WO2024059183A1 - Methods and compositions for modulation of piezo1 in the treatment of cancer - Google Patents

Methods and compositions for modulation of piezo1 in the treatment of cancer Download PDF

Info

Publication number
WO2024059183A1
WO2024059183A1 PCT/US2023/032718 US2023032718W WO2024059183A1 WO 2024059183 A1 WO2024059183 A1 WO 2024059183A1 US 2023032718 W US2023032718 W US 2023032718W WO 2024059183 A1 WO2024059183 A1 WO 2024059183A1
Authority
WO
WIPO (PCT)
Prior art keywords
piezo
agent
tumor
cells
subject
Prior art date
Application number
PCT/US2023/032718
Other languages
French (fr)
Inventor
Arlene H. Sharpe
Emily F. GAUDIANO
Marian KALOCSAY
Robyn J. EISERT
Gary A. BRADSHAW
Martin W. LAFLEUR
Original Assignee
President And Fellows Of Harvard College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by President And Fellows Of Harvard College filed Critical President And Fellows Of Harvard College
Publication of WO2024059183A1 publication Critical patent/WO2024059183A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • 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
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4637—Other peptides or polypeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • 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
    • A61K2239/50—Colon

Definitions

  • PD-1 The coinhibitory receptor Programmed Cell Death-1 (PD-1, CD279) plays a critical role in T cell exhaustion during chronic infection and cancer.
  • PD-1 pathway inhibitors are FDA-approved for treating over 20 cancer types and have shown tremendous success in a subset of patients.
  • most cancer patients do not exhibit durable responses and patients with specific cancer types such as glioblastoma experience little to no benefit from PD-1 pathway inhibitors. Consequently, efforts are currently focused on identifying potential combination treatments that can synergize with PD-1 blockade to increase the breadth and durability of response.
  • PD-1 Despite its widespread use in the clinic, there is a limited mechanistic understanding of PD-1 signaling. Ligation of PD-1 on CD8 + T cells by its ligands Programmed Death Ligand (PD-L)l and PD-L2 results in the attenuation of multiple cellular processes driven by engagement of the T cell receptor (TCR) and costimulatory receptor CD28. PD-1 plays a pivotal role in regulating the dephosphorylation of TCR-related proteins and in modulating T cell functions such as cytokine production, Ca 2+ flux, cytolysis, cytoskeletal rearrangements, migration and metabolism.
  • TCR T cell receptor
  • PD-1 exerts its inhibitory functions, at least in part, through the recruitment of SH2-containing tyrosine phosphatases SHP2 (PTPN11) and to a lesser extent, SHP1 (PTPN6), which bind phosphotyrosine residues within the immunoreceptor tyrosine-based switch motif (ITSM) and inhibition motif (ITIM) on the PD-1 cytoplasmic tail.
  • SHP2 SH2-containing tyrosine phosphatases SHP2
  • PTPN6 SHP1
  • ITIM inhibition motif
  • kits for treating cancer in a subject comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1.
  • the agent may be a small molecule agonist of PIEZO 1, such as Yodal, critic 1 , critic2, or a modulator of PIEZO1, such as Docosahexaenoic acid.
  • the method may further comprise administering an immune checkpoint inhibitor to the subject.
  • the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM- 4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof.
  • an immune checkpoint protein selected from CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM- 4,
  • the agent that increases or stabilizes the activity or expression of PIEZO 1 and the immune checkpoint inhibitor are administered conjointly. In some embodiments, the that agent increases or stabilizes the activity or expression of PIEZO 1 and the immune checkpoint inhibitor act synergistically when administered.
  • the agent is a gRNA fused to a transcription activator, such as a gRNA that comprises a region that is complementary to a portion of a gene that encodes a PIEZO 1 protein.
  • the agent is a vector encoding a PIEZO 1 protein, such as a viral vector encoding a PIEZO 1 protein.
  • the agent may be an agent is administered systemically, intravenously, subcutaneously, or intramuscularly.
  • the agent may be administered to the subject in a pharmaceutically acceptable formulation.
  • the method may further comprise administering to the subject an additional agent, such as a chemotherapeutic agent or a cancer vaccine.
  • the method may further comprise administering to the subject a cancer therapy, such as radiation.
  • the subject may be refractory for immune checkpoint inhibitory therapy.
  • kits for treating cancer in a subject unresponsive to immune checkpoint inhibitor therapy comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1 (e.g., any agent that increases or stabilizes the activity or expression of PIEZO 1 disclosed herein) and an immune checkpoint inhibitor.
  • the agent may be a small molecule agonist of PIEZO1, such as Yodal, critic 1 , critic2, or a modulator of PIEZO1, such as Docosahexaenoic acid.
  • the method may further comprise administering an immune checkpoint inhibitor to the subject, such an inhibitor of PD-1 or PD-L1, or another immune checkpoint inhibitor disclosed herein.
  • T-cells that have been treated ex vivo with an agent that increases or stabilizes the activity or expression of PIEZO1 (e.g., any agent that increases or stabilizes the activity or expression of PIEZO1 disclosed herein).
  • the T- cells may be tumor infiltrating lymphocytes.
  • T-cells are autologous.
  • T-cells are allogeneic.
  • the subject is a human.
  • FIG. 1A-1J show that quantitative proximity proteomics identifies PD-1 and PIEZO1 association following PD-L1 ligation.
  • Figure 1 A shows a diagram of PD-1- APEX2 proximity labeling dynamics in Jurkat cells treated with TCR-PD-L1 and TCR- control beads.
  • Figure IB shows an experimental schematic of PD-1-APEX2 proximity labeling time course with bead treatment. TMT ratios (TMT RA) over time (min) of (Fig. 1C) SHP2, (Fig. ID) PIEZO 1, (Fig. IE) SHP1, (Fig IF) CD3£ and (Fig.
  • Figure II shows that log-adjusted p-values of significantly enriched GO Terms in TCR-PD-L1 or TCR-control conditions identified from three independent experiments and calculated from fold changes comparing TCR-PD- L1 and TCR-control mean slope of each gene over time.
  • the top 50 genes were selected for analysis using gProfiler g:Ost.
  • Figure 1 J shows rank list of the top ten genes identified in TCR-PD-L1 or TCR-control conditions calculated from the mean slope of each gene in three independent experiments. Schematics created using BioRender.
  • FIG. 2A-Fig 2K shows that PD-1 inhibits TCR-induced PIEZO1 activity around F- actin rings.
  • Figure 2A shows a schematic of the working principle of GenEPi reporter in which GCaMP fluorescence is increased by PIEZO 1 -specific Ca 2+ influx.
  • Yellow arrows indicate F-actin rings and merged images contain GenEPi and PD-1 signal only.
  • Normalized fluorescence intensity for Fig. 2D shows that PD-1 expression and Fig. 2E shows PIEZO1 activity calculated from the sum of pixel values within each region of interest (ROI) at 200 seconds of stimulation. ROIs comprise single cells.
  • Statistical significance for a) and b) was measured using Student’s unpaired t-test and statistical significance for Figure 2C was measure using two- way ANOVA. * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • kymographs for Fig. 2G shows TCR-control and Fig. 2H shows TCR-PD-L1 conditions.
  • Quantification of kymographs for local Fig. 21 shows PD-1 expression and Fig. 2J shows PIEZO 1 activity during stimulation.
  • Fig. 2K shows normalized fluorescence intensity for local PIEZO 1 activity over 200 seconds calculated from the sum of pixel values within each ROI for TCR-control and TCR-PD-L1 conditions.
  • Statistical significance for Fig. 21 and Fig. 2J was measured using Student’s unpaired t-test and statistical significance for k) was measured using two-way ANOVA Schematic created using BioRender.
  • Fig. 3A-3N shows that PIEZO 1 activity in CD8 + T cells regulates antitumor immunity.
  • FIG. 3E shows CD8 + TILs and Fig. 3F and Fig. 3G shows frequencies of CD62L-expressing CD8 + TILs isolated from MC38 and B16-0VA tumors on day 15 post-implantation.
  • Ratio of Fig. 3H and Fig. 31 shows frequencies of TIM-3 and Slamf6-expressing CD8 + TILs.
  • Fig. 3 J shows a schematic of tumor growth experiment with PD-1 blockade.
  • Fig. 3K shows MC38 and Fig. 3L shows B16-0VA tumors in Cre+ and Cre- E8i-Cre-ER T2 Piezo l flx/flx mice with corresponding Fig. 3M shows MC38 and Fig. 3N shows B16-0VA survival analysis.
  • Statistical significance for tumor growth curves was calculated using two-way ANOVA. * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • Statistical significance of survival curves was measured using Log-rank Mantel Cox test. * p ⁇ 0.0332, ** p ⁇ 0.0021, *** p ⁇ 0.002.
  • Fig. 4A-4K shows combined PD-1 blockade and PIEZO1 agonism promote tumor control.
  • FIG. 4H shows a schematic of combined PD-1 blockade and PIEZO1 agonist Yodal administration to B16.F10 tumor-bearing mice.
  • Fig. 41 shows tumor volumes (mm 3 ) were measured over time (days) and
  • Figure 4J shows individual tumor growth curves from data depicted in Fig. 41.
  • Statistical significance of survival curves was measured using Log-rank Mantel Cox test. * p ⁇ 0.0332, ** p ⁇ 0.0021, *** p ⁇ 0.002.
  • Fig. 5A-5C shows PD-1-APEX2 construct design and cell line optimization.
  • Fig. 5 A Design of human and murine PD-1-APEX2 constructs.
  • hPGK denotes the human phosphoglycerate kinase promoter.
  • Fig. 5B PD-1-APEX2 expression in Jurkat cells was measured by flow cytometry and compared to unstimulated, non-lentivirally transduced Jurkat cells.
  • Fig. 5C Labelling efficiency of PD-1-APEX2 probe shown via Western blot stained with Ponceau to assess protein loading and probed with streptactin-HRP.
  • Fig. 6A-6P shows Dynabead optimization. Tosyl-activated Dynabeads coated with various ratios of crosslinking-CD3/CD28 antibodies and recombinant murine PD-L1 or human IgGiK (hlgGiK) control ligand (x-axis) were incubated with murine CD8 + T cells for 48 h to assess percentages of cells expressing Fig. 6A) CD8[3, Fig. 6B) CD44, Fig. 6C) PD- 1, Fig. 6D) granzyme B, and percentage of Fig. 6E) live cells via flow cytometry. Cell culture supernatants were collected to assess concentrations of excreted cytokines Fig.
  • Fig. 6F IL-2
  • Fig. 6G IFNy
  • Fig. 6H TNFoc using CBA assay.
  • the x-axis denotes specified protein ratios where “T” represents the combined percentage of TCR components CD3 and CD28 while the latter number represents percentage of murine PD-L1 or hlgGiK on the beads.
  • T represents the combined percentage of TCR components CD3 and CD28 while the latter number represents percentage of murine PD-L1 or hlgGiK on the beads.
  • Increasing concentrations of beads coated with 60% PD-L1 and 40% TCR components selected from the previous assay were incubated with primary murine CD8 + T cells for 48 h to assess percentages of cells expressing Fig. 61) CD8[3, Fig. 6J) CD44, Fig. 6K) PD-1, 1) granzyme B and percentage of Fig. 6M) live cells via flow cytometry.
  • Fig. 7A-7I shows APEX2 proximity labelling identifies PIEZO 1 as target of murine PD-1.
  • Fig. 8A-8E shows additional PD-1-APEX2 findings.
  • Data are presented as means from three independent experiments ⁇ SD.
  • Statistical significance was assessed using two-way ANOVA analysis comparing TCR-PD-L1 and TCR-control mlgGi bead- treated groups. Only significant differences are indicated.
  • Fig. 8E Hierarchical one-way clustering of the averaged TMT ratios of all identified proteins calculated from three independent experiments.
  • Fig. 9A-9G shows PD-L1 inhibits PIEZO1 activity in PD-1 -expressing GenEPi Jurkat cells in vitro
  • Fig. 9A Electroporated and blasticidin-selected GenEPi Jurkat cells were incubated with a dose titration of doxycycline (25-200ng/mL) for 24 h and treated with 1O
  • Fig. 9B Schematic of TIRF time lapse imaging experimental design. Raw integrated density reported as intensity for Fig. 9C) PD-1 expression and Fig.
  • PIEZO 1 activity calculated from the sum of pixel values within each region of interest (ROI) at 200 seconds of stimulation presented as normalized values in Fig. 2.
  • Fig. 9E 10 s or Fig. 9F
  • FIG. 10A-10G shows E8i-Cre-ER T2 PIEZO 1 KO is specific to CD8 + T cells.
  • Fig. 10A Schematic of E8i-Cre-ER T2 Piezo P' x f ' x mouse model.
  • Fig. 11A-11K shows E8i-Cre-ER T2 Piezo P lx flx CD8 + T cells show minimal phenotypic alterations at baseline.
  • CD8 + T cells isolated from spleens of Cre+ and Cre- E8i- Cre-ER T2 Piezo l flx/flx mice were stimulated for 24 h with increasing doses of anti- CD3/CD28 crosslinking antibodies (0-10 ug/mL, x-axis) and assessed for frequencies of CD8 + Fig. 11 A) live cells, Fig. 1 IB) PD-1, Fig. 11C) CTLA-4, Fig. 1 ID) CD44, Fig. 1 IE) CD62L, Fig.
  • Fig. 12A-12S shows flow cytometric analysis of TILs from tumor bearing E8i-Cre- ER T2 Pzezo/ ⁇ ’ ⁇ x mice.
  • Fig. 12A Gating strategy for CD8 TILs isolated from MC38 or B16- OVA tumors.
  • Frequencies of Fig. 12B and E CD3E + , Fig. 12C and F) total CD4 + T cells and Fig. 12D and G) CD8 + /CD4 + ratio (counts).
  • Fig. 13A-13B shows individual tumor growth curves of E8i-Cre-ER T2 mice. Individual tumor growth curves from data depicted in Figure 2.3k-l of Cre+ and Cre- E8i- Cre-ER T2 PIEZO l flx/flx mice treated with anti-PD-1 or isotype control antibody and bearing Fig. 13A) MC38 or Fig. 13B) Bl 6-0 VA tumors.
  • Fig. 14A-14D shows PIEZO agonism influences TIL phenotypes in WT mice.
  • Statistical significance was assessed using 2-way ANOVA analysis. Frequencies of CD8 + TILs that are Fig. 14C) Slamf6+ TIM-3- and Fig. 14D) Slamf6- TIM-3+ assessed by flow cytometry.
  • Statistical significance was assessed using Student’ s unpaired t test, ns, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • the PD-1 pathway plays a critical role in mediating T cell exhaustion, and blockade of this pathway can promote antitumor immunity. While PD-1 inhibitors are revolutionizing cancer therapy, only a subset of patients respond and show durable remission, highlighting the need to better understand how the PD-1 pathway suppresses T cell functions.
  • PD- 1/PD-L1 ligation-induced dynamic changes of the local proteome proximal to PD-1 were quantified using unbiased multiplexed proximity proteomics.
  • the mechanosensitive cation channel PIEZO 1 was identified as a primary target of PD-1 -mediated inhibition.
  • mice Stimulation of CD8 + T cells through TCR and CD28 engagement triggered the activation of PIEZO1, while simultaneous PD-1 ligation countered this activation.
  • Mice lacking PIEZO1 selectively on CD8 + T cells exhibited increased tumor growth marked by impaired CD8 + T cell function, which could not be rescued by PD-1 blockade.
  • mice treated with PIEZO 1 agonist showed increased numbers and function of CD8 + tumor-infiltrating lymphocytes compared to controls.
  • Combined administration of PIEZO 1 agonist and anti- PD-1 significantly reduced tumor burden and improved survival in a tumor model unresponsive to PD-1 blockade.
  • PIEZO 1 inhibition as an important mechanism by which PD-1 signaling regulates CD8 + T cell functions and suggest PIEZO1 agonism and/or modulation as a novel approach for augmenting cancer immunotherapy.
  • unbiased proximity-labeling as applied to the characterization of the dynamic behavior of proteins recruited to the cytoplasmic tail of PD-1 following ligation and identification of the mechanosensitive ion channel PIEZO 1 (FAM38a) as a primary target of PD-1 -mediated inhibition.
  • PIEZO1 is expressed on a variety of cancers, which include epithelial and immune cells, where its activity can be regulated by external forces such as shear stress, cyclical hydrostatic pressure, and membrane deformation (force- from-lipid), as well as actomyosin contractility and extracellular matrix tethering (force- from-filament). Both external and internal forces contribute to PIEZO 1 gating, a process by which an ion channel transitions between its open and closed conformations as a means of regulating the passage of electrical current through the ion-conducting pore; this, in turn, induces downstream signaling.
  • the stimulation of CD8 + T cells through TCR and CD28 engagement triggers the activation of PIEZO 1.
  • KO knockout mice deficient in PIEZO 1 only in CD8 + T cells exhibited markedly increased tumor growth.
  • CD8 + tumor infiltrating lymphocytes (TILs) isolated from these mice were less abundant and less activated.
  • CD8 + T cell-specific PIEZO 1 KO decreased the efficacy of PD-1 blockade in tumor-bearing mice.
  • an element means one element or more than one element.
  • agent is used herein to denote a chemical compound, a small molecule, a mixture of chemical compounds and/or a biological macromolecule (such as a nucleic acid, an antibody, an antibody fragment, a protein or a peptide). The activity of such agents may render them suitable as a “therapeutic agent” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
  • antibody broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site.
  • Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
  • antibody as used herein also includes an “antigen-binding portion” of an antibody (or simply “antibody portion”).
  • antigen-binding portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody.
  • binding fragments encompassed within the term “antigenbinding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • a F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778).
  • scFv single chain Fv
  • single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody.
  • Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes.
  • VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448; Poljak et al. (1994) Structure 2:1121- 1123).
  • An antibody for use in the instant invention may be a bispecific antibody.
  • a bispecific antibody has binding sites for two different antigens within a single antibody polypeptide. Antigen binding may be simultaneous or sequential.
  • Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecific antibodies produced by a hybrid hybridoma or a trioma are disclosed in U.S. Patent 4,474,893. Bispecific antibodies have been constructed by chemical means (Staerz et al. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci.
  • bispecific antibodies are also described in U.S. Patent 5,959,084. Fragments of bispecific antibodies are described in U.S. Patent 5,798,229.
  • Bispecific agents can also be generated by making heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identification of clones producing and co-assembling both antibodies. They can also be generated by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof such as Fab and Fv sequences.
  • an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, biomarker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al. ( ⁇ 99$)Mol.
  • Antibody portions such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies.
  • antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.
  • Antibodies may also be "humanized” which is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences.
  • the humanized antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
  • the term “humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • cancer or “tumor” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.
  • Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell.
  • cancer includes premalignant as well as malignant cancers.
  • Cancers include, but are not limited to, B cell cancer, e.g., myelomas like multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like.
  • cancers include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma,
  • human sarcomas and carcinomas e.g.,
  • cancers are epithlelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
  • the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.
  • the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma, or breast carcinoma.
  • the epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.
  • the cancer comprises a solid tumor.
  • the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a blood born tumor, a brain/CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing tumor, an eye tumor, a gallbladder tumor, a gastrointestinal, a kidney tumor, a laryngeal or hypopharyngreal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastom
  • the phrase “ conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include synergistic effects of the two agents).
  • the different therapeutic agents can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially.
  • the different therapeutic agents can be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about a week of one another.
  • a subject who receives such treatment can benefit from a combined effect of different therapeutic agents.
  • any two agents and/or additional agents may be conjointly administered according to the methods provided herein.
  • PIEZO 1 is a mechanosensitive ion channel protein that in humans is encoded by the gene PIEZ01. It is a mechanosensitive non-specific cation channel. It plays a key role in epithelial cell adhesion by maintaining integrin activation through R-Ras recruitment to the ER, likely in its activated state, and subsequent stimulation of calpain signaling.
  • Exemplary nucleotide and amino acid sequences of human PIEZO 1, which correspond to GenBank Accession numbers, are listed below in Table 1.
  • an agent described herein targets an amino acid sequence disclosed in Table 1 or at least partially encodes an sequence listed in Table 1.
  • an agent described herein targets a nucleic acid sequence described in Table 1 or at least partially encodes an sequence listed in Table 1.
  • PIEZO1. may comprise a sequence listed below in Table 1, or may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% homology to a sequence listed in Table 1.
  • CCTCCTCCTA ACCTCAATGCATCGCGTCTTCCAGCCCCCGGCTCCGAGGGCTCAGCCTCCAGGTGGTCTA
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • polynucleotide and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified, such as by conjugation with a labeling component.
  • the term “recombinant” polynucleotide means a polynucleotide of genomic, cDNA, semi synthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.
  • prevent, ” “preventing, ” “prevention, ” and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.
  • small molecule is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.
  • the term “subject” means a human or non-human animal selected for treatment or therapy.
  • tumor microenvironment is an art-recognized term and refers to the cellular environment in which the tumor exists, and includes, for example, interstitial fluids surrounding the tumor, surrounding blood vessels, immune cells, other cells, fibroblasts, signaling molecules, and the extracellular matrix.
  • therapeutically-effective amount and “effective amount” as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment.
  • “Treating” a disease in a subject or “treating” a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g, the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.
  • kits for treating cancer in a subject comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1.
  • the agent may be a small molecule agonist of PIEZO 1, such as Yodal, critic 1, romance2, or a modulator of PIEZO1, such as Docosahexaenoic acid.
  • the method may further comprise administering an immune checkpoint inhibitor to the subject.
  • the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM- 4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof.
  • the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1.
  • the agent that increases or stabilizes the activity or expression of PIEZO1 and the immune checkpoint inhibitor are administered conjointly In some embodiments, the agent increases or stabilizes the activity or expression of PIEZO 1 and the immune checkpoint inhibitor act synergistically when administered.
  • the agent is a gRNA fused to a transcription activator, such as a gRNA that comprises a region that is complementary to a portion of a gene that encodes a PIEZO 1 protein.
  • the agent is a vector encoding a PIEZO 1 protein, such as a viral vector encoding a PIEZO1 protein.
  • the agent that increases or stabilizes the activity or expression of PIEZO 1 may be a small molecule agonist of PIEZO 1, such as Yodal, critic 1 , critic2, a modulator of PIEZO1, such as Docosahexaenoic acid, a gRNA disclosed herein, or any combination thereof.
  • the agent may be administered systemically, intravenously, subcutaneously, or intramuscularly.
  • the agent may be administered to the subject in a pharmaceutically acceptable formulation.
  • the method may further comprise administering to the subject an additional agent, such as a chemotherapeutic agent or a cancer vaccine.
  • the method may further comprise administering to the subject a cancer therapy, such as radiation.
  • the subject may be refractory for immune checkpoint inhibitory therapy.
  • PIEZO 1 e.g., any agent that increases or stabilizes the activity or expression of PIEZO 1 disclosed herein
  • an immune checkpoint inhibitor e.g., any agent that increases or stabilizes the activity or expression of PIEZO 1 disclosed herein
  • the agent may be a small molecule agonist of PIEZO1, such as Yodal, critic 1 , critic2, a modulator of PIEZO1, such as Docosahexaenoic acid, or any combination thereof.
  • the method may further comprise administering an immune checkpoint inhibitor to the subject, such an inhibitor of PD-1 or PD-L1, or another immune checkpoint inhibitor disclosed herein.
  • T-cells that have been treated ex vivo with an agent that increases or stabilizes the activity or expression of PIEZO1 (e.g., any agent that increases or stabilizes the activity or expression of PIEZO1 disclosed herein).
  • the T- cells may be tumor infiltrating lymphocytes.
  • T-cells are autologous (z.e., derived from the subject).
  • T-cells are allogeneic (ie., derived from a doner).
  • the subject is a human.
  • compositions for preventing or treating cancer in a subject comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1.
  • the agent may increase the activity of PIEZO 1 by at least 5%, at least 10%, at least
  • the agent may increase the expression of PIEZO 1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • the agent may increase the expression of PIEZO 1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • An agent disclosed herein may increase PIEZO 1 mRNA by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • Measurement of PIEZO 1 can be done in a biological sample or multiple biological samples taken from the subject over a period of time.
  • the agent provided herein is a polypeptide agent (e.g., a polypeptide that binds to a PIEZO 1 protein).
  • a polypeptide agent disclosed herein may increase or stabilize the expression or activity of PIEZO 1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • the agent may be a chimeric or fusion polypeptide.
  • the agent may be a ligand or binding partner of PIEZO 1.
  • a fusion or chimeric polypeptide can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence.
  • polypeptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding a polypeptide(s). Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous polypeptides in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N. Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J.
  • agents that are vectors that contain the isolated nucleic acid molecules described herein, such as those that encode a PIEZO 1 peptide.
  • the term “vector,” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector is another type of vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non- episomal mammalian vectors
  • Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby be replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes.
  • Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors or simply, “expression vectors”.
  • cells that contain a nucleic acid described herein e.g., a nucleic acid encoding an antibody, antigen binding fragment thereof, antibody-like molecule, or polypeptide described herein.
  • the cell can be, for example, prokaryotic, eukaryotic, mammalian, avian, murine and/or human.
  • interfering nucleic acids described herein may be contacted with a cell or administered to an organism (e.g., a human).
  • constructs and/or vectors encoding the interfering RNA molecules may be contacted with or introduced into a cell or organism.
  • a viral, retroviral or lentiviral vector is used.
  • agents for increasing the expression or activity of PIEZO 1 are delivered to subjects by use of viral vectors.
  • viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.
  • adeno-associated virus vectors adeno-associated virus vectors
  • herpes simplex virus vectors SV 40 vectors
  • polyoma virus vectors polyoma virus vectors
  • papilloma virus vectors papilloma virus vectors
  • picornavirus vectors e.g., pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox
  • pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox
  • a helper-dependent or gutless adenovirus a helper-dependent or gutless adenovirus.
  • adenoviruses can be used to deliver nucleic acid agents for increasing the expression or activity of PIEZO 1.
  • Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy.
  • nucleic acid agents for increasing the expression or activity of PIEZO 1 can be delivered by adeno-associated virus (AAV) vectors.
  • AAV vector that expresses a nucleic acid agent for increasing the expression or activity of PIEZO 1 is a recombinant AAV vector having, for example, either an U6 or Hl RNA promoter, or a cytomegalovirus (CMV) promoter.
  • Suitable AAV vectors for use in agents, compositions, and methods described include, but are not limited to AAVs described in Passini et al., Methods Mol. Biol. 246: 225-36 (2004).
  • the agent comprises a CRISPR activation agent and/or a sgRNA.
  • the agent is an sgRNA.
  • An sgRNA combines tracrRNA and crRNA, which are separate molecules in the native CRISPR/Cas9 system, into a single RNA construct, simplifying the components needed to use CRISPR activation system.
  • the crRNA of the sgRNA has complementarity to PIEZO 1 DNA.
  • Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies may also be fully human.
  • a monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.
  • the methods and compositions provided herein relate to antibodies and antigen binding fragments thereof that bind specifically to a PIEZO 1 protein.
  • Such antibodies can be polyclonal or monoclonal and can be, for example, murine, chimeric, humanized or fully human.
  • the agent may be a recombinant antibodies specific for a PIEZO 1 protein, such as chimeric or humanized monoclonal antibodies, can be made using standard recombinant DNA techniques.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in US Pat No. 4,816,567; US Pat. No. 5,565,332; Better et al.
  • Human monoclonal antibodies specific for a PIEZO 1 protein can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system.
  • “HuMAb mice” which contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy (p and y) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous p and K chain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856 859).
  • mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal antibodies
  • the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal antibodies
  • HuMAb mice The preparation of HuMAb mice is described in Taylor, L. et al.
  • small molecule agents e.g., small molecule agents that increase or stabilize the expression or activity of a PIEZO 1 protein.
  • agents include those known in the art and those identified using the screening assays described herein.
  • a small molecule provided herein may have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% specificity for a PIEZO 1 protein.
  • assays used to identify agents include obtaining a population of cells and a small molecule agent, wherein the cells are incubated with a small molecule agent and the level of a PIEZO 1 protein is measured.
  • Agents identified via such assays may be useful, for example, for increasing the expression or the activity of a PIEZO 1 protein in a subject.
  • Agents useful in the methods disclosed herein may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Agents may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al, 1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145).
  • Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.
  • composition e.g., a pharmaceutical composition, containing at least one agent described herein together with a pharmaceutically acceptable carrier.
  • the composition includes a combination of multiple (e.g., two or more) agents described herein.
  • the pharmaceutical composition is delivered locally or systemically. In some embodiments, the pharmaceutical composition may be administered to a tumor present in the subject. In some embodiments, the agent or pharmaceutical composition is administered with an additional cancer therapeutic agent. In some embodiments, the additional cancer therapeutic agent is a chemotherapeutic agent. In some embodiments, the pharmaceutical composition further comprises an additional agent for treatment of cancer. In some embodiments, the additional agent is a tumor vaccine.
  • the additional therapeutic agent is a chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CytoxanTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; emylerumines and memylamelamines including alfretamine, triemylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimemylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (articularly cryptophycin 1 and crypto
  • chemotherapeutic agent include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NolvadexTM), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FarestonTM); inhibitors of the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MegaceTM), exemestane, formestane, fadrozole, vorozole (RivisorTM), letrozole (FemaraTM), and anastrozole (ArimidexTM); and antiandrogens such as flutamide, nilutamide,
  • SERMs selective estrogen
  • the additional cancer therapeutic agent is an immune checkpoint inhibitor.
  • Immune Checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response.
  • immune checkpoint proteins are CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM- 4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof.
  • compositions and/or agents disclosed herein may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; or (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous, intrathecal, intracerebral or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue
  • parenteral administration for example, by subcutaneous, intramuscular, intravenous, intrathecal, intracer
  • Methods of preparing pharmaceutical formulations or compositions include the step of bringing into association an agent described herein with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • compositions suitable for parenteral administration comprise one or more agents described herein in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, dimethyl sulfoxide (DMSO), polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • DMSO dimethyl sulfoxide
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • provided herein are methods of treating a cancer by administering to a subject (e.g., to a tumor present in a subject) an agent and/or a pharmaceutical composition described herein.
  • a subject e.g., to a tumor present in a subject
  • an agent and/or a pharmaceutical composition described herein e.g., an agent and/or a pharmaceutical composition described herein.
  • the subject is a human.
  • the methods described herein may be used to treat any cancerous or pre-cancerous tumor.
  • the cancer includes a solid tumor.
  • Cancers that may be treated by methods and compositions provided herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the subject has cancer.
  • the cancer comprises a solid tumor.
  • the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a blood born tumor, a brain/CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing tumor, an eye tumor, a gallbladder tumor, a gastrointestinal, a kidney tumor, a laryngeal or hypopharyngreal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penile tumor, a pituitary tumor
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions or agents to be administered may be varied so as to obtain an amount of the active ingredient (e.g., an agent described herein) which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • an amount of the active ingredient e.g., an agent described herein
  • compositions disclosed herein may be administered over any period of time effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the period of time may be at least 1 day, at least 10 days, at least 20 days, at least 30, days, at least 60 days, at least three months, at least six months, at least a year, at least three years, at least five years, or at least ten years.
  • the dose may be administered when needed, sporadically, or at regular intervals. For example, the dose may be administered monthly, weekly, biweekly, triweekly, once a day, or twice a day.
  • a dose of the composition is administered at regular intervals over a period of time.
  • a dose of the composition is administered at least once a week. In some embodiments, a dose of the composition is administered at least twice a week. In certain embodiments, a dose of the composition is administered at least three times a week. In some embodiments, a dose of the composition is administered at least once a day. In some embodiments, a dose of the composition is administered at least twice a day.
  • doses of the composition are administered for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 4 weeks, for at least 1 month, for at least 2 months, for at least 3 months, for at least 4 months, for at least 5 months, for at least 6 months, for at least 1 year, for at least two years, at least three years, or at least five years.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician could prescribe and/or administer doses of the compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • Example 1 Proximity proteomics identifies PD-1 and PIEZO1 association
  • PD- l-APEX2-expressing Jurkat cells were sorted and assessed for labeling efficiency using anti-streptavidin Western blotting (Fig. 5B,C).
  • Tosyl-activated Dynabeads were covalently coupled to CD3 and CD28 crosslinking antibodies using either control murine IgGi ligand (mlgGi, TCR-control) or recombinant PD-Ll-Fc fusion protein (TCR-PD-L1) to provide TCR and co-stimulatory signals for T cell activation while also driving strong inhibitory signals through PD-1, respectively (Fig. 6).
  • control murine IgGi ligand mlgGi, TCR-control
  • TCR-PD-L1 recombinant PD-Ll-Fc fusion protein
  • Biotinylation of proteins proximal to the PD-1 cytoplasmic tail was induced by the addition of H2O2 to the celkbead mixture during the last minute of each timepoint (Fig. 1 A, Fig. 5C).
  • Cell pellets were lysed and biotinylated proteins were enriched using streptavidin beads under denaturing conditions. Trypsin digest-derived peptides were labelled with tandem mass tags (TMT) and quantified by triple-stage mass spectrometry (MS 3 ) 31 .
  • TMT tandem mass tags
  • MS 3 triple-stage mass spectrometry
  • TMT RA scaled TMT ratios
  • CD3 subunits, CD28 and ZAP70 remain proximal to PD-1 at the immune synapse in the presence or absence of PD-L1 ligation (Fig. 1F-1G, Fig. 7D-I and Fig. 8A-D).
  • One-way hierarchical clustering confirmed different recruitment kinetics for SHP1, CD3 subunits, CD28 or Zap70 (Fig. 1H, Fig. 8E).
  • the limited changes in PD-1 binding by known mediators as compared to the robust, ligand-dependent association of PIEZO 1 and PD-1 implicates an uncharacterized mechanism of PD-1 that involves PIEZO1 function.
  • gProfiler g:OSt gene ontology (GO) term functional enrichment analysis was used to compare pathways that were enriched in TCR-PD-L1 or TCR-control stimulated conditions. It was found that clathrin-mediated endocytosis and membrane trafficking were among the most significantly enriched pathways in TCR-PD-L1 conditions as compared to TCR-controls, suggesting that PD-1 ligation may alter receptor clustering and turnover in the plasma membrane to prevent stable contact with antigen presenting cells at the immune synapse (Fig. II).
  • Example 2 PD-1 ligation inhibits PIEZOl-mediated Ca 2+ influx
  • GenEPi contains a low affinity and high dynamic range GFP, calmodulin and Ml 3 peptide fusion protein (GCaMP) engineered to ensure the specificity of sensing PIEZO- 1 -mediated Ca 2+ influx.
  • GenEPi reporter was expressed using a Tet-On system and is comprised of GCaMP fused to the cytoplasmic tail of PIEZO 1 using a flexible linker (Fig. 2A).
  • GenEPi Jurkat cells exhibited increasing levels of reporter expression and PIEZO 1 activity with increasing doses of doxycycline in the presence of the PIEZO 1 agonist Yodal, verifying the inducibility and functional specificity of GenEPi (Fig. 9A).
  • PD-1 -expressing GenEPi Jurkat cells were passed through a flow-cell chamber coated with crosslinking CD3 and CD28 antibodies and either PD-L1 or control mlgGi ligand (Fig. 9B).
  • TIRF Total Internal Reflection Fluorescence
  • GenEPi Jurkat cells ligated with PD-L1 was delayed and had lower intensity overall as compared to TCR-stimulated cells, while PD-1 intensity remained the same in both conditions, suggesting that PD-1 inhibits PIEZO 1 activation (Fig. 2B and 2E, Fig. 9C).
  • GenEPi Jurkat cells exhibited strong PIEZO 1 activity following 100 s of TCR stimulation that peaked at 200 s and persisted for 5 min (300 s), further confirming that TCR stimulation induces PIEZO1 activity (Fig. 2B, and 2E-2F, Fig. 9D, and captured via extended videos, incorporated herein by reference).
  • PIEZO 1 activity was significantly decreased upon PD-1 ligation in the presence of PD-L1 at PD-1 clustering sites during the process of immune synapse and F-actin ring formation implying that PD-1 ligation results in the attenuation of PIEZO 1 activity through physical association (Fig. 2C,2G-2K).
  • PIEZO 1 activity following PD-1 ligation was assessed by quantifying mean fluorescence intensity (MFI) of GCaMP fluorescence and frequency of GFP -positive, PD-1 -expressing GenEPi Jurkat cells treated with TCR-control or TCR-PD-L1 beads.
  • MFI mean fluorescence intensity
  • PD-1 -expressing GenEPi Jurkat cells exhibited a significant decrease in PIEZO1 activity following exposure to TCR-PD-L1 beads compared to GenEPi Jurkat cells treated with TCR-control beads or PIEZO 1 agonist Yodal (Fig. 9E-G).
  • Example 3 CD8 + -specific PIEZO1 KO impairs antitumor immunity
  • a tamoxifen- inducible CD8 + -specific PIEZO 1 KO mouse was developed by crossing Piezo l ⁇ x mice with E8i-Cre-ER T2 Rosa26 tdTomato reporter mice (Extended Data Fig. 6a). Following tamoxifen treatment, the efficacy of PIEZO 1 deletion was -70% in Cre+ CD8 + T cells as compared to Cre- controls, as assessed by RT-qPCR using probes specific for the loxP regions flanking exons 20-23 of PIEZO 1 (Fig. 10B).
  • the frequency of CD8 + T cells expressing Cre protein was consistent with decreased PIEZO 1 transcript levels assessed by RT-qPCR (Fig. 10B-D).
  • the level of Cre reporter in CD8 + splenocytes was assessed on day 15 following tumor implantation and observed sustained Cre activity in Cre+ CD8 + T cells, confirming the efficacy of our inducible Cre system (Fig. 10E,F).
  • a significant decrease in PIEZO- 1 -specific Ca 2+ influx induced by Yodal treatment (visualized using the cell permeable Ca 2+ dye Indo-1) was observed in Cre+ CD8 + T cells compared to Cre- CD8 + T cells; Fig. 10G).
  • E8i-CRE-ER T2 Pzezo7- /Zx ⁇ x Cre+ and Cre- tumor-bearing mice were treated with anti-PD-1 or isotype control antibody (Fig. 3J). Strikingly, administration of PD-1 blocking antibody on days 14 and 17 reduced clearance of MC38 tumors, which are highly sensitive to PD-1 blockade, in E8i-CRE-ER T2 Piezo P lx f ' x Cre+ mice compared to anti-PD-1 treated Cre- mice (Fig. 3K and Fig. 13A).
  • PIEZO1 KO in CD8 + T cells significantly impaired overall survival in both tumor models regardless of PD-1 blockade (Fig. 3M and 3N).
  • PD-l-mediated inhibition of PIEZO1 in CD8 + T cells contributes to immune evasion by tumors, underscoring the importance of PIEZO 1 function in shaping T cell responses.
  • Example 4 PIEZO1 agonism improves CD8 + T cell antitumor immunity
  • MC38 tumors were implanted subcutaneously in WT mice and administered Yodal (7.5mg/kg) intraperitoneally (i.p.) (Fig. 4A). While Yodal treatment had variable effects on MC38 tumor growth in WT mice (Fig.14A-B), phenotypic analysis of the TILs at day 15 following MC38 tumor implantation revealed increased percentages of CD8 + TILs in Yodal-treated mice compared to controls (Fig. 4B).
  • Yodal-treated CD8 + TILs expressed more activation and effector proteins, including PD-1, granzyme B and perforin and lower expression of CD62L (Fig. 4C-F).
  • PIEZO 1 -agonized CD8 + TILs expressed high levels of terminal exhaustion marker TIM-3 and low levels of progenitor stem-like marker Slamf6 compared to controls, further suggesting a role for PIEZO 1 in driving an effector-exhausted phenotype (Fig. 4G, Fig. l4C-D).
  • PIEZO 1 -agonism improves CD8 + T cell activation and function in the TME and has significant therapeutic benefit in mouse models when combined with PD-1 blockade to control tumor growth.
  • combining PD-1 blockade with PIEZO1 agonism improved tumor control in PD-1 blockade-unresponsive tumors (Fig. 4H-4K).
  • the effectiveness of this combination may result from 1) prevention of PIEZO 1 inactivation as well as inhibitory phosphatase recruitment by PD-1 blockade and 2) PIEZO1 agonism further increasing the number and function of cytotoxic CD8 + T cells in the tumor, leading to enhanced tumor control.
  • PIEZO1 activity may be regulated by TCR-induced cytoskeletal rearrangements, specifically retrograde actin flow, which is supported by the data capturing PIEZO 1 activity concentrated around F-actin ring structures.
  • the mechanism by which Ca 2+ influx is sustained to induce specific gene transcription following CRAC channel activation in T cells remains unclear.
  • Previous studies have proposed a positive feedback loop between F- actin polymerization and Ca 2+ influx, which persists over hours at the immune synapse to maintain TCR signaling 7 . It is possible that actomyosin contractile forces modulate PIEZO 1 to sustain Ca 2+ influx, resulting in downstream gene transcription.
  • PD-1 sequestration of actin cytoskeletal regulatory proteins and proteins that mediate membrane turnover may play a role in impairing actin cytoskeletal dynamics and stability at the immune synapse, thereby attenuating actomyosin contractile forces that activate PIEZO 1.
  • mice show that modulating PIEZO 1 activity influences the anti-tumor activity of CD8 + T cells, subsequently affecting tumor growth.
  • combined therapy using PIEZO1 agonist and PD-1 blockade improved tumor control in mice harboring tumors unresponsive to PD-1 blockade.
  • these studies expand our understanding of the diverse mechanisms by which PD-1 inhibits T cell function, identify a novel role for PIEZO 1 in controlling CD8 + T cell function and reveal a new combination therapeutic strategy using PD-1 blockade and PIEZO1 agonism. Further understanding of the mechanisms controlling PD-1 and PIEZO 1 association, the specific downstream pathways involved and the effects of external mechanical force on this biology should provide additional fundamental and therapeutic insights.
  • Jurkat E6.1 and human PD-1 -expressing Jurkat E6.1 cells 35.8 line transduced with an ORF-T2A/IRES-GFP reporter
  • ORF-T2A/IRES-GFP reporter a gift from N. Haining/K. Yates, Dana Farber Cancer Institute
  • RPMI media supplemented with 10% FBS, 1% penicillin/streptomycin, 1% HEPES and 0.1% BME (denoted RIO).
  • MC38-OVA (a gift from N. Haining/N. Collins, Dana-Farber Cancer Institute)
  • B16-OVA generated in collaboration with the N. Haining lab
  • MC38-WT a gift from D. Vignali, University of Pittsburgh School of Medicine
  • B16.F10 a gift from G.
  • MC38-OVA and B16-OVA cells were produced by transduction of parental MC38 and B16.F10 cells with the lentiviral vector TRC-pLX305 (Broad Institute) containing OVA protein.
  • PD-1 sequences were obtained from NCBI and were modified using the codon optimization tool from Integrated DNA Technologies (IDT).
  • APEX2 sequences have been previously described 17 .
  • PD-1-APEX2 fusion sequences contain human or murine PD-1 linked to APEX2 on the C-terminal tail by an 8-residue serine-glycine linker (GGSSGGSS) and a FLAG-TAG added to the C-terminal tail of APEX2. All human and murine PD-1- APEX2 double-stranded gene fragments (gBlocks) were generated by IDT. Cloning of gBlock gene fragments into the pXPR_053 plasmid (Addgene #113591) was completed by GenScript.
  • 293x cells were transfected with PD-1-APEX2 fusion plasmids along with PsPax2 and MD2G packaging plasmids suspended in Optimem (Life Technologies #31985-062) and polyethylamine “Max” (PEI, Polysciences #24765-2), and supplemented with fresh media after 24 h.
  • Lentivirus was harvested 48 h following media supplementation by ultracentrifugation for 2 h at 71,934.8 RCF at 4°C.
  • Viral titer was calculated using serial dilutions of the viral stock on 293x cells in DMEM complete media containing PEI “Max”.
  • a multiplicity of infection of 30 was used to spin transduce Jurkat cells for 2 h at 652 RCF at 37°C.
  • Jurkat cells were stained with human PD-1 surface antibody (BioLegend, clone EH12.2H7, #329936) and evaluated for PD-1 expression using flow cytometry 24 h and 48 h following transduction.
  • Highly expressing PD-1-APEX2 fusion clones were sorted on the BD Aria II. Stocks were frozen in RIO media containing 10% dimethyl sulfoxide (DMSO) for future experiments.
  • DMSO dimethyl sulfoxide
  • PD-l-APEX2-expressing Jurkat cells were cultured at a concentration of 1 million cells/mL in labeling media (RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% HEPES, 0.1% BME and 500
  • labeling media RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% HEPES, 0.1% BME and 500
  • TCR-PD-L1 or TCR-control Dynabeads were added to PD-l-APEX2-expressing Jurkat cells at a ratio of 1 :4 cells/beads for the specified timepoints. No beads were added to the 0 min control to assess protein localization at baseline.
  • H2O2 hydrogen peroxide
  • 2X quenching buffer DPBS supplemented with 20mM sodium ascorbate, lOmM trolox and 20mM sodium azide
  • cells were spun down immediately at 726 RCF for 2 min.
  • Cells were immediately washed three times with ice-cold IX quenching buffer and spun down at 726 RCF for 2 min after each wash.
  • IX quenching wash cells were washed with 10 mL ice-cold PBS and 1 million cells were taken for Western blotting analysis. Cells were then spun down a final time at 726 RCF for 2 min. All PBS was removed from cell pellets, which were flash-frozen on dry ice and stored at -80°C until processing and streptavidin pulldown.
  • PD-l-APEX2-expressing Jurkat cell pellets were processed using previously described methods with slight modification 3 .
  • Cell pellets were lysed in filtered 8M urea with 1% sodium dodecyl sulfate (SDS) at room temperature for 15 min.
  • 55% ice-cold trichloroacetic acid (TCA) was added to lysates at a 1 : 1 ratio and left on ice for 15 min to precipitate the protein. Lysates were spun down at 20,817 RCF for 15 min at room temperature to prevent urea from precipitating out of solution. The supernatant was discarded, and protein pellets were washed 4X with ice-cold acetone and spun down at 20,817 RCF for 10 min following each wash.
  • Protein pellets were then air-dried and resuspended in 8M urea lysis buffer supplemented with 1% SDS and lOmM tris(2- carboxyethyl)phosphine (TCEP, Sigma) and 100 mM NH4CO3. Pellets were water-bath sonicated for 3 cycles of 30 s and vortexed mixed at 37°C for 2 h until the pellets were completely resuspended. Redissolved pellets were checked for an alkaline pH and then spun down at 20,817 RCF for 15 min at room temperature.
  • 8M urea lysis buffer supplemented with 1% SDS and lOmM tris(2- carboxyethyl)phosphine (TCEP, Sigma) and 100 mM NH4CO3.
  • Pellets were water-bath sonicated for 3 cycles of 30 s and vortexed mixed at 37°C for 2 h until the pellets were completely resuspended. Redissolved pellets were checked for an
  • Streptavidin beads were digested with lysyl endopeptidase (LysC, 2mg/mL, Wako) in 50pL 200mM 3 -[4-(2-hydroxyethyl)piperazin-l-yl]propane-l -sulfonic acid (EPPS) pH 8.5 and 2% acetonitrile at 37°C. Trypsin (50pL stock, Promega #V5111, in EPPS buffer and at a final dilution of 1 : 100) was added for additional digest overnight at 37°C. Beads were removed with a magnetic rack and clear supernatants were transferred to new tubes.
  • Peptide digest reactions were directly labelled with TMT1 Iplex (Thermo Fisher Scientific #A34808) reagents in 200mM EPPS pH 8.5, 30% acetonitrile for 1 h at room temperature. Labeling efficiency was measured by MS of mixed small aliquots of the labeling reactions and was >95%. Frozen and thawed TMT labeling reactions were quenched with 0.3% hydroxylamine for 15 min at room temperature.
  • Fraction pairs 1+7, 2+8, 3+9, 4+10, 5+11, 6+12 were mixed, dried down to completion, desalted with Stage tips and run on Orbitrap Lumos mass spectrometers (Thermo Fisher Scientific).
  • Peptide-spectrum matches were obtained by a SEQUEST (v.28, rev.12) based software, searching a database with respective UniProt mouse and human reference proteomes with added common contaminants and reverse peptide sequences as decoy. After mzXML conversion of spectra, searches used a mass tolerance of 20 p.p.m.
  • Scaled quantification data were analyzed by one-way clustering (Ward’s method) using the JMP Pro statistical software package.
  • Proteomics raw data and search results were deposited in the PRIDE archive 51 and can be accessed under ProteomeXchange 52 accession numbers PXD036136, PXD036207 and PXD036147 for human PD-1-APEX2 experiments and PXD036218 and PXD036216 for murine PD-1- APEX2 experiments.
  • the top 50 proteins were selected for GO Term functional enrichment analysis using g-Profiler g:GOSt functional profiling 33 . Adjusted p- values of significantly enriched functions for TCR-control and TCR-PD-L1 conditions were reported in Fig. li.
  • GenEPi reporter plasmid (XLGenEPi) was generated by the Pantazis lab. GenEPi reporter expression was driven by a doxycycline-inducible Tet-On system. Jurkat cells were co-nucleofected with the XLGenEPi plasmid and pCMV_pBase at a molecular ratio of 1 : 1 using Lonza’s SE Cell line kit (cat. V4XC-2024) and corresponding CL-120 program on Lonza’s Amaxa-4D Nucleofector.
  • the pCMV_pBase plasmid is a non-viral vector that expresses the piggyBac transposase (pBASe) which, when co-transfected, allows for the integration of the GenEpi transgene into the genome using two terminal repeat domains flanking the GenEPi reporter 53 .
  • This integration into the genome allows for the generation of stable GenEPi-expressing clones that can be selected for using blasticidin (BSD).
  • BSD blasticidin
  • 2pg of XLGenEpi and equimolecular amounts of pCMV_pBase plasmids were used to nucleofect 1 million Jurkat cells in cuvettes.
  • GenEPi nucleofected cells were selected with 8pg/mL BSD for 7 days.
  • Cells were then rested in complete RPMI media for 24 h and treated with 200 ng/mL doxycycline for 24 h to induce GenEPi reporter expression.
  • Selected Jurkat cells were then treated with 5pM Yodal and GFP -positive clones were immediately sorted on the BD FACS Aria and cultured to generate stable cell lines.
  • GenEPi Jurkat cells were treated with ImmunoCult Human CD3/CD28 T cell activator (STEMCELL Technologies #10971) for 48 h prior to imaging to induce PD-1 expression.
  • GenEPi Jurkat cells were treated with doxycycline (200ng/mL) for 24 h prior to imaging to temporally control and induce GenEPi expression.
  • GenEPi Jurkat cells were then stained with F-actin SPY550-FastAct (1 :500, Cytoskeleton #CY-SC205) and antihuman PD-1 AF647 (1 :50, Biolegend #329910 clone EH12.2H7) for 1 h at 37°C in PBS supplemented with 10% FBS (PBS-F). Cells were washed twice with PBS-F and resuspended at a concentration of 1 million/mL in complete RPMI media.
  • TIRF imaging was performed using a fully motorized Nikon Ti inverted microscope equipped with a Nikon Ti-TIRF-EM Motorized Illuminator and a Nikon LUN-F Laser Launch with single fiber output (488nm, 90mW;561 nm, 70mW; 640nm, 65mW).
  • 80 pL of stained XLGenEPi Jurkat cells were flowed into one of the 6 coated
  • Signals from the different channels were acquired sequentially using a Chroma ZT 405/488/561/640 multi-band pass dichroic mirror mounted on a Nikon TIRF filter cube located in the filter cube turret, and band pass emission filters for GenEPi AF488 (Chroma ET525/50m), SPY5550-FastAct (Chroma ET 595/50m) and PD-1 AF647 (Chroma ET 6551p) channels, respectively, located on a Sutter emission filter wheel within the infinity space of the stand.
  • Time-lapse imaging was performed with a time interval of 20 s and total acquisition time of 5 min.
  • An ND 16 filter was introduced in the light-path to reduce irradiation. Imaging conditions were optimized to reduced photobleaching and phototoxicity.
  • TIRF image analysis was completed using a custom workflow built in Fiji/ImageJ 54 .
  • the TIRF images were opened in Fiji and split into the different fluorescent channels.
  • a 500 pixel rolling ball background subtraction was applied to reduce background intensities close to 0.
  • the individual PD-1 and PIEZO1 channels were processed by performing a Tophat/difference of Gaussian filter to emphasize the small puncta.
  • the processed images were segmented by intensity -based thresholding.
  • a close filter followed by a size filter was applied to the thresholded masks to generate the final masks for PD-1 and PIEZO1.
  • Regions of interest (ROI) were then drawn to demarcate cell boundaries to calculate the parameters on a per cell basis.
  • the mask areas, the intensities of the signal and all ROIs were measured.
  • Normalized fluorescence intensity for PIEZO 1 activity was calculated from the sum of pixel values within each ROI for TCR-control and TCR-PD-L1 conditions. Only cells expressing both PD-1 and the PIEZO1 GenEPi reporter were analyzed. Representative images were processed using Nikon Imaging Software (NIS) and Fiji/ImageJ. Al-denoise was applied to F-actin channels using NIS. A 25 pixel rolling ball background subtraction was then applied to F-actin channels in Fiji to reduce background. A 50 pixel rolling ball background subtraction was applied to GenEPi reporter and PD-1 channels in Fiji to reduce background. A median filter of 1 pixel was applied to GenEPi reporter and PD-1 channels in Fiji.
  • mice Seven- to ten-week-old age-matched female or male mice were used for all in vivo experiments and seven- to fourteen-week-old mice were used for CD8 + T cell isolation and in vitro experimentation.
  • Wild type C57BL/6J mice were purchased from Jackson Laboratories.
  • E8I-Cre-ER T2 mice were a generous gift from the Vignali lab.
  • Homozygous E8I-Cre-ER T2 mice were crossed with homozygous Piezol flx/flx mice purchased from Jackson Laboratories (Stock #029213). Mice were crossed until they were fixed for the Pi ezol flx/flx mutant allele as assessed by the Transnetyx genotyping service.
  • the Piezol-2 WT probe was used to detect the WT Piezol allele (forward primer:
  • the L1L2-Bact-P MD probe (forward primer: GCTGGCGCCGGAAC; reverse primer: GCGACTATAGAGATATCAACCACTTTGT) was used to detect the LlL2_Bact_P cassette, composed of an FRT site followed by a lacZ sequence and a loxP site, confirming the integration of the floxed Piezol mutant allele.
  • eGFP E8I-Cre-ER T2 and Rosa26-LSL TD tomato knock-in activity reporters were also quantified by Transnetyx using the eGFP (forward primer: CGTCGTCCTTGAAGAAGATGGT; reverse primer: CACATGAAGCAGCACGACTT) and tdRFP (forward primer: AGATCCACCAGGCCCTGAA; reverse primer: GTCTTGAACTCCACCAGGTAGTG) probes, respectively.
  • Heterozygous Cre mice were bred to generate both Cre+ and Cre- littermates. To induce Piezol deletion, Cre+ and Cre- mice were intraperitoneally injected with 8 doses of lOmg/mL tamoxifen daily.
  • mice were anesthetized with 2.5% 2,2,2-tribromoethanol (Avertin, Sigma-Aldrich catalog no. T48402-25G) and injected in the flank subcutaneously with 2.5 * 10 5 MC38, B16.F10 or B16-OVA tumor cells. Once palpable tumors were observed, tumors were measured every other day to calculate tumor volume over time. Tumor volume was calculated using the equation (L*W 2 )/2 where L denotes tumor length and W denotes tumor width. Mice were monitored for body condition and weight loss. Mice with tumors that exceeded 2000mm 3 , that were severely ulcerated or that infiltrated the i.p. cavity were sacrificed. Tumor infiltrating lymphocyte isolation
  • Tumors were harvested at day 15 following implantation and processed for analysis. Extracted tumors were mechanically chopped and treated with collagenase type 1 (Worthington Biochemical, #LS004194) and mixed for 25 min at 37°C. Lymphocytes were enriched using a 40/70% Percoll gradient that was centrifuged at 805 RCF for 20 min (no brake). Immune cells were harvested from the interface between 40% and 70% Percoll and resuspended in MACS buffer (PBS with 1 % FBS and 2 mM EDTA) for staining for flow cytometry analyses.
  • MACS buffer PBS with 1 % FBS and 2 mM EDTA
  • Cells were surface stained with antibodies listed below at a 1 : 100 dilution (unless otherwise noted) in 96-well V-bottom plates for 45 min in the dark on ice. Samples were washed twice with MACS buffer and fixed for 20 min in the dark at room temperature and permeabilized with FoxP3/Transcription Factor Staining Buffer Set Kit according to the manufacturer’s protocol (eBioscience #00-5523-00). Cells were stained with intracellular antibodies listed below at a 1 : 100 dilution (unless otherwise noted) for 1 h in the dark on ice and washed twice with eBioscience Permeabilization Buffer. All washes used 200p.L of the specified buffers and spins were performed at 726 RCF for 2 min at 4°C. Fixed and stained samples were acquired the following day on the BD FACSymphony and were analyzed using FlowJo software.
  • Flow cytometry analyses were performed on a BD FACSymphony and cell sorting was performed on a BD Aria II.
  • the following fluorescent antibodies were purchased from Biolegend for flow cytometry and cell sorting: human PD-1 (clone EH12.2H7, #329936) and murine CD45.2 (APC-Cy7, clone 104, #109824), CD8b (Alexa Fluor700, clone 53-6.7, #00730), CD44 (APC, clone IM7, #103012), PD-1 (PE-Cy7, RMP1-30, #109110), CD69 (BV421, H1.2F3, #104528), CD62L (BV605, PerCPCy5.5, clone MEL-14), CD3e (BUV395, PerCpCy55, FITC, clone 145-2C11), perforin (PE, clone S16009A), granzyme B (BV421, clone
  • LIVE/DEAD Fixable Aqua dead cell stain (1 :600, L34957) and LIVE/DEAD Fixable Near-IR dead cell stain (1 :600, L34976) were purchased from Thermo Fisher Scientific.
  • Jurkat cells or tumor cells were lysed in Pierce RIPA Buffer supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (100X) for 15 min on ice.
  • Whole cell lysates were spun down at 20,817 RCF at 4°C for 15 min. Supernatants were collected and transferred to new Eppendorf tubes and I OpL of each lysate was taken for protein estimation using the Pierce BCA Protein Assay Kit to normalize for protein loading. Lysates were denatured with 4X Laemmli Sample Buffer (BioRad) or 4X NuPAGE LDS (Invitrogen) containing beta mercaptoethanol (BME) and boiled for 5 min at 95°C.

Abstract

Provided herein are methods of treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1.

Description

METHODS AND COMPOSITIONS FOR MODULATION OF PIEZO1 IN THE TREATMENT OF CANCER
RELATED APPLICATIONS
This application claims priority to U.S. provisional application 63/406,506, filed September 14, 2022, hereby incorporated by reference in its entirety.
GOVERNMENT SUPPORT
This invention was made with government support under CA247073, CA224088, AI056299, and AI108545, awarded by National Institutes of Health (NIH). The government has certain rights in this invention.
BACKGROUND
The coinhibitory receptor Programmed Cell Death-1 (PD-1, CD279) plays a critical role in T cell exhaustion during chronic infection and cancer. PD-1 pathway inhibitors are FDA-approved for treating over 20 cancer types and have shown tremendous success in a subset of patients. However, most cancer patients do not exhibit durable responses and patients with specific cancer types such as glioblastoma experience little to no benefit from PD-1 pathway inhibitors. Consequently, efforts are currently focused on identifying potential combination treatments that can synergize with PD-1 blockade to increase the breadth and durability of response.
Despite its widespread use in the clinic, there is a limited mechanistic understanding of PD-1 signaling. Ligation of PD-1 on CD8+ T cells by its ligands Programmed Death Ligand (PD-L)l and PD-L2 results in the attenuation of multiple cellular processes driven by engagement of the T cell receptor (TCR) and costimulatory receptor CD28. PD-1 plays a pivotal role in regulating the dephosphorylation of TCR-related proteins and in modulating T cell functions such as cytokine production, Ca2+ flux, cytolysis, cytoskeletal rearrangements, migration and metabolism. PD-1 exerts its inhibitory functions, at least in part, through the recruitment of SH2-containing tyrosine phosphatases SHP2 (PTPN11) and to a lesser extent, SHP1 (PTPN6), which bind phosphotyrosine residues within the immunoreceptor tyrosine-based switch motif (ITSM) and inhibition motif (ITIM) on the PD-1 cytoplasmic tail. However, the precise mechanism by which PD-1 and SHP2/SHP1 counter T cell activation remain unclear. Recent studies have shown that T cell-specific SHP2 knockout (KO) mice respond to PD-1 blockade and T cells from these mice can become exhausted, showing that PD-1 inhibition can occur in the absence of SHP2. These data suggest that the consequences of phosphatase association with PD-1 are more complex than previously thought and that unidentified proteins and signaling pathways may play a role in mediating PD-1 function. Thus, it was hypothesized that additional unidentified proteins contribute to PD-1 -mediated CD8+ T cell inhibition and among these proteins potential targets for therapeutics may be identified that can augment PD-1 -based immunotherapy.
SUMMARY
In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1. The agent may be a small molecule agonist of PIEZO 1, such as Yodal, Jedi 1 , Jedi2, or a modulator of PIEZO1, such as Docosahexaenoic acid. The method may further comprise administering an immune checkpoint inhibitor to the subject.
In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1 and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM- 4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof.
In some embodiments, the agent that increases or stabilizes the activity or expression of PIEZO 1 and the immune checkpoint inhibitor are administered conjointly. In some embodiments, the that agent increases or stabilizes the activity or expression of PIEZO 1 and the immune checkpoint inhibitor act synergistically when administered.
In some embodiments, the agent is a gRNA fused to a transcription activator, such as a gRNA that comprises a region that is complementary to a portion of a gene that encodes a PIEZO 1 protein. In some embodiments, the agent is a vector encoding a PIEZO 1 protein, such as a viral vector encoding a PIEZO 1 protein.
The agent may be an agent is administered systemically, intravenously, subcutaneously, or intramuscularly. The agent may be administered to the subject in a pharmaceutically acceptable formulation. The method may further comprise administering to the subject an additional agent, such as a chemotherapeutic agent or a cancer vaccine. The method may further comprise administering to the subject a cancer therapy, such as radiation. The subject may be refractory for immune checkpoint inhibitory therapy.
In some aspects, provided herein are methods of treating cancer in a subject unresponsive to immune checkpoint inhibitor therapy, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1 (e.g., any agent that increases or stabilizes the activity or expression of PIEZO 1 disclosed herein) and an immune checkpoint inhibitor. The agent may be a small molecule agonist of PIEZO1, such as Yodal, Jedi 1 , Jedi2, or a modulator of PIEZO1, such as Docosahexaenoic acid. The method may further comprise administering an immune checkpoint inhibitor to the subject, such an inhibitor of PD-1 or PD-L1, or another immune checkpoint inhibitor disclosed herein.
In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject T-cells that have been treated ex vivo with an agent that increases or stabilizes the activity or expression of PIEZO1 (e.g., any agent that increases or stabilizes the activity or expression of PIEZO1 disclosed herein). The T- cells may be tumor infiltrating lymphocytes. In some embodiments, T-cells are autologous. In some embodiments, T-cells are allogeneic.
In some aspects, the subject is a human.
BRIEF DESCRIPTION OF THE FIGURES
Figs. 1A-1J show that quantitative proximity proteomics identifies PD-1 and PIEZO1 association following PD-L1 ligation. Figure 1 A shows a diagram of PD-1- APEX2 proximity labeling dynamics in Jurkat cells treated with TCR-PD-L1 and TCR- control beads. Figure IB shows an experimental schematic of PD-1-APEX2 proximity labeling time course with bead treatment. TMT ratios (TMT RA) over time (min) of (Fig. 1C) SHP2, (Fig. ID) PIEZO 1, (Fig. IE) SHP1, (Fig IF) CD3£ and (Fig. 1G) CD28 following stimulation of PD-l-APEX2-expressing Jurkat cells with TCR-PD-L1 (PD-L1) or TCR-control (mlgGl) beads. Data are presented as means from three independent experiments ± SD. Statistical significance was assessed using two-way ANOVA analysis comparing TCR-PD-L1 and TCR-control bead-treated groups. Only significant differences are indicated. Figure 1H shows
Figure imgf000004_0001
hierarchical one-way clustering of the averaged TMT ratios from the top 25 proteins calculated from three independent experiments. Figure II shows that log-adjusted p-values of significantly enriched GO Terms in TCR-PD-L1 or TCR-control conditions identified from three independent experiments and calculated from fold changes comparing TCR-PD- L1 and TCR-control mean slope of each gene over time. The top 50 genes were selected for analysis using gProfiler g:Ost. Figure 1 J shows rank list of the top ten genes identified in TCR-PD-L1 or TCR-control conditions calculated from the mean slope of each gene in three independent experiments. Schematics created using BioRender.
Fig. 2A-Fig 2K shows that PD-1 inhibits TCR-induced PIEZO1 activity around F- actin rings. Figure 2A shows a schematic of the working principle of GenEPi reporter in which GCaMP fluorescence is increased by PIEZO 1 -specific Ca2+ influx. Representative TIRF microscopy images of GenEPi Jurkat (AF488) cells stained with PD-1 (AF647) and F-actin dye (SPY555) and passed through flow cell chambers coated with anti-CD3/CD28 crosslinking antibodies and either Fig. 2b shows control mlgGi ligand or Fig. 2C shows PD-L1 to assess PIEZO 1 activity over 300 seconds of stimulation. Yellow arrows indicate F-actin rings and merged images contain GenEPi and PD-1 signal only. Normalized fluorescence intensity for Fig. 2D shows that PD-1 expression and Fig. 2E shows PIEZO1 activity calculated from the sum of pixel values within each region of interest (ROI) at 200 seconds of stimulation. ROIs comprise single cells. Figure 2F shows normalized PIEZO 1 activity over 200 seconds measured at intervals of 20 seconds. Data are presented as means of n=37-50 ROIs ± SEM. Statistical significance for a) and b) was measured using Student’s unpaired t-test and statistical significance for Figure 2C was measure using two- way ANOVA. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Representative kymographs for Fig. 2G shows TCR-control and Fig. 2H shows TCR-PD-L1 conditions. Quantification of kymographs for local Fig. 21 shows PD-1 expression and Fig. 2J shows PIEZO 1 activity during stimulation. Fig. 2K shows normalized fluorescence intensity for local PIEZO 1 activity over 200 seconds calculated from the sum of pixel values within each ROI for TCR-control and TCR-PD-L1 conditions. ROIs comprise PD-1 clustering sites. Data are presented as means of n=5-l 1 ROIs ± SEM. Statistical significance for Fig. 21 and Fig. 2J was measured using Student’s unpaired t-test and statistical significance for k) was measured using two-way ANOVA Schematic created using BioRender.
Fig. 3A-3N shows that PIEZO 1 activity in CD8+ T cells regulates antitumor immunity. Fig. 3 A shows a schematic of E8i-Cre-ERT2 Piezo l^x/:flx tumor growth experiment and TIL analysis. Tumor volumes (mm3) were measured over time (days) for Fig. 3B shows MC38 and Fig. 3C shows B16-0VA tumors in Cre+ and Cre- E8i-Cre-ERT2 Piezol^* mice. Data are presented as means of n=10-l l for MC38 and n=12-18 for B16-0VA ± SD from two independent experiments for each tumor type including male and female mice. Frequencies of Fig. 3D and Fig. 3E shows CD8+ TILs and Fig. 3F and Fig. 3G shows frequencies of CD62L-expressing CD8+ TILs isolated from MC38 and B16-0VA tumors on day 15 post-implantation. Ratio of Fig. 3H and Fig. 31 shows frequencies of TIM-3 and Slamf6-expressing CD8+ TILs. Data are presented as means of n=18-26 from three independent experiments ± SD. Statistical significance was measured using Student’s unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Non-parametric t tests were used for graphs that did not exhibit Gaussian distribution. Fig. 3 J shows a schematic of tumor growth experiment with PD-1 blockade. Tumor volumes (mm3) were measured over time (days) for Fig. 3K shows MC38 and Fig. 3L shows B16-0VA tumors in Cre+ and Cre- E8i-Cre-ERT2 Piezo lflx/flx mice with corresponding Fig. 3M shows MC38 and Fig. 3N shows B16-0VA survival analysis. Data are presented as means of n=5-8 ± SD from one experiment for each tumor type. Statistical significance for tumor growth curves was calculated using two-way ANOVA. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical significance of survival curves was measured using Log-rank Mantel Cox test. * p<0.0332, ** p<0.0021, *** p<0.002.
Fig. 4A-4K shows combined PD-1 blockade and PIEZO1 agonism promote tumor control. Fig. 4A shows a schematic of Yodal treatment in MC38 tumor bearing WT mice. Frequencies of b) CD8+ T cells, (Fig. 4C) CD62L-expressing, (Fig. 4D) granzyme B- expressing, (Fig. 4E) PD-1 -expressing and (Fig. 4F) perforin-expressing CD8+ T cells and (Fig. 4G) ratio of TIM-3/Slamf6 expressing CD8+ T cells in tumors assessed by flow cytometry. Data are presented as means of n=6-12 ± SD from one or two independent experiments. Statistical significance was measured using Student’s unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Fig. 4H shows a schematic of combined PD-1 blockade and PIEZO1 agonist Yodal administration to B16.F10 tumor-bearing mice. Fig. 41 shows tumor volumes (mm3) were measured over time (days) and Figure 4J shows individual tumor growth curves from data depicted in Fig. 41. Fig. 4K shows corresponding survival analysis. Data are combined from two independent experiments and are presented as means of n=13-15. Statistical significance of tumor growth was measured using 3-way ANOVA. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical significance of survival curves was measured using Log-rank Mantel Cox test. * p<0.0332, ** p<0.0021, *** p<0.002.
Fig. 5A-5C shows PD-1-APEX2 construct design and cell line optimization. Fig. 5 A. Design of human and murine PD-1-APEX2 constructs. hPGK denotes the human phosphoglycerate kinase promoter. Fig. 5B. PD-1-APEX2 expression in Jurkat cells was measured by flow cytometry and compared to unstimulated, non-lentivirally transduced Jurkat cells. Fig. 5C. Labelling efficiency of PD-1-APEX2 probe shown via Western blot stained with Ponceau to assess protein loading and probed with streptactin-HRP.
Fig. 6A-6P shows Dynabead optimization. Tosyl-activated Dynabeads coated with various ratios of crosslinking-CD3/CD28 antibodies and recombinant murine PD-L1 or human IgGiK (hlgGiK) control ligand (x-axis) were incubated with murine CD8+ T cells for 48 h to assess percentages of cells expressing Fig. 6A) CD8[3, Fig. 6B) CD44, Fig. 6C) PD- 1, Fig. 6D) granzyme B, and percentage of Fig. 6E) live cells via flow cytometry. Cell culture supernatants were collected to assess concentrations of excreted cytokines Fig. 6F) IL-2, Fig. 6G) IFNy and Fig. 6H) TNFoc using CBA assay. The x-axis denotes specified protein ratios where “T” represents the combined percentage of TCR components CD3 and CD28 while the latter number represents percentage of murine PD-L1 or hlgGiK on the beads. Increasing concentrations of beads coated with 60% PD-L1 and 40% TCR components selected from the previous assay were incubated with primary murine CD8+ T cells for 48 h to assess percentages of cells expressing Fig. 61) CD8[3, Fig. 6J) CD44, Fig. 6K) PD-1, 1) granzyme B and percentage of Fig. 6M) live cells via flow cytometry. Cell culture supernatants were collected to assess levels of excreted cytokines Fig. 6N) IL-2, Fig. 60) IFNy, Fig. 6P) TNFoc using CBA assay. Data are presented as means of n=5 biological replicates ± SD. Statistical significance was assessed between control and PD-L1 groups using two-way ANOVA analysis, ns, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig. 7A-7I shows APEX2 proximity labelling identifies PIEZO 1 as target of murine PD-1. TMT ratios over time (min) of Fig. 7A) SHP2, Fig. 7B) PIEZO1, Fig. 7C) SHP1, Fig. 7D) CD3£, Fig. 7E) CD28, Fig. 7F) ZAP70, Fig. 7G) CD35, Fig. 7H) CD3E and Fig. 71) CD3y following stimulation of murine PD-1-APEX2 with murine TCR-PD-L1 (mPD- Ll) or TCR-control hlgGiK beads at indicated timepoints. Data are represented as means ± SD from two independent experiments using two-way ANOVA analysis comparing PD-L1 and control hlgGiK bead-treated groups. Only significant differences are indicated. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig. 8A-8E shows additional PD-1-APEX2 findings. TMT ratios over time (min) of Fig. 8A) CD38, Fig. 8B) CD3E, Fig. 8C) CD3y and Fig. 8D) ZAP70 following stimulation of PD-1-APEX2 with TCR-PD-L1 or TCR-control mlgGi beads. Data are presented as means from three independent experiments ± SD. Statistical significance was assessed using two-way ANOVA analysis comparing TCR-PD-L1 and TCR-control mlgGi bead- treated groups. Only significant differences are indicated. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Fig. 8E) Hierarchical one-way clustering of the averaged TMT ratios of all identified proteins calculated from three independent experiments.
Fig. 9A-9G shows PD-L1 inhibits PIEZO1 activity in PD-1 -expressing GenEPi Jurkat cells in vitro Fig. 9A) Electroporated and blasticidin-selected GenEPi Jurkat cells were incubated with a dose titration of doxycycline (25-200ng/mL) for 24 h and treated with 1O|1M Yodal immediately prior to flow cytometric analysis. Numbers on plots indicate frequencies of FITC+ cells. Fig. 9B) Schematic of TIRF time lapse imaging experimental design. Raw integrated density reported as intensity for Fig. 9C) PD-1 expression and Fig. 9D) PIEZO 1 activity calculated from the sum of pixel values within each region of interest (ROI) at 200 seconds of stimulation presented as normalized values in Fig. 2. ROIs comprise single cells. Data are presented as means of n=37-50 ROIs ± SEM. Statistical significance was measured using Student’s unpaired t-test. ns, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Flow cytometric analysis of PIEZO1 reporter activity in PD-1 -expressing GenEPi Jurkat cells treated with RPMI media, Yodal (5|1M), TCR- control or TCR-PDL1 beads for Fig. 9E) 10 s or Fig. 9F) 30 s acquisition. Fig. 9G) MFI of XLGenEPi reporter in all conditions following 30 s acquisition. Cells were treated with beads 20 min prior to acquisition and with Yodal (5|1M) immediately prior to acquisition. Data are presented as means of n=7-12 technical replicates ± SD. Statistical significance was assessed using one-way ANOVA analysis, ns, * p<0.05, ** p<0.01, *** p<0.001, **** pO.OOOl.
Fig. 10A-10G shows E8i-Cre-ERT2 PIEZO 1 KO is specific to CD8+ T cells. Fig. 10A) Schematic of E8i-Cre-ERT2 Piezo P'x f'x mouse model. Fig. 10B) Relative fold change of PIEZO 1 expression in Cre+ and Cre- CD8+ T cells following 8 doses of lOmg/mL tamoxifen daily using qPCR and TaqMan probes recognizing specific Piezo 1 floxed regions. Data are presented as means of n=6 ± SD. Flow cytometric analysis comparing Cre expression using an eGFP reporter in Cre+ or Cre- Fig. IOC) murine CD8+, CD4+, CD1 lb+ and CD1 lc+ splenocytes following in vivo tamoxifen treatment and Fig. 10D) in murine CD8+, CD4+ and B220+ splenocytes following in vitro 4-hydroxy (4-OH) tamoxifen treatment. Numbers on plots indicate frequencies of eGFP-expressing cells. Fig. 10E) Cre expression measured by eGFP and Fig. 10F) Cre activity measured by TD-tomato frequencies in CD8+ T cells isolated from the spleens of MC38 and B16-0VA tumor bearing mice at day 15 after tumor implantation. Data are presented as means of n=22-34 ± SD from four independent experiments. Fig. 10G) Ratio of bound/unbound Ca2+ and fold change of Ca2+ influx in non-treated versus Yodal-treated CD8+ T cells from Cre+ and Cre- mice stained with Indo-1 following tamoxifen dosing. Data are presented as means of n=3 ± SD. Statistical significance was measured using Student’s unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig. 11A-11K shows E8i-Cre-ERT2 Piezo Plx flx CD8+ T cells show minimal phenotypic alterations at baseline. CD8+ T cells isolated from spleens of Cre+ and Cre- E8i- Cre-ERT2 Piezo lflx/flx mice were stimulated for 24 h with increasing doses of anti- CD3/CD28 crosslinking antibodies (0-10 ug/mL, x-axis) and assessed for frequencies of CD8+ Fig. 11 A) live cells, Fig. 1 IB) PD-1, Fig. 11C) CTLA-4, Fig. 1 ID) CD44, Fig. 1 IE) CD62L, Fig. 1 IF) granzyme B, Fig. 11G) Ki-67 and Fig. 11H) IFNy/TNFoc. Cytometric bead array assays were performed on the supernatants of stimulated Cre+ and Cre- CD8+ T cells to assess concentrations (pg/mL) of Fig. 1 II) IFNy, Fig. 11 J) TNFoc and Fig. 1 IK) IL- 2. Data are presented as means of n=3-4 ± SD. Statistical significance was assessed using Student’s unpaired t-test. Only significant differences are indicated. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig. 12A-12S shows flow cytometric analysis of TILs from tumor bearing E8i-Cre- ERT2Pzezo/^’^xmice. Fig. 12A) Gating strategy for CD8 TILs isolated from MC38 or B16- OVA tumors. Frequencies of Fig. 12B and E) CD3E+, Fig. 12C and F) total CD4+ T cells and Fig. 12D and G) CD8+/CD4+ ratio (counts). Frequencies of CD8+ cells expressing Fig. 12H and K) PD-1, Fig. 121 and J) CTLA4, Fig. 12J and M) CD69, Fig. 12N and O) granzyme B, Fig. 12P and Q) Slamf6 and Fig. 12R and S) TIM-3 isolated from Cre+ and Cre- mice bearing MC38 or Bl 6-0 VA tumors on day 15 after tumor implantation. Data are presented as means of n=18-26 ± SD from three independent experiments. Statistical significance was measured using Student’s unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001, **** pO.OOOl.
Fig. 13A-13B shows individual tumor growth curves of E8i-Cre-ERT2 mice. Individual tumor growth curves from data depicted in Figure 2.3k-l of Cre+ and Cre- E8i- Cre-ERT2 PIEZO lflx/flx mice treated with anti-PD-1 or isotype control antibody and bearing Fig. 13A) MC38 or Fig. 13B) Bl 6-0 VA tumors.
Fig. 14A-14D shows PIEZO agonism influences TIL phenotypes in WT mice. Tumor volume (mm3) measured over time (days) for Fig. 14A) experiment 1 and Fig. 14B) experiment 2 of WT mice bearing MC38 tumors and treated with Yodal (7.5 mg/kg). Data are presented as means of n=4-8 ± SD. Statistical significance was assessed using 2-way ANOVA analysis. Frequencies of CD8+ TILs that are Fig. 14C) Slamf6+ TIM-3- and Fig. 14D) Slamf6- TIM-3+ assessed by flow cytometry. Data are presented as means of n=12 ± SD from one or two independent experiments. Statistical significance was assessed using Student’ s unpaired t test, ns, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
DETAILED DESCRIPTION
The PD-1 pathway plays a critical role in mediating T cell exhaustion, and blockade of this pathway can promote antitumor immunity. While PD-1 inhibitors are revolutionizing cancer therapy, only a subset of patients respond and show durable remission, highlighting the need to better understand how the PD-1 pathway suppresses T cell functions. Here, PD- 1/PD-L1 ligation-induced dynamic changes of the local proteome proximal to PD-1 were quantified using unbiased multiplexed proximity proteomics. The mechanosensitive cation channel PIEZO 1 was identified as a primary target of PD-1 -mediated inhibition. Stimulation of CD8+ T cells through TCR and CD28 engagement triggered the activation of PIEZO1, while simultaneous PD-1 ligation countered this activation. Mice lacking PIEZO1 selectively on CD8+ T cells exhibited increased tumor growth marked by impaired CD8+ T cell function, which could not be rescued by PD-1 blockade. Conversely, mice treated with PIEZO 1 agonist showed increased numbers and function of CD8+ tumor-infiltrating lymphocytes compared to controls. Combined administration of PIEZO 1 agonist and anti- PD-1 significantly reduced tumor burden and improved survival in a tumor model unresponsive to PD-1 blockade. These findings identify PIEZO 1 inhibition as an important mechanism by which PD-1 signaling regulates CD8+ T cell functions and suggest PIEZO1 agonism and/or modulation as a novel approach for augmenting cancer immunotherapy. Provided herein is unbiased proximity-labeling as applied to the characterization of the dynamic behavior of proteins recruited to the cytoplasmic tail of PD-1 following ligation and identification of the mechanosensitive ion channel PIEZO 1 (FAM38a) as a primary target of PD-1 -mediated inhibition. PIEZO1 is expressed on a variety of cancers, which include epithelial and immune cells, where its activity can be regulated by external forces such as shear stress, cyclical hydrostatic pressure, and membrane deformation (force- from-lipid), as well as actomyosin contractility and extracellular matrix tethering (force- from-filament). Both external and internal forces contribute to PIEZO 1 gating, a process by which an ion channel transitions between its open and closed conformations as a means of regulating the passage of electrical current through the ion-conducting pore; this, in turn, induces downstream signaling. The stimulation of CD8+ T cells through TCR and CD28 engagement triggers the activation of PIEZO 1. Notably, it is also demonstrated that simultaneous ligation of PD-1 and engagement of TCR and CD28 significantly reduces PIEZO 1 -mediated Ca2+ influx. Moreover, knockout mice (KO) deficient in PIEZO 1 only in CD8+ T cells exhibited markedly increased tumor growth. CD8+ tumor infiltrating lymphocytes (TILs) isolated from these mice were less abundant and less activated. CD8+ T cell-specific PIEZO 1 KO decreased the efficacy of PD-1 blockade in tumor-bearing mice. In complementary studies, stimulating PIEZO 1 activity in wild-type (WT) tumor-bearing mice by systemic treatment with the PIEZO1 agonist Yodal resulted in increased activation of effector CD8+ T cells in the tumor microenvironment (TME). Importantly, combined administration of anti-PD-1 and Yodal decreased tumor burden and improved survival in WT mice bearing tumors unresponsive to PD-1 blockade. These summarized results identify reduction of PIEZO1 activity as a novel mechanism by which PD-1 mediates its inhibitory functions and pharmacological agonism of PIEZO 1 as a potentially effective combination therapy to improve PD-1 inhibitors in cancer patients.
Definitions
For convenience, certain terms employed in the specification, examples, and appended claims are collected here.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. The term “agent” is used herein to denote a chemical compound, a small molecule, a mixture of chemical compounds and/or a biological macromolecule (such as a nucleic acid, an antibody, an antibody fragment, a protein or a peptide). The activity of such agents may render them suitable as a “therapeutic agent” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
Unless otherwise specified here within, the terms “antibody” and “antibodies” broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
The term “antibody” as used herein also includes an “antigen-binding portion” of an antibody (or simply “antibody portion”). The term “antigen-binding portion”, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody. Examples of binding fragments encompassed within the term “antigenbinding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448; Poljak et al. (1994) Structure 2:1121- 1123).
An antibody for use in the instant invention may be a bispecific antibody. A bispecific antibody has binding sites for two different antigens within a single antibody polypeptide. Antigen binding may be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecific antibodies produced by a hybrid hybridoma or a trioma are disclosed in U.S. Patent 4,474,893. Bispecific antibodies have been constructed by chemical means (Staerz et al. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83: 1453, and Staerz and Bevan (1986) Immunol. Today 7:241). Bispecific antibodies are also described in U.S. Patent 5,959,084. Fragments of bispecific antibodies are described in U.S. Patent 5,798,229.
Bispecific agents can also be generated by making heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identification of clones producing and co-assembling both antibodies. They can also be generated by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof such as Fab and Fv sequences.
Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, biomarker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al. (\99$)Mol. Immunol. 31 : 1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.
Antibodies may also be "humanized” which is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The term “humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The terms “cancer” or “tumor” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.
Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. As used herein, the term “cancer” includes premalignant as well as malignant cancers. Cancers include, but are not limited to, B cell cancer, e.g., myelomas like multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), myeloma, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, cancers are epithlelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma, or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a blood born tumor, a brain/CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing tumor, an eye tumor, a gallbladder tumor, a gastrointestinal, a kidney tumor, a laryngeal or hypopharyngreal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penile tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a Rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma, a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymus tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms tumor.
As used herein, the phrase “ conjoint administration" refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include synergistic effects of the two agents). For example, the different therapeutic agents can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. In certain embodiments, the different therapeutic agents can be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic agents. As used herein, any two agents and/or additional agents may be conjointly administered according to the methods provided herein.
PIEZO 1 is a mechanosensitive ion channel protein that in humans is encoded by the gene PIEZ01. It is a mechanosensitive non-specific cation channel. It plays a key role in epithelial cell adhesion by maintaining integrin activation through R-Ras recruitment to the ER, likely in its activated state, and subsequent stimulation of calpain signaling. Exemplary nucleotide and amino acid sequences of human PIEZO 1, which correspond to GenBank Accession numbers, are listed below in Table 1. In some embodiments, an agent described herein targets an amino acid sequence disclosed in Table 1 or at least partially encodes an sequence listed in Table 1. In some embodiments, an agent described herein targets a nucleic acid sequence described in Table 1 or at least partially encodes an sequence listed in Table 1. As used herein, PIEZO1. may comprise a sequence listed below in Table 1, or may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% homology to a sequence listed in Table 1. Table 1
SEO ID NO: 1 : Human PIEZO1 Amino Acid Sequence; NP 001136336,2
MEPHVLGAVLYWLLLPCALLAACLLRFSGLSLVYLLFLLLLPWFPGPTRCGLQGHTGRLLRALLGLSLLF LVAHLALQICLHIVPRLDQLLGPSCSRWETLSRHIGVTRLDLKDIPNAIRLVAPDLGILWSSVCLGICG RLARNTRQSPHPRELDDDERDVDASPTAGLQEAATLAPTRRSRLAARFRVTAHWLLVAAGRVLAVTLLAL AGIAHPSALSSVYLLLFLALCTWWACHFPISTRGFSRLCVAVGCFGAGHLICLYCYQMPLAQALLPPAGI WARVLGLKDFVGPTNCSSPHALVLNTGLDWPVYASPGVLLLLCYATASLRKLRAYRPSGQRKEAAKGYEA RELELAELDQWPQERESDQHWPTAPDTEADNCIVHELTGQSSVLRRPVRPKRAEPREASPLHSLGHLIM DQSYVCALIAMMVWSITYHSWLTFVLLLWACLIWTVRSRHQLAMLCSPCILLYGMTLCCLRYVWAMDLRP ELPTTLGPVSLRQLGLEHTRYPCLDLGAMLLYTLTFWLLLRQFVKEKLLKWAESPAALTEVTVADTEPTR TQTLLQSLGELVKGVYAKYWIYVCAGMFIWSFAGRLWYKIVYMFLFLLCLTLFQVYYSLWRKLLKAFW WLWAYTMLVLIAVYTFQFQDFPAYWRNLTGFTDEQLGDLGLEQFSVSELFSSILVPGFFLLACILQLHY FHRPFMQLTDMEHVSLPGTRLPRWAHRQDAVSGTPLLREEQQEHQQQQQEEEEEEEDSRDEGLGVATPHQ ATQVPEGAAKWGLVAERLLELAAGFSDVLSRVQVFLRRLLELHVFKLVALYTVWVALKEVSVMNLLLWL WAFALPYPRFRPMASCLSTVWTCVI IVCKMLYQLKWNPQEYSSNCTEPFPNSTNLLPTEISQSLLYRGP VDPANWFGVRKGFPNLGYIQNHLQVLLLLVFEAIVYRRQEHYRRQHQLAPLPAQAVFASGTRQQLDQDLL GCLKYFINFFFYKFGLEICFLMAVNVIGQRMNFLVTLHGCWLVAILTRRHRQAIARLWPNYCLFLALFLL YQYLLCLGMPPALCIDYPWRWSRAVPMNSALIKWLYLPDFFRAPNSTNLISDFLLLLCASQQWQVFSAER TEEWQRMAGVNTDRLEPLRGEPNPVPNFIHCRSYLDMLKVAVFRYLFWLVLVWFVTGATRISIFGLGYL LACFYLLLFGTALLQRDTRARLVLWDCLILYNVTVI ISKNMLSLLACVFVEQMQTGFCWVIQLFSLVCTV KGYYDPKEMMDRDQDCLLPVEEAGI IWDSVCFFFLLLQRRVFLSHYYLHVRADLQATALLASRGFALYNA ANLKSIDFHRRIEEKSLAQLKRQMERIRAKQEKHRQGRVDRSRPQDTLGPKDPGLEPGPDSPGGSSPPRR QWWRPWLDHATVIHSGDYFLFESDSEEEEEAVPEDPRPSAQSAFQLAYQAWVTNAQAVLRRRQQEQEQAR QEQAGQLPTGGGPSQEVEPAEGPEEAAAGRSHWQRVLSTAQFLWMLGQALVDELTRWLQEFTRHHGTMS DVLRAERYLLTQELLQGGEVHRGVLDQLYTSQAEATLPGPTEAPNAPSTVSSGLGAEEPLSSMTDDMGSP LSTGYHTRSGSEEAVTDPGEREAGASLYQGLMRTASELLLDRRLRIPELEEAELFAEGQGRALRLLRAVY QCVAAHSELLCYFI I ILNHMVTASAGSLVLPVLVFLWAMLSIPRPSKRFWMTAIVFTEIAVWKYLFQFG FFPWNSHWLRRYENKPYFPPRILGLEKTDGYIKYDLVQLMALFFHRSQLLCYGLWDHEEDSPSKEHDKS GEEEQGAEEGPGVPAATTEDHIQVEARVGPTDGTPEPQVELRPRDTRRISLRFRRRKKEGPARKGAAAIE AEDREEEEGEEEKEAPTGREKRPSRSGGRVRAAGRRLQGFCLSLAQGTYRPLRRFFHDILHTKYRAATDV YALMFLADWDFI 11 IFGFWAFGKHSAATDITSSLSDDQVPEAFLVMLLIQFSTMWDRALYLRKTVLGK LAFQVALVLAIHLWMFFILPAVTERMFNQNWAQLWYFVKCIYFALSAYQIRCGYPTRILGNFLTKKYNH LNLFLFQGFRLVPFLVELRAVMDWVWTDTTLSLSSWMCVEDIYANIFI IKCSRETEKKYPQPKGQKKKKI VKYGMGGLI ILFLIAI IWFPLLFMSLVRSWGWNQPIDVTVTLKLGGYEPLFTMSAQQPSI IPFTAQAY EELSRQFDPQPLAMQFISQYSPEDIVTAQIEGSSGALWRISPPSRAQMKRELYNGTADITLRFTWNFQRD LAKGGTVEYANEKHMLALAPNSTARRQLASLLEGTSDQSWIPNLFPKYIRAPNGPEANPVKQLQPNEEA DYLGVRIQLRREQGAGATGFLEWWVIELQECRTDCNLLPMVIFSDKVSPPSLGFLAGYGIMGLYVSIVLV IGKFVRGFFSEISHSIMFEELPCVDRILKLCQDIFLVRETRELELEEELYAKLIFLYRSPETMIKWTREK E
SEO ID NO: 2: Human NM 001142864,4 Homo sapiens piezo type mechanosensitive ion channel component 1 (PIEZO 1\ mRNA
GGGAGCCGCCGTCCGGCCCAGCTCGGCCCCAGTGAGCCGAGCGCTGCGCTCCGCCGAGGGGCAGGGCGGT CGCCTGAGCGAGCGCGGGCCCGGGACGTCGGCACCGGCGGGGCGGCCGAAGGAGAAGGAGGAAGAGGAGA
AGGCGGCGCGCGGGTCCCCGCGGGTCAGCCATGGCGCGCCGGCCCCGGGGCCCCCGCACCGCCCCATAGC
GCCGCGGCGTCCGCTCGGTCTGGGCCGGGCCCTGGGCCCTCCAGCCATGGAGCCGCACGTGCTCGGCGCG
GTCCTGTACTGGCTGCTGCTGCCCTGCGCGCTGCTGGCTGCCTGCCTGCTCCGCTTCAGCGGACTCTCGC
TGGTCTACCTGCTCTTCCTGCTGCTGCTGCCCTGGTTCCCCGGCCCCACCCGATGCGGCCTCCAAGGTCA
CACAGGCCGCCTCCTGCGGGCATTGCTGGGCCTCAGCCTGCTCTTCCTGGTGGCCCATCTCGCCCTCCAG
ATCTGCCTGCATATTGTGCCCCGCCTGGACCAGCTCCTGGGACCCAGCTGCAGCCGCTGGGAGACCCTCT
CGCGACACATAGGGGTCACAAGGCTGGACCTGAAGGACATCCCCAACGCCATCCGGCTGGTGGCCCCTGA
CCTGGGCATCTTGGTGGTCTCCTCTGTCTGCCTCGGCATCTGCGGGCGCCTTGCAAGGAACACCCGGCAG
AGCCCACATCCACGGGAGCTGGATGATGATGAGAGGGATGTGGATGCCAGCCCGACGGCAGGGCTGCAGG
AAGCAGCAACGCTGGCCCCTACACGGAGGTCACGGCTGGCCGCTCGTTTCCGAGTCACGGCCCACTGGCT GCTGGTGGCGGCTGGGCGGGTCCTGGCCGTAACACTGCTTGCACTGGCAGGCATCGCCCACCCCTCGGCC CTCTCCAGTGTCTACCTGCTGCTCTTCCTGGCCCTCTGCACCTGGTGGGCCTGCCACTTTCCCATCAGCA
CTCGGGGCTTCAGCAGACTCTGCGTCGCGGTGGGGTGCTTCGGCGCCGGCCATCTCATCTGCCTCTACTG
CTACCAGATGCCCTTGGCACAGGCTCTGCTCCCGCCTGCCGGCATCTGGGCTAGGGTGCTGGGTCTCAAG
GACTTCGTGGGTCCCACCAACTGCTCCAGCCCCCACGCGCTGGTCCTCAACACCGGCCTGGACTGGCCTG
TGTATGCCAGCCCCGGCGTCCTCCTGCTGCTGTGCTACGCCACGGCCTCTCTGCGCAAGCTCCGCGCGTA
CCGCCCCTCCGGCCAGAGGAAGGAGGCGGCAAAGGGGTATGAGGCTCGGGAGCTGGAGCTAGCAGAGCTG
GACCAGTGGCCCCAGGAACGGGAGTCTGACCAGCACGTGGTGCCCACAGCACCCGACACCGAGGCTGATA
ACTGCATCGTGCACGAGCTGACCGGCCAGAGCTCCGTCCTGCGGCGGCCTGTGCGGCCCAAGCGGGCTGA
GCCCAGGGAGGCGTCTCCGCTCCACAGCCTGGGCCACCTCATCATGGACCAGAGCTATGTGTGCGCGCTC
ATTGCCATGATGGTATGGAGCATCACCTACCACAGCTGGCTGACCTTCGTACTGCTGCTCTGGGCCTGCC
TCATCTGGACGGTGCGCAGCCGCCACCAACTGGCCATGCTGTGCTCGCCCTGCATCCTGCTGTATGGGAT
GACGCTGTGCTGCCTACGCTACGTGTGGGCCATGGACCTGCGCCCTGAGCTGCCCACCACCCTGGGCCCC
GTCAGCCTGCGCCAGCTGGGGCTGGAGCACACCCGCTACCCCTGTCTGGACCTTGGTGCCATGTTGCTCT
ACACCCTGACCTTCTGGCTCCTGCTGCGCCAGTTTGTGAAAGAGAAGCTGCTGAAGTGGGCAGAGTCTCC
AGCTGCGCTGACGGAGGTCACCGTGGCAGACACAGAGCCCACGCGGACGCAGACGCTGTTGCAGAGCCTG
GGGGAGCTGGTGAAGGGCGTGTACGCCAAGTACTGGATCTATGTGTGTGCTGGCATGTTCATCGTGGTCA
GCTTCGCCGGCCGCCTCGTGGTCTACAAGATTGTCTACATGTTCCTCTTCCTGCTCTGCCTCACCCTCTT
CCAGGTCTACTACAGCCTGTGGCGGAAGCTGCTCAAGGCCTTCTGGTGGCTCGTGGTGGCCTACACCATG
CTGGTCCTCATCGCCGTCTACACCTTCCAGTTCCAGGACTTCCCTGCCTACTGGCGCAACCTCACTGGCT
TCACCGACGAGCAGCTGGGGGACCTGGGCCTGGAGCAGTTCAGCGTGTCCGAGCTCTTCTCCAGCATCCT
GGTGCCCGGCTTCTTCCTCCTGGCCTGCATCCTGCAGCTGCACTACTTCCACAGGCCCTTCATGCAGCTC
ACCGACATGGAGCACGTGTCCCTGCCTGGCACGCGCCTCCCGCGCTGGGCTCACAGGCAGGATGCAGTGA
GTGGGACCCCACTGCTGCGGGAGGAGCAGCAGGAGCATCAGCAGCAGCAGCAGGAGGAGGAGGAGGAGGA
GGAGGACTCCAGGGACGAGGGGCTGGGCGTGGCCACTCCCCACCAGGCCACGCAGGTGCCTGAAGGGGCA
GCCAAGTGGGGCCTGGTGGCTGAGCGGCTGCTGGAGCTGGCAGCCGGCTTCTCGGACGTCCTCTCACGCG
TGCAGGTGTTCCTGCGGCGGCTGCTGGAGCTTCACGTTTTCAAGCTGGTGGCCCTGTACACCGTCTGGGT
GGCCCTGAAGGAGGTGTCGGTGATGAACCTGCTGCTGGTGGTGCTGTGGGCCTTCGCCCTGCCCTACCCA
CGCTTCCGGCCCATGGCCTCCTGCCTGTCCACCGTGTGGACCTGCGTCATCATCGTGTGTAAGATGCTGT
ACCAGCTCAAGGTTGTCAACCCCCAGGAGTATTCCAGCAACTGCACCGAGCCCTTCCCCAACAGCACCAA
CTTGCTGCCCACGGAGATCAGCCAGTCCCTGCTGTACCGGGGGCCCGTGGACCCTGCCAACTGGTTTGGG
GTGCGGAAAGGGTTCCCCAACCTGGGCTACATCCAGAACCACCTGCAAGTGCTGCTGCTGCTGGTATTCG
AGGCCATCGTGTACCGGCGCCAGGAGCACTACCGCCGGCAGCACCAGCTGGCCCCGCTGCCTGCCCAGGC
CGTGTTTGCCAGCGGCACCCGCCAGCAGCTGGACCAGGATCTGCTCGGCTGCCTCAAGTACTTCATCAAC
TTCTTCTTCTACAAATTCGGGCTGGAGATCTGCTTCCTGATGGCCGTGAACGTGATCGGGCAGCGCATGA
ACTTTCTGGTGACCCTGCACGGTTGCTGGCTGGTGGCCATCCTCACCCGCAGGCACCGCCAGGCCATTGC
CCGCCTCTGGCCCAACTACTGCCTCTTCCTGGCGCTGTTCCTGCTGTACCAGTACCTGCTGTGCCTGGGG
ATGCCCCCGGCCCTGTGCATTGATTATCCCTGGCGCTGGAGCCGGGCCGTCCCCATGAACTCCGCACTCA
TCAAGTGGCTGTACCTGCCTGATTTCTTCCGGGCCCCCAACTCCACCAACCTCATCAGCGACTTTCTCCT
GCTGCTGTGCGCCTCCCAGCAGTGGCAGGTGTTCTCAGCTGAGCGCACAGAGGAGTGGCAGCGCATGGCT
GGCGTCAACACCGACCGCCTGGAGCCGCTGCGGGGGGAGCCCAACCCCGTGCCCAACTTTATCCACTGCA
GGTCCTACCTTGACATGCTGAAGGTGGCCGTCTTCCGATACCTGTTCTGGCTGGTGCTGGTGGTGGTGTT
TGTCACGGGGGCCACCCGCATCAGCATCTTCGGGCTGGGCTACCTGCTGGCCTGCTTCTACCTGCTGCTC
TTCGGCACGGCCCTGCTGCAGAGGGACACACGGGCCCGCCTCGTGCTGTGGGACTGCCTCATTCTGTACA
ACGTCACCGTCATCATCTCCAAGAACATGCTGTCGCTCCTGGCCTGCGTCTTCGTGGAGCAGATGCAGAC
CGGCTTCTGCTGGGTCATCCAGCTCTTCAGCCTTGTATGCACCGTCAAGGGCTACTATGACCCCAAGGAG
ATGATGGACAGAGACCAGGACTGCCTGCTGCCTGTGGAGGAGGCTGGCATCATCTGGGACAGCGTCTGCT
TCTTCTTCCTGCTGCTGCAGCGCCGCGTCTTCCTTAGCCATTACTACCTGCACGTCAGGGCCGACCTCCA
GGCCACCGCCCTGCTAGCCTCCAGGGGCTTCGCCCTCTACAACGCTGCCAACCTCAAGAGCATTGACTTT
CACCGCAGGATAGAGGAGAAGTCCCTGGCCCAGCTGAAAAGACAGATGGAGCGTATCCGTGCCAAGCAGG
AGAAGCACAGGCAGGGCCGGGTGGACCGCAGTCGCCCCCAGGACACCCTGGGCCCCAAGGACCCCGGCCT
GGAGCCAGGGCCCGACAGTCCAGGGGGCTCCTCCCCGCCACGGAGGCAGTGGTGGCGGCCCTGGCTGGAC
CACGCCACAGTCATCCACTCCGGGGACTACTTCCTGTTTGAGTCCGACAGTGAGGAAGAGGAGGAGGCTG
TTCCTGAAGACCCGAGGCCGTCGGCACAGAGTGCCTTCCAGCTGGCGTACCAGGCATGGGTGACCAACGC
CCAGGCGGTGCTGAGGCGGCGGCAGCAGGAGCAGGAGCAGGCAAGGCAGGAACAGGCAGGACAGCTACCC
ACAGGAGGTGGTCCCAGCCAGGAGGTGGAGCCAGCAGAGGGCCCCGAGGAGGCAGCGGCAGGCCGGAGCC
ATGTGGTGCAGAGGGTGCTGAGCACGGCGCAGTTCCTGTGGATGCTGGGGCAGGCGCTAGTGGATGAGCT
GACACGCTGGCTGCAGGAGTTCACCCGGCACCACGGCACCATGAGCGACGTGCTGCGGGCAGAGCGCTAC
CTCCTCACACAGGAGCTCCTGCAGGGCGGCGAAGTGCACAGGGGCGTGCTGGATCAGCTGTACACAAGCC
AGGCCGAGGCCACGCTGCCAGGCCCCACCGAGGCCCCCAATGCCCCAAGCACCGTGTCCAGTGGGCTGGG CGCGGAGGAGCCACTCAGCAGCATGACAGACGACATGGGCAGCCCCCTGAGCACCGGCTACCACACGCGC AGTGGCAGTGAGGAGGCAGTCACCGACCCCGGGGAGCGTGAGGCTGGTGCCTCTCTGTACCAGGGACTGA TGCGGACGGCCAGCGAGCTGCTCCTGGACAGGCGCCTGCGCATCCCAGAGCTGGAGGAGGCAGAGCTGTT TGCGGAGGGGCAGGGCCGGGCGCTGCGGCTGCTGCGGGCCGTGTACCAGTGTGTGGCCGCCCACTCGGAG CTGCTCTGCTACTTCATCATCATCCTCAACCACATGGTCACGGCCTCCGCCGGCTCGCTGGTGCTGCCCG TGCTCGTCTTCCTGTGGGCCATGCTGTCGATCCCGAGGCCCAGCAAGCGCTTCTGGATGACGGCCATCGT CTTCACCGAGATCGCGGTGGTCGTCAAGTACCTGTTCCAGTTTGGGTTCTTCCCCTGGAACAGCCACGTG GTGCTGCGGCGCTACGAGAACAAGCCCTACTTCCCGCCCCGCATCCTGGGCCTGGAGAAGACTGACGGCT ACATCAAGTACGACCTGGTGCAGCTCATGGCCCTTTTCTTCCACCGCTCCCAGCTGCTGTGCTATGGCCT CTGGGACCATGAGGAGGACTCACCATCCAAGGAGCATGACAAGAGCGGCGAGGAGGAGCAGGGAGCCGAG GAGGGGCCAGGGGTGCCTGCGGCCACCACCGAAGACCACATTCAGGTGGAAGCCAGGGTCGGACCCACGG ACGGGACCCCAGAACCCCAAGTGGAGCTCAGGCCCCGTGATACGAGGCGCATCAGTCTACGTTTTAGAAG AAGGAAGAAGGAGGGCCCAGCACGGAAAGGAGCGGCAGCCATCGAAGCTGAGGACAGGGAGGAAGAAGAG GGGGAGGAAGAGAAAGAGGCCCCCACGGGGAGAGAGAAGAGGCCAAGCCGCTCTGGAGGAAGAGTAAGGG CGGCCGGGCGGCGGCTGCAGGGCTTCTGCCTGTCCCTGGCCCAGGGCACATATCGGCCGCTACGGCGCTT CTTCCACGACATCCTGCACACCAAGTACCGCGCAGCCACCGACGTCTATGCCCTCATGTTCCTGGCTGAT GTTGTCGACTTCATCATCATCATTTTTGGCTTCTGGGCCTTTGGGAAGCACTCGGCGGCCACAGACATCA CGTCCTCCCTATCAGACGACCAGGTACCCGAGGCTTTCCTGGTCATGCTGCTGATCCAGTTCAGTACCAT GGTGGTTGACCGCGCCCTCTACCTGCGCAAGACCGTGCTGGGCAAGCTGGCCTTCCAGGTGGCGCTGGTG CTGGCCATCCACCTATGGATGTTCTTCATCCTGCCCGCCGTCACTGAGAGGATGTTCAACCAGAATGTGG TGGCCCAGCTCTGGTACTTCGTGAAGTGCATCTACTTCGCCCTGTCCGCCTACCAGATCCGCTGCGGCTA CCCCACCCGCATCCTCGGCAACTTCCTCACCAAGAAGTACAATCATCTCAACCTCTTCCTCTTCCAGGGG TTCCGGCTGGTGCCGTTCCTGGTGGAGCTGCGGGCAGTGATGGACTGGGTGTGGACGGACACCACGCTGT CCCTGTCCAGCTGGATGTGTGTGGAGGACATCTATGCCAACATCTTCATCATCAAATGCAGCCGAGAGAC AGAGAAGAAATACCCGCAGCCCAAAGGGCAGAAGAAGAAGAAGATCGTCAAGTACGGCATGGGTGGCCTC ATCATCCTCTTCCTCATCGCCATCATCTGGTTCCCACTGCTCTTCATGTCGCTGGTGCGCTCCGTGGTTG GGGTTGTCAACCAGCCCATCGATGTCACCGTCACCCTGAAGCTGGGCGGCTATGAGCCGCTGTTCACCAT GAGCGCCCAGCAGCCGTCCATCATCCCCTTCACGGCCCAGGCCTATGAGGAGCTGTCCCGGCAGTTTGAC CCCCAGCCGCTGGCCATGCAGTTCATCAGCCAGTACAGCCCTGAGGACATCGTCACGGCGCAGATTGAGG GCAGCTCCGGGGCGCTGTGGCGCATCAGTCCCCCCAGCCGTGCCCAGATGAAGCGGGAGCTCTACAACGG CACGGCCGACATCACCCTGCGCTTCACCTGGAACTTCCAGAGGGACCTGGCGAAGGGAGGCACTGTGGAG TATGCCAACGAGAAGCACATGCTGGCCCTGGCCCCCAACAGCACTGCACGGCGGCAGCTGGCCAGCCTGC TCGAGGGCACCTCGGACCAGTCTGTGGTCATCCCTAATCTCTTCCCCAAGTACATCCGTGCCCCCAACGG GCCCGAAGCCAACCCTGTGAAGCAGCTGCAGCCCAATGAGGAGGCCGACTACCTCGGCGTGCGTATCCAG CTGCGGAGGGAGCAGGGTGCGGGGGCCACCGGCTTCCTCGAATGGTGGGTCATCGAGCTGCAGGAGTGCC GGACCGACTGCAACCTGCTGCCCATGGTCATTTTCAGTGACAAGGTCAGCCCACCGAGCCTCGGCTTCCT GGCTGGCTACGGCATCATGGGGCTGTACGTGTCCATCGTGCTGGTCATCGGCAAGTTCGTGCGCGGATTC TTCAGCGAGATCTCGCACTCCATTATGTTCGAGGAGCTGCCGTGCGTGGACCGCATCCTCAAGCTCTGCC AGGACATCTTCCTGGTGCGGGAGACTCGGGAGCTGGAGCTGGAGGAGGAGTTGTACGCCAAGCTCATCTT CCTCTACCGCTCACCGGAGACCATGATCAAGTGGACTCGTGAGAAGGAGTAGGAGCTGCTGCTGGCGCCC GAGAGGGAAGGAGCCGGCCTGCTGGGCAGCGTGGCCACAAGGGGCGGCACTCCTCAGGCCGGGGGAGCCA CTGCCCCGTCCAAGGCCGCCAGCTGTGATGCATCCTCCCGGCCTGCCTGAGCCCTGATGCTGCTGTCAGA GAAGGACACTGCGTCCCCACGGCCTGCGTGGCGCTGCCGTCCCCCACGTGTACTGTAGAGTTTTTTTTTT AATTAAAAAATGTTTTATTTATACAAATGGACAATCAGA
SEO ID NO: 3 NG 042229, 1 Homo sapiens piezo type mechanosensitive ion channel component 1 (PIEZO1E RefSeqGene (LRG 1137) on chromosome 16
TAGTTTTATTTGAGATGAGGTTTTGCCATGTTGGCCAGGCTGGTCTCGAACTCGCCACCTCAGGTGATCC TCCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCACGAGCCACTGCACCCAGCCTGAAAATTGTTTT AACCACGTACATATTTCTTAAAATTGTCTATCATGGTCAAAGTACATAATATAAATGTACCTTTTAAATC ATTTTTAAGTGTCCAATTTGGAGGCATTAAGCACATTTACAATGTTGTGTCAACACTACTATTTCCAGAA CCTTTTTATCACCCCAAAGAGAAACTCTGCCACTGTTAAACAACAGCTGCCCACTTCCCCCTCTGGCCTG GTAACCACCCTCTGCTGTCTGTCTCCATAGACATTTCACGTACACAGAGCCACATGGTGTCTTCCTTCTG TGCCTGGCTCCCTTCCCTCCACGTCCCCCAGCCCCGTGCTGGCGCGGTGGCTCACACCTGTGATCCCAGC ACCTTGGGAGGCCAAGGCAGGAGGATCGCTTGAGCCTGGGAGTTTGAGACCAGCGTGGGCAATATAGCAA GACCCCATCTCTATGGGAAAAAAAATTGGCTGGGTGTGGCAGTGCATGCCTGTAGTTCCAGTTACTTGGG AGGCTGAGGTGGGAGGCTTCCTTGAGCCTGAGGTCCAGGTTACAGGGAGCCATGATTGCACCACTGCAAT CTAGCCTGGTAGCCTGGGTGACAGAGCGGGACCCTGTCTCACACACACACACACAAGTTCATCCCTGTTG TAGCGTACATCAGAGTTTCATTTCTTTTTATGGACAAATAATATCAGATTGTATGAACAGACCACATTTT GTTTCTACACTCCATTAACAGACTCTGGGGTTGCTGCCTCTTGAAATTATTATTTATTTTATTACTTTTT TTGAGACAGGGTCTTGCTCTTTCACCCAGGCAGGAGTGCAGTGGCATGATCTCAGCTCACTGCAATCTCC ACCTCCTGGGATCAAGCGATTCTCCCGCCTCAGCCTCCCGAGTAGCTGGAACCACAGGCATGCGCTACCA TGCCCGGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTTGAACCC CTGACCTCAAGTGATCCACCCACCTCAGCCTCCCAAAGTGCTGGTGTTGCAGGTGTGAGCCACAGTGCCC GGCCAAAATTATTATTATTTTTGAGACAGGGTCTTGGCCTGTTGTCCAGGCCGGAGTGCAGTGGCGCCAT CACTACTCACTGCAGCCTCCACCTCCTGGGCTCTAGAGATCCTCCCACCTCAGCCTCCTAAGTAGCCGGG ACCACAGGCAGGCACCACCACATCTGGCTAATTTTTTTTTTTTTTGAGGCGGAGTCTCGCTCTGTCACCC AGGCTGGAGTGTAATGGCATGGTCTTGGCTCACTGCAACCTCTGCCTCCCGGGTTCAAGCGATTCTGCCT CAGCTTCCCGAGTAGCTGGGATTACAGGTGCCTGCTGCCACGCCTGGCTAATTTTTGTATTTTAGTAGAG ACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCCTGTGATCCACCCGCCTCAGCCTCC CAAAGTGCTGGGATTACAGGCATGAGGCACCATGCCTGCCTGGCATGCCTGGCTAATTTTTATTTTTTGT ATAGATGGGGTCTTGCTGTGTTGTCCAGGCTGGTCTCCAACTCCTGGCCACAAGCCATTCTCCTGCCTTA GGC C C C C AAAGTGTTGGGATTAC AGACGTGAGC C AC C AC AC C C AGC CTGAAATTAATTTTTAAGTATAGA CAAGTAATGGTGACTATGTTATCATTGGGGAAAAAGTTACAACCTGCATGATAAAGCTTAAAGTCCCTGT GACCCCTCTTGCCCTGGCTTTTCCTCTCTCCCAAGGGGACCTGACCATTGTTGCTCTCCCAAGACTAAGT CACTGGCCGGGCATGGTGGCTCACACCTGTAATCCCAGCAGTTTGGGAGGTCAAGGCAGGCGGATCATTT GAGGTCAGGAGTTCAAGACCTGGCCAACATTTTGAAATCCCATCTCTACTAAAAATACAAAAGTTGGCCA GGTGTGGTGGCACACGCCTGTGATCCCAGCTACTTGGGAGGTTGAGGCAGGAGAATCACTAGAACTCAGG AGGCGGAGGGTGCAGTGAGCCGAGATTGCTCCACTGCACTCCAGCCTGGACAACAGTGGCAGACTTTGTC TCAAGAAAATACAATAAAATAAAAAATAAATCACTGTTTTAAACAGTGACAAAAATAAAAAGCAACAGCA AGTTGAGAAAGGGCTCCCAGCTGTGGGTCAGTGGGACAGGAGTGACTTTGGACTTGGAATCTGGAAAGCT GGGTGCAAAGGCCGGTCTATTCCTTTGGCAAATGGTTATCCCGCTCTGGCCCTTAGCGTCCTCTTCTGCA ACAGGGGTTGAGTTAGAACACGGTCCAAATTCCTGAACGGGCAGTGAACTGGGCACAGGTGGGCTCCCCT GCCCTTGCCCGCCCCACCTCTCCCTGGCTGGCAAGCAGAGATCTTTTTCAGAGTATTTGTCTCCATTTTG CTAATTTTATTTTGTGAATAAGTGAATTATGTATGAAGCAAAATATTAAAACAGCACCAAAGGGTATAGG TGAAGACCAGGTCTCCCTCCCACCCCAAGTGGGTGAGCGTGGCCCTGAGTGTAGCTGCTTGCTTTTCCCA ATTGTTAGCACTGTAACTTAGCAATTGTTCCTCCTTGATAACAGAGCAATTGCGGGATTCTCTCTAACCG CTGCTACAGAGTGCCATTGTATCTTAGCTTCATGTTGATACAAAACACCTAGCCGGATTCTGTTTTTTAC CTGCTGTGTAGCACTCCACTGTGAGACACGCTATCATTTATTTGACAAGCTTCTGTGGGCAGAGTTTTTC TGCTTTTTGCTGCTTGAACAATTCTAGGACACATACATCCTCTACCTTTGCAAGTCCATCTGCAGGGCAA GCTCCCAGACTTCCCCACCTCTGCAGCAACTTTTCAAAGCCTGACCAGAGGGTGAGACTTGCAGAGAAAG TAAATCAGCCTTTTTTTTTTTTCTTCTTTGAGATGGAGTCTCATTCTGTTGCCCAGGCTGGAGTGCAGTG GGGTGATCTCGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCCTGCCTCAGCCTTCCGAGT AGCTGGGACTACAGGCGCCCGCCACCGCGCCCGGCTAATTTTTTGTATTTTTAGTAGAGATGGGGTTTCA CCGTGTTAGCCAGGATGGTCTCCATCTGCTGACCTCGTGATCCACCCGCCTCGGCCTCCAAAAGTGCTGG GATTACAGGCGTGAGCCACCGCACCCGGCCTATATCAGCCTTTAAACACTAGACTTCCCATGAAAATCTC CATTTCTGGGTGGTTTTTTTTTTTTTAATGGAGTTTCGTTCTTGTCACTCAGGCTGGAGTGCAATGGCAT GATCTCGGCTCACGGCAACCTCTGCCTCCCAGGTTCAAGCGATTTTCCTGCCTCGGCCTCCTGAGTAGTT GGGAATACAGGCGCCTGCCATCACGCCCGGCTAATTTTTGTATATTTATTTATTTATTTATTTATTTATT TATTTATTTATTGAGACGGAGTCTGGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGCTATCTCGGCTCAC TGCAAGCTCCGCCTCCTGGGTTGATGCCATTCTCCTGCCTCAGCCTCCCGAGTAGTTGGGACTACAGGAG CCCGCCACCACGCCCGGCTAATTTTTTATGTTTTTAGTAGAGATGGGTTTTCACCGTGTTAGCCAGGATG GTCTCGATCTCCTGACCTGGTGATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCC ACCGCGCCCGGCCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTCG AACTCCTGACGTCAAGTGATCCCCCGACCCCCTACCCCCTTGCCTCAGCCCCGCAAAGTGCTGGGATTAC AGGCCTGAGCCACTGCCCCGCCATCTTTTTTTTTTTTTTTTTTTTTTGAGCTGGAGGGTGTGGGCTTGTG CTGGGCTCTCATGAGTTGCCAGTGCCTTGAGGCTCAGTGGCCACAGTCCCCCCAGCCCCACTCCCTGCCT TACGCAGCTGAGTTTGAGAGCCATGAACTGAGCCCAATTAAACGGTGTAGAGCCCTTTGCCCTCTTTCTG TGCCCATCTCAAGGGCAGACGAAGGCCAAACTGACGTGGCCTGGGTGAGGGTCCCGAACACCCTGGACTC AGACCCTACCTCTCCGTGGCAGGGCGCCGCGGCAGCAGGTGCACAGGCCGTGGCTCTCACTCTCACCGTC TCACGCCAGAGCGACCCGGGCAAAGCCGCGCGCGGTGGCCGGCGTGGCTGGGAGCAGCCCCCGAGCCCCC GTCAGACCAACGCGGATCGCGGCACTGCTGCGACCCGAGCATCCCGCGTGCTCCCTCCGGGACCACTAGG CCCGGCCGCCGCGGGAACTCCGAGCCTCCCCACGGTCCCCAGGGGGCGCTGCGTGCTCGCGCCTCGGGGA GCAAAGCGAAAGTCGCTCGGGCACGCGGGGTGCAGTTGCGCGTGGGGCGCTGCCTGGGGGAGCCAGAGGC CCCGGGACCCCTTATGTAGCGCCCGGGCCCCAGCCGCAGCCCACCCAGCGAGAAGCGCCCCTGCCCACCG CCGCGCACGCTCGGAGCCGCGGGGCCAAGACTGCGGGAGGGGAGGGGTCCGCTCCGGGCCAGCGCCAGTC GGGGGTCTCCAGGCCCCAACGCACCAGGGCGGGCCGGAGGCGGGACGGGCCGCGCGGGACAGCGGAGGGG GTGCCTGGGCGCGCCGGGCCGTCGGGGAAGCGCGGAGGCGGGCACGCCGGGGAGGGCGGGAGGGGGCCGA GCTTATAAAGGCCCGCGGGCGGAGGAGGGCGGGAGCCGCCGTCCGGCCCAGCTCGGCCCCAGTGAGCCGA
GCGCTGCGCTCCGCCGAGGGGCAGGGCGGTCGCCTGAGCGAGCGCGGGCCCGGGACGTCGGCACCGGCGG
GGCGGCCGAAGGAGAAGGAGGAAGAGGAGAAGGCGGCGCGCGGGTCCCCGCGGGTCAGCCATGGCGCGCC
GGCCCCGGGGCCCCCGCACCGCCCCATAGCGCCGCGGCGTCCGCTCGGTCTGGGCCGGGCCCTGGGCCCT
CCAGCCATGGAGCCGCACGTGCTCGGCGCGGTCCTGTACTGGCTGCTGCTGCCCTGCGCGCTGCTGGCTG
GTGAGTGGGGGGCGGGCGCCTGGGGGCGACGGGAGGGGGCTGCGTCTCGGCTCCCCACGGCCTGGACACC
GGACGACGCCGGCCGGGGCGAGGGCTGCGGGCGAGCGGGCGCGGAAATTCCCAGGGACGCGCGACCCGGG
CGCCCGCATTCCTGAAGCATGAGCGCGCCAGGCGGCGGCGGGGCTCCTGTCCCAGGGCCGGGCTGGAAGG
GCGGCGGCGGCTGGGGGAGACGGCACCGCGTGCCCACGGGGGCGGTCGAGCGAGCGCCGGGCATAGCGCG
GCTGGCGTCTCCGCCGGGGCGCTGCGGAGAGGAGGCCGCCGGGCGAGGCGGTGTTTGCCCCGGTGGGAAG
GGCCGCGGCGGTGGTGGGGGGAGCACGAATCTCTTTTTCTCTTTCGGGTTTAAAAAAAAAAGCGCAAAGT
TGCATCAGGACTTCCTGACAATCTGGGAGAAGGCGGGCTTCCTGCCTGGAGCTGTTTAATTTGGAGCTTC
CCGAGCCCAACGAACGTCCGTGCCCAGGGCCCAGCCCCGCTCACCGCTGCACCCCCCTCTGCCGGACTGA
GGCGGTCCCACACTTTGGAAAAAAATAGTGTGGGTTCCTCCCTGGTCCTCCCTTGCCCTACTGGGCTCAG
TTTCGCAGGGGCGGGGGCCGGCCTCTGCCCTGGTCTGGGGGAGGGGACACCCCCGGAGGCTGTGGCCTGG
TGTCAGGGCGGGGCAGGGGTCCCCAGTCCTGGCATCTGTGTTCCCTGCTTGCCGGGCAGTGGTGCCCCTT
TCGCGAAGCACACCCGGGTGGCTTGGTGCTGCACGGCCTGGCACCCCTACCCTTCCCCGACCCTGGCCTA
GCCGGGACCCAGGGTCCGCGCCCTCCGCCCGGGGGCTCCCCACGTGTGATTGATCTGGGAAGCAGTCGGA
TGGAATTAACCCACGGACAAGTGGGACGGTTTGCATTGGGAGTCCGCCATGGACACGGCAGGTGGGGCCT
TTTGATTGTAAAAGCCCTTTCGGAGCCCTTGCCTCGCTCCAGGTGGGAGCTCGCCCAGCGCTAGCTTTGG
GGATCTAGAGCCGCCTGCCTGAGGCTCCCAGACAGACTGCGTTTTGATCGGTCGCACAGAAAGGTGGTGA
AACTTGGGGAAGATTTTCTAGACAGGAATCAATGAAAACCATTGAGGCTGGAGAGGAGAGGTTTTGAGCA
ACTCTCTTCAGTGCGGTCAGCCCTGTGTGGACTGGGCAGCCTGGGACCTGCTCCCAGTGCAGGGTCAGAT
GGGCCGTAAACAGGGCCCGGCTGTGTTCCTTCCTGTGCCTTGAAAACAAGCAGGACAGCCTGGCACAGAG
GCAGAGTCTAGAGCTGACAGGCCTTAGAGAGGGAAACAGGAAAGCTTCTGAAACGTCCCGTTCACACTGA
TCGTTCCATTTCCTCTTGTGTCTGAGTGGGAGCGGGTGTCCTCCCTGCAGGGAATGCCCCCCCTCTCAGA
TGGCAGCTGCTCCTTGGGCAGAGTTGGCAATGTTTTTCTTTAAATGACCAGATGGTAAATATTTTCATGT
CACAAAATCTTCTTCTTCTGGTATTTTTCCAGCCATTAAATGTAAAAGCCCTCCTGAGTTCATGGGCTGT
ACCTAAACAGGTGTTGAGCCCGATTCTGTGGCACATGTGGTTTGCTGCCCCCTGGGCATTGGTCAGGGGG
CCTGGGTTCTGCCTTCTCGATTGCTATCCGCGTGGGGGATCTGGGGGAGGGATCACTGTTCTTCTTGCTT
TTGGCCTCCTTGGGGAGGATGGGGAGGTAGCCCAGGGGTGCTCACCCAGGCCCCGTGTCAGTCTTCTATG
AAACTTTTAAAGAATAGTGATGACTGACTGTCTGTCTGTATGGTACTTTCCTTAAACCTAAAACTGGTCC
CAAATAAAGTCTCTTAATTTGAAAGATGCTGAAGCCCGGGCCATACCCCACACTGATTCTGTGTCTGGGG
ATGGGGCGTGGGGCCTGGGCCTCACTCAGTGTTTTCTCTCAGTCACCTGGGGGAGATGGAAGTGGAGCCG
GCCAAGAACCCTGCCTGCCTGCCTGCTGGCCGGGACTCCTGAGTCAGGCTCTCTGGCCCTGGGGTGTGGG
CAGCTCCAGATGGACCCGCGATGTGCAGGTTCAGCTGGCCTGGCCGGAGGTGGGACACTGGCTTTGCTGT
CTTTGGAGTGCCCCCTCCCTCTCTGGCGAGCTTTGGCTGGAAGCAGTTCTACCGTGTTTTGGAAATGAAT
GAGGCCTTCAGAAGGCATTAGTCAGTGTGTGCCTGCGCTGGCTCAGACAGTGCCTGGTGAGGGTTTGAGT
CATCCTGGGGTGCCCCTGGCCCCCACGCCCTCCCTCTCCAGTGCAGGATCATTACCCAAAAATCTGGCAG
GGAGCTGCCCCACCCACAGGGAGCAGGGGCCTCCTTCAGCAGTCTCACCTAATGTTGCTGGAGCCTTGGG
GGATCAGGGCCCATCTCTTCTAGAGAGATGTCAGGGCAGGGCTGGGCGCGATGGCTCACACCTGTAATCC
CAGAGCTTCGGGAGGCCAAGGTGGGAGGATTGCTTGAGCGTAGCCATTCGAGAGCAGCCTGGGCAACGTA
GAGATCCCCATCTCTATGAAAAATATTTAAAAATTAGCTGGGCATGGTGGTAGTGCACCTGTAGTCCCAG
CTACTCAGGAGGCCAAGGTGGGATGATTGTTTGAGTCCAGGAGTTGGAGGCTACAGTGAGCCATGATTGT
ATGACTGCACTCCAGCCTGGGTGACAGACCCTGTCTCAAAAAAGAAAAAGGAAAAAAAAGGGCAGGACAT
TTGTGATTCTGATAATATGGGACCGCACCTCCAGTTCTATCAGTGGGAAATCTAGACAGGGCTGCGGAAA
GCCAGCTGGTGCGAGAGGAGCCACCGTGTCACTGACTGTGGGACACCCACGTGGGCTGACAATATGGCTT
CTGCTTTTCAGGGTGCCTCGTGGCACAGCCTAGGGGCACACCCACGGCCGGCAAGCTGGGCGTCACCTCC
TTCAGGCTGATTGTCACTGAGAAGTGTCACCATTTAGCATGAGGGATGCTGCCTCCTTTTTCAGAACATT
GTCACCATCAGGGTCTCACCACTCCTGGGAGGCGGCCGAGAAGCTGGGGAACAGCAGGCACTCGGCTCAC
AGTTGCTCAGCAGTGCAGACCCTCTGAGCTGAGCATGGCAGAGTCACCCTTCGGAGGCCTGTGCCCGGGT
CTCAGGACCTGCACAGAACCCTGGCCTGTCCCATCCGAGGGTGCTGGGAAGAGCATGGCCGTGGCAGAGT
AGGGTGGGAGCTGCTTTCCTCTGTGGCTTGGGGGCCCCTTCTGAGCATCAGCTCCCTGGTGTGACAGAGG
GGCGCACTCTGTCCCCATGCTGGGCCTGGAGGCTGGATGAGTCAGCAGGAGAGCCTGGGGCCTGCCTCAC
AGCACCAAGGGCTGCAGGTGTGAGTGTGCACATGTGTGCGTGTTTGGGGAAGGGGCCAGGGACTGCCCAG
GAGCTGAGGATGGGTCACAGCGGGTGCTCGTCCCGCAGCGGGTCACTGGTGCCCAGGACACAGGGAGCTC
CAGCCCCAGCTGCCAGGGTCCCACAGAGAGGAAGTTTCCTCTGGGGGTGGGTGGGGGCGCACAGTCTCTG
ATCCTGGCCCCAAGGCAGCTTCCTGGGCGGTGTCTCTCCTGTGCTGACTCGGCAGTGCATTTGCTTTCGG
TGCTCAAAGATGAAGGGGAACCACCGTGGGCCTTGACGGCCTCATCTGCCCGCTGCAGCCCACTCCTGAG ATGGGACCACCGCAGTCGTCAGGGTCCAGTGAGAGCCGCATCTTGCAGGAAGCCATTCCTGGCCTCTCTG
GCCTCAGAAATCCCCTTTTCAATTCAACAAAATGTTAGTCTTCTGTTTAGCTATTTTAGAAATAGACAGT
TAGGCTTTTTTCTCTTTTTTCTTAAAGACAGAGGCTCACTCTGTCGCCCAGGCTGGAGTGTAGCGGCGTG
ATTTTGGCTCCCTACAACTTCAGCCTCCAGGGCTCAAGCCATCCTCCTGCCTCAGCCTCCTGAGTAGCTG
AGATTACAGGTGTGTAGCACCATACCTAATTTTTGTATTTTTTGTAGAGACGGGGTTTCACCACTTTGGC
CAGGCTGGCCTTGAACTCCTGACCTTAAGCGATCTGCCCGCCTCAGCCTCCAAAAGTGCTGGGGTTACAG
GCATGAGCCACCGCGCCCGGCTGACAGTTAGACTTTTGCTTCTTTGTTTATATAAGCTTTTTCTTCTGGT
TCCAAAAGCAAGTTTGCCCTTCCTTGTGGTAGAGAATCCTAGCTCACAGAGCAGTTTAGAAGCCAGCACA
GTATCCCACACACACATCTGGCATGGACAGACCCTTCCTTGCTGGGTGTGGGTCCTGTGTTCTTCTGAAA
GGCAAGCTGTTCCCAGCCAACCCCTGCCCCTCTCTGCCTTAGCCTGCCTGGAGGCCTGAGTCTCCTGGGT
GACTGTGAGGTGGGACCCCCCCTTCCCCTACCCCCACCCACATCCTCTGTATCTGCCTTCTGTCCTGCTC
TTGACCTTTGAGCTCCTCTGCTGGCTTCAGGGTGCGGCTGTTGAGCCTATTTTTTGGATTAGGACTCTGG
GGTGAGGGAAGTTAATTCACACACCCAAGATCACACTGGTGGGAAGGGACAGGCCCGGGGTGAAGGCTTC
TCCTCTCCTTGGCGGTTGAGTCCCACACCTGCTGGCCGAGGCACCTGAAGGGGACTTGGGGTCCAGGGTC
ACTGGGAGGACGGGGGCAGGCAGAGGGGTGGCCGACCTGGTGGCGGCTCGTGGGCAGCAGCCGACCCTAT
CTTGCTCTGAACGTGTGGGGCCCTCACCCCCTTCTCTGGGTCTGGGTTTCCTCCCCTGTAAGTGACACCG
TAAAAGCTTCACAGCCGCTTCCAAGTCTCAGGTCTCTCGGGCTTTGGATCTCACACCCAGGCTGGGTGGG
GGTAGGGGCGGGACAGCCGTCCCCCCCGGAAGGCTCAGAATTCCTCGCACAATCGTGGGGCCAGGAGACC
CGCAACACAGGCTTTCCCAGCTGCGCTGAGTGCCGCGGTGGCCGGGGGTCCGTCGGGCCTCCATGGAGCT
GAGGGGAAGGGGCCACTCACCGCCTGGTCCCGGAGCACACAGGGCAGCTCCCAGGAGCACAGAGGCTCCT
TGGGACCTGTGGGGCTGTCGGCCTCCCTCATGCTGCACACACAGCGCGTCCCCAGGGGTGTCTGCAGCCC
AGCCCATCCCACTGCAGATTCCCGACCACTCAGATTCATTCATGCATCCTCTCACGCCGGTCCTGTGGGA
CAGAGCTCTGGGCAGCAGCCAGAAGTCCAAGTTCTGGTTCAGGGCCAGTGGAGGTGGGTGTTGGGGTGGG
GCTGGAGCTCCCTGCTCTCCCTCCCAAGCTAGCCAGGAAAGGAGGTTGGGGGCCCCGCACGGTCATTGCT
GTTTATTCACAAAGCGCGATGCTGAGCACAGGCGGGGAAAGAAAAGTGCGATCAGTGCCAGGAAAATGGG
GCTCCCCCGACGCCGTCCAAAATGGGATCCCTTGCCGGGCGCGGTGGCTCACACCTGTAATCCCAGCACT
TTGGGAAGCCGAGGCAGGTGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGACCAACATGGCAAAAC
TCCATCTCAGCTAAAAATACAAAAATTAGCTGGGCATGGTGGCGGGCACCTGTATTCCCAGCTGGGAAGG
CTGAGACAGGAGAATCGCCGGAACCCAGGAGGTAGAGGTTGCAGTGAGCCACAGTCGTGCCACTGCACTC
CAGCTTGGGGGCTTGGGGGACAGCAAGACTCCCTCTCAAAAAAAAAAAAAAAAAGTGAAGTCGTAAATCA
GATTAAATTCCCTTTTTAACCCTTTGAACCTCTGTCCTCCCCTGTTCCCAGCGGGAAGCCTCTGGTGAAC
GCGCCATGCACCCCACCTGCCCCCGCTCTCTGGGTCTCTCTCCCAGCTGGAACGGCCGCTTCCCCAGGTG
CCTTCCCTGGGCCACAGCCTTGTGCCTCGGCGGCTGCTGGATGCCTGGGTGTGTGGGTGGCTCCCAGTGT
GTGGGATGGCACACGAGCCTCTCGCCCTTCTGTGTGGGGTCGCACACCCACCGCAGGCCGTATTTTTGCT
CACGTTCATGTTTCTCCACGGTGGACGCTGGGTTGCAGGGACCCTTCCTGTGTGCGGGTGAGGATCTGGG
CAGCTGCTGGTGCCGGGCCCATGGGGACGCTGACCGTCCCGGGTGCCGGCTCTGAGGTGTGCAGTGGACG
GCTGTCCTGCCGGGCGCTGCCAGGGCCCCTTAGGCCGACGTGCGTGGCCACCCGATTCCCCGCCGTTGTC
TCAGGAACCTTTGCCGAGTGGGGTGGATCAATTTTTCGGGTGTGTTTTAATAGCATAATTACAGGGAGTA
TTTCAGGCTCCCTCTGACGGGCCGGCAGGGTTTGGCTGCCGGCTGTTTACCAGGCTCCAATCTGCACACT
ATTTTTCTGTGGGTATATATAGCTGGGGCTGCTTCTCCTTCCTCAGGTTCAGGCTAAAGAGGGACAGCAG
CCGCCTCAGCCACCCCCTGTGGTTTCCTTTGCCTGTGGATGGGCGGCTAAAATGGGCCCAGGAAGAGTCA
AGAACAAGGCCGGCTCTCGGTGCCACAGCTCTACCCCCAAAAGCAGGAAGGGGGCTCGGGCCATGCCCAT
CTGTGAGCTACACCGGTCCGGGAGCGGCATCAGGCAGGGGAGTCCTGGACCCCCGCAGTGCTGGGGTGTG
TTTGTCCGCCCTCCCTCCCGTGTGTCTAGAAGCCTCCAGCCTCGGGGAAAACAATGAAACTCAACTGTGA
CTTAAACAGATTCCCAGGCCCGCAGGAGCTCCCGGAGGCTTGTGGCTGTGGCGAGACCTGGAGGGCCATG
CGGGAGGGACAGACGCAGGTTTGCGGAGGCCGCCTGCCCAGGAGGGGCGTCAAAGGAGGGGACAGATGTG
GGTTTAGGGAGGCCACCTGCCCGGGAGGAGCCTCGAAGGAAGGCACAGGCGTGGGTTTGGGGCTGCCTGC
CCTGGAGGGGCCTCAAGGACCCCAGGTCTGGTCTTGGTCTCACTCACCTCCTGGACCCCCCAAGGCCTGC
AGTTTGCAATCTGTCGCCTGGGACCCCCACTGTCTGCCTTTACGCAGCTCAGCCACCACGCGGCCCTCGC
TCCCTCATTTACTTGATTTCTGTTTATGGTTAAAGTACCGTTTAAAACGACACATCATTAAAGCAACATG
AAAGGGAGTTTTGAAAAGGGAAGCCATCGTCCATCCCACTGCCCCTGCCTCAGCGGGGATTTACTTTTCC
TTTCCTGTCTGCGGGCCAGTGACAATGAGGACCCCGCAATGTGTCTGCGGGCCAGTGACAATGAGGACCC
CGCAATGTGTCTGCGGGCCAGTGACAGTGAGGACCCCGCAATGTGTCTGCGGGCCAGTGACAGTGAGGAC
CCTGCAATGTGTCTGCGGGCCAGTGACAATGAGGACCCTGCAATGGGCGGCCTGTAAGGCTCTGCCCTGG
CCTCCGCTGCCTTCGCTTTCTCCCTCCTTGGTGGGTGCACGCCCTTGTGCTTTTCCTAAAAGAGCAGGTC
CTCCGGGCATGGTGGCTCACGCCGGTAATCCCAGCACTTTGGGAGGCCGAGACGGGTGGATCCCAAGGCC
AGGAGTTCAAGACCAGCCTGGCCAACATGGCAAAACCCTGTCCCTACTGAAAATACAAAAATTAGCTGGG
TATGGTGACAGGCATCTGTAATCCTAGCTACTCGGGAGGCTGAGGCAGGAGAATCACTTGAATCCGGGAG
ACGGAGTTTGCAGTGAGCCGAGATCACACCATTGCACTCCAGCCTGGGCAACAAGAGCGAAACTCCATCT CAAAAAAGAAGAAGAAGAAAATCCTGACACTTCAGCCTGGTGCAGGCCCTTCCCTCCTTCTAGTCCCTGC
CAAGAAGTGAGCCGGGCCCAGATCTCCTGCCGGGCGGGGAATGAGCACACACATTCCCCTCTTGGGACAG
ACAGCAGCAGCAGCCCTGTTGCACACATGAGGACGTACAGGCTCAGGGGCCGTGGGTGGCAGAGAGGCTA
TCAGCGCCGGACTGGCCCGCCCCGAGCCAGGGTCCAGCCCCACAGTCCTGTCCCCAAGCCCTGGCCCTTC
TGCGGTCACTCCCGTCTGCTGAATCCCCTACTCTGCCCCTGGTGTGTGGCCCCCCAGTTCCCTCCTGTGT
TCATTCCCTGCTAACCTCCCGTGGCTTCGCCTCCCCAGATGCCCTGAGCACACAGCCTCTCCCCTTCCTC
CCCTCCTAGATGTGCACATAGGAGCCGCCCAAAGGCTGGGGCAGCTAGTGGGGCCCCTCCAAGGGAAGCT
GGGCCCCGGGCAATGCCCTGAGCCACCAGGTCCTGGCCCTGCGTCTCATCCCTTCTTTTTTTTTTTTGAG
ACAGAGTCTCGCTCTGTCGCCCTGGCTGGAGTGCAGTGGCCCAGTCTGGGCTCACTGCAAGCTCTGCCTC
CCAGGTTCACGCCATTCTCCTTCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGCCCACCACCACACTC
CGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAACCAGGATGGTCTCAATCTCCTGA
CCTCATGATCCACCCGCCTCGGCCTCCCAGAATGCTGGGATGACAGGCGTGAGCCACCGCGCCCGGCCTC
ATTTGGTTCCTTCTGTGCACCACAGTGTGGGATCTCGGGTCCTGGGTGGCACGTGTTTAACCTGAAAGGA
AACTGCCCCTGCGCTCCCCAGCTTGTAGCCCGTGGGCCTGGCTCTGAGGCCCCGACTGCCCCACGGCCTG
TGCTGCGCATGCTGGAGCCAGGCTCCGGCTCTGGTTGGCGCCTCCCGTGGCTTTAATCTGCAGTGACCTG
GGTGCTTAACGAGGGCCTTTGCCTCTGCGTGTGCACTGCCTTCTTCTGAGGAGTCTCCTCAAGTCTCCCA
GCTGTTTAAACAATGGGGTCTTTTGTCTTTTGACCTGTTGGTGCCATCAGCCAGCCTCCTGCACATACTC
TCCCTACCCTTGGCTGCCACTCCTGTCCTGCCCTTCGCCATGTCTTTTTTCTTTTCTTTTTTTTTTTTTT
TTTTGACAGCGTCTTATTCTGCCACCCAGGCTGTAGTGGAGTGGCACGATCTTGGCTCACTGCACCCTCC
GCCTCCCTGGTTCAACCGATTCTCCTGCCTTAGCCTCCCAAGTAGCTAGGACTACAGGTGCCCACCACCA
CACCCGGCTAATTTTTGTATTGTTAGTGGAGATGGAGTTTCACCATTTTGGCCAGGCTGGTCTCAAACTC
CTGACCTTAGGTGATCCGCCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGCGCCG
GGCCCCCGCCATGTGTTTTTAAGAGCAGGCATTTCATTCAGATGAAGCCCGGACACTCTTGGGGGTTCTG
CTCAGGGGCTCTACCTGCCACTGAACGTCCTCTCCCTGTGTGGGTGGCCATGGCATTCAGCCTGTGTTGG
GCCTTGTTCTGCCTGGCTACTGGCCGCTAGGTCTGCCCCGGCATCCTCTGTGTCCTGTGTGGGCGCCTCT
GCCTCCTGCTCCAGCCTGTGCCAGGCAATCCTGCTCACCTTCCAGGAGCCAGGCCTCTCCCCAGGGCCTG
CGTCCTGTCAGGGTCAGGGACGGCCCCTCTGCCATGCTCCGGAGTCCCTGGTCCCCTCACTCCGTTACGT
CCTGGGTGTCACGGGTACGGCCGGGAACTCTGTCGTCTTCACTGTCTGGGCCGGGGCCTCCCTGGGTGTC
TGCTGGATGGAGTGGGTGCCTTTGGGTCCCTGCAAAGTGAGCCTGCCTCCCAGCACCGCCCTGTCGTTAC
ATAGCCACTATCTTTGCGCCTGTTTTTCCTTCCTTTGACTGGTTCCTCTGGGGTTAATTCCCAGGCCTGG
TATTACCATCTCTGAATGCCTGGGTGGTTTCAGCGCCCAGGAGGCTGTGCTGAGGTATCTTAGACCATGT
GGGCACCGTTCGCTCCTGCGCAGCCGGCTCCGGGGTGCCCTGTTTTTGCGGAATCCTGCAGGGAACCCCA
TGTACCCTAAGAGTGCTCTCCCCAGCCACTGTGGCATAAGACAAGCGGTCTCTTTGCCCTTGGGCCCCAT
CCTTGTCTGGTCGGCCCTTCTTCATGGGTCCAGCGCGGGATTGCCGGCTTCCTTTTCAGGCTTCCTGGGA
CCCCCACTCAGACCTGCAGCTGGGCCAGCGATGCCCACCCGTTTCTCCTCCACGTGGTATACAGAGGTGC
CCAGGCTGCTGCTGGGGACTCTGGAGCCCAGGAGTGAGTCTCCTTGACCCTGAGCTGTCCTGGCTATCAC
AGCTGGGTCCTGTTTTCCCCTTCAGCACCCACGGGTGTCTTTCTCCAGTTTATATTGTTATTTTTATTTA
TTTACTTATCGAGACAGAGTCTCGCTCTGTCGCCCAAGCTGGTGTGCAGTGGCACAATCTCGACTCATCG
CAAACTCCGCCTCCCGGGTTCAAGCGATTCTCATGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCATG
TATCACCAGGCTGGACCTCTAGTTTATATTATTACAGCCTGGTCAGGGAGTCACTGGGCGTCGCCATCCT
GTGGGTGGGAAGGGGGCCCAGGCAGAGGCCAGGAGGAGGTGACAGTCATGCGCTTAGAAGCTCAGGCCAC
GCCCACCCAGCCCTGCCTGGTGACCGGTTCCTGCTGTGTTGGGAGCTGCATCCCAGACCTTATCGCCGGG
CACAGAACTTCCAAGCCAGGGGAGAGGGAGGCCTGGAGGGGCCCCAATCTCTGAAGGTCAGCAGTGGCGG
GGAGGAGGCTTGCGGTGCTGAAGGGACTCGGGGGACCTGCAGGGAGGTGTTGGTGGTCAGGGATGGTGGC
ACCTGAGGGGACTTTTGGCAGGGCCAGAAGTGCCCAGAGCAAGCTCCGGTGGGGCCCTGCACTGGAGGGC
TGGGGTCGAGTGACCCTCTTCCTAAGACCACCCAGGAGGACACTGGGTGCAGGGTGGCCGGAGCCCTCAC
CCCCAGTAGGCAGCTGCTGTCCACTCCGCCGACCTGCCTGTCACCCAGTAGGCAGCTGCTGTCCACTCCC
CCGACCTGCCTGTCACCCAGTAGGCAGCTGCTGTCCACTCCCCCGACCTGCCTGTCACCCAATAGGCAGC
TGCTGTCCAGTCCCCCGACCTGCCTGTCAGCCTTTTCCCTGTCAGGCCCTGTTCCTAGGAGCCTGGAGAC
CTCAGGGGTGGCCTTGAGCCCCCAGGGTTTTTTTGAGGGGAAGCGCCAGCTGCTGTCTTCACCCTTCCCC
TAGTGAGGCCAGGCTGTGCAGGGCCACGTGGAGGCAGTCTGTGCTGCGCCCATCGGTCGCCTGGCTTCCT
GCCGACCCTCGGCCCCCAGCCACCTCTGGTCTCGGGCAAGGCCCCTCCCCCTGCCCACCTCCTCCCTGGC
CCCCACGCCAGGTGGGCAGACCTCCTCTGCGGTTTTTATTCAGGGGGTCCCTCGTTGGCTGCCCACTCTT
GGAGGGCTGTCCTGACTCAGGCCTCCCCTCTACTCAGATCCCCTGAGCGAGGGCCTGGGCGTGACGCCGG
GAGTTACTGGGGGGCCAGAGGGGGAGGCTCAGCCTGAATCAATGAGACCCAGGAGGAAGGAGGCACGTGG
ACCTGAGGGCTGGCTGGAGCCGCTGGTGACAGATGGAGGAGTAATTGCTGCTTCCAGAGCACAGCGAGCT
CGAGCTCCCTGGAGTGCCAGAAGCTTCTGGGTGGACAGACAGGCCGCCTCACATTCCAGAGGCTGACACA
GTCTTCCAGGCACCCCTGGGGCCAGCTGGAAGCCATGTGCCTCCACTCACGTGTCCCGTGGGTGTTTGGA
GGGGAGGCTGGCCCTGCCATGGCCCACCCCAGCCTGCGGCCTCAGGAGCTTGCACATTTGCAAGGGGGTG ACATGAAGACTGAGCCAGGGCTGCGGCGGGCATCCCCCTCAGAGAACAGGGAGGAGGGGGCACAGGCCTT TT TTTTTTTTTTTTTGACAGAGTCTCACTCTTTGGCCAGGCTGGAATGCAGTGGCACGATCTCAGCTCAC TGCCACCTTCGCCTCCCAGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCCAGTAGCTGAGATTACAGGCA CCCACCATCACGCCCGGCTCATTTTTGTATTTTTAGTAGAGACGGGGTTTTACCATGTTGTCCAGGATGG TCTTGATCTGTTGACCTCGTGATCTGCTTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCA CTGTGCCCTGCCAGGCACGGGCCTTTTTATGAGGGAGGAAGCCGTGCCCTCTGCCACCTGCCTGTGGGCT GGGGCCTGCAGGCTCCCAGGGCCCTCAGCTCCAGCGTGGCTGTTGGTTATTGGCACCTGGGCCTCATGTC CACATGGAAAGTATATTCCGAGCTGGTGGCTAAGCCACAAGAGCCTGCTTACCTGTCCCAACCCCCGGCT CACTTGCTGCCATGATACGTCTGGGATTCTGGGCCCTCCTTCTGAGCTTGGACACAGACTTGGGACTTCA GCGCTGGACACAGGTGCTGGATCTGGGGTCTCAATGGCACCAGGGATGGTCATTCTGACAGCCATGCCCA GAGTCTCAGGAGGGCCTGGGGCCGCAGGTGGACAGGCATCCTGACCAGAGTCTCAGGAGGGCCTGGGGCC GCGGGTGGACAGGCATAGAGTCTCAGGTGGGCCTGGGGCCGCGGGTGGACGGGCATAGAGTCTCAGGAGG GCCTGGGGCCGCGGGTGGATGGGCATAGAGTCTCAGGAGGGCCTGGGGCCGCGGGTGGACGGGCATAGAG TCTCAGGTGGGCCTGGGGCCGCGGGTGGACGGGCATAGAGTCTCAGGAGGGCCTGGGGCCGCGGGTGGAC GGGCATAGAGTCTCAGGAGGGCCTGGGGCCGCGGGTGGACGGGCATAGAGTCTCAGGAGGGCCTGGGGCC GCGGGTGGACGGGCATAGAGTCTCAGGAGGGCCTGGGGCCGCGGGTGGACGGGCATAGAGTCTCAGGAGG GCCTGGGGCCGCGGGTGGACGGGCATCTGGACGTCGGCTCCACTAGTGTCACCTCTGCAACCTTGTCCCA CCCAGCACCTCCCTATGGCCCTGCTCTGACTGTGGGCTGAGGGGGACCCCCATGGCTAGCTTGGGACCTC CGCTGGACACTGTCATAGGGCACTTGTCGAGTGGCCCTGCTGAGGTTCAGGCTTGGGCGTGTGTGGCCCC TGAGCCCAGCAGCTGCGTGAGCCGTGGGGAGCCGAGCCCCCAGTCAAGAAAGCTGTGTGCTCCTTACCCT GCCTGCTGGGCTTGGGGCCTGCAGGGCAAGGGAGGCGGTGGGATGGGAGCCCCGCCTGGCCTGGTTTCTT GTTGTCGCCGCACTCCTAGCTGTATGTAGTCAGATGAGGCTCCCAGTCCTGGCTCTGCTGCTGAGCACCA TTGGGCCGGTGGTTGGGTGAATCTCTCAGCCTCTCTGAGCCTCCAGCACTTCGTCCTTAAGAGTGGAACT GGTCTGGACCTCCCAAGGGGGGGTCACTGGGTGCAGAGTTCCTGCAAGGCTGGGCCCGGTGCCAGTAGCG GTCGCTCTGTGGGGCCTTTCGCACGGTTGATTTTTATCTCCCTGTGAGATTAGCGCCTGCCCTGCTCTGC CTCACGGCTCTGGCTCTCCGGGACTGAGACCACCTGCTAAGTGACAGGGGCGGAAGGACCTTGCAACACA GAGCAAGACGTGATAGGGTGGAGCTGCTCCGCCCAGGCCCCGCCTGCAGATGAGAGGGCCCAGAGCCTCT TAATCCCCTGCGCTCAGGGCATGTGACGGCAGGGAGTCTCTCCAGTGCCGCCCACCGGATTCCTATAGTT AGAGCTCCTCGCCCAAGCCCCCAGGGCACCTCACTGGCCTCCCAGGTGAGGACATGAGGCCCAGCGACTC TGGTGGTGGCTGGTGTCACCTATGGCAGGGCCGCGCGGGGCCCAGTGGAGGACGCCTGGCCCGGACCTGA CTGCTCCGTGGGAACCGTGGGGCGTGCTCAGGGTGACTTGGTGCGGCTGGGCTGCCAGCCGGCCTGGCTG AGTCACTGGCAGTGAAGGCACCAGGTGGTGGACGTGCTCGGCAGCTGGCGGGAGAGTACCCCCACCCAGG ACCGTGTTCAGGCTGAGGGGGAAGGGAGCGGCCCAGGCAGGCACCGCCCATGGCGGGGCCCCCGCACTAA CGGCCACCTCTCCCGATGGACTGCAGTTCTGGAGCCGCCCCTGAGGAGAACGCCCTGCTCAGTGTCCTGG CAGACTCTGGACTCTTCACCGTGCCATCCTCGCTACAGCCTGGCCAGGCAGCCGCTCCCGTCCCCCCACC CCACAGACAGGAACTGCAGACTCCTAAAGGTCGAGCCAGCTCCCAAGTCCATAGCCAGAAAGTGATGCAG GCGGGGCCCGGCTGGGGGGCAGGTGTGACCGGGTCATGGGGGAGCGGGCTGTGCACAGGCATCAGGCTGA GAGGTCTCGGGGATACGTCCTGGGAGCCGCCTGCCAGGCGTCCTGTGGGGCTGCCTGCAGGCCTCCTCTG GCTTTGGCAGGTTCTGTCCCGACAGACCGAGGCTTGGGAAGACCGGATTGTGCACTCTCTGCCTGGAGAG AGGGTGGGGAGGGGCTGGGGCGTGTGGGCAGGGAGAACACCCTGGGAGGCATCAGTCCTCTTCCCTGGGG ACGGGCCATGCGTCAACAGTCCCTCACTTCCTCTCTCAGGGGCAGAGGCGTGGGGGTCCCGAGCCTCCAG GTTGGAGTAGGTTAGGAGGGCCTCAGAGCCCCCTGCTGCCCCGCCATGTGGCATTGGAAGGGGTGTGACC CCCTAGGGGATCTGTTAACCACACTCCGCCCCATGCCGGCCTCTCTGTTCCCTCTAACCTGCGCAGGAGC AGGGCCAGCTCTGAGCTAGCAGAGGTGACATGAGGCTCGTCCTGGCCCCGCCCACCCTGGCCCCTCCCTG TTTCCCACCACGCTCCGCCCACCAGAGCTACATGTTGTGTTTGAGCCTCAGCCCCAGACCTGGCTCAGGT CCTCAGGAGGAAGCAGTGACAGCCAGGCCCGGGGTCCTCTCCCTGCTCTGCTCCCTGCGTGCTGACTGGA GGCTGCAGGTCCCCAGGCTTGGCCCTGACCCTCAGGAATGGAGCCGGCCGATGTGAGGTGGGGGCTCCGG TCATGTGCAGTGGTAGGAGAGGAGGCGGGACCGGTCCCACAGCTCCATTGCTGCCGAGGCGTTCCGCAGG TCTGTCTTCATATTGGTAAGGAAAATGCGAGGATGGTGTAGCCTGGGCATCCCAGTCCCCAAGTCCGAAA TCTAAATCCAAGCGGAAAATTCCAGGCCTGACTTCATACGCCAGGTCACAGTCAGAAGCCAGAATTATTG AAAGTATGCTCTGGGCGTGGTGGCCTCACACCTGTAATCCCAGCACTTTGGGAGGCCATGGTGGGAGGAT CACTTGAGGCCAGGAGTTTGAGAGCAGCCTGGGTAACATAGTGAGACCCCCGTCATTATTTACACAAAAT TATGTGACTGCCTTTAGGGTATGTGTACGAGGTATATATTGAAATGTGAGTAGAATTTTGTGTTGAGTCC CATCCCCAAGATACCTCATTATGCGTGTGCAAATATTCCAAAATCCAGAAAATCCTAAACTGGAAATACT TCTGGCCCCTTTGGATGAGGGGCCCCCACTCTGCATAGCAAGTGTGGGCGCGGGTGCTGCTGTGTCCTAG CGTACTTCAGTGTGTGGCCTTTGCAGTGAATAGGGCTGGTGTCCTCACTGTACAGATGAGGAAACTGAGG CCCAGCTTGCTTTGCCAAGGTGCATCCGGCCCCGGCCATGGCTATTCTGGCTCCAGATCCCATGGTCTGC AGCCACAATACTGCTGTGCCCCGGACTGGGCCCTGCAGCTCGCGGTGTCGCTGGCCTGCCCATTGTGGGC ACCGCCCCCACCCCAGACTGGCCGAGGCCTAGAAGGGAGCAGGGCCTGGCTGAGGCTGCAGGGGTGGGGA CGGTCAGCCAGCCCCTCACTGCCAGGAAGGGCGCATCCATCCTGGCCTCTCCCCAGGGAGAAGGGAGGAG
CGGCTGAGAGGGAAGCGCTCTTGCCCTGTGGACGAGCTCCTGCCCCACGGACTAGGGAGCCCCCGCCCAC
AACCTGCTTGTCAGGGCCACCCGGGACCCCCGGGAGTTCGGCTGCTCGCTCTGCTGTTAGGAATTGGATT
AGTTTTCCATAAAAACAGGATGTGGTGGGTGAGAGGGCAGTGTGTCCGTCTTTCTCACTCCCCTTTTTCC
AGGAACTGAGCACGCGCATAGGTTTTAGCCAGGGCCGTCCAGTCCCCTCCCCACCCCCCACAGGGAACAA
TCCACTCTCTGCTCTTAAGTGGCCACTTAATCAGCTTCTCCTCCTGGCCCGGGGAGCTTCTTGGAGCCGG
CCTGCCGTGGTGGGAACAGCTATGGGGACACCCTGCCATAAGGTCCAGCAGCTAAGCTGGGATGTGGGGG
GAGGGGCTGCGAGGCCCAGGCAGTGTGCCAGGCCGCACAAGAGGAGCCCAGCTCTTGCCCCACCAGCTGG
CAGCCCTGGACCGAGGTTGGGCCCGTGAGGTTGGCTGGGCCCTGGGCCCTGGGCCCCCCTCCCCAGGACA
CGACTGTGGTGGCACATGGCTTTGGGGGCTCGTGGGTCCCACTTTGCAGACCTCTGCTTTAAGGGGTCTG
GTCCACGGGGTCCCCTCTGGAGGGCCTGGGGGAAATCTCAGGGACCTGGGGTCTGGACCCGGGGGAGGGA
GCGGGAGAAGCATGTGCGTGAGTCTCGTGCTGTCAGGGAGCCCGGGAAGTCTGCGAGGGCTTGGGGGTTG
TGTCAGGGAGTGTGGGTTTTGCCCTTCAGTGGTGGAAGCTGGCTTGAGTCCCCTCGATCCCTCAAGGCTG
TAGTCCTGACTCGGGGCTGTAGGGCAGGGCAGGGTGGGGTCACCCTGAGGCAGAAGGCTCAGCGGAGATG
TTCTGGCTTGGCTCTGCCCCTCCCTGGCTGGGTGGCCTCACCCTGTGACCTTTGGGGACCCTGGTTCCTC
TGAGCCAGGGGACAGCAGTGACTCCGCCTTCCTAGGTGGCTGAGGATGACATGGGCTCCCCCTGCAAATG
TGGGTTCTGGCTCAGTGGCCAAGTGTATGATGGTATGTGGCTCTTGGGGTCCTGAGAGAGATGGGAGAGG
AGCAGGGGTTTGTAGGGAAGCTGGCGGCTTCCACCCCAGCCAGTCACCTGCAGTGGGGGAGTTCCAAAGC
TGACTGAAGCTTCGACCTTGTGGCTGGTCCCCTTCCTTCCTGCCTCAGTCATTCTGGTCTCTGGGGGATC
AGGGCTGGGGGGCTCTGGGCTGTGGGGGCCTGTTTTTGTGACTTAAAGCTCTCCCAGCACAGCCCCCTGA
CCTCCTTCCTCATGGGCAGGACCTGGCCCAGGGGTCTCAGCACAGCCACAGGCCAGGGATGCCCTTGCAG
ATGGCCCTGGATGGAATTCCAGAACTCAGAAATGTCTCCTTCCCGTAAGGATGTCCCGAGACTCATGAGA
CCGTTTCCTTCTGGGAAGGGGGAGGAATGGGGAGATGAGTGAAGAGCGCACTGCAGCTCAATCCGGGAAG
AAGCTAATCAATCAATCAGGGAAGCCAATCGATCGGAGAAGCTGATGGGAAAGCTGTGCTTGGTAGAATA
GGCTCGTGCGGGCAGAGCAGTCGCGGCACTCACAGGCTGACTGTGAGGACCTTGGGTGTTACTTTGTGCT
TCTCTGCATTTTAAAAACTTTTGAAAGTGGAGAAGAGGAAAGTGAATGTTCTCTGAGATTTCATGGAAAG
GGGAAACTGAGGCCCACTCTAGCCAGTTTGGGCCCAGGGTTCCAACCTGGGGTGGCCCCGGCCCTCGTGG
CCTGAGGTGATCGTCCCTGTGGCTCTGAGAGCAGCTGGGGCCGGGTCCCCGTTCTGGGGCTGGTGATCCT
GGGGAAGAGCCAGGCAGTGCCCTGCCCACCTAGTGGTTATGAGCCCAGAATGTTGATTTTTTTCCCTTGG
TTGCTTCATGACTTTGTTGAATTTCCAGAGTATGTGTGGGGGCCCCCGGCGTCCCACTCGCCCCAGCCCT
GTGGCAGCAGAGCTGGCTGTCAAGCTCAGTCAGCTGGGCCCAGGGCCCCGGAGGTAGGTGGGTGTGTGCC
TGAGCTTCCCCTTTGGGCCCTGCCAGGTGCTGGGAGGGACCACACAGGCGGCAGGAACTCGGGGTCCCCA
GGCCTCGGCCACACCAGCCTGGTGCTTGTTATATATTGATATGTCTCTCTACCTGTGAAATGGGTATTTA
TTTTAAGGAGCTGACGCACGCGATTGTGGGGCTGTCACGTCTGAAGTTCGCAGGGCGCGCTGGCTGTCAA
GAGCCGCTGTTGCAGTCTCGAGTCCAGGCTGTGCGCTCAGGCCGGGCTTCTCTGTGGCGGTCCTGGGAAT
TCCTCCTCCGGGCCCTTAGGCGACACTCCCCTGCCTTCAGCTGATTGGATGAGGCCCTCATGCCGTCTGC
TGATTTAAATCAATCTCATTTTAAAAACACCTGCACACCAAATTGCGATTGGCGTTTGACTAAACTGGGC
AGCGTGGCCCAGCAGCGCTGACACATGAATCAACCCTCACACCCCACCCGCAGGCGTGACCACGGCACCC
ACGCTGCCGACTGAGAACACAGCCGTGCGCTGACTCGCATGTGATGTCTCTGGTCGCACCTCCTCCATCC
TCAGCACCCTGTTCAGAATGGAGATAGCGGCCGGGTGCAGTGCTCACACCTGTATCCCCAGCACCTTGGG
AGGCCTGGGTGGGCGGGTCACCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCAT
CTCTACTGAAAATAGAAAAATTAGCCGGGTGTGGTGGCAGGTGTCTGTAGTCCCAGCTACTCGGGAGGCT
GAGGCAGGAGAATCGCTTGAACCCAGGAGGTAGAGGTTGTAGTGCGCGAGATTGTGCCACTGCACTCCAG
CCTGGGCGGGCGACAGAGTGAGACTCTGTCTCAAAAAAAAAAAAAAAAAAAATTAGAGATAACGACTTGG
GCTCTCTAGAGACCAGGGAGCCAGGCTCCAGGCGCTGCTGTCCAGCCCCTTGCCAGCTATACGAGGTGCT
CACCTGGCCACGTGTCCGGGCAGTGCTTCTGGGGTGGCATCCACTGGGAGGGCAAGATGGTTCTGGTACA
GGTGCCCAATTCTGTCCCCATTTCACTTACTGGGAACTCAAGGCACAGAGAGGGGAGGGCTGAGCTAGGA
CCAGACCCCAGTCTCCTGCAGAAGTACACAGACATGATGTACGACCATCCTGGACACCTGCCCTGAGATT
CCCCTCCCTCTCCTGCCCTGTCCCAGTGGCCTGGGGGGAAGGGGAAGCAAGGTTCTGAAGGGAGTGTGAC
CAGACACCTGCCCGTGACACCCCCTCTCCAGGCTGCCTCCGAGTGGCTGGTGACTCCCCTCCTGCCTGCG
AGGGAGGTGGCCAGGTTGCATTCCTCTCTGAGTGCCGGGGAAGTCCCTAGAGAGCAGGCCAGCCTGTGAC
TGGGCCCTGGGGCAGTCTAGACAGGCCAGACTGGACAGGCCAGGGGGCTGGGTGCCGCTGGGTAAATCAC
AGGGTGAGGGCTCTGAGTCAGCACCCCATCTTCTGTCCTGGGTCCAGCACCGCTGAGGACACAGTGGGCA
GCCGGGTCTGCCAGGGCCAGGTAGCTGTGTTGAGAAGGCAGTGCTCCTGAGAGGCGGCTACCGGGAGGTT
TTCAATGGCCAGGCTTCTTAGGAAGCCCTTGTTGCCTCTCTGGGGTGAGTTGCTGGGGCCATGGTTGGAG
TGGTCGCCAGTGTCTGCCCCTGGTGCCGAGGGCGGAGTCCTCGTTTTGGGAGGTCACGGCATGATGCTGG
GAGTCAAAGGCAGGCCGTGGCAGGGGCATTCCTTTTTTCTTTCTTTTTTTTTTTTTTTTGAGACGGAGTT
TCACTCTTGTCGCCCAGGCTGGAGTGCAGTGGTGCGATCCCAGCTCACTGGGATCCCAGGCTCCCAGGTT
CAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGTACGTGCCACCATGCCCGGCTAAT TTTTGTATTTTTAGTAGCGACGGGGTTTCGTCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCATGA TCCACCCGCCTTAGCTTCCCAAAGTGTTGGCATTACAGGTGTGAGCCACCGCGCCCGGCCTGGCAGGGGA ATGTCGACGCGTGATCTCTGCCTGGAGAGCACGTTCATGTTTCCCAGAGGACACTTTAGAACATGGCGCC TGGGTTTGGATGAACCTCAGCCTAAGAATCTACCTGCTCAGGATCCAGCGACGCTGGTGGTGTGGACTTC AGCTCTGGAGAATGGGTTATATGGAACCTGGGCGCCGGGAGGGCATTGCCACGTGCTTGCTGCTGGGGCT TCAAGAGGACCCCATCTCCTGTGGCCGAGACCCCGTGTCTCAAGGCACATCCCCTTTCGTACCCCGCCCC ACCCTCCGCAGCTTCATGACCTCTGGGTTTCCCCCAGGACCTTCGCATCTGATGTTCCCAGATCCTTCCT GCCACTGGGTCCTGCTCTGGTGCCCCCCGGGGAAGCCTTCCCTGAGGACCCAGCTCAGCATCTGGTGCTA ACTGTGGCTCATCGTGCACTTTGGCCCCCAGGAGGCTGTGGGCTCCTGAAACCCCTCTGAACCACGGGCT TCCAACCACACCTGACCCCCTGCGTGCCGACCCTGTGTGCAGGTGTACAGGTGTGCCTGGGGCAGGATGG CTGTGACGGCCTCACAGAGCCCGGAGAGCTGCCTCCTAGCTTCCAAAGCCTTCATTTCAGGAATCTTACC CTCTCAATTAGTCTGGAATGCTGGGGGCGGGGCCAGCTCCAGGTCACAGAGCGACCTTGTTTACCCAGAC CTTAACATCGGCCCTTCCATGCTAATCAATGTAATCATTGTGGTCCATGCGGCCTCTGGAATGTGCTGTC CACTCCCTGGGTCAGCCAGAGAGTGTCAGGGAGCACCTACCGGCTGTTACCCAGTGCTGCCCTGACCTGG TTCTTCACTCTGCACATTTGTATCACGCCAGACCCTGGCTGGCAGCTCCAGGTGACGAGGCATGTCAGTG CCTTCCTGTTCTGTTCTGTTTTGTTTTGTTTTTTAAATCGGGATGAGGCCTTCCTGTGTTGCCCAGGTTG GTCTTGAACTCTCAGGGTCAAGCGAACCTTCTGCCTTGGCCTCATAAACTGCTGGATTACAGGCAGGAGT CACCATACCTGGCCCACTGCTACTTTCTAGATGAAGAGACAGAATCCCAGAGAAGAAGCAGGGGTTTGGC TGCTGGTCTGGAGCCGGTTCTGCTCACCTCCAGCTTCTGCCTTGGGCCGCCCTGTTCACACAGGAGCTGC TCACAGGCTGAGACCTCGAGCAGGGCCCTCCTAGAGGAACTGGGCCCCCGTAAGTGCCCTGAGCCGCCAG GAGCCGGCCCTGCGTCTCATCCCTATCTCCGGAGGACATTGGCTGCTAGCTCACCAGCTGGCCCCTGGGC AGGCTTGAATCATGACCTGGAACGCCAGGTGTCTCTGGCTCTACCCCTGGACCTGCACCCTGTCCAAGTG CCCCAGGGCCAGACTGTTTTGGTTGCACCCTCTGGACGGGCTACCCCCATGATGGCTGCTCATGGAAAGC TGTGGTTCTTAGGGAGCTGCCAATTCCTAGTCCTGCAGCCTGGAGCTCCTGGGTATAAGGTGGGGGCTCG GGCGTATTGGGGACTGGGGGTCTCAGGAAAGAGCCTGTGGGACCTGTGAACTCATAGCTGCTGGCCGAGG GACCACCTTGTGGCTGTCCTTCTCAGCTAGGCCTGGTCAGGGCTTGTGTGCAGGGCGGCTGAAGCTGTGG GAGGCCACACTGTCCACACAGTGCCCTGTAAGCCCACCGTGCCTCAGTTTCCCCGTCTGACAAGTGGCAC AACAGAAGCCACTTCCTGGCACACAGCTCAGGGTCAGGGCCGACACAGCACTTGTGGGCTGCCGGGAGAC CCGAGAGGCTGCCCCTTCCTTGCCTTGGCTGCCACGGGTGACCTGGCAAACCCCTCTGGCCGTGGCACCA CTGGGGGTCTACCCTTGGCAGTCAGGGTTGGCCGCTTGGCTGGGAGCCCCTTCTCCTCCCCAGACACATC CTCTCCTTGGGGCTGGAGGGGGTCCTGCCGTCCCCGGGATTGTCGAGCAGCAGGAATCCAGGAGGGCAGT GCCTGCAGCTCAGATGGGGGCCAGTGGGCAGGGCCGATCCAAGGGTGGCAGGAAAGTGCCCATCACTGAC CTCAGGTGGGGGAGGCCATGGTGTGTGAAGGAAGGAGAGCTGAGTGGGAGGTCTTACTTTGTCACCGCCC CTCTGAGTGCCTGCTGTGTGCAAAGGCCAGCGGGGGCCCTTTCTTCAGCTGGGCTCTGCCCAGACCCCTG AGCTCTGGGTGGGCCGGGAGGGAGACCTTGCTGCTCACAGAGGTGGCTCTGCCTGACCCAGCTCCCTTCC CAGGGCACAGTGGGGGCATAGGGTCGGCTCCGTCAGACATTCCGGGACCTGCGTCCTTCCTGGGGCCACA CCCTCTACCCACTGTCCCCCACCTATTTACCTGTATCTGCGCCAGAGATGGCTGCCCAGATAAGCCCTGG TTTCCTCCCTTTCTGGAGAGGCTGCGGGGGCTGGCGAGGAACCCACCTGCGCAGAGAGTCAGGGGATTGC TCTGTGTGGAACGCAGGCCTCACCCATGCCCTGGAATCTGTCCCCTTCTCTGTTGCTGAGGGATGAGTCC CAGATCCCTGACCCTGCAGGGGAGCCCAGCACAGAAAAACTCTGAGGCCTCCACACCCTGGCAGCGCTGC TGGTCGTCTGTGGGGAAGGACAGGCCCTGGGAGGGAGGGGGAGGTGCGGAGGGCAGTGGGGAGGGTCAGG AGGAAGTGGGGGAAGGGCCACCCAGGCCCCGATGTGGGGGATGTCTCACGCGTGGGGTGGGGCATTCTCA TCTCTGCTTGGTCTCCTGCCATGCTGGGGGTCGTTCACTGCGGACCCCAAGTACCATGAAGATGGGGATG GGATGCTGAGCAGCATCGGGGAGAACGCAAAGGCACCTCCCAGCTCACCCGCCCCCACCCCGCAAGCACA ACCATTGCCATGGTGTGGGGACCGGAAGGCGGCGGCTTTGGGACCAGACTGCTTCTGCCTCGGGCCGTGC CGCTGGGCCTTTGGTCAGCACCATCGTGCCCTGCAAGTCACTCTTAGCCTGGTGCCCTCCTGGGCAGGGC AGTGCCACAAGAGCTCAGGCCCAGATGTGCAGGTGCCACTGTTCCACCCACCAGCAGGTCACCTGGGGAA CCCTCCCCTCCTGCAGCCTCTGTGTGCTCATCTGTGAAATGGGCGTGGTGGAGTCACCCCTGGCTTGTGG GAGAATCCCAGGGGCTGATGCCTGCAGGACCCTGTGGGCTTTGCCCCGCTCCCTGGGCAGAGACAGTTCC CCCAGTCCCACCCACGTGGCTGTGTGCAGCAGGTGCCTGCTGACCCTCTGTTCCTGCACAGTCACCTTCT TCACAGACGGACCCCCACCCTGCCCTGCAAACCCCTCCAGGGGCTGCGGGCTGAGTGTGTCCGGGAGGGT GTCCTGACTCTCCACGCCAGCAGGTCTGAGAGCAGATGGCTGTGGCAGGTGCGGTGGGTGCCCAGCCCAC AGCAGCCACCAGGCCTGCAGGACCCTGCCCCGTGTAGGTCAGATGAGCCATAAAACTGAGTTTCCTGGAC ACTGAGCTAATTAAACCTGGACACCGAGCTAATTAAATGGTCCAGAAGCTCCTCAGTGCCCCAGGCTGCT GGCCGGGCTCCAAGTAGGTGAGGAACATTCCGTTTACCTCCTGCTGCGTCGAAGGCGGGTGGCTCCCCTC GGGCCCCTGCCTGTCCCGGGCCCCCTGGGTGCTGCTGCGTCAAAGGCGGGTGTCTCCGCTTGGGACCCTG CCTGTCCTGGGCTTCCTGGGTGCTGCTGCCTCGAAGACGGGTGGCTCCCCTCGGGCCCCTGCCTGTCCTG GGCTCCCTGGGTCCTGCTGCCTTAAAGGCGGGTCGTTCCCCTCGGATCCCTGCCTGTCTTGAGCTCCCTG GGTGCTGCCTGCCCTGTGGCCACGTCCCACTGTTGCCTTTGGACAAAGCTCTCCTAGGGCCTGGGGTTCT CCCATGAGATGACAGGGGTTGGTTCAAAGGTCTCTGAGGTTTTTAGAGCTTTGTGAAGTGTTCTAGAATC
CATTTTCTCTCTCATTGCTGGACAGAGAGTATAGACTGGGCTTTTTCTTGAGTTTCTCTCAACTCTTCTT
TGTCCATGAGGCAGGAAGAGGGGCTTCCTCAGTCCTCAGCTTGGGGTGGATTGGGATGGACAGAGAACCG
TGCAGGATCCCAGCCTGAGACGGTCCAGCGCCCCGGGGGAGCGGAGCCCGTGGCTCAGCCGTCTGTAGCC
ACGGCCGGGGTCACTGTCACTGAGGGCACGGAGGCCCCGCCGGCGAGGTCCGAGGTGGGCGTGTGGGGTG
GTGGGCGCTGGAGGCAGGACCTCTGCTTGTGGAGGGTGGGCCAGGCGTGGACCAGGTGTCACGTCCGCTG
GGGCCTTTGAGGGGCAGGTGGGGATTGGCGCGGAGAAGGAGTGAAGCAGCTGGGGTTTTGGGAGCCTGTG
CGGAGCAGGCCCGGATGCCAGGGTGGCTGGTGAAGGTGGGCCTGGCCGGGCTCGGCCTCCATTGGGGGAG
CCCCCCAGGTCCCCTCCCCACACAGCTTCCCCCTCTGTCTGCTGTCCAAGGCTCCTGTGCTGGCACTCTG
GGGTGGTAGGCCATGCAGCCCGTGTGAACCTCCATAGACCTTGCTGTGAACGCTGCACGGGGCGTCTGGG
GGCGGGCTGGCTTCCCCCCGTCCCCTGGGCCAACCTTGCCAGCCTTCTTCTTCCATAAAAGTGGGATCCG
TCTGAGCCCTCATGGCCCCTCTGGGTCTGGGTTTGCTTTTTGTGCTCTGCCTGGGGTCATGGCAGGGAAG
GCCCAGCAGGCTCCCTTGCAGAGCAGGGCAGAGCAGCGGCCGCCAGGAGCTGCCTGTACCACGTCTGCTC
TTCTTCCTCTCTTCCTCTGCCCGGCCCCGCCCACCGTCGAAAGCACTAGCACGGGAGTGTTTATCGGTTT
CTCTTCCAAGCCAAATAAGGCAGAGAGCGCTCTTGCAGGAGTTGGAGCAGGCCAGGGGGAGGGCAGCTGG
GGCTTGGACCAGGACCCCGGCTCCCTGTAGCTGCCCTGGCCTGGCAGCTGTCATCCGCGAACTCTGATCC
TCGCCTGCCCTCACCCGGCCCCTTTCAGAGCTTCCCATGCTCAGCTGGTAGCAAAAGAAGTCAAGACCTA
GCAAGGTGCCTGCCCCGGCCAGGAGGCCCAGGTTCTAGCCCTGCAGCTGCCGCTGTGAGCTCTGGGGACT
CAGACGACTGATGTCCTTCCCAGCTTCTGTTTCCTTTGATGAGAGGAAGGTTGGACCAAGCGACCTGCCC
TGCTTCTGTCTGTTTCCTCCAGTCCTTTTCCTTTTTGACCAGTTCACTTGTTGATAACTCCAGCAACGCA
TGAAAACATTCCCCTTGTAAAAAAGGGAAATGCCGTGGGTAGGGGGGACCAAGGACCTCTTCTGCAGGAG
CAGACGCAACAGCTTGGTGTGGATCTTCCTCAGCCTTTTTGTCCATCCTAGACATGTGGAATTTCAGTTT
TACACAAAAGGAATGGTAGGCGTCTGACTCTCTGTGGCTTGTATCACTTGGCAGTGCCACAGGGATTGGT
GCATGTTTGCTCTCTCATGCTATGTGTCAGGCACTGTTGTCTCTGCGACAATCCTGTGAGGAGGGAGTTA
TGGTTCTTGATTTATAGACGAACAAACTAAGACAGAGGAGATTGGGCTCATCTGTGGTCATCTCAGCAGT
TGTGAGAGGTCAGGGCCAGAGCCCAGTGGTGTCATTCTGCAGGGCACATACAGACACGACCCACAGGGTT
CTGCCGCACCCCCTGCCGCTTGGCCTGCTCTGCAGGTGGGTGCCGAGGCTGTTCCTGCATCTGCTGTGTC
TTGTGAGTCCGTGGGTGCCTGTGGAGTGGGGGCAGTGGGTTTGCATTTCCAAGCACTGCCTCTGTCATAG
GCTCAGGGAGAAGCAGTTACGCATGAGCTGCTATGAGCCACGGGTATGACTGGCATCACAGCAGGAGGAG
CCCTGGGGATGGCTGAGTGACGATGGGTGGGGACAGAGCTCACAGAAGCCAGGGTTGCCCCCAGGCCAGG
CCACTGCTGGGGCCAAGGCTGGGATGCAGCACGATGTTGGTCTGTCAGGGAGTGGCAGGAAGCCCACAGG
GTCAGCTGACACTTAACCCAGAGTGGTGGGAGCCTTGAATGCCAGACTGAGGGCCACACAGTTGGTCTCG
GTGCACAAGGACTTGGGCTGAGAGGGCTGTTGCTCCTCAATGGCCATCTCCTTGTGGGAGGAGAGTCCGG
GCAGCCGCTTCCAGGAACCCACCCATGCTTGAAATAGTGGGACCACAGGGCTGAGGAGGCTCCTGGGGAT
TCCTCCCAGGATTCAGCTGCTCTGTCCCCAACCCCGGAGGGCCTATTCGTCTAGCCCTATATCCTCTCTG
CCAGTTAGGTCAGGGCAGAGAAGTAGAGGCACAGAGGGTGGCGGAGGGGTGTCCAGCCAGGCCTTTCTGT
GGCACCTTCTGAGGCTGGCTGGAGTCTGTGAAATCTGTGATAACTGGCCCAGACCCTTCCTGCCTCCTGG
GGCATCTGCTGGAGCTGAGGCTGCTGGGGGAGTCTGCCTGTGACTTGAGTCTCTTGCTGGGCCATGCTGG
GCTCAGTTCGCCTGTCTGTGGGGTGGGATGGTGCCACCCTTAGGTTGTTGGGAGGACCCAAGGAGAGTGA
TGCCTCCAGCCATGGCAGCTGGCCCAGCTCCGGCCCGCAGCCTGGCTCCTTCAGGGCCAGGAACCCCCAG
GTCATGGACTCCAACCCTCGGGCTCTCCTGCTTCCCAGGTGACGAAGCTTCAACCCATGGGCTCTCCTTC
CTGGGTGTGGAAGCCCTGGAGAAGGTGGAGATGGGATCTCAGTCCAGCCCATTGGCTCCACTTCCTTTGA
GAAGCTCTTTCCCCCGTCTTGGCCCCCAGCCCTTGTTCAAGCACCTGTCCCTCTCCTGTCTCCTAGGCCC
CGTCAGTCCTTTTGGAGACCTGTTCCCACCCGCCCCTGCTCTCACAGGCTGCCAGTGTCCACACACTCTC
AGGCACTGTCATTGGAGCCTTTTAACCACACGGGAAAGCAGGCAGGTTAGGTAACTACCCCCCACCCCCC
CGCCAGCAACGCCCCCTGCATACCCAGGGCCTGCACCAGGTGCTGCTGCGCTGCCAGCTCTCTCGAGGTC
CCTCCTCCTAACCTCAATGCATCGCGTCTTCCAGCCCCCGGCTCCGAGGGCTCAGCCTCCAGGTGGTCTA
ACCTGGAGTCTAAACCTGGATTGCCATCTGCCTGCCTCAGCCCTTCCCTTCCCCAGGGACCGAGGGCCAG
TGCGGCTCCCCAGCCACCCATTTTTTGTTTGTTTTGAGACGGAGTCTCGCTCTGTCGCCCAGGCTGGAGT
GCAGTGTCCTGATCTCGGCTCACTGCAAGCTCCATCTCCTGGGTTCACGCCATTCTCCTGCCTCAGCCCC
CCAAGTAGCTGGGACTACAGGTGCCCGCCACCACACCCGGCTACTTTTTTGTATTTTTAGTAGAGACAGG
GTTTCACCATGTTGGCCAGGATGGTCTCGATCTCCTGACCTTCTGATCTGCCCGCCTCGGCCTCCCAAAG
TGCTGGGATTACAGGCATGAGCCACTGCACCTGGCTTGTTTGTTTTTATTTTTATTGTTTTTTGAGACGG
AATCTTGCTCTATTGCCCAGGCTGGAGTGCAGTGACATAATCTCAGCTCACTGCAACCTCCACCTCCCAG
GTTCAAGCGATTCTCTTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACCCACCACCACACCCAGAT
ACATTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTTTCGAACTCCTGACCATG
AGATGGCGGGCTTGAGCCATCTTCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACGG
TGCCCGGCCCCAGCCACCAGTTTTGTACAGCACTCAGGTGGGTGTGGTTCCTGAGGACATGTCACACGAG
ATCCTTCCGGTCCATTAGCCCACCCCGCCCCATCAGTGGCCAGTGCGGCCATTGTGGGGTCCAGAGTCCC GAGGCAGAACGACTGACTCCAGGTTGTGGAGGGGCCTCTTAGGATCCTTCTAGGCACCCAGGTTGGGCAG GTGCCTCCATGCCTAAGACTGAGCTCCTGGTGGACATCGGGCTGGGTGTGGGCGTCCTGCACACCCTCCC TGTGGCCTGGTGCCCGTGAGGAGTGGACAGTCACCCCGAGAGCATAGACCACCCTCGAGGCTCCCAGATG GCGGCCGCAGAGCCCCACTTCCCTGCCTGGTGTGTGATACCTTCCATGGCTGAGGACACCTCTGACTGCT CACTCCTGTGACCCTGGACCCAAGTGCTGGGCCACACCTGGACTCCAGGCCCCAGTCCTTCCTTCTTCTG CTGTCCTGGCTCCTGGGTGGCCCCCGCCCTCCCCGCTCCGAGGCTCTGCCCTGTGCTGGGAACCCAGGAA TTGCCACCCCCTGGCCCCGGTTTCTCATGTCAGGATCTGCTGGCCAGGTTGACTCCTGAGCGCCACTGTC TCCTCAGACTTGCTGGTCCCACCCGGTCCCCACCTGTCCACCGCCCGAGGCTCTCCTGTGAGGCCGCTCT GCAGCTCTTGGCCTGGGGGCCCCCGGCCTCTCATGGTGCCTTCTGCACTGTCCCCTTTGGAGATGGGAGC TTTGAGGGTAGGCGCCACCGTTCGGTGAAAAGGCGGTATAAGCAAAGCAACCCCCAGATACTGAAGGAGC CGGGAAATAAAAGGAGGCAGACAGAGCAAGTGTGTTGGTACCCGCCTATTTACTGGCAGGAACTTACAGA CGGAAACATGGCCTCAGGCAGCCTCCAGGCAGGTAGAGCTCTGAACCCAAACGCCCGATCTGGGGCTCGT ATCGTGCCCTGGGAGGAATGTGCAGGTGGCTGGGAACGTGGCGGGTGGGGTGGCCAGATTCCTGCTGCAG CACCGGGTTTGTTTAGGAAGAAACGTACGAGGACTAGACACTCTTTTTTTTCTTTCCTGCACGTGAATGC CAGGGAGTAGGTGCTCCTGCAGGAAGATGATGCATCAACCAGCCAGTCTGGAGGCATTCCGAGACCCGGG GTTAATCAGCAGATTAGCATTTAAATAAAGTTACTCTGTCCCCACAGCCAGCTAGGCCCGAGCCACATGG CGGGGGCGGCGGGGGCGGCTGTTCGCCGCATCCCAGGCTTGCTCCATCTCCTCGTCTGCAATGTGGAGGT GACAGTCGTGGTCCTCGGCTAGTGCCACAGGACTGTACGGGACAGATCACCCAGCCCCGTGCCTGCACTG GGGGCGGCGGTGACGTGATGGCCGCGGCGGTGACCTGATGGCCGCAGGTGTGTCCACCCTCTGGTTAGGC CTCTCCTCTCCCTGGGGCGCAACAGGTGTCCTCGGGTGGGAGGTTCAGCTGCTGGGCCTGGGGCCCCAGC TTTCAGCTCCTGTGCAGGCGTGGAGCGGGACGGCACGCCGGGGCGTTCGTTCACACGATGCCCCCAGCTC GTCTCTGCCTGCTCTGTGATGCAGCCTCAGCTCCTGTGGGTCCCGGGTCACCCACCCGTGTGGGCCGCAG TCTTTCTGCACCACGTCCTGCTCTCGGCCAGCGCTGGCCTGCGGGCCCCGGCAGTGTGGGGTCACACACA GGCCGCACCGGCTGTTTCACGGAGCCCCCCTGCTCAAAATCCTGGCCTTGGGTCTCTGTGTGGCTCCTGG AGGGAGCATGTATGAGGTGTCCAGGTTGTGAAAGCCCCATCCGGCAGCCCTCCCTGTGACAGTCCTTCAG GCCTCGTCAGGCTTGGTTCAGCCCGTGTTAGAGGAGGCCAGACAGCTCCTCCTCGATCACCACAGGGAAC GAGGGTTGGGCAGTTTCTCCTCTCCCACCCGGGCGTGGGTCAGCTGCAAGGGTCTCATCCCAGGAACTGG ATCCTGGCAGGGGGGCTCAGGCATCTGACATCCCCTGGCAGGTCTTCCTATGTGCCAGTGGGAGTGGGTG GTGTCCAGCCCTCTTTGTGGAATGTTCCGGTTGAGTGGTGTGTGGTCCTGGTTACCACCATGTGGCATTT GTGGCTTTCCTGGCCAGCACCCACTGAAAGCAGAGGCCAGCCCAGCCCTTGGACACCCAGGGGCCCAGCA GCTACCGTGGGGGCAGCCTCTCCCCTCCTGACTGTGCTCCTCTCAGCTGTAGGATGAAGGTCTGTGGATG GGTGATGGCTGGGCTCTCTGCTTCCCAAGGAGCTGGATGTGGGCCATAGTCAGGCGTGTGCTGGTGAGGC GAGGGGGTTGGGGGTTGGGAACCACAGCTCTCTCTGACCCCATCCCATTGCAGCTGACTCAGGAAAAGGC ATTACCCCCACGGGGTCACAGGCCCAGCCTGGGCAGCTCCTGATGGGAACATGGGAGCCACAGGGCAGGG CAGGCTCCTGTCCCCGCTCCTTGGATTGTCTCAGGCCCCGCCTCCCCTTTGGCTGAATTTCTTCCTTCTC TGCCTGAGCCTGATCTGCGAAGCTTCCGACCTGCCCTGAGTTGGAGCCAGTCTTCAGGGCCAAAAGCAAT GCCGTCTCCTCCAGGCAGCCTGCTCTGAAGTCTCCCCCTCTGGCTTTAGCCCACAAAGTCACAGCTGAGG CCACATCAACACACTTAGACCAAGAGCACTGCCGGCTGCATAGACAGCACAGGTAGATCCAAGGAGTGTT TCGAAAAGCCGTGGCCCTGCGGCCGGGATGCCCGGACCTGGGTCCTGACCACTCTTGGGAGCTCCTCCCC TCTCAGAGCCCATCCCATCTGAAAATGGGGGCATTGGAGCATTGGAGGAGTGCTCCTCACTCCTGCCCTG CTAGCCGGCTACTCCGAGGACCCCAAGACCAGATGATGGGAGAAGTGGTGGGCTGGGCTGTGACCCTGAG CCCCCTTCTCATCCCACCCGTGAGGCTGCCCCTGCTTGGTGTCTGTGGCAAGGCTGACAGAAGATGGCGC CAGGGACCAGCCAGGGCCCTCCTGTCTCAGCCCGTCCTTGGGTGAGTGGGTGTGCTCTGGGGGTGGCAGG TGTGCTCCGGGGGCGGGAGGTGTGCTGCGGGGGGCCGGGTACGCTCTGGGGGGGCGGGTGAGCTCTGGGG GGCTGGGTGCGCTCTGGGGGGGCTGGTGTGCTCTGGGGATGGACAGGTGTGCTCTGGGGGGCGGGGTGTG CTCTGGGGATGGATAGGTGTGCTCTGGGGGGGCGGTTGTGCTCTGGGGGGGCAGGGGTGCTCCGGGGGTG AGTGTGCTCTGGGGGGCGGTGGGTGTGCTCTGGGGGCGGTGGGTGGGCTTTGGGCTAAGTAGCCCTCTTC CTCTCTGAGCCTTTTCCTTGTCTGTGTTGTTGGTGAGTGGCCGCCACCTGGGACAAGCCTTAGCTGTCTG TCCAACTGTCCACCATGTCCCCGGACCCTAGACCAAGCCCGCTGCCTGGTGGGGCCTGCTTGGTATTGAA TGAATGAATGAATGGATGAATGGATGAATGGATGAATGAATGAGTGAACAAACTCTGGGGGGCGTGTTGC TGATCACCCCAGGGCAGGCCCGTGGTGGGTCATGGTCTCAGGCAGGGGACTTGCCCTGCCGGGTGACGTT GGCCTGCCGGGGAACCCCTGCCAAGGTGCCTCCCATGCCCCCATCGTCCTCTGCCTTTGAGTCCACTCTG CCCCTCAAGGTTCCCCTCTGGCTCGGCCAGGGGCAGGGCGGTCAGGGAGGCCTCCAGCTCCAGCCCCTCC CTCATCTCCATCCTCAGCCCCTGCACCCGCCTCATCTCCAGCCCCAGCCCCTTGCAGCCTCAGGCTCGGG CTTCTGAGAATCCTTTGCAGATTTCAGGGCTGGTTATTTTTTTATTCTGGCAAAATATACATCCATAAAG CTTTGTTTCCTGAGATGATCTCACCCTGTCGCCCAGGCTGGAGTGCGGCAGCGTGATCATGGCTCACTGC GGCCTCAGCCTCCTGGGCTCAAGTGATCCTCCTGCTCCAGCCTCCTGAGTAGCTGGGACCACAGGCATGC ACCATCATCCCCGGCTAATTTTTGAATTTTTTTGTAGAGATGGGGTCTCTGTGTTGCCTGGTCTGGTCTC GAACTCCTGGGCTCAAGTGATCCGCCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCCTCAGCCACC ACACCCGGCCTAAAGTTTGTCACGTTAGCCGCATTGAAGGGCACAGGTCAGCAGCGGTAAGCATGTTCGC
ATTGTTGTGCAGCCATCACTGACACCCATCTCTAGAACTTCTTCGTTTTCCCAAAGGAAACTGCACGCAC
GGCACCACCTCCCTGGCCTCCCCACAGCCTCAGGGTCTGGTGACCTTCATCCCCAGGACCTACGCTTCTG
ACTCCAGGAGCCCTGCCTGGTGTCCACACCACACTAAGGTGCCCTGCCACGGGCTTCGAACCCAACCCTG
GTGCACTTGTTGAGCATTGGACGTTCCTGGCAGGTCAGACCGTGAGCTCCTCTGCTCTGACGGATGCATG
GACGGCTGCCACCTGGCTGGACAGGTGCAGAGGGGAGGTGCCACCTTCTCTGGGGACAGGACTGGGGCAG
AACTCACCCGGCTGAAAAGTGCAGCTGTGGAGAACTTCAGAACTTCAGAAGCCTTTGCTGTGAATGCATT
TTCCTCCTGCCTCTTGGGCCAGATCTTAGGGATTTTTTTTTTTTTTAATGAAAATGTATAATCGTCAAAA
AACTTTAGATTTAAAAGCATGGAGCCGATGGCTATGCTTGGTGCGTTAATGGGAGGAAGAGGGGATCTTA
ACTTCAGGGAGGGCTCCTGGGGGAGCTGGGGGAGGCTGCTGTGTCCAGCAGGGCGGGCGGTGCCCCCCGG
AGCCCGGCACTCCGCGATGTGTGCGCTAAGCCGAGGCCGCCTTGAGCCCGTAGCAGCGCCGAGCGCGATT
CTTTCGTGTCTGCTTCCGGGAGGGTGGAAGGGTGAAGCTGTTGAGAGTGGGAAGGGAGGGGCTGTGCTTC
TTGAGTTTGCCGTGTGCCCCTGACCCTTTCAGGTGCAAGCGTCAGCTCCACGGTGCCAATGGGGAGATGG
GTCTGGAGGCCCCACCAGGCTGTGAAAGGCCCGGCCTCCTGTGCTGTCCGTGGAGGTCGGGGTCTCCCTC
TGCCCTGCGCCTCCTCTAACTTGCCCTGCGTTTTCCGTGCCCCTGGTCTAGGGTGAGGCTCGGGTCTTGG
GGACTGAGGGGCAGGTGGGTGTCAAGGTGAGAGCTGGGCTGGGCTGGGCCTCCCTCCAGAAGACTTCCCG
GGGCTCCTTTGGCACCAAGGATGGGGCAGATGGTGGCACGCATGGCCTCCCCTGGGCACAGAGCCAGACT
ATGGAGGGATCTCGTTGGCTACATCCTGGCTGGGGCTTTGGGGTCATGGTGGGAGCAGCAGCCAGGGGTC
TCGCTTGGACACGGGTCTGAGCTCACTGTGGCCTCCTTCCTCAGCCTCCTGACCGGGGTCCTGAGGCTGG
AGGGTGGGTGGAGGGTGGAGGGTGGGTGGATAGTGGGCTGGAGGATGGGCTGCTGGCGCCCCTTAGGCAG
CATGAGGCGCCTTTCCAGTCCCAGTGTCCTCCCTGGTAGCCCCGGAAAGGCTGAGTGGTCCCTCAGGTCC
CCTGCGAGGAGACGACATCAGGCCCCACCCCTCATCTCCTGACAAACCTCACATTCCTAGGAGGAAGCAG
GCCTGGCTAGCACGTGGCAGGCACAGCCTCCCTGACTCCAGACCTTATCAGATCAGTGCAAGTGCCAGGG
AGACCCCGGGTGGGAGCTGTGTGGTTGGGACTGATGCAGGCACTGGCCGGACTCTCAGGATGAGGGTGGG
AGGCCAGGCCCCGGGACCTTCTGGGACAGGCAGGAGGGCAGACACGGGACATGGTGTGGGGGCTGTGGCG
AGTGCGGCCACGCAGAGCATTTGGGCTGAAGGCGCGCTGTGTCCCACTGCCCCATACGTGTGCGGGGGCT
CCCAGACACGTGCTGGGGGCAGGAATCATAGCATTTTTTTGTTTGATTGTTTTTTGAGATGGAGTCTCGC
TCTGTCGCCCAGGCTGGAGTGCAGTGGCTCAATCTCGGCTCACTGCAACCTCCACCTCCCGGGTTCAAGC
GATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGAATTACAGGTGCCCGCCACCACACCCAGCTAATTTTTG
TATTTTTTTGTCAAGACAGGGATTCACCATATTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGGTGATC
CTCCTGCTGCAGCCTCCCAAACTGCTGGGATTACAGGCGTGAGCCACCACACCCAGCCAAGAATAGCATT
TTCTTTCTTTCTCTTTTTGAGGCGGAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCAGTGGTGCCATCTCA
GTGTAACCTCCGCCTCCCGGTTTCAAGCAATTCTTCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGT
GCCCGCCACCATGCCCGGCTAATTTTTGTATTTTCGGTGGAGACAGGGTTTCACCACGTTGGCCAGGCTG
GTCTTGAACTCCTGACCTCAAGTGATCCGCCCGCCTTGGCCTCCCAAAGTGCTGGGATGACAGGTGTGAA
CCACTGCACCCAGCCCAAGAATAGCATTTTTAAAGGAAAAAAAGCAAGGTAGAAAGATGGGTCAAAACCC
CAGCGCCCCGCTGGGAGGCTGGTGCCCGTGTCGTGGCGTGTGTGAGATATGTACGGAAGCGCGAGGCGGC
TTCCACGGCTCTGGCTCAGGGCAGCACTCACAGGGCACACGGCCTCGGCGGTGTGACGAGCTGGGCCGTG
GGTGCGCTGTCCTCTGGGGTGGGGCTGGCCATCCTCTGTTGGCGACCTAGTTAACGCCCATCTCCCCGCA
GCCTGCCTGCTCCGCTTCAGCGGACTCTCGCTGGTCTACCTGCTCTTCCTGCTGCTGCTGCCCTGGTTCC
CCGGCCCCACCCGATGCGGCCTCCAAGGTAAGGCCAGGGGACCCTGCCCACGCTCTCAGGGTGGGAGGGG
GTGGGCATTCGTTTGGGGCCAAAATTCGTACAGCTTTCCTCACCTTTAACAGCCCGGGGTGGAAGATGAG
GGGTCCCGAAATAAAATTTTTTTTTTTTTTTGAGACGGAGTCTCGCTCAGTCGCCCAGGCTGGAGTGCAG
TGGCGCGATCTCTGCTCACTGCAGGCTTCGCCTCCCAGGTTCACGCCATTCTCCTGCCTCAGCCTCCCGG
GTAGCTGGGACTACAGGCGCCCGCCACCACGCCTGGCTTATTTTTTTTTGTATTTTTAGTAGAGACGGGG
TTTCACCGTGATAGCCAGGATGGTCTCGATCTCCTGACCTTGTGATCCGCCCGCCTCGGCCTCCCAAAGT
GCTGGGATTACAGACGTGAGCCACCGCGCCCGGCTTTTTTTTTTTTTTTTTTTTTTTTGAGACCGAGTCT
CGCTCTATCTCCCAGGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAACCTCCGCATCCCAGGTTCA
AGTAATCCTCGCATCCCAGCCTCCCAAGTAGCTGCAATTACAGGTGTGAGCCACCACGCCCAGCCATCAA
AGTTTCTTATTTTCATTGTAGACCCCAAGCTAAGCTCAGAATCTTTTCTGATCCGTGTTGCAGGGGCTGT
GAGGGGCGAGGAGGGTCATGGGGGTGGAGTCTCTGTGTCCCTTGGGAGGCCACAGGGAGTGGATGGGCCC
CAACTGCCCACTCTCTGCAGGCTCCAGGCACGTTTCCTGCCTGCTGAGGCTCTGGCAGCCTGGGAGTTCC
AGCCGCCTGGACCGCCCTCCCGCTGCCCGCTGTGGGGTTGGACCTCAGTTGACTTTGATCCACTTGTGCG
GGGGGGGGGGGGAGCTGAGACCCCCCTTGGGAACCACCTTGGGGTCTGAGCTGGGCCAGGACTGTGTTGC
TCTCCCGTATCCTCCAAACAGAGGGCGGTGAGGCCACGTGCCAAGCCTGCAGCCTGGGTGACGAGGGTAG
CAGCAAACCCTCCATCCTGTGACATGTCACCTACCTGCCCTGTGGGGGCCATGTCCATTTTGTGCCCACG
TTTTAGGAGAGCGCGTTTTGGAGCAAACACTCAGAGACCTGTCCCCAAAGGCCATGGGCCAAACGTGGGT
GGGGCCGGGCCCTCCGCTGGTCTTTGGGTTTGGAGGGTGAAGAGCCACTGAAAGGAAGGAAGGTCCCCAG
GGAGGGCAAGTGTAGGCAGCAAGAGACCTTCCCCAGCAGAGGGCAGAGAGGAGGCCAAGCAGGACCAGGG GGCGTGACTGTGGAGACAGGCTCTTGGGTGCCGCCCTTCTGGGAGGTGCTGGACGCATCAGGCTGGCAGC
GTCGGGGCCTGTCCATGGCTACGAGGTGTCCACCGGGCTGCGTTTTTCCAGGGGACTCTGGGGAGGATCT
GCTTCCTCTTCCAGCTGCTCCAGGTGCCTACCTTCCTGGGCTTGGGGCCCCTCCCTCCATCTCCAGAGCC
GGTGGGGTGGGGTCAGGATCACACCCTGTCTCTCCTGCTCCCTCTCCCCTGCGGTAACCCTGTGGCTTCG
TCCCATCTGCCTCCCCGGCTAATCCAGGCCGACTTCCCTGGTTTAAGGTCAGCTGTTGGGCAGCCCCCAT
TTCCTCTGCTGCCTGGACTCGCCTCTGCATGTGAGCTTCTGCGTTCACAGATTCTGGGGACTGGGGTGCA
GGCATCATTGGGGTCCATCGTTCTGCCAGCTGCAGGTGGAAGGTCGCTCTCCGGCCTCGCGACATCTGGC
TGGAAGCATCCCAGAGGCTCCTCCTAGCTCGTGGTGCTGTGGGGTGGGCAATGGTCTTTTTTATTTATTT
TTTTTGAGATGGAGTCTCACTCTGTCACCCAGGCTGGAGTGCAATGGCACGATCTGGGCTCACTGCAACC
TCCGCCTCGCGGGTTCAAGCGATTCTCCCGCCTCGGCCTCCCGAGTAGCTGGGATTACAGGCGCCCACCA
TCATGCCTGGCTAATTTTTGTGTTTTTGTAGAGACAGGGGTTTTACCATGTTGGCCAGGCTGGTCTTGAA
CTCCTGACCTCAGGTGATCTGCCCACCTCGGCCGAGACTACAGGCATGAGCTACCGTGCTTGGCTTTTTT
TTTTTTTTTCGAGTGGGGGTCTTGCTTGGTTGCCCAGGCTGGAGAGCAGTGGCATGATCATGGCTCACTG
CAGCCTCCACCTCCTGGGCTCAAGCGATCCTCCTGCCTTAGCCTACCAAAGTGCTGAGATTACAGGTGTG
AGTCACTGTGCCCAGCGCAGGCACTGATCTTGGATCATGGGTAGAATTCAGGGGTGCAGAGAGGCATTTT
GGGAGGGGCCCAGTGCGGCCGAGTGCTGGGAGTCGCTGGGGCCCGAGGCCCAAACCCCTCCAGGATGCAT
GGCTGGGGTGGGTGGGCACCCCCACTCCCGGTCCCTATCCTGGGCCTCCCCTTTGCTCTGTGGAGCCGGT
TCTGTCTGTTCCCAGGCCCCTGGCGTTTCGCCGTTTGTTCCGTAAATATTTCATGCCCAGGGCGCAATTG
GAAACACTTTCCTTTAATCAGCAGTGGGGGAAGGCAGGCGCCCAGCCAGGCCAGGGGAGGAGCTGGGGTG
GGAAGATTTGGAGAGGACCCGGGAGGACTTCCCTGCCTGAGCCTCGTCAGAGGCCCTTCAGAGACGGAGA
TGCTGCCCAGTTTTCCGGGAGGGAGAGAGGAAAGTGTGAGGCTTGCCCGAGCCGAGTGCCCGGGGCTTTA
TGATTTGTGCAGCTGCTGGGCTTGGCGTGGCCCTGGTGGAAGCTCTGACGCCATCTGAGCCTTGGTCCCC
TTGTAGGCGTCAGCCTGATCCTTGGGGGGCTGGAGGTTGAGCTCAGACTGAACAGGGAGGAGAGGGGTAG
GGGCTGGGGCTGGGGCAGAGGGAACAGACCCTGGTGCTCCAGGCAGGGCTGCAGGCAGAGCCACAGGGGG
TGGCTCCCAGAGACCTGCTTGTCATTTAGGGACTCAGAAGCCCCATCCTGCCTTAGGATAAAACCCACCT
CCAAGACTGACCCTGCTGTCTGGAGAGAGATCACCCCCCACCTCTTACCATGCCCCGTGCAGGGAGGCCC
TGCCACCCTCTCCAGCTTCACTGGTCCCCCGGCTCTGCCAGCCTTGTGTAGTCTCCATCCTCTGTCTCAA
AAGGCCCCTCTCGTGGCCCTACCCCGCAGCTCAGCCCTAGCTGTCGCTGGGTGGGCCTCATGGCTCAGCC
TCAAGGCACCCTGGGTCCCTTGGCTGGTCCCATGCACACCCCATGGATCAGGTTCCAGGGGGCTCCCATG
CACACCCCACGGATCAGACTCAGGGCAACTCCACACTCCCCACAGAACAATCAGGCCCAGCCCTTGTCCG
GCAGGTGTGGGGATGGTGGCTATGGACCACGCATCAGGATGCTGCCGTGGTGGGGCCCCTCAGCCTTTGC
AGGCCGTGCCGGGGTCCAAGGGCTCCGGCTCTGGGGGAGGTCCATGCTCTGTCAACCATCCGGTCTGCAA
TACCCTGGGGCCTCCCCGCCCCTTCCTGTTTCCTCCTGAGAGCCCTGCAAGCTGGAGGGGGAGCCCCGGC
CCCTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTTGTTCTGTTGCCCAGGCTGGAGTGCAGTGGCGCGAT
CTCAACTCACTGCAACCTCCGCCTCCTGGGTTTAAGTGAAAGTGATTCTCCTGCCTCAGCCTCCTGAGTA
GCTGAGATTACAGGTGCCCATCACCACGCCTGGCTAATTTTTGTATTTTCAGTAGAAACAGGGTTTCACC
ATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTGAGGTGATCCACCCACCTCGGCCTCCCAAAGGTGTGA
ACCACCACGCCAGGCCCCCTGCACCTTCTTCTGGGTGGGCAAGAGCCATGCTGAGCCCCTCCCCTGCCAT
GGATAGGGGTGGCAAGAATAGTCCATTCCCCATGGGCTCCCTCCGGCAGGCAGCGGATGGGTTGGGGCCC
ACACAGTGGTTCAGGAAAGGCTGAAGGGCGGTTGCCGGCCAGAGCTGGGCGGGTCCCTGGCCTGAGCCTC
CTCTCACTCCCTTTCCCAGGATGGAAAGGCCACTGTGAGCTGGGTGTGTCCTGCCAGGAGGGAGTTGGAG
TCGGGGGCAGGAGACCCACTGGGTCTCCTGGGCTCCTGGAAGAGGCAGCAGTGGGTCCCTGCGGAGGTGG
CCTGATCCCCGGCTCAGCCTGGTTTTCCCAGCTTTGCATTTGGGGGTGGGACCTGCAGGGGGAGAGTGAC
GGGGTGGGCGGGGACAGGGGCTGCAGGTGGAGGCACGAGAAAGCCACCCGACCCCTTGAGGTCATGCTGT
TTGCGTCGGAGAAGAGGGCCGGTGTCAGGGTGCCCCCTCGGCCTCTGTGTGCACCTCCCCGCTCCCCGCC
CCGTGCAGGTCAGCAGAGCCTCGATCCCTCTGCACGTTCCGGCCCCCTCCCATCTCCAGCAGCGTCTCTT
CCCGGGGTGCCCCTCGGACTCTCAGCTCCCTAGTCTGATCCAGCCCAGGCAGGGAGCGGGCGTGCCACGT
GGCCAGGAGTGGCCTCCAGGCCCAGCTGCGGCCCCTTCGACCCTGCGGAGGGATCATGGCCCAGCTGTTC
TAACATGGCCAGACCAGGACACTCTGGCCGGCCCGAGAACTGAACCGGGAAGGAGGCAGGGAAAGGGGAG
GGAGGAAGACAATGGGGAAGCAGTGACATCCGAGATGTAGCCAGAGACGGACATCCTGGACTGTCGTGCA
GGGCAAGGCGGGTGGGCGGGAGCCAGGTGGCCTGAGAGCCCCTCCCTGCCCAGGGGTCTCTGGTCAGGCA
GTTCCTTCCCGGGCTGCTGGATCGTGTGTGCAGGGAACCCGTCAGCCTGGCTGCCCAAGGGCCCAGATGT
CCTCTCAGTGCCCGGGGGTCCTTGACAGCCCCAGCAGGAGCCCCACGTGCCTGTGGGGCAGGCCCCGCAG
GTCCCTCTCTGTAGGACTCAGAATACCTTCTCCAATGCCACGTGCTCTCCCTGAGTGCCCAGTGCCACAG
AGGGCCCCGCTGGCGAGGTTACTTCAACGTCAGGCTAGAGGGTGCAGCGAGCAGGACTCACAGCCCAGGT
TCCCAGGCAGGTGTGGCAGGAGCCCATTCCCTGGGTCGGTGTTCTCATGTCACCCCACGGTGACCCTGCT
GTACAGAGGCGGGGGCGCGGGGCCCCTGGCTGGTGCCCTCCTGCTGCCAGACTTGTGCTCTGCTGAAGAG
GGGCTGGCGCGGCAGGTATGAGCCCGGCACGGAGGTCGTGAGCAGTGAGAAGCCTGGCACCTATGGGTTC
GGGGCAGGGAGGCCCTGGAAGGTCCTGTCCTCCGGGAGCCCTGCACAGCACCCCCTGGTGGTGGCTCCTG TTCGTGTCGGGGCTGCAGCCTCCCTTCCTGGACGGTTTTCCATTCCTCCTTCCCACTCTCCCTGCTGCTC
ACCCCTCCCATCCTCCACAGGTCAGAGGTCATAGCTGCAGGGTCAGCAGGTCAGGGCCTGGGGCAACCCT
AGGAAGGTGTGAGTGTGAGAAGCTGGTCCAAACCTGCCTCGGCTGCCTGGTGGTGCGGACAGGGAGTCCT
GGGCATCCGTGAGGGCTGCTTCACGGTCAGGCTTAGGGATGTGCAGGGTGACTTGGACGTGGGTCATGAG
TCTTTGCTCCAGAAAGAGGGGGCTGAGTGTCCAAGGCCAATCCCGAGTCTGTCACCTAACACCATTTGTG
CTCAAAAAACTGAACAGAGTGGACACAGGCCCTGAGTTTGCGCCCCGAGGCCGAGAGAGGGCAGATGTGG
TGGTGACATTCACCACCTTGGACCCAGACCCAGATGCTACCTGTCCCTGAGCTCCAGGAAGTTGTGAGAA
GGGCCTGAGCTGTTCTGCACTTTCTCGTGGCCGGGCGTGGCTGGTGGTGCAGGAGTTGCTGCCCCAGGGT
GAGGGCCCGGAAGCTCCTGCCAGCACGTGCCGGGGTGGAAAGGGAAGCTGTCCACAGCCCTGTCAGGACT
CAGAACCCGGTGGGTCAAGGACTTTGGTCCGGACCCCTGCTGAAGGGTGAGCTGTCCACATGTGCGCCGA
GAGCAGAGGTGAAGCCAGGGCTCCTGAGTGCCCCCAGCCACAGGGTGCGCCCGCCCAGCCCCTGCCCTGC
AGCCGAAGGCCTCCCTGCTGGGGGGCTGAGTCCAGTGGGGCCACAGGCAGCTGGGAGCAGGACAAGGCTG
CCAGGCAACCAGATGGTGCTGCCGCTTCCTGCCAGGTGTGGGTGCACAGAGAGAGAGAGGATGCCGGTCT
GGGGCCTGACCTGGTGCACAGCAGGTGCCTGAAATGCCAGGGTGGCCATGGGGACTGGGTACCATGCATA
GGCCATGCATCGGGATGCAACTTCTCCTTGCAGCCCCTCAGCCCCAGGGAGGCAGCTGCCTGCCCCACTT
TTCTCCAGAGCCATCATGGCCCTGCTCCCACCCCAGCCACGGCTGCTCAGGGGCGCTCCGCATGCTCTGG
TCTCCATCCACCTGCAGCCCCCACTGGGGTGAGGTGGAGCTTCTTGCCTCTCCTTGTGTCTATTTCCTCT
GCTTCCACAACTGAACGGTGACAGGTATTTGCTGGATGAGGGAGCACACCCCAGGTGGTTTCCTCTGAGC
CTGGGAGGCCTTTTCCTGCCTGTGGGCCCCAGGCCCATCCTGCTGCCACCCCCAGGAGGATGCCCGGCTC
CTTGTGACAAGAGTGACCCTCGGGAGGCGTGGGGAGTGGGGCTGGCCGGCCTGCCTGATGGGGTCCTGAG
TCCATGGCGGGTTTGCATCTCAGGCCTCTGGGCTCTGGCCGGGCTGGGGGCTATTGTCCGGCTGAGCGGC
CTGGGCTGCGGCCCCTCCCCGTCCCCGGGACCAGCCTCACCCACTCGCTCTGCCGCAGGTCACACAGGCC
GCCTCCTGCGGGCATTGCTGGGCCTCAGCCTGCTCTTCCTGGTGGCCCATCTCGCCCTCCAGATCTGCCT
GCATATTGTGCCCCGCCTGGACCAGCTCCTGGGACCCAGCTGTGAGTCGCTGAGGGGGCGGGGTAGGGAT
AGCCATCCTGGGGGTCAGGGAGAGGGCCCTGCAGTGACCCCGAGTCTCCTGGGGGGGTTGACTCAGCCTG
ATTTATGTCTGGCCTGGATGGTCCAGGTGAAACGCTCCAGGGATGACCAGGCCACGGTGCTGGCTGGGCA
GAGCCTGACCTGGGTTCCCCCGTCTTTCTCTGCAGGCAGCCGCTGGGAGACCCTCTCGCGACACATAGGG
GTCACAAGGTAAGACCATTCCTCCCACCCCCAACCAGCAAGCCTCCCTTGGGGATTTCAGGCCCCAGGAA
GTGGGGGGACCCAGGAGGGACAGAGGGGGACCTGGAGACTCATCCACACTCCCACCCACACCTGGAGACC
CATCCACGCTCCCACCCACACCTGGAGACCCATCCACACTCTCACCCGCACCTGGAGACCCATCCACACT
CCCACCCGCACCTGGAGACCCATCCACACTCCCACCCGCACCTGCAGACCCATCCACACTCTCACCCGCA
CCTGGAGACCCATCCACACTCCCACCCGCACCTGGAGACCCATCCACACTCCCACCCGCACCTGGAGACC
CATCCACACTCCCACCCGCACCTGGAGACCCATCCACACTCACACCCACACCTGTCCTCTTGGTCTGACC
GCGGCTGCTCCCTGCTCTCCGCAGGCTGGACCTGAAGGACATCCCCAACGCCATCCGGCTGGTGGCCCCT
GACCTGGGCATCTTGGTGGTCTCCTCTGTCTGCCTCGGCATCTGCGGGCGCCTTGCAAGGAACACCCGGC
AGAGCCCACATCCACGGGAGCTGGTGAGGGCAGCTGCGTCACCCGTGTGTCAGGGAGGTCATTCGAGAGC
TGTGGTCTCAGCCATTTTGAGGGTTATTTTAATCTTTTTAAAACAGATGTAGACGTTTTGGTTGTAAGTT
GGTGTTAAAGAGAGGAGGAAGTTCCAAATCCCACCCCGGGGCCCAGCCTGCAGTTCCATCCGTTCAGACC
TGTTTCTACTCGGGCTCTGCCTCTAGTCAGAAACCTCCACGCCCCGACATGGCATCTGTGCCCTTAGGAA
CTCTTCACAGGGGAATTATTTGGGGCCACGCGGTGGTGGAAACCTGCAGTGCTGGGCAGTGGGTCTGGCT
GGAGAGCCACTGCAGAAGGGCTGAGAAGGGGCGGCCCCAGCAGGCCCCATGCACTTACAGGCAAACAGGC
TGTCGGCCCAGAGCCCCAGCAGGGGCCTGGACCCCAGGAGCGACGGGCCTGAAGCAGGGCCTCTGTCCTC
GGAGTGGGAGGCAGAGTGAACTTTAGCTGCTACAAGACTTGGAGGTCCGGCCCCGGGAATCTCTGAGCTA
TGGCCCCCTCACACGGACAGGGCATGAGCTGGGGCCTGCGACACCCAACGTTGACTGCTCAGACCCTCTC
GTCCCTGCCTGGGCCCACACTTGCCATCCCCAGGCTCAGCCAGGATTTATGGCCACCTGGGTGTCCTGGC
CCACGAACTCCCTGCCCCGGGCACCCAGCCTCCTCCCACCTCACCCTCCCAGTGCCCACTCGTCAGATAG
CTGATGCTCGCCACGCACATGGGCCTCAACTTCTAGAGGAGTCCCGAGAGGGAAAGGGGTTCCCCAAGCC
ACACGGTAGGTCGAGTACACCTGCTCAGCCTGCAGAGGCGGCCTGCCCTCCTGCCTGTCCTGTCGCCACC
CCATGGGGCAAGGCTGGTGCCCGCATTTCAGAAGTGGGAACGGGGACCTGGAGGTCAAGTGGCTTTTCCG
GGCATTTGAGGGGGTCCAGCGCTCATGAGGACTGTGCTGCTGCCTGTCCTCCAAGAGACCCGCCCCTCCC
TCGTCAGCCCCTCCATGCTGATGGGGGCATGGGGCATGAGCTTCCTAGAGTTTGGCTGCTGGGAAGGGGC
TGGGTGGCTGGACTCTGGTCATCTTCATCTTGGTGTGATGGGAGCCTGAGTGTGCAGCTGCGCCCTGGGC
GGCCAGCGTGGACAGACTTCAGAGTGAGCTGGGGGCACCAGGCACCTGCAGGATACCGTTCCGAAAGATC
TAGAAGCCCCATGACCCGGCCTGGCAGGGCCCCCGGGCGACCCTCACTCCCACATATGGGCAACACACAC
ACGTGTTTGCAGGCATGCGACGGAGTGTGCCAGCCAGCCCGAGGGGTGCTGGCCGCTAGAGGAGGGTGAG
GGGCGTCTGCAGCATGGCTGCGGCACGTGGGGCCCAAGTCAGGGCCGGGCCTGCCCCTCGGTGGCCTCAC
ACCCCAAGTCAGGGCCGGGCCTGCCCCTCGGTGGCCTCATACCCCAAGTCAGGGCCGGGCCTGCCCCTCG
GTGGCCTCACACCTGTTCTCTCACAGCAGCTGCCTCTGTTCTTTCCACTCCCAGAAATGCTCATTTTTCA
CGGCTCTGCTCAGAAGATTTCCCAGGGCCAGGGCCAGGGCCAGGCTGTGGGTGGGGTGAGCGATGGCGCC CTGCGTTCTGATCCAGCCTCGCCCCCAGCTGCTGTGTGGTCTCTGAGAGGGGTGGGCTCAGGGCCTGGGC CTCTCCTGCCCTGATCTCCCTGTTACCCTGGCCTCGGAGCCTCCCAGCAGCCCCTGCAGCCCTGGAGGCC TGTGAGGACAGGACTGGTCTGGTTCATCTGTCCCGTCAAGCCCCCATCCCATTTGACAGAAGGGGAATAG GCTCCGAGAGGCTGTGACTTGCCTCGGCCTGCAGAGCACGGAGACTGTCAGAGCTGGGGTGGGGCCTATC GTCCAGCCCCACAGCCTGCGGTCTCAGCCCGGGCACCTGGCGCCTCAGCCACCAGGAGAGCCCCCGAGTC ACAGATGGGGACACTGGGCCGGTGGCAGCCAGGCCAGAGCCAGGGCATCTGACGACAGCCGTGTTCTTTC CACCACACCTGGCTTTCCCTGGTGTTTGGGAAATGGCTGTGTTCTGGGAAATCTTTAGTCAGGTCCAGGG AAAACAGGCCCGGCAGGTCTCTCTCCCCAGCCCCAGGCCGGCCTGTCACTTCTTCTAGGCAGCTGGCCAC CTCTGTCCCCCCAGAGGCTTGGGGAGGGCAGGAGTACCACCCCTATCTTCCAGGCAGAGCCACCCAGAAG GCTGGCAGGGGTACACAGGAGACCCTGGAGCCTGGCCATGTCCCTCAGGCCCCCTTGGTCTCTAGAGACC CCCCGGGGTATAGACAGGGCCCCGCTGCTCCCTGGGTGCGTGGTTCGGGGAGATGAGGTGGTATAGGACA GTCTGTGGTCTGTCTGTACCTGGCAAGGTCATCACGTGCCTGGGCTTGGCAGGACAGACCCTGGGTCTTC GGCCAGGGTGGGAGCTGCTACCAGGAAGGCCTGCAGGAACTGTGAGCTTGAGTGAGGAAGTAGGAAGGTG TCAGGCAGACCTCAGGGACGGCTGGGGCCTGTGCCCGGGGAGGCTGTCCTGTGGCCCTCAGAGGAGCAGC TGTGGATGTGGCCGCCTCCCACGCTCCTGGCTGGGCAGGTGTGGGCTGGAGAGGTGGCGTCAGTGCGATA CACCTGACCTTGCCCCTGTCCGTGACCTTGGCAGGATGATGATGAGAGGGATGTGGATGCCAGCCCGACG GCAGGGCTGCAGGAAGCAGCAACGCTGGCCCCTACACGGAGGTCACGGCTGGCCGCTCGTTTCCGAGTCA CGGCCCACTGGCTGCTGGTGGCGGCTGGGCGGGTCCTGGCCGTAACACTGCTTGCACTGGCAGGTACGCA CCGAGGCAGGGGGCACTGGCAGTCACACTGGGAGGGGTCTTGGGAGTTCCCTGATGACTGTGGAGACAGC GGGACACATGGCACTGGCCAGGTACCACCCTGTGTGCCCCTGCCCCGCAGGCATCGCCCACCCCTCGGCC CTCTCCAGTGTCTACCTGCTGCTCTTCCTGGCCCTCTGCACCTGGTGGGCCTGCCACTTTCCCATCAGCA CTCGGGGCTTCAGCAGACTCTGCGTCGCGGTGGGGTGCTTCGGCGCCGGCCATCTCATCTGCCTCTACTG CTACCAGATGCCCTTGGCACAGGCTCTGCTCCCGCCTGCCGGCATCTGGGCTAGGTAACGGCTTGCCACA CAGCCCCTTTTTCCTGCCACCCTGGTCCCGCCCACCTGGCTCGTCTAGCCCCTGTGGCCCCACTGCCTCT GGGGTGGTAGGCTGTGACGGGTCTTCTCTGGACAGGGTGCTGGGTCTCAAGGACTTCGTGGGTCCCACCA ACTGCTCCAGCCCCCACGCGCTGGTCCTCAACACCGGCCTGGACTGGCCTGTGTATGCCAGCCCCGGCGT CCTCCTGCTGCTGTGCTACGCCACGGCCTCTCTGCGCAAGCTCCGCGCGTACCGCCCCTCCGGCCAGGTG AGCACCTGCCACCCATGGTGGGTGGGCTGAGGCCAGGCCATGGGGCTGGTCTCAGGACCTCCTGCCTCTG GGTGGGGTGTGGAGCTGGTTTGGGCTCAAGACGCTGGTCTCTGCAGAGGAAGGAGGCGGCAAAGGGGTAT GAGGCTCGGGAGCTGGAGCTAGCAGAGCTGGACCAGTGGCCCCAGGAACGGGAGTCTGACCAGGTGAGCA GCCAGGCAGGTGGAGACGCCAGCGTGGGGGGCGCCCGGCCAGCCCGTGCATGGCTCAGCGCTGCTTGCCC ACAGCACGTGGTGCCCACAGCACCCGACACCGAGGCTGATAACTGCATCGTGCACGAGCTGACCGGCCAG AGCTCCGTCCTGCGGCGGCCTGGTGAGTACCGCACACTGCAAGGTATGGCTGGGTGCGGGGGGCGGGGCG GAGGCCGGTGCTGCCCCCTGGTGGCCGCCTGGCGCTCTCGCATGCTCGCGCCGCACCTCTGCCTGCCGCC CCCTCGGGGGCCCAGGACATCCACGGGTCGGTGTCAGTGACCCCCGAGACCCCCAGGGCAGCCGAGTGGC CATGTCACTGACCAACCCCCAAGACCCCCAGGGCAGCTGAGTGGCCGCGTCGTTGATCCCCAAGACCCCA TGGGGGGCCTCCAGGTCCCCCAACCCCTCCCCAGAGAATGTGGCTATGCTGTCTTGTGCTGTTAGCTCTG GGAGCTGCTCCAGGTGGCCCAGTGGCCCCAGGAGGCCGCTCGTCCAGGGCAGGGGCTGGCCTGGGAACTC TGTGTTGGCCACGTCGCCTTGGGAGGGCCTGGGGGCTCTTTCTGGCTACTTTCTTTCTTTACCCTAACCC TTGATTTTCCATTTTGCAATGTGTTTCTGAATGAAGCAAATGAAGCCACGGCCCTGGGGTGGGGGTCCTG AGAGTCTTCAGGTGCGCAGAGCTGGAAAGGGGGTCAGGGCCACCTTTCCCACCCTTTCAAGGAAAGTGAG GCCCAGAGAACGGCAGGTGCTGGCAGGGCCATCCCTGACGCTCAGGGACGGTGTCAGCCCAATTGCCGGA GCCCTCGTGTTCTGCCCATAGCCCACCGGGGGCCTGTCTCTCCTGCTGTGTGCTTGCCCAGGGCCCAGAT TTTAGGGCATAGTCAGGGTGGGGAGGCCTGCAGATCAACCTGCCGAAGCTGACCGCTGTCCCCACCTGCA GTGCGGCCCAAGCGGGCTGAGCCCAGGGAGGCGTCTCCGCTCCACAGCCTGGGCCACCTCATCATGGACC AGAGCTATGTGTGCGCGCTCATTGCCATGATGGTAGGCGGCTGTGGGGGTTGGGGTGGGCGGCCCCCTCT GCCGCGCAGGTGTGGGGCATCGCCTGGGTGGGGTGCGCTGGGCAGCTGTGCAGCCCCCTCTGCCGCGCAG GTGTGGGGCATCGCCTGGGTGGGGTGCGCTGGGCAGCTGTGCAGCCCCCTCTGCCGCGCAGGTGTGGGGC ATCGCCTGGGTGGGGTGCGCTGGGCAGCTGTGCAGCCCTCTCTGCCGCGCAGGTATGGAGCATCACCTAC CACAGCTGGCTGACCTTCGTACTGCTGCTCTGGGCCTGCCTCATCTGGACGGTGCGCAGCCGCCACCAAC TGGCCATGCTGTGCTCGCCCTGCATCCTGCTGTATGGGATGACGCTGTGCTGCCTACGCTACGTGTGGGC CATGGACCTGCGCCCTGAGCTGCCCACCACCCTGGGCCCCGTCAGCCTGCGCCAGCTGGGGCTGGAGCAC ACCCGCTACCCCTGTCTGGACCTTGGTGCCATGGTGAGTGTGCACCACCACATCCGGGGGTGCCTGGGTG CGCAGACCCATCAGGGTTGTCGTCCTGTTCAATGTCCACTTGCCCGGGGGAGTGGCAGCGCCAAGAAGGC AGATGTGTCTGTCTGTCCCCTTCTGCCCACCCAGAGCCAGCCCAGAGTAGCTTCTCAGTGAGCGTTTGTT GACTGAATAAACAGACAACCTTGTGTTGGCACGGGCACCACCCCTGTGCCCTGACACTGTGTGAGCGTGG GCTCTGTTGGCACGGGCACCACCCCTGTGCCCCGACACTGTGTGTGAGCATGGGCTATGCCCATTGGCAC AGGCACCACCCCTGCGCCCCTGACAGTGTGTGTGAGCGTGGGCTCTGCCCATTGGCACCATGCACAGCCC TGGGTCTCAGTGACAAGCTGTGCAGGCCATGTGTTCACAGGGTGCCTGCGTGTCCATGTGAAACGGGTGC CAGCATCGTGTCTGGACACCTGTTTGCAGGCCAGTGGGTCTCATCTTGTGAAACTTGTGAGCCTGTGTGC
CAACATATGCACCTGTGAGCTTGTGTGCATATGTGAGCACGCATGTGGGCAAACATGCACTCCAGCATGT
GGACATGTGTGCAGGTGCGTGCATGGATCTGTGCCCACGTGAACTAGTGAACCCGTGTGTGACTCTGCGT
GTGAGCGCAAGTGAACCCATGCACTCATCCATGGATGTGAGAGTGTGTGCTCGTGTGCCTCTGAGTGGGT
GTGAGCGAGAGGGTGTTCGGTGCCTGTGGGGAGGCTGCGGTGGATGGGCTGGTGCCAGCCGCCTGAGAGC
TCTTGCCCCCTGCTATAGGAGGGTGCTGGGTCCCCCGGCTGTGGGAGGGGTGCTGGGCCCCCCGGCTGAC
TGTGACACCCTGCGCTTGTCACAGTTGCTCTACACCCTGACCTTCTGGCTCCTGCTGCGCCAGTTTGTGA
AAGAGAAGCTGCTGAAGTGGGCAGAGTCTCCAGCTGCGCTGACGGAGGTCACCGTGGCAGACACAGGTGA
GTGGTGGGCCAGAGGCGGGGGTTGCCCTCCTGCCTGCCCGCCCTGATGCCATCGCCTGCCCCTGGCTTGG
CCCACAGAGCCCACGCGGACGCAGACGCTGTTGCAGAGCCTGGGGGAGCTGGTGAAGGGCGTGTACGCCA
AGTACTGGATCTATGTGTGTGCTGGCATGTTCATCGTGGTCAGCTTCGCCGGCCGCCTCGTGGTCTACAA
GATTGTCTACATGTTCCTCTTCCTGCTCTGCCTCACCCTCTTCCAGGTGGCTGGGGGGCCGGGATGGGGG
CTGGGGCACGGACCCTCCCCGCGGTCCTCACCACCCCCACCTCACCCGGCAGGTCTACTACAGCCTGTGG
CGGAAGCTGCTCAAGGCCTTCTGGTGGCTCGTGGTGGCCTACACCATGCTGGTCCTCATCGCCGTCTACA
CCTTCCAGTTCCAGGACTTCCCTGCCTACTGGCGCAACCTCACTGGCTTCACCGACGAGCAGTGAGTCCA
GGCTGGGGCGGTGGGGCAGGGGCGCCGAAACCCCGTGCACTTCCCCGGGGCTGCAGCGGCTCTGCCGGGG
GCCGGGCCGGTGCTGATGCTGCCCCTCCACAGGCTGGGGGACCTGGGCCTGGAGCAGTTCAGCGTGTCCG
AGCTCTTCTCCAGCATCCTGGTGCCCGGCTTCTTCCTCCTGGCCTGCATCCTGCAGCTGCACTACTTCCA
CAGGCCCTTCATGCAGCTCACCGACATGGAGCACGTGTCCCTGCCTGGCACGCGCCTCCCGCGCTGGGCT
CACAGGTGCGGCCCCGCCCTCCCTGTCCGGCCCTGGAGAGGTGTAGCCTCCTGGGCCAGGGAGGGAGCCA
GGTGGGAGTTGGACAGGAGCCACATCTTCCACCTTCAGATCCCAAGGGGCATTTGCTCATACCAAGGGGA
TGGCAGTAGCGTGGAGGTCACAGGGACAGTGGGCATGAGTTGCGACACAGCTGTGCACCTGAACTGGCAG
CTGCAGCAGAAGCGGTGCCGACAGGGCTTCTTCCAGCCCCAGGAAATGAGGGGCAGGAACCCAGTTGGGA
GATGACATTTTCGGACCCTCTCCCAGGCAGGATGCAGTGAGTGGGACCCCACTGCTGCGGGAGGAGCAGC
AGGAGCATCAGCAGCAGCAGCAGGAGGAGGAGGAGGAGGAGGAGGACTCCAGGGACGAGGGGCTGGGCGT
GGCCACTCCCCACCAGGCCACGCAGGTGCCTGAAGGTGGGTTGGGCGGGCAGAGCACAGCTGCCACCCAG
TCTGCTGTGCCATGTCCCAGCTCGGGGGGCGTTGGCAGAGTCCCCTCTGGGCTCCAGAGCCTCTTCCTCA
CAGGGGACCCGGGAATCCCCGTTTGTGCCCCGCACTGACCCTCACACCATCACAGGGGCAGCCAAGTGGG
GCCTGGTGGCTGAGCGGCTGCTGGAGCTGGCAGCCGGCTTCTCGGACGTCCTCTCACGCGTGCAGGTGTT
CCTGCGGCGGCTGCTGGAGCTTCACGTTTTCAAGCTGGTGGCCCTGTACACCGTCTGGGTGGCCCTGAAG
GAGGTGAGTGTGGCAGGCAACTCAGCTTCCCATCTGGGGTGGGGTCGCTCTGGCCTGCCCAGCTGGCCTC
CCCAAGCCCAGCCCCACGTGCCCACTGCCCTCCCCAAGCCCAGCCCCACGTGCCCACTGCCCTCAGGTGT
CGGTGATGAACCTGCTGCTGGTGGTGCTGTGGGCCTTCGCCCTGCCCTACCCACGCTTCCGGCCCATGGC
CTCCTGCCTGTCCACCGTGTGGACCTGCGTCATCATCGTGTGTAAGATGCTGTACCAGCTCAAGGTTGTC
AACCCCCAGGAGTATTCCAGCAACTGCACCGAGGTACCGGCCCCCGAGGGCTGGGACGGGAGGAAGCTCC
AGGCAACTCTGTATTCGCAGCCCGACCCTCCTGGGGCAGCTGCCTCAGTGCAGTGGGGCCAGCAATGGAG
ATGGAGGACTCTCCCCTGGGGGCGCCAAGGGGGCTTCCTGGAGGCAGCATCCTTCGACCTCAACTGTGGA
CCAGGGGCGCACTCCCTGCACACAAGGGTGTCCAGTAGGGGCGGAGTCCCAGGGTCTCCGGCAGTGAGGA
CGGGAGGGCCCCACCCCTGGACAGGGAGAGACAGTCAGGCATCTCTGCCTGGGACCTTCTCGCACATCCC
TCCTTCTCCCTGGACCTCTCTTCACTCCCCCAGCCCCTGCCCGTGGTCTCCCTGTTTCTCAAACACCTGG
TCCCCTTCCCCGTGAAGGTGGCTCCAAGGCTGGCAGCCCCCGTGTCCCTGGCTGGGGAGCAGTGGACCTG
CCCCAGAGCTGTGGCTGTGGTGGGCTCCGGGCAGGGCCAGGGGGCACTGTGGCCTGGGAGGGGGCACTGA
TGCCTGGCCTCTTGCCAGCCCTTCCCCAACAGCACCAACTTGCTGCCCACGGAGATCAGCCAGTCCCTGC
TGTACCGGGGGCCCGTGGACCCTGCCAACTGGTTTGGGGTGCGGAAAGGGTTCCCCAACCTGGGCTACAT
CCAGGTGAGTTGAAGGGCTGGTGGGCGGCTGGGCGGGCGAGTACCCGGCTGCCCCCTGACCCTTGCCCTC
CGCAGAACCACCTGCAAGTGCTGCTGCTGCTGGTATTCGAGGCCATCGTGTACCGGCGCCAGGAGCACTA
CCGCCGGCAGCACCAGCTGGCCCCGCTGCCTGCCCAGGCCGTGTTTGCCAGCGGCACCCGCCAGCAGCTG
GACCAGGATCTGCTCGGCTGCCTCAAGTACTTCATCAACTTCTTCTTCTACAAATTCGGGCTGGAGGTGA
GGCAAGGACATTGCCTCCCCCTGGGGCAGGGCTTGGCCTTCGGGAGGGAGGGACGGCTGCACCGTGCAGG
CACCGCAAGCCTGGCCCCACCTGGGTTTGCCTGGGCCACAGAGGGTGGGGGACTCAGGGCCAGGCACGGC
TTCCCTGGACTCCTGTGGTGTGTCGGTGCTGACAACAGGCAGGGGGCCAAGTTAGATCTGCTCTACTGTA
CAGCCCACCTCCTGGAGCCTCAGTTTCCCCTGCACGATGGCAACTGCCAGCCACTCCTGCCCTCTTGACA
GCGCCGCTGGCCCTGTCCTTGCTTGATGCCCGCAGCCTCCAGGCAGGGCTGCTGCAAGCCTGAGGCCTGC
TGGGTGGGACAAGAAAGTCCCTCCCCCCAGACTCAGTGCATCCCCACACCCCGCCCTCTCCCCTCCCCAG
ATCTGCTTCCTGATGGCCGTGAACGTGATCGGGCAGCGCATGAACTTTCTGGTGACCCTGCACGGTTGCT
GGCTGGTGGCCATCCTCACCCGCAGGCACCGCCAGGCCATTGCCCGCCTCTGGCCCAACTACTGCCTCTT
CCTGGCGCTGTTCCTGCTGTACCAGTACCTGCTGTGCCTGGGGATGCCCCCGGCCCTGTGCATTGGTGAG
GGGCACGTGGCTTGGGTGGGAGTGGGCTTTGTGGCTTTGTGGATGCCCGTGGGGGTGTTTCCCGCCTGCC
CCAGACTCCTGTCCACCCTCCTAGACTTAGCCTTGGCCTCCTCCAGTCCCTCCTCTCTGCTCCACATCCT ACCCAGGCGCCATCACCTGCATCCTGTCTCTCGGGGGCGGCCTGGCCCCCTCCAGGCTCTGCTGTTCTCT
TTTCCTTTTTTGCCCAGTATTCTATCTTGAAATATTTCAAACCTTCAGGAAAGTTGTGAGTCACACAAGG
AGCACTGCGTCCTCTCCGGGCCCCGTGCGGGTCGGCCGTGATGCCTCACACCCAAGTGCTGCCTCGAGCA
TGCGTCTCCTGGGTGAGGGTGTCTGAGCCCACAGCCCCACACCCGTGGTCCCGTCTCCTGGCAGTGCCAT
CGTCAAACGTGTCCTCTGCATTCAAACAGCCCCGGCCTCAGCACCCTTCTTTGTGGCCATTTGGTTTTCA
GACGGGATCTGGTCTGATGTTTGCTCTAGTCTCTTGTCTTGGGTCTAAGCCGCCCCCGCCTCTCCTGTCT
CTTGGGAAGTTCCGGAGGGAGGCCGGTAGCGTTGCTGACGCCGTGAGACTGGATTTGCGTGGCTGTCCTG
GTGCTGCAGGTCTGCTCAAGGCACACAGCACCCTGCGGTCTGAGATGGGGAGTCACATTTGTGCACGTGG
CCGGCTCAGGGGCGTCCCACCTGCCCCACAGTGGCACCCAGCCTGTTGGCACTGGTGGGCTGTGTGGGGT
CAGCCTTGTGGTTTTACGAAACAGACTTTCTCTCTGCTGTCTCTGTGTGTCTGTCAGCTGGGATTCCCAT
CAAGGACAGCTGCCATTTGTTACTGGCTGCTTTCCAAGAATTCTGTCATCCGCAGACCCTGGGCCTCCCC
TCTGCTGAGTGGGTCCTGGCCCCTCCGGCCACACACTGTTACATCATCTCCCCGTATTTGGCTGGGCATG
GTGGTTCACGCCTGTGATGCCAGCACTTTGGGAGGCTGAGGCAGGAGCATCCCTTGAGGCCAGGAGTTTG
AAACCACTCTGGTCAACATAGCAAGAACCCTTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTCTTGCTC
TGTCACCCAGGCTGGAGTACACTGGCACAATCTCGGCTCACTGCAGCCTCCGCCTCCCGGGTTCAAGCAA
TTCTCCTGTCTCAGCCTCCCTCGTGGCTAGGATTACAGGCAAGCACCACCAGGCCCGGCTAATTTTTGTA
TTTTTAGTAGAGACGGGGTTTCACCATCTTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGGTGATCCAC
CCGCCTCCCAAATTACAGGCCTCCCTCCTGGGATTACAGGCGTGAGCTGCCACGCCCGGCCCCGTCTTGT
TTTCTGCTCCCAGGCGCTGCTGCCTCATCTTCTGCTACCCAGGCCCAGCCTTGTGCTCACAGCCATTGCT
CCAGGGAGCCCAATCGAGTTCTAGGAGCGTGAGGTTTAGAGCCCGGGGTCTGGGCGCTGGGTGTGCCTGT
TGCTACAGGGCTGCCTCAGCCTCTGGGCCCTCCAGCTCTTCCTTGTTGAAACATCTGCTTTCGAGCATCA
CCGAGGCCAGCTCCCCGTCTCCTGTCCACCTCTTCCTTGTTGAAATACCTGCTATCAAGCGTCACCTAGG
CCAGCTCCCCTTCTTCTGCCTCCTTCCACGCGGCTGCGCCATGCAGTCGCCATCCTGTGAGATCAGCATG
TCCTGGGTTCCCCAACATCGAGGGTAACTTTGTTTTTGTATCGTGAGGTTCCCTCTGTGGCAGATGGGGC
TGTGGGTTCAGCATGTCCTGGGTTCCCCAGCATCGAGGGTCACTTTGTTTTTGGGTCGCGAGGTTCCCTC
TGTGGCAGATGGGGCTGTGGGTTCAGCATGTCCTGGGTTCCCCAGCATCGAGGGTCACTTTGTTTTTGTG
TCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGGGTTCAGCATGTCCTGGGTTCCCCAGCATCGAGGGT
CACTTTGTTTTTGGGTCGCGAGGTTCCCTCTGTGGCAGATGGGGGCTGTGGGTTCAGAATGTCCTGGGTT
CCCCAGCATCGAGGGTCACTTTGTTTTTGGGTCACGAGGTTCCCTCTGTGGCAGATGGGGCTGTGGGTTC
AGCATGTCCTGGGTTCCCCAGCATCGAGGGTCACTTTGTTTTTGTGTCGCGAGGTTCCCTCTGTGGCAGA
TGGGGCTGTGAGATCAGCATGTCCTGGGTTCCCCAACATCGAGGGTCACTTTGTTTTTGGGTCGCGAGGT
TCCCTCTGTGGCAGATGGGGCTGTGGGTTCAGCATGTCCTGGGTTCCCCAGCATCGAGGGTCACTTTGTT
TTTGGGTCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGGGTTCAGCATGTCCTGGGTTCCCCAGCATC
GAGGGTCACTTTGTTTTTGGGTCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGGGTTCAGAATGTCCT
GGGTTCCCCAGCATCGAGGGTCACTTTGTTTTTGGGTCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTG
GGTTCAGCATGTCCTGGGTTCCCCAGCATCGAGGGTCACTTTGTTTTTGGGTCACGAGGTTCCCTCTGTG
GCAGATGGGGCTGTGGGTTCAGCATGTCCTGGGTTCCCCAACATCGAGGGTCACTTTGTTTTTGGGTCGC
GAGGTTCCCTCTGTGGCAGATGGGGCTGTGAGTTCAGCATGTCCTGGGTTCCCCAGCATGGAGGGTCACT
TTGTTTTTGTGTCGCAAGGTTCCCTCTGTGGCAGATGGGGCTGTGAGTTCAGCATGTCCTGGGTTCCCCA
GCATGGAGGGTCACTTTGTTTTTGTGTCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGAGTTCAGCAT
GTCCTGGGTTCCCCAGCATGGAGGGTCACTTTGTTTTTGTGTCGCGAGGTTCCCTCTGTGGCAGATGGGG
CTGTGAGTTCAGAATGTCCTGGGTTCCCCAGCATCGAGGGTCACTTTGTTTTTGTGTCGCGAGGTTCCCT
CTGTGGCAGATGGGGCTGTGAGATCAGCATGTCCTGGGTTCCCCAACATCGAGGGTCACTTTGTTTTTGG
GTCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGGGTTCAGCATGTCCTGGGTTCCCCAGCATCGAGGG
TCACTTTGCTTTTGGGTCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGAGTTCAGCATGTCCTGGGTT
CCCCAGCATGGAGGGTCACTTTGTTTTTGTGTCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGGGTTT
CGCAGATGCGTGGAGTCACATCCATGCCCTCAGTCCTTAGGGACCGACCCTCCCTGCCTCACACGCCTCC
CAGGAAGTGTGGCCGGGGGCCGGCAGTGCCACGGCTCCCTCCCCAGCAGGCCCCGGCCGCTCCCATCCCC
AGCACGTGGTCCTATCAGAACGCCACGTCAGCAGGACTCCCAGCAGGTGGCCTTTAGGTCTGGCTTCTTT
CACTTGGCAGAGCACACTGAGGTCTGTCTAGGCTGTCGCATGGATCCCGGTCCCACGTGCTGAGCAGCGC
GTTCCCAGCTGTGGTTGCTGCAGGTTGAACTTTTCCTGGCTGCAGGCGTCCGTGCAGCTTCTGGCCGTTG
TTTTCAGAGCTGTCCTATCACACGCACTGTCCTATCATGGAATATGACGCCGTGTGGGCCACAACTCAGG
CCCAGCAGCCCCCACCCCCGTGCTCTCTGGCCTCCTGCTCAGTTCCTTTGCCCCCAGGGGCTTGGTGCAG
AGTTGAAGGAATCTGTGTGTGTGAACACACAGGACACTAGAGCTGTCAGTTCTCGAGACACCAGGTGTGC
GCGAGGTGATTCCCATGGACCCTGAGGGCTGGTGATAGACTCGGGTCAACGGGTGGGGACCGGGTGTCTC
AGGCCCCAGGCAGGCCCGGCCCTTCCTGACATGACACCCCTTCCCCCAGATTATCCCTGGCGCTGGAGCC
GGGCCGTCCCCATGAACTCCGCACTCATCAAGTGGCTGTACCTGCCTGATTTCTTCCGGGCCCCCAACTC
CACCAACCTCATCAGTGAGTGCCCCCCACCACCCCCGCCTCTGCAGAGGACCCTCAGAGTACATTCACGC
CCCCAAATCTGCTCACAAGTGTGCACACAGGCGTGCACGGGCGGAGGTGTGGTCAGGCACATGGCGGCCT GCAGGCCCTGACCTCGCACGCACGCACGCAGACCTCAGCCTGTGTGCACGGCAGCCCTTGTGCAGATGCC
CTCACACCGGGGCTCCCCCAGGGACACCCGGCCACTCACCCAGGCAGACGTGTGTCCGCTCCCAGCGGCT
GCACGCCGACAGGCCTGGGGTGGGAGGTGGGATTTATGCGCCGTGCCCACCTCGTGTGGGTCCCCGTGTG
GCACAGCGGCGGCTCCTGTGTCCTGCAGGCGACTTTCTCCTGCTGCTGTGCGCCTCCCAGCAGTGGCAGG
TGTTCTCAGCTGAGCGCACAGAGGAGTGGCAGCGCATGGCTGGCGTCAACACCGACCGCCTGGAGCCGCT
GCGGGGGGAGCCCAACCCCGTGCCCAACTTTATCCACTGCAGGTGGGTTCCACGTCACCCTCCACGGGGA
ACCTTCTGGGAGGGGTGGCCGGGGCGCCCGCCCTGACGCTCCGGCCTGGCAGGTCCTACCTTGACATGCT
GAAGGTGGCCGTCTTCCGATACCTGTTCTGGCTGGTGCTGGTGGTGGTGTTTGTCACGGGGGCCACCCGC
ATCAGCATCTTCGGGCTGGGCTACCTGCTGGCCTGCTTCTACCTGCTGCTCTTCGGCACGGCCCTGCTGC
AGAGGGACACACGGGCCCGCCTCGTGCTGTGGGACTGCCTCATTCTGTACAACGTCACCGTCATCATCTC
CAAGAACATGCTGTCGGTGAGCCTCCGGCCCCCCCGCACCCACCGCCCTGGGGCCCCGCTGGCCCCGCTG
ACCCTGCTCTCCCCCAGCTCCTGGCCTGCGTCTTCGTGGAGCAGATGCAGACCGGCTTCTGCTGGGTCAT
CCAGCTCTTCAGCCTTGTATGCACCGTCAAGGGCTACTATGACCGTGAGTGGCCAGGACGGTGGCGGGGG
AGGGCGTGGGGAAGCCCCCTGCTCCTGGGCCCTGGGCCTGACCCTTGCCGGTGCCTGCCTTGCAGCCAAG
GAGATGATGGACAGAGACCAGGACTGCCTGCTGCCTGTGGAGGAGGCTGGCATCATCTGGGACAGCGTCT
GCTTCTTCTTCCTGCTGCTGCAGCGCCGCGTCTTCCTTAGCCATTACTACCTGCACGTCAGGGCCGACCT
CCAGGCCACCGCCCTGCTAGCCTCCAGGCAAGCTTGGGCCCAGACACAGCCCAGAGCTCCCGTCTTGGGG
CTGGGAGGGGGCAATGGGAGGTTCCTCACTGTCTCAGGCCCCGGCCCGTGGAGGGCAGGCTCTGCCACTC
TGTGACATGGGCGTGTCATCTAGAGGGAGAATGAAGGCCGGCAGATCCCCGGCACCATCACACTCTGCCC
CAGTGCTGGGTCTGTCAGAGACCACAGGCTGCAGTGCTGACGGTGGCTGGTGTCTCACCCCCAGCCAACT
TTCCCACTAAGGGCTAAGTTTCTCCACCAGCGGGAGGGCCACTGTGTGGTGTCACGACTGCCCCAGGGAG
GGGTTCTGGCTTGGGGCCAGCTTTGCCTTCTTCCCTGCAGCTGTGGTGGGGTGGGTGCCACCAGACGCCC
CTGCATCTGTACGGCAGAAGGGCCTGTCCTCGCCGCAGACAGCACGGAGGGTGGGGGCAGCAGATGCCTC
CCCCGTGGGTGCCTCTTGTCCAGCGTGGGCAGAGAGGAGCAGGCTGAGCTGTCCCGGGCTGAGCGGGGAG
CGGCGGCTGCCCATGTTGCTGGGGTCGAGTGCCTGGTGCTCACACCCCATCCCCGCCTCCCTACAGGGGC
TTCGCCCTCTACAACGCTGCCAACCTCAAGAGCATTGACTTTCACCGCAGGATAGAGGAGAAGTCCCTGG
CCCAGCTGAAAAGACAGTAGGTGCCTCTGGGGCGGGGACTCCCCGGCTCCTCCCCCCAATGCTCAGCATA
CCCCACCTTTCCCCACCACAGGATGGAGCGTATCCGTGCCAAGCAGGAGAAGCACAGGCAGGGCCGGGTG
GACCGCAGTCGCCCCCAGGACACCCTGGGCCCCAAGGACCCCGGCCTGGAGCCAGGTGAGTGCAGCTGGA
GTCGGGCACCCAGGGCCCCGTGTCCAGCATGTCTGTGCCTGCTGGCGTGTGCTGCGTCTGTGCCCATGTG
ACGTCCCACAGGGCTCCCAGCCCGCCTGTCCTGTCCGCATGATCACCCTCTGTCTGGCAGGCCCCATGGC
CGCCCTGTGACTGTCCGTCCACGCACATGGGCTCTGAGCCCCATGGCCCCACACGGCCCCCGTCACTGTG
GGTGTCCGTGTCTGTCTCCACCTATCCTGTCTCCAAGACGGGAGCACTCACAGCCCCGACCCCTCCTGGT
GGCTTGACTGCTGCCTCATGCTCACCCTGCCCCTCCACAGGGCCCGACAGTCCAGGGGGCTCCTCCCCGC
CACGGAGGCAGTGGTGGCGGCCCTGGCTGGACCACGCCACAGGTACCCCCAATTAGGCCGCCTGTGGCCA
CCCTCTCAGGCCCTCTGTGCCCCCATCTGTCCTCTGCCTGGCCTGCTATCTTCCCCTCCCTTCCCCCGAC
TCCCAGGCCCTGAGCGTCAGGACGTGCTCAGGCCTCCTGGGTCGGGGGGTGCCTCACTGGCTGCAGACCC
CTGGGCTGACTATGTCCTCTCCTGGCTATGCCCCAGCCCTTCCAACAGTGGGAGTCTCGGAGCTTGCCCC
GATGACACATGGTGGTCGAGCAGCGATCTCACCTGGGACCCAGCAGCACTGCGTTATTCTGTTTTTGTTT
CTTTTTGAGATGGAGTCTCGCTCTGTCACTGCAGGCTGGAGTGCAGTGGCATGATCTCAGCTCACTGCAA
TCTCTACCTCCCGGGTTCAAGTGATCCTCCTGCCTCAGCCTCCCAAGTAACTGAGACTACAAGCATGTGC
CCCACTCCAGGCCTTTTTTTTTTTTTTTTGGAGACGGTGTCTTGCCCTGTCGCCCAGGCTACAGTGCAAT
GGCGTGATTGCGGCTCACTACAACCCCCACCTCCCAGGTTCAAAGGATTCTCCTGCCTCAGCCTCCCAAG
TAGCTGGGATTACAGGTGCCCGCCACCACGCCCAGCTAATTTTCGTATTTTTAGTAGAGATGGGGTTTCA
CCATATTGGCCAGGCTGGTCTCGAACCTCTGACTTCAGGTGATCCGCCCGCCTCAGCCTCCCACAGTGCT
GGGATTACAGGCGTCAGCCACCGTGCCCGGCCTGTTCTGTTTTTCTAACTCTCACACAGCCTCCTGGGTT
TTCCCCGGTCCTCTGCAGTCGGCCCACTCTGCACCCCAGCCCGCGCTGGCTCTGCTCCTCAGCTGCCCTG
CCCACCTCTGTCTTGTCCCACCGCGCTGGCCTGTGTCTTGTGCCTGCACTGCTCCCGGCTACTCCGCATG
GGAAGGGTGGCTCTCGGGCCTTGGCCCATGCAGGCGGAGGGGGTCTGGCTGGGAGTCTCCCTGCATGGAA
GGCTGGCTCTCAGTGCTGCCTGCCCACAGTCATCCACTCCGGGGACTACTTCCTGTTTGAGTCCGACAGT
GAGGAAGAGGAGGAGGCTGTTCCTGAAGACCCGAGGCCGTCGGCACAGAGTGCCTTCCAGGTGAGGTGGG
AGAGCCCCGTCGGCCCCACTCCAACCACAGAGCTTGTGGTCCTGGACCAGGGCAGCATAGAGGGTGTCAG
ATGCCCCCAGGGCCTGGGAGCCGAGCTCCTCCACCTCCAGTTAGCCCACCCCGCCCCATCCAGGCCTCCC
AAGTCCCATGGGAAACCAGGCTACAGGGACATGGGTCATGTGTAGCCTGCTGCCCCACGGTCTTGGCTCT
GACCACCCAGGTTCTGGTGGCTGCCCGTGGCCTGACCTGTGAGACCGGCCCAACACCTTTGTGCTGGCCG
CCTGGCTGTCCTGGGTCCATCTTTGGGCCCCTGGCTCTTGGTGTTAGACCAGCCCACCCAACTCCTGAAT
GGGTGGGAGTCTTCCCCCACAGCCCCTCAGGGTCCCCATCCGGGAGGGGCTCAGGGACACGGAGGTCCCT
GGGAGACACAGAGCAGGGATCTGGATCTGGCGCCCGGCTTGCCCAACCCCAGCTTCCCGCCTGGGTCTGA
TGGCTCGGGAGGCCGGGTCCTAACCCGGGGGCTGGCCGACAGCTGGCGTACCAGGCATGGGTGACCAACG CCCAGGCGGTGCTGAGGCGGCGGCAGCAGGAGCAGGAGCAGGCAAGGCAGGAACAGGCAGGACAGCTACC
CACAGGTGAGCTGGGGGGCGTGGGGACTCTGAGGGGAAGCCGCGGGACTGCCAGTCACTCACCAGCATCC
TGTGCCCAGGAGGTGGTCCCAGCCAGGAGGTGGAGCCAGCAGAGGGCCCCGAGGAGGCAGCGGCAGGTAC
GTGGGCCCGGGGCTGGGGAGTGGGAGGTCTCTCTTGGCCCCACAGGCTGCCCCTCCAGCGCCCCCTCCCG
CCCTCCCGCAGGCCGGAGCCATGTGGTGCAGAGGGTGCTGAGCACGGCGCAGTTCCTGTGGATGCTGGGG
CAGGCGCTAGTGGATGAGCTGACACGCTGGCTGCAGGAGTTCACCCGGCACCACGGCACCATGAGCGACG
TGCTGCGGGCAGAGCGCTACCTCCTCACACAGGAGCTCCTGCAGGTGAGCCTGCCCGTGCACCACGCTCG
TCCCTGCTCTGCCTGACTACGCCCCTGCCTGCTTAACAGCCTAGTCCCGCGCCCACTGCACGAAACCCCG
TGTGGGGACAAGAGCTGGACGCAGCCCTGAGCCCCCTGCTGTGCCCTGCAGGGCGGCGAAGTGCACAGGG
GCGTGCTGGATCAGCTGTACACAAGCCAGGCCGAGGCCACGCTGCCAGGCCCCACCGAGGCCCCCAATGC
CCCAAGCACCGTGTCCAGGTAGGTGCGGGGGTGACCCGAGCCCCAGCTGCTGCCCCTGGTGTGTGGGCAT
CGCCTAGCCATCCCCGACCCTCGCCATTCCCTTGTACCCCAAAGGACCGTGGGCACTTTCCACCCTGACC
CTCCCTGTAGCCTGGGGTCAGGCCATAGAGCAGGATTCTCTGTGACTCGGCTTCCCTCCCCAGTGGGCTG
GGCGCGGAGGAGCCACTCAGCAGCATGACAGACGACATGGGCAGCCCCCTGAGCACCGGCTACCACACGC
GCAGTGGCAGTGAGGAGGCAGTCACCGACCCCGGGGAGCGTGAGGCTGGTGCCTCTCTGTACCAGGGACT
GATGCGGACGGCCAGCGAGCTGCTCCTGGACAGGTGGGGGCGGGACGCGCACAACACCAGCCTCACCATG
GCCCTCGGGGAGCAGCCGAACAGGGGCAGGAGACTGACTGTGACCGGCAACAGATCGGGCCGTCATGCCT
TCGGGCAGTCCCAGACTCCCCCAAACACGCGGGTCTCCCTGTAGGCGCCTGCGCATCCCAGAGCTGGAGG
AGGCAGAGCTGTTTGCGGAGGGGCAGGGCCGGGCGCTGCGGCTGCTGCGGGCCGTGTACCAGTGTGTGGC
CGCCCACTCGGAGCTGCTCTGCTACTTCATCATCATCCTCAACCACATGGTCACGGCCTCCGCCGGCTCG
CTGGTGCTGCCCGTGCTCGTCTTCCTGTGGGCCATGCTGTCGATCCCGAGGCCCAGCAAGCGCTTCTGGA
TGACGGCCATCGTCTTCACCGAGGTGGGCCGAGGCCGCGGGGGAGGGGGCGCCCGGCCCACCGCGCCGTG
ACCCTCCCCGCGTGCTGAGCCCCCTCCCCCACAGATCGCGGTGGTCGTCAAGTACCTGTTCCAGTTTGGG
TTCTTCCCCTGGAACAGCCACGTGGTGCTGCGGCGCTACGAGAACAAGCCCTACTTCCCGCCCCGCATCC
TGGGCCTGGAGAAGACTGACGGCTACATCAAGTACGACCTGGTGCAGCTCATGGCCCTTTTCTTCCACCG
CTCCCAGCTGCTGGTGAGTGTGAGCCTTGGCTGGCAATGCGGGGCTGGGCAGGCCCTCTGGGCACCTGTG
CTCTCCACCAGGGAGGCAAGGCCCCCTCACCACACCCTCCCGCCCCTCAGTGCTATGGCCTCTGGGACCA
TGAGGAGGACTCACCATCCAAGGAGCATGACAAGAGCGGCGAGGAGGAGCAGGGAGCCGAGGAGGGGCCA
GGGGTGCCTGCGGCCACCACCGAAGACCACATTCAGGTGGAAGCCAGGGTCGGACCCACGGACGGGACCC
CAGAACCCCAAGTGGAGCTCAGGCCCCGTGATACGAGGCGCATCAGTCTACGTTTTAGAAGAAGGAAGAA
GGAGGGCCCAGCACGGAAAGGAGCGGCAGCCATCGGTATAAGCGCCCTGCCTCACAACCTCCTGCCTACC
CAGTTTTCTGAGTGGGGCTACTGCAGGGAGGGTCTTTCTCAGATGAGACGGCCAAGCCCAGTGCGAGGCC
CACCTGGATCCCAGGAAGGTGCCACTTCTGAGCCACAGCTCCCGGCTCTGCCTACAGAGCCGTCCCTGAC
TGCTGCCCCCGGGGATGCTCCCCACGTGTAGGGTGACTGTTGGCCTGGGCTGGCCCCTCACAGTTGCCCC
AGACAGAGGACACAGCCCCAGCTGTCTCCTTGCCAGTGACACTGGGAGCTTTCCTGTGCTCCGTCTGCTT
GTCTGTCAAACAGGGAGAATGCCAGCCTCTTAGGGTGGTCAGGAGCCATGAGCCAGGCCCAGTCCCCAGG
GGGCCCAGGCAGAAGTCAGCTTTTCCCTACAGAAGCTGAGGACAGGGAGGAAGAAGAGGGGGAGGAAGAG
AAAGAGGCCCCCACGGGGAGAGAGAAGAGGCCAAGCCGCTCTGGAGGAAGAGTAAGGGCGGCCGGGCGGC
GGCTGCAGGGCTTCTGCCTGTCCCTGTGAGTGATGGCGGCCGGGGGCAGCTGGGGAGTGGGGGTGGGGAG
GCGGGTACTGGGCCCAGGCTGAGCGCCCCCTTCCGCAGGGCCCAGGGCACATATCGGCCGCTACGGCGCT
TCTTCCACGACATCCTGCACACCAAGTACCGCGCAGCCACCGACGTCTATGCCCTCATGTTCCTGGCTGA
TGTTGTCGACTTCATCATCATCATTTTTGGCTTCTGGGCCTTTGGGGTGAGCCAGGCCCGGGACCCAAAC
CCAGTGTACGCAGAGCTCAGCAGCCACCCACATCCCCTGGGCTTGGCTCCCCCTGACCTGTGCTCTCCTG
GCCACAGAAGCACTCGGCGGCCACAGACATCACGTCCTCCCTATCAGACGACCAGGTACCCGAGGCTTTC
CTGGTCATGCTGCTGATCCAGTTCAGTACCATGGTGGTTGACCGCGCCCTCTACCTGCGCAAGACCGTGC
TGGGCAAGCTGGCCTTCCAGGTGGCGCTGGTGCTGGCCATCCACCTATGGATGTTCTTCATCCTGCCCGC
CGTCACTGAGAGGTGGGCCCACGCGTGGGGGCGCTCGGTCTCCAGGGGCGGGGCAGTGCAGGCTGGGGGC
CCTGCGGGGCTGTTTCTGATGGGGTCCTTGACCTGGCCATCCCGCCCCAGGATGTTCAACCAGAATGTGG
TGGCCCAGCTCTGGTACTTCGTGAAGTGCATCTACTTCGCCCTGTCCGCCTACCAGATCCGCTGCGGCTA
CCCCACCCGCATCCTCGGCAACTTCCTCACCAAGAAGTACAATCATCTCAACCTCTTCCTCTTCCAGGGG
TGAGTGCAGGTCCGCCGGGGTGGGGGTCACGGCCCGGGCATGAGGGAGCCCACCTGACGGGAACCCTGGC
TGTGGGCAGGTTCCGGCTGGTGCCGTTCCTGGTGGAGCTGCGGGCAGTGATGGACTGGGTGTGGACGGAC
ACCACGCTGTCCCTGTCCAGCTGGATGTGTGTGGAGGACATCTATGCCAACATCTTCATCATCAAATGCA
GCCGAGAGACAGAGAAGGTGCCTGGGCCCAGGGCGGGGGCCGGGACAAGGGCCAGGGATATGCCCTCTCC
CTAAGACAGAGGCACTGCTGCCACGAGAACCCGTGGTGCTGGAGGCCCTCCCAGGGCTCGGAGCCCATGG
GGACATGAGGCGAGCCCACCCACTAGCTGATCACGAGGCCAGTGATCTTGGCAGCTGCGAGTGAGTGCTG
GGCGCAGAAGTGGGCAGCGGAGTTGGTCCTGTTCCAGGCAGGCTGGCAGCAGAGCAGGGCCTGGTGCAGG
GAGGACCGGACAGCCACTGTTTGCTGCATTCTTGTTTAATGGCCTTTCTCAGAGAGAATTCGTGCGTCAG
ACCACTCCCCCACGTAAAAAGTACAACTCAGGGGTTTCTAGTGGATTCACAGTTGAGCATCTGCCTTCTC ACCACTTCAAAAAGAAACCCCGGGCCGGGCACAGCGGCTCACCCCTATCATCCCAGCACTGTAAGAGACC CAGGAGGGAGGTACTGCTCAAGGCCAGGAGTTCAAGATCTGCACGGCCACAAGCGAGACCCTGCCTCAGC AAATGTAAAAATAAAACAATTAGCTGGCAATGGCAGCTCATGCCTATGGTCCCAGCACTTTGGGAGGCCA AAGGAGGAGGATCAGTTGAGGCCAGGAGTTTTAAGACCAGCCTGGGCAACATAGTGAGTGAGACTCTCTC TACAAAAAAATAAACGTTAGCTGGGTGTGATGGTGCACACTTGTGCTCCCAGCTACTCTGGAGGCTGAGG TGGGAGGATGGCTTGAGGCCAGGAGTACAGGGCTGCATTAAGCCTGATCACACCACTGCACTCCAGCTTG GGCAACAGAGTGAGACCCTGTCTCTAAAAAAGTAAAAAGAAGAAACCCAGTGCCCATTGGCTGTCACTCG GTTTCCCCTCCCCTGGCCCCTAGTAACCCCTTGTCTAGGTCCTGGTTCTATGAGATTTGCCTACAGTGGA TATTTCATGAAAACAGGCTCAGACAGCGTTTGTCCTTTTGTAATCAGCTTTCCTTGCTCAGCATGGTGTC TCTGAGGTCCACCCACGTGGCAGTATGGCCTTCCTGTTTATGGGCAAATGATATTCTGTGGCATGGATGG ACCACAACATGCTCATCTGTTGATGGGCTGTTGCCCGTGATGCTGCCAGGCACGCCGGTGTACACGTCTG TGTGCCCGTGCCCCTGGTCCTGTGGCTGCACGCCAGGGCCGGAAGTGCTGGTGGTGGTTTCCATCATGAG GAGCTGCTGGTTTTCCATAGCAGCTCCACCATCTAAGGTTCCCACCACCAACACGAGGTTGCGGTTTCTC CACATCCTCAACAACCTGTTACTATGTCTTTTTTGTTCTGGCCATGCTGCTGAACGGGTGGCTTGCTGTG GGCTCTTCTCAGTTCCCTGTTGACCGACGCTGAGCGTCTTTTCATGTGCTTGGCCATTTGTGTATCTTCT CTGGGGAAATGTCTATTCAAATCCTTTCTCCATAGTTTAGTTGGGCTTTTGAGATAGGATCTCAGGCTGA AGTGCAGTGGCATGACCTTCACTCACTGTAGCCTCTGCCTCCCAGGTTCAAGCGATTCTCCCACCTCAGC CTCCCGAGTAGCTGGGACTACAGGTGTGCACCACCAGGCCTGGCTAGTCTTTTGTATTTTTGGCCAGGTT CGTCTCAAACTCCTGACCTCAAAGTGCTGGGATGACAAGTGTGAGCCGCCACACCCAGCAGTTGGGTTGT TTTTTATTAGAGTTTTTTCTGTTTTTCTGCAGATACATTGGCTAGAAATGACTGAATTGGAAGAATTCTC TGTATTCTCTGGTTGCTAGAACCTTATCAATTAAAATTTGCAGAAAATTTCTCCAATTCTATGGACTGTC TCTTAAATTTTCTTGGTGTCTTTGGAAGCACAAAGTATTTATTTTGGTAATATCTGGTTTACTTTGTTTC CTTCGCCAAATCCAGGTTCATGAAGATTTGCCCGTTTTCTTCTAAAAGTTCTATAGTTTTAGCTCTGAAG TTTCGCTCTTTGATCCACTTTGAGGTAAATTTTGGCACATGGTATGAGGCAGGAGTTGCGTTTCATTCTC CTGCCTGGGGCGGTGCCTGCACCGTGTTGAAAAAGGTTGTCCGTTCCCACTGAACGGTCACAGCTCCCTT GTCTAAGATCAACGACCCCTGAACATGAGGGTTCCAACTGGACTCTTAGTTCTACTCCACTGGCCTGTGT CTGCCCACCATTACTACCGCTGTGCCATACTGAGGTCAGGCTGGGGCTTTTCTGGGCTGCTGGGTGAGCT GGAGAATGCGGTTGTGTGACCCGCAGGAAGGGCAGAGCTGAGCGTATGACCTGTGTCGTTCCCCTCCAGA AATACCCGCAGCCCAAAGGGCAGAAGAAGAAGAAGATCGTCAAGTACGGCATGGGTGGCCTCATCATCCT CTTCCTCATCGCCATCATCTGGTTCCCACTGCTCTTCATGTCGCTGGTGCGCTCCGTGGTTGGGGTTGTC AACCAGCCCATCGATGTCACCGTCACCCTGAAGCTGGGCGGCTATGAGGTGAGCATGTGTGGGTCCGCCT GTCCATTCCCATCCCCTGGGGGTTCTGGCCAAGGTGGTGCACCACCCCCAGCCGCTCCTCCACGCTCATC TTCGTGGCCCCGTGTCCCCGTGCCTGCCCCAGCCGCTGTTCACCATGAGCGCCCAGCAGCCGTCCATCAT CCCCTTCACGGCCCAGGCCTATGAGGAGCTGTCCCGGCAGTTTGACCCCCAGCCGGTAAGTGGCCTCTGC CCTGTGAAAGCTGGTGTGGGGAGGCGGCTGCAGTCACTGAGGGTGTCACTTGTACCCAGCTGGCCATGCA GTTCATCAGCCAGTACAGCCCTGAGGACATCGTCACGGCGCAGATTGAGGGCAGCTCCGGGGCGCTGTGG CGCATCAGTCCCCCCAGCCGTGCCCAGATGAAGCGGGAGCTCTACAACGGCACGGCCGACATCACCCTGC GCTTCACCTGGAACTTCCAGAGGTTCGTCCTGGACTTGGGGCAGTGCCTGGGTGGGTGGACCCACTACAG TGGGTCACGCTGTGTTCCCACCCCCAGGGACCTGGCGAAGGGAGGCACTGTGGAGTATGCCAACGAGAAG CACATGCTGGCCCTGGCCCCCAACAGCACTGCACGGCGGCAGCTGGCCAGCCTGCTCGAGGGCACCTCGG ACCAGTCTGTGTGAGTGAAGGGCCCGGGTGGTGGGCAGGAGGGCTGTGCCAGGTTGGCTGGGCCAGGCCT GACCTGCCAGCACCTCCCTGCAGGGTCATCCCTAATCTCTTCCCCAAGTACATCCGTGCCCCCAACGGGC CCGAAGCCAACCCTGTGAAGCAGCTGCAGCCCAGTGAGTATGGGCGTGGGGGTTGGGGGAGGCTAGAGAG GGGTGACCTGCGGCCTCAACGATCTTCTCCCTCCATCCCAGATGAGGAGGCCGACTACCTCGGCGTGCGT ATCCAGCTGCGGAGGGAGCAGGGTGCGGGGGCCACCGGCTTCCTCGAATGGTGGGTCATCGAGCTGCAGG AGTGCCGGACCGACTGCAACCTGCTGCCCATGGTCATTTTCAGTGACAAGGTCAGCCCACCGAGCCTCGG CTTCCTGGCTGGCTACGGGTGAGTGAGTGGCTGGGGGGGCACCCCGCAGCTCGGGGGGCTCCGGGCGGCC CCAGGACTCACCAGCTTCCCCCGCAGCATCATGGGGCTGTACGTGTCCATCGTGCTGGTCATCGGCAAGT TCGTGCGCGGATTCTTCAGCGAGATCTCGCACTCCATTATGTTCGAGGAGCTGCCGTGCGTGGACCGCAT CCTCAAGCTCTGCCAGGACATCTTCCTGGTGCGGGAGACTCGGGAGCTGGAGCTGGAGGAGGAGTTGTAC GCCAAGCTCATCTTCCTCTACCGCTCACCGGAGACCATGATCAAGTGGACTCGTGAGAAGGAGTAGGAGC TGCTGCTGGCGCCCGAGAGGGAAGGAGCCGGCCTGCTGGGCAGCGTGGCCACAAGGGGCGGCACTCCTCA GGCCGGGGGAGCCACTGCCCCGTCCAAGGCCGCCAGCTGTGATGCATCCTCCCGGCCTGCCTGAGCCCTG ATGCTGCTGTCAGAGAAGGACACTGCGTCCCCACGGCCTGCGTGGCGCTGCCGTCCCCCACGTGTACTGT AGAGTTTTTTTTTTAATTAAAAAATGTTTTATTTATACAAATGGACAATCAGAGGCCAGTCCCCCGTCCT TGCCTTCCGGCTCCAGTGTGGTGTACCAGGTGGCCACTGCCTGCTGTCCCGGCAAGCACGTCCTCACGCT CAGCTCTGGCCCGCCTCGTCGTCACTGTCCTCAATCAGACAGTCCCGGATCTCCTGCAAGCCCCAGGCCC TAGAGAGAAAGCTGCAGTGGGCACCAGCCCCGAGGCCCCTTACCCACGCGCCACGGCAGTGTGGGCCCCA GTACCTCTGTGTGCGGAGCTGGGCCTCAGCCAGGATGTACGGCGGCAGGGGGTCCAGTGAGGGCTGCAAC AGAGGCCGGTGTCGAGGGGCCAAGAAACCTGCCAGCACCCCCTCAAGCCACCCACGAGGTGACGGCCTTC CCGCGTCCCCAAGCCCGGCCCAGGACAGGTGGGCACGTACTCACCAAGTCCTTCATGTTCACGCGGCAGC TGTAGCACAGCTGCTCCTCAATGCAGGCTTGGGGGTCCTCCCTGCGGGAGCAGAGCTGGCTGTGTGCCCA CCTTGGCACCTCAATGCCGTGCAGTGTGGGGTAAGGACAGGTCCCCCTCCCGCCCCATGCAGCTGGCCAT GGGTGGCAGGGACAGGGACTCACCTCCTGCAGGCCCCCTGGCCACAGCGCTGGGCCCAGCCCACCCCTGG AGAACAGCAGGGCCCCGGGGGTGTCCGGGTCTCAGTCAGGGGGATGGGTGACTGCATCTGGGAGAGACGC GAGGAGGTCTGAGCCCCAAAAGCCGTGGCACTGTCTGCAAGCAGAGAGGGGGTGACGGAGCCTGGGGCTG CTGAGCCCCCACACCCACTCCCTGGCTCCCTCCCCGCATCACCCCAAGAGCATGAAACACAGACCAGCGG CGTCGACGTCCAGGGCACACATGCAGAGGAGGCAGCGGGGGCCGGAGTCGCCAGCAGCAGGGCCATCCCG GGGGCCCTTCACCAGCTTCTCACTTGTCCTGTGGCCGGATTGTGGGAGGAGCAGGTGAATCACACGGGCT GGGGCTCCACCCTAAGCCCTGGCCTGAGCACGAGTGGTGAGCTCACCACCCGCACACACCCGCGCACAGC ACACACCCGCGCACCCCCCGCACACACCCACACACACCCACACCTGTACACAGTGCTGACAGTGGAGGGG AACTGGGTCTGCAGCCTGAGGATGAAGGCCTCCATCAGCCGGTGGATGCTGGCCTTTTCAGGGGCCTAGG GACAATCAGATTGGAGGCTATGGGAAAGCCCAGAGGCCAGCCCCAGGCTGGGGCAGAGTCCCCAGGACAG GTGCCGTGGGTTCCACGCTGCTGGCCCACATGGGTCTGGCTTTGCTGCGACTGCTCAAGGCAGCCAGAGA ACCCGCTTCAGCAGTGCACCCACTGTGGGGTCCTCCTGTCACCCCCACACCCTCACGGCACAGGGTGGGC TCAGAGGATGCAGCACTGCTGGGGGCCACAGCAGGAGTCACTGAAGGTGTGCTGGTGACCTGAGCCTGTG AGAGGCTGCCCAGCCCACATGGCCCACCCCGGTGTCAATGGTGAGAGAGGTTGCCCAGCCCACAAGGCCC ACCTTGGTGTCGACGGCTGGTGTGAAGACAGAAGGAACGGAGAACAGGCGGTTGTAGAAAGCGACCTCCT TCAGGGTGTGGTCCCGCATGGGCCGCACCACCACCACGTCCCCGTGCCGCTCATCCGAGAAGCCCTGCGG GAGGCAGGGGCTGTGTGGTCCAGGTCAAGGCCCGAATGTGCTTGCCCCTCCTCCCCTGCCAGGTAGGGGA GGTCACAGACCCCACCACCATCCGAGGCCGCAGGACTGATACGCGAGAGGCTACTCTCCAGTAAAGGTGG CCGAAGGGGTGAAGGTGAGGATGGGCCTCCTGAACACCCAGGCCCCTGCCTACCGTATCCCAGGCCAGGA AGGCCCCTCGACCCAGCGCCAGGTTGGTCATGAGCTTGATAGCCAAGCGTGTGCAGCTGTCCCCAGTCAT GACCTTGGAGTAGCCGTGGGCTC
It will be appreciated that specific sequence identifiers (SEQ ID NOs) have been referenced throughout the specification for purposes of illustration and should therefore not be construed to be limiting. Any marker encompassed by the present invention, including, but not limited to, the markers described in the specification and markers described herein, are well-known in the art and may be used in the embodiments encompassed by the present invention.
The phrase “pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
The terms “polynucleotide”, and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are nonlimiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified, such as by conjugation with a labeling component. The term “recombinant” polynucleotide means a polynucleotide of genomic, cDNA, semi synthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.
The terms “prevent, ” “preventing, ” “prevention, ” and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.
The term “small molecule" is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.
As used herein, the term “subject” means a human or non-human animal selected for treatment or therapy.
The “tumor microenvironment” is an art-recognized term and refers to the cellular environment in which the tumor exists, and includes, for example, interstitial fluids surrounding the tumor, surrounding blood vessels, immune cells, other cells, fibroblasts, signaling molecules, and the extracellular matrix.
The phrases "therapeutically-effective amount" and “effective amount” as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment. “Treating" a disease in a subject or “treating" a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g, the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.
In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1. The agent may be a small molecule agonist of PIEZO 1, such as Yodal, Jedi 1, Jedi2, or a modulator of PIEZO1, such as Docosahexaenoic acid. The method may further comprise administering an immune checkpoint inhibitor to the subject.
In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1 and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM- 4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof. In some preferred embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1.
In some embodiments, the agent that increases or stabilizes the activity or expression of PIEZO1 and the immune checkpoint inhibitor are administered conjointly In some embodiments, the agent increases or stabilizes the activity or expression of PIEZO 1 and the immune checkpoint inhibitor act synergistically when administered. In some embodiments, the agent is a gRNA fused to a transcription activator, such as a gRNA that comprises a region that is complementary to a portion of a gene that encodes a PIEZO 1 protein. In some embodiments, the agent is a vector encoding a PIEZO 1 protein, such as a viral vector encoding a PIEZO1 protein. For example, without being bound by theory or methodology, the agent that increases or stabilizes the activity or expression of PIEZO 1 may be a small molecule agonist of PIEZO 1, such as Yodal, Jedi 1 , Jedi2, a modulator of PIEZO1, such as Docosahexaenoic acid, a gRNA disclosed herein, or any combination thereof.
The agent may be administered systemically, intravenously, subcutaneously, or intramuscularly. The agent may be administered to the subject in a pharmaceutically acceptable formulation. The method may further comprise administering to the subject an additional agent, such as a chemotherapeutic agent or a cancer vaccine. The method may further comprise administering to the subject a cancer therapy, such as radiation. The subject may be refractory for immune checkpoint inhibitory therapy.
In some aspects, provided herein are methods of treating cancer in a subject unresponsive to immune checkpoint inhibitor therapy, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1 (e.g., any agent that increases or stabilizes the activity or expression of PIEZO 1 disclosed herein) and an immune checkpoint inhibitor. The agent may be a small molecule agonist of PIEZO1, such as Yodal, Jedi 1 , Jedi2, a modulator of PIEZO1, such as Docosahexaenoic acid, or any combination thereof. The method may further comprise administering an immune checkpoint inhibitor to the subject, such an inhibitor of PD-1 or PD-L1, or another immune checkpoint inhibitor disclosed herein.
In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject T-cells that have been treated ex vivo with an agent that increases or stabilizes the activity or expression of PIEZO1 (e.g., any agent that increases or stabilizes the activity or expression of PIEZO1 disclosed herein). The T- cells may be tumor infiltrating lymphocytes. In some embodiments, T-cells are autologous (z.e., derived from the subject). In some embodiments, T-cells are allogeneic (ie., derived from a doner).
In some aspects, the subject is a human.
Modulators of PIEZO 1
Provided herein are methods and compositions for preventing or treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1. The agent may increase the activity of PIEZO 1 by at least 5%, at least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. The agent may increase the expression of PIEZO 1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
An agent disclosed herein may increase PIEZO 1 mRNA by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. Measurement of PIEZO 1 can be done in a biological sample or multiple biological samples taken from the subject over a period of time.
Polypeptide Agents
In some embodiments, the agent provided herein is a polypeptide agent (e.g., a polypeptide that binds to a PIEZO 1 protein). A polypeptide agent disclosed herein may increase or stabilize the expression or activity of PIEZO 1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, the agent may be a chimeric or fusion polypeptide. In some embodiments, the agent may be a ligand or binding partner of PIEZO 1. A fusion or chimeric polypeptide can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety.
The polypeptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding a polypeptide(s). Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous polypeptides in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N. Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc. 91 :501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem. 11 :255; Kaiser et al. (1989) Science 243: 187; Merrifield, B. (1986) Science 232:342; Kent, S. B. H. (1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which are incorporated herein by reference.
Nucleic Acids
In certain embodiments, provided herein are agents that are vectors that contain the isolated nucleic acid molecules described herein, such as those that encode a PIEZO 1 peptide. As used herein, the term “vector,” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby be replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In certain embodiments, provided herein are cells that contain a nucleic acid described herein (e.g., a nucleic acid encoding an antibody, antigen binding fragment thereof, antibody-like molecule, or polypeptide described herein). The cell can be, for example, prokaryotic, eukaryotic, mammalian, avian, murine and/or human.
The interfering nucleic acids described herein may be contacted with a cell or administered to an organism (e.g., a human). Alternatively, constructs and/or vectors encoding the interfering RNA molecules may be contacted with or introduced into a cell or organism. In certain embodiments, a viral, retroviral or lentiviral vector is used.
In some embodiments, agents for increasing the expression or activity of PIEZO 1, are delivered to subjects by use of viral vectors. Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc. \ (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus.
In some embodiments, adenoviruses can be used to deliver nucleic acid agents for increasing the expression or activity of PIEZO 1. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy.
In some embodiments, nucleic acid agents for increasing the expression or activity of PIEZO 1 can be delivered by adeno-associated virus (AAV) vectors. In some embodiments, a AAV vector that expresses a nucleic acid agent for increasing the expression or activity of PIEZO 1 is a recombinant AAV vector having, for example, either an U6 or Hl RNA promoter, or a cytomegalovirus (CMV) promoter. Suitable AAV vectors for use in agents, compositions, and methods described include, but are not limited to AAVs described in Passini et al., Methods Mol. Biol. 246: 225-36 (2004).
In another embodiments, the agent comprises a CRISPR activation agent and/or a sgRNA. In some embodiments, the agent is an sgRNA. An sgRNA combines tracrRNA and crRNA, which are separate molecules in the native CRISPR/Cas9 system, into a single RNA construct, simplifying the components needed to use CRISPR activation system. In some embodiments, the crRNA of the sgRNA has complementarity to PIEZO 1 DNA.
Antibody Agents
Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies may also be fully human. The terms “monoclonal antibodies” and “monoclonal antibody composition”, as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.
In certain embodiments, the methods and compositions provided herein relate to antibodies and antigen binding fragments thereof that bind specifically to a PIEZO 1 protein. Such antibodies can be polyclonal or monoclonal and can be, for example, murine, chimeric, humanized or fully human. In some embodiments, the agent may be a recombinant antibodies specific for a PIEZO 1 protein, such as chimeric or humanized monoclonal antibodies, can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in US Pat No. 4,816,567; US Pat. No. 5,565,332; Better et al. (1988) Science 240: 1041-1043; Liu et al. (\9KT) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999- 1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559); Morrison, S. L. (1985) Science 229: 1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature 321 :552- 525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J. Immunol. 141 :4053-4060.
Human monoclonal antibodies specific for a PIEZO 1 protein can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. For example, “HuMAb mice” which contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy (p and y) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous p and K chain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856 859). Accordingly, the mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49 101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65 93, and Harding, F. and Lonberg, N. (1995) Ann. N. Y Acad. Sci 764:536 546). The preparation of HuMAb mice is described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287 6295; Chen, J. et al. (1993) International Immunology 5: 647 656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci USA 90:3720 3724; Choi et al. (1993) Nature Genetics 4: 117 123; Chen, J. et al. (1993) EMBO J. 12: 821 830; Tuaillon et al. (1994) J. Immunol. 152:2912 2920; Lonberg et al, (1994) Nature 368(6474): 856 859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49 101; Taylor, L. et al. (1994) International Immunology 6: 579 591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65 93; Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536 546; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845 851. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and 5,545,807.
Small Molecule Agents
Certain embodiments of the methods and compositions disclosed herein relate to the use of small molecule agents e.g., small molecule agents that increase or stabilize the expression or activity of a PIEZO 1 protein. Such agents include those known in the art and those identified using the screening assays described herein. A small molecule provided herein may have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% specificity for a PIEZO 1 protein.
In some embodiments, assays used to identify agents (e.g., anti-cancer agents) in the methods described herein include obtaining a population of cells and a small molecule agent, wherein the cells are incubated with a small molecule agent and the level of a PIEZO 1 protein is measured. Agents identified via such assays, may be useful, for example, for increasing the expression or the activity of a PIEZO 1 protein in a subject.
Agents useful in the methods disclosed herein may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Agents may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al, 1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261 : 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37: 1233.
Libraries of agents may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/or spores, (Ladner, USP 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sci USA 89: 1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner, supra.).
Pharmaceutical Compositions
In certain embodiments, provided herein is a composition, e.g., a pharmaceutical composition, containing at least one agent described herein together with a pharmaceutically acceptable carrier. In one embodiment, the composition includes a combination of multiple (e.g., two or more) agents described herein.
In some embodiments, the pharmaceutical composition is delivered locally or systemically. In some embodiments, the pharmaceutical composition may be administered to a tumor present in the subject. In some embodiments, the agent or pharmaceutical composition is administered with an additional cancer therapeutic agent. In some embodiments, the additional cancer therapeutic agent is a chemotherapeutic agent. In some embodiments, the pharmaceutical composition further comprises an additional agent for treatment of cancer. In some embodiments, the additional agent is a tumor vaccine. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (Cytoxan™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; emylerumines and memylamelamines including alfretamine, triemylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimemylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (articularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, foremustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin phili); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carrninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (Adramycin™) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as demopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replinisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- tricUorotriemylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiopeta; taxoids, e.g., paclitaxel (Taxol™, Bristol Meyers Squibb Oncology, Princeton, N.J.) and docetaxel (Taxoteret™, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitroxantrone; vancristine; vinorelbine (Navelbine™); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in the definition of “chemotherapeutic agent” are anti -hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston™); inhibitors of the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprohde, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In some embodiments, the additional cancer therapeutic agent is an immune checkpoint inhibitor. Immune Checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response. Examples of immune checkpoint proteins are CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM- 4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof.
The pharmaceutical compositions and/or agents disclosed herein may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; or (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous, intrathecal, intracerebral or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation. Methods of preparing pharmaceutical formulations or compositions include the step of bringing into association an agent described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Pharmaceutical compositions suitable for parenteral administration comprise one or more agents described herein in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions include water, ethanol, dimethyl sulfoxide (DMSO), polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
Therapeutic Methods
In some aspects, provided herein are methods of treating a cancer by administering to a subject (e.g., to a tumor present in a subject) an agent and/or a pharmaceutical composition described herein. In some aspects, the subject is a human.
In some embodiments, the methods described herein may be used to treat any cancerous or pre-cancerous tumor. In some embodiments, the cancer includes a solid tumor. Cancers that may be treated by methods and compositions provided herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; mammary paget's disease; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; and malignant roblastoma; sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii; choriocarcinoma; malignant mesonephroma; hemangiosarcoma; malignant hemangioendothelioma; kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
In some embodiments, the subject has cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a blood born tumor, a brain/CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing tumor, an eye tumor, a gallbladder tumor, a gastrointestinal, a kidney tumor, a laryngeal or hypopharyngreal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penile tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a Rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma (such as uveal melanoma (UVM), a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymus tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms tumor. Actual dosage levels of the active ingredients in the pharmaceutical compositions or agents to be administered may be varied so as to obtain an amount of the active ingredient (e.g., an agent described herein) which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
The compositions disclosed herein may be administered over any period of time effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The period of time may be at least 1 day, at least 10 days, at least 20 days, at least 30, days, at least 60 days, at least three months, at least six months, at least a year, at least three years, at least five years, or at least ten years. The dose may be administered when needed, sporadically, or at regular intervals. For example, the dose may be administered monthly, weekly, biweekly, triweekly, once a day, or twice a day. In certain embodiments, a dose of the composition is administered at regular intervals over a period of time. In some embodiments, a dose of the composition is administered at least once a week. In some embodiments, a dose of the composition is administered at least twice a week. In certain embodiments, a dose of the composition is administered at least three times a week. In some embodiments, a dose of the composition is administered at least once a day. In some embodiments, a dose of the composition is administered at least twice a day. In some embodiments, doses of the composition are administered for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 4 weeks, for at least 1 month, for at least 2 months, for at least 3 months, for at least 4 months, for at least 5 months, for at least 6 months, for at least 1 year, for at least two years, at least three years, or at least five years.
The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could prescribe and/or administer doses of the compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
Exemplification
Example 1: Proximity proteomics identifies PD-1 and PIEZO1 association
Previous proteomic studies involving PD-1 pulldown have identified direct binding partners of PD-1 but have not been successful in tracking the time-resolved behavior of proteins recruited to PD-1 following PD-L1 ligation. To overcome these issues and to test if important factors in PD-1 -mediated CD8+ T cell inhibition remain unidentified, proximitylabeling combined with multiplexed quantitative mass spectrometry was utilized. This made possible time-resolved detection of proteins lying within ~10nm radius of the PD-1 cytoplasmic tail in the presence or absence of PD-L1 ligation (Fig. 1A). Jurkat cell lines stably expressing human or murine PD-1 fused to an ascorbate peroxidase 2 (APEX2) labeling probe via the C-terminus of the PD-1 cytoplasmic tail was utilized (Fig. 5A). PD- l-APEX2-expressing Jurkat cells were sorted and assessed for labeling efficiency using anti-streptavidin Western blotting (Fig. 5B,C). Tosyl-activated Dynabeads were covalently coupled to CD3 and CD28 crosslinking antibodies using either control murine IgGi ligand (mlgGi, TCR-control) or recombinant PD-Ll-Fc fusion protein (TCR-PD-L1) to provide TCR and co-stimulatory signals for T cell activation while also driving strong inhibitory signals through PD-1, respectively (Fig. 6). Following incubation in labeling media containing biotinyl tyramide, PD-l-APEX2-expressing Jurkat cells were co-cultured with TCR-PD-L1 or TCR-control beads for 2, 5, 10 and 20 min while the 0 min condition contained no beads (Fig. IB). Biotinylation of proteins proximal to the PD-1 cytoplasmic tail was induced by the addition of H2O2 to the celkbead mixture during the last minute of each timepoint (Fig. 1 A, Fig. 5C). Cell pellets were lysed and biotinylated proteins were enriched using streptavidin beads under denaturing conditions. Trypsin digest-derived peptides were labelled with tandem mass tags (TMT) and quantified by triple-stage mass spectrometry (MS3)31.
To quantify proteins co-localized with PD-1 over time following PD-L1 ligation relative to TCR/CD28 stimulation alone, scaled TMT ratios (TMT RA) of identified peptides for each condition were calculated. A strong 2-3 -fold, time-dependent enrichment of SHP2 following TCR-PD-L1 ligation was observed as compared to TCR-control stimulation. Enrichment was observed using either human or murine PD-1 peroxidase fusion proteins, confirming that SHP2 is recruited to PD-1 following ligation (Fig. 1C, Fig. 7 A). A robust 2-3-fold enrichment of the mechanosensitive ion channel PIEZO1 following PD-L1 ligation compared to TCR-control stimulation using both human and murine PD-1 peroxidase fusion proteins was observed (Fig. ID, Fig. 7B): among 1247 quantified proteins, the peptides corresponding to PIEZO 1 fell within a small group that associated with PD-1 in a time-dependent manner following PD-L1 ligation. One-way hierarchical data clustering confirmed near-identical recruitment kinetics for SHP2 and PIEZO 1 to PD-1 (Fig. lH). Thus, following PD-L1 binding to PD-1, PIEZO1 associates with PD-1.
The recruitment kinetics of previously reported mediators and targets of PD-1 signaling, including SHP1, CD3 subunits, CD28 and Zap70 were also identified. While significant enrichment of SHP1 following 10 and 20 min of PD-1 ligation (as compared to TCR stimulation alone) was detected, the degree of enrichment was markedly lower as compared to SHP2 or PIEZO1 (Fig. IE, Fig. 7C). Although CD3 subunits, CD28 and ZAP70 were identified as binding to PD-1, significant increases in association of these proteins with PD-1 following PD-L1 ligation were not observed. These data suggest that CD3 subunits, CD28 and ZAP70 remain proximal to PD-1 at the immune synapse in the presence or absence of PD-L1 ligation (Fig. 1F-1G, Fig. 7D-I and Fig. 8A-D). One-way hierarchical clustering confirmed different recruitment kinetics for SHP1, CD3 subunits, CD28 or Zap70 (Fig. 1H, Fig. 8E). The limited changes in PD-1 binding by known mediators as compared to the robust, ligand-dependent association of PIEZO 1 and PD-1 implicates an uncharacterized mechanism of PD-1 that involves PIEZO1 function.
To further assess the landscape of protein localization changes proximal to PD-1, gProfiler g:OSt gene ontology (GO) term functional enrichment analysis was used to compare pathways that were enriched in TCR-PD-L1 or TCR-control stimulated conditions. It was found that clathrin-mediated endocytosis and membrane trafficking were among the most significantly enriched pathways in TCR-PD-L1 conditions as compared to TCR-controls, suggesting that PD-1 ligation may alter receptor clustering and turnover in the plasma membrane to prevent stable contact with antigen presenting cells at the immune synapse (Fig. II). As anticipated, signaling pathways involved in TCR and immune response activation, as well as actin cytoskeleton organization, were enriched in TCR- control conditions compared to those treated with TCR-PD-L1 beads (Fig. II). When the mean slope for each gene across all time points was calculated in both TCR-PD-L1 and TCR-control conditions as a means to rank the most highly enriched proteins, SHP2 and PIEZO 1 ranked highest along with sorting nexins involved in intracellular trafficking (SNX12), actin polymerizing subunits (ARPC2, TFG) and membrane transporters (SLC34A1, SLC44A1) (Fig. 1J). In addition, the observed enrichment for PD-1 following PD-L1 ligation signifies an increase in local PD-1 concentration, indicating clustering of PD-1 molecules that undergo enhanced biotinylation (Fig. 1 J). As expected, various TCR- and CD28-related genes enriched in the TCR-control -treated samples, including CD28, CD3^, ZAP70, VAV3 and PI3K-related proteins were identified (Fig. 1 J). Collectively, these data highlight the changing membrane dynamics that occur following TCR stimulation and PD-L1 ligation in T cells, which not only confirm previous findings, but also identify the association between PD-1 and PIEZO1.
Example 2: PD-1 ligation inhibits PIEZOl-mediated Ca2+ influx
Since PIEZO 1 activation has been shown to promote optimal TCR signaling and PD-1 ligation attenuates T cell function, it was hypothesized that PD-1 association with PIEZO 1 would result in PIEZO 1 inhibition and thereby impair T cell activation. Upon TCR stimulation, there is a tightly regulated response to Ca2+ influx, with Ca2+ release-activated channels (CRAC) and voltage gated Ca2+ channels being necessary for the TCR signaling cascade. Due to the involvement of multiple Ca2+ channels, uncoupling the individual contribution of each ion channel during T cell activation is challenging. The role of PIEZO 1 in T cell activation and its regulation by PD-1 using a Jurkat cell line stably expressing an inducible, fluorescent PIEZO 1 -specific activity reporter (GenEPi) was exploreed. GenEPi contains a low affinity and high dynamic range GFP, calmodulin and Ml 3 peptide fusion protein (GCaMP) engineered to ensure the specificity of sensing PIEZO- 1 -mediated Ca2+ influx. The GenEPi reporter was expressed using a Tet-On system and is comprised of GCaMP fused to the cytoplasmic tail of PIEZO 1 using a flexible linker (Fig. 2A). GenEPi Jurkat cells exhibited increasing levels of reporter expression and PIEZO 1 activity with increasing doses of doxycycline in the presence of the PIEZO 1 agonist Yodal, verifying the inducibility and functional specificity of GenEPi (Fig. 9A).
To visualize and compare changes in PIEZO1 activity in the presence or absence of PD-1 ligation, PD-1 -expressing GenEPi Jurkat cells were passed through a flow-cell chamber coated with crosslinking CD3 and CD28 antibodies and either PD-L1 or control mlgGi ligand (Fig. 9B). Using Total Internal Reflection Fluorescence (TIRF) time lapse imaging, the intensity of PIEZO 1 activity and PD-1 expression were assessed at the ligandcell interface at various times after TCR-control and TCR-PD-L1 stimulation (Fig. 2B and 2C and Fig. 9B). It was found that PIEZO 1 activation in GenEPi Jurkat cells ligated with PD-L1 was delayed and had lower intensity overall as compared to TCR-stimulated cells, while PD-1 intensity remained the same in both conditions, suggesting that PD-1 inhibits PIEZO 1 activation (Fig. 2B and 2E, Fig. 9C). Specifically, GenEPi Jurkat cells exhibited strong PIEZO 1 activity following 100 s of TCR stimulation that peaked at 200 s and persisted for 5 min (300 s), further confirming that TCR stimulation induces PIEZO1 activity (Fig. 2B, and 2E-2F, Fig. 9D, and captured via extended videos, incorporated herein by reference). This activity coincided with F-actin ring formation, where PIEZO1 activity appeared to localize around F-actin ring structures (Fig. 2B). A significant decrease in PIEZO1 activity over time in the presence of PD-L1 was observed; PIEZO1 activity was visible starting at 140 s, but remained less intense than in TCR-stimulated conditions throughout the time course (Fig. 2C, and 2E-2F, and captured via extended videos, incorporated herein by reference). Interestingly, local PIEZO 1 activity was significantly decreased upon PD-1 ligation in the presence of PD-L1 at PD-1 clustering sites during the process of immune synapse and F-actin ring formation implying that PD-1 ligation results in the attenuation of PIEZO 1 activity through physical association (Fig. 2C,2G-2K).
In complementary studies flow cytometry was used to assess changes in PIEZO 1 activity following PD-1 ligation by quantifying mean fluorescence intensity (MFI) of GCaMP fluorescence and frequency of GFP -positive, PD-1 -expressing GenEPi Jurkat cells treated with TCR-control or TCR-PD-L1 beads. PD-1 -expressing GenEPi Jurkat cells exhibited a significant decrease in PIEZO1 activity following exposure to TCR-PD-L1 beads compared to GenEPi Jurkat cells treated with TCR-control beads or PIEZO 1 agonist Yodal (Fig. 9E-G). Together, these data imply that contractile F-actin rings formed at the immune synapse provide sufficient force to gate PIEZO1 in an open conformation, thereby sustaining Ca2+ influx for optimal TCR signaling (Fig. 2C). Importantly, it was demonstrated that this activity is inhibited by PD-1 ligation (Fig. 2C).
Example 3: CD8+-specific PIEZO1 KO impairs antitumor immunity
To investigate PD-1 and PIEZO1 function specifically in CD8+ T cells, a tamoxifen- inducible CD8+-specific PIEZO 1 KO mouse was developed by crossing Piezo l^x mice with E8i-Cre-ERT2 Rosa26 tdTomato reporter mice (Extended Data Fig. 6a). Following tamoxifen treatment, the efficacy of PIEZO 1 deletion was -70% in Cre+ CD8+ T cells as compared to Cre- controls, as assessed by RT-qPCR using probes specific for the loxP regions flanking exons 20-23 of PIEZO 1 (Fig. 10B). The frequency of CD8+ T cells expressing Cre protein was consistent with decreased PIEZO 1 transcript levels assessed by RT-qPCR (Fig. 10B-D). As an additional control, the level of Cre reporter in CD8+ splenocytes was assessed on day 15 following tumor implantation and observed sustained Cre activity in Cre+ CD8+ T cells, confirming the efficacy of our inducible Cre system (Fig. 10E,F). Functionally, a significant decrease in PIEZO- 1 -specific Ca2+ influx induced by Yodal treatment (visualized using the cell permeable Ca2+ dye Indo-1) was observed in Cre+ CD8+ T cells compared to Cre- CD8+ T cells; Fig. 10G). In vitro stimulation of tamoxifen-treated Cre+ E8i-Cre-ERT2 Piezo l^x/^x CD?>+ T cells with a titration of crosslinking CD3 and CD28 antibodies revealed slight decreases in viability, CD44, granzyme B and IFNy/TNFoc co-expression, as well as increased expression of CD62L at higher CD3/CD28 stimulation concentrations compared to controls (Extended Data Fig. 7a,d-f,h), suggesting that PIEZO 1 KO impairs CD8+ T cell activation and effector function. Significant differences were not observed in PD-1, CTLA4 and Ki-67 expression or production of IL-2, IFNy or TNFoc between the two groups (Fig. 11B-C, G, LK).
Given the increase in PIEZO 1 activity observed following TCR stimulation (Fig. 2D), it was hypothesized that loss of PIEZO 1 in CD8+ T cells would lead to more rapid tumor growth as a consequence of impaired T cell activation. Indeed, when MC38 or Bl 6- OVA tumor cells were implanted in tamoxifen-treated E8i-Cre-ERT2 Piezo
Figure imgf000058_0001
mice and tumor growth was monitored, loss of PIEZO 1 in CD8+ T cells alone was sufficient to drive significantly increased tumor burden in Cre+ mice as compared to Cre- controls in both tumor models (Fig. 3A-3C). There also was a significant decrease in CD8+ T cell frequencies (Fig. 3D-3E and Fig. 12A), an increase in total CD4+ T cell frequencies and lower CD8+/CD4+ T cell ratios in the tumor with no significant changes in CD3e frequencies (Fig. 12A-G). In addition, Cre+ CD8+ TILs from MC38 tumors expressed significantly more CD62L and significantly less PD-1, CTLA4 and CD69 compared to Cre- controls (Fig. 3F and Fig. 12H-J). Cre+ CD8+ TILs from B16-0VA tumors also expressed significantly more CD62L and significantly less CTLA4 as compared to Cre- controls but did not show significant differences in the frequencies of PD-1- or CD69-expressing cells although both trended toward decreased frequency (Fig. 3G and Fig. 12K-M). Moreover, Cre+ CD8+ TILs from B16-0VA tumors expressed significantly less granzyme B. CD8+ TILs from MC38 tumors also expressed less granzyme B, but this trend was not significant (Fig. 12N,O). Interestingly, PIEZO1 KO CD8+ TILs from MC38 and B16-0VA models consistently retained high expression of Slamf6 and had minimal expression of TIM-3 (Fig. 3H and 31, and Fig. 12P-S), suggesting that PIEZO1 is involved in regulating the transition of Slamf6 progenitor cells to TIM-3 effector-like CD8+ TILs, thereby controlling exhausted CD8+ T cell subpopulations. Together, these findings demonstrate that loss of PIEZO 1 activity in CD8+ T cells decreases their activation and acquisition of effector functions to impair antitumor immunity and promote tumor growth, similar to PD-1 -mediated inhibition of T cell activation.
To further study PD-1 regulation of PIEZO 1, E8i-CRE-ERT2Pzezo7-/Zx^x Cre+ and Cre- tumor-bearing mice were treated with anti-PD-1 or isotype control antibody (Fig. 3J). Strikingly, administration of PD-1 blocking antibody on days 14 and 17 reduced clearance of MC38 tumors, which are highly sensitive to PD-1 blockade, in E8i-CRE-ERT2 Piezo Plx f'x Cre+ mice compared to anti-PD-1 treated Cre- mice (Fig. 3K and Fig. 13A). Administration of PD-1 blockade on days 10 and 13 following tumor implantation did not significantly reduce growth of Bl 6-0 VA tumors in E8i-CRE-ERT2 Piezo l^x/:flx CXQ+ mice in contrast to marked tumor control in anti-PD-1 treated Cre- mice (Fig. 3J,L, and Fig. 13B). PIEZO1 KO in CD8+ T cells significantly impaired overall survival in both tumor models regardless of PD-1 blockade (Fig. 3M and 3N). Thus, PD-l-mediated inhibition of PIEZO1 in CD8+ T cells contributes to immune evasion by tumors, underscoring the importance of PIEZO 1 function in shaping T cell responses.
Example 4: PIEZO1 agonism improves CD8+ T cell antitumor immunity
The impaired antitumor immunity observed in CD8+-specific PIEZO 1 KO mice led us to ask whether PIEZO1 agonism would enhance antitumor immunity. To answer this question, MC38 tumors were implanted subcutaneously in WT mice and administered Yodal (7.5mg/kg) intraperitoneally (i.p.) (Fig. 4A). While Yodal treatment had variable effects on MC38 tumor growth in WT mice (Fig.14A-B), phenotypic analysis of the TILs at day 15 following MC38 tumor implantation revealed increased percentages of CD8+ TILs in Yodal-treated mice compared to controls (Fig. 4B). Additionally, Yodal-treated CD8+ TILs expressed more activation and effector proteins, including PD-1, granzyme B and perforin and lower expression of CD62L (Fig. 4C-F). PIEZO 1 -agonized CD8+ TILs expressed high levels of terminal exhaustion marker TIM-3 and low levels of progenitor stem-like marker Slamf6 compared to controls, further suggesting a role for PIEZO 1 in driving an effector-exhausted phenotype (Fig. 4G, Fig. l4C-D).
Next considered was if the increased CD8+ T cell effector function observed in PIEZO 1 -agonized CD8+ TILs would improve the efficacy of PD-1 blockade. To assess the effects of combined anti-PD-1 and PIEZO1 agonist Yodal treatment, the B16.F10 tumor model was chosen because it is unresponsive to PD-1 blockade (Fig. 4H). Mice treated with either PD-1 blockade or Yodal alone did not show decreased tumor growth (Fig. 41 and 4J). In marked contrast, tumor growth was significantly attenuated when PD-1 blockade and Yodal were administered together (Fig. 41 and 4J). Moreover, combined anti-PD-1 and Yodal treatment significantly improved survival in B16.F10 tumor-bearing mice as compared to single and negative controls, such that 40% of mice treated with combination therapy - but not mice treated with monotherapy - survived beyond 30 days (Fig. 4K). These findings point to PIEZO 1 agonism as a potential avenue for therapeutic intervention in cancer.
Example 5: Discussion
While PD-1 signaling counters T cell activation, the understanding of the signaling mechanisms by which PD-1 exerts its inhibitory functions is incomplete. Herein a novel function was identified for PD-1 in restraining T cell signals driven by a mechanosensitive ion channel. Specifically, PD-1 ligation induces co-localization of PD-1 with PIEZO 1 and this association impairs PIEZO 1 activation and subsequent Ca2+ influx that drives T cell activation, acquisition of effector functions and key cell fate decisions. Knockout of PIEZO 1 in murine CD8+ T cells alone is sufficient to impair CD8+ TIL-mediated tumor control and this cannot be overcome by PD-1 blockade. Conversely, PIEZO 1 -agonism improves CD8+ T cell activation and function in the TME and has significant therapeutic benefit in mouse models when combined with PD-1 blockade to control tumor growth. These findings are the first to document coinhibitory receptor-mediated negative regulation of a mechanosensor and highlight PIEZO1 agonism in combination with PD-1 blockade as a possible therapeutic approach for targeting immune checkpoint blockade-resistant cancers.
Recent studies have demonstrated that PIEZO 1 -driven Ca2+ influx in T cells leads to calpain activation and reorganization of cortical F-actin scaffolding, linking PIEZO 1 to optimal TCR signaling. In contrast, PD-1 ligation has been shown to impair F-actin clearance from the cellular interface, reduce Ca2+ signaling and abrogate tumor cell killing through decreased tumor-T cell couplings. These findings confirm these opposing functions of PD-1 and PIEZO 1. Based on the data, PD-1 targets PIEZO 1 to prevent optimal T cell activation through inhibition of Ca2+ influx and destabilization of the immune synapse (Fig. 2). This model is supported by the in vitro studies, which demonstrate that PD-1 ligation inhibits PIEZO 1 at the immune synapse, and the in vivo studies provided herein, which highlight the necessity of PIEZO 1 for promoting optimal CD8+ TIL function in tumor models and the inability of PD-1 blockade to reverse accelerated tumor growth resulting from PIEZO 1 KO in CD8+ T cells (Fig. 2, 3). These findings imply that expression of PIEZO1 is required for PD-1 to fully execute T cell inhibition (Fig. 3K-3N). Although PIEZO 1 agonism alone is not sufficient to consistently decrease tumor burden in WT mice (Fig. 14A,B), combining PD-1 blockade with PIEZO1 agonism improved tumor control in PD-1 blockade-unresponsive tumors (Fig. 4H-4K). The effectiveness of this combination may result from 1) prevention of PIEZO 1 inactivation as well as inhibitory phosphatase recruitment by PD-1 blockade and 2) PIEZO1 agonism further increasing the number and function of cytotoxic CD8+ T cells in the tumor, leading to enhanced tumor control.
PD-l-mediated inhibition of PIEZO1 is demonstrated herein (Fig. 2). PIEZO1 activity may be regulated by TCR-induced cytoskeletal rearrangements, specifically retrograde actin flow, which is supported by the data capturing PIEZO 1 activity concentrated around F-actin ring structures. The mechanism by which Ca2+ influx is sustained to induce specific gene transcription following CRAC channel activation in T cells remains unclear. Previous studies have proposed a positive feedback loop between F- actin polymerization and Ca2+ influx, which persists over hours at the immune synapse to maintain TCR signaling7. It is possible that actomyosin contractile forces modulate PIEZO 1 to sustain Ca2+ influx, resulting in downstream gene transcription. Moreover, the proximity proteomics experiments demonstrate PD-1 co-localization with various proteins involved in actin polymerization, membrane trafficking and endocytosis (Fig. 1H-J). Thus, PD-1 sequestration of actin cytoskeletal regulatory proteins and proteins that mediate membrane turnover may play a role in impairing actin cytoskeletal dynamics and stability at the immune synapse, thereby attenuating actomyosin contractile forces that activate PIEZO 1.
Although previous proteomic methods have been used to characterize PD-l- mediated T cell inhibition, PD-1 co-localization with PIEZO1 and subsequent inhibition of PIEZO1 have never been documented. However, rather than studying PD-1 in isolation, insight into the spatial and temporal behavior of proteins involved in PD-1 signaling at the membrane was gained by using a multiplexed proximity proteomic approach that assesses changes in membrane dynamics, receptor clustering and protein recruitment immediately proximal to PD-1. Given the dynamic nature of immune cell receptor signaling, high- resolution proximity proteomic methods may be instrumental in studying additional macromolecular immunoreceptor complexes for which, despite clinical relevance, signaling remains largely unclear.
In summary, a new mechanism by which PD-1 exerts its inhibitory signals through negative regulation of the mechanosensitive ion channel PIEZO 1 is described herein. These studies are the first to demonstrate PD-1 co-localization with PIEZO 1 following PD-L1 ligation. This association results in the inhibition of PIEZO 1 -mediated Ca2+ influx, while TCR/CD28 stimulation in the absence of PD-L1 ligation promotes PIEZO1 activity around F-actin rings. The selective inhibition of PIEZO 1 by PD-1 illuminates the significance of PIEZO 1 in regulating CD8+ T cell function and introduces a novel receptor target class. Moreover, these findings in mice show that modulating PIEZO 1 activity influences the anti-tumor activity of CD8+ T cells, subsequently affecting tumor growth. Strikingly, combined therapy using PIEZO1 agonist and PD-1 blockade improved tumor control in mice harboring tumors unresponsive to PD-1 blockade. Together, these studies expand our understanding of the diverse mechanisms by which PD-1 inhibits T cell function, identify a novel role for PIEZO 1 in controlling CD8+ T cell function and reveal a new combination therapeutic strategy using PD-1 blockade and PIEZO1 agonism. Further understanding of the mechanisms controlling PD-1 and PIEZO 1 association, the specific downstream pathways involved and the effects of external mechanical force on this biology should provide additional fundamental and therapeutic insights.
Example 6: Methods
Cell lines
Jurkat E6.1 and human PD-1 -expressing Jurkat E6.1 cells (35.8 line transduced with an ORF-T2A/IRES-GFP reporter) (a gift from N. Haining/K. Yates, Dana Farber Cancer Institute) were cultured in RPMI media supplemented with 10% FBS, 1% penicillin/streptomycin, 1% HEPES and 0.1% BME (denoted RIO). MC38-OVA (a gift from N. Haining/N. Collins, Dana-Farber Cancer Institute), B16-OVA (generated in collaboration with the N. Haining lab), MC38-WT (a gift from D. Vignali, University of Pittsburgh School of Medicine), B16.F10 (a gift from G. Dranoff, Novartis Institutes for Biomedical Research, Cambridge, MA) and 293x (a gift from C. Kadoch, Dana-Farber Cancer Institute) cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin and 20 pg/mL gentamicin. MC38-OVA and B16-OVA cells were produced by transduction of parental MC38 and B16.F10 cells with the lentiviral vector TRC-pLX305 (Broad Institute) containing OVA protein.
PD-1-APEX2 plasmid design, cloning and stable expression
PD-1 sequences were obtained from NCBI and were modified using the codon optimization tool from Integrated DNA Technologies (IDT). APEX2 sequences have been previously described17. PD-1-APEX2 fusion sequences contain human or murine PD-1 linked to APEX2 on the C-terminal tail by an 8-residue serine-glycine linker (GGSSGGSS) and a FLAG-TAG added to the C-terminal tail of APEX2. All human and murine PD-1- APEX2 double-stranded gene fragments (gBlocks) were generated by IDT. Cloning of gBlock gene fragments into the pXPR_053 plasmid (Addgene #113591) was completed by GenScript.
To generate stable PD-l-APEX2-expressing Jurkat cell lines, 293x cells were transfected with PD-1-APEX2 fusion plasmids along with PsPax2 and MD2G packaging plasmids suspended in Optimem (Life Technologies #31985-062) and polyethylamine “Max” (PEI, Polysciences #24765-2), and supplemented with fresh media after 24 h. Lentivirus was harvested 48 h following media supplementation by ultracentrifugation for 2 h at 71,934.8 RCF at 4°C. Viral titer was calculated using serial dilutions of the viral stock on 293x cells in DMEM complete media containing PEI “Max”. A multiplicity of infection of 30 was used to spin transduce Jurkat cells for 2 h at 652 RCF at 37°C. Jurkat cells were stained with human PD-1 surface antibody (BioLegend, clone EH12.2H7, #329936) and evaluated for PD-1 expression using flow cytometry 24 h and 48 h following transduction. Highly expressing PD-1-APEX2 fusion clones were sorted on the BD Aria II. Stocks were frozen in RIO media containing 10% dimethyl sulfoxide (DMSO) for future experiments.
APEX2 proximity labeling and sample processing
PD-l-APEX2-expressing Jurkat cells were cultured at a concentration of 1 million cells/mL in labeling media (RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% HEPES, 0.1% BME and 500|iM biotinyl tyramide purchased from Toronto Research Chemicals) for 2 h. TCR-PD-L1 or TCR-control Dynabeads (see supplemental methods for PD-L1 bead generation) were added to PD-l-APEX2-expressing Jurkat cells at a ratio of 1 :4 cells/beads for the specified timepoints. No beads were added to the 0 min control to assess protein localization at baseline. Following each timepoint, hydrogen peroxide (H2O2, Sigma Aldrich) was added at a final concentration of ImM for 1 min to each condition not including the no H 2 O2 -treated control used to quantify background labelling. The labeling reaction was quenched using 2X quenching buffer (DPBS supplemented with 20mM sodium ascorbate, lOmM trolox and 20mM sodium azide) and cells were spun down immediately at 726 RCF for 2 min. Cells were immediately washed three times with ice-cold IX quenching buffer and spun down at 726 RCF for 2 min after each wash. Following the third IX quenching wash, cells were washed with 10 mL ice-cold PBS and 1 million cells were taken for Western blotting analysis. Cells were then spun down a final time at 726 RCF for 2 min. All PBS was removed from cell pellets, which were flash-frozen on dry ice and stored at -80°C until processing and streptavidin pulldown.
PD-l-APEX2-expressing Jurkat cell pellets were processed using previously described methods with slight modification3. Cell pellets were lysed in filtered 8M urea with 1% sodium dodecyl sulfate (SDS) at room temperature for 15 min. 55% ice-cold trichloroacetic acid (TCA) was added to lysates at a 1 : 1 ratio and left on ice for 15 min to precipitate the protein. Lysates were spun down at 20,817 RCF for 15 min at room temperature to prevent urea from precipitating out of solution. The supernatant was discarded, and protein pellets were washed 4X with ice-cold acetone and spun down at 20,817 RCF for 10 min following each wash. Protein pellets were then air-dried and resuspended in 8M urea lysis buffer supplemented with 1% SDS and lOmM tris(2- carboxyethyl)phosphine (TCEP, Sigma) and 100 mM NH4CO3. Pellets were water-bath sonicated for 3 cycles of 30 s and vortexed mixed at 37°C for 2 h until the pellets were completely resuspended. Redissolved pellets were checked for an alkaline pH and then spun down at 20,817 RCF for 15 min at room temperature. Clear supernatants were transferred to new microcentrifuge tubes and freshly prepared 50mM ammonium bicarbonate (NH4CO3) containing 400mM iodoacetamide was added to each sample at a final concentration of 20mM iodoacetamide. Samples were immediately vortexed and incubated in the dark at room temperature for 25 min. During the incubation period, streptavidin beads (Pierce) were washed twice with 4M urea and 0.5% SDS. Alkylation of cysteine resides was quenched using a solution of lOOmM dithiothreitol (DTT) containing streptavidin beads such that each sample received l OOpL beads and reached a final concentration of 50mM DTT, 4M urea and 0.5% SDS (1 : 1). Samples containing streptavidin beads were rotated overnight at 4°C and the following day were applied to the Dyna-Mag-2 to remove the supernatant. Beads were washed with 4M urea and 0.5% SDS and transferred to new Eppendorf tubes to decrease background signal. Beads were washed 3X more with 4M urea and 0.5% SDS followed by 3 washes with 4M urea to remove all detergent.
Mass spectrometry sample preparation
Streptavidin beads were digested with lysyl endopeptidase (LysC, 2mg/mL, Wako) in 50pL 200mM 3 -[4-(2-hydroxyethyl)piperazin-l-yl]propane-l -sulfonic acid (EPPS) pH 8.5 and 2% acetonitrile at 37°C. Trypsin (50pL stock, Promega #V5111, in EPPS buffer and at a final dilution of 1 : 100) was added for additional digest overnight at 37°C. Beads were removed with a magnetic rack and clear supernatants were transferred to new tubes. Peptide digest reactions were directly labelled with TMT1 Iplex (Thermo Fisher Scientific #A34808) reagents in 200mM EPPS pH 8.5, 30% acetonitrile for 1 h at room temperature. Labeling efficiency was measured by MS of mixed small aliquots of the labeling reactions and was >95%. Frozen and thawed TMT labeling reactions were quenched with 0.3% hydroxylamine for 15 min at room temperature. Reactions were then mixed, dried in a speed vac centrifuge to near completion and subjected to alkaline reversed phase fractionation (Thermo Fisher Scientific #84868) with 12 elution subsequent fractions of 10%, 12.5%, 15%, 17.5%, 20%, 25%, 30%, 35%, 40%, 50%, 65% and 80% acetonitrile. Fraction pairs 1+7, 2+8, 3+9, 4+10, 5+11, 6+12 were mixed, dried down to completion, desalted with Stage tips and run on Orbitrap Lumos mass spectrometers (Thermo Fisher Scientific).
Mass spectrometry analysis
Data collection followed a synchronous precursor selection (SPS) MS3 TMT method49. Peptides were separated prior to the electrospray ionization with a Proxeon EASY-nLC1200 system over a ~35cm capillary column of 100pm inner diameter packed with Accucore C18 beads (2.6 pm, 150A, Thermo Fisher Scientific). Service MS1 scans were performed in the Orbitrap at a resolution of 120,000, mass range 400-1400 thomson (Th). After collision induced dissociation (CID, CE=35) and using an isolation window of 0.4 m/z, MS2 was performed in the ion-trap with maximum injection times of 150-400 ms. For MS3 quantification, precursors were selected following a Top 10 method followed by high-energy collision-induced dissociation (HCD, CE=65). Orbitrap MS3 analysis was done at a resolution of 60,000 at 200 Th with varying injection times of up to 650 ms and chargestate dependent variable isolation windows from 0.7 to 1.2 Da as described previously50. Peptide-spectrum matches (PSM) were obtained by a SEQUEST (v.28, rev.12) based software, searching a database with respective UniProt mouse and human reference proteomes with added common contaminants and reverse peptide sequences as decoy. After mzXML conversion of spectra, searches used a mass tolerance of 20 p.p.m. for precursors and a fragment-ion tolerance of 0.9 Da. Searches allowed for up to two missed trypsin cleavage sites with dynamic modification of oxidized methionines (+15.9949 Da) and static peptide N-terminal and lysine modifications with TMT11 (+229.1629 Da). PSM were filtered by linear discriminant analysis with a false discovery rate (FDR) of 1% and a following FDR of 1% for collapsed proteins. MS1 data were calibrated post search and searched again. TMT signal to noise quantification for peptides was filtered for an MS2 isolation specificity of 70% or greater and a summed signal to noise of 200 or greater for all TMT channels for each peptide. Details of TMT intensity quantification applied were described previously50. Scaled quantification data were analyzed by one-way clustering (Ward’s method) using the JMP Pro statistical software package. Proteomics raw data and search results were deposited in the PRIDE archive51 and can be accessed under ProteomeXchange52 accession numbers PXD036136, PXD036207 and PXD036147 for human PD-1-APEX2 experiments and PXD036218 and PXD036216 for murine PD-1- APEX2 experiments.
Summary statistics and GO Term analysis
Raw data from three independent proximity labeling time course experiments were normalized to ACACA. Proteins that were not identified in all three experiments were excluded from the analysis. Each protein was then normalized to its respective 0 min control condition and the mean slope over time for TCR-control and TCR-PD-L1 conditions was calculated for each protein, where larger slope values indicate a stronger association with PD-1. The top 10 mean slope values for TCR-control and TCR-PD-L1 conditions were reported in Fig. Ij. The mean slope ratios between TCR-PD-L1 /TCR- control and TCR-control/TCR-PD-Ll conditions were then calculated for all proteins to assess protein enrichment per condition. The top 50 proteins were selected for GO Term functional enrichment analysis using g-Profiler g:GOSt functional profiling33. Adjusted p- values of significantly enriched functions for TCR-control and TCR-PD-L1 conditions were reported in Fig. li.
Jurkat GenEPi nucleofection and stable clone selection
The GenEPi reporter plasmid (XLGenEPi) was generated by the Pantazis lab. GenEPi reporter expression was driven by a doxycycline-inducible Tet-On system. Jurkat cells were co-nucleofected with the XLGenEPi plasmid and pCMV_pBase at a molecular ratio of 1 : 1 using Lonza’s SE Cell line kit (cat. V4XC-2024) and corresponding CL-120 program on Lonza’s Amaxa-4D Nucleofector. The pCMV_pBase plasmid is a non-viral vector that expresses the piggyBac transposase (pBASe) which, when co-transfected, allows for the integration of the GenEpi transgene into the genome using two terminal repeat domains flanking the GenEPi reporter53. This integration into the genome allows for the generation of stable GenEPi-expressing clones that can be selected for using blasticidin (BSD). 2pg of XLGenEpi and equimolecular amounts of pCMV_pBase plasmids were used to nucleofect 1 million Jurkat cells in cuvettes. Cells were rested in complete RPMI media (RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% HEPES, 0.1% BME, ImM sodium pyruvate and 4.5g/L of glucose) for 24 h following nucleofection. To generate stable clones, GenEPi nucleofected cells were selected with 8pg/mL BSD for 7 days. Cells were then rested in complete RPMI media for 24 h and treated with 200 ng/mL doxycycline for 24 h to induce GenEPi reporter expression. Selected Jurkat cells were then treated with 5pM Yodal and GFP -positive clones were immediately sorted on the BD FACS Aria and cultured to generate stable cell lines.
Flow cell chamber preparation and TIRE imaging
6-channel |i-Slide VI Glass Bottom slides (Ibidi #80607) were coated with Poly-D- Lysine for 1 h at room temperature. Excess Poly-D-Lysine was removed from the channels and the channels were washed 4X with PBS. Chambers were then coated with 2pg/mL CD3 and CD28 crosslinking antibodies and 4pg/mL PD-L1 or mlgGi overnight at 4°C. Slides were then washed 4X with PBS and 80pL of PBS was added to the flow cell chamber to prevent drying out prior to use. Stable GenEPi Jurkat cells were treated with ImmunoCult Human CD3/CD28 T cell activator (STEMCELL Technologies #10971) for 48 h prior to imaging to induce PD-1 expression. GenEPi Jurkat cells were treated with doxycycline (200ng/mL) for 24 h prior to imaging to temporally control and induce GenEPi expression. GenEPi Jurkat cells were then stained with F-actin SPY550-FastAct (1 :500, Cytoskeleton #CY-SC205) and antihuman PD-1 AF647 (1 :50, Biolegend #329910 clone EH12.2H7) for 1 h at 37°C in PBS supplemented with 10% FBS (PBS-F). Cells were washed twice with PBS-F and resuspended at a concentration of 1 million/mL in complete RPMI media.
TIRF imaging was performed using a fully motorized Nikon Ti inverted microscope equipped with a Nikon Ti-TIRF-EM Motorized Illuminator and a Nikon LUN-F Laser Launch with single fiber output (488nm, 90mW;561 nm, 70mW; 640nm, 65mW). To immobilize the sample, 80 pL of stained XLGenEPi Jurkat cells were flowed into one of the 6 coated |i-Slide channels (Ibidi #80607). Flow was established by capillarity using a Kimwipe. Imaging was performed under static conditions (e.g., not flow). Images were collected using an Apo TIRF 100x/1.49 DIC oil immersion objective lens with Nikon NF immersion oil, adjusting the correction collar to minimize spherical aberration. Images were captured with an Andor Zyla 4.2 Plus sCMOS monochrome camera using the 16-bit dual gain digitizer mode, 540 MHz readout rate and 2x2 pixel binning (resulting pixel size 0.1 um/px) and Nikon Elements Acquisition Software AR 5.02. The TIR angle for each channel was adjusted using fluorescence beads in the same flow chambers as used in the experiments and verified with a control sample. Signals from the different channels were acquired sequentially using a Chroma ZT 405/488/561/640 multi-band pass dichroic mirror mounted on a Nikon TIRF filter cube located in the filter cube turret, and band pass emission filters for GenEPi AF488 (Chroma ET525/50m), SPY5550-FastAct (Chroma ET 595/50m) and PD-1 AF647 (Chroma ET 6551p) channels, respectively, located on a Sutter emission filter wheel within the infinity space of the stand. Time-lapse imaging was performed with a time interval of 20 s and total acquisition time of 5 min. An ND 16 filter was introduced in the light-path to reduce irradiation. Imaging conditions were optimized to reduced photobleaching and phototoxicity. Fiji analysis for TIRF imaging
TIRF image analysis was completed using a custom workflow built in Fiji/ImageJ54. The TIRF images were opened in Fiji and split into the different fluorescent channels. A 500 pixel rolling ball background subtraction was applied to reduce background intensities close to 0. The individual PD-1 and PIEZO1 channels were processed by performing a Tophat/difference of Gaussian filter to emphasize the small puncta. The processed images were segmented by intensity -based thresholding. A close filter followed by a size filter was applied to the thresholded masks to generate the final masks for PD-1 and PIEZO1. Regions of interest (ROI) were then drawn to demarcate cell boundaries to calculate the parameters on a per cell basis. The mask areas, the intensities of the signal and all ROIs were measured. Normalized fluorescence intensity for PIEZO 1 activity was calculated from the sum of pixel values within each ROI for TCR-control and TCR-PD-L1 conditions. Only cells expressing both PD-1 and the PIEZO1 GenEPi reporter were analyzed. Representative images were processed using Nikon Imaging Software (NIS) and Fiji/ImageJ. Al-denoise was applied to F-actin channels using NIS. A 25 pixel rolling ball background subtraction was then applied to F-actin channels in Fiji to reduce background. A 50 pixel rolling ball background subtraction was applied to GenEPi reporter and PD-1 channels in Fiji to reduce background. A median filter of 1 pixel was applied to GenEPi reporter and PD-1 channels in Fiji. Gamma adjustments of 0.8 for PD-1 and GenEPi channels and 1.0 for the F-actin channel were also applied to supplemental videos. Coloring for supplemental videos was adjusted such that PD-1 is displayed in magenta, GenEPi in yellow and F-actin in cyan to clearly convey changes in each channel when merged.
Mice
Seven- to ten-week-old age-matched female or male mice were used for all in vivo experiments and seven- to fourteen-week-old mice were used for CD8+ T cell isolation and in vitro experimentation. Wild type C57BL/6J mice were purchased from Jackson Laboratories. E8I-Cre-ERT2 mice were a generous gift from the Vignali lab. Homozygous E8I-Cre-ERT2 mice were crossed with homozygous Piezolflx/flxmice purchased from Jackson Laboratories (Stock #029213). Mice were crossed until they were fixed for the Pi ezolflx/flx mutant allele as assessed by the Transnetyx genotyping service. The Piezol-2 WT probe was used to detect the WT Piezol allele (forward primer:
CTGTCCCCTTCCCCATCAAG; reverse primer: GGGTCCAGGGTAGACAACAG). The L1L2-Bact-P MD probe (forward primer: GCTGGCGCCGGAAC; reverse primer: GCGACTATAGAGATATCAACCACTTTGT) was used to detect the LlL2_Bact_P cassette, composed of an FRT site followed by a lacZ sequence and a loxP site, confirming the integration of the floxed Piezol mutant allele. eGFP E8I-Cre-ERT2 and Rosa26-LSL TD tomato knock-in activity reporters were also quantified by Transnetyx using the eGFP (forward primer: CGTCGTCCTTGAAGAAGATGGT; reverse primer: CACATGAAGCAGCACGACTT) and tdRFP (forward primer: AGATCCACCAGGCCCTGAA; reverse primer: GTCTTGAACTCCACCAGGTAGTG) probes, respectively. Heterozygous Cre mice were bred to generate both Cre+ and Cre- littermates. To induce Piezol deletion, Cre+ and Cre- mice were intraperitoneally injected with 8 doses of lOmg/mL tamoxifen daily. Deletion efficiency was assessed using RT- qPCR with probes Mm01241547_gl spanning exons 21-22 (ThermoFisher #4351372) and Mm01241549_ml spanning exons 23-24 (ThermoFisher #4331182) to specifically assess the region containing loxP sites in exons 20 and 23. Results indicated that Cre+ mice required two copies of the Cre+ allele for -70% PIEZO 1 deletion. Cre+ and Cre- mice Piezolflx/flxmice were then bred separately to ensure the homozygous expression of Cre alleles for efficient gene deletion. All mice were housed in specific pathogen-free conditions and all animal experimentation was performed in accordance with regulations and animal care guidelines from the Harvard Medical School Standing Committee on Animals (IACUC) and the National Institute of Health.
Tumor implantation
Mice were anesthetized with 2.5% 2,2,2-tribromoethanol (Avertin, Sigma-Aldrich catalog no. T48402-25G) and injected in the flank subcutaneously with 2.5 * 105 MC38, B16.F10 or B16-OVA tumor cells. Once palpable tumors were observed, tumors were measured every other day to calculate tumor volume over time. Tumor volume was calculated using the equation (L*W2)/2 where L denotes tumor length and W denotes tumor width. Mice were monitored for body condition and weight loss. Mice with tumors that exceeded 2000mm3, that were severely ulcerated or that infiltrated the i.p. cavity were sacrificed. Tumor infiltrating lymphocyte isolation
Tumors were harvested at day 15 following implantation and processed for analysis. Extracted tumors were mechanically chopped and treated with collagenase type 1 (Worthington Biochemical, #LS004194) and mixed for 25 min at 37°C. Lymphocytes were enriched using a 40/70% Percoll gradient that was centrifuged at 805 RCF for 20 min (no brake). Immune cells were harvested from the interface between 40% and 70% Percoll and resuspended in MACS buffer (PBS with 1 % FBS and 2 mM EDTA) for staining for flow cytometry analyses.
Flow cytometry staining
Cells were surface stained with antibodies listed below at a 1 : 100 dilution (unless otherwise noted) in 96-well V-bottom plates for 45 min in the dark on ice. Samples were washed twice with MACS buffer and fixed for 20 min in the dark at room temperature and permeabilized with FoxP3/Transcription Factor Staining Buffer Set Kit according to the manufacturer’s protocol (eBioscience #00-5523-00). Cells were stained with intracellular antibodies listed below at a 1 : 100 dilution (unless otherwise noted) for 1 h in the dark on ice and washed twice with eBioscience Permeabilization Buffer. All washes used 200p.L of the specified buffers and spins were performed at 726 RCF for 2 min at 4°C. Fixed and stained samples were acquired the following day on the BD FACSymphony and were analyzed using FlowJo software.
Antibodies for flow cytometry and sorting
Flow cytometry analyses were performed on a BD FACSymphony and cell sorting was performed on a BD Aria II. The following fluorescent antibodies were purchased from Biolegend for flow cytometry and cell sorting: human PD-1 (clone EH12.2H7, #329936) and murine CD45.2 (APC-Cy7, clone 104, #109824), CD8b (Alexa Fluor700, clone 53-6.7, #00730), CD44 (APC, clone IM7, #103012), PD-1 (PE-Cy7, RMP1-30, #109110), CD69 (BV421, H1.2F3, #104528), CD62L (BV605, PerCPCy5.5, clone MEL-14), CD3e (BUV395, PerCpCy55, FITC, clone 145-2C11), perforin (PE, clone S16009A), granzyme B (BV421, clone GB11, #515408), CTLA4 (BV605, clone UC10-4B9, #106323), TIM-3 (BV711, BV421, clone RMT3-23, #119727), Slamf6 (PE, APC, clone 330-AJ), IFNy (APC, clone XMG1.2, #505810) and TNFoc (PerCP/Cy5.5, clone MP6-XT22, #506322). The following fluorescent antibodies were purchased from BD Biosciences: CD4 (BUV496, BUV737, clone GK1.5), LFA1 (BV786, clone M17/4) and Ki67 (PerCpCy55, clone B56). LIVE/DEAD Fixable Aqua dead cell stain (1 :600, L34957) and LIVE/DEAD Fixable Near-IR dead cell stain (1 :600, L34976) were purchased from Thermo Fisher Scientific.
In vivo PD-1 blockade and Piezo 1 agonist treatment
Two lOOpg doses of anti-PD-1 (Freeman lab or BioXcell, clone 29F.1A12, #BE0273) or rat IgG2a isotype control (BioXcell, clone 2A3, #BP0089) were administered interperitoneally to mice on days 10 and 13 (B16-OVA model) or 14 and 17 (MC38 model) following tumor implantation. Yodal (Tocris, #5586) was reconstituted in DMSO to reach a final stock concentration of 17 mg/mL. Reconstituted Yodal was freshly formulated with PBS and sonicated prior to each experiment to obtain a dose of 7.5mg/kg per mouse (250pg/200pL) administered interperitoneally 3-5 times between days 5 and 13.
Western blotting
Jurkat cells or tumor cells were lysed in Pierce RIPA Buffer supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (100X) for 15 min on ice. Whole cell lysates were spun down at 20,817 RCF at 4°C for 15 min. Supernatants were collected and transferred to new Eppendorf tubes and I OpL of each lysate was taken for protein estimation using the Pierce BCA Protein Assay Kit to normalize for protein loading. Lysates were denatured with 4X Laemmli Sample Buffer (BioRad) or 4X NuPAGE LDS (Invitrogen) containing beta mercaptoethanol (BME) and boiled for 5 min at 95°C. 15-40pg of protein per lysate was loaded and run on a NuPAGE 4-12% Bis-Tris protein gel and then transferred onto a nitrocellulose membrane. Ponceau staining was performed to check transfer efficiency and protein loading. Membranes were then blocked for 1 h in TBS supplemented with 1% Tween (TBS-T) and 5% milk supplemented with 0.2% Tween at room temperature. Membranes were then incubated with primary antibody Streptactin-HRP (Bio-Rad, 1 :50,000, #1610381) and rocked overnight in blocking buffer at 4°C. Membranes were washed 3X for 5 min in TBS-T buffer prior to imaging. Membranes were then treated with SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher #34580) and imaged using the Amersham Imager 600. PD-L1 bead generation
M-450 Tosyl-activated magnetic Dynabeads (Invitrogen #14013) were washed using the Dyna-Mag-2 in 0.1M sodium phosphate buffer, pH 7.4-8.0. Magnetic beads were then treated with a mixture of human or mouse CD3 crosslinking antibody (BioXcell, human clone OKT3 #BE0001-2, murine clone 145-2C11 #BE0001-l), CD28 (BioXcell, human clone 9.3 #BE0248, murine clone 37.51 #BE0015-l) crosslinking antibody and either recombinant human (for use in Jurkat cells, R&D Systems #156-B7-100) or murine (for use in primary murine CD8+ T cells, R&D Systems #1019-B7-100) PD-L1 fusion protein or recombinant human IgGiK (for use in primary murine CD8+ T cells, SouthernBiotech #015 lk-01) or mouse IgGi (for use in Jurkat cells, SouthernBiotech #0102-01) control protein depending on the PD-1 construct and origin of cells used. Multiple titrations of protein concentrations were tested to determine which ratio generated the most significant inhibition of T cells: 10/90%, 20/80%, 40/60%, 60/40% and 80/20% of TCR/CD28 antibodies and recombinant PD-L1 or control respectively. Antibodies and recombinant protein mixtures were crosslinked to beads using a total of 200pg of protein per ImL of beads. Beads were incubated and rotated for 16-24 h at room temperature. Beads were then applied to the Dyna-Mag-2 to remove supernatant and washed in PBS supplemented with 0.1% BSA and 2mM EDTA pH 7.4. To deactivate the remaining free tosyl groups on the beads, beads were rotated overnight at room temperature in 0.2M Tris- HCL supplemented with 0.1% BSA, pH 8.5. Beads were washed 3X more with PBS buffer supplemented with 0.1% BSA and 2mM EDTA pH 7.4 and reconstituted in this buffer at 100 million beads per ImL and stored at 4°C prior to use.
On-bead protein ratios and cell: bead ratios
Naive murine CD8+ T cells were isolated from spleens of wild type mice using negative selection magnetic-activated cell sorting (MACS) isolation (Miltenyi #130-096- 543). Naive CD8+ T cells were stimulated for 24 h on 96-well U-bottom plates coated with anti-CD3 and anti-CD28 crosslinking antibodies. After 24 h, cells were moved to a new U- bottom plate. Murine TCR-PD-L1 or TCR-control hlgGiK beads containing various protein ratios outlined above were added to CD8+ T cells at various celkbead ratios (1 :2, 1 :4, 1 :6) for 48 h. Supernatant was collected 48 h following the addition of beads for cytometric bead array (CBA) analysis (BD Biosciences). After 48 h, CD8+ T cells were stained and acquired the following day on the BD FACSymphony for flow cytometric analysis as described in Flow Cytometry Staining in Main Methods.
Indo-1 Ca2+ flux assay
Naive CD8+ T cells were isolated from the spleens of WT mice or E8i-Cre-ERT2 Piezolflx/flxCre+ or Cre- mice treated with lOmg/mL tamoxifen for 8 days using naive CD8+ T cell MACS. Purified CD8+ T cells were stimulated on 96-well U-bottom dishes coated with 4pg/mL of crosslinking murine CD3 and CD28 antibodies for 48 h. Stimulated CD8+ T cells were then rested for 4-12 h in RIO media and moved to a fresh, uncoated 96- well U-bottom plate. WT or E8i-Cre-ERT2Piezolflx/flxCD8+ T cells were then stained in PBS containing 1% FBS for 1 h at room temperature with near IR fixable LIVE/DEAD stain and 1|1M of Indo-1 (Thermo Fisher Scientific #11226). Cells were then washed twice with 1% FBS PBS and resuspended in RIO media in 5mL round-bottom polystyrene tubes. TCR-PD-L1 or TCR-control beads were added at a ratio of 1 :5 celkbead and Yodal was added to samples at a final concentration of 5|1M. Following addition of PIEZO1 agonist or beads for specified timepoints, cells were immediately acquired for 1 min using tube mode on the BD FACSymphony or BD LSRII and analyzed using FlowJo software. Ca2+ influx was quantified by calculating the ratio of bound (BUV395) to unbound (BUV496) Ca2+ using Indo-1.
RT-qPCR
Murine CD8+ T cells were isolated from the spleens and inguinal lymph nodes of Cre+ and Cre- naive mice treated with lOmg/mL tamoxifen for 8 days using CD8+ T cell positive selection MACS (Miltenyi #130-117-044) or cell sorting. Isolated CD8+ T cells were washed with PBS and spun down at 453 RCF for 5 min. PBS was removed from the cell pellets and cell pellets were frozen at -80°C or immediately processed for RNA. RNA was isolated from purified CD8+ T cell pellets using Qiagen’s RNAeasy Mini Kit (#74104). RNA was quantified using the Qubit RNA HS Assay Kit (#Q32852) and RNA concentration was normalized across samples. Reverse transcription was perform using the Superscript VILO cDNA Synthesis Kit (Thermo Fisher Scientific #11-754-050) to generate cDNA. cDNA was diluted and mixed with Pzezof specific fluorescein amidites (FAM) Taqman probes (Thermo Fisher Scientific, Assay ID # Mm01241547_gl and #MmO1241549_ml) and control eukaryotic 18s rRNA FAM probe (Thermo Fisher Scientific, Assay ID #Hs99999901_sl) according to the Fast Advanced Mastermix real time PCR protocol. PCR mixtures were plated in Roche LightCycler 480 96-well plates (#04729692001) and sealed. Plates were run on the Roche LightCycler 480 with a 20 s polymerase activation step at 95°C followed by 40 cycles of 1 s of denaturing at 95°C and 20 s of annealing and extending at 60°C.
PIEZO 1 KO CD8+ T cell stimulation and flow cytometry
E8I-Cre-ERT2 Cre+ and Cre- mice were treated with 8 doses of lOOmg/mL tamoxifen. Naive CD8+ T cells were isolated from spleens of tamoxifen-treated mice using negative selection MACS isolation (Miltenyi). Naive CD8+ T cells were stimulated for 48 h on 96-well U-bottom plates coated with a titration of anti-CD3 and anti-CD28 crosslinking antibodies (0, 0.1, 1.0, 5.0 and 10|ig/mL). Supernatant from each sample was collected 48 h following the addition of beads for CBA analysis (BD Biosciences). After 48 h, CD8+ T cells were stained as described in Flow Cytometry Staining in Main Methods and analyzed on the BD FACSymphony or CytoFLEX (Beckman Coulter).
Incorporation by Reference
All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:
1. A method of treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1.
2. The method of claim 1, wherein the agent is a small molecule agonist of PIEZO 1.
3. The method of claim 2, wherein the small molecule agonist is Yodal, Jedil or Jedi2.
4. The method of any one of claim 1 to 3, wherein the method further comprises administering an immune checkpoint inhibitor to the subject.
5. A method of treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1 and an immune checkpoint inhibitor.
6. The method of claim 4 or 5, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof.
7. The method of claim 6, wherein the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-Ll.
8. The method of any one of claims 4 to 7, wherein the agent that increases or stabilizes the activity or expression of PIEZO 1 and the immune checkpoint inhibitor are administered conjointly.
9. The method of claim 8, wherein the administration of the agent that increases or stabilizes the activity or expression of PIEZO 1 and the immune checkpoint inhibitor act synergistically.
10. The method of claim 1, wherein the agent is a gRNA fused to a transcription activator.
11. The method of claim 10, wherein the gRNA comprises a region that is complementary to a portion of a gene that encodes a PIEZO 1 protein.
12. The method of claim 1, wherein the agent is a vector encoding a PIEZO1 protein, such as a viral vector encoding a PIEZO 1 protein.
13. The method any one of claims 1 to 12, wherein the agent is administered systemically, intravenously, subcutaneously, or intramuscularly.
14. The method of any one of the previous claims, wherein the agent is administered to the subject in a pharmaceutically acceptable formulation.
15. The method of any one of claims 1 to 14, wherein the method further comprises administering an additional agent or cancer therapy.
16. The method of claim 15, wherein i) the additional agent is a chemotherapeutic agent or a cancer vaccine; and/or ii) the cancer therapy is radiation.
17. The method of any one of claims 1 to 16, wherein the subject is refractory for immune checkpoint inhibitory therapy.
18. A method of treating cancer in a subject unresponsive to immune checkpoint inhibitor therapy, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1 and an immune checkpoint inhibitor.
19. The method of claim 18, wherein the agent is a small molecule agonist of PIEZO 1.
20. The method of claim 19, wherein the small molecule agonist is Yodal, Jedi 1 or Jedi2.
21. The method of any one of claim 18 to 20, wherein the immune checkpoint inhibitor therapy comprises an inhibitor of PD-1 or PD-L1.
22. A method of treating cancer in a subject, the method comprising administering to the subject T-cells that have been treated ex vivo with an agent that increases or stabilizes the activity or expression of PIEZO 1.
23. The method of claim 22, wherein the T-cells are tumor infiltrating lymphocytes.
24. The method of claim 22 or claim 23, wherein the T-cells are autologous.
25. The method of claim 22 or claim 23, wherein the T-cells are allogeneic.
26. The method of any one of claims 1 to 25, wherein the subject is a human.
PCT/US2023/032718 2022-09-14 2023-09-14 Methods and compositions for modulation of piezo1 in the treatment of cancer WO2024059183A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263406506P 2022-09-14 2022-09-14
US63/406,506 2022-09-14

Publications (1)

Publication Number Publication Date
WO2024059183A1 true WO2024059183A1 (en) 2024-03-21

Family

ID=88241486

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/032718 WO2024059183A1 (en) 2022-09-14 2023-09-14 Methods and compositions for modulation of piezo1 in the treatment of cancer

Country Status (1)

Country Link
WO (1) WO2024059183A1 (en)

Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4474893A (en) 1981-07-01 1984-10-02 The University of Texas System Cancer Center Recombinant monoclonal antibodies
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5789650A (en) 1990-08-29 1998-08-04 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5798229A (en) 1993-08-02 1998-08-25 Merck Patent Gesellschaft Mit Beschrankter Haftung Bispecific molecules recognizing lymphocyte antigen CD2 and tumor antigens
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
US5874299A (en) 1990-08-29 1999-02-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5877397A (en) 1990-08-29 1999-03-02 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5959084A (en) 1990-10-29 1999-09-28 Chiron Corporation Bispecific antibodies, methods of production and uses thereof
WO2019202473A1 (en) * 2018-04-16 2019-10-24 Université de Lausanne Ucp2 inducing agents for the treatment of cancer resistant to immune checkpoint blockade
WO2020028686A1 (en) * 2018-08-01 2020-02-06 New York University Targeting piezo1 for treatment of cancer and infectious diseases

Patent Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4474893A (en) 1981-07-01 1984-10-02 The University of Texas System Cancer Center Recombinant monoclonal antibodies
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5789650A (en) 1990-08-29 1998-08-04 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5877397A (en) 1990-08-29 1999-03-02 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5874299A (en) 1990-08-29 1999-02-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
US5959084A (en) 1990-10-29 1999-09-28 Chiron Corporation Bispecific antibodies, methods of production and uses thereof
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5798229A (en) 1993-08-02 1998-08-25 Merck Patent Gesellschaft Mit Beschrankter Haftung Bispecific molecules recognizing lymphocyte antigen CD2 and tumor antigens
WO2019202473A1 (en) * 2018-04-16 2019-10-24 Université de Lausanne Ucp2 inducing agents for the treatment of cancer resistant to immune checkpoint blockade
WO2020028686A1 (en) * 2018-08-01 2020-02-06 New York University Targeting piezo1 for treatment of cancer and infectious diseases

Non-Patent Citations (55)

* Cited by examiner, † Cited by third party
Title
BEIDLER ET AL., J. IMMUNOL., vol. 141, 1988, pages 4053 - 4060
BERGERKIMMEL: "Guide to Molecular Cloning Techniques", vol. 152, 1987, ACADEMIC PRESS, INC., article "Methods in Enzymology"
CANE ET AL., SCIENCE, vol. 282, 1998, pages 63
CARRELL ET AL., ANGEW. CHEM. INT. ED. ENGL., vol. 33, 1994, pages 2061
CHAIKEN I. M., CRC CRIT. REV. BIOCHEM., vol. 11, 1981, pages 255
CHEN, J ET AL., EMBO J, vol. 12, 1993, pages 821 - 830
CHEN, J ET AL., INTERNATIONAL IMMUNOLOGY, vol. 5, 1993, pages 647 - 656
CHO ET AL., SCIENCE, vol. 261, 1993, pages 1303
CHOI ET AL., NATURE GENETICS, vol. 4, 1993, pages 117 - 123
CULL ET AL., PROC NATL ACAD SCI USA, vol. 89, 1992, pages 1865 - 1869
CWIRLA ET AL., PROC. NATL. ACAD. SCI., vol. 87, 1990, pages 6378 - 6382
DEWITT ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 90, 1993, pages 6909 - 6448
ERB, PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 11422
FELICI, J. MOL. BIOL., vol. 222, 1991, pages 301 - 310
FISHWILD, D ET AL., NATURE BIOTECHNOLOGY, vol. 14, pages 845 - 851
FODOR, NATURE, vol. 364, 1993, pages 555 - 556
HARDING, F.LONBERG, N., ANN. N. Y ACAD. SCI, vol. 764, 1995, pages 536 - 546
HARDING, FLONBERG, N, ANN. N.Y. ACAD. SCI, vol. 764, 1995, pages 536 - 546
HOUGHTEN, BIOTECHNIQUES, vol. 13, 1992, pages 412 - 421
HUSTON, PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883
JONES ET AL., NATURE, vol. 321, 1986, pages 552 - 525
KAISER ET AL., SCIENCE, vol. 243, 1989, pages 187
KENT, S. B. H., ANNU. REV. BIOCHEM., vol. 57, 1988, pages 957
KIPRIYANOV ET AL., HUMAN ANTIBODIES AND HYBRIDOMAS, vol. 6, 1995, pages 93 - 101
KIPRIYANOV ET AL., MOL. IMMUNOL., vol. 31, 1994, pages 1047 - 1058
KOZARSKYWILSON, CURRENT OPINION IN GENETICS AND DEVELOPMENT, vol. 3, 1993, pages 499 - 503
LAM, ANTICANCER DRUG DES, vol. 12, 1997, pages 145
LAM, NATURE, vol. 354, 1991, pages 82 - 84
LIU ET AL., J. IMMUNOL., vol. 139, 1987, pages 3521 - 3526
LIU ET AL., PROC. NATL. ACAD. SCI. USA, vol. 84, 1987, pages 3439 - 3443
LONBERG, N ET AL., NATURE, vol. 368, no. 6474, 1994, pages 856 - 859
LONBERG, NHUSZAR, D, INTERN. REV. IMMUNOL. VOL., vol. 13, 1995, pages 65 - 93
LONBERG, NHUSZAR, D, INTERN., vol. 13, 1995, pages 65 - 93
MERRIFIELD, B, SCIENCE, vol. 232, 1986, pages 342
MERRIFIELD, J, J. AM. CHEM. SOC., vol. 91, 1969, pages 501
MORRISON, S. L., SCIENCE, vol. 229, 1985, pages 1202 - 1207
NISHIMURA ET AL., CANCER RES, vol. 47, 1987, pages 999 - 1005
OFFORD, R. E.: "Semisynthetic Proteins", 1980, WILEY PUBLISHING
OI ET AL., BIOTECHNIQUES, vol. 4, 1986, pages 214
PASSINI ET AL., METHODS MOL. BIOL., vol. 246, 2004, pages 225 - 36
POLJAK ET AL., STRUCTURE, vol. 2, 1994, pages 1121 - 1123
SCOTTSMITH, SCIENCE, vol. 249, 1990, pages 404 - 406
SHAW ET AL., J. NATL. CANCER, vol. 80, 1988, pages 1553 - 1559
STAERZ ET AL., NATURE, vol. 314, 1985, pages 446 - 449
STAERZBEVAN, IMMUNOL. TODAY, vol. 7, 1986, pages 241
STAERZBEVAN, PROC. NATL. ACAD. SCI. USA, vol. 83, 1986, pages 1453
SUN ET AL., PROC. NATL. ACAD. SCI., vol. 84, 1987, pages 214 - 218
TAYLOR, L ET AL., NUCLEIC ACIDS RESEARCH, vol. 20, 1992, pages 6287 - 6295
TAYLOR, L, INTERNATIONAL IMMUNOLOGY, vol. 6, 1994, pages 579 - 591
TUAILLON ET AL., PROC. NATL. ACAD. SCI USA, vol. 90, 1993, pages 3720 - 3724
TUAILLON, J. IMMUNOL., vol. 152, 1994, pages 2912 - 2920
VERHOEYAN ET AL., SCIENCE, vol. 239, 1988, pages 1534 - 1043
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546
YU JIA-LIN ET AL: "Piezo-type mechanosensitive ion channel component 1 (Piezo1) in human cancer", BIOMEDICINE & PHARMACOTHERAPY, ELSEVIER, FR, vol. 140, 16 May 2021 (2021-05-16), XP086635114, ISSN: 0753-3322, [retrieved on 20210516], DOI: 10.1016/J.BIOPHA.2021.111692 *
ZUCKERMANN ET AL., J. MED. CHEM., vol. 37, 1994, pages 1233 - 85

Similar Documents

Publication Publication Date Title
US11065285B2 (en) Biomarkers and combination therapies using oncolytic virus and immunomodulation
WO2018112032A1 (en) Methods and compositions for targeting tumor-infiltrating tregs using inhibitors of ccr8 and tnfrsf8
US20200010552A1 (en) Therapies based on control of regulatory t cell stability and function via a neuropilin-1:semaphorin axis
CN106459989A (en) Human mesothelin chimeric antigen receptors and uses thereof
US20230190897A1 (en) Dickkopf2 (dkk2) inhibition suppresses tumor formation
US10753938B2 (en) Beta-catenin inhibitors in cancer immunotherapy
US20230340124A1 (en) Methods of treating an individual that has failed an anti-pd-1/anti-pd-l1 therapy
EP4059569A1 (en) Methods and pharmaceutical compositions for enhancing cd8+ t cell-dependent immune responses in subjects suffering from cancer
US20220213212A1 (en) Methods and compositions for treating cancer
JP2019503984A (en) Humanized anti-Dkk2 antibody and method of use thereof
EP3289104B1 (en) Method for treating high-grade gliomas
WO2024059183A1 (en) Methods and compositions for modulation of piezo1 in the treatment of cancer
JP2022500378A (en) Treatment of cancer by inhibiting the ubiquitin-conjugating enzyme E2K (UBE2K)
US20230266332A1 (en) Methods and compositions for preventing and treating a cancer
US20220144926A1 (en) Identification and targeting of pathogenic extracellular matrix for diagnosis and treatment of cancer and other diseases
US20200164034A1 (en) Methods for improving sex-dimorphic responses to targeted therapy in melanoma
WO2023150181A1 (en) Methods and compositions for treating cancer
CA3123046A1 (en) Phosphorylated dicer antibody and methods of use thereof
Varano THE ROLE OF THE B CELL ANTIGEN RECEPTOR IN A MOUSE MODEL OF NON-HODGKIN LYMPHOMA