WO2018112033A1 - Méthodes et compositions pour le ciblage de tregs infiltrant les tumeurs - Google Patents

Méthodes et compositions pour le ciblage de tregs infiltrant les tumeurs Download PDF

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WO2018112033A1
WO2018112033A1 PCT/US2017/066097 US2017066097W WO2018112033A1 WO 2018112033 A1 WO2018112033 A1 WO 2018112033A1 US 2017066097 W US2017066097 W US 2017066097W WO 2018112033 A1 WO2018112033 A1 WO 2018112033A1
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tumor
agent
subject
antibody
product
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Christophe Benoist
Diane Mathis
Angela MAGNUSON
Ayla Ergun
Ralph Weissleder
Evgeny KINER
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President And Fellows Of Harvard College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Treg regulatory T cells characterized by expression of transcription factor FOXP3 (Treg) are critical to the maintenance of immunologic homeostasis, the enforcement of tolerance to self, and the prevention of runaway immune responses.
  • Tregs regulate the activation and differentiation of conventional CD4 + T cells, as well as many other cells of the innate and adaptive immune systems, through a variety of mechanisms.
  • a subset of Tregs known as tumor infiltrating Tregs (TITRs) are known to infiltrate cancerous tumors and are believed to play a role in the suppression of a host's immune response against the infiltrated tumors. TITRs are therefore an attractive therapeutic target for the treatment of cancer.
  • TITR tumor infiltrating T regulatory cells
  • kits for increasing the amount of T effector cells in a tumor in a subject by administering to the subject an agent, or a pharmaceutical composition comprising the agent, that inhibits the activity or expression of a product of a gene listed in Table 1 or Table 2.
  • the agent inhibits the activity or expression of a product of C3AR1, CCL22, CSF2RB, CXCR3, CXCR6, FAM83G, IL12RB1, IL12RB2, IL1R2, IL1RL1, IL21R MIIP, RGS1, MMP9, PODNL1, RGS1, SAMSN1, or SH2D2A.
  • the agent may be an antibody (e.g., an antibody specific for a protein product of a gene listed in Table 1 or Table 2, such as a protein localized on the surface of a cell), peptide, small molecule, or an interfering nucleic acid.
  • the agent disclosed herein is an agent for genome editing (e.g., an agent used to delete at least a portion of a gene listed in Table 1 or Table 2, such as a gene listed in Table 1 or Table 2 that is localized intracellularly).
  • the methods describe administering a second agent (e.g., a chemotherapeutic agent, immune checkpoint inhibitor, or a tumor vaccine).
  • the methods provided herein relate to methods of targeting an agent to TITRs in a subject (e.g., a subject with cancer) by administering to the subject the agent (e.g., a drug, such as a toxin disclosed herein) that is conjugated to a polypeptide or protein that binds to the protein product of a gene listed in Table 1 or Table 2.
  • the agent e.g., a drug, such as a toxin disclosed herein
  • the polypeptide or protein is an antibody specific for a protein product of a gene listed in Table 1 or Table 2 (e.g., an antibody specific for a protein product of a gene listed in Table 1 or Table 2, such as a protein localized on the surface of a cell).
  • test agent may be a member of a library of test agents.
  • the agent may be, for example, an antibody, peptide, small molecule, a protein drug conjugate, or an interfering nucleic acid.
  • provided herein are methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject by administering to the subject an agent that induces cytotoxicity in cells that express a product of a gene listed in Table 1 or Table 2.
  • methods of treating a tumor in a subject by administering to the subject an agent that induces cytotoxicity in cells that express a product of a gene listed in Table 1 or Table 2.
  • methods of increasing the amount of T effector cells in a tumor in a subject by administering to the subject an agent that induces cytotoxicity in cells that express a product of a gene listed in Table 1 or Table 2.
  • the cytotoxicity may be antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).
  • the agent may be, for example, an antibody, peptide, small molecule, a protein drug conjugate, or an interfering nucleic acid.
  • TITRs in a subject by expression level of a gene in Table 1 or Table 2.
  • methods of targeting and killing TITRs by first measuring expression level of a gene listed in Table 1 or Table 2, and, if the expression level is above a determined threshold, targeting and killing the TITR.
  • a gene e.g., mRNA or a protein
  • Figure 1 has three Parts, 1-3, and shows a flow chart describing the generation of three independent and cross-confirming datasets.
  • Part 1 shows the purification and profiling of Treg cells infiltrating three different transplantable tumors in immunocompetent mice.
  • Part 2 shows the purification of TITR cells from patients with colorectal tumors, and comparison of their gene expression profiles with those of Treg cells purified from normal human colon (many from the same donors).
  • Part 3 shows the mining of large datasets from TCGA for genes whose expression correlated with that of the Treg-defining factor FOXP3.
  • Figure 2 has three Parts, A-C, and shows identification of TITR signature.
  • Part A shows fold change (FC).
  • FC x FC plots depicting the FC in expression of genes in tumor versus spleen for one tumor type versus another tumor type (i, ii) MC38 x B 16, (iii) B 16 x BP, (iv) MC38 x BP.
  • An additive, filtered gene-set (genes with FC in expression > 4 in TITRs versus splenic Tregs, for each of the three transplantable tumor models) is highlighted in red on Part Aii-iv.
  • Part B shows a heatmap depicting FC values for each gene in the TITR gene-set in tumor/spleen or tissue/spleen are depicted in the heatmap (color scale is blue to red; the darker the red, the greater the FC value).
  • Part C shows tumor-preferential genes (highlighted) were revealed by plotting the filtered TITR gene-set on a FC x FC plot where the x-axis is FC in expression of gene X in tissue Tregs compared to splenic Tregs (average FC across colon, pancreas, adipose tissue (fat) and muscle) and the y-axis is the FC in expression of gene X in TITRs vs splenic Tregs (average FC across MC38, B 16 and BP).
  • Figure 3 has four Parts, A-D, and shows the conservation and derivation of TITR signature across species and individual human colon cancer patients.
  • Part A shows the transcriptomic profile of human colon tumor Tregs versus normal colonic mucosa Tregs.
  • the plot shows FC and p values for the expression of each gene in tumor/standard Tregs.
  • Part B shows the comparison of batches 1 and 2 of the human tumor versus normal colonic Tregs. Genes up two-fold or greater in tumor versus standard Tregs in both human data sets are highlighted in red. Part C shows the FC values for these highlighted genes for individual patients are presented in the heatmap. Known targets are annotated. Part D shows the mouse TITR signature is highlighted in purple on a human tumor/standard Treg data set.
  • Figure 4 has four Parts, A-D, and shows the correlation to FOXP3 in TCGA.
  • Part A shows the correlation of genes to FOXP3 in raw data (x-axis) cf. data with immunocyte infiltrate regressed out (y-axis).
  • Part B shows a plot of how genes correlate with FOXP3 in two different cancer types (colon and breast) from TCGA (immunocyte infiltrate regressed). Genes that highly correlate with FOXP3 are circled in green.
  • Part C shows a heatmap with FOXP3 correlation coefficients for each of the four tumor types analyzed from TCGA: breast (BRCA), colon (COAD), lung (LUSC) and pancreas (PAAD).
  • Part D shows classic Treg signature highlighted in pink on breast and colon datasets.
  • Figure 5 has two Parts, A-B, and shows the combinatorial data integration.
  • Part A shows the overall score for human (x-axis) and mouse (y-axis) tumor Tregs. Highlighted are genes at the top of the ranking in both species (red), with high scores in the mouse and still in top 10% of differential transcripts in human (blue), or highest in the human ranking, but not in the mouse (green).
  • Part B shows a combination of overall score for over-expression in human tumor Tregs (x-axis) with average correlation score derived from the whole-tumor TCGA datasets (y-axis).
  • Figure 6 shows the examination of how the combined TITR signature looks in 4 different cancers (colon, breast, pancreas and lung) from TCGA.
  • Red or white clusters represent genes whose expression correlates with expression of the other genes in a given cluster in these TCGA data.
  • Known, validated targets and FOXP3, IL2RB are annotated in grey.
  • Select lesser known targets that made the final list are annotated in black.
  • Figure 7 has two Parts, A-B, and shows the technique used for CRISPR-based editing of targets in Treg cells.
  • Part A provides a schematic depiction of the protocol whereby loss of function (LOF) mutations were induced in TITR target genes, specifically in Treg cells, by utilizing the CRISPR/Cas9 system.
  • Treg cells were isolated from spleens and lymph nodes of Cas9 expressing Foxp3-Thyl . l mice, and sorted as CD4+Thyl . l+. Treg cells were then activated with anti CD3/CD28 beads and 2000 Units/ml IL-2 for -40 hours and infected with retroviruses that carried sgRNAs against the genes of interest.
  • Retroviral vectors were engineered to express one of three different fluorescent proteins: GFP, BFP and RFP (the latter always carrying the non-targeting control sgRNA).
  • GFP GFP
  • BFP BFP
  • RFP the latter always carrying the non-targeting control sgRNA
  • 0.6* 10 6 of these transduced Tregs were then transferred together with 2* 10 6 CD45.1-Foxp3-GFP marked "filler cells" (splenocytes) to RAG2 KO mice, which do not harbor T cells.
  • CD45.1-Foxp3-GFP marked "filler cells" splenocytes
  • Tregs transduced with control or sgRNA encoding vectors in different locations in the body were compared, and it was determined that the specific sgRNA had an effect on Treg accumulation in the tumor versus general lymphoid tissues (spleen and lymph nodes).
  • the data are summarized in Part B.
  • the percentage of cells with a given sgRNA in the tumors was divided by the average percentage of these cells in the lymph nodes and spleens to find sgRNAs that are underrepresented in the tumors. Such underrepresentation is because inactivation of the target gene hampers Treg ability to colonize tumors.
  • Figure 8 has 5 Parts, A-E, shows a summary of the data from single agent mAb treatments.
  • MC38 tumor-bearing mice received anti-CD30, CCR8, IL1RL1, IL21R, CXCR3 or CXCR6 mAb treatment on days 7, 10 and 13 or 10, 13 and 16 following tumor cell injection.
  • the delta in tumor volume from the time treatment began until experiment end (Day 21) is the metric plotted.
  • Excellent responses to treatment were observed following treatment with anti-CD30 or anti-CCR8 mAbs, with 63% or 47% mice responding to anti- CD30 or anti-CCR8 mAb treatment, respectively (panel A).
  • a modest response was observed when anti-ILlRLl (a.k.a.
  • provided herein are methods and compositions related to the treatment or prevention of cancer (e.g., by targeting a tumor in a subject with cancer) by administering to the subject an agent disclosed herein.
  • the invention relates to targeting tumors in a subject, and/or decreasing the number of tumor infiltrating T regulatory cells (TITRs) (e.g., TITRs present within a tumor) in a subject by administering to the subject an agent disclosed herein.
  • TITRs tumor infiltrating T regulatory cells
  • the methods include decreasing the number or activity of TITR cells in a tumor present in a subject and/or increasing the amount of T effector cells in a tumor in a subject by administering to the subject an agent that inhibits the activity or expression of a product of a gene listed in Table 1 or Table 2. Also provided herein are methods of determining whether a test agent is an anti-cancer agent (e.g., an agent that decreases the number and/or activity of TITRs). In some embodiments, methods disclosed herein relate to methods of determining whether a cell is a TITR cell.
  • provided herein are methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject by administering to the subject an agent that induces cytotoxicity in cells that express a product of a gene listed in Table 1 or Table 2.
  • methods of treating a tumor in a subject by administering to the subject an agent that that induces cytotoxicity in cells that express a product of a gene listed in Table 1 or Table 2.
  • methods of identifying a TITR in a subject by determining the expression level of a gene or gene product in Table 1 or Table 2 in the TITR.
  • methods of targeting and killing the TITR by first measuring the expression level of a gene listed in Table 1 or Table 2 in the TITR, and, if the expression level is above a determined threshold, targeting and killing the TITR.
  • the present disclosure relates, at least in part, to the discovery of new targets for tumor immunotherapy, namely genes specifically over-expressed in TITR versus Tregs from secondary lymphoid organs or normal tissues.
  • This Treg- specific gene signature is conserved across species (human and mouse), is consistently expressed in tumor Tregs across individual colon cancer patients, and is present in at least four different types of tumors in The Cancer Genome Atlas.
  • Novel immunotherapies can be developed based on these targets, aiming to reduce the function and/or number of Tregs in tumors, and hence alleviate their suppressive effects and unleash an efficient anti-tumor immune response.
  • This goal may be achieved by blocking the function of these molecules in TITR through, for example, infusion of antibodies, peptides, interfering nucleic acids, or small molecule inhibitors disclosed herein, hence inhibiting TITR homeostasis or function; by using these molecules, for example, as targets of lytic antibodies, via complement- or ADCC- mediated toxicity, to preferentially deplete TITRs; and/or, for example, by genetic means (e.g., RNAi) to perturb the expression of these genes in TITRs.
  • These agents may be delivered alone, or coupled to other molecules that enhance the specificity for TITRs over other Tregs or immunocytes.
  • the agents and methods described herein may be combined with other known agents and methods. Definitions
  • an element means one element or more than one element.
  • agent is used herein to denote a chemical compound, a small molecule, a mixture of chemical compounds and/or a biological macromolecule (such as a nucleic acid, an antibody, an antibody fragment, a protein or a peptide).
  • a biological macromolecule such as a nucleic acid, an antibody, an antibody fragment, a protein or a peptide.
  • An agent may be cytotoxic to a cell (e.g., TITR).
  • An agent disclosed herein may target cells for cytotoxicity via CDC or ADCC-mediated cytotoxicity, or as a drug-antibody conjugate where the drug is cytotoxic.
  • antibody' ' ' and “antibodies” broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site.
  • Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
  • antibody as used herein also includes an "antigen-binding portion" of an antibody (or simply “antibody portion”).
  • antigen-binding portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody.
  • binding fragments encompassed within the term "antigen- binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a disul
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778).
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequences of specific scFv can be linked to human
  • immunoglobulin constant region cDNA or genomic sequences in order to generate expression vectors encoding complete IgG polypeptides or other isotypes.
  • VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444- 6448; Poljak et a/. (1994) Structure 2: 1121-1123).
  • An antibody for use in the instant invention may be a bispecific antibody.
  • a bispecific antibody has binding sites for two different antigens within a single antibody polypeptide. Antigen binding may be simultaneous or sequential.
  • Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecific antibodies produced by a hybrid hybridoma or a trioma are disclosed in U.S. Patent 4,474,893. Bispecific antibodies have been constructed by chemical means (Staerz et al. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83 : 1453, and Staerz and Bevan (1986) Immunol. Today 7:241). Bispecific antibodies are also described in U.S.
  • Bispecific agents can also be generated by making heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identification of clones producing and co-assembling both antibodies. They can also be generated by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof such as Fab and Fv sequences.
  • an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, biomarker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al. (1994) Mol. Immunol. 31 : 1047-1058).
  • Antibody portions, such as Fab and F(ab')2 fragments can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies.
  • immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.
  • Antibodies may also be humanized which is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences.
  • the humanized antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
  • the term "humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • cancer includes, but is not limited to, solid tumors and blood borne tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term “cancer” further encompasses primary and metastatic cancers.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies that specifically bind to the same epitope, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • polynucleotide and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or
  • Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • the following are non- limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present,
  • nucleotide structure may be imparted before or after assembly of the polymer.
  • sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified, such as by conjugation with a labeling component.
  • recombinant polynucleotide means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.
  • prevent refers to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.
  • small molecule is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.
  • the term "subject' means a human or non-human animal selected for treatment or therapy.
  • therapeutically-effective amount and “effective amount' as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment.
  • Treating' a disease in a subject or “treating' a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.
  • TITRs Tumor Infiltrating Tregs
  • Tregs are often found at elevated frequencies in blood and tumors of human cancer patients and, for many cancers, a high density of Tregs correlates with poor prognosis.
  • the involvement of Tregs in tumors has been demonstrated in animal models where their depletion via administration of anti-CD25 antibody or transfer of cells depleted of CD25 + Tregs, mostly eliminated different types of tumors.
  • Treg depletion increased the number of CD4 + and/or CD8 + effector T cells (Teff) in the tumor, which exhibited robust tumor-specific killing activity. Removal of Treg-mediated suppression of the anti-tumor immune response can therefore result in tumor irradication.
  • compositions that relate, at least in part, to the targeting of a tumor, decreasing the number or activity of TITRs in a tumor, e.g., by inhibiting the expression of at least one product of a gene listed in Table 1 or Table 2.
  • accession targets number(s) targets number(s) number(s)
  • TITRs in a subject comprising administering to the subject an agent that inhibits the activity or expression of a product of a gene listed in Table 1 or Table 2.
  • methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject by administering to the subject an agent that induces cytotoxicity e.g., ADCC or
  • CDC cytotoxicity or a cytotoxic drug-protein conjugate in cells that express a product of a gene listed in Table 1 or Table 2.
  • methods of treating a tumor in a subject by administering to the subject an agent that induces cytotoxicity in cells that express a product of a gene listed in Table 1 or Table 2.
  • the agent inhibits the activity or expression of a product of C3AR1, CCL22, CSF2RB, CXCR3, CXCR6, FAM83G, IL12RB1, IL12RB2, IL1R2, IL1RL1, IL21R, MIIP, RGS1, MMP9, PODNL1, RGS1, SAMSN1, or SH2D2A.
  • TITRs Tumor Infiltrating Tregs
  • a tumor e.g., a tumor present in a subject with cancer
  • methods related to treating or preventing a tumor e.g., a tumor present in a subject with cancer
  • TITRs i.e., TITRs in the tumor
  • An agent disclosed herein may be an antibody, a small molecule, a peptide, or an interfering nucleic acid.
  • An agent may reduce the number of TITRs (e.g., TITRs in a tumor) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • TITRs e.g., TITRs in a tumor
  • An agent disclosed herein may reduce the activity or expression of a product of a gene in Table 1 or Table 2 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • An agent disclosed herein may reduce the mRNA of a product of a gene in Table 1 or Table 2 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • provided herein are methods related to decreasing the number of
  • TITRs in a tumor may be identified using any technique known in the art, including detecting the expression of a product of a gene listed in Table 1 or Table 2 by cells in the tumor, wherein expression of a product (e.g., an mRNA product or a protein product) of a gene listed in Table 1 or Table 2 by a cell in the tumor indicates that there is a TITR in the tumor.
  • a product e.g., an mRNA product or a protein product
  • methods of identifying TITRs in a tumor by expression level of a gene or gene product in Table 1 or Table 2.
  • the TITR is identified as a TITR if the TITR has an expression level of a gene or product of a gene in Table 1 or Table 2 that is above a certain threshold.
  • methods of targeting and killing a TITR by first measuring the expression level of a gene or gene product listed in Table 1 or Table 2, and, if the expression level is above a determined threshold, killing the TITR.
  • a gene product e.g., an mRNA product
  • a gene product may be detected by nucleic acid amplification, a nucleic acid probe, or through sequencing.
  • a protein product may be detected by using an antibody specific for the protein product, through immunocytochemistry (THC), or by flow cytometry (e.g., FACS).
  • test agent is an anti-cancer therapeutic agent by determining whether the test agent inhibits the expression or activity of a protein product of a gene listed in Table 1 or Table 2, wherein the test agent is an anti- cancer therapeutic agent if the test agent inhibits the expression or activity of a protein product of a gene listed in Table 1 or Table 2.
  • the test agent is a member of a library of test agents.
  • the test agent may be any agent disclosed herein, including an interfering nucleic acid, a peptide, a small molecule, an antibody, or a peptide- drug conjugate.
  • a test agent disclosed herein may inhibit the expression or activity of a protein product of a gene in Table 1 or Table 2 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • a method of determining whether a TITR is present in a tumor comprising detecting the expression of a product of a gene listed in Table 1 or Table 2 by cells in the tumor, wherein expression of a product (e.g., an mRNA product or a protein product) of a gene listed in Table 1 or Table 2 by a cell in the tumor indicates that there is a TITR in the tumor.
  • a gene product e.g., an mRNA product
  • a gene product may be detected by nucleic acid amplification, a nucleic acid probe, or through sequencing.
  • a protein product may be detected, for example, by using an antibody specific for the protein product, through immunocytochemistry (IHC), or by flow cytometry (e.g., FACS).
  • IHC immunocytochemistry
  • FACS flow cytometry
  • methods of treating a subject with a tumor comprising administering an agent described herein that inhibits the expression or activity of a product of a gene listed in Table 1 or Table 2.
  • the agent described herein is an antibody specific for a protein product of any one of the genes in Table 1 or Table 2.
  • the antibody induces cytotoxicity in cells that express a product of a gene listed in Table 1 or Table 2.
  • An antibody disclosed herein may inhibit expression or activity of a product of a gene in Table 1 or Table 2 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • An antibody disclosed herein may inhibit the binding of a protein product of a gene in Table 1 or Table 2 to another protein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%).
  • An antibody provided herein may have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%), at least 95%, or 100% specificity for a product of a gene in Table 1 or Table 2.
  • Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies may also be fully human.
  • a monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it
  • the methods and compositions provided herein relate to antibodies and antigen binding fragments thereof that bind specifically to a product of a gene in Table 1 or Table 2.
  • the antibodies inhibit the function of the protein, such as inhibiting the activity of the protein, or interfering with protein-protein interactions.
  • Such antibodies can be polyclonal or monoclonal and can be, for example, murine, chimeric, humanized or fully human.
  • the agent may be a recombinant antibodies specific for a product of a gene in Table 1 or Table 2, such as chimeric or humanized monoclonal antibodies, can be made using standard recombinant
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in US Pat No. 4,816,567; US Pat. No. 5,565,332; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.
  • Human monoclonal antibodies specific for a product of a gene in Table 1 or Table 2 can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system.
  • “HuMAb mice” which contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy ( ⁇ and ⁇ ) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and ⁇ chain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856 859).
  • mice exhibit reduced expression of mouse IgM or ⁇ , and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of
  • the agent provided herein is a polypeptide agent (e.g., a polypeptide that binds to a protein expressed by a gene listed in Table 1 or Table 2).
  • the polypeptide induces cytotoxicity in cells that express a product of a gene listed in Table 1 or Table 2.
  • a polypeptide agent disclosed herein may inhibit the expression or activity of a product of a gene in Table 1 or Table 2 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • a polypeptide agent disclosed herein may inhibit the binding of a product of a gene in Table 1 or Table 2 to another protein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • the agent may be a chimeric or fusion polypeptide.
  • a fusion or chimeric polypeptide can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992).
  • polypeptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding a polypeptide(s). Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous polypeptides in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N. Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J.
  • interfering nucleic acid molecules that selectively target and inhibit the activity or expression of a product (e.g., an mRNA product) of a gene listed in Table 1 or Table 2 are provided herein and/or used in methods described herein.
  • the interfering nucleic acid induces cytotoxicity in cells that express a product of a gene listed in Table 1 or Table 2.
  • An agent may inhibit the expression or activity of a product (e.g., an mRNA product) of a gene in Table 1 or Table 2 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%>, at least 90%, at least 95%, or 100%>.
  • a product e.g., an mRNA product of a gene in Table 1 or Table 2 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%>, at least 90%, at least 95%, or 100%>.
  • An agent disclosed herein may comprise at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%>, at least 85%>, at least 90%, at least 95%, or 100% complementarity to a product (e.g., an mRNA product) of a gene in Table 1 or Table 2.
  • a product e.g., an mRNA product
  • the inhibiting nucleic acid is a siRNA, a shRNA, a PNA, or a miRNA molecule.
  • Interfering nucleic acids generally include a sequence of cyclic subunits, each bearing a base-pairing moiety, linked by intersubunit linkages that allow the base- pairing moieties to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence.
  • Interfering RNA molecules include, but are not limited to, antisense molecules, siRNA molecules, single-stranded siRNA molecules, miRNA molecules and shRNA molecules.
  • the interfering nucleic acid molecule is double-stranded RNA.
  • the double-stranded RNA molecule may have a 2 nucleotide 3' overhang.
  • the two RNA strands are connected via a hairpin structure, forming a shRNA molecule.
  • shRNA molecules can contain hairpins derived from microRNA molecules.
  • an RNAi vector can be constructed by cloning the interfering RNA sequence into a pCAG-miR30 construct containing the hairpin from the miR30 miRNA.
  • RNA interference molecules may include DNA residues, as well as RNA residues.
  • Interfering nucleic acid molecules provided herein can contain RNA bases, non-RNA bases or a mixture of RNA bases and non-RNA bases.
  • interfering nucleic acid molecules provided herein can be primarily composed of RNA bases but also contain DNA bases or non-naturally occurring nucleotides.
  • the interfering nucleic acids can employ a variety of oligonucleotide chemistries.
  • oligonucleotide chemistries include, without limitation, peptide nucleic acid (PNA), linked nucleic acid (LNA), phosphorothioate, 2'0-Me-modified oligonucleotides, and morpholino chemistries, including combinations of any of the foregoing.
  • PNA and LNA chemistries can utilize shorter targeting sequences because of their relatively high target binding strength relative to 2'0-Me oligonucleotides.
  • Phosphorothioate and 2'0- Me-modified chemistries are often combined to generate 2'0-Me-modified oligonucleotides having a phosphorothioate backbone. See, e.g., PCT Publication Nos. WO/2013/112053 and WO/2009/008725, incorporated by reference in their entireties.
  • PNAs Peptide nucleic acids
  • the backbone of PNAs is formed by peptide bonds rather than phosphodiester bonds, making them well-suited for antisense applications (see structure below).
  • the backbone is uncharged, resulting in PNA/DNA or PNA/RNA duplexes that exhibit greater than normal thermal stability. PNAs are not recognized by nucleases or proteases.
  • PNAs are capable of sequence-specific binding in a helix form to DNA or RNA.
  • Characteristics of PNAs include a high binding affinity to complementary DNA or RNA, a destabilizing effect caused by single-base mismatch, resistance to nucleases and proteases, hybridization with DNA or RNA independent of salt concentration and triplex formation with homopurine DNA.
  • PANAGENE.TM. has developed its proprietary Bts PNA monomers (Bts; benzothiazole-2- sulfonyl group) and proprietary oligomerization process.
  • the PNA oligomerization using Bts PNA monomers is composed of repetitive cycles of deprotection, coupling and capping.
  • PNAs can be produced synthetically using any technique known in the art. See, e.g., U.S. Pat. Nos. 6,969,766, 7,211,668, 7,022,851, 7, 125,994, 7, 145,006 and 7,179,896. See also U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 for the preparation of PNAs. Further teaching of PNA compounds can be found in Nielsen et al., Science, 254: 1497-1500, 1991. Each of the foregoing is incorporated by reference in its entirety.
  • Interfering nucleic acids may also contain "locked nucleic acid” subunits (LNAs).
  • LNAs locked nucleic acid subunits
  • LNAs are a member of a class of modifications called bridged nucleic acid (BNA).
  • BNA is characterized by a covalent linkage that locks the conformation of the ribose ring in a C30- endo (northern) sugar pucker.
  • the bridge is composed of a methylene between the 2'-0 and the 4'-C positions. LNA enhances backbone preorganization and base stacking to increase hybridization and thermal stability.
  • LNAs The structures of LNAs can be found, for example, in Wengel, et al., Chemical Communications (1998) 455; Tetrahedron (1998) 54:3607, and Accounts of Chem. Research (1999) 32:301); Obika, et al., Tetrahedron Letters (1997) 38:8735; (1998) 39:5401, and Bioorganic Medicinal Chemistry (2008) 16:9230.
  • Compounds provided herein may incorporate one or more LNAs; in some cases, the compounds may be entirely composed of LNAs. Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligonucleotides are described, for example, in U.S. Pat. Nos.
  • intersubunit linkers include phosphodiester and phosphorothioate moieties; alternatively, non-phosphorous containing linkers may be employed.
  • One embodiment is an LNA containing compound where each LNA subunit is separated by a DNA subunit. Certain compounds are composed of alternating LNA and DNA subunits where the intersubunit linker is phosphorothioate.
  • Phosphorothioates are a variant of normal DNA in which one of the nonbridging oxygens is replaced by a sulfur.
  • the sulfurization of the internucleotide bond reduces the action of endo-and exonucleases including 5' to 3' and 3' to 5' DNA POL 1 exonuclease, nucleases SI and PI, RNases, serum nucleases and snake venom
  • Phosphorothioates are made by two principal routes: by the action of a solution of elemental sulfur in carbon disulfide on a hydrogen phosphonate, or by the method of sulfurizing phosphite triesters with either tetraethylthiuram disulfide (TETD) or 3H-1, 2- bensodithiol-3-one 1, 1-dioxide (BDTD) (see, e.g., Iyer et al., J. Org. Chem. 55, 4693-4699, 1990).
  • TETD tetraethylthiuram disulfide
  • BDTD 2- bensodithiol-3-one 1, 1-dioxide
  • the latter methods avoid the problem of elemental sulfur' s insolubility in most organic solvents and the toxicity of carbon disulfide.
  • the TETD and BDTD methods also yield higher purity phosphorothioates.
  • 2'0-Me oligonucleotides carry a methyl group at the 2' -OH residue of the ribose molecule.
  • 2'-0-Me-RNAs show the same (or similar) behavior as DNA, but are protected against nuclease degradation.
  • 2'-0-Me-RNAs can also be combined with phosphothioate oligonucleotides (PTOs) for further stabilization.
  • PTOs phosphothioate oligonucleotides
  • 2'0-Me oligonucleotides phosphodiester or phosphothioate
  • can be synthesized according to routine techniques in the art see, e.g., Yoo et al., Nucleic Acids Res. 32:2008-16, 2004).
  • interfering nucleic acids described herein may be contacted with a cell or administered to an organism (e.g., a human).
  • constructs and/or vectors encoding the interfering RNA molecules may be contacted with or introduced into a cell or organism.
  • a viral, retroviral or lentiviral vector is used.
  • the vector has a tropism for cardiac tissue.
  • the vector is an adeno-associated virus.
  • the interfering nucleic acid molecule is a siRNA molecule.
  • siRNA molecules should include a region of sufficient homology to the target region, and be of sufficient length in terms of nucleotides, such that the siRNA molecule down- regulate target RNA.
  • ribonucleotide or nucleotide can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.
  • the sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double-strand character of the molecule.
  • siRNA molecule may be modified or include nucleoside surrogates.
  • Single stranded regions of an siRNA molecule may be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3'- or 5 '-terminus of an siRNA molecule, e.g., against exonucleases, or to favor the antisense siRNA agent to enter into RISC are also useful.
  • Modifications can include C3 (or C6, C7, CI 2) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, CI 2, abasic, tri ethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.
  • a “small hairpin RNA” or “short hairpin RNA” or “shRNA” includes a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNAs provided herein may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • Non-limiting examples of shRNAs include a double-stranded polynucleotide molecule assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions.
  • the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 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, or more nucleotides.
  • miRNAs represent a large group of small RNAs produced naturally in organisms, some of which regulate the expression of target genes. miRNAs are formed from an approximately 70 nucleotide single-stranded hairpin precursor transcript by Dicer. miRNAs are not translated into proteins, but instead bind to specific messenger RNAs, thereby blocking translation. In some instances, miRNAs base-pair imprecisely with their targets to inhibit translation.
  • antisense oligonucleotides may be 100% complementary to the target sequence, or may include mismatches, e.g., to improve selective targeting of allele containing the disease-associated mutation, as long as a heteroduplex formed between the oligonucleotide and target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation which may occur in vivo.
  • certain oligonucleotides may have about or at least about 70% sequence complementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide and the target sequence.
  • 70% sequence complementarity e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide
  • Oligonucleotide backbones that are less susceptible to cleavage by nucleases are discussed herein. Mismatches, if present, are typically less destabilizing toward the end regions of the hybrid duplex than in the middle. The number of mismatches allowed will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.
  • Interfering nucleic acid molecules can be prepared, for example, by chemical synthesis, in vitro transcription, or digestion of long dsRNA by Rnase III or Dicer. These can be introduced into cells by transfection, electroporation, or other methods known in the art. See Hannon, GJ, 2002, RNA Interference, Nature 418: 244-251; Bernstein E et al., 2002, The rest is silence. RNA 7: 1509-1521; Hutvagner G et al., RNAi: Nature abhors a double- strand. Curr. Opin. Genetics & Development 12: 225-232; Brummelkamp, 2002, A system for stable expression of short interfering RNAs in mammalian cells.
  • Short hairpin RNAs induce sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul CP, Good PD, Winer I, and Engelke DR. (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnol. 20:505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester WC, and Shi Y. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99(6):5515-5520; Yu J-Y, DeRuiter SL, and Turner DL. (2002).
  • an interfering nucleic acid molecule or an interfering nucleic acid encoding polynucleotide can be administered to the subject, for example, as naked nucleic acid, in combination with a delivery reagent, and/or as a nucleic acid comprising sequences that express an interfering nucleic acid molecule.
  • the interfering nucleic acid is administered directly to a tumor in a subject.
  • the nucleic acid comprising sequences that express the interfering nucleic acid molecules are delivered within vectors, e.g. plasmid, viral and bacterial vectors.
  • Suitable delivery reagents include, but are not limited to, e.g., the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), atelocollagen, nanoplexes and liposomes.
  • atelocollagen as a delivery vehicle for nucleic acid molecules is described in Minakuchi et al.
  • liposomes are used to deliver an inhibitory oligonucleotide to a subject.
  • Liposomes suitable for use in the methods described herein can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which are herein incorporated by reference.
  • the liposomes for use in the present methods can also be modified so as to avoid clearance by the mononuclear macrophage system ("MMS") and reticuloendothelial system (“RES").
  • MMS mononuclear macrophage system
  • RES reticuloendothelial system
  • modified liposomes have opsonization-inhibition moieties on the surface or incorporated into the liposome structure.
  • the agent disclosed herein is an agent for genome editing (e.g., an agent used to delete at least a portion of a gene listed in Table 1 or Table 2, such as a gene listed in Table 1 or Table 2 that is localized intracellularly).
  • Deletion of DNA may be performed using gene therapy to knock-out or disrupt the target gene.
  • a "knock-out" can be a gene knock-down or the gene can be knocked out by a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques known in the art, including, but not limited to, retroviral gene transfer.
  • the agent is a nuclease (e.g., a zinc finger nuclease or a TALEN).
  • Zinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA- binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences, which enable zinc-finger nucleases to target unique sequence within a complex genome. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Other technologies for genome customization that can be used to knock out genes are meganucleases and TAL effector nucleases (TALENs).
  • a TALEN is composed of a TALE DNA binding domain for sequence-specific recognition fused to the catalytic domain of an endonuclease that introduces double-strand breaks (DSB).
  • the DNA binding domain of a TALEN is capable of targeting with high precision a large recognition site (for instance, 17 bp).
  • Meganucleases are sequence-specific endonucleases, naturally occurring "DNA scissors," originating from a variety of single-celled organisms such as bacteria, yeast, algae and some plant organelles. Meganucleases have long recognition sites of between 12 and 30 base pairs. The recognition site of natural meganucleases can be modified in order to target native genomic DNA sequences (such as endogenous genes).
  • the agent comprises a CRISPR-Cas9 guided nuclease and/or a sgRNA (Wiedenheft et al., "RNA-Guided Genetic Silencing Systems in Bacteria and Archaea,” Nature 482:331-338 (2012); Zhang et al., “Multiplex Genome Engineering Using CRISPR/Cas Systems," Science 339(6121): 819-23 (2013); and Gaj et al., "ZFN, TALEN, and CRISPR/Cas-based Methods for Genome Engineering," Cell 31(7): 397-405 (2013), which are hereby incorporated by reference in their entirety).
  • CRISPR-Cas9 interference is a genetic technique which allows for sequence-specific control of gene expression in prokaiyotic and eukaryotic cells by guided nuclease double- stranded DNA cleavage. It is based on the bacterial immune system - derived CRISPR (clustered regularly interspaced palindromic repeats) pathway.
  • the agent is an sg NA.
  • An sgR A combines tracrRNA and crRNA, which are separate molecules in the native CRISPR/Cas9 system, into a single RNA construct, simplifying the components needed to use CRISPR/Cas9 for genome editing.
  • the crRNA of the sgRNA has complementarity to at least a portion of a gene listed in Table 1 or Table 2 such as a portion of a gene listed in Table 1 or Table 2 that is localized
  • the sgRN A may target at least a portion of a gene listed in Table 1 or Table 2.
  • Certain embodiments of the methods and compositions disclosed herein relate to the use of small molecule agents e.g., small molecule agents that inhibit the expression or activity of a product of a gene listed in Table 1 or Table 2, for decreasing the number or activity of TITRs (e.g., TITRs in a tumor), in a subject, or increasing the number or activity of T effector cells in a tumor in a subject.
  • the small molecule induces cytotoxicity in cells that express a product of a gene listed in Table 1 or Table 2.
  • Such agents include those known in the art and those identified using the screening assays described herein.
  • a small molecule provided herein may have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%), at least 95%, or 100% specificity for a product of a gene in Table 1 or Table 2.
  • assays used to identify agents include obtaining a population of cells and a small molecule agent, wherein the cells are incubated with a small molecule agent and the number or activity of TITRs in the population of cells is subsequently measured.
  • Agents identified via such assays may be useful, for example, for decreasing the number or activity of TITRs (e.g., TITRs in a tumor), in a subject, or increasing the number or activity of T effector cells in a tumor in a subject.
  • Agents useful in the methods disclosed herein may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Agents may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al, 1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145).
  • Agents useful in the methods disclosed herein may be identified, for example, using assays for screening candidate or test agents e.g., agents that decrease the activity or expression of a product of a gene in Table 1 or Table 2 or that decrease the number or activity of TITRs in a sample or population of cells.
  • assays for screening candidate or test agents e.g., agents that decrease the activity or expression of a product of a gene in Table 1 or Table 2 or that decrease the number or activity of TITRs in a sample or population of cells.
  • Peptide-drug conjugates are an emerging class of anti-cancer therapeutics. Highly cytotoxic small molecule drugs are conjugated to proteins (e.g., antibodies, such as monoclonal antibodies) to create a single molecular entity. Peptide-drug conjugates combine the high efficacy of small molecules with the target specificity of antibodies to enable the selective delivery of drug payloads to cancerous tissues, which reduces the systematic toxicity of conventional small molecule drugs.
  • proteins e.g., antibodies, such as monoclonal antibodies
  • peptide-drug conjugates are prepared by conjugating small molecule drugs to either cysteines generated from reducing an internal disulfide bond or surface- exposed lysines. Because multiple lysines and cysteines are present in antibodies, these conventional approaches usually lead to heterogeneous products with undefined drug- antibody ratio. Each individual antibody-drug conjugate may exhibit different
  • peptide-drug conjugates are prepared using site-specific conjugation techniques.
  • provided herein are methods of targeting an agent to TITRs in a subject by administering to the subject the agent conjugated to a polypeptide or protein that binds to a product of a gene listed in Table 1 or Table 2.
  • the peptide- drug conjugate induces cytotoxicity in cells that express a product of a gene listed in Table 1 or Table 2.
  • the polypeptide or protein is an antibody specific for a protein product of the gene listed in Table 1 or Table 2.
  • the antibody is a polyclonal, monoclonal, chimeric, humanized, or an antibody fragment.
  • the agent is a drug (e.g., a cytotoxic agent)
  • cytotoxic agents include, but are not limited to, MMAE, DM-1, a maytansinoid, a doxorubicin derivative, a auristatin, a calcheamicin, CC-1065, aduocarmycin or a anthracycline.
  • peptide-drug conjugates with defined position of drug- attachment and defined drug to antibody ratio.
  • the drug-to-antibody ratio is about 2: 1, about 3 : 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 11 : 1, or about 12: 1.
  • TITR tumor infiltrating T regulatory cells
  • a tumor e.g., a tumor type disclosed herein
  • a subject e.g., a subject with cancer
  • an antibody e.g., a monoclonal antibody
  • an immune checkpoint inhibitor e.g., an immune checkpoint inhibitor disclosed herein
  • kits for treating a tumor in a subject comprising conjointly administering to the subject an antibody (e.g., a monoclonal antibody) that inhibits the activity or expression of a product of a gene listed in Table 1 or Table 2 and an immune checkpoint inhibitor (e.g., an immune checkpoint inhibitor disclosed herein).
  • an antibody e.g., a monoclonal antibody
  • an immune checkpoint inhibitor e.g., an immune checkpoint inhibitor disclosed herein.
  • kits for increasing the amount of T effector cells in a tumor in a subject by conjointly administering to the subject an antibody (e.g., a monoclonal antibody), or a pharmaceutical composition comprising the agent, that inhibits the activity or expression of a product of at least a portion of a gene listed in Table 1 or Table 2 and an immune checkpoint inhibitor (e.g., an immune checkpoint inhibitor disclosed herein).
  • an immune checkpoint inhibitor e.g., an immune checkpoint inhibitor disclosed herein.
  • the immune checkpoint inhibitor inhibits an immune checkpoint protein.
  • Immune checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response.
  • immune checkpoint proteins are CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof.
  • the immune checkpoint inhibitor is a PD-1 inhibitor.
  • agents of the invention may be used alone or conjointly administered with another type of therapeutic agent.
  • the phrase "conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include synergistic effects of the two agents).
  • the different therapeutic agents can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially.
  • the different therapeutic agents can be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about a week of one another.
  • a subject who receives such treatment can benefit from a combined effect of different therapeutic agents.
  • composition e.g., a pharmaceutical composition, containing at least one agent (e.g., a test agent, such as an antibody, an interfering nucleic acid, a peptide, or a small molecule) described herein together with a pharmaceutically acceptable carrier.
  • agent e.g., a test agent, such as an antibody, an interfering nucleic acid, a peptide, or a small molecule
  • the composition includes a combination of multiple (e.g., two or more) agents described herein.
  • the pharmaceutical composition is delivered locally or systemically. In some embodiments, the pharmaceutical composition may be administered to a tumor present in the subject. In some embodiments, the agent or pharmaceutical composition is administered with a second cancer therapeutic agent. In some embodiments, the second cancer therapeutic agent is a chemotherapeutic agent. In some embodiments, the pharmaceutical composition further comprises a second agent for treatment of cancer. In some embodiments, the second agent is a tumor vaccine. In some embodiments, the second agent is an agent that decreases the activity or expression of a gene listed in Figure 6, such as FOXP3 or IL2RB. In some embodiments, the second agent is an agent that decreases the activity or expression of a gene or gene product known to be upregulated in TITRs.
  • the second therapeutic agent is a chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CytoxanTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; emylerumines and memylamelamines including alfretamine, triemylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimemylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (articularly cryptophycin 1 and crypto
  • spongistatin nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;
  • nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;
  • nitrosoureas such as carmustine, chlorozotocin, foremustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin phili); dynemicin, including dynemicin A;
  • bisphosphonates such as clodronate; an esperamicin; as well as neocarzinostatin
  • aclacinomysins actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carrninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (AdramycinTM) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex
  • aminolevulinic acid aminolevulinic acid
  • eniluracil amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKTM;
  • razoxane rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- tricUorotriemylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
  • paclitaxel TexolTM, Bristol Meyers Squibb Oncology, Princeton, N.J.
  • docetaxel TaxoteretTM, Rhone-Poulenc Rorer, Antony, France
  • chlorambucil gemcitabine
  • mitroxantrone vancristine; vinorelbine (NavelbineTM); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluoromethylornithine
  • chemotherapeutic agent anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens and selective estrogen receptor modulators SERMs
  • SERMs selective estrogen receptor modulators
  • tamoxifen including NolvadexTM
  • raloxifene including NolvadexTM
  • droloxifene 4-hydroxytamoxifen
  • trioxifene keoxifene
  • LY117018 4-hydroxytamoxifen
  • toremifene FarestonTM
  • inhibitors of the enzyme aromatase which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MegaceTM), exemestane, formestane, fadrozole, vorozole (RivisorTM), letrozole (FemaraTM), and anastrozole (ArimidexTM)
  • anti-androgens such as flutamide
  • the second cancer therapeutic agent is an immune checkpoint inhibitor, e.g., inhibitors of immune checkpoint proteins such as CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof.
  • immune checkpoint inhibitor e.g., inhibitors of immune checkpoint proteins such as CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B,
  • compositions and/or agents disclosed herein may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; or (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous, intrathecal, intracerebral or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue
  • parenteral administration for example, by subcutaneous, intramuscular, intravenous, intrathecal, intracer
  • Methods of preparing pharmaceutical formulations or compositions include the step of bringing into association an agent described herein with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • compositions suitable for parenteral administration comprise one or more agents described herein in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, dimethyl sulfoxide (DMSO), polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • DMSO dimethyl sulfoxide
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • provided herein are methods of treating a cancer by administering to a subject (e.g., to a tumor present in a subject) an agent and/or a pharmaceutical composition described herein.
  • the methods described herein may be used to treat any cancerous or pre-cancerous tumor.
  • the cancer includes a solid tumor.
  • Cancers that may be treated by methods and compositions provided herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp;
  • adenocarcinoma familial polyposis coli
  • solid carcinoma carcinoid tumor, malignant
  • branchiolo-alveolar adenocarcinoma papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell
  • adenocarcinoma granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;
  • endometrioid carcinoma skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma;
  • cystadenocarcinoma papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma;
  • infiltrating duct carcinoma medullary carcinoma; lobular carcinoma; inflammatory carcinoma; mammary paget's disease; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; and malignant roblastoma;
  • Sertoli cell carcinoma malignant leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma;
  • glomangiosarcoma malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma;
  • myxosarcoma liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal
  • rhabdomyosarcoma alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma;
  • lymphangiosarcoma osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma;
  • ameloblastic fibrosarcoma malignant pinealoma; chordoma; malignant glioma;
  • ependymoma ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal;
  • cerebellar sarcoma cerebellar sarcoma; ganglion euroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophil
  • a TITR is present in a tumor by detecting the expression of a product of a gene listed in Table 1 or Table 2 by cells in the tumor.
  • expression of a product of a gene listed in Table 1 or Table 2 by a cell in the tumor indicates that there is a TITR in the tumor.
  • the gene product is an mRNA product.
  • the gene product is a protein product. The protein product can be detected using an antibody specific for a protein product, by IHC, or by flow cytometry (e.g., FACS).
  • provided herein are methods of identifying TITRs in a subject (e.g., a subject with a tumor) by determining the expression level of a gene in Table 1 or Table 2.
  • methods of targeting and killing TITRs by first measuring the expression level of a gene listed in Table 1 or Table 2 in the TITR, and, if the expression level is above a determined threshold, targeting and killing the TITR.
  • the threshold for a gene may be determined by a number of techniques, including, but not limited to, determining the expression of a gene or gene product in diseased tissues (e.g., tumor or cancerous tissues) versus healthy tissues (e.g., tissues not associated with a tumor or cancer). Healthy and diseased tissues may be taken from the subject or from different individuals.
  • the expression threshold of a gene or gene product is determined by examining the gene or gene product expression in tissues from a tissue bank or third party source.
  • the subject has cancer.
  • the cancer comprises a solid tumor.
  • the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a blood born tumor, a brain/CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing tumor, an eye tumor, a gallbladder tumor, a gastrointestinal, a kidney tumor, a laryngeal or hypopharyngreal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penile tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma,
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions or agents to be administered may be varied so as to obtain an amount of the active ingredient (e.g., an agent described herein) which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • an amount of the active ingredient e.g., an agent described herein
  • the selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could prescribe and/or administer doses of the compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • Example 2 Transcripts specific to mouse tumor Tregs.
  • MC38 transplantable MC38 colon adenocarcinoma
  • B16 B16-F10 melanoma
  • BP BRAF.PTEN melanoma
  • tumors were inoculated subcutaneously into immunocompetent Foxp3-GFP/C57BL6 reporter mice, in which Treg cells are uniquely identified and readily sorted on the basis of the fluorescent GFP reporter. After establishment and growth of these tumors (21 days), immunocytes were isolated from the tumors and spleens from these animals, stained with fluorochrome-conjugated antibodies, and 1,000 Tregs
  • RNAseq whole-transcriptome shotgun sequencing
  • the fold change (FC) in expression of each gene in tumor versus splenic Tregs was calculated.
  • a sizeable set of genes was overexpressed in tumor versus splenic Tregs.
  • the vast majority of genes overexpressed in TITRs from one mouse model were similarly upregulated in the other murine tumor models (e.g. MC38 and B 16 comparison shown in Figure 2, Part Ai).
  • the analyses also identified genes that were differentially downregulated in TITRs versus splenic Tregs. Given that such genes were under-expressed in TITRs (hence, expressed at very low levels), they offered less desirable targets and so exploration of these was paused here.
  • genes were selected with a FC in expression > 4 and a p value of ⁇ 10-3 in TITRs versus splenic Tregs, in either of the three transplantable tumor models.
  • a group of differential transcripts were typical of tumor infiltrating myeloid cells (identified using myeloid cell signatures from the ImmGen database) and were discounted as residual contamination. Overexpression of this filtered gene-set was strongly conserved across the three tumor types that were examined (filtered
  • tissue-Tregs include populations in the visceral adipose tissue or injured muscle (reviewed in (Panduro et al., 2016)).
  • Treg cells can adopt distinct subphenotypes, which differ by their preferential expression of chemokine receptors, effector molecules, and cofactors that collaborate with FoxP3 to drive these functional nuances (Feuerer et al., 2009b; Campbell and Koch, 2011).
  • chemokine receptors chemokine receptors
  • effector molecules effector molecules
  • cofactors that collaborate with FoxP3 to drive these functional nuances
  • the TITR gene-set was selected as described above, and analyzed for how these genes behaved in different tissue Treg subsets, namely those from colon, pancreas, adipose tissue and muscle. Although 2-3 clusters of genes were similarly upregulated in TITRs and Tregs from colon, fat, and muscle, there were large and distinct sets of genes which exhibited increased upregulation in TITRs versus tissue Tregs ( Figure 2, Part B; FC values for tumor/spleen or tissue/spleen are depicted in the heatmap).
  • Tumor- preferential overexpression was also revealed by plotting the filtered TITR gene-set on a FC x FC plot where the x-axis is the FC in expression in tissue Tregs compared to splenic Tregs (average FC across colon, pancreas, adipose tissue and muscle) and the y-axis is the FC in expression in TITRs versus splenic Tregs (average FC across MC38, B16 and BP) (Figure 2, Part C). Some transcripts (area A) are preferentially overexpressed in tissue Tregs e.g.
  • Example 4 Transcripts Specific to Human Colorectal Tumor Tregs.
  • Tregs were purified from freshly harvested human colorectal tumors or normal colon tissue and then cryopreserved. These were profiled by RNAseq. After dissociation, immunocytes from both tissues were stained with antibodies, and 1,000 Tregs were purified by flow cytometry (CD25+CD127-CD4+CD8-CD3+CD45+, double-sorting). RNAseq was performed on these purified Treg samples, and the data were processed, normalized and filtered as above, (the profiles were generated in two different batches). Next, to detect genes that were differentially expressed in tumor versus normal tissue Tregs, the FC was calculated in expression of each gene in tumor Tregs versus normal colon Tregs, and the p. value associated with this difference. As illustrated in Figure 3, Part A there was a significant bias in the transcriptome of these tumor tregs, with 38 transcripts induced relative to normal colon Tregs (at a threshold fold change of 2, and t.test
  • these over-expressed human transcripts included several genes previously recognized to have activity in Treg cells and/or costimulatory function (ENTPD1 (encodes CD39), DUSP4, TNFRSF4, TNFRSF9, TNFRSF18 (aka OX40, 4- IBB, GITR, respectively) and FOXP3 itself).
  • ENTPD1 encodes CD39
  • DUSP4 TNFRSF4
  • TNFRSF9 TNFRSF18
  • FOXP3 FOXP3 itself.
  • this same analysis identified genes that were downregulated in tumor verus control Tregs, perhaps surprisingly including genes known to be involved in Treg function, namely IL10, CCR7 and CXCR5 (highlighted in Fig.
  • FOXP3 is the key transcription factor that defines Treg cells, conditioning a substantial portion of their transcriptional identity (Josefowicz et al., 2012).
  • TCGA a publically available database of gene expression in 33 types of cancer from more than 11,000 patients was analyzed.
  • immunocytes that can be found in a tumor (namely granulocytes, dendritic cells,
  • Treg-specific genes such as those encoding costimulatory molecules CTLA4, CD80 and ICOS, but also chemokine and chemokine receptors like CCL22.
  • positive correlation to FOXP3 was similar in different types of tumors ( Figure 4, Part B, C), supporting the notion that these are not spurious correlations.
  • This sharing also indicated that the tumor- Treg specific transcriptome is largely shared between human tumor types.
  • these O P3-correlated transcripts were not simply a phenocopy of the classic Treg signature (Ferraro et al., 2014). Only a subset of the Treg-up signature was positively correlated with FOXP3, as illustrated for the breast and colon tumor datasets in Figure 4, Part D.
  • only a fraction of transcripts correlated to FOXP3 actually belong to the classic Treg signature.
  • the fold change in expression was ranked of each gene in TITR relative to the splenic Treg controls, and summed these ranks across the three datasets (MC38, B16 and BP infiltrating Tregs), to yield an overall rank of Treg genes preferentially induced in the mouse tumor models.
  • the correlation coefficients was averaged to yield a combined correlation to FOXP3 across these tumors.
  • Figure 5, Part A plots the overall score for human and mouse tumor Tregs (x and y axes, respectively).
  • Figure 5 combines the same overall score for over-expression in human tumor Tregs with the average correlation score derived from the TCGA datasets. Many, of the Treg transcripts with the highest rank for over-expression in human TITRs also showed significant association to FOXP3 in the TCGA datasets (e.g. TNFRSF9, IL21R), although this did not apply all (e.g. DUSP4, IRAK2 OR CCNG2).
  • TITR genes differentially expressed in TITR
  • Table 1 or Table 2 genes differentially expressed in TITR
  • This list combines (i) transcripts that ranked in the top 3% for differential expression in human colorectal TITRs; (ii) transcripts that ranked in the top 3% for differential expression in mouse TITRs and ranked in the top 10% for differential expression in human colorectal TITRs; (iii) transcripts that ranked in the top 2% for correlation with FOXP3 in TCGA tumor data (average of all 4 tumor-types) and ranked in the top 10% for differential expression in human colorectal TITRs.
  • Example 7 CRISPR-based editing of TITR targets in Treg cells.
  • LEF loss of function
  • a "retro-sgRNA" plasmid was cloned by Gibson assembly of U6- gRNA cassette and EFla promoter from the lentiCRISPR vector (Shalem et al, Science 2014) into a retroviral MSCV-GFP plasmid (Hoist et al, Nat. Protoc. 2006).
  • TagBFP (BFP) and mRFPl (RFP) were cloned in place of eGFP (GFP). Cloning of individual sgRNAs was done by utilizing Bbsl sites, as previously described (Shalem et al, Science 2014).
  • Retrovirus was prepared by transfecting Platinum-E cells at 70% confluency on a 60 mm plate with 2.3 ug of retro-sgRNA and 1.5 ug of pCL-Eco using Minis TransIT-293T reagent (Morita et al., Gene Therapy 2000). Medium was changed after 16 hours and the retrovirus was collected and passed through a 0.45 u filter 48 hours after media change.
  • panel A Treg cells were isolated from spleens and lymph nodes (axillary, brachial, cervical and inguinal) of 8-10 weeks old Cas9 expressing Foxp3 -Thy 1.1 mice (generated by crossing Foxp3-Thyl .
  • Treg cells were then activated with anti CD3/CD28 beads and 2000 Units/ml IL-2 for -40 hours, and infected with retroviruses that carried sgRNAs against the genes of interest (detailed earlier).
  • retroviral vectors were engineered to express one of three different fluorescent proteins: BFP, GFP and RFP (the latter always carrying the non- targeting control sgRNA). 0.6* 10 6 of these transduced Treg cells were then transferred together with 2* 10 6 CD45.
  • l-Foxp3-GFP marked "filler cells" (splenocytes) to RAG2 KO mice, which do not harbor T cells.
  • These mice were given a subcutaneous injection of 0.5* 10 6 MC38 tumor cells immediately after the cell transfer. After 16-19 days in the mice, the Treg cells expanded and migrated to different tissues as well as the tumor. The ratios of Tregs transduced with control or sgRNA encoding vectors in different locations of the body were compared and determine whether the specific sgRNA had an effect on Treg
  • Example 8 Preclinical testing of TITR targets - mAb treatments
  • Monoclonal antibodies specific for TITR targets were administered to tumor-bearing mice to pre-clinically test the effect of TITR target modulation.
  • the transplantable MC38 colon adenocarcinoma (MC38) was used. Tumors were inoculated subcutaneously into immunocompetent C57BL/6 mice and allowed to grow for 7 days before treatment groups were assigned according to tumor volume. Mice received 200 ug single agent mAb i.p. on day 7 or day 10 and this dose was repeated twice at 72 hour intervals, for a total of three doses. Control mice received either 200 ug of appropriate isotype control or an irrelevant mAb, or were left untreated. Tumor volumes were measured at regular intervals from Day 7 until Day 21.
  • any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web and/or the National Center for Biotechnology Information (NCBI) on the World wide Web.
  • TIGR The Institute for Genomic Research
  • NCBI National Center for Biotechnology Information

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Abstract

L'invention concerne des méthodes et des compositions associées au traitement ou à la prévention du cancer (par exemple, par ciblage d'une tumeur chez un sujet atteint d'un cancer) par l'administration à un sujet d'un agent décrit ici. Dans certains aspects, l'invention concerne le ciblage de tumeurs chez un sujet, et/ou la diminution du nombre de lymphocytes T régulateurs infiltrant les tumeurs (TITRs) (par exemple, TITRs présents dans une tumeur) chez un sujet par l'administration au sujet d'un agent décrit ici. L'invention concerne en outre des procédés d'identification d'agents anticancéreux qui réduisent le nombre ou l'activité de TITRs chez un sujet ou dans une tumeur présente chez un sujet. Selon certains aspects, l'invention concerne des procédés pour identifier des TITRs chez un sujet (par exemple, identification des TITRs dans une tumeur présente chez un sujet).
PCT/US2017/066097 2016-12-13 2017-12-13 Méthodes et compositions pour le ciblage de tregs infiltrant les tumeurs WO2018112033A1 (fr)

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