Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • 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
  • C12N5/0635—B lymphocytes
• • 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
  • C12N5/0693—Tumour cells; Cancer cells
  • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
• • 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
  • C12N5/10—Cells modified by introduction of foreign genetic material
  • C12N5/12—Fused cells, e.g. hybridomas
  • C12N5/16—Animal cells
  • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/567—Framework region [FR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
• • 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
  • C12N2510/04—Immortalised cells

Definitions

• ADP-ribosyl transferase 1 (ART1), an ARTC family mono-ADP-ribosyltransferase, functions extracellularly to ADP-ribosylate cell surface proteins or target soluble proteins in the local tumor microenvironment.
• Mono-ADP-ribosylation can be blocked by arginine analogues and nicotinamide mimics that act as competitive inhibitors.
• Such analogues include the antibiotic novobiocin, which has previously been utilized safely in lung cancer trials, based upon other non-targeted mechanisms.
• MIBG meta-iodobenzylguanidine
• MIBG may exert inhibitory effects on the metastatic properties of a hepatocellular carcinoma cell line, possibly through inhibition of mono-ADP-ribosylation.
• a tremendous effort has been undertaken by pharmaceutical companies to develop small molecule inhibitors of intracellular poly- and mono- ADP-ribosylation.
• these drugs are designed to compete with NAD + at the enzyme active site and because they are largely based on benzamide or purine structures, the agents also have the potential to inhibit other enzymes that utilize NAD + , including ART 1. However, they are not specific for ART 1 monoribosyltransferase activity.
• ART1 selective inhibitors of ART1 , e.g., inhibitors of mono-ADP-ribosylation, to suppress tumor growth and facilitate cytotoxicity of immune cells towards ART1 expressing cells such as cancer cells including lung cancer cells.
• the disclosure provides for antibodies, fragments thereof and single chain ART1 binding polypeptides, targeting ART1 , an extracellular mono-ADP ribosyltransferase, e.g., antibodies that bind ART1 , for the treatment of diseases including cancer.
• ART1 is highly expressed in multiple human non-small cell lung cancer (NSCLC) lines of distinct driver mutation status and strong ART1 protein expression was observed in over half of human lung adenocarcinomas.
• NSCLC non-small cell lung cancer
• ART1 overexpression plays an important role in survival and metastatic outgrowth of disseminated tumor cells, likely due to protection from immune cells in the tumor microevironment.
• compounds that specifically inhibit ART1 or its function such as anti-human ART1 specific antibodies or portions thereof, can be used as targeted therapeutics in ART1 -overexpressing cancers, such as in NSCLC patients, to limit metastatic spread of cancer by facilitating immune-mediated destruction of disseminated cells.
• ART1 is highly druggable by various therapeutic modalities including antibodies and fragments thereof.
• inhibitors of ADP-ribosylation may be used in a combination therapy with cytotoxic chemotherapy or with immune checkpoint inhibitors.
• the ART 1 inhibitors are useful in a wide-variety of cancers, including for example colon cancer or breast cancer.
• the inhibitors may be useful in inhibiting cancer progression and/or metastasis.
• the disclosure provides an isolated antibody that binds human ART1 and optionally murine ART1.
• the antibody may be produced from a vertebrate cell, e.g., one transfected with nucleic acid sequences encoding an anti-ART1 antibody or antigen binding fragment thereof, or a polypeptide, that inhibits human ART1 activity, from an immune cell or a hybridoma, e.g., which expresses a monoclonal antibody.
• an isolated monoclonal antibody that binds human and mouse ART1 is provided.
• the nucleic acid sequences encoding an anti-ART1 antibody or antigen binding fragment thereof, or the polypeptide may be operably linked to a promoter, such as a heterologous promoter.
• the cell may be a mammalian cell, a primate cell, an insect cell or a plant cell.
• an isolated nucleic acid comprising a promoter operably linked to a nucleotide sequence which encodes at least the variable region of a human or murine heavy or light chain that binds human and/or mouse ART1 is provided.
• an expression cassette comprising nucleic acid sequences encoding an anti-ART1 antibody or antigen binding fragment thereof, or a polypeptide, that inhibits human ART1 activity, which sequence encodes QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKD TSKSQVVLTMTNMDPVDTATYYCARIYGGDSWGYFDNWGQGTLVTVSS (SEQ ID NO:1); QIVLTQSPAIMSASLGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSL TISSMEAGDAATYYCQQWSSNPPTFGAGTKLELK (SEQ ID NO:2); QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRISTSGFTNYNPSLKSRVTMSVD SSKNQFSLKLSSLTAADTAV
• an expression cassette comprising nucleic acid sequences encoding an anti-ART1 antibody or antigen binding fragment thereof, or a polypeptide, that inhibits human ART1 activity, which sequence encodes a plurality of CDRs having S NARMGVS (SEQ ID NO:21), HIFSNDEKSYSTSLKS (SEQID NO:22), IYGGDSWGYFDN (SEQ ID NO:23), QVTLKESGPVLVKPTETLTLTCTVSGFSLS (SEQ ID NO:24); SSSVSY (SEQ ID NO:28), SSVSY (SEQ ID NO:81), DTS (SEQ ID NO:29), or QQWSSNPPT (SEQ ID NQ:30); or GFSLSNARMG (SEQ ID NO:66) IFSNDEK (SEQ ID NO:67), ARIYGGDSWGYFDN (SEQ ID NO:68); SSSVSY (SEQ ID NO:28) , DTS (SEQ ID NO:29), or QQQ
• an expression cassette comprising nucleic acid sequences encoding an anti-ART1 antibody or antigen binding fragment thereof, or a polypeptide, that inhibits human ART1 activity, which sequence encodes a plurality of CDRs having GGSISSYY (SEQ ID NO:35), ISTSGFT (SEQ ID NO:36), ARDGWGRVFDI (SEQ ID NO:37) or QSVSSSY (SEQ ID NO:42), GAS (SEQ ID NO:43) or QQYGSST (SEQ ID NO:44), optionally including one or more framework regions having QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO:38), WSWIRQPAGKGLEWIGR (SEQ ID NO:39), NYNPSLKSRVTMSVDSSKNQFSLKLSSLTAADTAVYYC (SEQ ID NQ:40) or WGLGTMVTVSS (SEQ ID NO:41), or EIVLTQSPGTLSLSPGERATLSCRAS (SEQ ID NO:35
• the antibody or antigen binding fragment thereof, or polypeptide comprises CDRs comprising SEQ ID NO:21 , SEQ ID NO:22 and SEQ ID NO:23, and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NQ:30.
• the antibody or antigen binding fragment thereof, or polypeptide comprises CDRs comprising SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37, and QSVSSSY (SEQ ID NO:42), GAS (SEQ ID NO:43) and SEQ ID NO:44.
• the antibody or antigen binding fragment thereof, or polypeptide comprises CDRs comprising SEQ ID NO:66, SEQ ID NO:67 and SEQ ID NO:68, and SEQ ID NO:81 , SEQ ID NO:29 and SEQ ID NO:30.
• a framework region in the antibody or antigen binding fragment thereof, or polypeptide comprises SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and/or WGQGTLVTVSS (SEQ ID NO:27), or a sequence having one, two, three, four, or five conservative amino acid substitutions, and optionally one two or three non-conservative substitutions.
• a framework region in the antibody or antigen binding fragment thereof, or polypeptide comprises SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, and/or SEQ ID NO:34, or a sequence having one, two, three, four or five conservative amino acid substitutions, and optionally one two or three nonconservative substitutions or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid seqeince identity thereto.
• a framework region in the antibody or antigen binding fragment thereof, or polypeptide comprises SEQ ID NO:38, SEQ ID NO:39, SEQ ID NQ:40, and/or SEQ ID NO:41 , or a sequence having one, two, three, four or five conservative amino acid substitutions, and optionally one two or three nonconservative substitutions or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid seqeince identity thereto...
• a framework region in the antibody or antigen binding fragment thereof, or polypeptide comprises SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 and/or SEQ ID NO:63, or a sequence having one, two, three, four or five conservative amino acid substitutions, and optionally one two or three nonconservative substitutions or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid seqeince identity thereto.
• the antibody or antigen binding fragment thereof, or polypeptide binds to a portion of human or mouse ART1 from residue 70 to 100, 170 to 182, 192 to 206 or 230 to 245, e.g., relative to, e.g. SEQ ID NQ:90.
• a CDR has one, two or three amino acid substitutions relative to NARMGVS (SEQ ID NO:21), HIFSNDEKSYSTSLKS (SEQ ID NO:22) or IYGGDSWGYFDN (SEQ ID NO:23).
• a CDR with one or two substitutions relative to NARMGVS(SEQ ID NO:21) has NAHMGVS (SEQ ID NO:93), QARMGIS (SEQ ID NO:94) or NGRMGVS (SEQ ID NO:95);
• a CDR with one, two or three substitutions relative to HIFSNDEKSYSTSLKS (SEQ ID NO:22) has HIFSNDEKSYSTSIKS (SEQ ID NO:96), HLFSNDEKSYSTSIKS (SEQ ID NO:97) or HIFTNDEKSYSSSLKS (SEQ ID NO:98);
• a CDR with one or a few substitutions relative to IYGGDSWGYFDN has IYGGADSWGYFEN (SEQ ID NO:99), IYGGDSWAYFDN (SEQ ID NQ:100), or LYGIDSWGYFDN (SEQ ID NQ:101)
• a CDR has one, two or three amino acid substitutions relative to GFSLSNARMG SEQ ID NO:66), IFSNDEK (SEQ ID NO:67) or ARIYGGDSWGYFDN (SEQ ID NO:68.
• a CDR with one or two substitutions relative to.GFSLSNARMG has GFSISNARMG (SEQ ID NQ:102), GFSASNTRMG (SEQ ID NQ:103) or GFSISNLRMA (SEQ ID NQ:104).
• a CDR with one or two substitutions relative to IFSNDEK has LFSNDEK (SEQ ID NQ:105) or IFSNEDK (SEQ ID NQ:106).
• a CDR with one, two or three substitutions relative to ARIYGGDSWGYFDN has GRIYGGDSWGYFDN (SEQ ID NQ:107), ARIYAADSWGYFDN (SEQ ID NO:108) or IRAYGGDSWLYFDN (SEQ ID NQ:109).
• a composition having an ART1 expression cassette e.g., in a gene expression vector, ART1 binding antibodies or antigen binding fragments thereof, or polypeptides that bind ART1 , may be employed in in vitro and in vivo methods.
• the composition may be employed to inhibit or treat cancer in a mammal, e.g., by administering to the mammal an effective amount of the composition.
• the mammal may have lung cancer, e.g., non-small cell lung cancer, colon cancer, melanoma, glioblastoma, breast cancer, or colorectal cancer.
• the mammal is a human.
• the amount is effective to inhibit ART1 enzymatic activity, decrease tumor burden, inhibit metastases, enhance immune-mediated anti-tumor activity, or increase survival.
• the mammal has an ART1 overepressing tumor.
• the composition may be employed to prevent or inhibit ART1 -mediated immunosuppression in a mammal or to enhance an immune response in a mammal in need thereof, e.g., a mammal having an ART1 overexpressing tumor, e.g., a mammal having NSCLC, colon cancer or melanoma..
• an anti-ART1 antibody that binds to and/or inhibits the activity of human ART1 and/or murine ART1 .
• an isolated cell comprising an expression cassette comprising a heterologous promoter operably linked to nucleic acid sequences encoding an anti-human ART1 antibody, or an antigen binding fragment thereof, or a polypeptide, that inhibits human ART1 activity, wherein the antibody, the antigen binding fragment thereof, or the polypeptide has: i) a variable region comprising a first complementarity determining region (CDR) comprising GFSLSNARM (SEQ ID NO:66) or NARMGVS (SEQ ID NO:21) operably linked to a second CDR comprising IFSNDEK (SEQ ID NO:67) or HIFSNDEKSYSTSLKS (SEQ ID NO:22) operably linked to a third CDR comprising ARIYGGDSWGYFDN (SEQ ID NO:68) or IYGGDSWGYFDN (SEQ ID NO:23); and/or ii) a variable region comprising a first CDR comprising SSVSY (SEQ ID NO:81) or
• the cell comprises or expresses a heavy Ig chain comprises a variable region comprising QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKD TSKSQVVLTMTNMDPVDTATYYCARIYGGDSWGYFDNWGQGTLVTVSS (SEQ ID NO:1) or QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAH IFSNDEK
• SYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARIYGGDSWGYFDNWGQGTLVTVSS (SEQ ID NO:82); or a light Ig chain ii) comprises a variable region comprising QIVLTQSPAIMSASLGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSL TISSMEAGDAATYYCQQWSSNPPTFGAGTKLELK (SEQ ID N0:2) or DIQLTQSPSFLSASVGDRVTITCRASSSVSYMHWYQQKPGTS PKRLIYDTS KLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCQQWSSNPPTFGQGTKLEIK (SEQ ID NO:83).
• the cell has or expressed a heavy Ig chain comprising iii) comprises a variable region comprising: QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRISTSGFTNYNPSLKSRVTMSVD SSKNQFSLKLSSLTAADTAVYYCARDGWGRVFDIWGLGTMVTVSS (SEQ ID NO:3); or a light Ig chain comprising iv) comprises a variable region comprising EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFT LTISRLEPEDFAVYYCQQYGSSTFGPGTKVDIK (SEQ ID NO:4).
• the cell has or expresses a light Ig chain comprising ii) comprises a variable region comprising DIQLTQSPSFLSASVGDRVTITCRASSSVSYMHWYQQKPGTSPKRLIYDTSKLASGVPSRFSGSGSGTEYTLT ISSLQPEDFATYYCQQWSSNPPTFGQGTKL (SEQ ID NO:69);
• the cell is a mammalian cell. In one embodiment, the cell is a primate cell. In one embodiment, the cell is a human cell.
• hybridoma comprising nucleic acid sequences encoding an anti-human ART1 monoclonal antibody that inhibits human ART1 activity, wherein the antibody has: i) a variable region comprising a first complementarity determining region (CDR) comprising GFSLSNARM (SEQ ID NO:66) or NARMGVS (SEQ ID NO:21) operably linked to a second CDR comprising IFSNDEK (SEQ ID NO:67) or HIFSNDEKSYSTSLKS (SEQ ID NO:22) operably linked to a third CDR comprising ARIYGGDSWGYFDN (SEQ ID NO:68) or IYGGDSWGYFDN (SEQ ID NO:23); and/or ii) a variable region comprising a first CDR comprising SSVSY (SEQ ID NO:81) or SSSVSY (SEQ ID NO:28) operably linked to a second CDR comprising DTS (SEQ ID NO:29) operably linked to
• an isolated nucleic acid comprising a promoter, e.g., a heterologous promoter, operably linked to a nucleotide sequence which encodes at least the variable region of a heavy or light Ig chain that binds human and/or mouse ART 1 , wherein the chain comprises: i) a variable region comprising a first complementarity determining region (CDR) comprising GFSLSNARM (SEQ ID NO:66) or NARMGVS (SEQ ID NO:21) operably linked to a second CDR comprising IFSNDEK (SEQ ID NO:67) or HIFSNDEKSYSTSLKS (SEQ ID NO:22) operably linked to a third CDR comprising ARIYGGDSWGYFDN (SEQ ID NO:68) or IYGGDSWGYFDN (SEQ ID NO:23); and/or ii) a variable region comprising a first CDR comprising SSVSY (SEQ ID NO:81) or SSSVS
• an isolated antibody or antigen fragment thereof that binds human and mouse ART1 wherein the antibody or the antigen binding fragment thereof have: i) a variable region comprising a first complementarity determining region (CDR) comprising GFSLSNARM (SEQ ID NO:66) or NARMGVS (SEQ ID NO:21) operably linked to a second CDR comprising IFSNDEK (SEQ ID NO:67) or HIFSNDEKSYSTSLKS (SEQ ID NO:22) operably linked to a third CDR comprising ARIYGGDSWGYFDN (SEQ ID NO:68) or IYGGDSWGYFDN (SEQ ID NO:23); and/or ii) a variable region comprising a first CDR comprising SSVSY (SEQ ID NO:81) or SSSVSY (SEQ ID NO:28) operably linked to a second CDR comprising DTS (SEQ ID NO:29) operably linked to a third CDR comprising QQWSSNPPT (
• variable region of i) in the antibody or fragment thereof further comprises one or more framework regions comprising one or more of: QVTLKESGPVLVKPTETLTLTCTVSGFSLS (SEQ ID NO:24), WIRQPPGKALEWLA (SEQ ID NO:25), RLTISKDTSKSQVVLTMTNMDPVDTATYYCAR (SEQ ID NO:26), WGQGTLVTVSS (SEQ ID NO:27), QVTLKESGPVLVKPTETLTLTCTVS (SEQ ID NO:71), VSWIRQP PGKALEWLAH (SEQ ID NO:72), SYSTSLKSRLTISKDTSKSQVVLTM TNMDPVDTATYYC (SEQ ID NO:73), or WGQGTLVTVSS (SEQ ID NO:74).
• QVTLKESGPVLVKPTETLTLTCTVSGFSLS SEQ ID NO:24
• WIRQPPGKALEWLA SEQ ID NO:25
• variable region of ii) of the anibody or fragment thereof further comprises one or more framework regions comprising one or more of: QIVLTQSPAIMSASLGEKVTMTCSA (SEQ ID NO:31), MHWYQQKSGTSPKRWIY (SEQ ID NO:32), KLASGVPARFSGSGSGTSYSLTISSMEAGDAATYYC (SEQ ID NO:33), FGAGTKLELK (SEQ ID NO:34), DIQLTQSPSFLSASVGDRVTITCRAS (SEQ ID NO:76), YMHWYQQKPGTS PKRLIY (SEQ ID NO:77), KLASGVPSRFSGSGSGTEYTLTISSLQPEDFATY YC (SEQ ID NO:78), FGQGTKLEIK (SEQ ID NO:79).
• QIVLTQSPAIMSASLGEKVTMTCSA SEQ ID NO:31
• MHWYQQKSGTSPKRWIY SEQ ID NO:32
• variable region of ii) further comprises one or more framework regions comprising one or more of: DIQLTQSPSFLSASVGDRVTITCRA (SEQ ID NO:51), MHWYQQKPGTSPKRLIY (SEQ ID NO:52), KLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYYC (SEQ ID NO:53), or FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:54).
• the variable region of ii) further comprises one or more framework regions comprising one or more of:
• DIQMTQSPSSLSASVGDRVTITCSA (SEQ ID NO:55), MHWYQQKPGTSPKRLIY (SEQ ID NO:56), KLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC (SEQ ID NO:57), or FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:58).
• variable region of ii) further comprises one or more framework regions comprising one or more of: EIVLTQSPATLSLSPGERATLSCRA (SEQ ID NO:59), MHWYQQKPGTSPRRLIY (SEQ ID NQ:60), KLATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYC (SEQ ID NO:61), or TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:62).
• EIVLTQSPATLSLSPGERATLSCRA SEQ ID NO:59
• MHWYQQKPGTSPRRLIY SEQ ID NQ:60
• KLATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYC SEQ ID NO:61
• variable region of iii) further comprises one or more framework regions comprising one or more of: QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO:38), WSWIRQPAGKGLEWIGR (SEQ ID NO:39), NYNPSLKSRVTMSVDSSKNQFSLKLSSLTAADTAVYYC (SEQ ID NQ:40), or WGLGTMVTVSS (SEQ ID NO:41).
• the variable region of iv) further comprises one or more framework regions comprising one or more of:
• EIVLTQSPGTLSLSPGERATLSCRAS SEQ ID NO:45
• LAWYQQKPGQAPRLLIY SEQ ID NO:46
• SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC SEQ ID NO:47
• FGPGTKVDIK SEQ ID NO:63
• a method to inhibit or treat cancer in a mammal comprising: administering to a mammal a composition comprising an effective amount of an anti-human ART1 antibody or an antigen binding fragment thereof, or a polypeptide that binds human ART 1 .
• the cancer is lung cancer, colon cancer, melanoma, glioblastoma, breast cancer or colorectal cancer.
• the mammal is a human.
• the amount is effective to decrease tumor burden, inhibit metastases, increase survival, or any combination thereof.
• the composition is intravenously or subcutaneously administered.
• the method further comprises administering a chemotherapeutic drug.
• the method further comprises administering an immune checkpoint inhibitor.
• the antibody, the antigen binding fragment thereof, or the polypeptide has: i) a variable region comprising a first complementarity determining region (CDR) comprising GFSLSNARM (SEQ ID NO:66) or NARMGVS (SEQ ID NO:21) operably linked to a second CDR comprising IFSNDEK (SEQ ID NO:67) or HIFSNDEKSYSTSLKS (SEQ ID NO:22) operably linked to a third CDR comprising ARIYGGDSWGYFDN (SEQ ID NO:68) or IYGGDSWGYFDN (SEQ ID NO:23); and/or ii) a variable region comprising a first CDR comprising SSVSY (SEQ ID NO:81) or SSSVSY (SEQ ID NO:28) operably linked to a second CDR comprising DTS (SEQ ID NO:29) operably linked to a third CDR comprising QQWS
• CDR complementar
• a method to prevent, inhibit or treat ART1 -mediated immunosuppression in a mammal comprising: administering to a mammal a composition comprising an effective amount of an antihuman ART 1 antibody or an antigen binding fragment thereof, or a polypeptide that binds human ART 1 .
• the mammal has cancer.
• the mammal is a human.
• the composition is intravenously administered.
• the composition is sucutanenously administered
• the method further comprises administering a chemotherapeutic drug.
• the method further comprises administering an immune checkpoint inhibitor.
• the antibody, the antigen binding fragment thereof, or the polypeptide has: i) a variable region comprising a first complementarity determining region (CDR) comprising GFSLSNARM (SEQ ID NO:66) or NARMGVS (SEQ ID NO:21) operably linked to a second CDR comprising IFSNDEK (SEQ ID NO:67) or HIFSNDEKSYSTSLKS (SEQ ID NO:22) operably linked to a third CDR comprising ARIYGGDSWGYFDN (SEQ ID NO:68) or IYGGDSWGYFDN (SEQ ID NO:23); and/or ii) a variable region comprising a first CDR comprising SSVSY (SEQ ID NO:81) or SSSVSY (SEQ ID NO:28) operably linked to a second CDR comprising DTS (SEQ ID NO:29) operably linked to a third CDR comprising QQWSSNPPT (SEQ ID NQ:30); or iii
• a method to enhance an immune response in a mammal having cancer comprising: administering to a mammal a composition comprising an effective amount of an anti-human ART1 antibody or an antigen binding fragment thereof, or a polypeptide that binds human ART1.
• the mammal is a human.
• the heavy chain is an IgG heavy chain.
• the light chain is an IgK light chain.
• the antibody, the antigen binding fragment thereof, or the polypeptide has: i) a variable region comprising a first complementarity determining region (CDR) comprising GFSLSNARM (SEQ ID NO:66) or NARMGVS (SEQ ID NO:21) operably linked to a second CDR comprising IFSNDEK (SEQ ID NO:67) or HIFSNDEKSYSTSLKS (SEQ ID NO:22) operably linked to a third CDR comprising ARIYGGDSWGYFDN (SEQ ID NO:68) or IYGGDSWGYFDN (SEQ ID NO:23); and/or ii) a variable region comprising a first CDR comprising SSVSY (SEQ ID NO:81 ) or SSSVSY (SEQ ID NO:28) operably linked to a second CDR comprising DTS (SEQ ID NO:29) operably linked to a third CDR comprising QQWSSNPPT (SEQ ID NQ:30); or ii
• an isolated antibody or fragment thereof is provided, wherein, when bound to human ART1 , the antibody binds to at least one of the following residues: S75, S77, T79, R80, R89, H92, or Y99 of human ART1 , e.g., SEQ ID NQ:90.
• an isolated antibody or fragment thereof binds to two, three, four, five or six of S75, S77, T79, R80, R89, H92, or Y99 of human ART1 , e.g., SEQ ID NQ:90.
• an isolated antibody or fragment thereof binds to S75, S77, T79, R80, R89, H92, and Y99 of human ART1 , e.g., SEQ ID NQ:90.
• FIGS 11A-11 C Identification of Functional ART1 Antibodies.
• Anti-ART1 hybridoma supernatants were also tested for inhibition of human ART1 transiently expressed in HEK293 cells. This method, which has previously been described for ART2 (Krebs et.
• Figures 12A-12D Affinity Measurement of 22C12 and 14G01 to Human and Mouse ART1 by Surface Plasmon Resonance. Purified antibodies from hybridoma clones 22C12 and 14G01 were captured on an antiMouse Fc surface and the indicated concentration of analyte was injected over the surface. A) 22C12 binding to human ART 1 . B) 22C12 binding to mouse ART 1 . C) 14G01 binding to human ART 1 . D) 14G01 binding to mouse ART1 . Sensorgrams are double-reference subtracted using a control surface and blank injections. Affinity constants were determined by kinetic fit using a 1 :1 binding model.
• FIG. 13 Dose-Dependent Inhibition of Cell-Surface ADP-Ribosylation by 22C12 and 14G01 .
• HEK293 cells transiently transfected with human ART1 were incubated with 22C12 or 14G01 at the indicated concentration followed by treatment with etheno-NAD + , staining with anti-etheno antibody and flow cytometry.
• % Inhibition was normalized to HEK293 cells transfected with human ART1 and stained with anti- etheno antibody without etheno-NAD + treatment.
• FIGS 14A-14G ART1 is overexpressed in a subset of human lung cancers.
• A-B ART1 Immunofluorescence staining of human lung cancer cell lines A549 and H1650 and benign bronchial epithelium cell line BEAS2B.
• C Ratio of ART1 surface MFI and ART1 total MFI (graph in Fig.
• Tumors were scored for infiltration of immune cells (listed in Table 4).
• E Representative IHC images and pie chart depicting percentage of tumors that stained positive for ART1 membranous expression or ART1 expression in the cytoplasm only
• F Percentage of tumors with a low or intermediate/high CD8 T cell score in tumors with membranous ART1 staining or cytoplasmic ART1 staining only. Chi-square test. Gene expression data were square root-transformed prior to statistical testing. *p ⁇ 0.05.
• FIGS 15A-15D ART1 expression promotes tumor growth in murine lung tumor models.
• KP1 cells were stably transduced with ART1 overexpression lentiviral vector (KP1 -ART1 0E ) and subsequently transduced with shRNA targeting ART 1 (shART 1 ). Where indicated mice were treated with doxycycline-water ad libitum to induce shART 1 .
• C) Representative images of lung sections stained with H&E (left panels) and enumeration of lung nodules (right panel) from mice sacrificed on day 14 after injection of KP1 -ART1 0E . (n 4-5 mice/group) Welch’s t-test.
• Lung nodules in H&E stainings are indicated by black arrows. Tumor nodule counts were determined using Image J software.
• FIGS 16A-16L ART1 blockade reduces lung tumor burden and promotes infiltration of P2X7R+ CD8 T cells
• G Counts of P2X7R+ CD8 T cells, PD-1 + CD8 T cells and KI67high CD8 T cells per gram of tumor-bearing lung tissue.
• H Counts of P2X7R+ CD8 TCM, P2X7R+ CD8 TEM, and P2X7R+ CD8 TRM per gram of tumor-bearing lung tissue. Box and whiskers plots indicate median and 10-90 percentiles. Welch’s t-test. Percentage and count data were square root transformed prior to statistical testing.
• ART1 blocking antibody 22C12 Ab
• iso Ctrl Ab iso Ctrl Ab
• CD8 depleting antibodies clone: 53-6.7
• CD4 depleting antibodies clone: GK1 .5
• FIGS 17A-17C ART1 -mediated ADP-ribosylation and NICD of lung tumor-infiltrating T cell subsets (A-C) ADP-ribosylation and NICD assay.
• T cells isolated from wild type KP1 tumor bearing lungs of C57BL/6 mice were incubated for two hours with ethano-NAD (eNAD) alone (-rART1 ) or with eNAD and recombinant mouse ART1 (rART1 ), CD38 blocking antibody (NIMR-5) (CD38 block) or ART1 blocking antibody (ART1 block (22C12)).
• the P2X7R+ and P2X7R- fractions of each T cell subset were analyzed separately for (B) ADP-ribosylation by total eNAD staining and (C) NICD based on co-staining with eNAD and DAPI. Repeated measures one-way ANOVA was used to determine statistically significant differences between treatments. Each connected line represents paired analysis of one mouse. Percentage data were square root-transformed prior to statistical testing *p ⁇ 0.05, **p ⁇ 0.01. ***p ⁇ 0.001 , ****p ⁇ 0.0001
• FIGS 18A-18H ART1 overexpression in human lung tumors is associated with low infiltration of P2X7R+ CD8 T cells.
• C Representative images of CD8 (red), P2X7R (green) and nuclear stain by DAPI (blue) immunofluorescence staining in lung tumor and matched normal tissue.
• F Representative dot plot of P2X7R and CD38 expression on CD8 T cells infiltrating normal lung tissue and lung tumor tissue.
• G Percentage of CD8 T cells expressing P2X7R. Paired t-test (H) Percentage of P2X7R+ CD8 T cells with high surface expression of CD38. Bars graphs indicate mean values. Paired t-test. Percentage and MFI data were log-transformed prior to statistical testing *p ⁇ 005, ***p ⁇ 0.001 , ****p ⁇ 0.0001
• FIGS 19A-19D ART1 and GPLD1 expression in human lung cancer cell lines and tumor samples, Expression of cytotoxicity and immunoregulatory genes in lung adenocarcinoma patients (TCGA).
• B) Violin plot depicting GPLD1 qPCR analysis of matched lung tumor tissue and normal lung tissue from patients with stage l-lll lung adenocarcinoma (n 40), Wilcoxon matched pairs signed rank test.
• FIGS 21 A-21 D CRISPR/Cas9-mediated knockout of ART1 in B16-F10 cells
• A-B Confirmation of ART1 knockout in CRISPR/Cas9 clones of B16-F10 mouse melanoma cells.
• B16-F10 scramble clone B16CONTROL (Scr-6) was transduced with a non-specific CRISPR gRNA and serves as a control.
• the B16- F10 ART 1 knockout (ART 1 KO) clones B16ART 1 KO (42-1 ) and B16ART 1 KO (63-1 ) were transduced with gRNA targeting two different regions in exon 1 of the ART1 gene.
• A) Flow cytometry cell surface staining of ART1 on B16 CRISPR clones B16CONTROL (Scr-6), B16ART1 KO (42-1 ) and B16ART1 KO (63-1 ).
• Orange histograms represent cells stained with ART1 primary antibody (rabbit IgG) and Cy5-conjugated secondary anti-rabbit antibody. Blue histograms represents cells stained with secondary antibody alone.
• FIGS 22A-22H Generation and functional testing of ART1 -blocking antibody 22C12.
• E Etheno-NAD (eNAD) ADP-ribosylation assays determining half-maximum inhibition (IC50) of ADP-ribosylation of 22C12 antibody clones, against HEK293 cells transfected with ART1 .
• Hu lgG1 was used as isotype control.
• 2E2 antibody was used as an irrelevant antibody.
• F Western blot analysis of ADP-ribosylated proteins (using MAR/PAR antibody) in KP1-ART1OE cells incubated for 2 hours with indicated concentrations of NAD+ treated with or without 20 ug/ml of 22c12 ART1 antibody. (G-H) Toxicity study.
• mice Tumor-naive mice were treated with intraperitoneal injections of 22C12 antibodies at 25mg/kg every three days for three weeks and monitored for weight loss and blood glucose levels at baseline and every week until the end of the study. Mice treated with 22c12 antibodies remained normal in appearance, activity, gait and alertness compared to mice treated with isotype control antibody.
• FIGS 23A-23G In vivo inhibition of ART1 reduces tumor burden and promotes P2X7R+ CD8 T cell tumor infiltration in KP1-ART1OE and LLC1 tumor models.
• A) Experiment schema for in vivo experiment studying tumor progression of KP1-ART1OE flank tumors following intratumoral (i.t.) treatment with anti-ART1 Ab (22C12 Ab) or lgG2A isotype control Ab (iso Ctrl Ab) (n 7-8 mice/group). Tumors were harvested on day 25 after tumor inoculation for weighing and flow cytometry analysis. The Experiment was repeated once with similar results.
• mice were treated with CD8 depleting antibodies (CD8 depl, clone: 53-6.7), or CD4 depleting antibodies (CD4 depl, clone: GK1 .5), or isotype control antibodies (iso Ctrl). 500 ug on day -2 followed by 250 ug every five days from day 2 until endpoint.
• Asterisks in graphs indicate number of tumor free mice at endpoint.
• FIGS 25A-25C RNAseq analysis of P2X7R expression and immunoregulatory genes in CD8 T cells from KP1 lung-tumor bearing mice, ART1 qPCR analysis of CD8 and CD4 T cells and B16-F10 and KP1-ART1 0E tumor cells (A-B) RNA sequencing of CD8 T cells isolated from spleens and lungs of naive mice and mice inoculated intravenously with KP1 tumor cells. Tumors were harvested for CD8 T cell isolation on day 7 and 17 after tumor injection.
• A) Heatmap depicts gene expression of P2RX7 and genes regulating CD8 T cell cytotoxicity (GZMA, GZMB, IFNG, PRF1) and immunoregulatory molecules (CTLA4, HAVCR2, LAG3, PDCD1 , TIGIT).
• B) Scatter plot depicts gene expression of P2RX7. Kruskal-Wallis test was used to determine statistical difference between P2RX7 expression levels at indicated time points of spleen-derived and lung- derived CD8 T cells separately. Scatter plot shoes mean with SEM.
• FIGS 26A-26G ADP-ribosylation test of recombinant ART1 (rART1), Inhibition of ADP-ribosylation by ART1 and ART2 blocking antibodies, P2RX7 splice variant expression in T cells and tumor cells, Proliferation of tumor cells in presence of NAD+ and ART1 blockade.
• rART1 recombinant ART1
• ART2 Inhibition of ADP-ribosylation by ART1 and ART2 blocking antibodies
• P2RX7 splice variant expression in T cells and tumor cells Proliferation of tumor cells in presence of NAD+ and ART1 blockade.
• Hist ADP-ribosylate histone
• ART1 was inactivated by boiling where indicated. Each dot represents a technical replicate.
• eNAD etheno-NAD assay measuring the blocking effect of ART1- and ART2 blockade on ADP-ribosylation of CD4 and CD8 T cells.
• One-way ANOVA with Tukey’s test for multiple comparisons.
• C-D Gene expression analysis by qPCR depicting expression level of (C) p2rx7-k and (D) p2rx7-a isoforms in CD4 Tconv cells and CD8 T cells isolated from KP1 tumor-bearing lungs on day 15 after tumor inoculation as well as in tumor cells KP1 , LLC1 and B16.
• E-G Proliferation assay testing the growth of (E) KP1 ART1OE lung cells, (F) LLC1 cells and (G) B16 CRISPR clones B16CONTROL (Scr-6), B16ART 1 KO (42-1 ), B16ART 1 KO (63-1 ) in vitro in the presence of NAD+ (20 uM) and/or ART 1 blocking antibody (22c12, 20 ug/ml).
• FIGS 27A-27B A) Wild type KP1 lung carcinoma cells were exposed to Thapsigargin for 24 hours and assessed for mRNA expression of ART1 by qPCR.
• CD8 T cells were isolated from wild type KP1 tumor-bearing lungs by magnetic bead sorting using CD8 (TIL) MicroBeads, mouse (Miltenyi, Catalog# 130-116-478) kit. Cells were added to 48 well plates pre-coated with mouse recombinant ART 1 (rART 1 ) (10 pg/ml) for 24 hours at 4°C. 1 x106 CD8 T cells were resuspended in serum-free RPMI 1640 medium (Gibco) containing 100 pM etheno-NAD and 5pg/ml anti-ART2.2 antibody (s+16a, Biolegend, Catalog# 149801) and added to wells.
• TIL CD8
• rART 1 mouse recombinant ART 1
• CD8 T cells were removed from plate by gentle pipetting and fixed with 3.7% formaldehyde for 5 minutes. Cells were spun down for 30 seconds in a microcentrifuge. The supernatant was discarded, and the cell pellet was resuspended in 1 mL of deionized H2O. The samples were then spun down for 30 seconds the pellets resuspended in 200 pL of deionized H2O. 5 pL of the cell suspension was added to each gelatin-coated slide (Gelatin-Coated Microscope Slides # 1178T40, Thomas Scientific). 3 spots were made per slide and each spot was smeared with the side of a pipette tip.
• Figure 29 Representative images of ART1 immunofluorescence staining in lung tumor and matched normal tissue from lung adenocarcinoma patients. Slides were counterstained with DAPI nuclear stain.
• FIGS 30A-30I Characterization of lung tumor-infiltrating dendritic cells (DCs) from mouse lungs orthotopically inoculated by tail vein injections with KP1 -ART1 OE tumor cells.
• Mice received intraperitoneal (i.p.) treatment with ART1 blocking antibody (22C12) or isotype matched control antibody (iso Ctrl) every three days starting on day 6 until day 18.
• ART1 blocking antibody 22C12
• iso Ctrl isotype matched control antibody
• mice were euthanized and lung were weighed and digested for flow cytometry analysis.
• FIGS 31A-31 D CRISPR/Cas9-mediated knockout of ART1 in B16-F10 cells and 22C12-mediated blockade of Art1 causes inhibition of murine tumor growth.
• B16-F10 scramble clone B16 CONTROL (Scr ' 6) was transduced with a non-specific CRISPR gRNA and serves as a control.
• the B16-F10 ART1 knockout (ART1 KO) clone and B16 AR TI KO (63-I) were transduced with gRNA targeting a region of exon 1 of the ART1 gene.
• FIGS 32A-32D 22C12-mediated blockade of ART1 in LLC1 causes inhibition of murine flank tumor growth.
• FIG. 33 Binding of TDI-Y-009 (22C12 hLC1 lgG4 )to ART1 and ART1 paralogs. Bound antibody was detected through colorimetric reaction mediated by the HRP-conjugated secondary antibody reagent.
• FIG. 34 Binding of the 22C12 hLC1 lgG4 (TDI-Y-009) antibody to ART1 over-expressing KP1 cells.
• the binding EC50 was determined to be 4.4nM.
• FIG. 35 Plasma concentrations of TDI-Y-009 in C57BL/6 mice following single IV bolus administration. Captured antibody was measured through colorimetric reaction mediated by the HRP- conjugated secondary antibody reagent.
• FIGS 36A-36C TDI-Y-009 suppresses tumor growth in an orthotopic lung tumor model.
• C) Nodule counts and (D) average lung nodule area on day 19 after tumor inoculation. Tumor nodule counts and area was determined using Image J software, a.u. arbitrary units. Welch’s t-test.
• Figures 37A-37B No adverse safety effects observed in mice treated with TDI-Y-009.
• FIG 39 ART1 expression following radiation and chemotherapy. Following treatment with radiation therapy (8 Gy x 3, top row) and cisplatin (bottom row) total and surface ART1 expression increased in KP1 (left panel) and A549 cells (right panel) as measured by RTPCR and by single cell immunofluorescence.
• FIG. 40 MARylation expression following radiation.
• radiation therapy 8 Gy x 3
• MARylation increased cell surface mono-ADP-ribosylation (MARylation) as measured by single cell immunofluorescence using a MAR specific antibody.
• the increase in MARylation was blocked by the addition of 22C12 to the cell culture.
• Immune checkpoint inhibitors alone or in combination with chemotherapy, have become the standard of care in patients with advanced non-small cell lung cancer (NSCLC) without targetable molecular alterations (Mok et al., 2019).
• NSCLC non-small cell lung cancer
• the majority of lung cancer patients either do not respond to or do not experience long-term benefit from ICI, including many of those patients with high tumor PD-L1 expression (Gandhi et al., 2018; Gandini et al., 2016).
• ICI Immune checkpoint inhibitors
• ARTs or ADPs Cell surface mono-ADP ribosyltransferases transfer the ADP-ribose moiety from NAD + to amino acid residues to post-translationally modify target proteins.
• ADP- ribosyltransferase-1 (ART1) is expressed at low levels in healthy tissues including the lung.
• ART1 is a GPI- anchored enzyme, with an extracellular catalytic domain. Therefore ART1 may mono-ADP-ribosylate extracellular proteins in the local microenvironment, altering their function (Stevens et al., 2009; Okazaki et al., 1994; Balducci et al., 1999).
• ART1 in lung cancer has not been investigated, but previous studies have suggested increased ART1 protein expression in colorectal cancer and in glioblastoma, where high expression was associated with a poor prognosis (Tang et al., 2013). In mouse models of colorectal cancer, ART1 expression was shown to promote a more aggressive phenotype with increased epithelial-to-mesenchymal transition and increased angiogenesis (Yang et al., 2016; Song et al., 2016). However, it has not been determined whether tumor ART1 expression could regulate tumor cross-talk with the immune microenvironment.
• P2X7R P2X7 receptor
• gene id P2rx7
• P2X7R P2X7 receptor
• P2X7R is an ATP-gated cation channel of the purinergic type 2 receptor family, with low affinity for extracellular ATP, that activates pro-inflammatory pathways (Burnstock & Knight, 2004). It is expressed on multiple immune cell subsets including T cells and its expression is essential for inflammatory responses and anti-tumor immunity (Adinolfi et al., 2015; Haag et al., 2007). In NSCLC, high P2X7R expression has been associated with improved overall and progression-free survival (Boldrini et al., 2015).
• cytosolic NAD + is released into the local extracellular environment where it may be used as a substrate by extracellular ADP-ribosyl transferases to catalyze the transfer of the ADP-ribose to P2X7R (Haag et al., 2007).
• This covalent modification results in constitutive activation of P2X7R leading to large pore formation, uncontrolled calcium influx, phosphatidylserine externalization, and ultimately a process described as NAD-induced cell death (NICD) (Scheuplein et al., 2009).
• extracellular NAD + concentrations are generally low and tightly regulated by the ADP-ribosyl cyclase CD38, which is expressed on activated immune cells as well as on cancer cells (Sandoval-Montes & Santos-Argumedo, 2005; Chen et al., 2018).
• extracellular NAD + concentrations can increase following rapid release from stressed or dying cells (Haag et al., 2007).
• ART-mediated NICD of T cells has been proposed as a homeostatic mechanism to eliminate naive and bystander T cells in inflamed tissues (Adriouch et al., 2007).
• NICD tissue-resident memory T cells
• NSCLC non-small cell lung cancer
• ICI therapeutic immune checkpoint inhibition
• ART1 Epithelial cells in the injured or inflamed lung may overexpress ART1 as a mechanism of cell survival to protect against cell clearance by inflammatory cells.
• An evolutionarily conserved parallel protective role was hypothesized to be provided by ART1 expression in lung cancer cells.
• An analogy may be drawn between ART1 and immune checkpoint pathways. In both cases, evolutionary mechanisms that exist to protect tissues from collateral damage at sites of inflammation are utilized by cancers to evade the immune response. Given the success of checkpoint inhibition as a strategy to overcome immune escape and effectively treat metastatic lung cancer, a similar strategy was envisioned for ART1 inhibition.
• ART1 is overexpressed in lung cancers, is cytoprotective, and facilitates metastatic growth.
• inhibitors of mono-ADP-ribosylation were identified to utilize for therapeutic inhibition of cancers.
• evidence of ART1 expression in human NSCLC tumors was found using whole tumor RT-PCR, immunofluorescence, and immunohistochemistry.
• Heterogeneous expression existed by RT-PCR, implying that ART1 tumor expression may be more apparent in distinct subgroups of patients.
• ART1 was knocked down in a KRAS ⁇ 12 ⁇ + /p53’ ⁇ cell line, KP1 (developed from a genetically engineered mouse model), using shRNA technology (sh175KP1 ).
• KP1 developed from a genetically engineered mouse model
• shRNA technology sh175KP1
• a highly significant decrease in metastasis was noted in the ART1 -knockdown cell lines compared to their parent lines.
• An in vitro model was employed to assess the ability of freshly procured neutrophils from immunocompetent mice to induce apoptosis in lung cancer cells.
• Chemical inhibition of mono-ADP-ribosylation in the parent KP1 cell line with two well established inhibitors facilitated neutrophil-induced apoptosis, implying that the enzymatic activity of ART1 is critical to the phenotype.
• ART1 expression is cytoprotective to lung cancer cells and facilitates metastatic outgrowth of circulating cells through its inhibitory actions on tumor suppressive immune cells or soluble proteins in the blood or metastatic niche. It is likely that mono-ADP ribosylation also affects other immune cells in the tumor microenvironment, particularly T cells. Because ART1 is an extracellular enzymatic target, it is highly druggable and thus susceptible to therapeutic intervention.
• a “vector” refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide, and which can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo.
• Illustrative vectors include, for example, plasmids, viral vectors, liposomes and other gene delivery vehicles.
• the polynucleotide to be delivered may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic interest), a coding sequence of interest in vaccine development (such as a polynucleotide expressing a protein, polypeptide or peptide suitable for eliciting an immune response in a mammal), and/or a selectable or detectable marker.
• Transduction are terms referring to a process for the introduction of an exogenous polynucleotide into a host cell leading to expression of the polynucleotide, e.g., the transgene in the cell, and includes the use of recombinant virus to introduce the exogenous polynucleotide to the host cell.
• Transduction, transfection or transformation of a polynucleotide in a cell may be determined by methods well known to the art including, but not limited to, protein expression (including steady state levels), e.g., by ELISA, flow cytometry and Western blot, measurement of DNA and RNA by heterologousization assays, e.g., Northern blots, Southern blots and gel shift mobility assays.
• Methods used for the introduction of the exogenous polynucleotide include well-known techniques such as viral infection or transfection, lipofection, transformation and electroporation, as well as other non-viral gene delivery techniques.
• the introduced polynucleotide may be stably or transiently maintained in the host cell.
• Gene delivery refers to the introduction of an exogenous polynucleotide into a cell for gene transfer, and may encompass targeting, binding, uptake, transport, localization, replicon integration and expression.
• Gene transfer refers to the introduction of an exogenous polynucleotide into a cell which may encompass targeting, binding, uptake, transport, localization and replicon integration, but is distinct from and does not imply subsequent expression of the gene.
• Gene expression or “expression” refers to the process of gene transcription, translation, and post- translational modification.
• polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof.
• a polynucleotide may comprise modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
• polynucleotide refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the disclosure described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
• Nucleic acid sequence is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides.
• nucleic acid and polynucleotide refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and singlestranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped polynucleotides.
• an “isolated” polynucleotide e.g., plasmid, virus, polypeptide or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Isolated nucleic acid, peptide or polypeptide is present in a form or setting that is different from that in which it is found in nature.
• a given DNA sequence e.g., a gene
• RNA sequences such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins.
• the isolated nucleic acid molecule may be present in single-stranded or double-stranded form.
• the molecule will contain at a minimum the sense or coding strand (i.e., the molecule may single-stranded), but may contain both the sense and antisense strands (i.e., the molecule may be double-stranded).
• Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this disclosure are envisioned. Thus, for example, a 2-fold enrichment, 10-fold enrichment, 100-fold enrichment, or a 1000-fold enrichment.
• TRS transcriptional regulatory sequence
• Transcriptional regulatory sequences of use in the present disclosure generally include at least one transcriptional promoter and may also include one or more enhancers and/or terminators of transcription.
• “Operably linked” refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner.
• a transcriptional regulatory sequence or a promoter is operably linked to a coding sequence if the TRS or promoter promotes transcription of the coding sequence.
• An operably linked TRS is generally joined in cis with the coding sequence, but it is not necessarily directly adjacent to it.
• Heterologous means derived from a genotypically distinct entity from the entity to which it is compared.
• a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).
• a transcriptional regulatory element such as a promoter that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous transcriptional regulatory element.
• a “terminator” refers to a polynucleotide sequence that tends to diminish or prevent read-through transcription (i.e., it diminishes or prevent transcription originating on one side of the terminator from continuing through to the other side of the terminator).
• the degree to which transcription is disrupted is typically a function of the base sequence and/or the length of the terminator sequence.
• transcriptional termination sequences are specific sequences that tend to disrupt read-through transcription by RNA polymerase, presumably by causing the RNA polymerase molecule to stop and/or disengage from the DNA being transcribed.
• sequence-specific terminators include polyadenylation (“polyA”) sequences, e.g., SV40 polyA.
• polyA polyadenylation
• insertions of relatively long DNA sequences between a promoter and a coding region also tend to disrupt transcription of the coding region, generally in proportion to the length of the intervening sequence. This effect presumably arises because there is always some tendency for an RNA polymerase molecule to become disengaged from the DNA being transcribed, and increasing the length of the sequence to be traversed before reaching the coding region would generally increase the likelihood that disengagement would occur before transcription of the coding region was completed or possibly even initiated.
• Terminators may thus prevent transcription from only one direction (“uni-directional” terminators) or from both directions (“bi-directional” terminators), and may be comprised of sequence-specific termination sequences or sequence-non-specific terminators or both.
• sequence-specific termination sequences or sequence-non-specific terminators or both.
• “Host cells,” “cell lines,” “cell cultures,” “packaging cell line” and other such terms denote higher eukaryotic cells, such as mammalian cells including human cells, useful in the present disclosure, e.g., to produce recombinant virus or recombinant fusion polypeptide. These cells include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell.
• Recombinant as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
• a recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
• control element or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature.
• Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
• a promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter. Promoters include AAV promoters, e.g., P5, P19, P40 and AAV ITR promoters, as well as heterologous promoters.
• An “expression vector” is a vector comprising a region which encodes a gene product of interest, and is used for effecting the expression of the gene product in an intended target cell.
• An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target.
• the combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art.
• the terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, acetylation, phosphonylation, lipidation, or conjugation with a labeling component.
• exogenous when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial or natural means.
• An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid which occurs naturally within the organism or cell.
• an exogenous nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature, e.g., an expression cassette which links a promoter from one gene to an open reading frame for a gene product from a different gene.
• Transformed or transgenic is used herein to include any host cell or cell line, which has been altered or augmented by the presence of at least one recombinant DNA sequence.
• the host cells of the present disclosure are typically produced by transfection with a DNA sequence in a plasmid expression vector, as an isolated linear DNA sequence, or infection with a recombinant viral vector.
• sequence homology means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of a selected sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred.
• the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); not less than 9 matches out of 10 possible base pair matches (90%), or not less than 19 matches out of 20 possible base pair matches (95%).
• Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred.
• two protein sequences or polypeptide sequences derived from them of at least 30 amino acids in length
• the two sequences or parts thereof are more homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.
• a polynucleotide sequence is structurally related to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is structurally related to all or a portion of a reference polypeptide sequence, e.g., they have at least 80%, 85%, 90%, 95% or more, e.g., 99% or 100%, sequence identity.
• the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
• the nucleotide sequence “TATAC” corresponds to a reference sequence “TATAC” and is complementary to a reference sequence “GTATA”.
• sequence identity means that two polynucleotide sequences are identical (i.e. , on a nucleotide-by-nucleotide basis) over the window of comparison.
• percentage of sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
• percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• the identical nucleic acid base e.g., A, T, C, G, U, or I
• substantially identical denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
• Constant amino acid substitutions are, for example, aspartic-glutamic as polar acidic amino acids; lysine/arginine/histidine as polar basic amino acids; leucine/isoleucine/methionine/valine/alanine/glycine/proline as non-polar or hydrophobic amino acids; serine/ threonine as polar or uncharged hydrophilic amino acids.
• Conservative amino acid substitution also includes groupings based on side chains.
• a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
• Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, He; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic; trp, tyr, phe.
• Non-conservative substitutions entail exchanging a member of one of the classes described above for another.
• ART1 ADP ribosyltransferase 1 , e.g., a mammalian ART1 having a sequence in Accesion Nos. NP_004305.2, XP_0115184161 , NP_033840.2, or XP_011239959.1 , the disclosures of which are incorporated by reference herein, or a protein with at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto, alternatively spliced isoforms thereof, peptide fragments thereof or post-translationally modified proteins or peptides thereof
• an antibody within the scope of this disclosure may bind human ART1 , e.g., MQMPAMMSLL LVSVGLMEAL QAQSHPITRR DLFSQEIQLD MALASFDDQY AGCAAAMTAA LPDLNHTEFQ ANQVYADSWT LASSQWQ
• antibody refers to a full-length immunoglobulin molecule or an immunologically-active fragment of an immunoglobulin molecule such as the Fab or F(ab’)2 fragment generated by, for example, cleavage of the antibody with an enzyme such as pepsin or co-expression of an antibody light chain and an antibody heavy chain in, for example, a mammalian cell, or ScFv.
• the antibody can also be an IgG, IgD, IgA, IgE or IgM antibody.
• Full-length immunoglobulin "light chains” (about 25 kD or 214 amino acids) are encoded by a variable region gene at the amino-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the carboxy-terminus.
• Full-length immunoglobulin "heavy chains” (about 50 kD or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
• An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
• VL variable light chain
• VH variable heavy chain
• immunoglobulins may exist in a variety of other forms including, for example, Fv, ScFv, Fab, and F(ab')2, as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al. (1987)) and in single chains (e.g., Huston et al. (1988) and Bird et al. (1988), which are incorporated herein by reference).
• bifunctional hybrid antibodies e.g., Lanzavecchia et al. (1987)
• single chains e.g., Huston et al. (1988) and Bird et al. (1988
• antibody includes antigen binding antibody fragments, as are known in the art, including Fab, Fabz, single chain antibodies (scFv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
• An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, also called CDR's.
• the extent of the framework region and CDR's have been precisely defined (see, "Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, (1983); which is incorporated herein by reference).
• the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
• a "human framework region” is a framework region that is substantially identical (about 85% or more, usually 90 to 95% or more) to the framework region of a naturally occurring human immunoglobulin.
• the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR's.
• the CDR's are primarily responsible for binding to an epitope of an antigen.
• Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species.
• the variable segments of the genes from a mouse monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3.
• One example of a chimeric antibody is one composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species may be used.
• humanized immunoglobulin refers to an immunoglobulin having a human framework region and one or more CDR's from a non-human (usually a mouse or rat) immunoglobulin.
• the non-human immunoglobulin providing the CDR's is called the "donor” and the human immunoglobulin providing the framework is called the “acceptor.”
• Constant regions need not be present, but if they are, they are generally substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, or about 95% or more identical.
• all parts of a humanized immunoglobulin, except possibly the CDR's are substantially identical to corresponding parts of natural human immunoglobulin sequences.
• a “humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
• the donor antibody has been “humanized”, by the process of "humanization”, because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDR's.
• humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab’)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
• Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• CDR complementary determining region
• donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
• Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• the humanized antibody has substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody optimally also will include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al. (1986); Riechmann et al. (1988); and Presta (1992)).
• humanized antibodies may have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
• conservative substitutions are intended combinations such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr.
• Humanized immunoglobulins including humanized antibodies, have been constructed by means of genetic engineering. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non- human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain.
• Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522 (1986); Riechmann et al., Nature, 332:323 (1988); Verhoeyen et al., Science, 239:1534 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• such "humanized" antibodies are chimeric antibodies that have substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.
• Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).
• the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147:86 (1991)).
• human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
• a framework may be one from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or a consensus framework derived from many human antibodies. For example, comparison of the sequence of a mouse heavy (or light) chain variable region against human heavy (or light) variable regions in a data bank (for example, the National Biomedical Research Foundation Protein Identification Resource) shows that the extent of homology to different human regions varies greatly, typically from about 40% to about 60-70%. By choosing one of the human heavy (respectively light) chain variable regions that is most homologous to the heavy (respectively light) chain variable region of the other immunoglobulin, fewer amino acids will be changed in going from the one immunoglobulin to the humanized immunoglobulin. The precise overall shape of a humanized antibody having the humanized immunoglobulin chain may more closely resemble the shape of the donor antibody, also reducing the chance of distorting the CDR's.
• one of the 3-5 most homologous heavy chain variable region sequences in a representative collection of at least about 10 to 20 distinct human heavy chains is chosen as acceptor to provide the heavy chain framework, and similarly for the light chain.
• One of the 1 to 3 most homologous variable regions may be used.
• the selected acceptor immunoglobulin chain may have at least about 65% homology in the framework region to the donor immunoglobulin.
• acceptor immunoglobulin it may be considered desirable to use light and heavy chains from the same human antibody as acceptor sequences, to be sure the humanized light and heavy chains will make favorable contacts with each other. Regardless of how the acceptor immunoglobulin is chosen, higher affinity may be achieved by selecting a small number of amino acids in the framework of the humanized immunoglobulin chain to be the same as the amino acids at those positions in the donor rather than in the acceptor.
• Humanized antibodies generally have advantages over mouse or in some cases chimeric antibodies for use in human therapy: because the effector portion is human, it may interact better with the other parts of the human immune system (e.g., destroy the target cells more efficiently by complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC)); the human immune system should not recognize the framework or constant region of the humanized antibody as foreign, and therefore the antibody response against such an antibody should be less than against a totally foreign mouse antibody or a partially foreign chimeric antibody.
• CDC complement-dependent cytotoxicity
• ADCC antibody-dependent cellular cytotoxicity
• DNA segments having immunoglobulin sequences typically further include an expression control DNA sequence operably linked to the humanized immunoglobulin coding sequences, including naturally-associated or heterologous promoter regions.
• the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the humanized light chains, heavy chains, light/heavy chain dimers or intact antibodies, binding fragments or other immunoglobulin forms may follow (see, S. Beychok, Cells of Immunoglobulin Synthesis, Academic Press, New York, (1979), which is incorporated herein by reference).
• substantially homologous modified immunoglobulins to the native sequences can be readily designed and manufactured utilizing various recombinant DNA techniques well known to those skilled in the art.
• the framework regions can vary at the primary structure level by several amino acid substitutions, terminal and intermediate additions and deletions, and the like.
• a variety of different human framework regions may be used singly or in combination as a basis for the humanized immunoglobulins of the present disclosure.
• modifications of the genes may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see, Gillman and Smith, Gene, 8:81 (1979) and Roberts et al., Nature, 328:731 (1987), both of which are incorporated herein by reference).
• Substantially homologous immunoglobulin sequences are those which exhibit at least about 85% homology, usually at least about 90%, or at least about 95% homology with a reference immunoglobulin protein.
• polypeptide fragments comprising only a portion of the primary antibody structure may be produced, which fragments possess one or more immunoglobulin activities (e.g., antigen binding).
• immunoglobulin activities e.g., antigen binding
• polypeptide fragments may be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by inserting stop codons at the desired locations in vectors known to those skilled in the art, using site-directed mutagenesis.
• the disclosure provides for an antibody, antigen binding fragment thereof, or a polypeptide, directed against ART 1 .
• the antibody, fragment thereof, or polypeptide binds both human and mouse ART 1 , and so are likely bind to conserved sequences in those proteins (see the alignment below), while antbodies that bind human but not mouse ART1 likely bind to non-conserved sequences (see the alignment below).
• VLIPPFETFQVINASRLAQGPARIYLRALGKHSTYNCEYIKDKKCKSGPCHLDNSAMG SEQ ID NO:90
• VLIPPFETFQVINTSRPTQGPARIYLRALGKRSTYNCEYIKEKKCRSGPCWLGSSAPG SEQ ID NO:92
• an antibody, antigen binding fragment thereof, or a polypeptide, directed against ART1 that binds to both human and mouse ART1 may bind to residues including those from position 170 to 185 (human numbering), 195 to 210, or 230 to 250, or a combination thereof.
• antibodies that bind to ART1 include those that bind to residues including those from positon 110 to 160, 185 to 225 or 245 to 275, or a combination thereof, in ART 1.
• antibodies that bind to ART 1 include those that bind to residues including those from positon 20 to 50, 80 to 100, 170 to 85, 225 to 245 or 275 to the C-terminus, or a combination thereof.
• an antibody, antigen binding fragment thereof, or a polypeptide binds to at least one of the following residues: S75, S77, T79, R80, R89, H92, or Y99 of human ART1 , e.g., SEQ ID NO:90.
• an isolated antibody or fragment thereof binds to two, three, four, five or six of S75, S77, T79, R80, R89, H92, or Y99 of human ART1 , e.g., SEQ ID NO:90.
• an isolated antibody or fragment thereof binds to S75, S77, T79, R80, R89, H92, and Y99 of human ART1 , e.g., SEQ ID NQ:90.
• an antibody consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two copies of a light (L) chain polypeptide.
• Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1 , CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region.
• the variable regions of each pair of light and heavy chains form the antigen binding site of an antibody.
• the nucleic acid sequence which encodes an antibody directed against ART1 can comprise one or more nucleic acid sequences, each of which encodes one or more of the heavy and/or light chain polypeptides of an anti-ART1 antibody.
• the nucleic acid sequence which encodes an antibody directed against ART1 can comprise a single nucleic acid sequence that encodes the heavy chain polypeptide and the light chain polypeptide of an anti-ART1 antibody.
• the nucleic acid sequence which encodes an antibody directed against ART1 can comprise a first nucleic acid sequence that encodes the heavy chain polypeptide of an anti-ART1 antibody, and a second nucleic acid sequence that encodes the light chain polypeptide of an anti-ART1 antibody.
• the nucleic acid sequence which encodes a fragment of an antibody directed against ART1 can comprise a nucleic acid sequence encoding a heavy chain variable region polypeptide of an anti-ART1 antibody, a nucleic acid sequence encoding a light chain variable region polypeptide of an anti-ART1 antibody, or a nucleic acid sequence encoding a heavy chain variable region and a light chain vasrtiabel region polypeptide of an anti-ART1 antibody.
• the nucleic acid sequence which encodes an antibody directed against ART1 encodes an antigen-binding fragment (also referred to as an “antibody fragment”) of an anti-ART1 antibody.
• antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., ART1) (see, generally, Holliger and Hudson 2005).
• antigenbinding fragments include but are not limited to (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody.
• the nucleic acid sequence which encodes an antibody directed against ART1 can comprise a nucleic acid sequence encoding a Fab fragment of an antiART 1 antibody.
• the nucleic acid sequence which encodes an antibody or fragment thereof directed against ART1 can comprise a nucleic acid sequence encoding a heavy chain variable region that binds ART1. In one embodiment, the nucleic acid sequence which encodes an antibody or fragment thereof directed against ART1 can comprise a nucleic acid sequence encoding a light chain variable region that binds ART1. In one embodiment, the nucleic acid sequence which encodes an antibody directed against ART1 can comprise a nucleic acid sequence encoding one, two or three CDRs, e.g., of a heavy chain variable region, that bind(s) ART1.
• the nucleic acid sequence which encodes an antibody directed against ART1 can comprise a nucleic acid sequence encoding one, two or three CDRs, e.g., of a light chain variable region, that bind(s) ART1.
• the antibody fragment may be a scFv antibody or a nanobody (VHH antibodies having a single variable domain in a heavy chain), Fab or F(ab’)2.
• the nucleic acid sequence can encode the ART1 -binding monoclonal antibody 22C12 or a fragment thereof. In one embodiment, the nucleic acid sequence can encode the ART1 -binding monoclonal antibody 14G01 or a fragment thereof.
• nucleic acid sequence which encodes an antibody against ART1 that recognizes (binds) human and mouse ART 1 . In an embodiment, the nucleic acid sequence which encodes an antibody against ART1 recognizes human but not mouse ART1.
• An antibody, or antigen-binding fragment thereof can be obtained by any means, including via in vitro sources (e.g., a hybridoma or a cell line producing an antibody recombinantly) and in vivo sources (e.g., rodents).
• in vitro sources e.g., a hybridoma or a cell line producing an antibody recombinantly
• in vivo sources e.g., rodents
• a human antibody or a chimeric antibody can be generated using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes.
• transgenic mice wherein endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, the AlivaMab® mouse, Veloclmmune mouse, Trianni® mouse, KymabTM mouse, HUMAB-MOUSETM , the Kirin TC MOUSETM, and the KM-MOUSETM (see, e.g., Lonberg, Nat. Biotechnol., 23(9):1117 (2005), and Lonberg, Handb. Exp. Pharmacol., 181 :69 (2008)).
• the nucleic acid sequence which encodes an antibody directed against ART 1 , an antigen-binding fragment thereof, or a polypeptide that binds ART1 can be generated using methods known in the art.
• polypeptides, and proteins can be recombinantly produced using standard recombinant DNA methodology (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001 ; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994).
• an antibody directed against ART 1 can be isolated and/or purified from a source, such as a bacterium, an insect, or a mammal, e.g., a rat, a human, etc., into which a synthetically produced nucleic acid sequences encoding such antibody or antigen-binding fragment was introduced. Methods of isolation and purification are well-known in the art.
• the nucleic acid sequences described herein can be commercially synthesized. In this respect, the nucleic acid sequence can be synthetic, recombinant, isolated, and/or purified.
• the nucleic acid sequence which encodes an antibody directed against ART1 may be identified by extracting RNA from the available antibody producing hybridoma cells.
• cDNA is produced by reverse transcription and PCR amplification of the light and heavy chains and is carried out using a rapid amplification of cDNA ends (RACE) strategy in combination with specific primers for conserved regions in the constant domains.
• RACE rapid amplification of cDNA ends
• nucleic acid sequence which encodes an antibody directed against ART1 may also be fully or partly humanized by means known in the art.
• an antibody chimera may be created by substituting DNA encoding the mouse Fc region of the antibody with that of cDNA encoding for human.
• the Fab portion of the molecule may also be humanized by selectively altering the DNA of non-CDR portions of the Fab sequence that differ from those in humans by exchanging the sequences for the appropriate individual amino acids.
• humanization may be achieved by insertion of the appropriate CDR coding segments into a human antibody "scaffold".
• Resulting antibody DNA sequences may be modified for high expression levels in mammalian cells through removal of RNA instability elements ans/or codon optimization, as is known in the art.
• nucleic acid sequences which encode the heavy chain and light chain of an antibody directed against ART1 may be expressed under the control of a single promoter in a 1 :1 ratio using a 2A sequence (a cis-acting hydrolase element) self-cleavable sequence.
• the 2A sequence self-cleaves during protein translation and leaves a short tail of amino acids in the C-terminus of the upstream protein.
• a Furin cleavage recognition site may be added between the 2A sequence and the upstream gene to assure removal of the remaining amino acids.
• Plasmids expressing the correct inserts may be identified by DNA sequencing and by antibody specific binding using western analysis and ELISA assays.
• the disclosure also provides a gene transfer vector comprising a nucleic acid sequence which encodes an antibody, an antigen binding fragment thereof, or a polypeptide, directed against ART1.
• the gene transfer vector is a virus.
• the disclosure further provides a method of using the gene transfer vector or encoded gene product against ART1 in a mammal, which method comprises administering to the mammal the above-described gene transfer vector or the encoded gene product.
• Various aspects of the gene transfer vector, antibody or antigen binding fragment thereof, and methods are discussed below. Although each parameter is discussed separately, the gene transfer vector, antibody or antigen binding fragment thereof, or polypeptide, and method, may comprise combinations of the parameters set forth below. Accordingly, any combination of parameters can be used according to the gene transfer vector, antibody or antigen binding fragment thereof, the polypeptide, and the method.
• a “gene transfer vector” is any molecule or composition that has the ability to carry and deliver a heterologous nucleic acid sequence into a suitable host cell where synthesis of the encoded protein takes place.
• a gene transfer vector is a nucleic acid molecule that has been engineered, using recombinant DNA techniques that are known in the art, to incorporate the heterologous nucleic acid sequence.
• the gene transfer vector is comprised of DNA.
• suitable DNA-based gene transfer vectors include plasmids and viral vectors.
• gene transfer vectors that are not based on nucleic acids, such as liposomes are also known and used in the art.
• the gene transfer vector can be based on a single type of nucleic acid (e.g., a plasmid) or non-nucleic acid molecule (e.g., a lipid or a polymer).
• the gene transfer vector can be integrated into the host cell genome, or can be present in the host cell in the form of an episome.
• the gene transfer vector is a viral vector.
• Suitable viral vectors include, for example, retroviral vectors, herpes simplex virus (HSV)-based vectors, parvovirus-based vectors, e.g., adeno- associated virus (AAV)-based vectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors.
• HSV herpes simplex virus
• AAV adeno- associated virus
• AAV-adenoviral chimeric vectors e.g., AAV-adenoviral chimeric vectors
• adenovirus-based vectors e.g., adeno-associated virus (AAV)-based vectors.
• AAV-adenoviral chimeric vectors e.g., AAV-adenoviral chimeric vectors
• adenovirus-based vectors e.g., adeno-associated virus
• These viral vectors can be prepared using standard recombin
• Any viral vector may be employed to deliver antibody encoding sequences to cells including mammalian cells, or to mammals, include but are not limited to adeno-associated virus, adenovirus, herpesvirus, retrovirus, aor lentivirus vectors.
• the viral vector may comprise expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the nucleic acid sequence in a host cell.
• expression control sequences such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the nucleic acid sequence in a host cell.
• Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, CA. (1990).
• promoters including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art.
• Representative sources of promoters include for example, virus, mammal, insect, plant, yeast, and bacteria, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available, for example, from depositories such as the ATCC as well as other commercial or individual sources.
• Promoters can be unidirectional (i.e. , initiate transcription in one direction) or bi-directional (i.e. , initiate transcription in either a 3’ or 5’ direction).
• Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter.
• Inducible promoters include, for example, the Tet system (U.S. Patent Nos. 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl. Acad.
• Enhancer refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoters (such as the commonly-used CMV promoter) also comprise enhancer sequences.
• Enhancers can be located upstream, within, or downstream of coding sequences.
• the nucleic acid sequence encoding an antibody against ART1 , or an antigen-binding fragment thereof is operably linked to a CMV enhancer/chicken beta-actin promoter (also referred to as a “CAG promoter”) (see, e.g., Niwa et al., Gene, 108:193 (1991); Daly et al., Proc. Natl. Acad. Sci. U.S.A., 96:2296 (1999); and Sondhi et al., Mol. Ther., 15:481 (2007)).
• CMV enhancer/chicken beta-actin promoter also referred to as a “CAG promoter”
• AAV vectors are produced using well characterized plasmids.
• human embryonic kidney 293T cells are transfected with one of the transgene specific plasmids and another plasmid containing the adenovirus helper and AAV rep and cap genes (specific to AAVrh.10, 8 or 9 as required).
• the cells are harvested and the vector is released from the cells by five freeze/thaw cycles.
• Subsequent centrifugation and benzonase treatment removes cellular debris and unencapsidated DNA.
• lodixanol gradients and ion exchange columns may be used to further purify each AAV vector.
• the purified vector is concentrated by a size exclusion centrifuge spin column to the required concentration.
• the buffer is exchanged to create the final vector products formulated (for example) in 1x phosphate buffered saline.
• the viral titers may be measured by TaqMan® real-time PCR and the viral purity may be assessed by SDS-PAGE.
• composition comprising, consisting essentially of, or consisting of the above-described antibody, antibody fragment, such as a single chain polypeptide, or gene transfer vector and a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, or an antibody or antigen binding fragment thereof optionally with a pharmaceutically acceptable (e.g., physiologically acceptable) carrier.
• a pharmaceutically acceptable carrier e.g., physiologically acceptable
• additional components can be included that do not materially affect the composition (e.g., adjuvants, buffers, stabilizers, anti-inflammatory agents, solubilizers, preservatives, etc.).
• the composition consists of the gene transfer vector and the pharmaceutically acceptable carrier, or the antibody, antigen binding fragment thereof or polypeptide optionally with a pharmaceutically acceptable carrier
• the composition does not comprise any additional components.
• Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition.
• the composition optionally can be sterile with the exception of the gene transfer vector or an antibody or antigen binding fragment thereof or polypeptide described herein.
• the composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use.
• the compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).
• Suitable formulations for the composition include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain anti-oxidants, buffers, and bacteriostats, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
• the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use.
• Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
• the carrier is a buffered saline solution.
• the gene transfer vector, antibody or antigen binding fragment thereof is administered in a composition formulated to protect the gene transfer vector or antibody or antigen binding fragment thereof from damage prior to administration.
• the composition can be formulated to reduce loss of the gene transfer vector on devices used to prepare, store, or administer the gene transfer vector, such as glassware, syringes, or needles.
• the composition can be formulated to decrease the light sensitivity and/or temperature sensitivity of the gene transfer vector or an antibody or antigen binding fragment thereof.
• the composition may comprise a pharmaceutically acceptable liquid carrier, such as, for example, those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.
• a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.
• Use of such a composition will extend the shelf life of the gene transfer vector, facilitate administration, and increase the efficiency of the method.
• Formulations for gene transfer vector-containing compositions are further described in, for example, Wright et al., Curr. Opin. Drug Discov. Devel., 6(2) 174-178 (2003) and Wright et al., Molecular Therapy, 12: 171-178 (2005))
• the composition also can be formulated to enhance transduction efficiency.
• the gene transfer vector or antibody or antigen binding fragment thereof can be present in a composition with other therapeutic or biologically-active agents.
• factors that control inflammation such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the gene transfer vector or the antibody or antigen binding fragment thereof.
• Immune system stimulators or adjuvants e.g., interleukins, lipopolysaccharide, and double-stranded RNA, can be administered to enhance or modify the anti-ART1 immune response.
• Antibiotics i.e., microbicides and fungicides, can be present to treat existing infection and/or reduce the risk of future infection, such as infection associated with gene transfer procedures.
• Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
• a formulation of the present disclosure comprises a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, celluloses, polypropylene, polyethylenes, polystyrene, polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.
• a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and
• the composition can be administered in or on a device that allows controlled or sustained release, such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant.
• Implants see, e.g., U.S. Patent No. 5,443,505
• devices see, e.g., U.S. Patent No. 4,863,457
• an implantable device e.g., a mechanical reservoir or an implant or a device comprised of a polymeric composition
• the composition also can be administered in the form of sustained-release formulations (see, e.g., U.S. Patent No.
• 5,378,475) comprising, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), and/or a polylactic-glycolic acid.
• a polyphosphoester such as bis-2-hydroxyethyl-terephthalate (BHET)
• BHET bis-2-hydroxyethyl-terephthalate
• compositions comprising the gene transfer vectors, antibody or antigen binding fragment thereof or polypeptide
• Delivery of the compositions may be intracerebral (including but not limited to intraparenchymal, intraventricular, or intracisternal), intrathecal (including but not limited to lumbar or cisterna magna), or systemic, including but not limited to intravenous, oral, or any combination thereof, using devices known in the art. Delivery may also be via surgical implantation of an implanted device.
• the method comprises administering a “therapeutically effective amount” of the composition comprising the gene transfer vector, antibody or antigen binding fragment thereof described herein.
• a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
• the therapeutically effective amount may vary according to factors such as the extent of pathology, age, sex, and weight of the individual, and the ability of the gene transfer vector, antibody or antigen binding fragemtn thereof to elicit a desired response in the individual.
• the dose of gene transfer vector in the composition required to achieve a particular therapeutic effect typically is administered in units of vector genome copies per cell (gc/cell) or vector genome copies/per kilogram of body weight (gc/kg).
• gc/cell vector genome copies per cell
• gc/kg vector genome copies/per kilogram of body weight
• a therapeutically effective amount may be between 1 x 1 O 10 genome copies to 1x 10 13 genome copies.
• a therapeutically effective amount may be between 1 x 10 12 genome copies to 1x 10 15 genome copies (total).
• a therapeutically effective amount may be between 1 x 10 12 genome copies/kg to 1x 10 15 genome copies/kg.
• the dose of antibody or antigen binding fragment thereof in the composition required to achieve a particular therapeutic effect typically is administered in units of antibody or antigen binding fragment per kg (mg/kg) or total dose (mg).
• a therapeutically effective amount of antibody or antigen binding fragment thereof may be between 25 to 200 mg, e.g., 50 to 100 mg, 25 to 50 mg, 50 to 75 mg, 100 to 150 mg, 150 to 200 mg, 200 mg to 300 mg, 300 mg to 400 mg, 400 mg to 500 mg, or 500 mg to 600 mg.
• a therapeutically effective amount of antibody or antigen binding fragment thereof may be between 1 mg/kg to 20 mg/kg, e.g., 2 to 5 mg/kg, 5 to 7 mg/kg or 10 to 15 mg/kg.
• the composition is administered once to the mammal. It is believed that a single administration of the composition will result in persistent expression of the anti-ART1 antibody in the mammal with minimal side effects. However, in certain cases, it may be appropriate to administer the composition multiple times during a therapeutic period to ensure sufficient exposure of cells to the composition. For example, the composition may be administered to the mammal two or more times (e.g., 2, 3, 4, 5, 6, 6, 8, 9, or 10 or more times) during a therapeutic period.
• compositions which comprise a therapeutically-effective amount of gene transfer vector comprising a nucleic acid sequence which encodes an antibody directed against ART1 , or a therapeutically effective amount of the antibody or antigen binding fragment thereof, as described above.
• diseases which may be prevented, inhibited or treated with the antibody or antigen binding fragment thereto includes but is not limited to neoplasms carcinomas (e.g., squamous-cell carcinomas, adenocarcinomas, hepatocellular carcinomas, and renal cell carcinomas), particularly those of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemias; benign and malignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign and malignant melanomas; myeloproliferative diseases; sarcomas, particularly Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, and synovial sarcoma; tumors of the central organ
• the disease is pancreatic cancer, lung cancer, liver cancer, skin cancer, colon cancer, breast cancer, prostate cancer, leukemia, Burkitt like lymphoma, acute lymphoblastic leukemia or melanoma.
• compositions are useful to treat a subject with a medical condition or disorder that involves overexpression of ART1 or treat changes in ART1 activity, e.g., cancer.
• a medical condition or disorder that involves overexpression of ART1 or treat changes in ART1 activity e.g., cancer.
• the subject may be any animal, including a human and non-human animal.
• Non-human animals includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are envisioned as subjects, such as non-human primates, sheep, dogs, cats, cows and horses.
• the subject may also be livestock such as, cattle, swine, sheep, poultry, and horses, or pets, such as dogs and cats.
• Exemplary subjects include human subjects suffering from or at risk for the medical diseases and conditions described herein.
• the subject is generally diagnosed with the condition of the subject disclosure by skilled artisans, such as a medical practitioner.
• the methods of the disclosure described herein can be employed for subjects of any species, gender, age, ethnic population, or genotype. Accordingly, the term subject includes males and females, and it includes elderly, elderly-to-adult transition age subjects adults, adult-to-pre-adult transition age subjects, and pre-adults, including adolescents, children, and infants.
• human ethnic populations include Caucasians, Asians, Hispanics, Africans, African Americans, Native Americans, Semites, and Pacific Islanders.
• the methods of the disclosure may be more appropriate for some ethnic populations such as Caucasians, especially northern European populations, as well as Asian populations.
• subject also includes subjects of any genotype or phenotype as long as they are in need of the disclosure, as described above.
• the subject can have the genotype or phenotype for any hair color, eye color, skin color or any combination thereof.
• subject includes a subject of any body height, body weight, or any organ or body part size or shape.
• NARMGVS SEQ ID NO:21
• WGLGTMVTVSS (SEQ ID NO:41 )
• LAWYQQKPGQAPRLLIY SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:47)
• VSWIRQP PGKALEWLAH (SEQ ID NO:72)
• VLIPPFETFQVIN SR QGPARIYLRALGK STYNCEYIK+KKC+SGPC L +SA G (SEQ ID N0:91) MKIPAMMSLLLVSVGLRDGVQVQSYSISQLDIFSQETPLDMAPASFDDQYAGCLADMTAA LPDLNHSEFQANKVYADGWAQANNQWQERRAWGSVWGSLP-PSPP-GFRDEHGVALLA YTANSPLHKEFNAAVREAGRSRAHYLHHFSFKTLHFLLTEALQLLRS-HRSRGCQQVYRG VHGLRFRPAGPGATVRLGGFASASLKNVAAQQFGEDTFFGIWTCLGAPIRGYSFFPEEEEEE VLIPPFETFQVINTSRPTQGPARIYLRALGKRSTYNCEYIKEKKCRSGPCWLGSSAPG (SEQ ID NO:92)
• HIFSNDEKSYSTSIKS (SEQ ID NO:96) HLFSNDEKSYSTSIKS (SEQ ID NO:97) HIFTNDEKSYSSSLKS (SEQ ID NO:98) IYGGADSWGYFEN (SEQ ID NO:99) IYGGDSWAYFDN (SEQ ID NQ:100) LYGIDSWGYFDN (SEQ ID NQ:101) GFSISNARMG (SEQ ID NQ:102) GFSASNTRMG (SEQ ID NQ:103) GFSISNLRMA (SEQ ID NQ:104).
• LFSNDEK (SEQ ID NQ:105) IFSNEDK (SEQ ID NQ:106).
• GRIYGGDSWGYFDN (SEQ ID NQ:107) ARIYAADSWGYFDN (SEQ ID NQ:108) IRAYGGDSWLYFDN (SEQ ID NQ:109) Exemplary Embodiments
• the disclosure provides an isolated cell comprising an expression cassette comprising a heterologous promoter operably linked to nucleic acid sequences encoding an anti-human ART1 antibody, or an antigen binding fragment thereof, or a polypeptide, that inhibits human ART1 activity, wherein the antibody, the antigen binding fragment thereof, or the polypeptide has: a variable heavy chain region comprising a first complementarity determining region (CDR) operably linked to a second CDR operably linked to a third CDR comprising; and/or a variable light region comprising a first CDR operably linked to a second CDR operably linked to a third CDR.
• the sequences in the CDRs may include any of SEQ ID Nos.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31- 34, 38-41 , 45-47, 51 -63, 71-74, or 76-79, or a sequence with 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions. In one embodiment, the majority or all of the substitutions are conversative substitutions. In one embodiment, the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos.
• the cell is a mammalian cell, e.g., a primate cell or a rodent cell, e.g., a CHO cell. In one embodiment, the cell is a human cell.
• hybridoma comprising nucleic acid sequences encoding an anti-human ART1 monoclonal antibody that inhibits human ART1 activity, wherein the antibody has: a variable heavy chain region comprising a first CDR operably linked to a second CDR operably linked to a third CDR comprising; and/or a variable light region comprising a first CDR operably linked to a second CDR operably linked to a third CDR.
• the sequences in the CDRs may include any of SEQ ID Nos. 21-23, 28-30, 35-37, 42-44, 66-68, or 81 , or a sequence with 1 , 2, 3, 4 or 5 substitutions. In one embodiment, the majority or all of the substitutions are conversative substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31-34, 38-41 , 45-47, 51-63, 71-74, or 76-79, or a sequence with 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24- 27, 31-34, 38-41 , 45-47, 51-63, 71-74, or 76-79, or a sequence having at least 80%, 85%, 90%, 92%, 95% or 99% amino acid sequience identity thereto.
• an isolated nucleic acid comprising a promoter operably linked to a nucleotide sequence which encodes at least the variable region of a heavy or light Ig chain that binds human and/or mouse ART1 , wherein the chain comprises: a variable heavy chain region comprising a first CDR operably linked to a second CDR operably linked to a third CDR comprising; and/or a variable light region comprising a first CDR operably linked to a second CDR operably linked to a third CDR.
• the sequences in the CDRs may include any of SEQ ID Nos. 21-23, 28-30, 35-36, 42-44, 66-68, or 81 , or a sequence with 1 , 2, 3, 4 or 5 substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31-34, 38-41 , 45-47, 51-63, 71-74, or 76-79, or a sequence with 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31-34, 38-41 , 45-47, 51-63, 71-74, or 76-79, or a sequence having at least 80%, 85%, 90%, 92%, 95% or 99% amino acid sequence identity thereto.
• an isolated antibody or antigen fragment thereof that binds human and mouse ART1 , wherein the antibody or the antigen binding fragment thereof has: a variable heavy chain region comprising a first CDR operably linked to a second CDR operably linked to a third CDR comprising; and/or a variable light region comprising a first CDR operably linked to a second CDR operably linked to a third CDR.
• the sequences in the CDRs may include any of SEQ ID Nos. 21-23, 28-30, 35-36, 42-44, 66-68, or 81 , or a sequence with 1 , 2, 3, 4 or 5 substitutions. In one embodiment, the majority or all of the substitutions are conversative substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31-34, 38-41 , 45-47, 51-63, 71-74, or 76-79, or a sequence with 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31-34, 38-41 , 45-47, 51-63, 71-74, or 76-79, or a sequence having at least 80%, 85%, 90%, 92%, 95% or 99% amino acid sequence identity thereto.
• Inhibitors of ART1 may be employed in vivo.
• a method to inhibit or treat cancer in a mammal in which a composition comprising an effective amount of an antihuman ART 1 antibody or an antigen binding fragment thereof, or a polypeptide that binds human ART 1 , is administered to a mammal.
• the cancer is lung cancer, colon cancer, melanoma, glioblastoma, breast cancer or colorectal cancer.
• the mammal is a human.
• the amount is effective to decrease tumor burden, inhibit metastases, increase survival, or any combination thereof.
• the composition is systemically administered.
• the mammal is also administered a chemotherapeutic drug.
• the mammal is administered an immune checkpoint inhibitor.
• the heavy chain of the antibody or fragment thereof is an IgG heavy chain.
• the light chain of the antibody or fragment thereof is an IgK light chain.
• the fragment is Fab', F(ab')2, scFv or a single domain.
• the antibody or the antigen binding fragment thereof, or the polypeptide has: a variable hevy chain region comprising a first CDR operably linked to a second CDR operably linked to a third CDR comprising; and/or a variable light region comprising a first CDR operably linked to a second CDR operably linked to a third CDR.
• the sequences in the CDRs may include any of SEQ ID Nos. 21-23, 28-30, 35-36, 42-44, 66-68, or 81 , or a sequence with 1 , 2, 3, 4 or 5 substitutions. In one embodiment, the majority or all of the substitutions are conversative substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31-34, 38-41 , 45-47, 51-63, 71-74, or 76-79, or a sequence with 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31-34, 38-41 , 45-47, 51-63, 71-74, or 76-79, or a sequence having at least 80%, 85%, 90%, 92%, 95% or 99% amino acid sequence identity thereto.
• a method to prevent, inhibit or treat ART1 -mediated immunosuppression in a mammal includes administering to a mammal a composition comprising an effective amount of an anti-human ART1 antibody or an antigen binding fragment thereof, or a polypeptide that binds human ART1.
• the composition is systemically administered.
• the mammal is also administered a chemotherapeutic drug and/or radiation therapy.
• the mammal is administered an immune checkpoint inhibitor.
• the heavy chain of the antibody or fragment thereof is an IgG heavy chain.
• the light chain of the antibody or fragment thereof is an IgK light chain.
• the fragment is Fab' or scFv.
• the antibody or the antigen binding fragment thereof, or the polypeptide has: a variable hevy chain region comprising a first CDR operably linked to a second CDR operably linked to a third CDR comprising; and/or a variable light region comprising a first CDR operably linked to a second CDR operably linked to a third CDR.
• the sequences in the CDRs may include any of SEQ ID Nos. 21-23, 28-30, 35-36, 42-44, 66-68, or 81 , or a sequence with 1 , 2, 3, 4 or 5 substitutions. In one embodiment, the majority or all of the substitutions are conversative substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31-34, 38-41 , 45-47, 51-63, 71-74, or 76-79, or a sequence with 1 , 2 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31 -34, 38-41 , 45-47, 51 -63, 71 -74, or 76-79, or a sequence having at least 80%, 85%, 90%, 92%, 95% or 99% amino acid sequence identity thereto.
• a method to enhance an immune response in a mammal having cancer comprising: administering to a mammal a composition comprising an effective amount of an anti-human ART1 antibody or an antigen binding fragment thereof, or a polypeptide that binds human ART1.
• the composition is systemically administered.
• the mammal is also administered a chemotherapeutic drug and/or radiation therapy.
• the mammal is administered an immune checkpoint inhibitor.
• the heavy chain of the antibody or fragment thereof is an IgG heavy chain.
• the light chain of the antibody or fragment thereof is an IgK light chain.
• the fragment is Fab' or scFv.
• the antibody or the antigen binding fragment thereof, or the polypeptide has: a variable hevy chain region comprising a first CDR operably linked to a second CDR operably linked to a third CDR comprising; and/or a variable light region comprising a first CDR operably linked to a second CDR operably linked to a third CDR.
• the sequences in the CDRs may include any of SEQ ID Nos. 21 -23, 28-30, 35-36, 42-44, 66-68, or 81 , or a sequence with 1 , 2, 3, 4 or 5 substitutions. In one embodiment, the majority or all of the substitutions are conversative substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31 -34, 38-41 , 45-47, 51 -63, 71 -74, or 76-79, or a sequence with 1 , 2 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.
• the heavy or light chain may include one or more framework regions including but not limited to one of SEQ ID Nos. 24-27, 31 -34, 38-41 , 45-47, 51 -63, 71 -74, or 76-79, or a sequence having at least 80%, 85%, 90%, 92%, 95% or 99% amino acid sequence identity thereto.
• ART1 To determine whether ART1 contributes to distinct phenotypic characteristics, ART1 expression was knocked down in murine KRAS G12D/+ /p53 /_ cell lines (KP1 ) using shRNAs. In both tail vein injection induced lung and subcutaneously injected flank tumor models in immunocompetent mice, a significant decrease in tumor burden was noted upon ART1 -knockdown and enzymatic inhibition. In the KP1 tail vein model, ART1 knockdown is associated (p ⁇ 0.001 ) with control of tumor progression and with long term survival (approximately 50%) of the mice, superior to that seen with immune checkpoint blockade (ICB) using a PD-1 antibody.
• ICB immune checkpoint blockade
• ART1 may specifically MAR P2RX7 on CD8+ T cells, in particular tissue resident memory T cells (Trm).
• Trm tissue resident memory T cells
• P2RX7 is a cytolytic ATP receptor that mediates apoptotic cell death of T cells, macrophages, and dendritic cells.
• MAR of P2RX7 on the surface of T cells results in prolonged receptor activation, Ca 2+ influx, and T cell apoptosis, a phenomenon termed NAD-induced cell death (NICD).
• NAD-induced cell death NAD-induced cell death
• ART 1 -induced MAR of P2RX7 on T cells could allow ART 1 -high cancer cells to blunt the T cell immune response against them by inducing T cell apoptosis.
• NAD + released during cell death and inflammation has previously been demonstrated to regulate homeostasis of cytotoxic CD8 T cells through extracellular ADP-ribosylation.
• RNAseq of lung -infiltrating CD8 T cell populations in murine models we showed that the relative expression of P2RX7 increases in CD8+ T cells with increasing tumor burden following tail vein injection in mice (p ⁇ 0.001) and that P2X7R+ CD8 T cells are preferentially ADP-ribosylated.
• ART1 should be highly druggable.
• MAR can be blocked by small molecule arginine analogues such as the antibiotic novobiocin, MIBG (a safe norepinephrine analogue imaging agent), and nonspecific PARP inhibitors (like EB-47). These drugs compete with NAD+ at the enzyme active site and have been shown to have anti-cancer effects in murine models or in untargeted patient populations.
• ART1 -overexpressing tumors can be targeted with therapeutic monoclonal antibodies, similar to ICB.
• a humanized therapeutic monoclonal antibody (22C12) which binds to ART1 and inhibits ADP-ribosylation on ART1 -expressing cancer cells was developed (see Examples 2 and 3).
• Preliminary data demonstrated a drastic reduction (-59%) of murine KP1 flank tumors when intratumorally treated with 22C12 compared to control antibody. This reduction was associated with significant enrichment of P2X7R+ CD8+ T cells and P2RX7+CD103+ DC within the tumors.
• ART1 expression may be upregulated by cell stress and its enzymatic activity increased by release of NAD+, both of which occur following commonly used cytotoxic therapies.
• increases of tumor ART1 expression posttreatment may serve as the basis for therapeutic inhibition of ART1 combined with chemotherapy, radiation therapy, or immune checkpoint blockade (ICB).
• Antibodies that specifically inhibit ART1 or its function may be used as targeted therapeutics in ART1- expressing, e.g., overexpressing, cancers such as in ART-1 overexpressing NSCLC patients to limit metastatic spread of cancer by facilitating immune-mediated destruction of disseminated cells.
• ART1 is highly druggable.
• ART1 antibodies may be used as combination therapy with cytotoxic chemotherapy or with immune checkpoint inhibitors.
• the antibody is reactive against both mouse and human ART1.
• Antibodies are enzymatically screened for inhibitors of mono-ADP-ribosylation utilizing ART1 , substrate proteins, and labeled NAD+.
• Potential therapeutic antibodies are tested utilizing in vitro neutrophil and lymphocyte cytotoxicity assays.
• therapeutic blockade of ART1 facilitates cytotoxicity of the immune cells towards the ART1 expressing lung cancer cells.
• ART1 expression (and a similar phenotype) was observed in murine and human breast cancer models and in clinical specimens, as well as in human colorectal cancer specimens.
• the anti-ART1 antibodies have broad anti-cancer applicability.
• ART1 To generate functional humanized antibodies against ART1 , purified recombinant human and mouse ART1 produced in HEK293 mammalian cells was used to immunize transgenic mice with a human immunoglobulin repertoire. Following immunization and test bleed analysis, spleens were harvested and fused with a myeloma fusion partner to generate hybridoma.
• hybridoma supernatants were screened on ELISA plates coated with purified human ART1 .
• Hybridoma cells from positive wells were frozen for later recovery.
• Anti-human ART1 positive hybridoma supernatants were then tested for inhibition of purified human ART1 by a fluorescent NAD + readout. This assay measures NAD + consumed from ADP-ribosylation of histone protein by ART1 .
• Anti-ART1 hybridoma supernatants were also tested for inhibition of human ART1 transiently expressed in HEK293 cells. This method, which has previously been described for ART2 (Krebs et al.), determines cell-surface ADP-ribosylation via an NAD + analogue (etheno-NAD + ) which is then detected by an anti-etheno antibody using flow cytometry. Two hybridoma supernatants (fusion wells 22C12 and 14G01 ) were positive for inhibition of human ART1 in the biochemical and cell-based assays. Supernatants from 22C12 and 14G01 were then also tested for binding to moARTI by ELISA and inhibition of purified mouse ART1 ( Figure 11 ).
• ART1 tilts the balance of life and death for anti-tumor T cells
• Elimination of T cells through NAD-induced cell death following mono-ADP-ribosylation of the P2X7- receptor constitutes a regulatory mechanism to maintain tissue immune homeostasis.
• the present disclosure demonstrates that, in non-small cell lung cancer, tumor cells overexpressing the mono-ADP- ribosyltransferase 1 , ART1 , coopt this mechanism to escape immune-mediated control by eliminating P2X7- receptor-positive CD8 T cells in the local microenvironment.
• Therapeutic targeting of ART1 with a novel monoclonal antibody improved tumor control in mouse models of lung cancer.
• ART1 was strongly expressed in the majority of lung adenocarcinomas and that its membrane expression was associated with lower CD8 T cell infiltration compared to ART1 negative tumors.
• a murine model of NSCLC it was demonstrated that genetic and pharmacologic targeting of ART1 inhibited tumor growth in immunocompetent but not in immunodeficient mice and increased infiltration of P2X7R + CD8 T cells in tumors in the immunocompetent mice.
• CD8 T cells isolated from wild type mice, but not P2X7R /_ mice were preferentially susceptible to ART1 -mediated ADP-ribosylation and NICD.
• ART1 -mediated NICD is a mechanism of immune resistance in NSCLC and other cancers.
• Immune checkpoint inhibitors alone or in combination with chemotherapy, have become the standard of care in patients with advanced non-small cell lung cancer (NSCLC) without targetable molecular alterations (Mok et al., 2019; Reck et al., 2016).
• NSCLC non-small cell lung cancer
• the majority of lung cancer patients either do not respond to or do not experience long-term benefit from ICI, including many of those patients with high tumor PD-L1 expression (Gandhi et al., 2018; Gandini et al., 2016).
• ICI Immune checkpoint inhibitors
• ART1 ADP-ribosyltransferase-1
• ART1 is a GPI-anchored enzyme, with an extracellular catalytic domain. Therefore, ART1 can mono-ADP-ribosylate extracellular proteins in the local microenvironment, altering their function (Stevens et al., 2009; Okazaki et al., 1994; Balducci et al., 1999).
• the expression of ART1 in lung cancer has not been investigated, but previous studies have suggested increased ART1 protein expression in colorectal cancer and in glioblastoma, where high expression was associated with a poor prognosis (Yang et al., 2013).
• ART1 expression was shown to promote a more aggressive phenotype with increased epithelial-to-mesenchymal transition, cellular proliferative signaling and increased angiogenesis (Yang et al., 2013; Song et al., 2016). However, it has not been determined whether tumor ART1 expression could regulate tumor crosstalk with the immune microenvironment.
• P2X7R P2X7 receptor
• P2X7R gene id: P2RX7
• P2X7R is an ATP-gated cation channel of the purinergic type 2 receptor family, with low affinity for extracellular ATP, that activates pro-inflammatory pathways (Burnstalk & Knight, 2004). It is expressed on multiple immune cell subsets including T cells and its expression is essential for inflammatory responses and anti-tumor immunity (Adinolfi et al., 2015; Haag et al., 2007). P2X7R can also be overexpressed on cancer cells where it may promote tumor progression.
• cytosolic NAD + is released into the local extracellular environment where it may be used as a substrate by extracellular ADP-ribosyl transferases to catalyze the transfer of the ADP-ribose to P2X7R (Haag et al., 2007).
• NAD-induced cell death results in constitutive activation of P2X7R triggering large pore formation, uncontrolled calcium influx, and phosphatidylserine externalization, which leads to a process known as NAD-induced cell death (NICD) (Scheuplein et al., 2009).
• NAD + concentrations are low and tightly regulated by the ADP-ribosyl cyclase CD38, which is expressed on activated immune cells as well as on cancer cells (Sandoval-Montes & Santos-Argumedo, 2005; Chen et al., 2018).
• extracellular NAD + concentrations can increase following rapid release from stressed or dying cells (Haag et al., 2007).
• NICD ART-mediated NICD of T cells has been proposed as a homeostatic mechanism to eliminate naive and bystander T cells in inflamed tissues (Adriouch et al., 2007). More recently, NICD was shown to regulate the homeostasis of CD4 regulatory T cells (CD4 Tregs) which have broad immunoregulatory function as well as tissue-resident memory T cells (TRMs), the presence of which in lung tumors has been associated with good prognosis (Stark et al., 2018; Nizard et al., 2017). These pre-clinical studies discern the role of ADP-ribosyltransferase-2 (ART2) in immune modulation through NICD.
• CD4 Tregs CD4 regulatory T cells
• TRMs tissue-resident memory T cells
• ART2 is expressed on murine lymphocytes where it can auto-ADP-ribosylate the P2X7R mediating NICD in cis.
• the ART2 gene contains premature stop codons rendering it a pseudogene while other ARTs like ART1 , ART3, ART4 and ART5 are transcriptionally active.
• ART1 is expressed on the surface of human lung cancer cells and that its expression is associated with reduced lung tumor infiltration of P2X7R + CD8 T cells.
• tumor cell ART1 expression promoted escape from CD8 T cell-mediated tumor control.
• ART1 -blockade with a therapeutic monoclonal antibody reduced the growth and dissemination of ART1 -expressing tumors in immunocompetent mice and promoted tumor infiltration of activated P2X7R + CD8 T cells.
• ART1 tumor expression is a unique mechanism of immune resistance and that ART1 is an actionable target to enhance T cell-mediated tumor rejection.
• ART1 is expressed in human NSCLC and associated with reduced CD8 T cell infiltration
• ART1 expression was assessed in human NSCLC lines A549 and H1650 and in a benign bronchial epithelial cell line (BEAS2B) by immunofluorescence.
• the tumor cell lines had heterogeneous expression of cell surface (Fig. 14A-B) and total cell ART1 (Fig. 14A, Fig. 19A). Both tumor cell lines had higher ratios of cell surface/total cell expression than did BEAS2B cells (59.5% and 55.4% vs. 29.2% respectively, Fig. 14C).
• ART1 gene expression by RT-qPCR in tumor and matched normal lung tissue from 40 patients with stage l-lll lung adenocarcinoma showed significantly higher mean expression in the cancer samples, driven by a fraction of the tumors with markedly higher expression (Fig.14D).
• the matched tumors also had significantly lower expression of glycosylphosphatidylinositol specific phospholipase D1 (GPLD1), the only well characterized mammalian phospholipase regulating cleavage of GPI anchors (Fig. 19B).
• GPLD1 glycosylphosphatidylinositol specific phospholipase D1
• Fig. 19B glycosylphosphatidylinositol specific phospholipase D1
• Cell- associated GPLD1 can release GPI-anchored proteins from the cell surface, but expression has been shown to be down-regulated with stress in lung cancer cells, suggesting that tumor cells are more likely to retain ART1 on the cell surface
• tissue microarray of 493 stage I lung adenocarcinomas was analyzed for ART1 expression by immunohistochemistry. Staining for ART1 in the cancer cells was strong, moderate, and weak in 55%, 42% and 3% of the tumors, respectively (Fig. 19C and Table 2).
• ART1 expression by IHC in the cancer cells was diffuse cytoplasmic but staining concentrated near the cell periphery and membrane (membranous) was identified in 10% of the tumors (Fig. 14E and Table 3).
• Tumors with a mucinous histologic subtype a rare tumor which only comprised 3.7% of the cohort, were particularly likely to express membranous ART1 compared to other histologic subtypes (44% vs. 8.4%).
• Tumors were also scored for infiltration of CD3, CD8, CD4 and FoxP3 T cells, CD20 (B cells), CD56 (natural killer (NK) cells) and CD68 or CD163 (macrophages) (Table 4).
• transcriptomic data from a lung adenocarcinoma cohort was analyzed using the cBioportal platform to assess whether ART1 tumor expression was associated with differential expression of genes associated with CD8 T cell cytotoxicity; IFNy (IFNG), Granzyme A (GZMA), Granzyme B (GZMB), Perforin 1 (PRF1) 41 BB (TNFRSF9) as well as genes associated with immunoregulation; CTLA-4 (CTLA4), PD-1 (PDCD1), Tim-3 (HAVCR2), Lag-3 (LAG3) and Tigit (TIGIT).
• IFNy IFNy
• GZMA Granzyme A
• GZMB Granzyme B
• PRF1 Perforin 1 41 BB
• CTLA-4 CTLA-4
• PD-1 PDCD1
• Tim-3 HAVCR2
• LAG3 Lag-3
• Tigit Tigit
• ART1 tumor expression exerts immune resistance in mouse lung tumor models
• ART1 expression protects tumors from T cell-mediated rejection
• a mouse model of ART1 over-expressing NSCLC was developed.
• An ART1 plasmid was introduced into a KP1 cells, which were originally derived from inducible KRAS G12D/+ /p53 /_ mice (KP1 -ART1 0E ).
• the parent wild type KP1 line has low level ART1 cell surface expression at baseline, while the engineered KP1 -ART1 0E line showed an approximately 9-fold increase in ART1 surface expression per cell by IF (Fig. 20A).
• KP1 -ART1 0E cells were transduced with a doxycycline-inducible short hairpin RNA (shRNA) targeting ART1 (shARTI ).
• shRNA doxycycline-inducible short hairpin RNA
• Doxycycline-induced ART1 -knockdown markedly reduced both ART1 cell surface expression (Fig. 20A) and ADP-ribosylation of tumor cell surface targets on the cancer cells themselves (Fig. 20B).
• Proliferation of KP1 -ART1 0E cells remained unaffected by ART1 knockdown (Fig. 20C).
• KP1 -ART1 0E cells were subcutaneously inoculated in immunocompetent wild-type and T cell-deficient nude C57BL/6 mice. Half of the mice in each group were given doxycycline to induce ART1 knockdown in vivo, which was confirmed by immunofluorescence staining of tumor specimens (Fig. 20D). In immunocompetent mice, KP1 -ART1 0E flank tumors grew rapidly while doxycycline-induced ART1 knockdown significantly delayed flank tumor growth (Fig. 15A, left panel). In T cell-deficient nude mice, KP1-ART1 0E flank tumors had a similar growth rate as in wild type mice.
• KP1 -ART1 0E lung tumors KP1 -ART1 0E cells were injected in the tail vein, and cohorts of the mice were given doxycycline to induce ART1 knockdown in vivo (Fig.15B).
• ART1 knockdown resulted in a significantly decreased lung tumor burden at day 14, assessed by nodule frequency count from hematoxylin and eosin (H&E) stained lung sections (Fig. 15C).
• Lung CD8 T cell infiltration was determined by flow cytometry at days 16 and 25.
• mice with induced ART1 knockdown had significantly higher frequency of CD8 T cells among total lung tumor-infiltrating leukocytes (CD45+ cells) than mice bearing ART 1 -expressing tumors (Fig. 15D).
• the percentage of lung tumor-infiltrating CD8 T cells was decreased at day 25 compared to day 16 in control mice, consistent with a loss of immune control associated with tumor progression.
• a melanoma line was chosen, as human melanomas are shown to strongly express ART1 in the Human Protein Atlas.
• B16-F10 mouse melanoma cells have high intrinsic ART1 cell surface expression and an ART1 -negative derivative was generated using CRISPR/Cas9 and two different guide RNA (Fig. 21A-B).
• ART1 -expressing B16-F10 cells in syngeneic immune competent mice resulted in formation of rapidly growing flank tumors, while ART1 -deficient B16-F10 cells showed markedly impaired tumor growth or failed to form palpable tumors (Fig. 21 C).
• ART1 - deficient B16-F10 cells The impaired growth of ART1 - deficient B16-F10 cells in vivo was not due to decreased fitness of the cancer cells themselves since, in vitro, ART 1 -deficient cells proliferated faster than ART 1 -expressing B16-F10 cells (Fig. 21 D).
• ART1 blockade reduces lung tumor burden and promotes infiltration of P2X7R + CD8 T cells
• Therapeutic targeting surface ART1 was investigated using a 22C12 monoclonal antibody targeting ART1.
• Therapeutic antibody candidates were initially developed through immunization of AlivaMab® Mouse transgenic mice with a human immunoglobulin repertoire utilizing human ART1.
• Candidate antibodies that bound to both human and mouse ART1 and inhibited mono-ADP-ribosylation were further developed (Fig. 22A-B).
• the lead candidate, 22C12 which potently inhibited ART1 enzymatic activity in the primary screening assay, was further developed.
• 22C12 antibody clones with mouse light chains (22C12 (mLC)) and human light chains (22C12 (HuLC)) were generated which were tested for activity in vitro and in vivo.
• Binding of 22C12 antibodies to HEK 293 cells transfected with ART1 was determined by NAD-Glo assay showing half-maximum binding (ECso) values in the range of 0.8-1 .5 nM (Fig. 22C). Binding of 22C12 to KP1 - ART1 0E cells was assessed by flow cytometry staining (Fig. 22D). Half-maximum inhibition of ADP- ribosylation (ICso), by 22C12 antibodies, as determined by cell surface ADP-ribosylation of HEK-ART1 0E cells, was achieved at 4.5 nM antibody concentration (Fig. 22E).
• ICso ADP- ribosylation
• ART1 blockade resulted in a significantly delayed tumor growth compared to tumors treated with isotype control antibody (Fig. 23B), with average tumor weight at day 25 significantly lower in the mice treated with 22C12 compared with isotype control antibody (Fig. 23C).
• mice were treated intraperitoneally with 22C12 antibody (25 mg/kg) or the equivalent dose of isotype control antibody starting on day 6 after tumor injection until day 18 (Fig. 16A).
• mouse lungs were fixed and stained with H&E to assess lung tumor burden, which showed fewer and significantly smaller tumor nodules in mice treated with 22C12 antibody compared with isotype-treated mice (Fig. 16B-D).
• endogenous surface ART1 expression on Lewis lung carcinoma (LLC1 ) cells was assessed (Fig. 20A).
• LLC1 orthotopic lung tumor model treatment strategy as in Fig. 16A
• mice treated with ART1 blockade had reduced lung tumor burden compared to control mice (Fig. 23D-E).
• ART1 expression modulates intratumoral CD8 T cells.
• KP1ART1 0E orthotopic lung tumor model was employed, where CD8 and CD4 T cells were depleted in the mice by administration of monoclonal antibodies.
• ART1 was blocked by i.p. injection of 22C12 starting on day 6 after tumor injection (Fig. 161). Lungs were harvested on day 19 after tumor inoculation to assess tumor burden by H&E staining.
• mice inoculated with ART1 -deficient B16-F10 (B16 ART1 K0 ) cells showed markedly impaired tumor growth or failed to form palpable tumors (3/7 mice tumor free at day 70).
• CD8 T cell-depletion resulted in higher tumor burden and significantly reduced survival compared to mice bearing B16 ART1 K0 tumors treated with isotype control antibody.
• Mice that received CD4 T cell depletion developed tumors albeit with slower tumor growth compared with CD8 depleted mice and no significant different in survival compared with isotype control treated animals (Fig. 24A, lower panel, 24B).
• CD8 T cells expressing P2X7R are eliminated by NICD.
• NICD In acutely inflamed tissues characterized by increased extracellular NAD + , CD8 T cells expressing P2X7R are eliminated by NICD.
• CD8 T cell P2X7R expression for anti-tumor immunity in lung cancer has not been well described. It was assessed whether P2RX7 expression was altered in murine CD8 T cells over the course of lung tumor progression.
• RNA sequencing analysis was performed on CD8 T cells isolated from lungs and spleens from mice orthotopically inoculated with wild type KP1 cells, which express low levels of ART1 .
• P2RX7 Gene expression of P2RX7 as well as genes associated with CD8 T cell cytotoxicity and immunoregulation was assessed in CD8 T cells isolated on day 7 and 17 after tumor inoculation as well as in CD8 T cells from naive non-tumor bearing mouse lungs.
• Etheno-tagged NAD + (eNAD) was added to the co-cultures which was detectable by flow cytometry to identify ADP-ribosylated cells, while DAPI (4',6- diamidino-2-phenylindole) staining was used to measure cell death.
• An ART2-blocking nanobody (s+16a), was used to block ART2, which is expressed on murine lymphocytes and can mediate auto-ADP-ribosylation of T cells in cis. Surface ART1 was blocked using the 22C12 monoclonal antibody. The ability of the ART2 and ART1 blocking antibodies to inhibit ADP-ribosylation was confirmed in an experiment where T cells were cultured in the presence of eNAD alone.
• ART2 blockade resulted in reduced ADP-ribosylation of CD8 T cells from 70.1 ⁇ 8.8% to 12.9 ⁇ 2.6% and of CD4 T cells from 54.6 ⁇ 9.6% to 9.3 ⁇ 7.2%.
• ART1 -blockade did not affect ADP-ribosylation of CD8 or CD4 T cells
• the CD38 blocking antibody (NIMR-5) was used to assess whether expression of CD38 could play a cytoprotective role by catabolizing free NAD + from the immediate micromilieu.
• CD4 Tregs CD4+ regulatory T cells
• NICD NICD via P2X7R
• ADP-ribosylation and NICD were separately analyzed on CD4 Tregs, CD4 Tconv cells and CD8 T cells as well as on P2X7R+ and P2X7R- fractions of the T cell subsets separately (Fig. 17).
• Average P2X7R expression was 9.3 ⁇ 2.4% of CD8 T cells, 21 ,6 ⁇ 3.9% of CD4 Tconv and 80.8 ⁇ 2.6% of CD4 Tregs.
• P2X7R+ but not P2X7R- CD8 T cells were sensitive to ART 1 - mediated ADP-ribosylation which was increased in the presence of CD8 blocking antibodies.
• Addition of ART1 blocking antibody (22c12) to the co-culture reduced ART1 -mediated ADP-ribosylation of CD8 T cells as well as CD4 Tconv and CD4 Tregs to baseline levels (Fig. 17B).
• NICD was measured which was determined by the frequency of cells that stained positive for both eNAD and DAPI (Fig. 17C).
• P2X7R+ CD8 T cells, but not P2X7R- CD8 T cells were susceptible to ART1 -mediated NICD, which was exacerbated by CD38 blockade.
• ART1 -blockade reduced NICD of P2X7R+ CD8 T cells to baseline levels. While we detected low NICD levels of CD4 Tconv cells, NICD of CD4 Tregs was significantly elevated in the presence of rART1 , primarily in the P2X7R+ subset and was reduced to baseline levels upon ART1 -blockade. In contrast to its effect on P2X7R+ CD8 T cells, CD38 blockade reduced ART1 -mediated NICD of P2X7R+ CD4 Tregs (Fig. 17C).
• CD8 T cells and CD4 Tregs are susceptible to ART1 -mediated ADP- ribosylation and NICD via the P2X7R.
• the significant ADP-ribosylation of P2X7R- CD4 Tregs, and to a lesser extent P2X7R- and P2X7R+ CD4 Tconv cells, following co-culture with rART1 and CD38 blockade indicate that additional targets exist on these cells, which are sensitive to ADP-ribosylation by ART1 in the absence of CD38 expression.
• the cytoprotective role of CD38 against ART1 -mediated NICD observed in P2X7R+ CD8 T cells was reversed in P2X7R+ Tregs.
• RNA isolated from KP1 , B16 and LLC1 mouse tumor cells was analyzed for expression of the P2RX7 splice variants. It was observed that CD8 T cells CD4 Tconv cells isolated from KP1 tumor-bearing lungs expressed comparable levels of P2RX7-k while all tumor cells expressed low levels of P2RX7-k (Fig. 26C).
• P2RX7-a expression was low in both CD8 T cells and CD4 Tconv cells while we detected expression in KP1 and LLC1 cells and highly expressed in B16 cells, which may protect ART1 -expressing tumor cells from NICD following auto-ADP-ribosylation (Fig. 26D).
• proliferation assays demonstrate that ART1- expressing tumor cells grown in the presence of NAD+ and/or ART1 blockade have no significant differences in cell growth (Fig.26E-G).
• ART1 expressing human lung tumors have reduced infiltration of P2X7R + CD8 T cells
• the tumor immune contexture is associated with prognosis and response to immunotherapy, with CD8 T cell infiltration generally serving as an indicator of an ongoing anti-tumor immune response which can be reinvigorated by ICI.
• CD8 T cell infiltration generally serving as an indicator of an ongoing anti-tumor immune response which can be reinvigorated by ICI.
• ICI anti-tumor immune response
• ART1 overexpression on human lung cancer cells was shown to be associated with poor survival and reduced intratumoral CD8 T cells, specifically a reduction in the P2X7R + CD8 T cell subset. Furthermore, expression of ART1 in mouse tumors promoted tumor growth in immune competent but not in T-cell deficient mice or following CD8 T cell depletion and is associated with a reduction in tumor-infiltrating P2X7R + CD8 T cells.
• P2X7R + but not P2X7R- CD8 T cells were susceptible to ART1 -mediated ADP-ribosylation and to NICD which was exacerbated upon blockade of CD38 indicating a potential cytoprotective role.
• ART1 is an actionable target to improve immune- mediated tumor control.
• ART1 should be highly druggable.
• treatment with a monoclonal antibody that binds to and inhibits ART 1 -induced ADP-ribosylation had therapeutic benefits in preclinical models, resulting in reduced growth of ART1 + lung cancer and increased tumor-infiltration of activated and proliferating P2X7R + CD8 T cells.
• ART1 has only recently been described to play a role in cancer progression.
• Xu et al. demonstrated that overexpression of ART1 facilitated tumor growth, while knockdown inhibited tumor growth in various immune competent models. This effect was attributed to cis- ADP-ribosylation of integrin and Rho effector family members, subsequently affecting downstream mediators of cellular migration.
• the anti-tumor effects of ART1 knockdown or blockade in mouse lung cancer models is dependent on CD8 T cells.
• ART1 In vitro, knockdown of ART1 had no effect on KP1 -ART1 0E cell proliferation and actually enhanced tumor cell proliferation of B16 mouse melanoma cells yet resulted in impaired tumor growth in vivo in immunocompetent mice. Thus, although ART1 has cancer cell-intrinsic effects that may be model- dependent, the immune suppressive effects of ART1 expression seem to dominate in the present in vivo models.
• ART1 was found to block increases infiltration of P2X7R+ CD8 TRMS in tumor-bearing lungs Together with the observation that lung tumor-infiltrating CD8 T cells have elevated expression of P2X7R and co-express cytotoxic and immunoregulatory markers, it is likely that P2X7R + CD8 T cells infiltrating lung tumors represent a critical tissue-resident subset of memory T cells with anti-tumor activity and that they are targeted by ART1 tumor expression.
• T cell expression of CD38 is also likely to be a critical component in determining whether cells undergo NICD.
• CD38 is upregulated on mouse and human T cells upon activation and differentiation and may represent a cytoprotective mechanism to avoid ADP-ribosylation and NICD in NAD + enriched inflamed tissues.
• the present results indicate that P2X7R + CD8 T cell,s and P2X7R+ CD4 Tregs to a lesser extent, are susceptible to ART1 -mediated ADP-ribosylation and NICD, and that CD38-blockade enhances ADP- ribosylation and NICD of P2X7R+ CD8 T cells in presence of ART 1 .
• treatment of ART1 -positive adenocarcinoma patients with CD38 blockade could have the dual effect of exacerbating NICD of CD8 T cells while protecting CD4 Tregs, thus skewing the CD8 T cell to CD4 Treg ratio which is associated with immunotherapy response and tumor rejection.
• CD38 is expressed by other immune cells and some cancer cells and has been shown in pre-clinical studies to contribute to acquired resistance to PD-1/PD-L1 blockade by converting NAD + into ADPR, a precursor of adenosine, which has broad immune suppressive function.
• the anti-CD38 antibody daratumumab was recently tested in combination with atezolizumab (an anti-PD-L1 antibody) in NSCLC patients in a clinical trial (NCT03023423). This study was terminated early because of increased mortality in the combination treatment arm.
• ART1 expression has been demonstrated to increase following cellular stress, so its expression in tumors may be highly dynamic and potentially fluctuate depending on the degree of inflammation in the TME as well as in response to treatment.
• cytotoxic agents such as chemotherapy and radiation.
• Such treatments will also contribute to increased levels of extracellular NAD + following cell death, potentially priming the local microenvironment for ART1- induced mono-ADP-ribosylation.
• ART1 as an actionable barrier to response to combinations of cytotoxic agents with immunotherapy are needed.
• ART1 -expressing tumor cells eliminate tumor-infiltrating CD8 T cells via NICD.
• NICD tumor-infiltrating CD8 T cells
• Pharmacologic targeting of ART1 may potentiate CD8 T cell- mediated immune responses in NSCLC patients.
• ART1 expression data were square root transformed, while percentages of tissue-infiltrating immune cells were log-transformed prior to statistical testing by paired t-test to ensure the underlying assumptions of the test were met. Statistically significant differences in ART1 MFI on human lung tumor cell lines was determined by one-way ANOVA with Tukey’s test for multiple comparisons.
• Mouse experiment data Data consisting of counts, percentages and expression data were log- transformed or square root transformed where indicated prior to statistical testing by Welch’s t-test.
• Tumor growth data comparing the effect of induced ART1 -knockdown in KP1-ART1 0E tumors or ART1 knockout in B16-F10 tumors were analyzed by repeated-measures ANOVA with Geisser-Greenhouse correction.
• TMA was scored for (1) low, (2) intermediate or (3) high infiltration of immune cell subsets; Pan T cells, CD4 T cells, CD8 T cells, Tregs, B cells and macrophages in tumor and stroma using markers; CD3, CD4, CD8, FoxP3, CD20, CD56, CD68 and CD163. NK cell infiltration in tumor and stroma was determined as absent or present using the CD56 marker. The scoring cell number cutoffs are described in Table 4.
• mice All animal work was done following a protocol approved by the Institutional Animal Care and Use Committee of New York Presbyterian Hospital/Weill Cornell Medical College (IACUC # 2010-0050, 2015- 0028).
• Wild type C57BL/6 mice (strain: C57BL/6NTac) and athymic nude mice (strain: B6.Cg/NTac-Foxn1 nu NE10) were purchased from Taconic Biosciences. All mice were maintained under pathogen-free conditions in the Weill Cornell Medicine animal facility.
• mice were sacrificed, parts of the tumor-bearing lungs were formalin-fixed, paraffin- embedded and sectioned for subsequent H&E stain and blinded enumeration of lung nodules. The remaining parts of the tumor-bearing lungs were weighed and dissociated into a single cell suspension and stained for analyzed by flow cytometery for characterization of CD8 T cells.
• tumor diameters were measured with digital calipers and the tumor volume determined by the formula (length x width 2 ) /2).
• the flank tumor model using B16-F10 CRISPR sublines it was observed that some mice died prior to reaching maximum tumor volumes with evidence of metastatic dissemination. Where indicated, tumors were excised, weighed, and processed for immunofluorescence and/or flow cytometry analysis.
• CD4 and CD8 T cell depletion For CD8 and CD4 T cell depletion, a-CD8 (clone: 53-6.7, Bioxcell # BP0004-1) and a-CD4 (clone: GK1.5, Bioxcell # BP0003-1) antibodies were ip injected per mouse according to the following regimen: day -1 and day 3 (500 ug), then every 72 hrs till experiment endpoint (250 ug) (38).
• mice from other groups received via ip injection InVivoPlus rat lgG2a isotype control, anti-trinitrophenol (clone 2A3, Bioxcell #BP0089) and InVivoPlus rat lgG2b isotype control, anti-keyhole limpet hemocyanin (clone LTF-2, Bioxcell # BP0090), respectively.
• 22C12 treatment of tumor bearing mice For flank tumors, Intra-tumoral injections started when KP1- ART1 0E tumors became palpable on day 11 and every 72hrs until day 23 after tumor inoculation. Mice were injected with 5mg/kg ART1 antibody Clone 22C12 for group ‘22C12 Ab’ or Mouse lgG1 isotype control (BioXcell, Cat# BP0297) for group ‘Iso Ctrl Ab’. Tumor sizes were measured every 72hrs and mice were sacrificed on day 25 after tumor inoculation when tumors were weighed and processed for flow cytometry staining. For the orthotopic lung tumor models, mice were injected i.v.
• mice were intra-peritoneally (i.p.) injected with 25mg/kg ART1 antibody Clone 22C12 for group ‘22C12 Ab’ or 25 mg/kg of humanized light chain 22C12 Ab 22C12 Ab (HuLC1) or Mouse lgG1 isotype control (BioXcell, Cat# BP0297) for group ‘Iso Ctrl Ab’, i.p. injections started from day 6 and continued every 72 hours till day 18 as indicated.
• the human cell lines H1650, A549, BEAS2B and HEK293 were obtained from ATCC and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin in a humidified 5% CO2 incubator at 37°C.
• the mouse NSCLC cell line KP1 was previously generated from lung tumors of KRAS G12D/+ /p53 /_ (KP1) mice.
• Mouse LLC1 lung cancer cells and B16-F10 melanoma cells were obtained from ATCC and cultured in DMEM medium supplemented with 10% FBS and 1% penicillin streptomycin in a humidified 5% CO2 incubator at 37°C.
• KP1-ART1 0E and inducible hairpin stable cell lines (I) Generation of KP1-ART1 0E cells:
• the pLVX-IRES-tdTomato vector is designed to constitutively coexpress the protein of interest and tdTomato from PQMV IE when transduced into mammalian cells. Before transduction, the vector was packaged into viral particles in HEK293T cells, using Lenti-XTM HT Packaging System (Catalog# 632160 and 632161 , Takara). The presence of tdTomato allows transductants to be visualized by fluorescence microscopy and sorted by flow cytometry.
• ART1 gene was overexpressed by using this construct (pLVX-IRES-td- tomato_ART1).
• Recombinant lentiviruses were generated from 293T cells (6 x 10 6 cells/100 mm plate) by transient transfection of 7ug of lentiviral short hairpin constructs (LT3GENIR) and the lentivirus packaging system (Clontech lenti-x single shot). LV particles were harvested 48 hours and 72 hours later, filtered through 0.45 pm filters, and concentrated by adding lenti-x concentrator (clontech). The LV were then incubated for 30 minutes at 4°C and centrifuging at 1 ,500 g for 45 minutes at 4 °C.
• LV particles were used to infect subconfluent cell cultures for 6 hours in the presence of 4 pg/mL polybrene (Sigma-Aldrich). Selection of viral infected cells expressing ART1 was done by sorting for tdtomato positive cells.
• (II) Generation of shARTI cells shRNA- LT3GENIR construct was used to knock down ART1 genes in KP1-ART1 0E cells. The custom designed lentiviral construct expressed short hairpins targeting the Art1 gene and had GFP expression for selection. LV were generated as described in the previous section. Selection of viral infected cells expressing the shRNA was done by using 1 mg/mL G418 (Neomycin analogue, Sigma-Aldrich) in the media.
• ART1 shRNA construct #1 Art1_87 LT3GENIR. Antisense. Guide. Sequence TTTGATGTATTCACAGTTGTAT. (SEQ ID NO:17) 97mer.construct TGCTGTTGACAGTGAGCGATAGACATCTTTTCTCAAGAAATAGTGAAGCCACAGATGTATTTCTTGAGAAA AGATGTCTAGTGCCTACTGCCTCGGA (SEQ ID NO:18).
• CRISPR-mediated gene knockout of ART 1 in B16-F10 cells CRISPR/Cas9 mediated knockout of ART1 in B16-F10 cells was performed using Sigma-Aldrich custom-made, ready-to-use DNA plasmids on the U6gRNA:CMV-CAS9-2A-tGFP backbone.
• B16-F10 clones B16 ART1 KO 63-1) (sequence 5’-3’: CCTGCGCTTTCGGCCAGCG; SEQ ID NO: 5) and B16 ART1 KO ⁇ 42 - 1 > (sequence 5’-3’: CCAACAAAGTATACGCGGA; SEQ ID NO:19).
• a negative control plasmid was used to create the B16-F10 clone B16 C0NTR0L ⁇ Scr - 6 > (sequence 5’-3’: CGCGATAGCGCGAATATATT; SEQ ID NQ:20).
• B16-F10 cells were seeded in 12 well-plates and incubated for 48 hours to reach 80% confluency.
• Each CRISPR plasmid (0.5 pg DNA) were mixed with 3 Di TransIT-CRISPR reagent (Sigma-Aldrich) in 100 pl Opti-MEM medium (Gibco) and incubated at room temperature for 30 minutes. Mixture was added to the B16-F10 cells and incubated in a humidified 5% CO2 incubator at 37°C for 24 hours.
• Flow cytometry activated cell sorting FACS was used to sort transfected GFP-positive single cells into flat-bottom 96 well-plates. Clones were expanded and tested for ART1 surface expression by flow cytometry and immunofluorescence staining ( Figure 21 ).
• Proliferation Assay 1 .4x10 4 cells were plated in a 6-well plate. Cells were trypsinized and counted using Cellometer cell counting chambers (Nexcelom Bioscience) every day for four days. For experiments where cells were treated with NAD (20 pM, Sigma Aldrich, Catalog# N8285) +/- 22C12 (20 pg/ml), FBS media was replenished with the mentioned reagents every 24 hours till endpoint.
• the cells were then treated with primary antibody ART1 (Purified ART1 antibody, Pocono, rabbit #2) (1 :200) or Poly/Mono-ADP Ribose (CST, Clone: E6F6A Rabbit mAb Catalog#83732) (1 :200) dissolved in 1% BSA (in 1xPBS, referred to as ‘cell IF antibody buffer’) for one hour in a 37°C air incubator.
• Cells were washed with PBS-CM and then incubated with anti-rabbit fluorescent secondary antibody 1 :500 (Thermofisher, #A10523) dissolved in ‘cell IF antibody buffer’ for 30 minutes.
• NAD NAD + (Sigma Aldrich, Catalog# N8285) using dose dependent serial dilution (range of 0 to 50 uM) without or with dox (Art1 KD) or with Art1 blocking antibody (22C12).
• mice were euthanized and tumor-bearing lungs were perfused by injection of 10 ml cold PBS through the right ventricle. Lungs and subcutaneous tumors were excised and chopped into small pieces using scalpels. Lung and tumor fragments were transferred to GentleMACS C tubes (Miltenyi, Catalog#130-096-334) after which lung dissociation mix (Miltenyi, Catalog#130-095-927) and mouse tumor dissociation mix (Miltenyi, Catalog#130-096-730) respectively was added to the tubes after preparation according to manufacturer’s instructions.
• GentleMACS C tubes Miltenyi, Catalog#130-096-334
• Tissue homogenates were resuspended in RPMI 1640 (Corning, Catalog#15-040-CV) supplemented with 10% FBS and passed through a 70 pM strainer (Corning, Catalog#431751) to obtain a single cell suspension. Cells were pelleted and washed once in cold PBS.
• Tissue was digested for 30 minutes at 37°C, filtered and centrifuged at 4°C to collect pellet which was resuspended in Ack lysis buffer that was deactivated using RBC lysis with ice cold DMEM + 10% FBS +1% p/s. Pellet was resuspended in FACS buffer to flow sorting staining.
• ADP-ribosylation and NICD assay Lung single cell suspensions were pelleted and resuspended in MACS buffer (AutoMACS Rinsing solution (Miltenyi, Catalog#130-091 -222) supplemented with 0.5% BSA stock solution (Miltenyi, Catalog#130-091 -376)). Isolation of T cells was performed by magnetic bead sorting using Pan T Cell Isolation Kit II, mouse (Miltenyi, Catalog# 130-095-130) according to manufacturer’s instructions. Cells were added to 48 well plates pre-coated with mouse ART1 (10 pg/ml) for 24 hours at 4°C 48 hours prior to co-culture.
• T cells were resuspended in serum-free RPMI 1640 medium (Corning, Catalog#15-040-CV) containing 100 pM eNAD (Sigma Aldrich, Catalog#N2630), 5 pg/ml anti-ART2.2 antibody (s+16a, Biolegend, Catalog# 149801), with or without 30 ug/ml anti-CD38 neutralizing antibody (Clone: NIMR-5, Novus Biologicals, Catalog# NBP2-59506) and with or without 20 ug/ml ART1 blocking 22C12 antibody added to wells. Cells were incubated at 37°C for 2 hours. T cells were removed from plate by gentle pipetting and transferred to staining plates.
• T cells were stained with PE-conjugated anti-etheno-NAD antibody for 30 minutes at 4°C (Clone: IG4, Santa Cruz, Catalog#sc-52666) followed by washing in FACS- buffer (PBS supplemented with 2mM EDTA and 0.2% BSA) and staining with surface antibodies CD3 AF594 (Biolegend, Catalog#100240), CD8a BV605 (Biolegend, Catalog#100743), CD4 APC-Cy7 ((Biolegend, Catalog#100413), CD25 AF488 ((Biolegend, Catalog#102018), and P2X7R PE-Cy7 ((Biolegend, Catalog#148707) for 20 minutes at 4°C.
• CD3 AF594 Biolegend, Catalog#100240
• CD8a BV605 Biolegend, Catalog#100743
• CD4 APC-Cy7 (Biolegend, Catalog#100413)
• CD25 AF488 (Biolegend, Catalog#102018)
• DAPI Biolegend, Catalog#422801
• FACSymphony Analyzer BD Biosciences
• Flow cytometry data was analyzed using the FlowJo software (FlowJo LLC, Becton Dickinson).
• Cells were washed in FACS buffer and resuspended in Alexa Fluor® 488-conjugated Goat anti-mouse IgG secondary antibody (Biolegend, Catalog#405319) for 20 min at 4°C.
• Cells were washed in FACS buffer and incubated with a master mix of fluorophore-conjugated surface marker antibodies; CD3-Viogreen (Miltenyi, Catalog#130-113-704), CD8-PerCP-Vio700 (Miltenyi, Catalog#103-113-723), CD38-PE (eBioscience, Catalog#12-0389-42) for 20 min, 4°C.
• CD45- VioBlue (Miltenyi, Catalog# 130-110-802), CD3-FITC (Miltenyi, Catalog# 130-119-798), CD8b-PerCPVio700 (Miltenyi, Catalog# 130-111-715), P2X7R-APC (Miltenyi, Catalog# 130-114-330), CD279 (PD1)-PE (Miltenyi, Catalog# 130-111-953).
• Ki67 PE-Vio770 (Miltenyi, Catalog# 130- 120-419).
• CD8 T cells the dispersed cells were surface stained in master mixes of following conjugated antibodies: CD69 PE (Miltenyi, Catalog# 130-115-575), CD3-FITC (Miltenyi, Catalog# 130-119-798), CD8b-PerCPVio700 (Miltenyi, Catalog# 130-111-715), P2X7R-APC (Miltenyi, Catalog# 130-114-330), CD62L-VioBlue (Miltenyi, Catalog# 130-112-841) and CD44 PE-Vio770 (Miltenyi, Catalog# 130-110-085).
• TCM CD62L+CD44+CD69-
• TEM CD62L-CD44+CD69-
• TRM CD62L-CD44+CD69+.
• human and mouse cells were washed in FACS buffer and fixed using IC fixation buffer (Invitrogen) for 30 min, 4°C.
• IC fixation buffer Invitrogen
• To get absolute counts of immune populations 30 ul counting beads (CountBrightTM Absolute Counting Beads, 0.52x105 beads/50 ul, Invitrogen # C36950) were added before data acquisition per sample.
• Absolute counts were calculated using the formula; ((cell event count x counting bead volume) / (counting bead event count x cell volume)) x counting bead concentration. Stained samples were acquired on a MACSQuant analyzer and flow cytometry data was analyzed using the FlowJo software (FlowJo LLC, Becton Dickinson).
• Frozen Tissue Immunofluorescence Mouse and biomarked patient samples were fixed in formaldehyde and kept 30% sucrose (in PBS) until the samples sink. Samples were embedded in OCT blocks and sectioned using cryostat (Leica). Sections were placed on charged slides, demarcated with barrier pen, and dehydrated in acetone. Sections were then blocked for 1 hr in blocking solution (0.25% Triton-x100+ 5% FBS in 1X PBS).
• ART1 antibody Purified ART1 antibody, Pocono, rabbit #2, 1 :100
• CD8 Polyclonal Antibody # PA5-88265 , 1 :100
• P2X7R P2RX7 antibody cat#113544, biolegend, 1 :100
• antibody buffer 5% FBS dissolved in 1X PBS.
• Purified ART1 antibody Purified ART1 antibody, Pocono, rabbit #2, 1 :100 was used for Art1 staining of human patient samples.
• CD8/P2X7R staining of human samples the following antibodies were used: CD8 Antibody (YTS105.18) (cat # NB200-578 Novus Biologicals, 1 :100) and P2X7/P2RX7 Antibody (7G1 D6) (NBP2-61748 Novus Biologicals, 1 :100). Multiple sections of matched tumor and normal lungs were stained. The samples were washed multiple times in blocking solution and incubated in respective secondary antibodies (1 :200 each secondary antibody) dissolved in antibody buffer for 1 hour in the dark. The samples were washed multiple times in blocking solution and incubated for 5 minutes with Hoechst (HOECHST3342, Thermofisher, 1 :1000 in 1X PBS,).
• Sections were mounted using prolong gold mounting media (# P36934, Thermofisher). Sections were cured overnight at 20°C in dark. Secondary only antibody stained sections were used to determine specificity of each primary antibody. Fluorescence microscopy was performed using Zeiss LSM 880 Laser Scanning Confocal Microscope. Multiple fields were acquired from multiple sections of each sample. Imaged (NIH) was used for image processing, background subtraction, quantification MFI calculations, and cell counting.
• NIR Imaged
• cellular proteins were resolved in 10% SDS/PAGE, transferred to nitrocellulose membranes, and probed with rabbit MAR/PAR antibody (CST #83732, 1 :1000). Blots were acquired using MyECL Imager (Thermofisher). Pageruler plus prestained protein ladder, (10 to 250 kDa, # 26619, Thermofisher) was used to determine weights of protein bands.
• RNA Extraction_(QIAGEN RNeasy® Mini Kit) Total RNA from cells was extracted with RNA Extraction_(QIAGEN RNeasy® Mini Kit). For initial studies with tumor cell lines, for each well 500 ng extracted RNA was reversely transcribed to cDNA using the RNA to cDNA EcoDryTM Premix (Random Hexamers) (catalogue # 639546, Takara). Quantitative PCR was carried out using SYBR green master mix (IQTM SYBR® Green Supermix, #1708884). The primer sequences for the human and mouse genes are listed in Table 5. C1000 Thermal Cycler (Bio-Rad) was used to perform real-time qPCR, and relative quantification performed using Bio-Rad CFX Manager software.
• RNA was plated into each well and FAM-cojugated mouse primers for ‘gene of interest’ [P2X7R (Thermofisher Assay ID: Mm01199500_m1 , targeting exon 2-3) or P2X7R-A (Thermofisher Assay ID: APXGWX4, targeting exon 1 , custom made) or P2X7R-K (Thermofisher Assay ID: APAAF6U, targeting exon 1 , custom made) and VIC conjugated GAPDH primers (Thermofisher Assay ID: Mm05724508_g1 , targeting exon 4) were added to the reaction mix (iTaq Universal Probes One-Step Kit, Biorad # 1725141). The relative mRNA expression levels were calculated using the 2" ⁇ ct method and normalized to relevant house-keeping gene (GAPDH).
• RNA sequencing and gene expression analysis CD8 T cells were isolated from untreated mice bearing KP1 lung tumors and RNA sequencing was performed as previously described (Markowitz et al., 2018). In order to display gene expression of select genes over the various treatment groups and cell types, FPKM for each treatment/cell type was imported into R (version 3.6.2). The function pheatmap was used to display gene expression as a heatmap and gene expression values were centered and scaled along rows by determining z-score for each value. Clustering was carried out using hierarchical clustering.
• TCGA data analysis cBioPortal was used to visualize and analyze transcriptomic data from the TCGA PanCancer Atlas (www.cbioportal.org). Gene expression and OS data of 503 lung adenocarcinoma samples were analyzed. Samples were stratified into mRNA expression data (Batch normalized from Illumina HiSeq_RNASeqV2) into ART 1 high and ART 1 low tumors using a z score threshold of ⁇ 1 .0.
• NAD-glo assay NAD/NADH-GloTM Assay (#G9071 , Promega) kit was used. Histone (1 .5mg/mL), NAD (200nM) (both from the kit) and recombinant ART1 (40nM) or denatured ART1 (40nM) enzymes (enzymes were cloned, expressed and purified by our collaborators at the Tri-lnstitutional Therapeutic Discovery Institute (TDI)) were added in a 96-well white opaque bottom plate. All the components were added to 1xPBS with a final reaction volume of 50pL/well and incubated on a shaker at at 37°C for 1 hour and equilibrated to room temperature for 5 minutes.
• TDI Tri-lnstitutional Therapeutic Discovery Institute
• the NAD/NADH-Glo Detection Reagent was prepared by mixing 1 mL reconstituted luciferin detection reagent, 5 uL reductase, 5 uL reductase substrate, 5 uL NAD cycling enzyme, and 5 uL NAD cycling substrate by gently inverting 5 times. 50 uL/well supernatant and 50 uL/well Detection Reagent were transferred to a new 96-well white luminometer plate, then incubated on a shaker in the dark at room temperature for 30 minutes. The luminescence of the samples was read on a luminometer.
• Anti-human ART1 positive hybridoma supernatants were then tested for inhibition of purified human ART1 by a fluorescent NAD + readout (Abeam, cat. ab176723). Anti-ART1 hybridoma supernatants were also tested for inhibition of human ART1 transiently expressed in HEK293 cells using the e-NAD-based ADP-ribosylation assay (Krebs et al., 2003). The hybridoma supernatants clone 22C12 was positive for inhibition of human ART1 in the biochemical and cell-based assays. Supernatants from 22C12 were then tested for binding to murine ART1 by ELISA and enzymatic inhibition of purified mouse ART1.
• Binding affinity testing of 22C12 to human and mouse ART1 Following hybridoma subcloning and expansion of clone 22C12, the antibody was purified from hybridoma supernatant for potency ranking and affinity determination by bioluminescence (BLI). Range of concentrations of purified mouse and human light chain 22C12 antibodies and purified human or mouse ART1 was used to determine KD.
• NAD-induced cell death of P2X7-receptor (P2X7R)-expressing T cells mediated by mono-ADP- ribosyltransferases (ARTs) regulates immune homeostasis in inflamed tissues.
• ARTs mono-ADP- ribosyltransferases
• ART1 -inhibition slowed tumor growth in a CD8 T cell dependent manner and promotes tumor infiltration of activated P2X7R + CD8 T cells.
• P2X7R + CD8 T cells were susceptible to ART1 -mediated ADP-ribosylation and NICD, which was exacerbated upon blockade of the NAD + -degrading ADP-ribosyl cyclase CD38.
• ART1 -mediated NICD provides for immune resistance in NSCLC and antibody- mediated targeting of ART1 can improve tumor control.
• CD163 (>150 vs ⁇ 150) 21 (8.2%) 27 (13.2%) 0.083
• ART1 staining intensity in a NSCLC tissue microarray (TMA). Clinical parameters and immune cell scoring of an adenocarcinoma tissue microarray. Table compares tumors with strong ART1 staining vs tumors with weak or moderate ART1 staining. Continuous variables are reported as median (interquartile range) and categorical variables are reported as number (percent). Chi-square test or Fisher’s exact test (**) were used for pairwise comparison of categorical variables. A Mann-Whitney U test was used for pairwise comparisons of continuous variables.
• Table 3 Localization of ART1 staining in a NSCLC tissue microarray (TMA). Clinical parameters and immune cell scoring of an adenocarcinoma tissue microarray. Table compares tumors with ART1 staining located to the cell surface or cell surface and cytoplasm vs tumors with ART1 staining located to the cytoplasm only. Continuous variables are reported as median (interquartile range) and categorical variables are reported as number (percent). Chi-square test or Fisher’s exact test (*) were used for pairwise comparison of categorical variables. A Mann-Whitney U test was used for pairwise comparisons of continuous variables.
• ART1 (Art1 KO) was knocked out using CRISPR/Cas9 in B16-F10 melanoma cells using a guide RNA ( Figures 31 A-B).
• cis-ADP ribosylation was also greatly inhibited in B16-F10 ‘Scr Control 6’ cells when co-treated with extracellular NAD+ and 22C12 antibody (Figure 31 C).
• the B16 and LLC1 cell lines were obtained from ATCC and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin in a humidified 5% CO2 incubator at 37°C.
• CRISPR-mediated gene knockout of ART 1 in B16-F10 cells CRISPR/Cas9 mediated knockout of ART 1 in B16-F10 cells was performed using Sigma-Aldrich custom-made, ready-to-use DNA plasmids on the U6gRNA:CMV-CAS9-2A-tGFP backbone.
• B16-F10 clones B16 ART1 KO 63-1) (sequence 5’-3’: CCTGCGCTTTCGGCCAGCG; SEQ ID NO:5) and B16 ART1 KO ⁇ 42 - 1 > (sequence 5’-3’: CCAACAAAGTATACGCGGA; SEQ ID N0:6).
• a negative control plasmid was used to create the B16-F10 clone B16 C0NTR0L ⁇ Scr - 6 > (sequence 5’-3’: CGCGATAGCGCGAATATATT; SEQ ID NO:7).
• B16-F10 cells were seeded in 12 well-plates and incubated for 48 hours to reach 80% confluency.
• Each CRISPR plasmid (0.5 pg DNA) were mixed with 3 pl TransIT-CRISPR reagent (Sigma-Aldrich) in 100 pl Opti-MEM medium (Gibco) and incubated at room temperature for 30 min. Mixture was added to the B16-F10 cells and incubated in a humidified 5% CO2 incubator at 37°C for 24 hours.
• Flow cytometry activated cell sorting FACS was used to sort transfected GFP-positive single cells into flat-bottom 96 well-plates. Clones were expanded and tested for ART1 surface expression by flow cytometry and immunofluorescence staining ( Figures 31 A-B).
• Adherent cells were plated in poly-D-lysine coated coverslips and were treated with serum free media for 12 hours before all experiments.
• Cells were washed with PBS-CM (1 mM MgClz, 0.1 mM CaCIz) and were fixed with 3.7% formaldehyde for 5 minutes (to prevent permeabilization) and incubated with blocking solution (5% BSA in 1xPBS) for 1 hour in a 37°C air incubator.
• the cells were then treated with primary antibody ART1 (Purified ART1 antibody, Pocono, rabbit #2) (1 :200) dissolved in 1% BSA (in 1xPBS, referred to as ‘cell IF antibody buffer’) for one hour in a 37°C air incubator.
• ART1 Purified ART1 antibody, Pocono, rabbit #2
• Cells were treated with serum free media O/N before all experiments. Cells were then treated with NAD + (Sigma Aldrich, Catalog# N8285) using dose dependent serial dilution (0 and 20 uM) without or with Art1 blocking antibody (20 ug/ml) (22C12). Cells were washed with PBS and lysed in a mixture of 1X lysis buffer (cat#9803, CST) and Halt Protease & Phosphatase Inhibitor Single-Use Cocktail (cat# 78442, Thermofisher). Cells were harvested by scraping, centrifuged to collect supernatant.
• NAD + Sigma Aldrich, Catalog# N8285
• cellular proteins were resolved in 10% SDS/PAGE, transferred to nitrocellulose membranes, and probed with rabbit MAR/PAR antibody (CST #83732, 1 :1000). Blots were acquired using MyECL Imager (Thermofisher). Pageruler plus prestained protein ladder (10 to 250 kDa, # 26619, Thermofisher) was used to determine weights of protein bands.
• Flow cytometry to detect binding of 22c12 Ab to LLC1 cells Dispersed LLC1 cells were incubated in AF780 viability dye for 10 minutes (1 :200 in PBS). Pellets were then washed and resuspended in FACS buffer. Cells were incubated with 22C12 (final concentration 20 ug/ml, in 4 degrees) for 30 minutes followed by anti-rabbit 568 (1 :200, in 4 degrees) for 30 minutes. Samples were washed three times in FACS buffer, filtered and analyzed via flow cytometer.
• B16-F10 cells and LLC1 were subcutaneously injected into the flank of C57BL/6 mice (1x10 5 cells in 0.05mL PBS for B16 and 5x10 5 cells in 0.05mL for LLC1). After tumors were palpable, tumor diameters were measured with digital calipers and the tumor volume determined by the formula (length x width 2 ) /2). 22C12 treatment of tumor bearing mice: For flank tumors, Intra-tumoral injections started when tumors became palpable on day 12 (for B16) and day 7 (for LLC1) and treated intratumorally every 72 hours from there onwards.
• mice were injected with 5mg/kg ART1 antibody Clone 22C12 for group ‘22C12 Ab’ or Mouse lgG2a isotype control (BioXcell, Cat# BE0085) for group ‘Iso Ctrl Ab’.
• Humanization of the murine 22C12 antibody light-chain variable region was carried out followed by assessment of the antigen binding and functional properties of a panel of humanized candidate molecules.
• TDI-Y-009 mAb The full-length protein sequences of the TDI-Y-009 mAb are shown in Table 9.
• IMGT complementary determining region (CDR) definitions are indicated in Table 10.
• the antibody TDI-Y-009 was characterized for binding, pharmacokinetics, safety and efficacy.
• the antibody was formatted with a mouse lgG1 Fc in order to lower the risk of eliciting anti-drug antibodies (ADA) as studies were run in immune competent mice for an extended time period.
• ART1 Human, Cynomolgus, Mouse
• Sequence identity of cynomolgus and mouse ART1 to human ART1 are 95% and 76% respectively.
• the mAb was diluted to 50 nM, 10 nM, 3.3 nM and 1.1 nM and captured for 60 seconds.
• the dissociation phase was monitored for 10 minutes followed by a regeneration step.
• a new aliquot of antibody was captured for each binding cycle.
• the processed sensorgram data were globally fit using a simple 1 :1 interaction model. Similar high affinity binding of the mAb to human and cynomolgus ART1 was observed with comparable on and off rates. Binding to mouse ART1 was about 2-fold lower relative to human ART (Table 11 ).
• ART1 paralogs ART3, ART4, ART5
• ART1 or the paralogs were plated on a high binding ELISA plate at 2.5 pg/mL in 1xPBS and incubated o/n at 4°C with shaking. After blocking, 1 :5 serial dilutions of TDI-Y-009 mAb were added to the plate and incubated at RT for 90’.
• KP1 -ART1 0E or KP1 -ART1 knockdown cells were seeded on plates at 100,000 cells/well.
• Test antibodies starting at 100 pg/mL were serially diluted 1 :5 then added to cells and incubated for 30 minutes on ice followed by washes. Cells were then stained with an anti-human IgG Fc and incubated for 30 minutes on ice followed by washes. Cells were resuspended in Flow buffer containing 1000X diluted SYTOX green dead stain and plates were read on Cytoflex flow cytometer.
• Dose-responsive binding to KP1 -ART1 0E cells by TDI- Y-009 was observed ( Figure 34), but not to KP1 -ART1 knockdown cells (data not shown).
• TDI-Y-009 cross-links with the following residues of huARTI : S75, S77, T79, R80, R89, H92, and Y99.
• Corresponding paratope amino acids for TDI-Y-009 included: S51 (CDR2), T96 (CDR3) in the light chain; heavy chain CDR2 residues S55, K59, T63 and heavy chain CDR3 residues S105, and Y108.
• the epitope is located in the N-terminal helical domain of huARTI and does not overlap with the ART1 active site or the NAD binding pocket located in the C-terminal beta-sheet domain of the enzyme. Engagement of the epitope residue Y99 (in helix 3 of the enzyme) may orient the antibody toward the NAD binding pocket possibly sterically hindering NAD binding. Alternatively, inhibition of ART1 activity may be due to conformational changes induced upon binding of Y- 009, restricting substrate engagement.
• TDI-Y-009 A study to assess the single-dose PK of TDI-Y-009 was performed in C57BL/6 mice.
• mice were administered TDI-Y-009 or an equivalent dose of isotype control antibody IP, starting on day 6 after tumor cell injection until day 18.
• Mice treated with the TDI-Y-009 antibody had significantly reduced lung tumor burden compared to the control mice, with reduced numbers of tumor nodules and reduction in tumor nodule area ( Figure 36).
• the use of mouse lgG1 Fc in reformatted TDI-Y-009 for this study ruled out a role for ADCC in eliciting anti-tumor activity.
• Figure 39 depicts the huART1/mAb interaction.
• a PDB structure of huArtl was generated using Swissmodel software and was colored in blue on the epitope site. Amino acids corresponding to 75-99 (SLSPTRPSPPPLGFRDEHGVALLAY;SEQ ID NO:70) of huARTI sequence provided.
• a framework region in the antibody or antigen binding fragment thereof, or polypeptide comprises QVTLKESGPVLVKPTETLTLTCTVS (SEQ ID NO:71 ), VSWIRQP PGKALEWLAH (SEQ ID NO:72), SYSTSLKSRLTISKDTSKSQVVLTM TNMDPVDTATYYC (SEQ ID NO:73), or WGQGTLVTVSS (SEQ ID NO:74), a sequence having one, two, three, four, or five conservative amino acid substitutions, and optionally one two or three non-conservative substitutions.
• a framework region in the antibody or antigen binding fragment thereof, or polypeptide comprises DIQLTQSPSFLSASVGDRVTITCRAS (SEQ ID NO:76), YMHWYQQKPGTS PKRLIY (SEQ ID NO:77), KLASGVPSRFSGSGSGTEYTLTISSLQPEDFATY YC (SEQ ID NO:78), FGQGTKLEIK (SEQ ID NO:79), or a sequence having one, two, three, four, or five conservative amino acid substitutions, and optionally one two or three non-conservative substitutions.
• an antibody or antigen binding fragment thereof, or polypeptide comprises AS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNKTTPPVLDSDGSFFLYSRLTVDKSRWQYEGNVFSC SVM HEALHNHYTQKSLSLSLGK (SEQ ID NO:75), RTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSG
• an antibody or antigen binding fragment thereof, or polypeptide comprises QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAH IFSNDEK SYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARIYGGDSWGYFDNWGQGTLVTVSS (SEQ ID NO:82), DIQLTQSPSFLSASVGDRVTITCRASSSVSYMHWYQQKPGTS PKRLIYDTS KLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCQQWSSNPPTFGQGTKLEIK (SEQ ID NO:83) or a sequence having one, two, three, four, or five conservative amino acid substitutions, and optionally one two or three non-conservative substitutions, or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%,

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Biomedical Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Biotechnology (AREA)
• Wood Science & Technology (AREA)
• General Health & Medical Sciences (AREA)
• Immunology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Cell Biology (AREA)
• Microbiology (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Oncology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biophysics (AREA)
• Animal Behavior & Ethology (AREA)
• Hematology (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Epidemiology (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (2)

Family

ID=87553072

Family Applications (1)

Country Status (6)

Family Cites Families (4)

• 2023
  • 2023-02-07 EP EP23750514.4A patent/EP4476266A4/en active Pending
  • 2023-02-07 CA CA3251163A patent/CA3251163A1/en active Pending
  • 2023-02-07 JP JP2024547005A patent/JP2025508690A/ja active Pending
  • 2023-02-07 US US18/836,705 patent/US20250382381A1/en active Pending
  • 2023-02-07 WO PCT/US2023/062151 patent/WO2023150797A2/en not_active Ceased
  • 2023-02-07 AU AU2023216372A patent/AU2023216372A1/en active Pending

Also Published As

Similar Documents

Legal Events