Patent Application
20200087625
Treatment with autologous CD8 positive patient-specific T cells has produced long term remissions in some patients with melanoma and other cancers (Rosenberg and Restifo, 2015). However, it has been very difficult to identify, and expand such cells. Usually, the so-called Tumor Infiltrating Lymphocytes (TILs) must be isolated from surgical or biopsy specimens, and expanded for weeks in tissue culture until a sufficiently large number of cells can be recovered for re-infusion (Rosenberg et al., 2008) (
Recently, the Rosenberg group at the NCI was successful in identifying neo-antigen specific T cells in the peripheral blood of some melanoma patients, using a combination of surface markers and functional assessment of reactivity to tumor antigens (Gros et al., 2016). However, the number of patients in which such cells can be recovered and expanded is meager. Accordingly, there is an unmet and critical need for simple and practical methods to increase the numbers of TILs in the blood, to demonstrate their tumor reactivity, and to expand them for re-administration.
A major goal of cancer immunotherapy is the expansion and/or reactivation of cytotoxic CD8+ T cell responses against malignant cells. Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), it was demonstrated that loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic acid-rich extracellular vesicles (EVs), which induce a type I interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. These observations uncover a role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.
In one embodiment, a protocol that allows maximizing the therapeutic effect of anti-PD1 therapy for the clonal expansion of tumor specific CD8+ T cells in TILs and spleen by local administration of an immune stimulating agent (e.g., a TLR7 or TLR9 agonist), is provided. This clonal expansion is a great predictor of efficacy of anti-PD1 therapy and hence a guiding indicator patients should be administered/treated with this therapy.
In one embodiment, a patient with cancer is administered specific drugs that promote the release of immunogenic tiny EVs from cancer cells, in the absence of cytotoxicity, which in turn induces clonal expansion of tumor specific CD8+ T cells, without knowing the exact antigen specific for the tumor cells. In one embodiment, after drug therapy, tumor specific CD8+ T cells are expanded in tissue culture, which can be made more efficient by purification of activated CD8+ T cells and by co-culture of the T cells with the immunogenic EVs derived from the blood of the same patient in the presence or absence of feeder cells. In one embodiment, the treatment of cancer patients with anti-PD1 and other checkpoint inhibitors should be undertaken specifically in those subjects who have circulating clonal T cells after drug treatment, and at the time when both EVs and TIL concentrations in the blood reach maximal levels. In one embodiment, antigen presenting cells, like dendritic cells and macrophages, release exosomes (EVs) containing antigen and immune stimulating DNA/RNA which may function as “artificial antigen presenting cells” that travel through the lymphatics to distant sites, in order to initiate a good immune response.
In one embodiment, a method to isolate and expand cancer-specific CD8+ T cells is provided. In one embodiment, the method includes administering to a mammal having a tumor an effective amount of a composition comprising an agent that promotes the release of extracellular vesicles from tumor cells; and collecting from the mammal extracellular vesicles and immune cells including tumor specific CD8+ T cells. In one embodiment, the composition comprises a TLR7 or TLR9 agonist. In one embodiment, the extracellular vesicles and the immune cells are collected from blood of the mammal. In one embodiment, the extracellular vesicles and the immune cells are collected contemporaneously. In one embodiment, the agent inhibits or inactivates LATS1 and/or LATS2. In one embodiment, the method further includes culturing the immune cells to expand and/or activate cancer-specific CD8+ T cells. In one embodiment, the collected extracellular vesicles are cultured with the immune cells. In one embodiment, the collected extracellular vesicles are less than about 0.5 microns in diameter. In one embodiment, the collected extracellular vesicles are isolated. In one embodiment, in the isolated extracellular vesicles are less than about 0.5 microns in diameter. In one embodiment, the cultured and expanded cancer-specific CD8+ T cells are isolated from non-cancer specific CD8+ T cells. In one embodiment, the method further includes culturing the immune cells in the presence of feeder cells. In one embodiment, the method further includes collecting the expanded, or activated and expanded, cancer-specific CD8+ T cells. In one embodiment, the composition is orally or parenterally administered or by intrapulmonary routes. In one embodiment, the composition is administered to the tumor by direct injection. In one embodiment, the composition is administered systemically using liposomes, antibodies or other targeting mechanisms. In one embodiment, the mammal is subjected to chemotherapy before the immune cells are collected. In one embodiment, the mammal is subjected to chemotherapy after the immune cells are collected. In one embodiment, the method further includes isolating the cultured cancer-specific CD8+ T cells. In one embodiment, the cancer-specific CD8+ T cells are administered to the mammal. In one embodiment, the mammal is subjected to chemotherapy after the cancer-specific CD8+ T cells are administered. In one embodiment, wherein the therapy is anti-PD therapy. In one embodiment, the therapy is a checkpoint inhibitor therapy. In one embodiment, the inhibitor comprises pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, or ipilimumab.
In one embodiment, a method to isolate and expand cancer-specific CD8+ T cells is provided. In one embodiment, the method includes collecting from a mammal having a tumor extracellular vesicles and immune cells including tumor specific CD8+ T cells; culturing the immune cells and enriching for cancer-specific CD8+ T cells; and culturing the enriched cancer-specific CD8+ T cells and the extracellular vesicles. In one embodiment, the mammal is subjected to chemotherapy before the immune cells are collected. In one embodiment, the mammal is subjected to chemotherapy after the immune cells are collected. In one embodiment, the method further includes isolating the cultured cancer-specific CD8+ T cells. In one embodiment, the cancer-specific CD8+ T cells are administered to the mammal. In one embodiment, the mammal is subjected to chemotherapy after the cancer-specific CD8+ T cells are administered. In one embodiment, wherein the therapy is anti-PD therapy. In one embodiment, the therapy is a checkpoint inhibitor therapy. In one embodiment, the inhibitor comprises pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, or ipilimumab.
In one embodiment, a method to enhance the immunogenicity of tumor cells is provided. In one embodiment, the method includes modifying ex vivo tumor cells of a mammal to provide for tumor cells that do not express or have reduced expression of LAT1 and/or LAT2; and administering to the mammal an amount of the modified cells effective to enhance the immune response to the tumor in the mammal.
Although direct injection of toll-like receptor 7 (TLR7) agonists into primary tumors can induce tumor-specific oligoclonal T cell responses whose magnitude correlates with therapeutic efficacy, tumors are not always accessible to local therapy. Herein below, it is demonstrated that a single systemic administration of a phospholipid conjugated TLR7 agonist can also expand tumor-specific cytotoxic T cells that are shared by different animals. The expansion can be achieved without causing apparent toxicity. Similar technology combining immune repertoire analysis and immunomodulatory drugs can help to guide the development of optimal immunotherapeutic regimens in cancer patients.
(C) Kaplan-Meier tumor-free survival curves for mice injected with WT or LATS1/2 dKO B16-OVA cells are shown (n=14 mice for each group). ***p<0.001, log-rank test. (D) WT or two independent clones of LATS1/2 dKO SCC7 cells were transplanted into C3H/HeOu mice and tumor growth was monitored after the indicated times. Data are represented as mean±SEM; n=8 tumors for each group. p values were determined using two-way ANOVA test, comparing each group to WT group. ***p<0.001.
(E) Kaplan-Meier tumor-free survival curves for mice injected with WT or LATS1/2 dKO SCC7 cells are shown (n=4 mice for each group). p values were determined using log-rank test, comparing each group to WT group. **p<0.01.
(F) BALB/c mice were injected with WT or LATS1/2 dKO 4T1 cells and primary tumor weight was determined 28 days after transplantation. (G) WT or LATS1/2 dKO 4T1 cells were transplanted into the mammary fat pad of BALB/c mice and lung metastasis of the primary tumor was determined 28 days after transplantation. Normal lung tissue was stained with black India ink, whereas tumor nodules remain white. The gross appearance of the lungs (left panel) and tumor nodules on lungs (right panel) were examined. Data are represented as mean±SEM; n=16 tumors (F) and n=8 mice (G) for each group. ***p<0.001, Mann-Whitney test.
A “phospholipid” as the term is used herein refers to a glycerol mono- or diester bearing a phosphate group bonded to a glycerol hydroxyl group with an alkanolamine group being bonded as an ester to the phosphate group, of the general formula
wherein R11 and R12 are each independently hydrogen or an acyl group, and R13 is a negative charge or a hydrogen, depending up on pH. When R13 is a negative charge, a suitable counterion, such as a sodium ion, can be present. For example, the alkanolamine moiety can be an ethanolamine moiety, such that m=1. It is also understood that the NH group can be protonated and positively charged, or unprotonated and neutral, depending up on pH. For example, the phospholipid can exist as a zwitterion with a negatively charged phosphate oxy anion and a positively charged protonated nitrogen atom. The carbon atom bearing OR12 is a chiral carbon atom, so the molecule can exist as an R isomer, an S isomer, or any mixture thereof. When there are equal amounts of R and S isomers in a sample of the compound of formula (II), the sample is referred to as a “racemate.” For example, in the commercially available product 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, as used in Example I below, the R3 group is of the chiral structure
which is of the R absolute configuration.
A phospholipid can be either a free molecule, or covalently linked to another group for example as shown
wherein a wavy line indicates a point of bonding.
Accordingly, when a substituent group, such as R3 of the compound of formula (I) herein, is stated to be a phospholipid what is meant that a phospholipid group is bonded as specified by the structure to another group, such as to an N-benzyl heterocyclic ring system as disclosed herein. The point of attachment of the phospholipid group can be at any chemically feasible position unless specified otherwise, such as by a structural depiction. For example, in the phospholipid structure shown above, the point of attachment to another chemical moiety can be via the ethanolamine nitrogen atom, for example as an amide group by bonding to a carbonyl group of the other chemical moiety, for example
wherein R represents the other chemical moiety to which the phospholipid is bonded. In this bonded, amide derivative, the R13 group can be a proton or can be a negative charge associated with a counterion, such as a sodium ion. The acylated nitrogen atom of the alkanolamine group is no longer a basic amine, but a neutral amide, and as such is not protonated at physiological pH.
An “acyl” group as the term is used herein refers to an organic structure bearing a carbonyl group through which the structure is bonded, e.g., to glycerol hydroxyl groups of a phospholipid, forming a “carboxylic ester” group. Examples of acyl groups include fatty acid groups such as oleoyl groups, that thus form fatty (e.g, oleoyl) esters with the glycerol hydroxyl groups. Accordingly, when R11 or R12, but not both, are acyl groups, the phospholipid shown above is a mono-carboxylic ester, and when both R11 and R12 are acyl groups, the phospholipid shown above is a di-carboxylic ester.
Within the present disclosure it is to be understood that a compound of the formula (I) or a salt thereof may exhibit the phenomenon of tautomerism whereby two chemical compounds that are cap able of facile interconversion by exchanging a hydrogen atom between two atoms, to either of which it forms a covalent bond. Since the tautomeric compounds exist in mobile equilibrium with each other they may be regarded as different isomeric forms of the same compound. It is to be understood that the formulae drawings within this specification can represent only one of the possible tautomeric forms. However, it is also to be understood that the disclosure encompasses any tautomeric form, and is not to be limited merely to any one tautomeric form utilized within the formulae drawings. The formulae drawings within this specification can represent only one of the possible tautomeric forms and it is to be understood that the specification encompasses all possible tautomeric forms of the compounds drawn not just those forms which it has been convenient to show graphically herein. For example, tautomerism may be exhibited by a pyrazolyl group bonded as indicated by the wavy line. While both substituents would be termed a 4-pyrazolyl group, it is evident that a different nitrogen atom bears the hydrogen atom in each structure.
Such tautomerism can also occur with substituted pyrazoles such as 3-methyl, 5-methyl, or 3,5-dimethylpyrazoles, and the like. Another example of tautomerism is amido-imido (lactam-lactim when cyclic) tautomerism, such as is seen in heterocyclic compounds bearing a ring oxygen atom adjacent to a ring nitrogen atom. For example, the equilibrium:
is an example of tautomerism.
Accordingly, a structure depicted herein as one tautomer is intended to also include the other tautomer.
It will be understood that when compounds of the present disclosure contain one or more chiral centers, the compounds may exist in, and may be isolated as pure enantiomeric or diastereomeric forms or as racemic mixtures. The present disclosure therefore includes any possible enantiomers, diastereomers, racemates or mixtures thereof of the compounds of the disclosure.
The isomers resulting from the presence of a chiral center comprise a pair of non-superimposable isomers that are called “enantiomers.” Single enantiomers of a pure compound are optically active, i.e., they are capable of rotating the plane of plane polarized light. Single enantiomers are designated according to the Cahn-Ingold-Prelog system. The priority of substituents is ranked based on atomic weights, a higher atomic weight, as determined by the systematic procedure, having a higher priority ranking Once the priority ranking of the four groups is determined, the molecule is oriented so that the lowest ranking group is pointed away from the viewer. Then, if the descending rank order of the other groups proceeds clockwise, the molecule is designated (R) and if the descending rank of the other groups proceeds counterclockwise, the molecule is designated (S). In the example in Scheme 14, the Cahn-Ingold-Prelog ranking is A>B>C>D. The lowest ranking atom, D is oriented away from the viewer.
The present disclosure is meant to encompass diastereomers as well as their racemic and resolved, diastereomerically and enantiomerically pure forms and salts thereof. Diastereomeric p airs may be resolved by known separation techniques including normal and reverse phase chromatography, and crystallization.
“Isolated optical isomer” means a compound which has been substantially purified from the corresponding optical isomer(s) of the same formula. In one embodiment, the isolated isomer is at least about 80%, e.g., at least 90%, 98% or 99% pure, by weight.
Isolated optical isomers may be purified from racemic mixtures by well-known chiral separation techniques. According to one such method, a racemic mixture of a compound of the disclosure, or a chiral intermediate thereof, is separated into 99% wt. % pure optical isomers by HPLC using a suitable chiral column, such as a member of the series of DAICEL® CHIRALPAK family of columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). The column is operated according to the manufacturer's instructions.
As used herein, “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, behenic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.
The pharmaceutically acceptable salts of the compounds useful in the present disclosure can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile may be employed. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985), the disclosure of which hereby incorporated by reference.
The compounds of the formulas described herein can be solvates, and in some embodiments, hydrates. The term “solvate” refers to a solid compound that has one or more solvent molecules associated with its solid structure. Solvates can form when a compound is crystallized from a solvent. A solvate forms when one or more solvent molecules become an integral part of the solid crystalline matrix up on solidification. The compounds of the formulas described herein can be solvates, for example, ethanol solvates. Another type of a solvate is a hydrate. A “hydrate” likewise refers to a solid compound that has one or more water molecules intimately associated with its solid or crystalline structure at the molecular level. Hydrates can form when a compound is solidified or crystallized in water, where one or more water molecules become an integral part of the solid crystalline matrix.
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.
The following definitions are used, unless otherwise described: halo or halogen is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups; but reference to an individual radical such as “propyl” embraces only the straight chain radical, a branched chain isomer such as “isopropyl” being specifically referred to. Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Het can be heteroaryl, which encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C1-C4)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
It will be appreciated by those skilled in the art that compounds of the disclosure having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present disclosure encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the disclosure, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine agonist activity using the standard tests described herein, or using other similar tests which are well known in the art. It is also understood by those of skill in the art that the compounds described herein include their various tautomers, which can exist in various states of equilibrium with each other.
Cellular transformation, tumor growth, and metastasis constitute a multistep process that requires the continuous rewiring of signaling pathway s and alterations of the reciprocal interaction between cancer cells and the tumor microenvironment, thereby allowing cells to acquire features to become fully neoplastic and eventually malignant (Hanahan and Weinberg, 2011). The Hippo pathway has gained great interest in recent y ears as being strongly involved in several of these key hallmarks of cancer progression (Harvey et al., 2013; Moroishi et al., 2015a) and, in general, serves important regulatory functions in organ development, regeneration, and stem cell biology (Johnson and Halder, 2014; Yu et al., 2015). The heart of the mammalian Hippo pathway is a kinase cascade involving mammalian STE20-like protein kinase 1 (MST1; also known as STK4) and MST2 (also known as STK3) (homologs of Drosophila Hippo), as well as two groups of MAP4Ks (mitogen-activated protein kinase kinase kinase kinases)-MAP4K1/2/3/5 (homologs of Drosophila Happy hour) and MAP4K4/6/7 (homologs of Drosophila Misshapen)—and the large tumor suppressor 1 (LATS1) and LATS2 (homologs of Drosophila Warts) (Meng et al., 2016). When the Hippo pathway is activated, MST1/2 or MAP4Ks phosphorylate and activate the LATS1/2 kinases, which, in turn, directly phosphorylate and inactivate Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ; also known as WWTR1), the two major downstream effectors that mediate transcriptional output of the Hippo pathway (Hansen et al., 2015). Activation of LATS1/2 kinases (and inactivation of YAP/TAZ) rep resents the major functional output of the Hippo pathway.
Previous studies have convincingly established the Hippo pathway as a suppressor signal for cellular transformation and tumorigenesis, though other studies revealed its oncogenic functions in certain contexts (Moroishi et al., 2015a; Wang et al., 2014). Deletion of MST1/2 in mouse liver results in tissue overgrowth and tumor development, demonstrating the tumor suppressor function of these kinases (Zhou et al., 2009). Complementarily, overexpression of YAP in mouse liver also promotes tissue overgrowth and tumorigenesis (Camargo et al., 2007; Dong et al., 2007). These studies have demonstrated an inhibitory role of the Hippo pathway in tumor initiation. However, effects of the Hippo pathway in tumor growth, especially in the context of reciprocal interactions between tumor cells and host anti-tumor immune responses, remain largely unknown.
Recent advances in cancer immunotherapy have improved patient survival. However, only a minority patients with pulmonary metastatic disease responds to treatment with checkpoint inhibitors. As an alternate approach, we have tested the ability of systemically administered 1V270, a toll-like receptor 7 (TLR7) agonist conjugated to a phospholipid, to inhibit lung metastases in two variant murine 4T1 breast cancer models, as well as in B16 melanoma, and Lewis lung c models. In the 4T1 breast cancer models, 1V270 therapy inhibited lung metastases if given up to a week after primary tumor initiation. The treatment protocol was facilitated by the minimal toxic effects exerted by the phospholipid TLR7 agonist, compared to the unconjugated agonist. The 1V270 therapy inhibited colonization by tumor cells in the lungs in a NK cell dependent manner. Additional experiments revealed that single administration of 1V270 led to tumor-specific CD8+ cell-dependent adaptive immune responses that suppressed late stage metastatic tumor growth in the lungs. T cell receptor (TCR) repertoire analyses showed that 1V270 therapy induced oligoclonal tumor-specific T cells in the lungs and regional lymph nodes. Different animals displayed commonly shared TCR clones following 1V270 therapy. Intranasal administration of 1V270 also suppressed lung metastasis and induced tumor-specific adaptive immune responses. These results indicate that systemic 1V270 therapy can induce tumor-specific cytotoxic T cell responses to pulmonary metastatic cancers, and that TCR repertoire analyses can be used to monitor, and to predict, the response to therapy.
Exemplary TLR7 ligands are shown below.
In one embodiment, the TLR7 ligand has formula (I):
wherein X1 is —O—, —S—, or —NRc—;
R1 is hydrogen, (C1-C10)alkyl, substituted (C1-C10)alkyl, C6-10aryl, or substituted C6-10aryl, C5-9heterocyclic, substituted C5-9heterocyclic;
Rc is hydrogen, C1-10alkyl, or substituted C1-10alkyl; or Rc and R1 taken together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring;
each R2 is independently —OH, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, —C(O)—(C1-C6)alkyl (alkanoyl), substituted —C(O)—(C1-C6)alkyl, —C(O)—(C6-C10)aryl (aroyl), substituted —C(O)—(C6-C10)aryl, —C(O)OH (carboxyl), —C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted —C(O)O(C1-C6)alkyl, —NRaRb, —C(O)NRaRb (carbamoyl), halo, nitro, or cyano, or R2 is absent;
each Ra and Rb is independently hydrogen, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het (C1-C6)alkyl, or (C1-C6)alkoxycarbonyl;
wherein the substituents on any alkyl, aryl or heterocycle groups are hydroxy, C1-6alkyl, hydroxy C1-6alkylene, C1-6alkoxy, C3-6cycloalkyl, C1-6alkoxyC1-6alkylene, amino, cyano, halo, or aryl;
n is 0, 1, 2, 3 or 4;
X2 is a bond or a linking group; and
R3 is a phospholipid comprising one or two carboxylic esters;
or a tautomer thereof;
or a pharmaceutically acceptable salt or solvate thereof.
In one embodiment, the composition of the disclosure comprises nanoparticles comprising a compound of formula (I). As used herein, a nanoparticle has a diameter of about 30 nm to about 600 nm, or a range with any integer between 30 and 600, e.g, about 40 nm to about 250 nm, including about 40 to about 80 or about 100 nm to about 150 nm in diameter. The nanoparticles may be formed by mixing a compound of formula (I), which may spontaneously form nanoparticles, or by mixing a compound of formula (I) with a preparation of lipids, such as phospholipids including but not limited to phosphatidylcholine, phosphatidylserine or cholesterol, thereby forming a nanoliposome. Optionally, a compound of formula (I), a lipid preparation and a glycol such as propylene glycol are combined.
In one embodiment, a composition comprises an amount of a compound of Formula (I):
wherein X1 is —O—, —S—, or —NRc—;
R1 is hydrogen, (C1-C10)alkyl, substituted (C1-C10)alkyl, C6-10aryl, or substituted C6-10 aryl, C5-9 heterocyclic, substituted C5-9 heterocydic;
Rc is hydrogen, C1-10alkyl, or substituted C1-10alkyl; or Rc and R1 taken together with the nitrogen to which they are attached form a heterocycle ring or a substituted heterocyclic ring;
each R2 is independently —OH, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, —C(O)—(C1-C6)alkyl (alkanoyl), substituted —C(O)—(C1-C6)alkyl, —C(O)—(C6-C10)aryl(aroyl), substituted —C(O)—(C6-C10)aryl, —C(O)OH (carboxyl), —C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted —C(O)O(C1-C6)alkyl, —NRaRb, —C(O)NRaRb (carbamoyl), halo, nitro, or cyano, or R2 is absent;
each Ra and Rb is independently hydrogen, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het (C1-C6)alkyl, or (C1-C6)alkoxycarbonyl; wherein the substituents on any alkyl, aryl or heterocyclic groups are hydroxy, C1-6alkyl, hydroxy C1-6alkylene, C1-6alkoxy, C3-6cycloalkyl, C1-6alkoxyC1-6alkylene, amino, cyano, halo, or aryl;
X2 is a bond or a linking group; and
R3 is a phospholipid comprising one or two carboxylic esters;
or a tautomer thereof;
or a pharmaceutically acceptable salt or solvate thereof. Optionally, the composition further comprises an antigen. In one embodiment, the composition having an antigen is administered concurrently, prior to or subsequent to administration of the composition having a compound of formula (I).
For example, R3 can comprise a group of formula
wherein R11 and R12 are each independently a hydrogen or an acyl group, R13 is a negative charge or a hydrogen, and m is 1 to 8, wherein a wavy line indicates a position of bonding, wherein an absolute configuration at the carbon atom bearing OR12 is R, S, or any mixture thereof.
For example, m can be 1, providing a glycerophosphatidylethanolamine. More specifically, R11 and R12 can each be oleoyl groups.
In various embodiments, the phospholipid of R3 can comprise two carboxylic esters and each carboxylic ester includes one, two, three or four sites of unsaturation, epoxidation, hydroxylation, or a combination thereof.
In various embodiments, the phospholipid of R3 can comprise two carboxylic esters and the carboxylic esters of are the same or different. More specifically, each carboxylic ester of the phospholipid can be a C17 carboxylic ester with a site of unsaturation at C8-C9. Alternatively, each carboxylic ester of the phospholipid can be a C18 carboxylic ester with a site of unsaturation at C9-C10.
In various embodiments, X2 can be a bond or a chain having one to about 10 atoms in a chain wherein the atoms of the chain are selected from the group consisting of carbon, nitrogen, sulfur, and oxygen, wherein any carbon atom can be substituted with oxo, and wherein any sulfur atom can be substituted with one or two oxo groups. The chain can be interspersed with one or more cycloalkyl, aryl, heterocyclyl, or heteroaryl rings.
In various embodiments, X2 can be C(O), or can be any of
In various embodiments, R3 can be dioleoylphosphatidyl ethanolamine (DOPE).
In various embodiments, R3 can be 1,2-dioleoyl-sn-glycero-3-phospho ethanolamine and X2 can be C(O).
In various embodiments, X1 can be oxygen.
In various embodiments, X1 can be sulfur, or can be —NRc— where Rc is hydrogen, C1-6 alkyl or substituted C1-6 alkyl, where the alkyl substituents are hydroxy, C3-6cycloalkyl, C1-6alkoxy, amino, cyano, or aryl. More specifically, X1 can be —NH—.
In various embodiments, R1 and Rc taken together can form a heterocyclic ring or a substituted heterocyclic ring. More specifically, R1 and Rc taken together can form a substituted or unsubstituted morpholino, piperidino, pyrrolidino, or piperazino ring.
In various embodiments R1 can be a C1-C10 alkyl substituted with C1-6 alkoxy.
In various embodiments, R1 can be hydrogen, C1-4alkyl, or substituted C1-4alkyl. More specifically, R1 can be hydrogen, methyl, ethyl, propyl, butyl, hydroxy C1-4alkylene, or C1-4alkoxy C1-4alkylene. Even more specifically, R1 can be hydrogen, methyl, ethyl, methoxyethyl, or ethoxy ethyl.
In various embodiments, R2 can be absent, or R2 can be halogen or C1-4alkyl. More specifically, R2 can be chloro, bromo, methyl, or ethyl.
In various embodiments, X1 can be O, R1 can be C1-4alkoxy-ethyl, n can be 1, R2 can be hydrogen, X2 can be carbonyl, and R3 can be 1,2-dioleoylphosphatidyl ethanolamine (DOPE).
In various embodiments, the compound of Formula (I) can be:
In various embodiments, the compound of formula (I) can be the R— enantiomer of the above structure:
In one embodiment, the TLR& ligand is:
X1═—O—, —S—, or —NRc—,
wherein Rc hydrogen, C1-10alkyl, or C1-10alkyl substituted by C3-6 cycloalkyl, or Rc and R1 taken together with the nitrogen atom can form a heterocyclic ring or a substituted heterocyclic ring wherein the substituents are hydroxy, C1-6 alkyl, hydroxy C1-6 alkylene, C1-6 alkoxy, C1-6 alkoxy C1-6 alkylene, or cyano;
wherein R1 is (C1-C10)alkyl, substituted (C1-C10)alkyl, C6-10 aryl, or substituted C6-10 aryl, C5-9 heterocyclic, substituted C5-9 heterocyclic; wherein the substituents on the alkyl, aryl or heterocyclic groups are hydroxy, C1-6 alkyl, hydroxy C1-6 alkylene, C1-6 alkoxy, C1-6 alkoxy C1-6 alkylene, amino, cyano, halogen, or aryl;
each R2 is independently —OH, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, —C(O)—(C1-C6)alkyl (alkanoyl), substituted —C(O)—(C1-C6)alkyl, —C(O)—(C6-C10)aryl(aroyl), substituted —C(O)—(C6-C10)aryl, —C(O)OH (carboxyl), —C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted —C(O)O(C1-C6)alkyl, —NRaRb, —C(O)NRaRb (carbamoyl), —O—C(O)NRaRb, —(C1-C6)alkylene-NRaRb, —(C1-C6)alkylene-C(O)NRaRb, halo, nitro, or cyano;
wherein each Ra and Rb is independently hydrogen, (C1-6)alkyl, (C3-C8)cycloalky, (C1-66)alkoxy, halo(C1-6)alkyl, (C3-C8)cycloalkyl(C1-6)alkyl, (C1-6)alkanoyl, hydroxy(C1-6)alkyl, aryl, aryl(C1-6)alkyl, aryl, aryl(C1-6)alkyl, Het, Het (C1-6)alkyl, or (C1-6)alkoxycarbonyl; wherein X2 is a bond or a linking group; wherein R3 is a phospholipid comprising one or two carboxylic esters wherein n is 0, 1, 2, 3, or 4; or a tautomer thereof; or a pharmaceutically acceptable salt thereof.
In cases where compounds are sufficiently basic or acidic to form acid or base salts, use of the compounds as salts may be appropriate. Examples of acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
Acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
Alkyl includes straight or branched C1-10 alkyl groups, e.g., methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, 1-methylpropyl, 3-methylbutyl, hexyl, and the like.
Lower alkyl includes straight or branched C1-6 alkyl groups, e.g., methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and the like.
The term “alkylene” refers to a divalent straight or branched hydrocarbon chain (e.g., ethylene: —CH2—CH2—).
C3-7 cycloalkyl includes groups such as, cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, and alkyl-substituted C3-7 cycloalkyl group, preferably straight or branched C1-6 alkyl group such as methyl, ethyl, propyl, butyl or pentyl, and C5-7 cycloalkyl group such as, cyclopentyl or cyclohexyl, and the like.
Lower alkoxy includes C1-6 alkoxy groups, such as methoxy, ethoxy or prop oxy, and the like.
Lower alkanoyl includes C1-6 alkanoyl groups, such as formyl, acetyl, propanoyl, butanoyl, pentanoyl or hexanoyl, and the like.
C7-11 aroyl, includes groups such as benzoyl or naphthoyl;
Lower alkoxy carbonyl includes C2-7 alkoxy carbonyl groups, such as methoxy carbonyl, ethoxy carbonyl or prop oxy carbonyl, and the like.
Lower alkylamino group means amino group substituted by C1-6 alkyl group, such as, methylamino, ethylamino, propylamino, butylamino, and the like.
Di(lower alkyl)amino group means amino group substituted by the same or different and C1-6 alkyl group (e.g., dimethylamino, diethylamino, ethylmethylamino).
Lower alkylcarbamoyl group means carbamoyl group substituted by C1-6 alkyl group (e.g., methylcarbamoyl, ethylcarbamoyl, propylcarbamoyl, butylcarbamoyl).
Di(lower alkyl)carbamoyl group means carbamoyl group substituted by the same or different and C1-6 alkyl group (e.g., dimethylcarbamoyl, diethylcarbamoyl, ethylmethylcarbamoyl).
Halogen atom means halogen atom such as fluorine atom, chlorine atom, bromine atom or iodine atom.
Aryl refers to a C6-10 monocyclic or fused cyclic aryl group, such as phenyl, indenyl, or naphthyl, and the like.
Heterocyclic or heterocycle refers to monocyclic saturated heterocyclic groups, or unsaturated monocyclic or fused heterocyclic group containing at least one heteroatom, e.g., 0-3 nitrogen atoms NRc, 0-1 oxygen atom (—O—), and 0-1 sulfur atom (—S—). Non-limiting examples of saturated monocyclic heterocyclic group includes 5 or 6 membered saturated heterocyclic group, such as tetrahydrofuranyl, pyrrolidinyl, morpholinyl, piperidyl, piperazinyl or pyrazolidinyl. Non-limiting examples of unsaturated monocyclic heterocyclic group includes 5 or 6 membered unsaturated heterocyclic group, such as furyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, thienyl, pyridyl or pyrimidinyl. Non-limiting examples of unsaturated fused heterocyclic groups includes unsaturated bicyclic heterocyclic group, such as indolyl, isoindolyl, quinolyl, benzothiazolyl, chromanyl, benzofuranyl, and the like. A Het group can be a saturated heterocyclic group or an unsaturated heterocyclic group, such as a heteroaryl group.
Rc and R1 taken together with the nitrogen atom to which they are attached can form a heterocyclic ring. Non-limiting examples of heterocyclic rings include 5 or 6 membered saturated heterocyclic rings, such as 1-pyrrolidinyl, 4-morpholinyl, 1-piperidyl, 1-piperazinyl or 1-pyrazolidinyl, 5 or 6 membered unsaturated heterocyclic rings such as 1-imidazolyl, and the like.
The alkyl, aryl, heterocyclic groups of R1 can be optionally substituted with one or more substituents, wherein the substituents are the same or different, and include lower alkyl; cycloalkyl, hydroxyl; hydroxy C1-6 alkylene, such as hydroxymethyl, 2-hydroxyethyl or 3-hydroxypropyl; lower alkoxy; C1-6 alkoxy C1-6 alkyl, such as 2-methoxyethyl, 2-ethoxyethyl or 3-methoxypropyl; amino; alkylamino; dialkyl amino; cyano; nitro; acyl; carboxyl; lower alkoxycarbonyl; halogen; mercapto; C1-6 alkylthio, such as, methylthio, ethylthio, propylthio or butylthio; substituted C1-6 alkylthio, such as methoxyethylthio, methylthioethylthio, hydroxyethylthio or chloroethylthio; aryl; substituted C6-10 monocyclic or fused-cyclic aryl, such as 4-hydroxyphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl or 3,4-dichlorophenyl; 5-6 membered unsaturated heterocyclic, such as furyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, thienyl, pyridyl or pyrimidinyl; and bicyclic unsaturated heterocyclic, such as indolyl, isoindolyl, quinolyl, benzothiazolyl, chromanyl, benzofuranyl or phthalimino. In certain embodiments, one or more of the above groups can be expressly excluded as a substituent of various other groups of the formulas.
The alkyl, aryl, heterocyclic groups of R2 can be optionally substituted with one or more substituents, wherein the substituents are the same or different, and include hydroxyl; C1-6 alkoxy, such as methoxy, ethoxy or propoxy; carboxyl; C2-7 alkoxycarbonyl, such as methoxycarbonyl, ethoxy carbonyl or propoxycarbonyl) and halogen.
The alkyl, aryl, heterocyclic groups of Rc can be optionally substituted with one or more substituents, wherein the substituents are the same or different, and include C3-6 cycloalkyl; hydroxyl; C1-6 alkoxy; amino; cyano; aryl; substituted aryl, such as 4-hydroxyphenyl, 4-methoxyphenyl, 4-chlorophenyl or 3,4-dichlorophenyl; nitro and halogen.
The heterocyclic ring formed together with Rc and R1 and the nitrogen atom to which they are attached can be optionally substituted with one or more substituents, wherein the substituents are the same or different, and include C1-6 alkyl; hydroxy C1-6 alkylene; C1-6 alkoxy C1-6 alkylene; hydroxyl; C1-6 alkoxy; and cyano. A specific value for X1 is a sulfur atom, an oxygen atom or —NRc—.
In other embodiments, the TLR7 ligand has formula (I) wherein R3 is hydrogen, (C1-C10)alkyl, substituted (C1-C10)alkyl, C6-10aryl, or substituted C6-10aryl, C5-9heterocyclic, substituted C5-9heterocyclic.
In other embodiments, the TLR7 ligand has formula (I), wherein R3 is independently —OH, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, —C(O)—(C1-C6)alkyl(alkanoyl), substituted —C(O)—(C1-C6)alkyl, —C(O)—(C6-C10)aryl (aroyl), substituted —C(O)—(C6-C10)aryl, —C(O)OH (carboxyl), —C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted —C(O)O(C1-C6)alkyl, —NRaRb, —C(O)NRaRb (carbamoyl), halo, nitro, or cyano, or R2 is absent; each Ra and Rb is independently hydrogen, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het (C1-C6)alkyl, or (C1-C6)alkoxycarbonyl; wherein the substituents on any alkyl, aryl or heterocyclic groups are hydroxy, C1-6alkyl, hydroxy C1-6 alkylene, C1-6alkoxy, C3-6cycloalkyl, C1-6alkoxy C1-6alkylene, amino, cyano, halo, or aryl.
The invention will be further described by the following non-limiting examples.
The present disclosure provides several discoveries. First, the local administration of an immune stimulating agent (specifically a TLR7 or TLR9 agonist) to a syngeneic cancer in a mouse can cause the clonal expansion of tumor specific CD8+ T cells in TILs and spleen, as detected by RNA sequencing of T cell receptor variable region genes (
In a separate series of experiments, it was found that inactivation in cancer cells of two enzymes, called LATS1 and LATS2, markedly increases the response of the immune system to the cancers, leading to tumor eradication in several examples (
After drug therapy, the isolation and expansion of the tumor specific CD8+ T cells in tissue culture can be made much more efficient by purification of activated CD8+ T cells, and by co-culture of the T cells with the immunogenic tiny EVs derived from the blood of the same patient in the presence or absence of feeder cells. Moreover, the treatment of cancer patients with anti-PD1 and other checkpoint inhibitors should be undertaken specifically in those subjects who have circulating clonal T cells after drug treatment, and at the time when both EVs and TIL concentrations in the blood reach maximal levels.
To identify drugs that can induce immunogenic EV release with discernible toxic effects, in one embodiment, a mouse melanoma cell line is contacted with the drugs, a maximal non-toxic concentration is identified, released EVs with less than 0.2 micron diameter into the medium are measured, and the ability of the released EVs to stimulate tumor reactive T cells and to cause clonal expansion is assessed.
In contrast to current methods for the isolation of tumor specific autologous CD8+ T cells, which typically requires the processing of surgical specimens or biopsies to release the Tumor Infiltrating Lymphocytes (TILs) (
Further, the current method for the expansion of TILs in tissue culture before re-infusion often leads to the co-expansion of non-specific T cells that can outgrow the effector T cells against the cancer. This problem can lead to therapeutic failure. In contrast, the presently disclosed methods involve a combination of drug treatments that enhances tumor specific T cells in the peripheral circulation before isolation and expansion of T cells in tissue culture, combined with stimulation with autologous immune EVs that contain tumor antigens and immunostimulatory molecules. This app roach renders TIL therapy much more efficient and less expensive.
In one embodiment, the following protocol may be employed:
The disclosure will significantly improve isolation and expansion of autologous tumor specific T cells for cancer immunotherapy. Biopsies or surgical resection will no longer be required. Patients who are likely to respond to the T cell therapy will be identified early, before expansion of autologous T cells. Then all patients with metastatic cancer will be potential candidates for treatment.
In the present study, the role of the LATS1/2 kinases in the growth of established tumors in the context of anti-tumor immunity was investigated. Surprisingly, inactivation of the “tumor suppressor” LATS1/2 in tumor cells strongly suppresses tumor growth in immune-competent, but not immune-compromised, mice due to the induction of host anti-tumor immune responses. The data indicate a new paradigm for how tumor immunogenicity is regulated through the Hippo signaling pathway in tumor cells and also have implications for targeting LATS1/2 in cancer immunotherapy.
C57BL/6, C3H/HeOu, or BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, Me., USA). Myd88, Tlr4, Tlr7, and Tlr9KO mice were kind gifts from Dr. Shizuo Akira (Osaka University, Osaka, Japan). Ticam1 (also known as TRIF) KO mice were kindly provided by Dr. Bruce Beutler (University of Texas Southwestern Medical Center, Dallas, Tex., USA). Casp1 (also known as Caspase-1) KO mice were kindly provided by Dr. Richard A. Flavell (Yale University School of Medicine, New Haven, Conn., USA). Rag1 KO mice, (also known as STING) KO mice, and OT-Itransgenic mice were purchased from The Jackson Laboratory. Ifnar1 KO mice were purchased from B&K Universal (East Yorkshire, United Kingdom). These mouse strains were backcrossed for 10 generations onto the C57BL/6 background at the University of California, San Diego. Mutant mice were bled by the University of California, San Diego Animal Care Program. 7-12 weeks old mice were used and all animal experiments were approved by the University of California, San Diego, Institutional Animal Care and Use Committee.
All cell lines were cultured under an atmosphere of 5% CO2 at 37° C. B16-OVA cells (B16F10 cells expressing ovalbumin) were cultured in DMEM (GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO), penicillin (100 U/ml), and streptomycin (100 mg/ml). SCC7, 4T1, EL4, bone marrow-derived dendritic cells (BMDCs), mouse primary lymph node cells and CD8+ T cells were cultured in RPMI 1640 (GIBCO) supplemented with 10% FBS (GIBCO), penicillin (100 U/ml), and streptomycin (100 mg/ml).
LATS1/2-deficient cells were created through the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system (Ran et al., 2013). We use a transient CRISPR strategy for the deletion of LATS1/2 to avoid any potential unspecific effects mediated by stable Cas9/sgRNA genome integration. Cells were transiently transfected with a Cas9 and single-guide RNA (sgRNA) expression plasmid encoding puromycin resistance (PX459; Addgene plasmid #48139). The CRISPR-transfected cells will thus acquire transient resistance to puromycin. The guide sequences were designed using the Optimized CRISPR Design at http://crispr.mit.edu. The guide sequences used are 5′-AGACGTTCTGCTCCGAAATC-3′ (SEQ ID NO:1) or 5′-ACGTTTCCATTGGCGAATGA-3′ (SEQ ID NO:2) for mouse Lats1; 5′-GAGTGTCCAGCTTACAAGCG-3′ (SEQ ID NO:3) or 5′-GCTGGGTGGTGCAAACTACG 3′ (SEQ ID NO:4) for mouse Lats2.
Following transfection and transient selection with puromycin for 3 days, cells were single-cell sorted by fluorescence-activated cell sorting (FACS) into 96-well plate without puromycin. Knockout clones were selected by immunoblot analysis for the lack of LATS1/2 proteins and YAP phosphorylation. LATS1/2 dKO cells were sensitive to puromycin after expansion, indicating a transient expression of CRISPR/Cas9 system in those cells. Two independent clones were analyzed as indicated and the parental LATS1/2 WT cells (not transfected with PX459) were used as control.
B16-OVA cells stably expressing empty vector, YAP(5SA), YAP(5SA/S94A), or TAZ(4SA) were generated by retroviral infection. 293 Phoenix retrovirus packaging cells were transfected with pBABE empty vector, pBABE YAP(5SA), pBABE YAP (5SA/S94A), or pBABE TAZ (4 SA) constructs. Forty-eight hours after transfection, retroviral supernatant was supplemented with 5 mg/mL polybrene, filtered through a 0.45 mm filter, and used for infection. Forty-eight hours after infection, cells were selected with 4 mg/mL puromycin in culture medium.
Equal amount of protein samples were resolved in SDS-PAGE in reducing conditions unless otherwise mentioned in the Figure Legends. Antibodies to YAP (#14074), pYAP (S127 in humans and S112 in mice; #4911), YAP/TAZ (#8418), LATS1 (#3477), CD81 (#10037), and ALIX (#2171) were obtained from Cell Signaling those to actin (# ab3280) and ovalbumin (OVA, # ab1221) were from Abcam; those to LATS2 (# A300-479A, also weakly recognize LATS1) were from Bethyl Laboratories; those to FLOT1 (#610821) and HSP90 (#610418) were from BD Biosciences. The phos-tag electrophoresis was performed as described previously (Moroishi et al., 2015b). YAP proteins can be separated into multiple bands in the presence of phos-tag depending on differential phosphorylation levels, with phosphorylated proteins migrating more slowly. Where indicated, cells were treated with serum starvation (DMEM or RPMI 1640 without other supplements), 1 mg/ml Latrunculin B (LatB), or 25 mM 2-deoxy-D-glucose (2-DG) for 1 hour before harvest.
Cells were treated with or without 1 mg/ml Latrunculin B (LatB) for 1 hour before harvest. Cells were then fixed for 10 minutes at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS) and were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature. Cells were then incubated consecutively with primary antibodies to YAP/TAZ (Santa Cruz, # sc-101199) (overnight at 4° C.) and Alexa Fluor 488-labeled goat secondary antibodies (for 90 minutes at room temperature) in PBS containing 1% bovine serum albumin (BSA). Cells were covered with a drop of ProLong Gold antifade reagent with DAPI (Invitrogen) for observation. Cells in five randomly selected views (about 100 cells) were selected for the quantification of YAP/TAZ localization.
Total RNA (500 ng) isolated from cells with the use of RNeasy Plus Mini Kit (QIAGEN) was reverse-transcribed to complementary DNA using iScript cDNA Synthesis Kit (Bio-Rad). Complementary DNA was then diluted and used for quantification by real-time PCR, which was performed using KAPA SYBR FAST qPCR Kit (Kapa Biosystems) and the 7300 real-time PCR system (Applied Biosystems). The sequences of the PCR primers (forward and reverse, respectively) are 5′-GCCTGGAGAAACCTGCCAAGTATG-3′ (SEQ ID NO:5) and 5′-GAGTGGGAGTTGCTGTTGAAGTCG-3′ (SEQ ID NO:6) for mouse Gapdh; 5′-AGCTGACCTGGAGGAAAACA-3′ (SEQ ID NO:7) and 5′-GACAGGCTTGGCGATTTTAG-3′ (SEQ ID NO:8) for mouse Ctgf; 5′-GCTCAGTCAGAAGGCAGACC-3′ (SEQ ID NO:9) and 5′-GTTCTTGGGGACACAGAGGA-3′ (SEQ ID NO:10) for mouse Cyr61; 5′-AGGAGAAGAGTTGCCCACCTATGAG-3′ (SEQ ID NO:11) and 5′-TCGAAGAGCTTCATCCTGTCGC-3′ (SEQ ID NO:12) for mouse Amotl2; 5′-CCTGAGAAAGAAGAAACACAGCCTC-3′ (SEQ ID NO:13) and 5′-GCAAGTTGGTTGAGGAAGAGAGGG-3′ (SEQ ID NO:14) for mouse Ifna4; 5′-GAAGAGTTACACTGCCTTTGCCATC-3′ (SEQ ID NO:15) and 5′-AAACACTGTCTGCTGGTGGAGTTC-3′ (SEQ ID NO:16) for mouse Ifnb1. Reactions for Gapdh mRNA were performed concurrently on the same plate as those for the test mRNAs, and results were normalized by the corresponding amount of Gapdh mRNA.
Each 6-well plate was coated with 1.5 mL of bottom agar (DMEM or RPMI 1640 containing 10% FBS and 0.5% Difco agar noble). Cells (5×103 cells for B16-OVA and SCC7, 2.5×103 cells for 4T1) were suspended in 1.5 mL of top agar (DMEM or RPMI 1640 containing 10% FBS and 0.35% Difco agar noble) into each well. Cells were cultured for approximately two weeks and replaced with fresh medium every three days. Colonies were stained using 0.005% crystal violet in 5% methanol and quantified using ImageJ software.
B16-OVA cells (2×105) were subcutaneously transplanted into both back flanks of C57BL/6 mice. Tumor height and width were measured with a caliper every 2-3 days to calculate tumor volume (=width2×height×0.523). Mice were sacrificed when tumors reached maximum allowed size (15 mm in diameter) or when signs of ulceration were evident. Likewise, 1×105 of SCC7 cells were subcutaneously transplanted into both back flanks of C3H/HeOu mice and 2.5×105 of 4T1 cells were transplanted into both mammary fat pads of BALB/c mice. For 4T1 lung metastasis assay, lungs were tracheally injected with India ink 28 days after transplantation, and then destained in Fekete's solution to count tumor nodules.
For tumor vaccination experiments, C57BL/6 mice were immunized intradermally at the base of the tail with irradiated B16-OVA cells (100 Gy, 1×106) 12 days prior to challenge with B16-OVA cells (one time vaccination, without any adjuvant). For immunization with EVs, C57BL/6 mice were inoculated with irradiated B16-OVA cells (100 Gy, 1×106) at the base of the tail and EVs freshly isolated from culture supernatants of B16-OVA cells (6×106) were injected every 3 days (days 0, 3, 6, and 9) into the same place until challenged with B16-OVA cells at day 12.
Tumors were fixed with 4% paraformaldehyde in PBS, embedded in paraffin, sectioned with a microtome, and stained with hematoxylin-eosin according to standard procedures. Immunostaining of tumors was performed with frozen cryostat sections with PE-conjugated antibodies to CD45 (eBioscience, #12-0451-82).
Serum anti-OVA IgG concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, half area 96-well plates (Corning) were coated with 5 mg/ml OVA protein (Worthington Biochemical, # LS003056) in PBS overnight at 4° C. Plates were washed and then blocked for 3 hours at room temperature with blocking buffer [1% BSA (bovine serum albumin) in PBS], followed by wash and incubation with serum samples tested at a 1:100 to 1:125,600 dilutions in blocking buffer overnight at 4° C. Plates were then washed and incubated with HRP-conjugated detecting antibody in blocking buffer at room temperature for 2 hours. Plates were washed and incubated with TMB substrate (KPL, #95059-286), and then read at 450 nm and 650 nm after stopping the development with 1 M phosphoric acid. Each ELISA plate contained a titration of a previously quantified serum to generate a standard curve. Anti-OVA IgG concentrations were determined from the lowest dilution of serum samples within a standard curve and reported as U/ml.
Flow cytometry was performed using a BD LSRFortessa and results were analyzed using FlowJo software (Treestar). Cell suspensions were incubated in mouse Fc block (anti CD16/CD32; BD Biosciences, #553142) prior to staining. Fluorochrome conjugated anti-mouse CD45 (clone 30F-11), CD3e (clone 145-2C11), CD8a (clone 53-6.7), Granzyme B (clone GB11), and IFNγ (clone XMG1.2) antibodies were used following the manufacturers protocol. Kb-SIINFEKL-tetramer was used for identifying OVA-specific CD8+ T cells. Propidium iodide (PI) was used to stain dead cells.
To analyze intracellular cytokine expression, cells were re-stimulated ex vivo with 10 mg/ml SIINFEKL peptide (AnaSpec, # AS-60193-1) for 5 hours in the presence of protein transport inhibitor (BD biosciences, #555029) for the last 4 hours. Intracellular cytokine staining was then performed using Fixation/Permeabilization Solution Kit (BD Biosciences, #554714).
EL4 cells were pulsed with 8 mg/ml SIINFEKL peptide or irrelevant peptides for 2 hours at 37° C., and then labeled with 0.25 mM or 2.5 mM CFSE (carboxyfluorescein succinimidyl ester; Thermo Fisher Scientific, # C34554) for minutes at 37° C., respectively. CFSElow (SIINFEKL loaded target) and CSFEhigh (irrelevant peptide control) EL4 cells were mixed at 1:1 ratio, and then co-cultured with CD8+ T cells isolated from splenocytes of C57BL/6 mice challenged (or not) with WT or LATS1/2 dKO B16-OVA cells at 8:1 effector to target cell ratio (E:T). CD8+ T cells were isolated using Easy Sep Mouse CD8a Positive Selection Kit (STEM CELL, #18753) from pooled splenocytes of 3-4 mice per group for each experiment. The frequencies of CFSElow and CSFEhigh EL4 cells in CFSE positive fraction were determined by flow cytometric analysis 18 hours after incubation and the percent of specific killing was calculated. Specific killing (%)=[1−“Sample ratio”/“Negative control ratio”]×100; “Sample ratio”=[CFSElow(target)/CSFEhigh(irrelevant)] value of each samples co-cultured with CD8+ T cells; “Negative control ratio”=[CFSElow(target)/CSFEhigh(irrelevant)] value of EL4 cells not cultured with CD8+ T cells.
For conditioned medium preparation, B16-OVA cells were seeded on culture plates and incubated in DMEM supplemented with 10% FBS for 24 hours at 37° C. to allow cell attachment. The cells were then washed with PBS, and culture medium was switched to DMEM without serum. After incubation for 48 hours, conditioned medium was collected and centrifuged at 2,000 g for 10 minutes at 4° C. to remove cell debris. The resulting supernatant was used for the experiment.
Naive CD8+ T cells were isolated from OVA-specific T cell receptor transgenic OT-I mice using Easy Sep Mouse CD8a Positive Selection Kit (STEM CELL) and labeled with 2 mM CFSE. Bone marrow derived dendritic cells (BMDCs) were generated by 6 days of GM-CSF (20 ng/ml; eBioscience, #14-8331-80) differentiation, and then incubated (or not) for 18 hours with conditioned medium (10% of the total volume) from WT or LATS1/2 dKO B16-OVA melanoma cells and pulsed with OVA protein (10 mg/ml) for the last 4 hours. The cells were washed and cultured with CFSE-labeled OT-ICD8+ T cells at 1:1 ratio for 3 days. OT-1 T cell proliferation was monitored by CFSE dilution using a flow cytometer and a division index was determined using FlowJo software (Treestar).
IFNγ or IL-12 levels in culture supernatants were determined by ELISA. For ex vivo IFNγ production from lymph node cells, draining lymph nodes (inguinal lymph nodes) were isolated from C57BL/6 mice challenged (or not) with B16-OVA cells and cultured with OVA protein (100 mg/ml) for 3 days. For IL-12 production from BMDCs, BMDCs were generated by 6 days of GM-CSF (20 ng/ml) differentiation and stimulated (or not) for 18 hours with conditioned medium (10% of the total volume) or EVs isolated from culture supernatants of equal numbers of WT or LATS1/2 dKO B16-OVA cells (EVs from 2×105 cells were used to stimulate 1×106 BMDCs). Both cultures were done in RPMI 1640 supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/ml) under an atmosphere of 5% CO2 at 37° C., and then aliquots of cell culture supernatants were used for cytokine ELISA. For cell conditioned medium preparation, B16-OVA cells were seeded on culture plates and incubated in DMEM supplemented with 10% FBS for 24 hours at 37° C. to allow cell attachment. The cells were then washed with PBS, and culture medium was switched to DMEM without serum. After incubation for 48 hours, conditioned medium was collected and centrifuged at 2,000 g for 10 minutes at 4° C. to remove cell debris. The resulting supernatant was used for EV isolation, which is described in the “EV isolation and analysis” section.
IFNγ concentrations were determined using Mouse IFN-gamma DuoSet ELISA (R&D Systems, # DY485-05) according to a manufacturer's protocol. For IL-12 ELISA, half area 96-well plates were coated with capture antibody (Purified Rat Anti-Mouse IL-12 p40/p70; BD Biosciences, #551219) in PBS overnight at 4° C. Plates were washed and then blocked for 3 hours at room temperature with blocking buffer (1% BSA in PBS), followed by wash and incubation with culture supernatants overnight at 4° C. Plates were then washed and incubated with biotinylated detection antibody (Biotin Rat Anti-Mouse IL-12 (p40/p70; BD Biosciences, #554476) in blocking buffer at room temperature for 1 hour, followed by wash and incubation with streptavidin-HRP conjugate for 20 minutes. Plates were washed and incubated with TMB substrate (KPL, #95059-286), and then read at 450 nm and 650 nm after stopping the development with 1 M phosphoric acid. Concentrations were determined by comparison to a standard curve.
B16-OVA cells were seeded in 150 mm culture plate and incubated in DMEM supplemented with 10% FBS for 24 hours at 37° C. to allow cell attachment. The cells were then washed with PBS twice, and culture medium was switched to 35 mL of DMEM without serum. After incubation for 48 hours, conditioned medium was collected and centrifuged at 2,000 g for 10 minutes at 4° C. to thoroughly remove cell debris. The resulting supernatant was then filtered through a 0.22 mm PVDF filter (Millip ore, # SLGV033RB) to remove cell debris and microvesicles (for the detergent treatment experiment, the resulting flow-through was treated with 1% Triton X-100 for 10 minutes at 4° C. prior to the ultracentrifugation). The flow-through was transferred into ultracentrifuge tubes (BECKMAN COULTER, #344058) and then ultracentrifuged in a Beckman SW32Ti rotor at 30,000 rpm for 90 minutes at 4° C. The resulting pellets were washed with 35 mL of PBS and then ultracentrifuged again at 30,000 rpm for 90 minutes at 4° C. The resulting EV pellets were re-suspended in PBS for experimental use. Protein concentrations of EVs were determined using Micro BCA Protein Assay Kit (Thermo, #23235). RNA in EVs was isolated using TRIzol reagents (Thermo, #15596026) according to the manufacturer's protocol and concentrations were determined using Agilent 2200 TapeStation (Agilent Technologies). For ribonuclease treatment, total RNA isolated from EVs was digested for 30 minutes at 37° C. with 100 mg/ml RNase A (Thermo, # EN0531) in a buffer comprising 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 300 mM NaCl. RNA was then resolved in agarose gels in non-denaturing conditions. Nanoparticle tracking analysis was performed using NanoSight NS300 system (Malvern Instruments, Ranch Cucamonga, Calif., USA) on isolated EVs diluted 5,000-fold with PBS for analysis.
EV samples were resolved in SDS-PAGE and the gels were cut into three regions, and then digested with try p sin. Extracted peptides were analyzed using a C18 column and an EASY-nLC-1000 (Thermo Scientific) coupled to a hybrid quadrupole-orbitrap Q-Exactive mass spectrometer (Thermo Scientific). A data-dependent, top 50 method was utilized for analysis. The resulting RAW files were analyzed with Proteome Discoverer 1.4 and MASCOT. Results were filtered with 1% FDR at the protein level and exported to our in-house FileMakerPro database iSPEC and analyzed with Align!, which calculated intensity based absolute quantification (iBAQ) values (Schwanha{umlaut over ( )}usser et al., 2011) that were used for subsequent analysis. The ratio of the iBAQ values for WT and LATS1/2 dKO EVs (DKO/WT ratio) in the individual experiments was calculated and scored according to the following criteria: score 2, >5-fold; score 1, 5- to 2-fold; score 0, 2- to 0.5-fold; corel, 0.5- to 0.2-fold; and score 2, <0.2-fold. We then added each scores from the individual experiments and set the threshold as a score of >3 for the top 100 most significantly increased proteins in LATS1/2 dKO EVs. Gene Ontology (GO) analysis was done using the PANTHER program (Mi et al., 2013). Heatmap s were generated using NetWalker (Komurov et al., 2012).
Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc, La Jolla, Calif., USA). Statistical parameters and methods are reported in the FIGS. and the Figure Legends. A value of p<0.05 was considered statistically significant. Epidemiological data are obtained using the PrognoScan database (Mizuno et al., 2009). Association of gene expression with the survival of patients was evaluated using log-rank test and a value of p<0.05 was considered statistically significant.
To elucidate the role of the Hippo pathway in anti-tumor immunity, murine syngeneic tumor models of three different cancer types in three different host genetic backgrounds were used; B16-OVA melanoma (B16F10 melanoma expressing ovalbumin [OVA]) in C57BL/6 mice, SCC7 head and neck squamous cell carcinoma in C3H/HeOu mice, and 4T1 breast cancer in BALB/c mice. These syngeneic allograft models have been well characterized and extensively used to study reciprocal interactions between tumor cells and host anti-tumor immune responses (Dranoff, 2011; Lei et al., 2016). Deletion of LATS1/2 almost completely abolished YAP/TAZ regulation by the Hippo pathway, while deletion of other components had only a partial or minor effect on YAP/TAZ activity (Meng et al., 2015). Therefore, LATS1/2 was deleted in B16-OVA melanoma cells using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome-editing technology (Ran et al., 2013). Multiple independent LATS1/2 double-knockout (dKO) clones were obtained as verified by the lack of protein expression of both LATS1 and LATS2 (
LATS1/2 was also deleted in SCC7 squamous cell carcinoma cells and found that LATS1/2 deficiency almost completely blocked YAP phosphorylation (
To investigate the role of the Hippo pathway in tumor growth in vivo, equal numbers of WT or LATS1/2 dKO B16-OVA cells were subcutaneously transplanted into the back flanks of C57BL/6 mice and monitored their growth. Unexpectedly, deletion of LATS1/2 in B16-OVA cells strongly inhibited tumor growth in vivo (
Since LATS1/2 deletion exerts completely opposite effects on tumor cell growth in vitro and in vivo (
Although WT and LATS1/2 dKO B16-OVA cells showed identical expression of OVA (
Given that LATS1/2 deletion in tumor cells enhances host antitumor immune responses, it was hypothesized that LATS1/2-null tumor cells, by stimulating anti-tumor immunity, may protect the host from challenge with the corresponding LATS1/2 WT tumor cells. To test this, we performed two sets of experiments: co-injection of LATS1/2 dKO and WT tumor cells into each side of the same mouse (
Next, it was tested whether adaptive immunity is required for tumor suppression by LATS1/2 deletion. WT or LATS1/2 dKO B16-OVA cells were subcutaneously transplanted into RAG-1 (recombination activating gene 1) knockout (KO) mice that are immune-compromised due to the lack of mature T and B cells. LATS1/2 dKO B16-OVA tumor cells grew similarly to WT cells (
YAP and TAZ are the most characterized downstream effectors of the Hippo pathway. LATS1/2 cells directly phosphorylate YAP/TAZ on multiple serine residues, leading to cytoplasmic retention, degradation, and thereby inactivation of YAP/TAZ. Because high YAP/TAZ activation was observed in LATS1/2 dKO B16-OVA tumors in vivo (
Extracellular Vesicles Released from LATS1/2-Null Tumor Cells Stimulate Immune Responses
Next it was explored how LATS1/2 deficiency in tumors stimulates host anti-tumor immune responses. Because we observed a preeminent CD8+ T cell cross-priming in mice injected with LATS1/2 dKO B16-OVA cells (
To elucidate the mechanistic basis for immunostimulatory effects of LATS1/2 dKO EVs, the nature of EVs released from WT or LATS1/2 dKO B16-OVA cells was characterized. It was found that LATS1/2 dKO B16-OVA cells produced slightly more EVs compared with WT cells, as assessed by nanoparticle tracking analysis (
EVs from LATS1/2 dKO Tumor Cells Stimulate the Toll-Like Receptors-Type I Interferon Pathway
To test the hypothesis that nucleic-acid-rich EVs released from LATS1/2-null tumors stimulate host anti-tumor immunity, it was examined whether alterations in the host nucleic-acid-sensing pathways imp air the tumor-protective effects of LATS1/2 deletion in vivo. Both microbial (non-self) and self nucleic acids can be recognized by distinct families of pattern recognition receptors, including endosomal Toll-like receptors (TLRs) and cytosolic non-TLR sensors (
Distinct types of TLRs utilize MYD88 or TRIF as adaptor proteins and specifically respond to a wide range of ligands on the cell surface, as well as in the endosome (
Activation of TLRs-MYD88/TRIF signaling results in pro-inflammatory cytokine production as well as type I IFN (in particular, IFNa and IFNb) production, which stimulates anti-tumor immune responses (
In this study, it was demonstrate that LATS1/2 deletion unmasks a malignant cell's immunogenic potential and restrains tumor growth due to the induction of anti-tumor immune responses. The effects of LATS1/2 deletion on tumor growth are striking insofar as LATS1/2 dKO completely abolishes the tumor growth potential of SCC7 and dramatically reduces tumor growth and the metastasis of B16 and 4T1 cells. LATS1/2-null B16 melanomas secrete nucleic-acid-rich EVs that stimulate the host TLRs-MYD88/TRIF-IFN pathway s to induce anti-tumor immunity and the eventual elimination of tumor cells (FIG. 7). LATS1/2 deletion similarly stimulates host immune responses in both SCC7 and 4T1 syngeneic models (
It is generally accepted that the Hippo pathway is a tumor suppressor that inhibits proliferation and survival of normal cells, preventing tumorigenesis (Harvey et al., 2013; Moroishi et al., 2015a; Wang et al., 2014), yet a few studies did suggest an oncogenic role of the Hippo pathway in certain contexts (Barry et al., 2013; Cottini et al., 2014). We have analyzed human epidemiological data using the PrognoScan database (Mizuno et al., 2009) to find any correlation between LATS1/2 mRNA expression levels and patient outcome in different types of human cancer. Among 107 epidemiological datasets available, 26 studies show significant (p<0.05) correlation between LATS2 mRNA levels and patient outcome, which includes 17 studies showing better patient survival with low LATS2 expression. Moreover, 12 studies show significant correlation between LATS1 mRNA levels and patient outcome, which includes 5 studies showing better patient survival with low LATS1 expression. In addition, low YAP expression predicted worse patient survival in human colorectal cancer (Barry et al., 2013) and multiple myeloma (Cottini et al., 2014). Therefore, although YAP/TAZ hyperactivation is frequently observed in human cancers (Harvey et al., 2013; Moroishi et al., 2015a), the precise role of the Hippo pathway in human cancer might be context dependent. In this study, we show that deletion of LATS1/2 in tumor cells strongly suppresses tumor growth in vivo. On the surface, the present data cannot be easily reconciled with the tumor suppressor model of LATS1/2 in the Hippo field.
The following model is proposed: LATS1/2 suppress tumor initiation as well as inhibit immunogenicity. These two activities are important for the physiological role of LATS1/2 in maintaining tissue homeostasis. LATS1/2 normally provide growth inhibitory signals to the cells; therefore, they function cell autonomously to limit tissue overgrowth. It is also proposed that LATS1/2 suppresses immunogenicity, serving as a built-in mechanism to prevent overgrowth of undesirable cells at the wrong places in the organism. For example, inactivation of LATS1/2 is needed to promote cell proliferation during wound healing and tissue regeneration. However, cells with imp aired LATS1/2 activity may over-proliferate and migrate to the wrong place. Such undesirable cells should be eliminated to maintain tissue homeostasis and integrity. This can be achieved because inactivation of LATS1/2 in these cells can induce a strong immune response. Therefore, the immunosuppressive function of LATS1/2 is consistent with its physiological roles in tissue homeostasis.
In the established tumor cell lines of B16, SCC7, and 4T1, YAP and TAZ are not constitutively active. In fact, YAP and TAZ are readily regulated (in B16 and 4T1 cells) or even largely inactive (in SCC7 cells). Therefore, the tumorigenicity of these cancer cell lines is independent of the Hippo pathway. Nevertheless, deletion of LATS1/2 causes a moderate increase of anchorage-independent growth of these tumor cells in vitro, consistent with the growth inhibitory effect of LATS1/2. However, the enhanced immunogenicity unmasked by the LATS1/2 deletion in these cells can induce strong immune responses and overwhelm any growth advantage that might be gained due to LATS1/2 deletion, leading to strong inhibition of tumor growth in the immune-competent mice. The dual functions of LATS1/2 in sup pressing cell growth and immunogenicity can explain previous observations along with the present data.
The present results indicate that inactivation of the Hippo pathway in tumor cells induces host inflammatory responses. Interestingly, recent studies revealed that the Hippo pathway can respond to (Nowell et al., 2016; Taniguchi et al., 2015) and mediate (Liu et al., 2016) inflammatory signals. This study, together with these recent findings, suggests a reciprocal interaction between the Hippo pathway and inflammatory responses. LATS1/2-deficient tumor-derived EVs contain higher amounts of nucleic acids, which stimulate the host TLRs-MYD88/TRIF nucleic-acid sensing pathway s, provoking a type I IFN response to establish robust anti-tumor immunity. Recent studies indicate that tumor cells themselves can produce type I IFN in response to chemotherapy, thus enhancing anti-tumor immune responses (Chiappinelli et al., 2015; Sistigu et al., 2014). Because WT and LATS1/2 dKO B16-OVA cells showed similar expression levels of type I IFN genes such as Ifna4 and Ifnb1 (
A series of unbiased Hippo pathway interactome studies have linked endosomal compartments to the Hippo pathway (Moya and Halder, 2014). It is possible that the Hippo pathway may regulate endocytic trafficking and, therefore, regulate EV biogenesis. Little is known about the signaling mechanisms involved in EV biogenesis and incorporation of proteins or nucleic acids into EVs. Given the known effect of YAP on global microRNA (miRNA) biogenesis (Mori et al., 2014) and the functional importance of miRNA in EVs (Yanez-Mo et al., 2015), one may speculate that the effect of YAP/TAZ on miRNA biogenesis may increase immunogenicity of LATS1/2-null cells. However, TEAD mediated transcription is required for tumor suppression by YAP (
Recent advances in cancer immunotherapy have provided new therapeutic approaches for cancer, and several immune checkpoint inhibitors indeed show impressive effects in the clinic (Sharma and Allison, 2015). However, individual immune response to cancer immunotherapy often relies on tumor immunogenicity that varies extensively between different cancer types and different individuals; therefore, immune checkpoint inhibitors may not work in cases where tumor immunogenicity is intrinsically limited (Pico de Coana et al., 2015). The present study revealed that inactivation of LATS1/2 in tumor cells increases tumor immunogenicity and enhances tumor vaccine efficacy. Therefore, it is speculated that inhibiting LATS1/2 may enhance anti-tumor immune response and, therefore, would be an attractive approach to treat cancer. Furthermore, LATS1/2 inhibition to improve immunogenicity of tumor cells may enhance immune checkpoint inhibitor efficacy. Thus, a combination of LATS1/2 inhibitors and immune checkpoint inhibitors would be a novel and exciting therapeutic app roach for poorly immunogenic cancers, especially in cases where malignancy is driven by oncogenic alterations that leave the Hippo signaling pathway intact. It is noteworthy that germline or somatic mutations affecting the core components of the Hippo pathway are uncommon in human cancers (Harvey et al., 2013; Moroishi et al., 2015a). Therefore, inhibition of LATS1/2 may enhance tumor immunity in most cancer types. However, the caveat remains that the immune system of mice is considerably different from that of humans, and whether our findings in mice can directly be translated to humans remains to be determined. Moreover, the effect of LATS1/2 inhibition as an intervention for established tumors needs to be explored. Nevertheless, future studies expanding the therapeutic potentials of the Hippo pathway will have important clinical implications.
Recent advances in cancer treatment have improved the survival and quality of life for patients, especially those who are in the early clinical stages (Steeg 2016). However, prolonged survival is still unachievable in most patients with advanced cancer that have distal metastases. In particular, metastases of primary tumors contribute to 90% of patient deaths (Lambert et al., 2017). The lungs are the second most frequent site of metastases from extra-thoracic malignancies (Mohammed et al., 2011). Cancer in the lungs can create an immunosuppressive and angiogenic microenvironment (Kitamura et al., 2015; Ostrand-Rosenberg and Fenselau, 2018). Recent studies suggest that immunotherapy with checkpoint inhibitors can overcome the immunosuppressive networks to prevent, and in some cases, to eradicate lung metastases (Sharma and Allison, 2015, Wolchok et al., 2013). However, checkpoint inhibitors achieve a progression-free survival in only 10-30% of patients with metastasis (Wolchok et al., 2017). Hence, there are strong demands for new immunotherapeutic approaches to improve survival rates of metastatic cancer patients.
A synthetic TLR7 agonist, imiquimod, has already been approved for human use and shows favorable clinical efficacy in patients with dermatological tumors, including basal cell carcinoma and actinic keratosis (Geisse et al., 2004). However, the drugs applications are limited to topical use because of immunotoxicity induced by systemic administration (Savage et al., 1996; Engel et al., 2011). To improve the pharmacokinetics and reduce severe immune adverse effects, our laboratory developed 1V270, a small molecule TLR7-specific ligand (1V136, SM360320) conjugated to a phospholipid moiety (Chan et al., 3009; Wu et al., 2014). Herein below it is demonstrated that intratumoral administration of 1V270 induces tumor-specific adaptive immune responses and inhibits primary tumor growth in murine syngeneic models of head and neck cancer and melanoma (Hayashi et al., 2011; Sato-Kaneko et al., 2017). The local 1V270 treatment activates tumor-associated macrophages and converts an immune-suppressive tumor microenvironment to a tumoricidal environment without causing systemic cytokine induction (Sato-Kaneko et al., 2017). Subsequently, 1V270 therapy induced tumor-specific adaptive immune responses that suppressed tumor growth in uninjected tumors. Reports by others have demonstrated that the use of local or systemic TLR 7 agonists, alone or as vaccine adjuvants, can induce tumor-specific immune responses and reduce the growth of colon, renal and mammary carcinomas (Wange et al., 2010; Koga-Yamakawa et al., 2013).
In contrast to the therapeutic advantages of using TLR 7 agonists on the innate immune cells in the tumor microenvironment, some recent reports indicate that TLR7 signaling pathway may promote tumor growth in primary lung carcinoma (Cherfils-Vicini et al., 2010; Chatterjee et al., 2014). This phenomenon is attributable to increased recruitment of myeloid-derived suppressor cells (MDSCs) to the tumor following TLR7 therapy (Chatterjee et al., 2014; Ochi et al., 2012). Thus, TLR7 therapy can be a double-edged sword depending on the type of tumor, the levels of receptor expression, and infiltration of suppressor cells in the tumor microenvironment (Dajon et al., 2015).
In this study, the effects of 1V270 therapy on metastatic lung tumors was evaluated. Since metastatic lung tumors are not readily accessible to intratumoral drug delivery, parenteral drug administration was analyzed. Therapeutic effects of 1V270 were evaluated in three murine syngeneic tumor models, 4T1 breast cancer, B16 melanoma, and Lewis lung carcinoma (LLC). The results showed that a single systemic dose of 1V270 reduced tumor lung colonization in all three models tested. Systemic 1V270 therapy activated local innate immune cells, including natural killer (NK) cells and antigen presenting dendritic cells. T cell receptor (TCR) repertoire analyses revealed that 1V270 therapy induced tumor-specific oligoclonal cytotoxic T cells, that were shared by different mice, and that suppressed the growth of metastatic tumors. Anti-metastatic effects of 1V270 were also achieved by intranasal drug administration. These document that both local and systemic therapy with a phospholipid-conjugated TLR 7 agonist can safely induce tumor-specific cytotoxic T cell responses in pulmonary metastatic cancer, that peripheral T cell repertoire analysis may be used to monitor the effects of therapy.
Observations support the concept that an immunomodulatory drug and TCR repertoire analysis can be applied for monitoring prognosis of metastatic lung cancer.
Wild-type female BALB/c mice, C57BL/6 and BG-albino mice were purchased from Jackson Laboratory (Bar Harbor, Mass.). The studies involving animals were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. A small molecule TLR7 ligand (1V136, SM360320) and phospholipid-conjugated TLR7 agonist, 1V270, were synthesized in our laboratory as described previously and was formulated in 20% hydroxypropylbeta-cyclodextrin (Chan et al., 2009). Endotoxin levels of these drugs and other reagents were determined by Endosafe® (Charles River Laboratory, Wilmington, Mass.) and were less than 15 EU/mg
The 4T1 (mouse breast cancer cell line), B16 melanoma cell line, and LLC cell line and BALB/3T3 fibroblast (Clone A31, CLL163) were obtained from American Type Culture Collection (Rockville, Md., USA). The cells were tested for murine pathogens and were confirmed negative prior to inoculation in mice. A GLF-expressing 4T1, B16 and LLC cells were prepared as described previously (Godebu et al., 2014). Two metastatic 4T1 models, spontaneous and IV metastasis models, were used in this study.
GFP and luciferase (GLF)-expressing tumor cells (2×104 of 4T1-GLF, 5×105 of B16-GLF, and 1×106 of LLC-GLF) were i.v. injected into BALB/c mice for 4T1 models, BG-albino or wild-type C57BU6 for B16 melanoma or LLC models). To generate bioluminescence signals, D-luciferin (3 mg/100 μL/mouse) was injected i.p. 12-15 minutes prior to the image acquisition. Image data were acquired by 1 Ss exposure using the IVIS Spectrum and analyzed using the Living Image software, version 4.5.2 (Perkin Elmer, Waltham, Mass.). We confirmed that the tumor signals in the lungs at day 10 correlated with the number of lung metastasis determined on day 21, as well as the overall survival (Supplemental figures 10).
The cells were labeled by incubating with cocktails of antibodies at 4° C. for 30 minutes (Table 1) to identify various cell types.
Anti-asialo GM1 rabbit polyclonal antibody (Waka, Richmond, Va.) or rabbit 1 gG polyclonal antibody (Millipore, Temecula, Calif.) was used for NK cell depletion. Mouse anti-CDS (clone 2.43), and isotype control Ab (clone LFA-2) were used for CD8+ cell depletion. We confirmed over 90% depletion of NK cells and CD8+ cells using flow cytometry (
Tumor-specific cytotoxicity was examined using 4T1 cells as target cells and BALB/3T3 cells as irrelevant cells. BALB/c mice were treated with 1V270 (200 μg/injection) on day −1 and 4T1 cells were inoculated on day 0. Three 4T1 cell lysate and 10 units/mL of IL-2 for 3 days. 4T1 and BALB/3T3 cells were labeled with 2.5 μM and 0.25 μM CFSE, respectively, for 12 minutes at 37° C. and were mixed at 1:1 ratio. Splenocytes cultured for 3 days were then cocultured with 4T1 and BALB/3T3 cells at 16:1 to 2:1 effector to target cell ratio (E:T) for 16 hours. The frequencies of 4T1 (CFSE high) and BALB/3T3 (CFSE low) cells were determined by flow cytometry, and the percent specific killing was calculated. Specific killing (%)=[1−“Sample ratio”/“Negative control ratio”)×100; “Sample ratio”=[4T1 (target)/BALB/3T3 (irrelevant)) value of each sample co-cultured with CD8+ T cells; “Negative control ratio”=14T1 (target)/BALB/3T3 (irrelevant)) value cultured without CD8+ T cells.
CD8+ T cells were isolated from single cell suspensions of tumors, or spleens using mouse CD8+ T cell isolation kit (Miltenyi Biotec). Total RNA was extracted from CD8+ T cells with RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Next-generation sequencing was performed with an unbiased TCR repertoire analysis technology (Repertoire Genesis Inc., Osaka, Japan) as described previously (Sato-Kaneko et al., 2017).
In the spontaneous metastasis model, othotopically implanted tumor cells spontaneously form lung metastases within three weeks after implantation. 5×105 4T1 cells were inoculated in both sides of the 4th mammary pads of female BALB/c mice. Tumor length and width were recorded, and tumor volumes were calculated using the formula: volume (mm3)=(width)2×length/2. On day 28, mice were euthanized and the lungs were stained with intratracheally injected India ink and destained in Fekete's solution to count tumor nodules. In the experimental metastasis model, 2×104 4T1 cells were i.v. injected. On day 21, mice were euthanized and tumor nodules in lungs were counted as described above. In the secondary challenge experiment, 1V270 treated-mice without tumor signals in the lungs on day 21 of the experimental metastasis protocol were included. 5×105 4T1 cells were inoculated in both sides of the 4th mammary p ads. Tumor length and width were recorded, and tumor volumes were calculated similar with
spontaneous metastasis model.
Lungs were fixed with 10% formalin for overnight, dehydrated and embedded in paraffin on the Excelsior ES tissue processor (Thermo Scientific) and sectioned at 5 um thickness on a rotary microtome. Antigen retrieval was performed in Citrate buffer, pH 6.0, heated to 98° C. for 8 minutes and cooled at room temperature for 20 minutes. The sections were stained on a Lab Vision 360 automated immunostaining instrument (Thermo Scientific) using a 2 step immunoperoxidase protocol. Briefly, the slides were blocked for endogenous peroxidase activity, washed and incubated with protein blocking buffer, 5% normal donkey serum in TBS for 10 minutes. The primary antibodies, both rabbit poly clonal, CD45 (ab10558, AbCam, used at 1 ug/ml diluted in 5% NDS) and CD3 (ab16669 from AbCam at a 1:100 dilution) were incubated for 1 hour at room temperature. After washing in TBS-tween, the slides were incubated with the secondary antibody—HRP-Donkey F(ab′)2 anti rabbit (Jackson ImmunoResearch Laboratories diluted 1:200) for 30 minutes at room temperature, washed and reacted with DAB as the brown color substrate, hematoxylin was used the counterstain. Antibody details are shown in Table 2. Images were acquired using Axio Imager Zeiss microscope (Zeiss, Thornwood, N.Y.).
The sera were collected from mice receiving 200 nmol of 1V136, 1V270 or vehicle 2, 4, 6, and 24 hours after i.p. administration (15). Cytokine levels in sera were determined by Luminex bead assays (MILLIPLEX™ MAP kit, Millip ore, Billerica, Mass.).
To make the single cell suspensions, tumors were dissociated using the mouse tumor dissociation kit (MiltenyiBiotec, San Diego, Calif.) and the gentle MACSOcto Dissociator according to the manufacture's protocol. Single cell suspensions of spleens, lungs and mLN were prepared in Hank's Balanced Salt Solution (HBSS) supplemented with 20 μg/mL DNaseI (Worthington, Lakewood, N.J.) and 0.6 mg/mL collagensse type I (Worthington). Total cell number was counted by the ViaCount assay (MilliporeSigma, Darmstadt, Germany). Dead cells were excluded by propidium iodide staining.
For NK cell depletion, 50 μL of anti-asialo GM1 rabbit poly clonal antibody (Wako, Richmond, Va.) or rabbit IgG polyclonal antibody (Millipore, Temecula, Calif.) was injected on days −1, 1, 5, 9, 13, and 17. Mouse anti-CDS (clone2.43) and isotype control Ab (clone LFA-2) were purchased from BioXcell (West Lebanon, N.H.). Anti-CDS and isotype control (200 μg/dose) were i.p administered on days 5, 8, 11, 14, 16, 19, and 23 to mice.
Means and standard errors of means (SEM) are shown in other analyses. In dot plots, each dot represents a tumor, a spleen, or a lymph node from an individual mouse and the horizontal and vertical bars indicate mean and mean±SEM. Mann-Whitney test was used to comp are two groups. Using tumor volumes collected over all time points, two-way repeated measures ANOVA was used to comp are different groups, with pair-wise contrasts made at the final time point using a Bonferroni post hoc test. To comp are cross-sectional outcomes among more than two groups, Kruskal-Wallis tests with Dunn's post hoc test were applied. Correlations between tumor volumes and TCR reportire analysis data were analyzed using a Pearson's correlation test, pooling data across the different treatment groups. Analysis of covariance (sometimes on the log scale) was used to test whether the correlation was mediated by differences among the treatment groups in both mean immune marker level and tumor volumes. p<0.05 were considered statistically significant. Prism 6 (GraphPad Software, San Diego, Calif.) statistical software was used to carry out these analyses.
Systemic Administration of IV270 Inhibits Spontaneous Lung Metastasis in a CD8+ Dependent Manner in a Murine 4T1 Orthotopic Breast Cancer Model
1V270 induces tumor-specific adaptive immune responses when administered intratumorally (Hayashi et al., 2011; Sato-Kaneko et al., 2017). However, recent reports claim that TLR7 activation in the lung can promote primary tumor growth (Cherfils-Vicini et al., 2010; Chatterjee et al., 2014). Thus, it was examined whether systemic 1V270 therapy would also promote tumor-specific adaptive T cell responses and if such responses could restrain pulmonary metastatic disease. For this purpose, the murine 4T1 breast cancer model that exhibits characteristics similar to the human disease, in which orthotopically implanted tumor cells spontaneously metastasize to the lungs (Pulaski and Ostrand-Rosenburg 2001), was employed.
4T1 cells (5×105) were inoculated into the 4th mammary pads on day 0, and 1V270 (20, 80, or 200 μg/injection) was injected intraperitoneally (i.p.) twice a week for three weeks, starting on day 7 (
To study the possible involvement of cytotoxic T cell immune responses in the anti-metastatic effects of 1V270, CD8+ cells were depleted with monoclonal antibody (mAbs) prior to treatment with the TLR agonist (
Systemic Administration of JV270 Induces Tumor-Specific CD8+ T Cells in an Intravenous Metastatic Model of 4T1 Breast Cancer
Intravenous (IV) lung metastasis models have been used to evaluate in more detail the immune responses to circulating tumor cells induced by 1V270 therapy. Each animal received 2×104 4T1 cells directly in the tail vein on day 0, and the numbers of lung nodules were counted on day 21 (
To examine the role of CD8+ T cells after systemic 1V270 treatment, mediastinal lymph node (mLN) cells, splenocytes, and lung tissues were analyzed in the IV metastasis model on day 21 (
Tumor-Infiltrating T Cells in IV270 Treated Mice Show High Clonalities and Intra- and Inter-Individual Commonality by TCR Repertoire Analysis
Increased clonality of CD8+ T cells has been associated with both a positive clinical outcome and immune-related adverse events after immune checkpoint therapy (Ikeda et al., 2017; Dubudhi et al., 2016). Other studies have also indicated that clonal expansion of tumor-specific T cells is a biomarker for suppression of tumor growth (Straten et al., 1998; Kim et al., 2004). Intratumoral treatment with 1V270 induces local expansion an systemic dispersion of oligoclonal tumor-specific T cells by TCR repertoire analysis using next generation RNAseq methodology (Sato-Kaneko et al., 2017). Thus, it was important to determine whether systemic 1V270 therapy also induced oligoclonal expansion of tumor-specific T cells.
To validate that 1V270 therapy induced tumor-specific adaptive immune responses, we monitored the growth of secondarily challenged tumors following prior 1V270 treatment. The mice treated with 1V270 using the IV metastasis protocol were orthotopically re-challenged with 4T1 cells on day 21 (
To examine clonal specificity of tumor-specific T cells, CD8+ cells were isolated from the spleens and the TILs of secondarily challenged tumors after initial 1V270 therapy. The TCR repertoires were assessed by next generation RNA sequencing of both TCRα and TCR β genes as previously described (Yoshida et al., 2000). The clonality indices of CD8+ T cells in TILs, as assessed by I-Shannon index, were negatively correlated with the volumes of the secondarily challenged tumors only in the mice treated with 1V270 and exposed to tumor cells (Pearson's correlation coefficient, r2=0.97, P=0.015,
Dendritic Cells in the Lungs and Draining Lymph Nodes are Activated, and CD S+ T Cells are Recruited to the Draining Lymph Nodes Following 1V270 Therapy
Previously it was demonstrated that 1V270 activates antigen presenting cells (APCs) and promotes cross-presentation of antigen to CD8+ T cells (Goff et al., 2015). Since the 1V270 therapy induced a tumor-specific CD8+ T cell response in the 4T1 model, we evaluated whether the therapy activated APCs in the lungs, and/or in the draining lymph nodes. BALB/c mice were i.p. administered with 1V270 on day −1, and 4T1 cells were injected the next day, and the dendritic cell populations in the draining mediastinal LNs (mLNs) and the lungs were examined on day 7 after the tumor injection. In the 1V270-treated mice, a population of CD11c+ dendritic cells was increased in both mLNs and the lungs (p<0.01,
1V270 Treatment Activates Innate Immunity and Inhibits Lung Colonization by Tumor Cells in a NKc-Cell Dependent Manner
In the IV metastasis model, the administration of 1V270 one day before IV injection of tumor cells was required to restrain lung colonization. Since adaptive immune responses require several days to develop, this observation indicated that one or more innate immune cell types in the lung mediated the early therapeutic effect. To enable the monitoring of the detailed kinetics of the colonization process of 4T1 cells expressing both green fluorescent protein (GFP) and luciferase (4T1-GLF) were prepared using lentivirus vectors (Godebu et al., 2014). Subsequently, tumor implantation and growth were monitored using an IVIS Spectrum® in vivo imaging system. In both vehicle- and 1V270-treated mice, tumor cells accumulated in the lungs quickly after the injection (at Oh,
To identify the types and functions of innate immune cells recruited into the lung following 1V270 administration, the mice were injected with 1V270 on day −1, 4T1 cells were i.v. administered on day 0, and the bronchioalveolar cells were isolated on day 7 after the tumor injection. A single cell suspension of lung cells was stained for natural killer (NK) cells and myeloid suppressor cells (MDSC) and analyzed by flow cytometry (
NK cells can be activated directly by TLR7 agonists, and indirectly by type I IFN which is secreted from accessory dendritic cells (Liu et al., 2007; Hart et al., 2005). To examine the role of NK cells in the early therapeutic efficacy of 1V270, this cell type was depleted by treatment with anti-asialo GM1 poly clonal antibody (Kasai et al., 1980). Over 90% of NK cells were depleted by antibody injection on days −4, −1, 3 and 10 (
Intranasal Administration of 1V270 is Also Effective in Preventing Metastasis and in Inducing Anti-Tumor Immunity
It was demonstrated that intranasal (i.n.) administration of 1V270 activates nasal and lung APCs, without causing systemic cytokine release (Wu et al., 2014). Therefore, we examined whether in 1V270 treatment could impair tumor growth in the IV metastasis model (
It was examined whether i.n. administration of 1V270 could induce tumor-specific adaptive immune responses similar to the effects of systemic administration. Mice were treated with i.n. 1V270 (200 or 500 μg/50 μL) and then received 4T1 cells by the i.v. route. The surviving mice on day 21 were orthotopically re-challenged with tumor cells. The re-challenged tumor growth was significantly inhibited in the mice which were i.n. treated with 1V270 (
Anti-Metastatic Effects of JV270 were Observed in Murine Syngeneic Melanoma and Lung Carcinoma Models
To evaluate whether the 1V270 therapy can be effective in other cancer types, we employed two additional murine syngeneic metastasis models; B16 melanoma and Lewis lung cancer (LLC). Luciferase and GFP expressing cells (B16-GLF and LLC-GLF) were prepared using a lentivirus vector for in vivo imaging analysis. Mice received systemic 1V270 treatment on day −1, and then B16-GLF and LLC-GLF cells were i.v. administered on day 0. In both metastasis models, 1V270 inhibited lung metastasis by day 14 (
In patients with an advanced stage of cancer, the development of metastasis is almost inevitable since the metastatic niches are seeded with tumor cells long before clinical presentation (Valastyan and Weinberg, 2011). The ability of immune-checkpoint inhibitors to reactivate tumor-specific cytotoxic T cells provided evidence that immunotherapy can overcome these limitations, at least in some patients. Thus, there is an unmet medical need for additional agents that can increase the frequency of cytotoxic T cells at metastatic sites. Because each immunotherapy type exploits a distinct biological mechanism, biomarkers that predict efficacy and adverse effects are required (Topalian et al., 2000). Systemic 1V270 treatment systemically induced cytotoxic tumor-specific CD8+ T cells, as assessed by both in vitro tumor-specific cytotoxicity assays, and tumor re-challenge experiments. TCR repertoire analyses of TILs in the secondarily challenged tumors indicated that 1V270 therapy strongly increased T cell clonality. The levels of clonality negatively correlated with the tumor volumes of secondarily challenged tumors. Of interest, the clonal similarity between tumor infiltrating and splenic T cells was increased in 1V270 treated and tumor-exposed animals. A recent paper demonstrated that antitumor immune cells proliferate in the secondary lymphoid organs, including draining LNs and spleen, and can be detected in the peripheral blood during tumor rejection (Spitzer et al., 2017). These findings suggested that immune monitoring should be possible by analyzing the TCR repertoire of peripheral T cells.
Theoretically, the TCR repertoire might be diverse among individual tumor-bearing mice, even though they share the same genetic background (Venturi et al., 2008). In a chronic virus infection, patients develop common clones which interact with highly immunodommant antigens (Cerundolo et al., 2016; Miyama et al., 2017). In the present study, an eight-fold higher number of shared clones in TILs was identified in the 1V270 treated and tumor-exposed group, compared to the no-tumor exposed group. As increased frequency of shared clones suggested that the systemic 1V270 treatment may skew the TCR repertoire toward tumor-specific clones, that may recognize the same tumor antigens.
When administrated locally, synthetic TLR7 and TLR9 agonists are potent immune adjuvants, that can induce Th1 and cytotoxic T cell responses over a period of week (Sato-Kaneko et al., 2017; Cho et al., 2002). When given systemically, however, some TLR agonists can cause a cytokine release syndrome that could potentially enhance metastatic growth by stimulating either angiogenesis or the development of M2 macrophages (Hageman et al., 2005; Sanmarco et al., 2017). Therefore, effective systemic TLR7 therapy must clearly demonstrate that CD8 responses are induced without toxicity to the host or adverse changes in the tumor microenvironment. The present data demonstrated that some monocyte linage, myeloid derived suppressor cells (MDSCs) were recruited to the lung after 1V270 administration. Immature MDSCs have the ability to suppress anti-tumor T cell responses (Quail and Joyce, 2013). Thus, we were concerned that systemic 1V270 treatment may promote tumor growth. However, other innate immune cells, including NK cells and dendritic cells, were also recruited to the lung after 1V270 administration, as reported previously in other models using TLR ligands (Smits et al., 2008). The recruitment and activation of the NK cells impeded tumor lung colonization, indicating that the NK cells could overcome the suppressive function of MDSC recruited by 1V270 administration. Another concern in immunotherapy using TLR7 ligands is that stimulation of a tumor TLR7 pathway could promote growth and chemo-resistance in some primary tumors expressing this receptor (Chrfils-Vincini et al., 2010; Chatterjee et al., 2014). In our study, 4T1, B16, and LLC cells did not express TLR7 by quantitative RT-PCR (Supplemental figures 8). We therefore conclude that systemic and i.n. TLR7 treatment is an effective therapy for TLR7 negative tumors.
1V270 inhibited the growth of small subcutaneous tumors when locally (intratumorally) injected (Hayashi et al., 2011; Sato-Kaneko et al., 2017). In the 4T1 metastatic model, orthotopically implanted primary tumors in the mammary gland were advanced at the time of initiation of 1V270 treatment. The ability of the TLR7 phospholipid agonist to prevent early lung metastasis may be attributable both to the lower tumor burden and to NK recruitment and activation. The in vivo imaging studies in NK cell depleted mice confirmed the critical role of this cell type in constraining tumor colonization, thus allowing for the development of a specific CD8 T cell response in the later phases of metastasis.
Intratracheal administration of a low molecular weight TLR7 agonist (SM276001) was reported to suppress metastatic lung tumors (Koga-Yamakawa et al., 2013). It was demonstrated that a low molecular weight TLR 7 agonist (SM360320)(1V136) induced high levels of systemic proinflammatory cytokines following parenteral and i.n. administration to mice and that conjugation of a TLR7 ligand to a phospholipid moiety could markedly reduce in vivo cytokine release (
In summary, single systemic administration of a phospholipid conjugated TLR7 agonist inhibited lung metastasis in three different murine syngeneic models of human malignancy, 4T1 breast cancer, B16 melanoma and Lewis lung carcinoma models. The drug quickly activated NK cells in the lung, and later induced a cytotoxic T cell response. These two different mechanisms, NK cell-mediated and tumor-specific adaptive T cell responses, were responsible for the early and late phases of tumor growth inhibition. The anti-tumor effects were achieved without significant systemic release of inflammatory cytokines following systemic administration. Furthermore, 1V270 therapy induced oligoclonal CD8 T cell responses as determined by TCR repertoire analyses of both spleen and mediastinal lymph nodes. The emergence of shared T cell clones correlated with the development of adaptive immunity against tumor cells. These results suggest that TCR repertoire analyses may be used to guide clinical trials of TLR and other immunotherapies in patients with metastatic cancer.
All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.
This application claims the benefit of the filing date of U.S. application Ser. No. 62/479,057, filed on Mar. 30, 2017, the disclosure of which is incorporated by reference herein.
This invention was made with government support under grant number R35CA196878 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/25522 | 3/30/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62479057 | Mar 2017 | US |
