WO2012078540A1 - Modulating immune cell activity using cytokine-induced src homology 2 and/or high temperature requirement a-1 - Google Patents

Modulating immune cell activity using cytokine-induced src homology 2 and/or high temperature requirement a-1 Download PDF

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WO2012078540A1
WO2012078540A1 PCT/US2011/063375 US2011063375W WO2012078540A1 WO 2012078540 A1 WO2012078540 A1 WO 2012078540A1 US 2011063375 W US2011063375 W US 2011063375W WO 2012078540 A1 WO2012078540 A1 WO 2012078540A1
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Prior art keywords
htral
cish
seq
nucleotide sequence
cells
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PCT/US2011/063375
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French (fr)
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Douglas C. Palmer
Nicholas P. Restifo
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Publication of WO2012078540A1 publication Critical patent/WO2012078540A1/en

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    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464492—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/73—Hydrolases (EC 3.)
    • C12N2501/734—Proteases (EC 3.4.)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 20,489 Byte ASCII (Text) file named "709100_ST25.TXT" dated October 25, 2011.
  • Adoptive cell therapy can be an effective treatment for cancer in some patients.
  • obstacles to the overall success of adoptive cell therapy still exist.
  • the adoptive transfer of T-cells specific for tumor antigens or self-antigens can, in some cases, result in toxicities (Palmer et al., PNAS 105(23):8061 -66 (2008); Johnson et al., Blood 114(3):535-46 (2009)).
  • the in vivo persistence, survival, and anti-tumor activity of T cells can, in some cases, decrease following adoptive transfer (Heemskerk et al. Hum. Gene Ther. 19(5):496-510 (2008)).
  • An embodiment of the invention provides a host cell comprising an high temperature requirement serine peptidase 1 (HTRAl)//tfra/ inhibitor and a cytokine-induced Src homology 2 protein (ClS)/cish activator.
  • HTRAl serine peptidase 1
  • ClS cytokine-induced Src homology 2 protein
  • Another embodiment of the invention provides a peripheral blood cell (PBC) comprising a HTRAl/htral inhibitor.
  • Still another embodiment of the invention provides a host cell comprising an anti- cish shMIR comprising SEQ ID NO: 3 or 4 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 3 or 4.
  • Another embodiment of the invention provides a PBC comprising a HTRAl /htral activator.
  • Still another embodiment of the invention provides a host cell comprising a ClS/cish inhibitor and a HTRAl/htral activator.
  • An embodiment of the invention provides a composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an HTRAl I htral inhibitor and/or a ClS/cish activator.
  • An embodiment of the invention provides a composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an HTRAl /htral activator and/or a ClS/cish inhibitor.
  • the invention further provides embodiments including populations of cells and pharmaceutical compositions relating to the host cells (e.g., PBCs and T cells) and populations of cells of the invention.
  • the host cells e.g., PBCs and T cells
  • Embodiments of methods of treating or preventing cancer or a chronic infectious disease in a mammal methods of treating or preventing an auto- and/or allo-immune disease in a mammal, methods of suppressing T cell activity in a mammal, methods of increasing T cell activity in a mammal, and methods of treating or preventing a disease selected from the group consisting of age-related macular degeneration, Alzheimer's disease, and familial ischemic cerebral small-vessel disease in a mammal are further provided by the invention.
  • Figure 1 A is a graph showing tumor size (mm 2 ) in tumor-bearing mice treated with interleukin (IL)-2 and cish '1' (P C ish-/-) (closed square ( ⁇ )), wild-type (PWT) (open square ( ⁇ )), or T cells that underwent no treatment (NT) (H ⁇ ) at days post-treatment.
  • IL interleukin
  • P WT wild-type
  • NT underwent no treatment
  • Figure IB is a graph showing tumor size (mm ) in tumor-bearing mice treated with cish '1' (J* cish-/-) T cells with (closed circle ( ⁇ )) or without interleukin (IL)-2 (closed triangle ( A)), WT (PWT) T cells with (open circle (O)) or without IL-2 (open triangle ( ⁇ )), or NT (*) T cells at days post-treatment.
  • Figure 1C is a graph showing survival of tumor-bearing mice treated with a retroviral vector encoding human gplOO (V), exogenous IL-2 (I), and cish '1' T cells (closed square ( ⁇ )), WT T cells (open square ( ⁇ )), or NT (H ⁇ ) T cells at days post-adoptive cell transfer (ACT).
  • Figure ID is a graph showing tumor size (mm ) in tumor-bearing mice treated with control shMIR-treated (open squares ( ⁇ )) or anti-cish shMIR-treated (closed squares ( ⁇ )) WT CD8 + pmel-l T cells, control shMIR-treated cish ' ' ' T cells (circles ( ⁇ )), or NT (*) T cells at days post-treatment.
  • Figure 2A is a graph showing Tbet expression of cish '1' (squares ( ⁇ )) or WT (circles (O)) T cells relative to ⁇ -actin (ACTb) expression at hours post stimulation.
  • Figure 2B is a graph showing cMyc expression of cish '1' (squares ( ⁇ )) or WT (circles (O)) T cells relative to ⁇ -actin (ACTb) expression at hours post stimulation.
  • Figure 2C is a graph showing cMyc activity in arbitrary units (AU) of cish '1' T cells untransduced (T) or transduced with a Myc (c-Myc) luciferase reporter (squares ( ⁇ )) or WT T cells T cells untransduced (A) or transduced with a Myc (c-Myc) luciferase reporter (circles (O)) relative to ⁇ -actin (ACTb) expression at hours post stimulation.
  • AU arbitrary units
  • Figure 2D is a graph showing Bcl2ll (Bcl-xL) expression of cish '1' (squares ( ⁇ )) or WT (circles (O)) T cells relative to ⁇ -actin (ACTb) expression at hours post stimulation.
  • Figure 2E is a graph showing ccnd2 (cyclin D2) expression of cish '1' (squares ( ⁇ )) or WT (circles (O)) T cells relative to ⁇ -actin (ACTb) expression at hours post stimulation.
  • Figure 3 A is a graph showing ID 1 expression of cish '1' (squares ( ⁇ )) or WT (circles (O)) T cells relative to ⁇ -actin (ACTb) expression at hours post stimulation.
  • Figure 3B is a graph showing Stral3 expression of cish '1' (squares ( ⁇ )) or WT (circles (O)) T cells relative to ⁇ -actin (ACTb) expression at hours post stimulation.
  • Figure 3C is a graph showing SMAD activity in arbitrary units (AU) of cish '1' T cells untransduced ( ⁇ ) or transduced with a SMAD luciferase reporter (squares ( ⁇ )) or WT
  • SMAD luciferase reporter circles (O)
  • ACTb ⁇ -actin
  • Figure 4A is a graph showing Tbet expression relative to ⁇ -actin (ACTb) expression of cish '1' T cells treated with control (closed circles ( ⁇ )) or ti-htral shMIR (open squares ( ⁇ )) or WT T cells treated with control (grey circles (O)) or anti-htr l shMIR (grey squares ( ⁇ )) at hours post stimulation.
  • ACTb ⁇ -actin
  • Figure 4B is an enlargement of Figure 4 A, and is a graph showing Tbet expression relative to ⁇ -actin (ACTb) expression of cish '1' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti- htral shMIR (grey squares) at hours post stimulation.
  • ACTb ⁇ -actin
  • Figure 4C is a graph showing myc (c-Myc) expression relative to ⁇ -actin (ACTb) expression of cish '1' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti-htral shMIR (grey squares) at hours post stimulation.
  • Figure 4D is a graph showing BcUll (Bcl-xL) expression relative to ⁇ -actin (ACTb) expression of cish "1' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti-htral shMIR (grey squares) at hours post stimulation.
  • BcUll Bcl-xL
  • ACTb ⁇ -actin
  • Figure 4E is a graph showing cyclin-El expression relative to ⁇ -actin (ACTb) expression of cish "1' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti-htral shMIR (grey squares) at hours post stimulation.
  • ACTb ⁇ -actin
  • Figure 4F is a graph showing CDC2a expression relative to ⁇ -actin (ACTb) expression of cish ' ' ' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti-htral shMIR (grey squares) at hours post stimulation.
  • ACTb ⁇ -actin
  • Figure 4G is a graph showing Ccndl (cyclin D2) expression relative to ⁇ -actin (ACTb) expression of cish '1' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti-htral shMIR (grey squares) at hours post stimulation.
  • Ccndl cyclin D2 expression relative to ⁇ -actin (ACTb) expression of cish '1' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti-htral shMIR (grey squares) at hours post stimulation.
  • Figure 5 A is a graph showing relative luminescence (RLU) of SBE-luc WT or cish-i- pmel-1 T cells after CD3 stimulation in the presence of exogenous HTRA1 or inactive HTRA1 (S328A) (50 ng/mL).
  • WT + HTRA1 S328A
  • WT + HTRA1 diamonds (O)
  • cish-/- + HTRA1 S328A
  • cish-/- + HTRA1 circles ( ⁇ )).
  • Figure 5B is a graph showing IFN- ⁇ secretion (ng/mL "1 ) after an overnight co-culture of WT or cish-/- pmel-l T cells with peptide-pulsed splenocytes in the presence of exogenous HTRA1 or inactive HTRAl (S328A) (50 g mL).
  • WT + HTRAl (S328A) open squares ( ⁇ )
  • WT + HTRAl diamonds ( )
  • cish-/- + HTRAl (S328A) closed squares ( ⁇ )
  • cish-/- + HTRAl (circles ( ⁇ )).
  • Figure 5C is a graph showing relative luminescence (RLU) of SBE-luc WT or cish-i- pmel-1 T cells after CD3 stimulation in the presence or absence of inactive HTRAl (S328A) (50 Mg mL).
  • WT + HTRAl (S328A) (diamonds ( ⁇ )); WT alone (open squares ( ⁇ )); cish-/- + HTRAl (S328A) (closed squares ( ⁇ )); cish-/- alone (circles ( ⁇ )).
  • Figures 6A-6C are graphs showing expression of gato3 (A), IL-4 (B), or IL-5 (C) (x 10 "3 ) relative to genes housekeeping (HKG) genes (gusb, hprtl, hsp90abl, gapdh, and actb) in WT (circles (o)) or cish-/- p-mel-1 (squares ( ⁇ )) T cells after CD3 stimulation.
  • HKG housekeeping
  • Figures 7A-7H are graphs showing expression of cd27 (A), cd28 (B), icos (C), IL-la (D), IFN-y (E), gzmb (F), prcdl (G), or tnfrsfi (H) (x 10 "3 ) relative to genes housekeeping (HKG) genes (gusb, hprtl, hsp90abl, gapdh, and actb) in WT (circles (o)) or cish-/- p-mel-1 (squares ( ⁇ )) T cells after CD3 stimulation.
  • Figure 8 is a graph showing tumor size (mm 2 ) in tumor-bearing ragl-/- mice days after treatment with nontransduced (NT) pmel-1 T cells (X), wild-type T cells (open circle (o)), or cish -I- T cells (closed circle ( ⁇ )).
  • NT nontransduced
  • X wild-type T cells
  • ⁇ closed circle
  • Figure 9 is a graph showing tumor size (mm ) in tumor-bearing ragl-/- mice days after treatment with nontransduced (NT) pmel-1 T cells (X), cish-/- T cells and antiCD8 antibody on day 38, (grey square), or cish -I- T cells and anti-IgG antibody on day 38 (black square).
  • An embodiment of the invention provides a host cell comprising a cytokine- induced Src homology 2 protein (CIS)/cish inhibitor and a high temperature requirement serine peptidase 1 (HTRAl )lhtral activator.
  • CIS cytokine- induced Src homology 2 protein
  • HTRAl serine peptidase 1
  • CIS also known as Gl 8, SOCS, CIS- 1 , and CISH
  • SOCS suppressor of cytokine signaling family of proteins.
  • the SOCS family also includes SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS 7.
  • SOCS proteins inhibit the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling pathway.
  • the ClS/cish inhibitor may be any suitable agent that inhibits the expression of a cish mRNA or CIS protein.
  • the ClS/cish inhibitor can be a nucleic acid at least about 10 nucleotides in length that specifically binds to and is complementary to a target nucleic acid encoding ClS/cish or a complement thereof.
  • the ClS/cish inhibitor may be introduced into a host cell, wherein the cell is capable of expressing ClS/cish, in an effective amount for a time and under conditions sufficient to interfere with expression of the CIS/ 'cish.
  • RNA interference RNA interference
  • the CIS/cish inhibitor may comprise an RNAi agent.
  • the RNAi agent may comprise a small interfering RNA (siRNA), a microRNA (miRNA), or an antisense nucleic acid.
  • siRNA small interfering RNA
  • miRNA microRNA
  • antisense nucleic acid e.g., siRNA, miRNA, and/or antisense nucleic acid can comprise overhangs. That is, not all nucleotides need bind to the target sequence.
  • RNA interference nucleic acids employed can be at least about 19, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 220, at least about 240, from about 19 to about 250, from about 40 to about 240, from about 60 to about 220, from about 80 to about 200, from about 60 to about 180, from about 80 to about 160, and/or from about 100 to about 140 nucleotides in length.
  • the RNAi agent e.g., siRNA or shRNA
  • a nucleotide sequence included in a cassette e.g., a larger nucleic acid construct such as an appropriate vector.
  • vectors include lentiviral and adenoviral vectors, as well as other vectors described herein with respect to other aspects of the invention.
  • An example of a suitable vector is described in Aagaard et al. Mol. Ther., 15(5): 938-45 (2007).
  • the resulting nucleic acid can be longer than the comprised RNAi nucleic acid, e.g., greater than about 70 nucleotides in length.
  • the RNAi agent employed cleaves the target mRNA. In other embodiments, the RNAi agent employed does not cleave the target mRNA.
  • the antisense nucleic acid comprises a nucleotide sequence complementary to at least about 8, at least about 15, at least about 19, or from about 19 to about 22 nucleotides of a nucleic acid encoding CIS or a complement thereof.
  • the siRNA may comprise, e.g., trans-acting siRNAs (tasiRNAs) and/or repeat- associated siRNAs (rasiRNAs).
  • the miRNA may comprise, e.g., a short hairpin miRNA (shMIR).
  • the CIS/cish inhibitor may inhibit or downregulate to some degree the expression of the protein encoded by a cish gene, e.g., at the DNA, RNA, or other level of regulation.
  • a host cell comprising a CIS/cish inhibitor expresses no CIS/cish or lower levels of CIS/cish as compared to a host cell that lacks a CIS/cish inhibitor.
  • the CIS/cish inhibitor such as an RNAi agent, such as a shMIR, can target a nucleotide sequence of a cish gene or mRNA encoded by the same.
  • the cish sequence is a human sequence.
  • human cish is assigned Gene NCBI Entrez Gene ID No. 1154, and an Online Mendelian Inheritance in Man (OMIM) No. 602441.
  • the human cish gene is found on chromosome 3 at 3p21.3.
  • Two transcriptional variants include mRNAs: NM_013324 and NM_145071, with corresponding protein sequences NP_037456 and NP_659508, respectively.
  • NM_013324 is provided as SEQ ID NO: 1
  • NM_145071 is provided as SEQ ID NO: 2.
  • Human genomic cish sequences include AC096920, AF035946, CH471055, and Z77852.
  • Human cish mRNA sequences also include AF035947, AF132297, AK313850, BC031590, BC064354,
  • Human CIS amino acid sequences include AAQ13420, EAW65127, EAW65128, EAW65129, EAW65130, AAF97410, AAD28471, BAG36578, AAH31590, AAH64354, and BAA92328.
  • Other human sequences, as well as other ClS/cish species can be employed in accordance with the invention.
  • the ClS/cish inhibitor such as an RNAi agent, such as a shMIR
  • RNAi agent such as a shMIR
  • the ClS/cish inhibitor can target a nucleotide sequence selected from the group consisting of the 5' untranslated region (5' UTR), the 3' untranslated region (3' UTR), and the coding sequence of cish, complements thereof, and any combination thereof. Any suitable cish target sequence can be employed.
  • the sequences of the ClS/cish inhibitor can be designed against a human cish with Accession No. NM_013324 (SEQ ID NO: 1) but also recognize NM_145071 (SEQ ID NO: 2).
  • the shMIR comprises SEQ ID NO: 3, encoded by nucleotide sequence SEQ ID NO: 12 and designed against the cish gene target GAGCCTGTTTCTGGGAGAA (SEQ ID NO: 9).
  • the shMIR comprises SEQ ID NO: 4, encoded by the nucleotide sequence SEQ ID NO: 13 and designed against the cish gene target GTCAACGCCTCTAGGTACA (SEQ ID NO: 10).
  • RNAi agents can be designed against any appropriate cish mRNA sequence. In this regard, an
  • embodiment of the invention provides a host cell comprising an anti-cish shMIR comprising SEQ ID NO: 3 or 4.
  • the host cell may or may not further comprise a high temperature requirement serine peptidase 1 (HTRAl )/ ztrai activator.
  • HTRAl also known as L56, ARMD7, ORF480, and PRSS 1 1
  • TGF transforming growth factor
  • the high temperature requirement serine peptidase 1 (KTRAl)/htral activator may comprise any suitable agent that provides the over-expression of a htral mRNA or HTRA1 protein.
  • a host cell comprising a HTRAl/Zztrai activator expresses higher levels of ETRAl/htral than host cells that lack a WTRAl/htral activator.
  • the KTRAl/htral activator may be introduced into a host cell, wherein the cell is capable of expressing KTRAl/htral, in an effective amount for a time and under conditions sufficient to cause the over-expression of HTRAl//ztr 7.
  • the KTRAl/htral activator may comprise a recombinant nucleic acid that encodes KTRAl/htral.
  • the recombinant polynucleotide that encodes KTRAl/htral may comprise a nucleotide sequence comprising SEQ ID NO: 5 (human htral) or SEQ ID NO: 11 (mouse htral).
  • the invention provides a PBC comprising a KTRAl/htral activator. Suitable KTRAl/htral activators are as described herein with respect to other aspects of the invention.
  • the PBC may or may not further comprise a CIS/cish inhibitor. Suitable CIS/cish inhibitors are as described herein with respect to other aspects of the invention.
  • a host cell comprising a KTRAl/htral activator and/or a CIS/cish inhibitor may further comprise and express a nucleic acid comprising a nucleotide sequence encoding a T-cell receptor (TCR) having antigenic specificity for a cancer antigen or a chronic infectious disease antigen.
  • TCR T-cell receptor
  • An immune response may be characterized by an increased production of cytokines such as, e.g., interferon gamma (IFNy), the stimulation of a cell- mediated immune response such as, e.g., the activation of T-cells and/or macrophages, and/or the destruction of cells expressing the cancer antigen or chronic infectious disease antigen.
  • cytokines such as, e.g., interferon gamma (IFNy)
  • IFNy interferon gamma
  • TCRs having antigenic specificity for a cancer antigen include those disclosed in U.S. Patent Application Publication Nos.
  • 2009/0053184 (disclosing TCRs having antigenic specificity for MART-1, NY-ESO-1, or g lOO); 2010/0015113 (disclosing TCRs having antigenic specificity for gplOO); 2010/0034834 (disclosing TCRs having antigenic specificity for NY-ESO-1, MART-1, TRP-1, TRP-2, gplOO, p53, or tyrosinase tumor antigen); and 2009/0304657 (disclosing TCRs having antigenic specificity for MART-1, NY-ESO-1, p53, or gplOO); U.S.
  • Patents 7,820,174 (disclosing TCRs having antigenic specificity for TRAIL presented by TRAIL-R1) and 5,830,755 (disclosing TCRs having antigenic specificity for MART-1 or gplOO); and International Publications WO 2009/042570 (disclosing TCRs having antigenic specificity for NY-ESO-1 , MART-1, TRP-1, TRP-2, gplOO, p53, or tyrosinase tumor antigen) and WO 2010/088160 (disclosing TCRs having antigenic specificity for tyrosinase tumor antigen), each of which are incorporated herein by reference.
  • any of the HTRAl/htral activators and/or CIS/cish inhibitors described herein can be loaded onto nanoparticles.
  • the loaded nanoparticles can be combined with T cells (hereinafter referred to as "nanoparticle composition") and adoptively transferred into patients.
  • the invention provides a composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an HTRAl/htral activator and/or a CIS/cish inhibitor.
  • an HTRAl/htral activator can be loaded on nanoparticles and combined with T cells.
  • the T cells After adoptive transfer of the T cells and loaded nanoparticles, the T cells would then traffic to the tumor, and the HTRAl/htral activator would enhance T cell-mediated tumor killing.
  • the HTRAl/htral activator is a HTRAl protein.
  • the HTRAl protein may comprise any of the human HTRAl amino acid sequences described herein.
  • the HTRAl protein comprises SEQ ID NO: 7.
  • the PBC may be any PBC.
  • the PBC is a T-cell.
  • a T cell comprising a HTRAl/htral activator and/or a CIS/cish inhibitor advantageously demonstrates increased T cell activity.
  • a composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an HTRAl/htral activator and/or a CIS/cish inhibitor also advantageously demonstrates increased T cell activity.
  • T cell activity is increased in accordance with the invention if T cell activity is greater, quantitatively or qualitatively, after administration of a HTRAl/htral activator and/or a CIS/cish inhibitor as compared to the immune response in the absence of the administration of a HTRAl/htral activator and/or a CIS/cish inhibitor.
  • a quantitative increase in T cell activity encompasses an increase in the magnitude or degree of the T cell activity.
  • the magnitude or degree of T cell activity can be measured on the basis of any number of known parameters, such as an increase in the level of antigen-specific cytokine production (cytokine concentration), an increase in the number of T cells activated (e.g., proliferation of antigen- specific T cells) or recruited, an increase in the persistence and/or survival of T cells, and/or a decrease in T-cell death (e.g., apoptosis).
  • Methods of detecting and measuring an increase in T cell activity are known in the art. For example, measuring the types and levels of cytokines produced can detect and measure an increase in T cell activity.
  • An increase in T cell activity may be characterized by an increase in the production of, e.g., IFNy, TNF- , GM-CSF, and/or interleukin (IL)-2, an increase in the production of Thl immunoglobulins, an increase in tumor destruction in vivo, a decrease in TGFpi, and/or an increase in the differentiation of Thl cells.
  • a qualitative increase in T cell activity encompasses any change in the nature of the T cell activity that renders the T cell more effective at combating a given antigen or disease.
  • Qualitative and quantitative increases in T cell activity can occur simultaneously, and are not mutually exclusive.
  • CIS/cish also increases the expression of signal transducer and activator of transcription 5 (STAT5) target genes such as, for example, v-myc,
  • myelocytomatosis viral oncogene homolog avian
  • cMYC Bcl-2-associated death promoter
  • bcl2ll Bcl-xL
  • Tbet T-cell-specific T-box transcription factor
  • Tbet T-cell-specific T-box transcription factor
  • cell cycle genes e.g., ccnd2 (cyclin D2), ccnel (cyclin El), and/or cdkl (cyclin CDC2a)
  • TCR T cell receptor
  • CIS/cish decreases TGF- ⁇ protein levels, decreases TGF- ⁇ signaling (e.g., decreases activation of the TGF- ⁇ signaling molecule, SMAD2), and decreases the expression of TGF- ⁇ target genes such as, for example, cyclin-dependent kinase inhibitor 2B (Cdkn2b) (believed to inhibit the cell cycle), deleted in esophageal cancer 1 (DEC1) (believed to inhibit cell function), inhibitor of DNA binding 1 (ID1) (believed to induce apoptosis), Stral3, and CD 103.
  • Cdkn2b cyclin-dependent kinase inhibitor 2B
  • DEC1 deleted in esophageal cancer 1
  • ID1 inhibitor of DNA binding 1
  • Stral3, and CD 103 apoptosis
  • compositions comprising a CIS/cish inhibitor may be the result of decreased degradation of HTRA1.
  • the invention provides a host cell comprising a HTRAl //ztra7 inhibitor and a CIS/cish activator.
  • the KTRAl/htral inhibitor may be any suitable agent that inhibits the expression of a htral mRNA or protein.
  • the KTRAl/htral inhibitor can be a nucleic acid that specifically binds to and is complementary to a target nucleic acid encoding HTRAl/Zztrai or a complement thereof.
  • the HTRAl /htral inhibitor may be introduced into a host cell, wherein the cell is capable of expressing HTRAl, in an effective amount for a time and under conditions sufficient to interfere with expression of HTRAl .
  • RNA interference RNA interference
  • the HTRAl/htral inhibitor may comprise an RNAi agent.
  • the antisense nucleic acid comprises a nucleotide sequence complementary to at least about 8, at least about 15, at least about 19, or from about 19 to about 22 nucleotides of a nucleic acid encoding HTRAl or a complement thereof.
  • the siRNA may comprise, e.g., trans-acting siRNAs (tasiRNAs) and/or repeat- associated siRNAs (rasiRNAs).
  • the miRNA may comprise, e.g., a short hairpin miRNA (shMIR).
  • the HTRAl/htral inhibitor may inhibit or downregulate to some degree the expression of the protein encoded by a htral gene, e.g., at the DNA, RNA, or other level of regulation.
  • a host cell comprising a HTRAl/htral inhibitor expresses no HTRAl/htral or lower levels of HTRAl /htral as compared to a host cell that lacks a HTRAl/htral inhibitor.
  • the HTRAl/htral inhibitor such as an RNAi agent, such as a shMIR, can target a nucleotide sequence of a htral gene or mRNA encoded by the same.
  • the htral sequence is a human sequence.
  • human htral is assigned Gene NCBI Entrez Gene ID No. 5654, and an Online Mendelian Inheritance in Man (OMIM) No. 602194.
  • the human htral gene is found on chromosome 10 at 10q26.3.
  • the htral mRNA sequence is NM_002775, with corresponding protein sequence NP_002766. Accordingly, NM 002775 is provided as SEQ ID NO: 5.
  • Human genomic htral sequences include AF157623, BX842242, and CH471066.
  • Human htral mRNA sequences include AF070555, AF097709, AI423369, AK091944, AK092476, AK290089, BC01 1352, BC031082, BM993893, CR590731, CR593682, CR596858, CR597732, CR603877, CR605013, CR61 1805, CR613636, CR623030, CR624458,
  • Mouse htral mRNA sequences include NM_019564.
  • Human HTRAl amino acid sequences include AAD41525, EAW49312, EAW49313, AAC97211, BAG52446, BAG52557, BAF82778, and AAH11352.
  • Other human sequences, as well as other HTRAl/htral species can be employed in accordance with the invention.
  • the HTRAl /htral inhibitor such as an RNAi agent, such as a shMIR
  • a nucleotide sequence selected from the group consisting of 5' untranslated region (5' UTR), the 3' untranslated region (3' UTR), and the coding sequence of htral, complements thereof, and any combination thereof.
  • Any suitable htral target sequence can be employed.
  • the sequences of the HTRAl/htral inhibitor can be designed against a human htral with Accession No. NM_002775 (SEQ ID NO: 5).
  • the shMIR comprises SEQ ID NO: 6, designed against the htral gene target GCTATCATCAATTATGGAA (SEQ ID NO: 15).
  • RNAi agents can be designed against any appropriate htral mRNA sequence.
  • HTRAl/Tztrai inhibitor comprising an RNAi agent
  • RNAi agent Other aspects of the HTRAl/Tztrai inhibitor comprising an RNAi agent are as described for the ClS/cish inhibitor.
  • the HTRAl/Zztrai inhibitor comprises NVP- LBP976.
  • NVP-LBP976 has the chemical structure:
  • the ClS/cish activator may comprise any suitable agent that provides the over- expression of a cish mRNA or protein.
  • a host cell comprising a ClS/cish activator expresses higher levels of ClS/cish than host cells that lack a ClS/cish activator.
  • the ClS/cish activator may be introduced into a host cell, wherein the cell is capable of expressing ClS/cish, in an effective amount for a time and under conditions sufficient to cause the over-expression of ClS/cish.
  • the ClS/cish activator may comprise a recombinant nucleic acid that encodes ClS/cish.
  • the recombinant polynucleotide that encodes ClS/cish may comprise a nucleotide sequence comprising SEQ ID NO: 1 or 2.
  • the invention provides a PBC comprising a HTRAl/htral inhibitor. Suitable HTRAl//ztra7 inhibitors are as described herein with respect to other aspects of the invention.
  • the PBC may or may not further comprise a ClS/cish activator. Suitable ClS/cish activators are as described herein with respect to other aspects of the invention.
  • the invention provides a T cell comprising a ClS/cish activator, wherein the T cell has suppressed T cell activity.
  • Suitable ClS/cish activators are as described herein with respect to other aspects of the invention.
  • the T cell may or may not further comprise a HTRAl I htral inhibitor.
  • Suitable HTRAl Ihtral inhibitors are as described herein with respect to other aspects of the invention.
  • any of the HTRAl Ihtral inhibitors and/or ClS/cish activators described herein can be loaded onto nanoparticles.
  • the loaded nanoparticles can be combined with T cells (hereinafter referred to as "nanoparticle composition") and adoptively transferred into patients.
  • the invention provides a composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an HTRAl/htral inhibitor and/or a ClS/cish activator.
  • the ClS/cish activator is a CIS protein.
  • the CIS protein may comprise any of the human CIS amino acid sequences described herein.
  • the CIS protein may comprise SEQ ID NO: 8.
  • the PBC may be any PBC.
  • the PBC is a T-cell.
  • a nanoparticle composition or T cell comprising a UTRAl/htral inhibitor and/or a ClS/cish activator advantageously demonstrates suppressed T cell activity.
  • T cell activity is suppressed in accordance with the invention if the T cell activity is diminished, quantitatively or qualitatively, after administration of a HTRAl/htral inhibitor and/or a ClS/cish activator as compared to the immune response in the absence of the administration of a HTRAl/htral inhibitor and/or a ClS/cish activator.
  • a quantitative decrease in T cell activity encompasses a decrease in the magnitude or degree of the activity.
  • the magnitude or degree of T cell activity can be measured on the basis of any number of known parameters, such as a decrease in the level of cytokine (e.g., antigen-specific cytokine) production (cytokine concentration), a decrease in the number of T cells activated (e.g., proliferation of T cells (e.g., antigen- specific T-cells)) or recruited, etc., an decrease in the persistence and/or survival of T cells, and/or an increase in T -cell death (e.g., apoptosis).
  • cytokine e.g., antigen-specific cytokine
  • T cells activated e.g., proliferation of T cells (e.g., antigen-specific T-cells)
  • T cells e.g., antigen-specific T-cells
  • Suppression of T cell activity may be characterized by a decrease in the production of, e.g., IFNy, TNF-a, GM-CSF, and/or IL-2; a decrease in the production of Thl immunoglobulins, a decrease in tumor destruction in vivo, an increase in TGFpi, and/or a decrease in the differentiation of Thl cells.
  • a qualitative decrease in T cell activity encompasses any change in the nature of the T cell activity that renders it less effective at mediating the destruction of a given antigen.
  • Qualitative and quantitative suppression of T cell activity can occur simultaneously, and are not mutually exclusive.
  • HTRA1 increases the expression of TGF- ⁇ target genes such as, for example, plasminogen activator inhibitor-1 (PAI-1); increases TGF- ⁇ levels; and decreases the expression of STAT5 target genes such as, for example, cMYC, bcUll (Bcl-xL), granzyme B, tbx21 (Tbet), and cell cycle genes (e.g., ccndl (cyclin D2), ccnel (cyclin El), and/or cdkl (cyclin CDC2a)).
  • TGF- ⁇ target genes such as, for example, plasminogen activator inhibitor-1 (PAI-1); increases TGF- ⁇ levels; and decreases the expression of STAT5 target genes such as, for example, cMYC, bcUll (Bcl-xL), granzyme B, tbx21 (Tbet), and cell cycle genes (e.g., ccndl (cyclin D2), ccnel (
  • nucleic acid includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides (e.g., ribonucleic acid nucleotides as well as deoxyribonucleic acid nucleotides), and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
  • nucleotide e.g., ribonucleic acid nucleotides as well as deoxyribonucleic acid nucleotides
  • nucleic acid refers generically to nucleic acids and includes DNA and RNA unless the recitation explicitly states that the nucleic acid is a specific one, e.g., DNA or RNA. If a nucleic acid refers to a sequence that contains thymine (t), that does not necessarily indicate that the nucleic acid is DNA; in some embodiments the nucleic acid is RNA and/or DNA. Similarly, if a nucleic acid refers to a sequence that contains uracil (u) that does not necessarily indicate that the nucleic acid is RNA; in some embodiments the nucleic acid is DNA and/or RNA.
  • t thymine
  • u uracil
  • the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
  • nucleic acid molecules relevant to the invention can readily be obtained in a variety of ways, including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA. These methods and others useful for isolating such DNA are set forth, for example, by Sambrook et al., "Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), by Ausubel, et al., eds., "Current Protocols In Molecular Biology,” Current Protocols Press (1994), and by Berger and Kimmel, "Methods In
  • nucleic acids larger than about 100 nucleotides in length can be synthesized as several fragments, each fragment being up to about 100 nucleotides in length. The fragments can then be ligated together to form a full length nucleic acid encoding the polypeptide.
  • One method is polymer-supported synthesis using standard phosphoramidite chemistry.
  • the nucleic acid can be obtained by screening an appropriate cDNA library prepared from one or more tissue source(s) that express the polypeptide, or a genomic library from any subspecies.
  • the source of the genomic library may be any tissue or tissues from a mammalian or other species believed to harbor a gene encoding a protein relevant to the invention (e.g., CIS and/or HTRA1).
  • the library can be screened for the presence of a cDNA/gene using one or more nucleic acid probes (oligonucleotides, cDNA or genomic DNA fragments that possess an acceptable level of homology to the gene or gene homologue cDNA or gene to be cloned) that will hybridize selectively with the gene or gene homologue cDNA(s) or gene(s) that is(are) present in the library.
  • the probes preferably are
  • the probes can be degenerate. After hybridization, the blot containing the library is washed at a suitable stringency, depending on several factors such as probe size, expected homology of probe to clone, type of library being screened, number of clones being screened, and the like.
  • Stringent washing solutions can be low in ionic strength and are used at relatively high temperatures.
  • PCR polymerase chain reaction
  • poly(A)+RNA or total RNA is extracted from a tissue that expresses the gene product.
  • cDNA is then prepared from the RNA using the enzyme reverse transcriptase.
  • Two primers typically complementary to two separate regions of the cDNA are then added to the cDNA along with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the two primers.
  • the invention provides for the use of isolated, purified or enriched nucleic acid sequences of, for example, about 15 to about 500 nucleotides in length, about 15 to about 100 nucleotides in length, about 15 to about 50 nucleotides in length, and about 15 to about 30 nucleotides in length, which have sequence that corresponds to a portion of one of the nucleotides described herein.
  • the nucleic acid can be at least about 17, about 20, about 22, or about 25 nucleotides in length.
  • the nucleic acid sequence can be about 30 to about 300 nucleotides in length, or about 45 to about 200 nucleotides in length, or about 45 to about 100 nucleotides in length.
  • the nucleic acid can be at least about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 17, about 20, about 22, about 25, about 30, about 35, about 40, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 1000, about 10,000, about 50,000, about 100,000 or more nucleotides in length, or about 100,000, about 75,000, about 50,000, about 10,000, about 5,000, about 1000, about 750, about 500, about 250, about 200, about 100, about 50, about 40, about 30, about 25, about 22, about 20, about 17, about 15, about 12, about 10, about 9, about 8, about 7, about 6, about 5, or fewer nucleotides in length.
  • the nucleic acid can have a length in a range from any one of the above lengths to any other of the above lengths including endpoints.
  • a nucleic acid or protein in accordance with the invention can be at least, e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identical to reference sequences provided herein.
  • a nucleotide that hybridizes under stringent conditions to a nucleotide described herein can be employed. Unless otherwise specified, percent identities for nucleic acids and amino acid sequences are determined as follows.
  • Percent identity of two nucleic acid sequences or two amino acid sequences is determined using the algorithm of Karlin and Altschul ⁇ Proc. Natl. Acad. Set USA, 87:2264-2268 (2002), modified as in Karlin and Altschul et al., Proc. Nat. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J Mol. Biol.
  • Gapped BLAST is utilized as described in Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST are used.
  • the term “specifically hybridizes” indicates that the probe hybridizes to a sufficiently greater degree to the target sequence than to a non-target sequence, e.g., at a level which allows ready identification of probe/target sequence hybridization under selective hybridization conditions.
  • Selective hybridization conditions refer to conditions that allow such differential binding.
  • specifically binds and selective binding conditions refer to such differential binding of any type of probe, and to the conditions that allow such differential binding.
  • the nucleotide sequence encoding CIS and/or HTRAl encodes any suitable CIS and/or HTRAl, respectively, including functional portions or functional variants thereof.
  • the term "functional portion” refers to any part or fragment of the CIS or HTRAl, which part or fragment retains the biological activity of the CIS or HTRAl, respectively, of which it is a part (the parent CIS and/or HTRAl, respectively).
  • the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%), 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more, of the parent CIS or HTRAl , respectively.
  • the term "functional variant” as used herein refers to CIS or HTRAl having substantial or significant sequence identity or similarity to a parent CIS or HTRAl, respectively, which functional variant retains the biological activity of the CIS and/or HTRAl, respectively, of which it is a variant.
  • the functional variant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical in amino acid sequence to the parent CIS or HTRAl, respectively.
  • HTRAl encompass, for example, those parts and variants, respectively, of HTRAl that retain the ability to induce the production of effector cytokines (e.g., IFN- ⁇ , TNF-a, and/or GM-CSF), enhance the production of Thl immunoglobulins, increase tumor destruction in vivo, decrease TGFpi levels, induce the differentiation of Thl cells, or treat or prevent cancer and/or a chronic infectious disease, to a similar extent, the same extent, or to a higher extent, as the parent HTRAl .
  • effector cytokines e.g., IFN- ⁇ , TNF-a, and/or GM-CSF
  • Functional portions and functional variants of CIS encompass, for example, those parts and variants, respectively, of CIS that retain the ability to reduce the production of effector cytokines (e.g., IFN- ⁇ , TNF-a, and/or GM-CSF), reduce the production of Thl immunoglobulins, decrease tumor destruction in vivo, increase TGFpi levels, reduce the differentiation of Thl cells, or treat or prevent auto and/or allo-immune diseases, to a similar extent, the same extent, or to a higher extent, as the parent CIS.
  • effector cytokines e.g., IFN- ⁇ , TNF-a, and/or GM-CSF
  • Thl immunoglobulins decrease tumor destruction in vivo
  • increase TGFpi levels reduce the differentiation of Thl cells
  • treat or prevent auto and/or allo-immune diseases to a similar extent, the same extent, or to a higher extent, as the parent CIS.
  • the nucleic acids described herein are re
  • the term "recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above.
  • the replication can be in vitro replication or in vivo replication.
  • the nucleic acids of the invention can be incorporated into a recombinant expression vector.
  • the term "recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell.
  • the vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring.
  • the recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vectors can comprise naturally-occurring, non-naturally-occurring internucleotide linkages, or both types of linkages.
  • the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.
  • the recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transduce any suitable host cell.
  • Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • the vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).
  • Bacteriophage vectors such as GTIO, XGTl 1, ⁇ (Stratagene), ⁇ 4, and ⁇ 149, also can be used.
  • plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19
  • the recombinant expression vector is a viral vector (e.g., adenoviral vector, adeno-associated viral (AAV) vector, herpes viral vector, retroviral vector, or lentiviral vector) or a transposon vector.
  • a viral vector e.g., adenoviral vector, adeno-associated viral (AAV) vector, herpes viral vector, retroviral vector, or lentiviral vector
  • AAV adeno-associated viral vector
  • the recombinant expression vectors described herein can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra.
  • Constructs of expression vectors which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell.
  • Replication systems can be derived, e.g., from ColEl, 2 ⁇ plasmid, ⁇ , SV40, bovine papilloma virus, and the like.
  • the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
  • regulatory sequences such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
  • the recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transduced host cells.
  • Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host cell to provide prototrophy, and the like.
  • Suitable marker genes for the expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
  • the term "host cell” refers to any type of cell that can contain the recombinant expression vector(s) described herein.
  • the host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa.
  • the host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human.
  • the host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5a E.
  • the host cell is preferably a prokaryotic cell, e.g., a DH5a cell.
  • the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a peripheral blood cell (PBC), peripheral blood leukocyte (PBL), or a peripheral blood mononuclear cell (PBMC).
  • PBC peripheral blood cell
  • PBL peripheral blood leukocyte
  • PBMC peripheral blood mononuclear cell
  • the host cell is a T cell.
  • the T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified.
  • the T cell is a human T cell. More preferably, the T cell is a T cell isolated from a human.
  • the T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4 + /CD8 + double positive T cells, CD4 + helper T cells, e.g., Thi and Th 2 cells, CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells, naive T cells, and the like.
  • CD4 + /CD8 + double positive T cells e.g., CD4 + helper T cells, e.g., Thi and Th 2 cells
  • CD8 + T cells e.g., cytotoxic T cells
  • TILs tumor infiltrating cells
  • memory T cells e.g., naive T cells, and the like.
  • the T cell is a CD8+ T cell or a CD4+ T cell.
  • the host cells can be transduced with the nucleic acids using any suitable method.
  • host cells can be transduced with viral vectors using viruses (e.g., retrovirus or lentivirus) and host cells can be transduced with transposon vectors using electroporation.
  • viruses e.g., retrovirus or lentivirus
  • a population of cells comprising at least one host cell (e.g., a PBC or a T cell) described herein.
  • the population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described herein, in addition to at least one other cell, e.g., a host cell (e.g., a T cell), which does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cells, a muscle cell, a brain cell, etc.
  • a host cell e.g., a T cell
  • a cell other than a T cell e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an end
  • the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) comprising the recombinant expression vector.
  • the population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector.
  • the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.
  • inventive host cells can be isolated and/or purified.
  • isolated as used herein means having been removed from its natural environment.
  • purified as used herein means having been increased in purity, wherein “purity” is a relative term, and not to be necessarily construed as absolute purity.
  • the purity can be at least about 50%, can be greater than 60%, 70% or 80%, or can be 100%.
  • inventive host cells (including populations thereof) and nanoparticle compositions can be formulated into a composition, such as a pharmaceutical composition.
  • a pharmaceutical composition comprising any of the host cells (including populations thereof) and/or nanoparticle compositions described herein, and a pharmaceutically acceptable carrier.
  • composition can comprise an inventive host cell or population thereof and/or nanoparticle composition in combination with other pharmaceutically active agents or drugs, such as a chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • chemotherapeutic agents e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • the carrier is a pharmaceutically acceptable carrier.
  • the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration.
  • the pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.
  • the choice of carrier will be determined in part by the particular host cell, population thereof, or nanoparticle composition, as well as by the particular method used to administer the inventive host cell, population thereof, or nanoparticle composition.
  • compositions of the invention there are a variety of suitable formulations of the pharmaceutical composition of the invention.
  • the following formulations for parenteral, intratumoral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, and interperitoneal administration are exemplary and are in no way limiting. More than one route can be used to administer the inventive host cell, population thereof, or nanoparticle composition, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the inventive host cell, population thereof, or nanoparticle composition can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents
  • Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-p-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.
  • parenteral formulations will typically contain from about 0.05% to about 50% by weight of the host cell, population thereof, or nanoparticle composition, in solution.
  • Preservatives and buffers may be used.
  • such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17.
  • HLB hydrophile-lipophile balance
  • Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • sterile liquid excipient for example, water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
  • injectable formulations are in accordance with the invention.
  • the requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).
  • administering cells e.g., T cells
  • the cells are administered via injection.
  • inventive host cell or population thereof can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, nanoparticles, or liposomes.
  • an assay which comprises comparing the extent to which target cells are lysed by or IFN- ⁇ secretion is increased or decreased in T cells comprising the nucleic acid or recombinant expression vectors described herein upon administration of a given dose of such T cells to a mammal among a set of mammals of which each is given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal.
  • the extent to which IFN- ⁇ is secreted upon administration of a certain dose can be assayed by methods known in the art, including, for instance, the methods described herein in the Examples (e.g., ELISA).
  • the dose of the inventive host cell, populations thereof, or nanoparticle composition also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inventive host cell, populations thereof, or nanoparticle composition.
  • the attending physician will decide the dosage of the inventive host cell, populations thereof, or nanoparticle composition, with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inventive host cell, populations thereof, or nanoparticle composition to be administered, route of administration, and the severity of the condition being treated.
  • the dose of the inventive host cell, populations thereof, or nanoparticle composition can be about 0.001 to about 1000 mg/kg body weight of the subject being treated/day, from about 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1 mg/kg body weight/day.
  • inventive host cell, population of cells, or nanoparticle composition comprising a CIS/cish inhibitor, KTRAl/htral activator, and/or pharmaceutical compositions comprising the same, can be used in methods of increasing T cell activity in a mammal or methods of treating or preventing cancer or a chronic infectious disease in a mammal.
  • nanoparticle compositions or cells comprising a CIS/cish inhibitor and/or a KTRAl/htral activator advantageously increase the production of effector cytokines (e.g., IFN- ⁇ , IL-2); increase T cell proliferation, persistence, survival, and cytolytic activity; decrease T cell apoptosis; and/or decrease tumor size in vivo as compared to nanoparticle compositions or cells that lack any one or more of a CIS/cish inhibitor and/or a HTRAl/ ztra7 activator.
  • effector cytokines e.g., IFN- ⁇ , IL-2
  • an embodiment of the invention provides a method of treating or preventing cancer or a chronic infectious disease in a mammal, comprising administering to the mammal a host cell, population of cells, or nanoparticle composition, comprising a CIS/cish inhibitor, a
  • KTRAl/htral activator and/or pharmaceutical compositions comprising the same, in an amount effective to treat or prevent cancer or the infectious disease in the mammal.
  • Another embodiment of the invention comprises a method of increasing T cell activity in a mammal, comprising administering to the mammal a host cell, population of cells, or nanoparticle composition, comprising a CIS/cish inhibitor, a KTRAl/htral activator, and/or pharmaceutical compositions comprising the same, in an amount effective to increase T cell activity in the mammal.
  • inventive host cell, population of cells, or nanoparticle composition comprising a CIS/cish activator, a KTRAl/htral inhibitor, and/or pharmaceutical compositions comprising the same, can be used in methods of suppressing T cell activity in a mammal or methods of treating or preventing an auto- and/or allo-immune disease in a mammal.
  • cells or nanoparticle compositions comprising a CIS/cish activator and/or a KTRAl/htral inhibitor advantageously decrease the production of effector cytokines (e.g., IFN- ⁇ , IL-2); decrease T cell proliferation, persistence, survival, and cytolytic activity; and/or increase T cell apoptosis as compared to nanoparticle compositions or cells that lack any one or more of a ClS/cish activator and/or a HTRAl/htral inhibitor.
  • effector cytokines e.g., IFN- ⁇ , IL-2
  • T cell proliferation, persistence, survival, and cytolytic activity e.g., T cell proliferation, persistence, survival, and cytolytic activity
  • T cell apoptosis compared to nanoparticle compositions or cells that lack any one or more of a ClS/cish activator and/or a HTRAl/htral inhibitor.
  • an embodiment of the invention provides a method of treating or preventing an auto- and/or allo-immune disease in a mammal comprising administering to the mammal a host cell, population of cells, or nanoparticle composition, comprising any one or more of a ClS/cish activator, a
  • HTRAl//ztra7 inhibitor and/or pharmaceutical compositions comprising the same, in an amount effective to treat or prevent an auto- and/or allo-immune disease in the mammal.
  • Another embodiment of the invention comprises a method of suppressing T cell activity in a mammal, comprising administering to the mammal a host cell, population of cells, or nanoparticle composition comprising a ClS/cish activator, a HTRAl/htral inhibitor, and/or pharmaceutical compositions comprising the same, in an amount effective to suppress T cell activity in the mammal.
  • Another embodiment of the invention comprises a method of treating or preventing a disease selected from the group consisting of age-related macular degeneration, Alzheimer's disease, and familial ischemic cerebral small- vessel disease in a mammal, comprising administering to the mammal a PBC, a host cell, population of cells, or nanoparticle composition, comprising a ClS/cish inhibitor, a HTRAl/htral activator, a ClS/cish activator, a HTRAl/htral inhibitor, and/or pharmaceutical compositions comprising the same, in an amount effective to treat or prevent the disease in the mammal.
  • a disease selected from the group consisting of age-related macular degeneration, Alzheimer's disease, and familial ischemic cerebral small- vessel disease in a mammal
  • the method comprises treating or preventing Alzheimer's disease or familial ischemic cerebral small-vessel disease comprising administering to the mammal a PBC, a host cell, population of cells, or nanoparticle composition, comprising a ClS/cish inhibitor or a HTRAl/htral activator, and/or pharmaceutical compositions comprising the same, in an amount effective to treat or prevent the disease in the mammal.
  • the method comprises treating or preventing age-related macular degeneration comprising administering to the mammal a PBC, a host cell, population of cells, or nanoparticle composition, comprising a ClS/cish activator or a HTRAl/htral inhibitor, and/or pharmaceutical compositions comprising the same, in an amount effective to treat or prevent the disease in the mammal.
  • inventive methods can provide any amount of any level of treatment or prevention of cancer, a chronic infectious disease, an allo-immune disease, and/or an autoimmune disease in a mammal.
  • the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, a chronic infectious disease, an allo-immune disease, and/or an autoimmune disease, being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • the cells can be cells that are allogeneic or autologous to the mammal.
  • the cells are autologous to the mammal.
  • the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,
  • the chronic infectious disease can be any chronic infectious disease, including any of HIV, influenza, herpes, and malaria.
  • the auto- or allo-immune disease that may be treated by the inventive methods may be any auto- or allo-immune disease.
  • the autoimmune diseases that may be treated by the inventive methods may be generally characterized by an inappropriate immune reaction against self cells, tissues, and/or organs.
  • autoimmune diseases include, but are not limited to: vitiligo, alopecia, autoimmune kidney disease, celiac disease, inflammatory bowel disease, hepatitis, Addison's disease, Hashimoto's disease, Graves disease, atrophic gastritis/pernicious anemia, acquired hypogonadism/infertility, hypoparathyroidism, multiple sclerosis, Myasthenia gravis, Coombs positive hemolytic anemia, systemic lupus
  • the allo-immune diseases that may be treated by the inventive methods may be generally characterized by an immune reaction against foreign or transplanted organs, tissues, and/or cells.
  • Exemplary allo-immune diseases include, but are not limited to: acute and chromic graft-versus-host disease (GVHD) and/or graft rejection that can occur, e.g., in the setting of solid organ transplantation (e.g., pancreatic, renal, cardiac, stem cell, or liver transplantation) and graft-versus-host disease that can occur in the setting of allogeneic hematopoietic stem cell transplantation (e.g., bone marrow, peripheral blood, or cord blood transplantation).
  • GVHD acute and chromic graft-versus-host disease
  • graft rejection that can occur, e.g., in the setting of solid organ transplantation (e.g., pancreatic, renal, cardiac, stem cell, or liver transplantation)
  • graft-versus-host disease e.g., bone marrow, peripheral blood, or cord blood transplantation
  • the mammal referred to in the inventive methods can be any mammal.
  • the term "mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order
  • Artiodactyla including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
  • Pmel-1 Thyl .l, Pmel-1 Ly5.1, pmel-1 cish- ⁇ -, RAG1-/-, C57BL/6 mice were bred, genotyped and housed according to the guidelines of the Animal Care and Use Committee at the National Institutes of Health.
  • B16 melanoma was obtained from the NCI Tumor Repository and grown in 10% FCS containing culture media.
  • CD8 + T cells were isolated from splenocytes by magnetic bead negative selection (Miltenyi Biotec or Stemcell Technologies). Primary stimulation was accomplished using plate-bound anti-CD3 (1 ⁇ g/mL) and soluble anti-CD28 (1 ⁇ g/mL) (BD Biosciences).
  • Pmel-1 splenocytes were stimulated with 1 ⁇ hgpl00 25 _33 peptide (Anaspec) in culture medium containing 2 ng ml "1 IL-2 (Chiron
  • mice were implanted with subcutaneous B16 melanoma (1 x 10 5 -5 x 10 s ).
  • mice 10-14 days post implantation, mice (n > 5 for all groups unless otherwise indicated) were injected intravenously (i.v.) with CD8-enriched naive or in vitro activated pmel-1 splenocytes (0.25 x 10 6 -1 x 10 6 CD8+ Vpl3+ T cells), and received 0.5 x 10 7 -2 x 10 7 plaque-forming units of rVV or fowlpox encoding hgplOO (Bernard Moss, National Institutes of Health).
  • mice received interperitoneal (i.p.) injections of hIL-2 (Palmer et al. PNAS 105:8061-66 (2008)) or 100 ⁇ g of CD8-depleting antibody (53.6.7, BD
  • PharMingen was injected i.p. Mice were randomized, and tumors were blindly measured by using digital calipers. The products of the perpendicular diameters are presented as mean ⁇ SEM. At times indicated after ACT spleens were harvested, ACK-lysed, enumerated, stained, and evaluated by flow cytometry. For time course experiments, splenocytes were enriched for the congenic marker using Miltenyi or Cell-Sep protocols, cyro-preserved, thawed simultaneously, enumerated and stimulated as indicated.
  • apoptosis evaluation following stimulation with aCD3/aCD28, cells were stained with annexin and 7AAD as per the manufacturer's instructions (BD Pharmigen). After surface staining, intracellular staining was accomplished by using Ctyofix/cytoperm, then Phosphobuffer II or III, and antibody as per the manufacturer's instructions (BD Pharmigen). For the co- immunoprecipitation protocol, cells were lysed, cleared, and normalized using a
  • BCA bicinchoninic acid
  • IP antibody- bonded immunoprecipitation
  • Western blotting was performed using Bio-Rad reagents and protocols on nitrocellulose paper, stained with aV5, aCIS, aHtrAl, (Santa Cruz), SMAD2, pSMAD2, pSTAT5 (Cell Signal Technologies). Blots were developed using chemiluminescence (Pierce) and acquired using ChemidocTM system (Bio-Rad).
  • iCIS and iRFP cell lines were developed by transducing 293T cells with a lentivirus containing CIS or RFP driven by a tetracycline promoter, and phoshoglycerate kinase promoter driven TetR and puromycin resistance genes. Cells were then selected using puromycin (3 ⁇ g/mL). Retroviral production and transduction were accomplished as previously described (Abad et al. J. Immunother. 31 : 1-6 (2008)). The shorthairpin microRNA sequences used are shown in Table 1. Knockdown was evaluated by taqman or western blotting.
  • Yeast two-hybrid screen was performed by ProteinLinks as previously described (Xu et al. PNAS 94:12473-78 (1997)). Briefly, CIS-bait was cloned into TetR or LexA vectors and screened against a murine spleen-derived cDNA library (5 x 10 or 1 x 10 clones). All positive interactions were confirmed using co-immunoprecipitation.
  • lymphopoeisis, memory state, proliferation, and cell surface marker expression of cish "1' CD8 + T cells is similar to that of wild-type cells.
  • This example also demonstrates that cish mRNA expression increases following stimulation of wild-type T cells but not cish '1' T cells.
  • CIS melanoma/melanocyte-specific T cell receptor
  • CIS has been shown to be induced by IL-2 and TCR stimulation (Aman et al. J. Biol. Chem. 274:30266-72 (1999)).
  • cish mRNA expression was found to increase in both naive and primed CD8 + pmel-1 T cells (WT) upon stimulation (or re-stimulation), but not cish knockouts ⁇ cish '1' ).
  • CD8 + WT CD8 + WT
  • CD28 + WT CD8 + WT
  • CD69, CD80, CD86, LFA-1, Sca-1, CD 127, KLRG1 and many others also yielded no significant differences.
  • cish '1' CD 8 + pmel-1 T cells acquired the memory marker CD44, diluted out carboxyfluorescein (CFSE), and proliferated similarly to pmel-1 WT CD8 + T cells after stimulation.
  • CFSE carboxyfluorescein
  • IFN- ⁇ secretion was also evaluated by ELISA after an overnight co-culture of primed WT or cish '1' pmel-1 T cells. Upon antigen re-encounter, previously primed cish " ' " pmel-1 T cells released dramatically more IFN- ⁇ than wild-type (WT) T cells (Table 2A).
  • IL-2 secretion was also evaluated by ELISA after an overnight co-culture of primed WT or cish-l- pmel-1 T cells. When assayed for IL-2 or TNF-a after a co-culture of primed CD8 + T cells, it was observed that cish " ' " CD8 + T cells also released more IL-2 or TNF-a (Table 2A).
  • PBL transduced with a T cell receptor also secrete greater levels of IFN- ⁇ in the absence of cish.
  • PBL that were mock-transduced or transduced with T cell receptor 1G4 and shRNA retrovirus targeting cish (or scramble as a control) were co-cultured with 624mel, 526mel, or 888mel overnight and selected for 4 days with puromycin (5 x 10 4 : 5 x 10 4 ). The results are shown in Table 2B. TABLE 2B
  • mice were implanted with non- immunogenic B16 melanoma cells. After the tumor was established and vascularized, the mice were treated with naive CD8 + mel-l T cells (cish ' ' ' or WT) and recombinant vaccine encoding gplOO with or without exogenous IL-2.
  • CIS has been implicated in the suppression of STAT5 signaling, presumably by blocking STAT5 docking to the activated cytokine receptor (Aman et al. J Biol. Chem.
  • STAT5 phosphorylation was measured by flow cytometric analysis in naive CD8 + WT or cish '1" pmel-1 T cells after the addition of 150 IU of IL-2. However, after careful analysis with a myriad of doses of cytokines, including IL-2, IL-7, IL-15, or IL-21, and time points, no discernable differences in STAT5 phosphorylation were observed in the absence of cish. Because increased functionality after TCR stimulation was observed, it was
  • a yeast-two hybrid screen was performed using CIS as bait and a mouse spleen cDNA library as prey.
  • CIS interacted with the ATP-independent serine protease HTRAl .
  • RFP red fluorescent protein
  • iRFP and iCIS CIS tet-inducible lentiviruses
  • iRFP and iCIS-transduced cells expressed their respective products, as confirmed by flow cytometric and immunoblot analyses. These lines were then transfected with Chloromphenicol R -V5 or htral -V 5 containing plasmids and confirmed for protein expression by immunoblot analysis. Upon immunoprecipation of CIS and immunoblotting for V5, it was found that CIS directly associated with HTRAl and not the control. Immunoprecipation of V5 and immunoblotting of CIS yielded a similar result.
  • PCR Real-time polymerase chain reaction
  • TGF- ⁇ evaluation by ELISA in an overnight co-culture with gpl00 25-33 revealed decreased TGF- ⁇ protein in cish '1' (80 pg ml/ 1 TGF- ⁇ ) as compared to WT (225 pg ml/ 1 TGF- ⁇ ) pmel-1 T cells, which was not attributable to decreased TGF- ⁇ ⁇ mRNA levels.
  • TGF- ⁇ 1 secretion was evaluated by ELIS A in an overnight culture of primed WT or cish '1' pmel-1 T cells in the presence or absence of NVP-LBP976. Inhibition of HTRAl proteolytic activity by NVP-LBP976 resulted in increased TGF- ⁇ protein levels in the absence of cish (Table 4).
  • TGF- ⁇ signaling has been implicated in the suppression of many pro- functional/survival genes such as tbx21 (T-bet), myc (c-Myc), Bcl-2/xL, and cyclins in CD8 + T cells (Li et al. Cell 134: 392-04 (2008); Sanjabi et al. Immunity 31 :131-44 (2009); Wan et al. Immunol. Rev. 220: 199-13 (2007); Wan et al. J Clin. Immunol. 2: 647-59 (2008)).
  • TGF- ⁇ has been implicated in the induction of the gut-homing receptor CD 103 in CD8 + T cells (El-Asady et al. J Exp. Med. 201 : 1647-57 (2005); Uss et al. J. Immunol. 177: 2775-83 (2006)).
  • flow cytometric analysis revealed reduced CD 103 expression in primed cish '1' pmel-1 T cells relative to WT pmel-1 T cells.
  • CD 103 was highly upregulated in the presence of cish after stimulation.
  • TGF- ⁇ signaling can induce the expression of genes such as ID1 and Stral3 that were found to be upregulated in WT pmel-1 T cells after stimulation ( Figure 3A-B). These findings correlated with increased SMAD reporter activity in lentiviral transduced WT pmel-1 T cells ( Figure 3C).
  • bromodeoxyuridine (BrdU) was injected. In vivo BrdU
  • phosphatidylserine normally expressed only on the cytoplasmic side of the cell membrane except in T cells experiencing apoptosis, revealed that the presence of cish was associated with increased staining and thus more apoptotic cell death.
  • the percentage of annexin V positive WT or cish '1' pmel-1 T cells was measured on days 5, 6, and 7 following adoptive transfer. The results are shown in Table 6.
  • Bcl-2 an anti-apoptotic factor suppressed by TGF- ⁇
  • MFI mean fluorescence intensity
  • control-transduced cish '1' CD8 + pmel-1 T cells experienced enhanced up-regulation of thxll (T-bet), myc (c-Myc), bcl2ll (Bcl-xL), ccnel (cyclin El), cdkl (cyclin CDC2a) and ccnd2 (cyclin D2) (Figure 4A-F).
  • the knockdown of htral in cish '1' CD8 + pmel-1 T cells significantly abrogated the enhanced expression of these target genes (Figure 4A-F).
  • WT CD8 + pmel-1 T cells experienced a significantly lower induction of these target genes and expressed low levels of HTRAL Nonetheless, the knockdown of htral in WT CD8 + pmel-1 T cells appeared to result in a significant (p ⁇ 0.0001) albeit modest, inhibition of several of these targets compared to control WT T cells.
  • cish expression in control or htral (SEQ ID NO: 6) shMIR transduced WT or cish '1" pmel-1 T cells was also measured by real-time PCR. There was no significant difference in the induction of cish expression with the knockdown of htral in WT CD8 + pmel-1 T cells. Knockdown of htral in cish '1' T cells abrogated the enhanced expression of known targets of TGF- ⁇ suppression and induced expression of TGF- ⁇ promoted factors. Thus, it appears that there exists a causal relationship between the enhanced functionality of cish '1' CD8 + T cells and the presence of htral. It appears that the presence of CIS might be involved in the removal or degradation of HTRAL
  • HtrAl V5 or Control- V5 was transfected into iCIS or iRFP cells. After induction of CIS or RFP by doxycycline, V5 levels were evaluated by immunoblotting. A densitometric evaluation of HtrAl V5 or control V5 staining relative to ⁇ -actin was also performed following the administration of doxycycline. The results are shown in Table 8. TABLE 8
  • proteasome inhibitor I (Calbiochem) were added after stimulation of primed WT or cish '1' CD8 + pmel-1 T cells.
  • a selective and titratable increase in HTRAl protein was observed as the concentration of proteasome inhibitor increased in the presence (WT) but not in the absence of cish.
  • titrated levels (0, 0.1, 1, or 10 ⁇ ) of proteasome inhibitor I were added and immunoblot analysis for SMAD2 phosphorylation was performed. Here, it was found that with increased protreasome inhibitor and
  • yc cytokines such as IL-2
  • TCR stimulation drive the expression of STAT5 target genes including cish.
  • TGF- ⁇ is degraded by HTRAl, but in the presence of CIS, TGF- ⁇ is preserved while HTRAl is degraded.
  • TGF- ⁇ then acts in a negative feed-back loop, inhibiting STAT5 target gene expression, and that TGF- ⁇ does not appear to negatively regulate CIS expression.
  • HtrA and control HtrA S328A were produced.
  • Micro-scale purification screening of HtrA and HtrA S328A from insect and mammalian cell culture was performed using 20 ml of transiently transfected HEK293E expression culture or 50 ml of Baculovirus Expression Vector System (BEVS) expression culture as source material. Gels are stained with Coomassie ® blue dye, which confirmed that HtrA and HtrA S328A were purified.
  • BEVS Baculovirus Expression Vector System
  • HTRAl or HTRAl (S328A) (1 ⁇ g) was cultured overnight with hTGFpl (2 ⁇ g) with or without 50mM Tris. The samples were run on a gel and stained with Coomassie ® blue dye. The gels showed that HTRAl modestly degraded TGFpi, but HTRAl (S328A) did not degrade TGFpi .
  • WT or cish-i- pmel-1 T cells were cultured overnight with peptide-pulsed splenocytes in the presence of exogenous HTRAl or inactive HTRAl (S328A) (50 ⁇ g/mL). IFN- ⁇ production was evaluated by ELISA.
  • Pmel-1 T cells were transduced with reporter-lentivirus after CD3 stimulation and the relative luminescence (RLU) was measured. An increase in NFKB expression was observed in cish deficient T cells as compared to WT cells. No significant difference in MAPK/ERK was observed in cish deficient T cells as compared to WT cells.
  • P-mel- 1 T cells 2.5 x 10 5 ) (nontransduced (NT), WT or cish-/-) were adoptively transferred into tumor-bearing ragl-/- mice (8 x 10 6 rVVgplOO, IL-2 (12 ⁇ g) BID x 3 days). Tumor size was measured. The removal of cish prolonged tumor therapy in the absence of host T cells (Figure 8).
  • P-mel-1 T cells 2.5 x 10 5 ) (nontransduced (NT), cish-/- with anti-CD8 on day 38, or cish-/- with anti-IgG on day 38) were adoptively transferred into tumor-bearing ragl-/- mice (8 x 10 6 rVVgplOO, IL-2 (12 ⁇ g) BID x 3 days). Tumor size was measured. The removal of cish prolonged tumor therapy in the absence of host T cells or host B cells ( Figure 9) ⁇
  • naive WT or cish '1' pmel-1 T cells were co-cultured with pulsed targets (gpl00 25-33 ).
  • IFN- ⁇ secretion was evaluated by ELISA after overnight co-culture. It was observed that removal of cish resulted in increased ex vivo function as measured by IFN- ⁇ secretion (Table 10).

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Abstract

The invention provides a peripheral blood cell (PBC) comprising a high temperature requirement serine peptidase 1 (HTRA1)/htra1 activator, a host cell comprising a cytokine-induced Src homology 2 protein (CIS)/cish inhibitor and a HTRA1/htra1 activator, a host cell comprising an anti-cish shMIR comprising SEQ ID NO: 3 or 4, and related populations of cells, pharmaceutical compositions, methods of treating or preventing cancer or a chronic infectious disease in a mammal, and methods of increasing T cell activity in a mammal. The invention also provides a host cell comprising a HTRA1/htra1 inhibitor and a CIS/cish activator, a PBC comprising a HTRA1/htra1 inhibitor, and related populations of cells, pharmaceutical compositions, methods of treating or preventing an auto- and/or allo-immune disease in a mammal, and methods of suppressing T cell activity in a mammal.

Description

MODULATING IMMUNE CELL ACTIVITY USING CYTOKINE-INDUCED SRC HOMOLOGY 2 AND/OR HIGH TEMPERATURE REQUIREMENT A-l
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 61/420,825, filed December 8, 2010, which is incorporated herein by reference in its entirety.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED
ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a computer-readable
nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 20,489 Byte ASCII (Text) file named "709100_ST25.TXT" dated October 25, 2011.
BACKGROUND OF THE INVENTION
[0003] Adoptive cell therapy can be an effective treatment for cancer in some patients. However, obstacles to the overall success of adoptive cell therapy still exist. For example, the adoptive transfer of T-cells specific for tumor antigens or self-antigens can, in some cases, result in toxicities (Palmer et al., PNAS 105(23):8061 -66 (2008); Johnson et al., Blood 114(3):535-46 (2009)). In addition, the in vivo persistence, survival, and anti-tumor activity of T cells can, in some cases, decrease following adoptive transfer (Heemskerk et al. Hum. Gene Ther. 19(5):496-510 (2008)).
[0004] In spite of considerable research into treatments for cancer, chronic infectious diseases, autoimmune diseases, and allo-immune diseases, there still exists a need for improved compositions and methods for treating and/or preventing cancer, chronic infectious diseases, autoimmune diseases, and allo-immune diseases.
BRIEF SUMMARY OF THE INVENTION
[0005] An embodiment of the invention provides a host cell comprising an high temperature requirement serine peptidase 1 (HTRAl)//tfra/ inhibitor and a cytokine-induced Src homology 2 protein (ClS)/cish activator. [0006] Another embodiment of the invention provides a peripheral blood cell (PBC) comprising a HTRAl/htral inhibitor.
[0007] Still another embodiment of the invention provides a host cell comprising an anti- cish shMIR comprising SEQ ID NO: 3 or 4 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 3 or 4.
[0008] Another embodiment of the invention provides a PBC comprising a HTRAl /htral activator.
[0009] Still another embodiment of the invention provides a host cell comprising a ClS/cish inhibitor and a HTRAl/htral activator.
[0010] An embodiment of the invention provides a composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an HTRAl I htral inhibitor and/or a ClS/cish activator.
[0011] An embodiment of the invention provides a composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an HTRAl /htral activator and/or a ClS/cish inhibitor.
[0012] The invention further provides embodiments including populations of cells and pharmaceutical compositions relating to the host cells (e.g., PBCs and T cells) and populations of cells of the invention.
[0013] Embodiments of methods of treating or preventing cancer or a chronic infectious disease in a mammal, methods of treating or preventing an auto- and/or allo-immune disease in a mammal, methods of suppressing T cell activity in a mammal, methods of increasing T cell activity in a mammal, and methods of treating or preventing a disease selected from the group consisting of age-related macular degeneration, Alzheimer's disease, and familial ischemic cerebral small-vessel disease in a mammal are further provided by the invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0014] Figure 1 A is a graph showing tumor size (mm2) in tumor-bearing mice treated with interleukin (IL)-2 and cish'1' (PCish-/-) (closed square (■)), wild-type (PWT) (open square (□)), or T cells that underwent no treatment (NT) (H<) at days post-treatment.
[0015] Figure IB is a graph showing tumor size (mm ) in tumor-bearing mice treated with cish'1' (J* cish-/-) T cells with (closed circle (·)) or without interleukin (IL)-2 (closed triangle ( A)), WT (PWT) T cells with (open circle (O)) or without IL-2 (open triangle (Δ)), or NT (*) T cells at days post-treatment. [0016] Figure 1C is a graph showing survival of tumor-bearing mice treated with a retroviral vector encoding human gplOO (V), exogenous IL-2 (I), and cish'1' T cells (closed square (■)), WT T cells (open square (□)), or NT (H<) T cells at days post-adoptive cell transfer (ACT).
[0017] Figure ID is a graph showing tumor size (mm ) in tumor-bearing mice treated with control shMIR-treated (open squares (□)) or anti-cish shMIR-treated (closed squares (■)) WT CD8+pmel-l T cells, control shMIR-treated cish''' T cells (circles (·)), or NT (*) T cells at days post-treatment.
[0018] Figure 2A is a graph showing Tbet expression of cish'1' (squares (■)) or WT (circles (O)) T cells relative to β-actin (ACTb) expression at hours post stimulation.
[0019] Figure 2B is a graph showing cMyc expression of cish'1' (squares (■)) or WT (circles (O)) T cells relative to β-actin (ACTb) expression at hours post stimulation.
[0020] Figure 2C is a graph showing cMyc activity in arbitrary units (AU) of cish'1' T cells untransduced (T) or transduced with a Myc (c-Myc) luciferase reporter (squares (■)) or WT T cells T cells untransduced (A) or transduced with a Myc (c-Myc) luciferase reporter (circles (O)) relative to β-actin (ACTb) expression at hours post stimulation.
[0021] Figure 2D is a graph showing Bcl2ll (Bcl-xL) expression of cish'1' (squares (■)) or WT (circles (O)) T cells relative to β-actin (ACTb) expression at hours post stimulation.
[0022] Figure 2E is a graph showing ccnd2 (cyclin D2) expression of cish'1' (squares (■)) or WT (circles (O)) T cells relative to β-actin (ACTb) expression at hours post stimulation.
[0023] Figure 3 A is a graph showing ID 1 expression of cish'1' (squares (■)) or WT (circles (O)) T cells relative to β-actin (ACTb) expression at hours post stimulation.
[0024] Figure 3B is a graph showing Stral3 expression of cish'1' (squares (■)) or WT (circles (O)) T cells relative to β-actin (ACTb) expression at hours post stimulation.
[0025] Figure 3C is a graph showing SMAD activity in arbitrary units (AU) of cish'1' T cells untransduced (Ξ) or transduced with a SMAD luciferase reporter (squares (■)) or WT
T cells untransduced (®) or transduced with a SMAD luciferase reporter (circles (O)) relative to β-actin (ACTb) expression at hours post stimulation.
[0026] Figure 4A is a graph showing Tbet expression relative to β-actin (ACTb) expression of cish'1' T cells treated with control (closed circles (·)) or ti-htral shMIR (open squares (□)) or WT T cells treated with control (grey circles (O)) or anti-htr l shMIR (grey squares (□)) at hours post stimulation. [0027] Figure 4B is an enlargement of Figure 4 A, and is a graph showing Tbet expression relative to β-actin (ACTb) expression of cish'1' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti- htral shMIR (grey squares) at hours post stimulation.
[0028] Figure 4C is a graph showing myc (c-Myc) expression relative to β-actin (ACTb) expression of cish'1' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti-htral shMIR (grey squares) at hours post stimulation.
[0029] Figure 4D is a graph showing BcUll (Bcl-xL) expression relative to β-actin (ACTb) expression of cish"1' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti-htral shMIR (grey squares) at hours post stimulation.
[0030] Figure 4E is a graph showing cyclin-El expression relative to β-actin (ACTb) expression of cish"1' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti-htral shMIR (grey squares) at hours post stimulation.
[0031] Figure 4F is a graph showing CDC2a expression relative to β-actin (ACTb) expression of cish''' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti-htral shMIR (grey squares) at hours post stimulation.
[0032] Figure 4G is a graph showing Ccndl (cyclin D2) expression relative to β-actin (ACTb) expression of cish'1' T cells treated with control (black circles) or anti-htral shMIR (white squares) or WT T cells treated with control (grey circles) or anti-htral shMIR (grey squares) at hours post stimulation.
[0033] Figure 5 A is a graph showing relative luminescence (RLU) of SBE-luc WT or cish-i- pmel-1 T cells after CD3 stimulation in the presence of exogenous HTRA1 or inactive HTRA1 (S328A) (50 ng/mL). WT + HTRA1 (S328A) (open squares (□)); WT + HTRA1 (diamonds (O)); cish-/- + HTRA1 (S328A) (closed squares (■)); cish-/- + HTRA1 (circles (·)).
[0034] Figure 5B is a graph showing IFN-γ secretion (ng/mL"1) after an overnight co-culture of WT or cish-/- pmel-l T cells with peptide-pulsed splenocytes in the presence of exogenous HTRA1 or inactive HTRAl (S328A) (50 g mL). WT + HTRAl (S328A) (open squares (□)); WT + HTRAl (diamonds ( )); cish-/- + HTRAl (S328A) (closed squares (■)); cish-/- + HTRAl (circles (·)). [0035] Figure 5C is a graph showing relative luminescence (RLU) of SBE-luc WT or cish-i- pmel-1 T cells after CD3 stimulation in the presence or absence of inactive HTRAl (S328A) (50 Mg mL). WT + HTRAl (S328A) (diamonds (♦)); WT alone (open squares (□)); cish-/- + HTRAl (S328A) (closed squares (■)); cish-/- alone (circles (·)).
[0036] Figures 6A-6C are graphs showing expression of gato3 (A), IL-4 (B), or IL-5 (C) (x 10"3) relative to genes housekeeping (HKG) genes (gusb, hprtl, hsp90abl, gapdh, and actb) in WT (circles (o)) or cish-/- p-mel-1 (squares (■)) T cells after CD3 stimulation.
[0037] Figures 7A-7H are graphs showing expression of cd27 (A), cd28 (B), icos (C), IL-la (D), IFN-y (E), gzmb (F), prcdl (G), or tnfrsfi (H) (x 10"3) relative to genes housekeeping (HKG) genes (gusb, hprtl, hsp90abl, gapdh, and actb) in WT (circles (o)) or cish-/- p-mel-1 (squares (■)) T cells after CD3 stimulation.
[0038] Figure 8 is a graph showing tumor size (mm2) in tumor-bearing ragl-/- mice days after treatment with nontransduced (NT) pmel-1 T cells (X), wild-type T cells (open circle (o)), or cish -I- T cells (closed circle (·)).
[0039] Figure 9 is a graph showing tumor size (mm ) in tumor-bearing ragl-/- mice days after treatment with nontransduced (NT) pmel-1 T cells (X), cish-/- T cells and antiCD8 antibody on day 38, (grey square), or cish -I- T cells and anti-IgG antibody on day 38 (black square).
DETAILED DESCRIPTION OF THE INVENTION
[0040] An embodiment of the invention provides a host cell comprising a cytokine- induced Src homology 2 protein (CIS)/cish inhibitor and a high temperature requirement serine peptidase 1 (HTRAl )lhtral activator.
[0041] CIS (also known as Gl 8, SOCS, CIS- 1 , and CISH) is a member of the suppressor of cytokine signaling (SOCS) family of proteins. In addition to CIS, the SOCS family also includes SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS 7. Without being bound to a particular theory, it is believed that SOCS proteins inhibit the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling pathway.
[0042] The ClS/cish inhibitor may be any suitable agent that inhibits the expression of a cish mRNA or CIS protein. The ClS/cish inhibitor can be a nucleic acid at least about 10 nucleotides in length that specifically binds to and is complementary to a target nucleic acid encoding ClS/cish or a complement thereof. The ClS/cish inhibitor may be introduced into a host cell, wherein the cell is capable of expressing ClS/cish, in an effective amount for a time and under conditions sufficient to interfere with expression of the CIS/ 'cish. In some embodiments, RNA interference (RNAi) is employed. In this regard, the CIS/cish inhibitor may comprise an RNAi agent. In an embodiment, the RNAi agent may comprise a small interfering RNA (siRNA), a microRNA (miRNA), or an antisense nucleic acid. The RNAi agent, e.g., siRNA, miRNA, and/or antisense nucleic acid can comprise overhangs. That is, not all nucleotides need bind to the target sequence. RNA interference nucleic acids employed can be at least about 19, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 220, at least about 240, from about 19 to about 250, from about 40 to about 240, from about 60 to about 220, from about 80 to about 200, from about 60 to about 180, from about 80 to about 160, and/or from about 100 to about 140 nucleotides in length.
[0043] The RNAi agent, e.g., siRNA or shRNA, can be encoded by a nucleotide sequence included in a cassette, e.g., a larger nucleic acid construct such as an appropriate vector. Examples of such vectors include lentiviral and adenoviral vectors, as well as other vectors described herein with respect to other aspects of the invention. An example of a suitable vector is described in Aagaard et al. Mol. Ther., 15(5): 938-45 (2007). When present as part of a larger nucleic acid construct, the resulting nucleic acid can be longer than the comprised RNAi nucleic acid, e.g., greater than about 70 nucleotides in length. In some embodiments, the RNAi agent employed cleaves the target mRNA. In other embodiments, the RNAi agent employed does not cleave the target mRNA.
[0044] Any type of suitable siRNA, miRNA, and/or antisense nucleic acid can be employed. In an embodiment, the antisense nucleic acid comprises a nucleotide sequence complementary to at least about 8, at least about 15, at least about 19, or from about 19 to about 22 nucleotides of a nucleic acid encoding CIS or a complement thereof. In an embodiment, the siRNA may comprise, e.g., trans-acting siRNAs (tasiRNAs) and/or repeat- associated siRNAs (rasiRNAs). In another embodiment, the miRNA may comprise, e.g., a short hairpin miRNA (shMIR).
[0045] The CIS/cish inhibitor may inhibit or downregulate to some degree the expression of the protein encoded by a cish gene, e.g., at the DNA, RNA, or other level of regulation. In this regard, a host cell comprising a CIS/cish inhibitor expresses no CIS/cish or lower levels of CIS/cish as compared to a host cell that lacks a CIS/cish inhibitor. In accordance with an embodiment of the invention, the CIS/cish inhibitor, such as an RNAi agent, such as a shMIR, can target a nucleotide sequence of a cish gene or mRNA encoded by the same. In an embodiment, the cish sequence is a human sequence. For example, human cish is assigned Gene NCBI Entrez Gene ID No. 1154, and an Online Mendelian Inheritance in Man (OMIM) No. 602441. The human cish gene is found on chromosome 3 at 3p21.3. Two transcriptional variants include mRNAs: NM_013324 and NM_145071, with corresponding protein sequences NP_037456 and NP_659508, respectively. Accordingly, NM_013324 is provided as SEQ ID NO: 1, and NM_145071 is provided as SEQ ID NO: 2. Human genomic cish sequences include AC096920, AF035946, CH471055, and Z77852. Human cish mRNA sequences also include AF035947, AF132297, AK313850, BC031590, BC064354,
BF511692, CA435538, CR594144, CR602225, D83532, and DA957793. Human CIS amino acid sequences include AAQ13420, EAW65127, EAW65128, EAW65129, EAW65130, AAF97410, AAD28471, BAG36578, AAH31590, AAH64354, and BAA92328. Other human sequences, as well as other ClS/cish species can be employed in accordance with the invention.
[0046] In accordance with an embodiment of the invention, the ClS/cish inhibitor, such as an RNAi agent, such as a shMIR, can target a nucleotide sequence selected from the group consisting of the 5' untranslated region (5' UTR), the 3' untranslated region (3' UTR), and the coding sequence of cish, complements thereof, and any combination thereof. Any suitable cish target sequence can be employed. The sequences of the ClS/cish inhibitor can be designed against a human cish with Accession No. NM_013324 (SEQ ID NO: 1) but also recognize NM_145071 (SEQ ID NO: 2). In an embodiment, the shMIR comprises SEQ ID NO: 3, encoded by nucleotide sequence SEQ ID NO: 12 and designed against the cish gene target GAGCCTGTTTCTGGGAGAA (SEQ ID NO: 9). In another embodiment, the shMIR comprises SEQ ID NO: 4, encoded by the nucleotide sequence SEQ ID NO: 13 and designed against the cish gene target GTCAACGCCTCTAGGTACA (SEQ ID NO: 10). RNAi agents can be designed against any appropriate cish mRNA sequence. In this regard, an
embodiment of the invention provides a host cell comprising an anti-cish shMIR comprising SEQ ID NO: 3 or 4.
[0047] The host cell may or may not further comprise a high temperature requirement serine peptidase 1 (HTRAl )/ ztrai activator. HTRAl (also known as L56, ARMD7, ORF480, and PRSS 1 1) is a serine protease that is a member of the trypsin family of serine proteases. Without being bound to a particular theory, it is believed that HTRAl inhibits the transforming growth factor (TGF)-p family of proteins. [0048] The high temperature requirement serine peptidase 1 (KTRAl)/htral activator may comprise any suitable agent that provides the over-expression of a htral mRNA or HTRA1 protein. In this regard, a host cell comprising a HTRAl/Zztrai activator expresses higher levels of ETRAl/htral than host cells that lack a WTRAl/htral activator. The KTRAl/htral activator may be introduced into a host cell, wherein the cell is capable of expressing KTRAl/htral, in an effective amount for a time and under conditions sufficient to cause the over-expression of HTRAl//ztr 7. In this regard, the KTRAl/htral activator may comprise a recombinant nucleic acid that encodes KTRAl/htral. In an embodiment of the invention, the recombinant polynucleotide that encodes KTRAl/htral may comprise a nucleotide sequence comprising SEQ ID NO: 5 (human htral) or SEQ ID NO: 11 (mouse htral).
[0049] In another embodiment, the invention provides a PBC comprising a KTRAl/htral activator. Suitable KTRAl/htral activators are as described herein with respect to other aspects of the invention. The PBC may or may not further comprise a CIS/cish inhibitor. Suitable CIS/cish inhibitors are as described herein with respect to other aspects of the invention.
[0050] In an embodiment of the invention, a host cell comprising a KTRAl/htral activator and/or a CIS/cish inhibitor may further comprise and express a nucleic acid comprising a nucleotide sequence encoding a T-cell receptor (TCR) having antigenic specificity for a cancer antigen or a chronic infectious disease antigen. The phrase "antigenic specificity" as used herein means that the TCR can specifically bind to, immunologically recognize, and/or mediate an immune response against a cancer antigen or a chronic infectious disease antigen. An immune response may be characterized by an increased production of cytokines such as, e.g., interferon gamma (IFNy), the stimulation of a cell- mediated immune response such as, e.g., the activation of T-cells and/or macrophages, and/or the destruction of cells expressing the cancer antigen or chronic infectious disease antigen. Exemplary TCRs having antigenic specificity for a cancer antigen include those disclosed in U.S. Patent Application Publication Nos. 2009/0053184 (disclosing TCRs having antigenic specificity for MART-1, NY-ESO-1, or g lOO); 2010/0015113 (disclosing TCRs having antigenic specificity for gplOO); 2010/0034834 (disclosing TCRs having antigenic specificity for NY-ESO-1, MART-1, TRP-1, TRP-2, gplOO, p53, or tyrosinase tumor antigen); and 2009/0304657 (disclosing TCRs having antigenic specificity for MART-1, NY-ESO-1, p53, or gplOO); U.S. Patents 7,820,174 (disclosing TCRs having antigenic specificity for TRAIL presented by TRAIL-R1) and 5,830,755 (disclosing TCRs having antigenic specificity for MART-1 or gplOO); and International Publications WO 2009/042570 (disclosing TCRs having antigenic specificity for NY-ESO-1 , MART-1, TRP-1, TRP-2, gplOO, p53, or tyrosinase tumor antigen) and WO 2010/088160 (disclosing TCRs having antigenic specificity for tyrosinase tumor antigen), each of which are incorporated herein by reference.
[0051] In an embodiment, any of the HTRAl/htral activators and/or CIS/cish inhibitors described herein can be loaded onto nanoparticles. The loaded nanoparticles can be combined with T cells (hereinafter referred to as "nanoparticle composition") and adoptively transferred into patients. In this regard, the invention provides a composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an HTRAl/htral activator and/or a CIS/cish inhibitor. For example, an HTRAl/htral activator can be loaded on nanoparticles and combined with T cells. After adoptive transfer of the T cells and loaded nanoparticles, the T cells would then traffic to the tumor, and the HTRAl/htral activator would enhance T cell-mediated tumor killing. Preferably, the HTRAl/htral activator is a HTRAl protein. The HTRAl protein may comprise any of the human HTRAl amino acid sequences described herein. Preferably, the HTRAl protein comprises SEQ ID NO: 7. The PBC may be any PBC. Preferably, the PBC is a T-cell.
[0052] A T cell comprising a HTRAl/htral activator and/or a CIS/cish inhibitor advantageously demonstrates increased T cell activity. A composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an HTRAl/htral activator and/or a CIS/cish inhibitor, also advantageously demonstrates increased T cell activity. T cell activity is increased in accordance with the invention if T cell activity is greater, quantitatively or qualitatively, after administration of a HTRAl/htral activator and/or a CIS/cish inhibitor as compared to the immune response in the absence of the administration of a HTRAl/htral activator and/or a CIS/cish inhibitor. A quantitative increase in T cell activity encompasses an increase in the magnitude or degree of the T cell activity. The magnitude or degree of T cell activity can be measured on the basis of any number of known parameters, such as an increase in the level of antigen-specific cytokine production (cytokine concentration), an increase in the number of T cells activated (e.g., proliferation of antigen- specific T cells) or recruited, an increase in the persistence and/or survival of T cells, and/or a decrease in T-cell death (e.g., apoptosis). Methods of detecting and measuring an increase in T cell activity are known in the art. For example, measuring the types and levels of cytokines produced can detect and measure an increase in T cell activity. An increase in T cell activity may be characterized by an increase in the production of, e.g., IFNy, TNF- , GM-CSF, and/or interleukin (IL)-2, an increase in the production of Thl immunoglobulins, an increase in tumor destruction in vivo, a decrease in TGFpi, and/or an increase in the differentiation of Thl cells. A qualitative increase in T cell activity encompasses any change in the nature of the T cell activity that renders the T cell more effective at combating a given antigen or disease. Qualitative and quantitative increases in T cell activity can occur simultaneously, and are not mutually exclusive.
[0053] The inhibition of CIS/cish also increases the expression of signal transducer and activator of transcription 5 (STAT5) target genes such as, for example, v-myc,
myelocytomatosis viral oncogene homolog (avian) (cMYC), Bcl-2-associated death promoter (bcl2ll (Bcl-xL)), granzyme B, T-cell-specific T-box transcription factor (Tbet)/tfoc2i and cell cycle genes (e.g., ccnd2 (cyclin D2), ccnel (cyclin El), and/or cdkl (cyclin CDC2a)), but does not increase STAT5 phosphorlyation and does not alter the expression of T cell receptor (TCR) signaling genes such as, for example, tumor necrosis factor receptor superfamily, member 9 (4- IBB) and programmed cell death 1 (PD1).
[0054] The inhibition of CIS/cish decreases TGF-β protein levels, decreases TGF-β signaling (e.g., decreases activation of the TGF-β signaling molecule, SMAD2), and decreases the expression of TGF-β target genes such as, for example, cyclin-dependent kinase inhibitor 2B (Cdkn2b) (believed to inhibit the cell cycle), deleted in esophageal cancer 1 (DEC1) (believed to inhibit cell function), inhibitor of DNA binding 1 (ID1) (believed to induce apoptosis), Stral3, and CD 103. Without being bound to a particular theory, it is believed that CIS interacts with HTRA1, a protein believed to degrade TGF-β family members. It is believed that the improved functionality of T cells or nanoparticle
compositions comprising a CIS/cish inhibitor may be the result of decreased degradation of HTRA1.
[0055] In still another embodiment, the invention provides a host cell comprising a HTRAl //ztra7 inhibitor and a CIS/cish activator.
[0056] The KTRAl/htral inhibitor may be any suitable agent that inhibits the expression of a htral mRNA or protein. The KTRAl/htral inhibitor can be a nucleic acid that specifically binds to and is complementary to a target nucleic acid encoding HTRAl/Zztrai or a complement thereof. The HTRAl /htral inhibitor may be introduced into a host cell, wherein the cell is capable of expressing HTRAl, in an effective amount for a time and under conditions sufficient to interfere with expression of HTRAl . In some embodiments, RNA interference (RNAi) is employed. In this regard, the HTRAl/htral inhibitor may comprise an RNAi agent.
[0057] Any type of suitable siRNA, miRNA, and/or antisense nucleic acid can be employed. In an embodiment, the antisense nucleic acid comprises a nucleotide sequence complementary to at least about 8, at least about 15, at least about 19, or from about 19 to about 22 nucleotides of a nucleic acid encoding HTRAl or a complement thereof. In an embodiment, the siRNA may comprise, e.g., trans-acting siRNAs (tasiRNAs) and/or repeat- associated siRNAs (rasiRNAs). In another embodiment, the miRNA may comprise, e.g., a short hairpin miRNA (shMIR).
[0058] The HTRAl/htral inhibitor may inhibit or downregulate to some degree the expression of the protein encoded by a htral gene, e.g., at the DNA, RNA, or other level of regulation. In this regard, a host cell comprising a HTRAl/htral inhibitor expresses no HTRAl/htral or lower levels of HTRAl /htral as compared to a host cell that lacks a HTRAl/htral inhibitor. In accordance with the invention, the HTRAl/htral inhibitor, such as an RNAi agent, such as a shMIR, can target a nucleotide sequence of a htral gene or mRNA encoded by the same. In an embodiment, the htral sequence is a human sequence. For example, human htral is assigned Gene NCBI Entrez Gene ID No. 5654, and an Online Mendelian Inheritance in Man (OMIM) No. 602194. The human htral gene is found on chromosome 10 at 10q26.3. The htral mRNA sequence is NM_002775, with corresponding protein sequence NP_002766. Accordingly, NM 002775 is provided as SEQ ID NO: 5. Human genomic htral sequences include AF157623, BX842242, and CH471066. Human htral mRNA sequences include AF070555, AF097709, AI423369, AK091944, AK092476, AK290089, BC01 1352, BC031082, BM993893, CR590731, CR593682, CR596858, CR597732, CR603877, CR605013, CR61 1805, CR613636, CR623030, CR624458,
CR624921, D87258, DA633502, and Y07921. Mouse htral mRNA sequences include NM_019564. Human HTRAl amino acid sequences include AAD41525, EAW49312, EAW49313, AAC97211, BAG52446, BAG52557, BAF82778, and AAH11352. Other human sequences, as well as other HTRAl/htral species can be employed in accordance with the invention.
[0059] In accordance with an embodiment of the invention, the HTRAl /htral inhibitor, such as an RNAi agent, such as a shMIR, can target a nucleotide sequence selected from the group consisting of 5' untranslated region (5' UTR), the 3' untranslated region (3' UTR), and the coding sequence of htral, complements thereof, and any combination thereof. Any suitable htral target sequence can be employed. The sequences of the HTRAl/htral inhibitor can be designed against a human htral with Accession No. NM_002775 (SEQ ID NO: 5). In an embodiment, the shMIR comprises SEQ ID NO: 6, designed against the htral gene target GCTATCATCAATTATGGAA (SEQ ID NO: 15). RNAi agents can be designed against any appropriate htral mRNA sequence.
[0060] Other aspects of the HTRAl/Tztrai inhibitor comprising an RNAi agent are as described for the ClS/cish inhibitor.
[0061] In an embodiment of the invention, the HTRAl/Zztrai inhibitor comprises NVP- LBP976. NVP-LBP976 has the chemical structure:
Figure imgf000013_0001
[0062] The ClS/cish activator may comprise any suitable agent that provides the over- expression of a cish mRNA or protein. A host cell comprising a ClS/cish activator expresses higher levels of ClS/cish than host cells that lack a ClS/cish activator. The ClS/cish activator may be introduced into a host cell, wherein the cell is capable of expressing ClS/cish, in an effective amount for a time and under conditions sufficient to cause the over-expression of ClS/cish. In this regard, the ClS/cish activator may comprise a recombinant nucleic acid that encodes ClS/cish. In an embodiment of the invention, the recombinant polynucleotide that encodes ClS/cish may comprise a nucleotide sequence comprising SEQ ID NO: 1 or 2.
[0063] In another embodiment, the invention provides a PBC comprising a HTRAl/htral inhibitor. Suitable HTRAl//ztra7 inhibitors are as described herein with respect to other aspects of the invention. The PBC may or may not further comprise a ClS/cish activator. Suitable ClS/cish activators are as described herein with respect to other aspects of the invention.
[0064] In yet another embodiment, the invention provides a T cell comprising a ClS/cish activator, wherein the T cell has suppressed T cell activity. Suitable ClS/cish activators are as described herein with respect to other aspects of the invention. The T cell may or may not further comprise a HTRAl I htral inhibitor. Suitable HTRAl Ihtral inhibitors are as described herein with respect to other aspects of the invention.
[0065] In an embodiment, any of the HTRAl Ihtral inhibitors and/or ClS/cish activators described herein can be loaded onto nanoparticles. The loaded nanoparticles can be combined with T cells (hereinafter referred to as "nanoparticle composition") and adoptively transferred into patients. In this regard, the invention provides a composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an HTRAl/htral inhibitor and/or a ClS/cish activator. Preferably, the ClS/cish activator is a CIS protein. The CIS protein may comprise any of the human CIS amino acid sequences described herein. The CIS protein may comprise SEQ ID NO: 8. The PBC may be any PBC. Preferably, the PBC is a T-cell.
[0066] A nanoparticle composition or T cell comprising a UTRAl/htral inhibitor and/or a ClS/cish activator advantageously demonstrates suppressed T cell activity. T cell activity is suppressed in accordance with the invention if the T cell activity is diminished, quantitatively or qualitatively, after administration of a HTRAl/htral inhibitor and/or a ClS/cish activator as compared to the immune response in the absence of the administration of a HTRAl/htral inhibitor and/or a ClS/cish activator. A quantitative decrease in T cell activity encompasses a decrease in the magnitude or degree of the activity. The magnitude or degree of T cell activity can be measured on the basis of any number of known parameters, such as a decrease in the level of cytokine (e.g., antigen-specific cytokine) production (cytokine concentration), a decrease in the number of T cells activated (e.g., proliferation of T cells (e.g., antigen- specific T-cells)) or recruited, etc., an decrease in the persistence and/or survival of T cells, and/or an increase in T -cell death (e.g., apoptosis). Methods of detecting and measuring suppression of T cell activity are known in the art. For example, measuring the types and levels of cytokines produced can detect and measure suppression of T cell activity.
Suppression of T cell activity may be characterized by a decrease in the production of, e.g., IFNy, TNF-a, GM-CSF, and/or IL-2; a decrease in the production of Thl immunoglobulins, a decrease in tumor destruction in vivo, an increase in TGFpi, and/or a decrease in the differentiation of Thl cells. A qualitative decrease in T cell activity encompasses any change in the nature of the T cell activity that renders it less effective at mediating the destruction of a given antigen. Qualitative and quantitative suppression of T cell activity can occur simultaneously, and are not mutually exclusive. [0067] Without being bound to a particular theory, it is believed that the inhibition of HTRA1 increases the expression of TGF-β target genes such as, for example, plasminogen activator inhibitor-1 (PAI-1); increases TGF-β levels; and decreases the expression of STAT5 target genes such as, for example, cMYC, bcUll (Bcl-xL), granzyme B, tbx21 (Tbet), and cell cycle genes (e.g., ccndl (cyclin D2), ccnel (cyclin El), and/or cdkl (cyclin CDC2a)).
[0068] By "nucleic acid" as used herein includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides (e.g., ribonucleic acid nucleotides as well as deoxyribonucleic acid nucleotides), and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. When a nucleic acid is recited it refers generically to nucleic acids and includes DNA and RNA unless the recitation explicitly states that the nucleic acid is a specific one, e.g., DNA or RNA. If a nucleic acid refers to a sequence that contains thymine (t), that does not necessarily indicate that the nucleic acid is DNA; in some embodiments the nucleic acid is RNA and/or DNA. Similarly, if a nucleic acid refers to a sequence that contains uracil (u) that does not necessarily indicate that the nucleic acid is RNA; in some embodiments the nucleic acid is DNA and/or RNA. It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
[0069] The nucleic acid molecules relevant to the invention can readily be obtained in a variety of ways, including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA. These methods and others useful for isolating such DNA are set forth, for example, by Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), by Ausubel, et al., eds., "Current Protocols In Molecular Biology," Current Protocols Press (1994), and by Berger and Kimmel, "Methods In
Enzymology: Guide To Molecular Cloning Techniques," vol. 152, Academic Press, Inc., San Diego, Calif. (1987). [0070] Chemical synthesis of a nucleic acid molecule can be accomplished using methods well known in the art, such as those set forth by Engels et al., Angew. Chem. Intl. Ed., 28:716-734 (1989). These methods include, inter alia, the phosphotriester,
phosphoramidite and H-phosphonate methods of nucleic acid synthesis. Nucleic acids larger than about 100 nucleotides in length can be synthesized as several fragments, each fragment being up to about 100 nucleotides in length. The fragments can then be ligated together to form a full length nucleic acid encoding the polypeptide. One method is polymer-supported synthesis using standard phosphoramidite chemistry.
[0071] Alternatively, the nucleic acid can be obtained by screening an appropriate cDNA library prepared from one or more tissue source(s) that express the polypeptide, or a genomic library from any subspecies. The source of the genomic library may be any tissue or tissues from a mammalian or other species believed to harbor a gene encoding a protein relevant to the invention (e.g., CIS and/or HTRA1). The library can be screened for the presence of a cDNA/gene using one or more nucleic acid probes (oligonucleotides, cDNA or genomic DNA fragments that possess an acceptable level of homology to the gene or gene homologue cDNA or gene to be cloned) that will hybridize selectively with the gene or gene homologue cDNA(s) or gene(s) that is(are) present in the library. The probes preferably are
complementary to or encode a small region of the DNA sequence from the same or a similar species as the species from which the library was prepared. Alternatively, the probes can be degenerate. After hybridization, the blot containing the library is washed at a suitable stringency, depending on several factors such as probe size, expected homology of probe to clone, type of library being screened, number of clones being screened, and the like.
Stringent washing solutions can be low in ionic strength and are used at relatively high temperatures.
[0072] Another suitable method for obtaining a nucleic acid for use in accordance with the invention is the polymerase chain reaction (PCR). In this method, poly(A)+RNA or total RNA is extracted from a tissue that expresses the gene product. cDNA is then prepared from the RNA using the enzyme reverse transcriptase. Two primers typically complementary to two separate regions of the cDNA (oligonucleotides) are then added to the cDNA along with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the two primers.
[0073] The invention provides for the use of isolated, purified or enriched nucleic acid sequences of, for example, about 15 to about 500 nucleotides in length, about 15 to about 100 nucleotides in length, about 15 to about 50 nucleotides in length, and about 15 to about 30 nucleotides in length, which have sequence that corresponds to a portion of one of the nucleotides described herein. The nucleic acid can be at least about 17, about 20, about 22, or about 25 nucleotides in length. The nucleic acid sequence can be about 30 to about 300 nucleotides in length, or about 45 to about 200 nucleotides in length, or about 45 to about 100 nucleotides in length. The nucleic acid can be at least about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 17, about 20, about 22, about 25, about 30, about 35, about 40, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 1000, about 10,000, about 50,000, about 100,000 or more nucleotides in length, or about 100,000, about 75,000, about 50,000, about 10,000, about 5,000, about 1000, about 750, about 500, about 250, about 200, about 100, about 50, about 40, about 30, about 25, about 22, about 20, about 17, about 15, about 12, about 10, about 9, about 8, about 7, about 6, about 5, or fewer nucleotides in length. The nucleic acid can have a length in a range from any one of the above lengths to any other of the above lengths including endpoints.
[0074] A nucleic acid or protein in accordance with the invention can be at least, e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identical to reference sequences provided herein. A nucleotide that hybridizes under stringent conditions to a nucleotide described herein can be employed. Unless otherwise specified, percent identities for nucleic acids and amino acid sequences are determined as follows. Percent identity of two nucleic acid sequences or two amino acid sequences is determined using the algorithm of Karlin and Altschul {Proc. Natl. Acad. Set USA, 87:2264-2268 (2002), modified as in Karlin and Altschul et al., Proc. Nat. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J Mol. Biol.
215:403-410 (1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=l, to obtain nucleotide sequences with a percent identity to a nucleic acid employed in the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences with a percent identity to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See <www.ncbi.nih.gov>. [0075] In connection with nucleic acid hybridization, the term "specifically hybridizes" indicates that the probe hybridizes to a sufficiently greater degree to the target sequence than to a non-target sequence, e.g., at a level which allows ready identification of probe/target sequence hybridization under selective hybridization conditions. "Selective hybridization conditions" refer to conditions that allow such differential binding. Similarly, the terms "specifically binds" and "selective binding conditions" refer to such differential binding of any type of probe, and to the conditions that allow such differential binding.
[0076] The nucleotide sequence encoding CIS and/or HTRAl encodes any suitable CIS and/or HTRAl, respectively, including functional portions or functional variants thereof. The term "functional portion" refers to any part or fragment of the CIS or HTRAl, which part or fragment retains the biological activity of the CIS or HTRAl, respectively, of which it is a part (the parent CIS and/or HTRAl, respectively). In reference to the parent CIS or HTRAl, the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%), 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more, of the parent CIS or HTRAl , respectively. The term "functional variant" as used herein refers to CIS or HTRAl having substantial or significant sequence identity or similarity to a parent CIS or HTRAl, respectively, which functional variant retains the biological activity of the CIS and/or HTRAl, respectively, of which it is a variant. In reference to the parent CIS or HTRAl, the functional variant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical in amino acid sequence to the parent CIS or HTRAl, respectively. Functional portions and functional variants of HTRAl encompass, for example, those parts and variants, respectively, of HTRAl that retain the ability to induce the production of effector cytokines (e.g., IFN-γ, TNF-a, and/or GM-CSF), enhance the production of Thl immunoglobulins, increase tumor destruction in vivo, decrease TGFpi levels, induce the differentiation of Thl cells, or treat or prevent cancer and/or a chronic infectious disease, to a similar extent, the same extent, or to a higher extent, as the parent HTRAl . Functional portions and functional variants of CIS encompass, for example, those parts and variants, respectively, of CIS that retain the ability to reduce the production of effector cytokines (e.g., IFN-γ, TNF-a, and/or GM-CSF), reduce the production of Thl immunoglobulins, decrease tumor destruction in vivo, increase TGFpi levels, reduce the differentiation of Thl cells, or treat or prevent auto and/or allo-immune diseases, to a similar extent, the same extent, or to a higher extent, as the parent CIS. [0077] Preferably, the nucleic acids described herein are recombinant. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.
[0078] The nucleic acids of the invention can be incorporated into a recombinant expression vector. For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring, non-naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.
[0079] The recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transduce any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as GTIO, XGTl 1, ΧΖεφΙΙ (Stratagene), λΕΜΒΕ4, and λΝΜΙ 149, also can be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19
(Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector (e.g., adenoviral vector, adeno-associated viral (AAV) vector, herpes viral vector, retroviral vector, or lentiviral vector) or a transposon vector. [0080] The recombinant expression vectors described herein can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, and the like.
[0081] Desirably, the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
[0082] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transduced host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host cell to provide prototrophy, and the like. Suitable marker genes for the expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
[0083] As used herein, the term "host cell" refers to any type of cell that can contain the recombinant expression vector(s) described herein. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5a E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., a DH5a cell. For purposes of producing cells for adoptive cell therapy, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a peripheral blood cell (PBC), peripheral blood leukocyte (PBL), or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell. [0084] For purposes herein, the T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. Preferably, the T cell is a human T cell. More preferably, the T cell is a T cell isolated from a human. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells, e.g., Thi and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells, naive T cells, and the like. Preferably, the T cell is a CD8+ T cell or a CD4+ T cell.
[0085] The host cells can be transduced with the nucleic acids using any suitable method. For example, host cells can be transduced with viral vectors using viruses (e.g., retrovirus or lentivirus) and host cells can be transduced with transposon vectors using electroporation.
[0086] Also provided by an embodiment of the invention is a population of cells comprising at least one host cell (e.g., a PBC or a T cell) described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described herein, in addition to at least one other cell, e.g., a host cell (e.g., a T cell), which does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cells, a muscle cell, a brain cell, etc.
Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) comprising the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.
[0087] The inventive host cells (including populations thereof) can be isolated and/or purified. The term "isolated" as used herein means having been removed from its natural environment. The term "purified" as used herein means having been increased in purity, wherein "purity" is a relative term, and not to be necessarily construed as absolute purity. For example, the purity can be at least about 50%, can be greater than 60%, 70% or 80%, or can be 100%.
[0088] The inventive host cells (including populations thereof) and nanoparticle compositions can be formulated into a composition, such as a pharmaceutical composition. In this regard, an embodiment of the invention provides a pharmaceutical composition comprising any of the host cells (including populations thereof) and/or nanoparticle compositions described herein, and a pharmaceutically acceptable carrier. The
pharmaceutical composition can comprise an inventive host cell or population thereof and/or nanoparticle composition in combination with other pharmaceutically active agents or drugs, such as a chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
[0089] Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.
[0090] The choice of carrier will be determined in part by the particular host cell, population thereof, or nanoparticle composition, as well as by the particular method used to administer the inventive host cell, population thereof, or nanoparticle composition.
Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. The following formulations for parenteral, intratumoral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, and interperitoneal administration are exemplary and are in no way limiting. More than one route can be used to administer the inventive host cell, population thereof, or nanoparticle composition, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
[0091] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The inventive host cell, population thereof, or nanoparticle composition can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.
[0092] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
[0093] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-p-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.
[0094] The parenteral formulations will typically contain from about 0.05% to about 50% by weight of the host cell, population thereof, or nanoparticle composition, in solution.
Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
[0095] Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). Preferably, when administering cells, e.g., T cells, the cells are administered via injection.
[0096] It will be appreciated by one of skill in the art that, in addition to the above- described pharmaceutical compositions, the inventive host cell or population thereof can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, nanoparticles, or liposomes.
[0097] Many assays for determining an administered dose are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which target cells are lysed by or IFN-γ secretion is increased or decreased in T cells comprising the nucleic acid or recombinant expression vectors described herein upon administration of a given dose of such T cells to a mammal among a set of mammals of which each is given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal. The extent to which IFN-γ is secreted upon administration of a certain dose can be assayed by methods known in the art, including, for instance, the methods described herein in the Examples (e.g., ELISA).
[0098] The dose of the inventive host cell, populations thereof, or nanoparticle composition also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inventive host cell, populations thereof, or nanoparticle composition. Typically, the attending physician will decide the dosage of the inventive host cell, populations thereof, or nanoparticle composition, with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inventive host cell, populations thereof, or nanoparticle composition to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the dose of the inventive host cell, populations thereof, or nanoparticle composition can be about 0.001 to about 1000 mg/kg body weight of the subject being treated/day, from about 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1 mg/kg body weight/day.
[0099] It is contemplated that the inventive host cell, population of cells, or nanoparticle composition, comprising a CIS/cish inhibitor, KTRAl/htral activator, and/or pharmaceutical compositions comprising the same, can be used in methods of increasing T cell activity in a mammal or methods of treating or preventing cancer or a chronic infectious disease in a mammal. Without being bound to a particular theory, it is believed that nanoparticle compositions or cells comprising a CIS/cish inhibitor and/or a KTRAl/htral activator advantageously increase the production of effector cytokines (e.g., IFN-γ, IL-2); increase T cell proliferation, persistence, survival, and cytolytic activity; decrease T cell apoptosis; and/or decrease tumor size in vivo as compared to nanoparticle compositions or cells that lack any one or more of a CIS/cish inhibitor and/or a HTRAl/ ztra7 activator. In this regard, an embodiment of the invention provides a method of treating or preventing cancer or a chronic infectious disease in a mammal, comprising administering to the mammal a host cell, population of cells, or nanoparticle composition, comprising a CIS/cish inhibitor, a
KTRAl/htral activator, and/or pharmaceutical compositions comprising the same, in an amount effective to treat or prevent cancer or the infectious disease in the mammal.
[0100] Another embodiment of the invention comprises a method of increasing T cell activity in a mammal, comprising administering to the mammal a host cell, population of cells, or nanoparticle composition, comprising a CIS/cish inhibitor, a KTRAl/htral activator, and/or pharmaceutical compositions comprising the same, in an amount effective to increase T cell activity in the mammal.
[0101] It is also contemplated that the inventive host cell, population of cells, or nanoparticle composition, comprising a CIS/cish activator, a KTRAl/htral inhibitor, and/or pharmaceutical compositions comprising the same, can be used in methods of suppressing T cell activity in a mammal or methods of treating or preventing an auto- and/or allo-immune disease in a mammal. Without being bound to a particular theory, it is believed that cells or nanoparticle compositions comprising a CIS/cish activator and/or a KTRAl/htral inhibitor advantageously decrease the production of effector cytokines (e.g., IFN-γ, IL-2); decrease T cell proliferation, persistence, survival, and cytolytic activity; and/or increase T cell apoptosis as compared to nanoparticle compositions or cells that lack any one or more of a ClS/cish activator and/or a HTRAl/htral inhibitor. In this regard, an embodiment of the invention provides a method of treating or preventing an auto- and/or allo-immune disease in a mammal comprising administering to the mammal a host cell, population of cells, or nanoparticle composition, comprising any one or more of a ClS/cish activator, a
HTRAl//ztra7 inhibitor, and/or pharmaceutical compositions comprising the same, in an amount effective to treat or prevent an auto- and/or allo-immune disease in the mammal.
[0102] Another embodiment of the invention comprises a method of suppressing T cell activity in a mammal, comprising administering to the mammal a host cell, population of cells, or nanoparticle composition comprising a ClS/cish activator, a HTRAl/htral inhibitor, and/or pharmaceutical compositions comprising the same, in an amount effective to suppress T cell activity in the mammal.
[0103] Another embodiment of the invention comprises a method of treating or preventing a disease selected from the group consisting of age-related macular degeneration, Alzheimer's disease, and familial ischemic cerebral small- vessel disease in a mammal, comprising administering to the mammal a PBC, a host cell, population of cells, or nanoparticle composition, comprising a ClS/cish inhibitor, a HTRAl/htral activator, a ClS/cish activator, a HTRAl/htral inhibitor, and/or pharmaceutical compositions comprising the same, in an amount effective to treat or prevent the disease in the mammal. Preferably, the method comprises treating or preventing Alzheimer's disease or familial ischemic cerebral small-vessel disease comprising administering to the mammal a PBC, a host cell, population of cells, or nanoparticle composition, comprising a ClS/cish inhibitor or a HTRAl/htral activator, and/or pharmaceutical compositions comprising the same, in an amount effective to treat or prevent the disease in the mammal. Preferably, the method comprises treating or preventing age-related macular degeneration comprising administering to the mammal a PBC, a host cell, population of cells, or nanoparticle composition, comprising a ClS/cish activator or a HTRAl/htral inhibitor, and/or pharmaceutical compositions comprising the same, in an amount effective to treat or prevent the disease in the mammal.
[0104] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect, which could include 100% or complete treatment or prevention. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of cancer, a chronic infectious disease, an allo-immune disease, and/or an autoimmune disease in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, a chronic infectious disease, an allo-immune disease, and/or an autoimmune disease, being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.
[0105] For purposes of the inventive methods, wherein host cells or populations of cells are administered, the cells can be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.
[0106] With respect to the inventive methods, the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer. Preferably, the cancer is skin cancer. More preferably, the cancer is melanoma.
[0107] With respect to the inventive methods, the chronic infectious disease can be any chronic infectious disease, including any of HIV, influenza, herpes, and malaria.
[0108] The auto- or allo-immune disease that may be treated by the inventive methods may be any auto- or allo-immune disease. The autoimmune diseases that may be treated by the inventive methods may be generally characterized by an inappropriate immune reaction against self cells, tissues, and/or organs. Exemplary autoimmune diseases include, but are not limited to: vitiligo, alopecia, autoimmune kidney disease, celiac disease, inflammatory bowel disease, hepatitis, Addison's disease, Hashimoto's disease, Graves disease, atrophic gastritis/pernicious anemia, acquired hypogonadism/infertility, hypoparathyroidism, multiple sclerosis, Myasthenia gravis, Coombs positive hemolytic anemia, systemic lupus
erythematosis, allergic diseases (such as asthma, hay fever, or allergic rhinitis), Sjogren's syndrome, rheumatoid arthritis, auto-immune thyroiditis, Crohn's disease, ulcerative colitis, cardiovascular disease (e.g., atherosclerosis), and immune mediated (type-1) diabetes mellitus. The allo-immune diseases that may be treated by the inventive methods may be generally characterized by an immune reaction against foreign or transplanted organs, tissues, and/or cells. Exemplary allo-immune diseases include, but are not limited to: acute and chromic graft-versus-host disease (GVHD) and/or graft rejection that can occur, e.g., in the setting of solid organ transplantation (e.g., pancreatic, renal, cardiac, stem cell, or liver transplantation) and graft-versus-host disease that can occur in the setting of allogeneic hematopoietic stem cell transplantation (e.g., bone marrow, peripheral blood, or cord blood transplantation).
[0109] The mammal referred to in the inventive methods can be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order
Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
[0110] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLES
Mice and Cell Lines
[0111] Pmel-1 Thyl .l, Pmel-1 Ly5.1, pmel-1 cish-Ι-, RAG1-/-, C57BL/6 mice (NCI- Frederick and The Jackson Laboratory) were bred, genotyped and housed according to the guidelines of the Animal Care and Use Committee at the National Institutes of Health. B16 melanoma was obtained from the NCI Tumor Repository and grown in 10% FCS containing culture media. Where indicated, CD8+ T cells were isolated from splenocytes by magnetic bead negative selection (Miltenyi Biotec or Stemcell Technologies). Primary stimulation was accomplished using plate-bound anti-CD3 (1 μg/mL) and soluble anti-CD28 (1 μg/mL) (BD Biosciences). To generate effector cells, Pmel-1 splenocytes were stimulated with 1 μΜ hgpl0025_33 peptide (Anaspec) in culture medium containing 2 ng ml"1 IL-2 (Chiron
Corporation) and cultured in 10% FCS containing RPMI for 1 week.
Adoptive Immunotherapy
[0112] Mice were implanted with subcutaneous B16 melanoma (1 x 105-5 x 10s). At the time of ACT, 10-14 days post implantation, mice (n > 5 for all groups unless otherwise indicated) were injected intravenously (i.v.) with CD8-enriched naive or in vitro activated pmel-1 splenocytes (0.25 x 106-1 x 106 CD8+ Vpl3+ T cells), and received 0.5 x 107-2 x 107 plaque-forming units of rVV or fowlpox encoding hgplOO (Bernard Moss, National Institutes of Health). Where indicated, mice received interperitoneal (i.p.) injections of hIL-2 (Palmer et al. PNAS 105:8061-66 (2008)) or 100 μg of CD8-depleting antibody (53.6.7, BD
PharMingen) was injected i.p. Mice were randomized, and tumors were blindly measured by using digital calipers. The products of the perpendicular diameters are presented as mean ± SEM. At times indicated after ACT spleens were harvested, ACK-lysed, enumerated, stained, and evaluated by flow cytometry. For time course experiments, splenocytes were enriched for the congenic marker using Miltenyi or Cell-Sep protocols, cyro-preserved, thawed simultaneously, enumerated and stimulated as indicated.
Flow Cytometry, ELISA, Western Blotting and Immunoprecipitation
[0113] For flow cytometry cells were stained with antibodies acquired from BD
Pharmigen or eBioscience and processed as previously described (Palmer et al. PNAS 105:8061-66 (2008)). Intracellular staining was performed per manufacturer protocol (BD Biosciences). Carboxyfluorescein succinimidyl ester (CFSE) labeling was per manufacturer protocol (Invitrogen). Cytokine quantities were determined by ELISA (R&D Systems). Real-time PCR was conducted using commercially available primer/probe sets (Applied Biosystems) and a Prism® 7900HT (Applied Biosystems). Gene expression levels were calculated relative to the housekeeping gene encoding beta-actin (Actb). For apoptosis evaluation, following stimulation with aCD3/aCD28, cells were stained with annexin and 7AAD as per the manufacturer's instructions (BD Pharmigen). After surface staining, intracellular staining was accomplished by using Ctyofix/cytoperm, then Phosphobuffer II or III, and antibody as per the manufacturer's instructions (BD Pharmigen). For the co- immunoprecipitation protocol, cells were lysed, cleared, and normalized using a
bicinchoninic acid (BCA) assay (Pierce). Cleared supernatants were applied to antibody- bonded immunoprecipitation (IP) direct columns and treated as per the manufacturer's instructions (Pierce). Western blotting was performed using Bio-Rad reagents and protocols on nitrocellulose paper, stained with aV5, aCIS, aHtrAl, (Santa Cruz), SMAD2, pSMAD2, pSTAT5 (Cell Signal Technologies). Blots were developed using chemiluminescence (Pierce) and acquired using Chemidoc™ system (Bio-Rad).
Transfections and Transductions
[0114] For transient transfection, 293T cells were transfected with Lipofectamine™ 2000 as per the manufacturer's instructions (Invitrogen). iCIS and iRFP cell lines were developed by transducing 293T cells with a lentivirus containing CIS or RFP driven by a tetracycline promoter, and phoshoglycerate kinase promoter driven TetR and puromycin resistance genes. Cells were then selected using puromycin (3 μg/mL). Retroviral production and transduction were accomplished as previously described (Abad et al. J. Immunother. 31 : 1-6 (2008)). The shorthairpin microRNA sequences used are shown in Table 1. Knockdown was evaluated by taqman or western blotting.
TABLE 1
Figure imgf000030_0001
Yeast two hybrid
[0115] Yeast two-hybrid screen was performed by ProteinLinks as previously described (Xu et al. PNAS 94:12473-78 (1997)). Briefly, CIS-bait was cloned into TetR or LexA vectors and screened against a murine spleen-derived cDNA library (5 x 10 or 1 x 10 clones). All positive interactions were confirmed using co-immunoprecipitation.
EXAMPLE 1
[0116] This example demonstrates that the lymphopoeisis, memory state, proliferation, and cell surface marker expression of cish"1' CD8+ T cells is similar to that of wild-type cells. This example also demonstrates that cish mRNA expression increases following stimulation of wild-type T cells but not cish'1' T cells.
[0117] To evaluate the role of CIS in CD8+ T cell function, a cish knockout mouse was developed, cish'1' mice were then backcrossed with C57BL/6 mice and bred with the melanoma/melanocyte-specific T cell receptor (TCR) a transgenic mouse, termed pmel-1. CIS has been shown to be induced by IL-2 and TCR stimulation (Aman et al. J. Biol. Chem. 274:30266-72 (1999)). cish mRNA expression was found to increase in both naive and primed CD8+ pmel-1 T cells (WT) upon stimulation (or re-stimulation), but not cish knockouts {cish'1').
[0118] To further characterize cish'1' CD8+ T cells, extensive phenotyping using flow cytometric analysis was performed. Here, lymphopoeisis in cish'1' pmel-1 mice was found to be similar to that of wild-type (WT) littermates in splenic CD8+ and CD4+ ratios. A comparable memory state as indicated by CD44 and CD62L expression was also found in CD8+ T cells from WT and cish'1' pmel-1 mice. Expression analysis of CD8, clonotypic V l3, tetramer-binding, and IL-2 receptor (CD25, CD122 and CD132) yielded no discernable differences between CD8+ WT and cish'1' pmel-1 T cells. Analysis of other markers such as CD27, CD28, CD69, CD80, CD86, LFA-1, Sca-1, CD 127, KLRG1 and many others also yielded no significant differences. Furthermore, cish'1' CD 8+ pmel-1 T cells acquired the memory marker CD44, diluted out carboxyfluorescein (CFSE), and proliferated similarly to pmel-1 WT CD8+ T cells after stimulation. In addition, the onset of skin depigmentation, vitiligo, which is typically observed in pmel-1 transgenic mice, was not altered in any of the genotypes pmel-1 WT, cish+/~, and cish'1'.
EXAMPLE 2
[0119] This example demonstrates that cish-defici t T cells secrete higher levels of IFN- γ and IL-2 upon antigen encounter as compared to WT cells. [0120] To assess if there were any functional differences in cish"1' CD8+ T cells, CD8+ enriched naive WT or cish'1" pmel-1 T cells were co-cultured with pulsed targets (gpl0025-33). IFN-γ secretion was evaluated by ELISA after the overnight co-culture. Interestingly, cish- deficient pmel-1 T cells released significantly more IFN-γ upon antigen encounter (Table 2A). IFN-γ secretion was also evaluated by ELISA after an overnight co-culture of primed WT or cish'1' pmel-1 T cells. Upon antigen re-encounter, previously primed cish"'" pmel-1 T cells released dramatically more IFN-γ than wild-type (WT) T cells (Table 2A). IL-2 secretion was also evaluated by ELISA after an overnight co-culture of primed WT or cish-l- pmel-1 T cells. When assayed for IL-2 or TNF-a after a co-culture of primed CD8+ T cells, it was observed that cish"'" CD8+ T cells also released more IL-2 or TNF-a (Table 2A).
TABLE 2A
Figure imgf000032_0001
[0121] PBL transduced with a T cell receptor also secrete greater levels of IFN-γ in the absence of cish. PBL that were mock-transduced or transduced with T cell receptor 1G4 and shRNA retrovirus targeting cish (or scramble as a control) were co-cultured with 624mel, 526mel, or 888mel overnight and selected for 4 days with puromycin (5 x 104: 5 x 104). The results are shown in Table 2B. TABLE 2B
Figure imgf000033_0001
EXAMPLE 3
[0122] This example demonstrates that cw z-deficient T cells mediate tumor regression in vivo, and that T cells treated with anti-m/z shMIR increases the in vitro and in vivo functionality of T cells as compared to WT cells.
[0123] To evaluate the in vivo functionality of cish''' CD8+ T cells, the pmel-l tumor model (Palmer et al. PNAS 105:8061-66 (2008)) was employed. C57BL/6 mice were implanted with non- immunogenic B16 melanoma cells. After the tumor was established and vascularized, the mice were treated with naive CD8+ mel-l T cells (cish''' or WT) and recombinant vaccine encoding gplOO with or without exogenous IL-2. Here, a strong and durable regression of melanoma was observed after the adoptive transfer of cish'1' pmel-l T cells that was not observed after the adoptive transfer of WT pmel-l T cells (Figure 1 A). Adoptive transfer of cish''" pmel-l T cells enhanced the survival of tumor-bearing mice as compared to the adoptive transfer of WT pmel-l T cells (Figure 1C). Exogenous IL-2 significantly augmented the in vivo functionality of cish''" pmel-l T cells (Figure IB).
[0124] To obviate any potential confounding factors associated with the cish"'" mice, cish was knocked down in WT CD8+ pmel-l T cells using a retrovirus shMIR targeting cish (cish shMIR D2 SEQ ID NO: 3) and knockdown was confirmed. Here, it was confirmed that the cish depletion resulted in increased in vitro (Table 3) and in vivo (Figure ID) functionality of CD8+ T cells. Similar results were also obtained using cish shMIR F2 SEQ ID NO: 4. TABLE 3
Figure imgf000034_0001
EXAMPLE 4
[0125] This example demonstrates that CIS associates with HTRA1.
[0126] CIS has been implicated in the suppression of STAT5 signaling, presumably by blocking STAT5 docking to the activated cytokine receptor (Aman et al. J Biol. Chem.
274:30266-72 (1999); Matsumoto et al. Blood 89:3148-54 (1997); Yoshimura et al. EMBO J. 14:2816-26 (1995)). These findings have been observed in ectopic expression models, but have not been evaluated in the complete absence of cish. Based on the increased in vitro and in vivo functionality observed in the CD8+ cish'1' pmel-1 T cells, it was hypothesized that cish depletion would result in enhanced STAT5 phosphorylation following exposure to common gamma chain cytokines (yc).
[0127] STAT5 phosphorylation was measured by flow cytometric analysis in naive CD8+ WT or cish'1" pmel-1 T cells after the addition of 150 IU of IL-2. However, after careful analysis with a myriad of doses of cytokines, including IL-2, IL-7, IL-15, or IL-21, and time points, no discernable differences in STAT5 phosphorylation were observed in the absence of cish. Because increased functionality after TCR stimulation was observed, it was
hypothesized that perhaps STAT5 phosphorylation was enhanced in this setting. However, after TCR stimulation and cish upregulation, STAT5 phosphorylation appeared similar in both primed cish'1' and primed WT CD8+ T cells after CD3 stimulation. Phosphorylation of several other signal transduction pathways, including pERKl/2, pSTATl , pSTAT4, pSTAT4 and p38, was measured by flow cytometric analysis after CD3 stimulation of primed WT or cish'1' pmel-1 T cells, but failed to reveal insights into the observed enhanced functionality of the cish''' C + T cells. [0128] In order to investigate what accounted for the functional differences in cisti ' CD8+ T cells, a yeast-two hybrid screen was performed using CIS as bait and a mouse spleen cDNA library as prey. Here, it was found that CIS interacted with the ATP-independent serine protease HTRAl . In order to confirm this interaction in mammalian cells, a model was developed in which 293 cells were stably transduced with red fluorescent protein (RFP) or CIS tet-inducible lentiviruses termed iRFP and iCIS, respectively. After the addition of doxycyline, iRFP and iCIS-transduced cells expressed their respective products, as confirmed by flow cytometric and immunoblot analyses. These lines were then transfected with ChloromphenicolR-V5 or htral -V 5 containing plasmids and confirmed for protein expression by immunoblot analysis. Upon immunoprecipation of CIS and immunoblotting for V5, it was found that CIS directly associated with HTRAl and not the control. Immunoprecipation of V5 and immunoblotting of CIS yielded a similar result. Co-immunoprecipitation of endogenous HtrAl and CIS in primed WT CD8+ pmel-1 T cells (and vice-versa), but not in the absence of cish, also suggested a direct association of CIS and HtrAl .
EXAMPLE 5
[0129] This example demonstrates that the absence of cish results in increased HTRAl protein expression and decreased TGF-β signaling. This example also demonstrates that pharmacological blocking of HTRAl may reverse this decrease in TGF-β signaling.
[0130] Real-time polymerase chain reaction (PCR) revealed similar expression of htral mRNA in both primed WT and cish'1' pmel-1 T cells after CD3 stimulation. Interestingly, immunoblotting revealed elevated HTRAl protein in the cish'1' CD8+ T cells. Transduction of htral resulted in 2-3 logs higher levels of htral mRNA but not HTRAl protein in cish- replete T cells. HTRAl is a serine protease and has been implicated in the degradation of TGF-β family members (Launay et al. Cell Death Differ. 15: 1408-16 (2008); Tocharus et al. Dev. Growth Differ. 46:257-74 (2004); Tsuchiya et al. Bone: 37: 323-36 (2005)). TGF-βΙ evaluation by ELISA in an overnight co-culture with gpl0025-33 revealed decreased TGF-βΙ protein in cish'1' (80 pg ml/1 TGF-βΙ) as compared to WT (225 pg ml/1 TGF-βΙ) pmel-1 T cells, which was not attributable to decreased TGF-β Ι mRNA levels.
[0131] Phosphorylation of SMAD2 (s465/467) (and control beta-actin) after CD3 stimulation of primed WT or cish'1' pmel-1 T cells was measured by immunoblot analysis. Markedly enhanced TGF-β signaling, as assessed by SMAD2 phosphorylation, in CD8+ T cells was found in WT mice but not in cish-l- mice. To more accurately quantify TGF- β/SMAD activity, transgenic mice containing the firefly luciferase gene under the control of 12 SMAD-binding element repeats, called SBE-luc (Lin et al., J Immunol, 175: 547-54 (2005)) were used, which were crossed with cish-l- mice. Significantly more SMAD reporter activity was observed in cw/z-replete versus cw z-deficient CD8+ T cells. These results were consistent with the hypothesis that CIS mediates the destruction of the TGF-β protease, HTRAl . This notion was further confirmed when it was observed that this SMAD2
phosphorylation was inhibited by a cell-permeable small molecule, 2-(3-(6-methylpyridin-2-yl)- lH-pyrazol-4-yl)-l,5-naphthyridine ("compound 19") (Gellibert et al., J. Med. Chem., 47: 4494- 506 (2004)) capable of potent, selective, reversible, and ATP-competitive inhibition of TGF-β receptor phosphorylation.
[0132] In addition, TGF-β 1 secretion was evaluated by ELIS A in an overnight culture of primed WT or cish'1' pmel-1 T cells in the presence or absence of NVP-LBP976. Inhibition of HTRAl proteolytic activity by NVP-LBP976 resulted in increased TGF-βΙ protein levels in the absence of cish (Table 4).
TABLE 4
Figure imgf000036_0001
EXAMPLE 6
[0133] This example demonstrates that the absence of cish results in decreased TGF-β signaling, including reduced repression of tbx21 (T-bet), myc (c-Myc), and survival genes such as bcUll (Bcl-xL) and cyclins.
[0134] TGF-β signaling has been implicated in the suppression of many pro- functional/survival genes such as tbx21 (T-bet), myc (c-Myc), Bcl-2/xL, and cyclins in CD8+ T cells (Li et al. Cell 134: 392-04 (2008); Sanjabi et al. Immunity 31 :131-44 (2009); Wan et al. Immunol. Rev. 220: 199-13 (2007); Wan et al. J Clin. Immunol. 2: 647-59 (2008)). Using real-time PCR analysis, it was observed that tbx21 (T-bet) and myc (c-Myc) expression increased in cish'1" pmel-1 T cells after TCR stimulation (Figure 2A-B). Increased myc (c- Myc) expression correlated to elevated myc (c-Myc) reporter activity in lentiviral transduced cish'1' pmel-1 T cells (Figure 2C). In addition, real-time PCR analysis revealed increased expression of bcUll (Bcl-xL) and cyclin in cish'1' CD8+ T cells (Figure 2D-E).
[0135] TGF-β has been implicated in the induction of the gut-homing receptor CD 103 in CD8+ T cells (El-Asady et al. J Exp. Med. 201 : 1647-57 (2005); Uss et al. J. Immunol. 177: 2775-83 (2006)). In accordance with decreased TGF-β signaling, flow cytometric analysis revealed reduced CD 103 expression in primed cish'1' pmel-1 T cells relative to WT pmel-1 T cells. CD 103 was highly upregulated in the presence of cish after stimulation. Conversely, TGF-β signaling can induce the expression of genes such as ID1 and Stral3 that were found to be upregulated in WT pmel-1 T cells after stimulation (Figure 3A-B). These findings correlated with increased SMAD reporter activity in lentiviral transduced WT pmel-1 T cells (Figure 3C).
EXAMPLE 7
[0136] This example demonstrates that in the absence of cish, there is decreased TGF-β signaling, increased tbx21 (T-bet), myc (c-Myc), bcl2ll (Bcl-xL), and Bcl-2 expression, and increased in vivo survival and function of T cells, as measured by flow cytometric analysis.
[0137] Recent work has implicated TGF-β in CD8+ T cell apoptosis (Sanjabi et al.
Immunity 31 : 131-44 (2009); Tinoco et al. Immunity 31 : 145-57 (2009)). After an acute stimulation of primed CD8+ pmel-1 T cells, cish'1' T cells accumulated about half as much annexin V as WT T cells.
[0138] Gene-marked CD8+ T cells were adoptively transferred into unmanipulated mice. The in vivo survival of WT or cish'1' pmel-1 T cells was measured after the adoptive transfer of 5 x 105 WT or cish'1' cells. The results are shown in Table 5. Increased numbers of CD8+ T cells in the absence of cish compared to cish-inXaci littermates were observed. TABLE 5
Figure imgf000038_0001
[0139] To evaluate if these increased numbers of cish-l- CD8+ T cells were attributable to enhanced proliferation, bromodeoxyuridine (BrdU) was injected. In vivo BrdU
incorporation or annexin V staining on congenically marked WT or cish'1' pmel-1 T cells was measured five days after adoptive transfer, cish'1" pmel-1 T cells demonstrated decreased ex vivo annexin V uptake as compared to WT cells. However, the proliferation of cish-l- pmel-1 T cells, as measured by BrdU incorporation, was similar to that of WT cells.
[0140] Ex vivo staining of T cells with annexin V, which binds an epitope of
phosphatidylserine normally expressed only on the cytoplasmic side of the cell membrane except in T cells experiencing apoptosis, revealed that the presence of cish was associated with increased staining and thus more apoptotic cell death. The percentage of annexin V positive WT or cish'1' pmel-1 T cells was measured on days 5, 6, and 7 following adoptive transfer. The results are shown in Table 6.
TABLE 6
Figure imgf000038_0002
[0141] Accordingly, the reduced contraction of cish' " pmel- 1 T cells after vaccination (Table 5) appeared to be attributable to reduced apoptosis (Table 6) and not proliferation of the cells.
[0142] Expression of Bcl-2, an anti-apoptotic factor suppressed by TGF-β, was measured by mean fluorescence intensity (MFI) and found to be higher in cish'1' (900 MFI) than in WT cells (550 MFI). Thus, Bcl-2 was highly expressed in cish" ' pmel-1 T cells during the peak of the response, perhaps accounting for the delayed in vivo contraction.
[0143] This example demonstrated that the absence of CIS is associated with decreased TGF-β signaling and an increase in expression of tbx21 (T-bet), myc (c-Myc), bcl2ll (Bcl- xL), and Bcl-2, as well as reduced apoptosis of T cells.
EXAMPLE 8
[0144] This example demonstrates that the knockdown of htral mitigates the augmented function of cz's/z-deficient CD8+ T cells.
[0145] To evaluate the role of HTRA1 in cish'1' T cell function, WT and cish'1' CD8+ pmel-1 T cells were transduced with retroviruses encoding shMIR targeting htral (SEQ ID NO: 6) or a scramble sequence (control) and knockdown of htral was confirmed. Similar to previous observations, control-transduced cish'1' CD8+ pmel-1 T cells experienced enhanced up-regulation of thxll (T-bet), myc (c-Myc), bcl2ll (Bcl-xL), ccnel (cyclin El), cdkl (cyclin CDC2a) and ccnd2 (cyclin D2) (Figure 4A-F). In contrast to control-transduced cish'1" CD8+ pmel-1 T cells, the knockdown of htral in cish'1' CD8+ pmel-1 T cells significantly abrogated the enhanced expression of these target genes (Figure 4A-F). As previously shown, WT CD8+ pmel-1 T cells experienced a significantly lower induction of these target genes and expressed low levels of HTRAL Nonetheless, the knockdown of htral in WT CD8+ pmel-1 T cells appeared to result in a significant (p < 0.0001) albeit modest, inhibition of several of these targets compared to control WT T cells.
[0146] Five days after control or htral (SEQ ID NO: 6) shMIR transduction of WT or cish'1" pmel-1 T cells, CD 103 expression was measured by flow cytometric evaluation. In addition, annexin V staining was measured four hours after restimulation five days after htral or control shMIR transduction of WT or cish'1" pmel-1 T cells. The knockdown of htral in cish''" CD8+ pmel-1 T cells resulted in the selective induction of CD 103 and increased annexin V staining that resembled control and htral shMIR transduced WT CD8+ pmel-1 T cells.
[0147] To assess the functional significance of htral knockdown in cish'1' T cells, an overnight co-culture with peptide-pulsed target cells was performed and IFN-γ levels were determined by ELISA. The results are set forth in Table 7. TABLE 7
Figure imgf000040_0001
[0148] Here, it was found that the knockdown of htral in cish'1" CD8+ pmel-1 T cells significantly (p < 0.04) abrogated IFN-γ production (Table 7). No significant changes in IFN-γ production were observed in control or htral shMIR (SEQ ID NO: 6) transduced WT CD8+ pmel-1 T cells (Table 7).
[0149] cish expression in control or htral (SEQ ID NO: 6) shMIR transduced WT or cish'1" pmel-1 T cells was also measured by real-time PCR. There was no significant difference in the induction of cish expression with the knockdown of htral in WT CD8+ pmel-1 T cells. Knockdown of htral in cish'1' T cells abrogated the enhanced expression of known targets of TGF-β suppression and induced expression of TGF-β promoted factors. Thus, it appears that there exists a causal relationship between the enhanced functionality of cish'1' CD8+ T cells and the presence of htral. It appears that the presence of CIS might be involved in the removal or degradation of HTRAL
[0150] This example demonstrated that the knockdown of htral decreases the enhanced function of cw 2-deficient CD8+ T cells.
EXAMPLE 9
[0151] This example demonstrates that CIS degrades HTRA1, and proteasomal blockage of CIS results in the accumulation of HTRAl in the presence of CIS.
[0152] In order to evaluate if CIS is involved in HTRAl degradation, the model described in Example 4 was used. HtrAl V5 or Control- V5 was transfected into iCIS or iRFP cells. After induction of CIS or RFP by doxycycline, V5 levels were evaluated by immunoblotting. A densitometric evaluation of HtrAl V5 or control V5 staining relative to β-actin was also performed following the administration of doxycycline. The results are shown in Table 8. TABLE 8
Figure imgf000041_0001
[0153] Here, a selective decrease in HTRAl was found only in the presence of CIS and not RFP or CIS transfected with control (Table 8).
[0154] To determine if the presence of CIS in T cells results in HTRAl degradation, titrated amounts (0, 0.1, 1, or 10 μΜ) of proteasome inhibitor I (Calbiochem) were added after stimulation of primed WT or cish'1' CD8+ pmel-1 T cells. Here, a selective and titratable increase in HTRAl protein was observed as the concentration of proteasome inhibitor increased in the presence (WT) but not in the absence of cish. To determine if increased HTRAl levels correlated with decreased TGF-β signaling, titrated levels (0, 0.1, 1, or 10 μΜ) of proteasome inhibitor I were added and immunoblot analysis for SMAD2 phosphorylation was performed. Here, it was found that with increased protreasome inhibitor and
subsequently increased HTRAl, decreased SMAD2 phosphorylation was observed that was similar to cish"1' T cells after stimulation. Thus, it appears that the presence of CIS results in proteasome-mediated destruction of HTRAl .
[0155] Based on these findings, without being bound to a particular theory, it is believed that yc cytokines such as IL-2, presumably through TCR stimulation, drive the expression of STAT5 target genes including cish. It is believed that TGF-β is degraded by HTRAl, but in the presence of CIS, TGF-β is preserved while HTRAl is degraded. It is also believed that TGF-β then acts in a negative feed-back loop, inhibiting STAT5 target gene expression, and that TGF-β does not appear to negatively regulate CIS expression.
EXAMPLE 10
[0156] This example demonstrates that recombinant HtrA degrades ΤΰΡβΙ in vitro.
[0157] Recombinant HtrA and control HtrA S328A (deleteriously mutated) were produced. Micro-scale purification screening of HtrA and HtrA S328A from insect and mammalian cell culture was performed using 20 ml of transiently transfected HEK293E expression culture or 50 ml of Baculovirus Expression Vector System (BEVS) expression culture as source material. Gels are stained with Coomassie® blue dye, which confirmed that HtrA and HtrA S328A were purified.
[0158] HTRAl or HTRAl (S328A) (1 μg) was cultured overnight with hTGFpl (2 μg) with or without 50mM Tris. The samples were run on a gel and stained with Coomassie® blue dye. The gels showed that HTRAl modestly degraded TGFpi, but HTRAl (S328A) did not degrade TGFpi .
[0159] The relative density of the TGFpi bands in the gel were measured and quantified. The results are shown in Table 9.
TABLE 9
Figure imgf000042_0001
[0160] The addition of exogenous HTRAl significantly reduced TGF-p/SMAD signaling in WT CD8+ T cells after stimulation as compared to the mutant HTRAl (Figure 5A). As a control in the same experiment, the impact of the presence or absence of mutant HTRAl was measured and no change was found in TGF-p/SMAD signaling, indicating that the mutant HTRAl protein lacked enzymatic activity (Figure 5C).
EXAMPLE 1 1
[0161] This example demonstrates that the function of wild-type T cells is enhanced in the presence of recombinant HTRAl .
[0162] WT or cish-i- pmel-1 T cells were cultured overnight with peptide-pulsed splenocytes in the presence of exogenous HTRAl or inactive HTRAl (S328A) (50 μg/mL). IFN-γ production was evaluated by ELISA.
[0163] The addition of HTRAl to an overnight co-culture of pmel-1 T cells and peptide- pulsed targets significantly enhanced the production of IFN-γ in WT CD8+ T cells compared to that of inactive HTRAl (Figure 5B). There was no significant difference in cish-i- pmel-1 T cell production of IFN-γ in the presence of either intact or mutant htral, perhaps indicating saturation of endogenous HTRA1 in the absence of cish. Taken together, these data revealed that the removal of cish dramatically enhanced CD8+ T cell functionality and tumor killing.
EXAMPLE 12
[0164] This example demonstrates increased NFKB transcriptional activity in the absence of cish.
[0165] Pmel-1 T cells were transduced with reporter-lentivirus after CD3 stimulation and the relative luminescence (RLU) was measured. An increase in NFKB expression was observed in cish deficient T cells as compared to WT cells. No significant difference in MAPK/ERK was observed in cish deficient T cells as compared to WT cells.
EXAMPLE 13
[0166] This example demonstrates increased GATA3 target expression in the absence of cish.
[0167] Real-time PCR analysis of gata3, IL-4, or IL-5 normalized to five housekeeping (HKG) genes (gusb, hprtl, hsp90abl, gapdh, and actb) was performed with respect to primed WT or cish-/- pmel-1 T cells after CD3 stimulation. An increase in expression of each of gataS, IL-4, and IL-5 was observed in cish deficient T cells as compared to WT cells (Figures 6A-6C).
EXAMPLE 14
[0168] This example demonstrates increased pro-stimulatory/pro-inflammatory target expression in the absence oicish.
[0169] Real-time PCR analysis of cd27, cd28, icos, IL-la, IFN-y, gzmb, prcdl, and tnfrsfi) normalized to five housekeeping (HKG) genes (gusb, hprtl, hsp90abl, gapdh, and actb) was performed with respect to primed WT or cish-/- pmel-1 T cells after CD3 stimulation. An increase in expression of each of cd27, cd28, icos, IL-la, IFN-y, gzmb, and tnfrsf9 was observed in cish deficient T cells as compared to WT cells (Figures 7A-7H). EXAMPLE 15
[0170] This example demonstrates that the removal of cish prolongs tumor therapy in the absence of host T and B cells.
[0171] P-mel- 1 T cells (2.5 x 105) (nontransduced (NT), WT or cish-/-) were adoptively transferred into tumor-bearing ragl-/- mice (8 x 106 rVVgplOO, IL-2 (12 μg) BID x 3 days). Tumor size was measured. The removal of cish prolonged tumor therapy in the absence of host T cells (Figure 8).
[0172] P-mel-1 T cells (2.5 x 105) (nontransduced (NT), cish-/- with anti-CD8 on day 38, or cish-/- with anti-IgG on day 38) were adoptively transferred into tumor-bearing ragl-/- mice (8 x 106 rVVgplOO, IL-2 (12 μg) BID x 3 days). Tumor size was measured. The removal of cish prolonged tumor therapy in the absence of host T cells or host B cells (Figure 9)·
[0173] Four to seven days after adoptive transfer, naive WT or cish'1' pmel-1 T cells were co-cultured with pulsed targets (gpl0025-33). IFN-γ secretion was evaluated by ELISA after overnight co-culture. It was observed that removal of cish resulted in increased ex vivo function as measured by IFN-γ secretion (Table 10).
TABLE 10
Figure imgf000044_0001
[0174] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [0175] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0176] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):
1. A peripheral blood cell (PBC) comprising a high temperature requirement serine peptidase 1 (HTRAl )lhtral activator.
2. The PBC according to claim 1, wherein the HTRAl Ihtral activator comprises a recombinant nucleic acid that encodes HTRAl .
3. The PBC according to claim 2, wherein the HTRAl Ihtral activator that comprises a recombinant nucleic acid that encodes HTRAl comprises a nucleotide sequence comprising SEQ ID NO: 5 or 11 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 5 or 1 1.
4. The PBC according to claims 1-3, further comprising a cytokine-induced Src homology 2 protein (ClS)/cish inhibitor.
5. The PBC according to claim 4, wherein the CIS/cish inhibitor comprises a small interfering RNA (siR A), a microRNA, or an antisense nucleic acid.
6. The PBC according to claim 5, wherein the CIS/cish inhibitor comprises siRNA complementary to a nucleotide sequence encoding cish.
7. The PBC according to claim 6, wherein the siRNA is complementary to a nucleotide sequence comprising SEQ ID NO: 1 or 2.
8. The PBC according to claim 5, wherein the CIS/cish inhibitor comprises an antisense nucleic acid complementary to a nucleotide sequence encoding cish.
9. The PBC according to claim 8, wherein the antisense nucleic acid is
complementary to a nucleotide sequence comprising SEQ ID NO: 1 or 2.
10. The PBC according to claim 5, wherein the CIS/cish inhibitor comprises an miRNA and the miRNA is a short hairpin miRNA (shMIR).
11. The PBC according to claim 10, wherein the shMIR comprises SEQ ID NO: 3 or 4 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 3 or 4.
12. A host cell comprising a CIS/cish inhibitor and a HTRAl/htral activator.
13. The host cell according to claim 12, wherein the HTRAl Ihtral activator comprises a recombinant nucleic acid that encodes HTRAl .
14. The host cell according to claim 13, wherein the recombinant nucleic acid that encodes HTRAl comprises a nucleotide sequence comprising SEQ ID NO: 5 or 11 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 5 or 11.
15. The host cell according to any one of claims 12-14, wherein the CIS/cish inhibitor comprises a small interfering RNA (siRNA), a microRNA, or an antisense nucleic acid.
16. The host cell according to claim 15, wherein the CIS/cish inhibitor comprises siRNA complementary to a nucleotide sequence encoding cish.
17. The host cell according to claim 16, wherein the siRNA is complementary to a nucleotide sequence comprising SEQ ID NO: 1 or 2.
18. The host cell according to claim 15, wherein the CIS/cish inhibitor comprises an antisense nucleic acid complementary to a nucleotide sequence encoding cish.
19. The host cell according to claim 18, wherein the antisense nucleic acid is complementary to a nucleotide sequence comprising SEQ ID NO: 1 or 2.
20. The host cell according to claim 15, wherein the CIS/cish inhibitor comprises an miRNA and the miRNA is a short hairpin miRNA (shMIR).
21. The host cell according to claim 20, wherein the shMIR comprises SEQ ID NO: 3 or 4 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 3 or 4.
22. A host cell comprising an mti-cish shMIR comprising SEQ ID NO: 3 or 4 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 3 or 4.
23. The host cell of claim 22, wherein the host cell is a PBC.
24. The host cell of claim 23, wherein the PBC is a T cell.
25. A host cell comprising a HTRAl/htral inhibitor and a CIS/cish activator.
26. The host cell according to claim 25, wherein the CIS/cish activator comprises a recombinant nucleic acid that encodes CIS.
27. The host cell according to claim 25 or 26, wherein the HTRAl/htral inhibitor comprises a small interfering RNA (siR A), a microRNA (miRNA), or an antisense nucleic acid.
28. The host cell according to claim 27, wherein the HTRAl/htral inhibitor comprises siRNA complementary to a nucleotide sequence encoding HTRAl .
29. The host cell according to claim 28, wherein the siRNA is complementary to a nucleotide sequence comprising SEQ ID NO: 5 or 1 1.
30. The host cell according to claim 27, wherein the HTRAl/htral inhibitor comprises an antisense nucleic acid complementary to a nucleotide sequence encoding HTRAl .
31. The host cell according to claim 30, wherein the antisense nucleic acid is complementary to a nucleotide sequence comprising SEQ ID NO: 5.
32. The host cell according to claim 27, wherein the HTRAl/htral inhibitor comprises an miRNA and the miRNA is a short hairpin miRNA (shMIR).
33. The host cell according to claim 32, wherein the shMIR comprises SEQ ID NO: 6 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 6.
34. The host cell according to claim 25 or 26, wherein the HTRAl/htral inhibitor comprises NVP-LBP976.
35. The host cell according to claim 26, wherein the recombinant nucleic acid that encodes CIS comprises a nucleotide sequence comprising SEQ ID NO: 1 or 2 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 1 or 2.
36. A peripheral blood cell (PBC) comprising a HTRAl/htral inhibitor.
37. The PBC according to claim 36 further comprising a CIS/cish activator.
38. The PBC according to claim 37, wherein the CIS/cish activator comprises a recombinant nucleic acid that encodes CIS.
39. The PBC according to any one of claims 36-38, wherein the HTRAl/htral inhibitor comprises a small interfering RNA (siRNA), a microRNA, or an antisense nucleic acid.
40. The PBC according to any one of claims 36-39, wherein the HTRAl/htral inhibitor comprises siRNA complementary to a nucleotide sequence encoding HTRAl .
41. The PBC according to claim 40, wherein the siRNA is complementary to a nucleotide sequence comprising SEQ ID NO: 5.
42. The PBC according to claim 39, wherein the HTRAl/htral inhibitor comprises an antisense nucleic acid complementary to a nucleotide sequence encoding HTRAl .
43. The PBC according to claim 42, wherein the antisense nucleic acid is
complementary to a nucleotide sequence comprising SEQ ID NO: 5.
44. The PBC according to claim 39, wherein the WTRAl/htral inhibitor comprises an miRNA and the miRNA is a short hairpin miRNA (shMIR).
45. The PBC according to claim 44, wherein the shMIR comprises SEQ ID NO: 6 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 6.
46. The PBC according to any one of claims 36-38, wherein the HTRAl/htral inhibitor comprises NVP-LBP976.
47. The PBC according to claim 38, wherein the recombinant nucleic acid that encodes CIS comprises a nucleotide sequence comprising SEQ ID NO: 1 or 2 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 1 or 2.
48. A population of cells comprising at least one PBC of any one of claims 1-1 1 or at least one host cell of any one of claims 12-24.
49. A population of cells comprising at least one host cell of any one of claims 25-35 or at least one PBC of any one of claims 36-47.
50. A composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an HTRAl/htral activator and/or a CIS/cish inhibitor.
51. The composition according to claim 50, wherein the HTRAl/htral activator comprises a recombinant nucleic acid that encodes HTRAl .
52. The composition according to claim 51, wherein the HTRAl Ihtral activator that comprises a recombinant nucleic acid that encodes HTRAl comprises a nucleotide sequence comprising SEQ ID NO: 5 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 5.
53. The composition according to claim 50, wherein the HTRAl/htral activator comprises a HTRAl protein.
54. The composition according to claim 53, wherein the HTRAl protein comprises SEQ ID NO: 7 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 7.
55. The composition according to any one of claims 50-54, wherein the CIS/cish inhibitor comprises a small interfering RNA (siRNA), a microRNA, or an antisense nucleic acid.
56. The composition according to claim 55, wherein the CIS/cish inhibitor comprises siRNA complementary to a nucleotide sequence encoding CIS.
57. The composition according to claim 56, wherein the siRNA is complementary to a nucleotide sequence comprising SEQ ID NO: 1 or 2.
58. The composition according to claim 55, wherein the CIS/cish inhibitor comprises an antisense nucleic acid complementary to a nucleotide sequence encoding CIS.
59. The composition according to claim 58, wherein the antisense nucleic acid is complementary to a nucleotide sequence comprising SEQ ID NO: 1 or 2.
60. The composition according to claim 55, wherein the CIS/cish inhibitor comprises an miRNA and the miRNA is a short hairpin miRNA (shMIR).
61. The composition according to claim 60, wherein the shMIR comprises SEQ ID NO: 3 or 4 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 3 or 4.
62. The composition according to any one of claims 50-61, wherein the PBCs are T cells.
63. A composition comprising nanoparticles and PBCs, wherein the nanoparticles comprise an WTRAl/htral inhibitor and/or a CIS/cish activator.
64. The composition according to claim 63, wherein the CIS/cish activator comprises a recombinant nucleic acid that encodes CIS.
65. The composition according to claim 63, wherein the CIS/cish activator comprises a CIS protein.
66. The composition according to claim 65, wherein the CIS protein comprises SEQ ID NO: 8 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 8.
67. The composition according to any one of claims 63-66, wherein the
WTRAl/htral inhibitor comprises a small interfering RNA (siRNA), a microRNA (miRNA), or an antisense nucleic acid.
68. The composition according to claim 67, wherein the WTRAl/htral inhibitor comprises siRNA complementary to a nucleotide sequence encoding HTRAl .
69. The composition according to claim 67, wherein the siRNA is complementary to a nucleotide sequence comprising SEQ ID NO: 5.
70. The composition according to claim 67, wherein the WTRAl/htral inhibitor comprises an antisense nucleic acid complementary to a nucleotide sequence encoding HTRAl .
71. The composition according to claim 67, wherein the antisense nucleic acid is complementary to a nucleotide sequence comprising SEQ ID NO: 5.
72. The composition according to claim 67, wherein the WTRAl/htral inhibitor comprises an miRNA and the miRNA is a short hairpin miRNA (shMIR).
73. The composition according to claim 72, wherein the shMIR comprises SEQ ID NO: 6 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 6.
74. The composition according to any one of claims 63-66, wherein the
ETRAVhtral inhibitor comprises NVP-LBP976.
75. The composition according to claim 64, wherein the recombinant nucleic acid that encodes CIS comprises a nucleotide sequence comprising SEQ ID NO: 1 or 2 or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 1 or 2.
76. A pharmaceutical composition comprising the PBC of any one of claims 1-11, the host cell of any one of claims 12-24, the population of cells of claim 48, or the composition of any one of claims 50-62, and a pharmaceutically acceptable carrier.
77. A pharmaceutical composition comprising the host cell of any one of claims 25-35, the PBC of any one of claims 36-47, the population of cells of claim 49, or the composition of any one of claims 63-75, and a pharmaceutically acceptable carrier.
78. A method of treating or preventing cancer or a chronic infectious disease in a mammal, comprising administering to the mammal the PBC of any one of claims 1-1 1, the host cell of any one of claims 12-24, the population of cells of claim 48, the composition of any one of claims 50-62, or the pharmaceutical composition of claim 76 in an amount effective to treat or prevent cancer or a chronic infectious disease in the mammal.
79. A method of treating or preventing an auto- and/or allo-immune disease in a mammal, comprising administering to the mammal the host cell of any one of claims 25-35, the PBC of any one of claims 36-47, or the population of cells of claim 49, the composition of any one of claims 63-75, or the pharmaceutical composition of claim 77 in an amount effective to treat or prevent an auto- and/or allo-immune disease in the mammal.
80. A method of suppressing T cell activity in a mammal, comprising
administering to the mammal the host cell of any one of claims 25-35, the PBC of any one of claims 36-47, or the population of cells of claim 49, the composition of any one of claims 63- 75, or the pharmaceutical composition of claim 77 in an amount effective to suppress T cell activity in the mammal.
81. A method of increasing T cell activity in a mammal, comprising administering to the mammal the PBC of any one of claims 1-1 1, the host cell of any one of claims 12-24, the population of cells of claim 48, the composition of any one of claims 50-62, or the pharmaceutical composition of claim 76 in an amount effective to increase T cell activity in the mammal.
82. A method of treating or preventing a disease selected from the group consisting of age-related macular degeneration, Alzheimer's disease, and familial ischemic cerebral small-vessel disease in a mammal, comprising administering to the mammal the PBC of any one of claims 1-11 and 36-47, the host cell of any one of claims 12-35, the population of cells of claim 48 or 49, the composition of any one of claims 50-75, or the pharmaceutical composition of claim 76 or 77, in an amount effective to treat or prevent the disease in the mammal.
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