Patent Application
20200377599
Targeting immune checkpoints, such as programmed cell death protein 1 (PD-1) and its ligand PD-L1, have been approved for treating multiple types of human cancer. However, many cancer patients fail to respond to anti-PD-1/PD-L1 treatment and the underlying mechanism(s) for this resistance is not well understood. Recent studies revealed that response to PD-L1 blockade might correlate with PD-L1 expression levels on tumor cells.
Thus, it is important to mechanistically understand the pathways controlling PD-L1 protein expression and stability, which can offer a molecular basis to improve the clinical response rate and efficacy of PD-1/PD-L1 blockade in cancer patients. Accordingly, improved methods for determining the underlying mechanism(s) of PD-L1 expression levels on tumor cells are needed.
As described below, the present disclosure features compositions and methods of treating a subject, particularly a mammalian subject, and more particularly, a human subject, who has a cancer (e.g., colon cancer, breast cancer, melanoma, lung cancer, head and neck cancer, prostate cancer). The described compositions and methods embrace the use of an anti-PD-L1 or an anti-PD-1 antibody and an inhibitor of cyclin D kinase 4/6 (CDK4/6) to treat the cancer.
In one aspect, the present invention provides a therapeutic combination comprising a cyclin D kinase 4/6 (CDK4/6) inhibitor and an anti-PD-L1 antibody and/or an anti-PD-1 antibody.
In another aspect, the present invention provides a method of reducing tumor growth, the method involving contacting a tumor cell with a cyclin D kinase 4/6 (CDK4/6) inhibitor and an anti-PD-L1 and/or an anti-PD-1 antibody, thereby reducing tumor growth.
In another aspect, the present invention provides a method of treating cancer in a subject, the method comprising administering to the subject a cyclin D kinase 4/6 (CDK4/6) inhibitor and an anti-PD-L1 and/or an anti-PD-1 antibody, thereby treating cancer in the subject.
In another aspect, the invention features a kit comprising a cyclin D kinase 4/6 (CDK4/6) inhibitor, an anti-PD-L1 and/or an anti-PD-1 antibody.
In various embodiments of any of the above aspects or any other aspect of the invention delineated herein, the CDK4/6 inhibitor is palbociclib, ribociclib, abemaciclib or trilaciclib. In some embodiments of the above aspects, the anti-PD-1 antibody is nivolumab, pembrolizumab, or pidilizumab. In some embodiments of the above aspects, the anti-PD-L1 antibody is MPDL3280A, MEDI4736, BMS-936559, or MSB0010718C. In some embodiments of the above aspects, the combination comprises a CDK4/6 inhibitor and an anti-PD-L1 or an anti-PD-1 antibody. In some embodiments of the above aspects, the combination is formulated in a single composition or is formulated and administered separately. In some embodiments, the combination comprises a CDK4/6 inhibitor (e.g., palbociclib) and an anti-PD1 antibody. In various embodiments, the cancer is colon cancer, breast cancer, melanoma, prostate cancer, lung cancer, and head and neck cancer. In some embodiments of the above aspects, the treatment reduces tumor growth relative to a reference. In some embodiments of the above aspects, the treatment increases survival of the subject.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By “cyclin D kinase 4 (CDK4)” is meant a protein or fragment thereof having serine/threonine kinase activity, and having at least about 85% identity to NCBI Ref. Seq. NP_000066, which functions in cell cycle regulation.
By “CDK4/6 Inhibitor” is meant an agent that inhibits CDK4 and/or 6 expression, function or activity. Exemplary CDK4/6 inhibitors include, but are not limited to, palbociclib, ribociclib, abemaciclib and trilaciclib.
By “anti-PD-L1 antibody” is meant an antibody, or fragment thereof, that selectively binds a PD-L1 polypeptide. Exemplary anti-PD-L1 antibody is MPDL3280A, MEDI4736, BMS-936559, or MSB0010718C.
By “PD-L1 polypeptide” is meant a polypeptide or fragment thereof having at least about 85%, or greater, amino acid identity to NCBI Accession No. NP_001254635 (SEQ ID NO: 1, below) and having PD-1 binding activity.
By “PD-L1 nucleic acid molecule” is meant a polynucleotide encoding a PD-L1 polypeptide. An exemplary PD-L1 nucleic acid molecule sequence is provided at NCBI Accession No. NM_001267706 and in SEQ ID NO: 2, below.
NCBI ACCESSION NO. NM_001267706 mRNA
By “anti-PD-1 antibody” is meant an antibody, or fragment thereof, that selectively binds a PD-1 polypeptide. In one embodiment, the anti-PD-1 antibody is nivolumab, pembrolizumab, or pidilizumab.
By “PD-1 polypeptide” is meant a polypeptide or fragment thereof having at least about 85%, or greater, amino acid identity to NCBI Accession No. NP_005009.2 (SEQ ID NO: 3, below) and having PD-L1 binding activity.
By “PD-1 nucleic acid molecule” is meant a polynucleotide encoding a PD-1 polypeptide. An exemplary PD-1 nucleic acid molecule sequence is provided at NCBI Accession No. NG_012110.1 and in SEQ ID NO: 4, below)
By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease, such as cancer.
By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression or activity levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.”
By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.
In this disclosure, “comprises,” “comprising,” “containing,” and “having” and the like can have the meaning ascribed to them in U.S. Patent law, and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In a disease, such as cancer (e.g., colon cancer, breast cancer, melanoma, lung cancer, head and neck cancer, prostate cancer), the normal function of a cell tissue or organ is subverted to enable immune evasion and/or escape.
By “effective amount” is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. In one embodiment, an effective amount of an agent defined herein is sufficient to reduce or stabilize the proliferation of a cancer cell. In another embodiment, an effective amount of an agent defined herein is sufficient to kill a cancer cell.
The invention provides a number of targets that are useful for the development of highly specific drugs to treat a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
By “reference” is meant a standard or control condition.
By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
IB of WCL derived from HeLa cells pre-treated with/without IFNγ (10 ng/ml) for 12 hours and then synchronized in M phase by nocodazole treatment prior to releasing back into the cell cycle for the indicated times.
The present disclosure features compositions and methods of treating a cancer in a subject by administering to the subject an anti-PD-L1 or an anti-PD-1 antibody and an inhibitor of cyclin D kinase (CDK)4/6 to treat the cancer.
The invention is based, at least in part, on the discovery that PD-L1 protein abundance fluctuates during the cell cycle and is negatively regulated during cell cycle progression by cyclin D/CDK4 and the Cullin 3SPOP E3 ligase. This fluctuation is mediated by proteasome degradation. Treatment with an inhibitor of CDK4/6, Palbociclib, elevated PD-L1 protein levels largely through inhibiting cyclin D/CDK4-mediated phosphorylation of SPOP to promote its degradation by APC/Ccdh1. Loss-of-function mutations in SPOP compromised ubiquitination-mediated PD-L1 degradation, and led to increased PD-L1 expression and reduced tumor-infiltrating lymphocytes (TILs) in mouse tumors and human prostate cancer specimens. Upregulation of PD-L1 by CDK4/6 inhibition helped tumors evade immune surveillance, which could be one possible underlying reason for acquired drug resistance to CDK4/6 inhibitors in clinical therapy. Combining CDK4/6 inhibitor treatment with anti-PD-1 immunotherapy enhanced tumor regression and dramatically improved overall survival rates in mouse tumor models. The present invention uncovers a novel molecular mechanism for regulating PD-L1 protein stability during cell cycle progression, and shows that using combination treatment with CDK4/6 inhibitors and PD-1/PD-L1 immune checkpoint blockade enhances the therapeutic efficacy for human cancers.
The invention provides compositions comprising a CDK4/6 inhibitor and an anti-PD-1 and/or anti-PD-L1 antibody.
Antibodies that target PD-1 include, but are not limited to, nivolumab, Bristol-Myers Squibb; pembrolizumab, Merck, Whitehouse Station, N.J.; pidilizumab, CureTech, Yavne, Israel).
Antibodies that target PD-L1 include, but are not limited to MPDL3280A, Genentech, South San Francisco, Calif.; MEDI4736, MedImmune/AstraZeneca; BMS-936559, Bristol-Myers Squibb; MSB0010718C, EMD Serono, Rockland, Mass.).
Examples of CDK4/6 inhibitors include palbociclib (PD0332991), ribociclib (LEE011), abemaciclib (LY2835219) and trilaciclib (G1T28).
The methods and compositions provided herein can be used to treat or prevent progression of a cancer (e.g., colon cancer, breast cancer, melanoma, prostate cancer, lung cancer, head and neck cancer). In general, methods of the invention involve the administration of therapeutic combinations comprising an agent that inhibits the expression or activity of cyclin-dependent kinase 4/6 (CDK4/6); and an agent that targets PD-L1 or programmed cell death-1 (PD-1) Compositions of the invention are administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk of developing cancer (e.g., colon cancer, breast cancer, melanoma, prostate cancer, lung cancer, head and neck cancer). Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, family history, and the like). Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g., measurable by a test or diagnostic method).
In one embodiment, a therapeutic combination of the invention comprises a CDK4/6 inhibitor, an anti-PD-L1 antibody, and/or an anti-PD-1 antibody. In particular embodiments, a therapeutic combination of the invention comprises a CDK4/6 inhibitor and an anti-PD-L1 antibody or an anti-PD-1 antibody. The CDK4/6 inhibitor may be administered prior to, concurrent with, or subsequent to administration of the anti-PD-L1 antibody or an anti-PD-1 antibody. In particular embodiments, the CDK4/6 inhibitor and the anti-PD-L1 antibody and/or anti-PD-1 antibody administration is conducted within about 1-3 hours, 4-6 hours, 7-12 hours, or 13-24 hours. In other embodiments, the administration occurs within about 1-3 days, 3-5 days, or 7-10 days. If desired, such therapeutic combinations are administered in combination with standard chemotherapeutics. Methods for administering combination therapies (e.g., concurrently or otherwise) are known to the skilled artisan and are described for example in Remington's Pharmaceutical Sciences by E. W. Martin.
The present invention features compositions comprising an agent that inhibits the function, expression or activity of cyclin-dependent kinase 4/6 (CDK4/6); and an agent that targets PD-L1 or programmed cell death-1 (PD-1), which are useful for treating cancer (e.g., Typically, such compositions comprise an effective amount of an agent that inhibits the expression or activity of cyclin-dependent kinase 4/6 (CDK4/6); and an effective amount of an agent that targets PD-L1 or programmed cell death-1 (PD-1) in a physiologically acceptable carrier. Therapeutic combinations of the invention are typically formulated and administered separately, but may also be combined and administered in a single formulation.
Typically, the carrier or excipient for the composition provided herein is a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof. The preparation of such solutions ensuring sterility, pH, isotonicity, and stability is effected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, and the like.
The administration may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing the disease symptoms in a subject. The composition may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, intraperitoneally, intramuscular, intrathecal, or intradermal injections that provide continuous, sustained levels of the agent in the patient. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the cancer. Generally, amounts will be in the range of those used for other agents used in the treatment of cancer, although in certain instances lower amounts will be needed because of the increased specificity of the agent. A composition is administered at a dosage that ameliorates or decreases effects of the cancer as determined by a method known to one skilled in the art.
The therapeutic or prophylactic composition may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, intrathecally, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
Pharmaceutical compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with an organ, such as the heart; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a disease using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.
Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the agent in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic agent is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic agent in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.
Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates a cardiac dysfunction or disease, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) (e.g., a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor and anti-PD-L1 or PD-1 antibody described herein) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
In some embodiments, the composition comprising the active therapeutic agent is formulated for intravenous delivery. As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
The invention provides kits for the treatment or prevention of cancer. In some embodiments, the kit includes a therapeutic composition containing an agent that inhibits the expression or activity of cyclin-dependent kinase 4/6 (CDK4/6); and an agent that targets PD-L1 or programmed cell death-1 (PD-1) in unit dosage form. In other embodiments, the kit includes an agent that inhibits the expression or activity of cyclin-dependent kinase 4/6 (CDK4/6); and an agent that targets PD-L1 or programmed cell death-1 (PD-1) in unit dosage form in a sterile container. Such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired a pharmaceutical composition of the invention is provided together with instructions for administering the pharmaceutical composition to a subject having or at risk of contracting or developing cancer. The instructions will generally include information about the use of the composition for the treatment or prevention of cancer. In other embodiments, the instructions include at least one of the following: description of the therapeutic/prophylactic agent; dosage schedule and administration for treatment or prevention of cancer or symptoms thereof; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987);
“Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
Dysregulated cell cycle progression is one of the hallmarks of human cancers, and targeting cyclin-dependent kinases (CDKs) to block cell cycle transitions has been validated as an effective therapy for cancer patients. Although it has been reported that PD-L1 expression can be regulated at both transcriptional and post-translational levels, it remains largely unknown whether PD-L1 stability is regulated under physiological conditions such as during cell cycle progression. To address this question, multiple cell lines, including HeLa, MDA-MB-231 and HCC1954, were synchronized at M phase using nocodazole treatment, and then released back into the cell cycle (
Cyclins and cyclin-dependent kinases play crucial roles in regulating the stability of cell cycle-related proteins during cell cycle progression. Thus, a genetic method was adopted to deplete each major cyclin to explore their potential involvement in regulating the protein stability of PD-L1. Notably, it was found that depleting all three cyclin D isoforms (D1, D2 and D3), dramatically elevated PD-L1 protein abundance in mouse embryonic fibroblasts (MEFs), whereas neither cyclin A (A1 and A2) nor cyclin E (E1 and E2) depletion had this effect (
Consistent with the findings that cyclin D, but not cyclin A or cyclin E, suppressed the PD-L1 protein abundance in cells, depletion of the cyclin D-binding partner CDK4 increased PD-L1 protein abundance whereas depletion of CDK2, a binding partner of cyclin A and cyclin E, did not (
The retinoblastoma protein (Rb) is a major target of CDK4/6, whose tumor suppressive function is frequently compromised in human cancer. In keeping with previous studies, a panel of 19 human cancer cell lines was examined and it was found that Rb loss typically led to elevation of CDK4 endogenous inhibitor, p16, which further positively correlated with increased PD-L1 expression levels (
Physiologically, it was found that palbociclib treatment significantly elevated PD-L1 protein levels in all of the 14 different mouse tissues that were examined, including lung, heart, pancreas, bone marrow, spleen, kidney, stomach, large intestine, brain, cerebellum, liver, mammary gland, uterus and ovary (
As a functional consequence, it was found that palbociclib-induced upregulation of PD-L1 was coupled with a concurrent decrease in absolute number of CD3+ TILs in tumors derived from transplanted MC38 and B16-F10 mouse cancer cell lines, as well as in autochthonous mice mammary tumors induced by MMTV-ErbB2 (
The ubiquitin-proteasome system (UPS) is the most important pathway for regulating protein stability and is responsible for controlling multiple cellular processes including cell cycle progression. To further explore whether the UPS is involved in suppressing PD-L1 stability, cells were treated with the proteasome inhibitor, MG132, and cullin-based ubiquitin E3 ligase inhibitor, MLN4924, and it was found that both inhibitors stabilized PD-L1 protein in cells (
Cullin 3-based E3 ubiquitin ligases recognize their downstream substrates through one of several adaptor proteins, which typically contain one BTB domain to interact with Cullin 3 and one substrate-recognizing motif to recruit the specific substrate. It was found that SPOP, but not any of the other examined adaptor proteins, including Keap1, KLHL2, KLHL3, KLHL12, KLHL20, or KLHL37, specifically interacted with PD-L1 in cells (
The last eight amino acids of PD-L1 (283-290) were further identified as the potential binding motif (also called degron) for SPOP, as the PD-L1 (Δ283-290) mutant failed to bind with SPOP and became resistant to SPOP-mediated degradation (
In keeping with the result that SPOP specifically interacts with PD-L1 in cells, ectopic expression of SPOP, but not Keap1 or hCop1, markedly reduced PD-L1 protein abundance in cells (
Although it has been reported that SPOP mutations occur in approximately 10-15% of human prostate cancers (PrCa), SPOP mutations were analyzed in all cancer types from the TCGA database and it was found that recurrent hotspot mutations in SPOP largely occur in prostate adenocarcinoma (PRAD, 11%), uterine corpus endometrial carcinoma (UCEC, 14%), and uterine carcinosarcoma (UCS, 7%) (
Furthermore, it was explored whether loss-of-function SPOP mutations in PrCa correlated with elevated PD-L1 and decreased TILs in human PrCa. To this end, anti-PD-L1 and anti-CD8 antibodies were first validated through several methods, including immunoblot, immunofluorescence, and immunohistochemistry (IHC) (
To further explore the physiological function of SPOP in regulating PD-L1 stability during cell cycle progression, it was confirmed that SPOP protein abundance also fluctuated during the cell cycle and displayed an inverse correlation with PD-L1 protein levels (
In keeping with this hypothesis, the interaction between SPOP and Cdh1 was detected at the endogenous level in cells (
To further gain insight into how cyclin D/CDK4 regulates PD-L1 during cell cycle progression through disrupting the Cdh1/SPOP axis, it was found that cyclin D1/CDK4 directly phosphorylates SPOP at Ser6, but not Ser222, the only two conserved SP sites in SPOP (
Interestingly, it was noticed that one canonical 14-3-3 protein binding motif (RxxpS/pTxP) was located at Ser6 in SPOP (
Although the phosphorylation-deficient S6A mutant of SPOP did not affect its ability to interact with Cullin 3 and self-dimerize (
Recent clinical studies revealed that resistance to CDK4/6 inhibitor treatment often develops during cancer therapy. However, the molecular mechanism(s) for CDK4/6 inhibitor resistance remains largely unknown. The results herein demonstrate that CDK4/6 inhibitor treatment could elevate PD-L1 protein levels, allowing tumor cells to evade cancer immune-surveillance. Given this scenario, it was hypothesized that the combination of CDK4/6 inhibitor and anti-PD-1/PD-L1 antibody treatment may overcome CDK4/6 inhibitor-mediated cancer immune evasion by eliciting immune-mediated anti-tumor immunity. To examine this hypothesis, the combination treatment of MC38 subcutaneous tumor mouse model with anti-PD-1 (1A12) antibody and the CDK4/6 inhibitor palbociclib was assessed (see treatment regimen in
Given that the PD-1/PD-L1 pathway plays a crucial role in tumor immune evasion, but only about 20-30% of tumors respond to PD-1/PD-L1 antibody treatment, it is promising to explore combinations of PD-1/PD-L1 immune checkpoint blockade with targeted and conventional cancer therapies to enhance response rates and benefit more cancer patients. The examples of this disclosure showed that PD-L1 protein abundance is negatively regulated during cell cycle progression by cyclin D/CDK4 and the Cullin 3SPOP E3 ligase in a proteasome-mediated degradation manner. Importantly, CDK4/6 inhibitors treatment could elevate PD-L1 protein levels largely through inhibiting cyclin D/CDK4-mediated phosphorylation of SPOP to promote its degradation by APC/CCdh1 (
PD-L1 stability during cell cycle progression and shows the potential for combination treatment with CDK4/6 inhibitors and PD-1/PD-L1 immune checkpoint blockade to enhance therapeutic efficacy for human cancers.
The results described herein above, were obtained using the following methods and materials.
HEK293T, HEK293, HeLa, MDA-MB-231, MCF7, Hs578T, WT MEFs, cyclin D1−/− MEFs, cyclin D2−/−MEFs, cyclin D3−/− MEFs, cyclin D1−/−D2−/−D3−/−MEFs, cyclin D1fl/flD2−/−D3fl/fl, Cdk4−/− and Cdk4−/− MEFs, cyclin A1+/+A2+/+ and cyclin A1−/−A2−/− MEFs, cyclin E1+/+E2+/+ and cyclin E1−/−E2−/−, Spop+/+ and Spop−/−MEFs (a kind gift of Dr. Nicholas Mitsiadesa, Baylor College of Medicine, Houston, Tex.) were cultured in DMEM medium supplemented with 10% FBS (Gibco), 100 units of penicillin and 100 μg/ml streptomycin (Gibco). HLF, HepG2, Huh1 and Huh7 were cultured in RPMI medium supplemented with 10% FBS. MDA-MB-231 PD-L1 WT and PD-L1 KO cells are kind gift from Dr. Mien-Chie Hung. BT549, T47D, ZR75-1, HCC1954, HCC1937, MDA-MB436, MDA-MB468 and SKBR3 cells were from Dr. Alex Toker laboratory at BIDMC, Harvard
Medical School, and cultured in RPMI medium or McCoy′s5A (Corning, N.Y.) medium supplemented with 10% FBS. PC3, DU145, 22RV1, LNCaP and C42 were kind gifts from Dr. Pier Paolo Pandolfi group at BIDMC, Harvard Medical School, and cultured in RPMI medium (Corning, N.Y.) with 10% FBS. Mouse tumor derived MC38 cell line was a kind gift from Dr. Arlene Sharpe at Harvard Medical School. Mouse tumor derived 4T1 and B16-F10 cell lines were routinely cultured in Gordon Freeman's laboratory in DMEM medium supplemented with 10% FBS (Gibco), 100 units of penicillin and 100 μg/ml streptomycin (Gibco). All cell lines were routinely tested to be negative for mycoplasma contamination.
Cells with 80% confluence were transfected using lipofectamine plus reagents in Opti-MEM medium (Invitrogen). 293FT cells were used for packaging of lentiviral and retroviral cDNA expressing viruses, as well as subsequent infection of various cell lines were performed according to the protocols described previously37. Briefly, medium with secreted viruses were collected twice at 48 hours and 72 hours after transfection. After filtering through 0.45 μM filters, viruses were used to infect cells in the presence of 4 μg/mL polybrene (Sigma-Aldrich). 48 hours post-infection, cells were split and selected using hygromycin B (200 μg/mL) or puromycin (1 μg/mL) for 3 days. Cells were harvested and lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP40) supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Calbiochem) for immunoblot analysis.
Nocodazole (M1404) and Taxol were purchased from Sigma. Thymidine (CAS: 50-89-5) and cycloheximide (66-81-9) were purchased from Acros organics. PD0332991 (S1116) was purchased from Selleckchem. MG132 (BML-PI102-0005) was purchased from Enzo life science. MLN4924 was a kind gift from Dr. William Kaelin (Dana-Farber cancer institute).
Myc-tagged Cullin 1, Cullin 2, Cullin 3, Cullin 4A, Cullin 4B, Cullin 5, Flag-tagged SPOP WT, Y87C, F102C, W131G, delta MATH, delta BTB, pLenti-HA-SPOP WT, Y87C,
F102C, W131G, pGEX-4T-1-SPOP, Flag-Keap1, Flag-Cop1, shScramble, shCullin 3, shSPOP, and His-ubiquitin constructs were described previously38. Myc-Cullin 7 construct was kindly offered by Dr. James A. DeCaprio (Dana-Farber Cancer Institute). KLHL2 and KLHL3 constructs were generous gifts from Dr. Shinichi Uchida (Tokyo Medical and Dental University). KLHL12 and KLHL37 constructs were purchased from Addgene. KLHL20 construct was offered by Dr. Ruey-Hwa Chen (Institute of Biological Chemistry, Academia Sinica, Taiwan). The construct of HA-PD-L1 (HA tag in the N-terminus of PD-L1) was kindly provided by Dr. Mien-Chie Hung (The University of Texas MD Anderson Cancer Center). HA-Cdh1, HA-Cdc20, shCdh1 and shCdc20 were described previously39. HA-14-3-3 isoform constructs were described previously (Gao et al. Nat Cell Biol 11, 397-408 (2009)). pcDNA3-PD-L1, pCMV-GST-PD-L1-tail (cytoplasmic amino acids), Flag-SPOP with delta D-Box (RxxL), Flag-SPOP S6A, HA-tagged CDK2, CDK4 and CDK6 were generated in this study.
PD-L1 (E1L3N) rabbit mAb (13684), anti-pS10-H3 (3377), anti-pS780-Rb (8180), anti-pS807/811-Rb (8516), anti-Rb (9309), anti-cyclin D1 (2978), anti-cyclin D2 (3741), anti-CDK4, anti-CDK6 (3136), anti-cullin 3 (2759), anti-GST (2625), rabbit polyclonal anti-Myc-Tag antibody (2278) and mouse monoclonal anti-Myc-Tag (2276) antibodies were purchased from Cell Signaling Technology. Mouse PD-L1 antibody (MAB90781-100) was purchased from R&D systems. Anti-mPD-L1 for immunoblotting (clone 298B.8E2), anti-mPD-L1 (clone 298B.3G6) for immunohistochemistry, and anti-human PD-L1 for immunoprecipitation (clone 29E.12B1) were generated in the laboratory of Dr. Gordon J. Freeman. Anti-SPOP antibody (16750-1-AP) was purchased from Proteintech. Anti-cyclin A antibody (sc-751), anti-cyclin B antibody (sc-245), anti-cyclin E (SC-247), anti-cyclin D3 (sc-182), anti-Cdh1 antibody (sc56312), anti-Cdc20 antibody (sc-8358), anti-Cdc20 antibody (sc-13162), anti-Plk1 antibody (sc-17783), anti-TRIM24 (TIF1α, SC-271266), and anti-HA antibody (sc-805, Y-11) and anti-GST (sc-459) were obtained from Santa Cruz. Anti-GFP (8371-2) antibody was purchased from Clontech. Anti-Flag (F-2425), anti-Flag (F-3165, clone M2), anti-Vinculin (V9131), anti-Flag agarose beads (A-2220), anti-HA agarose beads (A-2095), peroxidase-conjugated anti-mouse secondary antibody (A-4416) and peroxidase-conjugated anti-rabbit secondary antibody (A-4914) were purchased from Sigma. Anti-HA (MMS-101P) was obtained from BioLegend.
Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). Protein concentrations were measured by the Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent as described previously (Gao et al. Nat Cell Biol 11, 397-408 (2009)). Equal amounts of protein were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitations analysis, 1000 pg total cell lysates were incubated with the primary antibody-conjugated beads for 4 hours at 4° C. The recovered immunocomplexes were washed four times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies.
The prostate tumor specimens were obtained from Shanghai Changhai Hospital in China. Usage of these specimens was approved by the Institute Review Board of Shanghai Changhai Hospital. For IHC, the paraformaldehyde fixed paraffin embedded prostate tumor samples were deparaffinized in xylene (3×10 min), rehydrated through a series of graded alcohols (100%, 95%, 85%, and 75%) to water. Samples were then subjected to heat-mediated antigen retrieval at 95° C. for 20 min. For IHC analysis, we used UltraSensitive™ SP (Mouse) IHC Kit (KIT-9701, Fuzhou Maixin Biotech) following the manufacturer's instructions with minor modification. The sections were incubated with 3% H2O2 for 15 min at room temperature to block endogenous peroxidase activity. After incubating in normal goat serum for 1 hour to block non-specific binding of IgG, sections were treated with primary antibody (PD-L1, 298B.3G6, 18 μg/ml; CD8a, sc-53212, clone C8/144B, dilution 1:40) at 4° C. overnight. Sections were then incubated for 30 min with biotinylated goat-anti-mouse IgG secondary antibodies (Fuzhou Maixin Biotech), followed by incubation with streptavidin-conjugated HRP (Fuzhou Maixin Biotech). Specific samples were developed with 3′3-diaminobenzidine (DAB-2031,Fuzhou Maixin Biotech). Images were taken using an Olympus microscopic camera and matched software.
The expression level of PD-L1 in prostate cancer tumor samples was determined according to the intensity of the staining as 0, negative; 1, weak expression; 2, moderate expression and 3, strong expression. The numbers of intraepithelial CD8+ tumor-infiltrating T lymphocytes (TILs) was counted. Briefly, three independent areas with the most abundant infiltration were selected under a microscopic field at 200× magnification (0.0625 mm2). The number of intraepithelial CD8+ TILs was counted manually and calculated as cells per mm2. The Mann-Whitney test was used to compare the difference in PD-L1 expression between SPOP mutated and wide type cases. The Student's t test was used to determine p values of the difference in CD8+ TILs between SPOP mutated and wide type cases. p<0.05 was considered as significant.
Cyclin D1/CDK4 in vitro kinase assays were performed as described (Phelps et al. Methods In Enzymology 283, 194-205 (1997)). Briefly, bacterially purified His-SPOP WT,
S6A, S222A, and S6A/S222A were incubated with recombinant human Cdk4/Cyclin D1 protein (ab55695) in kinase buffer (50 mM HEPES, pH 7.0, 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT). ATP mix with γ-32P-ATP was added at a 100 μM final concentration. The reaction was initiated by the addition of Cdk4/Cyclin D1 in a volume of 30 μl for 30 min at 30° C. followed by adding 3×SDS-PAGE sample buffer to stop the reaction before resolution by SDS-PAGE and subsequent autoradiography.
PC3 or HeLa cells with 80% confluence were transfected with His-ubiquitin and the indicated constructs. 36 hours post-transfection, cells were treated with 30 mM MG132 for 6 hours and lysed in buffer A (6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4, and 10 mM imidazole [pH 8.0]). After sonication, the lysates were incubated with nickel-nitrilotriacetic acid (Ni-NTA) beads (QIAGEN) for 3 hours at room temperature. Subsequently, the His pull-down products were washed twice with buffer A, twice with buffer ANTI (1 volume buffer A and 3 volumes buffer TI), and one time with buffer TI (25 mM Tris-HCl and 20 mM imidazole [pH 6.8]). The pull-down proteins were resolved by 2×SDS-PAGE for immunoblotting.
Cells were transfected or treated under indicated conditions. For half-life studies, cycloheximide (20 μg/ml, Sigma) was added to the medium. At indicated time points thereafter, cells were harvested and protein abundances were measured by immunoblot analysis.
Cells were synchronized with nocodazole arrest and double thymidine treatment as described previously (Wan et al. Developmental cell 29, 377-391 (2014)). Cells synchronized with nocodazole or double thymidine-arrest and release were collected at the indicated time points and stained with propidium iodide (Roche) according to the manufacturer's instructions. Cells were fixed by 70% ethanol at −20° C. overnight and washed 3 times using cold PBS. The samples were digested with RNase for 30 minutes at 37° C. and stained with propidium iodide (Roche) according to the manufacturer's instructions. Stained cells were sorted with BD FACSCanto™ II Flow Cytometer. The results were analyzed by ModFit LT 4.1 and FSC express 5 softwares.
Total RNAs were extracted using the QIAGEN RNeasy mini kit, and reverse transcription reactions were performed using the ABI Taqman Reverse Transcription Reagents (N808-0234). After mixing the generated cDNA templates with primers/probes and ABI Taqman Fast Universal PCR Master Mix (4352042), reactions were performed with the ABI-7500 Fast Real-time PCR system and SYBR green qPCR Mastermix (600828) from Agilent Technologies Stratagene.
Cyclin D1−/−, D2−/−, D3−/− and D1F/FD2−/−D3F/FMEFs were derived from E13.5 mouse embryos as described previously.
Cyclin D1−/− mice were mated with MMTV-c-Myc or MMTV-Wnt1 mice (from the Jackson Laboratory) yielding cyclin D1−/−/MMTV-c-Myc or cyclin D1−/−/MMTV-Wnt1, as well as control cyclin D1+/+/MMTV-c-Myc or D1+/+/MMTV-Wnt1 mice. Mammary tumors were dissected from multiparous females and snap-frozen.
MMTV-ErbB2 female mice (from the Jackson Laboratory), bred into a mixed C57BL/6 and 129Sv background, were treated with palbociclib or vehicle only for 6 weeks after detection of palpable tumors. Palbociclib was administered daily by gastric gavage (150 mg/kg of body weight); every two weeks the daily dose was lowered to 100 mg/kg for 2-3 days. Control mice were treated with vehicle (10% 0.1N HCl, 10% Cremaphor EL, 20% PEG300, 60% 50 mM citrate buffer pH 4.5) 10 ml/kg by gastric gavage. After 6 weeks, tumors were collected and snap-frozen in OCT.
Treatment of Wild-Type Mice with Palbociclib
6-weeks old C57BL/6 female mice (from the Jackson Laboratory) were treated with palbociclib (150 mg/kg body weight, by gastric gavage) or vehicle only for 7 days. Subsequently, organs were collected and analyzed by immunoblotting.
1×105 B16-F10 or 2×105 MC38 cells were injected subcutaneously into 6-weeks old C57BL/6 female mice (from the Jackson Laboratory). Starting one week later, mice were treated daily with palbociclib (150 mg/kg body weight, by gastric gavage) or vehicle only, for 7 days. Subsequently, tumors were collected and analyzed by FACS or immunoblotting. 1×105 B16-F10 cells stably expressing SPOP WT or F102C mutant were injected subcutaneously into 6-weeks old C57BL/6 female mice (from the Jackson Laboratory). On day 3 after tumor cells were injected, control and PD-L1 mAb treatments were conducted by intra-peritoneal injection (200 μg/mouse in 200 μl HBSS saline buffer) every three days for a total of 3 injections. Subsequently, tumors were collected and analyzed by FACS.
TFM-embedded 10 μM-thick tumor tissue sections were fixed with 2% paraformaldehyde/PBS for 30 min, and permeabilized in 0.1% Triton X-100/PBS for 10 min. Tumor tissue sections were pre-blocked with 2% BSA/PBS for 45 min, then incubated with primary antibodies against PD-L1 (1:200), CD3 (Abcam, 1:250) for 2.5 hours at room temperature and followed with secondary anti-mouse antibodies conjugated with Alexa-fluor-568 (Invitrogen, 1:250) and anti-rabbit antibodies conjugated with Alexa-fluor-488 (Invitrogen, 1:250). Hoechst (life technology, 1:10,000) was used to stain nucleus. Tumor tissues were mounted with fluoromount-G® (SouthernBiotech) at 4° C. overnight. Tissue sections were examined with fluorescent microscope under a 20 x objective lens. CD3+ cell numbers were counted in an area of 5.95×105 μm2.
Single Cell Generation from Tumor Tissue and Flow Cytometry Analysis
Tumor tissues were minced and digested with 5 ml of 2 mg/ml collagenase (Sigma) in DMEM for 1 hour at 37° C. Cells were then collected by centrifuge and filtered through a 70 μm strainer in DMEM. Cell pellets were suspended and lysed in red blood cell lysis buffer for 5 min. The cells were then filtered through a 40 μm strainer in 1 x PBS with 2% BSA. 1 million cells were incubated with antibodies against PD-L1 (BD Biosciences, 1:100) conjugated with APC or antibodies against CD3 (Biolegend, 1:100) conjugated with APC or corresponding isotype IgG1 control at room temperature for 30 min. Cells were washed by 1×PBS with 2% BSA and analyzed by flow cytometry.
Animal studies were approved by Dana-Farber Cancer Institute Institutional Animal Care and Use Committee (IACUC; protocol number 04-047), and performed in accordance with guidelines established by NIH Guide for the care and use of laboratory animals. MC38 or CT26 tumors were established by subcutaneously injecting 1×105 MC38 or CT26 tumor cells in 100 μl HBSS into the right flank of 6-week old C57BL/6 or BALB/c female mice (Jackson Lab, ME). Tumor sizes were measured every three days by caliper after implantation and tumor volume was calculated by length×width2×0.5. On day 7 after tumor cells were injected, animals were pooled and randomly divided into four groups with comparable average tumor size. Moreover, the lab members who measured the mice were blinded to the treatment groups. Mice were grouped into control antibody treatment, PD-1 mAb treatment, CDK4/6 inhibitor treatment, and PD-1 mAb plus CDK4/6 inhibitor treatment. As illustrated in
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
This application claims the benefit of the following U.S. Provisional Application No. 62/592,655, filed Nov. 30, 2017, the entire contents of which are incorporated herein by reference.
This invention was made with government support under Grant No. GM094777, CA177910, and P50CA101942 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/062746 | 11/28/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62592655 | Nov 2017 | US |
