Patent Application
20240358705
The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jul. 11, 2024, is named 2932719-000182-US4_SL.xml and is 23,409 bytes in size.
This invention is directed to compositions and methods for treating or preventing GPR84 responsive conditions. For example, the invention provides a method of preventing or treating cancer in a subject by administering to the subject a therapeutically effective amount of a GPR84 antagonist. Also provided is a method of preventing recurrence of cancer in a subject by administering to the subject a therapeutically effective amount of a GPR84 antagonist. Further, the invention provides a method of modulating the immune system of a subject afflicted with or at risk of cancer by administering to the subject a therapeutically effective amount of a GPR84 antagonist. The invention provides a method of preventing or treating an inflammatory response in a subject by administering to the subject a therapeutically effective amount of a GPR84 agonist. Also provided is a method of preventing an immune system disease or disorder in a subject by administering to the subject a therapeutically effective amount of a GPR84 agonist. Further, the invention provides a method treating an immune system disease or disorder in a subject by administering to the subject a therapeutically effective amount of a GPR84 agonist. Also, the invention provides a method of modulating the immune system of a subject afflicted with or at risk of an immune system disease or disorder by administering to the subject a therapeutically effective amount of a GPR84 agonist.
GPR84 is a medium-chain fatty acid receptor. It was found through a data mining strategy searching for GPCRs. Still defined as an orphan receptor, its ligand, MCFAs, was not discovered until years later. Further examination found that GPR84 is Gi/o GPCR, and interaction with its ligand leads to an intracellular reduction of cyclic adenosine monophosphate (cAMP). GPR84 is distributed among various tissues in the body, such as adipocytes, but also, GPR84 is found to have expression in immune cells. In studies of adipogenesis, GPR84 was upregulated in human adipocytes stimulated with inflammatory cytokines and mice fed with high-fat diet. In the immune tissue, GPR84 is expressed in monocytes, macrophages, granulocytes, T cells, and B cells.
Aspects of the invention are drawn to a method of preventing cancer in a subject. In embodiments, the method comprises administering to the subject a therapeutically effective amount of a GPR84 antagonist.
Aspects of the invention are also drawn towards a method of preventing recurrence of cancer in a subject. In embodiments, the method comprises administering to the subject a therapeutically effective amount of a GPR84 antagonist.
Still further, aspects of the invention are drawn towards a method treating cancer in a subject. In embodiments, the method comprising administering to the subject a therapeutically effective amount of a GPR84 antagonist.
Also, aspects of the invention are drawn towards a method of modulating the immune system of a subject afflicted with or at risk of cancer. In embodiments, the method comprises administering to the subject a therapeutically effective amount of a GPR84 antagonist.
In embodiments, modulating comprises increasing the cytotoxicity of T-cells, decreasing immunosuppressive effects of myeloid-derived-suppressor cells, or both.
In embodiments, the GPR84 antagonist comprises a compound according to:
In embodiments, the therapeutically effective amount comprises about 10 mg/kg to about 90 mg/kg.
In embodiments, the GPR84 antagonist is administered daily, weekly, biweekly, or monthly.
In embodiments, the method further comprises administering to the subject at least one additional anti-cancer agent. For example, the at least one additional anti-cancer agent comprises an immunotherapeutic agent, a chemotherapeutic agent, radiation therapy, or any combination thereof. For example, the immunotherapeutic agent comprises an immune checkpoint inhibitor, an adoptive T-cell therapy, a monoclonal antibody, an oncolytic virus therapy, a cancer vaccine, an immune system modulator, or any combination thereof. For example, the monoclonal antibody comprises an immune checkpoint inhibitor. For example, the monoclonal antibody comprises anti-OX-40, anti-PD-1, anti-PD-L1, anti-LAG3, or anti-CTLA-4. For example, the anti-PD-1 antibody comprises a pembrolizumab, nivolumab, or cemiplimab. For example, the anti-CTLA-4 antibody comprises Ipilimumab. For example, the adoptive T-cell therapy comprises tumor-infiltrating lymphocyte (TIL) therapy, engineered T-cell receptor (TCR) therapy, CAR T-cell therapy, or natural killer (NK) cell therapy. For example, the at least one chemotherapeutic agent comprises cyclophosphamide, gemcitabine, 5-fluorouracil, docetaxel, doxorubicin, oxaliplatin, mitoxantrone, melphalan, or anthracyclines.
In embodiments, the cancer comprises breast cancer, prostate cancer, colorectal cancer, cervical cancer, lung cancer, lymphoma, leukemia, pancreatic cancer, liver cancer, brain cancer, or skin cancer.
In embodiments, the subject has a familial history of cancer, a chronic inflammatory condition, or a genomic mutation. For example, the inflammatory condition comprises colitis, chronic prostatitis. For example, the genomic mutation comprises a mutation of BRCA1, BRCA2, MLH1, MSH2, MSH6, PMS2, EPCAM, APC, or MUTYH.
Aspects of the invention are further drawn to a method of activating a T cell or NK cell. For example, the method comprising contacting a T cell or NK cell with a GPR84 antagonist, wherein the GPR84 antagonist activates the T cell or NK cell.
Embodiments further comprise the step of activating the T cell or NK cell with an additional stimuli. For example, the stimuli comprise anti-CD3, anti-CD28, PSA, Muc-1, MAGE, carcinoembryonic antigen (CEA) or a combination thereof.
In embodiments, the method is an ex vivo method.
In embodiments, the cell comprises a genetically engineered T cell or genetically engineered NK cell. For example, the cell comprises a CAR T cell or a CAR NK cell.
Embodiments further comprise the step of obtaining a T cell or NK cell from a subject and culturing the T cell or NK cell in a medium comprising a GPR84 antagonist.
Embodiments further comprise the step of administering to a subject a therapeutically effective amount of the GPR84 antagonist.
In embodiments, the T cell or NK cell is dormant prior to culturing.
Embodiments further comprise administering the activated T cell or activated NK cell to a subject.
Aspects of the invention are also drawn towards a pharmaceutical composition comprising a GPR84 antagonist, at least one additional anti-cancer agent, and a pharmaceutically acceptable carrier, excipient, or diluent. In embodiments, the GPR84 antagonist comprises a compound according to:
In embodiments, the antagonist comprises a compound as described in U.S. Pat. No. 11,098,071.
In embodiments, the at least one additional anti-cancer agent comprises an immunotherapeutic agent or a chemotherapeutic agent. For example, the immunotherapeutic agent comprises a CAR T-cell, an anti-cancer antibody, or both. For example, the anti-cancer antibody comprises an anti-checkpoint inhibitor antibody. For example, the at least one chemotherapeutic agent comprises cyclophosphamide, gemcitabine, 5-fluorouracil, docetaxel, doxorubicin, oxaliplatin, mitoxantrone, melphalan, or anthracyclines.
Aspects of the invention are drawn to a method of preventing an inflammatory response in a subject.
Further, aspects of the invention are drawn to a method of preventing an immune system disease or disorder in a subject.
Still further, aspects of the invention are drawn to a method of treating or ameliorating a symptom of an immune system disease or disorder in a subject.
Also, aspects of the invention are drawn to a method of modulating the immune system of a subject afflicted with or at risk of an immune system disease or disorder. In embodiments, modulating comprises increasing immunosuppressive effects of myeloid-derived-suppressor cells, decreasing the pro-inflammatory and cytotoxicity of T-cells, or both.
In embodiments, the GPR84 agonist comprises a compound according to:
In embodiments, the therapeutically effective amount comprises about 10 mg/kg to about 90 mg/kg.
In embodiments, the GPR84 agonist is administered daily, weekly, biweekly, or monthly.
In embodiments, the method further comprises administering to the subject at least one additional immunomodulatory or anti-inflammatory agent. For example, the at least one additional anti-inflammatory agent or immunomodulatory treatment comprises an antibody, a corticosteroid (e.g., prednisone, prednisolone, methylprednisolone, dexamethasone, or triamcinolone), a non-steroidal anti-inflammatory, or any combination thereof. For example, the immunomodulatory treatment comprises methotrexate, a non-steroidal anti-inflammatory, or sulfasalazine. For example, the antibody comprises an anti-TNF antibody (e.g., etanercept, infliximab, adalimumab, golimumab, or certolizumab), an anti-IL-5 antibody, an anti-IL4/IL5/IL13 antibody (e.g., dupilumab), an anti-1b antibody, an anti-IL-6 antibody, an anti-IL-17 antibody, or an anti-IL-12/IL-23 antibody (e.g., ustekinumab).
In embodiments, the inflammatory response comprises inflammation.
In embodiments, the disease or disorder comprises psoriasis, psoriatic arthritis, an auto-immune disease, colitis, asthma, glomerulonephritis, hepatitis, myocarditis, transplant rejection, pre-diabetes, rheumatoid arthritis, lupus, Crohn's disease, graph versus host disease, or a combination thereof.
Aspects of the invention are also drawn to a method of culturing a population of T cells in the presence of a GPR84 agonist. For example, the T cell comprises a CD4 T cell, a CD8 T cell, or a combination thereof. In embodiments, the GPR84 agonist maintains the sternness of the population of T cells. In embodiments, the GPR84 agonist prevents the activation of the population of T cells. In embodiments, the GPR84 agonist comprises a compound according to:
In embodiments, the population of T cells and/or NK cells comprises a population of CAR T cells and/or CAR NK cells.
Embodiments further comprise a step of obtaining a T cell or population of T cells from a subject.
Embodiments further comprise a step of administering the cultured population of T cells to a subject.
Aspects of the invention are also drawn towards a pharmaceutical composition comprising a GPR84 agonist, at least one additional anti-inflammatory or immunomodulatory agent, and a pharmaceutically acceptable carrier, excipient, or diluent. In embodiments, the at least one anti-inflammatory agent or at least one immunomodulatory agent comprises an antibody, a corticosteroid (e.g., prednisone, prednisolone, methylprednisolone, dexamethasone, or triamcinolone), a non-steroidal anti-inflammatory agent, or any combination thereof. For example, the immunomodulatory treatment comprises methotrexate, a non-steroidal anti-inflammatory, or sulfasalazine.
In embodiments, the antibody comprises an anti-TNF antibody (e.g., etanercept, infliximab, adalimumab, golimumab, or certolizumab), an anti-IL-5 antibody, an anti-IL4/IL5/IL13 antibody (e.g., dupilumab), an anti-1b antibody, an anti-IL-6 antibody, an anti-IL-17 antibody, or an anti-IL-12/IL-23 antibody (e.g., ustekinumab).
Other objects and advantages of this invention will become readily apparent from the ensuing description.
Aspects of the invention are drawn towards compositions and methods for modulating GPR84.
For example, aspects of the invention are drawn towards compositions and methods for increasing the efficacy of anti-tumor immune responses using GPR84 antagonists. Antagonism of the GPR84 receptor promotes the development of increased cytotoxicity in T cells while inducing a decrease in the immunosuppressive function of MDSC. This observation is surprising, for example, because we can modulate the immune response by targeting one receptor that is present in myeloid cells (macrophages, granulocytes, eosinophils, myeloid-derived suppressor cells), T cells (CD8+ killer T cells, CD4+ helper cells and CD4+FoxP3+ regulatory T cells) and Natural Killer cells (NK). Such modulation can cause a beneficial effect. This type of immune modulation can be used to increase the response to a vaccine (such as an anti-cancer vaccine), or to improve the efficacy of an anti-cancer treatment (such as chemotherapy, radiation therapy or immunotherapy).
Also, aspects of the invention are drawn towards compositions and methods for modulating inflammatory diseases using a GPR84 agonist.
By using an agonist for GPR84, immune cells can enter an anti-inflammatory state which could be of benefit in diseases associated with an uncontrolled inflammatory response such as colitis, pancreatitis, hepatitis, myocarditis, rheumatoid arthritis, lupus, early stage of diabetes (pre-diabetic conditions or insulitis) and others. The use of an agonist stimulates the myeloid cells to express arginase 1 and iNOS and causes T cells to decrease their activation as shown by a decrease in the expression of granzyme B and perform (in CD8T cells) and the production of inflammatory cytokines such as IFNg, IL2 and others (in CD4T cells).
Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the advantageous methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and cannot be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided can be different from the actual publication dates that can need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other reasonable order.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, toxicology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.
The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.
The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.
As used herein the term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
The term “subject” or “patient” can refer to any organism to which aspects of the invention can be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. For example, subjects to which compounds of the disclosure can be administered include animals, such as mammals. Non-limiting examples of mammals include primates, such as humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals for example pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. The term “living subject” can refer to a subject noted above or another organism that is alive. The term “living subject” can refer to the entire subject or organism and not just a part excised (e.g., a liver or other organ) from the living subject. As used herein, “pharmaceutically acceptable derivatives” of a compound can include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof. Such derivatives can be readily prepared by those of skill in this art using known methods for such derivatization. The compounds produced can be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs. As used interchangeably herein, “subject,” “individual,” or “patient,” can refer to a vertebrate, such as a mammal, for example a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. The term “pet” includes a dog, cat, guinea pig, mouse, rat, rabbit, ferret, and the like. The term farm animal includes a horse, sheep, goat, chicken, pig, cow, donkey, llama, alpaca, turkey, and the like.
The term “administering” can refer to introducing a substance into a subject. Any route of administration can be utilized including, for example, intranasal, topical, oral, parenteral, intravitreal, intraocular, ocular, subretinal, intrathecal, intravenous, subcutaneous, transcutaneous, intracutaneous, intracranial and the like administration. For example, “parenteral administration” can refer to administration via injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, and intramuscular administration.
In embodiments, “administering” can also refer to providing a therapeutically effective amount of a formulation or pharmaceutical composition to a subject. The formulation or pharmaceutical compound can be administered alone, but can be administered with other compounds, excipients, fillers, binders, carriers or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice. Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; and the like.
The term “simultaneous administration” can refer to a first agent and a second agent, when together in the therapeutic combination therapy, are administered either less than about 15 minutes, e.g., less than about 10, 5, or 1 minute. When the first agent and the second agent are administered simultaneously, the first and second treatments can be in the same composition (e.g., a composition comprising both the first and second therapeutic agents) or separately (e.g., the first therapeutic agent is contained in one composition and the second treatment is contained in another composition).
The term “sequential administration” can refer to a first agent and a second agent administered to a subject greater than about 15 minutes apart, such as greater than about 20, 30, 40, 50, 60 minutes, or greater than 60 minutes apart. Either agent can be administered first. For example, the first agent and the second agent can be included in separate compositions, which can be included in the same or different packages or kits.
The term “simultaneous administration” can refer to when the administration of a first agent and a second agent administered to a subject overlap each other.
The terms “co-administration” or the like, as used herein, can refer to the administration of a first active agent and at least one additional active agent to a single subject, and is intended to include treatment regimens in which the compounds and/or agents are administered by the same or different route of administration, in the same or a different dosage form, and at the same or different time.
The term “in combination” can refer to the use of more than one therapies (e.g., one or more prophylactic and/or therapeutic agents). The use of the term “in combination” does not restrict the order in which therapies are administered to a subject with a disease or disorder, or the route of administration.
The term “therapeutically effective amount” can refer to that amount of an embodiment of the composition or pharmaceutical composition being administered that will relieve to some extent one or more of the symptoms of the disease or condition being treated, and/or that amount that will prevent, to some extent, one or more of the symptoms of the condition or disease that the subject being treated has or is at risk of developing.
The phrase “pharmaceutical composition” or a “pharmaceutical formulation” can refer to a composition or pharmaceutical composition suitable for administration to a subject, such as a mammal, especially a human and that can refer to the combination of an active agent(s), or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo. A “pharmaceutical composition” can be sterile and can be free of contaminants that can elicit an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, intranasal, topical, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, by stent-eluting devices, catheters-eluting devices, intravascular balloons, inhalational and the like.
A “pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” or “pharmaceutically acceptable adjuvant” can refer to an excipient, diluent, carrier, and/or adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use and/or human pharmaceutical use. “A pharmaceutically acceptable excipient, diluent, carrier and/or adjuvant” as used herein can include one and more such excipients, diluents, carriers, and adjuvants.
“Modulating” can refer to regulating or adjusting the degree of activity of a process or the degree of an effect. “Modulating” includes activation, amplification, attenuation, and suppression, for example.
“Treatment” and “treating” can refer to the management and care of a subject for the purpose of combating a condition, disease or disorder, such as an inflammatory response, in any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. The term can include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, disease or disorder; curing or eliminating the condition, disease or disorder; and/or preventing the condition, disease or disorder, wherein “preventing” or “prevention” can refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder, and includes the administration of the active compounds to prevent or reduce the risk of the onset of symptoms or complications.
“Preventing” and “prevention” can refer to the prevention of the occurrence of a disease or disorder. In embodiments, the preventative treatment can reduce the recurrence of a disease or disorder.
The phrase “preventing cancer” can refer to prevention of cancer occurrence. In embodiments, the preventative treatment reduces the recurrence of the cancer. In other embodiments, preventative treatment decreases the risk of a patient from developing a cancer or inhibits progression of a pre-cancerous state (e.g., a colon polyp) to actual malignancy.
The term “cancer” can refer to the spectrum of pathological symptoms associated with the initiation or progression, as well as metastasis, of malignant tumors. The term “tumor” can refer to a new growth of tissue in which the multiplication of cells is uncontrolled and progressive. In embodiments, the tumor can be a malignant tumor, one in which the primary tumor has the properties of invasion or metastasis or which shows a greater degree of anaplasia than do benign tumors. Thus, “treatment of cancer” or “treating cancer” can refer to an activity that prevents, alleviates or ameliorates any of the primary phenomena (initiation, progression, metastasis) or secondary symptoms associated with the disease.
Treating cancer can be indicated by, for example, inhibiting or delaying invasiveness of a cancer. “Cancer invasion” can refer to the movement caused by cancer cells in vivo, into or through biological tissue or the like. For example, movements caused by cancer cells into or through barriers formed by special cell-based proteins, such as collagen and Matrigel, and other substances.
The term “anti-cancer” can refer to an action of suppressing the growth of cancer cells or killing cancer cells and an action of suppressing or blocking metastasis of cancer cells in connection with prevention and treatment of cancer. For example, anti-cancer can refer to inhibition of formation, infiltration, metastasis, and growth of cancers.
The term “modulating” can refer to an increase, decrease, or otherwise change an activity. For example, to “modulate the immune system” can refer to stimulating certain responses of the immune system by making it more reactive, for example intervening, via the production of specific cytokines, in development of the cells involved in the immune response. “Modulating the immune system” can also refer to depressing certain responses of the immune system by making it less reactive.
The term “immune system” can refer to the bodily system that protects the body from foreign substances, cells, and tissues by producing the immune response. For example, the immune system can comprise the thymus, spleen, lymph nodes, lymphoid tissue, lymphocytes, and antibodies. As used herein, the term “immune response” can refer to a bodily response to an antigen that occurs when lymphocytes identify the antigenic molecule as foreign and induce the formation of antibodies and lymphocytes.
The term “immune response” can refer to a bodily response to an antigen that occurs when lymphocytes identify the antigenic molecule as foreign and induce the formation of antibodies and lymphocytes.
The terms “inflammation” and “inflammatory reaction” can be used interchangeably. As used herein, the term “inflammation” can refer to a response to cellular injury which can be marked by capillary dilatation, leukocytic infiltration, redness, heat, pain, swelling, and loss of function and serves as a mechanism initiating the elimination of noxious agents and of damaged tissue.
The term “cytokine” can refer to any class of immunoregulatory proteins that are secreted by cells. For example, the cells are cells off the immune system. For example, the cytokine can be produced in the presence of disease or immunization and contribute to immune responses, inflammation, and endothelial cell activation. As used herein, the terms “pro-inflammatory cytokine” and “inflammatory cytokine” can be used interchangeably. In embodiments, pro-inflammatory cytokines can be produced by activated macrophages and are involved in the up-regulation of inflammatory reactions.
The term “cell of the immune system” or “immune cells” can be used interchangeably. For example, cells of the immune system can comprise lymphocytes (T-cells, B-cells, and NK cells), neutrophils, and monocytes/macrophages.
An “autologous cell” can refer to a cell which was derived from the same individual that is being treated by cell therapy.
A “donor cell” can refer to a cell that was derived from an individual other than the individual being treated by cell therapy.
An “allogeneic cell” can refer to a genetically distinct cell.
The term “immunotherapy” can refer to a treatment or prevention of disease that involves stimulation, enhancement, suppression, or desensitization of the immune system. For example, the disease can comprise an autoimmune disorder or cancer.
The term “fatty acid” can refer to a carboxylic acid with an aliphatic chain. For example, the aliphatic chain can be saturated or unsaturated.
The term “antagonist” can refer to a compound or composition that can decrease, block, inhibit, abrogate, or interfere with a biological response by binding to or blocking a cellular constituent. As used herein, the term “agonist” can refer to a compound or composition that interacts with a cellular constituent and elicits an observable response. For example, the cellular constitute comprises a receptor. For example, the receptor can comprise GPR84.
The term “agonist” can refer to any compound that stimulates activity at a receptor or receptors normally stimulated by naturally occurring substances, thus triggering a response.
The term “ligand” can refer to a molecule or agent that interacts with another species. For example, the ligand can comprise an agonist or an antagonist. For example, the species can comprise a receptor. For example, the receptor is can comprise GPR84.
The term “immunotherapeutic agent” can refer to an agent that can be used on or used to modify an immune mechanism or immune response.
The term “chemotherapeutic agent” can refer to any chemical compound useful in the treatment of a neoplastic disease, such as cancer.
The term “in vivo” can refer to an event that takes place in a subject's body.
The term “in vitro” can refer to an event that takes places outside of a subject's body.
The term “ex vivo” can refer to outside a living subject. Examples of ex vivo cell populations include in vitro cell cultures and biological samples such as fluid or tissue samples from humans or animals. Such samples can be obtained by methods well known in the art. Exemplary biological fluid samples include blood, cerebrospinal fluid, urine, saliva. Exemplary tissue samples include tumors and biopsies thereof. In this context, the present compounds can be in numerous applications, both therapeutic and experimental.
The term “genetically engineered cell” can refer to any cell of any organism that is modified, transformed, or manipulated by addition or modification of a gene, a DNA or RNA sequence, or protein or polypeptide. Isolated cells, host cells, and genetically engineered cells include isolated immune cells, such as NK cells and T cells, that contain the DNA or RNA sequences encoding a chimeric receptor or chimeric receptor complex and express the chimeric receptor on the cell surface. Isolated host cells and genetically engineered cells can be used, for example, for enhancing an NK cell activity or a T lymphocyte activity, treatment of cancer, and treatment of infectious diseases.
Aspects of the invention are drawn to pharmaceutical compositions comprising a GPR84 antagonist. Non-limiting examples of GPR84 antagonists comprise a compound according to:
In embodiments, the antagonist comprises a compound as described in U.S. Pat. No. 11,098,071.
Aspects of the invention are drawn to pharmaceutical compositions comprising a GPR84 agonist. Non-limiting examples of GPR84 agonists comprise a compound according to:
In embodiments, the pharmaceutical composition can comprise at least one additional active agent. Non-limiting examples of the at least one additional active include an anti-cancer agent, an anti-inflammatory agent, a pain reliever, or any combination thereof.
In embodiments, the anti-cancer agent comprises an immunotherapeutic agent. For example, the immunotherapeutic agent can be a genetically engineered cell (e.g., CAR T-cell or CAR NK cell), a monoclonal antibody (e.g., anti-cancer antibody), an adoptive T-cell therapy, an oncolytic virus therapy, a cancer vaccine, an immune system modulator, or any combination thereof. For example, the at least one additional active agent comprises an immunomodulatory agent or an anti-inflammatory agent. For example, the at least one additional anti-inflammatory agent or immunomodulatory treatment comprises an immunosuppressant (e.g., methotrexate), an anti-inflammatory (e.g., sulfasalazine), an antibody, a corticosteroid (e.g., prednisone, prednisolone, methylprednisolone, dexamethasone, or triamcinolone), a non-steroidal anti-inflammatory (e.g., ibuprofen, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin), or any combination thereof. For example, the antibody comprises an anti-TNF antibody (e.g., etanercept, infliximab, adalimumab, golimumab, or certolizumab), an anti-IL-5 antibody, an anti-IL4/IL5/IL13 antibody (e.g., dupilumab), an anti-1b antibody, an anti-IL-6 antibody, an anti-IL-17 antibody, or an anti-IL-12/IL-23 antibody (e.g., ustekinumab).
In embodiments, the monoclonal antibody comprises a full-length monoclonal antibody, a fragment antigen binding (Fab) fragment, a single-chain variable fragment (scFv), single-domain antibody (sdAb), a bispecific antibody, or a trispecific antibody.
In embodiments the anti-cancer antibody comprises anti-OX-40, or an anti-checkpoint inhibitor. The term “checkpoint inhibitor” can refer to any molecule, including antibodies and small molecules, that block the immunosuppression pathway induced by one or more checkpoint proteins. For example, the anti-checkpoint inhibitor comprises anti-PD-1 (e.g., pembrolizumab, nivolumab, or cemiplimab), anti-PD-L1, anti-LAG3, or anti-CTLA4 (e.g., ipilimumab).
In embodiments, the anti-cancer agent comprises a chemotherapeutic agent. For example, the chemotherapeutic agent comprises cyclophosphamide, gemcitabine, 5-fluorouracil, docetaxel, doxorubicin, oxaliplatin, mitoxantrone, melphalan, or anthracyclines.
In embodiments, the adoptive T-cell therapy comprises tumor-infiltrating lymphocyte (TIL) therapy, engineered T-cell receptor (TCR) therapy, CAR T-cell therapy, or natural killer (NK) cell therapy.
In embodiments, the anti-cancer agent comprises sipuleucel-T or bacillus Calmette-Guerin.
In embodiments, the oncolytic virus therapy comprises talimogene laherparepvec.
In embodiments, the immune system modulator comprises an interleukin (e.g., IL-2, IL-7, IL-12, or IL-21), an interferon, or an immunomodulators (IMiDs).
Pharmaceutical composition can also be included, or packaged, with other non-toxic compounds, such as pharmaceutically acceptable carriers, excipients, diluents, binders and fillers including, but not limited to, glucose, lactose, gum acacia, gelatin, mannitol, xanthan gum, locust bean gum, galactose, oligosaccharides and/or polysaccharides, starch paste, magnesium trisilicate, talc, com starch, starch fragments, keratin, colloidal silica, potato starch, urea, dextrans, dextrins, and the like. For example, the pharmaceutically acceptable carriers, excipients, binders, and fillers for use in the practice of the invention are those which render the compounds of the invention amenable to intranasal delivery, oral delivery, parenteral delivery, intravitreal delivery, intraocular delivery, ocular delivery, subretinal delivery, intrathecal delivery, intravenous delivery, subcutaneous delivery, transcutaneous delivery, intracutaneous delivery, intracranial delivery, topical delivery and the like. Moreover, the packaging material can be biologically inert or lack bioactivity, such as plastic polymers or silicone, and can be processed internally by the subject without affecting the effectiveness of the composition/formulation packaged and/or delivered therewith.
In embodiments, the pharmaceutical compositions can comprise pharmaceutically acceptable salts. Pharmaceutically acceptable salts can include, but are not limited to, amine salts, such as but not limited to N,N′-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl) aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates.
Different forms of the pharmaceutical composition can be calibrated in order to adapt both to different subjects and to the different needs of a single subject. However, the pharmaceutical composition need not counter every cause in every subject. Rather, by countering the necessary causes, the pharmaceutical composition will restore the body to its normal function. Then the body will correct the remaining deficiencies.
For oral preparations, the pharmaceutical composition can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as com starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
Embodiments of the pharmaceutical composition can be formulated into preparations for injection by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
Embodiments of the composition or pharmaceutical composition can be utilized in aerosol formulation to be administered via inhalation. Embodiments of the composition or pharmaceutical composition can be formulated into pressurized acceptable propellants such as dichiorodifluoromethane, propane, nitrogen and the like.
Unit dosage forms for oral administration, such as syrups, elixirs, and suspensions, can be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compositions. Similarly, unit dosage forms for injection or intravenous administration can comprise the pharmaceutical composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
Aspects of the invention are drawn to methods of preventing an inflammatory response in a subject (e.g., inflammation).
Also, aspects of the invention are drawn to methods for preventing, treating, or ameliorating a symptom of an immune system disease or disorder in a subject. For example, the immune system disease or disorder comprises psoriasis, psoriatic arthritis, an auto-immune disease, colitis, asthma, glomerulonephritis, hepatitis, myocarditis, transplant rejection, pre-diabetes, rheumatoid arthritis, lupus, Crohn's disease, graph versus host disease, or a combination thereof.
Further, aspects of the invention are drawn to methods of modulating the immune system of a subject afflicted with or at risk of an immune system disease or disorder. In embodiments, modulating comprises increasing immunosuppressive effects of myeloid-derived-suppressor cells (MDSC), decreasing the pro-inflammatory and cytotoxic effects of inflammatory cells, such as T-cells and NK cells. The term “T cell” can refer to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of T cell receptors on the cell surface. T cells can be isolated or obtained from commercial sources. “T cells” are all types of immune cells that express CD3, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, regulatory T cells (Tregs), and immune cells including gamma-delta T cells. “Cytotoxic cells” include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are cells that can mediate a cytotoxic response. The term “NK cells”, also known as natural killer cells, can refer to a type of lymphocyte that is derived from the bone marrow and plays a vital role in the innate immune system. NK cells provide a rapid immune response against virus-infected cells, tumor cells, or other stressed cells, even in the absence of antibodies and major histocompatibility complexes on the cell surface. The term “myeloid-derived suppressor cell” or “MDSC” can refer to a cell of the immune system that modulates the activity of a variety of effector cells and antigen-presenting cells, such as T cells, NK cells, dendritic cells, and macrophages, among others.
In embodiments, the method comprises administering to the subject a therapeutically effective amount of a GPR84 agonist. Non-limiting examples of GPR84 agonists comprise a compound according to:
In embodiments, the GPR84 agonist can be provided in a pharmaceutical composition. Non-limiting examples of the GPR84 agonist comprises a compound according to:
Aspects of the invention are drawn to methods for preventing cancer, preventing the recurrence of cancer, and/or treating cancer in a subject. For example, an embodiment comprises a method of preventing cancer in a subject comprising administering to the subject a therapeutically effective amount of a GPR84 antagonist.
“Cancer” can refer to diseases in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord. Also called malignancy.
In embodiments, the cancer can comprise a solid tumor or a liquid cancer. A “solid tumor” can refer to an abnormal mass of tissue that usually does not contain cysts or liquid. A “non-solid tumor”, which can be referred to as a “liquid cancer”, can refer to neoplasia of the hemopoietic system, such as lymphoma, myeloma, and leukemia, or neoplasia without solid formation generally and with spread substantially.
In some embodiment, the solid tumors include but not limited to brain cancer, lung cancer, liver cancer, hepatocellular carcinoma (HCC), esophageal cancer, cholangiocarcinoma, gallbladder carcinoma, stomach cancer, abdominal cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, renal cancer, bone cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, colorectal cancer, colon cancer, rectal cancer, bladder cancer, superficial bladder cancer, prostate cancer, adrenal tumors, squamous cell carcinoma, neuroma, malignant neuroma, myoepithelial carcinoma, synovial sarcoma, rhabdomyosarcoma, gastrointestinal interstitial cell tumor, skin cancer, basal cell carcinoma, malignant melanoma, thyroid cancer, nasopharyngeal carcinoma, hemangioma, epidermoid carcinoma, head and neck cancer, glioma, or Kaposi's sarcoma.
In some embodiments, the non-solid tumors include but not limited to leukemia, acute leukemia, chronic leukemia, chronic myelocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, T-cell leukemia, hairy cell leukemia, polycythemia, myelodysplastic syndrome, multiple myeloma, lymphadenoma, Hodgkin's lymphoma, and Non-Hodgkin's lymphoma.
In some embodiments, the cancer comprises breast cancer, prostate cancer, colorectal cancer, cervical cancer, lung cancer, lymphoma, leukemia, pancreatic cancer, liver cancer, brain cancer, or skin cancer.
In embodiments, the cancer comprises a cancer that is resistant to immune checkpoint inhibitors. For example, aspects of the invention can sensitize checkpoint resistant tumors to treatment, such as anti-cancer antibodies, by inhibiting suppressive myeloid cells (e.g., MDSC, tumor associated macrophages (TAM) or M2 macrophages) and regulatory-T cells, while at the same time activating T cells (e.g., killer and helper) and NK cells.
In embodiments, the subject afflicted with cancer or at risk of being afflicted with cancer has a familial history of cancer, a chronic inflammatory condition (e.g., colitis, chronic prostatitis), or a genomic mutation that predisposes that subject to the development of cancer. For example, the genomic mutation can be a mutation of BRCA1, BRCA2, MLH1, MSH2, MSH6, PMS2, EPCAM, APC, MUTYH, or OGG1. For example, Lynch syndrome, also known as hereditary non-polyposis colorectal cancer (HNPCC), is the most common cause of hereditary colorectal (colon) cancer. People with Lynch syndrome can get colorectal cancer and other cancers, and at a younger age (before 50), including uterine (endometrial), stomach, liver, kidney, brain, and certain types of skin cancers. Lynch syndrome is due to inherited changes (mutations) in genes that affect DNA mismatch repair, a process that fixes mistakes made when DNA is copied. These genes (MLHL, MSH2, MSH6, PMS2, and EPCAM) normally protect you from getting certain cancers, but some mutations in these genes prevent them from working properly. As another example, the adenomatous polyposis coli (APC) gene is a tumor suppressor gene, and mutations resulting in loss of APC protein function are associated with carcinogenesis. Another familial form of colorectal cancer, MUTYH-associated polyposis (MAP), was first described in families with multiple colorectal adenomas or carcinomas who lacked inherited APC mutations. Also, OGG1 acts together with MYH and MTH1 to identify and remove 8-oxoguanine that has been incorporated into DNA. OGG1 variants have been reported in association with colorectal cancer, alone or in combination with mutations in other genes.
Aspects of the invention are also drawn towards methods of modulating the immune system of a subject. For example, the subject is afflicted with cancer, or at risk of being afflicted with cancer. In embodiments, the method comprises administering to the subject a therapeutically effective amount of a GPR84 antagonist as described herein. In embodiments, the GPR84 antagonist can be provided in a pharmaceutical composition.
In embodiments, modulating comprises increasing the cytotoxicity of T-cells, decreasing immunosuppressive effects of myeloid-derived-suppressor cells, or both.
The term “T cell” can refer to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of T cell receptors on the cell surface. T cells can be isolated or obtained from commercial sources. “T cells” are all types of immune cells that express CD3, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, regulatory T cells (Tregs), and immune cells including gamma-delta T cells. “Cytotoxic cells” include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are cells that can mediate a cytotoxic response.
The term “NK cells”, also known as natural killer cells, can refer to a type of lymphocyte that is derived from the bone marrow and plays a vital role in the innate immune system. NK cells provide a rapid immune response against virus-infected cells, tumor cells, or other stressed cells, even in the absence of antibodies and major histocompatibility complexes on the cell surface.
The term “myeloid-derived suppressor cell” or “MDSC” can refer to a cell of the immune system that modulates the activity of a variety of effector cells and antigen-presenting cells, such as T cells, NK cells, dendritic cells, and macrophages, among others.
In embodiments, the GPR84 antagonist can be provided in a pharmaceutical composition. Non-limiting examples of GPR84 antagonists comprise a compound according to.
In embodiments, the antagonist comprises a compound as described in U.S. Pat. No. 11,098,071
In embodiments, a therapeutically effective amount can comprise less than about 0.1 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 120 mg/kg, about 135 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 225 mg/kg, about 250 mg/kg, about 275 mg/kg, about 300 mg/kg, about 325 mg/kg, about 350 mg/kg, about 375 mg/kg, about 400 mg/kg, about 425 mg/kg, about 450 mg/kg, about 475 mg/kg, about 500 mg/kg, about 525 mg/kg, about 550 mg/kg, about 575 mg/kg, about 600 mg/kg, about 625 mg/kg, about 650 mg/kg, about 675 mg/kg, about 700 mg/kg, about 725 mg/kg, about 750 mg/kg, about 775 mg/kg, about 800 mg/kg, about 825 mg/kg, about 850 mg/kg, about 875 mg/kg, about 900 mg/kg, about 1.0 g/kg, about 1.5 g/kg, about 2.0 g/kg, about 2.5 g/kg, about 5 g/kg, about 10 g/kg, about 25 g/kg, about 50 g/kg, or more than 50 g/kg of compound per body weight of a subject.
In embodiments, the therapeutically effective amount comprises less than about 0.1 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 120 mg, about 135 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 1.0 g, about 1.5 g, about 2.0 g, about 2.5 g, about 5 g, about 10 g, about 25 g, about 50 g, or more than 50 g.
In embodiments, the compound can be administered alone, or can be administered as a pharmaceutical composition together with other compounds, excipients, carriers, diluents, fillers, binders, or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice. Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; and the like.
Embodiments can be administered to a subject in one or more doses. The dose level can vary as a function of the specific composition or pharmaceutical composition administered, the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for a given compound are readily determinable by a variety of means. For example, dosages can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can be employed to help identify optimal dosage ranges. The precise dose to be employed can also depend on the route of administration and can be decided according to the judgment of the practitioner and each patient's circumstances.
In an embodiment, multiple doses of the pharmaceutical composition can be administered. The frequency of administration and the duration of administration of the pharmaceutical composition can vary depending on any of a variety of factors, e.g., patient response, severity of the symptoms, and the like. For example, in an embodiment, the pharmaceutical composition can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (ad), twice a day (qid), three times a day (tid), or four times a day. In an embodiment, the pharmaceutical composition can be administered 1 to 4 times a day over a period of time, such as 1 to 10-day time period, or longer than a 10-day period of time.
In embodiments, the GPR84 antagonist can be administered in combination with one or more additional active agents. For example, a first agent (e.g., a prophylactic or therapeutic agent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second agent (e.g., a prophylactic or therapeutic agent) to a subject with a disease or disorder or a symptom thereof.
In embodiments, the pharmaceutical composition comprising the GPR84 antagonist and the second active agent can be administered sequentially, such as one before the other, or concurrently or simultaneously, such as at about the same time.
Aspects of the invention are drawn towards compositions and methods for manufacturing cellular therapies. For example, aspects of the invention comprise methods of culturing a population of cells, and subsequently activating the population of cells (e.g., a T cell, or an NK cell). For example, the method can comprise culturing the population of cells (e.g., T cells or NK cells) in the presence of a GPR84 agonist, whereby the GPR84 agonist maintains the sternness (i.e., non-activated state). Keeping CAR cells in a state of stemness (i.e., not fully mature or active) increases their survival and their therapeutic function once administered to a subject. The use of a GPR84 agonist during the in vitro generation and culture of CAR cells will maintain sternness, while still being able to divide and increase in number.
Aspects of the invention are drawn towards compositions and methods for manufacturing cellular therapies. For example, aspects of the invention comprise methods of activating a cell (e.g., a T cell). To “activate” T cells and Natural killer (“NK”) cells can refer to induce a change in their biologic state by which the cells express activation markers, produce cytokines, proliferate and/or become cytotoxic to target cells. All these changes can be produced by primary stimulatory signals. Co-stimulatory signals amplify the magnitude of the primary signals and suppress cell death following initial stimulation resulting in a more durable activation state and thus a higher cytotoxic capacity.
In embodiments, the T cell comprises a CD4 T cell, a CD8 T cell, or a combination thereof.
Embodiments can comprise the step of obtaining a population of cells (e.g., T cells or NK cells) from a subject, and culturing the population of cells in a medium comprising a GPR84 agonist. For example, the population of cells can be maintained in their dormant state by culturing the cells in a medium comprising a GPR84 agonist.
In embodiments, the GPR84 agonist comprises a compound according to:
Activating the dormant cells can be accomplished by culturing the cells in a medium comprising a GPR84 antagonist. In embodiments, the GPR84 antagonist comprises a compound according to:
In embodiments, the antagonist comprises a compound as described in U.S. Pat. No. 11,098,071.
Activating the cells can be supplemented by additional stimuli, such as anti-CD3, anti-CD28, PSA, Muc-1, MAGE, carcinoembryonic antigen (CEA) or a combination thereof.
For example, the method comprises contacting a cell (e.g., a T cell) with a GPR84 antagonist, wherein the GPR84 antagonist activates the cell. For example, the method can comprise obtaining a population of cells (e.g., T cells) from a subject, and culturing the population of cells in a medium comprising a GPR84 antagonist. For example, the population of cells can be largely dormant prior to culturing in a medium comprising a GPR84 antagonist, which are then activated subsequent to the culturing step.
In embodiments, the method comprises contacting a cell (e.g., a T cell) with a GPR84 antagonist, wherein the GPR84 antagonist activates the cell. For example, the method can comprise obtaining a population of cells (e.g., T cells) from a subject, and culturing the population of cells in a medium comprising a GPR84 antagonist. For example, the population of cells can be largely dormant prior to culturing in a medium comprising a GPR84 antagonist, which are then activated subsequent to the culturing step.
In embodiments, the GPR84 antagonist comprises a compound according to:
Embodiments can further comprise a step of activating the cell (e.g., T cell) with an additional stimuli. For example, the additional stimuli comprises anti-CD3, anti-CD28, PSA, Muc-1, MAGE, carcinoembryonic antigen (CEA) or a combination thereof.
In embodiments, the method is an ex vivo method.
In embodiments, the cell comprises a genetically engineered cell, such as a genetically engineered T cell or a genetically engineered NK cell.
CAR T-cell therapies redirect a patient's T-cells to kill tumor cells by the exogenous expression of a CAR on a T-cell, for example. A CAR can be a membrane spanning fusion protein that links the antigen recognition domain of an antibody to the intracellular signaling domains of the T-cell receptor and co-receptor. A suitable cell can be used, for example, that can secrete an antibody. The antibody “payloads” to be secreted, can be, for example, minibodies, VHH, scFvs, IgG molecules, bispecific fusion molecules, and other antibody fragments. Upon contact or engineering, the cell described herein can then be introduced to a patient in need of a treatment by infusion therapies known to one of skill in the art.
Exemplary CARs and CAR factories useful in aspects of the invention include those disclosed in, for example, PCT/US2015/067225 and PCT/US2019/022272, each of which are hereby incorporated by reference in their entireties. In embodiments, the combination of an agonist and an antagonist of the GPR84 FFAR can be used in the generation of T cells for immunotherapy purposes, in particular CAR-T cells. For example, it has been shown that keeping CAR-T cells in a state of “stemness” (i.e., not fully mature and active), increases their survival and their therapeutic function when transferred in vivo into mice with tumors or in cancer patients. Without being bound by theory, the use of an agonist during the in vitro generation and culture of CAR-T cells can keep them in a state of “stemness”, while still being able to divide and increase in number. Once the CAR-T cells are harvested and infused into a patient with cancer, then one can treat the patient with an antagonist which can increase the maturation and cytotoxic function of the CAR-T cells as they reach the tumor. In addition the antagonist can decrease the immunosuppressive function of the myeloid cells in the tumor, allowing the CAR-T cells to fully execute their anti-tumor effect.
In embodiments, the activated cell (e.g. T cell) can be administered to a subject.
Aspects of the invention are directed towards kits, such as kits comprising compositions as described herein. For example, the kit can comprise therapeutic combination compositions described herein.
In one embodiment, the kit includes (a) a container that contains a composition, such as that described herein, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the agents for therapeutic benefit.
In an embodiment, the kit includes two or more agents. For example, the kit includes a container comprising a GPR84 antagonist, and a second container comprising a second active agent.
In an embodiment, the kit includes two or more agents. For example, the kit includes a container comprising a GPR84 antagonist, and a second container comprising a second active agent.
In an embodiment, the kit includes two or more agents. For example, the kit includes a containing comprising a GPR84 antagonist, and a second container comprises a GPR84 agonist. In embodiments, the kit further comprises a third container comprising a third active agent.
The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods of administering the therapeutic combination composition, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein), to treat a subject who has a nerve disconnectivity disorder). The information can be provided in a variety of formats, include printed text, computer readable material, video recording, or audio recording, or information that provides a link or address to substantive material.
The composition in the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. The GPR84 antagonist can be provided in any form, e.g., liquid, dried or lyophilized form, or for example, substantially pure and/or sterile. When the agents are provided in a liquid solution, the liquid solution is an aqueous solution. When the agents are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.
The kit can include one or more containers for the composition or compositions containing the agents. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents. The containers can include a combination unit dosage, e.g., in a desired ratio. For example, the kit includes a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kits can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight. The kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.
Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
Cells of the immune system use a variety of energy sources to support its functions, including sugars and fats. Fats contain fatty acids and are used in fatty acid oxidation, while sugars are the substrate used in glycolysis. Fatty acids are made of various carbon lengths and degrees of saturation. These variations can change the way and where fatty acids are used in tissue. Currently, there are four families of fatty acids. These are short-chain fatty acids (<5 carbons), medium-chain fatty acids (6-12 carbons), long-chain fatty acids (13-21 carbons) and very-long-chain fatty acids (>22 carbons). Short-chain fatty acids are synthesized by gut intestinal fermentation of indigestible foods. Medium, long, and very-long chains can either be synthesized from cells or obtained through the diet. Recently, FFAs have been more extensively studied as multiple reports have found that they play a role in regulating metabolic disorders. (1, 2)
Fatty acids are not only a component of cellular energy, but also serve as signaling ligands. Free fatty acids (FFA) bind to a family of G-protein coupled receptors (GPCRs), entitled free fatty acid receptors (FFAR) that are found on various cells in the body. Ligation with these GPCRs commits an intracellular signaling cascade which can have global effects on the phenotype, physiology, and action of the cell. Without wishing to be bound by theory, the presence of FFARs can act as an energy sensor and contribute towards regulation of cellular metabolism. FFARs structures restrict the length of the FFA that can bind to them. Currently, there are five main FFARs. These are FFAR1, FFAR2, FFAR3, FFAR4, and GPR84. FFAR1 and FFAR4 only bind long-chain FFA. FFAR2 and FFAR3 can bind to short-chain FFA. The fifth, and least explored FFAR, GPR84, has greatest affinity for medium-chain FFAs. We are interested in the role that the medium chain FFAR GPR84 has on the immune system.(1-4)
GPR84 is a medium-chain fatty acid receptor. It was found through a data mining strategy searching for GPCRs. [5] Still defined as an orphan receptor, its ligand, MCFAs, was not discovered until years later.[6] Further examination found that GPR84 is Gi/o GPCR, and interaction with its ligand leads to an intracellular reduction of cyclic adenosine monophosphate (cAMP). GPR84 is distributed among various tissues in the body, such as adipocytes, but also, GPR84 is found to have high expression in immune cells. In studies of adipogenesis, GPR84 was upregulated in human adipocytes stimulated with inflammatory cytokines and mice fed with high-fat diet. (7) In the immune tissue, GPR84 is expressed in monocytes, macrophages, granulocytes, T cells, and B cells. (8, 9) These studies found that immune cells exposed to a pro-inflammatory stimulus induces high expression of GPR84.[10] Other metabolically contributing factors which increase GPR84 expression in macrophages include hyperglycemia hypercholesterolemia, and oxidized LDL.[6, 11] Studies examining the role of GPR84 in T cells found that GPR84 deficient mice increased IL-4, IL-5 and IL-13, but had no effect on IL-2 or IFN-gamma production.[9]
Without being bound by theory, GPR84 acted as a feed-forward mechanism during inflammation. In this mechanism, a pro-inflammatory stimulus increases the expression of GPR84, and further signaling through GPR84 exacerbates this inflammatory response. This pathway was shown in one study where immune cells exposed to LPS induced GPR84 and that supplementing with MCFA increased the production of IL-12p40.[6] These feed-forward pro-inflammatory findings developed a therapeutic void and lead to the development of GPR84 agonists.
The synthetic agonist, 6-OAU, was found the increase chemotaxis of human polymorphonuclear leukocytes and macrophages and increase proinflammatory cytokine production.[12] In a separate study, GPR84 agonism under inflammatory conditions elevated pro-inflammatory cytokines and increased bacterial phagocytosis and this elevated response was abrogated in GPR84 deficient mice or in macrophages treated with a GPR84 antagonist.[11] A separate natural GPR84 agonist, Embelin, was also examined. Here, studies found that treatment with Embelin lead to neutrophil chemotaxis and primed these cells for oxidative burst and that loss of GPR84 abrogated the pro-inflammatory cytokine secretion.[13]A synthesized GPR84 agonist, DL175, was also found to promote phagocytosis of in human myeloid cells. [14]
The agonist used in vivo was conducted in nude mice, which lack the immune system and cannot provide any evidence that GPR84 agonists modulate the immune system. These controlled systems have raised skepticism that in these studies the agonists used can or cannot be acting through the GPR84 receptor at all. [19-22]
These findings regarding myeloid chemotaxis and metabolic regulation lead researchers to examine GPR84's anti-inflammatory and metabolic regulation. In studies using antagonists, a goal was to prevent chemotaxis and to elicit and anti-inflammatory response. The first antagonist created, PBl-4050, was found to be both an agonist of GPR40 and an antagonist of GPR84. PBl-4050 was found to affect macrophages, fibroblasts, myofibroblasts, and epithelial cells. Antagonism reduced fibrotic capacity of these cells while also dampening macrophage activation and secretion of pro-inflammatory and pro-fibrotic molecules. In addition, macrophage recruitment was prevented, which are contributors to fibrotic disease.[15] The use of GLPG1205, a GPR84 specific receptor antagonist was used in the reduction of inflammation and fibrosis. This study also found that antagonism of GPR84 led reductions in neutrophils, monocytes, and macrophage migration. When examined in three separate models of non-alcoholic steato-hepatitis (NASH), there was a significant decrease in macrophage accumulation as well as reductions in both inflammation and fibrosis.[16] Both studies showed similar outcomes and indicated that the use of antagonisms as an anti-inflammatory and anti-fibrotic drug can be therapeutic.
A study examined in depth the role of GPR84 in metabolic deregulation. Supplementing previous GPR84 antagonist literature, this study examined PBI-4547, a GPR40 and PPAR gamma agonist with some GPR84 antagonist, was studied in Non-alcoholic fatty liver disease (NAFLD). A model of diet-induced obesity was used and antagonism of GPR84 with PBI-4547 improved metabolic dysregulation through reductions in hyperglycemia and hypertriglyceridemia. When examining the effects in GPR84 deficient mice, this ability to regulate the metabolism was absent indicating GPR84 receptor can play a role. The mechanistic ability to regulate metabolism was due to an ability to modulate glycolysis and fatty acid oxidation. Researchers found increases in glucose transporters and upregulations of enzymes which leading to improved glycolysis. In addition, increases in fatty acid oxidation led to improve metabolism of fatty acid oxidation. Both metabolic adjustments reduced overall metabolic dysregulation and returned the system back to physiological homeostasis.[17] Supplementing these findings, a separate study found that the use of a separate GPR84 modulator, PBI-4050, also improved glycemic control, indicating that GPR84 can have a role in metabolic regulation. [18]
Our results are different in that they indicate that GPR84 acts as regulator of functions in T lymphocytes and myeloid cells (macrophages, myeloid derived suppressor cells and others) that will enhance the antitumor effect of immunotherapy. Most tumors are poorly immunogenic, i.e., the protein or markers they express are not strongly antigenic and therefore the immune response generated by T cells can be weak. Even when a strong response is generated the tumor microenvironment (TME) creates a major barrier for T cells to function appropriately. The TME impairs the normal protective immune responses by decreasing the ability of T cells to kill tumor cells and by increasing the presence of myeloid cells able to suppress T cell function (myeloid-derived suppressor cells or M2 macrophages). T cells kill tumor cells through the production of proteins including Granzyme B and perform, which when released in the proximity of a tumor cell, cause the formation of pores or holes in the membrane of the tumor cell and effectively kill it. In contrast myeloid-derived suppressor cells or M2 macrophages produce Arginase 1, nitric oxide or reactive oxygen and nitrogen species, which impair the function of T cells. This allows tumor cells to escape the innate (Natural Killer and macrophages) and adaptive (T cells and B cells) responses and continue to grow and metastasize. The mechanisms by which tumors impair the immune response are multiple and include the expression of checkpoint inhibitors such as PD-1, PD-L1, and CTLA-4, the depletion of amino-acids such as arginine by Arginase 1, the killing of T cells by the production of reactive oxygen species or reactive nitrogen species (Nitric Oxide), and changes in the metabolic microenvironment.
Our results indicate that the use of antagonists of GPR84 dramatically increase the expression of perform and granzyme B in T cells (both CD8+ cytotoxic T cells and CD4+ helper T cells), making them more cytotoxic against tumor cells. At the same time, the use of GPR84 antagonists decreases the expression of Arginase 1 and Nitric oxide synthase (NOS2) making MDSC less immunosuppressive. A surprising result described herein is a single agent, an antagonist to GPR84 FFAR, simultaneously increases the ability of T cells to kill tumors while decreasing the ability of MDSC to suppress the protective T cell response. Since T cells and MDSC are found to infiltrate tumor tissues, without wishing to be bound by theory, the use of a GPR84 antagonist can reverse the immunosuppressive tumor microenvironment and potentiate the therapeutic anti-tumor immune response. Non-limiting, exemplary uses of these observations comprise:
1. The use of GPR84 antagonists in the prevention of cancer: GPR84 antagonists can be given to patients with a high risk of developing cancer including (but not limited to) patients with a strong family history of cancer (breast, prostate, colorectal or other), patients with a chronic inflammatory condition such as colitis, chronic prostatitis, and patients with genomic mutations that place them at high risk for developing cancer.
2. The use of GPR84 antagonists to prevent recurrence of cancer: GPR84 can be given to patients who have undergone chemotherapy or the resection of a primary tumor such as colorectal or lung cancer and have a high risk of having a recurrence.
3. The use of GPR84 antagonists in the treatment of cancer: Since the immune system can initiate a natural response against certain tumor for example melanoma, or cervical cancer, then GPR84 antagonists can be used to enhance the function of the cytotoxic T cells and decrease the suppressive functions of myeloid cells.
4. The use of GPR84 antagonists in the treatment of cancer in conjunction with immunotherapy, chemotherapy, or radiation therapy.
5. The use of GPR84 antagonists in the generation of chimeric antigen receptor-T cells (CAR-T) for their use in adoptive cellular therapy.
Our data indicates the use of GPR84 antagonists can be effective in enhancing the treatment of cancer.
The use of GPR84 antagonists as an anti-tumor immune response can have broad implications in the treatment of cancer. Without wishing to be bound by theory, the use of GPR84 antagonists can enhance the efficacy of anti-tumor immune cells. This therapy can be useful in any cancer where an insufficient anti-tumor immune response occurs, and in situations where GPR84 antagonism elicits a strong anti-tumor response, antagonism can be used as a single agent anti-cancer agent. In addition, in situations where the goal is to enhance a muted anti-tumor response, due to the strong safety profile of GPR84 antagonists, combination therapies can be warranted. Some examples include combination therapies with immune checkpoint inhibitors and enhancing CAR-T cell therapy. The use a GPR84 antagonist to boost the immune activity can also switch immune cells which have entered a ‘dormant’ or ‘immunosuppressive’ state to an activated proinflammatory immune cell. In these situations, GPR84 antagonism can be beneficial in tumors which have high degrees of senescent anti-tumor immune cells or tumors which contain high amounts of immunosuppressive cells such as the myeloid derived suppressor cell.
Technologies used to enhance the immune response to cancer use cytokines such as interleukin 2, interferon gamma or interleukin 12. Even though these are naturally produced by the immune system, their use in the clinical setting requires the infusion of large doses of these cytokines which results in high levels of toxicity. Furthermore, these cytokines have an impact on T cells or NK cells or macrophages, but not on all the cells at the same time. Other forms of increasing the immune response to cancer use adjuvants such as Montanide ISA-51 or ISA-720, carboxymethylcellulose (Poly-lCLC), monophosphoryl lipid A (MPLA), imiquimod, CpG oligodeoxynucleotide (CpG ODN) and others, which are used by mixing them with vaccines. These can increase the presentation of the vaccine antigen to T cells by dendritic cells, but have little impact themselves on the tumor microenvironment (TME).
Approaches have been used to target MDSC. Among these is the use of retinoic acid which promotes the maturation of MDSC into granulocytes, or arginase inhibitors which block the function of arginase and thus prevent the depletion of the amino-acid arginine.
The use of FFARs in metabolism has been studied. Dietary supplementation of omega-3-fatty acids has been studied as a way of decreasing inflammation, but these approaches have remained in the category of dietary supplements rather than in the form of therapies. Some recent studies have begun which explore the modulation of the immune system through FFARs.[23] However, the focus of these therapies have been on FFAR1-4, while limited studies have begun on the less studied GPR84 FFAR. Most of this literature points to the effect that agonists of the GPR84 contribute to a pro-inflammatory response. In contrast, our results indicate instead that antagonism of the GPR84 receptor promotes the development of increased cytotoxicity in T cells while inducing a decrease in the immunosuppressive function of MDSC. Therefore, this unexpected and simultaneous function has been undescribed and therefore represents a different approach to cancer therapy. Some differences are that it is a report on the less-studied GPR84 FFAR as a contributor in modulating the immune response in cancer. It also indicates that the results are obtained by using antagonists rather than agonists of the GPR84 FFAR. Non-limiting, exemplary advantages of this mechanism are that antagonism of GPR84 can act as both a stand-alone treatment in the therapy of certain cancers and as a combination treatment with existing therapies such as approved immunotherapies and CAR-T cell therapies. Without wishing to be bound by theory, antagonism of GPR84 can modulate the immune system in cancer, allowing us to apply this therapy in any cancers which are susceptible to the anti-cancer properties of the immune system.
Various GPR84 antagonists which have been brought to clinical trials. PBl-4050 and GLPG1205, the currently two known GPR84 antagonists have both successfully been tested in phase II clinical trials and have strong safety profiles.
In regard to the safety profile, PBI-4050 is being tested as a treatment for idiopathic pulmonary fibrosis. Results found that GPR84 antagonist is well tolerated both as a single agent and in combination with nintedanib or pirfenidone in a phase II clinical trial. Results found that the ability to increase forced vital capacity was unsuccessful in patients both as a single agent and in combination with nintedanib. More so, without wishing to be bound by theory, combination therapy of PBI-4050 with pirfenidone worsened forced vital capacity (FVC), which indicates acceleration of disease. Arguments for a reduction in FVC and acceleration in disease can be due to a drug-drug interaction between PBI-4050 and pirfenidone. [25, 26] When further examining the effects of PBI-4050 on inflammatory and fibrotic biomarkers, results found that patients treated with PBI-4050 had significant increase in IL-9, IL-7, MIP-Ibeta, with near-significant increases in IL-IRa, IL-13, and G-CSF.[27] While the argument of reduction of inflammation occurs, some of these markers such as MIP-Ibeta and G-CSF are markers of inflammation. Such inflammatory increases can support the position whereby an unknown proinflammatory pathway can be stimulated with GPR84 antagonism.[27]
PBI-4050 has also been examined as a treatment in type II diabetic patients with metabolic syndrome. A phase II study found that treatment with PBl-4050 can reduce HbAlc in type II diabetic patients with metabolic syndrome.
The other GPR84 antagonist that has been used in clinical trials is GLPG1205. GLPG1205 is a negative allosteric modulator of GPR84 that has progressed through phase II clinical trials. Initial phase I studies found that GLPG1205 occupied the human GPR84 receptor, was well tolerated and had a terminal half-life of 30.1 to 140 hours. [29] Additionally, when studying the interaction of GLPG1205 with the cytochrome family of enzymes and were found to have strong safety profiles ensuring that this drugs administration can be used concomitantly with other therapies. [30]
GLPG1205 has also been studied in the treatment of interstitial pulmonary fibrosis. Results provided a non-significant reduction in fibrosis in all groups that were supplemented with GLPG1205 over the standard of care, which warranted further studies to determine if GLPG1205 does have an effect in this disease.
GLPG1205 use was also explored as a treatment for ulcerative colitis. Results from this study found that GLPG1205 was unable to improve the inflammatory response. In fact, GLPG1205 was found to lead to worsening of colitis in certain patients in the treatment study which led to the drug's discontinuation.[31] Without wishing to be bound by theory, the antagonism of the GPR84 receptor can lead to a pro-inflammatory response.
Cells of the immune system use a variety of energy sources to support its functions, including sugars and fats. Fats contain fatty acids and are used in fatty acid oxidation, while sugars are the substrate used in glycolysis. Fatty acids are made of various carbon lengths and degrees of saturation. These variations can change the way and where fatty acids are used in tissue. Currently, there are four families of fatty acids. These are short-chain fatty acids (<5 carbons), medium-chain fatty acids (6-12 carbons), long-chain fatty acids (13-21 carbons) and very-long-chain fatty acids (>22 carbons). Short-chain fatty acids are synthesized by gut intestinal fermentation of indigestible foods. Medium, long, and very-long chains can either be synthesized from cells or obtained through the diet.
Fatty acids are not only a component of cellular energy, but also serve as signaling ligands. Free fatty acids (FFA) bind to a family of G-protein coupled receptors (GPCRs), entitled free fatty acid receptors (FFAR) that are found on various cells in the body. Ligation with these GPCRs commits an intracellular signaling cascade which can have global effects on the phenotype, physiology, and action of the cell. Without wishing to be bound by theory, the presence of FFARs can act as an energy sensor and contribute towards regulation of cellular metabolism. FFARs structures restrict the length of the FFA that can bind to them. Currently, there are five main FFARs. These are FFAR1, FFAR2, FFAR3, FFAR4, and GPR84. FFAR1 and FFAR4 only bind long-chain FFA. FFAR2 and FFAR3 can bind to short-chain FFA. The fifth, and least explored FFAR, GPR84, has greatest affinity for medium-chain FFAs.
Aspects of the invention are drawn towards treating or preventing GPR84 responsive conditions. In embodiments, antagonism of the GPR84 receptor promotes the development of increased cytotoxicity in T cells while inducing a decrease in the immunosuppressive function of myeloid-derived-suppressor cells (MDSC).
Aspects of the invention are directed towards modulating the immune system by administering a GPR84 antagonist. As used herein, the term “modulating the immune system” can refer to increasing T-cells and decreasing immunosuppressive effects of myeloid-derived-suppressor cells, or both. Aspects of the invention are drawn towards administering a GPR84 antagonist to promote the development of increased cytotoxicity in T cells while inducing a decrease in the immunosuppressive function of MDSC.
Antagonists of GPR84 dramatically increases the expression of perform and granzyme B in T cells (both CD8+ cytotoxic T cells and CD4+ helper T cells), making them more cytotoxic against tumor cells. At the same time, the use of GPR84 antagonists decreases the expression of Arginase 1 and Nitric oxide synthase (NOS2) making MDSC less immunosuppressive. A surprising result described herein is a single agent, an antagonist to GPR84 FFAR, simultaneously increases the ability of T cells to kill tumors while decreasing the ability of MDSC to suppress the protective T cell response. Since T cells and MDSC are found to infiltrate tumor tissues, the use of a GPR84 antagonist can reverse the immunosuppressive tumor microenvironment and potentiate the therapeutic anti-tumor immune response.
Immune cells use a variety of metabolites to produce energy needed to carry out their respective functions. Research explores the effects that different energy sources can have on the function of immune cells. Many immune cells will use metabolite sensing receptors to detect different energy sources found in the extracellular environment. This mechanism is useful because it allows these cells to optimize their energy usage. Herein, the medium chain free fatty acid receptor (FFAR), GPR84 is described. Research has begun describing how FFARs can play a role in metabolic diseases. However, it remains to be explored how these FFARs can directly work in the role of both cancer and inflammation.
Described herein is modulation of the immune system using antagonists to the GPR84 FFAR. This observation is surprising, including but not limited to, that by targeting one receptor that is present in myeloid cells (macrophages, granulocytes, eosinophils, myeloid-derived suppressor cells), T cells (CD8+ killer T cells, CD4+ helper cells and CD4+FoxP3+ regulatory T cells) and Natural Killer cells (NK) we can regulate the immune response to cause a beneficial therapeutic effect.
A GPR84 antagonist increases the activity and inflammatory phenotype. This can be useful in diseases that require a highly activated and protective T cell response such as cancer, infectious diseases, improving the efficacy of vaccines or cancer immunotherapy, and others. This is caused by an increase in the use of glucose by T cells and the increased expression of granzyme B and perform (in CD8 T cells), an increase in inflammatory helper cytokines IL2 and IFNγ in CD4 T cells and a decrease in the expression of arginase 1 and iNOS in myeloid cells.
Without wishing to be bound by theory, a mechanism to modulate the immune response through GPR84 is due to control on the metabolic state of the immune cells. Immune cells that are more glycolytic have a more inflammatory phenotype while immune cells that use fatty acid oxidation are found to be more anti-inflammatory. The use of a GPR84 antagonists can lead cells to rely greater on glycolysis and therefore obtain a more pro-inflammatory phenotype. In contrast, GPR84 agonists can lead to less of a reliance on glycolysis and therefore present with a more anti-inflammatory phenotype. Our results indicate that this reprogramming can occur in both anti-cancer T cells as well as pro-cancer myeloid derived suppressor cells (MDSCs). In T cells, use of an agonist was found to reduce glycolysis and prevent T cells activation, proliferation and maturation. Use of an antagonist lead to an opposite effect, stimulating glycolysis and exhibiting a stronger cytotoxic phenotype. In the fatty acid oxidation reliant MDSC, agonism enhanced immunosuppressive phenotypes while antagonism reduced these phenotypes and immunosuppressive capacity. These results indicate that reprogramming immune cell metabolism is an effective strategy in enhancing anti-tumor capacity and provides a new approach in the treatment of diseases.
1.5×105 MC38 colon adenocarcinoma were subcutaneously inoculated into 7-8 week old C57BL/6 mice. Mice were randomized into four treatment groups: vehicle control, anti-CTLA4, 30 mg GLPG1205, or anti-CTLA4+30 mg GLPG1205. Checkpoint inhibitor therapy was administered on day 5, 8, and 11. First dose was 200 μg anti-CTLA4, and subsequent doses were 100 μg anti-CTLA4, administered by intraperitoneal injection. 30 mg GLPG1205 was suspended in 0.5% methylcellulose and administered twice daily by oral gavage. Tumor size and mouse weight were measured three times per week for 21 days.
Other checkpoint inhibitors to use include anti-PD-1 and anti-PD-L1. Other cancers to use include B16 melanoma, 3LL lung carcinoma, and mWnt triple-negative breast cancer.
Wojciechowicz, Megan L., and Avi Ma'ayan. “GPR84: an immune response dial?.” Nature reviews. Drug discovery 6 (2020): 374 for example, shows a schematic of the impact of GPR84 can have on modulating the immune system. However, it describes a dial in the opposite direction of our observations. They indicate that using a GPR84 agonist will act as a pro-inflammatory signal and will be useful in the treatment of diseases that require an active immune response, such as cancer. In contrast using an antagonist will lead to an anti-inflammatory response and can be useful in the treatment of certain inflammatory diseases, such as ulcerative colitis, Crohn's disease, and arthritis. This is opposite our observations where using an agonist stimulates anti-inflammatory responses and using and antagonist stimulates an inflammatory protective response.
The immortalized cell lines B16-F10 (ATCC CRL-6475), EL-4 (ATCC #TIB-39), E.G7-OVA (ATCC #CRL-2113) were purchased from the American Type Culture Collection. B16-OVA was kindly gifted from the laboratory of Dr. Paulo Rodriguez. Cells were subcultured when growth reached 80-90% confluency. For adherent cell lines, cells were detached using Trypsin-EDTA (0.25%) (Gibco #25200056) for three minutes at 37° C., 5% CO2.
Complete RPMI-1640 (cRPMI-1640) was used as the base for all cell cultures. Media was prepared using RPMI-1640 (Lonza BioWhittaker #12167F) with 10% heat-inactivated fetal bovine serum (FBS) (Gemini #100106), 25 mM HEPES (Gibco #15630080), 1% penicillin-streptomycin (Gibco #15140122), 3 mM UltraGlutamine I (Lonza #BE17-605E/U1), and 0.05 mM 2-mercaptoethanol (MP Biomedicals, #194834). Culture media for T lymphoma EL-4, E.G7-OVA, and gp100 pulsed EL-4 consisted of cRPMI-1640 supplemented with 1 mM sodium pyruvate (Gibco #11360070). Culture media for B16-OVA consisted of cRPMI-1640 supplemented with 0.8% G418 (Gibco #10131035)
Lymph nodes and spleen were isolated from wild-type C57BL/6N mice. Tissue was mechanically dissociated with the back of a syringe plunger and passed through a 70 M filter. CD3 T cells (Invitrogen #8804-6820-74) or CD8 T cells (Invitrogen #8804-6822-74) were enriched using a negative magnetic bead selection. 24-well plates (Corning #3524) were coated with either 0.1, 0.5, or 1 g/ml anti-mouse CD3e (BD Biosciences #553058, clone 145-2C11) and anti-mouse CD28 (BD Biosciences #553294, clone 37.51) suspended in PBS for two hours at 37° C., 5% C02. PBS containing coating antibodies were then removed and 1×106 T cells in 2 ml cRPMI-1640 was added to each well. GPR84 antagonist GLPG1205 was purchased (MedChemExpress #HY-135303) or provided by Galapagos NV. GPR84 agonist DL175 was purchased from Tocris Bioscience (#7082). For treatment, drugs were dissolved in DMSO (Fisher #BP231-100), and then diluted with cRPMI-1640 to an in vitro working concentration of 3 μM or 10 μM. Treated cells were incubated at 37° C., 5% CO2 for the designed study duration.
Bone marrow cells were isolated by flushing the tibia and femurs of wild-type C57BL/6N mice. Bone marrow was pelleted and resuspended in ACK lysis buffer (Quality Biological #118156101) for three minutes to lyse red blood cells. Purified leukocytes were then pelleted and resuspended at a concentration of 1×106 cells/ml in cRPMI-1640 containing 40 ng/ml G-CSF (Gemini #300-307P), GM-CSF (Gemini #300-308P), and IL-6 (Gemini #300-327P) to stimulate bone marrow myeloid derived suppressor cell (BM-MDSC) differentiation. To generate BM-MDSCs, 3×106 cells were plated in a 6-well dish and incubated at 37° C., 5% CO2 for 96 hours. For treatment, drugs were administered at either 3 μM or 10 μM concentrations and added to the BM-MDSC cell culture well 24 hours after plating.
Harvested cells were washed 1× with cold PBS and lysed in egg lysis buffer with 1× Halt protease and phosphatase inhibitor cocktail (Thermo #1861280) for 15 minutes on ice. Lysates were clarified by centrifugation at 12,500 RPM for 10 minutes, transferred to a clean 1.5 ml Eppendorf tube, and stored at −80° C. until analysis. Protein concentration was quantified using a BCA protein assay kit (Thermo #A53225). 20 g of protein diluted with 1×LDS sample buffer (Life Technologies #B0008) and 1×sample reducing agent (Life Technologies #B0009), were denatured at 95° C. for 5 minutes. Denatured T cell protein was resolved using a 10% Bolt Bis-Tris plus protein gel (Thermo #NW00102BOX) and denatured BM-MDSC protein was resolved using an 8% Bolt Bis-Tris plus protein gel (Thermo #NW00082BOX). Gels were transferred to a PVDF membrane (Invitrogen #IB401002) using a seven-minute P3 program on an Invitrogen iBlot dry transfer system. Membranes were then blocked for one hour using 5% nonfat dry milk in TBS-T, washed three times with TBS-T and probed with primary antibody diluted with 5% BSA in TBS-T overnight at 4° C. with gentle rocking. Primary antibody was removed, and membranes were washed three times with TBS-T followed by secondary antibody diluted in TBS-T at room temperature for one hour with gentle rocking. Membranes were washed three times with TBS-T and film developed with ECL western blotting substrate (Thermo #32106). Membranes were reprobed after stripping for seven minutes at room temperature (Thermo #21059) or after inactivation of horseradish peroxidase with 30% hydrogen peroxide for 15 minutes at room temperature (Fisher #H323-500). Primary antibodies used were rabbit anti-GPR84 (1:300) (Thermo #BS-15353R), rabbit anti-granzyme B (1:10,000) (CST #4275S), rabbit anti-perforin (1:1000) (CST #62550S), mouse anti-arginase-I (BD Biosciences #610708), mouse anti-iNOS (BD Biosciences #610432), and mouse anti-R-actin (1:500,000) (Sigma #A228). Secondary antibodies used were anti-mouse-HRP (Kindle Biosciences #R1005) and anti-rabbit-HRP (Kindle Biosciences #R1006). Analysis was conducted using ImageJ (NIH Bethesda, MD) and normalized to the housekeeping protein β-actin.
Single cell suspensions were either stained with Fc block (BD Biosciences #553142) for in vitro assays or Fc block in combination with Live/Dead Near-IR (Invitrogen #L10119) for in vivo assays for 15 minutes at room temperature. Cells were pelleted and subsequently stained with a selected panel from the antibodies anti-CD69 (BD Biosciences #746813, clone H1.2F3), anti-CD25 (BD Biosciences #553072, clone 7D4), anti-CTLA-4 (BD Biosciences #564332, clone UC10-4F10-11), anti-LAG-3 (BD Biosciences #745214, clone C9B7W), anti-PD-1 (BD Biosciences #566514, clone J43), anti-CD62L (BD Biosciences #612833, clone MEL-14), anti-CD44 (BD Biosciences #741057, clone IM7), anti-CD45 (BD Biosciences #612924, clone 30-F11), anti-CD3 (BD Biosciences #557984, clone 500A2), anti-CD4 (BD Biosciences #612900, clone GK1.5), anti-CD8 (BD Biosciences #563786, clone 53-6.7), SIINFEKL Tetramer (SEQ ID NO: 1) (NIH Tetramer Facility Atlanta, GA), anti-Gr-1 (BD Biosciences #562709, clone RB6-8C5), anti-Ly-6G (BD Biosciences #560599, clone 1A8), anti-Ly-6C (BD Biosciences #560592, clone AL-21), anti-CD11b (BD Biosciences #552850, clone M1/70), or anti-F4/80 (BD Biosciences #565612, clone T45-2342) for 30 minutes at 4° C. protected from light. Cells were washed pelleted and fixed with 4.2% formaldehyde (BD Biosciences #554722) for 10 minutes at 4° C. After fixation, cells were pelleted and permeabilized with perm/wash buffer (BD Biosciences #554723) for 20 minutes at 4° C. Cells were pelleted and intracellularly stained with anti-IFNγ (BD Biosciences #563773, clone XMG1.2) for 30 minutes at 4° C. protected from light. After staining, cells were pelleted, resuspended and run by a BD FACSymphony A3 flow cytometer.
Isolated CD3 T cells were labeled with 1 μM CFDA SE (Thermo #V12883) for 15 minutes at 37° C. protected from light. After incubation, equal parts PBS was added, cells were pelleted, resuspended in cRPMI-1640, and further incubated for 20 minutes on ice to quench remaining CFDA. Cells were pelleted, resuspended and plated in an antibody coated well with the indicated drug concentrations at time zero. Activated T cells were incubated at 37° C., 5% CO2 for 72 hours. After study duration, T cells were stained with anti-CD4, anti-CD8, anti-CD3 following the immune profiling by flow cytometry method and proliferation was measured by CFSE dilution.
Treated OT-I CD8 T cells were prepared following the in vitro T cell activation method. Non-target EL4 cells (CFSE low—0.2 μM) and target E.G7-OVA cells (CFSE high—1 M) were prepared following the CFSE labeling protocol. Cells were subsequently washed, pelleted, resuspended at 1×106 cells/ml, and mixed at 1:1 ratio. 100 μl of the mixed cells were plated into a 96-well flat bottom plate. In technical duplicate or triplicate OT-I T cells were added to wells to create an effector to target ratio of 3:1, 1:1, and 0.3:1 and incubated for 24 hours at 37° C., 5% C02. Cells were then harvested, CFSE positive cells were identified, and the percent specific lysis captured by flow cytometry and determined using the formula [1-(no effector control ratio/experimental ratio)]×100.
CRISPR-Cas9 GPR84 pmel-I T cells were prepared following the CRISPR-Cas9 method. Non-target EL4 cells (CFSE low—0.1 μM) and target EL4gp100 cells (CFSE high—1 M) were prepared. EL4gp100 CFSE high cells were prepared by pulsing EL4 with 1 g/ml gp100 (Anaspec #AS62589) and incubating for 30 minutes at 37° C., 5% C02. Cells were subsequently washed, pelleted, resuspended at 1×106 cells/ml, and mixed at 1:1 ratio. 1×105 of the mixed cells were plated into a 96-well round-bottom plate. In technical duplicate or triplicate GPR84 knockout or no target control CD8 μmel-I T cells were added to wells to create an effector to target ratio of 0.5:1, 0.25:1, and 0.12:1 and incubated for 24 hours at 37° C., 5% C02. Cells were then harvested, stained with anti-CD8 to remove the T cell population and the percent specific lysis was captured by flow cytometry.
24-well plates were coated with 1 g/ml anti-mouse CD3e and anti-mouse CD28 resuspended in PBS for two hours at 37° C., 5% C02. PBS containing coating antibodies were then removed and 1×106 naïve CFSE CD3 T cells in 1 ml was added to each well. Treated BM-MDSCs were removed using a rubber policeman. Cells were pelleted, resuspended in cRPMI-1640 and added to each T cell containing well at 1.25×105 cells in 1 ml, generating an 8:1 CD3 to MDSC ratio. Co-cultures were incubated for 72 hours at 37° C., 5% C02. Cells were harvested and stained following the immune profiling by flow cytometry method. MDSC suppressive capacity was measured by their ability to inhibit CD3 T cell proliferation measured by CFSE dilution.
Isolated pmel-I splenocytes were plated at a density of 2.5×106 in 1 ml of cRPMI-1640 containing 300 ng/ml anti-mouse CD3 (Biolegend #100340, clone 145-2C11), 500 IU/ml IL-2, 5 ng/ml IL-7 (PreproTech #217-17), and 100 ng/ml IL-15 (PreproTech #210-15) into a 24-well plate. Cells were incubated at 37° C., 5% CO2 for 48 hours. Cells were then pelleted and suspended to a concentration of 4×105 cells/ml in fresh cRPMI-1640 with 500 IU/ml IL-2 and cultured for an additional 24 hours at 37° C., 5% CO2. Before electroporation, T media was prepared consisting of modified cRPMI-1640 (20% FBS, 500 IU/ml IL-2, and no antibiotics). 615 μl of fresh T media was pipetted into 24-well plates. Preparation of CRISPR-Cas9 complexes and electroporation of primary T cells were conducted following the Alt-R CRISPR-Cas9 system using the Neon Transfection System instructions provided by Integrated DNA Technologies (IDT Coralville, IO). The program used for electroporation consisted of 1600V, 10 ms, and three pulses. Electroporated cells were transferred to 24 well plates containing fresh T media and an additional 525 μl of T media was added after 24 incubation at 37° C., 5% CO2. Cells were counted 48 hours after electroporation and cRPMI-1640 supplemented with 500 IU/ml IL-2 was added to adjust the cell density to 4×105 cells/ml. CRISPR-Cas9 knockout cells were expanded every 48 hours by adjusting cell density to 4×105 cells/ml in 24-well plates. Alt-R CRISPR-Cas9 no target control crRNA and GPR84 crRNA were purchased from IDT. GPR84 crRNA sequence is listed in Table 1.
Treated CD3 T cells were prepared following the in vitro T cell activation method and incubated for 72 hours at 37° C., 5% CO2. Culture supernatant was harvested and clarified by centrifugation at 2000 RPMI for five minutes. Supernatant was then transferred to a clean 1.5 ml Eppendorf tube and stored at −80° C. until analysis. Cytokines and chemokines were quantified using a Milliplex mouse cytokine/chemokine magnetic bead panel (Millipore MCYTMAG-70K-PX32) on a Luminex MAGPIX system with undiluted samples following the manufacturer's instructions.
Treated CD8 T cell were prepared following the in vitro T cell activation method and incubated for 72 hours at 37° C., 5% CO2. Treated BM-MDSCs were prepared followed the in vitro BM-MDSCs differentiation method. The XFe ATP rate assay preparation and analysis was conducted following the manufacturer's user guide and instructions. (Agilent #103592-100) On day of analysis, XFe cell culture microplate was coated with 22.4 g/ml CellTak (Corning #354240) for 20 minutes at room temperature. Treated cells were removed from culture, pelleted and resuspended in warm assay medium at a concentration of 3×106 cells/ml (1.5×105 cells/well) for CD8 T cells and 2.6×106 cells/ml (1.3×105 cells/well) for BM-MDSCs. Cells were adhered to the culture plate by centrifugation 200×G, for one minute with zero brake per the manufacturer's instructions for immobilization of non-adherent cells (Agilent, Santa Clara, CA). After XFe ATP rate assay was complete, wells that did not receive injections of oligomycin and/or rotenone/antimycin A were removed before analysis. Results were procured using the Seahorse XF real-time ATP rate assay report generator.
Treated CD3 T cells were prepared following the in vitro T cell activation method and incubated for 72 hours at 37° C., 5% CO2. Cells were washed, pelleted and resuspended in cRPMI-1640 with 30 M 2-NBDG. (Thermo #N13195) Cells were then incubated for one hour at 37° C., 5% CO2. After incubation, T cells were washed, pelleted and stained with anti-CD4, anti-CD8, anti-CD3 following the immune profiling by flow cytometry method. Percent uptake was calculated using mean fluorescent intensity (MFI).
Treated CD8 T cells were prepared following the in vitro T cell activation method and incubated for 48 hours at 37° C., 5% CO2. Cells were washed, pelleted, and lysed with buffer RLT containing 2-mercaptoethanol (Sigma #M7154-24ML). Lysed cells were then added to a QIAshredder (Qiagen #79656) column and tissue was homogenized by centrifugation at 12,000 RPM for two minutes at room temperature. RNA was extracted using a RNeasy mini kit (Qiagen #74106) following the manufacturer's instructions with an on-column DNase digestion step (Qiagen #79254). 1 g RNA was converted to cDNA using a Verso cDNA synthesis kit (Thermo #AB1453/B) with a MJ Research peltier thermocycler following the manufacturer's instructions. Quantitative PCR was conducted using a 5 μl reaction volume in a 384-well plate (Applied Biosystems #4309849) containing 10 ng of template cDNA, lx SYBR green PCR master mix (Applied Biosystems #A25742), 0.1 μM forward primer (IDT Coralville, IO), 0.1 M reverse primer (IDT Coralville, IO), and nuclease free water. (Invitrogen #AM9938) PCR amplification was performed in technical triplicate using an Applied Biosystems QuantStudio 12K flex system. Assay parameters used a hold stage followed by a PCR stage. The hold stage parameters were 50° C. for two minutes followed by 95° C. for 10 minutes. The three-step PCR stage consisted of 95° C. for 15 seconds, 58° C. for 15 seconds, and 72° C. for one minute for 40 cycles. Relative fold changes were calculated using ΔΔCt normalized to the housekeeping gene P3-actin. List of mouse PCR primers for enzymes in glycolysis are listed in Table 2.
Treated CD8 T cells were prepared following the in vitro T cell activation method and incubated for 16 hours at 37° C., 500 CO2. Treated T cells were mixed with treated BM-MDSCs at a 70% o (T cell): 30% (BM-MDSC) ratio moments before analysis. 10,000 cells with greater than 90% viability were used in the analysis at a concentration of 1,000 cells/μl. Barcoded cDNA libraries were prepared using a chromium next GEM single-cell 3′ reagent kit v3.1 (10× Genomics #1000127) following the manufacturer's instructions. Libraries were validated using a high sensitivity DNA kit (Agilent #50674626) using a 2100 bioanalyzer. (Agilent, Santa Clara, CA) DNA was subsequently quantified by fluorometry using a Qubit dsDNA high sensitivity assay kit (Invitrogen #Q32854). DNA libraries were pooled in equimolar ratios and sequenced using an Illumina NextSeq 500 (Illumina, San Diego, CA) following 10× Genomics' instructions. For analysis, dying cells (>12% o mitochondrial gene expression) and cells with >6000 or <200 unique genes expressed were removed. Doublets were subsequently removed using DoubletFinder (https://github.com/chris-mcginnis-ucsf/DoubletFinder). An unsupervised clustering and plotting using UMAPs was completed on the remaining cells. CD8 T cells were identified by first conducting CD3 followed by CD8 refinement and reclustering. To characterize the CD8 subsets, the top 50 differentially expressed genes between each subcluster were compared against publicly available datasets, and known genes associated with activation, exhaustion, and memory T cell states. To determine changes in metabolism and cytotoxicity, a curated list of genes associated with glucose, fatty acid, lipid metabolism, and cytotoxicity were generated. Average gene expression by treatment group was determined for each gene and visualized by heatmap.
C57BL/6N mice were bred in-house or purchased from Envigo (Indianapolis, IN). OT-I mice were bred in-house, and pmel-I mice were purchase from Jackson Laboratory (Bar Harbor, ME). Purchased mice were held for one week prior to use.
C57BL/6N mice were injected with 3×105 B16-OVA or B16 subcutaneously in the right flank. Five days after injection, mice were administered 200 μg cyclophosphamide monohydrate (Sigma #C73971G) dissolved in PBS by intraperitoneal injection to induce minor lymphodepletion. 48 hours after lymphodepletion, mice were intravenously adoptively transferred 2.5×106 CD8 OT-I or CD8 μmel-I pre-treated ex vivo following the in vitro T cell activation method. Tumor growth rates were determined by tumor volume measurement beginning on day four and continuing three times per week for the study duration. Tumor volumes were determined by the ellipsoid formula: [(Length×Width2)/2]. At the end of study, tumors were either fixed in 10% formalin or dissociated into a single-cell suspension for flow cytometry. Single-cell suspension was performed by chopping tumor tissue with scissors followed by a 30-minute incubation at 37° C. with 100 g/ml DNase I (Roche #10104159001) and 0.5 U/ml Liberase (Roche #05401127001) in PBS. Solution was then diluted with cRPMI-1640 and passed through a 70 μm cell strainer, pelleted and resuspended in ACK lysis buffer to remove erythrocytes. Cells were then counted, filtered through a 70 μm cell strainer, and 10×106 cells were aliquoted and stained following the immune profiling by flow cytometry method.
C57BL/6N mice were first administered 200 μg cyclophosphamide dissolved in PBS by intraperitoneal injection to induce minor lymphodepletion. 48 hours after lymphodepletion, mice were intravenously adoptively transferred 2×106 CFSE labeled CD8 OT-I T cells. Five hours later, mice were vaccinated by 100 μl subcutaneous injection with reducing doses of SIINFEKL (SEQ ID NO: 1) (Bachem #4033142.0001) in incomplete Freund's adjuvant (IFA) (Thermo #77145). Study ended 96 hours post-vaccination and lymph node tissue was isolated for analysis. Single-cell suspensions of lymph node were stained following the immune profiling by flow cytometry method. Vaccine effectiveness was determined by the percent proliferation of adoptively transferred cells. The sub-optimal vaccine dose was then selected as the amount of antigen that elicited a 50% response.
C57BL/6N mice were first administered 200 μg cyclophosphamide dissolved in PBS by intraperitoneal injection to induce minor lymphodepletion. 48 hours after lymphodepletion, mice were intravenously adoptively transferred 2×106 CFSE labeled CD8 OT-I T cells. Five hours later, mice were vaccinated by subcutaneous injection with 0.07 g SIINFEKL (SEQ ID NO: 1) in IFA. 16 hours after vaccination, mice were administered 30 mg/kg or 90 mg/kg GLPG1205 suspended in 0.5% methylcellulose (Sigma #M0430100G) with 0.5% Tween-80 (Sigma #P4780100ML) (MC) orally once daily for three days. 96 hours post-vaccination, lymph node and spleen tissue were isolated for analysis. Single-cell suspensions were treated with ACK lysis to remove red blood cells, and then stained following the immune profiling by flow cytometry method. Treatment effectiveness was determined by the percent proliferation and PD-1 expression of adoptively transferred cells.
C57BL/6N mice were injected with 3×105 B16-OVA subcutaneously in the right flank. Five days after injection, mice were administered 200 μg cyclophosphamide dissolved in PBS by intraperitoneal injection to induce minor lymphodepletion. 48 hours after lymphodepletion, mice were intravenously adoptively transferred 2.5×106 labeled CD8 OT-I T cells. Five hours later, mice were vaccinated by subcutaneous injection with 0.07 g SIINFEKL (SEQ ID NO: 1) in IFA. 16 hours after vaccination, mice were administered 30 mg/kg GLPG1205 suspended in MC orally once daily for three days. 96 hours post-vaccination, mice were subsequently administered two more series consisting of a 0.07 μg SIINFEKL (SEQ ID NO: 1) in IFA vaccination boost followed by three days of oral 30 mg/kg GLGP1205. Tumor growth rates were determined by tumor volume measurement beginning on day four and continuing three times per week for the study duration. Tumor volumes were determined by the ellipsoid formula: [(Length×Width2)/2].
Treated CD3 T cells were prepared following the in vitro T cell activation method and incubated for 72 hours at 37° C., 5% CO2. Cells were washed, pelleted and stained with anti-CD4, anti-CD8, anti-CD3, annexin V (BD Biosciences #550474), and propidium iodide (BD Biosciences #556463) in Annexin V binding buffer following the manufacturer's instructions (BD Biosciences #556547)
Statistical significance was determined using either an unpaired two-tailed t-test or a two-way ANOVA with or without Dunnett's post-hoc test in Graphpad Prism 6 (Graphpad San Diego, CA). Results are displayed as the mean with standard deviation or standard error described in the figure legend. The degrees of significance are defined by: *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.
GPR84 can have the ability to both modulate myeloid activity and regulate metabolic homeostasis.3,14,15 These results indicate how GPR84 affects the differentiation, activation, and function of immune cells. We show that these changes are in part due to GPR84's ability to modulate the metabolic state. We show how GPR84 mediated metabolic reprogramming leads to multiple new immunotherapies for cancer treatment.
Without wishing to be bound by theory, GPR84 can metabolically affect the T cell compartment and regulate activity. We determined GPR84 expression on both naïve and activated CD3 T cells. We found that basal expression of GPR84 on naïve T cells dramatically upregulated after anti-CD3/anti-CD28 stimulation. Without wishing to be bound by theory, pharmacological modulation with GPR84 antagonist GLGP1205 or agonist DL175 can affect expression. To assess this, we treated activated CD3 T cells with 3 μM or 10 μM GPR84 antagonist GLPG1205 or 3 μM or 10 μM GPR84 agonist DL175. While 10 M of the agonist DL175 led to a reduced trend in GPR84 expression, repeated biological experiments found that modulation of the receptor with antagonist or agonist did not significantly affect GPR84 expression. (
To determine how GPR84 affected CD8 cytotoxicity, we examined the effect that GPR84 modulation had on cytotoxic protein production. Once again, we activated T cells in the presence of low and high concentrations GPR84 antagonist GLPG1205 and agonist DL175. After treatment, we determined granzyme B and perforin by western blot. (
We conducted Annexin/PI apoptosis staining to ensure that the DL175 agonist induced reduction in proliferation was not due to T cell toxicity. Interestingly, using Annexin/PI staining we found that treatment with the antagonist GLPG1205 significantly reduced CD8 apoptosis, while treatment with the agonist DL175 displayed no measurable differences. (
Since the tumor microenvironment does not only consist of T cells, we investigated the effect of GPR84 regulation on the important tumor infiltrating MDSC. MDSC's have an immunosuppressive characteristic, and so their presence is considered pro-tumorigenic. Since we observed the activating properties of GPR84 antagonism on CD8 T cells, we observed if the effect of GPR84 modulation can elicit a similar effect on a different immune cell type. Like T cells, we first had to determine GPR84 expression on MDSCs. To test this, we looked at GPR84 expression in fresh bone marrow and differentiated BM-MDSCs. We found that GPR84 expression was absent in fresh bone marrow, but expression levels significantly increased after differentiation into BM-MDSCs. Like T cells, we wanted to see if pharmacological modulation with GPR84 antagonist GLPG1205 or agonist DL175 can affect expression. We treated differentiating BM-MDSCs with 3 μM or 10 μM GPR84 antagonist GLPG1205 or 3 μM or 10 μM GPR84 agonist DL175. While treatment with both the antagonist GLPG1205 and agonist DL175 led to a reduced intensity in GPR84 expression, repeated biological experiments found that modulation only neared statistical significance. (
Knowing that GPR84 receptor was present on BM-MDSCs, we next wanted to examine the effect that GPR84 modulation had on immunosuppressive protein production. To test this, BM-MDSCs were subsequently treated with low and high dose antagonist GLPG1205 and agonist DL175. After treatment, we determined the amount of immunosuppressive arginase-I and inducible nitric oxide synthase (iNOS) by western blot. (
GPR84 receptor modulation regulates CD8 T cell activation
Since increased cytotoxic capacity can be associated with overall CD8 T cell activation, we explored the activity profile of these cytotoxic T lymphocytes. As T cells activate, they upregulate various intracellular and extracellular surface markers which aid in their expansion and overall activity. Using flow cytometry, we examined the various CD8 T cells markers of activation, and explored how GPR84 antagonist GLPG1205 and agonist DL175 affected overall expression. We examined the effect of GPR84 modulation on the early activation marker CD69. Both 3 μM and 10 μM GLPG1205 significantly increased CD69 expression by 11.24% and 14.59% respectively. Treatment with 3 μM DL175 did not lead to a significant reduction, however 10 μM was found to significantly reduce CD69 expression by 6.47%. (
Since a robust anti-tumor effect also relies on an effective CD4 response, we investigated how GPR84 regulation also affected this T cell subset. Using flow cytometry, we examined various markers which aid in helper CD4 expansion and overall activity. We found that only two markers in CD4 T cells were significantly changed after GPR84 modulation. Compared to DMSO control, both 3 μM and 10 μM GLPG1205 significantly increased CD25 expression by 14.28% and 11.37% respectively. Using the agonist DL175 did not provide a significant difference at 3 μM but did significantly reduce CD25 expression by 16.15% at the higher 10 μM dose. (
With evidence indicating that GPR84 can regulate T cell activation, we determined if this activation or suppression associated with changes in inflammatory, mitogenic, and T cell differentiating cytokine production. To do this, we looked at the mitogenic and T cells differentiating cytokines interleukin-2 (IL-2), interleukin-15 (IL-15), interleukin-9 (IL-9), interleukin-12 subunit p40 (IL-12(p40)) and tumor necrosis factor alpha (TNFα). IL-2, a cytokine which promotes the proliferation of T and B cells, GLPG1205 treatment significantly increased production by 51.36% at 3 M and 31.1% at 10 μM compared to DMSO control. In contrast, treatment with DL175 did not significantly reduce IL-2 at either dose. (
Beyond cytokines highly effective for T cell activation and proliferation, we investigated if any other inflammatory cytokines and chemokines were affected by GPR84 regulation. In the same multiplex panel, we found that a large number of additional cytokines were directly affected by GPR84 modulation. G-CSF, a cytokine which promotes granulocyte production significantly increased 16.64% when treated with 3 μM antagonist GLPG1205 and by 26.52% at the 10 μM dose compared to DMSO control. In contrast, there was no significant reduction in G-CSF at either DL175 agonist dose. (
There is ample literature evidence to demonstrate that upregulated markers and cytokines secreted play an important role in T cells differentiation, activation, and proliferation. We showed that GPR84 regulation can affect activation marker expression and inflammatory cytokine production, leading to a functionally more cytotoxic CD8 T cell. However, the ability for GPR84 to modulate CD8 T cell differentiation remained unexplored. To address this, we differentiated naïve CD8 T cells with anti-CD3/anti-CD28 in the presence or absence of GPR84 receptor antagonist GLPG1205 or agonist DL175. We identified naïve T cells by CD62L+, CD44− and effector or memory CD8 T cells by CD62L−, CD44+. (
GPR84 receptor modulation regulates CD4 T cell differentiation
Like CD8, we determined how GPR84 modulation affected T cell differentiation in the CD4 T cell compartment. We identified naïve (CD62L+, CD44−) and effector/memory (CD62L−, CD44+) CD4 T cells in the same manner as the CD8 compartment. (
GPR84 receptor knockout by CRISPR-Cas9 enhances CD8 T cell cytotoxic capacity
To test that our enhanced cytotoxic capacity with GPR84 receptor antagonism GLPG1205 was not due to a pharmacological off-target effect, we compared the cytotoxic capacity of CRISPR-Cas9 GPR84 knockout primary pmel-I CD8 T cells (crGPR84) compared to a negative no target control (crNTC). Using gp100 pulsed EL4 tumor cells as the target cell, removal of GPR84 by CRISPR-Cas9 significantly improved the cytotoxic capacity. Referencing the representative
Using the research demonstrating GPR84's role in metabolic homeostasis as evidence, we explored if these changes in T cell activity and function can be a result from alterations in effector T cell metabolism. To do this, we used seahorse to measure the oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and proton efflux rate (PER). We found that treated CD8 T cells with antagonist GLPG1205 raises the overall OCR, ECAR, and PER. In contrast, treatment with DL175 agonist had an opposite effect with overall reductions. (
After determining that GPR84 did affect the total ATP production, the next step was to stratify the total ATP generation by either glycolysis or mitochondrial OXPHOS. After separating these values, we found that both ATP generating processes were significantly affected by GPR84 modulation. Use of the GPR84 antagonist GLPG1205 significantly increased ATP production through glycolysis compared to DMSO control (737.62 vs. 409.61 pmol/min). Additionally, a significant increase was also observed in ATP production through mitochondrial OXPHOS (486.75 vs. 392.74 pmol/min). The use of the GPR84 agonist DL175 had an opposite effect with a significant reduction in ATP rate through glycolysis (242.85 vs. 409.61 pmol/min) and mitochondrial OXPHOS (259.85 vs. 392.74 pmol/min) compared the DMSO baseline. (
Now knowing that CD8 T cells after treatment with antagonist GLPG1205 relied on glycolysis as their mode of ATP generation, we wanted to see if there was an increase in the uptake of glucose from the surrounding environment. Using the fluorescent glucose analog, 2-NBDG, we found that T cells treated with the antagonist GLPG1205 led to a significant uptake at both the 3 μM (0.19 Log2 fold change, or 13.92%) and 10 μM (0.24 Log2 fold change, or 17.74%) concentrations compared to DMSO control. Use of the agonist DL175 at 3 μM did not yield a significant change, but at the higher 10 μM dose, 2-NBDG uptake was significantly reduced by −0.07 Log2 fold change, or 5.01% compared to DMSO control. (
To link the increased uptake in glucose with the identified increase in glycolysis generated ATP, we looked at the relative gene expression fold-change of multiple enzymes in glycolytic pathway. We then compared the effect of GPR84 modulation on gene expression to the DMSO treated control. Referencing
Weshow that GPR84 receptor can modulate CD8 T cell metabolism. Building on this finding, we also wanted to see if our observed changes in BM-MDSCs activity was also attributed to metabolic modulation. Like CD8 T cells, we addressed these questions using seahorse to measure the oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and proton efflux rate (PER). Surprisingly, we found a different metabolic profile compared to the previously examined CD8 T cells. In this case, we found that both GLPG1205 and DL175 lead to an overall reduction in OCR. Subsequently, we did not observe any changes with either drug on ECAR and PER. (
After determining that GPR84 did affect the total ATP production, we wanted to know the amount of ATP generated from either glycolysis or mitochondrial OXPHOS. To do this, we stratified the ATP production rate and interestingly found that the observed changes in ATP production were only due to an effect on mitochondrial OXPHOS. When examining the rate of ATP production through mitochondrial OXPHOS, we found significant reductions with GPR84 antagonist GLPG1205 (537.83 vs. 690.95 pmol/min) and GPR84 agonist DL175 (461.66 vs. 690.95 pmol/min) treatment compared to DMSO control. (
We wanted to see if these changes in the ATP production landscape associated with a change in the energetic phenotype. Using an energetic map, we found that DMSO plotted in the upper right corner, indicating the baseline activity state. Treatment of BM-MDSCs with antagonist GLPG1205 and agonist DL175 exhibited movement from an aerobic-predominant energetic phenotype to a more glycolytic-predominant quiescent phenotype. (
GPR84 receptor modulation of CD8 T cells reveals differentiation, metabolic and effector genomic changes by single-cell RNA sequencing
Building on the evidence that GPR84 regulation is a metabolic regulator of CD8 T cells, we wanted to have a understanding of how GPR84 receptor modulation alters the CD8 genomic landscape. To do this, we analyzed a heterogenous mixture of immune cells treated with GLPG1205 antagonist, DL175 agonist, or DMSO control by single-cell RNA sequencing (scRNA-seq). First, cells underwent an unsupervised clustering and graphed on a UMAP plot. (
In addition, we wanted to see if the genetic profile of treated CD8 T cells can expand on the metabolic and cytotoxic changes elucidated. To do this, we generated a curated list of genes associated with glucose metabolism, fatty acid metabolism, lipid metabolism, and cytotoxicity. We then plotted the average gene expression as a heatmap. In all curated gene lists we found similar gene expression changes. The was a dramatic increase in gene expression in the 10 μM GLPG1205 antagonist group, and a subsequent reduction in the 10 μM DL175 agonist group. (
The effect of GPR84 modulation on anti-tumor immunity
Pre-treatment of adoptively transferred CD8 T cells with GPR84 antagonist GLPG1205 confers an enhanced in vivo anti-tumor response
After determining the increased activity in CD8 T cells after GPR84 antagonism with GLPG1205, we wanted to see if these changes can have any anti-tumor implications. To examine this, we used the OT-I and pmel-I adoptive immunotherapy models. Mice were inoculated in the flank with B16-OVA for the OT-I, or B16 for the pmel-I transfers. We then lymphodepleted mice using low-dose cyclophosphamide and then adoptively transferred activated CD8 T cells pre-treated with either 10 μM GLPG1205 or DMSO. Starting from day four, tumor volumes were measured for the study duration. (
Pre-treatment of adoptively transferred CD8 T cells with GPR84 agonist DL175 confers an enhanced in vivo pro-tumor response
By determining the beneficial anti-tumor effects of pre-treating CD8 T cells with GPR84 antagonist GLPG1205, we were interested in understanding the effect on tumor growth after pre-treatment with GPR84 agonist DL175. Using the same experimental design, agonist pre-treated CD8 OT-I T cells were adoptively transferred into B16-OVA tumor bearing mice. (
In vivo titration of SIINFEKL (SEQ ID NO: 1) to achieve a suboptimal vaccination response
After we successfully determined that GLPG1205 can elicit an enhanced anti-tumor response through adoptive cellular transfer, we wanted to see if we can adapt GLPG1205 treatment for use in other anti-tumor therapies. Using data as evidence, we determined that GLPG1205 promotes the differentiation, proliferation and survival of CD8 T cells. Due to the strong observed effect that GLPG1205 antagonism has on enhancing T cell differentiation into the effector state, we selected a therapy that can align with these characteristics. Using these criteria, we selected a vaccine model, as exposure to antigen also induces reactive T cells to move from a naïve to an effector phenotype. To test GLPG1205's ability to enhance a vaccine response, we first needed to select and use a model which elicited a sub-optimal response. For these reasons, we selected a sub-optimal SIINFEKL (SEQ ID NO: 1) vaccination system with naïve OT-I adoptive transfer. Using this system allowed us to have a consistent number of adoptively transferred naïve OT-I CD8 T cells while simultaneously capable of titrating a well-characterized and responsive antigen. To identify a sub-optimal response, we reduced the amount of SIINFEKL antigen (SEQ ID NO: 1) used in the vaccine until we achieved a 50% OT-I proliferation response. To test this and optimize this system, we first lymphodepleted wild-type mice 48 hours prior to adoptive transfer. On the day of transfer, we intravenously injected 2×106 naïve CD8 CFSE labeled OT-I T cells. We subsequently vaccinated mice with titrated doses of SIINFEKL (SEQ ID NO: 1) in incomplete Freund's adjuvant (IFA) until we observed a sub-optimal response. We used IFA to promote an immune response and mimic the adjuvant used in traditional clinical vaccines. After vaccination mice were monitored for the full 96-hour study duration. (
GPR84 antagonist treatment improves in vivo vaccination response
By finding the sup-optimal antigen dose for vaccination, we were able to test if we can improve the vaccination response with GPR84 antagonist GLPG1205. For these experiments, we lymphodepleted mice for 48 hours prior to adoptive transfer. We then adoptively transferred 2×106 naïve CD8 CFSE labeled OT-I T cells followed by vaccination with 0.07 g SIINFEKL (SEQ ID NO: 1) in IFA. 24 hours after vaccination, we orally gavaged mice with either 30 mg/kg or 90 mg/kg GLPG1205 once daily for 3 days. We collected lymph node and spleens four days after transfer for analysis. (
GPR84 antagonist treatment enhances T cell differentiation after in vivo vaccination
After we saw a similar proliferative effect in vivo that we previously observed in vitro, we wanted to know if we can also see similar changes in T cell differentiation. As before, we examined the naïve and effector/memory CD8 OT-I T cells in the secondary lymphoid organs. In the lymph node compartment, treatment with MC after adoptively transferred naïve OT-I T cells with sub-optimal vaccination kept 32.4% in the naïve state. This was in comparison to GLPG1205 treated mice, which exhibited significant reductions in naïve CD8 OT-I T cells at both oral doses (30 mg/kg, 12.87%; 90 mg/kg, 9.48%). (
Since we demonstrated that orally administered GLPG1205 enhanced both the proliferation and differentiation of antigen specific T cells after vaccination, we wanted to see if these effects can have therapeutic efficacy. To test this, we set up a therapeutic anti-tumor vaccination model. We first injected mice with 3×105 B16-OVA tumor cells into the flank of wild-type mice. Five days after tumor seeding, we injected mice with 200 g of cyclophosphamide to induce lymphodepletion. Two days after lymphodepletion we adoptively transferred 2.5×106 naïve OT-I CD8 T cells and subsequently vaccinated them with 0.07 g SIINFEKL (SEQ ID NO: 1) in an IFA emulsion. We then orally administered control MC or 30 mg/kg GLPG1205 for three consecutive days. Following treatment, mice received two consecutive series of a boost containing 0.07 g SIINFEKL (SEQ ID NO: 1) in IFA emulsion and three days of treatment. (
Described herein is the use of an agonist to the medium chain free fatty acid receptor (FFAR) GPR84 to modify the immune response in both T cells and myeloid cells, to provide clinical benefit such that it treats an existing disease and improves the survival and outcomes. The simultaneous immune modulation of T cell and myeloid cell function is surprising and as shown herein can have multiple therapeutic applications.
For example, the use of an agonist to GPR84 FFAR can simultaneously affect myeloid and T cells by increasing the immunosuppressive functions of myeloid cells (monocytes, macrophages, granulocytes—including eosinophils, and myeloid-derived suppressor cells) and decreasing the cytotoxic activity and cytokine production of T cells. The decrease in T cell function is represented by a decrease in the expression of perform or granzyme B in CD8 T cells and a decreased cytokine production inf CD4T cells. The decrease in immunosuppressive functions in myeloid cells is shown by a decrease in arginase 1 and iNOS. This type of immune modulation can be beneficial in diseases with inflammation (chronic or acute) including colitis, asthma, glomerulonephritis, hepatitis, myocarditis, transplant rejection and others where the immune system is attacking one's organs. In a similar way one can treat patients in a pre-diabetic stage when the immune response is attacking the beta-cells of the pancreas and eventually leads to the development of full-blown diabetes. The opposite effect occurs when using an antagonist to GPR84 FFAR. Immune cells show an increase the cytotoxic (increased in granzyme B and perform) in CD8 T cells and in helper function (cytokine production) in the CD4 T cells. The antagonist also causes a decrease the immunosuppressive function of myeloid cells by diminishing the expression of arginase 1 and iNOS. This type of immune modulation can be used to increase the response to a vaccine, to increase the response to an infectious agent such as COVID-19 or influenza, to improve the efficacy of an anti-cancer treatment, be it chemotherapy, radiation therapy or immunotherapy.
The combination of an agonist and an antagonist of the GPR84 FFAR can be used in the generation of T cells for immunotherapy purposes, such as CAR-T cells. For example, it has been shown that keeping CAR-T cells in a state of “stemness”. i.e. not fully mature and active, increases their survival and their therapeutic function when transferred in vivo into mice with tumors or in cancer patients. Without wishing to be bound by theory, the use of an agonist during the in vitro generation and culture of CAR-T cells can keep them in a state of “stemness”, while still being able to divide and increase in number. Once the CAR-T cells are harvested and infused into a patient with cancer, then one can treat the patient with an antagonist which can increase the maturation and cytotoxic function of the CAR-T cells as they reach the tumor. In addition, the antagonist can decrease the immunosuppressive function of the myeloid cells in the tumor, allowing the CAR-T cells to fully execute their anti-tumor effect.
There are a variety of energy sources that immune cells use to carry out their function. Two drivers of cellular energy are derived from fats and sugars. Fats which contain fatty acids and are used in fatty acid oxidation, while sugars are the substrate used in glycolysis. Fatty acids are made of various carbon lengths and degrees of saturation. These variations can change the way and where fatty acids are used in tissue. Currently, there are four families of fatty acids. These are short-chain fatty acids (<5 carbons), medium-chain fatty acids (MCFA) (6-12 carbons), long-chain fatty acids (13-21 carbons) and very-long-chain fatty acids (>22 carbons). Short-chain fatty acids are synthesized by gut intestinal fermentation of indigestible foods. Medium, long, and very-long chains can be synthesized from cells or obtained through the diet. Recently, FFAs have been more extensively studied as multiple reports have found that they play an important role in regulating metabolic disorders.
This regulatory role does not solely rely on fatty acids as a component of cellular energy, but also through their use as a signaling ligands. Free fatty acids (FFA) bind to a family of G-protein coupled receptors (GPCRs), entitled free fatty acid receptors (FFAR). FFARs are found on various cells in the body and ligation with these GPCRs commits an intracellular signaling cascade which can have global effects on the phenotype, physiology, and action of the cell. Without wishing to be bound by theory, the presence of FFARs can act as an energy sensor and contribute towards regulation of cellular metabolism. FFARs structures restrict the length of the FFA that can bind to them. Currently, there are five main FFARs. These are FFAR1, FFAR2, FFAR3, FFAR4, and GPR84. FFAR1 and FFAR4 only bind long-chain FFA. FFAR2 and FFAR3 can bind to short-chain FFA. The fifth, and least explored FFAR, GPR84, has greatest affinity for medium-chain FFAs. Herein, we will validate the role that the medium chain FFAR GPR84 has on the immune system.
GPR84 is a medium-chain fatty acid receptor. It was found through a data mining strategy searching for GPCRs. Still defined as an orphan receptor, its ligand, MCFAs, was not discovered until years later. Further examination found that GPR84 is Gi/o GPCR, and interaction with its ligand leads to an intracellular reduction of cyclic adenosine monophosphate (cAMP). GPR84 is distributed among various tissues in the body, such as adipocytes, but also, GPR84 is found to have high expression in immune cells. In studies of adipogenesis, GPR84 was upregulated in human adipocytes stimulated with inflammatory cytokines and mice fed with high-fat diet.
In the immune tissue, GPR84 is expressed in monocytes, macrophages, granulocytes, T cells, and B cells. These studies found that immune cells exposed to a pro-inflammatory stimulus induce high expression of GPR84. Other metabolically contributing factors which increase GPR84 expression in macrophages include hyperglycemia, hypercholesterolemia, and oxidized LDL. Studies examining the role of GPR84 in T cells found that GPR84 deficient mice increased IL-4, IL-5 and IL-13, but had no effect on IL-2 or IFN-gamma production.
Without wishing to be bound by theory, these studies indicate that GPR84 acted as a feed-forward mechanism during inflammation. In this mechanism, a pro-inflammatory stimulus increases the expression of GPR84, and further signaling through GPR84 exacerbates this inflammatory response. This pathway was shown in one study where immune cells exposed to LPS induced GPR84 and that supplementing with MCFA increased the production of IL-12p40. These feed-forward pro-inflammatory findings developed a therapeutic void and lead to the development of GPR84 agonists. The synthetic agonist, 6-OAU, was found the increase chemotaxis of human polymorphonuclear leukocytes and macrophages and increase proinflammatory cytokine production. In a separate study, GPR84 agonism under inflammatory conditions elevated pro-inflammatory cytokines and increased bacterial phagocytosis and this elevated response was abrogated in GPR84 deficient mice or in macrophages treated with a GPR84 antagonist. A separate natural GPR84 agonist, Embelin, was also examined. Here, studies found that treatment with Embelin lead to neutrophil chemotaxis and primed these cells for oxidative burst and that loss of GPR84 abrogated the pro-inflammatory cytokine secretion. Also, a synthesized GPR84 agonist, DL175, was also found to promote phagocytosis of in human myeloid cells. In addition, there have been studies which have examined the effect of GPR84 agonists in cancer cells. However, most were conducted by examining the direct effect of agonist on cancerous cell lines in vitro. The sole agonist used in vivo was conducted in nude mice, which lack the immune system and cannot provide any evidence that GPR84 agonists modulate the immune system. These controlled systems have raised skepticism that in these studies the agonists used cannot be acting through the GPR84 receptor at all.
These findings regarding myeloid chemotaxis and metabolic regulation lead researchers to examine GPR84's anti-inflammatory and metabolic regulation. In studies using antagonists, a goal was to prevent chemotaxis and to elicit and anti-inflammatory response. The first antagonist created, PBI-4050, was found to be both an agonist of GPR40 and an antagonist of GPR84. PHI-4050 was found to be both an agonist of GPR40 and an antagonist of GPR84. PBI-4050 was found to affect macrophages, fibroblasts, myofibroblasts, and epithelial cells. Antagonism reduced fibrotic capacity of these cells while also dampening macrophage activation and secretion of pro-inflammatory and pro-fibrotic molecules. In addition, macrophage recruitment was prevented, known contributors to fibrotic disease. In a similar approach, the use of GLPG1205, a GPR84 specific receptor antagonist was used in the reduction of inflammation and fibrosis. This study also found that antagonism of GPR84 led reductions in neutrophils, monocytes, and macrophage migration. When examined in three separate models of NASH, there was a significant decrease in macrophage accumulation as well as reductions in both inflammation and fibrosis. Both studies showed similar outcomes, and both indicated that the use of antagonisms as an anti-inflammatory and anti-fibrotic drug can be therapeutic.
Another study examined the role of GPR84 in metabolic deregulation. Supplementing previous GPR84 antagonist literature, this study examined PBI-4547, a GPR40 and PPAR gamma agonist with some GPR84 antagonist, was studied in Non-alcoholic fatty liver disease (NAFLD). A model of diet-induced obesity was used and antagonism of GPR84 with PBI-4547 improved metabolic dysregulation through reductions in hyperglycemia and hypertriglyceridemia. Interestingly, when examining the effects in GPR84 deficient mice, this ability to regulate the metabolism was absent suggesting GPR84 receptor can play a major role. The mechanistic ability to regulate metabolism was due to an ability to modulate glycolysis and fatty acid oxidation. Researchers found increases in glucose transporters and upregulations of enzymes which leading to improved glycolysis. In addition, increases in fatty acid oxidation led to improve metabolism of fatty acid oxidation. Both metabolic adjustments reduced overall metabolic dysregulation and returned the system back to physiological homeostasis. A separate study found that the use of a separate GPR84 modulator, PBI-4050, also improved glycemic control, suggesting that GPR84 can have an important role in metabolic regulation.
Our data indicates that by using an antagonist or an agonist of GPR84 FFAR one can manipulate the inflammatory response such that it can cause a therapeutic effect in diseases associated with inflammation. Inflammation can refer to the effect caused by T lymphocytes and myeloid cells (macrophages and myeloid-derived suppressor cells). For example, one can want to increase (strengthen) the protective response of the immune system in an infectious disease such as COVID-19 or in cancer by increasing the ability of T cells to kill cells infected by the virus or tumor cells. Data described herein indicate that the use of an antagonist to GPR84 FFAR causes an increase in the proteins granzyme Band perform which are used by T cells to kill infected cells or tumor cells. This happens while simultaneously inhibiting the myeloid cells that decrease the function of T cells. Furthermore, in a disease such as COVID-19, one of the mechanisms that causes severe pulmonary complications, is the invasion of the lungs by myeloid-derived suppressor cells producing high levels of arginase 1. Data described herein indicate that the use of a GPR84 FFAR antagonist decreases the expression and production of arginase 1 and decreases its ability to block T cell function.
When using a GPR84 FFAR agonist, stimulation of immune cells with a GPR84 agonist causes a decrease in T cell cytotoxic mechanisms and increases the immunosuppressive mechanisms (arginase 1 and nitric oxide synthase) in MDSC. This leads to a decrease in T cell mediated inflammation by the increased function of MDSC. This approach can be useful in diseases mediated by inflammation such as autoimmune diseases including colitis, rheumatoid arthritis, lupus, Crohn's disease and others. Furthermore, it can be used to prevent diseases where the immune system causes damage to important structures including, but not limited to, diabetes mellitus (diabetes Type I), myocarditis, and the rejection of transplanted organs including solid organs (kidneys, liver, lung, heart) or bone marrow transplants.
Modulating immune cells in inflammatory diseases can play a pivotal role in managing disease progression. The value of the GPR84 receptor is its duality of function. We found that the use of a GPR84 agonist exhibits an anti-inflammatory immune profile, while a GPR84 antagonist was found to be pro-inflammatory. This contradicts current literature which suggests that GPR84 agonists are pro-inflammatory and antagonists are anti-inflammatory. The benefit of this duality is that in diseases in which the immune system is over activated, such as ulcerative colitis, Crohn's disease, Rheumatoid arthritis, Lupus, having the ability to modulate the immune system towards an anti-inflammatory state with a GPR84 agonist can be therapeutic. In contrast, the use of a GPR84 antagonist in diseases where the immune response is underactive, like cancer, can be beneficial
Immunomodulatory treatments to block inflammation can be used more often in medicine than those that increase the inflammatory response. Treatments to block inflammation can make use of drugs or antibodies that block the production of inflammatory cytokines or block the binding of these cytokines to their receptors. Examples include monoclonal antibodies directed against TNF (infliximab, adalimumab, golimumab and certolizumab), inhibitors of ILS (etanercept), inhibitors of IL4/IL13 (Dupilumab). There are the use of potent overall inhibitors including corticosteroids such as prednisone, triamcinolone and many others, or even chemotherapeutic agents. However, the use of modulators that can simultaneously modulate the function of T cells and myeloid cells to achieve a therapeutic response have not been reported.
The use of FFARs in metabolism has been studied. The modulation of immune cells through FFARs can provide opportunities to modulate inflammatory diseases. While most of the therapies have been studied in the more well studied FFARI-4, pharmacologic approaches have been made against the MCFA receptor GPR84. Literature has indicated that the use GPR84 agonists contributes to a pro-inflammatory response, while antagonists are suggested to be anti-inflammatory. Our alternative mechanism is in which the agonism of the GPR84 receptor leads to a dampening and anti-inflammatory response, while antagonism leads to a proinflammatory response. Our mechanism is different because it describes the use of GPR84 opposite of what has been reported. This surprising ability to modulate the function of inflammatory cells can have broad implications in the treatment of inflammatory diseases. In the case of diseases associated with inflammation such as ulcerative colitis, the use of a GPR84 agonist to promote an anti-inflammatory response can be beneficial. On the other side, diseases or circumstances where inflammation is desired, such as boosting the immune response after vaccination with weak antigens, the use of a GPR84 antagonist can be beneficial. Such an ability to modulate the activity of the immune system can be an integral component in inflammatory based diseases.
There are no known GPR84 agonists which have successfully made it to clinical trials. There are explanations for this which and can require further research before clinical trials and an approved therapy is provided. DIM, which is debated as a known allosteric modulator versus a direct agonist has been studied in vivo. However, these studies have been conducted in nude mice which lack an immune system, and therefore its direct role on immune tissue is unknown. Additional studies have examined this phenomenon, and even with synthesized agonists of GPR84, off-target effects were still found, muddying the ability to conclude that therapeutic effects were directly due to agonism of the GPR84 receptor. More research can be required to identify and examine a stable direct GPR84 agonist pre-clinically in vivo before movement into clinical trials can be warranted. A study which examined how quickly a GPR84 specific agonists (DL-175) was metabolized found that when exposed to mouse hepatocytes, this pharmacological agent was rapidly degraded. Such a finding can pose limitations as direct specific agonists of GPR84 can be structurally prone to rapid metabolism and clearance and can prevent their use for in vivo/clinical studies.
Various GPR84 antagonists have been brought to clinical trials. PBI-4050 and GLPG1205, the two known GPR84 antagonists have both been tested in phase II clinical trials and have strong safety profiles. In regard to the safety profile, PBI-4050 is used as a treatment for idiopathic pulmonary fibrosis. Preliminary results found that the GPR84 antagonist was well tolerated both as a single agent and in combination with nintedanib or pirfenidone in a phase II clinical trial. Results found that the ability to increase forced vital capacity was unsuccessful in patients both as a single agent and in combination with nintedanib. More so, without wishing to be bound by theory, combination therapy of PBl-4050 with pirfenidone worsened forced vital capacity, which can indicate acceleration of disease. Arguments for a reduction in FVC and acceleration in disease can be due to a drug-drug interaction between PBI-4050 and pirfenidone. When further examining the effects of PBI-4050 on inflammatory and fibrotic biomarkers, results found that patients treated with PBI-4050 had increase in IL-9, IL-7, MIP-1beta, with increases in IL-1Ra, IL-13, and G-CSF. While the argument of reduction of inflammation occurs, some of these markers such as MIP-1beta and G-CSF are common markers of inflammation. Such inflammatory increases can support the position whereby a currently unknown pro-inflammatory pathway can be stimulated with GPR84 antagonism.
PBI-4050 has also been examined as a treatment in type II diabetic patients with metabolic syndrome. A phase II study found that treatment with PBl-4050 can reduce HbAlc in type II diabetic patients with metabolic syndrome. Such results can pose to be promising in our proposal of modulation the immune system, as immune cell activity is able to be manipulated by metabolic regulation.
The other GPR84 antagonist that has been used in clinical trials is GLPG1205. GLPG1205 is a negative allosteric modulator of GPR84 that has progressed through phase II clinical trials. Initial phase I studies found that GLPG1205 occupied the human GPR84 receptor, was tolerated and had a terminal half-life of 30.1 to 140 hours. Additionally, when studying the interaction of GLPG1205 with the cytochrome family of enzymes and were found to have strong safety profiles indicating that this drugs administration can be used concomitantly with other therapies.
GLPG1205 has also been studied in the treatment of interstitial pulmonary fibrosis. Results provided a non-significant reduction in fibrosis in all groups that were supplemented with GLPG1205 over the standard of care, which warranted further studies to determine if GLPG1205 does have an effect in this disease.
GLPG1205 use was also explored as a treatment for ulcerative colitis. Results from this study found that GLPG1205 was unable to improve the inflammatory response. In fact, GLPG1205 was found to lead to worsening of colitis in certain patients in the treatment study which led to the drug's discontinuation. Without wishing to be bound by theory, the antagonism of the GPR84 receptor can lead to a pro-inflammatory response.
We do not foresee limitations with the use of GPR84 antagonists for implementation. Both drugs studied in the clinic have been touted to influence the immune system and inflammation. In addition, GPR84 antagonists have been used in diseases of metabolic dysregulation which supports that these drugs can be used in diseases of inflammation and can be excellent candidates as immune modulators in inflammatory diseases.
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, and are covered by the following claims.
This application is a Continuation-in-Part of International Application PCT/US2022/048410, filed on Oct. 31, 2022, which claims priority from U.S. Provisional Patent Application No. 63/273,691, filed on Oct. 29, 2021; U.S. Provisional Patent Application No. 63/273,698, filed on Oct. 29, 2021; U.S. Provisional Patent Application No. 63/337,230, filed on May 2, 2022; and U.S. Provisional Application No. 63/346,098, filed on May 26, 2022, the contents of each of which are incorporated herein by reference in their entireties. All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
This invention was made with government support under Grant No. P20 CA233374 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63273691 | Oct 2021 | US | |
| 63273698 | Oct 2021 | US | |
| 63337230 | May 2022 | US | |
| 63346098 | May 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2022/048410 | Oct 2022 | WO |
| Child | 18649074 | US |
