122] Reactions were allowed to continue through 40 cycles on a CFX96 (Bio-Rad), data acquired with CFX Manager Software 3.1 (BioRad) and analyzed using the 2(-Delta Delta C(T)) method with test genes compared relative to GAPDH33. For Nanostring analyses, the myeloid gene panel V2 was (UCSD Sanford Stem Cell Consortium Core Facility) used and analyzed using nCounter Analysis to identify changes in changes in expression levels of myeloid genes from CD45- enriched tumor infdtrating leukocytes.

[0123] DC activity assays: To identify changes in the phosphorylation status of STAT3, splenocytes were prepared from naive mice by mechanical dissociation and fdtration through a 70 pm strainer. Cells were stimulated with 1 pM AR-R17779 (Sigma, #SML2049) and 100 ng / mL LPS from E. coli 0111:B4 (Sigma, #L4130) for 15 minutes at 37 °C. Cells were then fixed in 4% paraformaldehyde (Electron Microscopy Sciences, #15710), permeabilized in 90% methanol, stained with anti- phosphorylated STAT3Tyr795(CST, #8119), and anti-CDl 1c followed by analysis by flow cytometry. For allogenic mixed leukocyte reactions, dendritic cells from CHRNA7KOand CHRNA7WTmice were prepared by differentiation of bone marrow isolated by centrifugation of femurs34and cultured in GM-CSF as previously described for 6 days35. CD4 or CD8 T cells were isolated from naive BALB / c mice (Jackson Labs, #000651) using magnetic bead isolation per manufacturer’s recommendation with anti-CD4 beads (MB, #130-116-480) or anti CD8 beads (MB, #130-090-318), respectively. Isolated T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) according to the manufacturer’s recommendation (Thermo-Fisher, #C34554) and incubated in round bottom 96 well plates in various ratios of DC:T cells (10: 1, 5: 1, 2: 1 for CD4 assays and 1 : 1 for CD8 assays) for 5 days. CFSE incorporation was assessed by flow cytometry using CD4 gating to measure the Division Index and the Biology algorithm available in FlowJo V10. The CD8 response was determined based on the number of CD8+cells under the gate shown in the representative histogram.

[0124] Example 11

[0125] A mouse model of triple negative breast cancer (TNBC) was used to test the efficacy of an agonist of the nicotinic acetylcholine receptor (nAchR), AR- R17779, that is expressed on tumor-associated myeloid immune cells. This drug activates otherwise tumor-suppressed / inactive myeloid immune cells to stimulate immune responses and attack tumor cells, then it was combined with immune checkpoint inhibitors to develop a more potent anti-tumor therapy. This approach addresses a major limitation to date of therapies based on immune checkpoint inhibitors alone which do not work in triple negative breast cancer cells (TNBC). Therefore, w demonstrated herein in an immune competent mouse tumor model is that while administration of AR-R17779 and anti-PD-Ll singly each have anti-tumor effects, the combination is vastly superior to either one alone. These findings demonstrate a very large advance in TNBC treatment by establishing a novel combination therapy.

[0126] Breast tumor cells were implanted into mice with knockout for the a7nAchR and an increase in tumor burden was observed. It was observed that bone marrow dendritic cells (DCs) lacking a7nAchR were less active and had a gene expression profile that was distinct from wildtype DCs. Therefore, the data showed that the loss of nAchR reduced the activity myeloid immune cells such as DCs. It was then hypothesized that if loss of a7nAchR blocked DC activity and increased tumor burden, then stimulating the a7nAchR, one could determine if tumor burden was reduced. The effect of AR-R17779 was tested in a mouse TNBC model, and an increased survival as a single agent was observed, and when co-administered with anti-PD-Ll there was a substantially increased survival compared to single agent administration and reduced lung metastasis.

[0127] A mouse model of TNBC was established using the 4T1 mouse breast tumor cell line which is a well-recognized TNBC that grows aggressively in balb / c female mice. Survival studies of mice that had been allowed to grow breast tumors to a specified size were performed, and then a treatment course was initiated. Treatments were performed with the following arms: 1) vehicle, 2) AR-R17779 alone, 3) PD-L1 alone, and 4) AR-R17779+PD-L1 together. Mice were followed for progression based on primary tumor burden with calipers, survival evaluated, and the number of lung tumor nodules quantified.

[0128] The combination of the a7nAchR agonist AR-R17779 when added in combination with the immune checkpoint inhibitor anti-PD-Ll is superior to either agent administered alone in a mouse model of TNBC. Anti-PD-Ll is a member of a class of clinically approved anti -cancer treatments, but none are effective in the treatment of TNBC. Herein is demonstrated the ability of increasing the efficacy of immune checkpoint inhibitors with a7nAchR agonists. This finding is especially attractive because clinical trials are more likely to enroll patients as an arm in a randomized control study where immune checkpoint inhibitors are already being tested based on established safety profiles.

[0129] There are no reports to date of using a7nAchR agonists as an anticancer therapy, and none that use an a7nAChR agonists in combination with immune checkpoint inhibitors.

[0130] For example, in embodiments AR-R17779 is administered as a combination with an immune checkpoint inhibitor to reduce lung tumor metastasis and greatly increase survival in an immune competent mouse model of TNBC. Further, the data demonstrates that a7nAchR is an important activator of myeloid immune cells such as tumor-associated macrophages and dendritic cells. Bibliography Hanahan, D. & Weinberg, R.A. Hallmarks of cancer: the next generation. Cell 144, 646-674 (2011). Speiser, D.E., Ho, P.C. & Verdeil, G. Regulatory circuits of T cell function in cancer . Nat Rev Immunol 16, 599-611 (2016). de Jonge, W. J., et al. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway. Nature Immunology 6, 844-851 (2005). Wang, H., et al. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 421, 384-388 (2003). Costantini, T.W., et al. Uniquely human CHRFAM7A gene increases the hematopoietic stem cell reservoir in mice and amplifies their inflammatory response. Proc Natl Acad Sci U SA 116, 7932-7940 (2019). Shi, F.D., et al. Nicotinic attenuation of central nervous system inflammation and autoimmunity. J Immunol 182, 1730-1739 (2009). Hao, J., et al. Attenuation of CNS inflammatory responses by nicotine involves alpha7 and non-alpha7 nicotinic receptors. Exp Neurol 227, 110-119 (2011). Kawashima, K., Fujii, T., Moriwaki, Y ., Misawa, H. & Horiguchi, K. Reconciling neuronally and nonneuronally derived acetylcholine in the regulation of immune function. Annals of the New York Academy of Sciences 1261, 7-17 (2012). Olofsson, P.S., et al. alpha7 nicotinic acetylcholine receptor (alpha7nAChR) expression in bone marrow-derived non-T cells is required for the inflammatory reflex. Molecular medicine (Cambridge, Mass.) 18, 539-543 (2012). Wilund, K.R., et al. Macrophages from alpha 7 nicotinic acetylcholine receptor knockout mice demonstrate increased cholesterol accumulation and decreased cellular paraoxonase expression: a possible link between the nervous system and atherosclerosis development. Biochemical and biophysical research communications 390, 148-154 (2009). van Maanen, M.A., Stoof, S.P., Larosa, G.J., Vervoordeldonk, M.J. & Tak, P.P. Role of the cholinergic nervous system in rheumatoid arthritis: aggravation of arthritis in nicotinic acetylcholine receptor alpha7 subunit gene knockout mice. Annals of the rheumatic diseases 69, 1717-1723 (2010). Bruchfeld, A., et al. Whole blood cytokine attenuation by cholinergic agonists ex vivo and relationship to vagus nerve activity in rheumatoid arthritis. J Intern Med 268, 94-101 (2010). van Maanen, M.A., et al. Two novel alpha7 nicotinic acetylcholine receptor ligands: in vitro properties and their efficacy in collagen-induced arthritis in mice. PLoS One 10, eOl 16227 (2015). Wang, H., et al. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 10, 1216-1221 (2004). Simard, A.R., et al. Differential modulation of EAE by alpha9*- and beta2*- nicotinic acetylcholine receptors. Immunol Cell Biol 91, 195-200 (2013). Pavlov, V.A., Chavan, S.S. & Tracey, K.J. Molecular and Functional Neuroscience in Immunity. Annu Rev Immunol 36, 783-812 (2018). Ewens, A., Mihich, E. & Ehrke, M.J. Distant metastasis from subcutaneously grown E0771 medullary breast adenocarcinoma. Anticancer Res 25, 3905-3915 (2005). Christowitz, C., et al. Mechanisms of doxorubicin-induced drug resistance and drug resistant tumour growth in a murine breast tumour model. BMC Cancer 19, 757 (2019). Hoshino, A., et al. Tumour exosome integrins determine organotropic metastasis. Nature 527, 329-335 (2015). Gray, M.J., et al. Phosphatidylserine-targeting antibodies augment the anti- tumorigenic activity of anti-PD-1 therapy by enhancing immune activation and downregulating pro- oncogenic factors induced by T-cell checkpoint inhibition in murine triple-negative breast cancers. Breast Cancer Res 18, 50 (2016). Bottos, A., et al. Decreased NK-cell tumour immunosurveillance consequent to JAK inhibition enhances metastasis in breast cancer models. Nat Commun 7, 12258 (2016). Vila-Leahey, A., et al. Ranitidine modifies myeloid cell populations and inhibits breast tumor development and spread in mice. Oncoimmunology 5, el 151591 (2016). Chan, T., et al. CHRFAM7A alters binding to the neuronal alpha-7 nicotinic acetylcholine receptor. Neurosci Lett 690, 126-131 (2019). Mullen, G., et al. (-)-Spiro[l-azabicyclo[2.2.2]octane-3,5'-oxazolidin-2'-one], a conformationally restricted analogue of acetylcholine, is a highly selective full agonist at the alpha 7 nicotinic acetylcholine receptor. J Med Chem 43, 4045- 4050 (2000). The, F.O., et al. Activation of the cholinergic anti-inflammatory pathway ameliorates postoperative ileus in mice. Gastroenterology 133, 1219-1228 (2007). van Maanen, M.A., et al. Stimulation of nicotinic acetylcholine receptors attenuates collagen-induced arthritis in mice. Arthritis Rheum 60, 114-122 (2009). Tracey, K.J. Reflex control of immunity. Nat Rev Immunol 9, 418-428 (2009). Reardon, C., et al. Lymphocyte-derived ACh regulates local innate but not adaptive immunity. Proc Natl Acad Sci USA 110, 1410-1415 (2013). Rosas-Ballina, M., et al. Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit. Science 334, 98-101 (2011). Erin, N., Duymus, O., Ozturk, S. & Demir, N. Activation of vagus nerve by semapimod alters substance P levels and decreases breast cancer metastasis. RegulPept 179, 101-108 (2012). Orr-Urtreger, A., et al. Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alpha-bungarotoxin binding sites and hippocampal fast nicotinic currents. J Neurosci 17, 9165-9171 (1997). Jensen, M.M., Jorgensen, J.T., Binderup, T. & Kjaer, A. Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18F-FDG-microPET or external caliper. BMC Med Imaging #, 16 (2008). Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408 (2001). Amend, S.R., Valkenburg, K.C. & Pienta, K.J. Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation. J Vis Exp (2016). Helft, J., et al. GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD1 lc(+)MHCII(+) Macrophages and Dendritic Cells. Immunity 42, 1197- 1211 (2015). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0131] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention. All publications, patents, and patent applications, Genbank sequences, websites and other published materials referred to throughout the disclosure herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application, Genbank sequences, websites and other published materials was specifically and individually indicated to be incorporated by reference. In the event that the definition of a term incorporated by reference conflicts with a term defined herein, this specification shall control.

Claims

WHAT IS CLAIMED IS:

1. A composition, or a pharmaceutically acceptable salt thereof, comprising an al nicotinic acetylcholine receptor agonist and an immune checkpoint inhibitor.

2. The composition of claim 1, wherein the al nicotinic acetylcholine receptor agonist is selected from acetylcholine; choline; anabasine (2-(3- pyridyl)-3,4,5,6-tetrahydropyridine); (3-(2,4-dimethoxybenzylidene) anabasine); (3E)-3-[(2,4-dimethoxyphenyl)methylidene]-3,4,5,6-tetrahydro-2,3'-bipyridine; (2'R)-spiro[l-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine];2-(3-pyridyl)-l-azabicyclo[3.2.2]nonane; (R)-7-chloro-N-(quinuclidin-3- yl)benzo[b]thiophene-2 -carboxamide, (2R)-N-(6-( IH-imidazol- 1 -yl)-4- pyrimidinyl)-4'H-spiro [4-azabicyclo[2.2.2] octane-2, 5 '-oxazol] -2'-amine, N- [(3S)-l-azabicyclo[2.2.2]oct-3-yl]-lH-indazole-3-carboxamide; (2- ((3r,4s,5s,7s)-l-azaadamantan-4-yloxy)-5-phenyl-l,3,4-thiadiazole; N-[(2S,3R)- 2-(pyridin-3 -ylmethyl)- 1 -azabicyclo [2 ,2.2]octan-3 -yl] - 1 -benzofuran-2- carboxamide ; N- [(3 R)- 1 -azabicyclo [2.2.2] octan-3 -yl] -4-chlorobenzamide hydrochloride; N-(3R)- 1 -Azabicyclo [2.2.2] oct-3 -yl-furo [2,3 -c]pyridine-5- carboxamide hydrochloride; (lR,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl lH-indole-3-carboxylate hydrochloride; 4-(4-Bromophenyl)-3a,4,5,9b- tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide; Nicotine; (lR,2R,4S)-2- (6-chloropyridin-3-yl)-7-azabicyclo[2.2.1]heptane; 2-[(2R,6S)-6-[(2S)-2- hydroxy-2 -phenylethyl] - 1 -methylpiperidin-2-yl] - 1 -phenylethanone; (3 S)- Spiro[l-azabicyclo[2.2.2]octane-3,5'-oxazolidine]-2'-one hydrochloride; a salt or a combination thereof.

3. The composition of claim 1, wherein the al nicotinic acetylcholine receptor agonist comprises the compound AR-R17779, wherein AR-R17779 has the structure of Formula I:

4. The composition of claim 1, wherein the immune checkpoint inhibitor inhibits PD-1, PD-L1, CTLA-4 or a combination thereof.

5. The composition of claim 1, wherein the immune checkpoint inhibitor comprises anti-PD-Ll .

6. The composition of claim 1, wherein the immune checkpoint inhibitor comprises pembrolizumab, nivolumab, ipilimumab, atezolizumab, durvalumab, avelumab, cemiplimab, penpulimab, retifanlimab, tiragolumab, relatlimab or a combination thereof.

7. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

8. A method of treating cancer in a subject in need thereof comprising administering to the subject a composition comprising a al nicotinic acetylcholine receptor agonist and an immune checkpoint inhibitor.

9. The method of claim 8, wherein the cancer is selected from skin cancer (including melanoma), lung cancer (including, Non-Small Cell Lung Cancer (NSCLC)), cervical cancer, colon cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, gastric cancer, breast cancer (including triple negative breast cancer), colorectal cancer, renal cancer, bladder cancer, Merkel cell carcinoma, urothelial carcinoma, stomach cancer, rectal cancer, cutaneous squamous cell carcinoma, nasopharyngeal carcinoma and other cancers, such as those with DNA repair deficiencies. For example, the compositions can reduce the severity of breast cancer, such as triple negative breast cancer.

10. The method of claim 8, wherein the cancer is breast cancer.

11. The method of claim 11 , wherein the breast cancer is triple negative breast cancer.

12. The method of claim 8, wherein survival of the subject treated with the composition is longer relative to survival of the subject treated with the al nicotinic acetylcholine receptor agonist or the immune checkpoint inhibitor alone.

13. The method of claim 8, wherein a tumor burden of the subject treated with the composition is decreased relative to a subject treated with the al nicotinic acetylcholine receptor agonist or the immune checkpoint inhibitor alone.

14. The method of claim 8, wherein the immune checkpoint inhibitor inhibits PD-1, PD-L1, CTLA-4 or a combination thereof.

15. The method of claim 8, wherein the immune checkpoint inhibitor comprises anti-PD-Ll.

16. The method of claim 8, wherein the immune checkpoint inhibitor comprises pembrolizumab, nivolumab, ipilimumab, atezolizumab, durvalumab, avelumab, cemiplimab, penpulimab, retifanlimab, tiragolumab, relatlimab or a combination thereof.

17. The method of claim 8, wherein the al nicotinic acetylcholine receptor agonist is selected from acetylcholine; choline; anabasine (2-(3-pyridyl)-3, 4,5,6- tetrahydropyridine); (3-(2,4-dimethoxybenzylidene) anabaseine); (3E)-3-[(2,4- dimethoxyphenyl)methylidene]-3,4,5,6-tetrahydro-2,3'-bipyridine; (2'R)-spiro[l- azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine];2-(3-pyridyl)-l-azabicyclo[3.2.2]nonane; (R)-7-chloro-N-(quinuclidin-3- yl)benzo[b]thiophene-2-carboxamide, (2R)-N-(6-(lH-imidazol-l-yl)-4- pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-oxazol]-2'-amine, N- [(3 S)- 1 -azabicyclo[2.2.2] oct-3 -yl] - lH-indazole-3 -carboxamide; (2- ((3r,4s,5s,7s)-l-azaadamantan-4-yloxy)-5-phenyl-l,3,4-thiadiazole; N-[(2S,3R)- 2-(pyridin-3 -ylmethyl)- 1 -azabicyclo [2.2 ,2]octan-3 -yl] - 1 -benzofuran-2- carboxamide ; N- [(3 R) - 1 -azabicyclo [2.2.2] octan-3 -yl] -4-chlorobenzamide hydrochloride; N-(3R)-l-Azabicyclo[2.2.2]oct-3-yl-furo[2,3-c]pyridine-5-carboxamide hydrochloride; (lR,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl lH-indole-3-carboxylate hydrochloride; 4-(4-Bromophenyl)-3a,4,5,9b- tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide; Nicotine; (lR,2R,4S)-2- (6-chloropyridin-3-yl)-7-azabicyclo[2.2.1]heptane; 2-[(2R,6S)-6-[(2S)-2- hydroxy-2 -phenylethyl] - 1 -methylpiperidin-2-yl] - 1 -phenylethanone; (3 S)- Spiro[l-azabicyclo[2.2.2]octane-3,5'-oxazolidine]-2'-one hydrochloride; a salt or a combination thereof.

18. The method of claim 8, wherein the al nicotinic acetylcholine receptor agonist comprises the compound AR-R17779, wherein AR-R17779 has the structure of Formula I:

19. The method of claim 8, wherein the composition activates tumor- associated myeloid immune cells.

20. The method of claim 8, wherein route of administration is selected from oral, topical, subcutaneous, intramuscular, intraperitoneal, intrathecal, transdermal, or intravenous injection.

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