WO2020174472A1 - Cells and methods for improved immunotherapy - Google Patents

Cells and methods for improved immunotherapy Download PDF

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Publication number
WO2020174472A1
WO2020174472A1 PCT/IL2020/050218 IL2020050218W WO2020174472A1 WO 2020174472 A1 WO2020174472 A1 WO 2020174472A1 IL 2020050218 W IL2020050218 W IL 2020050218W WO 2020174472 A1 WO2020174472 A1 WO 2020174472A1
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WIPO (PCT)
Prior art keywords
cell
sh2b3
immune
cells
protein
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PCT/IL2020/050218
Other languages
French (fr)
Inventor
Igal LOURIA-HAYON
Yishai OFRAN
Michal HAYUN
Original Assignee
Rambam Med-Tech Ltd.
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Publication date
Application filed by Rambam Med-Tech Ltd. filed Critical Rambam Med-Tech Ltd.
Priority to EP20763331.4A priority Critical patent/EP3930731A4/en
Publication of WO2020174472A1 publication Critical patent/WO2020174472A1/en
Priority to IL285877A priority patent/IL285877A/en
Priority to US17/412,572 priority patent/US20210386784A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention is in the field of immunotherapy.
  • the present invention provides cells comprising decreased SH2B adapter protein 3 (SH2B3) protein expression and/or function.
  • the present invention further provides methods of treating a disease in a subject comprising administering a cell comprising decreased SH2B3 expression and/or function, and methods of determining suitability to be so treated.
  • a method of treating a disease in a subject in need thereof comprising administering to the subject an immune cell comprising decreased SH2B adapter protein 3 (SH2B3) protein expression and/or function, thereby treating the disease.
  • SH2B3 SH2B adapter protein 3
  • a method of determining suitability of a subject in need thereof to receive a treatment comprising an immune cell comprising decreased SH2B3 protein expression and/or function the method comprising
  • composition comprising an immune cell comprising decreased SH2B adapter protein 3 (SH2B3) protein expression and/or function.
  • SH2B3 SH2B adapter protein 3
  • the disease is an immune-associated disease.
  • the immune-associated disease is selected from cancer, Myelodysplastic syndrome (MDS), bacterial infection, fungal infection, viral infection and parasitic infection.
  • MDS Myelodysplastic syndrome
  • the cell is allogenic to the subject.
  • the method further comprises providing an immune cell from the subject and decreasing SH2B3 protein expression and/or function in the provided cell.
  • the decreased SH2B3 protein expression and/or function comprises decreased SH2B3 mRNA levels, SH2B3 protein levels, SH2B3 protein function, or a combination thereof.
  • the decreased SH2B3 protein expression and/or function comprises expression of an SH2B3 inhibitor molecule.
  • the SH2B3 inhibitor molecule is selected from a small nucleic acid molecule that hybridizes to and inhibits SH2B3 mRNA transcription and/or translation, a small molecule inhibitor of SH2B3 protein, an anti-SH2B3 antibody or antigen binding fragment, and a genome editing agent for mutation of a SH2B3 genetic locus.
  • the small molecule inhibitor, antibody or antigen binding fragment is a SH2B3 antagonist.
  • the small molecule inhibitor, antibody, or antigen binding fragment binds to and occludes at least one of a Pleckstrin Homolog (PH) domain, and a Src Homolog 2 (SH2) domain of SH2B3.
  • PH Pleckstrin Homolog
  • SH2 Src Homolog 2
  • the small nucleic acid molecule that hybridized to and inhibits SH2B3 is an anti-sense RNA. ⁇ 9, ?) ⁇ 2 / LI ⁇ 17,3 ⁇ 4 ⁇ ng to some embodiments, the immune cell is selected P ⁇ T/A 2 'i'L 2 l?-ccl 1 , a natural killer (NK) cell, a neutrophil, a dendritic cell, a myeloid derived suppressor cell and a macrophage.
  • NK natural killer
  • the T-cell is a CD8+ T-cell, CD4+T-cell or both.
  • the immune cell further comprises a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the CAR binds to a target antigen on a cell associated with the disease.
  • the cell is selected from a cancer cell, a bacterial cell, a fungal cell, a parasite cell and a cell infected with a vims.
  • the method further comprises administering an anticancer or anti-pathogen therapeutic treatment.
  • the treatment comprising an immune cell comprising decreased SH2B3 protein expression and/or function is a method of the invention.
  • the assaying chemotaxis is performed ex vivo.
  • the method further comprises treating the subject suitable for treatment by a method of the invention.
  • the decreasing occurs ex vivo.
  • the decreasing comprises introducing an SH2B3 inhibitor molecule into the immune cell.
  • the immune cell is an immune cell to be used for adoptive immunotherapy.
  • the enhancing an immune response comprises at least one of: increasing cytotoxicity of the immune cell and increasing migration of the immune cell.
  • the decreased SH2B3 protein expression and/or function comprises decreased SH2B3 mRNA levels, SH2B3 protein levels, SH2B3 protein function, or a combination thereof.
  • the decreased SH2B3 pro ⁇ T ⁇ 3 ⁇ 4 2 93 ⁇ 4(il3 ⁇ 4?3 ⁇ 4d/or function comprises expression of an SH2B3 inhibitor molecule.
  • the SH2B3 inhibitor molecule is selected from a small nucleic acid molecule that hybridizes to and inhibits SH2B3 mRNA transcription and/or translation, a small molecule inhibitor of SH2B3 protein, an anti-SH2B3 antibody or antigen binding fragment, and a genome editing agent for mutation of SH2B3.
  • the small molecule inhibitor, antibody or antigen binding fragment is a SH2B3 antagonist.
  • the small molecule inhibitor, antibody, or antigen binding fragment binds to and occludes at least one of a Pleckstrin Homolog (PH) domain, and a Src Homolog 2 (SH2) domain of SH2B3.
  • PH Pleckstrin Homolog
  • SH2 Src Homolog 2
  • the small nucleic acid molecule that hybridized to and inhibits SH2B3 is an anti-sense RNA.
  • the immune cell is selected from a T-cell, a B-cell, a natural killer (NK) cell, a neutrophil, a dendritic cell, a myeloid derived suppressor cell and a macrophage.
  • NK natural killer
  • the T-cell is a CD8+ T-cell, CD4+T-cell or both.
  • the cell comprises a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the CAR binds to a target antigen on a cell associated with a disease.
  • the cell associated with a disease is selected from a cancer cell, a bacterial cell, a fungal cell, a parasite cell and a cell infected with a virus.
  • the cell is produced by providing a cell from a subject and decreasing SH2B3 protein expression and/or function in the extracted cell in vitro.
  • FIGS 1A-1R (1A) A representative photograph of subcutaneous tumors growth in WT and Lnk KO mice.
  • C Representative FACS size analyses of single cells from tumors from WT and Lnk KO mice.
  • IE Representative micrographs of tumor interface from WT and Lnk KO mice.
  • IQ A representative bar chart of cytokine levels in the TME as assessed by the Pro teome -Profiler Mouse XL Cytokine Array kit (R&D Systems).
  • Figures 2A-2F (2A) Photographs of immunoblotting of pSTAT3 and STAT3 from lysates of CD8+ splenocytes. (2B) A bar chart quantifying the relative phosphorylation of STAT3 from Figure 2A. (2C) Photographs of immunoblotting of pSTAT3 and STAT3 from lysates of CD8+ splenocytes from tumor bearing mice. (2D) A bar chart quantifying the relative phosphorylation of STAT3 from Figure 2C. (2E) Photographs of immunoblotting of pErk and Erk from lysates of CD8+ splenocytes. (2F) A bar chart quantifying the relative phosphorylation of Erk from Figure 2E.
  • FIGS 3A-C (3A) A bar chart of fold induction of INFy geometric mean fluorescence intensity (gMFI) in CD8 + T-cell populations of WT and Lnk KO naive and tumor bearing mice. The gMFI of activated cells was normalized to the gMFI of non- activated cells and is expressed as fold induction. (3B) Representative histograms of INFy ?n ten s i t y in CD8 + T-cell populations of tumor bearing nE£J(3 ⁇ 43 ⁇ 4! ⁇ / g 3 ⁇ 4?u?/pes. FMO - Fluorescence minus one control.
  • gMFI geometric mean fluorescence intensity
  • 5D Pie graphs of the percent of cells with a given surface marker that also expression CXCR2.
  • 5E A bar graph of the percent of CD8+ infiltrating cells that express CXCR1.
  • FIGS 6A-6B (6A) Representative images of LLC subcutaneous tumors grown in WT host (left) and in Lnk _/ host (right) mice. The image was taken after tumor harvest at day 14 of tumor growth. (6B) A scatter plot of LLC tumor weights from WT and Lnk KO mice.
  • the present invention in some embodiments, provides cells comprising an exogenous protein and decreased SH2B adapter protein 3 (SH2B3) protein expression and/or function.
  • the present invention further concerns pharmaceutical composition comprising those cells as well as a method of treating a disease in a subject comprising administering those cells or compositions.
  • Uses of the cells of the invention, as well as methods of determining suitability to be treated by a method of the invention are also provided.
  • a cell comprising decreased SH2B adapter protein 3 (SH2B3) expression and/or function.
  • SH2B3 SH2B adapter protein 3
  • the cell is a mammalian cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is a mouse cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is from a subject. In some embodiments, ⁇ ,O 3 ⁇ 4 IZ ⁇ 2 i n need of immunotherapy. In some embodiments, th? ⁇ Ju j t3 ⁇ 4? 2 3 ⁇ 4 ⁇ ?°? 3 ⁇ 4 d of adoptive immunotherapy. In some embodiments, the subject comprises healthy immune cells. In some embodiments, the subject comprises immune cells with reduced ability to induce an immune response. In some embodiments, the cells are allogenic to the subject. In some embodiments, the cells are autologous to the subject. In some embodiments, the cells are syngeneic to the subject.
  • the cell is an immune cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is not a hematopoietic cell. In some embodiments, the immune cell is a T-cell. In some embodiments, the T-cell is a CD8 positive T-cell. In some embodiments, the T-cell is a cytotoxic CD8 positive T-cell. In some embodiments, the T-cell is a CD4 positive T-cell. In some embodiments, the T-cell is a CD4 positive helper T-cell. In some embodiments, the immune cell is a cytotoxic immune cell.
  • the T-cell is selected from a CD8 positive and a CD4 positive T-cell. In some embodiments, the T-cell is a CD8 positive T-cell, a CD4 positive T-cell or both. In some embodiments, the immune cell is a B-cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a neutrophil. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the immune cell is a myeloid derived suppressor cell (MDSC).
  • MDSC myeloid derived suppressor cell
  • the cell is selected from a T-cell, a B-cell, an NK cell, a neutrophil, a dendritic cell, an MDSC and a macrophage. In some embodiments, the cell is selected from a T-cell, a B-cell, an NK cell, a neutrophil, a dendritic cell, and a macrophage.
  • SH2B3 is mammalian SH2B3. In some embodiments, SH2B3 is rodent SH2B3. In some embodiments, SH2B3 is mouse SH2B3. In some embodiments, SH2B3 is human SH2B3.
  • mouse SH2B3 is SH2B3 isoform 1. In some embodiments, mouse SH2B3 is SH2B3 isoform 2. In some embodiments, mouse SH2B3 is SH2B3 isoform XI. In some embodiments, mouse SH2B3 comprises and/or consists of an amino acid sequence selected from those provide in RefSeq IDs NP_032533.1, NP_001293055.1, NP_001293056.1, NP_001293057.1 and XP_006530233.1.
  • mouse SH2B3 comprises and/or consists of an amino acid sequence provide in RefSeq IDs NP_032533.1, NP_001293055.1, NP_001293056.1, NP_001293057.1 or XP_006530233.1. Each possibility represents a separate embodiment of the invention.
  • mouse SH2B3 comprises and/or consists of the amino acid sequence provided in RefSeq ID NP_032533.1.
  • mouse SH2B3 comprises and/or consists of the amino WO 2020/174472 PCT/IL2020/05 a02 ⁇ u18umenC cont ⁇ C
  • mouse SH2B3 is encoded by a nucleic acid molecule comprising or consisting of the nucleotide sequence selected from those provided in a RefSeq IDs NM_008507.4, NM 001306126.1, NM 001306127.1, NM_001306128.1 and XM_006530170.3.
  • mouse SH2B3 is encoded by a nucleic acid molecule comprising or consisting of the nucleotide sequence provided in a RefSeq ID NM_008507.4, NM 001306126.1, NM 001306127.1, NM_001306128.1 or
  • mouse SH2B3 is encoded by a nucleic acid molecule that comprises and/or consists of the nucleotide sequence provided in RefSeq ID NM_008507.4. In some embodiments, mouse SH2B3 is encoded by a nucleic acid molecule that comprises and/or consists of the nucleotide sequence
  • the nucleic acid molecule encoding SH2B3 is selected from a cDNA and an mRNA. In some embodiments, the nucleic acid molecule encoding SH2B3 is a cDNA. In some embodiments, the nucleic acid molecule encoding SH2B3 is an mRNA. It will be understood by a skilled artisan that when the molecule is an mRNA the thymines will be replaced by uracil.
  • human SH2B3 is SH2B3 isoform 1. In some embodiments, human SH2B3 is SH2B3 isoform 2. In some embodiments, human SH2B3 is SH2B3 isoform XI. In some embodiments, human SH2B3 is SH2B3 isoform X2. In some embodiments, human SH2B3 is SH2B3 isoform X3. In some embodiments, human SH2B3 is SH2B3 isoform X4. In some embodiments, human SH2B3 is SH2B3 isoform X5. In some embodiments, human SH2B3 is SH2B3 isoform X6.
  • human SH2B3 comprises and/or consists of an amino acid sequence selected from those provide in RefSeq IDs NP_005466.1, NP_001278353.1, XP_011536021.1, XP_011536022.1,
  • human SH2B3 comprises p £i 3 ⁇ 4? 0 v?v9/il3 ⁇ 4?s l 3 ⁇ 4f an amino acid sequence provide in RefSeq IDs NP_005466.1, NP_001278353.1, XP_011536021.1, XP_011536022.1, XPJ305253875.1, XP_005253876.1,
  • human SH2B3 comprises and/or consists of the amino acid sequence provided in RefSeq ID NP_005466.1.
  • human SH2B3 comprises and/or consists of the amino acid sequence MN GP ALQPS SPSS APS AS P A A APRGWSEFCELH A V A A AREL ARQ YWLFAREHPQH APLRAELVSLQFTDLFQRYFCREVRDGRAPGRDYRDTGRGPPAKAEASPEPGPGP A APGLPKARS S EEL APPRPPGPCS F QHFRRS LRHIFRRRS AGELP A AHT AA APGTPG E A AETP ARPGLAKKFLPW S L AREPPPE ALKE A VLRY S LADE AS MDS G ARW QRGRL ALRRAPGPDGPDRVLELFDPPKSSRPKLQAACSSIQEVRWCTRLEMPDNLYTFVLK VKDRTDIIFE V GDEQQLNS WM AELS ECT GRGLES TE AEMHIPS ALEPS TS S S PRGS T DS LN QG AS PGGLLDP ACQKTDHFLS C YP
  • human SH2B3 is encoded by a nucleic acid molecule comprising or consisting of the nucleotide sequence selected from those provided in a RefSeq IDs NM 005475.2, NM_001291424.1, XM_011537719.2, XM_011537720.3, XMJ305253818.4, XMJ305253819.4, XRJ301748535.1, XM_011537721.3,
  • human SH2B3 is encoded by a nucleic acid molecule comprising or consisting of the nucleotide sequence provided in a RefSeq ID NM_005475.2, NM_001291424.1, XM_011537719.2, XM_011537720.3, XMJ305253818.4, XMJ305253819.4,
  • human SH2B3 is encoded by a nucleic acid molecule that comprises and/or consists of the nucleotide sequence provided in RefSeq ID NM_005475.2.
  • human SH2B3 is encoded by a nucleic acid molecule that comprises and/or consists of the nucleotide sequence ATGAACGGGCCTGCCCTGCAGCCCTCCTCGCCCTCTTCCGCGCCCTCAGCCTCC CCGGCGGCGGCCCCGCGGGGCTGGAGCGAGTTCTGTGAGTTGCACGCCGTAGC '3 ⁇ 4L3 ⁇ 43 ⁇ 4 ) J 1 Zi1 ⁇ 2Z?GGGAGCTGGCCCGCCAGTACTGGCTGTTCGCC££ / 0 3 ⁇ , ⁇ 3 ⁇ 4 ⁇ GC AGCACGCCGCTGCGCGCCGAGCTGGTGTCGCTGCAGTTCACCGACCTCTTC CAGCGCTACTTCTGCCGCGAGGTGCGCGACGGACGGGCGCCGGGCCGCGACTA CCGGGACACAGGCCGTGGGCCCCCAGCCAAGGCCGAGGCGTCCCCGGAGCCA GGCCCCGGCCCCGCCCTGGCCTGCCCAAGGCCCGCAGCTGAGGAGCT GGCC
  • the nucleic acid molecule encoding SH2B3 is selected from a cDNA and an mRNA. In some embodiments, the nucleic acid molecule encoding SH2B3 is a cDNA. In some embodiments, the nucleic acid molecule encoding SH2B3 is an mRNA. It will be understood by a skilled artisan that when the molecule is an mRNA the thymidines will be replaced by uracil. 74472
  • decreased SH2B3 protein expression and/or function is decreased SH2B3 protein expression, decreased SH2B3 protein function or both.
  • the cell comprises decreased SH2B3 expression.
  • the cell comprises decreased SH2B3 protein expression.
  • the cell comprises decreased SH2B3 protein levels.
  • the cell comprises decreased SH2B3 mRNA expression.
  • the cell comprises decreased SH2B3 mRNA levels.
  • the cell comprises decreased SH2B3 protein function.
  • a decreased is with respect to an unmodified cell.
  • an increase is with respect to an unmodified cell.
  • the unmodified cell and the cell of the invention are the same cell type and the cell of the invention has the decrease.
  • the cell lacks SH2B3 protein expression.
  • the cell is an SH2B3 knockout.
  • cell comprises a DNA alternation that results in reduced or non-existent SH2B3 protein expression.
  • the DNA alteration results in reduced or non-existent functional SH2B3 protein expression.
  • the DNA alteration is selected from a mutation, a deletion and an insertion.
  • the DNA alteration is a gene trap. In some embodiments, the DNA alteration results in negligible or no protein expression.
  • decreased SH2B3 function comprises increased C-X-C Motif Chemokine Receptor (CXCR) signaling.
  • the CXCR is CXCR1, CXCR2 and/or CXCR3.
  • decreased SH2B3 function comprises increased CXCR1 signaling.
  • decreased SH2B3 function comprises increased CXCR2 signaling.
  • decreased SH2B3 function comprises increased CXCR1 and/or CXCR2 signaling.
  • decreased SH2B3 function comprises increased Osteopontin (OPN) receptor signaling.
  • OPN receptor is CD44.
  • the OPN receptor is an integrin receptor.
  • the integrin receptor is alpha 4/beta 1 (a4b1), a9b1 or a9b4. Each possibility represents a separate embodiment of the invention.
  • the signaling is downstream signaling.
  • decreased SH2B3 function does not comprise increased receptor expression.
  • receptor expression is total expression.
  • receptor expression is surface expression.
  • decreased SH2B3 function comprises increased phosphorylated effector protein.
  • the effector protein is Signal Transducer and Activator of ⁇ 92P3 ⁇ 4 ⁇ 7 7 2 3 (STAT3).
  • the effector protein is a mitogen- activated protein kinase (MAPK).
  • the effector protein is Erkl and/or ERK2.
  • the ERK is ERK1.
  • the ERK is ERK2.
  • the ERK is selected from ERK1 and ERK2.
  • the effector protein is MAPK3.
  • the effector protein is STAT3, STAT5, AKT, MEK, ERK, MAPK, JAK2, JAK3, JAK, FLT3, P38, SRC, Paxillin, FAK, PLC or PKC.
  • the effector protein is STAT3, STAT5, AKT, MEK, ERK, MAPK, JAK2, JAK3, JAK, FLT3, P38, SRC, Paxillin, FAK, PLC or PKC.
  • the cell comprises a SH2B3 inhibitor molecule.
  • decreased SH2B3 protein expression and/or function comprises expression of an SH2B3 inhibitor molecule.
  • the SH2B3 inhibitor molecule is selected from a small nucleic acid molecule that hybridizes to and inhibits SH2B3 mRNA transcription and/or translation, a small molecule inhibitor of SH2B3 protein, an anti-SH2B3 antibody or antigen binding fragment, and a genome editing agent for mutation of SH2B3.
  • the SH2B3 inhibitor molecule is a small nucleic acid molecule that hybridizes to and inhibits SH2B3 mRNA transcription and/or translation, a small molecule inhibitor of SH2B3 protein, an anti-SH2B3 antibody or antigen binding fragment, or a genome editing agent for mutation of SH2B3.
  • the SH2B3 inhibitor molecule is a small nucleic acid molecule.
  • the small nucleic acid molecule hybridizes to and inhibits SH2B3 mRNA transcription and/or translation.
  • the small nucleic acid molecule binds to the SH2B3 genomic locus and inhibits transcription.
  • the small nucleic acid molecule binds to SH2B3 mRNA and inhibits translation.
  • inhibiting translation comprises inducing mRNA degradation.
  • the small nucleic acid molecule is single stranded. In some embodiments, the small nucleic acid molecule is double stranded.
  • the small nucleic acid molecule is single stranded or double stranded.
  • the small nucleic acid molecule that hybridized to and inhibits SH2B3 is selected from a microRNA (miR), small interfering RNA (siRNA), an RNAi agent, an iRNA agent and a short hairpin RNA (shRNA).
  • the small nucleic acid molecule that hybridized to and inhibits SH2B3 is a miR, a siRNA, an RNAi agent, an iRNA or an shRNA. Each possibility represents a separate embodiment of the invention. In some !3 ⁇ 4 7 -,?
  • the small nucleic acid molecule induces RNA interl££jyifc3 ⁇ 4?i ⁇ S5921?3.
  • the small nucleic acid molecule that hybridized to and inhibits SH2B3 is an anti-sense RNA.
  • the anti-sense RNA is an RNA comprising sequence that is antisense to a SH2B3 mRNA. Examples of such nucleic acid molecules can be found, for example, in WO2013/019857, the contents of which are incorporated herein by reference in their entirety.
  • the small nucleic acid molecule comprises at least 5, 7, 10, 13, 15, 17, 19, 21, 23 or 25 nucleotides. Each possibility represents a separate embodiment of the invention. In some embodiments, the small nucleic acid molecule comprises at most 15, 17, 19, 21, 23, 24, 25, 26, 27, 29, 31, 33, 35, 37, 39 or 40 nucleotides. Each possibility represents a separate embodiment of the invention.
  • the small nucleic acid molecule comprises between 13 and 35, 13 and 33, 13 and 31, 13 and 29, 13 and 27, 13 and 26, 13 and 25, 13 and 24, 13 and 23, 13 and 21, 13 and 19, 15 and 35, 15 and 33, 15 and 31, 15 and 29, 15 and 27, 15 and 26, 15 and 25, 15 and 24, 15 and 23, 15 and 21, 15 and 19, 17 and 35, 17 and 33, 17 and 31, 17 and 29, 17 and 27, 17 and 26, 17 and 25, 17 and 24, 17 and 23, 17 and 21, 17 and 19, 19 and 35, 19 and 33, 19 and 31, 19 and 29, 19 and 27, 19 and 26, 18 and 25, 19 and 24, 19 and 23, or 19 and 21.
  • Each possibility represents a separate embodiment of the invention.
  • the nucleic acid molecule is brought into the cell as part of a plasmid and/or vector that expresses the nucleic acid molecule.
  • Methods of expressing nucleic acids in a cell include, for example, electroporation, liposomal transfection, viral transfection, lentiviral infection, and calcium-phosphate transfection.
  • the nucleic acid molecules such as for example shRNAs, can be directly transferred to the cell, or an expression vector can be used to cause transcription of the molecule with the cell.
  • the cell comprises the small nucleic acid molecule.
  • the cell comprises an expression vector comprising or encoding the small nucleic acid molecule.
  • the expression vector is a mammalian expression vector.
  • a vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
  • 7 ⁇ lecto may be a DNA plasmid delivered via non-vira? i3 ⁇ 3 ⁇ 4?Si 0 S 5( v 3 ⁇ 4 8 viral methods.
  • the viral vector may be a lentiviral vector, retroviral vector, a herpes viral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector.
  • the promoters may be active in mammalian cells.
  • the promoters may be a viral promoter.
  • the small nucleic acid molecule, or a sequence that encodes the small nucleic acid molecule or a polypeptide is operably linked to a promoter.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), Heat shock, liposomes, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et ah, Nature 327. 70-73 (1987)), and/or the like.
  • electroporation e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82, 5824 (1985)
  • Heat shock e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82, 5824 (1985)
  • liposomes e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82, 58
  • promoter refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II).
  • RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 ( ⁇ ), pGL3, pZeoSV2( ⁇ ), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK- RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • expression vectors containing regulatory elements from eukaryotic viruses are used by the present invention.
  • SV40 vectors include pSVT7 and pMT2.
  • vectors derived from bovine papilloma virus include pBV-lMTHA
  • vectors derived from Epstein Bar virus include pHEBO
  • exemplary vectors include pMSG, pAV009/A+, pM?&i i1 ⁇ 23 ⁇ 4 0 3 ⁇ 43 ⁇ 43 ⁇ 43 ⁇ 4neo- 5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • recombinant viral vectors which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression.
  • lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells.
  • the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles.
  • viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • the expression construct can also include sequences engineered to optimize stability, production, purification, yield, protein translation or activity of the nucleic acid molecule or polypeptide.
  • nucleic acid construct administered to the cell employing any suitable mode of administration, described hereinabove.
  • the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) ⁇ 9u3 ⁇ 4?° 3 ⁇ 4117A ⁇ oh system as needed and then the modified cells are and optionally administered to an individual.
  • the decrease is at least a 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 100% decrease.
  • Detecting expression of protein and mRNA is well known in the art, and any known method can be used for determining a decrease. Examples of assays that may be used include, but are not limited to, rtPCR, real-time PCR, qPCR, immuno staining, in situ hybridization, western-blotting and ELISA.
  • the SH2B3 inhibitor molecule is a small molecule inhibitor of SH2B3.
  • the SH2B3 inhibitor molecule is an anti-SH2B3 antibody or antigen binding fragment thereof.
  • Anti-SH2B3 antibodies are well known in the art and may be purchased from retailers such as Thermo Fisher, Sigma Aldrich, R&D Systems and Millipore.
  • the small molecule inhibitor is an SH2B3 antagonist.
  • the antibody and/or antigen binding fragment is an SH2B3 antagonist.
  • the small molecule inhibitor, antibody, or antigen binding fragment binds to at least one of protein interacting domain of SH2B3.
  • the small molecule inhibitor, antibody, or antigen binding fragment occludes a protein interacting domain of SH2B3.
  • the small molecule inhibitor, antibody, or antigen binding fragment is a competitive antagonist.
  • the protein interacting domain of SH2B3 is selected from a Pleckstrin Homolog (PH) domain, and a Src Homolog 2 (SH2) domain of SH2B3.
  • the PH domain comprises and/or consists of amino acids 194-307 of SEQ ID NO: 3.
  • the PH domain comprises and/or consists of amino acids 206-307 of SEQ ID NO: 3.
  • the SH2 domain comprises and/or consists of amino acids 364-462 of SEQ ID NO: 3.
  • Suitable antibodies may be generated by techniques known in the art. Animals may be immunized with the domains listed above, or with fragments of those domains or peptides containing those domains.
  • antagonist generally refers to a molecule, compound or agent that binds to a target protein at the same site as an agonist, ligand or interacting protein but which does not activate the intracellular response initiated by the active form of the protein, and as such an antagonist can inhibit the intracellular responses by agonists.
  • an antibody refers to a polypeptide or group of polypeptides that include at least one binding domain that is formed from the folding of polypeptide three-dimensional binding spaces with internal surfiK£ T 73 ⁇ 4 P3 ⁇ 4 0 ?3 ⁇ 4 2 i?arge distributions complementary to the features of an antigenic determinant of an antigen.
  • An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light” and one "heavy” chain. The variable regions of each light/heavy chain pair form an antibody binding site.
  • An antibody may be oligoclonal, polyclonal, monoclonal, chimeric, camelised, CDR-grafted, multi- specific, bi-specific, catalytic, humanized, fully human, anti- idiotypic and antibodies that can be labeled in soluble or bound form as well as fragments, including epitope-binding fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences.
  • An antibody may be from any species.
  • the term antibody also includes binding fragments, including, but not limited to Fv, Fab, Fab', F(ab')2 single stranded antibody (svFC), dimeric variable region (Diabody) and disulphide-linked variable region (dsFv).
  • antibodies include immunoglobulin molecules and immunologic ally active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.
  • Antibody fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof.
  • fusion products may be generated including but not limited to, scFv- Fc fusions, variable region (e.g., VL and VH) ⁇ Fc fusions and scFv-scFv-Fc fusions.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.
  • the SH2B3 inhibitor molecule is a genome editing agent.
  • the genome editing agent is for excision of SH2B3.
  • the genome editing agent is for mutation of SH2B3.
  • mutation of SH2B3 is mutation of the SH2B3 genomic locus.
  • mutation of the SH2B3 genomic locus results in reduced expression of SH2B3 protein.
  • mutation of the SH2B3 genomic locus results in no expression of SH2B3 protein.
  • Genome editing agents are well known in the art. Examples include, but are not limited to CRISPR/Cas9, transcription activator-like effector nucleases (TALEN), zinc finger nucleases, and meganucleases. These agents can be employed for specific modification of a given genomic locus, or even nucleotide. This can be made specific to SH2B3, for examples with a guide RNA specific to a location in the SH2B3 gene. Examples of agents for genomic editing of SH2B3 to decrease its expression can be found for example in WO2016/085934, the contents of which are herein incorporated by reference in their 3J ⁇ 7 iiZ?
  • gRNAs for targeting SH2B3 and decr£SI( L 3 ⁇ 4!3 ⁇ 4]/ ® 3 ⁇ 4?iS are commercially available from vendors such as GenScript and plasmids with the gRNA can be obtained from abmXchange for example.
  • the cell further comprises an exogenous protein.
  • the exogenous protein does not decrease SH2B3 protein and/or function.
  • the exogenous protein is a synthetic protein.
  • the exogenous protein is a chimeric protein.
  • the exogenous protein is a receptor.
  • the exogenous protein is a surface protein.
  • the exogenous protein is a chimeric antigen receptor (CAR).
  • the exogenous protein is a targeting moiety.
  • the exogenous protein is expressed on the surface of the cell.
  • the protein is a cytotoxic protein.
  • the protein is an anti-cancer protein. In some embodiments, the protein is an immune stimulating protein. In some embodiments, the protein binds to a target antigen. In some embodiments, the protein binds to a target antigen on a pathogen. In some embodiments, the protein binds to a target antigen on a pathological cell. In some embodiments, the protein binds to a target antigen on a cell associated with a disease.
  • the cell is a CAR cell. In some embodiments, the cell is a CAR-T cell. In some embodiments, the cell is a CAR-NK cell.
  • the terms "CAR-T cell” and“CAR-NK cell” refer to an engineered receptor which has specificity for at least one protein of interest (for example an immunogenic protein with increased expression following treatment with an epigenetic modifying agent) and is grafted onto an immune effector cell (a T cell or NK cell).
  • the CAR- T cell has the specificity of a monoclonal antibody grafted onto a T-cell.
  • the CAR-NK cell has the specificity of a monoclonal antibody grafted onto a NK-cell.
  • the T cell is selected from a cytotoxic T lymphocyte and a regulatory T cell.
  • CAR-T and CAR-NK cells and their vectors are well known in the art. Such cells target and are cytotoxic to the protein for which the receptor binds.
  • a CAR-T or CAR-NK cell targets at least one viral protein.
  • a CAR-T or CAR-NK cell targets a plurality of viral proteins.
  • a CAR-T or CAR-NK cell targets a viral protein with increased expression due to contact with an epigenetic modifying agent.
  • ⁇ Poo® 20 d3 ⁇ 4ioT?uction °f CAR-T cells is well known in the art. In one? u! ⁇ ?S?
  • a monoclonal antibody to a viral protein can be made and then a vector coding for the antibody will be constructed.
  • the vector will also comprise a costimulatory signal region.
  • the costimulatory signal region comprises the intracellular domain of a known T cell or NK cell stimulatory molecule.
  • the intracellular domain is selected from at least one of the following: CD3Z, CD27, CD28, 4-l BB, 0X40, CD30, CD40, PD- 1, ICOS, lymphocyte function-associated antigen- 1 (LFA- 1), CD2, CD 7, LIGHT, NKG2C, B7- H3, and a ligand that specifically binds with CD83.
  • the vector also comprises a CD3Z signaling domain. This vector is then transfected, for example by lentiviral infection, into a T-cell.
  • the term“moiety” as used herein refers to a part of a molecule, which lacks one or more atom(s) compared to the corresponding molecule.
  • the term“moiety”, as used herein, further relates to a part of a molecule that may include either whole functional groups or parts of functional groups as substructures.
  • the term“moiety” further means part of a molecule that exhibits a particular set of chemical and/or pharmacologic characteristics which are similar to the corresponding molecule, i.e. fluorescence.
  • a fluorescent moiety is part or all of a fluorescent molecule that retains the ability to fluoresce.
  • a“targeting moiety” is a moiety that binds to or interacts with a target cell, and thereby can bring the cell of the invention to the target cell.
  • a targeting moiety is an antibody or antigen binding fragment thereof that binds a surface protein on the target cell.
  • the targeting moiety is a ligand or binding partner of a receptor on the target cell.
  • the targeting moiety binds to a target antigen on a target cell.
  • the target cell is a cancer cell.
  • the target cell is a pathogen.
  • the pathogen is selected from a bacterium, a fungus, a virus, a parasite and a cancer.
  • the cell of the invention is produced by extracting a cell from a subject and decreasing SH2B3 protein expression and/or function in the extracted cell in vitro.
  • the immune cell is obtained from the subject.
  • SH2B3 protein expression and/or function is decreased in the obtained cell.
  • the production further comprises expressing in the cell the exogenous protein.
  • the cell is expanded in vitro.
  • compositions comprising a cell of the invention. 'YS1 ⁇ 2 ⁇ j 20 x3 ⁇ 47t‘tZ? e embodiments, the composition is a pharmaceutical ⁇ ( ⁇ feTSi ⁇ P ⁇ ome embodiments, the composition is configured for administration to a subject. In some embodiments, the composition is configured for systemic administration. In some embodiments, the composition is configured for intravenous administration. In some embodiments, the composition is configured for intratumoral administration.
  • a method of treating a disease in a subject in need thereof comprising administering to the subject a cell or composition of the invention, thereby treating the disease.
  • a method of treating a disease in a subject in need thereof comprising administering to the subject a cell comprising decreased SH2B3 protein expression and/or function, thereby treating the disease.
  • a cell of the invention for use in treating a disease in a subject in need thereof.
  • a cell with decreased SH2B3 protein expression and/or function for use in treating a disease in a subject in need thereof.
  • the disease is a pathogenic disease. In some embodiments, the disease is a disease treatable by immunotherapy. In some embodiments, the disease is an immune-associated disease. In some embodiments, an immune-associated disease is a disease treatable by immunotherapy. In some embodiments, the disease is a disease that induces IL-8 expression. In some embodiments, the induced IL-8 expression is proximal to the site of disease. In some embodiments, the disease comprises a cell with targetable protein expression. In some embodiments, the disease comprises a cell associated with the disease. In some embodiments, the targetable protein expression is surface protein expression. In some embodiments, the disease is cancer. In some embodiments, the disease is Myelodysplastic syndrome.
  • the disease is a bacterial infection. In some embodiments, the disease is a fungal infection. In some embodiments, the disease is a viral infection. In some embodiments, the disease is a parasitic infection. It will be understood by one skilled in the art that the exogenous protein may be tailor based on the disease to be treated.
  • a targeting moiety may be selected to target the cell of the invention to a pathogen or cancer cell. Bacterial, viral and fungal proteins are well known in the art, as are cancer specific surface markers. Antibodies, and binding molecules that can be used as targeting moieties are also well known. CARs may be used for targeting cancer cells, and the like.
  • the cell associated with the disease is ? ⁇ i ⁇ 3 ⁇ 42 0 A3 ⁇ 4R ⁇ ?3 ⁇ 4? lancer cell, a precancerous cell, a bacterial cell, a fungal cell, a parasite cell, and a cell infected with a virus.
  • the cell associated with the disease is selected from a cancer cell, a bacterial cell, a fungal cell, a parasite cell, and a cell infected with a virus.
  • the cell associated with the disease is a cancer cell.
  • the cell associated with the disease is a bacterial cell.
  • the cell associated with the disease is fungal cell.
  • the cell associated with the disease is a parasite cell.
  • the cell associated with the disease is a cell infected with a virus.
  • the cell associated with the disease is an IL-8 expressing cell.
  • the method further comprises providing the cell.
  • the cell is an immune cell.
  • the cell is from the subject.
  • the cell is autologous to the subject.
  • the cell is allogenic to the subject.
  • the cell is syngeneic to the subject.
  • the cell is from a healthy donor.
  • the provided cell is cultured in vitro.
  • the provided cell is expanded in vitro.
  • the cell is further enhanced in vitro to improve its immunotherapeutic potential.
  • Such alterations may include gene knockout, or genetic modification or expression of a protein or polypeptide.
  • the cell is returned to the subject.
  • the cell is administered to the subject.
  • the method is a method of immunotherapy.
  • immunotherapy is adoptive immunotherapy.
  • the method is a method of adoptive immunotherapy.
  • the method is combined with another method of immunotherapy.
  • cells to be used for immunotherapy are modified by decreasing SH2B3 protein expression and/or functional levels in the cells.
  • decreasing SH2B3 levels enhances the therapeutic potential of the cells.
  • the therapeutic potential is immunotherapeutic potential.
  • the term“therapeutic potential” refers to a cells ability to treat and or prevent a disease, disorder or pathological condition as well as improve a subj ecf s overall condition.
  • the terms “immunotherapeutic potential” and “therapeutic immune cell potential” are synonymous and refer to the qualitative and/or quantitative therapeutic potential of a beneficial T cell to be, or to become, therapeutic toward a subject’s Q J ⁇ some embodiments, the therapeutic potential is any P u!iP ⁇ ?®3 ⁇ 4® g 3 ⁇ 43 ⁇ 43 ⁇ 4;ing, repairing, replacing, preventing, or protecting potential or a combination thereof.
  • administering is administering at least 100, 500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 10000000, 50000000, 100000000, 500000000, 1000000000, or 5000000000 cells. Each possibility represents a separate embodiment of the invention.
  • the administering is repeated. In some embodiments, administering is administering at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. Each possibility represents a separate embodiment of the invention.
  • the terms“administering,”“administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.
  • One aspect of the present subject matter provides for oral administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof.
  • Other suitable routes of administration can include parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal.
  • the dosage administered 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 method further comprises administering to the subject another therapeutic treatment.
  • the therapeutic treatment is an anti cancer therapeutic treatment.
  • the therapeutic treatment is an anti pathogen therapeutic treatment.
  • a pathogen is selected from a bacterium, a fungus, a virus and a parasite.
  • a pathogen is a bacterium.
  • a pathogen is a virus.
  • a pathogen is a fungus.
  • a pathogen is a parasite.
  • the method further comprises administering to the subject an anticancer or anti-pathogen therapeutic treatment.
  • the method is a combined treatment with a second therapeutic treatment.
  • the second therapeutic treatment is a different therapeutic modality from an immunotherapy treatment. In some embodiments, the second therapeutic treatment is a different therapeutic modality from a treatment with immune cells. In some embodiments, the therapeutic treatment is selected from the group consisting of: irradiation, chemotherapy, immune checkpoint modulation, CAR therapy, adoptive T cell therapy, small molecule biologies, surgery, bone marrow transplant, targeted drug therapy, cellular ablation ⁇ ai v?3 ⁇ 4P ai3 ⁇ 4iZ? ⁇ tic. In some embodiments, the therapeutic treatment P Ti3 ⁇ 4?2?3 ⁇ 4P? 0 ? 1 ⁇ lorne embodiments, the therapeutic treatment is chemotherapy. In some embodiments, the therapeutic treatment is immune checkpoint modulation.
  • the therapeutic treatment is CAR therapy. In some embodiments, the therapeutic treatment is adoptive T cell therapy. In some embodiments, the therapeutic treatment is a small molecule biologic. In some embodiments, the therapeutic treatment is surgery. In some embodiments, the therapeutic treatment is bone marrow transplant. In some embodiments, the therapeutic treatment is targeted drug therapy. In some embodiments, the therapeutic treatment is cellular ablation. In some embodiments, the therapeutic treatment is an antibiotic.
  • immune checkpoint modulation comprises immune checkpoint blockade.
  • immune checkpoint blockade comprises administering an immune checkpoint inhibitor.
  • immune checkpoint blockade is intrinsic checkpoint blockade.
  • the immune checkpoint is selected from PD-1, PD-L1, CTLA-4 and HVEM.
  • the immune checkpoint is selected from PD-1, PD-L1, and CTLA-4.
  • Immune checkpoint inhibitors are well known in the art and include, but are not limited to, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab.
  • the other therapeutic treatment is administered before a cell of the invention. In some embodiments, the other therapeutic treatment is administered after a cell of the invention. In some embodiments, the other therapeutic treatment is administered concomitantly with a cell of the invention. In some embodiments, the other therapeutic treatment enhances the effect of a cell of the invention. In some embodiments, the other therapeutic treatment has an additive effect on the efficacy of a cell of the invention.
  • a method for improving a cell to be used for immunotherapy comprising decreasing SH2B3 expression and/or function in the cell, thereby improving the cell to be used for immunotherapy.
  • a method for enhancing an immune response of an immune cell comprising decreasing SH2B3 expression and/or function in the immune cell, thereby enhancing an immune response of an immune cell.
  • the method is performed in vitro. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in culture. In some embodiments, the method is performed in vivo. In some embodiments, the immune ⁇ 9 9?3 ⁇ 4 1 Z5A 7 ?o be used for immunotherapy. In some embodiments, ⁇ ?vJ 3 ⁇ 4?3 ⁇ 4?/5u93A?py is adoptive immunotherapy.
  • enhancing an immune response comprises increasing cytotoxicity of the immune cell.
  • enhanced cytotoxicity comprises enhanced phosphorylation of STAT3 in the cell.
  • enhanced cytotoxicity comprises enhanced expression of a proinflammatory cytokine.
  • the proinflammatory cytokine is interferon gamma.
  • Proinflammatory cytokines are well known in the art, and include, but are not limited to 11-2, interferon gamma, TNF alpha, IL- 1 and granulocyte-macrophage colony stimulation factor. Increases expression of any proinflammatory cytokine known in the art may be a readout for enhanced cytotoxicity.
  • enhancing an immune response comprises increasing migration of an immune cell.
  • enhancing an immune response comprises increasing invasion of an immune cell.
  • increasing migration comprises increasing invasion.
  • migration is to a site of disease.
  • a site of a disease is a tumor.
  • a site of disease is a site of infection.
  • the site of disease is a site with enhanced IL-8 expression.
  • the site of the disease comprises IL-8 expression.
  • invasion is to a site of disease.
  • enhancing an immune response comprises increasing proliferation of the immune cell.
  • enhancing an immune response comprises increasing the ratio of CD8 T cells to CD4 T cells present in a population of immune cells.
  • the immune cell is a population of immune cells.
  • a method for determining suitability of a subject to receive treatment comprising: a. receiving a cell from the subject; and b. assaying chemotaxis of an unmodified immune cell and an immune cell comprising decreased SH2B3 protein expression and/or function to the pathological cell; wherein improved chemotaxis of the immune cell comprising decreased SH2B3 protein expression and/or function as compared to the unmodified immune cell indicates the subject is suitable for a treatment of the invention, thereby determining suitability of a subject to receive treatment.
  • the treatment is a treatment compi?9T g I 3 ⁇ 4?3 ⁇ 4 ⁇ ??PulS cell comprising decreased SH2B3 protein expression and/or function.
  • the treatment is a treatment comprising administering an immune cell comprising decreased SH2B3 protein expression and/or function.
  • the treatment is a treatment comprising decreasing SH2B3 protein expression or function in an immune cell.
  • the immune cell is an immune cell of the subject.
  • the treatment is a treatment method of the invention.
  • the cell is a tumor cell and a tumor biopsy is received. In some embodiments, the cell is put in culture. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in a laboratory animal. Examples of laboratory animals include, but are not limited to, mice, rats, rabbits, and cats. In some embodiments, the laboratory animal is a mammal. In some embodiments, the laboratory animal is a mouse. In some embodiments, the laboratory animal is immune comprised. In some embodiments, the immune compromised animal is a SCID mouse. In some embodiments, the immune compromised animal is a NOD mouse. In some embodiments, the immune compromised animal is a SCID/NSG mouse.
  • the assaying chemotaxis is chemotaxis in cell culture. In some embodiments, the assaying chemotaxis is chemotaxis in a laboratory animal. In some embodiments, assaying chemotaxis is a migration assay. In some embodiments, assaying chemotaxis comprises a Transwell assay. In some embodiments, assaying chemotaxis comprises culturing the cell in one compartment of a Transwell and culturing the immune cell in another well of the Transwell dish. In some embodiments, the assaying chemotaxis comprises inserting the cell in a laboratory animal and administering the immune cells to the animal and assaying the ability of the immune cells to reach the cell in the animal.
  • the cell is injected. In some embodiments, the cell is injected subcutaneously. In some embodiments, the administered immune cells are injected. In some embodiments, a plurality of cells is received and at least one cell is used in a chemotaxis assay with the unmodified cell and at least one cell is used in a chemotaxis assay with the cell comprising decreased SH2B3 protein expression and/or function. In some embodiments, the chemotaxis assay comprises the setup of the“in vitro invasion assay” described herein. In some embodiments, the chemotaxis assay comprises the setup of the“in vitro Matrigel chemotaxis and invasion assay” described herein.
  • the in assay does not comprise addition of an inhibitor and/or a test molecule.
  • improved chemotaxis is increased ⁇ X H ⁇ SK ⁇ ome embodiments, an increase is at least a 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500% increase.
  • a decrease is at least a 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99 or 100% decrease.
  • improved chemotaxis is an increase in the number of cells that reach the cell.
  • the method further comprises treating the suitable subject.
  • the treatment is by any one of the methods of the invention.
  • a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.
  • mice model Lnk _/_ mice (a gift from Tony Pawson's lab) were backcrossed 10 generations into C57BL/6J-Ly5.2-Gpil b/b [1], [2].
  • Wield-type (WT) C57BL/6J01aHsd mice were obtained from the Pre-Clinical Research Authority at the Technion Faculty of Medicine. Experiments were approved and mice were housed and manipulated according to the guidelines of the Animal Care and Use Committee of the Technion.
  • B 16-F10 murine melanoma cells were cultured in RPMI 1640 supplemented with 10% FCS, 1% L-glutamine and 1% Penicillin-Streptomycin.
  • LL/2 (LLC1) murine Lewis Lung Carcinoma cells were cultured in DMEM supplemented with 10% FCS, 1% L-glutamine and 1% Penicillin-Streptomycin.
  • Tumor model For the subcutaneous melanoma model, 1.4 x 10 6 B 16-F10 cancer cells were subcutaneously injected into WT or Lnk _/ mice at the rear back. After 14 days mice were euthanized and tumors, spleens and inguinal lymph nodes were harvested. The length and width of tumors were measured with a manual caliper prior to harvest and tumors were weighted post-harvest.
  • Tumors and Matrigel plugs were cut into small pieces and digested with Collagenase IV, DNase IV and Hyaluronidase V (Sigma- Aldrich) in HBSS for 60 min at 37°C with stirring [3]. Supernatants were collected and stored in -20°C for cytokine analysis. Cells were washed and RBCs were lysed when necessary with ammonium chloride lysis buffer. Recovered cells were used for flow cytometry analysis.
  • ⁇ - S 7 ⁇ PC- anti mouse CD 19 (clone 6D5); PerCP/Cy5.5-anti mouse CD182(CXCR2) (clone SA044G4); Alexa Fluor700- or PE-anti mouse CD3 (clone 17A2); FITC- or PE- anti mouse/human CD45R/B220 (clone RA3-6B2);
  • fluorochrome-conjugated antibodies were purchased from Miltenyi Biotec: PE-Vio770 anti mouse NK1.1 (clone PK136); FITC- anti mouse CD19 (clone 6D5).
  • the following fluorochrome-conjugated antibodies were purchased from BD Biosciences: PE-anti mouse CD181(CXCR1) (cloneU45-632).
  • Fluorescent data was collected on LSR Fortessa cell analyzer (BD Biosciences). Fluorescent data of the modified chemotaxis assay was collected on Attune NxT acoustic focusing cytometer (Thermo Fisher Scientific). Results were analyzed using FlowJo software (Tree Star).
  • Cytokine analysis Adjacent to the process of tumor dismantling into single-cell suspension, primary supernatants were collected and stored in -20°C for further analysis.
  • the cytokine profile at the tumor microenvironment fluid was screened using Proteome Profiler Mouse XL Cytokine Array (R&D Systems; Cat. ARY028). The presence of selected cytokines was verified by ELISA, and their concentration was determined as well. Results were obtained and quantified using pixel density measurements using ImageJ software.
  • the tumor- supernatant were collected and evaluated using ELISA for Mip-2(mouse MIP-2 ELISA; RayBiotech), IL-6, and IL-10 (ELISA MAX standard kit; BioLegend). The data was acquired and processed using ELISA reader and Gene5 software (biotek).
  • In-vivo Matrigel chemotaxis and invasion assay An In-vivo study of adoptive cell transfer and Matrigel plug assay to determine the role of Lnk regulation in the migratory and invasion capacities of CD8+ T-cells. Experimental design: B 16-F10 melanoma cells were transplanted subcutaneously into WT and Lnk-/- donors. Spleen of tumor bearing donor (WT or Lnk-/-) was harvested on day 14, and CD8+ T-cells were isolated from the total splenocyte population.
  • the isolated CD8+ T-cells (WT or Lnk-/-) were then covalently labeled with CellTrace Violet (C34557, Thermo Fisher Scientific), and transferred to WT recipient.
  • Each WT recipient was transferred by spleen derived CD8+ T-cells from a single tumor bearing donor, either WT or Lnk-/-.
  • 'YS > ⁇ 3 ⁇ 4° 2 j ° 1 ui1 wing cell transfer
  • recipient was subcutaneously i njcctcK ⁇ v T J ? nc carrying Matrigel-plugs at the rear flank, every plug containing a single chemokine on each flank, as illustrated.
  • Matrigel (Corning, 354234): The bilateral combination of chemokine containing plugs on a single recipient was either of the following [left plug:right plug] - watenwater; Mip-2:water; SDF-la:Mip-2. On day 15, chemokine boost was given to each Matrigel plug. On day 16, plugs were harvested, dissolved and analyzed by flow cytometry for the amount of fluorescent labeled CD8+ T-cells in each plug.
  • In vitro invasion assay The role of Lnk regulation in the CXCRl/2-mediated migration of T-cells to melanoma was examined in vitro by a chemotaxis assay modified with Matrigel. 0.25 x 106 B16-F10 cells were cultured and incubated in a 24-well Cell Culture Receiver plate (M8812, Sigma- Aldrich), that was used as the lower wells of a Transwell systems. Inguinal lymph nodes were isolated from either WT or Lnk-/- melanoma tumor bearing mice and were either treated or not treated with the CXCRl/2 inhibitor SCH563705 (cat.
  • Intracellular INFy staining Inguinal lymph nodes (FN) were harvested from either naive or tumor bearing mice, and single cell suspensions were prepared as described herein. A total of lxlO 5 isolated FN cells were incubated ex-vivo for a total time of 6 hours in 5% FCS RPMI with 50U/ml IF-2 (PEPROTECH) and 5ug/ml Brefeldin A (BFA, BioFegend) alone, or were concurrently stimulated by adding PMA (Santa cruz) and Ionomycin (CAYMAN chemicals) to a final concentration of 50ng/ml and 750ng/ml, respectively.
  • PMA Purified IF-2
  • BFA BioFegend
  • Example 1 Lnk KO mice show decreased tumor size
  • SH2B adapter protein 3 also known as lymphocyte adapter protein LNK, is a small adapter protein of molecular weight 66 kilodaltons and comprising 575 amino acids.
  • the protein has two major domains, a 3’ phoshpotyrosine binding domain (Src homology 2, SH2) and a more 5’ membrane localization domain (pleckstrin homology domain, PH).
  • Lnk regulates intracellular cytokine signaling by binding phosphorylated tyrosine residues on activated target proteins through its SH2 domain.
  • Fnk functions broadly as a hub-inhibitor, by binding a variety of activated intracellular signaling molecules.
  • the tumors extracted from Lnk KO mice were visibly smaller than those from WT mice (Fig. 1A), with the average weight of tumors from KO mice being about one-third of the weight from WT mice (Fig. IB).
  • FACS analysis of the single cell suspension from the tumors indicated that tumors from Lnk KO mice had a statistically significant increase in tumor infiltrating lymphocytes (TIL) as compared to tumors from WT mice (Fig. ID). This increase was confirmed by FACS with lymphocyte- specific markers. Histological analysis confirmed the increased cellular infiltration into the interphase of tumors from Lnk KO mice (Fig. IE).
  • the population of infiltrating cells was further assayed in order to determine the identity of the cells.
  • CD3-zeta chain positive T cells (Fig. IF) were stained for CD8a and CD4 surface expression (Fig. 1G). More than twice as many infiltrating cells were CD3-zeta positive in the tumors from Lnk KO mice.
  • the ratio of CD8+ cytotoxic T-cells to CD4 helper cells was more than 3 times higher in tumors from Lnk KO mice. Staining with an anti-CD8a antibody confirmed that the infiltrating cells seen in the interphase of the tumor from Lnk KO mice were indeed cytotoxic T cells (Fig. 1H, left).
  • the infiltrating cell population comprises increased numbers of total T-cells (CD3) (Fig. II), T-Helper cells (CD4) (Fig. 1J), Cytotoxic T cells (CD8) (Fig. IK), B-cells (CD45R) (Fig. 1L), NK cells (CD161b/c) (Fig. 1M), neutrophils (CDl lb) (Fig. IN), macrophages (F4/80) (Fig. lO), and dendritic cells (CDl lc) (Fig. IP).
  • MIP-2, and IL-8 both signal through the CXCR1 and CXCR2 receptors.
  • LNK LNK in CXCR receptor signal transduction.
  • Spleen-derived CD8 + T-cells of naive mice of both genotypes WT or Lnk 7
  • the phosphorylation of STAT3, as a readout for signal transduction was measured using anti p-STAT3 antibody, and compared to the total level of STAT3 in the lysate (Fig. 2A).
  • lymph node cells were harvested from inguinal lymph nodes of WT and Lnk KO mice. Cells were acquired from both naive and tumor bearing mice of both genotypes. Following treatment with IL-2, and activation with PMA and Ionomycin in the presence of Brefeldin A for 6 hours, intracellular interferon gamma was measured in CD8a positive cytotoxic T cells (Fig. 3A). Fold induction of INFy expression was calculated over the expression in non-activated cells of the same phenotype, and a statistically significant increase was observed in the LnkKO cells of tumor bearing mice as compared to their WT counterparts (Fig. 3B). When the total number of INFy positive cells was counted the same increase in cells from tumor bearing mice was also observed (Fig. 3C and 3D).
  • Example 3 In-vivo adoptive cell transfer of Lnk KO cytotoxic T-cells
  • B 16-F10 melanoma cells were transplanted subcutaneously into WT and Lnk 7 donor mice. Spleen of tumor bearing donor mice (WT or Lnk 7 ) was harvested on day 14, and CD8 + T-cells were isolated from the total splenocyte population. The isolated CD8 + T-cells (WT or Lnk 7 ) were covalently labeled using the fluorescent dye CellTrace Violet (C34557, Thermo Fisher Scientific) and transferred to WT recipient mice.
  • recipient mice were subcutaneously injected with two chemokine carrying Matrigel-plugs, one plug into each of their rear flanks.
  • the two plugs each contained a single chemokine.
  • the bilateral combination of chemokine containing plugs on a single recipient was one of the following [left plug:right plug]: - watenwater; Mip-2:water; SDF-la:Mip-2. 24 hours later the plugs were harvested and dissolved into single cell suspensions which were analyzed by FACS for the presence of the fluorescently labeled CD8+ cells.
  • the number of detected T-cells derived from Lnk KO donors was significantly increased in Mip-2 containing plugs (Fig. 4). No significant difference was seen in the number of detected T-cells, of both genotypes, in plugs containing water, or plugs containing SDF-la.
  • the exterior portion of the membrane on the upper well was pre-coated with a thin layer of Matrigel to better mimic in vivo infiltration of tumors.
  • the Transwell system was co-cultured for 16 hours, and cells were then collected from the lower well. These cells were stained for CD3, CD8 and CD4 markers and counted using an acoustic flow -cytometer to determine the number of migrating lymph node cells.
  • Example 5 Lnk KO mice show decreased tumor size in a lung model

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Abstract

Cells comprising an exogenous protein and decreased SH2B adapter protein 3 (SH2B3) protein expression and/or function are provided. Pharmaceutical compositions comprising the cells of the invention as well as methods of treating a disease in a subject in need thereof comprising administering a cell and/or a pharmaceutical composition of the invention, and of determining suitability for treatment, are also provided.

Description

WO 2020/17447¾ AND METHODS FOR IMPROVED IMMUNOT1P.£T^.2 .20/050218
CROSS REFERENCE TO RELATED APPLICATIONS
[001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/810,438, filed February 26, 2019 titled: "CELLS AND METHODS FOR IMPROVED IMMUNOTHERAPY", the contents of which are all incorporated herein by reference in their entirety.
FIELD OF INVENTION
[002] The present invention is in the field of immunotherapy.
SUMMARY OF THE INVENTION
[003] The present invention provides cells comprising decreased SH2B adapter protein 3 (SH2B3) protein expression and/or function. The present invention further provides methods of treating a disease in a subject comprising administering a cell comprising decreased SH2B3 expression and/or function, and methods of determining suitability to be so treated.
[004] According to a first aspect, there is provided a method of treating a disease in a subject in need thereof, the method comprising administering to the subject an immune cell comprising decreased SH2B adapter protein 3 (SH2B3) protein expression and/or function, thereby treating the disease.
[005] According to another aspect, there is provided a method of determining suitability of a subject in need thereof to receive a treatment comprising an immune cell comprising decreased SH2B3 protein expression and/or function, the method comprising
a. receiving a cell associated with a disease from the subject; and
b. assaying chemotaxis of an unmodified immune cell and an immune cell comprising decreased SH2B3 protein expression and/or function to the pathological cell; wherein improved chemotaxis of the immune cell comprising decreased SH2B3 protein expression and/or function as compared to the unmodified immune cell indicates the subject is suitable for the treatment. 'Yw¾j 20 ¾Ili ¾ing to another aspect, there is provided a method for tfiTu 112’n nunc response of an immune cell, the method comprising decreasing SH2B3 protein expression and/or function in the cell, thereby enhancing an immune response of an immune cell.
[007] According to another aspect, there is provided a pharmaceutical composition comprising an immune cell comprising decreased SH2B adapter protein 3 (SH2B3) protein expression and/or function.
[008] According to some embodiments, the disease is an immune-associated disease.
[009] According to some embodiments, the immune-associated disease is selected from cancer, Myelodysplastic syndrome (MDS), bacterial infection, fungal infection, viral infection and parasitic infection.
[010] According to some embodiments, the cell is allogenic to the subject.
[Oi l] According to some embodiments, the method further comprises providing an immune cell from the subject and decreasing SH2B3 protein expression and/or function in the provided cell.
[012] According to some embodiments, the decreased SH2B3 protein expression and/or function comprises decreased SH2B3 mRNA levels, SH2B3 protein levels, SH2B3 protein function, or a combination thereof.
[013] According to some embodiments, the decreased SH2B3 protein expression and/or function comprises expression of an SH2B3 inhibitor molecule.
[014] According to some embodiments, the SH2B3 inhibitor molecule is selected from a small nucleic acid molecule that hybridizes to and inhibits SH2B3 mRNA transcription and/or translation, a small molecule inhibitor of SH2B3 protein, an anti-SH2B3 antibody or antigen binding fragment, and a genome editing agent for mutation of a SH2B3 genetic locus.
[015] According to some embodiments, the small molecule inhibitor, antibody or antigen binding fragment is a SH2B3 antagonist.
[016] According to some embodiments, the small molecule inhibitor, antibody, or antigen binding fragment binds to and occludes at least one of a Pleckstrin Homolog (PH) domain, and a Src Homolog 2 (SH2) domain of SH2B3.
[017] According to some embodiments, the small nucleic acid molecule that hybridized to and inhibits SH2B3 is an anti-sense RNA. Ύ9, ?){2 /LIί17,¾ί ng to some embodiments, the immune cell is selected P^T/A 2 'i'L2 l?-ccl 1 , a natural killer (NK) cell, a neutrophil, a dendritic cell, a myeloid derived suppressor cell and a macrophage.
[019] According to some embodiments, the T-cell is a CD8+ T-cell, CD4+T-cell or both.
[020] According to some embodiments, the immune cell further comprises a chimeric antigen receptor (CAR).
[021] According to some embodiments, the CAR binds to a target antigen on a cell associated with the disease.
[022] According to some embodiments, the cell is selected from a cancer cell, a bacterial cell, a fungal cell, a parasite cell and a cell infected with a vims.
[023] According to some embodiments, the method further comprises administering an anticancer or anti-pathogen therapeutic treatment.
[024] According to some embodiments, the treatment comprising an immune cell comprising decreased SH2B3 protein expression and/or function is a method of the invention.
[025] According to some embodiments, the assaying chemotaxis is performed ex vivo.
[026] According to some embodiments, the method further comprises treating the subject suitable for treatment by a method of the invention.
[027] According to some embodiments, the decreasing occurs ex vivo.
[028] According to some embodiments, the decreasing comprises introducing an SH2B3 inhibitor molecule into the immune cell.
[029] According to some embodiments, the immune cell is an immune cell to be used for adoptive immunotherapy.
[030] According to some embodiments, the enhancing an immune response comprises at least one of: increasing cytotoxicity of the immune cell and increasing migration of the immune cell.
[031] According to some embodiments, the decreased SH2B3 protein expression and/or function comprises decreased SH2B3 mRNA levels, SH2B3 protein levels, SH2B3 protein function, or a combination thereof. 'YS < j 20 ¾Ili ¾ing to some embodiments, the decreased SH2B3 pro^T^¾29¾(il¾?¾d/or function comprises expression of an SH2B3 inhibitor molecule.
[033] According to some embodiments, the SH2B3 inhibitor molecule is selected from a small nucleic acid molecule that hybridizes to and inhibits SH2B3 mRNA transcription and/or translation, a small molecule inhibitor of SH2B3 protein, an anti-SH2B3 antibody or antigen binding fragment, and a genome editing agent for mutation of SH2B3.
[034] According to some embodiments, the small molecule inhibitor, antibody or antigen binding fragment is a SH2B3 antagonist.
[035] According to some embodiments, the small molecule inhibitor, antibody, or antigen binding fragment binds to and occludes at least one of a Pleckstrin Homolog (PH) domain, and a Src Homolog 2 (SH2) domain of SH2B3.
[036] According to some embodiments, the small nucleic acid molecule that hybridized to and inhibits SH2B3 is an anti-sense RNA.
[037] According to some embodiments, the immune cell is selected from a T-cell, a B-cell, a natural killer (NK) cell, a neutrophil, a dendritic cell, a myeloid derived suppressor cell and a macrophage.
[038] According to some embodiments, the T-cell is a CD8+ T-cell, CD4+T-cell or both.
[039] According to some embodiments, the cell comprises a chimeric antigen receptor (CAR).
[040] According to some embodiments, the CAR binds to a target antigen on a cell associated with a disease.
[041] According to some embodiments, the cell associated with a disease is selected from a cancer cell, a bacterial cell, a fungal cell, a parasite cell and a cell infected with a virus.
[042] According to some embodiments, the cell is produced by providing a cell from a subject and decreasing SH2B3 protein expression and/or function in the extracted cell in vitro.
[043] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes ^9u2P?S(i¾iZ?ons within the spirit and scope of the invention will b c 7,1} it 2 2u (P 12! h ose skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[044] Figures 1A-1R: (1A) A representative photograph of subcutaneous tumors growth in WT and Lnk KO mice. (IB) A scatter plot of tumor weight from WT and Lnk KO mice. WT n=23, KO n=25, Error bars indicate ± SEM. (1C) Representative FACS size analyses of single cells from tumors from WT and Lnk KO mice. (ID) A scatter plot of the percentage of TILs in the tumors from WT and Lnk KO mice. WT n=15, KO n=15, Error bars indicate ± SEM. (IE) Representative micrographs of tumor interface from WT and Lnk KO mice. (1F-G) Representative FACS analysis of (IF) CD3-zeta expression and (1G) CD8 and CD4 expression by lymphocytes from tumors from WT and Lnk KO mice. (1H) Representative micrographs of immunohistochemistry staining for the cytotoxic T-cell marker CD8a in tumors of WT host (upper) and Lnk 7 host (lower). Black arrow - positive CD8a T-cells; Asterix— blood vessel. (1I-P) Scatter plots of the percentage of infiltrating cells that are (II) Total T- cells (CD3), (1J) T-Helper cells (CD4), (IK) Cytotoxic T cells (CD8), (1L) B-cells (CD45R), (1M) NK cells (CD161b/c), (IN) neutrophils (CDl lb), (lO) macrophages (F4/80), and (IP) dendritic cells (CD1 lc). (IQ) A representative bar chart of cytokine levels in the TME as assessed by the Pro teome -Profiler Mouse XL Cytokine Array kit (R&D Systems). The supernatants from three tumors from mice of each genotype were pooled. (1R) A representative bar chart of cytokine levels in the TME as assessed by ELISA. The supernatants from three tumors from mice of each genotype were pooled.
[045] Figures 2A-2F: (2A) Photographs of immunoblotting of pSTAT3 and STAT3 from lysates of CD8+ splenocytes. (2B) A bar chart quantifying the relative phosphorylation of STAT3 from Figure 2A. (2C) Photographs of immunoblotting of pSTAT3 and STAT3 from lysates of CD8+ splenocytes from tumor bearing mice. (2D) A bar chart quantifying the relative phosphorylation of STAT3 from Figure 2C. (2E) Photographs of immunoblotting of pErk and Erk from lysates of CD8+ splenocytes. (2F) A bar chart quantifying the relative phosphorylation of Erk from Figure 2E.
[046] Figures 3A-C: (3A) A bar chart of fold induction of INFy geometric mean fluorescence intensity (gMFI) in CD8+ T-cell populations of WT and Lnk KO naive and tumor bearing mice. The gMFI of activated cells was normalized to the gMFI of non- activated cells and is expressed as fold induction. (3B) Representative histograms of INFy ?n ten s i t y in CD8+ T-cell populations of tumor bearing nE£J(¾¾!^/g¾?u?/pes. FMO - Fluorescence minus one control. (3C) Bar chart of the percent of CD8+ T-cells that produced INFy (of naive and tumor bearing mice of both genotypes). Naive n=3 mice/group; Tumor bearing n=7 mice/group. The error bar indicates mean with SEM. Significance was determined by two-way ANOVA (Sidak's posttest) ns - nonsignificant, ***0.001>p,
****0.0001>p.
[047] Figure 4: A bar graph of the number of fluorescent CD8+ T-cells from WT and Lnk KO donor mice in various plugs inserted into WT mice. ns= non-significant, **** p=Value <0.0001, n=50, Error bars indicates Mean with SEM.
[048] Figures 5A-5E: (5A-C) Bar graphs of the number of (5A) CD3+ T-cells, (5B) CD8+ T-cells and (5C) CD4+ T-cells that migrated across the transwell to reach cocultured melanoma cells. * p-Value<0.05, ** p-Value<0.01, *** p-Value<0.001, **** p- Value<0.0001, WT n=3, KO n=6, lnh=CXCRl/2 inhibitor, Error bars indicate mean with SEM. (5D) Pie graphs of the percent of cells with a given surface marker that also expression CXCR2. (5E) A bar graph of the percent of CD8+ infiltrating cells that express CXCR1.
[049] Figures 6A-6B: (6A) Representative images of LLC subcutaneous tumors grown in WT host (left) and in Lnk_/ host (right) mice. The image was taken after tumor harvest at day 14 of tumor growth. (6B) A scatter plot of LLC tumor weights from WT and Lnk KO mice.
DETAILED DESCRIPTION OF THE INVENTION
[050] The present invention, in some embodiments, provides cells comprising an exogenous protein and decreased SH2B adapter protein 3 (SH2B3) protein expression and/or function. The present invention further concerns pharmaceutical composition comprising those cells as well as a method of treating a disease in a subject comprising administering those cells or compositions. Uses of the cells of the invention, as well as methods of determining suitability to be treated by a method of the invention are also provided.
[051] By a first aspect, there is provided a cell comprising decreased SH2B adapter protein 3 (SH2B3) expression and/or function.
[052] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is a mouse cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is from a subject. In some embodiments, Ύ,O ¾ IZΉ 2 i n need of immunotherapy. In some embodiments, th?^Jujt¾?2¾^?°? ¾ d of adoptive immunotherapy. In some embodiments, the subject comprises healthy immune cells. In some embodiments, the subject comprises immune cells with reduced ability to induce an immune response. In some embodiments, the cells are allogenic to the subject. In some embodiments, the cells are autologous to the subject. In some embodiments, the cells are syngeneic to the subject.
[053] In some embodiments, the cell is an immune cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is not a hematopoietic cell. In some embodiments, the immune cell is a T-cell. In some embodiments, the T-cell is a CD8 positive T-cell. In some embodiments, the T-cell is a cytotoxic CD8 positive T-cell. In some embodiments, the T-cell is a CD4 positive T-cell. In some embodiments, the T-cell is a CD4 positive helper T-cell. In some embodiments, the immune cell is a cytotoxic immune cell. In some embodiments, the T-cell is selected from a CD8 positive and a CD4 positive T-cell. In some embodiments, the T-cell is a CD8 positive T-cell, a CD4 positive T-cell or both. In some embodiments, the immune cell is a B-cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a neutrophil. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the immune cell is a myeloid derived suppressor cell (MDSC). In some embodiments, the cell is selected from a T-cell, a B-cell, an NK cell, a neutrophil, a dendritic cell, an MDSC and a macrophage. In some embodiments, the cell is selected from a T-cell, a B-cell, an NK cell, a neutrophil, a dendritic cell, and a macrophage.
[054] In some embodiments, SH2B3 is mammalian SH2B3. In some embodiments, SH2B3 is rodent SH2B3. In some embodiments, SH2B3 is mouse SH2B3. In some embodiments, SH2B3 is human SH2B3.
[055] In some embodiments, mouse SH2B3 is SH2B3 isoform 1. In some embodiments, mouse SH2B3 is SH2B3 isoform 2. In some embodiments, mouse SH2B3 is SH2B3 isoform XI. In some embodiments, mouse SH2B3 comprises and/or consists of an amino acid sequence selected from those provide in RefSeq IDs NP_032533.1, NP_001293055.1, NP_001293056.1, NP_001293057.1 and XP_006530233.1. In some embodiments, mouse SH2B3 comprises and/or consists of an amino acid sequence provide in RefSeq IDs NP_032533.1, NP_001293055.1, NP_001293056.1, NP_001293057.1 or XP_006530233.1. Each possibility represents a separate embodiment of the invention. In some embodiments, mouse SH2B3 comprises and/or consists of the amino acid sequence provided in RefSeq ID NP_032533.1. In some embodiments, mouse SH2B3 comprises and/or consists of the amino WO 2020/174472 PCT/IL2020/05 a02^u18u„C„ll^C
MNEPT V QPS RTS S APAS PAS PRGW S DFCEQH A A A A AREL ARQ YWLF ARAHPQPPR ADLVSLQFAELFQRHFCREVRESLAGPPGHDYRATAPPRPALPKARSSEDLGPRPA C ALQHLRRGLRQLFRRRS AGELPG AT S DTNDIDTT A AS RPGP ARKLLPW GLREPPT E ALKE V VLRY S L ADE A AMDS G ARW QRGRL VLRS PGPGHS HFLQLFDPPKS S KPKL QE AC S S IRE VRPCTRLEMPDNLYTFVLKV QDQTDIIFE V GDEQQLN S WLAELR AS T GLGLEHPDTELPLSLAAEPGPARSPRGSTDSLDQGASPGVLLDPACQKTDHFLSCY PWFHGPIS RVRA AQLV QLQGPD AHG VFLVRQS ES RRGE Y VLTFNLQGRAKHLRL V LTERGQCR V QHLHFPS V VDMLRHF QRS PIPLEC G A ACD VRLS G Y V V VLS Q APGS S NT VLFPF S LPHWD SELGHPHLS S V GCPPS HGAE ALPGQ VTPPEQIFHLVPS PEELAN SLRQLELESVSSARDSDYDMDSSSRGHLRAIDNQYTPLSQLCREADV (SEQ ID NO:
1).
[056] In some embodiments, mouse SH2B3 is encoded by a nucleic acid molecule comprising or consisting of the nucleotide sequence selected from those provided in a RefSeq IDs NM_008507.4, NM 001306126.1, NM 001306127.1, NM_001306128.1 and XM_006530170.3. In some embodiments, mouse SH2B3 is encoded by a nucleic acid molecule comprising or consisting of the nucleotide sequence provided in a RefSeq ID NM_008507.4, NM 001306126.1, NM 001306127.1, NM_001306128.1 or
XM_006530170.3. Each possibility represents a separate embodiment of the invention. In some embodiments, mouse SH2B3 is encoded by a nucleic acid molecule that comprises and/or consists of the nucleotide sequence provided in RefSeq ID NM_008507.4. In some embodiments, mouse SH2B3 is encoded by a nucleic acid molecule that comprises and/or consists of the nucleotide sequence
ATGAACGAGCCCACCGTGCAGCCGTCCCGCACATCCTCCGCACCCGCCTCGCC GGCATCCCCACGCGGCTGGAGCGACTTCTGCGAGCAGCACGCAGCAGCGGCG GCCCGGGAGCTGGCCCGCCAGTACTGGTTGTTTGCGCGCGCGCACCCACAGCC GCCGCGCGCGGACCTGGTGTCGCTGCAGTTCGCGGAGCTCTTCCAGCGCCACT TCTGCCGGGAGGTGCGCGAGAGCCTCGCAGGACCGCCGGGTCACGACTACCGC GCCACTGCTCCGCCCCGCCCCGCGCTGCCCAAGGCACGCAGCTCCGAGGACCT GGGCCCGCGGCCCGCCTGTGCCCTGCAGCACCTGCGCCGCGGCCTGCGCCAGC TCTTCCGCCGCCGCTCGGCAGGGGAGCTGCCCGGGGCTACCAGTGACACCAAT GACATCGACACCACCGCAGCCAGCAGGCCGGGCCCGGCCCGCAAGTTGCTACC CTGGGGCCTGCGAGAGCCGCCCACTGAGGCGCTCAAGGAGGTCGTATTGCGCT ATAGCCTGGCGGACGAGGCAGCAATGGACAGCGGCGCACGCTGGCAGCGGGG ^^^i^GCTTCGGTCTCCAGGTCCGGGCCACAGCCACl^WSS^W^TT CGATCCGCCCAAGAGCTCAAAGCCCAAGCTCCAAGAGGCCTGTTCCAGCATCC GGGAGGTCCGACCATGTACACGCCTGGAGATGCCTGACAACCTCTACACCTTT GTGTTGAAGGTGCAGGACCAGACAGACATCATCTTTGAGGTGGGAGATGAACA GCAGCTGAACTCATGGCTGGCAGAGCTCAGGGCAAGCACAGGCCTTGGGCTGG AGCACCCGGACACCGAGTTACCTCTTTCCTTAGCGGCAGAGCCTGGCCCAGCT AGATCCCCAAGGGGAAGCACTGACTCCCTGGACCAAGGTGCTTCACCTGGGGT GTTGCTGGACCCAGCCTGCCAGAAAACAGATCACTTCCTATCCTGCTACCCCTG GTTCCACGGCCCCATCTCCAGGGTGAGGGCTGCACAGCTGGTCCAGCTCCAGG GCCCTGATGCCCACGGCGTGTTCCTGGTGCGGCAGAGTGAGTCCCGGAGAGGA GAGTATGTACTCACATTCAACTTACAGGGCAGAGCCAAGCACTTACGCCTGGT GCTCACAGAGCGTGGACAGTGCCGGGTGCAACACCTGCACTTCCCCTCGGTGG TAGATATGCTCCGCCACTTCCAGCGTTCTCCTATCCCACTGGAATGTGGAGCAG CTTGTGACGTCCGACTCTCTGGCTATGTGGTAGTCCTCTCTCAGGCACCAGGTT CCTCCAACACCGTCCTCTTCCCTTTTTCCCTTCCTCACTGGGATTCGGAGCTGGG TCATCCCCACCTCAGCTCTGTTGGCTGTCCCCCCAGCCATGGTGCAGAGGCTCT CCCTGGCCAAGTGACACCACCTGAGCAGATCTTCCACCTGGTGCCTTCTCCTGA GGAACTGGCCAACAGTCTGCGGCAGCTGGAGCTCGAGTCTGTGAGCAGTGCCC GGGACTCGGACTATGACATGGACTCCTCTTCACGGGGCCACCTTCGGGCCATT GACAACCAGTACACCCCTCTCTCACAGCTGTGCAGAGAGGCAGACGTGTGA
(SEQ ID NO: 2). In some embodiments, the nucleic acid molecule encoding SH2B3 is selected from a cDNA and an mRNA. In some embodiments, the nucleic acid molecule encoding SH2B3 is a cDNA. In some embodiments, the nucleic acid molecule encoding SH2B3 is an mRNA. It will be understood by a skilled artisan that when the molecule is an mRNA the thymines will be replaced by uracil.
[057] In some embodiments, human SH2B3 is SH2B3 isoform 1. In some embodiments, human SH2B3 is SH2B3 isoform 2. In some embodiments, human SH2B3 is SH2B3 isoform XI. In some embodiments, human SH2B3 is SH2B3 isoform X2. In some embodiments, human SH2B3 is SH2B3 isoform X3. In some embodiments, human SH2B3 is SH2B3 isoform X4. In some embodiments, human SH2B3 is SH2B3 isoform X5. In some embodiments, human SH2B3 is SH2B3 isoform X6. In some embodiments, human SH2B3 comprises and/or consists of an amino acid sequence selected from those provide in RefSeq IDs NP_005466.1, NP_001278353.1, XP_011536021.1, XP_011536022.1,
XP_005253875.1, XP_005253876.1, XP_011536023.1, XP_024304558.1, and Ύίί In some embodiments, human SH2B3 comprisesp£i ¾?0v?v9/il¾?sl¾f an amino acid sequence provide in RefSeq IDs NP_005466.1, NP_001278353.1, XP_011536021.1, XP_011536022.1, XPJ305253875.1, XP_005253876.1,
XP_011536023.1, XP_024304558.1, or XP_006719243.1. Each possibility represents a separate embodiment of the invention. In some embodiments, human SH2B3 comprises and/or consists of the amino acid sequence provided in RefSeq ID NP_005466.1. In some embodiments, human SH2B3 comprises and/or consists of the amino acid sequence MN GP ALQPS SPSS APS AS P A A APRGWSEFCELH A V A A AREL ARQ YWLFAREHPQH APLRAELVSLQFTDLFQRYFCREVRDGRAPGRDYRDTGRGPPAKAEASPEPGPGP A APGLPKARS S EEL APPRPPGPCS F QHFRRS LRHIFRRRS AGELP A AHT AA APGTPG E A AETP ARPGLAKKFLPW S L AREPPPE ALKE A VLRY S LADE AS MDS G ARW QRGRL ALRRAPGPDGPDRVLELFDPPKSSRPKLQAACSSIQEVRWCTRLEMPDNLYTFVLK VKDRTDIIFE V GDEQQLNS WM AELS ECT GRGLES TE AEMHIPS ALEPS TS S S PRGS T DS LN QG AS PGGLLDP ACQKTDHFLS C YPWFHGPIS RVKA AQLV QLQGPD AHG VFL VRQS ETRRGE Y VLTFNF QGI AKHLRLS LTERGQCRV QHLHFPS V VDMLHHF QRS PI PLECGAACDVRLSSYVVVVSQPPGSCNTVLFPFSLPHWDSESLPHWGSELGLPHLS S S GCPRGLS PEGLPGRS S PPEQIFHLVPS PEEL AN S LQHLEHEPVNR ARDS D YEMDS S S RS HLRAIDN Q YTPL (SEQ ID NO: 3).
[058] In some embodiments, human SH2B3 is encoded by a nucleic acid molecule comprising or consisting of the nucleotide sequence selected from those provided in a RefSeq IDs NM 005475.2, NM_001291424.1, XM_011537719.2, XM_011537720.3, XMJ305253818.4, XMJ305253819.4, XRJ301748535.1, XM_011537721.3,
XRJ301748536.1, XRJ302957278.1, XMJ324448790.1 and XMJ306719180.4. In some embodiments, human SH2B3 is encoded by a nucleic acid molecule comprising or consisting of the nucleotide sequence provided in a RefSeq ID NM_005475.2, NM_001291424.1, XM_011537719.2, XM_011537720.3, XMJ305253818.4, XMJ305253819.4,
XRJ301748535.1, XM_011537721.3, XRJ301748536.1, XRJ302957278.1,
XM_024448790.1 or XM_006719180.4. Each possibility represents a separate embodiment of the invention. In some embodiments, human SH2B3 is encoded by a nucleic acid molecule that comprises and/or consists of the nucleotide sequence provided in RefSeq ID NM_005475.2. In some embodiments, human SH2B3 is encoded by a nucleic acid molecule that comprises and/or consists of the nucleotide sequence ATGAACGGGCCTGCCCTGCAGCCCTCCTCGCCCTCTTCCGCGCCCTCAGCCTCC CCGGCGGCGGCCCCGCGGGGCTGGAGCGAGTTCTGTGAGTTGCACGCCGTAGC '¾L¾¾) J 1Zi½Z?GGGAGCTGGCCCGCCAGTACTGGCTGTTCGCC££ / 03^,}¾^GC AGCACGCGCCGCTGCGCGCCGAGCTGGTGTCGCTGCAGTTCACCGACCTCTTC CAGCGCTACTTCTGCCGCGAGGTGCGCGACGGACGGGCGCCGGGCCGCGACTA CCGGGACACAGGCCGTGGGCCCCCAGCCAAGGCCGAGGCGTCCCCGGAGCCA GGCCCCGGCCCCGCCGCCCCTGGCCTGCCCAAGGCCCGCAGCTCTGAGGAGCT GGCCCCGCCGCGGCCGCCCGGGCCCTGCTCCTTCCAGCACTTTCGCCGCAGCCT CCGCCACATCTTCCGCCGCCGCTCGGCCGGGGAGCTGCCAGCGGCCCACACCG CTGCCGCCCCCGGGACCCCCGGAGAGGCTGCTGAGACCCCCGCCCGGCCTGGC CTGGCCAAGAAGTTCCTGCCCTGGAGCCTGGCCCGGGAGCCGCCACCCGAGGC GCTGAAGGAGGCGGTGCTGCGCTACAGCCTGGCCGACGAGGCCTCCATGGACA GCGGGGCACGCTGGCAGCGCGGGAGGCTGGCGCTGCGCCGGGCCCCGGGCCC CGATGGCCCCGACCGCGTGCTGGAGCTCTTCGACCCACCCAAGAGTTCAAGGC CCAAGCTACAAGCAGCTTGCTCCAGCATCCAGGAGGTCCGGTGGTGCACACGG CTTGAGATGCCTGACAACCTTTACACCTTTGTGCTGAAGGTGAAGGACCGGAC AGACATCATCTTTGAGGTGGGAGACGAGCAGCAGCTGAATTCATGGATGGCTG AGCTCTCGGAGTGCACAGGCCGAGGGCTGGAGAGCACAGAAGCAGAGATGCA TATTCCCTCAGCCCTAGAGCCTAGCACGTCCAGCTCCCCAAGGGGCAGCACAG ATTCCCTTAACCAAGGTGCTTCTCCTGGGGGGCTGCTGGACCCGGCCTGCCAG AAGACGGACCATTTCCTGTCCTGCTACCCCTGGTTCCACGGCCCCATCTCCAGA GTGAAAGCAGCTCAGCTGGTTCAGCTGCAGGGCCCTGATGCTCATGGAGTGTT CCTGGTGCGGCAGAGCGAGACGCGGCGTGGGGAATACGTGCTCACTTTCAACT TTCAGGGGATAGCCAAGCACCTGCGCCTGTCGCTGACAGAGCGGGGCCAGTGC CGTGTGCAGCACCTCCACTTTCCCTCGGTCGTGGACATGCTCCACCACTTCCAG CGCTCGCCCATCCCACTCGAGTGCGGCGCCGCCTGTGATGTCCGGCTCTCCAGC TACGTGGTAGTCGTCTCCCAACCACCAGGTTCCTGCAACACGGTCCTCTTCCCT TTCTCCCTTCCTCACTGGGATTCAGAGTCCCTTCCTCACTGGGGTTCAGAGTTG GGCCTTCCCCACCTTAGTTCTTCTGGCTGTCCCCGGGGGCTCAGCCCAGAGGGT CTCCCAGGGCGATCCTCACCCCCCGAGCAGATCTTCCACCTGGTGCCTTCGCCC GAAGAACTGGCCAACAGCCTGCAGCACCTGGAGCATGAGCCTGTGAATCGAG CCCGGGACTCGGACTACGAAATGGACTCATCCTCCCGGAGCCACCTGCGGGCC ATAGACAATCAGTACACACCTCTCTGA (SEQ ID NO: 4). In some embodiments, the nucleic acid molecule encoding SH2B3 is selected from a cDNA and an mRNA. In some embodiments, the nucleic acid molecule encoding SH2B3 is a cDNA. In some embodiments, the nucleic acid molecule encoding SH2B3 is an mRNA. It will be understood by a skilled artisan that when the molecule is an mRNA the thymidines will be replaced by uracil.
Figure imgf000014_0001
74472 |C embodiments, the cell comprises decreased SH2£S1 ¾?>92?^?54?¾sion and/or function. In some embodiments, decreased SH2B3 protein expression and/or function is decreased SH2B3 protein expression, decreased SH2B3 protein function or both. In some embodiments, the cell comprises decreased SH2B3 expression. In some embodiments, the cell comprises decreased SH2B3 protein expression. In some embodiments, the cell comprises decreased SH2B3 protein levels. In some embodiments, the cell comprises decreased SH2B3 mRNA expression. In some embodiments, the cell comprises decreased SH2B3 mRNA levels. In some embodiments, the cell comprises decreased SH2B3 protein function. In some embodiments, a decreased is with respect to an unmodified cell. In some embodiments, an increase is with respect to an unmodified cell. In some embodiments, the unmodified cell and the cell of the invention are the same cell type and the cell of the invention has the decrease.
[060] In some embodiments, the cell lacks SH2B3 protein expression. In some embodiments, the cell is an SH2B3 knockout. In some embodiments, cell comprises a DNA alternation that results in reduced or non-existent SH2B3 protein expression. In some embodiments, the DNA alteration results in reduced or non-existent functional SH2B3 protein expression. In some embodiments, the DNA alteration is selected from a mutation, a deletion and an insertion. In some embodiments, the DNA alteration is a gene trap. In some embodiments, the DNA alteration results in negligible or no protein expression.
[061] In some embodiments, decreased SH2B3 function comprises increased C-X-C Motif Chemokine Receptor (CXCR) signaling. In some embodiments, the CXCR is CXCR1, CXCR2 and/or CXCR3. In some embodiments, decreased SH2B3 function comprises increased CXCR1 signaling. In some embodiments, decreased SH2B3 function comprises increased CXCR2 signaling. In some embodiments, decreased SH2B3 function comprises increased CXCR1 and/or CXCR2 signaling. In some embodiments, decreased SH2B3 function comprises increased Osteopontin (OPN) receptor signaling. In some embodiments, the OPN receptor is CD44. In some embodiments, the OPN receptor is an integrin receptor. In some embodiments, the integrin receptor is alpha 4/beta 1 (a4b1), a9b1 or a9b4. Each possibility represents a separate embodiment of the invention. In some embodiments, the signaling is downstream signaling. In some embodiments, decreased SH2B3 function does not comprise increased receptor expression. In some embodiments, receptor expression is total expression. In some embodiments, receptor expression is surface expression. In some embodiments, decreased SH2B3 function comprises increased phosphorylated effector protein. In some embodiments, the effector protein is Signal Transducer and Activator of ^92P¾ ^7 723 (STAT3). In some embodiments, the effector protE£Ti¾L2P2S 5¾¾¾ular signal-regulated kinas (ERK). In some embodiments, the effector protein is a mitogen- activated protein kinase (MAPK). In some embodiments, the effector protein is Erkl and/or ERK2. In some embodiments, the ERK is ERK1. In some embodiments, the ERK is ERK2. In some embodiments, the ERK is selected from ERK1 and ERK2. In some embodiments, the effector protein is MAPK3. In some embodiments, the effector protein is STAT3, STAT5, AKT, MEK, ERK, MAPK, JAK2, JAK3, JAK, FLT3, P38, SRC, Paxillin, FAK, PLC or PKC. Each possibility represents a separate embodiment of the invention. In some embodiments, decreased SH2B3 function does not comprise increased total effector protein levels.
[062] In some embodiments, the cell comprises a SH2B3 inhibitor molecule. In some embodiments, decreased SH2B3 protein expression and/or function comprises expression of an SH2B3 inhibitor molecule. In some embodiments, the SH2B3 inhibitor molecule is selected from a small nucleic acid molecule that hybridizes to and inhibits SH2B3 mRNA transcription and/or translation, a small molecule inhibitor of SH2B3 protein, an anti-SH2B3 antibody or antigen binding fragment, and a genome editing agent for mutation of SH2B3. In some embodiments, the SH2B3 inhibitor molecule is a small nucleic acid molecule that hybridizes to and inhibits SH2B3 mRNA transcription and/or translation, a small molecule inhibitor of SH2B3 protein, an anti-SH2B3 antibody or antigen binding fragment, or a genome editing agent for mutation of SH2B3.
[063] In some embodiments, the SH2B3 inhibitor molecule is a small nucleic acid molecule. In some embodiments, the small nucleic acid molecule hybridizes to and inhibits SH2B3 mRNA transcription and/or translation. In some embodiments, the small nucleic acid molecule binds to the SH2B3 genomic locus and inhibits transcription. In some embodiments, the small nucleic acid molecule binds to SH2B3 mRNA and inhibits translation. In some embodiments, inhibiting translation comprises inducing mRNA degradation. In some embodiments, the small nucleic acid molecule is single stranded. In some embodiments, the small nucleic acid molecule is double stranded. In some embodiments, the small nucleic acid molecule is single stranded or double stranded. In some embodiments, the small nucleic acid molecule that hybridized to and inhibits SH2B3 is selected from a microRNA (miR), small interfering RNA (siRNA), an RNAi agent, an iRNA agent and a short hairpin RNA (shRNA). In some embodiments, the small nucleic acid molecule that hybridized to and inhibits SH2B3 is a miR, a siRNA, an RNAi agent, an iRNA or an shRNA. Each possibility represents a separate embodiment of the invention. In some !¾ 7-,? the small nucleic acid molecule induces RNA interl££jyifc¾?i^S5921?3. In some embodiments, the small nucleic acid molecule that hybridized to and inhibits SH2B3 is an anti-sense RNA. In some embodiments, the anti-sense RNA is an RNA comprising sequence that is antisense to a SH2B3 mRNA. Examples of such nucleic acid molecules can be found, for example, in WO2013/019857, the contents of which are incorporated herein by reference in their entirety.
[064] In some embodiments, the small nucleic acid molecule comprises at least 5, 7, 10, 13, 15, 17, 19, 21, 23 or 25 nucleotides. Each possibility represents a separate embodiment of the invention. In some embodiments, the small nucleic acid molecule comprises at most 15, 17, 19, 21, 23, 24, 25, 26, 27, 29, 31, 33, 35, 37, 39 or 40 nucleotides. Each possibility represents a separate embodiment of the invention. In some embodiments, the small nucleic acid molecule comprises between 13 and 35, 13 and 33, 13 and 31, 13 and 29, 13 and 27, 13 and 26, 13 and 25, 13 and 24, 13 and 23, 13 and 21, 13 and 19, 15 and 35, 15 and 33, 15 and 31, 15 and 29, 15 and 27, 15 and 26, 15 and 25, 15 and 24, 15 and 23, 15 and 21, 15 and 19, 17 and 35, 17 and 33, 17 and 31, 17 and 29, 17 and 27, 17 and 26, 17 and 25, 17 and 24, 17 and 23, 17 and 21, 17 and 19, 19 and 35, 19 and 33, 19 and 31, 19 and 29, 19 and 27, 19 and 26, 18 and 25, 19 and 24, 19 and 23, or 19 and 21. Each possibility represents a separate embodiment of the invention.
[065] In some embodiments, the nucleic acid molecule is brought into the cell as part of a plasmid and/or vector that expresses the nucleic acid molecule. Methods of expressing nucleic acids in a cell are well known in the art and include, for example, electroporation, liposomal transfection, viral transfection, lentiviral infection, and calcium-phosphate transfection. The nucleic acid molecules, such as for example shRNAs, can be directly transferred to the cell, or an expression vector can be used to cause transcription of the molecule with the cell. In some embodiments, the cell comprises the small nucleic acid molecule. In some embodiments, the cell comprises an expression vector comprising or encoding the small nucleic acid molecule. In some embodiments, the expression vector is a mammalian expression vector.
[066] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence. 7ί lecto may be a DNA plasmid delivered via non-vira? i3^¾?Si0 S5( v¾8 viral methods. The viral vector may be a lentiviral vector, retroviral vector, a herpes viral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector. The promoters may be active in mammalian cells. The promoters may be a viral promoter.
[068] In some embodiments, the small nucleic acid molecule, or a sequence that encodes the small nucleic acid molecule or a polypeptide is operably linked to a promoter. The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
[069] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), Heat shock, liposomes, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et ah, Nature 327. 70-73 (1987)), and/or the like.
[070] The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
[071] In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.
[072] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK- RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
[073] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, exemplary vectors include pMSG, pAV009/A+, pM?&i i½¾ 0 ¾¾¾¾neo- 5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
[074] In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
[075] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et ah, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et ah, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et ah, Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et ah, Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
[076] It will be appreciated that other than containing the necessary elements for the transcription, the expression construct can also include sequences engineered to optimize stability, production, purification, yield, protein translation or activity of the nucleic acid molecule or polypeptide.
[077] A person with skill in the art will appreciate that a nucleic acid construct administered to the cell employing any suitable mode of administration, described hereinabove. In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) ^9u¾?° ¾117Aίoh system as needed and then the modified cells are and optionally administered to an individual.
[078] In some embodiments, the decrease is at least a 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 100% decrease. Each possibility represents a separate embodiment of the invention. Detecting expression of protein and mRNA is well known in the art, and any known method can be used for determining a decrease. Examples of assays that may be used include, but are not limited to, rtPCR, real-time PCR, qPCR, immuno staining, in situ hybridization, western-blotting and ELISA.
[079] In some embodiments, the SH2B3 inhibitor molecule is a small molecule inhibitor of SH2B3. In some embodiments, the SH2B3 inhibitor molecule is an anti-SH2B3 antibody or antigen binding fragment thereof. Anti-SH2B3 antibodies are well known in the art and may be purchased from retailers such as Thermo Fisher, Sigma Aldrich, R&D Systems and Millipore. In some embodiments, the small molecule inhibitor is an SH2B3 antagonist. In some embodiments, the antibody and/or antigen binding fragment is an SH2B3 antagonist. In some embodiments, the small molecule inhibitor, antibody, or antigen binding fragment binds to at least one of protein interacting domain of SH2B3. In some embodiments, the small molecule inhibitor, antibody, or antigen binding fragment occludes a protein interacting domain of SH2B3. In some embodiments, the small molecule inhibitor, antibody, or antigen binding fragment is a competitive antagonist. In some embodiments, the protein interacting domain of SH2B3 is selected from a Pleckstrin Homolog (PH) domain, and a Src Homolog 2 (SH2) domain of SH2B3. In some embodiments, the PH domain comprises and/or consists of amino acids 194-307 of SEQ ID NO: 3. In some embodiments, the PH domain comprises and/or consists of amino acids 206-307 of SEQ ID NO: 3. In some embodiments, the SH2 domain comprises and/or consists of amino acids 364-462 of SEQ ID NO: 3. Suitable antibodies may be generated by techniques known in the art. Animals may be immunized with the domains listed above, or with fragments of those domains or peptides containing those domains.
[080] The term“antagonist” generally refers to a molecule, compound or agent that binds to a target protein at the same site as an agonist, ligand or interacting protein but which does not activate the intracellular response initiated by the active form of the protein, and as such an antagonist can inhibit the intracellular responses by agonists.
[081] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that include at least one binding domain that is formed from the folding of polypeptide three-dimensional binding spaces with internal surfiK£T7¾ P¾0 ?¾2i?arge distributions complementary to the features of an antigenic determinant of an antigen. An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light/heavy chain pair form an antibody binding site. An antibody may be oligoclonal, polyclonal, monoclonal, chimeric, camelised, CDR-grafted, multi- specific, bi-specific, catalytic, humanized, fully human, anti- idiotypic and antibodies that can be labeled in soluble or bound form as well as fragments, including epitope-binding fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences. An antibody may be from any species. The term antibody also includes binding fragments, including, but not limited to Fv, Fab, Fab', F(ab')2 single stranded antibody (svFC), dimeric variable region (Diabody) and disulphide-linked variable region (dsFv). In particular, antibodies include immunoglobulin molecules and immunologic ally active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Antibody fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof. The skilled artisan will further appreciate that other fusion products may be generated including but not limited to, scFv- Fc fusions, variable region (e.g., VL and VH)~ Fc fusions and scFv-scFv-Fc fusions.
[082] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.
[083] In some embodiments, the SH2B3 inhibitor molecule is a genome editing agent. In some embodiments, the genome editing agent is for excision of SH2B3. In some embodiments, the genome editing agent is for mutation of SH2B3. In some embodiments, mutation of SH2B3 is mutation of the SH2B3 genomic locus. In some embodiments, mutation of the SH2B3 genomic locus results in reduced expression of SH2B3 protein. In some embodiments, mutation of the SH2B3 genomic locus results in no expression of SH2B3 protein.
[084] Genome editing agents are well known in the art. Examples include, but are not limited to CRISPR/Cas9, transcription activator-like effector nucleases (TALEN), zinc finger nucleases, and meganucleases. These agents can be employed for specific modification of a given genomic locus, or even nucleotide. This can be made specific to SH2B3, for examples with a guide RNA specific to a location in the SH2B3 gene. Examples of agents for genomic editing of SH2B3 to decrease its expression can be found for example in WO2016/085934, the contents of which are herein incorporated by reference in their 3J} 7iiZ? C RNAs (gRNAs) for targeting SH2B3 and decr£SI( L¾!¾]/®¾?iS are commercially available from vendors such as GenScript and plasmids with the gRNA can be obtained from abmXchange for example.
[085] In some embodiments, the cell further comprises an exogenous protein. In some embodiments, the exogenous protein does not decrease SH2B3 protein and/or function. In some embodiments, the exogenous protein is a synthetic protein. In some embodiments, the exogenous protein is a chimeric protein. In some embodiments, the exogenous protein is a receptor. In some embodiments, the exogenous protein is a surface protein. In some embodiments, the exogenous protein is a chimeric antigen receptor (CAR). In some embodiments, the exogenous protein is a targeting moiety. In some embodiments, the exogenous protein is expressed on the surface of the cell. In some embodiments, the protein is a cytotoxic protein. In some embodiments, the protein is an anti-cancer protein. In some embodiments, the protein is an immune stimulating protein. In some embodiments, the protein binds to a target antigen. In some embodiments, the protein binds to a target antigen on a pathogen. In some embodiments, the protein binds to a target antigen on a pathological cell. In some embodiments, the protein binds to a target antigen on a cell associated with a disease.
[086] In some embodiments, the cell is a CAR cell. In some embodiments, the cell is a CAR-T cell. In some embodiments, the cell is a CAR-NK cell. As used herein, the terms "CAR-T cell” and“CAR-NK cell” refer to an engineered receptor which has specificity for at least one protein of interest (for example an immunogenic protein with increased expression following treatment with an epigenetic modifying agent) and is grafted onto an immune effector cell (a T cell or NK cell). In some embodiments, the CAR- T cell has the specificity of a monoclonal antibody grafted onto a T-cell. In some embodiments, the CAR-NK cell has the specificity of a monoclonal antibody grafted onto a NK-cell. In some embodiments, the T cell is selected from a cytotoxic T lymphocyte and a regulatory T cell.
[087] CAR-T and CAR-NK cells and their vectors are well known in the art. Such cells target and are cytotoxic to the protein for which the receptor binds. In some embodiments, a CAR-T or CAR-NK cell targets at least one viral protein. In some embodiments, a CAR-T or CAR-NK cell targets a plurality of viral proteins. In some embodiments, a CAR-T or CAR-NK cell targets a viral protein with increased expression due to contact with an epigenetic modifying agent. ^Poo®20d¾ioT?uction °f CAR-T cells is well known in the art. In one? u!^?S? ig552i?iple, a monoclonal antibody to a viral protein can be made and then a vector coding for the antibody will be constructed. The vector will also comprise a costimulatory signal region. In some embodiments, the costimulatory signal region comprises the intracellular domain of a known T cell or NK cell stimulatory molecule. In some embodiments, the intracellular domain is selected from at least one of the following: CD3Z, CD27, CD28, 4-l BB, 0X40, CD30, CD40, PD- 1, ICOS, lymphocyte function-associated antigen- 1 (LFA- 1), CD2, CD 7, LIGHT, NKG2C, B7- H3, and a ligand that specifically binds with CD83. In some embodiments, the vector also comprises a CD3Z signaling domain. This vector is then transfected, for example by lentiviral infection, into a T-cell.
[089] The term“moiety” as used herein refers to a part of a molecule, which lacks one or more atom(s) compared to the corresponding molecule. The term“moiety”, as used herein, further relates to a part of a molecule that may include either whole functional groups or parts of functional groups as substructures. The term“moiety” further means part of a molecule that exhibits a particular set of chemical and/or pharmacologic characteristics which are similar to the corresponding molecule, i.e. fluorescence. For example, a fluorescent moiety is part or all of a fluorescent molecule that retains the ability to fluoresce.
[090] As used herein, a“targeting moiety” is a moiety that binds to or interacts with a target cell, and thereby can bring the cell of the invention to the target cell. In some embodiments, a targeting moiety is an antibody or antigen binding fragment thereof that binds a surface protein on the target cell. In some embodiments, the targeting moiety is a ligand or binding partner of a receptor on the target cell. In some embodiments, the targeting moiety binds to a target antigen on a target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a pathogen. In some embodiments, the pathogen is selected from a bacterium, a fungus, a virus, a parasite and a cancer.
[091] In some embodiments, the cell of the invention is produced by extracting a cell from a subject and decreasing SH2B3 protein expression and/or function in the extracted cell in vitro. In some embodiments, the immune cell is obtained from the subject. In some embodiments, SH2B3 protein expression and/or function is decreased in the obtained cell. In some embodiments, the production further comprises expressing in the cell the exogenous protein. In some embodiments, the cell is expanded in vitro.
[092] By another aspect, there is provided a composition comprising a cell of the invention. 'YS½< j 20x¾7t‘tZ? e embodiments, the composition is a pharmaceutical ^^({feTSi^P^^^ome embodiments, the composition is configured for administration to a subject. In some embodiments, the composition is configured for systemic administration. In some embodiments, the composition is configured for intravenous administration. In some embodiments, the composition is configured for intratumoral administration.
[094] By another aspect, there is provided a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a cell or composition of the invention, thereby treating the disease.
[095] By another aspect, there is provided a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a cell comprising decreased SH2B3 protein expression and/or function, thereby treating the disease.
[096] By another aspect, there is provided a cell of the invention for use in treating a disease in a subject in need thereof.
[097] By another aspect, there is provided a cell with decreased SH2B3 protein expression and/or function for use in treating a disease in a subject in need thereof.
[098] In some embodiments, the disease is a pathogenic disease. In some embodiments, the disease is a disease treatable by immunotherapy. In some embodiments, the disease is an immune-associated disease. In some embodiments, an immune-associated disease is a disease treatable by immunotherapy. In some embodiments, the disease is a disease that induces IL-8 expression. In some embodiments, the induced IL-8 expression is proximal to the site of disease. In some embodiments, the disease comprises a cell with targetable protein expression. In some embodiments, the disease comprises a cell associated with the disease. In some embodiments, the targetable protein expression is surface protein expression. In some embodiments, the disease is cancer. In some embodiments, the disease is Myelodysplastic syndrome. In some embodiments, the disease is a bacterial infection. In some embodiments, the disease is a fungal infection. In some embodiments, the disease is a viral infection. In some embodiments, the disease is a parasitic infection. It will be understood by one skilled in the art that the exogenous protein may be tailor based on the disease to be treated. A targeting moiety may be selected to target the cell of the invention to a pathogen or cancer cell. Bacterial, viral and fungal proteins are well known in the art, as are cancer specific surface markers. Antibodies, and binding molecules that can be used as targeting moieties are also well known. CARs may be used for targeting cancer cells, and the like. 7 W c embodiments, the cell associated with the disease is ?ίiϊ ¾20A¾R±?¾? lancer cell, a precancerous cell, a bacterial cell, a fungal cell, a parasite cell, and a cell infected with a virus. In some embodiments, the cell associated with the disease is selected from a cancer cell, a bacterial cell, a fungal cell, a parasite cell, and a cell infected with a virus. In some embodiments, the cell associated with the disease is a cancer cell. In some embodiments, the cell associated with the disease is a bacterial cell. In some embodiments, the cell associated with the disease is fungal cell. In some embodiments, the cell associated with the disease is a parasite cell. In some embodiments, the cell associated with the disease is a cell infected with a virus. In some embodiments, the cell associated with the disease is an IL-8 expressing cell.
[0100] In some embodiments, the method further comprises providing the cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is from the subject. In some embodiments, the cell is autologous to the subject. In some embodiments, the cell is allogenic to the subject. In some embodiments, the cell is syngeneic to the subject. In some embodiments, the cell is from a healthy donor. In some embodiments, the provided cell is cultured in vitro. In some embodiments, the provided cell is expanded in vitro. In some embodiments, the cell is further enhanced in vitro to improve its immunotherapeutic potential. Such alterations may include gene knockout, or genetic modification or expression of a protein or polypeptide. In some embodiments, the cell is returned to the subject. In some embodiments, the cell is administered to the subject.
[0101] In some embodiments, the method is a method of immunotherapy. In some embodiments, immunotherapy is adoptive immunotherapy. In some embodiments, the method is a method of adoptive immunotherapy. In some embodiments, the method is combined with another method of immunotherapy. In some embodiments, cells to be used for immunotherapy are modified by decreasing SH2B3 protein expression and/or functional levels in the cells. In some embodiments, decreasing SH2B3 levels enhances the therapeutic potential of the cells. In some embodiments, the therapeutic potential is immunotherapeutic potential.
[0102] As used herein, the term“therapeutic potential” refers to a cells ability to treat and or prevent a disease, disorder or pathological condition as well as improve a subj ecf s overall condition. As used herein, the terms “immunotherapeutic potential” and “therapeutic immune cell potential” are synonymous and refer to the qualitative and/or quantitative therapeutic potential of a beneficial T cell to be, or to become, therapeutic toward a subject’s Q J } some embodiments, the therapeutic potential is anyPu!iP<?®¾®g¾¾¾;ing, repairing, replacing, preventing, or protecting potential or a combination thereof.
[0103] In some embodiments, administering is administering at least 100, 500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 10000000, 50000000, 100000000, 500000000, 1000000000, or 5000000000 cells. Each possibility represents a separate embodiment of the invention. In some embodiments, the administering is repeated. In some embodiments, administering is administering at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. Each possibility represents a separate embodiment of the invention.
[0104] As used herein, the terms“administering,”“administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for oral administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration can include parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal.
[0105] The dosage administered 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.
[0106] In some embodiments, the method further comprises administering to the subject another therapeutic treatment. In some embodiments, the therapeutic treatment is an anti cancer therapeutic treatment. In some embodiments, the therapeutic treatment is an anti pathogen therapeutic treatment. In some embodiments, a pathogen is selected from a bacterium, a fungus, a virus and a parasite. In some embodiments, a pathogen is a bacterium. In some embodiments, a pathogen is a virus. In some embodiments, a pathogen is a fungus. In some embodiments, a pathogen is a parasite. In some embodiments, the method further comprises administering to the subject an anticancer or anti-pathogen therapeutic treatment. In some embodiments, the method is a combined treatment with a second therapeutic treatment. In some embodiments, the second therapeutic treatment is a different therapeutic modality from an immunotherapy treatment. In some embodiments, the second therapeutic treatment is a different therapeutic modality from a treatment with immune cells. In some embodiments, the therapeutic treatment is selected from the group consisting of: irradiation, chemotherapy, immune checkpoint modulation, CAR therapy, adoptive T cell therapy, small molecule biologies, surgery, bone marrow transplant, targeted drug therapy, cellular ablation ^ai v?¾P ai¾iZ?<tic. In some embodiments, the therapeutic treatment P Ti¾?2?¾P?0?1{lorne embodiments, the therapeutic treatment is chemotherapy. In some embodiments, the therapeutic treatment is immune checkpoint modulation. In some embodiments, the therapeutic treatment is CAR therapy. In some embodiments, the therapeutic treatment is adoptive T cell therapy. In some embodiments, the therapeutic treatment is a small molecule biologic. In some embodiments, the therapeutic treatment is surgery. In some embodiments, the therapeutic treatment is bone marrow transplant. In some embodiments, the therapeutic treatment is targeted drug therapy. In some embodiments, the therapeutic treatment is cellular ablation. In some embodiments, the therapeutic treatment is an antibiotic.
[0107] In some embodiments, immune checkpoint modulation comprises immune checkpoint blockade. In some embodiments, immune checkpoint blockade comprises administering an immune checkpoint inhibitor. In some embodiments, immune checkpoint blockade is intrinsic checkpoint blockade. In some embodiments, the immune checkpoint is selected from PD-1, PD-L1, CTLA-4 and HVEM. In some embodiments, the immune checkpoint is selected from PD-1, PD-L1, and CTLA-4. Immune checkpoint inhibitors are well known in the art and include, but are not limited to, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab.
[0108] In some embodiments, the other therapeutic treatment is administered before a cell of the invention. In some embodiments, the other therapeutic treatment is administered after a cell of the invention. In some embodiments, the other therapeutic treatment is administered concomitantly with a cell of the invention. In some embodiments, the other therapeutic treatment enhances the effect of a cell of the invention. In some embodiments, the other therapeutic treatment has an additive effect on the efficacy of a cell of the invention.
[0109] By another aspect, there is provided a method for improving a cell to be used for immunotherapy, the method comprising decreasing SH2B3 expression and/or function in the cell, thereby improving the cell to be used for immunotherapy.
[0110] By another aspect, there is provided a method for enhancing an immune response of an immune cell, the method comprising decreasing SH2B3 expression and/or function in the immune cell, thereby enhancing an immune response of an immune cell.
[0111] In some embodiments, the method is performed in vitro. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in culture. In some embodiments, the method is performed in vivo. In some embodiments, the immune ^9 9?¾1Z5A7?o be used for immunotherapy. In some embodiments, ^?vJ ¾?¾?/5u93A?py is adoptive immunotherapy.
[0112] In some embodiments, enhancing an immune response comprises increasing cytotoxicity of the immune cell. In some embodiments, enhanced cytotoxicity comprises enhanced phosphorylation of STAT3 in the cell. In some embodiments, enhanced cytotoxicity comprises enhanced expression of a proinflammatory cytokine. In some embodiments, the proinflammatory cytokine is interferon gamma. Proinflammatory cytokines are well known in the art, and include, but are not limited to 11-2, interferon gamma, TNF alpha, IL- 1 and granulocyte-macrophage colony stimulation factor. Increases expression of any proinflammatory cytokine known in the art may be a readout for enhanced cytotoxicity. In some embodiments, enhancing an immune response comprises increasing migration of an immune cell. In some embodiments, enhancing an immune response comprises increasing invasion of an immune cell. In some embodiments, increasing migration comprises increasing invasion. In some embodiments, migration is to a site of disease. In some embodiments, a site of a disease is a tumor. In some embodiments, a site of disease is a site of infection. In some embodiments, the site of disease is a site with enhanced IL-8 expression. In some embodiments, the site of the disease comprises IL-8 expression. In some embodiments, invasion is to a site of disease. In some embodiments, enhancing an immune response comprises increasing proliferation of the immune cell. In some embodiments, enhancing an immune response comprises increasing the ratio of CD8 T cells to CD4 T cells present in a population of immune cells. In some embodiments, the immune cell is a population of immune cells.
[0113] By another aspect, there is provided a method for determining suitability of a subject to receive treatment, the method comprising: a. receiving a cell from the subject; and b. assaying chemotaxis of an unmodified immune cell and an immune cell comprising decreased SH2B3 protein expression and/or function to the pathological cell; wherein improved chemotaxis of the immune cell comprising decreased SH2B3 protein expression and/or function as compared to the unmodified immune cell indicates the subject is suitable for a treatment of the invention, thereby determining suitability of a subject to receive treatment. Ύ92 \ 7 ¾?2,c embodiments, the treatment is a treatment compi?9Tg I¾?¾{??PulS cell comprising decreased SH2B3 protein expression and/or function. In some embodiments, the treatment is a treatment comprising administering an immune cell comprising decreased SH2B3 protein expression and/or function. In some embodiments, the treatment is a treatment comprising decreasing SH2B3 protein expression or function in an immune cell. In some embodiments, the immune cell is an immune cell of the subject. In some embodiments, the treatment is a treatment method of the invention.
[0115] In some embodiments, the cell is a tumor cell and a tumor biopsy is received. In some embodiments, the cell is put in culture. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in a laboratory animal. Examples of laboratory animals include, but are not limited to, mice, rats, rabbits, and cats. In some embodiments, the laboratory animal is a mammal. In some embodiments, the laboratory animal is a mouse. In some embodiments, the laboratory animal is immune comprised. In some embodiments, the immune compromised animal is a SCID mouse. In some embodiments, the immune compromised animal is a NOD mouse. In some embodiments, the immune compromised animal is a SCID/NSG mouse. In some embodiments, the assaying chemotaxis is chemotaxis in cell culture. In some embodiments, the assaying chemotaxis is chemotaxis in a laboratory animal. In some embodiments, assaying chemotaxis is a migration assay. In some embodiments, assaying chemotaxis comprises a Transwell assay. In some embodiments, assaying chemotaxis comprises culturing the cell in one compartment of a Transwell and culturing the immune cell in another well of the Transwell dish. In some embodiments, the assaying chemotaxis comprises inserting the cell in a laboratory animal and administering the immune cells to the animal and assaying the ability of the immune cells to reach the cell in the animal. In some embodiments, the cell is injected. In some embodiments, the cell is injected subcutaneously. In some embodiments, the administered immune cells are injected. In some embodiments, a plurality of cells is received and at least one cell is used in a chemotaxis assay with the unmodified cell and at least one cell is used in a chemotaxis assay with the cell comprising decreased SH2B3 protein expression and/or function. In some embodiments, the chemotaxis assay comprises the setup of the“in vitro invasion assay” described herein. In some embodiments, the chemotaxis assay comprises the setup of the“in vitro Matrigel chemotaxis and invasion assay” described herein. In some embodiments, the in assay does not comprise addition of an inhibitor and/or a test molecule. Ύ9 79n72 lc embodiments, improved chemotaxis is increased ^X H^SK ^^ome embodiments, an increase is at least a 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500% increase. Each possibility represents a separate embodiment of the invention. In some embodiments, a decrease is at least a 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99 or 100% decrease. Each possibility represents a separate embodiment of the invention. In some embodiments, improved chemotaxis is an increase in the number of cells that reach the cell.
[0117] In some embodiments, the method further comprises treating the suitable subject. In some embodiments, the treatment is by any one of the methods of the invention.
[0118] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.
[0119] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.
[0120] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B".
[0121] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described ^Y9 ?S?9uZii ? of a single embodiment, may also be provided separafir ^fc^'i ii^li&able sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
[0122] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
[0123] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
[0124] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course 'Yv9a /L¾1/1 ¾7.2. L Press (1996); all of which are incorporated by re£9vJ^t?° ¾iS50 5 1v?ieral references are provided throughout this document.
Materials and Methods:
[0125] Mice model: Lnk_/_ mice (a gift from Tony Pawson's lab) were backcrossed 10 generations into C57BL/6J-Ly5.2-Gpilb/b [1], [2]. Wield-type (WT) C57BL/6J01aHsd mice were obtained from the Pre-Clinical Research Authority at the Technion Faculty of Medicine. Experiments were approved and mice were housed and manipulated according to the guidelines of the Animal Care and Use Committee of the Technion.
[0126] Cell lines: B 16-F10 murine melanoma cells were cultured in RPMI 1640 supplemented with 10% FCS, 1% L-glutamine and 1% Penicillin-Streptomycin. LL/2 (LLC1) murine Lewis Lung Carcinoma cells were cultured in DMEM supplemented with 10% FCS, 1% L-glutamine and 1% Penicillin-Streptomycin.
[0127] Tumor model: For the subcutaneous melanoma model, 1.4 x 106 B 16-F10 cancer cells were subcutaneously injected into WT or Lnk_/ mice at the rear back. After 14 days mice were euthanized and tumors, spleens and inguinal lymph nodes were harvested. The length and width of tumors were measured with a manual caliper prior to harvest and tumors were weighted post-harvest.
[0128] For the subcutaneous Lewis Lung Carcinoma model, 1.4 x 106 LL/2 (LLC1) cancer cells were used in the same manner.
[0129] Preparation of cells: Single cell suspensions of each organ were prepared as follows. Briefly, spleens and lymph nodes were ground and filtered through a 40pm cell strainer. Cells were washed and RBCs were lysed when necessary with ammonium chloride lysis buffer (0.15M NH4C1, O. lmM Na2EDTA, and lOmM KHC03). Cells were cultured in the proper media for the following experiment.
[0130] Tumors and Matrigel plugs were cut into small pieces and digested with Collagenase IV, DNase IV and Hyaluronidase V (Sigma- Aldrich) in HBSS for 60 min at 37°C with stirring [3]. Supernatants were collected and stored in -20°C for cytokine analysis. Cells were washed and RBCs were lysed when necessary with ammonium chloride lysis buffer. Recovered cells were used for flow cytometry analysis.
[0131] Flow Cytometry: Single cell suspensions (1-5 x 106 cells) were washed and suspended in cold PBS supplemented with 2% FCS, and ImM EDTA. The following fluorochrome-conjugated antibodies were purchased from BioLegend: Pacific Blue-, PE- or Ύί? £9-¾ίίί7ήί72se CD8a (clone 53-6.7); Pacific Blue- anti mouse CD4p£^J¾¾??^-S 7^PC- anti mouse CD 19 (clone 6D5); PerCP/Cy5.5-anti mouse CD182(CXCR2) (clone SA044G4); Alexa Fluor700- or PE-anti mouse CD3 (clone 17A2); FITC- or PE- anti mouse/human CD45R/B220 (clone RA3-6B2);
[0132] The following fluorochrome-conjugated antibodies were purchased from Miltenyi Biotec: PE-Vio770 anti mouse NK1.1 (clone PK136); FITC- anti mouse CD19 (clone 6D5). The following fluorochrome-conjugated antibodies were purchased from BD Biosciences: PE-anti mouse CD181(CXCR1) (cloneU45-632).
[0133] Fluorescent data was collected on LSR Fortessa cell analyzer (BD Biosciences). Fluorescent data of the modified chemotaxis assay was collected on Attune NxT acoustic focusing cytometer (Thermo Fisher Scientific). Results were analyzed using FlowJo software (Tree Star).
[0134] Histology and immunohistochemistry: Hematoxylin/eosin staining of representative tumors derived from WT or Lnk—/— and CD8a (D4W2Z) XP Rabbit mAh (#98941, Cell Signaling Technologies) immunohistochemistry on Tumor section is shown.
[0135] Cytokine analysis: Adjacent to the process of tumor dismantling into single-cell suspension, primary supernatants were collected and stored in -20°C for further analysis. The cytokine profile at the tumor microenvironment fluid was screened using Proteome Profiler Mouse XL Cytokine Array (R&D Systems; Cat. ARY028). The presence of selected cytokines was verified by ELISA, and their concentration was determined as well. Results were obtained and quantified using pixel density measurements using ImageJ software. The tumor- supernatant were collected and evaluated using ELISA for Mip-2(mouse MIP-2 ELISA; RayBiotech), IL-6, and IL-10 (ELISA MAX standard kit; BioLegend). The data was acquired and processed using ELISA reader and Gene5 software (biotek).
[0136] In-vivo Matrigel chemotaxis and invasion assay: An In-vivo study of adoptive cell transfer and Matrigel plug assay to determine the role of Lnk regulation in the migratory and invasion capacities of CD8+ T-cells. Experimental design: B 16-F10 melanoma cells were transplanted subcutaneously into WT and Lnk-/- donors. Spleen of tumor bearing donor (WT or Lnk-/-) was harvested on day 14, and CD8+ T-cells were isolated from the total splenocyte population. The isolated CD8+ T-cells (WT or Lnk-/-) were then covalently labeled with CellTrace Violet (C34557, Thermo Fisher Scientific), and transferred to WT recipient. Each WT recipient was transferred by spleen derived CD8+ T-cells from a single tumor bearing donor, either WT or Lnk-/-. 'YS> ±¾°2 j° 1ui1 wing cell transfer, recipient was subcutaneously i njcctcK^vT J ? nc carrying Matrigel-plugs at the rear flank, every plug containing a single chemokine on each flank, as illustrated.
[0138] Matrigel (Corning, 354234): The bilateral combination of chemokine containing plugs on a single recipient was either of the following [left plug:right plug] - watenwater; Mip-2:water; SDF-la:Mip-2. On day 15, chemokine boost was given to each Matrigel plug. On day 16, plugs were harvested, dissolved and analyzed by flow cytometry for the amount of fluorescent labeled CD8+ T-cells in each plug.
[0139] In vitro invasion assay: The role of Lnk regulation in the CXCRl/2-mediated migration of T-cells to melanoma was examined in vitro by a chemotaxis assay modified with Matrigel. 0.25 x 106 B16-F10 cells were cultured and incubated in a 24-well Cell Culture Receiver plate (M8812, Sigma- Aldrich), that was used as the lower wells of a Transwell systems. Inguinal lymph nodes were isolated from either WT or Lnk-/- melanoma tumor bearing mice and were either treated or not treated with the CXCRl/2 inhibitor SCH563705 (cat. A15234, AdooQ Bioscience) at a final concentration of 500nM/ 0.1 x 106 Cells. Lymph node cells were then placed into inserts with 3.0pm pore polyethylene terephthalate (PET) membrane (MCSP24H48, Millicell), that were used as the upper wells of the Transwell systems. Prior to incubation, the exterior portion of the membrane on the upper well was pre-coated with a thin layer of Matrigel (Corning, 354234).
[0140] The cells in the upper and lower wells were co-cultured for 16 hours. Cells were then collected off the lower well, stained for CD3, CD8 and CD4 markers and counted using an acoustic flow-cytometer.
[0141] Western blot: Western blot analysis of purified WT and KO Lnk cells. ERK and STAT3 phosphorylation and total ERK STAT3protein content were analyzed before and after stimulation with Mip2, IL-8 cytokines for 5 minutes. Obtained bends were quantified using ImageJ software (NIH). Columns of the left graph represent the ratio of p-STAT3 to total STAT3 over the background.
[0142] Statistical analysis: To compare tumor mass, size of infiltrating cell population, and surface marker expression we performed unpaired t test with Welch's correction. To compare in-vitro chemotaxis under CXCRl/2 inhibition we performed unpaired t test between treated and not-treated cell groups. To compare in-vivo cell infiltration into Matrigel plugs we performed ordinary one-way ANOVA with multiple comparisons post hoc. 'YS> ±¾¾0(J7AiZ?cally significant p values were labeled as follows: *pi> aT^LS? 9{050l1-8 al < 0.01; *** P-val < 0.001; **** P-val < 0.0001. The error bars indicate mean ± SEM. The data was analyzed using Prism software (GraphPad).
[0144] Intracellular INFy staining: Inguinal lymph nodes (FN) were harvested from either naive or tumor bearing mice, and single cell suspensions were prepared as described herein. A total of lxlO5 isolated FN cells were incubated ex-vivo for a total time of 6 hours in 5% FCS RPMI with 50U/ml IF-2 (PEPROTECH) and 5ug/ml Brefeldin A (BFA, BioFegend) alone, or were concurrently stimulated by adding PMA (Santa cruz) and Ionomycin (CAYMAN chemicals) to a final concentration of 50ng/ml and 750ng/ml, respectively. Following stimulation, cells were FC blocked (anti-CD 16/32; BioFegend), surface stained with CD3, CD8a and CD4 (17A2, 53-6.7 and RM4-5; BioFegend), fixed and permeabilized with CytoFix/CytoPerm (BD Biosciences), and stained for intracellular INFy (XMG1.2; BioFegend). Samples that were not stimulated with PMA and Ionomycin were used to asses specific individual cytokine production. Fluorescence minus one (FMO) controls excluding INFy were used in all experiments. Cells were acquired by flow cytometry (BD Biosciences HTS FSR Fortessa II) and analyzed using FlowJo (Tree Star). Geometric mean fluorescence intensity (gMFI) was normalized to the average expression of un-stimulated cells and expressed as fold induction.
Example 1: Lnk KO mice show decreased tumor size
[0145] SH2B adapter protein 3 (SH2B3), also known as lymphocyte adapter protein LNK, is a small adapter protein of molecular weight 66 kilodaltons and comprising 575 amino acids. The protein has two major domains, a 3’ phoshpotyrosine binding domain (Src homology 2, SH2) and a more 5’ membrane localization domain (pleckstrin homology domain, PH). Lnk regulates intracellular cytokine signaling by binding phosphorylated tyrosine residues on activated target proteins through its SH2 domain. Examples of such proteins include MAPK3, FLT3, KIT, JAK2, JAK3, LCK, PDGRFB, FMS, MPF, TRKA, PDGRFA and TCR-zeta. Binding of Fnk inhibits the signaling cascades downstream to the protein target. Moreover, Fnk functions broadly as a hub-inhibitor, by binding a variety of activated intracellular signaling molecules.
[0146] It was hypothesized that FNK may play a role in regulation of anti-tumor immune response. To test this hypothesis, wild-type (WT) and Fnk knockout (KO) mice (Fnk-/-) were subcutaneously injected with B 16-F10 melanoma cells and tumor growth was '¾L???v9 iZ4fu? 14 days. After 2 weeks, the animals were sacrificed STu¾S2?u?¾?J8 >vere harvested and measured. Tumor sections were fixed in 4% formaldehyde solution for histological and immunohistochemical analysis, while other sections of tumor were enzymatically dissolved into single-cell suspensions for flow cytometric analysis. Following the dissolution into single cells the supernatant was also collected in order to screen for the cytokine profile of the tumor microenvironment (TME). Cytokine measurement was performed by ELISA.
[0147] The tumors extracted from Lnk KO mice were visibly smaller than those from WT mice (Fig. 1A), with the average weight of tumors from KO mice being about one-third of the weight from WT mice (Fig. IB). FACS analysis of the single cell suspension from the tumors (Fig. 1C) indicated that tumors from Lnk KO mice had a statistically significant increase in tumor infiltrating lymphocytes (TIL) as compared to tumors from WT mice (Fig. ID). This increase was confirmed by FACS with lymphocyte- specific markers. Histological analysis confirmed the increased cellular infiltration into the interphase of tumors from Lnk KO mice (Fig. IE).
[0148] The population of infiltrating cells was further assayed in order to determine the identity of the cells. CD3-zeta chain positive T cells (Fig. IF) were stained for CD8a and CD4 surface expression (Fig. 1G). More than twice as many infiltrating cells were CD3-zeta positive in the tumors from Lnk KO mice. Similarly, the ratio of CD8+ cytotoxic T-cells to CD4 helper cells was more than 3 times higher in tumors from Lnk KO mice. Staining with an anti-CD8a antibody confirmed that the infiltrating cells seen in the interphase of the tumor from Lnk KO mice were indeed cytotoxic T cells (Fig. 1H, left). These cells were also seen in the core of the tumor (Fig 1H, right). The infiltrating cell population comprises increased numbers of total T-cells (CD3) (Fig. II), T-Helper cells (CD4) (Fig. 1J), Cytotoxic T cells (CD8) (Fig. IK), B-cells (CD45R) (Fig. 1L), NK cells (CD161b/c) (Fig. 1M), neutrophils (CDl lb) (Fig. IN), macrophages (F4/80) (Fig. lO), and dendritic cells (CDl lc) (Fig. IP).
[0149] Cytokine analysis in the TME did not show any obvious changes in secretion between tumors from WT and Lnk KO mice (Fig. IQ). The largest change observed was a non-significant increase in MIP-2 (IL-8) (Fig. 1R). It was hypothesized that LNK may function in CD8+ T cells downstream of CXCRl/2 activation, and act as a repressor of cellular activity. Thus, in the KO mice increased cellular activation would occur leading to the observed increase in cytotoxic T cell migration to the tumor and the resultant decrease in tumor size. increased cellular activation in CD8+ Lnk KO T celli*CT/IL2020/050218
[0150] MIP-2, and IL-8 both signal through the CXCR1 and CXCR2 receptors. To test the role of LNK in CXCR receptor signal transduction. Spleen-derived CD8+ T-cells of naive mice of both genotypes (WT or Lnk 7 ) were stimulated for 5 minutes with Mip-2 or human IL-8 prior to lysis and blotting. The phosphorylation of STAT3, as a readout for signal transduction, was measured using anti p-STAT3 antibody, and compared to the total level of STAT3 in the lysate (Fig. 2A). A significant increase in the phosphorylation of STAT3 was detected in Lnk 7 CD8+ T-cells after stimulation with Mip-2, a mouse homolog for human IL-8 (Fig. 2B). Next, spleen-derived CD8+ T-cells from mice of both genotypes bearing melanoma tumors were stimulated for 5 minutes with IL-8 prior to lysis and blotting (Fig. 2C). An increased basal phosphorylation of STAT3 was detected in Lnk 7 spleen- derived CD8+ T-cells of tumor bearing Lnk KO mice, and a further increase in the phosphorylation of STAT3 was seen after stimulation of these cells with IL-8 (Fig. 2D).When another signaling molecule, Erk (MAPK3), was examined (Fig. 2E), no basal increase was observed, but increased phosphorylation was observed in response to IL-8 in the Lnk KO splenic CD8+ cells (Fig. 2F).
[0151] In order to further test activation differences between Lnk KO and WT cells, lymph node cells were harvested from inguinal lymph nodes of WT and Lnk KO mice. Cells were acquired from both naive and tumor bearing mice of both genotypes. Following treatment with IL-2, and activation with PMA and Ionomycin in the presence of Brefeldin A for 6 hours, intracellular interferon gamma was measured in CD8a positive cytotoxic T cells (Fig. 3A). Fold induction of INFy expression was calculated over the expression in non-activated cells of the same phenotype, and a statistically significant increase was observed in the LnkKO cells of tumor bearing mice as compared to their WT counterparts (Fig. 3B). When the total number of INFy positive cells was counted the same increase in cells from tumor bearing mice was also observed (Fig. 3C and 3D).
Example 3: In-vivo adoptive cell transfer of Lnk KO cytotoxic T-cells
[0152] Next the ability to transfer Lnk KO cytotoxic T-cells with enhanced cellular activation was assessed. B 16-F10 melanoma cells were transplanted subcutaneously into WT and Lnk 7 donor mice. Spleen of tumor bearing donor mice (WT or Lnk 7 ) was harvested on day 14, and CD8+ T-cells were isolated from the total splenocyte population. The isolated CD8+ T-cells (WT or Lnk 7 ) were covalently labeled using the fluorescent dye CellTrace Violet (C34557, Thermo Fisher Scientific) and transferred to WT recipient mice. Each WT Ύίί.2020/174472 vccj spleen derived CD8+ T-cells from a single t u i o ip Ja i i ¾' ¾ ' ) ί ? i 2,’Either WT or Lnk KO.
[0153] Following cell transfer, recipient mice were subcutaneously injected with two chemokine carrying Matrigel-plugs, one plug into each of their rear flanks. The two plugs each contained a single chemokine. The bilateral combination of chemokine containing plugs on a single recipient was one of the following [left plug:right plug]: - watenwater; Mip-2:water; SDF-la:Mip-2. 24 hours later the plugs were harvested and dissolved into single cell suspensions which were analyzed by FACS for the presence of the fluorescently labeled CD8+ cells. The number of detected T-cells derived from Lnk KO donors was significantly increased in Mip-2 containing plugs (Fig. 4). No significant difference was seen in the number of detected T-cells, of both genotypes, in plugs containing water, or plugs containing SDF-la.
Example 4: Modified chemotaxis assay
[0154] The role of Lnk regulation in the CXCRl/2-mediated migration of T-cells to melanoma was examined in vitro by a chemotaxis assay modified with Matrigel. B16-F10 melanoma cells were cultured and incubated in the lower wells of a Transwell systems (control Transwells contained no cells). Cells of inguinal lymph nodes were isolated from WT or Lnk KO melanoma tumor bearing mice and were either pretreated or not pretreated with a CXCRl/2 inhibitor SCH563705. The lymph node cells were then placed into the upper wells of the Transwell systems. Prior to incubation, the exterior portion of the membrane on the upper well was pre-coated with a thin layer of Matrigel to better mimic in vivo infiltration of tumors. The Transwell system was co-cultured for 16 hours, and cells were then collected from the lower well. These cells were stained for CD3, CD8 and CD4 markers and counted using an acoustic flow -cytometer to determine the number of migrating lymph node cells.
[0155] The migration of CD3+ T-cells to the B 16-F10 melanoma cells was found to be increased for Lnk deficient cells as compared to WT (Fig. 5A). Furthermore, inhibition of the CXCRl/2 receptors (+lnh, right most columns of Fig. 4A) significantly decreased the migration of Lnk KO T-cells to the cancer cells, suggesting that both the CXCRl/2 pathway and Lnk regulation are involved in the regulation of T-cell migration to cancer. CXCRl/2 inhibition had no effect on the migration of WT T-cells. Similarly, CD8+ T-cells that were Lnk deficient also displayed increased migration that was CXCRl/2 dependent (Fig. 5B), however, no such increase was observed for CD4+ T-cells (Fig. 5C). Ύ9 on this observed dependence on CXCRl/2 s i g n a 1 ni ! ? ' 12i 1 ?c i c n t increased cell migration, the relative expression of CXCR2 and CXCR1 was measured in WT and Lnk KO cells. Tumor infiltrating lymphocytes were isolated from melanoma tumors in WT and Lnk KO mice, and the percentage of CD8, CD4, CD19 and CD8-CD4-CD19 positive cells that were also positive for CXCR2 was assessed (Fig. 5D). No significant difference in the expression of CXCR2 was observed on the infiltrating lymphocytes based on their genotype. Similarly, infiltrating CD8+ T-cells from both genotypes were assessed for CXCR1 expression and no significant difference was observed (Fig. 5E). All this suggests that Lnk regulation occurs at the signal transduction level and is not due to fluctuations in ligand or receptor expression.
Example 5: Lnk KO mice show decreased tumor size in a lung model
[0157] In a model similar to the melanoma model, cells of a Lewis Lung Carcinoma (LLC) cell-line were subcutaneously injected into WT and Lnk 7 mice at the rear back. Tumor growth was monitor for 14 days at which time tumors were then harvested and weighed. Similar to the results from melanoma cells, tumors from Lnk KO mice were visibly smaller (Fig. 6A) and had a decreased weight of on average about half of the weight of tumors from WT mice (Fig. 6B). These results show that the efficacy of Lnk KO T-cells against cancer cells is not limited by the type of cancer, but rather is a universal phenomenon.
[0158] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

WOu 2m02i0T/i1i74 · 472 PCT/IL2020/050218
1. A method of treating a disease in a subject in need thereof, the method comprising administering to said subject an immune cell comprising decreased SH2B adapter protein 3 (SH2B3) protein expression and/or function, thereby treating said disease.
2. The method of claim 1, wherein said disease is an immune-associated disease.
3. The method of claim 2, wherein said immune- associated disease is selected from cancer, Myelodysplastic syndrome (MDS), bacterial infection, fungal infection, viral infection and parasitic infection.
4. The method of any one of claims 1 to 3, wherein said cell is allogenic to said subject.
5. The method of claim 4, wherein said immune cell is obtained from said subject and SH2B3 protein expression and/or function is decreased in said obtained cell before said administering.
6. The method of any one of claims 1 to 5, wherein said decreased SH2B3 protein expression and/or function comprises decreased SH2B3 mRNA levels, SH2B3 protein levels, SH2B3 protein function, or a combination thereof.
7. The method of any one of claims 1 to 6, wherein said decreased SH2B3 protein expression and/or function comprises expression of an SH2B3 inhibitor molecule.
8. The method of claim 7, wherein said SH2B3 inhibitor molecule is selected from a small nucleic acid molecule that hybridizes to and inhibits SH2B3 mRNA transcription and/or translation, a small molecule inhibitor of SH2B3 protein, an anti- SH2B3 antibody or antigen binding fragment, and a genome editing agent for mutation of a SH2B3 genetic locus.
9. The method of claim 8, wherein said small molecule inhibitor, antibody or antigen binding fragment is a SH2B3 antagonist.
10. The method of claim 8 or 9, wherein said small molecule inhibitor, antibody, or antigen binding fragment binds to and occludes at least one of a Pleckstrin Homolog (PH) domain, and a Src Homolog 2 (SH2) domain of SH2B3.
11. The method of claim 8, wherein said small nucleic acid molecule that hybridized to and inhibits SH2B3 is an anti-sense RNA.
12. The method of any one of claims 1 to 11, wherein said immune cell is selected from a T-cell, a B-cell, a natural killer (NK) cell, a neutrophil, a dendritic cell, a myeloid derived suppressor cell and a macrophage.
13. The method of claim 12, wherein said T-cell is a CD8+ T-cell, CD4+T-cell or both.
14. The method of any one of claims 1 to 13, wherein said immune cell further comprises a chimeric antigen receptor (CAR).
WO 2020/174472 ethod of claim 14, wherein said CAR binds to a t¾gE(¾¾i?g£9?0 A8a cell associated with said disease.
16. The method of claim 15, wherein said cell is selected from a cancer cell, a bacterial cell, a fungal cell, a parasite cell and a cell infected with a virus.
17. The method of any one of claims 1 to 16, further comprising administering an anticancer or anti-pathogen therapeutic treatment.
18. A method of determining suitability of a subject in need thereof to receive a treatment comprising an immune cell comprising decreased SH2B3 protein expression and/or function, the method comprising
a. receiving a cell associated with a disease from said subject; and
b. assaying chemotaxis of an unmodified immune cell and an immune cell comprising decreased SH2B3 protein expression and/or function to said pathological cell; wherein improved chemotaxis of said immune cell comprising decreased SH2B3 protein expression and/or function as compared to said unmodified immune cell indicates said subject is suitable for said treatment.
19. The method of claim 18, wherein said treatment comprising an immune cell comprising decreased SH2B3 protein expression and/or function is a method of any one of claims 1 to 17.
20. The method of claim 18 or 19, wherein said assaying chemotaxis is performed ex vivo.
21. The method of any one of claims 18 to 20, further comprising treating said subject suitable for treatment by the method of any one of claims 1 to 17.
22. A method for enhancing an immune response of an immune cell, the method comprising decreasing SH2B3 protein expression and/or function in said cell, thereby enhancing an immune response of an immune cell.
23. The method of claim 22, wherein said decreasing occurs ex vivo.
24. The method of claim 22 or 23, wherein said decreasing comprises introducing an SH2B3 inhibitor molecule into said immune cell.
25. The method of any one of claims 22 to 24, wherein said immune cell is an immune cell to be used for adoptive immunotherapy.
26. The method of any one of claims 22 to 25, wherein said enhancing an immune response comprises at least one of: increasing cytotoxicity of said immune cell and increasing migration of said immune cell.
WO 2020/P4472.|T| accutjcal composition comprising an immune cel?^i(ii ¾?i g 5¾?v? eased SH2B adapter protein 3 (SH2B3) protein expression and/or function.
28. The pharmaceutical composition of claim 27, wherein said decreased SH2B3 protein expression and/or function comprises decreased SH2B3 mRNA levels, SH2B3 protein levels, SH2B3 protein function, or a combination thereof.
29. The pharmaceutical composition of claim 27 or 28, wherein said decreased SH2B3 protein expression and/or function comprises expression of an SH2B3 inhibitor molecule.
30. The pharmaceutical composition of claim 29, wherein said SH2B3 inhibitor molecule is selected from a small nucleic acid molecule that hybridizes to and inhibits SH2B3 mRNA transcription and/or translation, a small molecule inhibitor of SH2B3 protein, an anti-SH2B3 antibody or antigen binding fragment, and a genome editing agent for mutation of SH2B3.
31. The pharmaceutical composition of claim 30, wherein said small molecule inhibitor, antibody or antigen binding fragment is a SH2B3 antagonist.
32. The pharmaceutical composition of claim 30 or 31, wherein said small molecule inhibitor, antibody, or antigen binding fragment binds to and occludes at least one of a Pleckstrin Homolog (PH) domain, and a Src Homolog 2 (SH2) domain of SH2B3.
33. The pharmaceutical composition of claim 30, wherein said small nucleic acid molecule that hybridized to and inhibits SH2B3 is an anti-sense RNA.
34. The pharmaceutical composition of any one of claims 27 to 33, wherein said immune cell is selected from a T-cell, a B-cell, a natural killer (NK) cell, a neutrophil, a dendritic cell, a myeloid derived suppressor cell and a macrophage.
35. The pharmaceutical composition of claim 34, wherein said T-cell is a CD8+ T-cell, CD4+T-cell or both.
36. The pharmaceutical composition of any one of claims 27 to 35, wherein said cell comprises a chimeric antigen receptor (CAR).
37. The pharmaceutical composition of claim 36, wherein said CAR binds to a target antigen on a cell associated with a disease.
38. The pharmaceutical composition of claim 37, wherein said cell associated with a disease is selected from a cancer cell, a bacterial cell, a fungal cell, a parasite cell and a cell infected with a virus.
39. The pharmaceutical composition of any one of claims 27 to 38, wherein said cell is produced by providing a cell from a subject and decreasing SH2B3 protein expression and/or function in said extracted cell in vitro.
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