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WO2019102456A1 - Immunotoxins for treating cancer - Google Patents

Immunotoxins for treating cancer Download PDF

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Publication number
WO2019102456A1
WO2019102456A1 PCT/IL2018/051245 IL2018051245W WO2019102456A1 WO 2019102456 A1 WO2019102456 A1 WO 2019102456A1 IL 2018051245 W IL2018051245 W IL 2018051245W WO 2019102456 A1 WO2019102456 A1 WO 2019102456A1
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WO
WIPO (PCT)
Prior art keywords
seq
antibody
cancer
sequence
nsc
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IL2018/051245
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French (fr)
Inventor
Stipan Jonjic
Hrvoje Simic
Ofer Mandelboim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rijeka Faculty Of Medicine, University of
Yissum Research Development Co of Hebrew University of Jerusalem
Original Assignee
Rijeka Faculty Of Medicine, University of
Yissum Research Development Co of Hebrew University of Jerusalem
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Application filed by Rijeka Faculty Of Medicine, University of, Yissum Research Development Co of Hebrew University of Jerusalem filed Critical Rijeka Faculty Of Medicine, University of
Publication of WO2019102456A1 publication Critical patent/WO2019102456A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • A61K47/6825—Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • 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
    • C07K2317/50—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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention is in the field of immunotherapy and relates to antibodies that bind to a protein on the cancer cell and immunotoxins thereof, for use in treating cancer.
  • the present invention provides immunotoxins comprising anti-poliovirus receptor antibodies conjugated with a toxin, polynucleotide sequences encoding these antibodies and immunotoxins and methods of treating and diagnosing cancer, such as glioblastoma.
  • Immunotoxins are a class of cancer therapeutics that contains a cytotoxic agent fused to a targeting moiety, typically an antibody that binds antigens present on cancer cells.
  • a targeting moiety typically an antibody that binds antigens present on cancer cells.
  • Various toxic agents from different sources are used in immunotoxins, including bacterial, plant and human origin cytotoxic elements. Although bacterial and plant-derived toxins are commonly used in immunotoxins due to their high toxicity, their immunogenicity for human restricted their application in cancer therapy.
  • GBM Glioblastoma multiforme
  • Poliovirus receptor also termed CD155, is a transmembrane glycoprotein involved in mediating cell adhesion to extracellular matrix molecules. It was previously described as a tumor antigen and as a potential target for therapeutic intervention as its expression is up-regulated in neuroectodermal cancers, including GBM, medulloblastoma, and colorectal carcinoma (Solecki et al., J. Biol. Chem. 2002, 277: 25697-700) as well as in pancreatic cancer (Nishiwada et al., Anticancer Res. 2015, 35(4): 2287-97).
  • T cell immu noreceptor with Ig and ITIM domains is a co-inhibitory molecule expressed on various immune cells including T cells and Natural Killer cells (NK cells). TIGIT binds with high affinity to PVR. The PVR- TIGIT interaction inhibits cytotoxic activity of NK cells and T cells activity.
  • PVR has also a critical role in angiogenesis and is suggested to regulate the VEGF- induced angiogenesis by controlling the interaction of VEGFR2 with integrin a(n)b(3), and the VEGFR2-mediated Rapl-Akt signaling pathway (Kinugasa et al., 2012, Circ Res. 2012, 110,5,716-26).
  • PCT Application Publication No. WO 2004/074324 discloses molecules that specifically bind to PVR or to a derivative thereof.
  • the molecules have the ability to modulate a receptor mediated adhesion, trafficking and/or invasion behavior of a cell expressing the PVR or any derivative thereof.
  • PCT Application Publication No. WO 2013/184912 discloses agents that specifically bind the extracellular domain of one or more cell adhesion molecules of the immunoglobulin superfamily (IgCAM), wherein the IgCAM is inter alia PVR.
  • PCT Application Publication No. WO 2017/021526 discloses inhibitors against CD112
  • U.S. Patent Application Publication No. 20070041985 discloses molecules specifically binding to at least one intra- or extracellular domain of the PVR or any derivative thereof, wherein the molecule has the ability to modulate a receptor mediated adhesion, trafficking and/or invasion behavior of a cell expressing the PVR or any derivative thereof.
  • U.S. Patent Application Publication No. 20090215175 provides molecules (e.g. small chemical compounds, oligonucleotides, polypeptides, antibodies, and antibody fragments) which modulate the PVR functions necessary for adhesion, trafficking, invasion and/or metastatic potential of cells.
  • the molecules can be used for the treatment of cells having a metastatic potential, metastasis and cancer.
  • PCT Application Publication No. WO 2006/124667 discloses modulation of the protein zB7Rl (TIGIT) by monoclonal antibodies that block TIGIT binding to its ligand PVR.
  • PCT Application Publication No. WO 2017/149538 discloses monoclonal antibodies that recognise PVR and inhbit its binding to TIGIT.
  • the present invention provides antibody molecules and fragments that recognize the poliovirus receptor (PVR), immu notoxins comprising them and toxins, that kill tumor cells.
  • PVR poliovirus receptor
  • the anti-PVR antibodies disclosed herein are characterized by having unique sets of complementarity-determining regions (CDR) sequences, high affinity and high specificity to PVR and are useful in targeting toxins, radioactive molecules, or identifiable moieties to target cells, tissues and biological samples.
  • CDR complementarity-determining regions
  • the immunotoxins disclosed herein are useful as stand-alone therapy and in combination with other anti-cancer agents.
  • immunotoxins of anti PVR immunoglobulins injected to mice in a tumor in vivo model are effective in tumor regression and mice survival. It is further shown that the immunotoxins disclosed herein bind to glioma cancer cells and kill them. This property of specifically targeting a toxin moiety to cancer cells enables administration of lower doses with fewer side effects.
  • the anti-PVR Abs according to some embodiments of the invention were found to be highly efficient in targeting two different toxins, saporin and diphtheria toxin, to PVR expressing tumor cell lines. A cytotoxicity bioassay was used to show that the toxins were capable of killing glioma cells.
  • the anti- PVR mAbs according to the invention do not block TIGIT-
  • PVR interaction This property may prevent undesired side effects such as increasing systemic immune response.
  • the present invention provides an antibody which specifically binds to poliovirus receptor (PVR), or an antibody fragment thereof comprising at least the antigen binding portion, wherein the antibody or antibody fragment, comprising three complementarity determining regions (CDRs) of a heavy-chain variable region set forth in SEQ ID NO: 2 and three CDRs of a light-chain variable region set forth in SEQ ID NO: 4, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence.
  • PVR poliovirus receptor
  • the antibody comprises three complementarity determining regions (CDRs) of a heavy-chain variable region set forth in SEQ ID NO: 2 and three CDRs of a light-chain variable region set forth in SEQ ID NO: 4, or an analog or derivative thereof having at least 95% sequence identity with said antibody or fragment sequence.
  • CDRs complementarity determining regions
  • a selected set of CDRs may include sequences identified by more than one method, namely, some CDR sequences may be determined using RABAT and some using IMGT, for example.
  • the antibody or fragment comprises the CDR sequences contained in the variable regions of a single-chain monoclonal antibody (scFv) denoted PVR.16 (pOPE scFv#7).
  • the antibody fragment comprises at least one Fab.
  • the antibody fragment is selected from the group consisting of Fab, is F(ab’) 2 .
  • the antibody or the antibody fragment comprises heavy-chain CDR1 comprising the sequence SYW.
  • the antibody or the antibody fragment comprises heavy-chain CDR2 comprising the sequence IF1PNSGST (SEQ ID NO: 12).
  • the antibody or the antibody fragment comprises heavy-chain CDR3 comprising the sequence EGYVYYAMDY (SEQ ID NO: 7).
  • the antibody or antibody fragment comprises heavy- chain CDR1 comprising the sequence SYWMF1 (SEQ ID NO: 5).
  • the antibody or the antibody fragment comprises heavy-chain CDR1 comprising the sequence GYSFTSYW (SEQ ID NO: 11).
  • the antibody or the antibody fragment comprises heavy-chain CDR2 comprising the sequence IIF1PNSGSTIYNEKFK (SEQ ID NO: 6). According to some embodiments, the antibody or the antibody fragment comprises heavy-chain CDR3 comprising the sequence GREGYVYY AMD Y (SEQ ID NO: 13).
  • the antibody or the antibody fragment comprises: (i) F1C CDR1 comprising the sequence SYW; (ii) F1C CDR2 comprising the sequence: IF1PNSGST (SEQ ID NO: 12); and (iii) F1C CDR3 comprising the sequence: EGYVYYAMDY (SEQ ID NO: 7).
  • the antibody or the antibody fragment comprises light-chain CDR1 comprising the sequence QSLLYSGDQRNY (SEQ ID NO: 14). According to some embodiments, the antibody or the antibody fragment comprises light- chain CDR2 comprising the sequence WASX (SEQ ID NO: 15, wherein X is T or is absent). According to some embodiments, the antibody or the antibody fragment comprises light- chain CDR3 comprising the sequence QQYSRYPYT (SEQ ID NO: 16).
  • the antibody or the antibody fragment comprises: (i) LC CDR1 comprising the sequence QSLLYSGDQRNY (SEQ ID NO: 14); (ii) LC CDR2 comprising the sequence WAS; and (hi) LC CDR3 comprising the sequence: QQYSRYP (SEQ ID NO: 16).
  • the antibody or fragment comprises heavy chain CDR1 sequence comprising the sequence SYW, heavy chain CDR2 comprising the sequence: IHPNSGST (SEQ ID NO: 12), heavy chain CDR3 comprising the sequence: EGYVYYAMDY (SEQ ID NO: 7), light chain CDR1 comprising the sequence: QSLLYSGDQRNY (SEQ ID NO: 14), light chain CDR2 comprising the sequence WAS, and light chain CDR3 comprising the sequence: QQYSRYPYT (SEQ ID NO: 16), or analogs thereof comprising no more than 5% amino acid substitution, deletion and/or insertion in the hypervariable region (HVR) sequence.
  • HVR hypervariable region
  • HC CDR1 comprises the sequence SYW.
  • LC CDR2 sequence is WAS.
  • the antibody or fragment comprises a set of six CDR sequences consisting of: i. heavy chain CDR1 having a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 11 , SEQ ID NO: 17, and SEQ ID NO: 18, ii. heavy chain CDR2 having a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 12,
  • heavy chain CDR3 having a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13,
  • v. light chain CDR2 having a sequence selected from the group consisting of SEQ ID NO: 9, and SEQ ID NO 15, and
  • light chain CDR3 having a sequence selected from the group consisting of SEQ ID NO: 10, and SEQ ID NO: 16.
  • the antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • the antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
  • the antibody or fragment thereof comprises heavy chain variable region set forth in SEQ ID NO: 2, or an analog or derivative thereof having at least 90% or at least 95% sequence identity with the heavy chain variable region sequence.
  • the antibody or fragment thereof comprises light chain variable region set forth in SEQ ID NO: 4, or an analog thereof having at least 90%, or at least 95% sequence identity with the light chain variable region sequence.
  • the antibody or fragment thereof comprises a heavy chain variable region having a sequence set forth in SEQ ID NO: 2, and a light chain variable region having a sequence set forth in SEQ ID NO: 4, or an analog thereof having at least 95% sequence identity with the light and/or heavy chain sequence.
  • the antibody or fragment thereof comprises a heavy chain variable region having a sequence set forth in SEQ ID NO: 2, and a light chain variable region having a sequence set forth in SEQ ID NO: 4.
  • the invention also encompasses antibody or antibody fragment capable of binding to an epitope within the human PVR protein to which pOPE scFv#7 single chain antibody binds.
  • the antibody or antibody fragment analog have at least 95% sequence identity with the hypervariable region (HVR) of the reference antibody sequence.
  • the analog or derivative of the antibody or fragment thereof has at least 96, 97, 98 or 99% sequence identity with a variable region of the reference antibody sequence.
  • Each possibility represents a separate embodiment of the invention.
  • the analog comprises no more than one amino acid substitution, deletion or addition to one or more CDR sequences of the HVR, namely, any one of the CDR sequences set forth in SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, and 18.
  • the amino acid substitution is a conservative substitution.
  • the antibody or antibody fragment comprises a HVR having light and heavy chain regions defined above, in which one amino acid was substituted.
  • the antibody or antibody fragment comprises a CDR as defined above, in which one amino acid was substituted.
  • the present invention thus provides an antibody that specifically binds the human protein PVR, or a binding fragment thereof, wherein said antibody or fragment comprises a set of six CDR sequences wherein the set is selected from the group consisting of:
  • the present invention also provides monoclonal antibodies and binding fragments thereof, comprising a heavy chain and a hght chain, wherein said chains comprises a set of a heavy chain variable region sequence and a hght chain variable region sequence, said set comprises the sequences set forth in SEQ ID NO: 2 and 4.
  • the antibody is an isolated monoclonal antibody. According to some embodiments, the antibody is single-chain variable fragment (scFv).
  • scFv single-chain variable fragment
  • the scFv comprises a sequence according to SEQ ID NO: 20.
  • the antibody is selected from the group consisting of: chimeric antibody, and an antibody fragment comprising at least the antigen-binding portion of an antibody.
  • the antibody is a chimeric antibody comprising human constant region.
  • the antibody fragment is selected from the group consisting of: Fab, Fab', F(ab') 2 , Fd, Fd', Fv, dAb, isolated CDR region, single chain antibody (scab), "diabodies", and "linear antibodies".
  • Fab fragment of: Fab, Fab', F(ab') 2 , Fd, Fd', Fv, dAb, isolated CDR region, single chain antibody (scab), "diabodies", and "linear antibodies”.
  • the antibody fragment is F(ab’) 2 .
  • a diagnostic composition comprising the antibody or fragment thereof as described above is provided.
  • a conjugate comprising the antibody or fragment thereof as described above is provided.
  • the conjugate comprises an anti PVR antibody, fragment or scFv and at least one moiety selected from the group consisting of: a drug moiety, a toxin and a detectable probe.
  • the conjugate comprises a drug moiety. According to specific embodiments, the conjugate comprises a toxin.
  • Polynucleotide sequences encoding antibodies, having high specificity for PVR, as well as vectors and host cells carrying these polynucleotide sequences, are provided according to another aspect of the present invention.
  • polynucleotide sequences encoding the amino acid sequences of F1C variable region and light LC variable region described above are provided.
  • the polynucleotide sequence encodes an antibody or antibody fragment or chain capable of binding to an epitope within the human PVR protein to which binds an antibody having a heavy chain variable region of SEQ ID NO: 2 and a light chain variable region of SEQ ID NO: 4.
  • the polynucleotide comprises the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.
  • the polynucleotide sequence according to the invention encodes an antibody or antibody fragment or chain comprising the six CDR sequences: (i) heavy chain CDR1 having the sequence set for the in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17 or SEQ ID NO: 18, (ii) heavy chain CDR2 having the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 12, (iii) heavy chain CDR3 having the sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 13, (iv) light chain CDR1 having the sequence set forth in SEQ ID NO: 8 or SEQ ID NO 14, (v) light chain CDR2 having the sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 15, and (vi) light chain CDR3 having the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16.
  • Each combination of CDRs set is a separate embodiment.
  • the polynucleotide sequence encodes a scFv comprising a sequence set forth in SEQ ID NO: 20.
  • the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one antibody chain or fragment thereof according to the present invention.
  • the nucleic acid construct is a plasmid.
  • the plasmid comprises a polynucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.
  • the present invention provides a hybridoma cell capable of producing a monoclonal antibody or an antibody fragment comprising the specific CDR sequences and/or specific heavy and light chain variable regions defined above.
  • a hybridoma cell comprising at least one polynucleotide sequence disclosed above.
  • the antibody or fragment thereof according to the invention is attached to a cytotoxic moiety.
  • the present invention provides an immunotoxin comprising the antibody or fragments thereof as described hereinabove and a toxin.
  • the immunotoxin comprises a scFv sequence having a CDR set of six CDRs wherein: F1C CDR1 is selected from SYWMF1 (SEQ ID NO: 5) and GYSFTSYW (SEQ ID NO: 11); HC CDR2 is selected from IIHPNSGSTIYNEKFK (SEQ ID NO: 6) and IHPNSGST (SEQ ID NO: 12); HC CDR3 is selected from EGYVYYAMDY (SEQ ID NO: 7) and GREGYVY Y AMD Y (SEQ ID NO: 13); LC CDR1 is selected from KS SQSLLYSGDQRNYLA (SEQ ID NO: 8) and QSLLYSGDQRNY (SEQ ID NO: 14); LC CDR2 is selected from the group consisting of: WASTRES (SEQ ID NO: 9) and WAS; and LC CDR3 is selected from QQYYNYPRT (SEQ ID NO: 10) and Q
  • the immunotoxin comprises the scFv sequence set forth in SEQ ID No: 20.
  • the antibody may be linked to the toxin by, for example, chemical linking using a crosslinker and/or a peptide bond.
  • the toxin is a peptide-based toxin.
  • the antibody is connected to the toxin through a linker.
  • the linker comprises a stretch of 1-10 amino acid residues.
  • the toxin is selected from the group consisting of diphtheria toxin or a subunit thereof, saporin, ricin A, abrin, pseudomonas exotoxin or a portion thereof, restrictocin and gelonin.
  • saporin diphtheria toxin or a subunit thereof
  • ricin A saporin
  • abrin abrin
  • pseudomonas exotoxin or a portion thereof
  • restrictocin and gelonin are examples of the invention.
  • the toxin is a diphtheria toxin or an active fragment thereof.
  • the immunotoxin comprises a diphtheria toxin fragment is which domain R that provides binding to diphtheria toxin receptor, is absent.
  • the immunotoxin comprises a scFv molecule according to SEQ ID No: 20, a diphtheria toxin sequence and optionally at least one amino acid linker.
  • the immunotoxin sequence further comprises a His-tag sequence.
  • the immunotoxin comprises a scFv comprising a sequence set forth in SEQ ID No: 19 or an analog thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the immunotoxin comprises scFv comprising a sequence set forth in SEQ ID No: 19.
  • the toxin is saporin.
  • the toxin is selected from the group consisting of a saporin, anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC- 1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, and a PDB, or an analog, derivative, or prodrug of any thereof.
  • the present invention provides an immunotoxin comprising a diphtheria toxin and an scFV antibody comprising three CDRs of a heavy-chain variable region set forth in SEQ ID NO: 2 and three CDRs of a light-chain variable region set forth in SEQ ID NO: 4, or an analog or derivative thereof having at least 95% sequence identity with said antibody or fragment sequence.
  • the diphtheria toxin and the scFV are linked with a short peptide of 1-5 amino acid residues. According to certain embodiments, the diphtheria toxin and scFV are linked by a Ser-Gly linker.
  • the present invention provides an immunotoxin comprising saporin linked to an antibody comprising three CDRs of a heavy-chain variable region set forth in SEQ ID NO: 2 and three CDRs of a light-chain variable region set forth in SEQ ID NO: 4, or an analog or derivative thereof having at least 90% or at least 95% sequence identity with said antibody or fragment sequence.
  • the antibody is as described hereinabove.
  • the immunotoxin or antibody does not block TIGIT- PVR interaction.
  • the present invention provides, according to another aspect, a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient, at least one immunotoxin or antibody as described hereinabove, and optionally at least one pharmaceutical acceptable excipient, diluent, salt or carrier.
  • the pharmaceutical composition comprises an immunotoxin comprising an antibody or fragment thereof which is capable of binding to an epitope within the human PVR protein to which binds an antibody herein identified as PVR.16 (pOPE scFv#7), having a heavy chain variable region of SEQ ID NO: 2 and a light chain variable region of SEQ ID NO: 4.
  • the pharmaceutical composition comprises an immunotoxin comprising an antibody or antibody fragment thereof comprising the six CDRs: i. heavy chain CDR1 having a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 11 , SEQ ID NO: 17, and SEQ ID NO: 18, ii. heavy chain CDR2 having a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 12,
  • heavy chain CDR3 having a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13,
  • v. light chain CDR2 having a sequence selected from the group consisting of SEQ ID NO: 9, and SEQ ID NO 15, and
  • light chain CDR3 having a sequence selected from the group consisting of SEQ ID NO: 10, and SEQ ID NO: 16.
  • Each combination of CDRs set represents a separate embodiment of the invention.
  • the pharmaceutical composition according to the present invention is for use in treating cancer.
  • the cancer can be any cancer that expresses PVR.
  • the cancer overexpresses PVR.
  • the cancer is a malignant cancer.
  • the immunotoxin of the invention reduces or eliminates the tumor.
  • the cancer is a metastatic cancer.
  • the pharmaceutical composition according to the present invention is for use in inhibiting formation or distribution of metastases or reducing the total number of metastases in a subject.
  • the cancer is selected from the group consisting of a brain cancer, melanoma, a breast cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, a colon cancer, a cervical cancer, a kidney cancer, a lung cancer, a thyroid cancer, a prostate cancer, a renal cancer, a throat cancer, a laryngeal carcinoma, a bladder cancer, a hepatic cancer, a fibrosarcoma, an endometrial cells cancer, sarcoma, a myeloid, a leukemia and a lymphoma.
  • a brain cancer melanoma
  • a breast cancer an ovarian cancer
  • a pancreatic cancer a colorectal cancer
  • a colon cancer a cervical cancer, a kidney cancer, a lung cancer, a thyroid cancer, a prostate cancer, a renal cancer, a throat cancer, a laryngeal carcinoma, a bladder cancer, a hepatic cancer, a
  • the cancer is a solid cancer.
  • the solid cancer is selected from the group consisting of brain, melanoma (skin), lung, colon, breast, uterine, and renal cancer.
  • the cancer is glioblastoma.
  • the cancer that over-expresses PVR is selected from the group consisting of: GBM, medulloblastoma, colorectal carcinoma and pancreatic cancer.
  • the present invention provides a method of treating cancer comprising administering to a subject in need thereof an effective amount of the immunotoxin as described hereinabove.
  • the immunotoxin is administered directly to the cancer site.
  • the pharmaceutical composition is administered directly to the brain.
  • the pharmaceutical composition is administered intrathecally.
  • the subject is human.
  • the method comprises administering directly to the surgical site an effective amount of the immunotoxin before, during, and/or after surgery for cancer.
  • the treating comprises decreasing the size, growth rate, invasiveness, malignancy grade and/or risk of recurrence of a tumor associated with the cancer.
  • the therapeutically effective amount results in a decrease in tumor size or in the number of metastases in the subject.
  • the method of treating cancer comprises administering or performing at least one additional anti-cancer therapy.
  • the additional anticancer therapy is surgery, chemotherapy, radiotherapy, or immunother ap y.
  • the immunotoxin is co-administered, concurrently administered, and/or sequentially administered with one or more other anti-cancer agents, and/or in conjunction with radiation or surgery.
  • the additional anti-cancer agent is selected from the group consisting of: immune-modulator, activated lymphocyte cell, kinase inhibitor and chemotherapeutic agent.
  • the immune-modulator is an antibody, antibody fragment or antibody conjugate that binds to an antigen other than human PVR.
  • the use further comprises the use of an agent that downregulates the activity or expression of an immune inhibitory receptor.
  • the immune inhibitory receptor is selected from the group consisting of PD-l, TIGIT, DNAM-l, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55, LAIR1 , SIGLEC10, and 2B4. Each possibility represents a separate embodiment of the invention.
  • the present invention further comprises, according to another aspect, a method of determining or quantifying the expression or presence of PVR, the method comprising contacting a biological sample with an antibody or antibody fragment, and measuring the level of complex formation, wherein the antibody or antibody fragment comprises the complementarity determining regions (CDRs) of: (i) heavy chain CDR1 having the sequence set for the in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17 or SEQ ID NO: 18, (ii) heavy chain CDR2 having the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 12, (iii) heavy chain CDR3 having the sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 13, (iv) light chain CDR1 having the sequence set forth in SEQ ID NO: 8 or SEQ ID NO 14, (v) light chain CDR2 having the sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 15, and (vi) light chain CDR3 having the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16.
  • Determining and quantifying methods may be performed in-vitro or ex-vivo according to some embodiments or may be used in diagnosing conditions associated with expression of PVR.
  • the antibody or fragment thereof according to the present invention may be also used to configure screening methods.
  • an enzyme-linked immunosorbent assay (ELISA), or a radioimmunoassay (RIA) can be constructed for measuring levels of secreted or cell- associated polypeptide using the antibodies and methods known in the art.
  • the sample is a body fluid. According to other embodiments, the sample is a tissue sample or a biopsy.
  • the method is performed in-vitro or ex-vivo.
  • a kit for measuring the expression of PVR in biological sample is also provided comprising at least one antibody or antibody fragment comprising the complementarity determining regions (CDRs) of the antibody or fragment thereof as described hereinabove.
  • the present invention provides a kit for detecting cancer, the diagnostic kit comprises an antibody of fragment thereof as disclosed herein.
  • the invention provides a method of diagnosing, assessing the severity or staging of a cancer comprising determining the expression or activity of PVR in a sample from a subject using an antibody according to the present invention or a fragment or conjugate thereof, and comparing the expression or activity of PVR to a reference amount of PVR expression or activity.
  • Said reference amount may be obtained from a sample taken from a normal subject, from the same subject while being in a different stage of the disease or is determined from clinical data of a large population of subjects.
  • FIGS 1A-1B PVR expression on U-87 MG (Fig. 1 A) and U-87 MG PVRhi (Fig. 1 B) cell lines. Fluman glioblastoma cells U-87 MG were transfected to express high levels of hPVR (U-87 MG PVRhi). To determine level of PVR expression on indicated cell lines, cells were stained with aPVR antibody (clone CD155.16) or control isotype mAh and analyzed by flow cytometry.
  • aPVR antibody clone CD155.16
  • control isotype mAh were analyzed by flow cytometry.
  • FIG. 1 Efficacy of antiCDl55-saporin in killing PVR expressing tumor cells.
  • U87MG glioma cell line and U87PVRhi were treated with different anti-CDl55.l6 KAC and SA- saporin mixtures. Puromycin was used as a positive control (100 % killing). Release of adenylate cyclase was measured to determine the amount of cell death. Experiment was performed using ToxiLightTM non-destructive Cytotoxicity BioAssay Kit, Lonza. aCDl55.
  • l6 b+ designates biotinylated aCDl55.l6 antibody
  • aCDl55.l6b + Fab2 designates F(ab’) 2 fragments derived from aCDl55.l6 antibody
  • amouse b+ designates goat anti mouse biotinylated antibody
  • SA- saporin designates streptavidin saporin conjugate.
  • FIG. 3 Schematic presentation of aPVR-DT immunotoxin.
  • PVR-specific antibody fragments scFv
  • scFv diphtheria toxin subunits C and T to form new molecule: PVR- DT immunotoxin.
  • Domain R which provides binding to diphtheria toxin receptor is deleted from the final construct.
  • FIG. 4 Cytotoxic effect of aPVR-DT on human GBM cells.
  • Human glioblastoma transfectants U-87 MG PVRhi
  • aPVR-DT or parental, control aPVR antibody clone CD 155.16
  • Cytotoxic effect was determined by ToxyLight kit (Lonza) according to manufacturer’s instructions.
  • FIGS 5A-5B Antitumor effect of aPVR-DT in mouse model. NSG mice were inoculated h
  • mice received 100 pg (50 pg i.p. + 50 pg i.t.) of aPVR-DT or control aPVR mAh (clone CD155.16). Tumor growth (Fig. 5A) and mice survival (Fig. 5B) were monitored.
  • FIGS. 6A-6B Susceptibility to aPVR-DT is related to PVR expression.
  • NSG mice were injected with 10 7 human glioblastoma cells U-87 MG PVRhi (Fig. 6A) or U-87 MG (Fig. 6B) transfectants subcutaneously at right flank.
  • Fig. 6A human glioblastoma cells
  • Fig. 6B U-87 MG
  • mice received 100 pg (50 pg i.p. + 50 pg i.t.) of aPVR-DT or PBS and tumor growth was monitored.
  • the present invention provides antibodies specific to human poliovirus receptor (PVR) and immunotoxins thereof.
  • the immunotoxin comprises the anti PVR antibody that binds specifically to tumor cells to deliver a conjugated toxin for efficient tumor cell- killing.
  • the present invention provides an anti-PVR antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug or a radioisotope.
  • a therapeutic moiety such as a cytotoxin, a drug or a radioisotope.
  • these antibody conjugates are referred to as “immunotoxins.
  • immunotoxins refers to complexes of antibodies or their fragments with toxins, chemically bound. The antibody is directed against structures on the tumor cell surface.
  • the PVR molecules presented on the cancer cells serve as an antigen for the antibody portion of the immunotoxin.
  • the term "antigen” as used herein refers to a molecule or a portion of a molecule capable of eliciting antibody formation and being specifically bound by an antibody.
  • An antigen may have one or more than one epitope. The specific binding referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
  • PVR refers to the poliovirus receptor, also known as CD155 (cluster of differentiation 155).
  • the PVR is a transmembrane glycoprotein with an N-terminal signal sequence, three extracellular immunoglobulin (Ig)-like domains, a transmembrane domain and a cytoplasmic tail. It has a molecular size of approximately 80 kDa and a structure composed of three Ig-like domains, specifically an outermost V-like domain followed by two C2-like domains.
  • An exemplary human PVR according to the invention is set forth in GenBank accession numbers: NP_00l 129240.1 , NP_00l 129241.1, NP_00l 129242.2 and NP_006496.4. These poliovirus receptors share the sequence of the extracellular domain and therefore can be targeted by the affinity binding moiety of the invention.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental (e.g., kills) to cells.
  • Non-limiting examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, anti metabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti -mitotic agents (e.
  • An antibody of the present invention can be conjugated to a radioisotope, e.g., radioactive iodine, to generate cytotoxic radiopharmaceuticals for beating an overexpressed PVR-related disorder, such as a cancer.
  • a radioisotope e.g., radioactive iodine
  • the toxin portion of an immunotoxin of the invention may comprise an alkylating agent including, without limitation, Asaley NSC
  • NSC 348948 dianhydrogalactitol NSC 132313, fluorodopan NSC 73754, hepsulfam NSC
  • NSC 95466 piperazine NSC 344007, piperazinedione NSC 135758, pipobroman NSC 25154, porfiromycin NSC 56410, spirohydantoin mustard NSC 172112, teroxirone NSC 296934, tetraplatin NSC 363812, thio-tepa NSC 6396, triethylenemelamine NSC 9706, uracil nitrogen mustard NSC 34462, and Yoshi-864 NSC 102627.
  • the toxin portion of an immunotoxin of the invention may comprise an antimitotic agent including, without limitation, allocolchicine NSC 406042, Halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC 376128 (NG-auristatin derived), maytansine NSC 153858, rhizoxin NSC 332598, taxol NSC 125973, taxol derivative NSC 608832, thiocolchicine NSC 361792, trityl cysteine NSC 83265, vinblastine sulfate NSC 49842, and vincristine sulfate NSC 67574.
  • an antimitotic agent including, without limitation, allocolchicine NSC 406042, Halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC 376128 (NG-auristatin derived), maytans
  • the toxin portion of an immunotoxin of the invention may comprise a topoisomerase I inhibitor including, without limitation, camptothecin NSC 94600, camptothecin, Na salt NSC 100880, aminocamptothecin NSC 603071, camptothecin derivative NSC 95382, camptothecin derivative NSC 107124, camptothecin derivative NSC 643833, camptothecin derivative NSC 629971 , camptothecin derivative NSC 295500, camptothecin derivative NSC 249910, camptothecin derivative NSC 606985, camptothecin derivative NSC 374028, camptothecin derivative NSC 176323, camptothecin derivative NSC 295501 , camptothecin derivative NSC 606172, camptothecin derivative NSC 606173, camptothecin derivative NSC 610458, camptothecin derivative NSC 618939, camptothecin derivative NSC 6104
  • the toxin portion of an immunotoxin of the invention may comprise an topoisomerase II inhibitor including, without limitation, doxorubicin NSC 123127, amonafide NSC 308847, m-AMSA NSC 249992, anthrapyrazole derivative NSC 355644, pyrazoloacridine NSC 366140, bisantrene HCL NSC 337766, daunorubicin NSC 82151, deoxydoxorubicin NSC 267469, mitoxantrone NSC 301739, menogaril NSC 269148, N,N-dibenzyl daunomycin NSC 268242, oxanthrazole NSC 349174, rubidazone NSC 164011 , VM-26 NSC 122819, and VP-16 NSC 141540.
  • an topoisomerase II inhibitor including, without limitation, doxorubicin NSC 123127, amonafide NSC 308847,
  • the toxin portion of an immunotoxin of the invention may comprise an RNA or DNA antimetabolite including, without limitation, L- alanosine (NSC 153353), 5-azacytidine (NSC 102816), 5-fluorouracil (NSC 19893), acivicin (NSC 163501), aminopterin derivative (NSC 132483), aminopterin derivative (NSC 184692), aminopterin derivative (NSC 134033), an antifol (NSC 633713), an antifol NSC (623017), Baker's soluble antifol (NSC 139105), dichlorallyl lawsone (NSC 126771), brequinar (NSC 368390), ftorafur (pro-drag) (NSC 148958), 5,6-dihydro-5-azacytidine (NSC 264880), methotrexate (NSC 740), methotrexate derivative (NSC 174121), N-(
  • the antibody conjugates of the invention can be used to modify a given biological response, and the toxin or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-.
  • gamma gamma.
  • biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1 "), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • the antibody and toxin are both proteins and can be conjugated using techniques well known in the art. There are many crosslinkers available that can conjugate two proteins.
  • crosslinker is generally chosen based on the reactive functional groups available or inserted on the antibody or toxin. In addition, if there are no reactive groups a photoactivatible crosslinker can be used.
  • Crosslinking agents known to the art include the homobifunctional agents: glutaraldehyde, dimethyladipimidate and Bis(diazobenzidine) and the heterobifunctional agents: m maleimidobenzoyl-N-hydroxysuccinimide and sulfo-m maleimidobenzoyl-N- hydroxysuccinimide.
  • a spacer between the antibody molecule or fragment and the toxin e.g. a peptidic linker or spacer.
  • the antibody-toxin protein fusion is prepared using recombinant DNA techniques.
  • a DNA sequence encoding the ligand is fused to a DNA sequence encoding the toxin, resulting in a chimeric DNA molecule.
  • the chimeric DNA sequence is transfected into a host cell that expresses the ligand-toxin fusion protein.
  • the fusion protein can be recovered from the cell culture and purified using techniques known in the art.
  • the immunotoxin consists of a single polypeptide chain, wherein the PVR-binding portion is a single chain antibody.
  • the PVR-binding portion comprises a complete immunoglobulin molecule.
  • the PVR-binding portion is a dimer of Fab, Fab', scFv, single-domain antibody fragments, or disulfide-stabilized Fv fragments.
  • the PVR-binding portion comprises a variable heavy chain, variable light chain, Fab, Fab', scFv, single-domain antibody fragment, or disulfide-stabilized Fv fragment.
  • Portions of the PVR-binding molecule may be derived from one or more species, preferably comprising portions derived from the human species, and most preferably are completely human or humanized. Regions designed to facilitate purification or for conjugation to toxin may also be included in or added to the PVR-binding portion.
  • Antibodies, or immunoglobulins comprise two heavy chains linked together by disulfide bonds and two light chains, each light chain being linked to a respective heavy chain by disulfide bonds in a "Y" shaped configuration.
  • Proteolytic digestion of an antibody yields Fv (Fragment variable) and Fc (Fragment crystalline) domains.
  • the antigen binding domains, Fab include regions where the polypeptide sequence varies.
  • F(ab') 2 represents two Fab' arms linked together by disulfide bonds.
  • the central axis of the antibody is termed the Fc fragment.
  • Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (CH).
  • Each light chain has a variable domain (V L ) at one end and a constant domain (CL) at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain (CF11).
  • the variable domains of each pair of light and heavy chains form the antigen-binding site.
  • the domains on the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, joined by three hyper- variable domains known as complementarity determining regions (CDRs 1-3). These domains contribute specificity and affinity of the antigen-binding site.
  • CDR identification or determination from a given heavy or light chain variable sequence is typically made using one of few methods known in the art. For example, such determination is made according to the Rabat (Wu T.T and Rabat E.A., J Exp Med, 1970; 132:211-50), Chothia, Martin, and IMGT (Lefranc M-P, et al., Dev Comp Immunol, 2003, 27:55-77).
  • CDR having a sequence When the term“CDR having a sequence”, or a similar term is used, it includes options wherein the CDR comprises the specified sequences and also options wherein the CDR consists of the specified sequence.
  • the antigen specificity of an antibody is based on the hyper variable region (F1VR), namely the unique CDR sequences of both light and heavy chains that together form the antigen-binding site.
  • F1VR hyper variable region
  • the isotype of the heavy chain determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively).
  • the light chain is either of two isotypes (kappa, k or lambda, l). Both isotopes are found in all antibody classes.
  • antibody is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, and antibody fragments long enough to exhibit the desired biological activity, namely binding to human PVR.
  • Antibody or antibodies according to the invention include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof, such as the Fab or F(ab')2 fragments. Single chain antibodies also fall within the scope of the present invention.
  • Antibody fragments comprise only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen.
  • Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VF1 and CF11 domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CF11 domain; (iii) the Fd fragment having VF1 and CF11 domains; (iv) the Fd' fragment having VF1 and CF11 domains and one or more cysteine residues at the C-terminus of the CF11 domain; (v) the Fv fragment having the VL and VF1 domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 1989, 341, 544-546) which consists of a VF1 domain; (vii) isolated CDR regions; (viii) F(
  • antibody fragments Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology 70:163- 167 (1992)).
  • F(ab') 2 fragments can be isolated directly from recombinant host cell culture.
  • Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • the antibody of choice is a single chain Fv fragment (scFv).
  • Single chain antibodies can be single chain composite polypeptides having antigen binding capabilities and comprising amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain i.e. linked VH-V L or single chain Fv (scFv).
  • Techniques for the production of single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single-chain antibodies to PVR.
  • scFv refers to a single chain Fv antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain.
  • the chains are joined by a linker peptide.
  • the linker peptide can be from about 5 to 40 amino acids or from about 10 to 30 amino acids or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length.
  • mAb monoclonal antibody
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” is not to be construed as requiring production of the antibody by any particular method. mAbs may be obtained by methods known to those skilled in the art.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
  • Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 1991 , 352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222:581-597.
  • VH variable heavy
  • VL variable light
  • the invention also provides conservative amino acid variants of the antibody molecules according to the invention. Variants according to the invention also may be made that conserve the overall molecular structure of the encoded proteins. Given the properties of the individual amino acids comprising the disclosed protein products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., "conservative substitutions,” may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • the term“antibody analog” as used herein refers to an antibody derived from another antibody by one or more conservative amino acid substitutions.
  • antibody variant refers to any molecule comprising the antibody of the present invention.
  • fusion proteins in which the antibody or an antigen-binding-fragment thereof is linked to another chemical entity is considered an antibody variant.
  • Analogs and variants of the antibody sequences are also within the scope of the present application. These include, but are not limited to, conservative and non-conservative substitution, insertion and deletion of amino acids within the sequence. Such modification and the resultant antibody analog or variant are within the scope of the present invention as long as they confer, or even improve the binding of the antibody to the human PVR.
  • Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention.
  • Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, penetration, and targeting to specific cell populations, immunogenicity, and the like.
  • One of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, according to one table known in the art, the following six groups each contain amino acids that are conservative substitutions for one another:
  • the active agent is preferably utilized together with one or more pharmaceutically acceptable carrier(s) and optionally any other therapeutic ingredients.
  • the carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof.
  • the active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired exposure.
  • the immunotoxins will be suspended in a sterile saline solution for therapeutic uses.
  • the pharmaceutical compositions may alternatively be formulated to control release of active ingredient or to prolong its presence in a patient's system.
  • suitable drug delivery systems include, e.g., implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the molecule according to the present invention.
  • biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebaric acid.
  • the rate of release of the molecule according to the present invention, i.e., of an antibody or antibody fragment, from such a matrix depends upon the molecular weight of the molecule, the amount of the molecule within the matrix, and the size of dispersed particles.
  • the molecules of the present invention as active ingredients are dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known.
  • Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof.
  • PBS phosphate buffered saline
  • Other suitable carriers are well known to those skilled in the art.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.
  • composition of this invention may be administered by any suitable means, such as intrathecally, intravenously, orally, topically, intranasally, subcutaneously, intramuscularly, intra-arterially, intraarticularly, intralesionally, intratumorally or parenterally.
  • the immunotoxin is administered directly to the cancer site.
  • the cancer site is the in the brain.
  • Such administration may be aimed at bypassing the blood brain barrier.
  • the systemic administration is intrathecal administration, i.e. such composition is administered intrathecally.
  • the phrase "the immunotoxin is administered directly to the cancer site” refers to direct or substantially direct introduction including, without limitation, single or multiple injections of the immunotoxin directly into the tumor or peritu morally, continuous or discontinuous perfusion into the tumor or peritumorally, introduction of a reservoir into the tumor or peritumorally, introduction of a slow-release apparatus into the tumor or peritumorally, introduction of a slow-release formulation into the tumor or peritumorally, or direct application onto the tumor.
  • the therapeutically effective amount of the molecule according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered, its persistence in the blood circulation, and the judgment of the treating physician.
  • therapeutically effective amount refers to an amount of a drug effective to treat a disease or disorder in a mammal.
  • the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
  • the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life.
  • the cancer amendable for treatment by the present invention includes, but is not limited to: blastoma, carcinoma, lymphoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include glioblastoma, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma
  • the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non- Hodgkin’s lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft- tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma.
  • the cancerous conditions amendable for treatment of the invention include metastatic cancers.
  • the cancer is glioblastoma.
  • the pharmaceutical compositions according to the invention are for use in treating cancer characterized by PVR overexpression.
  • PVR overexpression related cancer types can be identified using known data bases such as The Cancer Genome Atlas (TCGA).
  • the cancer is selected from the group consisting of glioblastoma, adrenocortical carcinoma (ACC), chromophobe renal cell carcinoma (RICH), liver hepatocellular carcinoma (LIHC), colon and rectal adenocarcinoma (COAD, READ), pancreatic ductal adenocarcinoma (PAAD), pheochromocytoma & paraganglioma (PCPG), papillary kidney carcinoma (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), uterine corpus endometrial carcinoma (UCEC), cervical cancer (CESC), cutaneous melanoma (SKCM), meso
  • the pharmaceutical composition according to the present invention may be administered together with an anti- neoplastic composition.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cancer include but are not limited to, blastoma, carcinoma, lymphoma, sarcoma, and leukemia. More particular examples of such cancers include melanoma, brain, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, or endometrial cancer.
  • the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen comprising administration of at least one additional anti-cancer agent.
  • the anti-cancer agent is selected from the group consisting of an antimetabolite, a mitotic inhibitor, a taxane, a topoisomerase inhibitor, a topoisomerase II inhibitor, an asparaginase, an alkylating agent, an antitumor antibiotic, and combinations thereof.
  • an antimetabolite a mitotic inhibitor, a taxane, a topoisomerase inhibitor, a topoisomerase II inhibitor, an asparaginase, an alkylating agent, an antitumor antibiotic, and combinations thereof.
  • the antimetabolite is selected from the group consisting of cytarabine, gludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea.
  • the mitotic inhibitor is selected from the group consisting of vincristine, vinblastine, and vinorelbine.
  • the topoisomerase inhibitor is selected from the group consisting of topotecan and irenotecan.
  • the alkylating agent is selected from the group consisting of busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosamide, mechlorethamine, melphalan, thiotepa, dacarbazine, and procarbazine.
  • the antitumor antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, and plicamycin.
  • the topoisomerase II is selected from the group consisting of etoposide and teniposide. Each possibility represents a separate embodiment of the present invention.
  • the additional anti-cancer agent is selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof.
  • bevacizumab carboplatin
  • cyclophosphamide doxorubicin hydrochloride
  • gemcitabine hydrochloride gemcitabine hydrochloride
  • topotecan hydrochloride thiotepa
  • combinations thereof are selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof.
  • Monoclonal antibodies according to the present invention may be used as part of combined therapy with at least one anti-cancer agent.
  • the additional anti-cancer agent is an immuno-modulator, an activated lymphocyte cell, a kinase inhibitor or a chemotherapeutic agent.
  • the anti-cancer agent is an immuno-modulator, whether agonist or antagonist, such as antibody against an immune checkpoint molecule.
  • the additional anti-cancer agent is a chemotherapeutic agent.
  • the chemotherapy agent which could be administered together with the antibody according to the present invention, or separately, may comprise any such agent known in the art exhibiting anticancer activity, including but not limited to: mitoxantrone, topoisomerase inhibitors, spindle poison vincas: vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine, gemcitabin; podophyllo toxins: etoposide, irinotecan, topotecan, dacarbazin; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin; nitrosoureas: car
  • the chemotherapeutic agent is selected from alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressant, adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone analog.
  • the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel and docetaxel.
  • 5-fluorouracil 5-FU
  • leucovorin LV
  • irinotecan irinotecan
  • oxaliplatin oxaliplatin
  • capecitabine paclitaxel
  • docetaxel docetaxel.
  • One or more chemotherapeutic agents can be used.
  • the method of treating cancer comprises administration of an immunotoxin according to the invention and an additional anti-cancer agent.
  • the additional anti-cancer agent is selected from the group consisting of: immune-modulator, activated lymphocyte cell, kinase inhibitor and chemotherapeutic agent.
  • the immune-modulator is an antibody, antibody fragment or antibody conjugate that binds to an antigen other than human PVR.
  • the immune-modulator is an antibody against an immune checkpoint molecule.
  • the additional immune modulator is an antibody against an immune checkpoint molecule selected from the group consisting of human programmed cell death protein 1 (PD-l), PD-L1 and PD-L2, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, 0X40 (also referred to as CD134), killer cell immunoglobulin-like receptors (KIR), TIGIT and any combination thereof.
  • PD-l human programmed cell death protein 1
  • PD-L1 and PD-L2 carcinoembryonic antigen-related cell adhesion molecule 1
  • LAG3 lymphocyte activation gene 3
  • CD137 0X40
  • KIR killer cell immunoglobulin-like receptors
  • TIGIT TIGIT
  • the anti-cancer agent is selected from the group consisting of: Erbitux, cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine, vinblastine, vinorelbine, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide, teniposide and any combination thereof.
  • Erbitux Erbitux
  • cytarabine fludarabine
  • fluorouracil fluorouracil
  • mercaptopurine methotrexate
  • the anti-cancer agent is epidermal growth factor receptor (EGFR) inhibitor.
  • the EGFR inhibitor is selected from the group consisting of: Cetuximab (Erbitux®), Panitumumab (Vectibix®), and necitumumab (Portrazza®).
  • the EGFR inhibitor is Cetuximab (Erbitux®).
  • the present invention provides a method of treating cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of an immunotoxin according to the present invention.
  • Toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition) and the maximal tolerated dose for a subject compound.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in humans.
  • the dosage may vary depending inter alia upon the dosage form employed, the dosing regimen chosen, the composition of the agents used for the treatment and the route of administration utilized, among other relevant factors.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors.
  • a compound or an agent can be administered enterally or parenterally.
  • Enterally refers to administration via the gastrointestinal tract including per os, sublingually or rectally.
  • Parenteral administration includes administration intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct).
  • a compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug.
  • a physician who instructs a patient to self- administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.
  • the effective dose of a specific immunotoxin construct may depend on additional factors, including the type of cancer, the size of the tumor, the stage of the cancer, the immunotoxin' s toxicity to the patient, the specificity of targeting to cancer cells, as well as the age, weight, and health of the patient.
  • the effective dose by direct administration of immunotoxin may range from about 10 to 3000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 60 to 500, 70 to 400, 80 to 300, 90 to 200, or 100 to 150 micrograms/tumor/day.
  • the dose may range from approximately 10 to 20, 21 to 40, 41 to 80, 81 to 100, 101 to 130, 131 to 150, 151 to 200, 201 to 280, 281 to 350, 351 to 500, 501 to 1000, 1001 to 2000, or 2001 to 3000 micro grams/tumor/day.
  • the dose may be at least approximately 20, 40, 80, 130, 200, 280, 400, 500, 750, 1000, 2000, or 3000 micrograms/tu mor/day.
  • the effective dose of immunotoxin may range from about 100 to 5000, 200 to 4000, 300 to 3000, 400 to 2000, 500 to 1000, 600 to 900, or 700 to 1500 micrograms/tumor/month. In other embodiments, the dose may range from approximately 100 to 199, 200 to 399, 400 to 649, 650 to 999, 1000 to 1799, 1800 to 2499, 2500 to 3499, 3500 to 4999, 5000 to 7499, 7500 to 10000, or 10001 to 20000 micrograms/tumor/month.
  • the dose may be at least approximately 100, 200, 400, 650, 1000, 1400, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 7500, 8000, or 20000 micrograms/tumor/month.
  • the effective dose of immunotoxin results in an intratumoral concentration of at least approximately 5, 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 100, 200, 300, 400, or 500 micrograms/cm 3 of the immunotoxin.
  • the resulting intratumoral concentration of immunotoxin is approximately 5 to 500, 10 to 400, 15 to 300, 20 to 200, 25 to 100, 30 to 90, 35 to 80, 40 to 70, 45 to 60, or 50 to 55 micrograms/cm 3 .
  • the resulting intratumoral concentration of immunotoxin is approximately 10 to 15, 16 to 20, 21 to 25, 26 to 30, 31 to 35, 36 to 40, 41 to 45, 46 to 50, 51 to 55, 56 to 60, 61 to 65, 66 to 70, 71 to 75, 76 to 80, 81 to 85, 86 to 90, 91 to 95, 96 to 100, or 100 to 200 micrograms/cm 3 .
  • the effective dose of immunotoxin results in a plasma concentration of less than approximately 0.1, 1, 2.5, 5, 7.5, 10, 15, 20, 30, 40, or 50 micrograms/liter.
  • the resulting circulating concentration of immunotoxin is approximately 0.1 to 50, 1 to 40, 2.5 to 30, 5 to 20, or 7.5 to 10 micrograms/liter.
  • the resulting circulating concentration of immunotoxin is approximately 0.1 to 1 , 1.1 to 2.4, 2.5 to 5, 5.1 to 7.4, 7.5 to 10, 11 to 15, 16 to 20, 21 to 30, 31 to 40, or 41 to 50 micrograms/liter.
  • the effective dose of the immunotoxin is between about 100 and 3000 micrograms/tumor/month, for example approximately 100, 200, 300, 400, 750, or 1000 micrograms/tumor/month, wherein the patient is administered a single dose per day.
  • the single dose is administered approximately every month for approximately 1 , 2, 3, 4, 5, or 6 consecutive months. After this cycle, a subsequent cycle may begin approximately 1, 2, 4, 6, or 12 months later.
  • the treatment regime may include 1 , 2, 3, 4, 5, or 6 cycles, each cycle being spaced apart by approximately
  • the effective dose of the immunotoxin is between about 20 and 1240 micrograms/tumor/day, for example approximately 20, 40, 80, 130, 200, or 280 micrograms/tumor/day or approximately 100, 200, 330, 500, 700, 930, 1240 micrograms/tumor/day, wherein the patient is administered a single dose per day.
  • the single dose is administered approximately every day (one or more days may optionally be skipped) for approximately 1 , 2, 3, 4, 5, 6 or 7 consecutive days. After this cycle, a subsequent cycle may begin approximately 1 , 2, 3, 4, 5, or 6 weeks later.
  • the treatment regime may include 1,
  • the present invention further discloses methods for diagnosing and prognosing cancer.
  • the present invention provides a diagnostic and/or prognostic method of cancer disease in a subject, the method comprises the step of determining the expression level of PVR in a biological sample of said subject using at least one antibody as described herein.
  • biological sample encompasses a variety of sample types obtained from an organism that may be used in a diagnostic or monitoring assay.
  • the term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen, or tissue cultures or cells derived there from and the progeny thereof. Additionally, the term may encompass circulating tumor or other cells.
  • the term specifically encompasses a clinical sample, and further includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, biological fluids including aqueous humour and vitreous for eyes samples, and tissue samples.
  • the term also encompasses samples that have been manipulated in any way after procurement, such as treatment with reagents, solubilisation, or enrichment for certain components. Determining the expression level of PVR can be performed by a labeled anti-PVR antibody as described herein. Determining the expression can be performed, for example, by ELISA.
  • Determining and quantifying methods may be performed in-vitro or ex-vivo according to some embodiments or may be used in diagnosing conditions associated with expression of PVR.
  • the antibody or fragment thereof according to the present invention may be also used to configure screening methods.
  • an enzyme-linked immunosorbent assay (ELISA), or a radioimmunoassay (RIA) can be constructed for measuring levels of secreted or cell- associated polypeptide using the antibodies and methods known in the art.
  • the method for detecting or quantifying the presence of PVR comprises the steps of: i. incubating a sample with an antibody or an antibody fragment thereof as described hereinabove;
  • the method further comprises the steps of: iii. comparing the amount of (ii) to a standard curve obtained from a reference sample containing a known amount of PVR; and
  • the sample is a body fluid.
  • the method is performed in-vitro or ex-vivo.
  • the method of the invention can further comprise the step of comparing said level of expression to a control level.
  • the cell lines used were U-87 MG (ATCC® HTB-14TM) and U-87 MG PVRhi (human PVR transfectant of U-87 MG). Cells were cultivated at 37°C, >95% humidity and 5% CO2 in DMEM supplemented with 10% heat inactivated FCS (media and sera from Sigma-Aldrich). Flow cytometry
  • Flow cytometry was performed using aPVR mAh (clone CD155.16). Cells were incubated on ice for 30 minutes with 0.2 pg of mAh per 100,000 cells. Detection was performed with a secondary goat a-mouse Ab coupled to PE (Jackson ImmunoResearch) for 30 min on ice. Analysis was performed using the FACS-ARIA flow cytometer (BD Biosciences) and CellQuest software.
  • a monoclonal antibody anti-PVR (anti CD 155.16 KAC) that does not block binding of TIGIT to PVR, was generated in KAC mouse.
  • KAC mouse is a genetically engineered mouse strain that expresses BAP (Biotin Accepting Peptide) sequence on constant part of kappa chain and when the fusion is performed with SP2/0-3A4 cell line (Expressing BirA enzyme), the resulted m Ahs are produced as a biotinylated protein.
  • BAP Biotin Accepting Peptide
  • SP2/0-3A4 cell line Expressing BirA enzyme
  • This technology enabled the screen for potential antibody cytotoxic effect using Streptavidin-saporin conjugate (ATS Bio).
  • the amino acid and nucleic acid sequences of the mAh produced by this clone are as following: heavy chain variable region nucleic acid sequence - SEQ ID NO: 1, heavy chain variable region amino acid sequence - SEQ ID NO: 2, light chain variable region nucleic acid sequence - SEQ ID NO: 3, light chain variable region amino acid sequence - SEQ ID NO: 4, CDR sequences (according to RABAT): HC CDR 1 - SEQ ID NO: 5, HC CDR 2 - SEQ ID NO: 6, HC CDR 3 - SEQ ID NO: 7, LC CDR 1 - SEQ ID NO: 8, LC CDR 2 - SEQ ID NO: 9, and LC CDR 3 - SEQ ID NO: 10.
  • the antibody was used for producing a variety of immunoglobulin molecules and immunotoxins, including scFV molecules and recombinant immunotoxins, based on its variable region sequences.
  • Anti-PVR - saporin immunotoxins were tested for potential antitumor activity.
  • the immunotoxins were prepared by mixing biotinylated anti-PVR antibody with StreptAvidin- saporin (SA-saporin, ATS bio, Cat NO: IT-27) in equal molar concentrations.
  • SA-saporin StreptAvidin- saporin
  • the cytotoxicity test was performed using 10000 cells/50 pi in 96 well plates. Stocks containing 1 mM/ml of immunotoxins were made and 100 m ⁇ /well of sample or controls were added to the cells and incubated for 48h at 37°C. The Supernatant was then collected and analyzed using Lonza ToxiLightTM non-destructive Cytotoxicity BioAssay Kit.
  • the adenylate kinase release indicates cell death.
  • the antibodies used were: biotinylated (XCD155.16 antibody (aCDl55T6 b+) and F(ab’)2 fragments (Fab2) derived from (XCD155.16 antibody (aCDl55. 16 b+ Fab2).
  • F(ab’)2 antibodies were prepared by pepsin enzyme digestion of the monoclonal antibody anti CD155.16 (using regular protocol, Current protocol in Immunology) and purified with HiTrap protein L purification columns (product no: 29048665) on ACTA prime.
  • Immunotoxin was designed as a fusion protein containing the first 389 amino acid of diphtheria toxin and anti CD 155 scFV linked by Ser-Gly linker.
  • the amino acid sequence of the immunotoxin set forth in SEQ ID NO:l9 and includes the DT (first 389 AA), a Ser-Gly linker, scFv heavy chain, scFv light chain, a short linker and HIS tag.
  • Example 4 Antitumor effect of aPVR-DT in mouse model.
  • the efficacy of the aPVR-DT was determined in vivo in animal model.
  • U-87 MG PVRhi cells (1X10 per mouse) were injected subcutaneously into NSG mice (lacking NK, B and T cells). After establishment of tumor, aPVR-DT or control aPVR mAh (clone CD155.16) were injected i.p. or i.t. (50 pg/mouse) on days 3, 6, 9, 12 and 16. Where indicated, PBS alone was used as negative control. Tumor growth was monitored daily and measured by caliper. As shown in Figures 5 and 6, treatment with aPVR-DT leaded to tumor regression which is dependent on PVR expression. Example 5. PVR expression on human GBM samples.
  • CDR Complementary determining region
  • the CDR segments were identified using different algorithm methods.
  • the CDRs may be identified using IMGT algorithm (Lefranc et al., 1999, Nucleic Acids Research, 27, 209-212); RABAT algorithm (Wu TT and Rabat E.A., 1970, J. Exp. Med. 132, 211-250); Chothia (Chothia and Lesk, J Mol Biol. 1987 Aug 20;196(4):901-17); or enhanced
  • Table 1 summarizes the determined CDR sequences using two alternative methods as well as the minimal consensus sequence and combined sequence of sequences identified using both methods.

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Abstract

The present invention provides antibody molecules that recognize poliovirus receptor (PVR) and immunotoxins thereof comprising a toxin for treating cancer. The present invention further provides pharmaceutical compositions comprising the immunotoxins and methods for their use in treating cancer such as glioblastoma.

Description

IMMUNOTOXINS FOR TREATING CANCER
FIELD OF THE INVENTION
The invention is in the field of immunotherapy and relates to antibodies that bind to a protein on the cancer cell and immunotoxins thereof, for use in treating cancer. In particular, the present invention provides immunotoxins comprising anti-poliovirus receptor antibodies conjugated with a toxin, polynucleotide sequences encoding these antibodies and immunotoxins and methods of treating and diagnosing cancer, such as glioblastoma.
BACKGROUND OF THE INVENTION
Immunotoxins are a class of cancer therapeutics that contains a cytotoxic agent fused to a targeting moiety, typically an antibody that binds antigens present on cancer cells. Various toxic agents from different sources are used in immunotoxins, including bacterial, plant and human origin cytotoxic elements. Although bacterial and plant-derived toxins are commonly used in immunotoxins due to their high toxicity, their immunogenicity for human restricted their application in cancer therapy.
Glioblastoma multiforme (GBM) is the most dangerous type of brain tumors. It affects an enormous number of patients every year and the survival is approximately 14 to 16 months. GBM is driven by complex signaling pathways and considered as a most challenging to treat. The standard treatment includes surgical resection followed by radiation therapy and administration of the drug temozol amide (Temodal). Complete surgical excision is made impossible by the tumor's ability to disseminate outside its visible borders. Only 4 medicines are currently approved for GBM treatment by the US Food and Drug Administration and neither improves the survival for more than several months (Readron et al., 2013, Expert Rev Vaccines, 12(6): 597-615).
Poliovirus receptor (PVR), also termed CD155, is a transmembrane glycoprotein involved in mediating cell adhesion to extracellular matrix molecules. It was previously described as a tumor antigen and as a potential target for therapeutic intervention as its expression is up-regulated in neuroectodermal cancers, including GBM, medulloblastoma, and colorectal carcinoma (Solecki et al., J. Biol. Chem. 2002, 277: 25697-700) as well as in pancreatic cancer (Nishiwada et al., Anticancer Res. 2015, 35(4): 2287-97). PVR is also known to enhance the serum-induced activation of the Ras-Raf-MEK-ERK signaling, up- regulating cyclins D2 and E, and down-regulated p27Kipl, eventually shortening the period of the G0/G1 phase of the cell cycle (Kakunaga 2004, J. Biological Chemistry, 279, 36419- 36425). For that reason, blocking of PVR is anticipated to reduce viability of tumor cells. T cell immu noreceptor with Ig and ITIM domains (TIGIT) is a co-inhibitory molecule expressed on various immune cells including T cells and Natural Killer cells (NK cells). TIGIT binds with high affinity to PVR. The PVR- TIGIT interaction inhibits cytotoxic activity of NK cells and T cells activity.
PVR involvement in metastasis was demonstrated by injecting cancer cells to the tail of mice and measuring metastasis to the lungs. It has been shown that the upregulated PVR in cancer cells transinteracts with its counter-receptor TIGIT in platelets, and that this trans interaction enhances the metastasis of the cancer cells to the lungs (Morimoto et al., Oncogene, 2008, 27, 264—273).
PVR has also a critical role in angiogenesis and is suggested to regulate the VEGF- induced angiogenesis by controlling the interaction of VEGFR2 with integrin a(n)b(3), and the VEGFR2-mediated Rapl-Akt signaling pathway (Kinugasa et al., 2012, Circ Res. 2012, 110,5,716-26).
PCT Application Publication No. WO 2004/074324 discloses molecules that specifically bind to PVR or to a derivative thereof. The molecules have the ability to modulate a receptor mediated adhesion, trafficking and/or invasion behavior of a cell expressing the PVR or any derivative thereof.
PCT Application Publication No. WO 2013/184912 discloses agents that specifically bind the extracellular domain of one or more cell adhesion molecules of the immunoglobulin superfamily (IgCAM), wherein the IgCAM is inter alia PVR. PCT Application Publication No. WO 2017/021526 discloses inhibitors against CD112
(Nectin-2, PVRL2), CD155 (PVR), Galectin-9, TIM-3 and TIGIT for use in treating blood- borne cancers, in particular acute myeloid leukemia (AML).
U.S. Patent Application Publication No. 20070041985 discloses molecules specifically binding to at least one intra- or extracellular domain of the PVR or any derivative thereof, wherein the molecule has the ability to modulate a receptor mediated adhesion, trafficking and/or invasion behavior of a cell expressing the PVR or any derivative thereof.
U.S. Patent Application Publication No. 20090215175 provides molecules (e.g. small chemical compounds, oligonucleotides, polypeptides, antibodies, and antibody fragments) which modulate the PVR functions necessary for adhesion, trafficking, invasion and/or metastatic potential of cells. The molecules can be used for the treatment of cells having a metastatic potential, metastasis and cancer.
PCT Application Publication No. WO 2006/124667 discloses modulation of the protein zB7Rl (TIGIT) by monoclonal antibodies that block TIGIT binding to its ligand PVR.
PCT Application Publication No. WO 2017/149538 discloses monoclonal antibodies that recognise PVR and inhbit its binding to TIGIT.
There is an unmet need to provide additional and more effective, specific, safe and/or stable agents that alone or in combination with other agents, utilize the PVR ligand to target and kill tumor cells by localizing toxins to the cells.
SUMMARY OF THE INVENTION
The present invention provides antibody molecules and fragments that recognize the poliovirus receptor (PVR), immu notoxins comprising them and toxins, that kill tumor cells. The anti-PVR antibodies disclosed herein are characterized by having unique sets of complementarity-determining regions (CDR) sequences, high affinity and high specificity to PVR and are useful in targeting toxins, radioactive molecules, or identifiable moieties to target cells, tissues and biological samples. The immunotoxins disclosed herein are useful as stand-alone therapy and in combination with other anti-cancer agents.
It is now shown that immunotoxins of anti PVR immunoglobulins injected to mice in a tumor in vivo model, are effective in tumor regression and mice survival. It is further shown that the immunotoxins disclosed herein bind to glioma cancer cells and kill them. This property of specifically targeting a toxin moiety to cancer cells enables administration of lower doses with fewer side effects. Advantageously, the anti-PVR Abs according to some embodiments of the invention, were found to be highly efficient in targeting two different toxins, saporin and diphtheria toxin, to PVR expressing tumor cell lines. A cytotoxicity bioassay was used to show that the toxins were capable of killing glioma cells. Without wishing to be bound to any mechanism of action, it is suggested that this activity results from the ability of the antibody to bind the PVR expressing tumor cells and enable the internalization of the toxin into the tumor cell. Advantageously, the anti- PVR mAbs according to the invention do not block TIGIT-
PVR interaction. This property may prevent undesired side effects such as increasing systemic immune response.
According to one aspect, the present invention provides an antibody which specifically binds to poliovirus receptor (PVR), or an antibody fragment thereof comprising at least the antigen binding portion, wherein the antibody or antibody fragment, comprising three complementarity determining regions (CDRs) of a heavy-chain variable region set forth in SEQ ID NO: 2 and three CDRs of a light-chain variable region set forth in SEQ ID NO: 4, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence. According to some embodiments, the antibody comprises three complementarity determining regions (CDRs) of a heavy-chain variable region set forth in SEQ ID NO: 2 and three CDRs of a light-chain variable region set forth in SEQ ID NO: 4, or an analog or derivative thereof having at least 95% sequence identity with said antibody or fragment sequence. There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard unequivocal method. Determination of CDR sequences from antibody heavy and light chain variable regions can be made according to any method known in the art, including but not limited to the methods known as RABAT, Chothia, Martin and IMGT. A selected set of CDRs may include sequences identified by more than one method, namely, some CDR sequences may be determined using RABAT and some using IMGT, for example.
According to some embodiments, the antibody or fragment comprises the CDR sequences contained in the variable regions of a single-chain monoclonal antibody (scFv) denoted PVR.16 (pOPE scFv#7). According to some embodiments, the antibody fragment comprises at least one Fab. According to some specific embodiment, the antibody fragment is selected from the group consisting of Fab, is F(ab’)2.
According to some embodiments, the antibody or the antibody fragment comprises heavy-chain CDR1 comprising the sequence SYW. According to some embodiments, the antibody or the antibody fragment comprises heavy-chain CDR2 comprising the sequence IF1PNSGST (SEQ ID NO: 12). According to some embodiments, the antibody or the antibody fragment comprises heavy-chain CDR3 comprising the sequence EGYVYYAMDY (SEQ ID NO: 7). According to some embodiments, the antibody or antibody fragment comprises heavy- chain CDR1 comprising the sequence SYWMF1 (SEQ ID NO: 5). According to some embodiments, the antibody or the antibody fragment comprises heavy-chain CDR1 comprising the sequence GYSFTSYW (SEQ ID NO: 11). According to some embodiments, the antibody or the antibody fragment comprises heavy-chain CDR2 comprising the sequence IIF1PNSGSTIYNEKFK (SEQ ID NO: 6). According to some embodiments, the antibody or the antibody fragment comprises heavy-chain CDR3 comprising the sequence GREGYVYY AMD Y (SEQ ID NO: 13).
According to certain embodiments, the antibody or the antibody fragment comprises: (i) F1C CDR1 comprising the sequence SYW; (ii) F1C CDR2 comprising the sequence: IF1PNSGST (SEQ ID NO: 12); and (iii) F1C CDR3 comprising the sequence: EGYVYYAMDY (SEQ ID NO: 7).
According to some embodiments, the antibody or the antibody fragment comprises light-chain CDR1 comprising the sequence QSLLYSGDQRNY (SEQ ID NO: 14). According to some embodiments, the antibody or the antibody fragment comprises light- chain CDR2 comprising the sequence WASX (SEQ ID NO: 15, wherein X is T or is absent). According to some embodiments, the antibody or the antibody fragment comprises light- chain CDR3 comprising the sequence QQYSRYPYT (SEQ ID NO: 16).
According to certain embodiments, the antibody or the antibody fragment comprises: (i) LC CDR1 comprising the sequence QSLLYSGDQRNY (SEQ ID NO: 14); (ii) LC CDR2 comprising the sequence WAS; and (hi) LC CDR3 comprising the sequence: QQYSRYP (SEQ ID NO: 16).
According to some embodiments the antibody or fragment comprises heavy chain CDR1 sequence comprising the sequence SYW, heavy chain CDR2 comprising the sequence: IHPNSGST (SEQ ID NO: 12), heavy chain CDR3 comprising the sequence: EGYVYYAMDY (SEQ ID NO: 7), light chain CDR1 comprising the sequence: QSLLYSGDQRNY (SEQ ID NO: 14), light chain CDR2 comprising the sequence WAS, and light chain CDR3 comprising the sequence: QQYSRYPYT (SEQ ID NO: 16), or analogs thereof comprising no more than 5% amino acid substitution, deletion and/or insertion in the hypervariable region (HVR) sequence.
According to some embodiments, HC CDR1 comprises the sequence SYW. According to additional embodiments, LC CDR2 sequence is WAS.
According to some embodiments the antibody or fragment comprises a set of six CDR sequences consisting of: i. heavy chain CDR1 having a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 11 , SEQ ID NO: 17, and SEQ ID NO: 18, ii. heavy chain CDR2 having a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 12,
iii. heavy chain CDR3 having a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13,
iv. light chain CDR1 having a sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 14,
v. light chain CDR2 having a sequence selected from the group consisting of SEQ ID NO: 9, and SEQ ID NO 15, and
vi. light chain CDR3 having a sequence selected from the group consisting of SEQ ID NO: 10, and SEQ ID NO: 16.
According to some specific embodiments the antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. According to other specific embodiments the antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
According to some embodiments, the antibody or fragment thereof comprises heavy chain variable region set forth in SEQ ID NO: 2, or an analog or derivative thereof having at least 90% or at least 95% sequence identity with the heavy chain variable region sequence.
According to some embodiments, the antibody or fragment thereof comprises light chain variable region set forth in SEQ ID NO: 4, or an analog thereof having at least 90%, or at least 95% sequence identity with the light chain variable region sequence. According to some embodiments, the antibody or fragment thereof comprises a heavy chain variable region having a sequence set forth in SEQ ID NO: 2, and a light chain variable region having a sequence set forth in SEQ ID NO: 4, or an analog thereof having at least 95% sequence identity with the light and/or heavy chain sequence. According to a specific embodiment, the antibody or fragment thereof comprises a heavy chain variable region having a sequence set forth in SEQ ID NO: 2, and a light chain variable region having a sequence set forth in SEQ ID NO: 4.
The invention also encompasses antibody or antibody fragment capable of binding to an epitope within the human PVR protein to which pOPE scFv#7 single chain antibody binds.
Analogs and derivatives of the antibody, and the antibody fragments described above, are also within the scope of the invention.
According to some embodiments, the antibody or antibody fragment analog have at least 95% sequence identity with the hypervariable region (HVR) of the reference antibody sequence.
According to certain embodiments, the analog or derivative of the antibody or fragment thereof has at least 96, 97, 98 or 99% sequence identity with a variable region of the reference antibody sequence. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the analog comprises no more than one amino acid substitution, deletion or addition to one or more CDR sequences of the HVR, namely, any one of the CDR sequences set forth in SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, and 18. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the amino acid substitution is a conservative substitution.
According to some embodiments, the antibody or antibody fragment comprises a HVR having light and heavy chain regions defined above, in which 1 , 2, 3, 4, or 5 amino acids were substituted, deleted and/or added. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the antibody or antibody fragment comprises a HVR having light and heavy chain regions defined above, in which one amino acid was substituted. According to specific embodiments, the antibody or antibody fragment comprises a CDR as defined above, in which one amino acid was substituted.
The present invention thus provides an antibody that specifically binds the human protein PVR, or a binding fragment thereof, wherein said antibody or fragment comprises a set of six CDR sequences wherein the set is selected from the group consisting of:
1. SEQ ID NOs. 5, 6, 7, 8, 9 and 10;
ii. SEQ ID NOs. 11 , 12, 13, 14, 15 and 16;
iii. SEQ ID NOs. 17, 6, 7, 8, 9 and 10; and
IV. SEQ ID NOs. 18, 12, 13, 14, 15 and 16. The present invention also provides monoclonal antibodies and binding fragments thereof, comprising a heavy chain and a hght chain, wherein said chains comprises a set of a heavy chain variable region sequence and a hght chain variable region sequence, said set comprises the sequences set forth in SEQ ID NO: 2 and 4.
According to some embodiments, the antibody is an isolated monoclonal antibody. According to some embodiments, the antibody is single-chain variable fragment (scFv).
According to some embodiments, the scFv comprises a sequence according to SEQ ID NO: 20.
According to other embodiments, the antibody is selected from the group consisting of: chimeric antibody, and an antibody fragment comprising at least the antigen-binding portion of an antibody. According to specific embodiments, the antibody is a chimeric antibody comprising human constant region. According to a specific embodiment, the antibody fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fd, Fd', Fv, dAb, isolated CDR region, single chain antibody (scab), "diabodies", and "linear antibodies". Each possibility represents a separate embodiment of the present invention.
According to some embodiments, the antibody fragment is F(ab’)2.
According to some embodiments, a diagnostic composition comprising the antibody or fragment thereof as described above is provided.
According to some embodiments, a conjugate comprising the antibody or fragment thereof as described above is provided.
According to some embodiments, the conjugate comprises an anti PVR antibody, fragment or scFv and at least one moiety selected from the group consisting of: a drug moiety, a toxin and a detectable probe.
According to some embodiments, the conjugate comprises a drug moiety. According to specific embodiments, the conjugate comprises a toxin.
Polynucleotide sequences encoding antibodies, having high specificity for PVR, as well as vectors and host cells carrying these polynucleotide sequences, are provided according to another aspect of the present invention.
According to some embodiments, polynucleotide sequences encoding the amino acid sequences of F1C variable region and light LC variable region described above are provided.
According to some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain capable of binding to an epitope within the human PVR protein to which binds an antibody having a heavy chain variable region of SEQ ID NO: 2 and a light chain variable region of SEQ ID NO: 4. According to some embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.
According to some embodiments, the polynucleotide sequence according to the invention encodes an antibody or antibody fragment or chain comprising the six CDR sequences: (i) heavy chain CDR1 having the sequence set for the in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17 or SEQ ID NO: 18, (ii) heavy chain CDR2 having the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 12, (iii) heavy chain CDR3 having the sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 13, (iv) light chain CDR1 having the sequence set forth in SEQ ID NO: 8 or SEQ ID NO 14, (v) light chain CDR2 having the sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 15, and (vi) light chain CDR3 having the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16. Each combination of CDRs set is a separate embodiment.
According to some embodiments, the polynucleotide sequences defined above encodes a molecule selected from the group consisting of: an antibody, an antibody fragment comprising at least an antigen-binding portion, and an antibody conjugate comprising said antibody or antibody fragment. Each possibility represents a separate embodiment of the present invention.
According to some specific embodiments, the polynucleotide sequence encodes a scFv comprising a sequence set forth in SEQ ID NO: 20.
In a further aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one antibody chain or fragment thereof according to the present invention. According to some embodiments the nucleic acid construct is a plasmid. According to some embodiments the plasmid comprises a polynucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.
According to another aspect the present invention provides a hybridoma cell capable of producing a monoclonal antibody or an antibody fragment comprising the specific CDR sequences and/or specific heavy and light chain variable regions defined above. According to some embodiments, a hybridoma cell is provided comprising at least one polynucleotide sequence disclosed above.
According to some embodiments, the antibody or fragment thereof according to the invention is attached to a cytotoxic moiety. According to an additional aspect, the present invention provides an immunotoxin comprising the antibody or fragments thereof as described hereinabove and a toxin.
According to some embodiments, the immunotoxin comprises a scFv sequence having a CDR set of six CDRs wherein: F1C CDR1 is selected from SYWMF1 (SEQ ID NO: 5) and GYSFTSYW (SEQ ID NO: 11); HC CDR2 is selected from IIHPNSGSTIYNEKFK (SEQ ID NO: 6) and IHPNSGST (SEQ ID NO: 12); HC CDR3 is selected from EGYVYYAMDY (SEQ ID NO: 7) and GREGYVY Y AMD Y (SEQ ID NO: 13); LC CDR1 is selected from KS SQSLLYSGDQRNYLA (SEQ ID NO: 8) and QSLLYSGDQRNY (SEQ ID NO: 14); LC CDR2 is selected from the group consisting of: WASTRES (SEQ ID NO: 9) and WAS; and LC CDR3 is selected from QQYYNYPRT (SEQ ID NO: 10) and QQYYNYP (SEQ ID NO: 16).
According to some embodiments, the immunotoxin comprises the scFv sequence set forth in SEQ ID No: 20.
The antibody may be linked to the toxin by, for example, chemical linking using a crosslinker and/or a peptide bond.
According to some embodiments, the toxin is a peptide-based toxin. According to specific embodiments, the antibody is connected to the toxin through a linker. According to some embodiments, the linker comprises a stretch of 1-10 amino acid residues.
According to some embodiments, the toxin is selected from the group consisting of diphtheria toxin or a subunit thereof, saporin, ricin A, abrin, pseudomonas exotoxin or a portion thereof, restrictocin and gelonin. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the toxin is a diphtheria toxin or an active fragment thereof. According to some specific embodiments, the immunotoxin comprises a diphtheria toxin fragment is which domain R that provides binding to diphtheria toxin receptor, is absent.
According to some embodiments, the immunotoxin comprises a scFv molecule according to SEQ ID No: 20, a diphtheria toxin sequence and optionally at least one amino acid linker. According to yet other embodiments, the immunotoxin sequence further comprises a His-tag sequence.
According to some embodiments, the immunotoxin comprises a scFv comprising a sequence set forth in SEQ ID No: 19 or an analog thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the immunotoxin comprises scFv comprising a sequence set forth in SEQ ID No: 19.
According to some embodiments, the toxin is saporin.
According to additional embodiments, the toxin is selected from the group consisting of a saporin, anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC- 1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, and a PDB, or an analog, derivative, or prodrug of any thereof.
According to an aspect, the present invention provides an immunotoxin comprising a diphtheria toxin and an scFV antibody comprising three CDRs of a heavy-chain variable region set forth in SEQ ID NO: 2 and three CDRs of a light-chain variable region set forth in SEQ ID NO: 4, or an analog or derivative thereof having at least 95% sequence identity with said antibody or fragment sequence.
According to some embodiments, the diphtheria toxin and the scFV are linked with a short peptide of 1-5 amino acid residues. According to certain embodiments, the diphtheria toxin and scFV are linked by a Ser-Gly linker.
According to an aspect, the present invention provides an immunotoxin comprising saporin linked to an antibody comprising three CDRs of a heavy-chain variable region set forth in SEQ ID NO: 2 and three CDRs of a light-chain variable region set forth in SEQ ID NO: 4, or an analog or derivative thereof having at least 90% or at least 95% sequence identity with said antibody or fragment sequence. The antibody is as described hereinabove.
According to some embodiments, the immunotoxin or antibody does not block TIGIT- PVR interaction.
The present invention provides, according to another aspect, a pharmaceutical composition comprising as an active ingredient, at least one immunotoxin or antibody as described hereinabove, and optionally at least one pharmaceutical acceptable excipient, diluent, salt or carrier.
According to some embodiments, the pharmaceutical composition comprises an immunotoxin comprising an antibody or fragment thereof which is capable of binding to an epitope within the human PVR protein to which binds an antibody herein identified as PVR.16 (pOPE scFv#7), having a heavy chain variable region of SEQ ID NO: 2 and a light chain variable region of SEQ ID NO: 4.
According to some embodiments, the pharmaceutical composition comprises an immunotoxin comprising an antibody or antibody fragment thereof comprising the six CDRs: i. heavy chain CDR1 having a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 11 , SEQ ID NO: 17, and SEQ ID NO: 18, ii. heavy chain CDR2 having a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 12,
iii. heavy chain CDR3 having a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13,
iv. light chain CDR1 having a sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 14,
v. light chain CDR2 having a sequence selected from the group consisting of SEQ ID NO: 9, and SEQ ID NO 15, and
vi. light chain CDR3 having a sequence selected from the group consisting of SEQ ID NO: 10, and SEQ ID NO: 16.
Each combination of CDRs set represents a separate embodiment of the invention.
According to some embodiments, the pharmaceutical composition according to the present invention is for use in treating cancer. The cancer can be any cancer that expresses PVR. According to some embodiments, the cancer overexpresses PVR.
According to some embodiments, the cancer is a malignant cancer. According to some embodiments, the immunotoxin of the invention reduces or eliminates the tumor. According to other embodiments, the cancer is a metastatic cancer. According to some embodiments, the pharmaceutical composition according to the present invention is for use in inhibiting formation or distribution of metastases or reducing the total number of metastases in a subject.
According to some embodiments of the invention, the cancer is selected from the group consisting of a brain cancer, melanoma, a breast cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, a colon cancer, a cervical cancer, a kidney cancer, a lung cancer, a thyroid cancer, a prostate cancer, a renal cancer, a throat cancer, a laryngeal carcinoma, a bladder cancer, a hepatic cancer, a fibrosarcoma, an endometrial cells cancer, sarcoma, a myeloid, a leukemia and a lymphoma. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the cancer is a solid cancer. According to some specific embodiments, the solid cancer is selected from the group consisting of brain, melanoma (skin), lung, colon, breast, uterine, and renal cancer. According to certain embodiments, the cancer is glioblastoma.
According to yet other embodiments, the cancer that over-expresses PVR is selected from the group consisting of: GBM, medulloblastoma, colorectal carcinoma and pancreatic cancer.
According to yet another aspect, the present invention provides a method of treating cancer comprising administering to a subject in need thereof an effective amount of the immunotoxin as described hereinabove.
According to some embodiments, the immunotoxin is administered directly to the cancer site. According to some embodiments, the pharmaceutical composition is administered directly to the brain. According to some embodiments, the pharmaceutical composition is administered intrathecally.
According to some embodiments, the subject is human.
According to specific embodiments, the method comprises administering directly to the surgical site an effective amount of the immunotoxin before, during, and/or after surgery for cancer. According to some embodiments, the treating comprises decreasing the size, growth rate, invasiveness, malignancy grade and/or risk of recurrence of a tumor associated with the cancer.
According to some embodiments of the invention, the therapeutically effective amount results in a decrease in tumor size or in the number of metastases in the subject.
According to some embodiments, the method of treating cancer comprises administering or performing at least one additional anti-cancer therapy. According to certain embodiments, the additional anticancer therapy is surgery, chemotherapy, radiotherapy, or immunother ap y. According to some embodiments, the immunotoxin is co-administered, concurrently administered, and/or sequentially administered with one or more other anti-cancer agents, and/or in conjunction with radiation or surgery.
According to some embodiments, the additional anti-cancer agent is selected from the group consisting of: immune-modulator, activated lymphocyte cell, kinase inhibitor and chemotherapeutic agent.
According to other embodiments, the immune-modulator is an antibody, antibody fragment or antibody conjugate that binds to an antigen other than human PVR.
According to some embodiments of the invention, the use further comprises the use of an agent that downregulates the activity or expression of an immune inhibitory receptor. According to some embodiments of the invention, the immune inhibitory receptor is selected from the group consisting of PD-l, TIGIT, DNAM-l, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55, LAIR1 , SIGLEC10, and 2B4. Each possibility represents a separate embodiment of the invention.
The present invention further comprises, according to another aspect, a method of determining or quantifying the expression or presence of PVR, the method comprising contacting a biological sample with an antibody or antibody fragment, and measuring the level of complex formation, wherein the antibody or antibody fragment comprises the complementarity determining regions (CDRs) of: (i) heavy chain CDR1 having the sequence set for the in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17 or SEQ ID NO: 18, (ii) heavy chain CDR2 having the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 12, (iii) heavy chain CDR3 having the sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 13, (iv) light chain CDR1 having the sequence set forth in SEQ ID NO: 8 or SEQ ID NO 14, (v) light chain CDR2 having the sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 15, and (vi) light chain CDR3 having the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16.
Determining and quantifying methods may be performed in-vitro or ex-vivo according to some embodiments or may be used in diagnosing conditions associated with expression of PVR. The antibody or fragment thereof according to the present invention may be also used to configure screening methods. For example, an enzyme-linked immunosorbent assay (ELISA), or a radioimmunoassay (RIA) can be constructed for measuring levels of secreted or cell- associated polypeptide using the antibodies and methods known in the art.
According to some particular embodiments the sample is a body fluid. According to other embodiments, the sample is a tissue sample or a biopsy.
According to some embodiments, the method is performed in-vitro or ex-vivo. A kit for measuring the expression of PVR in biological sample is also provided comprising at least one antibody or antibody fragment comprising the complementarity determining regions (CDRs) of the antibody or fragment thereof as described hereinabove.
According to an aspect, the present invention provides a kit for detecting cancer, the diagnostic kit comprises an antibody of fragment thereof as disclosed herein. According to some embodiments, the invention provides a method of diagnosing, assessing the severity or staging of a cancer comprising determining the expression or activity of PVR in a sample from a subject using an antibody according to the present invention or a fragment or conjugate thereof, and comparing the expression or activity of PVR to a reference amount of PVR expression or activity. Said reference amount may be obtained from a sample taken from a normal subject, from the same subject while being in a different stage of the disease or is determined from clinical data of a large population of subjects.
Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-1B. PVR expression on U-87 MG (Fig. 1 A) and U-87 MG PVRhi (Fig. 1 B) cell lines. Fluman glioblastoma cells U-87 MG were transfected to express high levels of hPVR (U-87 MG PVRhi). To determine level of PVR expression on indicated cell lines, cells were stained with aPVR antibody (clone CD155.16) or control isotype mAh and analyzed by flow cytometry.
Figure 2. Efficacy of antiCDl55-saporin in killing PVR expressing tumor cells. U87MG glioma cell line and U87PVRhi were treated with different anti-CDl55.l6 KAC and SA- saporin mixtures. Puromycin was used as a positive control (100 % killing). Release of adenylate cyclase was measured to determine the amount of cell death. Experiment was performed using ToxiLight™ non-destructive Cytotoxicity BioAssay Kit, Lonza. aCDl55. l6 b+ designates biotinylated aCDl55.l6 antibody; aCDl55.l6b + Fab2 designates F(ab’)2 fragments derived from aCDl55.l6 antibody; amouse b+ designates goat anti mouse biotinylated antibody, SA- saporin designates streptavidin saporin conjugate.
Figures 3. Schematic presentation of aPVR-DT immunotoxin. PVR-specific antibody fragments (scFv) are fused to diphtheria toxin subunits C and T to form new molecule: PVR- DT immunotoxin. Domain R which provides binding to diphtheria toxin receptor is deleted from the final construct.
Figure 4. Cytotoxic effect of aPVR-DT on human GBM cells. Human glioblastoma transfectants (U-87 MG PVRhi) were exposed to different concentration of aPVR-DT or parental, control aPVR antibody (clone CD 155.16) for 48h in vitro. Cytotoxic effect was determined by ToxyLight kit (Lonza) according to manufacturer’s instructions.
Figures 5A-5B. Antitumor effect of aPVR-DT in mouse model. NSG mice were inoculated h
with 10 human glioblastoma transfectants U-87 MG PVRhi subcutaneously at right flank. On days 3, 6, 9, 12 and 16 mice received 100 pg (50 pg i.p. + 50 pg i.t.) of aPVR-DT or control aPVR mAh (clone CD155.16). Tumor growth (Fig. 5A) and mice survival (Fig. 5B) were monitored.
Figures 6A-6B. Susceptibility to aPVR-DT is related to PVR expression. NSG mice were injected with 107 human glioblastoma cells U-87 MG PVRhi (Fig. 6A) or U-87 MG (Fig. 6B) transfectants subcutaneously at right flank. On days 3, 6, 9, 12 and 16 mice received 100 pg (50 pg i.p. + 50 pg i.t.) of aPVR-DT or PBS and tumor growth was monitored.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides antibodies specific to human poliovirus receptor (PVR) and immunotoxins thereof. The immunotoxin comprises the anti PVR antibody that binds specifically to tumor cells to deliver a conjugated toxin for efficient tumor cell- killing.
According to an aspect, the present invention provides an anti-PVR antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins. " The term "immunotoxins" as used herein refers to complexes of antibodies or their fragments with toxins, chemically bound. The antibody is directed against structures on the tumor cell surface.
As disclosed herein the PVR molecules presented on the cancer cells serve as an antigen for the antibody portion of the immunotoxin. The term "antigen" as used herein refers to a molecule or a portion of a molecule capable of eliciting antibody formation and being specifically bound by an antibody. An antigen may have one or more than one epitope. The specific binding referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
The term "PVR" as used herein refers to the poliovirus receptor, also known as CD155 (cluster of differentiation 155). The PVR is a transmembrane glycoprotein with an N-terminal signal sequence, three extracellular immunoglobulin (Ig)-like domains, a transmembrane domain and a cytoplasmic tail. It has a molecular size of approximately 80 kDa and a structure composed of three Ig-like domains, specifically an outermost V-like domain followed by two C2-like domains. An exemplary human PVR according to the invention is set forth in GenBank accession numbers: NP_00l 129240.1 , NP_00l 129241.1, NP_00l 129242.2 and NP_006496.4. These poliovirus receptors share the sequence of the extracellular domain and therefore can be targeted by the affinity binding moiety of the invention.
A cytotoxin or cytotoxic agent includes any agent that is detrimental (e.g., kills) to cells. Non- limiting examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, anti metabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti -mitotic agents (e.g., vincristine and vinblastine). Other examples of therapeutic cytotoxins that can be conjugated to an antibody of the invention include calicheamicin and duocarmycin.
An antibody of the present invention can be conjugated to a radioisotope, e.g., radioactive iodine, to generate cytotoxic radiopharmaceuticals for beating an overexpressed PVR-related disorder, such as a cancer.
In additional non-limiting embodiments, the toxin portion of an immunotoxin of the invention may comprise an alkylating agent including, without limitation, Asaley NSC
167780, AZQ NSC 182986, BCNU NSC 409962, Busulfan NSC 750, carboxyphthalatoplatinum NSC 271674, CBDCA NSC 241240, CCNU NSC 79037, CHIP
NSC 256927, chlorambucil NSC 3088, chlorozotocin NSC 178248, cis-platinum NSC
119875, clomesone NSC 338947, cyanomorpholinodoxorubicin NSC 357704, cyclodisone
NSC 348948, dianhydrogalactitol NSC 132313, fluorodopan NSC 73754, hepsulfam NSC
329680, hycanthone NSC 142982, melphalan NSC 8806, methyl CCNU NSC 95441, mitomycin C NSC 26980, mitozolamide NSC 353451, nitrogen mustard NSC 762, PCNU
NSC 95466, piperazine NSC 344007, piperazinedione NSC 135758, pipobroman NSC 25154, porfiromycin NSC 56410, spirohydantoin mustard NSC 172112, teroxirone NSC 296934, tetraplatin NSC 363812, thio-tepa NSC 6396, triethylenemelamine NSC 9706, uracil nitrogen mustard NSC 34462, and Yoshi-864 NSC 102627. In other nonlimiting embodiments, the toxin portion of an immunotoxin of the invention may comprise an antimitotic agent including, without limitation, allocolchicine NSC 406042, Halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC 376128 (NG-auristatin derived), maytansine NSC 153858, rhizoxin NSC 332598, taxol NSC 125973, taxol derivative NSC 608832, thiocolchicine NSC 361792, trityl cysteine NSC 83265, vinblastine sulfate NSC 49842, and vincristine sulfate NSC 67574.
In additional non-limiting embodiments, the toxin portion of an immunotoxin of the invention may comprise a topoisomerase I inhibitor including, without limitation, camptothecin NSC 94600, camptothecin, Na salt NSC 100880, aminocamptothecin NSC 603071, camptothecin derivative NSC 95382, camptothecin derivative NSC 107124, camptothecin derivative NSC 643833, camptothecin derivative NSC 629971 , camptothecin derivative NSC 295500, camptothecin derivative NSC 249910, camptothecin derivative NSC 606985, camptothecin derivative NSC 374028, camptothecin derivative NSC 176323, camptothecin derivative NSC 295501 , camptothecin derivative NSC 606172, camptothecin derivative NSC 606173, camptothecin derivative NSC 610458, camptothecin derivative NSC 618939, camptothecin derivative NSC 610457, camptothecin derivative NSC 610459, camptothecin derivative NSC 606499, camptothecin 20 derivative NSC 610456, camptothecin derivative NSC 364830, camptothecin derivative NSC 606497, and morpholinodoxorubicin NSC 354646.
In additional non-limiting embodiments, the toxin portion of an immunotoxin of the invention may comprise an topoisomerase II inhibitor including, without limitation, doxorubicin NSC 123127, amonafide NSC 308847, m-AMSA NSC 249992, anthrapyrazole derivative NSC 355644, pyrazoloacridine NSC 366140, bisantrene HCL NSC 337766, daunorubicin NSC 82151, deoxydoxorubicin NSC 267469, mitoxantrone NSC 301739, menogaril NSC 269148, N,N-dibenzyl daunomycin NSC 268242, oxanthrazole NSC 349174, rubidazone NSC 164011 , VM-26 NSC 122819, and VP-16 NSC 141540.
In additional non-limiting embodiments, the toxin portion of an immunotoxin of the invention may comprise an RNA or DNA antimetabolite including, without limitation, L- alanosine (NSC 153353), 5-azacytidine (NSC 102816), 5-fluorouracil (NSC 19893), acivicin (NSC 163501), aminopterin derivative (NSC 132483), aminopterin derivative (NSC 184692), aminopterin derivative (NSC 134033), an antifol (NSC 633713), an antifol NSC (623017), Baker's soluble antifol (NSC 139105), dichlorallyl lawsone (NSC 126771), brequinar (NSC 368390), ftorafur (pro-drag) (NSC 148958), 5,6-dihydro-5-azacytidine (NSC 264880), methotrexate (NSC 740), methotrexate derivative (NSC 174121), N-(phosphonoacetyl)-L- aspartate (PALA) (NSC 224131), pyrazofurin (NSC 143095), trimetrexate (NSC 352122), 3- HP (NSC 95678), 2’-deoxy-5-fluorouridine (NSC 27640), 5-HP (NSC 107392), alpha-TGDR (NSC 71851), aphidioclin glycinate (NSC 303812), ara-C (NSC 63878), 5-aza-2'- deoxycytidine (NSC 127716), beta-TGDR (NSC 71261), cyclocytidine (NSC 145668), guanazole (NSC 1895), hydroxyurea (NSC 32065), inosine glycodialdehyde (NSC 118994), macbecin II (NSC 330500), pyrazoloimidazole (NSC 51143), thioguanine (NSC 752), and thiopurine (NSC 755).
The antibody conjugates of the invention can be used to modify a given biological response, and the toxin or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-. gamma.; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
Techniques for conjugating such therapeutic moiety to antibodies are well known, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Flellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", and in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985). In some embodiments, the antibody and toxin are both proteins and can be conjugated using techniques well known in the art. There are many crosslinkers available that can conjugate two proteins. (See for example "Chemistry of Protein Conjugation and Crosslinking". 1991, Shans Wong, CRC Press, Ann Arbor). The crosslinker is generally chosen based on the reactive functional groups available or inserted on the antibody or toxin. In addition, if there are no reactive groups a photoactivatible crosslinker can be used. Crosslinking agents known to the art include the homobifunctional agents: glutaraldehyde, dimethyladipimidate and Bis(diazobenzidine) and the heterobifunctional agents: m maleimidobenzoyl-N-hydroxysuccinimide and sulfo-m maleimidobenzoyl-N- hydroxysuccinimide.
In certain instances, it may be desirable to include a spacer between the antibody molecule or fragment and the toxin, e.g. a peptidic linker or spacer.
In additional embodiments, the antibody-toxin protein fusion is prepared using recombinant DNA techniques. In such case a DNA sequence encoding the ligand is fused to a DNA sequence encoding the toxin, resulting in a chimeric DNA molecule. The chimeric DNA sequence is transfected into a host cell that expresses the ligand-toxin fusion protein. The fusion protein can be recovered from the cell culture and purified using techniques known in the art.
According to some embodiments, the immunotoxin consists of a single polypeptide chain, wherein the PVR-binding portion is a single chain antibody. According to some embodiments, the PVR-binding portion comprises a complete immunoglobulin molecule. According to other embodiments, the PVR-binding portion is a dimer of Fab, Fab', scFv, single-domain antibody fragments, or disulfide-stabilized Fv fragments. According to other embodiments, the PVR-binding portion comprises a variable heavy chain, variable light chain, Fab, Fab', scFv, single-domain antibody fragment, or disulfide-stabilized Fv fragment. Portions of the PVR-binding molecule may be derived from one or more species, preferably comprising portions derived from the human species, and most preferably are completely human or humanized. Regions designed to facilitate purification or for conjugation to toxin may also be included in or added to the PVR-binding portion.
Antibodies, or immunoglobulins, comprise two heavy chains linked together by disulfide bonds and two light chains, each light chain being linked to a respective heavy chain by disulfide bonds in a "Y" shaped configuration. Proteolytic digestion of an antibody yields Fv (Fragment variable) and Fc (Fragment crystalline) domains. The antigen binding domains, Fab, include regions where the polypeptide sequence varies. The term F(ab')2 represents two Fab' arms linked together by disulfide bonds. The central axis of the antibody is termed the Fc fragment. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain (CF11). The variable domains of each pair of light and heavy chains form the antigen-binding site. The domains on the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, joined by three hyper- variable domains known as complementarity determining regions (CDRs 1-3). These domains contribute specificity and affinity of the antigen-binding site.
CDR identification or determination from a given heavy or light chain variable sequence, is typically made using one of few methods known in the art. For example, such determination is made according to the Rabat (Wu T.T and Rabat E.A., J Exp Med, 1970; 132:211-50), Chothia, Martin, and IMGT (Lefranc M-P, et al., Dev Comp Immunol, 2003, 27:55-77).
When the term“CDR having a sequence”, or a similar term is used, it includes options wherein the CDR comprises the specified sequences and also options wherein the CDR consists of the specified sequence.
The antigen specificity of an antibody is based on the hyper variable region (F1VR), namely the unique CDR sequences of both light and heavy chains that together form the antigen-binding site.
The isotype of the heavy chain (gamma, alpha, delta, epsilon or mu) determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively). The light chain is either of two isotypes (kappa, k or lambda, l). Both isotopes are found in all antibody classes.
The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, and antibody fragments long enough to exhibit the desired biological activity, namely binding to human PVR.
Antibody or antibodies according to the invention include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof, such as the Fab or F(ab')2 fragments. Single chain antibodies also fall within the scope of the present invention.
Antibody Fragments
"Antibody fragments" comprise only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VF1 and CF11 domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CF11 domain; (iii) the Fd fragment having VF1 and CF11 domains; (iv) the Fd' fragment having VF1 and CF11 domains and one or more cysteine residues at the C-terminus of the CF11 domain; (v) the Fv fragment having the VL and VF1 domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 1989, 341, 544-546) which consists of a VF1 domain; (vii) isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment including two Fab' fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g. single chain Fv; scFv) (Bird et al., Science 1988, 242, 423-426; and Fluston et al., Proc. Natl. Acad. Sci. (USA) 1988, 85,5879-5883); (x) "diabodies" with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93/11161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1 -VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng., 1995, 8, 1057-1062; and U.S. Pat. No. 5,641,870).
Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 70:163- 167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv).
Single chain antibodies can be single chain composite polypeptides having antigen binding capabilities and comprising amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain i.e. linked VH-VL or single chain Fv (scFv). Techniques for the production of single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single-chain antibodies to PVR.
The term“scFv” refers to a single chain Fv antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain. In some embodiments, the chains are joined by a linker peptide. The linker peptide can be from about 5 to 40 amino acids or from about 10 to 30 amino acids or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length.
The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. mAbs may be obtained by methods known to those skilled in the art. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 1991 , 352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222:581-597. The design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies through rapid identification and cloning of the functional variable heavy (VH) and variable light (VL) genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria are described in Fields et al. Nat Protoc. 2013, 8(6): 1125 -48.
The invention also provides conservative amino acid variants of the antibody molecules according to the invention. Variants according to the invention also may be made that conserve the overall molecular structure of the encoded proteins. Given the properties of the individual amino acids comprising the disclosed protein products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., "conservative substitutions," may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. The term“antibody analog” as used herein refers to an antibody derived from another antibody by one or more conservative amino acid substitutions.
The term“antibody variant” as used herein refers to any molecule comprising the antibody of the present invention. For example, fusion proteins in which the antibody or an antigen-binding-fragment thereof is linked to another chemical entity is considered an antibody variant.
Analogs and variants of the antibody sequences are also within the scope of the present application. These include, but are not limited to, conservative and non-conservative substitution, insertion and deletion of amino acids within the sequence. Such modification and the resultant antibody analog or variant are within the scope of the present invention as long as they confer, or even improve the binding of the antibody to the human PVR.
Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, penetration, and targeting to specific cell populations, immunogenicity, and the like. One of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, according to one table known in the art, the following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Pharmacology
In pharmaceutical and medicament formulations, the active agent is preferably utilized together with one or more pharmaceutically acceptable carrier(s) and optionally any other therapeutic ingredients. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof. The active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired exposure.
Typically, the immunotoxins will be suspended in a sterile saline solution for therapeutic uses. The pharmaceutical compositions may alternatively be formulated to control release of active ingredient or to prolong its presence in a patient's system. Numerous suitable drug delivery systems are known and include, e.g., implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the molecule according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebaric acid. The rate of release of the molecule according to the present invention, i.e., of an antibody or antibody fragment, from such a matrix depends upon the molecular weight of the molecule, the amount of the molecule within the matrix, and the size of dispersed particles. The molecules of the present invention as active ingredients are dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.
The pharmaceutical composition of this invention may be administered by any suitable means, such as intrathecally, intravenously, orally, topically, intranasally, subcutaneously, intramuscularly, intra-arterially, intraarticularly, intralesionally, intratumorally or parenterally.
According to some embodiments, the immunotoxin is administered directly to the cancer site. According to some embodiments, the cancer site is the in the brain. Such administration may be aimed at bypassing the blood brain barrier. In one specific embodiment the systemic administration is intrathecal administration, i.e. such composition is administered intrathecally.
As used herein, the phrase "the immunotoxin is administered directly to the cancer site" refers to direct or substantially direct introduction including, without limitation, single or multiple injections of the immunotoxin directly into the tumor or peritu morally, continuous or discontinuous perfusion into the tumor or peritumorally, introduction of a reservoir into the tumor or peritumorally, introduction of a slow-release apparatus into the tumor or peritumorally, introduction of a slow-release formulation into the tumor or peritumorally, or direct application onto the tumor.
It will be apparent to those of ordinary skill in the art that the therapeutically effective amount of the molecule according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered, its persistence in the blood circulation, and the judgment of the treating physician. As used herein the term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life.
The cancer amendable for treatment by the present invention includes, but is not limited to: blastoma, carcinoma, lymphoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include glioblastoma, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non- Hodgkin’s lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft- tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. The cancerous conditions amendable for treatment of the invention include metastatic cancers. According to specific embodiments, the cancer is glioblastoma.
According to other embodiments, the pharmaceutical compositions according to the invention are for use in treating cancer characterized by PVR overexpression. PVR overexpression related cancer types can be identified using known data bases such as The Cancer Genome Atlas (TCGA). According to certain embodiments, the cancer is selected from the group consisting of glioblastoma, adrenocortical carcinoma (ACC), chromophobe renal cell carcinoma (RICH), liver hepatocellular carcinoma (LIHC), colon and rectal adenocarcinoma (COAD, READ), pancreatic ductal adenocarcinoma (PAAD), pheochromocytoma & paraganglioma (PCPG), papillary kidney carcinoma (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), uterine corpus endometrial carcinoma (UCEC), cervical cancer (CESC), cutaneous melanoma (SKCM), mesothelioma (MESO), urothelial bladder cancer (BLCA), clear cell kidney carcinoma (KIRC), lung squamous cell carcinoma (LUSC), uterine carcinosarcoma (UCS), sarcoma (SARC), ovarian serous cystadenocarcinoma (OV), papillary thyroid carcinoma (THCA), glioblastoma multiforme (GBM), breast cancer (BRCA), lower grade glioma (LGG), and diffuse large B-cell lymphoma (DLBC). Each possibility represents a separate embodiment of the invention.
The pharmaceutical composition according to the present invention may be administered together with an anti- neoplastic composition.
The term "treatment" as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, blastoma, carcinoma, lymphoma, sarcoma, and leukemia. More particular examples of such cancers include melanoma, brain, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, or endometrial cancer. According to some embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen comprising administration of at least one additional anti-cancer agent.
According to some embodiments, the anti-cancer agent is selected from the group consisting of an antimetabolite, a mitotic inhibitor, a taxane, a topoisomerase inhibitor, a topoisomerase II inhibitor, an asparaginase, an alkylating agent, an antitumor antibiotic, and combinations thereof. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the antimetabolite is selected from the group consisting of cytarabine, gludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea. According to some embodiments, the mitotic inhibitor is selected from the group consisting of vincristine, vinblastine, and vinorelbine. According to some embodiments, the topoisomerase inhibitor is selected from the group consisting of topotecan and irenotecan. According to some embodiments, the alkylating agent is selected from the group consisting of busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosamide, mechlorethamine, melphalan, thiotepa, dacarbazine, and procarbazine. According to some embodiments, the antitumor antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, and plicamycin. According to some embodiments, the topoisomerase II is selected from the group consisting of etoposide and teniposide. Each possibility represents a separate embodiment of the present invention.
According to some particular embodiments, the additional anti-cancer agent is selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof. Each possibility represents a separate embodiment of the present invention.
Monoclonal antibodies according to the present invention may be used as part of combined therapy with at least one anti-cancer agent. According to some embodiments, the additional anti-cancer agent is an immuno-modulator, an activated lymphocyte cell, a kinase inhibitor or a chemotherapeutic agent. According to some embodiments, the anti-cancer agent is an immuno-modulator, whether agonist or antagonist, such as antibody against an immune checkpoint molecule.
According to other embodiments the additional anti-cancer agent is a chemotherapeutic agent. The chemotherapy agent, which could be administered together with the antibody according to the present invention, or separately, may comprise any such agent known in the art exhibiting anticancer activity, including but not limited to: mitoxantrone, topoisomerase inhibitors, spindle poison vincas: vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine, gemcitabin; podophyllo toxins: etoposide, irinotecan, topotecan, dacarbazin; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxifen, leuprolide, flutamide, and megestrol acetate.
According to some embodiments, the chemotherapeutic agent is selected from alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressant, adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone analog. According to another embodiment, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel and docetaxel. One or more chemotherapeutic agents can be used.
According to some embodiments, the method of treating cancer comprises administration of an immunotoxin according to the invention and an additional anti-cancer agent. According to some embodiments, the additional anti-cancer agent is selected from the group consisting of: immune-modulator, activated lymphocyte cell, kinase inhibitor and chemotherapeutic agent.
According to other embodiments, the immune-modulator is an antibody, antibody fragment or antibody conjugate that binds to an antigen other than human PVR. According to some embodiments, the immune-modulator is an antibody against an immune checkpoint molecule. According to some embodiments, the additional immune modulator is an antibody against an immune checkpoint molecule selected from the group consisting of human programmed cell death protein 1 (PD-l), PD-L1 and PD-L2, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, 0X40 (also referred to as CD134), killer cell immunoglobulin-like receptors (KIR), TIGIT and any combination thereof. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the anti-cancer agent is selected from the group consisting of: Erbitux, cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine, vinblastine, vinorelbine, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide, teniposide and any combination thereof. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the anti-cancer agent is epidermal growth factor receptor (EGFR) inhibitor. According to some embodiments, the EGFR inhibitor is selected from the group consisting of: Cetuximab (Erbitux®), Panitumumab (Vectibix®), and necitumumab (Portrazza®). According to certain embodiments, the EGFR inhibitor is Cetuximab (Erbitux®).
According to still another aspect the present invention provides a method of treating cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of an immunotoxin according to the present invention.
Toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition) and the maximal tolerated dose for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage may vary depending inter alia upon the dosage form employed, the dosing regimen chosen, the composition of the agents used for the treatment and the route of administration utilized, among other relevant factors. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors.
The term“administering” or“administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered enterally or parenterally. Enterally refers to administration via the gastrointestinal tract including per os, sublingually or rectally. Parenteral administration includes administration intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some embodiments, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self- administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.
The effective dose of a specific immunotoxin construct may depend on additional factors, including the type of cancer, the size of the tumor, the stage of the cancer, the immunotoxin' s toxicity to the patient, the specificity of targeting to cancer cells, as well as the age, weight, and health of the patient.
According to some embodiments, the effective dose by direct administration of immunotoxin may range from about 10 to 3000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 60 to 500, 70 to 400, 80 to 300, 90 to 200, or 100 to 150 micrograms/tumor/day. In other embodiments, the dose may range from approximately 10 to 20, 21 to 40, 41 to 80, 81 to 100, 101 to 130, 131 to 150, 151 to 200, 201 to 280, 281 to 350, 351 to 500, 501 to 1000, 1001 to 2000, or 2001 to 3000 micro grams/tumor/day. In specific embodiments, the dose may be at least approximately 20, 40, 80, 130, 200, 280, 400, 500, 750, 1000, 2000, or 3000 micrograms/tu mor/day.
According to other embodiments, the effective dose of immunotoxin may range from about 100 to 5000, 200 to 4000, 300 to 3000, 400 to 2000, 500 to 1000, 600 to 900, or 700 to 1500 micrograms/tumor/month. In other embodiments, the dose may range from approximately 100 to 199, 200 to 399, 400 to 649, 650 to 999, 1000 to 1799, 1800 to 2499, 2500 to 3499, 3500 to 4999, 5000 to 7499, 7500 to 10000, or 10001 to 20000 micrograms/tumor/month. In specific embodiments, the dose may be at least approximately 100, 200, 400, 650, 1000, 1400, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 7500, 8000, or 20000 micrograms/tumor/month.
According to other embodiments, the effective dose of immunotoxin results in an intratumoral concentration of at least approximately 5, 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 100, 200, 300, 400, or 500 micrograms/cm3 of the immunotoxin. In other embodiments, the resulting intratumoral concentration of immunotoxin is approximately 5 to 500, 10 to 400, 15 to 300, 20 to 200, 25 to 100, 30 to 90, 35 to 80, 40 to 70, 45 to 60, or 50 to 55 micrograms/cm3. In other embodiments, the resulting intratumoral concentration of immunotoxin is approximately 10 to 15, 16 to 20, 21 to 25, 26 to 30, 31 to 35, 36 to 40, 41 to 45, 46 to 50, 51 to 55, 56 to 60, 61 to 65, 66 to 70, 71 to 75, 76 to 80, 81 to 85, 86 to 90, 91 to 95, 96 to 100, or 100 to 200 micrograms/cm3.
According to other embodiments, the effective dose of immunotoxin results in a plasma concentration of less than approximately 0.1, 1, 2.5, 5, 7.5, 10, 15, 20, 30, 40, or 50 micrograms/liter. In other embodiments, the resulting circulating concentration of immunotoxin is approximately 0.1 to 50, 1 to 40, 2.5 to 30, 5 to 20, or 7.5 to 10 micrograms/liter. In other embodiments, the resulting circulating concentration of immunotoxin is approximately 0.1 to 1 , 1.1 to 2.4, 2.5 to 5, 5.1 to 7.4, 7.5 to 10, 11 to 15, 16 to 20, 21 to 30, 31 to 40, or 41 to 50 micrograms/liter.
According to particular non-limiting embodiments, the effective dose of the immunotoxin is between about 100 and 3000 micrograms/tumor/month, for example approximately 100, 200, 300, 400, 750, or 1000 micrograms/tumor/month, wherein the patient is administered a single dose per day. The single dose is administered approximately every month for approximately 1 , 2, 3, 4, 5, or 6 consecutive months. After this cycle, a subsequent cycle may begin approximately 1, 2, 4, 6, or 12 months later. The treatment regime may include 1 , 2, 3, 4, 5, or 6 cycles, each cycle being spaced apart by approximately
1 , 2, 4, 6, or 12 months.
According to other non-limiting embodiments, the effective dose of the immunotoxin is between about 20 and 1240 micrograms/tumor/day, for example approximately 20, 40, 80, 130, 200, or 280 micrograms/tumor/day or approximately 100, 200, 330, 500, 700, 930, 1240 micrograms/tumor/day, wherein the patient is administered a single dose per day. The single dose is administered approximately every day (one or more days may optionally be skipped) for approximately 1 , 2, 3, 4, 5, 6 or 7 consecutive days. After this cycle, a subsequent cycle may begin approximately 1 , 2, 3, 4, 5, or 6 weeks later. The treatment regime may include 1,
2, 3, 4, 5, or 6 cycles, each cycle being spaced apart by approximately 1, 2, 3, 4, 5, or 6 weeks.
The term "about" means that an acceptable error range, e.g., up to 5% or 10%, for the particular value should be assumed.
Diagnosis
The present invention further discloses methods for diagnosing and prognosing cancer.
According to an aspect, the present invention provides a diagnostic and/or prognostic method of cancer disease in a subject, the method comprises the step of determining the expression level of PVR in a biological sample of said subject using at least one antibody as described herein.
The term "biological sample" encompasses a variety of sample types obtained from an organism that may be used in a diagnostic or monitoring assay. The term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen, or tissue cultures or cells derived there from and the progeny thereof. Additionally, the term may encompass circulating tumor or other cells. The term specifically encompasses a clinical sample, and further includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, biological fluids including aqueous humour and vitreous for eyes samples, and tissue samples. The term also encompasses samples that have been manipulated in any way after procurement, such as treatment with reagents, solubilisation, or enrichment for certain components. Determining the expression level of PVR can be performed by a labeled anti-PVR antibody as described herein. Determining the expression can be performed, for example, by ELISA.
Determining and quantifying methods may be performed in-vitro or ex-vivo according to some embodiments or may be used in diagnosing conditions associated with expression of PVR. The antibody or fragment thereof according to the present invention may be also used to configure screening methods. For example, an enzyme-linked immunosorbent assay (ELISA), or a radioimmunoassay (RIA) can be constructed for measuring levels of secreted or cell- associated polypeptide using the antibodies and methods known in the art.
According to some embodiments, the method for detecting or quantifying the presence of PVR comprises the steps of: i. incubating a sample with an antibody or an antibody fragment thereof as described hereinabove;
ii. detecting the bound PVR using a detectable probe.
According to some embodiments, the method further comprises the steps of: iii. comparing the amount of (ii) to a standard curve obtained from a reference sample containing a known amount of PVR; and
iv. calculating the amount of the PVR in the sample from the standard curve.
According to some particular embodiments the sample is a body fluid.
According to some embodiments, the method is performed in-vitro or ex-vivo.
The method of the invention can further comprise the step of comparing said level of expression to a control level.
The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed as limiting the scope of the invention. EXAMPLES
Methods
Cell lines
The cell lines used were U-87 MG (ATCC® HTB-14™) and U-87 MG PVRhi (human PVR transfectant of U-87 MG). Cells were cultivated at 37°C, >95% humidity and 5% CO2 in DMEM supplemented with 10% heat inactivated FCS (media and sera from Sigma-Aldrich). Flow cytometry
Flow cytometry was performed using aPVR mAh (clone CD155.16). Cells were incubated on ice for 30 minutes with 0.2 pg of mAh per 100,000 cells. Detection was performed with a secondary goat a-mouse Ab coupled to PE (Jackson ImmunoResearch) for 30 min on ice. Analysis was performed using the FACS-ARIA flow cytometer (BD Biosciences) and CellQuest software.
Killing assay
For evaluation of immunotoxin cytotoxic activity against targets cells U-87 MG PVRhi cells were incubated with different concentrations of aPVR-DT or control aPVR antibody (clone CD 155.16). Cells were incubated for 48h and cytotoxic activity was determined with ToxyFight kit (Fonza).
Example 1. Producing of anti-PVR antibody
A monoclonal antibody anti-PVR (anti CD 155.16 KAC) that does not block binding of TIGIT to PVR, was generated in KAC mouse.
KAC mouse is a genetically engineered mouse strain that expresses BAP (Biotin Accepting Peptide) sequence on constant part of kappa chain and when the fusion is performed with SP2/0-3A4 cell line (Expressing BirA enzyme), the resulted m Ahs are produced as a biotinylated protein. This technology enabled the screen for potential antibody cytotoxic effect using Streptavidin-saporin conjugate (ATS Bio).
Immunization was performed using standard protocols as known in the art. In short, PVR-Fc fusion protein was emulsified in Freund adjuvant and injected subcutaneously into KAC mouse. Then, the sera were tested for developing sufficient antibody response (Figure 1). The fusion was performed with BirA-SP2/0 line using PEG. The hybridoma cells were tested by Eliza. All these candidates that showed the specific signal in EFISA were tested for their ability to recognize a native human PVR protein on transfectant cell lines U-87 MG PVRhi. The clone with the highest binding signal, aPVR mAh clone CD155.16 was chosen for next step. The amino acid and nucleic acid sequences of the mAh produced by this clone are as following: heavy chain variable region nucleic acid sequence - SEQ ID NO: 1, heavy chain variable region amino acid sequence - SEQ ID NO: 2, light chain variable region nucleic acid sequence - SEQ ID NO: 3, light chain variable region amino acid sequence - SEQ ID NO: 4, CDR sequences (according to RABAT): HC CDR 1 - SEQ ID NO: 5, HC CDR 2 - SEQ ID NO: 6, HC CDR 3 - SEQ ID NO: 7, LC CDR 1 - SEQ ID NO: 8, LC CDR 2 - SEQ ID NO: 9, and LC CDR 3 - SEQ ID NO: 10.
The antibody was used for producing a variety of immunoglobulin molecules and immunotoxins, including scFV molecules and recombinant immunotoxins, based on its variable region sequences.
Example 2. Cytotoxicity of aPVR - Saporin immunotoxins
Anti-PVR - saporin immunotoxins were tested for potential antitumor activity. The immunotoxins were prepared by mixing biotinylated anti-PVR antibody with StreptAvidin- saporin (SA-saporin, ATS bio, Cat NO: IT-27) in equal molar concentrations. The cytotoxicity test was performed using 10000 cells/50 pi in 96 well plates. Stocks containing 1 mM/ml of immunotoxins were made and 100 mΐ/well of sample or controls were added to the cells and incubated for 48h at 37°C. The Supernatant was then collected and analyzed using Lonza ToxiLight™ non-destructive Cytotoxicity BioAssay Kit. The adenylate kinase release, measured by its activity, indicates cell death. The antibodies used were: biotinylated (XCD155.16 antibody (aCDl55T6 b+) and F(ab’)2 fragments (Fab2) derived from (XCD155.16 antibody (aCDl55. 16 b+ Fab2). F(ab’)2 antibodies were prepared by pepsin enzyme digestion of the monoclonal antibody anti CD155.16 (using regular protocol, Current protocol in Immunology) and purified with HiTrap protein L purification columns (product no: 29048665) on ACTA prime.
As shown in Figure 2, the immunotoxins killed target cells regardless whether the whole antibody or Fab2 fragment was used. Example 3. Cytotoxicity of aPVR - diphtheria immunotoxins
The sequence of the antibody described in Example 1 was used to design aPVR - diphtheria immunotoxins (aPVR-DT; DT-CD155 scFv fusion protein) (Fig. 3). Immunotoxin was designed as a fusion protein containing the first 389 amino acid of diphtheria toxin and anti CD 155 scFV linked by Ser-Gly linker. The amino acid sequence of the immunotoxin set forth in SEQ ID NO:l9 and includes the DT (first 389 AA), a Ser-Gly linker, scFv heavy chain, scFv light chain, a short linker and HIS tag. The sequence of the scFv heavy + light chain as cloned set forth in SEQ ID NO: 20. Sequences were aligned in silico and DNA synthesis together with protein expression was ordered from Genscript, USA. Immunotoxin produced by Genscipt was used for all following experiments. An in vitro assay was conducted to examine the immunotoxin cytotoxicity toward tumor cells. Human glioblastoma cells U-87 MG PVRhi were seeded in 96 well plate at density of 5000 cells/well. After cell adhesion to the plate, different concentration of aPVR-DT or control aPVR mAh (clone CD 155.16) were added to the cells. Cytotoxic effect was determined by ToxiLight kit (Lonza; Catalog #: LT17-217). As shown in Figure 4, DT-anti CD155 efficiently kills PVR expressing tumor cells.
Example 4. Antitumor effect of aPVR-DT in mouse model. The efficacy of the aPVR-DT was determined in vivo in animal model. U-87 MG and h
U-87 MG PVRhi cells (1X10 per mouse) were injected subcutaneously into NSG mice (lacking NK, B and T cells). After establishment of tumor, aPVR-DT or control aPVR mAh (clone CD155.16) were injected i.p. or i.t. (50 pg/mouse) on days 3, 6, 9, 12 and 16. Where indicated, PBS alone was used as negative control. Tumor growth was monitored daily and measured by caliper. As shown in Figures 5 and 6, treatment with aPVR-DT leaded to tumor regression which is dependent on PVR expression. Example 5. PVR expression on human GBM samples.
Expression of PVR was determined by immunohistochemistry on human GBM samples from 36 donors using aPVR mAh (clone CD155-KLH.03). The samples were prepared according to the following protocol: · Paraffin slide insert in PT Link system to a temperature of 65 °C and then stick to a temperature of 97°C for 20 minutes. PT Link is an easy-to-use pre-treatment system that optimizes staining consistency. PT Link allows the entire pre-treatment process of deparaffinization, rehydration and epitope retrieval. Antigen retrieval -Target Retrieval Solution, pH 9.0 (DAKO Kit K 8000).
Cool 20 minutes in Washing buffer.
DAKO Autostainer:
Incubation primary antibodies (CD155-KLH.03) for 30 min
Wash in Washing buffer.
- EnVision FLEX PEROXIDASE BLOCKING REAGENT (6 Min)
- Wash in Washing buffer.
- EnVision FLEX/HRP DAKO (30 Min)
Wash in Washing buffer.
- DAB DAKO (10 Min).
• Counterstain with DAKO Mayer’s hematoxylin S3309 (1 Min).
• Rinse in running water for 10 min.
• Wash in distilled water for 5 min.
• Dehydrate through 70% ethanol for 2 Min, 96% ethanol for 2x3 min, 100% ethanol for 2x3 min.
• Clear in xylene for 2x5 min.
• Cover and seal with Entelan mounting medium.
The samples were analyzed for: 1 - weak expression; 2- moderate expression; or 3 - strong expression. PVR was found to be highly expressed in these samples. Example 6. Complementary determining region (CDR) sequences
The CDR segments were identified using different algorithm methods. For example, the CDRs may be identified using IMGT algorithm (Lefranc et al., 1999, Nucleic Acids Research, 27, 209-212); RABAT algorithm (Wu TT and Rabat E.A., 1970, J. Exp. Med. 132, 211-250); Chothia (Chothia and Lesk, J Mol Biol. 1987 Aug 20;196(4):901-17); or enhanced
Chothia method (MacCallum, R. M., Martin, A. C. R. et al. J. Mol. Biol. 1996. 262, 732-745)
Table 1. summarizes the determined CDR sequences using two alternative methods as well as the minimal consensus sequence and combined sequence of sequences identified using both methods.
Table 1. CDR sequences
Figure imgf000043_0002
Figure imgf000043_0001
method.
** wherein Xi is absent or Thr (T); X2 is absent or Met (M)
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.

Claims

1. An antibody which binds to human poliovirus receptor (PVR), or an antibody fragment thereof comprising at least the antigen binding portion, wherein the antibody or antibody fragment comprises three complementarity determining regions (CDRs) of a heavy-chain (HC) variable region comprising SEQ ID NO: 2 and three CDRs of a light- chain (LC) variable region comprising SEQ ID NO: 4, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence.
2. The antibody or the antibody fragment according to claim 1 , comprising a CDR set of six CDRs, wherein HC CDR1 comprising the sequence SYW, HC CDR2 comprising IHPNSGST (SEQ ID NO: 12), HC CDR3 comprising EG Y V Y Y AMD Y (SEQ ID NO: 7), LC CDR1 comprising QSLLYSGDQRNY (SEQ ID NO: 14), LC CDR2 comprising WAS, and LC CDR3 comprising QQYYNYP (SEQ ID NO: 16).
3. The antibody or the antibody fragment according to any one of claims 1 or 2, comprising a CDR set of six CDRs wherein: HC CDR1 is selected from SYWMH (SEQ ID NO: 5) and GYSFTSYW (SEQ ID NO: 11); HC CDR2 is selected from IIHPNSGSTIYNEKFK (SEQ ID NO: 6) and IHPNSGST (SEQ ID NO: 12); HC CDR3 is selected from EGY VY Y AMD Y (SEQ ID NO: 7) and GREGY VYY AMD Y (SEQ ID NO: 13); LC CDR1 is selected from KSSQSLLYSGDQRNYLA (SEQ ID NO: 8) and QSLLYSGDQRNY (SEQ ID NO: 14); LC CDR2 is selected from the group consisting of: WASTRES (SEQ ID NO: 9) and WAS; and LC CDR3 is selected from QQYYNYPRT (SEQ ID NO: 10) and QQYYNYP (SEQ ID NO: 16).
4. The antibody or the antibody fragment according to claim 3, wherein HC CDR1 comprises the sequence SYWMH (SEQ ID NO: 5); HC CDR2 comprises the sequence IIHPNSGSTIYNEKFK (SEQ ID NO: 6); and HC CDR3 comprises the sequence EGY VYY AMD Y (SEQ ID NO: 7).
5. The antibody or the antibody fragment according to any one of claims 3 or 4, wherein LC CDR1 comprises the sequence KSSQSLLYSGDQRNYLA (SEQ ID NO: 8); LC CDR2 comprises the sequence WASTRES (SEQ ID NO: 9); and LC CDR2 comprises the sequence QQYYNYPRT (SEQ ID NO: 10).
6. The antibody or the antibody fragment according to any one of claims 3 to 5, wherein HC CDR1 comprises the sequence SYWMH (SEQ ID NO: 5); HC CDR2 comprises the sequence IIHPNSGSTIYNEKFK (SEQ ID NO: 6); HC CDR3 comprises the sequence EGYVYYAMDY (SEQ ID NO: 7); LC CDR1 comprises the sequence KS S QSLL Y SGDQRN YLA (SEQ ID NO: 8); LC CDR2 comprises the sequence WASTRES (SEQ ID NO: 9); and LC CDR2 comprises the sequence QQYYNYPRT (SEQ ID NO: 10).
7. The antibody or the antibody fragment according to any one of claims 1 to 6, comprising the heavy chain variable region set forth in SEQ ID NO: 2, or an analog having at least 95% sequence similarity with said heavy chain variable region sequence.
8. The antibody or the antibody fragment according to any one of claims 1 to 7, comprising the light chain variable region set forth in SEQ ID NO: 4, or an analog having at least 95% sequence similarity with said heavy chain variable region sequence.
9. A single chain variable fragment (scFv) having an antigen binding site of an antibody according to any one of claims 1 to 8.
10. The single chain variable fragment of claim 9, comprising the sequence set forth in SEQ ID NO: 20.
11. The scFv of claim 9, comprising three CDRs of a heavy-chain variable region comprising SEQ ID NO: 2 and three CDRs of a light-chain variable region comprising SEQ ID NO: 4, or an analog or derivative thereof having at least 90% sequence identity with said scFv sequence.
12. The scFv of claim 9, comprising a CDR set of six CDRs, wherein HC CDR1 comprising SYW, HC CDR2 comprising IHPNSGST (SEQ ID NO: 12), HC CDR3 comprising EGYVYYAMDY (SEQ ID NO: 7), LC CDR1 comprising QSLLYSGDQRNY (SEQ ID NO: 14), LC CDR2 comprising WAS, and LC CDR3 comprising QQYYNYP (SEQ ID NO: 16).
13. A plasmid comprising at least one polynucleotide sequence encoding an antibody sequence according to any one of claims 1 to 12.
14. A hybridoma cell capable of producing an antibody according to any one of claims 1 to
10.
15. A conjugate comprising an antibody, antibody fragment or scFv according to any one of claims 1 to 12.
16. The conjugate of claim 15, said conjugate is an immunotoxin comprising the antibody, antibody fragment or scFv of any one of claims 1 to 12 linked to a toxin agent.
17. The immunotoxin of claim 16, wherein the toxin is selected from the group consisting of diphtheria toxin or a subunit thereof, saporin, ricin A, abrin, pseudomonas exotoxin or a portion thereof, restrictocin and gelonin.
18. The immunotoxin of claim 17, wherein the toxin is saporin.
19. The immunotoxin of claim 17, wherein the toxin is a diphtheria toxin.
20. An immunotoxin comprising a scFv sequence having a CDR set of six CDRs wherein:
HC CDR1 is selected from SYWMH (SEQ ID NO: 5) and GYSFTSYW (SEQ ID NO: 11); HC CDR2 is selected from IIHPNSGSTIYNEKFK (SEQ ID NO: 6) and IHPNSGST (SEQ ID NO: 12); HC CDR3 is selected from EGYVYYAMDY (SEQ ID NO: 7) and GREGYVY Y AMD Y (SEQ ID NO: 13); LC CDR1 is selected from KS S QSLL Y SGDQRN YLA (SEQ ID NO: 8) and QSLLYSGDQRNY (SEQ ID NO: 14); LC CDR2 is selected from the group consisting of: WASTRES (SEQ ID NO: 9) and WAS; and LC CDR3 is selected from QQYYNYPRT (SEQ ID NO: 10) and QQYYNYP (SEQ ID NO: 16).
21. The immunotoxin according to claim 20, comprising an scFv sequence set forth in SEQ ID NO: 20 or an analog thereof having at least 95% sequence identity.
22. The immunotoxin according to claim 20, having a sequence set forth in SEQ ID NO: 19 or an analog thereof having at least 95% sequence identity.
23. A pharmaceutical composition comprising as an active ingredient, at least one antibody, antibody fragment or scFv, according to any one of claims 1 to 12, and a pharmaceutical acceptable excipient, diluent, salt or carrier.
24. A pharmaceutical composition comprising as an active ingredient, at least one immunotoxin according to any one of claims 16 to 22, and a pharmaceutical acceptable excipient, diluent, salt or carrier.
25. The pharmaceutical composition of claim 24 for use in treating cancer.
26. The pharmaceutical composition of claim 25, wherein the cancer is PVR-expressing cancer.
27. The pharmaceutical composition of claim 26, wherein the PVR-expressing cancer is selected from the group consisting of: GBM, medulloblastoma, colorectal carcinoma and pancreatic cancer.
28. The pharmaceutical composition of any one of claims 25 or 26, wherein the cancer is selected from the group consisting of brain cancer, melanoma, a breast cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, a colon cancer, a cervical cancer, a kidney cancer, a lung cancer, a thyroid cancer, a prostate cancer, a brain cancer, a renal cancer, a throat cancer, a laryngeal carcinoma, a bladder cancer, a hepatic cancer, a fibrosarcoma, an endometrial cells cancer, sarcoma, a myeloid, a leukemia and a lymphoma.
29. The pharmaceutical composition of any one of claims 25 to 28 wherein the cancer is glioblastoma.
30. A method of treating cancer, comprising administering to a subject in need thereof, a pharmaceutical composition according to any one of claims 23 or 24.
31. The method of claim 30, wherein the pharmaceutical composition is administered directly to the cancer site.
32. The method of claim 31 , wherein the cancer is glioblastoma.
33. The method of claim 31, wherein the pharmaceutical composition is administered directly to the brain.
34. The method of claim 31, wherein the pharmaceutical composition is administered intrathecally.
35. The method of any one of claims 30 to 34, wherein the subject is human.
36. The method of any one of claims 30 to 34, further comprising an additional anti-cancer therapy selected from surgery, chemotherapy, radiotherapy, and immunotherapy.
37. The method of any one of claims 30 to 36 further comprising administering to said subject an immuno-modulator, activated lymphocyte cell, kinase inhibitor, chemotherapeutic agent or any other anti-cancer agent.
38. The method of claim 37, wherein the immune- modulator is an antibody against an immune checkpoint molecule selected from the group consisting of PD-l, CTLA-4, PDL-l , CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R, lymphocyte activation gene 3 (LAG3), CD137, 0X40 (also referred to as CD134), killer cell immunoglobulin-like receptors (KIR), TIGIT, and any combination thereof.
39. The method of any one of claims 29 to 38, wherein treating results in decreasing the size, growth rate, invasiveness, malignancy grade and/or risk of recurrence of a tumor associated with the cancer.
40. A method of diagnosing a cancer in a subject, the method comprising contacting a biological sample with an antibody, antibody fragment or scFv according to any one of claims 1 to 12.
41. A kit for diagnosing a cancer in a subject comprising at least one antibody, antibody fragment, or scFv according to any one of claims 1 to 12.
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