Patent Application
20190060361
The technology described herein relates to increasing the level, activity and/or lifespan of regulatory T cells, and treatments for diseases related thereto.
Autoimmune diseases can arise when the immune system becomes overactive. Instead of recognizing and destroying foreign entities, an unrestrained immune system will begin to attack a subject's own body. In a healthy subject, such diseases are prevented by regulatory T cells (Tregs), which act as a brake on the rest of the immune system, limiting its activity. If the Tregs themselves malfunction or prove insufficient to control the immune system, autoimmune diseases may arise. Accordingly, therapies have been developed for such conditions that attempt to increase the levels or activity of Tregs.
Existing therapies that target Tregs are only effective for very short periods of time. Tregs have a short lifespan after such treatments, and the subject must be continually re-treated in order to benefit from the treatment. As described herein, the inventors have discovered methods for engineering Tregs to display high levels of activity and strikingly long lifespan. Accordingly, provided herein are engineered Tregs as well as methods of making them and methods of treating disease.
Regulatory T cells (Tregs) are a specialized type of immune cell that control the overall immune response, for example, by suppressing the activity of other aspects of the immune system. Thus, Tregs normally act to downregulate the immune system, preventing overreactions that can result in autoimmune diseases. This natural role of Tregs can be exploited therapeutically, and a number of diseases can be treated by activating or stimulating a patient's Tregs. However, the existing methods of stimulating Tregs are only effective for exceptionally short periods of time, requiring constant redosing of the patient to provide a therapeutic effect.
Described herein is a method by which Tregs can be engineered to assume an activated status that persists for an execeptionally long time. Current methods of Treg activation result in Tregs that are active for periods of several days. The methods described herein provide Tregs that demonstrate activity over the course of several months.
In one aspect, described herein is a method of engineering a Treg cell, e.g., a Treg cell with a long-lived phenotype, the method comprising: contacting a Treg cell ex vivo with an inhibitor of cathepsin S, cathepsin K, and/or cathepsin L. In one aspect, described herein is a method of treating a Treg-mediated disease in a subject in need of treatment thereof, the method comprising: contacting a Treg cell ex vivo with an inhibitor of cathepsin S, cathepsin K, and/or cathepsin L; and administering the cell to the subject.
As used herein “cathepsin S” refers to a cysteine protease active at neutral pH which cleaves laminin, fibronectin elastin, osteocalcin, some collagens, chondroitin sulfate, heparan sulfate and proteoglycans of the basal membrane. Sequences for cathepsin S are known for a number of species, e.g., human cathepsin S (NCBI Gene ID: 1520). As used herein “cathepsin K” refers to a cysteine protease with a high specificity for kinins. Sequences for cathepsin K are known for a number of species, e.g., human cathepsin K (NCBI Gene ID: 1513). As used herein “cathepsin L” refers to a cysteine protease which cleaves collagen, elastin, and alpha-1 protease inhibitor. Sequences for cathepsin L are known for a number of species, e.g., human cathepsin L (NCBI Gene ID: 1514).
As used herein, the term “inhibitor” refers to an agent which can decrease the expression and/or activity of the targeted expression product, e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more. The efficacy of an inhibitor of a particular target e.g. its ability to decrease the level and/or activity of the target can be determined, e.g. by measuring the level of an expression product and/or the activity of the target. Methods for measuring the level of a given mRNA and/or polypeptide are known to one of skill in the art, e.g. RT-PCR with primers can be used to determine the level of RNA and Western blotting with an antibody (e.g. an anti-cathepsin S antibody, e.g. Cat No. ab134157; Abcam; Cambridge, Mass.) can be used to determine the level of a polypeptide. The activity of a cathepsin can be determined using methods known in the art, e.g. measuring the level of one or more of its enzymatics targets. Changes in the molecular weights of one or more targets, indicating cleavage of the target, are readily detected by western blot. In some embodiments, the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule.
In some embodiments, the inhibitor is an antibody reagent that binds specifically to cathepsin S, cathepsin K, and/or cathepsin L.
The inhibitors described herein can be inhibitors of cathepsin S, cathepsin K, and/or cathepsin L. In some embodiments, an inhibitor described herein can be a pan-cathepsin inhibitor, e.g. it can inhibit at least cathepsin S, K, and L and optionally, other cathepsin polypeptides. In some embodiments, an inhibitor described herein can inhibit cathepsin S, cathepsin K, and cathepsin L, but not other cathepsins. In some embodiments, an inhibitor described herein can inhibit cathepsin S and cathepsin K, but not other cathepsins. In some embodiments, an inhibitor described herein can be specific for cathepsin S, e.g., it inhibits only cathepsin S. In some embodiments, an inhibitor described herein can be specific for cathepsin K, e.g., it inhibits only cathepsin K. In some embodiments, an inhibitor described herein can be specific for cathepsin L, e.g., it inhibits only cathepsin L.
Inhibitors of cathepsins, including cathepsins S, K, and L are known in the art. Non-limiting examples of inhibitors specific for one or more of cathepsins S, K, and L can include, Fsn0503h humanize antibody; LY3000328; odancatib; balicatib; calpeptin; L006235; SID 26681509; VBY-891; VBY-129; VBY-825; and VBY-036. Further descriptions of cathepsin inhibitors and how to make them can be found, e.g., in U.S. Pat. Nos. 8,227,468; 8,975,296; 7,326,719; 8,877,967; 8,722,734; 8,293,722; and 8,680,152; US Patent Publications US2014/0155383; US2012/0329837; and US2006/0287402; International Patent Publications WO2000/049008; WO20012/156311; WO2005/019161; WO2005/040142; WO2012/151319; WO2014/164844; and WO2009/055467; EP Patent Publication 2635562; and Canadian Patent 2547591; each of which is incorporated by reference herein in its entirety.
In some embodiments, the methods described herein relate to contacting the cell with the inhibitor for a period of at least 2 hours. In some embodiments, the methods described herein relate to contacting the cell with the inhibitor for a period of at least 4 hours. In some embodiments, the methods described herein relate to contacting the cell with the inhibitor for a period of at least 6 hours. In some embodiments, the methods described herein relate to contacting the cell with the inhibitor for a period of no more than 24 hours. In some embodiments, the methods described herein relate to contacting the cell with the inhibitor for a period of no more than 12 hours. In some embodiments, the contact of the cell and the inhibitor can be stopped by, e.g., changing the media and/or isolating the cells from the media.
In some embodiments, the methods of treatment described herein can related to administering an autologous cell to the subject, e.g., a Treg cell can be isolated from the subject, engineered and/or contacted according to the methods described herein, and then administered to the subject.
As described elsewhere herein, the Treg cells described herein provide the surprising advantage of retaining their activity for a period of up to several months, as contrasted with current therapies where the cells lose their activity after a period of several days. Accordingly, the presently described methods and compositions permit much less frequent dosing of the subject. In some embodiments, the cells are administered no more frequently than once a month. In some embodiments, the cells are administered no more frequently than once every two months. In some embodiments, the cells are administered no more frequently than once every three months.
Additionally, because of the surprising efficacy of the cells and methods described herein, it is not necessary to treat the subject with compounds that activate Tregs. In some embodiments, the subject is not administered IL-2 or TGF-beta. In some embodiments, the subject is not administered an inhibitor of cathepsin S, cathepsin K, and/or cathepsin L.
In one aspect, described herein is a composition comprising a Treg cell and an inhibitor of cathepsin S, cathepsin K, and/or cathepsin L. In some embodiments, the inhibitor is present at a concentration sufficient to increase the activity, proliferation, and/or lifespan of the Treg cell. Methods of measuring Treg activity are described elsewhere herein. For example, Treg differentiation can be measured by detecting the expression of CD4 and Foxp3, or the immunosuppression of co-cultured Teff cells can be determined by measuring the level of IFN-gamma produced by the Teff cells. In some embodiments, the Treg cell is ex vivo and/or isolated. In some embodiments, the composition can further comprise, e.g. cell culture media.
Tregs which are engineered, e.g., treated and/or activated, according to the methods described herein are demonstrably different from naturally-occurring Tregs, as well as Tregs treated with, e.g., IL-2 or TGF-beta. For example, the Tregs described herein have a significantly longer lifespan and retain their activity for a markedly longer period of time. This difference in behavior is also reflected by structural differences that differentiate the presently described Tregs from previously-described (including naturally-occurring Tregs). In one aspect, described herein is an engineered Treg cell, the cell having a level of TLR7 polypeptide which is less than 50% of the level found in a naturally-occurring Treg cell, e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less. In one aspect, described herein is an engineered Treg cell, the cell having a level of activated TLR7 polypeptide which is less than 50% of the level found in a naturally-occurring Treg cell, e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less.
As used herein, “TLR7” or “Toll-like receptor 7” refers to a receptor protein that recognizes ssRNA in endosomes, e.g., recognition of viral infection. Sequences of TLR7 are known for a number of species, e.g., human TLR7 (NCBI Gene ID: 51284). Unactivated TRLR7 is present in a Treg as a polypeptide of about 125 kDa. TLR7 is activated by cleavage to form a polypeptide of about 70 kDa. In some embodiments, the level of activated TLR7 polypeptide is the level of the processed form of TLR7 having a molecular weight of about 70 kDa.
In some embodiments, cell has a level of unactivated TLR7 polypeptide which is 150% or greater than the level found in a naturally-occurring Treg cell, e.g. 150%, 200%, 300% or greater. In some embodiments, the level of unactivated TLR7 polypeptide is the level of TLR7 having a molecular weight of about 125 kDa.
Additional structural differences that distinguish the presently described Tregs can include increased levels of IL-2 (e.g., NCBI Gene ID: 3558) and TGF-beta (e.g., NCBI Gene ID: 7040) as well as decreased levels of IFN-γ (e.g., NCBI Gene ID: 3458) and IL-6 (e.g., NCBI Gene ID: 3569). In some embodiments, the cell described herein can further express a level of IFN-γ which is less than 50% of the level found in a naturally-occurring Treg cell (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less); a level of IL-6 which is less than 50% of the level found in a naturally-occurring Treg cell (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less); a level of IL-2 which is more than 150% of the level found in a naturally-occurring Treg cell (e.g. 150%, 200%, 300% or greater); and/or a level of TGF-β which is more than 150% of the level found in a naturally-occurring Treg cell (e.g. 150%, 200%, 300% or greater). In some embodiments, the cell described herein can further express a level of IFN-γ which is less than 50% of the level found in a naturally-occurring Treg cell (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less); a level of IL-6 which is less than 50% of the level found in a naturally-occurring Treg cell (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less); a level of IL-2 which is more than 150% of the level found in a naturally-occurring Treg cell (e.g. 150%, 200%, 300% or greater); and a level of TGF-β which is more than 150% of the level found in a naturally-occurring Treg cell (e.g. 150%, 200%, 300% or greater).
In one aspect, described herein is an engineered Treg cell, the cell expressing: a level of IFN-γ which is less than 50% of the level found in a naturally-occurring Treg cell; a level of IL-6 which is less than 50% of the level found in a naturally-occurring Treg cell; a level of IL-2 which is more than 150% of the level found in a naturally-occurring Treg cell; and/or a level of TGF-β which is more than 150% of the level found in a naturally-occurring Treg cell. In some embodiments, the cell expresses a level of IFN-γ which is less than 50% of the level found in a naturally-occurring Treg cell; a level of IL-6 which is less than 50% of the level found in a naturally-occurring Treg cell; a level of IL-2 which is more than 150% of the level found in a naturally-occurring Treg cell; and a level of TGF-β which is more than 150% of the level found in a naturally-occurring Treg cell.
Methods to measure gene expression products are known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents.
In some embodiments, a cell described herein has been contacted with an inhibitor of cathepsin S, cathepsin K, and/or cathepsin L. In some embodiments, a cell described herein can be an isolated cell. In some embodiments, a cell described herein can be an ex vivo cell.
In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having a Treg-mediated disease. A Treg-mediated disease can be any condition which is caused by, exacerbated by, and/or characterized by abnormally low levels of Tregs and/or Treg activity. Non-limiting examples of Treg-mediated diseases can include autoimmune disease; a cancer; a cardiovascular disease; a metabolic disease; systemic lupus erthythematosus; type I diabetes; arthritis; Sjoren's syndrome; type-II diabetes; obesity; atherosclerosis; abdominal aortic aneurysm; and/or transplant rejection (including organ transplantation, e.g., heart, liver, kidney, skin, lung, etc).
Subjects having a Treg disease, e.g., an autoimmune disease such as lupus can be identified by a physician using current methods of diagnosis. For example, symptoms and/or complications of lupus which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, fever, joint pain, muscle pain, fatigue, low white blood cell counts, peripheral neuropathy, skin lesions anemia, etc. Tests that may aid in a diagnosis of, e.g. lupus include, but are not limited to, antinuclear antibody testing, anti-extractable nuclear antigen, complement level testing, kidney function testing, liver enzyme testing, and complete blood count. A family history of lupus, or exposure to risk factors for lupus can also aid in determining if a subject is likely to have lupus or in making a diagnosis of lupus.
The compositions and methods described herein can be administered to a subject having or diagnosed as having a Treg-mediated disease. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. a Treg as described herein to a subject in order to alleviate a symptom of a Treg-mediated disease. As used herein, “alleviating a symptom” is ameliorating any condition or symptom associated with the disease. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
The term “effective amount” as used herein refers to the amount of a composition, e.g., a Treg as described herein, needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of a composition that is sufficient to provide a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the active ingredient, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in blood can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for Treg activity, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
In some embodiments, the technology described herein relates to a pharmaceutical composition comprising a Treg as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise a Treg as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of a Treg as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of a Treg as described herein. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. a Treg as described herein.
In some embodiments, the pharmaceutical composition comprising a Treg as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.
Suitable vehicles that can be used to provide parenteral dosage forms of a Treg as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a composition as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.
The methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. By way of example, if the subject is a subject in need of treatment for cancer, non-limiting examples of a second agent and/or treatment can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.
In certain embodiments, an effective dose of a composition comprising a Treg as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising a Treg can be administered to a patient repeatedly. In some embodiments the dosage can be from about 1×10̂5 cells to about 1×10̂8 cells per kg of body weight. In some embodiments, the dosage can be from about 1×10̂6 cells to about 1×10̂7 cells per kg of body weight. In some embodiments, the dosage can be about 1×10̂6 cells per kg of body weight. In some embodiments, one dose of cells can be administered. In some embodiments, the dose of cells can be repeated, e.g., once, twice, or more.
In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.
The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the treatement. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising a Treg as described herein can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
The dosage ranges for the administration of a Treg as described herein according to the methods described herein depend upon, for example, the form of the composition, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for symptoms or the extent to which, for example, Treg activity is desired to be induced. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.
The efficacy of a Treg as described herein in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. Treg activity. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of a mouse model of lupus. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. Treg activity.
In vitro and animal model assays are provided herein which allow the assessment of a given dose of a Treg as described herein.
By way of non-limiting example, the effects of a dose of a Treg as described herein can be assessed by administering a Treg as described herein to a B6-Faslpr mouse and measuring the level of serum IL6 and IL17, where a decrease in IL6 and IL17 indicates an improvement in the condition of the mice. Alternatively, the level of autoantibody production can be measured.
For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.
The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.
As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.
Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a Treg-mediated disease, e.g. an autoimmune disease. A subject can be male or female.
A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. a Treg-mediated disease) or one or more complications related to such a condition, and optionally, have already undergone treatment for the disease or the one or more complications related to the disease. Alternatively, a subject can also be one who has not been previously diagnosed as having the disease or one or more complications related to the disease. For example, a subject can be one who exhibits one or more risk factors for the disease or one or more complications related to the disease or a subject who does not exhibit risk factors.
A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
As used herein, “regulatory T cell” or “Treg” refers to T cells that suppress the function of other immune system cells.
As used herein, “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a cell is considered to be “engineered” when at least one aspect of the cell has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny an engineered cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
The term “isolated” or “partially purified” as used herein refers, in the case of a cell, to a cell separated from at least one other component that is present with cell as found in its natural source and/or that would be present with the cell when found in vitro. A cultured cell, or a cell existing ex vivo is considered “isolated.”
As used herein, the terms “protein” and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
In some embodiments, an inhibitor of a given polypeptide can be an antibody reagent specific for that polypeptide. As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F(ab′)2, Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
As described herein, an “antigen” is a molecule that is bound by a binding site on an antibody agent. Typically, antigens are bound by antibody ligands and are capable of raising an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof. The term “antigenic determinant” refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen-binding site of said molecule.
As used herein, the term “antibody reagent” refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody reagent” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.
The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (“FR”). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated by reference herein in their entireties). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
The terms “antigen-binding fragment” or “antigen-binding domain”, which are used interchangeably herein are used to refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term “antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546; which is incorporated by reference herein in its entirety), which consists of a VH or VL domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality.
As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
Additionally, and as described herein, a recombinant humanized antibody can be further optimized to decrease potential immunogenicity, while maintaining functional activity, for therapy in humans. In this regard, functional activity means a polypeptide capable of displaying one or more known functional activities associated with a recombinant antibody or antibody reagent thereof as described herein. Such functional activities include, e.g. the ability to bind to a target.
As used herein, “expression level” refers to the number of mRNA molecules and/or polypeptide molecules encoded by a given gene that are present in a cell or sample. Expression levels can be increased or decreased relative to a reference level.
As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.
Inhibitors of the expression of a given gene can be an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is an inhibitory RNA (iRNA). As used herein, the term “iRNA” refers to any type of interfering RNA, including but are not limited to RNAi, siRNA, shRNA, endogenous microRNA and artificial microRNA. Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part the targeted mRNA transcript. The use of these iRNAs enables the targeted degradation of mRNA transcripts, resulting in decreased expression and/or activity of the target.
As used herein, the term “iRNA” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein effects inhibition of the expression and/or activity of a target gene described herein. In certain embodiments, contacting a cell with the inhibitor (e.g. an iRNA) results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the cell without the presence of the iRNA.
In some embodiments, the iRNA can be a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.
In yet another embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.
Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, each of which is herein incorporated by reference
Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2—[known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2, also described in examples herein below.
Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.
An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Representative U.S. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.
Another modification of the RNA of an iRNA as described herein involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As used herein, the term “administering,” refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean±1%.
As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.
The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.
The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”
Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.
Other terms are defined herein within the description of the various aspects of the invention.
All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
Methods.
Using the lupus-prone Faslpr mice and human peripheral blood mononuclear cells, the role of CatS in mouse and human regulatory T-cell (Treg) toll-like receptor 7 (TLR7) expression and activation, Treg survival, immunosuppression, proliferation, and lupus mitigation was investigated.
Results.
CatS increases the expression of the active form of TLR7 in immune cells in mice and humans and in murine kidneys and enhances MyD88 and NF-κB activation. CatS deficiency or pharmacological inhibition enhanced splenic CD4+CD25highFoxp3+ Treg cell differentiate, their lifespan and T effector cell immunosuppressive function. Transfer of in vitro prepared Treg cells into lupus-prone B6-Faslpr mice reduced serum autoantibody titers and more interestingly, transfer of Treg cells isolated from CatS-deficient mice or from normal mice and treated overnight with a CatS inhibitor reduced further serum autoantibody levels. Also, such transferred Treg cells could be found at increased numbers fifteen weeks later in the spleens and kidneys of B6-Faslpr mice with sustained high immunosuppressive activity.
Conclusion.
CatS is involved in the propagation of the autoimmune response through a previously unidentified mechanism, which involves the activation of TLR7 and the suppression of Treg cell function. This new H-2-independent mechanism indicates the use of CatS inhibitor-treated Treg cells to treat patients with systemic autoimmunity who are MHC polymorphic.
Introduction.
Cathepsin S (CatS) mediates invariant chain (CD74) stepwise proteolysis in antigen presenting cells (APCs) and is important in major histocompatibility complex (MHC) class-II antigenic peptide loading and antigen presentation (1, 2).
It has been shown that CatS activates Toll-like receptors (TLRs) TLR7 and TLR9 (11, 12) which bind viral nucleic acids and activate the NF-κB and interferon (IFN) regulatory factor (IRF) signaling pathways, thereby triggering the production of inflammatory cytokines (e.g., IL6) and type-1 IFNs (e.g., IFN-α and IFN-β), respectively (13). Genetic absence of TLR7 diminishes autoantibodies against Smith antigen (Sm) or ribonucleoprotein (RNP)-Sm complex (RNP/Sm) and reduces kidney glomerular IgG and C3 deposition and glomerulonephritis (14), whereas TLR9 function in lupus remains controversial (14, 15). Patients with systemic lupus erythematosus (SLE) have increased IL6, IL17, and IFN-α levels which correlate with disease activity and manifestations (16). These cytokines interfere or diminish regulatory T-cell (Treg) suppression of CD4+ T-effector cells (Teff) (12, 17), leading to Teff resistance among SLE patients. Lastly, genetic duplication of TLR7 leads to lupus-like disease and autoimmunity in humans and the BXSB mice (18-20).
It was investigated whether CatS activates TLR7 and interferes with Treg function. Such an effect would explain why its inhibition mitigates lupus-related manifestations in both H-2b and H-2k mice. Indeed, it is demonstrated herein that CatS inhibition in mice and humans promotes Treg function and at least in mice transfer of Treg cells from CatS-deficient or those pre-treated with a CatS inhibitor mitigates lupus-like autoimmunity in the B6-Faslpr mice.
Materials and Methods
Mice.
B6-Faslpr (N11), B6-Tlr7−/− (N10), MRL/MpJ-Faslpr, and B6-CD45.1 transgenic mice (N25) were purchased from the Jackson Laboratory. B6-Tlr9−/− mice were purchased from the Mutant Mouse Regional Resource Centers at the Jackson Laboratory. B6-Ctss−/− mice (N15) were described previously (3). B6-Faslpr mice were crossbred with B6-Ctss−/− mice to generate B6-Faslpr Ctss−/− mice.
To perform Treg adoptive transfer in B6-Faslpr mice, spleen CD4+CD25+ Treg cells were purified from B6-WT, B6-Ctss−/−, or B6-CD45.1 mice according to the manufacturer's instructions (Miltenyi Biotec, Inc., Auburn, Calif.). The resulting CD45.1+CD4+CD25+ Treg cells were also further purified with cell sorter (The BD FACSAira™ Cell Sorter, BD Biosciences, San Jose, Calif.). Treg purity was confirmed by FACS and anti-Foxp3 antibody-mediated immunofluorescent staining. WT Treg cells were incubated with a CatS inhibitor (10 μg/mL, clinicaltrials.gov/show/NCT01515358) overnight before adoptive transfer. Each 9-week-old female B6-Faslpr recipient mouse received intravenous injection of 5×106 donor Treg cells. Blood samples and urine samples were collected biweekly three weeks after the adoptive transfer for 12 weeks. At the age of 24 weeks, mice were sacrificed and splenocytes were analyzed for CD4, CD25, and Foxp3 by FACS and kidneys were collected for CD4+Foxp3+ Treg immunofluorescent staining.
Immunofluorescent Staining.
Frozen kidney and spleen sections (5 μm), splenocytes, and purified Tregs were prepared for immunofluorescent staining using anti-CD45.1 (1:100, BioLegend, San Diego, Calif.), Ki67 (1:100, BioLegend), CD4 (1:250, Abnova, Walnut Calif.), and Foxp3 (1:100, eBioscience, San Diego, Calif.) monoclonal antibodies (mAb).
ELISA.
Serum autoantibodies were assessed by ELISA as described (21). NUNC maxisorp ELISA plates were pre-coated with ssDNA (100 μg/ml), dsDNA (100 μg/ml), histone (20 μg/ml) and RNP/Sm (20 μg/ml) in PBS at 4° C. overnight. Plates were blocked with 3% FCS for 1 h at 37° C., washed, and incubated with 1/300˜1/1000 dilutions of mouse sera for 1 h at 37° C. Anti-ssDNA antibody (clone TNT-3, IgG2a, Abcam, Cambridge, Mass.), anti-dsDNA antibody (clone HYB331-01, IgG2a, Abcam), anti-histones (clone 2Q2205, IgG2a, Abcam), and anti-RNP/Sm antibody (clone NB600-546, IgG3 Kappa, NOVUS Biologicals, Littleton, Colo.) were used as standards. Plates were washed, and a 1/1000 dilution of alkaline phosphatase-linked corresponding goat anti-mouse IgG2a or IgG3 secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) in PBS were added for 1 hour at 37° C., and developed with a phosphatase substrate for 30 minutes at 37° C. Mouse serum total CatS, pro-CatS levels were determined according to the manufacturer's instructions, including total CatS DuoSet (DY1183, R&D systems, Minneapolis, Minn.), pro-CatS DuoSet (DY2227, R&D systems). Active CatS levels were determined by subtracting Pro-CatS from total CatS.
Antibodies and Flow Cytometry Analysis.
The following antibodies were used for FACS analysis: FcR-blocking antibody anti-CD16/32 (eBioscience), anti-CD4-Alexa Fluor 488, anti-CD25-PE, anti-mFoxp3-Alexa Fluor 647, anti-mouse-PE and all isotype controls (all from BD Biosciences). To determine the proportion of CD4+CD25highFoxp3+ Treg cells in splenocytes, 100 μl of splenocyte suspension (˜1×107 cell) was incubated at 4° C. in a phosphate buffered saline containing 2% fetal calf serum (FCS) with the Alexa Fluor 488-conjugated anti-CD4 and PE-conjugated anti-CD25 fluorescent antibodies followed by Alexa Fluor 647-conjugated FoxP3 intracellular staining. Staining for intracellular Foxp3 was performed using the fixation/permeabilization solution kit (BD Biosciences). Isotype controls were used for each antibody. Cells were acquired and analyzed with a FACSCalibur™ flow cytometer using CellQuest™ research software (version 3.3, BD Biosciences).
Real-Time PCR, Western Blot Analysis, and JPM Labeling.
Total RNA was prepared from kidney tissue or Treg cells using the Qiagen mini kit (Qiagen Inc., Valencia, Calif.). RNA concentration and quality were evaluated using the Agilent 2100™ bioanalyzer (Nano LabChip, Agilent Technologies, Santa Clara Calif.). After the cDNA synthesis, gene expression was quantified by real-time PCR on the ABI Prism 7900™ sequence detection system (Taqman, Applied Biosystems Inc., Foster City, Calif.). The low-density array detected 5 genes in one run in triplicates including endogenous controls (β-actin) and the mRNA levels of TLR7.
For immunoblot analysis, an equal amount of protein from each cell type or kidney lysate preparation was separated on a SDS-PAGE, blotted, and detected with different antibodies, including TLR7 (1:500, Abcam), phosphor-MyD88 (1:500, Cell Signaling Technology, Danvers, Mass.), phosphor-p65 NF-κB (1:500, Cell Signaling Technology), β-actin (1:3,000, Santa Cruz Biotechnology Inc.), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:2000, Cell Signaling Technology).
The JPM probe was used to label and detect active cathepsins in kidney tissue extracts or Treg cell lysate. Tissues or cells were lysed in a pH 5.5 lysis buffer containing 1% Triton X-100, 40 mM sodium acetate, and 1 mM EDTA. Cathepsin active site JPM labeling was performed according to our prior studies (9, 22).
Teff and Treg Cell Preparation from Spleen Cells.
Mouse CD4+CD25− Teff cells and CD4+CD25−/− Treg cells in this study were purified from splenocytes. Briefly, splenocytes were collected from 6˜10-week-old mice or 24-week-old B6-Faslpr mice that received adoptive transfer of in vitro prepared Treg cells. Single cell suspensions were incubated with biotinylated-antibody cocktail containing antibodies against CD8a, CD11b, CD45R, CD49b and Ter-119 to deplete macrophages, granulocytes, B cells, and CD8+ T cells by negative selection. CD25+ cells were isolated from the CD4+ cell population by staining with PE-conjugated anti-CD25 antibody followed by incubation with magnetic-activated cell sorting (MACS) anti-PE microbeads (Miltenyi Biotec, Inc.). CD4+CD25−/− T cells were then positively selected on a MACS mini-separation magnetic column, and the flow through fraction containing CD4+CD25− T cells were collected. More than 90% of these cells were Treg cells that are positive for CD4 and CD25, as confirmed by FACS. Negative selection of CD25+ cells yielded CD4+CD25− Teff cells. In some experiments, we performed a second round of CD4+CD25+ Treg purification using a cell sorter with magnetic bead-purified Treg cells as starting material.
Treg Activity Assay in Suppressing Teff Cells.
To test the function of Treg cells from B6-WT, B6-Faslpr, B6-Ctss−/−, and B6-FaslprCtss−/− mice and those from B6-Faslpr recipient mice that received donor Treg cells, a CD4+CD25+ Treg isolation kit with or without a second round of further purification with a cell sorter was used to isolate Treg cells. CD4+CD25− Teff cells (3×104 cells) were used as responder T cells and co-cultured with or without CD4+CD25+ Treg cells (3×104 cells) with or without anti-CD3 mAb (2 μg/mL, clone OKT3, eBioscience) and anti-CD28 mAb (2.5 μg/ml, clone L293, BD Biosciences) or Treg cells pre-treated overnight with a CatS inhibitor (10 μg/mL) (23). Co-cultures were maintained in complete RPMI 1640 medium for 2 days. Culture media were collected for ELISA (eBioscience) to determine IFN-γ. All experiments were performed in triplicates.
In Vitro Treg Differentiations.
Naïve spleen cells or naïve CD4+ T cells purified from spleen cells (Miltenyi Biotec, Inc.) were cultured on an anti-CD3 mAb-pre-coated (5 μg/mL, eBioscience) 96-well plate in 200 mL complete RPMI 1640 containing 5 μg/mL anti-CD28 mAb (eBioscience) and stimulated with or without TGF-β (5 ng/mL, R&D Systems) and IL2 (200 U/mL, R&D System) for 2 days. Splenocytes and CD4+ T cells were collected and analyzed for CD4 and Foxp3 expression by FACS.
CatS Activity in Human Peripheral Blood Mononuclear Cells (PBMC), TLR7 Expression, and Treg Differentiation, Survival, and Immunosuppression.
Human PBMCs were purified from whole blood from three normal individuals from the blood bank of the Massachusetts General Hospital. Partners Human Research Committee approved the use of unidentified human blood specimens (protocol #2010-P-001930/2). PBMCs were incubated with and without a CatS inhibitor (10 μg/mL) overnight and their lysates were subjected to immunoblot analysis to detect both full-length and processed human TLR7. To purify human Treg and Teff cells, we first depleted non-CD4+ T cells using a cocktail of biotin-conjugated antibodies against CD8, CD14, CD15, CD16, CD19, CD36, CD56, CD123, TCRγ/δ and CD235a (glycophorin A), followed by anti-biotin MicroBeads separation. CD4+CD25+ Treg and CD4+CD25− Teff cells were separated from purified CD4+ T cells using the CD25 MicroBeads for subsequent positive selection, according to the manufacturer (Miltenyi Biotec, Inc.). Secondary purification was performed using the cell sorter and the magnetic bead-purified Treg cells as starting material.
Purified CD4+ T cells from human PBMCs (Miltenyi Biotec, Inc.) were cultured on an anti-CD3 mAb-pre-coated (5 μg/mL, eBioscience) 96-well plate in 200 μL complete RPMI 1640 containing 5 μg/mL anti-CD28 mAb (eBioscience) and stimulated with or without TGF-β (5 ng/mL, R&D Systems), IL2 (200 U/mL, R&D System), and CatS inhibitor (10 ug/mL) for 1, 2, or 5 days, or additional 5 days without treatment. CD4+ T cells were collected and analyzed for CD4, CD25, and Foxp3 expression by FACS.
To test the function of human Treg cells, we incubated CD4+CD25− Teff cells (1×104) with or without purified human CD4+CD25high Treg cells (1×104) pre-treated with and without CatS inhibitor (10 μg/mL) overnight in the presence of anti-CD3 (2 μg/mL, eBioscience) and anti-CD28 (2.5 μg/mL, BD Biosciences) mAbs. All incubations were run in triplicates in 96-well plates with a final volume of 200 μl. At 3 days, supernatants were evaluated for IFN-γ and IL2 production by ELISA (BD Pharmingen).
Statistical Analysis.
Data were analyzed using non-parametric Mann-Whitney U test followed by Bonferroni corrections due to small sample sizes and often skewed data distributions. To simplify the data presentation of autoantibodies and serum CatS levels, we did not compare data from each time point, but rather compared all time point together as a whole from each group of mice using repeated-measures ANOVA. All data are presented as mean±SEM. P<0.05 is considered statistically significant.
Results
CatS Mediates TLR7 Expression and Activation in the Kidney and Treg Cells.
B6-Faslpr mice develop spontaneous SLE-like manifestations after 13-14 weeks with lupus nephritis in a manner similar to that observed in patients with SLE (14). JPM probe (targeting cysteine protease active site) labeled kidney tissue lysates from 24-week-old B6-Faslpr mice and revealed elevated CatS activity (3, 22). CatS activity disappeared in kidney extracts from CatS-deficient B6-FaslprCtss−/− mice (
Similar observations were made when lysates from spleen Treg cells from B6-Faslpr and B6-FaslprCtss−/− mice were used. CD4+CD25highFoxp3+ Treg cells were purified from crude splenocytes using magnetic beads. FACS analysis showed that the purity of these cells reached to 90% (
CatS Controls Treg Differentiation and Function.
Increased TLR7 expression and activation, elevated MyD88 and NF-κB phosphorylation in kidneys and Treg cells from lupus-prone B6-Faslpr mice are consistent with increased IL6 (30) and IL17 (31) in SLE patients and lupus-prone mice. Elevated serum IL6 and IL17 and reduced serum IL2 and TGF-β were found in B6-Faslpr mice but reduced serum IL6 and IL17 and increased serum IL2 and TGF-β were found in B6-FaslprCtss−/− mice further indicating a role of CatS in Treg biology (data not shown). CatS deficiency and inhibition resulted in enhanced Treg differentiation as determined by the expression of Foxp3 when total spleen cells were cultured in the presence of IL2 and TGF-β (
CD4+CD25highFoxp3+ Treg cells prepared from splenocytes from B6-Ctss−/− or B6-FaslprCtss−/− mice using magnetic beads were 50%˜60% more potent than their WT counterparts in suppressing CD4+CD25− Teff cell function as estimated by IFN-γ production (
Reduced spleen Treg numbers in B6-Faslpr mice increased 15 weeks after adoptive transfer of in vitro prepared spleen Treg cells from WT mice. When the same numbers of donor Treg cells from Ctss−/− mice or WT Treg cells pre-incubated overnight with a CatS-selective inhibitor were transferred into B6-Faslpr mice, elevated spleen Treg cells were found 15 weeks later (
Starting at 12 weeks of age, B6-Faslpr mice developed autoantibodies against histone, ssDNA, dsDNA, and RNP/Sm and probably other autoantigens (
Spleen Treg cells purified in two steps—magnetic bead separation followed by secondary cell sorting purification (
To trace the survival and immunosuppressive activity of Treg cells in B6-Faslpr mice, CD45.1+ Treg cells were purified using the two-step approach (magnetic beads and cell sorter) to ensure Treg purity (
TLR7 immunoblot analysis demonstrated that cleaved TLR7 was significantly reduced in kidney lysates from B6-Faslpr mice receiving WT Treg cells and further reduced in kidneys from those receiving Treg cells from CatS-deficient or CatS inhibitor pre-treated Treg cells, reaching the levels recorded for lysates from B6-FaslprCtss−/− mice (
CatS Controls Human Treg Differentiation, Survival, and Immunosuppressive Activity.
CatS inhibitor-treated Treg cells can provide effective cell therapy for human SLE and other autoimmune diseases associated with Treg insufficiency. Naïve CD4+ T cells were purified from PBMCs obtained from healthy individuals and cultured in vitro in the presence of IL2, TGF-β, and a CatS inhibitor. As shown in
Discussion
Described herein is the demonstration that CatS is involved in the regulation of the immune system though yet another distinct mechanism, which involves the deterioration of Treg function by activating TLR7. Previously, it was shown that CatS mediates CD74 processing and MHC-II peptide loading in APC endolysosomes to control CD4+ T-cell activation and antibody production (1-3). While MHC class-II-antigenic peptide complex formation and antigen presentation in APCs from mice with the H-2b (e.g. C57BL/6 mice) and H-2d (e.g. Balb/C mice) haplotypes depend on CatS activity in CD74 processing (3, 35), the same action does not take place in APCs from mice with the H-2k (e.g. C3H/He and MRL/MpJ mice), H-2s (e.g. SJL/J mice), and H-2U (e.g. PL/J mice) haplotypes (9, 10). After noticing that CatS deficiency (accompanying manuscript) or pharmacologic inhibition (ref. 36 and accompanying manuscript) suppresses disease in lupus-prone mice irrespectively of their H-2 haplotypes, described herein is another mechanism whereby this protease interferes with immunoregulation: it deteriorates Treg function by activating TLR7.
Like antigen processing, CatS-mediated TLR7 activation, initially reported in macrophages (11, 12), occurs also in the endolysosomes. It is demonstrated herein that this activity takes place in mouse and human Treg cells as well as murine kidneys. The identification of non-H-2-restricted mechanism of action of CatS has significant implications in the treatment of autoimmune diseases in patients who have an obviously polymorphic MHC class-II system (37).
One unexpected discovery of this study is that in human and mouse naïve CD4+ T cells and splenocytes, CatS deficiency or inhibition enhanced IL2/TGF-β-induced Treg differentiation by 40% to 100% and prolonged Treg lifespan. A brief (overnight) inhibition in vitro with a CatS inhibitor prolonged Treg survival in lupus-prone mice for 15 weeks or possibly longer, without loosing their greater than 50% more potent immunosuppressive activity against Teff cells. CD4+CD25highFoxp3+ Treg cells mediate peripheral tolerance and suppress excessive immune responses (32). Besides CD4+CD25− Teff cells, Treg cells also suppress macrophages, B cells, NK cells, and CD8+ T cells (38, 39). Patients with active SLE have fewer Treg cells in lymphoid organs, the kidneys, or the blood. In addition to number changes, Treg cells in the peripheral tissues from SLE patients also show reduced immunosuppressive activity (34, 40). The lupus-prone mice (BZB×ZNW)F1 and MRL/MpJ-Faslpr are also known to have similar reductions in Treg numbers and immunosuppressive activity (41, 42). Treg numbers increase in patients with inactive disease (40) or after treatment (43, 44), indicating that increasing their numbers and function can be therapeutic.
It is demonstrated herein that long-lived effective Treg cells can be generated in vitro and infused into patients to control disease. It is specifically contemplated herein that Treg cells harvested from patients with autoimmune diseases can be treated in vitro with a CatS inhibitor and reinfused to control disease. Such an approach would obviate the administration of CatS inhibitors to patients and save them from potential side effects.
Regulatory T cells (Tregs) are immune suppressing cells, but their roles in tumor growth have been elusive, depending on tumor type or site. As described in Example 1, demonstrated herein is a role of cathepsin S (CatS) in reducing Treg immunosuppressive activity. The effect of CatS inhibition in Tregs on tumors was investigated. Mice receiving inhibitor-treated Tregs had fewer splenic and tumor Tregs, and lower levels of tumor and splenic cell proliferation than those in mice received saline-treated Tregs. In vitro, inhibitor-treated Tregs showed lower proliferation and higher apoptosis than saline-treated Tregs when cells were exposed to mouse bladder carcinoma MB49 cells. In contrast, both types of Tregs showed no difference in proliferation when they were co-cultured with normal splenocytes. Inhibitor-treated Tregs had less apoptosis in splenocytes, but more apoptosis in splenocytes with MB49 conditioned media than saline-treated Tregs. In turn, less proliferation and more apoptosis of MB94 cells was detected after co-culture with inhibitor-treated Tregs, compared with saline-treated Tregs. B220+ B-cell, CD4+ T-cell, and CD8+ T-cell proliferation and apoptosis were also lower in splenocytes co-cultured with inhibitor-treated Tregs than with saline-treated Tregs. Under the same condition, addition of cancer cell conditioned media greatly increased CD8+ T-cell proliferation and reduced CD8+ T-cell apoptosis. These observations indicate that CatS inhibition of Tregs reduces overall T-cell immunity under normal conditions, but enhances CD8+ T-cell immunity in the presence of cancer cells.
CD4+CD25+Foxp3+ regulatory T cells (Tregs) are immunosuppressive cells that play protective role in inflammatory diseases, such as atherosclerosis, abdominal aortic aneurysms, obesity and diabetes (1-7). In contrast, Tregs suppress cytotoxic CD8+ T cells in many solid tumors, thereby having a significant negative effect on tumor-associated overall survival (8). In patients with ovarian cancers, tumor Tregs exhibited more potent suppression of CD8+ T cells than those in the peripheral (9). Reduced tumor Treg contents were associated with improved overall survival of these patients (10). Tregs in gastric tumors (11), in the peripheral blood from patients with B cell lymphoma (12) or acute B lymphoblastic leukemia (13) were all elevated. In patients with breast cancer, tumor Treg contents and expression of PD-L1 (programmed death ligand 1) were positively correlated (14). In a mouse model of lung adenocarcinoma, Tregs in the advanced lung tumors suppressed the anti-tumor T-cell responses. Depletion of Tregs caused immune-mediated tumor destruction (15). In mouse melanoma cell tumor model, Treg-specific depletion of PTEN (phosphatase and tensin homolog), which stabilizes Tregs, reduced tumor growth and inflammation (16). In B-cell acute lymphoblastic leukemia or 4T1 mammary carcinoma mice, Treg ablation led to CD8+ T-cell generation, tumor regression, and extended survival (17, 18). All these studies point to a detrimental role of Tregs in cancers. However, Tregs also exert no role or even opposite role in different types of tumors. For example, in patients with multiple myeloma, the effect of anti-tumor drug bortezomib treatment was associated with Treg expansion. Treg ex vivo expansion decreased multiple myeloma viability (19). In colorectal cancers, Treg infiltration indicated better prognosis (20-23). In head and neck or oesophageal cancers, the progonostic role of Tregs was highly influenced by tumor site, and correlated with the molecular subtype and tumor stage (8). Therefore, the role of Tregs in tumors can be complicated and may depend on the types of the tumors.
Bladder carcinoma is the fifth most common cancer with increased incidence worldwide (24, 25). Patients with high content of tumor infiltration of Tregs may have elevated incidence of recurrence (26), although a direct role of Tregs in bladder cancer has not been tested. Of note, the role of Tregs in inflammatory diseases or in cancers may vary depending on the subtypes of Tregs. A recent study reported a detrimental role of fat-resident Tregs that contribute to age-associated insulin resistance. Selective depletion of this Treg population increased adipose tissue insulin sensitivity (27). In colorectal cancers, tumor infiltration of non-immunosuppressive Foxp3lo Tregs with no expression of the naive T cell marker CD45RA and instability of Foxp3 showed better prognosis than immunosuppression-competent Foxp3hi Tregs (16). Therefore, either depletion of Foxp3hi Tregs or local increase of Foxp3lo Tregs suppressed or prevented tumor formation.
It is described herein that Tregs had increased immunosuppressive activity after a brief treatment with a small molecule inhibitor of cathepsin S (CatS), a lysosomal cyeteine protease that mediates lysosomal protein proteolysis. CatS participates in toll-like receptor-7 (TLR7) activation in Tregs, thereby changing the TLR7 downstream signaling and cytokine profile leading to elevated immunosuppressive activity. It was investigated whether Tregs with or without CatS inhibition affect bladder tumor cells in a mouse bladder cancer MB49 cell subcutaneous implantation model.
Materials and Methods
Mice, Tumor Cell Culture, and Tumor Model
Wild-type (WT) C57BL/6 mice and CD45.1 transgenic mice (C57BL/6) were purchased from the Jackson Laboratory (Bar Harbor, Me.). MB49 cells are chemically induced murine bladder carcinoma cell line derived from C57BL/6 male mice (28) (American Type Culture Collection, ATCC, Manassas, Va.). MB49 cells were maintained in RPMI 1640 medium (Gibco, Big Cabin, Okla.) supplemented with 10% fetal calf serum, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), penicillin (100 IU/ml) and streptomycin (100 μg/ml), 5×10−5 M 2-mecaptoethnal, and 2 mM L-glutamine. Each WT recipient mouse received subcutaneous implantation of 2×106 MB49 cells on the right flank.
Treg Purification and Adoptive Transfer.
To perform Treg adoptive transfer in mice with tumor, splenic CD4+CD25+ Treg cells were purified from C57BL/6 WT or C57BL/6 CD45.1 transgenic mice according to the manufacturer's instructions (Miltenyi Biotec, Inc., Auburn, Calif.). The resulting CD45.1+CD4+CD25+ Treg cells were also further purified with cell sorter (The BD FACSAira™ Cell Sorter, BD Biosciences, San Jose, Calif.). Treg purity was confirmed by FACS and anti-Foxp3 antibody-mediated immunofluorescent staining. WT Treg cells were incubated with a CatS inhibitor (10 μg/mL, see, e.g., data available on the world wide web at clinicaltrials.gov/show/NCT01515358) overnight before adoptive transfer. Each 9-week-old male C57BL/6 WT mouse received intravenous injection of 5×106 donor Treg cells three days after mice received MB49 cell subcutaneous implantation. On day 7, mice were sacrificed, splenocytes and tumor tissue single cell preparation were analyzed for CD4, CD25 and Foxp3 by FACS. Spleen and tumor tissue were also collected to prepare 5 μm frozen sections for immunohistochemical analysis.
Immunohistochemistry.
Frozen tumor and spleen sections were prepared for immunohistochemical staining using FITC-conjugated anti-mouse CD45.1 monoclonal antibody (1:1000, Abcam, Cambridge, Mass.), anti-mouse Ki67 monoclonal antibody (1:400, Thermo Fisher Scientific, Waltham, Mass.), CD31 (1:1500, BD Biosciences). Frozen tumor and spleen sections were also prepared for histological detection of apoptotic cells using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit according to the manufacturer (EMD Millipore, Billerica, Mass., USA).
Treg Cell Co-Cultures.
CD4+CD25+ Tregs were isolated from total CD4+ T cells in spleens from CD45.1+ transgenic mice using a CD4+CD25+ Treg isolation kit according to the manufacturer's instructions (Miltenyi Biotec, Cambridge, Mass.). FACS analysis confirmed the purity of each preparation of greater than 93%. CD4+CD25+ Tregs (1×106) were treated with a CatS inhibitor (10 ug/ml) or phosphate-buffered saline (PBS) for 24 hours. Mouse bladder carcinoma MB49 cells were cultured in RPMI 1640 complete medium to 90% confluence. Splenocytes were isolate from C57BL/6 WT mice. To assess the interactions between Tregs and MB49 tumor cells or with TW splenocytes, tumor cells and WT splenocytes were collected and added to Tregs on a 6-well plate at a 1:1 ratio. For splenocyte and Treg co-cultures, MB49 tumor cell conditioned media was also added. After 24 hours of co-culture, Tregs or MB49 tumor cells were collected, washed, and used for FACS analysis.
Flow Cytometry of Tregs in Tumors and Spleens from MB49-Implanted Mice.
Mouse splenocytes were prepared by removing red blood cells as described previously (29). To determine the proportion of CD4+CD25+Foxp3+ Treg cells in splenocytes and tumor cell preparation, 100 μl of splenocyte or tumor total cell suspension (˜1×107 cell) was incubated at 4° C. in PBS containing 2% FCS with the Alexa Fluor 488-conjugated anti-CD4 and PE-conjugated anti-CD25 fluorescent monoclonal antibodies (mAb), followed by Alexa Fluor 647-conjugated Foxp3 intracellular staining. Staining for intracellular Foxp3 was performed using the fixation/permeabilization solution kit (BD Biosciences). Isotype controls were used for each antibody. The following antibodies were used for FACS analysis: FcR-blocking antibody anti-CD16/32 mAb (eBioscience, San Diego, Calif.), Alexa Fluor 488-conjugated anti-CD4 mAb, PE-conjugated anti-CD25 mAb, Alexa Fluor 647-conjugated anti-mFoxp3 mAb, anti-mouse-PE and all isotype controls (all from BD Biosciences).
Flow Cytometry of Co-Cultured Cells.
For the analysis of splenocytes that were co-cultured with different CD45.1+ Tregs, total cell mixtures, containing splenocytes, Tregs, with and without MB49 conditioned media were incubated with APC-conjugated anti-mouse CD45.1 mAb and PE-conjugated anti-CD4 mAb, PE-conjugated anti-CD8 mAb, or PE-conjugated anti-B220 mAb at 4° C. for 30 min for cell surface staining then suspended at a density of 2×106 cells/mL in PBS containing 1% FBS. For Treg analysis, cells were incubated with APC-conjugated anti-mouse CD45.1 mAb.
Cell proliferation was detected by Ki67 staining. To a cell pellet (1-5×107 cells), 5 ml of cold 70%-80% ethanol was added drop wise, followed by incubation at −20° C. for 2 hours. A 30-40 ml of a wash buffer (PBS with 1% FBS and 0.09% NaN3, pH7.2) was added to the fixed cells. Cells were precipitated by centrifugation for 10 minutes at 1000 rpm, washed once with 30˜40 ml of a wash buffer. Cells were resuspended to a concentration of 1×107/ml (1×106/100 μl) and transferred 100 μl of cell suspension into a fresh tube, followed by adding 20 μl of properly diluted antibody and 10 μl of propidium iodide staining solution (BD Bioscience). After a gentle mix, tubes were incubated at room temperature for 20-30 minutes in dark, washed with 2 ml of a washing buffer at 1000 rpm for 5 minutes, and suspended into 0.5 ml of PBS for FACS analysis.
Cell apoptosis was assessed using a FITC Annexin V Apoptosis Detection Kit I (RUO) according to the manufacturer (BD Bioscience). Cells were washed twice with a cold PBS and then suspended in lx binding buffer, at a concentration of 1×106 cells/ml. Each 100 μl of the suspension containing 1×105 cells was transferred to a 5-ml culture tube, followed by adding 5 μl of FITC-Annexin V and 10 μl of propidium iodide staining solution. After a gentle vortex, cells were incubated for 15 min at room temperature in dark, followed by adding 400 μl of 1× binding buffer for FACS analysis within 1 hour. Flow cytometric acquisition was performed using a FACSCalibur™ (BD Immunocytometry Systems), and all analyses were performed using Flowjo™ software (Tree Star Inc, Ashland, Oreg.).
Statistics.
Because of relatively small sample sizes and sometime skewed data distribution, the non-parametric Mann-Whitney U test was selected for paired data sets and one-way ANOVA with post-hoc Bonferroni test was used for comparison among three or more groups to examine statistical significance for all data from cultured cells and mouse model. P<0.05 was considered statistically significant. All analyses were performed using R software, version 3.0.1.
Results
Treg adoptive transfer has been used to assess Treg immunobiology in multiple mouse disease models (5, 7). To mice that received subcutaneous implantation of MB49 bladder cancer cells, intravenous Tregs from CD45.1 transgenic mice were also given to trace donor cells. Four days after Treg adoptive transfer, splenic and tumor total CD4+CD25+ Tregs and donor CD45.1+Foxp3+ cells were assessed by FACS analysis. Treg adoptive transfer did not change significantly splenic and tumor total CD4+CD25+ Tregs (
CatS Inhibitor-Treated Tregs Reduced Recipient Mouse Splenic and Tumor Cell Tregs and Total Cell Proliferation.
FACS analysis of splenocytes from recipient mice with subcutaneous implantation of MB49 tumor cells did not reveal significant differences in CD4+CD25+Foxp3+ total Treg contents between mice received with or without donor PBS-treated Tregs. However, splenic CD4+CD25+Foxp3+ Tregs were significantly reduced in mice received inhibitor-treated Tregs (
To test why inhibitor-treated donor Treg cells reduced splenic and tumor Tregs, immunohistochemical analysis of both spleens and tumors from control mice without Treg adoptive transfer and from mice received with PBS- or inhibitor-treated Tregs were performed. Total TUNEL-positive areas in percentage in spleens did not differ among all three groups of mice (
Differential Roles of CatS Inhibitor-Treated Tregs in the Presence and Absence of Tumor Cells.
CatS inhibitor-treated Tregs showed much stronger immunosuppressive activity than untreated or PBS-treated Tregs to T effector cells. Inhibitor-treated Tregs reduced splenic and tumor CD4+CD25+Foxp3+ Tregs in recipient mice (
In turn, different Tregs also affected differently tumor cell proliferation and apoptosis. Co-culture of inhibitor-treated Tregs showed significantly more suppression of tumor cell proliferation (Ki67+ MB49 cells) (
When inhibitor-treated Tregs and PBS-treated Tregs were co-cultured with WT splenocytes, the activities of these Tregs in regulating B-cell, CD4+ T-cell, and CD8+ T-cell proliferation and apoptosis also differed, depending on the Treg type and the presence of tumor cell conditioned media. In the absence of tumor cells, inhibitor-treated Tregs showed less activity on the proliferation and apoptosis of B220+ B cells, CD4+ T cells, and CD8+ T cells in WT splenocytes (
Discussion
No role for Tregs in promoting tumor growth was detected in mouse bladder carcinoma cell subcutaneous implant tumor model, affirming a tumor type-dependent role of Tregs in affecting tumor growth. Demonstrated herein are unexpected observations that may change the view of Tregs and their immunobiological activities.
Adoptive transfer of CatS inhibitor-treated Tregs reduced recipient spleen and tumor Treg contents. These observations may be explained by overall reduced spleen and tumor cell proliferation. In vitro, a role of CatS inhibition of Tregs in reducing tumor cell proliferation was confirmed. Tumor cells, in turn, reduced Treg proliferation and increased Treg apoptosis after Tregs were pre-treated with a CatS inhibitor.
In vitro studies revealed a role of tumor cells in enhancing Treg apoptosis after Tregs were pre-treated with a CatS inhibitor. In turn, inhibitor-treated Tregs were more potent than PBS-treated Tregs in promoting tumor cell apoptosis. These in vitro data indicate more cell apoptosis in spleens and tumors from mice received inhibitor-treated Tregs than those from mice received PBS-treated Tregs.
Interaction between Tregs and other immune cells has been a major focus of Treg studies. Under normal conditions, Tregs play an important role in suppressing T effector cells. CatS inhibition enhances this activity of Tregs. Indeed, significantly lower levels of B220+ B-cell, CD4+ T-cell, and CD8+ T-cell proliferation were demonstrated when WT splenocytes were co-cultured with CatS inhibitor-treated Tregs than in those were co-cultured with PBS-treated Tregs. These observations agree with the observation that CatS inhibition can increase Treg immunosuppressive activity, possibly by reducing the numbers of B cells and T cells via reduced proliferation and/or increased apoptosis of these lymphocytes. However, in the presence of tumor cell conditioned media, such activity of inhibitor-treated Tregs became reversed. Although the proliferations of B cells and CD4+ T cells were comparable whether WT splenocytes were co-cultured with PBS- or inhibitor-treated Tregs in the presence of tumor cell conditioned media. Under this condition, CatS inhibitor-treated Tregs greatly enhanced CD8+ T-cell proliferation and reduced CD8+ T-cell apoptosis. Without wishing to be bound by theory, it is possible that CatS inhibition may increase CD8+ T-cell immunity in mice with tumors because of increased proliferation and decreased apoptosis of CD8+ T cells.
Together, this study demonstrates that CatS inhibition can turn Tregs into potent immunosuppressive cells in the absence of tumor cells. However, in the presence of tumor cells or even tumor cell conditioned media, CatS inhibition can turn Tregs into immunoactive or immunocompetent cells by enhancing the number of cytotoxic CD8+ T cells and reducing the apoptosis of this T-cell population. It is contemplated herein that CatS inhibitor-treated Tregs can be utilized in a new regimen for therapy of tumors.
Tregs are immunosuppressive cells that control inflammatory diseases, including autoimmune diseases, such as SLE. CatS inhibitor-treated Tregs showed elevated differentiation, immunosuppressive activity, and extended life span. All these activities of Treg are beneficial to SLE, or other autoimmune diseases.
However, since Tregs generally are immunosuppressive cells, some Tregs can promote tumor growth. Therefore, as a therapeutic regimen, some Tregs could be beneficial to autoimmune diseases, but detrimental to cancers. Therefore, it was tested in Example 2 whether elevated immunosuppressive activity of CatS inhibitor-treated Tregs promotes tumor growth. A mouse bladder cancer cell subcutaneous model was used to test this hypothesis. This work demonstrated that CatS inhibitor-treated Tregs have higher immunosuppressive activity than PBS-treated Tregs under normal conditions (co-cultured with wild-type splenocytes). However, under cancer conditions, i.e. co-culture of Tregs with wild-type splenocytes and tumor cells or tumor cell conditioned media, CatS inhibitor-treated Tregs become more immunocompetent than PBS-treated Tregs. CatS inhibitor-treated Tregs showed elevated activity to promote CD8+ T-cell proliferation. These T cells are required to fight cancers.
In conclusion, these findings indicate that, under normal conditions, or non-tumor conditions, CatS inhibitor-treated Tregs are immunosuppressive to autoimmune diseases (as well as other inflammatory diseases such as atherosclerosis, AAA, obesity, and diabetes). However, under tumorous conditions, these cells change their nature into immunocompetent cells against tumor cells by increasing CD8+ T-cell proliferation. Therefore, it contemplated that as a therapeutic regimen, CatS inhibitor-treated Tregs can be beneficial to patients with cancer and/or can be used to treat autoimmune diseases/inflammatory diseases in patients with cancer without exacerbating the cancer.
This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/246,388 filed Oct. 26, 2016, the contents of which are incorporated herein by reference in their entirety.
This invention was made with government support under Grant Nos. HL81090, HL60942, and HL123568 awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/058775 | 10/26/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62246388 | Oct 2015 | US |
