Patent Application
20210401931
This document relates to materials and methods for treating cancer (e.g., PD-L1+ cancers). For example, this document provides methods and materials for using compositions (e.g., compositions containing a small bioactive S249/T252 phospho-mimicking polypeptide of an RB polypeptide) to reduce PD-L1 expression within cancer cells. This document also provides methods and materials for using compositions (e.g., compositions containing a small bioactive S249/T252 phospho-mimicking polypeptide of an RB polypeptide) in combination with other cancer treatment methods or agents to increase the effectiveness exhibited against the cancer within a mammal (e.g., a human).
Evasion of immune surveillance is a hallmark of human cancers (Hanahan and Weinberg, Cell, 144:646-674 (2011)). Neoantigens generated by cancerous cells are potentially recognizable by the immune system. However, tumors can often escape from immune attack via various distinct mechanisms, including the aberrant activation of immune checkpoints that terminates immune responses (Sharma and Allison, Cell, 161:205-214 (2015)).
Programmed death 1 (PD-1) is a major immune checkpoint inhibitory molecule expressed in activated T cells (Ishida et al., EMBO J 11:3887-3895 (1992)). Expression of its ligand PD-L1 (also known as B7-H1) in cancer cells triggers the engagement of PD-L1 with the PD-1 receptor on T cells, causing T cell apoptosis and decreased cytotoxic T cell function (Dong et al., Nat. Med., 8:793-800 (2002); and Dong et al., Nat. Med., 5:1365-1369 (1999)). Given that blockade of the PD-1/PD-L1 interaction can reactivate T-cell responses (Topalian et al., Curr. Opin. Immunol., 24:207-212 (2012)), several antibodies against PD-1 and PD-L1 molecules have been approved for treatment of human cancers in clinic. Although the PD-1/PD-L1 blockade can improve patient progress-free survival, the response rate among all patients is relatively low. Increasing evidence indicates that response to immune checkpoint therapies appears to highly correlate with PD-L1 expression in tumor cells (Topalian et al., N. Engl. J. Med., 366:2443-2454 (2012)).
The retinoblastoma protein RB is a well-studied tumor suppressor. It is a multi-functional protein that regulates a number of critical cellular activities, which include late G1 restriction point control and cell cycle progression, DNA damage response checkpoint activation, cell cycle exit and senescence, and differentiation (Manning and Dyson, Nat. Rev. Cancer, 12:220-226 (2012)). RB, along with its homolog proteins p107 and p130, belongs to the “pocket” protein family, which plays important roles in regulation of cell proliferation. It is generally accepted that RB protein exists in two function-related statuses, one is un- or hypo-phosphorylated state and the other is hyper-phosphorylated state (Narasimha et al., Elife 3 (2014)). In the underphosphorylated form, RB interacts through the pocket domain with E2F transcription factors and represses E2F transcription factors and thereby blocks G1/S transition. During the late G1 or upon mitogen stimulation, RB becomes phosphorylated at multiple sites mediated by CYCLIN D/CDK4/6 and CYCLIN E/CDK2 complexes, which causes disassociation of RB from E2F factors and allows cell-cycle progression (Sherr and Roberts, Genes Dev., 18:2699-2711 (2004)). Thus, it is well established that RB functions as a tumor suppressor by repressing the activities of E2F transcription factors and this function is abolished due to CDK-mediated phosphorylation. However, increasing evidence suggests that there exists E2F-independent tumor suppressor function of RB that can promote cancer progression, although the signaling pathways responsible for such function remains to be identified (Sun et al., Proc. Natl. Acad. Sci. USA, 108:704-709 (2011)).
This document provides materials and methods for treating cancer (e.g., PD-L1+ cancers). For example, this document provides methods and materials for using compositions (e.g., compositions containing a small bioactive S249/T252 phospho-mimicking polypeptide of an RB polypeptide) to reduce PD-L1 expression within cancer cells. This document also provides methods and materials for using compositions (e.g., compositions containing a small bioactive S249/T252 phospho-mimicking polypeptide of an RB polypeptide) in combination with other cancer treatment methods or agents to increase the effectiveness exhibited against the cancer within a mammal (e.g., a human).
As demonstrated herein, RB protein directly binds to an FxxxV motif in the DNA binding domain of p65 NFκB protein and inhibits expression of a subset of NFκB target genes including PD-L1 polypeptides. This effect is mediated by the arginine-rich linker (R-linker) region in the N-terminal segment of RB polypeptides, but not the pocket domain, and CDK4/6 phosphorylation of the S249 and T252 residues in the R-linker largely enhances RB interaction with and inhibition of p65. As also described herein, a small bioactive S249/T252 phospho-mimicking polypeptide of an RB polypeptide was found to not only block irradiation-induced PD-L1 expression, but also to increase the anti-cancer efficacy of irradiation in vivo. These results demonstrate that the compositions provided herein can be used to reduce PD-L1 polypeptide expression in cancer cells within a mammal (e.g., a human) having cancer. These results also demonstrate that the compositions provided herein can be used in combination with other cancer treatment methods (e.g., radiation) and/or cancer treatment agents (e.g., chemotherapeutic agents) to increase cancer treatment effectiveness as compared to treatments not involving such a combination. Having the ability to reduce PD-L1 polypeptide expression in cancer cells as described herein and/or to improve cancer treatment effectiveness as described herein can allow patients and clinicians to improve patient treatment and improve patient outcomes.
In general, one aspect of this document features a method for treating a mammal having cancer. The method comprises, or consists essentially of, administering a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:1 to the mammal, wherein the level of PD-L1 expression of the cancer is reduced. The mammal can be a human. The cancer can be prostate cancer. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:2. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:3. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:4. The can comprise polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:5. The polypeptide can be less than 75 amino acid residues in length. The polypeptide can be less than 50 amino acid residues in length. The polypeptide can be less than 25 amino acid residues in length. The polypeptide can comprise an amino acid sequence as set forth in any one of SEQ ID NOs:6-179. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:6. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:7. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:8. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:9. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:10. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:11. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:12. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:13. The polypeptide can be administered to the mammal as the sole active ingredient. The level of PD-L1 expression of the cancer can be reduced by at least about 5 percent. The level of PD-L1 expression of the cancer can be reduced by at least about 10 percent. The level of PD-L1 expression of the cancer can be reduced by at least about 25 percent. The level of PD-L1 expression of the cancer can be reduced by at least about 50 percent. The level of PD-L1 expression of the cancer can be reduced to a level not detectable on cancer cells present within the mammal. The number of cancer cells present within the mammal can be reduced. The number of cancer cells present within the mammal can be reduced by at least about 10 percent. The number of cancer cells present within the mammal can be reduced by at least about 25 percent. The number of cancer cells present within the mammal can be reduced by at least about 50 percent. The method can further comprise administering radiation to the mammal. The number of cancer cells within the mammal can be reduced as compared to the number of cancer cells present in a comparable mammal having cancer administered the radiation and not administered the polypeptide. The cancer-free survival of the mammal can be increased as compared to the cancer-free survival of a comparable mammal having cancer administered the radiation and not administered the polypeptide. The method can further comprise administering a chemotherapeutic agent to the mammal. The chemotherapeutic agent can be camptothecin, taxane, a kinase inhibitor, gemcitabine, or a combination thereof. The number of cancer cells within the mammal can be reduced as compared to the number of cancer cells present in a comparable mammal having cancer administered the chemotherapeutic agent and not administered the polypeptide. The cancer-free survival of the mammal can be increased as compared to the cancer-free survival of a comparable mammal having cancer administered the chemotherapeutic agent and not administered the polypeptide.
In another aspect, this document features a polypeptide comprising, or consisting essentially of, an amino acid sequence as set forth in SEQ ID NO:1. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:2. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:3. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:4. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:5. The polypeptide can be less than 75 amino acid residues in length. The polypeptide can be less than 50 amino acid residues in length. The polypeptide can be less than 25 amino acid residues in length. The polypeptide can comprise an amino acid sequence as set forth in any one of SEQ ID NOs:6-179. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:6. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:7. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:8. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:9. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:10. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:11. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:12. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:13.
In another aspect, this document features a composition comprising a polypeptide, wherein the polypeptide comprises, or consists essentially of, an amino acid sequence as set forth in SEQ ID NO:1. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:2. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:3. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:4. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:5. The polypeptide can be less than 75 amino acid residues in length. The polypeptide can be less than 50 amino acid residues in length. The polypeptide can be less than 25 amino acid residues in length. The polypeptide can comprise an amino acid sequence as set forth in any one of SEQ ID NOs:6-179. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:6. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:7. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:8. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:9. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:10. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:11. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:12. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:13. The polypeptide can be the sole active ingredient of the composition.
In another aspect, this document features a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide, wherein the polypeptide comprises, or consists essentially of, an amino acid sequence as set forth in SEQ ID NO:1. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:2. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:3. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:4. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:5. The polypeptide can be less than 75 amino acid residues in length. The polypeptide can be less than 50 amino acid residues in length. The polypeptide can be less than 25 amino acid residues in length. The polypeptide can comprise an amino acid sequence as set forth in any one of SEQ ID NOs:6-179. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:6. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:7. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:8. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:9. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:10. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:11. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:12. The polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:13. The molecule can be an expression vector. The expression vector can be a plasmid. The molecule can be a viral vector. The viral vector can be a pTsin lentiviral vector.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials for treating cancer. For example, this document provides polypeptides, isolated nucleic acids, vectors (e.g., viral vectors), host cells, compositions containing polypeptides, compositions containing vectors, methods for reducing PD-L1 expression within cancer cells, methods for treating cancer, and methods for increasing the effectiveness that a cancer treatment method and/or cancer agent exhibits against cancer within a mammal (e.g., a human).
In some cases, a polypeptide provided herein for reducing PD-L1 expression within cancer cells, for treating cancer, and/or for increasing the effectiveness of another cancer treatment method and/or cancer agent against cancer within a mammal as described herein can include the following amino acid sequence: NGX1PRX2PR, where X1 is D or E, and X2 is D or E (SEQ ID NO:1). For example, a composition provided herein that can be administered to a mammal to reduce PD-L1 expression within cancer cells and/or to treat cancer as described herein can include a polypeptide having NGDPRDPR (SEQ ID NO:2), NGDPREPR (SEQ ID NO:3), NGEPRDPR (SEQ ID NO:4), or NGEPREPR (SEQ ID NO:5). In some cases, a composition provided herein can include a polypeptide that is from about 8 to about 200 (e.g., from 8 to 180, from 8 to 170, from 8 to 160, from 8 to 150, from 8 to 140, from 8 to 130, from 8 to 120, from 8 to 110, from 8 to 100, from 8 to 90, from 8 to 80, from 8 to 70, from 8 to 60, from 8 to 50, from 8 to 40, from 8 to 30, from 8 to 20, or from 8 to 10) amino acid residues in length. For example, a composition provided herein can include a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NOs:2-179 with the total amino acid length of the polypeptide being from about 8 to about 50 (e.g., from 8 to 45, from 8 to 40, from 8 to 35, from 8 to 30, from 8 to 25, from 8 to 24, from 8 to 23, from 8 to 22, from 8 to 21, from 8 to 20, from 8 to 19, from 8 to 18, from 8 to 17, from 8 to 16, from 8 to 15, from 8 to 14, from 8 to 13, from 8 to 12, from 8 to 11, from 8 to 10, from 8 to 9). Examples of polypeptides that can be used to treat cancer as described herein include, without limitation, polypeptides that include an amino acid sequence set forth in Table 1.
In some cases, a polypeptide provided herein for reducing PD-L1 expression within cancer cells, for treating cancer, and/or for increasing the effectiveness of another cancer treatment method and/or cancer agent against cancer within a mammal as described herein can include the following amino acid sequence: NGX1PRX2PR, where X1 is D or E, and X2 is D or E (SEQ ID NO:1) with an N-terminal and/or C-terminal cell targeting sequence. An N-terminal and/or C-terminal cell targeting sequence such as eight D-arginine residues (SEQ ID NO: 91) can act as a signal peptide as described elsewhere (see, e.g., Jameson, Nature Medicine, 19:626-630 (2013)) to facilitate the entry of a polypeptide (e.g., an RB-phospho-mimicking polypeptide) provided herein into cells (e.g., cancer cells). Examples of cell targeting sequences that can be located at the N-terminus and/or C-terminus of a polypeptide provided herein (e.g., a polypeptide having an amino acid sequence set forth in Table 1) can be six, seven, eight, nine, ten, or more consecutive D-arginine residues (e.g., RRRRRRRR (SEQ ID NO:91)). Examples of such polypeptides are set forth in Table 2.
In some cases, a polypeptide provided herein that includes SEQ ID NO:1 (NGX1PRX2PR, where X1 is D or E, and X2 is D or E) with an N-terminal and/or C-terminal cell targeting sequence (e.g., SEQ ID NO:91) can include one or more peptidase cleavage sites located between the cell targeting sequence(s) and SEQ ID NO:1. For example, using SEQ ID NO:92 as an example, a polypeptide can be designed to include one or more peptidase cleavage sites at the position marked with an “X”: RRRRRRRRXNGDPRDPR (SEQ ID NO:180). Any appropriate peptidase cleavage site can be used. An example of a peptidase cleavage site that can be used as described herein include, without limitation, an AFK sequence of D amino acids, which is a tripeptide recognized by plasmin. For example, a polypeptide having SEQ ID NO:180 where the X represents AFK can have the following amino acid sequence: RRRRRRRRAFKNGDPRDPR (SEQ ID NO:181). In these cases, cleavage at the cleavage site via a protease within a cell can liberate the peptide containing SEQ ID NO:1 from the amino acid sequence of an N-terminal and/or C-terminal cell targeting sequence. As described herein, local tumor injection and/or use of nanoparticles that incorporate one or more tumor-specific antibodies to direct a polypeptide provided herein (e.g., a polypeptide having an amino acid sequence set forth in any of the sequences of Table 1). See, e.g., Cully, Nature Rev. Drug Discovery, 15:231 (2016); and Davis, Nature Rev. Drug Discovery, 7:771-82 (2008)). As also described herein, viral vectors such as adenoviral vectors can be used to deliver nucleic acid designed to express a polypeptide provided herein (e.g., a polypeptide having an amino acid sequence set forth in any of the sequences of Table 1) to cells (e.g., cancer cells).
A polypeptide provided herein (e.g., a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NOs:1-179) can be a substantially pure polypeptide. As used herein, the term “substantially pure” with reference to a polypeptide means that the polypeptide is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid. In some cases, a substantially pure polypeptide can be a polypeptide that is at least 60 percent pure or is any chemically synthesized polypeptide. A substantially pure polypeptide can be at least about 60, 65, 70, 75, 80, 85, 90, 95, or 99 percent pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.
A polypeptide provided herein (e.g., a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NOs:1-179) can be produced using any suitable method including, without limitation, solid phase synthesis, manual techniques, or automated techniques such as those involving the use of an Applied BioSystems (Foster City, Calif.) Peptide Synthesizer, a Biosearch Inc. (San Rafael, Calif.) automatic peptide synthesizer, a Biotage peptide synthesis instrument, or a CSBio Peptide Synthesizer. In some cases, a polypeptide provided herein can be produced recombinantly using nucleic acid as described herein.
In some cases, a polypeptide provided herein (e.g., a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NOs:1-179) can be prepared to include one or more salt, ester, amide, N-acyl, and/or O-acyl moieties. For example, salts of carboxyl groups of a polypeptide provided herein can be prepared by contacting the polypeptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base (e.g., sodium hydroxide), a metal carbonate or bicarbonate base (e.g., sodium carbonate or sodium bicarbonate), or an amine base (e.g., triethylamine or triethanolamine). Acid addition salts of a polypeptide provided herein can be prepared by contacting the polypeptide with one or more equivalents of an inorganic or organic acid (e.g., hydrochloric acid). Esters of carboxyl groups of a polypeptide provided herein can be prepared using any suitable means for converting a carboxylic acid or precursor to an ester. For example, one method for preparing esters of a polypeptide provided herein, when using the Merrifield synthesis technique, is to cleave the completed polypeptide from the resin in the presence of the desired alcohol under either basic or acidic conditions, depending upon the resin. The C-terminal end of the polypeptide then can be directly esterified when freed from the resin, without isolation of the free acid. Amides of a polypeptide provided herein can be prepared using techniques for converting a carboxylic acid group or precursor to an amide. One method for amide formation at the C-terminal carboxyl group includes cleaving the polypeptide from a solid support with an appropriate amine, or cleaving in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine. N-acyl derivatives of an amino group of a polypeptide provided herein can be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected polypeptide. O-acyl derivatives can be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation can be carried out using a standard acylating reagent such as an acyl halide, anhydride, and/or acyl imidazole. Both N- and O-acylation can be carried out together, if desired.
In some cases, a polypeptide provided herein (e.g., a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NOs:1-179) can be modified by linkage to a polymer such as polyethylene glycol (PEG), or by fusion to another polypeptide such as albumin. For example, one or more PEG moieties can be conjugated to a polypeptide provided herein via lysine residues or other linkages. Linkage to PEG or another suitable polymer, or fusion to albumin or another suitable polypeptide, can result in a modified polypeptide having an increased half-life as compared to an unmodified polypeptide. Without being bound by a particular mechanism, an increased serum half-life can result from reduced proteolytic degradation, immune recognition, or cell scavenging of the modified polypeptide. Any appropriate method can be used to modify a polypeptide provided herein by linkage to PEG (also referred to as “PEGylation”) or other polymers including, without limitation, those described elsewhere (U.S. Pat. No. 6,884,780; Cataliotti et al., Trends Cardiovasc. Med., 17:10-14 (2007); Veronese and Mero, BioDrugs, 22:315-329 (2008); Miller et al., Bioconjugate Chem., 17:267-274 (2006); and Veronese and Pasut, Drug Discov. Today, 10:1451-1458 (2005)). Examples of methods for modifying a polypeptide provided herein by fusion to albumin include, without limitation, those described elsewhere (U.S. Patent Publication No. 2004/0086976, and Wang et al., Pharm. Res., 21:2105-2111 (2004)).
This document also provides isolated nucleic acid molecules encoding a polypeptide provided herein as well as expression vectors containing such nucleic acids and host cells containing such nucleic acids and/or expression vectors. As used herein, the term “nucleic acid” refers to both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. A nucleic acid molecule can be double-stranded or single-stranded (i.e., a sense or an antisense single strand). Nucleic acids include, for example, cDNAs encoding a polypeptides provided herein.
An “isolated nucleic acid” as used herein refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a vertebrate genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a vertebrate genome. The term “isolated” as used herein with respect to nucleic acids also includes any non-naturally-occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.
An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not considered an isolated nucleic acid.
Isolated nucleic acid molecules can be produced using standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence that encodes a polypeptide provided herein (e.g., a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NOs:1-179). PCR refers to a procedure or technique in which target nucleic acids are enzymatically amplified. Sequence information from the ends of the region of interest or beyond typically is employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase can be used to synthesize complementary DNA (cDNA) strands. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis, Genetic Engineering News, 12:1 (1992); Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874-1878 (1990); and Weiss, Science, 254:1292 (1991)).
Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to 5′ direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
Isolated nucleic acids (e.g., nucleic acids encoding a polypeptide provided herein) also can be obtained by mutagenesis. For example, a reference sequence can be mutated using standard techniques including oligonucleotide-directed mutagenesis and site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology, Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al., 1992.
Sources of nucleotide sequences from which nucleic acid molecules encoding a polypeptide provided herein, or the nucleic acid complement thereof, can be obtained include total or polyA+ RNA from any eukaryotic source, including mammalian (e.g., human, rat, mouse, canine, bovine, equine, ovine, caprine, or feline) cellular source from which cDNAs can be derived. Other sources of the nucleic acid molecules include genomic libraries derived from any eukaryotic cellular source, including mammalian sources.
Nucleic acid molecules encoding a polypeptide provided herein can be identified and isolated using molecule cloning techniques, e.g., as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989). For example, reverse-transcriptase PCR (RT-PCR) can be used to isolate and clone cDNAs from isolated RNA that contains RNA sequences of interest (e.g., total RNA isolated from human tissue). Other approaches to identify, isolate, and clone cDNAs encoding a polypeptide provided herein include, for example, screening cDNA libraries.
Vectors containing nucleic acids such as those described herein also are provided. A “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
In expression vectors, a nucleic acid (e.g., a nucleic acid encoding a polypeptide provided herein) can be operably linked to one or more expression control sequences. As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 to 500 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.
Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are available commercially. In some cases, a viral vector such as a pTsin lentiviral vector can be designed to encode and express a polypeptide provided herein (e.g., a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NOs:1-179).
An expression vector including a nucleic acid sequence that encodes a polypeptide provided herein (e.g., a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NOs:1-179) can include a tag sequence designed to facilitate subsequent manipulation or localization of the expressed nucleic acid sequence (e.g., for purification). In some cases, tag sequences such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequences can be expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.
This document also provides host cells containing a nucleic acid or vector provided herein. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant nucleic acid or vector (e.g., an expression vector) can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. In some cases, host cells can be transformed or transfected use methods described elsewhere (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, New York (1989)). For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer can be used introduce nucleic acid encoding a polypeptide described herein into cells. In addition, naked DNA can be delivered directly to cells in vivo as described elsewhere (U.S. Pat. Nos. 5,580,859 and 5,589,466).
As described herein, the document also provides methods for reducing PD-L1 expression within cancer cells, methods for treating cancer, and/or methods for increasing the effectiveness that another cancer treatment method and/or cancer agent exhibits against cancer within a mammal (e.g., a human). In some cases, this document provides methods and materials for using compositions (e.g., compositions containing polypeptide provided herein) to reduce PD-L1 expression within cancer cells. This document also provides methods and materials for using compositions (e.g., compositions containing polypeptide provided herein) in combination with other cancer treatment methods or agents to increase the effectiveness exhibited against the cancer within a mammal (e.g., a human).
Any appropriate mammal (e.g., a human) having cancer can be treated as described herein. For example, a human having cancer can be treated using a composition containing a polypeptide provided herein (e.g., a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NOs:1-179) and/or a nucleic acid encoding a polypeptide provided herein. Other examples of mammals that can be treated as described herein include, without limitation, non-human primates, monkeys, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats.
Any type of cancer can be treated as described herein. For example, prostate cancer, pancreatic cancer, lung cancer, liver cancer, or breast cancer can be treated as described herein. In some cases, a mammal (e.g., a human) having a PD-L1+ cancer can be treated with a composition provided herein (e.g., a composition containing a polypeptide provided herein and/or a nucleic acid encoding a polypeptide provided herein) to reduce the level of PD-L1 expression in cancer cells within the mammal and/or to reduce the number of cancer cells within the mammal. In some cases, a mammal (e.g., a human) suspected to develop cancer can be treated with a composition provided herein (e.g., a composition containing a polypeptide provided herein and/or a nucleic acid encoding a polypeptide provided herein) to slow or reduce the likelihood of the progression of and/or development of cancer.
Any appropriate method can be used to identify a mammal as having cancer or as being at risk for developing cancer. For example, tissue biopsy and/or imaging techniques (e.g., CT or MM) can be used to identify a human or other mammal as having cancer. In some cases, a human's family health history or genetic markers (e.g., BRCA1 and BRCA2) can be evaluated to determine if the human is at risk of developing cancer.
Once identified as having cancer or as being at risk for developing cancer, the mammal can be administered or instructed to self-administer a composition provided herein such as a composition formulated to include a polypeptide provided herein and/or formulated to include a nucleic acid provided herein (e.g., a viral vector containing a nucleic acid sequence encoding a polypeptide provided herein). In some cases, a composition containing a polypeptide provided herein administered to a mammal as described herein can include that polypeptide as the sole active ingredient. For example, a mammal having cancer or at risk for developing cancer can be administered a composition containing a polypeptide provided herein as the sole active ingredient. In some cases, a composition containing a nucleic acid provided herein administered to a mammal as described herein can include that nucleic acid as the sole active ingredient. For example, a mammal having cancer or at risk for developing cancer can be administered a composition containing a nucleic acid provided herein as the sole active ingredient.
A polypeptide provided herein or a nucleic acid encoding such a polypeptide can be used to reduce the level of PD-L1 expression by cancer cells within a mammal, to reduce the number of cancer cells within a mammal, and/or to increase the effectiveness of other cancer treatment methods and/or agents. In some cases, a composition containing a polypeptide provided herein (and/or a nucleic acid provided herein) can be administered to a mammal having cancer or at risk of developing cancer as a combination therapy with one or more additional cancer treatment methods or agents to treat cancer. In those cases where a polypeptide provided herein (and/or a nucleic acid provided herein) are used in combination with one or more additional cancer treatment methods and/or agents to treat cancer as described herein, the one or more additional cancer treatment methods and/or agents can be administered at the same time or independently. For example, a composition containing a polypeptide provided herein (and/or a nucleic acid provided herein) can be administered first, and the one or more additional cancer treatment methods and/or agents can be administered second, or vice versa. As described herein, a polypeptide provided herein (and/or a nucleic acid provided herein) such as a polypeptide having any one of the amino acid sequences set forth in SEQ ID NO:1-179 can be used to increase the effectiveness of another cancer treatment method or agent (when compared to the effectiveness observed with that other cancer treatment method or agent in the absence of the polypeptide (or nucleic acid)). Examples of other cancer treatment methods and/or agents that can be used as described herein include, without limitation, radiation treatments, chemotherapies such as camptothecin therapy, taxane therapy, kinase inhibitor therapy, and gemcitabine therapy, and surgery.
In some cases, a polypeptide provided herein (and/or a nucleic acid provided herein) can be formulated into a pharmaceutically acceptable composition for administration to a mammal having cancer or at risk of developing cancer. For example, a therapeutically effective amount of a polypeptide provided herein (and/or a nucleic acid provided herein) can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. A pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.
Pharmaceutically acceptable carriers, fillers, and vehicles that may be used in a pharmaceutical composition described herein include, without limitation, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
A pharmaceutical composition containing a polypeptide provided herein (and/or a nucleic acid provided herein) can be designed for oral, parenteral (e.g., subcutaneous, intramuscular, intravenous, or intradermal administration), or inhaled administration. When being administered orally, a pharmaceutical composition containing a polypeptide provided herein (and/or a nucleic acid provided herein) can be in the form of a pill, tablet, or capsule. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Compositions for inhalation can be delivered using, for example, an inhaler, a nebulizer, and/or a dry powder inhaler. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
In some cases, a pharmaceutically acceptable composition including a polypeptide provided herein (and/or a nucleic acid provided herein) can be administered locally or systemically. For example, a composition containing a polypeptide provided herein (and/or a nucleic acid provided herein) can be administered systemically by an oral administration to or inhalation by a mammal (e.g., a human).
Effective doses can vary depending on the severity of the cancer and/or risk of cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician.
An effective amount of a composition containing a polypeptide provided herein (and/or a nucleic acid provided herein) can be any amount that reduces the level of PD-L1 expression by cancer cells within a mammal and/or reduces the number of cancer cells within the mammal without producing significant toxicity to the mammal. For example, an effective amount of a polypeptide provided herein (and/or a nucleic acid provided herein) can be from about 0.5 mg/kg to about 50 mg/kg (e.g., from about 1 mg/kg to about 50 mg/kg, from about 2.5 mg/kg to about 50 mg/kg, from about 5 mg/kg to about 50 mg/kg, from about 0.5 mg/kg to about 25 mg/kg, from about 0.5 mg/kg to about 15 mg/kg, from about 0.5 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg, or from about 2.5 mg/kg to about 7.5 mg/kg). In one example, 800 mg of a polypeptide having any one of the amino acid sequences set forth in SEQ ID NOs:1-179 can be administered once a day to a 80 kg human. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., cancer) may require an increase or decrease in the actual effective amount administered.
The frequency of administration of a composition containing a polypeptide provided herein (and/or a nucleic acid provided herein) can be any frequency that reduces the level of PD-L1 expression by cancer cells within a mammal and/or reduces the number of cancer cells within the mammal without producing significant toxicity to the mammal. For example, the frequency of administration can be from about three times a day to about once a day, from about once a week to about three times a month, from about twice a month to about six times a month, or from about twice a week to about once a month. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing a polypeptide provided herein (and/or a nucleic acid provided herein) can include rest periods. For example, a composition containing a polypeptide provided herein (and/or a nucleic acid provided herein) can be administered daily over a two-week period followed by a two-week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., cancer) may require an increase or decrease in administration frequency.
An effective duration for administering a composition containing a polypeptide provided herein (and/or a nucleic acid provided herein) can be any duration that reduces the level of PD-L1 expression by cancer cells within a mammal and/or reduces the number of cancer cells within the mammal without producing significant toxicity to the mammal. For example, the effective duration can vary from several days to several weeks, months, or years. In some cases, the effective duration for the treatment of cancer can range in duration from about one week to about 10 years. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition being treated.
In some cases, a course of treatment, the severity of one or more symptoms related to the condition being treated (e.g., cancer), and/or the number of cancer cells within a mammal can be monitored. Any appropriate method can be used to determine whether or not the severity of a symptom is reduced. For example, the severity of a symptom of cancer can be assessed using imaging techniques at different time points. In some cases, a scoring system can be used to assess the severity of cancer.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
LNCaP, PC-3, 22Rv1, DU145, TRAMP-C2, and human embryonic kidney cell line 293T cell lines were purchased from American Type Culture Collection (Manassas). C4-2 CRPC cell line was purchased from Uro Corporation. PTEN-CaP8 murine PTEN-deficient CRPC cell line was obtained from Dr. Hong Wu at UCLA. The androgen-refractory LNCaP subline, RF, was established as described elsewhere (Murillo et al., Endocrinology, 142: 4795-4805 (2001)). LNCaP, PC-3, 22Rv1, and DU145 were cultured in RPMI 1640 supplemented with 10% FBS. LNCaP-RF were cultured in RPMI 1640 supplemented with 10% charcoal-stripped FBS (CSS). PTEN-CaP8 and 293T cells were maintained in DMEM supplemented with 10% FBS. Cells were cultured at 37° C. supplied with 5% CO2. LAPC-4 cells were obtained from C. L. Sawyers and maintained in Iscove's Modified Dulbecco's Media with 10% FBS. RB-deficient mouse prostate epithelial cells (RB−/− PrE) were obtained from M. L. Day and S. W. Hayward and maintained in RPMI 1640 containing 200 μg/mL G418, 5% FBS, 100 m/mL streptomycin, 100 U/mL penicillin, and 0.25 μg/mL amphotericin B. SKO(Ptenf/fRb1+/+) and DKO cr (Ptenf/fRb1f/f) was obtained from David W. Goodrich and maintained in DMEM supplemented with 2.5% charcoal-stripped FBS, 5 μg/mL of insulin/transferring/selenium (Collaborative Research), 10 μg/mL of bovine pituitary extract (Sigma), 10 μg/mL of epidermal growth factor (Collaborative Research), and 1 μg/mL of cholera toxin (Sigma). All cell lines were kept in a 37° C. incubator at 5% CO2. Transfections were performed by using Lipofectamine 2000 (Thermo Fisher Scientific).
The advanced prostate cancer dataset was generated from patients undergoing standard of care clinical biopsies at Mayo Clinic. A tissue microarray was constructed from the formalin-fixed, paraffin-embedded (FFPE) samples of metastatic prostate cancer, identified after a search of pathologic and clinical databases of archival tissues. The human tissue microarray (TMA) contained 157 cores (16 0.6 mm and 141 1.0 mm cores) resulting from 53 samples (20 bone metastases and 33 non-bone metastases) from 51 patients. FFPE tissue was used for IHC analysis. 145 cores were used for IHC data analysis when cores with lost tissue greater than 50% were excluded.
Lentivirus-based control and gene-specific small hairpin RNAs (shRNAs) were purchased from Sigma-Aldrich. Viral packaging plasmids (pEXQV and pVSV-G) and shRNA plasmid were transfected to 293T cells by using Lipofectamine 2000. After 24 hours, virus culture medium was replaced with DMEM supplemented with 10% FBS and 1:100 of sodium Pyruvate. 48 hours post transfection, medium was collected and added to various cancer cells supplemented with 12 μg/mL of polybrene. Cancer cells were harvested 48 hours after puromycin selection. shRNA sequence information are provided in Table 3.
Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific). The NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) was used to assess RNA yield and quality. RNA was reversely transcribed using Superscript reverse transcriptase (Thermo Fisher Scientific) following manufacturer's instructions. Quantitative real-time PCR was performed by mixing cDNA, gene-specific primers, and IQ SYRB Green Supermix and detected by iCycler QTX detection system (Bio-Rad). The 2-ΔCt method was used to quantitate fold changes by normalizing to GAPDH. Primers for RT-qPCR are provided Table 4.
pCMV4 p65, pCMV HA hRB-wt, and pCMV HA hRb delta CDK were purchased from Addgene (Cambridge, Mass.). Expression vectors for GST-FBP1 and GST-TRIM28 recombinant proteins were constructed using the pGEX-4T-1 backbone vector. RL S249A/T252A polypeptide, RL S249D/T252D polypeptide, and HA-RB1 661W mutants were generated with the KOD Plus Mutagenesis Kit (Toyobo) following the manufacturer's instructions. The following antibodies were used: RB (BD Biosciences, 554136, working dilution 1:1000), RB (Cell Signaling, 9309, working dilution 1:1000), p65 (Cell Signaling, 8242S, working dilution 1:1000), p65 (Cell Signaling, 6956, working dilution 1:1000), PD-L1 (Cell Signaling, 13684S, working dilution 1:1000), PD-L1 (Proteintech, 17952-1-AP, working dilution 1:1000), p50 (Cell Signaling, 135865, working dilution 1:1000), Phospho-RB (Ser795) (Cell Signaling, 93015, working dilution 1:1000), Phospho-RB (Ser249, Thr252) (Thermo scientific, 701059, working dilution 1:1000), P107 (Santa Cruz Biotechnology, SC-318, working dilution 1:1000), P130 (Santa Cruz Biotechnology, SC-317, working dilution 1:1000), ERK1/2 (Santa Cruz Biotechnology, sc-135900, working dilution 1:5000), anti-Flag (Sigma-Aldrich, F-3165, working dilution 1:3000), and anti-HA (Covance, MMS-101R, working dilution 1:3000), anti-light chain specific rabbit IgG secondary antibody (211-032-171), and anti-light chain specific mouse IgG secondary antibody (115-035-174) (Jackson Immuno Research laboratories). Palbociclib (PD0332991) was purchased from Selleckchem. Helenalin was purchased from Abcam.
Cells were harvested and lysed by IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 1% protease inhibitor cocktails) on ice for more than 30 minutes. Cell lysate was centrifuged for 15 minutes at 13,000 rpm at 4° C., and the supernatant was incubated with primary antibodies and protein A/G agarose beads (Thermo Fisher Scientific) with rotating at 4° C. overnight. The next day, the beads were washed at least six times with IP buffer on ice, and then subjected to western blotting analysis.
Cells were harvested and lysed by IP buffer, and the supernatant was quantified by BCA protein quantification assay. Equal amounts of protein sample were added into 4× sample buffer and boiled for 5 minutes. The sample was subjected to SDS-PAGE analysis and transferred to nitrocellulose membrane. The membrane was blocked by 5% milk for 1 hour at room temperature and incubated with primary antibody at 4° C. overnight. The next day, the membrane was washed three times with 1×TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The protein bands were visualized by SuperSignal West Pico Stable Peroxide Solution (Thermo Fisher Scientific).
Immunofluorescent cytochemistry was performed as described elsewhere (Pan et al., EMBO J., 36:995-1010 (2017)). Briefly, cells were fixed in 4% paraformaldehyde for 15 minutes. After washed in PBS three times, fixed cells were permeabilized with 0.2% Triton X-100 for 20 minutes, washed in PBS, and then blocked in PBS supplemented with 10% goat serum. Cells were incubated with indicated primary antibody at 4° C. overnight. After washed three times with PBS, cells were incubated with secondary antibody that was conjugated with Alexa Fluor (Thermo Fisher Scientific) for 1 hour at room temperature. After washed three times with PBS, cells were counterstained with Vectashield (Vector Laboratories) containing DAPI (4′, 6-diamidino-2-phenylindole). Images were captured using Zeiss laser confocal microscope (LSM780).
Cells were lysed with IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 1% protease inhibitor cocktails) on ice for more than 30 minutes. GST fusion proteins were immobilized on glutathione-Sepharose beads (GE Healthcare Lifesciences). After washing with lysis buffer, the beads were incubated with cell lysates overnight at 4° C. overnight. The beads were then washed six times with binding buffer and re-suspended in sample buffer. The bound proteins were subjected to western blotting analysis.
Plasmid DNA (V5-CDK4 or pCMV4 p65) was add to the TNT® T7 Quick Master Mix and add 1 μL methionine (1 mM), by following the manufacturer's instruction of TNT® Quick Coupled Transcription/Translation Systems (Promega). GST or GST-RB-N recombinant proteins (GST-RB-N, GST-RB-N S249A/T252A, or GST-RB-N ΔS249/T252) were immobilized on glutathione-Sepharose beads. After washing with PBS, the beads were incubated with in vitro transcribed and translated CDK4, human recombinant CDK6/Cyclin D3 (Promega), and reaction buffer (40 mM Tris 7.5; 20 mM MgCl2; 0.1 mg/mL BSA; 50 μM DTT) at room temperature for 60 minutes. After washing with PBS, the beads were incubated with in vitro transcribed and translated p65 for 4 hours. The beads were then washed six times with PBS and re-suspended in sample buffer. The bound proteins were subjected to western blotting analysis.
PC-3 cells were treated with or without Palbociclib (5 μM) for 24 hours or PC-3 cells were infected with lentivirus expressing control shRNA or DUB3-specific shRNA following puromycin selection after 48 hours infection. Total RNA was isolated from cells using the RNeasy Plus Mini Kit (Qiagen). High-quality (Agilent Bioanalyzer RIN>7.0) total RNA was employed for the preparation of sequencing libraries using the Illumina TruSeq Stranded Total RNA/Ribo-Zero Sample Prep Kit. A total of 500-1,000 ng of riboRNA-depleted total RNA was fragmented by RNase III treatment at 37° C. for 10-18 minutes, and RNase III was inactivated at 65° C. for 10 minutes. Size selection (50- to 150-bp fragments) was performed using the FlashPAGE denaturing PAGE-fractionator (Thermo Fisher Scientific) before ethanol precipitation overnight. The resulting RNA was directionally ligated, reverse-transcribed, and treated with RNase H.
ChIP was performed as described elsewhere (Zhao et al., Cell Reports, 15:599-610 (2016)). For ChIP-reChIP assay DNA, cell lysates were sonicated and subjected to immunoprecipitation using p65 antibody. After being washed by RIPA buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, and 0.7% Na-Deoxycholate), the protein-DNA complexes were eluted by elution buffer (10 mM Tris, 1 mM EDTA, 2% SDS, and 20 mM DTT, PH 7.5) for 30 minutes at 37° C. Then, the supernatant was diluted 20 times and subjected to the second ChIP using IgG or RB antibodies (Li et al., Nature, 513:251-255 (2014)). DNA pulled down by antibodies or nonspecific IgG was amplified by real-time PCR. The ChIP primers are provided in Table 5.
Cells were collected and resuspend cell pellet in 1 mL of Buffer A (10 mM Hepes-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and 0.1% NP-40) to lyse the cells on ice for 10 minutes. Centrifuge sample at 6,500 rpm 4° C. for 3 minutes to pellet the nuclei. Wash nuclei pellet with Buffer A. Spin samples 3,500 rpm for 5 minutes at 4° C. Cell pellet was lysed by IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 1% protease inhibitor cocktails) on ice for more than 30 minutes. Protein concentration was determined by BCA protein quantification assay.
DNA oligonucleotide was labeled with biotin by following the manufacturer's instruction of Pierce™ Biotin 3′ End DNA Labeling Kit (Thermo Fisher Scientific). Complimentary 3′ end-labeled oligos were annealed prior to use. Five micrograms nuclear extracts were added to the binding reaction system (10× Binding Buffer, 1 μg/μL Poly (dI·dC), 1% NP-40, and 100 mM MgCl2) and incubated at room temperature for 5 minutes. Then, biotin-labeled doublestrand PD-L1-prom oligonucleotide DNA (5′-GGTCA-GGAAAGTCCAACGCC-3′; SEQ ID NO:255) was added to binding reactions and incubated at room temperature for 20 minutes. Competitive assays also were performed by addition of 200-fold excess of unlabeled probe (5′-GGTCAGGAAAGTCCAACGCC-3′; SEQ ID NO:256) or unlabeled mutant probe (5′-GGTCATTCCCTGAACACGCC-3′; SEQ ID NO:257) to nuclear extracts at room temperature for 5 minutes before the addition of the labeled probe. The shift was performed by following the manufacturer's instruction of LightShift™ Chemiluminescent EMSA Kit (Thermo Fisher Scientific). Briefly, bound complexes were separated on 6% nondenaturating polyacrylamide gels and transferred to nylon membrane. The transferred DNA was crosslinked to membrane using UV-light and detected by chemiluminescence.
6-week-old C57BL/6 mice (Jackson Lab) were used for animal experiments. All mice were housed in standard conditions with a 12-hour light/dark cycle and access to food and water ad libitum. PTEN-CaP8 cells (5×106) infected with lentivirus expression Tsin control or Tsin-RL S249D/T252D polypeptide (in 50 μL 1×PBS plus 50 μL Matrigel (BD Biosciences)) were injected s.c. into the right flank of mice. The volume of xenografts was measured every other day and calculated using the formula L×W2×0.5. After xenografts reached a size of approximately 40 mm3, mice were randomized into different groups and treated with IR (12 Gy initiated at day 1) and anti-PD-L1 (200 i.p., given at days 0, 3, 6, and 9) alone or combination of IR. Mice were euthanized, and tumors collected from all animals once tumors reached a volume of 200 mm3.
PC-3 cells were harvested and washed with PBS. Cells were fixed in 4% paraformaldehyde for 15 minutes. After washed with PBS, cells were incubated with ice-cold 100% methanol 30 minutes on ice. Cells were washed with PBS and incubated with PD-L1 antibody (Cell Signaling, 13684S, working dilution 1:400) for 1 hour at room temperature. Then, cells were washed with PBS and incubated with secondary antibody that was conjugated with Alexa Fluor (Thermo Fisher Scientific) for 1 hour at room temperature. After washed three times with PBS, cells were resuspended with PBS and analyzed on flow cytometer.
For flow cytometry analysis of mouse tissue samples, tumors were cut into small pieces and digested with 2 mg/mL collagenase (Sigma) in DMEM for 1 hour at 37° C. Cells were filtered through 70 μm nylon strainer and resuspended in red blood cell lysis buffer (Biolegend) for 3 minutes at room temperature. The cells were then suspended in PBS with 2% BSA and co-stained with the following antibodies: CD45 (Biolegend, 103112, APC conjugated), CD4 (Biolegend, 100510, FITC conjugated), CD8 (Biolegend, 100708, PE conjugated), CD11b (Biolegend, 101212, APC conjugated), and Gr1 (Biolegend, 108406, FITC conjugated). After incubated with antibody for 30 minutes, cells were washed with PBS and analyzed on a flow cytometer.
Statistical analyses were performed with two sided paired Student t test for single comparison and one-way ANOVA and a post hoc test for multiple comparisons. P values<0.05 are considered statistically significant. All values were expressed as means±SD. Pearson's product-moment correlation was used to calculate the correlation between PD-L1 and pRB-S249/T252 staining index in prostate cancer TMAs.
RB Suppresses PD-L1 Expression at the mRNA Level
Increasing evidence indicates that aberrant elevation of PD-L1 allows cancer cells to escape from immune surveillance. An array of small molecule inhibitors that are either in clinical use or potential agents for cancer therapy was surveyed to determine which compounds could trigger undesired upregulation of PD-L1 expression. In good agreement with previous reports (Dorand et al., Science, 353:399-403 (2016); and Zhu et al., Cell Reports, 16:2829-2837 (2016)), treatment with flavopiridol (effective inhibitor of CDK9 of the P-TEFb complex), roscovitine (nonselective inhibitor of CDKs 1, 2, 5, 7, and 9), and JQ1 (BET protein inhibitor), resulted in dramatic downregulation of PD-L1 mRNA in PC-3 prostate cancer cells (
Given that RB is a major downstream effector of CDK4/6 signaling, the following was performed to determine whether RB regulates PD-L1 expression. Transient knockdown of endogenous RB1 gene by two independent shRNAs invariably increased PD-L1 expression at the levels of mRNA, protein, and cell surface (
RB Phosphorylation by CDK4/6 Enhances RB Interaction with p65 NFκB Protein
Nuclear factor-κB (NFκB) plays a role in regulating PD-L1 transcription in response to various stimuli in different cancer types (Peng Jin et al., Cancer Research, 75:5034-5045 (2015); and Bouillez et al., Oncogene, 36:4037-4046 (2017)). Treatment of PC-3 cells with the NFκB inhibitor helenalin decreased PD-L1 mRNA expression (
The following was performed to further characterize how NFκB inhibition specifically impacts palbociclib-induced expression of PD-L1. Similar to palbociclib, RB knockdown-induced PD-L1 upregulation also was completely blocked by helenalin (
Serine-249 and Threonine-252 (S249/T252) Phosphorylation of RB and 161FQVTV165 Motif (SEQ ID NO: 258) in p65 are Required for p65-RB Interaction
There are four major CDK4 phosphorylation sites present in RB-N, two in the arginine-rich linker (R-linker) region and another two in the C-terminus (Zarkowska et al., J. Biol. Chem., 272:12738-12746 (1997)) (
It was determined that the amino acid sequence in the R-linker region that binds to p65 is evolutionally conserved from human to mouse (
An FxxxV (166FSLMV170) motif (SEQ ID NO: 286)-centered region in E1A-like inhibitor of differentiation-1 (EID1) is involved for its interaction with RB-N and S249/T252 phosphorylation by CDKs in the R-linker region abrogates in RB-N interaction with E1D1 (Hassler et al., Molecular Cell, 28:371-385 (2007)). It was determined that there are several acidic amino acids (negative charge) in the 166FSLMV170 (SEQ ID NO: 286)-centered region of EID1 whereas the R-linker in RB-N is an arginine (positive charge)-rich region (
Different from an acidic FxxxV-containing region in EID1, an evolutionally conserved 161FQVTV165 (SEQ ID NO: 258) (FxxxV)-centered motif in the RB-binding region of p65 that contains several basic (positive charge) amino acids (
Experiments were performed to determine whether the NFκB transcriptional program is globally regulated by RB phosphorylation. Endogenous RB was knocked down in PC-3 cells or cells were treated with the CDK4/6 inhibitor palbociclib, and RNA was isolated for high throughput sequencing (RNA-seq). A large set of genes was identified whose expression was invariably downregulated or upregulated by both RB knockdown and palbociclib among three replicates (one inconsistent replicate from control knockdown cells was removed for further analysis) (
Chromatin immunoprecipitation coupled quantitative PCR (ChIP-qPCR) analysis revealed that p65 protein readily bound to the genomic loci of the RB affected genes including PD-L1, and p65 binding was substantially increased by palbociclib treatment or RB knockdown in PC-3 cells (
In keeping with the finding that RB knockdown-induced PD-L1 expression was inhibited by the NFκB inhibitor helenalin (
Consistent with the finding in cultured cancer cells that S249/T252 phosphorylation is involved in RB binding of p65 and RB-mediated repression of PD-L1 expression, RB phosphorylation, especially S249/T252, but not total RB protein, was generally corrected with PD-L1 protein expression in an array of prostate cancerous and noncancerous cell lines (
Irradiation can cause cell cycle arrest and downregulation of RB phosphorylation (Abraham, Genes Dev., 15:2177-2196 (2001)). Based upon the data presented herein, it was hypothesize that PD-L1 expression can be induced by irradiation, and this effect is likely reversed by treatment of S249/T252 phosphorylation-mimicking peptide. Gamma irradiation inhibited RB phosphorylation at S249/T252 and markedly increased PD-L1 mRNA and protein expression in a time-dependent manner in PC-3 cells (
Induction of PD-L1 expression by gamma irradiation suggested that immune checkpoint blockade and the bioactive S249/T252-phosphorylation-mimicking peptide (RL-S249D/T252D peptide) of RB might enhance the anti-tumor efficacy of radiotherapy. PTEN-CaP8 mouse prostate tumor-bearing mice were treated with gamma irradiation (12 Gy) or mock treated in combination with anti-Pd-l1 or a non-specific control IgG (
In summary, the results provided herein demonstrate that CDK4/6 inhibitor-induced upregulation of PD-L1 expression occurs at mRNA level, and this effect is RB-dependent. Mechanistically, RB directly interacts with the NFκB protein p65, and the interaction is largely enhanced by CDK4/6 phosphorylation of S249/T252 sites in the N-terminal portion of RB-N. The results provided herein also demonstrate the development of a small RB-derived S249/T252 phospho-mimicking peptide that not only inhibits the basal level of PD-L1, but almost completely blocks irradiation-induced upregulation of PD-L1. This document also identifies a previously uncharacterized tumor suppressor function of phosphorylated RB that suppresses NFκB transcription activity, PD-L1 expression, and tumor immune evasion. Taken together, these results suggest that this activity of RB can be exploited to overcome immune destruction resistance associated with current therapeutics including radio- and chemo-therapy and CDK4/6 small molecule inhibitors in the clinic.
This example builds on and includes results from Example 1.
A recent study identifies the chromatin remodeling factor CHD1 as a positive regulator of NFκB and shows CHD1 and PTEN tumor suppressor gene are deleted mutually exclusively in human prostate cancers and their co-deletion is synthetic lethal (Zhao et al., Nature, 542:484-488 ((2017)). The MAP3K7 gene (encoding a kinase also known as TAK1, an upstream activator of NFκB) and PTEN were almost mutually exclusively deleted in multiple cancer types examined (
The following was performed to determine whether RB affects the synthetic lethality caused by PTEN/MAP3K7 or PTEN/CHD1 co-deficiency. RB KD blocked MAP3K7 or CHD1 KD-induced PARP and caspase-3 cleavage, apoptotic cell death, and growth inhibition in PTEN-null PC-3 cells (
Rescue experiments revealed that similar to RB-WT, restored expression of R661W (R654W in mouse Rb, an E2F1-binding deficient mutant (Sun et al., Proc. Natl. Acad. Sci. USA, 108:704-709 (2011)) largely blocked MAP3K7 or CHD1 KD-induced inhibition of cell growth and NFκB target gene expression although as expected, the growth inhibitory effect of R661W was not as robust as RB-WT in cell culture and in mice (
RB Interacts with p65 and the Interaction is Enhanced by RB Phosphorylation
To elucidate the molecular mechanisms by which RB regulates NFκB function, whether RB interacts with NFκB was examined. Co-IP demonstrated that endogenous RB interacted with endogenous p65, but not other NFκB/Rel family proteins RelB, c-Rel, p52, and p50 (
GST pull down assay revealed that RHD, the DNA binding domain in the N-terminal, but not the C-terminal of p65 specifically bound to RB protein (
While palbociclib treatment substantially reduced the level of hyperphosphorylated RB, it largely decreased RB-p65 interaction but substantially increased RB-E2F1 interaction (
Although RB-p65 interaction was largely enhanced by RB phosphorylation, certain basal level interaction between nonphosphorylatable RB and p65 was detectable (
RB S249/T252 Phosphorylation and 161FQVTV165 Motif (SEQ ID NO: 258) in p65 are Important for their Interaction
RB-p65 interaction was dependent on RB phosphorylation, and the interaction was largely diminished by CDK4/6 inhibitors or knockdown (
The amino acids surrounding S249/T252 in the R linker region are evolutionally conserved from human to mouse (
A previous study identified an FxxxV (166FSLMV170 (SEQ ID NO: 286)) motif-centered region in a protein termed E1A-like inhibitor of differentiation-1 (EID1) that is important for its interaction with RB-N(Hassler et al., Mol. Cell, 28:371-385 (2007)). p65 was found to also harbor an evolutionally conserved FxxxV (161FQVTV165 (SEQ ID NO: 258)) motif within the RB-N-binding region (
Specific interaction of RB with p65, but not other NFκB proteins suggests that RB may partially regulate NFκB transcription program. RNA-seq analysis was performed in RB-knockdown and palbociclib-treated PC-3 cells. A subset of genes whose expression was commonly down- or up-regulated by RB knockdown and palbociclib in three replicates was identified (one inconsistent replicate in shControl cells was excluded for analysis) (
Chromatin immunoprecipitation-coupled quantitative PCR (ChIP-qPCR) analysis revealed that p65 protein readily bound to the genomic loci of the RB-affected NFκB target genes including PD-L1, and p65 occupancy at these loci was substantially increased by RB knockdown or palbociclib in PC-3 cells (
T-cell responses can be reactivated by blockade of PD-1/PD-L1 interaction with agents such as PD-1 and PD-L1 antibodies and utilized for cancer treatment (Iwai et al., Proc. Natl. Acad. Sci. USA, 99:12293-12297 (2002); Topalian et al., Curr. Opin. Immunol., 24:207-212 (2012); Zhu and Chen, Curr. Opin. Investig. Drugs., 4:691-695 (2003)). The following was performed to determine whether RB regulation of PD-L1 expression is affected by RB phosphorylation and whether such regulatory mechanism can be harnessed for cancer therapy. Consistent with the finding that RB depletion upregulated PD-L1 mRNA expression (
The following was performed to examine whether PD-L1 expression is regulated by RB phosphorylation. Ectopic expression of RB WT substantially decreased PD-L1 expression, and this effect was largely attenuated in cells expressing the phosphorylation-resistant mutant RBΔCDK (
RB phosphorylation at S249/T252, but not total RB protein was generally corrected with PD-L1 protein expression in an array of prostatic cell lines (
Given that radiation inhibits RB phosphorylation by inducing cell cycle arrest, it was examined if radiation increased PD-L1 expression and if this effect can be reversed by RL-S249/T252D treatment. Gamma radiation inhibited RB phosphorylation at S249/T252 and markedly increased PD-L1 expression in a time-dependent manner (
The following was performed to examine whether administration of the bioactive RL-S249/T252D peptide of RB could enhance the anti-tumor efficacy of radiotherapy. PTEN-CaP8 murine prostate cancer cells infected with lentivirus of Tsin empty vector (EV) or Tsin-RL-S249D/T252D peptide were injected subcutaneously into immune-proficient mice. PTEN-CaP8 tumor-bearing mice were treated with gamma radiation (12 Gy) in combination with anti-PD-L1 antibody or non-specific control IgG (
A human identified as having cancer (e.g., PD-L1+ cancer such as PD-L1+ pancreatic cancer, PD-L1+ prostate cancer, PD-L1+ lung cancer, or PD-L1+ liver cancer) is administered a polypeptide that includes an amino acid sequence as set forth in any one of SEQ ID NOs: 1-179 (e.g., SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5) at least one time a week for one to six months. After this administration is initiated, a reduction in the number of cancer cells within the human is confirmed.
A human identified as having cancer (e.g., PD-L1+ cancer such as PD-L1+ pancreatic cancer, PD-L1+ prostate cancer, PD-L1+ lung cancer, or PD-L1+ liver cancer) is administered a polypeptide that includes an amino acid sequence as set forth in any one of SEQ ID NOs:1-179 (e.g., SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5) at least three times a week for one to six months. Radiation therapy to treat the cancer also is administered to the human. After this administration is initiated and after the radiation therapy is initiated, a reduction in the number of cancer cells within the human is confirmed.
A human identified as having cancer (e.g., PD-L1+ cancer such as PD-L1+ pancreatic cancer, PD-L1+ prostate cancer, PD-L1+ lung cancer, or PD-L1+ liver cancer) is administered a polypeptide that includes an amino acid sequence as set forth in any one of SEQ ID NOs:1-179 (e.g., SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5) at least seven times a week for one to six months. Chemotherapy to treat the cancer (e.g., treatment with camptothecin, taxane, gemcitabine, or a combination thereof) also is administered to the human. After this administration is initiated and after the chemotherapy is initiated, a reduction in the number of cancer cells within the human is confirmed.
A human identified as having cancer (e.g., PD-L1− cancer such as PD-L1− pancreatic cancer, PD-L1− prostate cancer, PD-L1− lung cancer, or PD-L1− liver cancer) is administered radiotherapy and/or chemotherapy, after which cancer cells within the human express an increased level of PD-L1. After this occurs, a polypeptide that includes an amino acid sequence as set forth in any one of SEQ ID NOs:1-179 (e.g., SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5) is administered to the human at least three times a week for one to six months. After this administration is initiated and after the radiotherapy and/or chemotherapy is initiated, a reduction in the number of cancer cells within the human is confirmed.
It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims priority to U.S. Application Ser. Nos. 62/758,429, filed on Nov. 9, 2018 and 62/636,734, filed on Feb. 28, 2018. The disclosures of the prior applications are considered part of the disclosure of this application, and are incorporated in its entirety into this application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/20073 | 2/28/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62636734 | Feb 2018 | US | |
| 62758429 | Nov 2018 | US |
