WO2023230632A2 - Traitement et détection de cancers ayant un phénotype de type neuronal progéniteur, squamoïde/basaloïde/mésenchymateux ou classique - Google Patents

Traitement et détection de cancers ayant un phénotype de type neuronal progéniteur, squamoïde/basaloïde/mésenchymateux ou classique Download PDF

Info

Publication number
WO2023230632A2
WO2023230632A2 PCT/US2023/067607 US2023067607W WO2023230632A2 WO 2023230632 A2 WO2023230632 A2 WO 2023230632A2 US 2023067607 W US2023067607 W US 2023067607W WO 2023230632 A2 WO2023230632 A2 WO 2023230632A2
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptides
gene
cells
sequence
cell
Prior art date
Application number
PCT/US2023/067607
Other languages
English (en)
Other versions
WO2023230632A3 (fr
Inventor
William Hwang
James Guo
Andrew Aguirre
Tyler Jacks
Jennifer Su
Carina SHIAU
Original Assignee
The General Hospital Corporation
Dana-Farber Cancer Institute, Inc.
Massachusetts Institute Of Technology
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 The General Hospital Corporation, Dana-Farber Cancer Institute, Inc., Massachusetts Institute Of Technology filed Critical The General Hospital Corporation
Publication of WO2023230632A2 publication Critical patent/WO2023230632A2/fr
Publication of WO2023230632A3 publication Critical patent/WO2023230632A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the subject matter disclosed herein is generally directed to methods of detecting treatment refractory cancer and treatment thereof.
  • Pancreatic ductal adenocarcinoma is an aggressive and treatment-refractory malignancy without adequate clinically-relevant biomarkers to guide patient-specific management. While genetic aberrations often inform treatment in other cancer types, there is mounting evidence that gene expression profiles (e.g., neural-like progenitor (NRP), squamoid/basaloid/mesenchymal (SBM), classical) may be more robust biomarkers for stratifying pancreatic cancer patients by survival and treatment resistance. However, diagnostic and therapeutic strategies tailored to these transcriptional subtypes remain absent.
  • NRP neural-like progenitor
  • SBM squamoid/basaloid/mesenchymal
  • the present invention provides for a method of treating cancer comprising administering to a subject in need thereof one or more therapeutic agents that target one or more cancer-specific genes or gene expression products selected from the group consisting of: a) TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, SCTR, PDGFD, C6, CRISP3 RELN, SERPINA6, CRP, FGG, APCS, CFH, HABP2, KLKB1, REGIA, and SPP1; b) MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, NECTIN4, COL17A1, MUC16, FGF19, IGF2, NPC2, SAA1, SRGN, CIS, and CST4; or c) CEACAM5, CLDN18, CEACAM6, CDH17,
  • the method further comprises detecting a neural-like progenitor phenotype (NRP), a squamoid/basaloid/mesenchymal (SBM), or a classical phenotype, and wherein (i) if a NRP phenotype is detected, administering one or more therapeutic agents that target one of the gene or gene expression products of group (a); (ii) if a SBM phenotype is detected, administering one or more therapeutic agents that target one or more of the gene or gene expression products of group (b); and (iii) if a classical phenotype is detect, administering one or more therapeutic agents that target one or more of the gene or gene expression products of group (c) or administer a standard of care therapeutic.
  • NRP neural-like progenitor phenotype
  • SBM squamoid/basaloid/mesenchymal
  • the gene encodes a cell-surface polypeptide and wherein the cell-surface polypeptides for group (a) are TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; for group (b) are MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4; and for group (c) are CEACAM5, CLDN18, CEACAM6, CDH17, TSPAN8, MUC13, and MUC17.
  • group (a) are TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; for group (b) are MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A,
  • the present invention provides for a method of treating cancer comprising administering to a subject in need thereof one or more therapeutic agents that target one or more receptor-ligand pairs selected from the group consisting of LAMA5-SDC1, TGFB2- TGFBR2, IL1A-IL1R1, IGF2-IGF1R, EFNA1-EPHA2, TNF-TNFRSF21, GDNF-GFRA1, CXCL8-CXCR2, CXCL2-CXCR2, IL2-IL2RG, CXCL12-CXCR4, IL2-IL2RA, HMGB1-SDC1, and GDNF-RET.
  • the one or more therapeutic agents comprises an adoptive cell therapy (ACT) engineered to bind the one or more cell-surface polypeptides.
  • ACT adoptive cell therapy
  • the ACT is a CAR-T cell or CAR NK cell comprising an engineered CAR receptor capable of binding the one or more cell-surface polypeptides.
  • the one or more agents comprises one or more antibodies specific for one of the one or more polypeptides.
  • the one or more antibodies is an antibody drug conjugate.
  • the one or more antibodies is configured to induce ADCC.
  • the one or more antibodies is a bi-specific antibody targeting a malignant surface marker and an immune cell.
  • the one or more therapeutic agents is a transcriptional repressor system comprising a DNA binding element connected to or otherwise capable of complexing with a transcriptional repressor and configured to bind an enhancer of a gene encoding the one or more polypeptides.
  • the one or more agents is a gene editing system configured to modify a gene encoding the one or more polypeptides, an enhancer associated with the gene encoding one or more polypeptides, or a mRNA encoding the one or more polypeptides such that expression or activity of the one or more polypeptides is reduced.
  • the gene editing system comprises a Cas polypeptide, a guide molecule capable of forming a complex with the Cas polypeptide and directing sequence-specific binding of the complex to a target sequence on the gene or enhancer associated with the gene such that one or more indels or insertions that reduce expression or activity of the one or more polypeptides is introduced into the gene or the enhancer associated with the gene encoding the one or more polypeptides.
  • the gene editing is a CRISPR-associated transposase (CAST) system comprising: (i) a catalytically inactive Cas polypeptide and a transposase linked to or otherwise capable of associating with the Cas polypeptide; (ii) a guide molecule capable of forming a complex with the Cas polypeptide and directing the complex to a target sequence in the gene or enhancer associated with the gene; and (iii) a donor construct comprising a donor sequence for insertion at the target sequence such that insertion of the donor sequence reduced expression or activity of the one or more polypeptides.
  • CAST CRISPR-associated transposase
  • the gene editing system is a prime editing system comprising: (i) a Cas polypeptide having nickase activity and a reverse transcriptase linked to or otherwise capable of associating with the Cas polypeptide; (ii) a prime editing guide RNA (pegRNA) capable of forming a complex with the Cas polypeptide and directing sequence-specific binding of the complex to a target sequence in the gene or enhancer associated with the gene, and wherein the pegRNA comprises a primer binding site configured to hybridize to a portion of a nicked strand of the gene or enhancer associated with the gene, and a reverse transcriptase template comprising a donor sequence encoding one or more edits or insertions that once incorporated into the gene or enhancer associated with the gene reduces expression or activity of the one or more polypeptides.
  • pegRNA prime editing guide RNA
  • the gene editing system is a base editing system comprising: a catalytically inactive Cas polypeptide linked to or otherwise capable of associating with a nucleobase deaminase and a guide molecule capable of forming a complex with the Cas polypeptide and directing sequence-specific binding of the complex to a target sequence on a gene, an enhancer associated with the gene, or a mRNA encoding the one or more polypeptides such that introduction of one or more base edits by the base editing system reduce expression or activity of the one or more polypeptides.
  • the one or more agents is an epigenetic modification polypeptide comprising a DNA binding domain linked to or otherwise capable of associating with an epigenetic modification domain such that binding of the DNA binding domain at target sequence on gDNA results in one or more epigenetic modifications by the epigenetic modification domain that decreases expression of the one or more polypeptides.
  • the one or more agents is an RNAi or antisense oligonucleotide (ASO). In certain embodiments, the one or more agents is a small molecule. In certain embodiments, the one or more agents is a degrader molecule.
  • ASO RNAi or antisense oligonucleotide
  • the subject is resistant to chemotherapy/radiation treatment (CRT).
  • the method further comprises administering the one or more therapeutic agents in combination with or following CRT.
  • the present invention provides for a non-invasive method for detecting expression of one or more genes in a subject having a cancer characterized by a NRP, SBM, or classical phenotype, said method comprising detecting whether one or more cell free transcripts for the one or more genes are present in a biological sample or circulating tumor cells obtained from the subject, wherein the one or more genes are selected from the group consisting of: i) CEACAM5, CLDN18, CEACAM6, CDH17, TSPAN8, MUC13, MUC17, REG4, PLA2G10, TFF1, and NRG4; ii) TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, SCTR, PDGFD, C6, CRISP3, RELN, SERPINA6, CRP, FGG, APCS, CFH, HABP2, KLKB1, REGIA, and SPP1
  • the biological sample is a blood sample
  • the one or more secreted polypeptides are encoded by one or more genes selected from the group consisting of: i) PDGFD, C6, CRISP3, RELN, SERPINA6, CRP, FGG, APCS, CFH, HABP2, and KLKB1, SPP1, CIS, and CST4 and/or ii) C0L17A1, MUC16, FGF19, IGF2, NPC2, SAA1, SRGN, and CIS.
  • the biological sample is a stool sample
  • the one or more secreted polypeptides are encoded by one or more genes selected from the group consisting of: i) PDGFD, CRISP3, RELN, REG11A, and SPP1; ii) C0L17A1, MUC16, and CST4; and/or iii) REG4, PLA2G10, TFF1, and NRG4.
  • the present invention provides for a non-invasive method for detecting expression of one or more polypeptides in a subject having pancreatic cancer, said method comprising detecting whether one or more polypeptides are present on circulating tumor cells obtained from the subject, wherein the one or more polypeptides are encoded by one or more genes selected from the group consisting of: i) CEACAM5, CLDN18, CEACAM6, CDH17, TSPAN8, MUC13, and MUC17; ii) TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; and/or iii) MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4.
  • the circulating tumor cells are obtained using one or more affinity agents specific for one or more polypeptides encoded for by one or more genes selected from the group consisting of: i) CEACAM5, CLDN18, CEACAM6, CDH17, TSPAN8, MUC13, and MUC17; ii) TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; and/or iii) MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4.
  • the present invention provides for a method of treating treatment resistant cancers comprising detecting in a sample obtained from the subject one or more cancer subtypes selected from the group consisting of neural-like progenitor (NRP), squamoid/basaloid/mesenchymal (SBM), and classical subtypes; and if the classical subtype is detected treating the subject with CRT and if the NRP and/or SBM subtype is detected treating with one or more agents according to any embodiment herein, wherein the classical subtype is detected by detecting one or more genes or polypeptides selected from the group consisting of: CEACAM5, CLDN18, CEACAM6, CDH17, TSPAN8, MUC13, and MUC17; or REG4, PLA2G10, TFF1, and NRG4; or TFF1, MUC5AC, LYZ, and MUCl; orHNF4A, GATA6, TFF1, CEACAM5, TSPAN8, FM05, BTNL8, REG4, SYTL2 and CLDN
  • NRP neural-like
  • the present invention provides for a method of stratifying a cancer in a subject in need thereof comprising detecting in a sample obtained from the subject one or more cancer subtypes selected from the group consisting of neural-like progenitor (NRP), squamoid/basaloid/mesenchymal (SBM), and classical subtypes, wherein detection of NRP or SBM subtypes indicates poor prognosis and/or resistance to CRT therapy, wherein the classical subtype is detected by detecting one or more genes or polypeptides selected from the group consisting of: CEACAM5, CLDN18, CEACAM6, CDH17, TSPAN8, MUC13, and MUC17; or REG4, PLA2G10, TFF1, and NRG4; or TFF1, MUC5AC, LYZ, and MUC1; or HNF4A, GATA6, TFF1, CEACAM5, TSPAN8, FM05, BTNL8, REG4, SYTL2 and CLDN18; or wherein the NRP sub
  • NRP neural-like
  • the one or more genes or polypeptides are detected in blood, stool, tumor biopsy, or resected tumor.
  • the one or more genes are detected in cell free RNA (cfRNA).
  • the one or more genes or polypeptides are detected in exosomes, ribonucleoprotein (RNP) complexes, or circulating tumor cells (CTCs).
  • C6, SERPINA6, CRP, FGG, APCS, CFH, HABP2, KLKB1, FGF19, IGF2, NPC2, SAA1, SRGN, and/or CIS are detected in blood.
  • REG4, TFF1, NRG4, REGIA, and/or CST4 are detected in stool.
  • the one or more genes or polypeptides are detected by RNA-seq, PCR, immunohistochemistry (IHC), ELISA, or western blot.
  • the cancer is selected from the group consisting of pancreatic, bladder, ovarian, lung adenocarcinoma, esophagus, colon, lung squamous, cervix, prostate, and head and neck.
  • the cancer subtype is detected before treatment and/or at any time point after treatment.
  • the present invention provides for a kit comprising reagents for detecting a panel of polypeptides for subtyping pancreatic cancer comprising: one or more polypeptides selected from the group consisting of C6, SERPINA6, CRP, FGG, APCS, CFH, HABP2, and KLKB1; and one or more polypeptides selected from the group consisting of FGF19, IGF2, NPC2, SAA1, SRGN, and CIS.
  • the present invention provides for a kit comprising reagents for detecting a panel of polypeptides for subtyping pancreatic cancer comprising: REGIA, and CST4; and one or more polypeptides selected from the group consisting of REG4, TFF1, and NRG4.
  • the present invention provides for a kit comprising reagents for detecting a panel of polypeptides for subtyping pancreatic cancer comprising: one or more polypeptides selected from the group consisting of PDGFD, C6, CRISP3, RELN, SERPINA6, CRP, FGG, APCS, CFH, HABP2, KLKB1, REGIA, SPP1, C7, HDGF, MAPT, REGIB, SERPINA4, PVR, DKK4, DPP7, NPNT, and TAFA1; and one or more polypeptides selected from the group consisting of COL17A1, MUC16, FGF19, IGF2, NPC2, SAA1, SRGN, CIS, CST4, SFN, FGFBP1, MUC16 (CA-125), HDGF, COPA, and CFB; and one or more polypeptides selected from the group consisting of REG4, PLA2G10, TFF1, NRG4, TFF1, MUC5AC, LYZ,
  • the present invention provides for a kit comprising reagents for detecting a panel of genes or polypeptides for subtyping pancreatic cancer comprising: one or more genes or polypeptides selected from the group consisting of TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; and one or more genes or polypeptides selected from the group consisting of MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4; and one or more genes or polypeptides selected from the group consisting of CEACAM5, CLDN18, CEACAM6, CDH17, TSPAN8, MUC13, and MUC17.
  • the panel comprises TM4SF4, CDH17, and CEACAM5.
  • the present invention provides for a kit comprising reagents for detecting a panel of genes or polypeptides for subtyping pancreatic cancer comprising: one or more genes or polypeptides selected from the group consisting of CTNND2, CRP, SERPINA6, NRXN3, PDGFD, C6, RELN, SPP1, GLIS3, NR5A2, DCDC2, DPYD, HNF1B, ZBTB16, KCNJ16, SLC4A4, and BICC1; and one or more genes or polypeptides selected from the group consisting of S100A2, KRT17, KRT7, TACSTD2, TP63, MSLN, SCEL, COL17A1, VIM, FN1, CST6, CST4, TUBB, and LY6D; and one or more genes or polypeptides selected from the group consisting of HNF4A, GATA6, TFF1, CEACAM5, TSPAN8, FMO5, BTNL8, REG4, SYTL2, and CL
  • FIG. 1 Cell surface targets are shared by esophagus, colon, pancreas, and lung cancers. Degree of overlap (hypergeometric test) among top 100 ranked cell surface targets for each of 16 cancer types using RNA-seq data from TCGA.
  • FIG. 2 Computed overlaps between transcriptional program signature genes and predicted secreted proteins from the Human Protein Atlas.
  • FIG. 3 Top 200 weighted consensus NMF genes for each transcriptional program, with blood-secreted proteins labeled.
  • Y-axis represents cNMF weights;
  • x-axis depicts transcriptional subtypes.
  • FIG. 4 Schematic of antibody -based therapeutic strategies against cancer cell surface antigens.
  • FIG. 5A-5B - Fig. 5A Scatterplot of TM4SF4 expression vs. NRP expression.
  • Fig. 5B Scatterplot ofPDAC vs. toxicity-prone tissue scores (y-axis) against PD AC vs. non-malignant pancreas scores (x-axis) (Methods).
  • Variance of gene’s expression transcripts per million, TPM) in TCGA PAAD cohort colored in correspondence with color legend.
  • FIG. 6 Immunohistochemistry of select subtype-specific targets (TM4SF4: NRP; CDH17: classical; CEACAM5: classical) in tumor and non-malignant, toxicity-prone tissues. Representative images of TM4SF4, CDH17, and CEACAM5 staining in pancreatic cancer; of ERBB2 and NECTIN4 in breast and urothelial cancer, respectively, for reference (top row). Target staining in other nonmalignant tissues (rows) also shown. Images provided by the Human Protein Atlas. [0036] FIG. 7A-7B - Spatially correlated receptor ligand pairs across compartments. Spearman rank correlation coefficient of expression of receptor-ligand pairs (gray dots) across paired epithelial: Fig.
  • FIG. 7A epithelial :CAF (left), epithelial :immune (middle), or CAF:immune (right) segments within the same region of interest (ROI) across all ROIs in CRT -treated (y axis) or untreated (x axis) tumors;
  • FIG. 8 Demonstration of individual cell segmentation with the Nanostring CosMx Spatial Molecular Imager platform on untreated PDAC tumor section. Individually localized mRNA transcripts are denoted by colored dots (CEACAM6, LCN2, COL11A1, COL12A1, APOD). Receptors and ligands within the high-plex panel can be evaluated for their expression between neighboring cells of interest. Image from Nanostring CosMx.
  • FIG. 9 Heatmap exhibiting ability of program gene markers (y-axis) to separate/stratify subtypes (x-axis).
  • FIG. 10 The neural-like progenitor program includes “brain tissue enhanced” genes from the Human Protein Atlas (HP A). Left: Overlap between the program (blue) and HPA brain enhanced (orange) genes. Right: HPA expression categories (color code) for select genes (columns) across brain regions (rows).
  • FIG. 11 Multiplexed immunofluorescence images of independent PDAC specimen showing absence of NRXN3 expression (top) and heterogeneous NRXN3 expression (bottom) in malignant cells/glands from two separate regions of the same tumor. Color legend indicates target of fluorophore-conjugated antibodies.
  • FIG. 12 Whole transcriptome spatial profiling as a discovery tool for media growth factors to enhance subtype stability and reconstruct ex vivo milieus.
  • FIG. 13 Whole transcriptome spatial profiling as a discovery tool for media growth factors to enhance subtype stability and reconstruct ex vivo milieus.
  • a “biological sample” may contain whole cells and/or live cells and/or cell debris.
  • the biological sample may contain (or be derived from) a “bodily fluid”.
  • the present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof.
  • Biological samples include cell cultures, bodily fluids,
  • the terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • therapeutic agent refers to a molecule or compound that confers some beneficial effect upon administration to a subject.
  • the beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
  • treatment or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
  • treating includes ameliorating, curing, preventing it from becoming worse, slowing the rate of progression, or preventing the disorder from re-occurring (i.e., to prevent a relapse).
  • the present invention provides for one or more therapeutic agents against combinations of targets identified. Targeting the identified combinations may provide for enhanced or otherwise previously unknown activity in the treatment of disease.
  • the term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g., physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human animals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver’s experience, but that include the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the compositions and therapeutic agents described herein.
  • a caregiver e.g., physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human animals
  • adjuvant therapy refers to any treatment given after primary therapy to increase the chance of long-term disease-free survival.
  • nonadjuvant therapy refers to any treatment given before primary therapy.
  • primary therapy refers to the main treatment used to reduce or eliminate the cancer.
  • Pancreatic ductal adenocarcinoma remains a treatment-refractory disease.
  • Embodiments disclosed herein provide for methods of treating cancers and non-invasive methods for detecting the same, in particular PDAC.
  • Applicants identified pancreatic ductal adenocarcinoma (PDAC) malignant cell programs using single nuclei sequencing (snRNA-seq) of tumors obtained from treatment naive subjects and subjects treated with standard therapies (chemotherapy/radiation or CRT).
  • neural-like progenitor (NRP), squamoid/basaloid/mesenchymal (SBM), and classical gene expression profiles (programs) are robust biomarkers for stratifying pancreatic cancer patients by survival and treatment resistance.
  • Applicants identified a set of specific biomarkers, including cell surface and secreted markers, expressed in malignant cells and tumor microenvironment (TME) cells, that can be used to detect the subtypes and stratify a subject’s cancer.
  • the biomarkers include ligand receptor pairs.
  • the biomarkers are spatially resolved, such that the spatial location of expression in malignant cells and the tumor microenvironment is known. The spatial location can indicate biomarkers that have an effect on specific cancer subtypes.
  • the biomarkers can be targeted with therapeutic agents to treat the cancer based on the subtypes detected.
  • the methods of stratifying a subjects cancer includes detecting one or more of the biomarkers.
  • the biomarkers are detected non- invasively, such as detected in blood or stool.
  • the biomarkers can be targeted with one or more therapeutic agents.
  • the biomarkers can be detected in samples obtained by biopsy.
  • spatially resolved biomarkers are detected in tissue samples obtained by biopsy.
  • the cancers that can be treated and stratified by the methods of the present invention include cancers that are refractory to the standard of care treatments.
  • the cancers express shared surface markers.
  • the cancer includes pancreatic, ovarian, lung adenocarcinoma, esophagus, colon, lung squamous, cervix, head and neck, prostate, and bladder cancer (see, e.g., Robertson, et al., Cell 171, 540-556 (2017), describing a poor-survival ‘neuronal’ subtype).
  • the cancerthat is refractory to a standard treatment expresses a neural-like progenitor (NRP) gene signature or has a NRP phenotype.
  • the cancer that responds to a standard treatment expresses a classical gene signature or has a classic phenotype.
  • NRP neural-like progenitor
  • spatially resolved growth factors such that the growth factors are expressed in cells in spatial proximity are provided.
  • the growth factors can shift cancer subtypes or maintain the subtypes that are in spatial proximity.
  • in vitro pancreatic cell lines, multicellular systems or complex cell populations that faithfully recapitulate a pancreatic cancer subtype in vivo are provided.
  • the cell lines, multicellular systems or complex cell populations can be used as models for screening for vulnerabilities in pancreatic cancer.
  • therapeutic agents are screened.
  • embodiments disclosed herein are directed to methods of treating distinct transcriptional cancer sub-types.
  • Applicant provides herein cell-surface targets associated with each distinct transcriptional sub-types that are expressed at a lower level in non-malignant “normal” tissues.
  • the transcriptional sub-types are neural-like progenitor (NRP), squamoid/basaloid/mesenchymal (SBM) and classical.
  • NTP neural-like progenitor
  • SBM squamoid/basaloid/mesenchymal
  • Gene expression signatures defining each sub-type are described in further detail under the section titled “Gene Signatures and Programs.”
  • a method of treating cancer may comprise administering one or more therapeutic agents that target one or more cancer-specific genes and/or expression products thereof to a subject in need thereof.
  • the one or more cancerspecific genes encode a polypeptide at least partially exposed on the cell surface (i.e., a cell-surface polypeptide).
  • the one or more therapeutic agents may specifically bind the cell surface polypeptide, either to reduce expression or activity thereof, or to deliver a therapeutic payload to the cancer cell.
  • the one or more therapeutic agents may reduce expression and/or activity of the one or more cancer-cell specific polypeptides.
  • the subject may suffer from a cancer having a NRP phenotype, a SBM phenotype, or a classical phenotype.
  • the subject may suffer from pancreatic, bladder, ovarian, lung, adenocarcinoma, esophagus, colon, lung, squamous cell, cervix, prostate, or head and neck cancer.
  • the above embodiments may further comprise assaying a sample from the subject to be treated to determine if the subject has a NRP phenotype, a SBM phenotype and a classical phenotype and selecting the appropriate therapeutic agent for gene or gene expression products specific for the detected phenotype.
  • Example methods for detecting a NRP, a SBM, and a classical phenotype are disclosed in further detail below, all of which may be used in combination with the treatment methods described in this section.
  • the subject to be treated has already been treated with a standard of care treatment and a treatment refractory subtype is enriched (e.g., NRP subtype).
  • a treatment refractory subtype is enriched (e.g., NRP subtype).
  • aspects of the invention involve modifying the therapy within a standard of care based on the detection of any of the biomarkers as described herein.
  • therapeutic agent targeting a biomarker is administered in combination with CRT.
  • CRT is used to reduce tumor size and one or more therapeutic agents targeting the residual tumor are administered (i.e., targeting biomarkers).
  • therapy comprising an agent is administered within a standard of care where addition of the agent is synergistic within the steps of the standard of care.
  • the agent targets and/or shifts a tumor to a treatment responsive phenotype. In one embodiment, the agent targets tumor cells expressing a gene program.
  • standard of care refers to the current treatment that is accepted by medical experts as a proper treatment for a certain type of disease and that is widely used by healthcare professionals. Standard of care is also called best practice, standard medical care, and standard therapy.
  • Standards of care for PDAC generally include surgery (e.g., tumor resection or palliative surgery), radiation, chemotherapy (e.g., Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Afinitor (Everolimus), Everolimus, 5-FU (Fluorouracil Injection), Fluorouracil Injection, Gemcitabine Hydrochloride, Gemzar (Gemcitabine Hydrochloride), Infugem (Gemcitabine Hydrochloride), Irinotecan Hydrochloride Liposome, Lynparza (Olaparib), Mitomycin, Onivyde (Irinotecan Hydrochloride Liposome), Paclitaxel Albumin-stabilized Nanoparticle Formulation, FOLFIRINOX, GEMCITABINE-CISPLATIN, GEMCITABINEOXALIPLATIN, OFF, Afinitor Disperz (Everolimus), Lanreotide Acetate, Lutathera (Lutetium Lu
  • a treatment clinical trial is a research study meant to help improve current treatments or obtain information on new treatments for patients with cancer.
  • the new treatment may be considered the new standard treatment.
  • cancer-specific genes or gene expression products for subjects having a NRP phenotype include TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, SCTR, PDGFD, C6, CRISP3, RELN, SERPINA6, CRP, FGG, APCS, CFH, HABP2, KLKB1, REGIA, and SPP1.
  • therapeutic targets for subjects having a cancer with a SBM phenotype include MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, NECTIN4, COL17A1, MUC16, FGF19, IGF2, NPC2, SAA1, SRGN, CIS, and CST4.
  • therapeutic targets for subjects having a classical phenotype include CEACAM5, CLDN18, CEACAM6, CDH17, TSPAN8, MUC13, MUC17, REG4, PLA2G10, TFF1, and NRG4.
  • the cancer-specific genes or gene expression products are cell-surface polypeptides.
  • the cell-surface polypeptides for a NRP phenotype are TM4SF4, CNTN4, NXRN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR.
  • the cell-surface polypeptides for a SBM phenotype are MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4.
  • the cell-surface polypeptides for a classical phenotype are CEACAM5, CLDN18, CEACAM6, CDH17, TSPAN8, MUC13, and MUC17.
  • genes therapeutically targeted or detected according to the present invention include genes for the distinct transcriptional cancer sub-types.
  • the therapeutic and diagnostic targets are NRP targets.
  • the therapeutic and diagnostic targets are SBM targets.
  • the therapeutic and diagnostic targets are Classical targets. All gene name symbols refer to the gene as commonly known in the art. The examples described herein that refer to the human gene names are to be understood to also encompasses genes in any other organism, for example mouse genes or any other gene used in a model of disease (e.g., homologous, orthologous genes). Any reference to the gene symbol is a reference made to the entire gene or variants of the gene.
  • any reference to the gene symbol is also a reference made to the gene product (e.g., protein).
  • the term, homolog may apply to the relationship between genes separated by the event of speciation (e.g., ortholog).
  • Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution.
  • Gene symbols may be those referred to by the HUGO Gene Nomenclature Committee (HGNC) or National Center for Biotechnology Information (NCBI).
  • HGNC HUGO Gene Nomenclature Committee
  • NCBI National Center for Biotechnology Information
  • a signature as described herein may encompass any of the genes described herein.
  • the example DNA, RNA, and protein can be used for the design of gene editing agents, antisense oligonucleotides (ASO), antibodies, small molecules, and RNAi.
  • the NRP target is TM4SF4 (also known as, Transmembrane 4 L Six Family Member 4, Il-TMP, Intestine And Liver Tetraspan Membrane Protein, Transmembrane 4 Superfamily Member 4, Transmembrane 4 L6 Family Member 4, ILTMP, Intestinal And Liver (II) Tetraspan Membrane Protein, and IL-TMP).
  • TM4SF4 is a cell surface glycoprotein that can regulate cell proliferation.
  • External Ids for TM4SF4 include: HGNC: 11856, NCBI Entrez Gene: 7104, NCBI Reference Sequence: NM_004617.4, NP_004608.1, NC-000003.12: 149474697-149503394, Ensembl: ENSG00000169903, OMIM®: 606567, and UniProtKB/Swiss-Prot: P48230.
  • the NRP target is CNTN4 (also known as, Contactin 4, BIG-2, Brain-Derived Immunoglobulin Superfamily Protein 2, Contactin-4, Axonal-Associated Cell Adhesion Molecule, Neural Cell Adhesion Protein BIG-2, AXCAM).
  • CNTN4 is a glycosylphosphatidylinositol-anchored neuronal membrane protein that may play a role in the formation of axon connections in the developing nervous system.
  • External Ids for CNTN4 include: HGNC: 2174, NCBI Entrez Gene: 152330, NCBI Reference Sequence: NM_001206955.2, NM_001206956.2, NM_001350095.2, NMJ75607.3, NMJ75613.3, NP_001193884.1, NP_001193885.1, NP_001337024.1, NP_783200.1, NP_783302.1, NC_000003.12:2098866- 3057959, Ensembl: ENSG00000144619, OMIM®: 607280, and UniProtKB/Swiss-Prot: Q8IWV2.
  • the NRP target is NRXN3 (also known as, Neurexin 3, KIAA0743, C14orf60 3, Neurexin-3 -Alpha, Chromosome 14 Open Reading Frame 60, Neurexin Ill-Alpha, Neurexin Ill-Beta, Neurexin-3-Beta, Neurexin III, Neurexin-3).
  • NRXN3 is a member of a family of proteins that function in the nervous system as receptors and cell adhesion molecules.
  • External Ids for NRXN3 include: HGNC: 8010, NCBI Entrez Gene: 9369, NCBI Reference Sequence: NM_001105250.3, NM_001272020.2, NM_001330195.2, NM_001366425.1, NM_001366426.1, NM_004796.6, NMJ38970.5, NP_001098720.1, NP_001258949.1, NP_001317124.1, NP_001353354.1, NP_001353355.1, NP_004787.2, NP_620426.2, NC_000014.9:78170373-79868291, Ensembl: ENSG00000021645, OMIM®: 600567, UniProtKB/Swiss-Prot: Q9Y4C0, and UniProtKB/Swiss-Prot: Q9HDB5.
  • the NRP target is SLC4A4 (also known as, Solute Carrier Family 4 Member 4, NBC1, HNBC1, HhNMC, NBC2, PNBC, Solute Carrier Family 4 (Sodium Bicarbonate Cotransporter), Member 4, Electrogenic Sodium Bicarbonate Cotransporter 1, Na(+)/HCO3(-) Cotransporter, SLC4A5, KNBC1, Sodium Bicarbonate Cotransporter 1 (Sodium Bicarbonate Cotransporter, Kidney; Sodium Bicarbonate Cotransporter, Pancreas), Solute Carrier Family 4, Sodium Bicarbonate Cotransporter, Member 4, Brain Type, Solute Carrier Family 4, Sodium Bicarbonate Cotransporter, Member 4, Solute Carrier Family 4, Sodium Bicarbonate Cotransporter, Member 5, Sodium Bicarbonate Cotransporter, NBCel-A, NBCE1, KNBC, NBC).
  • Solute Carrier Family 4 also known as, Solute Carrier Family 4 Member 4, NBC1, HNBC1, HhNMC, NBC2, PNBC, Solute Carrier Family 4 (Sodium Bicarbonate
  • SLC4A4 encodes a sodium bicarbonate cotransporter (NBC) involved in the regulation of bicarbonate secretion and absorption and intracellular pH.
  • External Ids for SLC4A4 include: HGNC: 11030, NCBI Entrez Gene: 8671, NCBI Reference Sequence: NM_001098484.3, NM_001134742.2, NM_003759.4, NP_001091954.1, NP_001128214.1, NP_003750.1, NC-000004.12:71062660-71572083, Ensembl: ENSG00000080493, OMIM®: 603345, and UniProtKB/Swiss-Prot: Q9Y6R1.
  • the NRP target is CSMD2 (also known as, CUB And Sushi Multiple Domains 2, KIAA1884, CUB And Sushi Domain-Containing Protein 2, CUB And Sushi Multiple Domains Protein 2, CUB And Sushi (SCR Repeat) Domain, DJ1007G16.1, DJ1007G16.2, DI947L8.1).
  • CSMD2 is thought to be involved in the control of complement cascade of the immune system.
  • External Ids for CSMD2 include: HGNC: 19290, NCBI Entrez Gene: 114784, Ensembl: ENSG00000121904, OMIM®: 608398, UniProtKB/Swiss-Prot: Q7Z408, and NCBI Reference Sequence: NM_001281956.2, NP_001268885.1, NM_052896.5, NP_443128.2, NC_000001.11:c34165842-33513998.
  • the NRP target is DSCAML1 (also known as, DS Cell Adhesion Molecule Like 1, KIAA1132, Down Syndrome Cell Adhesion Molecule-Like Protein 1, Down Syndrome Cell Adhesion Molecule 2, DSCAM2, Downs Syndrome Cell Adhesion Molecule Like 1, Down Syndrome Cell Adhesion Molecule Like 1, DSCAM-Like 1).
  • DSCAML1 is a member of the Ig superfamily of cell adhesion molecules and is involved in neuronal differentiation.
  • External Ids for DSCAMLl include: HGNC: 14656, NCBI Entrez Gene: 57453, Ensembl: ENSG00000177103, OMIM®: 611782, UniProtKB/Swiss-Prot: Q8TD84, and NCBI Reference Sequence: NM_001367904.1, NP_001354833.1, NM_001367905.1, NP_001354834.1, NM_020693.4, NP_065744.3, NC_000011.10:cl 17817514-117427772.
  • the NRP target is SLC3A1 (also known as, Solute Carrier Family 3 Member 1, RBAT, NBAT, CSNU1, ATR1, D2H, Solute Carrier Family 3 (Cystine, Dibasic And Neutral Amino Acid Transporters, Activator Of Cystine, Dibasic And Neutral Amino Acid Transport), Member 1, Solute Carrier Family 3 (Amino Acid Transporter Heavy Chain), Member 1, Neutral And Basic Amino Acid Transport Protein RBAT, B(0,+)-Type Amino Acid Transport Protein, Solute Carrier Family 3 (Cystine, Dibasic And Neutral Amino Acid Transporters), Member 1, Amino Acid Transporter 1, SLC3A1 Variant B, SLC3A1 Variant C, SLC3A1 Variant D, SLC3A1 Variant E, SLC3A1 Variant F, SLC3A1 Variant G, D2h).
  • Solute Carrier Family 3 also known as, Solute Carrier Family 3 Member 1, RBAT, NBAT,
  • SLC3A1 encodes a type II membrane glycoprotein which is one of the components of the renal amino acid transporter which transports neutral and basic amino acids in the renal tubule and intestinal tract.
  • External Ids for SLC3A1 include: HGNC: 11025, NCBI Entrez Gene: 6519, Ensembl: ENSG00000138079, OMIM®: 104614, UniProtKB/Swiss-Prot: Q07837, and NCBI Reference Sequence: NM_000341.4, NP_000332.2, NC_000002.12:44275480-44322437.
  • the NRP target is TRPV6 (also known as, Transient Receptor Potential Cation Channel Subfamily V Member 6, Epithelial Calcium Channel 2, ECAC2, CaTl, Calcium Transport Protein 1, Transient Receptor Potential Cation Channel, Subfamily V, Member 6, Epithelial Apical Membrane Calcium Transporter/Channel CaTl, Alu- Binding Protein With Zinc Finger Domain, Calcium Transporter-Like Protein, HSA277909, CaT- Like, ABP/ZF, HRPTTN, LP6728, TrpV6, CaT-L, ECaC2, CAT1, CATL, ZFAB).
  • TRPV6 encodes a member of a family of multipass membrane proteins that functions as calcium channels.
  • the encoded protein contains N-terminal ankyrin repeats, which are required for channel assembly and regulation.
  • External Ids for TRPV6 include: HGNC: 14006, NCBI Entrez Gene: 55503, Ensembl: ENSG00000165125, OMIM®: 606680, UniProtKB/Swiss-Prot: Q9H1D0, and NCBI Reference Sequence: NM_018646.6, NP_061116.5, NC_000007.14:cl42885745-142871208.
  • the NRP target is ABCB1 (also known as, ATP Binding Cassette Subfamily B Member 1, Multidrug Resistance Protein 1, CD243, GP170, ABC20, P-170, MDR1, PGY1, ATP -Binding Cassette, Sub-Family B (MDR/TAP), Member 1, ATP -Dependent Translocase ABCB1, Phospholipid Transporter ABCB1, Colchicin Sensitivity, P-Glycoprotein 1, P-Gp, CLCS, ATP -Binding Cassette Sub-Family B Member 1, Doxorubicin Resistance, CD243 Antigen, EC 3.6.3.44, EC 7.6.2.2, EC 7.6.2.1, EC 3.6.3, P-GP).
  • ABCB1 also known as, ATP Binding Cassette Subfamily B Member 1, Multidrug Resistance Protein 1, CD243, GP170, ABC20, P-170, MDR1, PGY1, ATP -Binding Cass
  • ABCB1 is a member of the superfamily of ATP -binding cassette (ABC) transporters. External Ids for ABCB 1 include:HGNC: 40, NCBI Entrez Gene: 5243, Ensembl: ENSG00000085563, OMIM®: 171050, UniProtKB/Swiss-Prot: P08183, and NCBI Reference Sequence: NM_000927.5, NP_000918.2, NM_001348944.2, NP_001335873.1, NM_001348945.2, NP_001335874.1, NM_001348946.2, NP_001335875.1, NC_000007.14:c87713295-87503017.
  • the NRP target is NLGN4Y (also known as, Neuroligin 4 Y-Linked, KIAA0951, Neuroligin-4, Y-Linked, Neuroligin 4, Y Linked, Alternative HNL4Y, Neuroligin Y, HNL4Y).
  • NLGN4Y encodes a type I membrane protein that belongs to the family of neuroligins, which are cell adhesion molecules present at the postsynaptic side of the synapse and may be essential for the formation of functional synapses.
  • External Ids for NLGN4Y include: HGNC: 15529, NCBI Entrez Gene: 22829, Ensembl: ENSG00000165246, OMIM®: 400028, UniProtKB/Swiss-Prot: Q8NFZ3, and NCBI Reference Sequence: NM_001164238.1, NP_001157710.1, NM_001206850.2, NP_001193779.1, NM_001365584.1, NP_001352513.1, NM_001365588.1, NP_001352517.1, NM_001365591.1, NP_001352520.1, NM_001365593.1, NP_001352522.1, NM_001394830.1, NP_001381759.1, NM_001394831.1, NP_001381760.1, NM_014893.5, NP_055708.3, NC_000024.10: 14522616-14845654.
  • the NRP target is SCTR (also known as, Secretin Receptor, Pancreatic Secretin Receptor, SCT-R, SR).
  • SCTR is a G protein-coupled receptor and belongs to the glucagon- VIP-secretin receptor family. It binds secretin which is the most potent regulator of pancreatic bicarbonate, electrolyte and volume secretion.
  • External Ids for SCTR include: HGNC: 10608, NCBI Entrez Gene: 6344, Ensembl: ENSG00000080293, OMIM®: 182098, UniProtKB/Swiss-Prot: P47872, and NCBI Reference Sequence: NM_002980.3, NP_002971.2, NC_000002.12:cl 19524483-119439843.
  • the SBM target is MSLN (also known as, Mesothelin, MPF, Pre-Pro-Megakaryocyte-Potentiating Factor, CAK1 Antigen, CAK1, Soluble MPF Mesothelin Related Protein, Megakaryocyte Potentiating Factor, SMRP).
  • MSLN encodes a preproprotein that is proteolytically processed to generate two protein products, megakaryocyte potentiating factor and mesothelin.
  • Megakaryocyte potentiating factor functions as a cytokine that can stimulate colony formation of bone marrow megakaryocytes.
  • Mesothelin is a glycosylphosphatidylinositol-anchored cell-surface protein that may function as a cell adhesion protein.
  • External Ids for MSLN include: HGNC: 7371, NCBI Entrez Gene: 10232, Ensembl: ENSG00000102854, OMIM®: 601051, UniProtKB/Swiss-Prot: Q13421, and NCBI Reference Sequence: NM_001177355.3, NP_001170826.1 , NM_005823.6, NP_005814.2 , NM_013404.4, NP_037536.2, NC_000016.10:760734-768865.
  • the SBM target is GPR87 (also known as, G Protein- Coupled Receptor 87, GPR95, G-Protein Coupled Receptor 87, G-Protein Coupled Receptor 95, Orphan GPCR 87, KPG 002, FKSG78).
  • GPR87 encodes a G protein-coupled receptor and is located in a cluster of G protein-couple receptor genes on chromosome 3.
  • External Ids for GPR87 include: HGNC: 4538, NCBI Entrez Gene: 53836, Ensembl: ENSG00000138271, OMIM®: 606379, UniProtKB/Swiss-Prot: Q9BY21, and NCBI Reference Sequence: NM_023915.4, NP_076404.3, NC_000003.12:cl51316820-151294086.
  • the SBM target is SLC2A1 (also known as, Solute Carrier Family 2 Member 1, DYT18, GLUT1, DYT9, Choreoathetosis/Spasticity, Episodic (Paroxysmal Choreoathetosis/Spasticity), Solute Carrier Family 2 (Facilitated Glucose Transporter), Member 1, Solute Carrier Family 2, Facilitated Glucose Transporter Member 1, Human T-Cell Leukemia Virus (I And II) Receptor, Glucose Transporter Type 1, Erythrocyte/Brain, HepG2 Glucose Transporter, GLUT-1, HTLVR, GLUT, CSE, Receptor For HTLV-1 And HTLV-2, GLUT1DS, SDCHCN, DYT17, EIG12, PED).
  • Solute Carrier Family 2 Member 1 also known as, Solute Carrier Family 2 Member 1, DYT18, GLUT1, DYT9, Choreoa
  • SLC2A1 encodes a major glucose transporter in the mammalian blood-brain barrier.
  • External Ids for SLC2A1 include: HGNC: 11005, NCBI Entrez Gene: 6513, Ensembl: ENSG00000117394, OMIM®: 138140, UniProtKB/Swiss-Prot: Pl 1166, and NCBI Reference Sequence: NM_006516.4, NP_006507.2, NC_000001.11 42958868-42925353.
  • the SBM target is LY6D (also known as, Lymphocyte Antigen 6 Family Member D, E48, Lymphocyte Antigen 6 Complex, Locus D, Lymphocyte Antigen 6D, E48 Antigen, Ly-6D).
  • LY6D may act as a specification marker at earliest stage specification of lymphocytes between B- and T-cell development.
  • External Ids for LY6D include: HGNC: 13348, NCBI Entrez Gene: 8581, Ensembl: ENSG00000167656, OMIM®: 606204, UniProtKB/Swiss-Prot: Q14210, and NCBI Reference Sequence: NM_003695.3, NP_003686.1, NC-000008.11 :c 142786539- 142784882.
  • the SBM target is PSCA (also known as, Prostate Stem Cell Antigen, LncPSCA, PRO232).
  • PSCA encodes a glycosylphosphatidylinositol-anchored cell membrane glycoprotein.
  • External Ids for PSCA include: HGNC: 9500, NCBI Entrez Gene: 8000, Ensembl: ENSG00000167653, OMIM®: 602470, UniProtKB/Swiss-Prot: 043653, and NCBI Reference Sequence: NM_005672.5, NP_005663.2, NC_000008.11 :142670297-142682725.
  • the SBM target is GPRC5A (also known as, G Protein- Coupled Receptor Class C Group 5 Member A, PEIG-1, RAIG1, TIG1, RAI3, G-Protein Coupled Receptor Family C Group 5 Member A, Retinoic Acid-Induced Gene 1 Protein, Retinoic Acid- Induced Protein 3, Phorbol Ester Induced Gene 1, Retinoic Acid Induced 3, GPCR5A, G Protein- Coupled Receptor, Class C, Group 5, Member A, Orphan G-Protein-Coupling Receptor PEIG-1, Phorbol Ester Induced Protein-1, Retinoic Acid Responsive, TPA Induced Gene 1, RAIG-1).
  • GPCR5A G Protein- Coupled Receptor, Class C, Group 5, Member A, Orphan G-Protein-Coupling Receptor PEIG-1, Phorbol Ester Induced Protein-1, Retinoic Acid Responsive, TPA Induced Gene 1, RAIG
  • GPRC5A encodes a member of the type 3 G protein-coupling receptor family, characterized by the signature 7-transmembrane domain motif.
  • External Ids for GPRC5A include: HGNC: 9836, NCBI Entrez Gene: 9052, Ensembl: ENSG00000013588, OMIM®: 604138, UniProtKB/Swiss- Prot: Q8NFJ5, and NCBI Reference Sequence: NM_003979.4, NP_003970.1, NC-000012.12:12891562-12917937.
  • the SBM target is ALPP (also known as, Alkaline
  • ALPP encodes an alkaline phosphatase, a metalloenzyme that catalyzes the hydrolysis of phosphoric acid monoesters. It belongs to a multigene family composed of four alkaline phosphatase isoenzymes. External Ids for ALPP include: HGNC: 439, NCBI Entrez Gene: 250, Ensembl: ENSG00000163283, OMIM®: 171800, UniProtKB/Swiss-Prot: P05187, and NCBI Reference Sequence: NM_001632.5, NP_001623.3, NC-000002.12:232378751-232382889.
  • the SBM target is MET (also known as, MET ProtoOncogene, Receptor Tyrosine Kinase, Hepatocyte Growth Factor Receptor, DFNB97, RCCP2, HGFR, Tyrosine-Protein Kinase Met, Scatter Factor Receptor, Proto-Oncogene C-Met, HGF/SF Receptor, HGF Receptor, SF Receptor, EC 2.7.10.1, Met Proto-Oncogene, EC 2.7.10, AUTS9, C- Met).
  • MET encodes a member of the receptor tyrosine kinase family of proteins and the product of the proto-oncogene MET.
  • External Ids for MET include: HGNC: 7029, NCBI Entrez Gene: 4233, Ensembl: ENSG00000105976, OMIM®: 164860, UniProtKB/Swiss-Prot: P08581, and NCBI Reference Sequence: NM_000245.4, NP_000236.2, NM_001127500.3, NP_001120972.1, NM_001324401.3, NP_001311330.1, NM_001324402.2, NP_001311331.1 ,
  • the SBM target is TACSTD2 (also known as, Tumor Associated Calcium Signal Transducer 2, GA733-1, TROP2, EGP-1, M1S1, Membrane Component Chromosome 1 Surface Marker 1, Tumor- Associated Calcium Signal Transducer 2, Pancreatic Carcinoma Marker Protein GA733-1, Trophoblast Cell Surface Antigen 2, Cell Surface Glycoprotein Trop-2, Epithelial Glycoprotein- 1, 40kD Glycoprotein, Identified By Monoclonal Antibody GA733, Membrane Component, Chromosome 1, Surface Marker 1, Gastrointestinal Tumor-Associated Antigen GA7331, Pancreatic Carcinoma Marker Protein GA7331, Cell Surface Glycoprotein TROP2, Truncated TACSTD2, GA7331, EGP1, GP50).
  • TACSTD2 also known as, Tumor Associated Calcium Signal Transducer 2, GA733-1, TROP2, EGP-1, M1S1, Membrane Component Chromosome 1 Surface Marker 1, Tumor- Associated
  • TACSTD2 encodes a carcinoma-associated antigen. This antigen is a cell surface receptor that transduces calcium signals. External Ids for TACSTD2 include: HGNC: 11530, NCBI Entrez Gene: 4070, Ensembl: ENSGOOOOO 184292, OMIM®: 137290, UniProtKB/Swiss-Prot: P09758, and NCBI Reference Sequence: NM_002353.3, NP_002344.2, NC_000001.11:c58577252-58575433.
  • the SBM target is NECTIN4 (also known as, Nectin Cell Adhesion Molecule 4, Nectin-4, PRR4, LNIR, PVRL4, Poliovirus Receptor-Related Protein 4, Poliovirus Receptor-Related 4, Ig Superfamily Receptor LNIR, Nectin 4, EDSS1).
  • NECTIN4 encodes a member of the nectin family. The encoded protein contains two immunoglobulin-like (Ig-like) C2-type domains and one Ig-like V-type domain. It is involved in cell adhesion through trans-homophilic and -heterophilic interactions.
  • NECTIN4 External Ids for NECTIN4 include: HGNC: 19688, NCBI Entrez Gene: 81607, Ensembl: ENSG00000143217, OMIM®: 609607, UniProtKB/Swiss-Prot: Q96NY8, and NCBI Reference Sequence: NM_030916.3, NP_112178.2, NC 000001.11 :cl61089558-161070998.
  • the classical target is CEACAM5 (also known as, CEA Cell Adhesion Molecule 5, Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5, CD66e, CEA, Carcinoembryonic Antigen Related Cell Adhesion Molecule 5, Meconium Antigen 100, Carcinoembryonic Antigen, CD66e Antigen).
  • CEACAM5 encodes a cell surface glycoprotein that represents the founding member of the carcinoembryonic antigen (CEA) family of proteins.
  • External Ids for CEACAM5 include: HGNC: 1817, NCBI Entrez Gene: 1048, Ensembl: ENSGOOOOO 105388, OMIM®: 114890, UniProtKB/Swiss-Prot: P06731, and NCBI Reference Sequence: NM_001291484.3, NP_001278413.1, NM_001308398.2, NP_001295327.1, NM_004363.6, NP_004354.3, NC_000019.10:41708626-41730433.
  • the classical target is CLDN18 (also known as, Claudin 18, Surfactant Associated Protein J, Claudin- 18, SFTPI, Surfactant, Pulmonary Associated Protein J, Surfactant Associated 5, SFTA5).
  • CLDN18 encodes a member of the claudin family. Claudins are integral membrane proteins and components of tight junction strands.
  • External Ids for CLDN18 include: HGNC: 2039, NCBI Entrez Gene: 51208, Ensembl: ENSG00000066405, OMIM®: 609210, UniProtKB/Swiss-Prot: P56856, and NCBI Reference Sequence: NM_001002026.3, NP_001002026.1, NM_016369.4, NP_057453.1, NC_000003.12: 137998816-138033649.
  • the classical target is CEACAM6 (also known as, CEA Cell Adhesion Molecule 6, CD66c, NCA, Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6 (Non-Specific Cross-Reacting Antigen), Carcinoembryonic Antigen Related Cell Adhesion Molecule 6, Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6, Normal Cross-Reacting Antigen, Non-Specific Crossreacting Antigen, Cluster Of Differentiation 66c, CD66c Antigen, CEAL).
  • CEACAM6 also known as, CEA Cell Adhesion Molecule 6, CD66c, NCA, Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6 (Non-Specific Cross-Reacting Antigen), Carcinoembryonic Antigen Related Cell Adhesion Molecule 6, Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6, Normal Cross-Reacting Antigen,
  • CEACAM6 encodes a protein that belongs to the carcinoembryonic antigen (CEA) family whose members are glycosyl phosphatidyl inositol (GPI) anchored cell surface glycoproteins.
  • External Ids for CEACAM6 include: HGNC: 1818, NCBI Entrez Gene: 4680, Ensembl: ENSG00000086548, OMIM®: 163980, UniProtKB/Swiss-Prot: P40199, and NCBI Reference Sequence: NM_002483.7, NP_002474.4, NC_000019.10:41755530-41772211.
  • the classical target is CDH17 (also known as, Cadherin 17, HPT-1, Intestinal Peptide-Associated Transporter HPT-1, Cadherin 17, LI Cadherin (Liver- Intestine), Liver-Intestine Cadherin, Cadherin- 17, Cadherin, Human Intestinal Peptide- Associated Transporter HPT-1, Human Peptide Transporter 1, HPT-1 Cadherin, LI Cadherin, Cadherin-16, Ll-Cadherin, CDH16, HPT1).
  • CDH17 is a member of the cadherin superfamily, genes encoding calcium-dependent, membrane-associated glycoproteins.
  • CDH17 External Ids for CDH17 include: HGNC: 1756, NCBI Entrez Gene: 1015, Ensembl: ENSG00000079112, OMIM®: 603017, UniProtKB/Swiss-Prot: Q12864, and NCBI Reference Sequence: NM_001144663.2, NP_001138135.1, NM_004063.4, NP_004054.3, NC_000008.11:c94217278-94127162.
  • the classical target is TSPAN8 (also known as, Tetraspanin 8, Transmembrane 4 Superfamily Member 3, CO-029, TM4SF3, Tumor-Associated Antigen CO-029, Tetraspanin-8, Tspan-8).
  • TSPAN8 is a member of the transmembrane 4 superfamily, also known as the tetraspanin family.
  • External Ids for TSPAN8 include: HGNC: 11855, NCBI Entrez Gene: 7103, Ensembl: ENSG00000127324, OMIM®: 600769, UniProtKB/Swiss-Prot: P19075, and NCBI Reference Sequence: NM_001369760.1, NP_001356689.1, NM_004616.3, NP_004607.1, NC_000012.12:c71157999-71125096.
  • the classical target is MUC13 (also known as, Mucin 13, Cell Surface Associated, Down-Regulated In Colon Cancer 1, DRCC1, Mucin 13, Epithelial Transmembrane, Mucin-13, MUC-13, RECC).
  • MUC13 also known as, Mucin 13, Cell Surface Associated, Down-Regulated In Colon Cancer 1, DRCC1, Mucin 13, Epithelial Transmembrane, Mucin-13, MUC-13, RECC.
  • Epithelial mucins, such as MUC13 are a family of secreted and cell surface glycoproteins expressed by ductal and glandular epithelial tissues.
  • External Ids for MUC13 include: HGNC: 7511, NCBI Entrez Gene: 56667, Ensembl: ENSG00000173702, OMIM®: 612181, UniProtKB/Swiss-Prot: Q9H3R2, and NCBI Reference Sequence: NM_033049.4, NPJ49038.3, NC_000003.12:cl24934751-124905442.
  • the classical target is MUC17 (also known as, Mucin 17, Cell Surface Associated, Small Intestinal Mucin-3, Mucin-17, MUC-17, MUC-3, MUC3, Small Intestinal Mucin MUC3, Membrane Mucin MUC17, Secreted Mucin MUC17, EC 6.3.2.8, EC 3.1.3).
  • MUC17 is a membrane-bound mucin that provides protection to gut epithelial cells.
  • External Ids for MUC17 include: HGNC: 16800, NCBI Entrez Gene: 140453, Ensembl: ENSG00000169876, OMIM®: 608424, UniProtKB/Swiss-Prot: Q685I3, and NCBI Reference Sequence: NM_001040105.2, NP_001035194.1, NC_000007.14: 101020081-101058859.
  • a method of treating cancer comprises administering an adoptive cell therapy (ACT) to a subject in need thereof.
  • the ACT comprises tumor infiltrating lymphocytes (TILs) specific for a tumor.
  • the ACT comprises immune cells expressing a T cell receptor (TCR) specific for a tumor.
  • the ACT comprises immune cells expressing a chimeric antigen receptor (CAR) specific for a tumor.
  • the ACT comprises a CAR-T cell or CAR NK cell specific for a tumor.
  • the ACT comprises cells specific for a surface marker described herein.
  • Adoptive cell therapy can refer to the transfer of cells, most commonly immune-derived cells (e.g., T cells or NK cells), back into the same patient or into a new recipient host with the goal of transferring the immunologic functionality and characteristics into the new host. If possible, use of autologous cells helps the recipient by minimizing GVHD issues.
  • TIL tumor infiltrating lymphocytes
  • allogenic cells immune cells are transferred (see, e.g., Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266). As described further herein, allogenic cells can be edited to reduce alloreactivity and prevent graft-versus-host disease. Thus, use of allogenic cells allows for cells to be obtained from healthy donors and prepared for use in patients as opposed to preparing autologous cells from a patient after diagnosis.
  • aspects of the invention involve the adoptive transfer of immune system cells, such as T cells or NK cells, specific for selected antigens, such as tumor associated antigens or tumor specific neoantigens (see, e.g., Maus et al., 2014, Adoptive Immunotherapy for Cancer or Viruses, Annual Review of Immunology, Vol. 32: 189-225; Rosenberg and Restifo, 2015, Adoptive cell transfer as personalized immunotherapy for human cancer, Science Vol. 348 no. 6230 pp. 62-68; Restifo et al., 2015, Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol.
  • TCR T cell receptor
  • Various strategies may for example be employed to genetically modify T cells by altering the specificity of the T cell receptor (TCR) for example by introducing new TCR a and 0 chains with selected peptide specificity (see U.S. Patent No. 8,697,854; PCT Patent Publications: W02003020763, W02004033685, W02004044004, W02005114215, W02006000830, W02008038002, W02008039818, W02004074322, WO2005113595, WO2006125962, WO2013166321, WO2013039889, WO2014018863, WO2014083173; U.S. Patent No. 8,088,379).
  • TCR T cell receptor
  • CARs chimeric antigen receptors
  • TCRs T cells or natural killer cells
  • NK natural killer cells
  • a wide variety of receptor chimera constructs having been described (see U.S. Patent Nos. 5,843,728; 5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014; 6,753,162; 8,211,422; and, PCT Publication WO9215322).
  • CARs are comprised of an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen-binding domain that is specific for a predetermined target (see, e.g., Gong Y, Klein Wolterink RGJ, Wang J, Bos GMJ, Germeraad WTV. Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. J Hematol Oncol. 2021; 14(1):73; Guedan S, Calderon H, Posey AD Jr, Maus MV. Engineering and Design of Chimeric Antigen Receptors. Mol Ther Methods Clin Dev.
  • the antigen-binding domain of a CAR is often an antibody or antibody fragment (e.g., a single chain variable fragment, scFv), the binding domain is not particularly limited so long as it results in specific recognition of a target.
  • the antigen-binding domain may comprise a receptor, such that the CAR is capable of binding to the ligand of the receptor.
  • the antigen-binding domain may comprise a ligand, such that the CAR is capable of binding the endogenous receptor of that ligand.
  • the antigen-binding domain of a CAR is generally separated from the transmembrane domain by a hinge or spacer.
  • the spacer is also not particularly limited, and it is designed to provide the CAR with flexibility.
  • a spacer domain may comprise a portion of a human Fc domain, including a portion of the CH3 domain, or the hinge region of any immunoglobulin, such as IgA, IgD, IgE, IgG, or IgM, or variants thereof.
  • the hinge region may be modified so as to prevent off-target binding by FcRs or other potential interfering objects.
  • the hinge may comprise an IgG4 Fc domain with or without a S228P, L235E, and/or N297Q mutation (according to Kabat numbering) in order to decrease binding to FcRs.
  • Additional spacers/hinges include, but are not limited to, CD4, CD8, and CD28 hinge regions.
  • the transmembrane domain of a CAR may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane bound or transmembrane protein.
  • Transmembrane regions of particular use in this disclosure may be derived from CD8, CD28, CD3, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, TCR.
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
  • a glycine-serine doublet provides a particularly suitable linker.
  • First-generation CARs typically consist of a single-chain variable fragment of an antibody specific for an antigen, for example comprising a VL linked to a VH of a specific antibody, linked by a flexible linker, for example by a CD8a hinge domain and a CD8a transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3( ⁇ or FcRy (scFv-CD3( ⁇ or scFv-FcRy; see U.S. Patent No. 7,741,465; U.S. Patent No. 5,912,172; U.S. Patent No. 5,906,936).
  • Second-generation CARs incorporate the intracellular domains of one or more costimulatory molecules, such as CD28, 0X40 (CD134), or 4-1BB (CD137) within the endodomain (for example scFv-CD28/OX40/4-lBB-CD3 ⁇ ; see U.S. Patent Nos. 8,911,993; 8,916,381; 8,975,071; 9,101,584; 9,102,760; 9,102,761).
  • Third-generation CARs include a combination of costimulatory endodomains, such a CD3 ⁇ -chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, 0X40, 4-1BB, CD2, CD7, LIGHT, LFA-1, NKG2C, B7-H3, CD30, CD40, PD-1, or CD28 signaling domains (for example scFv-CD28-4-lBB-CD3 ⁇ or scFv-CD28- OX40-CD3 ; see U.S. Patent No. 8,906,682; U.S. Patent No. 8,399,645; U.S. Pat. No. 5,686,281; PCT Publication No.
  • the primary signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcRbeta (Fc Epsilon Rib), CD79a, CD79b, Fc gamma Rlla, DAP10, and DAP12.
  • the primary signaling domain comprises a functional signaling domain of CD3( ⁇ or FcRy.
  • the one or more costimulatory signaling domains comprise a functional signaling domain of a protein selected, each independently, from the group consisting of: CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, I
  • the one or more costimulatory signaling domains comprise a functional signaling domain of a protein selected, each independently, from the group consisting of: 4-1BB, CD27, and CD28.
  • a chimeric antigen receptor may have the design as described in U.S. Patent No. 7,446,190, comprising an intracellular domain of CD3( ⁇ chain (such as amino acid residues 52-163 of the human CD3 zeta chain, as shown in SEQ ID NO: 14 of US 7,446,190), a signaling region from CD28 and an antigen-binding element (or portion or domain, such as scFv).
  • the CD28 portion when between the zeta chain portion and the antigen-binding element, may suitably include the transmembrane and signaling domains of CD28 (such as amino acid residues 114-220 of SEQ ID NO: 10, full sequence shown in SEQ ID NO: 6 of US 7,446,190; these can include the following portion of CD28 as set forth in Genbank identifier NM_006139 (sequence version 1, 2 or 3): IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS)) (SEQ ID NO: 20).
  • intracellular domain of CD28 can be used alone (such as amino sequence set forth in SEQ ID NO: 9 of US 7,446,190).
  • a CAR comprising (a) a zeta chain portion comprising the intracellular domain of human CD3 ⁇ chain, (b) a costimulatory signaling region, and (c) an antigen-binding element (or portion or domain), wherein the costimulatory signaling region comprises the amino acid sequence encoded by SEQ ID NO: 6 of US 7,446,190.
  • co- stimulation may be orchestrated by expressing CARs in antigenspecific T cells, chosen so as to be activated and expanded following engagement of their native a0TCR, for example by antigen on professional antigen-presenting cells, with attendant costimulation.
  • additional engineered receptors may be provided on the immunoresponsive cells, for example to improve targeting of a T-cell attack and/or minimize side effects.
  • Kochenderfer et al. (2009) J Immunother. 32 (7): 689-702 described anti-CD19 chimeric antigen receptors (CAR).
  • FMC63- 28Z CAR contained a single chain variable region moiety (scFv) recognizing CD 19 derived from the FMC63 mouse hybridoma (described in Nicholson et al., (1997) Molecular Immunology 34: 1157-1165), a portion of the human CD28 molecule, and the intracellular component of the human molecule.
  • FMC63-CD828BBZ CAR contained the FMC63 scFv, the hinge and transmembrane regions of the CD8 molecule, the cytoplasmic portions of CD28 and 4-1BB, and the cytoplasmic component of the molecule.
  • CD28 molecule included in the FMC63-28Z CAR corresponded to Genbank identifier NM_006139; the sequence included all amino acids starting with the amino acid sequence IEVMYPPPY (SEQ ID NO: 21) and continuing all the way to the carboxy -terminus of the protein.
  • IEVMYPPPY amino acid sequence IEVMYPPPY
  • This sequence encoded the following components in frame from the 5’ end to the 3’ end: an Xhol site, the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor a-chain signal sequence, the FMC63 light chain variable region (as in Nicholson et al., supra), a linker peptide (as in Cooper et al., supra), the FMC63 heavy chain variable region (as in Nicholson et al., supra), and a Notl site.
  • GM-CSF human granulocyte-macrophage colony-stimulating factor
  • a plasmid encoding this sequence was digested with Xhol and Noth
  • the Xhol and Notl-digested fragment encoding the FMC63 scFv was ligated into a second Xhol and Notl-digested fragment that encoded the MSGV retroviral backbone (as in Hughes et al., (2005) Human Gene Therapy 16: 457-472) as well as part of the extracellular portion of human CD28, the entire transmembrane and cytoplasmic portion of human CD28, and the cytoplasmic portion of the human TCR.- molecule (as in Maher et al., 2002) Nature Biotechnology 20: 70-75).
  • the FMC63-28Z CAR is included in the KTE-C19 (axicabtagene ciloleucel) anti-CD19 CAR-T therapy product in development by Kite Pharma, Inc. for the treatment of inter alia patients with relapsed/refractory aggressive B-cell non-Hodgkin lymphoma (NHL).
  • KTE-C19 axicabtagene ciloleucel
  • Kite Pharma, Inc. for the treatment of inter alia patients with relapsed/refractory aggressive B-cell non-Hodgkin lymphoma (NHL).
  • cells intended for adoptive cell therapies may express the FMC63-28Z CAR as described by Kochenderfer et al. (supra).
  • cells intended for adoptive cell therapies may comprise a CAR comprising an extracellular antigen-binding element (or portion or domain, such as scFv) that specifically binds to an antigen, an intracellular signaling domain comprising an intracellular domain of a CD3( ⁇ chain, and a costimulatory signaling region comprising a signaling domain of CD28.
  • a CAR comprising an extracellular antigen-binding element (or portion or domain, such as scFv) that specifically binds to an antigen, an intracellular signaling domain comprising an intracellular domain of a CD3( ⁇ chain, and a costimulatory signaling region comprising a signaling domain of CD28.
  • the CD28 amino acid sequence is as set forth in Genbank identifier NM_006139 (sequence version 1, 2 or 3) starting with the amino acid sequence IEVMYPPPY (SEQ ID NO: 21) and continuing all the way to the carboxy-terminus of the protein. The sequence is reproduced herein:
  • the antigen is CD19, more preferably the antigen-binding element is an anti-CD19 scFv, even more preferably the anti-CD19 scFv as described by Kochenderfer et al. supra).
  • Example 1 and Table 1 of WO2015187528 demonstrate the generation of anti-CD19 CARs based on a fully human anti-CD19 monoclonal antibody (47G4, as described in US20100104509) and murine anti-CD19 monoclonal antibody (as described in Nicholson et al. and explained above).
  • a signal sequence human CD8-alpha or GM-CSF receptor
  • extracellular and transmembrane regions human CD8- alpha
  • intracellular T-cell signaling domains CD28-CD3 ⁇ ; 4-lBB-CD3( ⁇ ; CD27-CD3( ⁇ ; CD28-CD27-CD3 ⁇ , 4-lBB-CD27-CD3 ; CD27-4-lBB-CD3 ⁇ ; CD28-CD27-FceRI gamma chain; or CD28-FcsRI gamma chain
  • cells intended for adoptive cell therapies may comprise a CAR comprising an extracellular antigen-binding element that specifically binds to an antigen, an extracellular and transmembrane region as set forth in Table 1 of WO2015187528 and an intracellular T-cell signaling domain as set forth in Table 1 of WO2015187528.
  • the antigen is CD19
  • the antigen-binding element is an anti-CD19 scFv, even more preferably the mouse or human anti-CD19 scFv as described in Example 1 of WO2015187528.
  • the CAR comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 as set forth in Table 1 of WO2015187528.
  • chimeric antigen receptor that recognizes the CD70 antigen is described in W02012058460A2 (see also, Park et al., CD70 as a target for chimeric antigen receptor T cells in head and neck squamous cell carcinoma, Oral Oncol. 2018 Mar;78:145-150; and Jin et al., CD70, a novel target of CAR T-cell therapy for gliomas, Neuro Oncol. 2018 Jan 10;20(l):55-65).
  • CD70 is expressed by diffuse large B-cell and follicular lymphoma and also by the malignant cells of Hodgkins lymphoma, Waldenstrom's macroglobulinemia and multiple myeloma, and by HTLV-1- and EBV-associated malignancies.
  • CD70 is expressed by non-hematological malignancies such as renal cell carcinoma and glioblastoma.
  • non-hematological malignancies such as renal cell carcinoma and glioblastoma.
  • Physiologically, CD70 expression is transient and restricted to a subset of highly activated T, B, and dendritic cells.
  • the immune cell may, in addition to a CAR or exogenous TCR as described herein, further comprise a chimeric inhibitory receptor (inhibitory CAR) that specifically binds to a second target antigen and is capable of inducing an inhibitory or immunosuppressive or repressive signal to the cell upon recognition of the second target antigen.
  • a chimeric inhibitory receptor inhibitory CAR
  • the chimeric inhibitory receptor comprises an extracellular antigenbinding element (or portion or domain) configured to specifically bind to a target antigen, a transmembrane domain, and an intracellular immunosuppressive or repressive signaling domain.
  • the second target antigen is an antigen that is not expressed on the surface of a cancer cell or infected cell or the expression of which is downregulated on a cancer cell or an infected cell.
  • the second target antigen is an MHC-class I molecule.
  • the intracellular signaling domain comprises a functional signaling portion of an immune checkpoint molecule, such as for example PD-1 or CTLA4.
  • an immune checkpoint molecule such as for example PD-1 or CTLA4.
  • the inclusion of such inhibitory CAR reduces the chance of the engineered immune cells attacking non-target (e.g., non-cancer) tissues.
  • T-cells expressing CARs may be further modified to reduce or eliminate expression of endogenous TCRs in order to reduce off-target effects. Reduction or elimination of endogenous TCRs can reduce off-target effects and increase the effectiveness of the T cells (U.S. 9,181,527).
  • T cells stably lacking expression of a functional TCR may be produced using a variety of approaches. T cells internalize, sort, and degrade the entire T cell receptor as a complex, with a half-life of about 10 hours in resting T cells and 3 hours in stimulated T cells (von Essen, M. et al. 2004. J. Immunol. 173:384-393).
  • TCR complex Proper functioning of the TCR complex requires the proper stoichiometric ratio of the proteins that compose the TCR complex.
  • TCR function also requires two functioning TCR zeta proteins with IT AM motifs.
  • the activation of the TCR upon engagement of its MHC-peptide ligand requires the engagement of several TCRs on the same T cell, which all must signal properly.
  • the T cell will not become activated sufficiently to begin a cellular response.
  • TCR expression may eliminated using RNA interference (e.g., shRNA, siRNA, miRNA, etc.), CRISPR, or other methods that target the nucleic acids encoding specific TCRs (e.g., TCR-a and TCR-0) and/or CD3 chains in primary T cells.
  • RNA interference e.g., shRNA, siRNA, miRNA, etc.
  • CRISPR CRISPR
  • TCR-a and TCR-0 CD3 chains in primary T cells.
  • CAR may also comprise a switch mechanism for controlling expression and/or activation of the CAR.
  • a CAR may comprise an extracellular, transmembrane, and intracellular domain, in which the extracellular domain comprises a targetspecific binding element that comprises a label, binding domain, or tag that is specific for a molecule other than the target antigen that is expressed on or by a target cell.
  • the specificity of the CAR is provided by a second construct that comprises a target antigen binding domain (e.g., an scFv or a bispecific antibody that is specific for both the target antigen and the label or tag on the CAR) and a domain that is recognized by or binds to the label, binding domain, or tag on the CAR.
  • a target antigen binding domain e.g., an scFv or a bispecific antibody that is specific for both the target antigen and the label or tag on the CAR
  • a domain that is recognized by or binds to the label, binding domain, or tag on the CAR See, e.g., WO 2013/044225, WO 2016/000304, WO 2015/057834, WO 2015/057852, WO 2016/070061, US 9,233,125, US 2016/0129109.
  • Switch mechanisms include CARs that require multimerization in order to activate their signaling function (see, e.g., US 2015/0368342, US 2016/0175359, US 2015/0368360) and/or an exogenous signal, such as a small molecule drug (US 2016/0166613, Yung et al., Science, 2015), in order to elicit a T-cell response.
  • Some CARs may also comprise a “suicide switch” to induce cell death of the CAR T-cells following treatment (Buddee et al., PLoS One, 2013) or to downregulate expression of the CAR following binding to the target antigen (WO 2016/011210).
  • a wide variety of vectors may be used, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids or transposons, such as a Sleeping Beauty transposon (see U.S. Patent Nos. 6,489,458; 7,148,203; 7,160,682; 7,985,739; 8,227,432), may be used to introduce CARs, for example using 2nd generation antigen-specific CARs signaling through CD3 ⁇ and either CD28 or CD137.
  • Viral vectors may for example include vectors based on HIV, SV40, EBV, HSV or BPV.
  • inducible gene switches are used to regulate expression of a CAR or TCR (see, e.g., Chakravarti, Deboki et al. “Inducible Gene Switches with Memory in Human T Cells for Cellular Immunotherapy.” ACS synthetic biology vol. 8,8 (2019): 1744-1754).
  • Cells that are targeted for transformation may for example include T cells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, human embryonic stem cells, tumor-infiltrating lymphocytes (TIL) or a pluripotent stem cell from which lymphoid cells may be differentiated.
  • T cells expressing a desired CAR may for example be selected through co-culture with y-irradiated activating and propagating cells (AaPC), which co-express the cancer antigen and co-stimulatory molecules.
  • AaPC y-irradiated activating and propagating cells
  • the engineered CAR T-cells may be expanded, for example by coculture on AaPC in presence of soluble factors, such as IL-2 and IL -21.
  • This expansion may for example be carried out so as to provide memory CAR+ T cells (which may for example be assayed by non-enzymatic digital array and/or multi-panel flow cytometry).
  • CAR T cells may be provided that have specific cytotoxic activity against antigen-bearing tumors (optionally in conjunction with production of desired chemokines such as interferon-y).
  • CAR T cells of this kind may for example be used in animal models, for example to treat tumor xenografts.
  • ACT includes co-transferring CD4+ Thl cells and CD8+ CTLs to induce a synergistic anti-tumour response (see, e.g., Li et al., Adoptive cell therapy with CD4+ T helper 1 cells and CD8+ cytotoxic T cells enhances complete rejection of an established tumour, leading to generation of endogenous memory responses to non-targeted tumour epitopes. Clin Transl Immunology. 2017 Oct; 6(10): el 60).
  • Thl7 cells are transferred to a subject in need thereof.
  • Thl7 cells have been reported to directly eradicate melanoma tumors in mice to a greater extent than Thl cells (Muranski P, et al., Tumor-specific Thl7-polarized cells eradicate large established melanoma. Blood. 2008 Jul 15; 112(2):362-73; and Martin-Orozco N, et al., T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity. 2009 Nov 20; 31(5):787-98).
  • ACT adoptive T cell transfer
  • ACT adoptive T cell transfer
  • ACT may include autologous iPSC-based vaccines, such as irradiated iPSCs in autologous anti-tumor vaccines (see e.g., Kooreman, Nigel G. et al., Autologous iPSC-Based Vaccines Elicit Anti-tumor Responses In Vivo, Cell Stem Cell 22, 1-13, 2018, doi.org/10.1016/j. stem.2018.01.016).
  • autologous iPSC-based vaccines such as irradiated iPSCs in autologous anti-tumor vaccines (see e.g., Kooreman, Nigel G. et al., Autologous iPSC-Based Vaccines Elicit Anti-tumor Responses In Vivo, Cell Stem Cell 22, 1-13, 2018, doi.org/10.1016/j. stem.2018.01.016).
  • CARs can potentially bind any cell surface-expressed antigen and can thus be more universally used to treat patients (see Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don’t Forget the Fuel, Front. Immunol., 03 April 2017, doi.org/10.3389/fimmu.2017.00267).
  • the transfer of CAR T-cells may be used to treat patients (see, e.g., Hinrichs CS, Rosenberg SA. Exploiting the curative potential of adoptive T-cell therapy for cancer. Immunol Rev (2014) 257(1):56— 71. doi: 10.1111/ imr.12132).
  • Approaches such as the foregoing may be adapted to provide methods of treating and/or increasing survival of a subject having a disease, such as a neoplasia, for example by administering an effective amount of an immunoresponsive cell comprising an antigen recognizing receptor that binds a selected antigen, wherein the binding activates the immunoresponsive cell, thereby treating or preventing the disease (such as a neoplasia, a pathogen infection, an autoimmune disorder, or an allogeneic transplant reaction).
  • a disease such as a neoplasia
  • a pathogen infection such as a neoplasia, a pathogen infection, an autoimmune disorder, or an allogeneic transplant reaction.
  • the treatment can be administered after lymphodepleting pretreatment in the form of chemotherapy (typically a combination of cyclophosphamide and fludarabine) or radiation therapy.
  • chemotherapy typically a combination of cyclophosphamide and fludarabine
  • ACT cyclophosphamide and fludarabine
  • Immune suppressor cells like Tregs and MDSCs may attenuate the activity of transferred cells by outcompeting them for the necessary cytokines. Not being bound by a theory lymphodepleting pretreatment may eliminate the suppressor cells allowing the TILs to persist.
  • the treatment can be administrated into patients undergoing an immunosuppressive treatment (e.g., glucocorticoid treatment).
  • the cells, or population of cells may be made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent.
  • the immunosuppressive treatment provides for the selection and expansion of the immunoresponsive T cells within the patient.
  • the treatment can be administered before primary treatment (e.g., surgery or radiation therapy) to shrink a tumor before the primary treatment.
  • the treatment can be administered after primary treatment to remove any remaining cancer cells.
  • immunometabolic barriers can be targeted therapeutically prior to and/or during ACT to enhance responses to ACT or CAR T-cell therapy and to support endogenous immunity (see, e.g., Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don’t Forget the Fuel, Front. Immunol., 03 April 2017, doi.org/10.3389/fimmu.2017.00267).
  • cells or population of cells such as immune system cells or cell populations, such as more particularly immunoresponsive cells or cell populations, as disclosed herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • the cells or population of cells may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intrathecally, by intravenous or intralymphatic injection, or intraperitoneally.
  • the disclosed CARs may be delivered or administered into a cavity formed by the resection of tumor tissue (i.e., intracavity delivery) or directly into a tumor prior to resection (i.e., intratumoral delivery).
  • the cell compositions of the present invention are preferably administered by intravenous injection.
  • the administration of the cells or population of cells can consist of the administration of 10 4 - 10 9 cells per kg body weight, preferably 10 5 to 10 6 cells/kg body weight including all integer values of cell numbers within those ranges.
  • Dosing in CAR T cell therapies may for example involve administration of from 10 6 to 10 9 cells/kg, with or without a course of lymphodepletion, for example with cyclophosphamide.
  • the cells or population of cells can be administrated in one or more doses.
  • the effective amount of cells are administrated as a single dose.
  • the effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient.
  • the cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions are within the skill of one in the art.
  • An effective amount means an amount which provides a therapeutic or prophylactic benefit.
  • the dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.
  • the effective amount of cells or composition comprising those cells are administrated parenterally.
  • the administration can be an intravenous administration.
  • the administration can be directly done by injection within a tumor.
  • engineered immunoresponsive cells may be equipped with a transgenic safety switch, in the form of a transgene that renders the cells vulnerable to exposure to a specific signal.
  • TK herpes simplex viral thymidine kinase
  • the herpes simplex viral thymidine kinase (TK) gene may be used in this way, for example by introduction into allogeneic T lymphocytes used as donor lymphocyte infusions following stem cell transplantation (Greco, et al., Improving the safety of cell therapy with the TK-suicide gene. Front. Pharmacol.
  • nucleoside prodrug such as ganciclovir or acyclovir causes cell death.
  • Alternative safety switch constructs include inducible caspase 9, for example triggered by administration of a small-molecule dimerizer that brings together two nonfunctional icasp9 molecules to form the active enzyme.
  • a wide variety of alternative approaches to implementing cellular proliferation controls have been described (see U.S. Patent Publication No. 20130071414; PCT Patent Publication WO2011146862; PCT Patent Publication W02014011987; PCT Patent Publication W02013040371; Zhou et al.
  • genome editing may be used to tailor immunoresponsive cells to alternative implementations, for example providing edited CAR T cells (see Poirot et al., 2015, Multiplex genome edited T-cell manufacturing platform for "off-the-shelf 1 adoptive T-cell immunotherapies, Cancer Res 75 (18): 3853; Ren et al., 2017, Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition, Clin Cancer Res. 2017 May l;23(9):2255-2266. doi: 10.1158/1078-0432.CCR-16-1300.
  • Cells may be edited using any CRISPR system and method of use thereof as described herein.
  • CRISPR systems may be delivered to an immune cell by any method described herein.
  • cells are edited ex vivo and transferred to a subject in need thereof.
  • Immunoresponsive cells, CAR T cells or any cells used for adoptive cell transfer may be edited. Editing may be performed for example to insert or knock-in an exogenous gene, such as an exogenous gene encoding a CAR or a TCR, at a preselected locus in a cell (e.g.
  • TRAC locus to eliminate potential alloreactive T-cell receptors (TCR) or to prevent inappropriate pairing between endogenous and exogenous TCR chains, such as to knock-out or knock-down expression of an endogenous TCR in a cell; to disrupt the target of a chemotherapeutic agent in a cell; to block an immune checkpoint, such as to knock-out or knock-down expression of an immune checkpoint protein or receptor in a cell; to knock-out or knock-down expression of other gene or genes in a cell, the reduced expression or lack of expression of which can enhance the efficacy of adoptive therapies using the cell; to knock-out or knock-down expression of an endogenous gene in a cell, said endogenous gene encoding an antigen targeted by an exogenous CAR or TCR; to knock-out or knock-down expression of one or more MHC constituent proteins in a cell; to activate a T cell; to modulate cells such that the cells are resistant to exhaustion or dysfunction; and/or increase the differentiation and/or proliferation of functionally exhausted
  • editing may result in inactivation of a gene.
  • inactivating a gene it is intended that the gene of interest is not expressed in a functional protein form.
  • the CRISPR system specifically catalyzes cleavage in one targeted gene thereby inactivating said targeted gene.
  • the nucleic acid strand breaks caused are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ).
  • NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions (Indel) and can be used for the creation of specific gene knockouts.
  • HDR homology directed repair
  • editing of cells may be performed to insert or knock-in an exogenous gene, such as an exogenous gene encoding a CAR or a TCR, at a preselected locus in a cell.
  • an exogenous gene such as an exogenous gene encoding a CAR or a TCR
  • nucleic acid molecules encoding CARs or TCRs are transfected or transduced to cells using randomly integrating vectors, which, depending on the site of integration, may lead to clonal expansion, oncogenic transformation, variegated transgene expression and/or transcriptional silencing of the transgene.
  • suitable ‘ safe harbor’ loci for directed transgene integration include CCR5 or AAVS 1.
  • Homology- directed repair (HDR) strategies are known and described elsewhere in this specification allowing to insert transgenes into desired loci (e.g., TRAC locus).
  • transgenes in particular CAR or exogenous TCR transgenes
  • loci comprising genes coding for constituents of endogenous T-cell receptor, such as T-cell receptor alpha locus (TRA) or T-cell receptor beta locus (TRB), for example T-cell receptor alpha constant (TRAC) locus, T-cell receptor beta constant 1 (TRBC1) locus or T-cell receptor beta constant 2 (TRBC1) locus.
  • TRA T-cell receptor alpha locus
  • TRB T-cell receptor beta locus
  • TRBC1 locus T-cell receptor beta constant 1 locus
  • TRBC1 locus T-cell receptor beta constant 2 locus
  • T cell receptors are cell surface receptors that participate in the activation of T cells in response to the presentation of antigen.
  • the TCR is generally made from two chains, a and P, which assemble to form a heterodimer and associates with the CD3 -transducing subunits to form the T cell receptor complex present on the cell surface.
  • Each a and P chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region.
  • variable region of the a and P chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within the population of T cells.
  • T cells are activated by processed peptide fragments in association with an MHC molecule, introducing an extra dimension to antigen recognition by T cells, known as MHC restriction.
  • MHC restriction Recognition of MHC disparities between the donor and recipient through the T cell receptor leads to T cell proliferation and the potential development of graft versus host disease (GVHD).
  • GVHD graft versus host disease
  • the inactivation of TCRa or TCR0 can result in the elimination of the TCR from the surface of T cells preventing recognition of alloantigen and thus GVHD.
  • TCR disruption generally results in the elimination of the CD3 signaling component and alters the means of further T cell expansion.
  • editing of cells may be performed to knock-out or knock-down expression of an endogenous TCR in a cell.
  • NHEJ-based or HDR-based gene editing approaches can be employed to disrupt the endogenous TCR alpha and/or beta chain genes.
  • gene editing system or systems such as CRISPR/Cas system or systems, can be designed to target a sequence found within the TCR beta chain conserved between the beta 1 and beta 2 constant region genes (TRBC1 and TRBC2) and/or to target the constant region of the TCR alpha chain (TRAC) gene.
  • Allogeneic cells are rapidly rejected by the host immune system. It has been demonstrated that, allogeneic leukocytes present in non-irradiated blood products will persist for no more than 5 to 6 days (Boni, Muranski et al. 2008 Blood 1;112(12):4746-54). Thus, to prevent rejection of allogeneic cells, the host's immune system usually has to be suppressed to some extent. However, in the case of adoptive cell transfer the use of immunosuppressive drugs also have a detrimental effect on the introduced therapeutic T cells. Therefore, to effectively use an adoptive immunotherapy approach in these conditions, the introduced cells would need to be resistant to the immunosuppressive treatment.
  • the present invention further comprises a step of modifying T cells to make them resistant to an immunosuppressive agent, preferably by inactivating at least one gene encoding a target for an immunosuppressive agent.
  • An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action.
  • An immunosuppressive agent can be, but is not limited to a calcineurin inhibitor, a target of rapamycin, an interleukin-2 receptor a-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, an inhibitor of dihydrofolic acid reductase, a corticosteroid or an immunosuppressive antimetabolite.
  • targets for an immunosuppressive agent can be a receptor for an immunosuppressive agent such as: CD52, glucocorticoid receptor (GR), a FKBP family gene member and a cyclophilin family gene member.
  • editing of cells may be performed to block an immune checkpoint, such as to knock-out or knock-down expression of an immune checkpoint protein or receptor in a cell.
  • Immune checkpoints are inhibitory pathways that slow down or stop immune reactions and prevent excessive tissue damage from uncontrolled activity of immune cells.
  • the immune checkpoint targeted is the programmed death-1 (PD-1 or CD279) gene (PDCD1) (see, e.g, Rupp LJ, Schumann K, Roybal KT, et al.
  • the immune checkpoint targeted is cytotoxic T-lymphocyte-associated antigen (CTLA-4).
  • CTLA-4 cytotoxic T-lymphocyte-associated antigen
  • the immune checkpoint targeted is another member of the CD28 and CTLA4 Ig superfamily such as BTLA, LAG3, ICOS, PDL1 or KIR.
  • the immune checkpoint targeted is a member of the TNFR superfamily such as CD40, 0X40, CD137, GITR, CD27 or TIM-3.
  • Additional immune checkpoints include Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) (Watson HA, et al., SHP-1: the next checkpoint target for cancer immunotherapy? Biochem Soc Trans. 2016 Apr 15;44(2):356-62).
  • SHP-1 is a widely expressed inhibitory protein tyrosine phosphatase (PTP).
  • PTP inhibitory protein tyrosine phosphatase
  • T-cells it is a negative regulator of antigendependent activation and proliferation. It is a cytosolic protein, and therefore not amenable to antibody-mediated therapies, but its role in activation and proliferation makes it an attractive target for genetic manipulation in adoptive transfer strategies, such as chimeric antigen receptor (CAR) T cells.
  • CAR chimeric antigen receptor
  • Immune checkpoints may also include T cell immunoreceptor with Ig and ITIM domains (TIGIT/Vstm3/WUCAM/VSIG9) and VISTA (Le Mercier I, et al, (2015) Beyond CTLA-4 and PD-1, the generation Z of negative checkpoint regulators. Front. Immunol. 6:418).
  • WO2014172606 relates to the use of MT1 and/or MT2 inhibitors to increase proliferation and/or activity of exhausted CD8+ T-cells and to decrease CD8+ T-cell exhaustion (e.g., decrease functionally exhausted or unresponsive CD8+ immune cells).
  • metallothioneins are targeted by gene editing in adoptively transferred T cells.
  • targets of gene editing may be at least one targeted locus involved in the expression of an immune checkpoint protein.
  • targets may include, but are not limited to CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4), TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL 1 ORA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF,
  • WO2016196388 concerns an engineered T cell comprising (a) a genetically engineered antigen receptor that specifically binds to an antigen, which receptor may be a CAR; and (b) a disrupted gene encoding a PD-L1, an agent for disruption of a gene encoding a PD- LI, and/or disruption of a gene encoding PD-L1, wherein the disruption of the gene may be mediated by a gene editing nuclease, a zinc finger nuclease (ZFN), CRISPR/Cas9 and/or TALEN.
  • a genetically engineered antigen receptor that specifically binds to an antigen, which receptor may be a CAR
  • a disrupted gene encoding a PD-L1
  • an agent for disruption of a gene encoding a PD- LI and/or disruption of a gene encoding PD-L1
  • the disruption of the gene may be mediated by a gene editing nuclease,
  • WO2015142675 relates to immune effector cells comprising a CAR in combination with an agent (such as CRISPR, TALEN or ZFN) that increases the efficacy of the immune effector cells in the treatment of cancer, wherein the agent may inhibit an immune inhibitory molecule, such as PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, or CEACAM-5.
  • an agent such as CRISPR, TALEN or ZFN
  • an immune inhibitory molecule such as PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, or CEACAM-5.
  • cells may be engineered to express a CAR, wherein expression and/or function of methylcytosine dioxygenase genes (TET1, TET2 and/or TET3) in the cells has been reduced or eliminated, such as by CRISPR, ZNF or TALEN (for example, as described in W0201704916).
  • a CAR methylcytosine dioxygenase genes
  • editing of cells may be performed to knock-out or knock-down expression of an endogenous gene in a cell, said endogenous gene encoding an antigen targeted by an exogenous CAR or TCR, thereby reducing the likelihood of targeting of the engineered cells.
  • the targeted antigen may be one or more antigen selected from the group consisting of CD38, CD138, CS-1, CD33, CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, CD362, human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms’ tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin (DI), B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI), and B-cell activating factor receptor (BAFF-R) (for example, as described in W02016011210 and W02017011804).
  • hTERT human
  • editing of cells may be performed to knock-out or knock-down expression of one or more MHC constituent proteins, such as one or more HLA proteins and/or beta-2 microglobulin (B2M), in a cell, whereby rejection of non-autologous (e.g., allogeneic) cells by the recipient’s immune system can be reduced or avoided.
  • one or more HLA class I proteins such as HLA- A, B and/or C, and/or B2M may be knocked-out or knocked-down.
  • B2M may be knocked-out or knocked-down.
  • Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, P-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.
  • at least two genes are edited.
  • Pairs of genes may include, but are not limited to PD1 and TCRa, PD1 and TCR , CTLA-4 and TCRa, CTLA-4 and TCRP, LAG3 and TCRa, LAG3 and TCR , Tim3 and TCRa, Tim3 and TCR , BTLA and TCRa, BTLA and TCR , BY55 and TCRa, BY55 and TCRP, TIGIT and TCRa, TIGIT and TCRP, B7H5 and TCRa, B7H5 and TCR0, LAIR1 and TCRa, LAIR1 and TCR0, SIGLEC10 and TCRa, SIGLEC1O and TCRP, 2B4 and TCRa, 2B4 and TCR0, B2M and TCRa, B2M and TCR0.
  • a cell may be multiply edited (multiplex genome editing) as taught herein to (1) knock-out or knock-down expression of an endogenous TCR (for example, TRBC1, TRBC2 and/or TRAC), (2) knock-out or knock-down expression of an immune checkpoint protein or receptor (for example PD1, PD-L1 and/or CTLA4); and (3) knock-out or knock-down expression of one or more MHC constituent proteins (for example, HLA-A, B and/or C, and/or B2M, preferably B2M).
  • an endogenous TCR for example, TRBC1, TRBC2 and/or TRAC
  • an immune checkpoint protein or receptor for example PD1, PD-L1 and/or CTLA4
  • MHC constituent proteins for example, HLA-A, B and/or C, and/or B2M, preferably B2M.
  • the T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631.
  • T cells can be expanded in vitro or in vivo.
  • Immune cells may be obtained using any method known in the art.
  • allogenic T cells may be obtained from healthy subjects.
  • T cells that have infiltrated a tumor are isolated.
  • T cells may be removed during surgery.
  • T cells may be isolated after removal of tumor tissue by biopsy.
  • T cells may be isolated by any means known in the art.
  • T cells are obtained by apheresis.
  • the method may comprise obtaining a bulk population of T cells from a tumor sample by any suitable method known in the art. For example, a bulk population of T cells can be obtained from a tumor sample by dissociating the tumor sample into a cell suspension from which specific cell populations can be selected.
  • Suitable methods of obtaining a bulk population of T cells may include, but are not limited to, any one or more of mechanically dissociating (e.g., mincing) the tumor, enzymatically dissociating (e.g., digesting) the tumor, and aspiration (e.g., as with a needle).
  • mechanically dissociating e.g., mincing
  • enzymatically dissociating e.g., digesting
  • aspiration e.g., as with a needle
  • the bulk population of T cells obtained from a tumor sample may comprise any suitable type of T cell.
  • the bulk population of T cells obtained from a tumor sample comprises tumor infdtrating lymphocytes (TILs).
  • TILs tumor infdtrating lymphocytes
  • the tumor sample may be obtained from any mammal.
  • mammal refers to any mammal including, but not limited to, mammals of the order Logomorpha, such as rabbits; the order Carnivora, including Felines (cats) and Canines (dogs); the order Artiodactyla, including Bovines (cows) and Swines (pigs); or of the order Perssodactyla, including Equines (horses).
  • the mammals may be non-human primates, e.g., of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • the mammal may be a mammal of the order Rodentia, such as mice and hamsters.
  • the mammal is a non-human primate or a human.
  • An especially preferred mammal is the human.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, spleen tissue, and tumors.
  • PBMC peripheral blood mononuclear cells
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation.
  • cells from the circulating blood of an individual are obtained by apheresis or leukapheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium lead to magnified activation.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor) according to the manufacturer's instructions.
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS.
  • a variety of biocompatible buffers such as, for example, Ca-free, Mg-free PBS.
  • the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
  • a specific subpopulation of T cells such as CD28+, CD4+, CDC, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques.
  • T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, or XCYTE DYNABEADSTM for a time period sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation time period is 24 hours.
  • use of longer incubation times such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells.
  • TIL tumor infiltrating lymphocytes
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • a preferred method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA- DR, and CD8.
  • monocyte populations may be depleted from blood preparations by a variety of methodologies, including anti-CD14 coated beads or columns, or utilization of the phagocytotic activity of these cells to facilitate removal.
  • the invention uses paramagnetic particles of a size sufficient to be engulfed by phagocytotic monocytes.
  • the paramagnetic particles are commercially available beads, for example, those produced by Life Technologies under the trade name DynabeadsTM.
  • other non-specific cells are removed by coating the paramagnetic particles with “irrelevant” proteins (e.g., serum proteins or antibodies).
  • Irrelevant proteins and antibodies include those proteins and antibodies or fragments thereof that do not specifically target the T cells to be isolated.
  • the irrelevant beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin.
  • depletion of monocytes is performed by preincubating T cells isolated from whole blood, apheresed peripheral blood, or tumors with one or more varieties of irrelevant or non-antibody coupled paramagnetic particles at any amount that allows for removal of monocytes (approximately a 20:1 bead:cell ratio) for about 30 minutes to 2 hours at 22 to 37 degrees C., followed by magnetic removal of cells which have attached to or engulfed the paramagnetic particles.
  • Such separation can be performed using standard methods available in the art.
  • any magnetic separation methodology may be used including a variety of which are commercially available, (e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®)).
  • DYNAL MPC® Magnetic Particle Concentrator
  • Assurance of requisite depletion can be monitored by a variety of methodologies known to those of ordinary skill in the art, including flow cytometric analysis of CD14 positive cells, before and after depletion.
  • the concentration of cells and surface can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used.
  • concentrations can result in increased cell yield, cell activation, and cell expansion.
  • use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28- negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • the concentration of cells used is 5 x 10 6 /ml. In other embodiments, the concentration used can be from about 1 x 10 5 /ml to 1 x 10 6 /ml, and any integer value in between.
  • T cells can also be frozen.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media, the cells then are frozen to -80° C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C. or in liquid nitrogen.
  • T cells for use in the present invention may also be antigen-specific T cells.
  • tumor-specific T cells can be used.
  • antigen-specific T cells can be isolated from a patient of interest, such as a patient afflicted with a cancer or an infectious disease.
  • neoepitopes are determined for a subject and T cells specific to these antigens are isolated.
  • Antigen-specific cells for use in expansion may also be generated in vitro using any number of methods known in the art, for example, as described in U. S. Patent Publication No. US 20040224402 entitled, Generation and Isolation of Antigen-Specific T Cells, or in U.S. Pat. Nos. 6,040,177.
  • Antigen-specific cells for use in the present invention may also be generated using any number of methods known in the art, for example, as described in Current Protocols in Immunology, or Current Protocols in Cell Biology, both published by John Wiley & Sons, Inc., Boston, Mass.
  • sorting or positively selecting antigen-specific cells can be carried out using peptide- MHC tetramers (Altman, et al., Science. 1996 Oct. 4; 274(5284):94-6).
  • the adaptable tetramer technology approach is used (Andersen et al., 2012 Nat Protoc. 7:891-902). Tetramers are limited by the need to utilize predicted binding peptides based on prior hypotheses, and the restriction to specific HLAs.
  • Peptide-MHC tetramers can be generated using techniques known in the art and can be made with any MHC molecule of interest and any antigen of interest as described herein. Specific epitopes to be used in this context can be identified using numerous assays known in the art. For example, the ability of a polypeptide to bind to MHC class I may be evaluated indirectly by monitoring the ability to promote incorporation of 125 I labeled 02- microglobulin (02m) into MHC class I/02m/peptide heterotrimeric complexes (see Parker et al., J. Immunol. 152: 163, 1994).
  • 02m 02- microglobulin
  • cells are directly labeled with an epitope-specific reagent for isolation by flow cytometry followed by characterization of phenotype and TCRs.
  • T cells are isolated by contacting with T cell specific antibodies. Sorting of antigenspecific T cells, or generally any cells of the present invention, can be carried out using any of a variety of commercially available cell sorters, including, but not limited to, MoFlo sorter (DakoCytomation, Fort Collins, Colo.), FACSAriaTM, FACSArrayTM, FACSVantageTM, BDTM LSR II, and FACSCaliburTM (BD Biosciences, San Jose, Calif.).
  • the method comprises selecting cells that also express CD3.
  • the method may comprise specifically selecting the cells in any suitable manner.
  • the selecting is carried out using flow cytometry.
  • the flow cytometry may be carried out using any suitable method known in the art.
  • the flow cytometry may employ any suitable antibodies and stains.
  • the antibody is chosen such that it specifically recognizes and binds to the particular biomarker being selected.
  • the specific selection of CD3, CD8, TIM-3, LAG-3, 4-1BB, or PD-1 may be carried out using anti-CD3, anti-CD8, anti-TIM-3, anti-LAG-3, anti-4-lBB, or anti-PD-1 antibodies, respectively.
  • the antibody or antibodies may be conjugated to a bead (e.g., a magnetic bead) or to a fluorochrome.
  • the flow cytometry is fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • TCRs expressed on T cells can be selected based on reactivity to autologous tumors.
  • T cells that are reactive to tumors can be selected for based on markers using the methods described in patent publication Nos. WO2014133567 and WO2014133568, herein incorporated by reference in their entirety.
  • activated T cells can be selected for based on surface expression of CD 107a.
  • the method further comprises expanding the numbers of T cells in the enriched cell population.
  • the numbers of T cells may be increased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), more preferably at least about 10- fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold), more preferably at least about 100-fold, more preferably at least about 1,000 fold, or most preferably at least about 100,000-fold.
  • the numbers of T cells may be expanded using any suitable method known in the art. Exemplary methods of expanding the numbers of cells are described in patent publication No. WO 2003057171, U.S. Patent No. 8,034,334, and U.S. Patent Application Publication No. 2012/0244133, each of which is incorporated herein by reference.
  • ex vivo T cell expansion can be performed by isolation of T cells and subsequent stimulation or activation followed by further expansion.
  • the T cells may be stimulated or activated by a single agent.
  • T cells are stimulated or activated with two agents, one that induces a primary signal and a second that is a co-stimulatory signal.
  • Ligands useful for stimulating a single signal or stimulating a primary signal and an accessory molecule that stimulates a second signal may be used in soluble form.
  • Ligands may be attached to the surface of a cell, to an Engineered Multivalent Signaling Platform (EMSP), or immobilized on a surface.
  • ESP Engineered Multivalent Signaling Platform
  • both primary and secondary agents are co-immobilized on a surface, for example a bead or a cell.
  • the molecule providing the primary activation signal may be a CD3 ligand
  • the co-stimulatory molecule may be a CD28 ligand or 4- IBB ligand.
  • T cells comprising a CAR or an exogenous TCR may be manufactured as described in WO2015120096, by a method comprising: enriching a population of lymphocytes obtained from a donor subject; stimulating the population of lymphocytes with one or more T-cell stimulating agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule which encodes the CAR or TCR, using a single cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the population of transduced T cells for a predetermined time to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium.
  • T cells comprising a CAR or an exogenous TCR may be manufactured as described in WO2015120096, by a method comprising: obtaining a population of lymphocytes; stimulating the population of lymphocytes with one or more stimulating agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule which encodes the CAR or TCR, using at least one cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the population of transduced T cells to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium.
  • the predetermined time for expanding the population of transduced T cells may be 3 days.
  • the time from enriching the population of lymphocytes to producing the engineered T cells may be 6 days.
  • the closed system may be a closed bag system. Further provided is population of T cells comprising a CAR or an exogenous TCR obtainable or obtained by said method, and a pharmaceutical composition comprising such cells.
  • T cell maturation or differentiation in vitro may be delayed or inhibited by the method as described in W02017070395, comprising contacting one or more T cells from a subject in need of a T cell therapy with an AKT inhibitor (such as, e.g., one or a combination of two or more AKT inhibitors disclosed in claim 8 of W02017070395) and at least one of exogenous Interleukin-7 (IL-7) and exogenous Interleukin- 15 (IL-15), wherein the resulting T cells exhibit delayed maturation or differentiation, and/or wherein the resulting T cells exhibit improved T cell function (such as, e.g., increased T cell proliferation; increased cytokine production; and/or increased cytolytic activity) relative to a T cell function of a T cell cultured in the absence of an AKT inhibitor.
  • an AKT inhibitor such as, e.g., one or a combination of two or more AKT inhibitors disclosed in claim 8 of W02017070395
  • IL-7 exogenous Interleuk
  • a patient in need of a T cell therapy may be conditioned by a method as described in WO2016191756 comprising administering to the patient a dose of cyclophosphamide between 200 mg/m2/day and 2000 mg/m2/day and a dose of fludarabine between 20 mg/m2/day and 900 mg/m 2 /day.
  • a patient in need of adoptive cell transfer may be administered a TLR agonist to enhance anti -tumor immunity (see, e.g., Urban-Wojciuk, et al., The Role of TLRs in Anti-cancer Immunity and Tumor Rejection, Front Immunol. 2019; 10: 2388; and Kaczanowska et al., TLR agonists: our best frenemy in cancer immunotherapy, J Leukoc Biol. 2013 Jun; 93(6): 847-863).
  • TLR agonists are delivered in a nanoparticle system (see, e.g., Buss and Bhatia, Nanoparticle delivery of immunostimulatory oligonucleotides enhances response to checkpoint inhibitor therapeutics, Proc Natl Acad Sci USA. 2020 Jun 3;202001569).
  • the agonist is a TLR9 agonist. Id.
  • surface or secreted markers are targeted using antibodies (e.g., NRP: TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; or SBM: MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4; or NRP: PDGFD, C6, CRISP3, RELN, SERPINA6, CRP, FGG, APCS, CFH, HABP2, KLKB1, REGIA and SPP1; or SBM: COL17A1, MUC16, FGF19, IGF2, NPC2, SAA1, SRGN, CIS, and CST4; or NRP TME: C7, HDGF, MAPT, REGIB, SERPINA4, PVR, CRISP3, DKK4, DPP7, NPNT
  • antibody is used interchangeably with the term “immunoglobulin” herein, and includes intact antibodies, fragments of antibodies, e.g., Fab, F(ab')2 fragments, and intact antibodies and fragments that have been mutated either in their constant and/or variable region (e.g., mutations to produce chimeric, partially humanized, or fully humanized antibodies, as well as to produce antibodies with a desired trait, e.g., enhanced binding and/or reduced FcR binding).
  • fragment refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain.
  • Fragments can be obtained via chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. Exemplary fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, VHH and scFv and/or Fv fragments.
  • a preparation of antibody protein having less than about 50% of nonantibody protein (also referred to herein as a “contaminating protein”), or of chemical precursors, is considered to be “substantially free.” 40%, 30%, 20%, 10% and more preferably 5% (by dry weight) of non-antibody protein or of chemical precursors is considered to be substantially free.
  • the antibody protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume or mass of the protein preparation.
  • antigen-binding fragment refers to a polypeptide fragment of an immunoglobulin or antibody that binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding).
  • antigen binding i.e., specific binding
  • antibody encompass any Ig class or any Ig subclass (e.g., the IgGl, IgG2, IgG3, and IgG4 subclasses of IgG) obtained from any source (e.g., humans and non-human primates, and in rodents, lagomorphs, caprines, bovines, equines, ovines, etc.).
  • Ig class or “immunoglobulin class”, as used herein, refers to the five classes of immunoglobulin that have been identified in humans and higher mammals, IgG, IgM, IgA, IgD, and IgE.
  • Ig subclass refers to the two subclasses of IgM (H and L), three subclasses of IgA (IgAl, IgA2, and secretory IgA), and four subclasses of IgG (IgGl, IgG2, IgG3, and IgG4) that have been identified in humans and higher mammals.
  • the antibodies can exist in monomeric or polymeric form; for example, IgM antibodies exist in pentameric form, and IgA antibodies exist in monomeric, dimeric or multimeric form.
  • IgG subclass refers to the four subclasses of immunoglobulin class IgG - IgGl, IgG2, IgG3, and IgG4 that have been identified in humans and higher mammals by the heavy chains of the immunoglobulins, VI - y4, respectively.
  • single-chain immunoglobulin or “single-chain antibody” (used interchangeably herein) refers to a protein having a two- polypeptide chain structure consisting of a heavy and a light chain, said chains being stabilized, for example, by interchain peptide linkers, which has the ability to specifically bind antigen.
  • domain refers to a globular region of a heavy or light chain polypeptide comprising peptide loops (e.g., comprising 3 to 4 peptide loops) stabilized, for example, by pleated sheet and/or intrachain disulfide bond. Domains are further referred to herein as “constant” or “variable”, based on the relative lack of sequence variation within the domains of various class members in the case of a “constant” domain, or the significant variation within the domains of various class members in the case of a “variable” domain.
  • Antibody or polypeptide “domains” are often referred to interchangeably in the art as antibody or polypeptide “regions”.
  • the “constant” domains of an antibody light chain are referred to interchangeably as “light chain constant regions”, “light chain constant domains”, “CL” regions or “CL” domains.
  • the “constant” domains of an antibody heavy chain are referred to interchangeably as “heavy chain constant regions”, “heavy chain constant domains”, “CH” regions or “CH” domains).
  • the “variable” domains of an antibody light chain are referred to interchangeably as “light chain variable regions”, “light chain variable domains”, “VL” regions or “VL” domains).
  • the “variable” domains of an antibody heavy chain are referred to interchangeably as “heavy chain constant regions”, “heavy chain constant domains”, “VH” regions or “VH” domains).
  • region can also refer to a part or portion of an antibody chain or antibody chain domain (e.g., a part or portion of a heavy or light chain or a part or portion of a constant or variable domain, as defined herein), as well as more discrete parts or portions of said chains or domains.
  • light and heavy chains or light and heavy chain variable domains include “complementarity determining regions” or “CDRs” interspersed among “framework regions” or “FRs”, as defined herein.
  • the term “conformation” refers to the tertiary structure of a protein or polypeptide (e.g., an antibody, antibody chain, domain or region thereof).
  • the phrase “light (or heavy) chain conformation” refers to the tertiary structure of a light (or heavy) chain variable region
  • the phrase “antibody conformation” or “antibody fragment conformation” refers to the tertiary structure of an antibody or fragment thereof.
  • antibody-like protein scaffolds or “engineered protein scaffolds” broadly encompasses proteinaceous non-immunoglobulin specific-binding agents, typically obtained by combinatorial engineering (such as site-directed random mutagenesis in combination with phage display or other molecular selection techniques).
  • Such scaffolds are derived from robust and small soluble monomeric proteins (such as Kunitz inhibitors or lipocalins) or from a stably folded extra-membrane domain of a cell surface receptor (such as protein A, fibronectin or the ankyrin repeat).
  • Curr Opin Biotechnol 2007, 18:295-304 include without limitation affibodies, based on the Z-domain of staphylococcal protein A, a three- helix bundle of 58 residues providing an interface on two of its alpha-helices (Nygren, Alternative binding proteins: Affibody binding proteins developed from a small three-helix bundle scaffold. FEBS J 2008, 275:2668-2676); engineered Kunitz domains based on a small (ca.
  • anticalins derived from the lipocalins, a diverse family of eight-stranded beta-barrel proteins (ca. 180 residues) that naturally form binding sites for small ligands by means of four structurally variable loops at the open end, which are abundant in humans, insects, and many other organisms (Skerra, Alternative binding proteins: Anticalins — harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities.
  • DARPins designed ankyrin repeat domains (166 residues), which provide a rigid interface arising from typically three repeated beta-turns
  • avimers multimerized LDLR-A module
  • avimers Smallman et al., Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol 2005, 23:1556-1561
  • cysteine-rich knottin peptides Kolmar, Alternative binding proteins: biological activity and therapeutic potential of cystine-knot miniproteins.
  • “Specific binding” of an antibody means that the antibody exhibits appreciable affinity for a particular antigen or epitope and, generally, does not exhibit significant cross reactivity. “Appreciable” binding includes binding with an affinity of at least 25 pM. Antibodies with affinities greater than 1 x 10 7 M' 1 (or a dissociation coefficient of IpM or less or a dissociation coefficient of Inm or less) typically bind with correspondingly greater specificity.
  • antibodies of the invention bind with a range of affinities, for example, lOOnM or less, 75nM or less, 50nM or less, 25nM or less, for example lOnM or less, 5nM or less, InM or less, or in embodiments 500pM or less, lOOpM or less, 50pM or less or 25pM or less.
  • An antibody that “does not exhibit significant crossreactivity” is one that will not appreciably bind to an entity other than its target (e.g., a different epitope or a different molecule).
  • an antibody that specifically binds to a target molecule will appreciably bind the target molecule but will not significantly react with non-target molecules or peptides.
  • An antibody specific for a particular epitope will, for example, not significantly crossreact with remote epitopes on the same protein or peptide.
  • Specific binding can be determined according to any art-recognized means for determining such binding. Preferably, specific binding is determined according to Scatchard analysis and/or competitive binding assays.
  • affinity refers to the strength of the binding of a single antigen-combining site with an antigenic determinant. Affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, on the distribution of charged and hydrophobic groups, etc. Antibody affinity can be measured by equilibrium dialysis or by the kinetic BIACORETM method. The dissociation constant, Kd, and the association constant, Ka, are quantitative measures of affinity.
  • the term “monoclonal antibody” refers to an antibody derived from a clonal population of antibody-producing cells (e.g., B lymphocytes or B cells) which is homogeneous in structure and antigen specificity.
  • the term “polyclonal antibody” refers to a plurality of antibodies originating from different clonal populations of antibody-producing cells which are heterogeneous in their structure and epitope specificity, but which recognize a common antigen.
  • Monoclonal and polyclonal antibodies may exist within bodily fluids, as crude preparations, or may be purified, as described herein.
  • binding portion of an antibody includes one or more complete domains, e.g., a pair of complete domains, as well as fragments of an antibody that retain the ability to specifically bind to a target molecule. It has been shown that the binding function of an antibody can be performed by fragments of a full-length antibody. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, Fv, single chains, single-chain antibodies, e.g., scFv, and single domain antibodies.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • portions of antibodies or epitope-binding proteins encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VH and CHI domains; (iv) the Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., 341 Nature 544 (1989)) which consists of a VH domain or a VL domain that binds antigen; (vii) isolated CDR regions or isolated CDR regions presented in a functional framework; (viii) F(ab')2 fragments which are bivalent fragments including two
  • a “blocking” antibody or an antibody “antagonist” is one which inhibits or reduces biological activity of the antigen(s) it binds.
  • the blocking antibodies or antagonist antibodies or portions thereof described herein completely inhibit the biological activity of the antigen(s).
  • Antibodies may act as agonists or antagonists of the recognized polypeptides.
  • the present invention includes antibodies which disrupt receptor/ligand interactions either partially or fully.
  • the invention features both receptor-specific antibodies and ligandspecific antibodies.
  • the invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation.
  • Receptor activation i.e., signaling
  • receptor activation can be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or of one of its down-stream substrates by immunoprecipitation followed by western blot analysis.
  • antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
  • receptors are targeted with antibodies that block ligand binding.
  • the invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex.
  • receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex.
  • neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
  • antibodies which activate the receptor are also act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor.
  • the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides disclosed herein.
  • the antibody agonists and antagonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6): 1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4): 1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J.
  • the antibodies as defined for the present invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti -idiotypic response.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • Simple binding assays can be used to screen for or detect agents that bind to a target protein, or disrupt the interaction between proteins (e.g., a receptor and a ligand). Because certain targets of the present invention are transmembrane proteins, assays that use the soluble forms of these proteins rather than full-length protein can be used, in some embodiments. Soluble forms include, for example, those lacking the transmembrane domain and/or those comprising the IgV domain or fragments thereof which retain their ability to bind their cognate binding partners. Further, agents that inhibit or enhance protein interactions for use in the compositions and methods described herein, can include recombinant peptido-mimetics.
  • Detection methods useful in screening assays include antibody-based methods, detection of a reporter moiety, detection of cytokines as described herein, and detection of a gene signature as described herein.
  • Another variation of assays to determine binding of a receptor protein to a ligand protein is through the use of affinity biosensor methods. Such methods may be based on the piezoelectric effect, electrochemistry, or optical methods, such as ellipsometry, optical wave guidance, and surface plasmon resonance (SPR).
  • surface markers are targeted with antibodies capable of inducing antibody-dependent cellular cytotoxicity (ADCC) (e.g., NRP: TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; or SBM: MSLN, GPR87, SLC2A1, LY6D, and PSCA).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCC also called antibody-dependent cell-mediated cytotoxicity, is an immune mechanism through which Fc receptor-bearing effector cells can recognize and kill antibody-coated target cells expressing tumor- or pathogen-derived antigens on their surface.
  • surface markers are targeted with bi-specific antigen-binding constructs, e.g., bi-specific antibodies (bsAb) or BiTEs, that bind two antigens (see, e.g., Suurs et al., A review of bispecific antibodies and antibody constructs in oncology and clinical challenges. Pharmacol Ther. 2019 Sep;201 : 103-119; and Huehls, et al., Bispecific T cell engagers for cancer immunotherapy. Immunol Cell Biol.
  • bi-specific antigen-binding constructs e.g., bi-specific antibodies (bsAb) or BiTEs, that bind two antigens (see, e.g., Suurs et al., A review of bispecific antibodies and antibody constructs in oncology and clinical challenges. Pharmacol Ther. 2019 Sep;201 : 103-119; and Huehls, et al., Bispecific T cell engagers for cancer immunotherapy. Immunol Cell Biol.
  • NRP TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; or SBM: MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4).
  • the bi-specific antigen-binding construct includes two antigen-binding polypeptide constructs, e.g., antigen binding domains, wherein at least one polypeptide construct specifically binds to a tumor surface protein (e.g., NRP: TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; or SBM: MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4).
  • the antigen-binding construct is derived from known antibodies or antigen-binding constructs.
  • the antigen- binding polypeptide constructs comprise two antigen binding domains that comprise antibody fragments.
  • the first antigen binding domain and second antigen binding domain each independently comprises an antibody fragment selected from the group of: an scFv, a Fab, and an Fc domain.
  • the antibody fragments may be the same format or different formats from each other.
  • the antigen-binding polypeptide constructs comprise a first antigen binding domain comprising an scFv and a second antigen binding domain comprising a Fab.
  • the antigenbinding polypeptide constructs comprise a first antigen binding domain and a second antigen binding domain, wherein both antigen binding domains comprise an scFv.
  • the first and second antigen binding domains each comprise a Fab.
  • the first and second antigen binding domains each comprise an Fc domain. Any combination of antibody formats is suitable for the bi-specific antibody constructs disclosed herein.
  • immune cells can be engaged to tumor cells.
  • tumor cells are targeted with a bsAb having affinity for both the tumor and a payload.
  • two targets are disrupted on a tumor cell by the bsAb (e.g., NRP: TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; or SBM: MSLN, GPR87, SLC2A1, LY6D, and PSCA).
  • an agent such as a bi-specific antibody, capable of specifically binding to a gene product expressed on the cell surface of the immune cells (e.g., CD3, CD8, CD28, CD16) and a tumor cell (e g., NRP: TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; or SBM: MSLN, GPR87, SLC2A1, LY6D, and PSCA) may be used for targeting polyfunctional immune cells to tumor cells.
  • Immune cells targeted to a tumor may include T cells or NK cells.
  • surface markers are targeted with antibody-drug conjugates (e g., NRP: TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; or SBM: MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4).
  • antibody-drug conjugates e g., NRP: TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR
  • SBM MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4
  • an ADC refers to a binding protein, such as an antibody or antigen binding fragment thereof, chemically linked to one or more chemical drug(s) (also referred to herein as agent(s)) that may optionally be therapeutic or cytotoxic agents.
  • an ADC includes an antibody, a cytotoxic or therapeutic drug, and a linker that enables attachment or conjugation of the drug to the antibody.
  • An ADC typically has anywhere from 1 to 8 drugs conjugated to the antibody, including drug loaded species of 2, 4, 6, or 8.
  • the ADC specifically binds to a gene product expressed on the cell surface of a tumor cell.
  • an agent such as an antibody, capable of specifically binding to a gene product expressed on the cell surface of the tumor cells may be conjugated with a therapeutic or effector agent for targeted delivery of the therapeutic or effector agent to the immune cells.
  • therapeutic or effector agents include immunomodulatory classes as discussed herein, such as without limitation a toxin, drug, radionuclide, cytokine, lymphokine, chemokine, growth factor, tumor necrosis factor, hormone, hormone antagonist, enzyme, oligonucleotide, siRNA, RNAi, photoactive therapeutic agent, anti-angiogenic agent and pro- apoptotic agent.
  • immunomodulatory classes such as without limitation a toxin, drug, radionuclide, cytokine, lymphokine, chemokine, growth factor, tumor necrosis factor, hormone, hormone antagonist, enzyme, oligonucleotide, siRNA, RNAi, photoactive therapeutic agent, anti-angiogenic agent and pro- apoptotic agent.
  • Non-limiting examples of drugs that may be included in the ADCs are mitotic inhibitors (e.g., maytansinoid DM4), antitumor antibiotics, immunomodulating agents, vectors for gene therapy, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, antihormone agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, and radiosensitizers.
  • mitotic inhibitors e.g., maytansinoid DM4
  • antitumor antibiotics e.g., antitumor antibiotics
  • immunomodulating agents e.g., antitumor antibiotics
  • vectors for gene therapy alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, antihormone agents, cor
  • Example toxins include ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, or Pseudomonas endotoxin.
  • RNase ribonuclease
  • DNase I DNase I
  • Staphylococcal enterotoxin-A Staphylococcal enterotoxin-A
  • pokeweed antiviral protein pokeweed antiviral protein
  • gelonin gelonin
  • diphtheria toxin diphtheria toxin
  • Pseudomonas exotoxin Pseudomonas exotoxin
  • Pseudomonas endotoxin Pseudomonas endotoxin.
  • Example radionuclides include 103m Rh, 103 Ru, 105 Rh, 105 Ru, 107 Hg, 109 Pd, 109 Pt, n i Ag, 153 Sm, 15 O, 161 Ho, 161 Tb, 165 Tm, 166 Dy, 166 Ho, 167 Tm, 168 Tm, 169 Er, 169 Yb, 177 Lu, 186 Re, 188 Re, 189m Os, 189 Re, 192 Ir, 194 Ir, 197 Pt, 198 Au, 199 Au, 2O1 T1, 203 Hg, 211 At, 211 Bi, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 215 Po, 217 At, 219 Rn, 221 Fr, 223 Ra, 224 Ac, 225 Ac, 225 Fm, 32 P, 33 P, 47 Sc, 51 Cr, 57 Co, 58 Co, 59 Fe, 62 Cu, 67 Cu, 67 Ga, 75 Br, 75 Se
  • Example enzymes include malate dehydrogenase, staphylococcal nuclease, delta-V- steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase or acetylcholinesterase.
  • Such enzymes may be used, for example, in combination with prodrugs that are administered in relatively non-toxic form and converted at the target site by the enzyme into a cytotoxic agent.
  • a drug may be converted into less toxic form by endogenous enzymes in the subject but may be reconverted into a cytotoxic form by the therapeutic enzyme.
  • surface or secreted markers are targeted using aptamers (e.g., NRP: TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; or SBM: MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4; or NRP: PDGFD, C6, CRISP3, RELN, SERPINA6, CRP, FGG, APCS, CFH, HABP2, KLKB1, REGIA and SPP1; or SBM: COL17A1, MUC16, FGF19, IGF2, NPC2, SAA1, SRGN, CIS, and CST4; or NRP TME: C7, HDGF, MAPT, REGIB, SERPINA4, PVR, CRISP3, DKK4, DPP7,
  • aptamers e
  • Nucleic acid aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, cells, tissues and organisms. Nucleic acid aptamers have specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing. Aptamers are useful in biotechnological and therapeutic applications as they offer molecular recognition properties similar to antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • RNA aptamers may be expressed from a DNA construct.
  • a nucleic acid aptamer may be linked to another polynucleotide sequence.
  • the polynucleotide sequence may be a double stranded DNA polynucleotide sequence.
  • the aptamer may be covalently linked to one strand of the polynucleotide sequence.
  • the aptamer may be ligated to the polynucleotide sequence.
  • the polynucleotide sequence may be configured, such that the polynucleotide sequence may be linked to a solid support or ligated to another polynucleotide sequence.
  • Aptamers like peptides generated by phage display or monoclonal antibodies (“mAbs”), are capable of specifically binding to selected targets and modulating the target's activity, e.g., through binding, aptamers may block their target's ability to function.
  • a typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind other proteins from the same gene family).
  • aptamers are capable of using the same types of binding interactions (e.g., hydrogen bonding, electrostatic complementarity, hydrophobic contacts, steric exclusion) that drives affinity and specificity in antibody-antigen complexes.
  • binding interactions e.g., hydrogen bonding, electrostatic complementarity, hydrophobic contacts, steric exclusion
  • Aptamers have a number of desirable characteristics for use in research and as therapeutics and diagnostics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over antibodies and other protein biologies. Aptamers are chemically synthesized and are readily scaled as needed to meet production demand for research, diagnostic or therapeutic applications. Aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to factors such as heat and denaturants and can be stored for extended periods (>1 yr) at room temperature as lyophilized powders. Not being bound by a theory, aptamers bound to a solid support or beads may be stored for extended periods.
  • Oligonucleotides in their phosphodiester form may be quickly degraded by intracellular and extracellular enzymes such as endonucleases and exonucleases.
  • Aptamers can include modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX identified nucleic acid ligands containing modified nucleotides are described, e.g., in U.S. Pat. No.
  • Modifications of aptamers may also include modifications at exocyclic amines, substitution of 4- thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or allyl phosphate modifications, methylations, and unusual base-pairing combinations such as the isobases isocytidine and isoguanosine. Modifications can also include 3' and 5' modifications such as capping. As used herein, the term phosphorothioate encompasses one or more non-bridging oxygen atoms in a phosphodiester bond replaced by one or more sulfur atoms.
  • the oligonucleotides comprise modified sugar groups, for example, one or more of the hydroxyl groups is replaced with halogen, aliphatic groups, or functionalized as ethers or amines.
  • the 2'-position of the furanose residue is substituted by any of an 0- methyl, O-alkyl, O-allyl, S-alkyl, S-allyl, or halo group.
  • aptamers include aptamers with improved off-rates as described in International Patent Publication No. WO 2009012418, “Method for generating aptamers with improved off-rates,” incorporated herein by reference in its entirety.
  • aptamers are chosen from a library of aptamers.
  • Such libraries include, but are not limited to, those described in Rohloff et al., “Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents,” Molecular Therapy Nucleic Acids (2014) 3, e201. Aptamers are also commercially available (see, e.g., SomaLogic, Inc., Boulder, Colorado). In certain embodiments, the present invention may utilize any aptamer containing any modification as described herein.
  • tumor subtype specific biomarkers are targeted with a genetic modifying agent (e.g., NRP: TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; or SBM: MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4; or NRP: PDGFD, C6, CRISP3, RELN, SERPINA6, CRP, FGG, APCS, CFH, HABP2, KLKB1, REGIA and SPP1; or SBM: COL17A1, MUC16, FGF19, IGF2, NPC2, SAA1, SRGN, CIS, and CST4; or NRP TME: C7, HDGF, MAPT, REGIB, SERPINA4, PVR, CRISP3, DKK4, D
  • the genetic modifying agent may comprise a programmable nuclease, such as, a CRISPR system, a zinc finger nuclease system, a TALEN, or a meganuclease.
  • a polynucleotide of the present invention described elsewhere herein can be modified using a genetic modifying agent.
  • the genetic modifying agent is a CRISPR-Cas system.
  • CRISPR-Cas systems comprise a Cas polypeptide and a guide sequence, wherein the guide sequence is capable of forming a CRISPR-Cas complex with the Cas polypeptide and directing site-specific binding of the CRISPR-Cas sequence to a target sequence.
  • the Cas polypeptide may induce a double- or single-stranded break at a designated site in the target sequence.
  • the site of CRISPR-Cas cleavage, for most CRISPR-Cas systems, is dictated by distance from a protospacer- adjacent motif (PAM), discussed in further detail below.
  • PAM protospacer- adjacent motif
  • a guide sequence may be selected to direct the CRISPR-Cas system to a desired target site at or near the one or more target genes.
  • CRISPR systems can be used in vivo (see, e.g., Chen H, Shi M, Gilam A, et al. Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII. Sci Rep. 2019;9(l): 16838; Hana S, Peterson M, McLaughlin H, et al. Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice. Gene Ther.
  • a CRISPR-Cas or CRISPR system as used in herein and in documents, such as International Patent Publication No. WO 2014/093622 (PCT/US2013/074667), refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (transactivating CRISPR) sequence (e g.
  • RNA(s) as that term is herein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus.
  • Cas9 e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). See, e.g., Shmakov et al. (2015) “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell, DOI: dx.doi.org/10.1016/j.molcel.2015.10.008.
  • CRISPR-Cas systems can generally fall into two classes based on their architectures of their effector molecules, which are each further subdivided by type and subtype. The two class are Class 1 and Class 2. Class 1 CRISPR-Cas systems have effector modules composed of multiple Cas proteins, some of which form crRNA-binding complexes, while Class 2 CRISPR-Cas systems include a single, multi-domain crRNA-binding protein.
  • the CRISPR-Cas system that can be used to modify a polynucleotide of the present invention described herein can be a Class 1 CRISPR-Cas system. In some embodiments, the CRISPR-Cas system that can be used to modify a polynucleotide of the present invention described herein can be a Class 2 CRISPR-Cas system.
  • the CRISPR-Cas system that can be used to modify a polynucleotide of the present invention described herein can be a Class 1 CRISPR-Cas system.
  • Class 1 CRISPR-Cas systems are divided into types I, II, and IV. Makarova et al. 2020. Nat. Rev. 18: 67-83., particularly as described in Figure 1.
  • Type I CRISPR-Cas systems are divided into 9 subtypes (I-A, LB, I-C, I-D, I-E, I-Fl, I-F2, 1-F3, and IG). Makarova et al., 2020.
  • Type I CRISPR-Cas systems can contain a Cas3 protein that can have helicase activity.
  • Type III CRISPR- Cas systems are divided into 6 subtypes (III-A, III-B, III-C, III-D, III-E, and III-F).
  • Type III CRISPR-Cas systems can contain a Cas 10 that can include an RNA recognition motif called Palm and a cyclase domain that can cleave polynucleotides.
  • Type IV CRISPR- Cas systems are divided into 3 subtypes. (IV-A, IV-B, and IV-C). Makarova et al., 2020.
  • Class 1 systems also include CRISPR-Cas variants, including Type I-A, I-B, I-E, I-F and I-U variants, which can include variants carried by transposons and plasmids, including versions of subtype I- F encoded by a large family of Tn7-like transposon and smaller groups of Tn7-like transposons that encode similarly degraded subtype LB systems.
  • CRISPR-Cas variants including Type I-A, I-B, I-E, I-F and I-U variants, which can include variants carried by transposons and plasmids, including versions of subtype I- F encoded by a large family of Tn7-like transposon and smaller groups of Tn7-like transposons that encode similarly degraded subtype LB systems.
  • the Class 1 systems typically comprise a multi-protein effector complex, which can, in some embodiments, include ancillary proteins, such as one or more proteins in a complex referred to as a CRISPR-associated complex for antiviral defense (Cascade), one or more adaptation proteins (e.g., Cast, Cas2, RNA nuclease), and/or one or more accessory proteins (e.g., Cas 4, DNA nuclease), CRISPR associated Rossman fold (CARF) domain containing proteins, and/or RNA transcriptase.
  • CRISPR-associated complex for antiviral defense Cascade
  • adaptation proteins e.g., Cast, Cas2, RNA nuclease
  • accessory proteins e.g., Cas 4, DNA nuclease
  • CARF CRISPR associated Rossman fold
  • the backbone of the Class 1 CRISPR-Cas system effector complexes can be formed by RNA recognition motif domain-containing protein(s) of the repeat-associated mysterious proteins (RAMPs) family subunits (e.g., Cas 5, Cas6, and/or Cas7).
  • RAMP proteins are characterized by having one or more RNA recognition motif domains. In some embodiments, multiple copies of RAMPs can be present.
  • the Class I CRISPR-Cas system can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more Cas5, Cas6, and/or Cas 7 proteins.
  • the Cas6 protein is an RNAse, which can be responsible for pre-crRNA processing. When present in a Class 1 CRISPR-Cas system, Cas6 can be optionally physically associated with the effector complex.
  • Class 1 CRISPR-Cas system effector complexes can, in some embodiments, also include a large subunit.
  • the large subunit can be composed of or include a Cas8 and/or CaslO protein. See, e.g., Figures 1 and 2. Koonin EV, Makarova KS. 2019. Phil. Trans. R. Soc. B 374: 20180087, DOI: 10.1098/rstb.2018.0087 and Makarova et al. 2020.
  • Class 1 CRISPR-Cas system effector complexes can, in some embodiments, include a small subunit (for example, Casl l). See, e.g., Figures 1 and 2. Koonin EV, Makarova KS. 2019 Origins and Evolution of CRISPR-Cas systems. Phil. Trans. R. Soc. B 374: 20180087, DOI: 10.1098/rstb.2018.0087.
  • the Class 1 CRISPR-Cas system can be a Type I CRISPR-Cas system.
  • the Type I CRISPR-Cas system can be a subtype I-A CRISPR-Cas system.
  • the Type I CRISPR-Cas system can be a subtype LB CRISPR-Cas system.
  • the Type I CRISPR-Cas system can be a subtype LC CRISPR-Cas system.
  • the Type I CRISPR-Cas system can be a subtype I-D CRISPR-Cas system.
  • the Type I CRISPR-Cas system can be a subtype I-E CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-Fl CRISPR- Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-F2 CRISPR- Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-F3 CRISPR- Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-G CRISPR- Cas system.
  • the Type I CRISPR-Cas system can be a CRISPR Cas variant, such as a Type I-A, I-B, I-E, I-F and I-U variants, which can include variants carried by transposons and plasmids, including versions of subtype I-F encoded by a large family of Tn7-like transposon and smaller groups of Tn7-like transposons that encode similarly degraded subtype I- B systems as previously described.
  • CRISPR Cas variant such as a Type I-A, I-B, I-E, I-F and I-U variants, which can include variants carried by transposons and plasmids, including versions of subtype I-F encoded by a large family of Tn7-like transposon and smaller groups of Tn7-like transposons that encode similarly degraded subtype I- B systems as previously described.
  • the Class 1 CRISPR-Cas system can be a Type III CRISPR-Cas system.
  • the Type III CRISPR-Cas system can be a subtype IILA CRISPR- Cas system.
  • the Type III CRISPR-Cas system can be a subtype III-B CRISPR-Cas system.
  • the Type III CRISPR-Cas system can be a subtype
  • the Type III CRISPR-Cas system can be a subtype III-D CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype IILE CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-F CRISPR-Cas system.
  • the Class 1 CRISPR-Cas system can be a Type IV CRISPR- Cas-system.
  • the Type IV CRISPR-Cas system can be a subtype IV-A CRISPR-Cas system.
  • the Type IV CRISPR-Cas system can be a subtype
  • Type IV CRISPR-Cas system can be a subtype IV-C CRISPR-Cas system.
  • the effector complex of a Class 1 CRISPR-Cas system can, in some embodiments, include a Cas3 protein that is optionally fused to a Cas2 protein, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8, a Cas 10, a Casl 1, or a combination thereof.
  • the effector complex of a Class 1 CRISPR-Cas system can have multiple copies, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, of any one or more Cas proteins.
  • Class 2 CRISPR-Cas Systems can, in some embodiments, include a Cas3 protein that is optionally fused to a Cas2 protein, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8, a Cas 10, a Casl 1, or a combination thereof.
  • the effector complex of a Class 1 CRISPR-Cas system can have multiple copies, such as 1, 2, 3, 4, 5, 6, 7,
  • the CRISPR-Cas system is a Class 2 CRISPR-Cas system.
  • Class 2 systems are distinguished from Class 1 systems in that they have a single, large, multi-domain effector protein.
  • the Class 2 system can be a Type II, Type V, or Type VI system, which are described in Makarova et al. “Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants” Nature Reviews Microbiology, 18:67-81 (Feb 2020), incorporated herein by reference.
  • Class 2 system Each type of Class 2 system is further divided into subtypes. See Markova et al. 2020, particularly at Figure. 2.
  • Class 2 Type II systems can be divided into 4 subtypes: II-A, II-B, II-C1, and II-C2.
  • Class 2 Type V systems can be divided into 17 subtypes: V-A, V-Bl, V-B2, V-C, V-D, V-E, V-Fl, V-F1(V-U3), V-F2, V-F3, V-G, V-H, V-I, V-K (V-U5),
  • Type IV systems can be divided into 5 subtypes: VI-A, VI-B1,
  • VI-B2, VI-C, and VI-D are VI-B2, VI-C, and VI-D.
  • Type V systems differ from Type II effectors (e.g., Cas9), which contain two nuclear domains that are each responsible for the cleavage of one strand of the target DNA, with the HNH nuclease inserted inside the Ruv-C like nuclease domain sequence.
  • the Type V systems e.g., Casl2 only contain a RuvC-like nuclease domain that cleaves both strands.
  • Type VI (Casl3) are unrelated to the effectors of Type II and V systems and contain two HEPN domains and target RNA. Casl3 proteins also display collateral activity that is triggered by target recognition. Some Type V systems have also been found to possess this collateral activity with two single-stranded DNA in in vitro contexts.
  • the Class 2 system is a Type II system.
  • the Type II CRISPR-Cas system is a II-A CRISPR-Cas system.
  • the Type II CRISPR-Cas system is a II-B CRISPR-Cas system.
  • the Type II CRISPR- Cas system is a II-C1 CRISPR-Cas system.
  • the Type II CRISPR-Cas system is a II-C2 CRISPR-Cas system.
  • the Type II system is a Cas9 system.
  • the Type II system includes a Cas9.
  • the Class 2 system is a Type V system.
  • the Type V CRISPR-Cas system is a V-A CRISPR-Cas system.
  • the Type V CRISPR-Cas system is a V-Bl CRISPR-Cas system.
  • the Type V CRISPR-Cas system is a V-B2 CRISPR-Cas system.
  • the Type V CRISPR- Cas system is a V-C CRISPR-Cas system.
  • the Type V CRISPR-Cas system is a V-D CRISPR-Cas system.
  • the Type V CRISPR-Cas system is a V-E CRISPR-Cas system.
  • the Type V CRISPR-Cas system is a V-Fl CRISPR-
  • the Type V CRISPR-Cas system is a V-Fl (V-U3) CRISPR- Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-F2 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-F3 CRISPR-Cas system.
  • the Type V CRISPR-Cas system is a V-G CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-H CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-I CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-K (V-U5) CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-Ul CRISPR-Cas system.
  • the Type V CRISPR-Cas system is a V-U2 CRISPR-Cas system. In some embodiments, the Type CRISPR-Cas system is a V-U4 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system includes a Casl2a (Cpf 1 ), Casl2b (C2cl), Casl2c
  • C2c3 Casl2d (CasY), Casl2e (CasX), Casl4, and/or Cas .
  • the Class 2 system is a Type VI system.
  • the Type VI CRISPR-Cas system is a VI-A CRISPR-Cas system.
  • the Type VI CRISPR-Cas system is a VI-B1 CRISPR-Cas system.
  • the Type VI CRISPR-Cas system is a VI-B2 CRISPR-Cas system.
  • the Type VI CRISPR-Cas system is a VI-C CRISPR-Cas system.
  • the Type VI CRISPR- Cas system is a VI-D CRISPR-Cas system.
  • the Type VI CRISPR-Cas system includes a Casl3a (C2c2), Casl3b (Group 29/30), Casl3c, and/or Casl3d.
  • guide molecule refers to polynucleotides capable of guiding Cas to a target genomic locus and are used interchangeably as in foregoing cited documents such as International Patent Publication No. WO 2014/093622 (PCT/US2013/074667).
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • the guide molecule can be a polynucleotide.
  • a guide sequence within a nucleic acid-targeting guide RNA
  • a guide sequence may direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence
  • the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid-targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay (Qui et al. 2004.
  • preferential targeting e.g., cleavage
  • cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • Other assays are possible and will occur to those skilled in the art.
  • the guide molecule is an RNA.
  • the guide molecule(s) (also referred to interchangeably herein as guide polynucleotide and guide sequence) that are included in the CRISPR-Cas or Cas based system can be any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence.
  • the degree of complementarity when optimally aligned using a suitable alignment algorithm, can be about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA),
  • a guide sequence and hence a nucleic acid-targeting guide, may be selected to target any target nucleic acid sequence.
  • the target sequence may be DNA.
  • the target sequence may be any RNA sequence.
  • the target sequence may be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), and small cytoplasmatic RNA (scRNA).
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • miRNA micro-RNA
  • siRNA small interfering RNA
  • snRNA small nuclear RNA
  • snoRNA small nucle
  • the target sequence may be a sequence within an RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNA, and IncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule.
  • a nucleic acid-targeting guide is selected to reduce the degree secondary structure within the nucleic acid-targeting guide. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148).
  • Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm see e.g., A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
  • a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence.
  • the guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence.
  • the direct repeat sequence may be located upstream (i.e., 5’) from the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., 3’) from the guide sequence or spacer sequence.
  • the crRNA comprises a stem loop, preferably a single stem loop.
  • the direct repeat sequence forms a stem loop, preferably a single stem loop.
  • the spacer length of the guide RNA is from 15 to 35 nt. In another example embodiment, the spacer length of the guide RNA is at least 15 nucleotides. In another example embodiment, the spacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27 nt, from 27 to 30 nt, e.g., 27, 28, 29, or 30 nt, from 30 to 35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.
  • the “tracrRNA” sequence or analogous terms includes any polynucleotide sequence that has sufficient complementarity with a crRNA sequence to hybridize.
  • the degree of complementarity between the tracrRNA sequence and crRNA sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
  • the tracr sequence and crRNA sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin.
  • degree of complementarity is with reference to the optimal alignment of the sea sequence and tracr sequence, along the length of the shorter of the two sequences.
  • Optimal alignment may be determined by any suitable alignment algorithm and may further account for secondary structures, such as self-complementarity within either the sea sequence or tracr sequence.
  • the degree of complementarity between the tracr sequence and sea sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the degree of complementarity between a guide sequence and its corresponding target sequence can be about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%;
  • a guide or RNA or sgRNA can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA or sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length; and tracr RNA can be 30 or 50 nucleotides in length.
  • the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or 99% or 99.5% or 99.9%, or 100%.
  • Off target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementarity between the sequence and the guide, with it being advantageous that off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between the sequence and the guide.
  • the guide RNA (capable of guiding Cas to a target locus) may comprise (1) a guide sequence capable of hybridizing to a genomic target locus in the eukaryotic cell; (2) a tracr sequence; and (3) a tracr mate sequence. All of (1) to (3) may reside in a single RNA, i.e., an sgRNA (arranged in a 5’ to 3’ orientation), or the tracr RNA may be a different RNA than the RNA containing the guide and tracr sequence. The tracr hybridizes to the tracr mate sequence and directs the CRISPR/Cas complex to the target sequence.
  • each RNA may be optimized to be shortened from their respective native lengths, and each may be independently chemically modified to protect from degradation by cellular RNase or otherwise increase stability.
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • the target polynucleotide can be a polynucleotide or a part of a polynucleotide to which a part of the guide sequence is designed to have complementarity with and to which the effector function mediated by the complex comprising the CRISPR effector protein and a guide molecule is to be directed.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • PAM elements are sequences that can be recognized and bound by Cas proteins. Cas proteins/effector complexes can then unwind the dsDNA at a position adjacent to the PAM element. It will be appreciated that Cas proteins and systems target RNA do not require PAM sequences (Marraffini et al. 2010. Nature. 463:568-571). Instead, many rely on PFSs, which are discussed elsewhere herein.
  • the target sequence should be associated with a PAM (protospacer adjacent motif) or PFS (protospacer flanking sequence or site), that is, a short sequence recognized by the CRISPR complex.
  • the target sequence should be selected, such that its complementary sequence in the DNA duplex (also referred to herein as the non-target sequence) is upstream or downstream of the PAM.
  • the complementary sequence of the target sequence is downstream or 3’ of the PAM or upstream or 5’ of the PAM.
  • the precise sequence and length requirements for the PAM differ depending on the Cas protein used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). Examples of the natural PAM sequences for different Cas proteins are provided herein below and the skilled person will be able to identify further PAM sequences for use with a given Cas protein.
  • the CRISPR effector protein may recognize a 3’ PAM.
  • the CRISPR effector protein may recognize a 3’ PAM which is 5’H, wherein H is A, C or U.
  • engineering of the PAM Interacting (PI) domain on the Cas protein may allow programing of PAM specificity, improve target site recognition fidelity, and increase the versatility of the CRISPR-Cas protein, for example as described for Cas9 in Kleinstiver BP et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jul 23;523(7561):481-5. doi: 10.1038/naturel4592. As further detailed herein, the skilled person will understand that Casl3 proteins may be modified analogously.
  • Gao et al “Engineered Cpfl Enzymes with Altered PAM Specificities,” bioRxiv 091611; doi: http://dx.doi.org/10.1101/091611 (Dec. 4, 2016).
  • Doench et al. created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. The authors showed that optimization of the PAM improved activity and also provided an on-line tool for designing sgRNAs.
  • PAM sequences can be identified in a polynucleotide using an appropriate design tool, which are commercially available as well as online.
  • Such freely available tools include, but are not limited to, CRISPRFinder and CRISPRTarget. Mojica et al. 2009. Microbiol. 155(Pt. 3):733-740; Atschul et al. 1990. J. Mol. Biol. 215:403-410; Biswass et al. 2013 RNA Biol. 10:817-827; and Grissa et al. 2007. Nucleic Acid Res. 35:W52-57.
  • Experimental approaches to PAM identification can include, but are not limited to, plasmid depletion assays (Jiang et al. 2013. Nat.
  • CRISPR-Cas systems that target RNA do not typically rely on PAM sequences. Instead, such systems typically recognize protospacer flanking sites (PFSs) instead of PAMs.
  • Type VI CRISPR-Cas systems typically recognize protospacer flanking sites (PFSs) instead of PAMs.
  • PFSs represents an analogue to PAMs for RNA targets.
  • Type VI CRISPR-Cas systems employ a Casl3.
  • Some Casl3 proteins analyzed to date, such as Casl3a (C2c2) identified from Leptotrichia shahii (LShCAsl3a) have a specific discrimination against G at the 3 ’end of the target RNA.
  • RNA Biology. 16(4): 504-517 The presence of a C at the corresponding crRNA repeat site can indicate that nucleotide pairing at this position is rejected.
  • some Casl3 proteins e.g., LwaCAsl3a and PspCasl3b
  • Type VI proteins such as subtype B have 5 '-recognition of D (G, T, A) and a 3'-motif requirement of NAN or NNA.
  • D D
  • NAN NNA
  • Casl3b protein identified in Bergeyella zoohelcum BzCasl3b. See e.g., Gleditzsch et al. 2019. RNA Biology. 16(4): 504-517.
  • Type VI CRISPR-Cas systems appear to have less restrictive rules for substrate (e.g., target sequence) recognition than those that target DNA (e.g., Type V and type II).
  • one or more components (e.g., the Cas protein) in the composition for engineering cells may comprise one or more sequences related to nucleus targeting and transportation. Such sequences may facilitate the one or more components in the composition for targeting a sequence within a cell.
  • NLSs nuclear localization sequences
  • the NLSs used in the context of the present disclosure are heterologous to the proteins.
  • Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: I) or PKKKRKVEAS (SEQ ID NO:2); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:3)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:4) or RQRRNELKRSP (SEQ ID NO: 5); the hRNPAl M9 NLS having the sequence
  • NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY SEQ ID NO:6; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 7) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:8) and PPKKARED (SEQ ID NON) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 10) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 11) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO:12) and PKQKKRK (SEQ ID NO: 13) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 14) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 15) of the mouse Mxl protein; the sequence KRKGD
  • the one or more NLSs are of sufficient strength to drive accumulation of the DNA-targeting Cas protein in a detectable amount in the nucleus of a eukaryotic cell.
  • strength of nuclear localization activity may derive from the number of NLSs in the CRISPR-Cas protein, the particular NLS(s) used, or a combination of these factors.
  • Detection of accumulation in the nucleus may be performed by any suitable technique.
  • a detectable marker may be fused to the nucleic acid-targeting protein, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g., a stain specific for the nucleus such as DAPI).
  • Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of nucleic acidtargeting complex formation (e.g., assay for deaminase activity) at the target sequence, or assay for altered gene expression activity affected by DNA-targeting complex formation and/or DNA- targeting), as compared to a control not exposed to the Cas protein, or exposed to a Cas protein lacking the one or more NLSs.
  • nucleic acidtargeting complex formation e.g., assay for deaminase activity
  • the Cas proteins may be provided with 1 or more, such as with, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous NLSs.
  • the proteins comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g., zero or at least one or more NLS at the amino-terminus and zero or at one or more NLS at the carboxy terminus).
  • each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies.
  • an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.
  • an NLS attached to the C- terminal of the protein.
  • the CRISPR-Cas protein and a functional domain protein are delivered to the cell or expressed within the cell as separate proteins.
  • each of the CRISPR-Cas and functional domain protein can be provided with one or more NLSs as described herein.
  • the CRISPR-Cas and functional domain protein are delivered to the cell or expressed with the cell as a fusion protein. In these embodiments one or both of the CRISPR-Cas and functional domain protein is provided with one or more NLSs.
  • the one or more NLS can be provided on the adaptor protein, provided that this does not interfere with aptamer binding.
  • the one or more NLS sequences may also function as linker sequences between the functional domain protein and the CRISPR-Cas protein.
  • guides of the disclosure comprise specific binding sites (e.g., aptamers) for adapter proteins, which may be linked to or fused to a functional domain protein or catalytic domain thereof.
  • the adapter proteins bind, and the functional domain protein or catalytic domain thereof associated with the adapter protein is positioned in a spatial orientation which is advantageous for the attributed function to be effective.
  • the one or more modified guide may be modified at the tetra loop, the stem loop 1, stem loop 2, or stem loop 3, as described herein, preferably at either the tetra loop or stem loop 2, and in some cases at both the tetra loop and stem loop 2.
  • a component in the systems may comprise one or more nuclear export signals (NES), one or more nuclear localization signals (NLS), or any combinations thereof.
  • the NES may be an HIV Rev NES.
  • the NES may be MAPK NES.
  • the component is a protein, the NES or NLS may be at the C terminus of component. Alternatively, or additionally, the NES or NLS may be at the N terminus of component.
  • the Cas protein and optionally said functional domain protein or catalytic domain thereof comprise one or more heterologous nuclear export signal(s) (NES(s)) or nuclear localization signal(s) (NLS(s)), preferably an HIV Rev NES or MAPK NES, preferably C-terminal.
  • NES(s) heterologous nuclear export signal(s)
  • NLS(s) nuclear localization signal(s)
  • HIV Rev NES or MAPK NES preferably C-terminal.
  • the CRISPR-Cas system may induce a double- or singlestranded break at a designated site in the target sequence.
  • the CRISPR-Cas system may introduce an indel, which, as used herein, refers to insertions or deletions of the DNA at particular locations on the chromosome.
  • the site of CRISPR-Cas cleavage, for most CRISPR-Cas systems, is dictated by distance from a protospacer-adjacent motif (PAM).
  • a guide sequence may be selected to direct the CRISPR-Cas system to induce cleavage at a desired target site at or near the one or more variants.
  • the CRISPR-Cas system is used to introduce one or more insertions or deletions to a target sequence on the gene or enhancer associated with the gene such that one or more indels or insertions reduce expression or activity of the one or more polypeptides.
  • More than one guide sequence may be selected to insert multiple insertion, deletions, or combination thereof.
  • more than one Cas protein type may be used, for example, to maximize targets sites adjacent to different PAMs.
  • a guide sequence is selected that directs the CRISPR-Cas system to make one or more insertions or deletions within the enhancer region.
  • a guide is selected that directs the CRISPR-Cas system to make an insertion 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs upstream of an enhancer controlling expression of a target gene.
  • a guide sequence is selected to that directs the CRISPR-Cas system to make an insertion 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs downstream of an enhancer controlling expression of a target gene.
  • a guide sequence is selected that directs the CRISPR-Cas system to make a deletion 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs downstream of an enhancer controlling expression of a target gene. In one example embodiment, a guide sequence is selected that directs the CRISPR-Cas system to make a deletion 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs downstream of an enhancer controlling expression of a target gene.
  • a donor template is provided to replace a genomic sequence in a target gene or sequence controlling expression of the target gene.
  • a donor template may comprise an insertion sequence flanked by two homology regions.
  • the insertion sequence comprises an edited sequence to be inserted in place of the target sequence (e.g., a portion of genomic DNA to be edited).
  • the homology regions comprise sequences that are homologous to the genomic DNA strands at the site of the CRISPR-Cas induced double-strand break. Cellular HDR mechanisms then facilitate insertion of the insertion sequence at the site of the DSB.
  • a donor template and guide sequence are selected to direct excision and replacement of a section of genome DNA comprising an enhancer controlling expression of a target gene or a section of genome DNA within the gene that is required for activity of the target gene.
  • the insertion sequence comprises a transcription factor binding site that recruits a repressor to the gene.
  • the donor template may include a sequence which results in a change in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides of the target sequence.
  • a donor template may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length.
  • the template nucleic acid may be 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/- 10, 70+/- 10, 80+/- 10, 90+/- 10, 100+/- 10, 1 10+/- 10, 120+/- 10, 130+/- 10, 140+/- 10, 150+/- 10, 160+/- 10, 170+/- 10, 1 80+/- 10, 190+/- 10, 200+/- 10, 210+/-10, of 220+/- 10 nucleotides in length.
  • the template nucleic acid may be 30+/-20, 40+/-20, 50+/-20, 60+/-20, 70+/- 20, 80+/-20, 90+/-20, 100+/-20, 1 10+/-20, 120+/-20, 130+/-20, 140+/-20, I 50+/-20, 160+/-20, 170+/-20, 180+/-20, 190+/-20, 200+/-20, 210+/-20, of 220+/-20 nucleotides in length.
  • the template nucleic acid is 10 to 1 ,000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to300, 50 to 200, or 50 to 100 nucleotides in length.
  • the homology regions of the donor template may be complementary to a portion of a polynucleotide comprising the target sequence.
  • a donor template might overlap with one or more nucleotides of a target sequences (e.g., about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides).
  • the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.
  • the donor template comprises a sequence to be integrated (e.g., a mutated gene).
  • the sequence for integration may be a sequence endogenous or exogenous to the cell. Examples of a sequence to be integrated include polynucleotides encoding a protein or a non-coding RNA (e.g., a microRNA).
  • the sequence for integration may be operably linked to an appropriate control sequence or sequences.
  • the sequence to be integrated may provide a regulatory function.
  • Homology arms of the donor template may comprise from about 20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp.
  • the exemplary upstream or downstream sequence have about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000.
  • one or both homology arms may be shortened to avoid including certain sequence repeat elements.
  • a 5' homology arm may be shortened to avoid a sequence repeat element.
  • a 3' homology arm may be shortened to avoid a sequence repeat element.
  • both the 5' and the 3' homology arms may be shortened to avoid including certain sequence repeat elements.
  • the donor template may further comprise a marker.
  • a marker may make it easy to screen for targeted integrations. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers.
  • the donor template of the disclosure can be constructed using recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996).
  • a donor template is a single-stranded oligonucleotide. When using a single-stranded oligonucleotide, 5' and 3' homology arms may range up to about 200 base pairs (bp) in length, e g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length.
  • Suzuki et al. describe in vivo genome editing via CRISPR/Cas9 mediated homologyindependent targeted integration (2016, Nature 540:144-149).
  • a composition for engineering cells comprises a template, e.g., a recombination template.
  • a template may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide.
  • a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a nucleic acid-targeting effector protein as a part of a nucleic acid-targeting complex.
  • the template nucleic acid alters the sequence of the target position. In an embodiment, the template nucleic acid results in the incorporation of a modified, or non- naturally occurring base into the target nucleic acid.
  • the template sequence may undergo a breakage mediated or catalyzed recombination with the target sequence. In an embodiment, the template nucleic acid may include sequence that corresponds to a site on the target sequence that is cleaved by a Cas protein mediated cleavage event.
  • the template nucleic acid may include a sequence that corresponds to both, a first site on the target sequence that is cleaved in a first Cas protein mediated event, and a second site on the target sequence that is cleaved in a second Cas protein mediated event.
  • the template nucleic acid can include a sequence which results in an alteration in the coding sequence of a translated sequence, e.g., one which results in the substitution of one amino acid for another in a protein product, e.g., transforming a mutant allele into a wild type allele, transforming a wild type allele into a mutant allele, and/or introducing a stop codon, insertion of an amino acid residue, deletion of an amino acid residue, or a nonsense mutation.
  • the template nucleic acid can include a sequence which results in an alteration in a non-coding sequence, e.g., an alteration in an exon or in a 5' or 3' non-translated or non-transcribed region.
  • alterations include an alteration in a control element, e.g., a promoter, enhancer, and an alteration in a cis-acting or trans-acting control element.
  • a template nucleic acid having homology with a target position in a target gene may be used to alter the structure of a target sequence.
  • the template sequence may be used to alter an unwanted structure, e.g., an unwanted or mutant nucleotide.
  • the template nucleic acid may include a sequence which, when integrated, results in decreasing the activity of a positive control element; increasing the activity of a positive control element; decreasing the activity of a negative control element; increasing the activity of a negative control element; decreasing the expression of a gene; increasing the expression of a gene; increasing resistance to a disorder or disease; increasing resistance to viral entry; correcting a mutation or altering an unwanted amino acid residue conferring, increasing, abolishing or decreasing a biological property of a gene product, e.g., increasing the enzymatic activity of an enzyme, or increasing the ability of a gene product to interact with another molecule.
  • the template nucleic acid may include a sequence which results in a change in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12 or more nucleotides of the target sequence.
  • a template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length.
  • the template nucleic acid may be 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/- 10, 70+/- 10, 80+/- 10, 90+/- 10, 100+/- 10, 1 10+/- 10, 120+/- 10, 130+/- 10, 140+/- 10, 150+/- 10, 160+/- 10, 170+/- 10, 1 80+/- 10, 190+/- 10, 200+/- 10, 210+/-10, of 220+/- 10 nucleotides in length.
  • the template nucleic acid may be 30+/-20, 40+/-20, 50+/-20, 60+/-20, 70+/- 20, 80+/-20, 90+/-20, 100+/-20, 1 10+/-20, 120+/-20, 130+/-20, 140+/-20, 150+/-20, 160+/- 20, 170+/-20, 180+/-20, 190+/-20, 200+/-20, 210+/-20, of 220+/-20 nucleotides in length.
  • the template nucleic acid is 10 to 1 ,000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to300, 50 to 200, or 50 to 100 nucleotides in length.
  • the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence.
  • a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g., about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides).
  • the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.
  • the exogenous polynucleotide template comprises a sequence to be integrated (e.g., a mutated gene).
  • the sequence for integration may be a sequence endogenous or exogenous to the cell. Examples of a sequence to be integrated include polynucleotides encoding a protein or a noncoding RNA (e.g., a microRNA).
  • the sequence for integration may be operably linked to an appropriate control sequence or sequences.
  • the sequence to be integrated may provide a regulatory function.
  • An upstream or downstream sequence may comprise from about 20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp.
  • the exemplary upstream or downstream sequence have about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000.
  • An upstream or downstream sequence may comprise from about 20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp.
  • the exemplary upstream or downstream sequence have about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000
  • one or both homology arms may be shortened to avoid including certain sequence repeat elements.
  • a 5' homology arm may be shortened to avoid a sequence repeat element.
  • a 3' homology arm may be shortened to avoid a sequence repeat element.
  • both the 5' and the 3' homology arms may be shortened to avoid including certain sequence repeat elements.
  • the exogenous polynucleotide template may further comprise a marker.
  • a marker may make it easy to screen for targeted integrations. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers.
  • the exogenous polynucleotide template of the disclosure can be constructed using recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996).
  • a template nucleic acid for correcting a mutation may designed for use as a single-stranded oligonucleotide.
  • 5' and 3' homology arms may range up to about 200 base pairs (bp) in length, e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length.
  • Suzuki et al. describe in vivo genome editing via CRISPR/Cas9 mediated homologyindependent targeted integration (2016, Nature 540:144-149).
  • the system is a Cas-based system that is capable of performing a specialized function or activity.
  • the Cas protein may be fused, operably coupled to, or otherwise associated with one or more functionals domains.
  • the Cas protein may be a catalytically dead Cas protein (“dCas”) and/or have nickase activity.
  • dCas catalytically dead Cas protein
  • a nickase is a Cas protein that cuts only one strand of a double stranded target.
  • the dCas or nickase provide a sequence specific targeting functionality that delivers the functional domain to or proximate a target sequence.
  • Example functional domains that may be fused to, operably coupled to, or otherwise associated with a Cas protein can be or include, but are not limited to a nuclear localization signal (NTS) domain, a nuclear export signal (NES) domain, a translational activation domain, a transcriptional activation domain (e.g.
  • VP64, p65, MyoDl, HSF1, RTA, and SET7/9) a translation initiation domain, a transcriptional repression domain (e.g., a KRAB domain, NuE domain, NcoR domain, and a SID domain such as a SID4X domain), a nuclease domain (e.g., FokI), a histone modification domain (e.g., a histone acetyltransferase), a light inducible/controllable domain, a chemically inducible/controllable domain, a transposase domain, a homologous recombination machinery domain, a recombinase domain, an integrase domain, and combinations thereof.
  • a transcriptional repression domain e.g., a KRAB domain, NuE domain, NcoR domain, and a SID domain such as a SID4X domain
  • a nuclease domain e.g
  • the functional domains can have one or more of the following activities: methylase activity, demethylase activity, translation activation activity, translation initiation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, molecular switch activity, chemical inducibility, light inducibility, and nucleic acid binding activity.
  • the one or more functional domains may comprise epitope tags or reporters.
  • epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • reporters include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and auto-fluorescent proteins including blue fluorescent protein (BFP).
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-galactosidase
  • beta-glucuronidase beta-galactosidase
  • luciferase green fluorescent protein
  • GFP green fluorescent protein
  • HcRed HcRed
  • DsRed cyan fluorescent protein
  • the one or more functional domain(s) may be positioned at, near, and/or in proximity to a terminus of the effector protein (e.g., a Cas protein). In embodiments having two or more functional domains, each of the two can be positioned at or near or in proximity to a terminus of the effector protein (e.g., a Cas protein). In some embodiments, such as those where the functional domain is operably coupled to the effector protein, the one or more functional domains can be tethered or linked via a suitable linker (including, but not limited to, GlySer linkers) to the effector protein (e g., a Cas protein). When there is more than one functional domain, the functional domains can be same or different.
  • a suitable linker including, but not limited to, GlySer linkers
  • all the functional domains are the same. In some embodiments, all of the functional domains are different from each other. In some embodiments, at least two of the functional domains are different from each other. In some embodiments, at least two of the functional domains are the same as each other.
  • the CRISPR-Cas system is a split CRISPR-Cas system. See e.g., Zetche et al., 2015. Nat. Biotechnol. 33(2): 139-142 and International Patent Publication WO 2019/018423, the compositions and techniques of which can be used in and/or adapted for use with the present invention.
  • Split CRISPR-Cas proteins are set forth herein and in documents incorporated herein by reference in further detail herein.
  • each part of a split CRISPR protein is attached to a member of a specific binding pair, and when bound with each other, the members of the specific binding pair maintain the parts of the CRISPR protein in proximity.
  • each part of a split CRISPR protein is associated with an inducible binding pair.
  • An inducible binding pair is one which is capable of being switched “on” or “off’ by a protein or small molecule that binds to both members of the inducible binding pair.
  • CRISPR proteins may preferably split between domains, leaving domains intact.
  • said Cas split domains e.g., RuvC and HNH domains in the case of Cas9
  • the reduced size of the split Cas compared to the wildtype Cas allows other methods of delivery of the systems to the cells, such as the use of cell penetrating peptides as described herein.
  • the gene editing system configured to modify the gene encoding the one or more polypeptides disclosed herein is a base editing system.
  • a Cas protein is connected or fused to a nucleotide deaminase.
  • base editing refers generally to the process of polynucleotide modification via a CRISPR-Cas-based or Cas-based system that does not include excising nucleotides to make the modification. Base editing can convert base pairs at precise locations without generating excess undesired editing byproducts that can be made using traditional CRISPR-Cas systems.
  • the nucleotide deaminase may be a DNA base editor used in combination with a DNA binding Cas protein such as, but not limited to, Class 2 Type II and Type V systems.
  • a DNA binding Cas protein such as, but not limited to, Class 2 Type II and Type V systems.
  • Two classes of DNA base editors are generally known: cytosine base editors (CBEs) and adenine base editors (ABEs).
  • CBEs convert a C*G base pair into a T*A base pair
  • ABEs convert an A»T base pair to a G*C base pair.
  • CBEs and ABEs can mediate all four possible transition mutations (C to T, A to G, T to C, and G to A).
  • the base editing system includes a CBE and/or an ABE.
  • a polynucleotide of the present invention described elsewhere herein can be modified using a base editing system. Rees and Liu. 2018. Nat. Rev. Gent. 19(12):770-788. Base editors also generally do not need a DNA donor template and/or rely on homology-directed repair.
  • the catalytically disabled Cas protein can be a variant or modified Cas can have nickase functionality and can generate a nick in the non-edited DNA strand to induce cells to repair the non-edited strand using the edited strand as a template.
  • Example Type V base editing systems are described in International Patent Publication Nos. WO 2018/213708, WO 2018/213726, and International Patent Applications No. PCT/US2018/067207, PCT/US2018/067225, and PCT/US2018/067307, each of which is incorporated herein by reference.
  • the base editing system may be an RNA base editing system.
  • a nucleotide deaminase capable of converting nucleotide bases may be fused to a Cas protein.
  • the Cas protein will need to be capable of binding RNA.
  • Example RNA binding Cas proteins include, but are not limited to, RNA- binding Cas9s such as Francisella novicida Cas9 (“FnCas9”), and Class 2 Type VI Cas systems.
  • the nucleotide deaminase may be a cytidine deaminase or an adenosine deaminase, or an adenosine deaminase engineered to have cytidine deaminase activity.
  • the RNA base editor may be used to delete or introduce a post-translation modification site in the expressed mRNA.
  • RNA base editors can provide edits where finer, temporal control may be needed, for example in modulating a particular immune response.
  • Example Type VI RNA- base editing systems are described in Cox et al. 2017. Science 358: 1019-1027, International Patent Publication Nos.
  • the gene editing system configured to modify a gene encoding the one or more polypeptides disclosed herein is a prime editing system. See e.g., Anzalone et al. 2019. Nature. 576: 149-157.
  • a genomic sequence in a target gene or sequence controlling expression of the target gene is replaced or deleted using a prime editing system.
  • prime editing systems can be capable of targeted modification of a polynucleotide without generating double stranded breaks. Further prime editing systems are capable of all 12 possible combination swaps.
  • Prime editing may operate via a“search- and-replace” methodology and can mediate targeted insertions, deletions, of all 12 possible base- to-base conversion and combinations thereof.
  • a prime editing system as exemplified by PEI, PE2, and PE3 (Id can include a reverse transcriptase fused or otherwise coupled or associated with an RNA-programmable nickase and a prime-editing extended guide RNA (pegRNA) to facility direct copying of genetic information from the extension on the pegRNA into the target polynucleotide.
  • pegRNA prime-editing extended guide RNA
  • Embodiments that can be used with the present invention include these and variants thereof.
  • Prime editing can have the advantage of lower off-target activity.
  • the prime editing guide molecule can specify both the target polynucleotide information (e.g., sequence) and contain a new polynucleotide cargo that replaces target polynucleotides.
  • the PE system can nick the target polynucleotide at a target side to expose a 3 ’hydroxyl group, which can prime reverse transcription of an edit-encoding extension region of the guide molecule (e.g., a prime editing guide molecule or peg guide molecule) directly into the target site in the target polynucleotide. See e.g., Anzalone et al. 2019. Nature. 576: 149-157, particularly at Figures lb, 1c, related discussion, and Supplementary discussion.
  • a prime editing system can be composed of a Cas polypeptide having nickase activity, a reverse transcriptase, and a guide molecule.
  • the Cas polypeptide can lack nuclease activity.
  • the guide molecule can include a target binding sequence as well as a primer binding sequence and a template containing the edited polynucleotide sequence.
  • the guide molecule, Cas polypeptide, and/or reverse transcriptase can be coupled together or otherwise associate with each other to form an effector complex and edit a target sequence.
  • the Cas polypeptide is a Class 2, Type V Cas polypeptide.
  • the Cas polypeptide is a Cas9 polypeptide (e.g., is a Cas9 nickase). In some embodiments, the Cas polypeptide is fused to the reverse transcriptase. In some embodiments, the Cas polypeptide is linked to the reverse transcriptase.
  • the prime editing system can be a PEI system or variant thereof, a PE2 system or variant thereof, or a PE3 (e.g., PE3, PE3b) system. See e.g., Anzalone et al. 2019. Nature. 576: 149-157, particularly at pgs. 2-3, Figs. 2a, 3a-3f, 4a-4b, Extended data Figs. 3a-3b, 4.
  • the peg guide molecule can be about 10 to about 200 or more nucleotides in length, such as 10 to/or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
  • the gene editing system configured to modify a gene encoding the one or more polypeptides disclosed herein is a CRISPR associated transposase system (CAST).
  • CAST CRISPR associated transposase system
  • a CAST system is used to replace all or a portion of an enhancer controlling the target gene expression.
  • CAST system can include a Cas protein that is catalytically inactive, or engineered to be catalytically active, and further comprises a transposase (or subunits thereof) that catalyze RNA-guided DNA transposition.
  • Such systems are able to insert DNA sequences at a target site in a DNA molecule without relying on host cell repair machinery.
  • CAST systems can be Classi or Class 2 CAST systems.
  • An example Class 1 system is described in Klompe et al. Nature, doi: 10.1038/s41586-019-1323, which is in incorporated herein by reference.
  • An example Class 2 system is described in Strecker et al. Science. 10/1126/science. aax9181 (2019), and PCT/US2019/066835 which are incorporated herein by reference.
  • the one or more agents is an epigenetic modification polypeptide comprising a DNA binding domain linked to or otherwise capable of associating with an epigenetic modification domain such that binding of the DNA binding domain at target sequence on genomic DNA (e.g., chromatin) results in one or more epigenetic modifications by the epigenetic modification domain that increases or decreases expression of the one or more polypeptides disclosed herein.
  • linked to or otherwise capable of associating with refers to a fusion protein or a recruitment domain or an adaptor protein, such as an aptamer (e.g., MS2) or an epitope tag.
  • the recruitment domain or the adaptor protein can be linked to an epigenetic modification domain or the DNA binding domain (e.g., an adaptor for an aptamer).
  • the epigenetic modification domain can be linked to an antibody specific for an epitope tag fused to the DNA binding domain.
  • An aptamer can be linked to a guide sequence.
  • the DNA binding domain is a programmable DNA binding protein linked to or otherwise capable of associating with an epigenetic modification domain.
  • Programmable DNA binding proteins for modifying the epigenome include, but are not limited to CRISPR systems, transcription activator-like effectors (TALEs), Zn finger proteins and meganucleases (see, e.g, Thakore PI, Black JB, Hilton IB, Gersbach CA. Editing the epigenome: technologies for programmable transcription and epigenetic modulation. Nat Methods. 2016; 13(2): 127-137; and described further herein).
  • the DNA binding domain is a nuclease-deficient RNA-guided DNA endonuclease enzyme or a nuclease-deficient endonuclease enzyme.
  • a CRISPR system having an inactivated nuclease activity e.g., dCas is used as the DNA binding domain.
  • the epigenetic modification domain is a functional domain and includes, but is not limited to a histone methyltransferase (HMT) domain, histone demethylase domain, histone acetyltransferase (HAT) domain, histone deacetylation (HD AC) domain, DNA methyltransferase domain, DNA demethylation domain, histone phosphorylation domain (e.g., serine and threonine, or tyrosine), histone ubiquitylation domain, histone sumoylation domain, histone ADP ribosylation domain, histone proline isomerization domain, histone biotinylation domain, histone citrullination domain (see, e.g., Epigenetics, Second Edition, 2015, Edited by C.
  • HMT histone methyltransferase
  • HAT histone acetyltransferase
  • HD AC histone deacetylation
  • DNA methyltransferase domain DNA
  • Example epigenetic modification domains can be obtained from, but are not limited to chromatin modifying enzymes, such as, DNA methyltransferases (e.g., DNMT1, DNMT3a and DNMT3b), TET1, TET2, thymine-DNA glycosylase (TDG), GCN5-related N- acetyltransferases family (GNAT), MYST family proteins (e.g., MOZ and MORF), and CBP/p300 family proteins (e.g., CBP, p300), Class I HDACs (e.g., HD AC 1-3 and HDAC8), Class II HDACs (e.g., HDAC 4-7 and HDAC 9-10), Class III HDACs (e.g, sirtuins), HDAC11, SET domain containing methyltransferases (e.g., SET7/9 (KMT7, NCBI Entrez Gene: 80854), KMT5A (SET8), MMSET, EZH2, and MLL
  • histone acetylation is targeted to a target sequence using a CRISPR system (see, e.g., Hilton IB, et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol. 2015).
  • histone deacetylation is targeted to a target sequence (see, e.g., Cong et al., 2012; and Konermann S, et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature. 2013;500:472-476).
  • histone methylation is targeted to a target sequence (see, e.g., Snowden AW, Gregory PD, Case CC, Pabo CO. Genespecific targeting of H3K9 methylation is sufficient for initiating repression in vivo. Curr Biol. 2002;12:2159-2166; and Cano-Rodriguez D, Gjaltema RA, Jilderda LJ, et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat Commun. 2016;7: 12284).
  • histone demethylation is targeted to a target sequence (see, e.g., Kearns NA, Pham H, TabakB, et al. Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nat Methods. 2015; 12(5):401-403).
  • histone phosphorylation is targeted to a target sequence (see, e.g., Li J, Mahata B, Escobar M, et al. Programmable human histone phosphorylation and gene activation using a CRISPR/Cas9-based chromatin kinase. Nat Commun. 2021 ; 12(1):896).
  • DNA methylation is targeted to a target sequence (see, e.g., Rivenbark AG, et al. Epigenetic reprogramming of cancer cells via targeted DNA methylation. Epigenetics. 2012;7:350-360; Siddique AN, et al. Targeted methylation and gene silencing of VEGF-A in human cells by using a designed Dnmt3a-Dnmt3L single-chain fusion protein with increased DNA methylation activity. J Mol Biol. 2013;425:479-491; Bernstein DL, Le Lay JE, Ruano EG, Kaestner KH. TALE- mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts.
  • a target sequence see, e.g., Rivenbark AG, et al. Epigenetic reprogramming of cancer cells via targeted DNA methylation. Epigenetics. 2012;7:350-360; Siddique AN, et al. Target
  • a modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs. Genome Res. 2018;28:1193-1206).
  • DNA demethylation is targeted to a target sequence using a CRISPR system (see, e.g., TET1, see Xu et al, Cell Discov.
  • DNA demethylation is targeted to a target sequence (see, e.g., TDG, see, Gregory DJ, Zhang Y, Kobzik L, Fedulov AV. Specific transcriptional enhancement of inducible nitric oxide synthase by targeted promoter demethylation. Epigenetics. 2013;8: 1205-1212).
  • Example epigenetic modification domains can be obtained from, but are not limited to transcription activators, such as, VP64 (see, e.g., Ji Q, et al. Engineered zinc-finger transcription factors activate OCT4 (POU5F1), SOX2, KLF4, c-MYC (MYC) and miR302/367. Nucleic Acids Res. 2014;42:6158-6167; Perez-Pinera P, et al. Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nat Methods. 2013;10:239-242; Farzadfard F, Perli SD, Lu TK.
  • transcription activators such as, VP64 (see, e.g., Ji Q, et al. Engineered zinc-finger transcription factors activate OCT4 (POU5F1), SOX2, KLF4, c-MYC (MYC) and miR302/367. Nucleic Acids Res. 2014;42:6158-6167
  • Example epigenetic modification domains can be obtained from, but are not limited to transcription repressors, such as, KRAB (see, e.g., Beerli RR, Segal DJ, Dreier B, Barbas CF., 3rd Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc Natl Acad Sci U S A. 1998;95:14628-14633; Cong L, Zhou R, Kuo YC, Cunniff M, Zhang F. Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. Nat Commun. 2012;3 :968; Gilbert LA, et al.
  • KRAB transcription repressors
  • the epigenetic modification domain linked to a DNA binding domain recruits an epigenetic modification protein to a target sequence.
  • a transcriptional activator recruits an epigenetic modification protein to a target sequence.
  • VP64 can recruit DNA demethylation, increased H3K27ac and H3K4me.
  • a transcriptional repressor protein recruits an epigenetic modification protein to a target sequence.
  • KRAB can recruit increased H3K9me3 (see, e.g., Thakore PI, D'lppolito AM, Song L, et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements.
  • methyl-binding proteins linked to a DNA binding domain such as MBD1, MBD2, MBD3, and MeCP2 recruits an epigenetic modification protein to a target sequence.
  • MBD1, MBD2, MBD3, and MeCP2 recruits an epigenetic modification protein to a target sequence.
  • Mi2/NuRD, Sin3 A, or Co-REST recruit HDACs to a target sequence.
  • the epigenetic modification domain can be a eukaryotic or prokaryotic (e.g., bacteria or Archaea) protein.
  • the eukaryotic protein can be a mammalian, insect, plant, or yeast protein and is not limited to human proteins (e.g., a yeast, insect, plant chromatin modifying protein, such as yeast HATs, HDACs, methyltransferases, etc.
  • a fusion protein comprising from N-terminus to C-terminus, an epigenetic modification domain, an XTEN linker, and a nuclease-deficient RNA-guided DNA endonuclease enzyme or a nuclease- deficient endonuclease enzyme.
  • the epigenetic modification polypeptide further comprises a transcriptional activator.
  • the transcriptional activator is VP64, p65, RTA, or a combination of two or more thereof.
  • the epigenetic modification polypeptide further comprises one or more nuclear localization sequences.
  • the epigenetic modification polypeptide comprises the nuclease-deficient RNA-guided DNA endonuclease enzyme.
  • the fusion protein comprises the nuclease-deficient DNA endonuclease enzyme.
  • the functional domains associated with the adaptor protein or the CRISPR enzyme is a transcriptional activation domain comprising VP64, p65, MyoDl, HSF1, RTA or SET7/9.
  • activation (or activator) domains in respect of those associated with the adaptor protein(s) include any known transcriptional activation domain and specifically VP64, p65, MyoDl, HSF1, RTA or SET7/9 (see, e.g., US Patent, US11001829B2).
  • the present invention provides a fusion protein comprising from N-terminus to C-terminus, an RNA-binding sequence, an XTEN linker, and a transcriptional activator.
  • the transcriptional activator is VP64, p65, RTA, or a combination of two or more thereof.
  • the fusion protein further comprises a demethylation domain, a nuclease- deficient RNA-guided DNA endonuclease enzyme or a nuclease-deficient endonuclease enzyme, a nuclear localization sequence, or a combination of two or more thereof.
  • the fusion protein comprises the nuclease-deficient RNA-guided DNA endonuclease enzyme.
  • the fusion protein comprises the nuclease-deficient DNA endonuclease enzyme.
  • the present invention provides a method of activating a target nucleic acid sequence in a cell, the method comprising: (i) delivering a first polynucleotide encoding a epigenetic modification polypeptide described herein including embodiments thereof to a cell containing the silenced target nucleic acid; and (ii) delivering to the cell a second polynucleotide comprising: (a) a sgRNA or (b) a crtracrRNA; thereby reactivating the silenced target nucleic acid sequence in the cell.
  • the sgRNA comprises at least one MS2 stem loop.
  • the second polynucleotide comprises a transcriptional activator.
  • the second polynucleotide comprises two or more sgRNA.
  • the polynucleotide is modified using a Zinc Finger nuclease or system thereof.
  • a Zinc Finger nuclease or system thereof One type of programmable DNA-binding domain is provided by artificial zinc- finger (ZF) technology, which involves arrays of ZF modules to target new DNA-binding sites in the genome. Each finger module in a ZF array targets three DNA bases. A customized array of individual zinc finger domains is assembled into a ZF protein (ZFP).
  • ZFP ZF protein
  • ZFPs can comprise a functional domain.
  • the first synthetic zinc finger nucleases (ZFNs) were developed by fusing a ZF protein to the catalytic domain of the Type IIS restriction enzyme Fokl. (Kim, Y. G. et al., 1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. U.S.A. 91, 883-887; Kim, Y. G. et al., 1996, Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. U.S.A. 93, 1156-1160).
  • ZFPs can also be designed as transcription activators and repressors and have been used to target many genes in a wide variety of organisms. Exemplary methods of genome editing using ZFNs can be found for example in U.S. Patent Nos.
  • a TALE nuclease or TALE nuclease system can be used to modify a polynucleotide.
  • the methods provided herein use isolated, non- naturally occurring, recombinant or engineered DNA binding proteins that comprise TALE monomers or TALE monomers or half monomers as a part of their organizational structure that enable the targeting of nucleic acid sequences with improved efficiency and expanded specificity.
  • Naturally occurring TALEs or “wild type TALEs” are nucleic acid binding proteins secreted by numerous species of proteobacteria.
  • TALE polypeptides contain a nucleic acid binding domain composed of tandem repeats of highly conserved monomer polypeptides that are predominantly 33, 34 or 35 amino acids in length and that differ from each other mainly in amino acid positions 12 and 13.
  • the nucleic acid is DNA.
  • polypeptide monomers “TALE monomers” or “monomers” will be used to refer to the highly conserved repetitive polypeptide sequences within the TALE nucleic acid binding domain and the term “repeat variable di-residues” or “RVD” will be used to refer to the highly variable amino acids at positions 12 and 13 of the polypeptide monomers.
  • the amino acid residues of the RVD are depicted using the IUPAC single letter code for amino acids.
  • a general representation of a TALE monomer which is comprised within the DNA binding domain is Xi-n-(Xi2Xi3)-Xi4-33 or 34 or 35, where the subscript indicates the amino acid position and X represents any amino acid.
  • X12X13 indicate the RVDs.
  • the variable amino acid at position 13 is missing or absent and in such monomers, the RVD consists of a single amino acid.
  • the RVD may be alternatively represented as X*, where X represents X12 and (*) indicates that X13 is absent.
  • the DNA binding domain comprises several repeats of TALE monomers and this may be represented as (Xi-n-(Xi2Xi3)-Xi4- 33 or 34 or 35) z , where in an advantageous embodiment, z is at least 5 to 40. In a further advantageous embodiment, z is at least 10 to 26.
  • the TALE monomers can have a nucleotide binding affinity that is determined by the identity of the amino acids in its RVD.
  • polypeptide monomers with an RVD of NI can preferentially bind to adenine (A)
  • monomers with an RVD of NG can preferentially bind to thymine (T)
  • monomers with an RVD of HD can preferentially bind to cytosine (C)
  • monomers with an RVD of NN can preferentially bind to both adenine (A) and guanine (G).
  • monomers with an RVD of IG can preferentially bind to T.
  • the number and order of the polypeptide monomer repeats in the nucleic acid binding domain of a TALE determines its nucleic acid target specificity.
  • monomers with an RVD of NS can recognize all four base pairs and can bind to A, T, G or C.
  • the structure and function of TALEs is further described in, for example, Moscou et al., Science 326:1501 (2009); Boch et al., Science 326: 1509-1512 (2009); and Zhang et al., Nature Biotechnology 29: 149-153 (2011).
  • polypeptides used in methods of the invention can be isolated, non-naturally occurring, recombinant or engineered nucleic acid-binding proteins that have nucleic acid or DNA binding regions containing polypeptide monomer repeats that are designed to target specific nucleic acid sequences.
  • polypeptide monomers having an RVD of HN or NH preferentially bind to guanine and thereby allow the generation of TALE polypeptides with high binding specificity for guanine containing target nucleic acid sequences.
  • polypeptide monomers having RVDs RN, NN, NK, SN, NH, KN, HN, NQ, HH, RG, KH, RH and SS can preferentially bind to guanine.
  • polypeptide monomers having RVDs RN, NK, NQ, HH, KH, RH, SS and SN can preferentially bind to guanine and can thus allow the generation of TALE polypeptides with high binding specificity for guanine containing target nucleic acid sequences.
  • polypeptide monomers having RVDs HH, KH, NH, NK, NQ, RH, RN and SS can preferentially bind to guanine and thereby allow the generation of TALE polypeptides with high binding specificity for guanine containing target nucleic acid sequences.
  • the RVDs that have high binding specificity for guanine are RN, NH RH and KH.
  • polypeptide monomers having an RVD of NV can preferentially bind to adenine and guanine.
  • monomers having RVDs of H*, HA, KA, N*, NA, NC, NS, RA, and S* bind to adenine, guanine, cytosine and thymine with comparable affinity.
  • the predetermined N-terminal to C-terminal order of the one or more polypeptide monomers of the nucleic acid or DNA binding domain determines the corresponding predetermined target nucleic acid sequence to which the polypeptides of the invention will bind.
  • the monomers and at least one or more half monomers are “specifically ordered to target” the genomic locus or gene of interest.
  • the natural TALE-binding sites always begin with a thymine (T), which may be specified by a cryptic signal within the non- repetitive N-terminus of the TALE polypeptide; in some cases, this region may be referred to as repeat 0.
  • TALE binding sites do not necessarily have to begin with a thymine (T) and polypeptides of the invention may target DNA sequences that begin with T, A, G or C.
  • T thymine
  • the tandem repeat of TALE monomers always ends with a half-length repeat or a stretch of sequence that may share identity with only the first 20 amino acids of a repetitive full-length TALE monomer and this half repeat may be referred to as a half-monomer. Therefore, it follows that the length of the nucleic acid or DNA being targeted is equal to the number of full monomers plus two.
  • TALE polypeptide binding efficiency may be increased by including amino acid sequences from the “capping regions” that are directly N-terminal or C-terminal of the DNA binding region of naturally occurring TALEs into the engineered TALEs at positions N-terminal or C-terminal of the engineered TALE DNA binding region.
  • the TALE polypeptides described herein further comprise an N-terminal capping region and/or a C-terminal capping region.
  • N-terminal capping region An exemplary amino acid sequence of a N-terminal capping region is:
  • An exemplary amino acid sequence of a C-terminal capping region is:
  • the DNA binding domain comprising the repeat TALE monomers and the C-terminal capping region provide structural basis for the organization of different domains in the d-TALEs or polypeptides of the invention.
  • N-terminal and/or C-terminal capping regions are not necessary to enhance the binding activity of the DNA binding region. Therefore, in certain embodiments, fragments of the N-terminal and/or C-terminal capping regions are included in the TALE polypeptides described herein.
  • the TALE polypeptides described herein contain a N-terminal capping region fragment that included at least 10, 20, 30, 40, 50, 54, 60, 70, 80, 87, 90, 94, 100, 102, 110, 117, 120, 130, 140, 147, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or 270 amino acids of an N-terminal capping region.
  • the N-terminal capping region fragment amino acids are of the C-terminus (the DNA-binding region proximal end) of an N-terminal capping region.
  • N-terminal capping region fragments that include the C-terminal 240 amino acids enhance binding activity equal to the full length capping region, while fragments that include the C-terminal 147 amino acids retain greater than 80% of the efficacy of the full length capping region, and fragments that include the C-terminal 117 amino acids retain greater than 50% of the activity of the full- length capping region.
  • the TALE polypeptides described herein contain a C-terminal capping region fragment that included at least 6, 10, 20, 30, 37, 40, 50, 60, 68, 70, 80, 90, 100, 110, 120, 127, 130, 140, 150, 155, 160, 170, 180 amino acids of a C-terminal capping region.
  • the C-terminal capping region fragment amino acids are of the N-terminus (the DNA-binding region proximal end) of a C-terminal capping region.
  • C-terminal capping region fragments that include the C-terminal 68 amino acids enhance binding activity equal to the full-length capping region, while fragments that include the C-terminal 20 amino acids retain greater than 50% of the efficacy of the full-length capping region.
  • the capping regions of the TALE polypeptides described herein do not need to have identical sequences to the capping region sequences provided herein.
  • the capping region of the TALE polypeptides described herein have sequences that are at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or share identity to the capping region amino acid sequences provided herein. Sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs.
  • the capping region of the TALE polypeptides described herein have sequences that are at least 95% identical or share identity to the capping region amino acid sequences provided herein.
  • Sequence homologies can be generated by any of a number of computer programs known in the art, which include but are not limited to BLAST or FASTA. Suitable computer programs for carrying out alignments like the GCG Wisconsin Bestfit package may also be used. Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
  • the TALE polypeptides of the invention include a nucleic acid binding domain linked to the one or more effector domains.
  • effector domain or “regulatory and functional domain” refer to a polypeptide sequence that has an activity other than binding to the nucleic acid sequence recognized by the nucleic acid binding domain.
  • the polypeptides of the invention may be used to target the one or more functions or activities mediated by the effector domain to a particular target DNA sequence to which the nucleic acid binding domain specifically binds.
  • the activity mediated by the effector domain is a biological activity.
  • the effector domain is a transcriptional inhibitor (i.e., a repressor domain), such as an mSin interaction domain (SID). SID4X domain or a Kriippel-associated box (KRAB) or fragments of the KRAB domain.
  • the effector domain is an enhancer of transcription (i.e., an activation domain), such as the VP 16, VP64 or p65 activation domain.
  • the nucleic acid binding is linked, for example, with an effector domain that includes but is not limited to a transposase, integrase, recombinase, resolvase, invertase, protease, DNA methyltransferase, DNA demethylase, histone acetylase, histone deacetylase, nuclease, transcriptional repressor, transcriptional activator, transcription factor recruiting, protein nuclear-localization signal or cellular uptake signal.
  • an effector domain that includes but is not limited to a transposase, integrase, recombinase, resolvase, invertase, protease, DNA methyltransferase, DNA demethylase, histone acetylase, histone deacetylase, nuclease, transcriptional repressor, transcriptional activator, transcription factor recruiting, protein nuclear-localization signal or cellular uptake signal.
  • the effector domain is a protein domain which exhibits activities which include but are not limited to transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, DNA methyltransferase activity, DNA demethylase activity, histone acetylase activity, histone deacetylase activity, nuclease activity, nuclear-localization signaling activity, transcriptional repressor activity, transcriptional activator activity, transcription factor recruiting activity, or cellular uptake signaling activity.
  • Other preferred embodiments of the invention may include any combination of the activities described herein. Meganucleases
  • a meganuclease or system thereof can be used to modify a polynucleotide.
  • Meganucleases which are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs). Exemplary methods for using meganucleases can be found in US Patent Nos. 8,163,514, 8,133,697, 8,021,867, 8,119,361, 8,119,381, 8,124,369, and 8,129,134, which are specifically incorporated herein by reference.
  • a target gene is modified with an ARCUS base editing system.
  • ARCUS base editing system Exemplary methods for using ARCUS can be found in US Patent No. 10,851,358, US Publication No. 2020-0239544, and WIPO Publication No. 2020/206231 which are incorporated herein by reference.
  • RNAi and antisense oligonucleotides ASO
  • tumor subtype specific biomarkers are targeted with RNAi or antisense oligonucleotides (ASO).
  • ASO antisense oligonucleotides
  • siRNA or miRNA refers to a decrease in the mRNA level in a cell for a target gene by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without the presence of the miRNA or RNA interference molecule.
  • the mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.
  • inhibitory nucleic acid molecules such as RNAi and ASOs can be used in vivo (see, e.g., Yan Y, Liu XY, Lu A, Wang XY, Jiang LX, Wang JC. Non-viral vectors for RNA delivery. J Control Release. 2022;342:241-279).
  • RNAi refers to any type of interfering RNA, including but not limited to, siRNAi, shRNAi, endogenous microRNA and artificial microRNA. For instance, it includes sequences previously identified as siRNA, regardless of the mechanism of down-stream processing of the RNA (i.e., although siRNAs are believed to have a specific method of in vivo processing resulting in the cleavage of mRNA, such sequences can be incorporated into the vectors in the context of the flanking sequences described herein).
  • the term “RNAi” can include both gene silencing RNAi molecules, and also RNAi effector molecules which activate the expression of a gene.
  • a “siRNA” refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is present or expressed in the same cell as the target gene.
  • the double stranded RNA siRNA can be formed by the complementary strands.
  • a siRNA refers to a nucleic acid that can form a double stranded siRNA.
  • the sequence of the siRNA can correspond to the full-length target gene, or a subsequence thereof.
  • the siRNA is at least about 15- 50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferably about 19-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).
  • shRNA small hairpin RNA
  • stem loop is a type of siRNA.
  • these shRNAs are composed of a short, e.g., about 19 to about 25 nucleotide, antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand.
  • the sense strand can precede the nucleotide loop structure and the antisense strand can follow.
  • microRNA or “miRNA” are used interchangeably herein are endogenous RNAs, some of which are known to regulate the expression of protein-coding genes at the posttranscri phonal level. Endogenous microRNAs are small RNAs naturally present in the genome that are capable of modulating the productive utilization of mRNA.
  • artificial microRNA includes any type of RNA sequence, other than endogenous microRNA, which is capable of modulating the productive utilization of mRNA. MicroRNA sequences have been described in publications such as Lim, et al., Genes & Development, 17, p.
  • miRNA-like stem-loops can be expressed in cells as a vehicle to deliver artificial miRNAs and short interfering RNAs (siRNAs) for the purpose of modulating the expression of endogenous genes through the miRNA and or RNAi pathways.
  • siRNAs short interfering RNAs
  • double stranded RNA or “dsRNA” refers to RNA molecules that are comprised of two strands. Double-stranded molecules include those comprised of a single RNA molecule that doubles back on itself to form a two-stranded structure. For example, the stem loop structure of the progenitor molecules from which the single-stranded miRNA is derived, called the pre-miRNA (Bartel et al. 2004. Cell 1 16:281 -297), comprises a dsRNA molecule.
  • the pre-miRNA Bartel et al. 2004. Cell 1 16:281 -297
  • Antisense therapy is a form of treatment that uses antisense oligonucleotides (ASOs) to target messenger RNA (mRNA).
  • ASOs are capable of altering mRNA expression through a variety of mechanisms, including ribonuclease H mediated decay of the pre-mRNA, direct steric blockage, and exon content modulation through splicing site binding on pre-mRNA (see, e.g., Crooke ST, Liang XH, Baker BF, Crooke RM. Antisense technology: A review. J Biol Chem. 2021;296: 100416. doi:10.1016/j .jbc.2021.100416).
  • Antisense oligonucleotides generally inhibit their target by binding target mRNA and sterically blocking expression by obstructing the ribosome. ASOs can also inhibit their target by binding target mRNA thus forming a DNA-RNA hybrid that can be a substance for RNase H. Commonly used antisense mechanisms to degrade target RNAs include RNase Hl -dependent and RISC-dependent mechanisms. Preferred ASOs include Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), and morpholinos.
  • LNA Locked Nucleic Acid
  • PNA Peptide Nucleic Acid
  • morpholinos morpholinos.
  • tumor subtype specific biomarkers are targeted with a small molecule inhibitor or small molecule degrader (e.g., ATTEC, AUTAC, LYTAC, or PROTAC).
  • receptors are targeted with small molecules that block ligand binding.
  • tumor subtype specific surface biomarkers are targeted with a degrader molecule.
  • small molecule refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, peptides, nucleic acids, etc.).
  • Preferred small organic molecules range in size up to about 5000 Da, e.g., up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g., up to about 900, 800, 700, 600 or up to about 500 Da.
  • the small molecule may act as an antagonist or agonist (e.g., blocking an enzyme active site or activating a receptor by binding to a ligand binding site).
  • degrader refers to all compounds capable of specifically targeting a protein for degradation (e.g., ATTEC, AUTAC, LYTAC, or PROTAC, reviewed in Ding, et al. 2020).
  • Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs.
  • PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome (see, e.g., Zhou et al., Discovery of a Small-Molecule Degrader of Bromodomain and Extra- Terminal (BET) Proteins with Picomolar Cellular Potencies and Capable of Achieving Tumor Regression. J. Med. Chem. 2018, 61, 462-481; Bondeson and Crews, Targeted Protein Degradation by Small Molecules, Annu Rev Pharmacol Toxicol. 2017 Jan 6; 57: 107-123; and Lai et al., Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL Angew Chem Int Ed Engl.
  • LYTACs are particularly advantageous for cell surface proteins as described herein (e.g., TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR; or MSLN, GPR87, SLC2A1, LY6D, and PSCA).
  • Described in certain example embodiments herein are methods of detecting cancers having a NRP, SBM, or classical phenotype.
  • the cancer is a pancreatic ductal adenocarcinoma (PDAC).
  • methods for detecting subtypes in a subject in need thereof by detecting subtype specific genes or polypeptides, detecting surface markers specific for a subtype, detecting secreted proteins specific for a subtype (e.g., on malignant cells or in the TME), or detecting ligand receptor interactions specific for a subtype (e.g., on malignant cells or in the TME).
  • biomarkers e.g., phenotype specific or cell type
  • Biomarkers in the context of the present invention encompasses, without limitation nucleic acids, proteins, reaction products, and metabolites, together with their polymorphisms, mutations, variants, modifications, subunits, fragments, and other analytes or sample-derived measures.
  • biomarkers include signature genes or signature gene products, and/or cells as described herein.
  • Biomarkers are useful in methods of monitoring, diagnosing, prognosing and/or staging treatment response in a subject by detecting a first level of expression, activity and/or function of one or more biomarker and comparing the detected level to a control of level wherein a difference in the detected level and the control level indicates that the presence of a treatment response in the subject (e.g., NRP, SBM and classical subtypes).
  • a treatment response in the subject e.g., NRP, SBM and classical subtypes.
  • diagnosis generally refers to the process or act of recognising, deciding on or concluding on a disease or condition in a subject on the basis of symptoms and signs and/or from results of various diagnostic procedures (such as, for example, from knowing the presence, absence and/or quantity of one or more biomarkers characteristic of the diagnosed disease or condition).
  • prognosing or “prognosis” generally refer to an anticipation on the progression of a disease or condition and the prospect (e.g., the probability, duration, and/or extent) of recovery.
  • a good prognosis of the diseases or conditions taught herein may generally encompass anticipation of a satisfactory partial or complete recovery from the diseases or conditions, preferably within an acceptable time period.
  • a good prognosis of such may more commonly encompass anticipation of not further worsening or aggravating of such, preferably within a given time period.
  • a poor prognosis of the diseases or conditions as taught herein may generally encompass anticipation of a substandard recovery and/or unsatisfactorily slow recovery, or to substantially no recovery or even further worsening of such.
  • the term “monitoring” generally refers to the follow-up of a disease or a condition in a subject for any changes which may occur over time. For example, changes in cancer subtype can be monitored during treatment, which can then direct treatment as described herein.
  • the biomarkers of the present invention are useful in methods of identifying patient populations at risk or suffering from an adverse tumor response based on a detected level of expression, activity and/or function of one or more biomarkers. These biomarkers are also useful in monitoring subjects undergoing treatments and therapies for suitable or aberrant response(s) to determine efficaciousness of the treatment or therapy and for selecting or modifying therapies and treatments that would be efficacious in treating, delaying the progression of or otherwise ameliorating a symptom.
  • the biomarkers provided herein are useful for selecting a group of patients at a specific state of a disease with accuracy that facilitates selection of treatments.
  • Distinct reference values may represent the prediction of a risk (e.g., an abnormally elevated risk) of having a given disease or condition as taught herein.
  • distinct reference values may represent the diagnosis of such disease or condition of varying severity.
  • distinct reference values may represent a good prognosis for a given disease or condition as taught herein vs. a poor prognosis for said disease or condition.
  • distinct reference values may represent varyingly favourable or unfavourable prognoses for such disease or condition. Reference values may be established according to known procedures previously employed for other cell populations, biomarkers and gene or gene product signatures.
  • a reference value may be established in an individual or a population of individuals characterised by a particular diagnosis, prediction and/or prognosis of said disease or condition (i.e., for whom said diagnosis, prediction and/or prognosis of the disease or condition holds true).
  • Such population may comprise without limitation 2 or more, 10 or more, 100 or more, or even several hundred or more individuals.
  • a “deviation” of a first value from a second value may generally encompass any direction (e.g., increase: first value > second value; or decrease: first value ⁇ second value) and any extent of alteration.
  • a deviation may encompass a decrease in a first value by, without limitation, at least about 10% (about 0.9-fold or less), or by at least about 20% (about 0.8-fold or less), or by at least about 30% (about 0.7-fold or less), or by at least about 40% (about 0.6-fold or less), or by at least about 50% (about 0.5-fold or less), or by at least about 60% (about 0.4-fold or less), or by at least about 70% (about 0.3-fold or less), or by at least about 80% (about 0.2-fold or less), or by at least about 90% (about 0.1 -fold or less), relative to a second value with which a comparison is being made.
  • a deviation may encompass an increase of a first value by, without limitation, at least about 10% (about 1.1-fold or more), or by at least about 20% (about 1 ,2-fold or more), or by at least about 30% (about 1.3-fold or more), or by at least about 40% (about 1.4-fold or more), or by at least about 50% (about 1.5-fold or more), or by at least about 60% (about 1.6- fold or more), or by at least about 70% (about 1.7-fold or more), or by at least about 80% (about 1.8-fold or more), or by at least about 90% (about 1.9-fold or more), or by at least about 100% (about 2-fold or more), or by at least about 150% (about 2.5-fold or more), or by at least about 200% (about 3-fold or more), or by at least about 500% (about 6-fold or more), or by at least about 700% (about 8-fold or more), or like, relative to a second value with which a comparison is being made.
  • a deviation may refer to a statistically significant observed alteration.
  • a deviation may refer to an observed alteration which falls outside of error margins of reference values in a given population (as expressed, for example, by standard deviation or standard error, or by a predetermined multiple thereof, e g., ilxSD or ⁇ 2xSD or ⁇ 3xSD, or ⁇ lxSE or ⁇ 2xSE or ⁇ 3xSE).
  • Deviation may also refer to a value falling outside of a reference range defined by values in a given population (for example, outside of a range which comprises >40%, > 50%, >60%, >70%, >75% or >80% or >85% or >90% or >95% or even >100% of values in said population).
  • a deviation may be concluded if an observed alteration is beyond a given threshold or cut-off.
  • threshold or cut-off may be selected as generally known in the art to provide for a chosen sensitivity and/or specificity of the prediction methods, e.g., sensitivity and/or specificity of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%.
  • receiver-operating characteristic (ROC) curve analysis can be used to select an optimal cut-off value of the quantity of a given immune cell population, biomarker or gene or gene product signatures, for clinical use of the present diagnostic tests, based on acceptable sensitivity and specificity, or related performance measures which are well-known per se, such as positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-), Youden index, or similar.
  • PV positive predictive value
  • NPV negative predictive value
  • LR+ positive likelihood ratio
  • LR- negative likelihood ratio
  • Youden index or similar.
  • neural-like progenitor (NRP) tumors are tumors that comprise malignant cells expressing a neural-like progenitor (NRP) gene signature or a TME gene signature associated with an NRP signature in malignant cells.
  • NRP neural-like progenitor
  • the NRP gene signature includes differential expression of one or more of KCNJ16, ZBTB16, CTNND2, PDE3A, PDGFD, CNTN4, CFTR, FLRT2, ADCY5, C6, CRISP3, RALYL, NR1H4, BCL2, ESRRG, SLC4A4, CSMD2, RGS17, CRP, SLC17A4, RELN, PAH, PKHD1, LINC01320, ACSM3, DSCAML1, AR, ZNF208, TTLL7, S0X6, SPP1, ASXL3, POU6F2, DZIP1, ITIH5, PRKG1, IL1R1, SEMA3E, GUCY1A2, AC092535.3, RCAN2, SLC3A1, MAN1A1, WDR72, ACSS3, 0NECUT1, NRP1, AKAP7, LDLRAD4, AC012593.1, CALN1, UGT2B15, AGBL4, GLIS3, TRPV6, ABCB1, PLXDC
  • the NRP gene signature as compared to other malignant cells, includes expression of one or more brain tissue enhanced genes, such as DLG2, NRCAM, NRXN3 and MAPK10.
  • the NRP gene signature as compared to other malignant cells and normal, toxicity- prone tissues, includes enhanced expression of one or more surface markers selected from TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, and SCTR.
  • the NRP gene signature as compared to other malignant cells, includes expression of one or more secreted markers selected from PDGFD, C6, CRISP3, RELN, SERPINA6, CRP, FGG, APCS, CFH, HABP2, KLKB1, REGIA and SPP1.
  • NRP tumors as compared to other tumor subtypes, express in the TME, one or more receptor ligand pairs correlated in CRT-treated subjects shown in Fig. 7.
  • NRP tumors as compared to other tumor subtypes, express in the TME, one or more secreted proteins selected from C7, HDGF, MAPT, REGIB, SERPINA4, PVR, CRISP3, DKK4, DPP7, NPNT and TAFA1.
  • the NRP gene signature as compared to other malignant cells, includes expression of one or more genes selected from CTNND2, CRP, SERPINA6, NRXN3, PDGFD, C6, RELN, SPP1, GLIS3, NR5A2, DCDC2, DPYD, HNF1B, ZBTB16, KCNJ16, SLC4A4, and BICCl.
  • squamoid/basaloid/mesenchymal (SBM) tumors are tumors that comprise malignant cells expressing a squamoid/basaloid/mesenchymal (SBM) gene signature or a TME gene signature associated with an SBM signature in malignant cells.
  • SBM squamoid/basaloid/mesenchymal
  • the SBM gene signature includes differential expression of one or more of IGF2, CST6, CRYAB, CST4, FBXO2, CHPF, LGALS1, ALDOA, MT1E, ISG15, CCDC85B, LY6K, KRTAP2-3, MT2A, CKAP4, PRNP, IFI27, DKK3, C9orfl6, GJA1, IFI6, CRIP1, POLR2L, THBS1, LGALS7, TNNC2, PTMS, R0M01, IFI27L2, CD81, TUBB, LY6E, C12orf57, PSAP, PDLIM4, C19orf53, CTSZ, SNRPD2, GPS2, OST4, PRDX1, NPC2, VIM, TRMT112, MZT2B, SCAND1, MMP2, CD59, MT1A, CFL1, SEC61G, TMSB10, BANF1, SOSTDC1, VAT1, MRPS12, PPDPF, NPB, TIMM8B, UBA
  • the SBM gene signature as compared to other malignant cells and normal, toxicity-prone tissues, includes enhanced expression of one or more surface markers selected from MSLN, GPR87, SLC2A1, LY6D, PSCA, GPRC5A, ALPP, MET, TACSTD2, and NECTIN4.
  • the SBM gene signature as compared to other malignant cells, includes expression of one or more secreted markers selected from COL17A1, MUC16, FGF19, IGF2, NPC2, SAA1, SRGN, CIS, and CST4.
  • SBM tumors as compared to other tumor subtypes, express in the TME, one or more receptor ligand pairs correlated in CRT -treated or untreated subjects shown in Fig. 7.
  • SBM tumors as compared to other tumor subtypes, express in the TME, one or more secreted proteins selected from SFN, COL17A1, FGFBP1, MUC16 (CA-125), HDGF, COP A, and CFB.
  • the SBM gene signature as compared to other malignant cells, includes expression of one or more genes selected from S100A2, KRT17, KRT7, TACSTD2, TP63, MSLN, SCEL, COL17A1, VIM, FN1, CST6, CST4, TUBB, and LY6D.
  • classical tumors are tumors that comprise malignant cells expressing a classical gene signature or a TME gene signature associated with a classical signature in malignant cells.
  • the classical gene signature includes differential expression of one or more of PTH2R, SULT1C2, ANXA10, HEPH, WDR72, FMO5, SULT IB 1, SYTL2, BTNL8, PLAC8, STXBP6, FER1L6, TM4SF20, ETNK1, KCNJ3, CPS1, CASR, THRB, PIP5K1B, REG4, BCAS1, NR3C2, SIPA1L2, CLDN18, PELI2, TMEM45B, SLC3A1, CBLB, PRKG1, GPC5, ARHGEF38, SLC41A2, ATP10B, NR5A2, PLD1, CYP3A5, TSPAN8, RAB27B, LRRC66, MCU, SLC40A1, XRCC4, CAPN8, ABHD2, ANPEP
  • the classical gene signature as compared to other malignant cells and normal, toxicity -prone tissues, includes enhanced expression of one or more surface markers selected from CEACAM5, CLDN18, CEACAM6, CDH17, TSPAN8, MUC13, and MUC17.
  • the classical gene signature as compared to other malignant cells, includes expression of one or more secreted markers selected from REG4, PLA2G10, TFF1, and NRG4.
  • classical tumors as compared to other tumor subtypes, express in the TME, one or more receptor ligand pairs correlated in untreated subjects shown in Fig. 7.
  • classical tumors as compared to other tumor subtypes, express in the TME, one or more secreted proteins selected from TFF1, MUC5AC, LYZ, and MUC1.
  • the classical gene signature as compared to other malignant cells, includes expression of one or more genes selected from HNF4A, GATA6, TFF1, CEACAM5, TSPAN8, FM05, BTNL8, REG4, SYTL2 and CLDN18.
  • PDAC tumor signatures and/or programs including, but not limited to, a malignant signature and/or program, a tumor microenvironment signature and/or program (e.g., CAF signature and/or program, an immune signature and/or program, a tumor spatial neighborhood); one or more co-expressed receptor-ligand pairs or a combination thereof.
  • the PDAC tumor signatures and/or programs include a neoadjuvant treated tumor expression program (“a treated program”); or a neoadjuvant untreated tumor expression program (an “untreated program”).
  • the neoadjuvant treatment includes chemotherapy and/or radiation, described further herein.
  • the PDAC tumor signature and/or program is a malignant cell signature and/or program. In some embodiments, the PDAC tumor signature and/or program is a CAF signature and/or program. In some embodiments, the PDAC tumor signature and/or program is an immune signature and/or program.
  • a “signature” may encompass any gene or genes, protein or proteins, or epigenetic element(s) whose expression profile or whose occurrence is associated with a specific cell type, subtype, or cell state of a specific cell type or subtype within a population of cells.
  • any of gene or genes, protein or proteins, or epigenetic element(s) may be substituted.
  • the terms “signature”, “gene program”, “expression profile”, or “expression program” may be used interchangeably. It is to be understood that also when referring to proteins (e.g., differentially expressed proteins), such may fall within the definition of “gene” signature.
  • Levels of expression or activity or prevalence may be compared between different cells in order to characterize or identify for instance signatures specific for cell (sub)populations.
  • Increased or decreased expression or activity or prevalence of signature genes may be compared between different cells in order to characterize or identify for instance specific cell (sub)populations.
  • the detection of a signature in single cells may be used to identify and quantitate for instance specific cell (sub)populations.
  • a signature may include a gene or genes, protein or proteins, or epigenetic element(s) whose expression or occurrence is specific to a cell (sub)population, such that expression or occurrence is exclusive to the cell (sub)population.
  • a gene signature as used herein, may thus refer to any set of up- and down-regulated genes that are representative of a cell type or subtype.
  • a gene signature as used herein may also refer to any set of up- and down-regulated genes between different cells or cell (sub)populations derived from a gene-expression profile.
  • a gene signature may comprise a list of genes differentially expressed in a distinction of interest.
  • the signature as defined herein can be used to indicate the presence of a cell type, a subtype of the cell type, the state of the microenvironment of a population of cells, a particular cell type population or subpopulation, and/or the overall status of the entire cell (sub)population. Furthermore, the signature may be indicative of cells within a population of cells in vivo. The signature may also be used to suggest for instance particular therapies, or to follow up treatment, or to suggest ways to modulate immune systems.
  • the signatures of the present invention may be discovered by analysis of expression profiles of single-cells within a population of cells from isolated samples (e.g., tumor samples), thus allowing the discovery of novel cell subtypes or cell states that were previously invisible or unrecognized.
  • the presence of subtypes or cell states may be determined by subtype specific or cell state specific signatures.
  • the presence of these specific cell (sub)types or cell states may be determined by applying the signature genes to bulk sequencing data in a sample.
  • the signatures of the present invention may be microenvironment specific, such as their expression in a particular spatio-temporal context.
  • signatures as discussed herein are specific to a particular pathological context.
  • a combination of cell subtypes having a particular signature may indicate an outcome.
  • the signatures can be used to deconvolute the network of cells present in a particular pathological condition.
  • the presence of specific cells and cell subtypes are indicative of a particular response to treatment, such as including increased or decreased susceptibility to treatment.
  • the signature may indicate the presence of one particular cell type.
  • the novel signatures are used to detect multiple cell states or hierarchies that occur in subpopulations of cancer cells that are linked to particular pathological condition (e.g., cancer grade), or linked to a particular outcome or progression of the disease (e.g., metastasis), or linked to a particular response to treatment of the disease.
  • the signature according to certain embodiments of the present invention may comprise or consist of one or more genes, proteins and/or epigenetic elements, such as for instance 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • the signature may comprise or consist of two or more genes, proteins and/or epigenetic elements, such as for instance 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • the signature may comprise or consist of three or more genes, proteins and/or epigenetic elements, such as for instance 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • the signature may comprise or consist of four or more genes, proteins and/or epigenetic elements, such as for instance 4, 5, 6, 7, 8, 9, 10 or more.
  • the signature may comprise or consist of five or more genes, proteins and/or epigenetic elements, such as for instance 5, 6, 7, 8, 9, 10 or more. In certain embodiments, the signature may comprise or consist of six or more genes, proteins and/or epigenetic elements, such as for instance 6, 7, 8, 9, 10 or more. In certain embodiments, the signature may comprise or consist of seven or more genes, proteins and/or epigenetic elements, such as for instance 7, 8, 9, 10 or more. In certain embodiments, the signature may comprise or consist of eight or more genes, proteins and/or epigenetic elements, such as for instance 8, 9, 10 or more. In certain embodiments, the signature may comprise or consist of nine or more genes, proteins and/or epigenetic elements, such as for instance 9, 10 or more.
  • the signature may comprise or consist of ten or more genes, proteins and/or epigenetic elements, such as for instance 10, 11, 12, 13, 14, 15, or more. It is to be understood that a signature according to the invention may for instance also include genes or proteins as well as epigenetic elements combined.
  • a signature is characterized as being specific for a particular tumor cell or tumor cell (sub)population if it is upregulated or only present, detected or detectable in that particular tumor cell or tumor cell (sub)population, or alternatively is downregulated or only absent, or undetectable in that particular tumor cell or tumor cell (sub)population.
  • a signature consists of one or more differentially expressed genes/proteins or differential epigenetic elements when comparing different cells or cell (sub)populations, including comparing different tumor cells or tumor cell (sub)populations, as well as comparing tumor cells or tumor cell (sub)populations with non-tumor cells or non-tumor cell (sub)populations.
  • genes/proteins include genes/proteins which are up- or down-regulated as well as genes/proteins which are turned on or off.
  • up- or down-regulation in certain embodiments, such up- or down-regulation is preferably at least twofold, such as two-fold, three-fold, four-fold, five-fold, or more, such as for instance at least tenfold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more.
  • differential expression may be determined based on common statistical tests, as is known in the art.
  • differentially expressed genes/proteins, or differential epigenetic elements may be differentially expressed on a single cell level or may be differentially expressed on a cell population level.
  • the differentially expressed genes/ proteins or epigenetic elements as discussed herein, such as constituting the gene signatures as discussed herein, when as to the cell population level refer to genes that are differentially expressed in all or substantially all cells of the population (such as at least 80%, preferably at least 90%, such as at least 95% of the individual cells). This allows one to define a particular subpopulation of tumor cells.
  • a “subpopulation” of cells preferably refers to a particular subset of cells of a particular cell type which can be distinguished or are uniquely identifiable and set apart from other cells of this cell type.
  • the cell subpopulation may be phenotypically characterized and is preferably characterized by the signature as discussed herein.
  • a cell (sub)population as referred to herein may constitute of a (sub)population of cells of a particular cell type characterized by a specific cell state.
  • induction or alternatively suppression of a particular signature preferable is meant induction or alternatively suppression (or upregulation or downregulation) of at least one gene/protein and/or epigenetic element of the signature, such as for instance at least two, at least three, at least four, at least five, at least six, or all genes/proteins and/or epigenetic elements of the signature.
  • Signatures may be functionally validated as being uniquely associated with a particular phenotype. Induction or suppression of a particular signature may consequentially be associated with or causally drive a particular phenotype.
  • Various aspects and embodiments of the invention may involve analyzing gene signatures, protein signature, and/or other genetic or epigenetic signature based on single cell analyses (e.g., single cell RNA sequencing) or alternatively based on cell population analyses, as is defined herein elsewhere.
  • the invention relates to gene signatures, protein signature, and/or other genetic or epigenetic signature of particular tumor cell subpopulations, as defined herein elsewhere.
  • the invention hereto also further relates to particular tumor cell subpopulations, which may be identified based on the methods according to the invention as discussed herein; as well as methods to obtain such cell (sub)populations and screening methods to identify agents capable of inducing or suppressing particular tumor cell (sub)populations.
  • the invention further relates to various uses of the gene signatures, protein signature, and/or other genetic or epigenetic signature as defined herein, as well as various uses of the tumor cells or tumor cell (sub)populations as defined herein.
  • Particular advantageous uses include methods for identifying agents capable of inducing or suppressing particular tumor cell (sub)populations based on the gene signatures, protein signature, and/or other genetic or epigenetic signature as defined herein.
  • the invention further relates to agents capable of inducing or suppressing particular tumor cell (sub)populations based on the gene signatures, protein signature, and/or other genetic or epigenetic signature as defined herein, as well as their use for modulating, such as inducing or repressing, a particular gene signature, protein signature, and/or other genetic or epigenetic signature.
  • genes in one population of cells may be activated or suppressed in order to affect the cells of another population.
  • modulating, such as inducing or repressing, a particular a particular gene signature, protein signature, and/or other genetic or epigenetic signature may modify overall tumor composition, such as tumor cell composition, such as tumor cell subpopulation composition or distribution, or functionality.
  • the signature genes of the present invention were discovered by analysis of expression profiles of single-cells within a population of cells from freshly isolated tumors, thus allowing the discovery of novel cell subtypes that were previously invisible in a population of cells within a tumor.
  • the presence of subtypes may be determined by subtype specific signature genes.
  • the presence of these specific cell types may be determined by applying the signature genes to bulk sequencing data in a patient tumor.
  • a tumor is a conglomeration of many cells that make up a tumor microenvironment, whereby the cells communicate and affect each other in specific ways.
  • specific cell types within this microenvironment may express signature genes specific for this microenvironment.
  • the signature genes of the present invention may be microenvironment specific, such as their expression in a tumor.
  • signature genes determined in single cells that originated in a tumor are specific to other tumors.
  • a combination of cell subtypes in a tumor may indicate an outcome.
  • the signature genes can be used to deconvolute the network of cells present in a tumor based on comparing them to data from bulk analysis of a tumor sample.
  • the presence of specific cells and cell subtypes may be indicative of tumor growth, invasiveness and resistance to treatment.
  • the signature gene may indicate the presence of one particular cell type.
  • the signature genes of the present invention are applied to bulk sequencing data from a tumor sample obtained from a subject, such that information relating to disease outcome and personalized treatments is determined.
  • the novel signature genes are used to detect multiple cell states that occur in a subpopulation of tumor cells that are linked to resistance to targeted therapies and progressive tumor growth.
  • gene program can be used interchangeably with “biological program”, “expression program”, “transcriptional program”, “expression profile”, or “expression program” and may refer to a set of genes that share a role in a biological function (e.g., an activation program, cell differentiation program, proliferation program).
  • Biological programs can include a pattern of gene expression that result in a corresponding physiological event or phenotypic trait.
  • Biological programs can include up to several hundred genes that are expressed in a spatially and temporally controlled fashion. Expression of individual genes can be shared between biological programs.
  • Expression of individual genes can be shared among different single cell types; however, expression of a biological program may be cell type specific or temporally specific (e.g., the biological program is expressed in a cell type at a specific time). Multiple biological programs may include the same gene, reflecting the gene’s roles in different processes. Expression of a biological program may be regulated by a master switch, such as a nuclear receptor or transcription factor. As used herein, the term “topic” refers to a biological program. The biological program can be modeled as a distribution over expressed genes.One method to identify cell programs is non-negative matrix factorization (NMF) (see, e.g., Lee DD and Seung HS, Learning the parts of objects by non-negative matrix factorization, Nature.
  • NMF non-negative matrix factorization
  • Topic modeling is a statistical data mining approach for discovering the abstract topics that explain the words occurring in a collection of text documents. Originally developed to discover key semantic topics reflected by the words used in a corpus of documents (Dumais, S.T., Furnas, G.W., Landauer, T.K., and Harshman, R. (1990). Indexing by Latent Semantic Analysis.
  • topic modeling can be used to explore gene programs (“topics”) in each cell (“document”) based on the distribution of genes (“words”) expressed in the cell.
  • a gene can belong to multiple programs, and its relative relevance in the topic is reflected by a weight.
  • a cell is then represented as a weighted mixture of topics, where the weights reflect the importance of the corresponding gene program in the cell.
  • Topic modeling using LDA has recently been applied to scRNA-seq data (see, e.g., Bielecki, Riesenfeld, Kowalczyk, et al., 2018 Skin inflammation driven by differentiation of quiescent tissue-resident ILCs into a spectrum of pathogenic effectors.
  • the biomarkers, and/or cells may be detected or isolated by immunoassays (described further herein), immunofluorescence (IF), immunohistochemistry (IHC), fluorescence activated cell sorting (FACS), mass spectrometry (MS), mass cytometry (CyTOF), any gene or transcript sequencing method including, but not limited to, RNA-seq, single cell RNA-seq, single nucleus RNA-seq, spatial transcriptomics, spatial proteomics, quantitative RT-PCR, single cell qPCR, FISH, RNA-FISH, MERFISH (multiplex (in situ) RNA FISH), Nanostring, in situ hybridization (ISH), CRISPR-effector system mediated screening assay (e.g.
  • SHERLOCK assay compressed sensing, and any combination thereof.
  • Other methods including absorbance assays and colorimetric assays are known in the art and may be used herein, detection may comprise primers and/or probes or fluorescently bar-coded oligonucleotide probes for hybridization to RNA (see e.g., Geiss GK, et al., Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol. 2008 Mar;26(3):317-25).
  • Other methods include microfluidics/nanotechnology sensors, and aptamer capture assay.
  • the present invention also may comprise a kit with a detection reagent that binds to one or more biomarkers or can be used to detect one or more biomarkers.
  • the methods of detection are non-invasive.
  • non-invasive refers to a non-surgical procedure (i.e., no cutting or entering a body part using medical instruments).
  • a blood test taking a stool sample, or taking a sample with a swab is considered non-invasive herein.
  • biomarkers are measured in the blood, serum or plasma of the subject.
  • biomarkers are measured in the stool of the subject.
  • blood includes any blood fraction, for example serum, that can be analyzed according to the methods described herein. Serum is a standard blood fraction that can be tested.
  • any appropriate blood fraction can be tested to determine blood levels and that data can be reported as a value present in that fraction.
  • the blood levels of a marker can be presented as pg/mL serum.
  • the biomarker is detected as a cell free transcript present in a blood sample (see, e.g., Larson MH, Pan W, Kim HI, et al.
  • a comprehensive characterization of the cell-free transcriptome reveals tissue- and subtype-specific biomarkers for cancer detection. Nat Commun. 2021; 12(1):2357; Koh W, Pan W, Gawad C, et al. Noninvasive in vivo monitoring of tissue-specific global gene expression in humans. Proc Natl Acad Sci U S A. 2014; 11 l(20):7361-7366; Qin J, Williams TL, Fernando MR.
  • a novel blood collection device stabilizes cell-free RNA in blood during sample shipping and storage.
  • the biomarker is detected as a secreted protein present in a blood sample (see, e.g., Cui J, Liu Q, Puett D, Xu Y. Computational prediction of human proteins that can be secreted into the bloodstream. Bioinformatics. 2008;24(20):2370-2375; Jeun M, Lee HJ, Park S, et al . A Novel Blood-Based Colorectal Cancer Diagnostic Technology Using Electrical Detection of Colon Cancer Secreted Protein-2. Adv Sci (Weinh). 2019;6(l 1): 1802115; and US patent application publication US20050069963A1).
  • the biomarker is detected as a secreted protein present in a stool sample (e.g., mass spectrometry proteomics on stool samples) (see, e.g., Komor MA, Bosch LJ, Coupe VM, et al. Proteins in stool as biomarkers for non-invasive detection of colorectal adenomas with high risk of progression. J Pathol. 2020;250(3):288-298; and Wang HP, Wang YY, Pan J, Cen R, Cai YK. Evaluation of specific fecal protein biochips for the diagnosis of colorectal cancer. World J Gastroenterol. 2014;20(5): 1332-1339).
  • a stool sample e.g., mass spectrometry proteomics on stool samples
  • the biomarker is detected in circulating tumor cells present in a blood sample.
  • circulating tumor cell or “CTC” refers to tumor cells which are shed from a tumor and present in the blood, i.e., in circulation.
  • Cell markers e.g., marker genes
  • a CTC can be a pancreatic cancer CTC (see, e.g., US Patent US 10900083B2).
  • the biomarker is detected in exosomes or ribonucleoprotein (RNP) complexes present in a blood sample.
  • exosomes refer to extracellular vesicles, which are generally of between 30 and 200 nm, for example in the range of 50-100 nm in size.
  • the extracellular vesicles can be in the range of 20-300 nm in size, for example 30-250 nm in size, for example 50-200 nm in size.
  • the extracellular vesicles are defined by a lipidic bilayer membrane.
  • Exosomes may be purified as described (see, e.g., US Patent application publication US20180066307A1; and US Patent application publication US20190285618A1).
  • RNPs ribonucleoprotein particles
  • RNA including proteincoding mRNAs and non-protein-coding RNAs
  • RBPs RNA-binding proteins
  • Immunoassay methods are based on the reaction of an antibody to its corresponding target or analyte and can detect the analyte in a sample depending on the specific assay format.
  • monoclonal antibodies are often used because of their specific epitope recognition.
  • Polyclonal antibodies have also been successfully used in various immunoassays because of their increased affinity for the target as compared to monoclonal antibodies
  • Immunoassays have been designed for use with a wide range of biological sample matrices. Immunoassay formats have been designed to provide qualitative, semi-quantitative, and quantitative results. In example embodiments, immunoassays can be used for non-invasive detection.
  • Quantitative results may be generated through the use of a standard curve created with known concentrations of the specific analyte to be detected.
  • the response or signal from an unknown sample is plotted onto the standard curve, and a quantity or value corresponding to the target in the unknown sample is established.
  • ELISA or EIA can be quantitative for the detection of an analyte/biomarker. This method relies on attachment of a label to either the analyte or the antibody and the label component includes, either directly or indirectly, an enzyme. ELISA tests may be formatted for direct, indirect, competitive, or sandwich detection of the analyte. Other methods rely on labels such as, for example, radioisotopes (I 125 ) or fluorescence.
  • Additional techniques include, for example, agglutination, nephelometry, turbidimetry, Western blot, immunoprecipitation, immunocytochemistry, immunohistochemistry, flow cytometry, Luminex assay, and others (see ImmunoAssay: A Practical Guide, edited by Brian Law, published by Taylor & Francis, Ltd., 2005 edition).
  • Other advanced techniques such as nonradioactive in situ hybridization (ISH), can be combined with immunochemistry to identify specific DNA or RNA molecules with fluorescent probes or tags that can be used for immunofluorescence and enzyme-linked fluorescence amplification.
  • ISH nonradioactive in situ hybridization
  • Exemplary assay formats include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluorescent, chemiluminescence, and fluorescence resonance energy transfer (FRET) or time resolved-FRET (TR-FRET) immunoassays.
  • ELISA enzyme-linked immunosorbent assay
  • FRET fluorescence resonance energy transfer
  • TR-FRET time resolved-FRET
  • biomarkers include biomarker immunoprecipitation followed by quantitative methods that allow size and peptide level discrimination, such as gel electrophoresis, capillary electrophoresis, planar electrochromatography, and the like.
  • Exemplary assay formats also include ELISA and Luminex LabMAP immunoassays.
  • the ELISA and Luminex LabMAP immunoassays are examples of sandwich assays.
  • sandwich assay refers to an immunoassay where the antigen is sandwiched between two binding reagents, which are typically antibodies. The first binding reagent/antibody being attached to a surface and the second binding reagent/antibody comprising a detectable group.
  • detectable groups include, for example and without limitation: fluorochromes, enzymes, epitopes for binding a second binding reagent (for example, when the second binding reagent/antibody is a mouse antibody, which is detected by a fluorescently-labeled anti-mouse antibody), for example an antigen or a member of a binding pair, such as biotin.
  • the surface may be a planar surface, such as in the case of a typical grid-type array (for example, but without limitation, 96-well plates and planar microarrays), as described herein, or a non-planar surface, as with coated bead array technologies, where each “species” of bead is labeled with, for example, a fluorochrome (such as the Luminex technology described herein and in U.S. Pat. Nos. 6,599,331, 6,592,822 and 6,268,222), or quantum dot technology (for example, as described in U.S. Pat. No. 6,306,610).
  • a fluorochrome such as the Luminex technology described herein and in U.S. Pat. Nos. 6,599,331, 6,592,822 and 6,268,222
  • quantum dot technology for example, as described in U.S. Pat. No. 6,306,610.
  • the system incorporates polystyrene microspheres that are dyed internally with two spectrally distinct fluorochromes. Using precise ratios of these fluorochromes, an array is created consisting of 100 different microsphere sets with specific spectral addresses. Each microsphere set can possess a different reactant on its surface. Because microsphere sets can be distinguished by their spectral addresses, they can be combined, allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel. A third fluorochrome coupled to a reporter molecule quantifies the biomolecular interaction that has occurred at the microsphere surface.
  • Microspheres are interrogated individually in a rapidly flowing fluid stream as they pass by two separate lasers in the Luminex analyzer.
  • High-speed digital signal processing classifies the microsphere based on its spectral address and quantifies the reaction on the surface in a few seconds per sample.
  • the bead-type immunoassays are preferable for a number of reasons. As compared to ELISAs, costs and throughput are far superior.
  • the beads are far superior for quantitation purposes because the bead technology does not require pre-processing or titering of the plasma or serum sample, with its inherent difficulties in reproducibility, cost and technician time. For this reason, although other immunoassays, such as, without limitation, ELISA, RIA and antibody microarray technologies, are capable of use in the context of the present invention, but they are not preferred.
  • Methods of detecting and/or quantifying a detectable label or signal generating material depend on the nature of the label.
  • the products of reactions catalyzed by appropriate enzymes can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light.
  • detectors suitable for detecting such detectable labels include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.
  • Any of the methods for detection can be performed in any format that allows for any suitable preparation, processing, and analysis of the reactions. This can be, for example, in multiwell assay plates (e.g., 96 wells or 384 wells) or using any suitable array or microarray. Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting a detectable label.
  • multiwell assay plates e.g., 96 wells or 384 wells
  • Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting a detectable label.
  • Histology also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope. Although one may divide microscopic anatomy into organology, the study of organs, histology, the study of tissues, and cytology, the study of cells, modern usage places these topics under the field of histology. In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. Biological tissue has little inherent contrast in either the light or electron microscope. Staining is employed to give both contrast to the tissue as well as highlighting particular features of interest.
  • the term histochemistry is used.
  • Antibodies can be used to specifically visualize proteins, carbohydrates, and lipids. This process is called immunohistochemistry, or when the stain is a fluorescent molecule, immunofluorescence. This technique has greatly increased the ability to identify categories of cells under a microscope.
  • Other advanced techniques such as nonradioactive in situ hybridization (ISH), can be combined with immunochemistry to identify specific DNA or RNA molecules with fluorescent probes or tags that can be used for immunofluorescence and enzyme-linked fluorescence amplification.
  • ISH nonradioactive in situ hybridization
  • biomarkers are detected using a spatial detection method, in particular, for identifying subtypes in a tissue sample or for identifying genes or polypeptides for use in screening methods described further herein.
  • An example spatial detection platform includes the digital spatial profiler (DSP), GeoMx DSP, which is built on Nanostring’s digital molecular barcoding core technology and is further extended by linking the target complementary sequence probe to a unique DSP barcode through a UV cleavable linker (see, e.g., Li X, Wang CY. From bulk, single-cell to spatial RNA sequencing. Int J Oral Sci. 2021 ; 13(1):36).
  • a pool of such barcode-labeled probes is hybridized to mRNA targets that are released from fresh or FFPE tissue sections mounted on a glass slide.
  • the slide is also stained using fluorescent markers (i.e., fluorescently conjugated antibodies) and imaged to establish tissue “geography” using the GeoMx DSP instrument.
  • fluorescent markers i.e., fluorescently conjugated antibodies
  • ROIs regions-of-interest
  • the DSP barcodes are released via UV exposure and collected from the ROIs on the tissue.
  • These barcodes are sequenced through standard NGS procedures. The identity and number of sequenced barcodes can be translated into specific mRNA molecules and their abundance, respectively, and then mapped to the tissue section based on their geographic location.
  • the DSP barcode can also be linked to antibodies to detect proteins.
  • An example spatial detection platform includes the CosMx Spatial Molecular Imager (Nanostring) platform, which enables high-plex (-1,000 genes) spatial transcriptomics and proteomics at single cell and subcellular resolution (see, e.g., He, et al., High-plex Multi omic Analysis in FFPE at Subcellular Level by Spatial Molecular Imaging, bioRxiv 2021.11.03.467020).
  • Other spatial detection methods or platform applicable to the present invention have been described (see, e.g., Li X, Wang CY. From bulk, single-cell to spatial RNA sequencing. Int J Oral Sci. 2021; 13(1):36. Published 2021 Nov 15. doi: 10.1038/s41368-021- 00146-0).
  • the spatial data used in the present invention can be any spatial data.
  • Methods of generating spatial data of varying resolution are known in the art, for example, ISS (Ke, R. et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nat. Methods 10, 857-860 (2013)), MERFISH (Chen, K. H., Boettiger, A. N., Moffitt, J. R , Wang, S. & Zhuang, X. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, (2015)), smFISH (Codeluppi, S. et al. Spatial organization of the somatosensory cortex revealed by cyclic smFISH.
  • proteomics and spatial patterning using antenna networks is used to spatially map a tissue specimen and this data can be further used to align single cell data to a larger tissue specimen (see, e.g., US20190285644A1).
  • the spatial data can be immunohistochemistry data or immunofluorescence data. MS methods
  • Biomarker detection may also be evaluated using mass spectrometry (MS) methods.
  • MS is used to detect biomarkers in non-invasive samples (e.g., blood or stool).
  • a variety of configurations of mass spectrometers can be used to detect biomarker values.
  • Several types of mass spectrometers are available or can be produced with various configurations.
  • a mass spectrometer has the following major components: a sample inlet, an ion source, a mass analyzer, a detector, a vacuum system, and instrument-control system, and a data system. Difference in the sample inlet, ion source, and mass analyzer generally define the type of instrument and its capabilities.
  • an inlet can be a capillary-column liquid chromatography source or can be a direct probe or stage such as used in matrix-assisted laser desorption.
  • Common ion sources are, for example, electrospray, including nanospray and microspray or matrix-assisted laser desorption.
  • Common mass analyzers include a quadrupole mass filter, ion trap mass analyzer and time-of-flight mass analyzer. Additional mass spectrometry methods are well known in the art (see Burlingame et al., Anal. Chem. 70:647 R-716R (1998); Kinter and Sherman, New York (2000)).
  • Protein biomarkers and biomarker values can be detected and measured by any of the following: electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), tandem time-of-flight (TOF/TOF) technology, called ultraflex III TOF/TOF, atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI- MS/MS, APCI-(MS).sup.N, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS
  • Labeling methods include, but are not limited to, isobaric tag for relative and absolute quantitation (iTRAQ) and stable isotope labeling with amino acids in cell culture (SILAC).
  • Capture reagents used to selectively enrich samples for candidate biomarker proteins prior to mass spectroscopic analysis include but are not limited to aptamers, antibodies, nucleic acid probes, chimeras, small molecules, an F(ab')2 fragment, a single chain antibody fragment, an Fv fragment, a single chain Fv fragment, a nucleic acid, a lectin, a ligand-binding receptor, affybodies, nanobodies, ankyrins, domain antibodies, alternative antibody scaffolds (e.g., diabodies etc.) imprinted polymers, avimers, peptidomimetics, peptoids, peptide nucleic acids, threose nucleic acid, a hormone receptor, a cytokine receptor, and synthetic receptors, and modifications and fragments of these.
  • aptamers antibodies, nucleic acid probes, chimeras, small molecules, an F(ab')2 fragment, a single chain antibody fragment, an Fv fragment,
  • the invention involves single cell RNA sequencing (see, e.g., Kalisky, T., Blainey, P. & Quake, S. R. Genomic Analysis at the Single-Cell Level. Annual review of genetics 45, 431-445, (2011); Kalisky, T. & Quake, S. R. Single-cell genomics. Nature Methods 8, 311-314 (2011); Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Research, (2011); Tang, F. et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nature Protocols 5, 516-535, (2010); Tang, F. et al.
  • the invention involves plate based single cell RNA sequencing (see, e.g., Picelli, S. et al., 2014, “Full-length RNA-seq from single cells using Smart-seq2” Nature protocols 9, 171-181, doi: 10.1038/nprot.2014.006).
  • the invention involves high-throughput single-cell RNA-seq.
  • Macosko et al. 2015, “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets” Cell 161, 1202-1214; International patent application number PCT/US2015/049178, published as W02016/040476 on March 17, 2016; Klein et al., 2015, “Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells” Cell 161, 1187-1201; International patent application number PCT/US2016/027734, published as WO2016168584A1 on October 20, 2016; Zheng, et al., 2016, “Haplotyping germline and cancer genomes with high-throughput linked-read sequencing” Nature Biotechnology 34, 303-311; Zheng, et al., 2017, “Massively parallel digital transcriptional profiling of single cells” Nat.
  • the invention involves single nucleus RNA sequencing.
  • Swiech et al., 2014 “In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9” Nature Biotechnology Vol. 33, pp. 102-106; Habib et al., 2016, “Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons” Science, Vol. 353, Issue 6302, pp. 925-928; Habib et al., 2017, “Massively parallel single-nucleus RNA-seq with DroNc-seq” Nat Methods. 2017 Oct;14(10):955-958; International Patent Application No.
  • Such applications are hybridization assays in which a nucleic acid that displays "probe" nucleic acids for each of the genes to be assayed/profiled in the profile to be generated is employed.
  • a sample of target nucleic acids is first prepared from the initial nucleic acid sample being assayed, where preparation may include labeling of the target nucleic acids with a label, e.g., a member of a signal producing system.
  • a label e.g., a member of a signal producing system.
  • the sample is contacted with the array under hybridization conditions, whereby complexes are formed between target nucleic acids that are complementary to probe sequences attached to the array surface.
  • the presence of hybridized complexes is then detected, either qualitatively or quantitatively.
  • an array of "probe" nucleic acids that includes a probe for each of the biomarkers whose expression is being assayed is contacted with target nucleic acids as described above. Contact is carried out under hybridization conditions, e.g., stringent hybridization conditions as described above, and unbound nucleic acid is then removed.
  • hybridization conditions e.g., stringent hybridization conditions as described above
  • unbound nucleic acid is then removed.
  • the resultant pattern of hybridized nucleic acids provides information regarding expression for each of the biomarkers that have been probed, where the expression information is in terms of whether or not the gene is expressed and, typically, at what level, where the expression data, i.e., expression profile, may be both qualitative and quantitative.
  • Optimal hybridization conditions will depend on the length (e.g., oligomer vs. polynucleotide greater than 200 bases) and type (e.g., RNA, DNA, PNA) of labeled probe and immobilized polynucleotide or oligonucleotide.
  • length e.g., oligomer vs. polynucleotide greater than 200 bases
  • type e.g., RNA, DNA, PNA
  • hybridization conditions are hybridization in 5xSSC plus 0.2% SDS at 65C for 4 hours followed by washes at 25 °C in low stringency wash buffer (IxSSC plus 0.2% SDS) followed by 10 minutes at 25°C in high stringency wash buffer (0.1 SSC plus 0.2% SDS) (see Shena etal., Proc. Natl. Acad. Sci. USA, Vol. 93, p. 10614 (1996)).
  • Useful hybridization conditions are also provided in, e.g., Tijessen, Hybridization With Nucleic Acid Probes", Elsevier Science Publishers B.V. (1993) and Kricka, "Nonisotopic DNA Probe Techniques", Academic Press, San Diego, Calif. (1992).
  • a further aspect of the invention relates to a method for identifying an agent capable of modulating a pancreatic cancer tissue system in a subject (i.e., a complex tumor model including malignant cells and tumor microenvironment cells).
  • a subject i.e., a complex tumor model including malignant cells and tumor microenvironment cells.
  • the invention provides for identifying an agent capable of modulating one or more phenotypic aspects of a cell or complex cell population (e.g., multicellular systems, such as, organoid, tissue explant, or organ on a chip) and translating the agent to an in vivo system (e.g., therapeutic agents).
  • the biomarkers for cancer subtypes described herein are detected.
  • modulating agents capable of maintaining a cancer subtype or shifting a complex system to a specific subtype are identified.
  • a cellular system that recapitulates an in vivo tumor can be used to screen for vulnerability to agents (e.g., therapeutic agents).
  • agents e.g., therapeutic agents.
  • Non-limiting functional measures that can be assayed include measures of tumor organoids, such as secreted growth factors (tumor microenvironment), released antigens, and metabolites.
  • tumor organoids such as secreted growth factors (tumor microenvironment), released antigens, and metabolites.
  • Non-limiting examples also include inducing tumor cell differentiation.
  • the pancreatic cancer tissue system (cell line) is of a specific subtype observed in vivo (e.g., NRP, SBM, classical).
  • the utility of using complex cell systems to identify modulators has been shown previously, as described further herein.
  • the complex cell system can be any system known in the art (e.g., such as, organoid, tissue explant, organ on a chip) and any detectable phenotype can be screened.
  • the pancreatic cancer tissue system is grown in the presence of growth factors or cells that are observed to be present in spatial proximity to a subtype in vivo.
  • the pancreatic cell line is grown in vitro in media comprising one or more growth factors selected from TFF1, MUC16, REGIB, NPNT, MAPT, HDGF, HCRT, LAMA5, SPON2, NELL2, and EREG; or one or more cells expressing a receptor selected from TMPRSS2, NRP2, IL1R1, and PVR.
  • growth factors selected from TFF1, MUC16, REGIB, NPNT, MAPT, HDGF, HCRT, LAMA5, SPON2, NELL2, and EREG
  • 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more growth factors are added to the pancreatic cancer tissue system, such as spatially identified growth factors.
  • a framework utilizing complex cellular models can be used to identify translatable tissue-modifying small molecules.
  • the framework can include: 1) choose a specific tumor physiological process that is well-modeled by an organoid or multicellular system and perform a phenotypic screen for marker(s) of the desired effect; 2) prioritize lead compound(s) through a rigorous statistical approach and validate compound(s) in orthogonal assays; 3) explore compound-mediated biology in the model with a high-content assay (e.g., single-cell RNA-seq, spatial assays) to examine putative mechanism of action; and, 4) where cellular mechanisms dictate potential for translation, test select compound(s) in vivo to validate the intended effect.
  • candidate agents are assayed using more than one multicellular platform described herein.
  • the pancreatic cancer tissue system is an organoid system (see, e.g., Yin X, Mead BE, Safaee H, Langer R, Karp JM, Levy 0. Engineering Stem Cell Organoids. Cell Stem Cell. 2016; 18(l):25-38).
  • organoid refers to a three-dimensional ex vivo tissue culture, cell cluster, or aggregate grown from embryonic stem cells, induced pluripotent stern cells or tissue-resident progenitor cells that resembles an organ, or part of an organ, and possesses cell types relevant to that particular organ.
  • Organoid systems have been described previously, for example, for brain, retinal, stomach, lung, thyroid, small intestine, colon, liver, kidney, pancreas, prostate, mammary gland, fallopian tube, taste buds, salivary glands, and esophagus (see, e.g., Clevers, Modeling Development and Disease with Organoids, Cell. 2016 Jun 16; 165(7): 1586-1597).
  • Tumor organoid systems have also been described (see, e.g., Porter, R.J., Murray, G.I. & McLean, M.H. Current concepts in tumour-derived organoids. Br J Cancer 123, 1209-1218 (2020). doi.org/10.1038/s41416-020-0993-5).
  • Organoids develop by selforganization, and can accurately represent the diverse genetic, cellular and pathophysiological hallmarks of cancer. Id. In addition, co-culture methods and the ability to genetically manipulate these organoids have widened their utility in cancer research (e.g., co-culture of cancer organoids with immune cells). Id.
  • the pancreatic cancer tissue system is an organ-on-chip platform.
  • Organ-on-a-chip technology refers to a multichannel microfluidic perfusion culture system, made from glass, plastic or a flexible polymer, that is lined with living human cells (see, e.g., Sontheimer-Phelps, A., Hassell, B. A. & Ingber, D. E. Modelling cancer in microfluidic human organs-on-chips. Nat. Rev. Cancer 19, 65-81 (2019); and Wu, Q., Liu, J., Wang, X. et al. Organ-on-a-chip: recent breakthroughs and future prospects. BioMed Eng OnLine 19, 9 (2020)).
  • This system allows more accurate modelling of organ-system physiology: for example, it facilitates the establishment of tissue-tissue interfaces, has separate vascular, extracellular and parenchymal compartments and allows for physiologically representative co-culture with TME and immune cells (see, e.g., Ingber, D. E. Developmentally inspired human ‘organs on chips’. Development 145, pii:devl56125 (2016)).
  • High-throughput organ-on-chip platforms applicable to the present invention have been described (see, e.g., Azizgolshani H, Coppeta JR, Vedula EM, et al. High-throughput organ-on-chip platform with integrated programmable fluid flow and realtime sensing for complex tissue models in drug development workflows. Lab Chip.
  • the pancreatic cancer tissue system is a tissue system or tissue explant (see, e.g., Ghosh S, Prasad M, Kundu K, et al. Tumor Tissue Explant Culture of Patient- Derived Xenograft as Potential Prioritization Tool for Targeted Therapy. Front Oncol. 2019;9: 17; Neil JE, Brown MB, Williams AC. Human skin explant model for the investigation of topical therapeutics. Sci Rep. 2020; 10(l):21192; and Grivel JC, Margolis L. Use of human tissue explants to study human infectious agents. Nat Protoc. 2009;4(2):256-269).
  • tissues are obtained from a subject, cut into individual explants, and transferred to tissue culture plates or culture slides.
  • patient derived xenografts PDXs
  • tissues are dissected into small blocks or biopsies and cultured at the liquid-air interface on collagen rafts.
  • high throughput platforms or formats are used.
  • high throughput format refers to a format where a complex cellular system can be grown in discrete volumes or wells that are amenable to screening with existing automation equipment (e.g., from 24 well tissue culture plates to 384 well) or any method that reduces the complexity in handling and growing the models.
  • existing automation equipment e.g., from 24 well tissue culture plates to 384 well
  • Non-limiting examples of high throughput screening methods applicable to present invention have been described (see, e.g., Langhans, S. A. Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning. Front. Pharmacol. 9, 1- 14 (2018); and Gunasekara, D. B. etal. Development of Arrayed Colonic Organoids for Screening of Secretagogues Associated with Enterotoxins. Anal. Chem. 90, 1941-1950 (2018)).
  • the method comprises detecting modulation of one or more phenotypic aspects of the cell or complex cell population by the candidate agent, thereby identifying the agent.
  • the phenotypic aspects of the cell or complex cell population that is modulated may be a gene signature or biological program specific to a cell type or cell phenotype or phenotype specific to a population of cells (e.g., NRP, SBM, classical subtype).
  • steps can include administering candidate modulating agents to cells, detecting identified cell (sub)populations for changes in signatures, or identifying relative changes in cell (sub) populations which may comprise detecting relative abundance of particular gene signatures.
  • modulate broadly denotes a qualitative and/or quantitative alteration, change or variation in that which is being modulated. Where modulation can be assessed quantitatively - for example, where modulation comprises or consists of a change in a quantifiable variable such as a quantifiable property of a cell or where a quantifiable variable provides a suitable surrogate for the modulation - modulation specifically encompasses both increase (e.g., activation) or decrease (e.g., inhibition) in the measured variable.
  • the term encompasses any extent of such modulation, e.g., any extent of such increase or decrease, and may more particularly refer to statistically significant increase or decrease in the measured variable.
  • modulation may encompass an increase in the value of the measured variable by at least about 10%, e.g., by at least about 20%, preferably by at least about 30%, e.g., by at least about 40%, more preferably by at least about 50%, e.g., by at least about 75%, even more preferably by at least about 100%, e.g., by at least about 150%, 200%, 250%, 300%, 400% or by at least about 500%, compared to a reference situation without said modulation; or modulation may encompass a decrease or reduction in the value of the measured variable by at least about 10%, e.g., by at least about 20%, by at least about 30%, e.g., by at least about 40%, by at least about 50%, e.g., by at least about 60%, by at least about 70%, e.g., by at least about 80%, by at least about 90%, e.g., by at least about 95%, such as by at least about 96%, 97%, 98%
  • agent broadly encompasses any condition, substance or agent capable of modulating one or more phenotypic aspects of a cell or cell population as disclosed herein. Such conditions, substances or agents may be of physical, chemical, biochemical and/or biological nature.
  • candidate agent refers to any condition, substance or agent that is being examined for the ability to modulate one or more phenotypic aspects of a cell or cell population as disclosed herein in a method comprising applying the candidate agent to the cell or cell population (e.g., exposing the cell or cell population to the candidate agent or contacting the cell or cell population with the candidate agent) and observing whether the desired modulation takes place.
  • Agents may include any potential class of biologically active conditions, substances or agents, such as for instance antibodies, proteins, peptides, nucleic acids, oligonucleotides, small molecules, genetic modifying agents or combinations thereof, as described herein.
  • the methods of phenotypic analysis can be utilized for evaluating environmental stress and/or state, for screening of chemical libraries, and to screen or identify structural, syntenic, genomic, and/or organism and species variations.
  • a culture of cells can be exposed to an environmental stress, such as but not limited to heat shock, osmolarity, hypoxia, cold, oxidative stress, radiation, starvation, a chemical (for example a therapeutic agent or potential therapeutic agent) and the like.
  • a representative sample can be subjected to analysis, for example at various time points, and compared to a control, such as a sample from an organism or cell, for example a cell from an organism, or a standard value.
  • screening of test agents involves testing a combinatorial library containing a large number of potential modulator compounds.
  • a combinatorial chemical library may be a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library, is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (for example the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • screening of test agents involves testing a library of known compounds, such as an FDA approved library.
  • the present invention provides for gene signature screening.
  • signature screening was introduced by Stegmaier et al. (Gene express! on -based high-throughput screening (GE-HTS) and application to leukemia differentiation. Nature Genet. 36, 257-263 (2004)), who realized that if a gene-expression signature was the proxy for a phenotype of interest, it could be used to find small molecules that effect that phenotype without knowledge of a validated drug target.
  • the signatures or biological programs of the present invention may be used to screen for drugs that reduce the signature or biological program in cells as described herein.
  • the signature or biological program may be used for GE-HTS.
  • pharmacological screens may be used to identify drugs that are selectively toxic to cells having a signature.
  • the Connectivity Map is a collection of genome-wide transcriptional expression data from cultured human cells treated with bioactive small molecules and simple pattern-matching algorithms that together enable the discovery of functional connections between drugs, genes and diseases through the transitory feature of common gene-expression changes (see, Lamb et al., The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease. Science 29 Sep 2006: Vol. 313, Issue 5795, pp. 1929-1935, DOI: 10.1126/science.1132939; and Lamb, J., The Connectivity Map: a new tool for biomedical research. Nature Reviews Cancer January 2007: Vol. 7, pp. 54-60).
  • Cmap can be used to screen for small molecules capable of modulating a signature or biological program of the present invention in silico.
  • Example 1 - Cancer types that show NRP signal [0409] Applicants identified the NRP subtype in pancreatic cancer expressing a signature enriched after treatment with chemotherapy and radiation (CRT).
  • the NRP subtype can be found in other treatment resistant cancers. For example, esophagus, colon, pancreas, and lung cancers all share cell surface markers associated with the NRP subtype (Fig. 1).
  • Non-invasive profiling of transcripts or proteins prior to, during and after treatment can inform prognoses and treatment strategies aimed at transcriptional subtypes, particularly for a) patients who do not receive resections or biopsies and b) when routine monitoring of disease progression is desired.
  • the NRP, SBM and classical transcriptional programs are each associated with distinct expression patterns, many of which overlap with genes encoding secreted proteins (Fig. 2). Applicants determined that many of these secreted proteins are predicted to be detectable in blood as well as the digestive tract/stool (Fig. 3).
  • Genes encoding secreted proteins in these respective program signatures and the biological sample they can be detected in include, but are not limited to:
  • NRP PDGFD, C6 (blood), CRISP3, RELN, SERPINA6 (blood), CRP (blood), FGG (blood), APCS (blood), CFH (blood), HABP2 (blood), KLKB1 (blood), REGIA (stool), CRISP3, SPP1, SBM: COL17A1, MUC16, FGF19 (blood), IGF2 (blood), NPC2 (blood), SAA1 (blood), SRGN (blood), CIS (blood), CST4 (stool),
  • NRP C7, HDGF, MAPT, REGIB, SERPINA4, PVR, CRISP3, DKK4, DPP7, NPNT, TAFA1 SBM: SFN, COL17A1, FGFBP1, MUC16 (CA-125), HDGF, COP A, CFB
  • HDGF is a general treatment resistant marker (see, e.g., Chen YT, et al. Hepatoma- derived growth factor supports the antiapoptosis and profibrosis of pancreatic stellate cells. Cancer Lett. 2019;457: 180-190).
  • cfRNA cell-free RNA
  • cfRNA cell-free RNA
  • Example 3 Cell surface targets for therapeutics and circulating tumor cell (CTC) detection
  • Pancreatic cancer development is associated with dramatic rewiring of the cell surfaceome, spawning highly-expressed tumor specific antigens that are relatively lowly expressed in non-malignant ‘normal’ tissues.
  • Transcriptional subtypes are associated with distinct cell surface profiles. These present an opportunity for therapeutics and diagnostics leveraging the specificity of antibody -based approaches.
  • ADCs antibody-drug conjugates
  • CTCs circulating tumor cells
  • Applicants have performed comprehensive computational analyses of whole transcriptome and proteomic datasets for pancreatic cancer and normal tissues to determine cancer- and subtype-specific cell surface targets/markers with enhanced expression relative to normal, toxicity-prone tissues (Fig. 5). These include the following:
  • NRP TM4SF4, CNTN4, NRXN3, SLC4A4, CSMD2, DSCAML1, SLC3A1, TRPV6, ABCB1, NLGN4Y, SCTR,
  • SBM MSLN, GPR87, SLC2A1, LY6D, PSCA,
  • Applicants can also target cell surface proteins which are involved in tumor-stromal interactions.
  • Pancreatic cancer is distinct from most other cancer types in that the microenvironment exhibits a strong desmoplastic reaction and is therefore deeply fibrotic.
  • Malignant cells in PDAC have been described as ‘tumor islands’ within a ‘sea of CAFs.’
  • the PDAC TME is immunosuppressive and anti-cancer immunomodulatory strategies which have been successful in other cancer types have not been broadly applicable to this disease.
  • the example receptor-ligand pairs shown include: Epithelial and CAF: SEMA7A and ITGB1, LAMA5 and ITGB1, AGTRAP and RACK1, ILIA and IL1R1, FGF21 and EPHA2, CALR and SCARF1, EFNB2 and RHBDL2, SEMA3A and NRP2, IGF2 and IGF1R, LAMA5 and SDC1, TNF and TNFRSF21, GDNF and.
  • Applicants can also use refined receptor-ligand interactions using the CosMx Spatial Molecular Imager (Nanostring) platform (Fig. 8), which enables high-plex (—1,000 genes) spatial transcriptomics at single cell and subcellular resolution (see, e.g., He, et al., High-plex Multiomic Analysis in FFPE at Subcellular Level by Spatial Molecular Imaging, bioRxiv 2021.11.03.467020).
  • the extent of receptor-ligand interactions between neighboring cells can be directly quantified and used to validate previously -identified candidates.
  • Example 5 Multiplexed RNA ISH or immunofluorescence panel for subtype determination
  • Applicants can use a multiplexed RNA ISH and/or protein panel (up to 9-plex in a single cycle using an instrument such as TissueFAXS) for subtype determination on tissue sections using the following markers, which have been selected based on intrinsic program specificity as well as performance in machine learning subtype classification models.
  • NRP CTNND2, CRP, SERPINA6, NRXN3, PDGFD, C6, RELN, SPP1, GLIS3, NR5A2, DCDC2, DPYD, HNF1B, ZBTB16, KCNJ16, SLC4A4, SBM: S100A2, KRT17, KRT7, TACSTD2, TP63, MSLN, SCEL, C0L17A1, VIM, FN1, CST6, CST4, TUBB,
  • HNF4A HNF4A
  • GATA6 GATA6, TFF1, CEACAM5, TSPAN8, FM05, BTNL8, REG4, SYTL2, CLDN18.
  • Example 6 Ex vivo microenvironment reconstruction using spatial transcriptome analyses
  • a detailed understanding of transcriptional subtypes and their vulnerabilities requires the ability to maintain phenotypes ex vivo and perform manipulations/perturbations in the ex vivo models thereafter.
  • standard 2D and 3D organoid conditions are not perfectly tailored to subtypes and may contain or lack growth factors essential to subtype stability.
  • Applicants propose strategies and present results to reconstruct the native microenvironmental milieu ex vivo leveraging custom spatial transcriptomic techniques (Fig. 12), innovative analyses, and supplementation of media formulations.
  • Figure 13 shows expression of growth factors and receptors in epithelial cells, immune cells and CAFs from PDAC tumors that can be spatially correlated to regions of a tumor specific to a PDAC subtype.
  • Growth factors that can be used to recapitulate the PDAC tumor subtypes ex vivo include TFF1, MUC16, REGIB, NPNT, MAPT, HDGF, HCRT, LAMA5, SPON2, NELL2, and EREG.
  • Receptors on tumor microenvironment cells that can be used to recapitulate the PDAC tumor subtypes ex vivo include TMPRSS2, NRP2, IL1R1, and PVR.
  • cells expressing the endogenous or recombinant receptors can be co-cultured with the tumor subtype models.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne de manière générale des méthodes de détection de cancer réfractaire au traitement et de traitement de celui-ci. Selon des modes de réalisation donnés à titre d'exemple, l'invention concerne des biomarqueurs spécifiques exprimés spécifiquement dans des sous-types de cancer du pancréas. Les biomarqueurs peuvent être utilisés pour le ciblage thérapeutique de cancers du pancréas. Les biomarqueurs peuvent être utilisés pour des méthodes de diagnostic non invasives.
PCT/US2023/067607 2022-05-27 2023-05-30 Traitement et détection de cancers ayant un phénotype de type neuronal progéniteur, squamoïde/basaloïde/mésenchymateux ou classique WO2023230632A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263346670P 2022-05-27 2022-05-27
US63/346,670 2022-05-27

Publications (2)

Publication Number Publication Date
WO2023230632A2 true WO2023230632A2 (fr) 2023-11-30
WO2023230632A3 WO2023230632A3 (fr) 2024-01-18

Family

ID=88920128

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/067607 WO2023230632A2 (fr) 2022-05-27 2023-05-30 Traitement et détection de cancers ayant un phénotype de type neuronal progéniteur, squamoïde/basaloïde/mésenchymateux ou classique

Country Status (1)

Country Link
WO (1) WO2023230632A2 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG11201406020WA (en) * 2012-03-30 2014-10-30 Univ Singapore Dual function markers for diagnostics and therapeutics for upper gastrointestinal tract precancer
WO2018089386A1 (fr) * 2016-11-11 2018-05-17 The Broad Institute, Inc. Modulation de la différenciation, du maintien et/ou de la fonction de cellules épithéliales intestinales par l'action des lymphocytes t
US20240043934A1 (en) * 2020-08-22 2024-02-08 The Broad Institute, Inc. Pancreatic ductal adenocarcinoma signatures and uses thereof

Also Published As

Publication number Publication date
WO2023230632A3 (fr) 2024-01-18

Similar Documents

Publication Publication Date Title
US20210047694A1 (en) Methods for predicting outcomes and treating colorectal cancer using a cell atlas
US20200147210A1 (en) Methods and compositions of use of cd8+ tumor infiltrating lymphocyte subtypes and gene signatures thereof
US20200347456A1 (en) Methods and compositions for detecting and modulating an immunotherapy resistance gene signature in cancer
US20200157633A1 (en) Methods and compositions for detecting and modulating an immunotherapy resistance gene signature in cancer
US20190263912A1 (en) Modulation of intestinal epithelial cell differentiation, maintenance and/or function through t cell action
US20220282333A1 (en) Methods for predicting outcomes of checkpoint inhibition and treatment thereof
US20210040442A1 (en) Modulation of epithelial cell differentiation, maintenance and/or function through t cell action, and markers and methods of use thereof
US20200149009A1 (en) Methods and compositions for modulating cytotoxic lymphocyte activity
US11630103B2 (en) Product and methods useful for modulating and evaluating immune responses
US20210130438A1 (en) Pan-cancer t cell exhaustion genes
US11994512B2 (en) Single-cell genomic methods to generate ex vivo cell systems that recapitulate in vivo biology with improved fidelity
US20210130776A1 (en) Methods and compositions for modulating suppression of lymphocyte activity
US11957695B2 (en) Methods and compositions targeting glucocorticoid signaling for modulating immune responses
US11793787B2 (en) Methods and compositions for enhancing anti-tumor immunity by targeting steroidogenesis
US11981922B2 (en) Methods and compositions for the modulation of cell interactions and signaling in the tumor microenvironment
WO2020191079A1 (fr) Compositions et méthodes de modulation de régulateurs métaboliques de pathogénicité de lymphocytes t
US20240043934A1 (en) Pancreatic ductal adenocarcinoma signatures and uses thereof
US20210263012A1 (en) Methods and compositions for modulating immune responses and lymphocyte activity
US20240156816A1 (en) Methods and compositions for predicting and preventing relapse of acute lymphoblastic leukemia
US20220154282A1 (en) Detection means, compositions and methods for modulating synovial sarcoma cells
US20210347847A1 (en) Therapeutic targeting of malignant cells using tumor markers
US20240108689A1 (en) Modulation of a pathogenic phenotype in th1 cells
WO2022256620A1 (fr) Nouvelles cibles pour améliorer l'immunité antitumorale
US20220033464A1 (en) Methods and compositons for modulations of immune response
US20210358573A1 (en) Molecular spatial mapping of metastatic tumor microenvironment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23812829

Country of ref document: EP

Kind code of ref document: A2