WO2019241847A1 - A therapeutic gpiib/iiia binding-protein drug conjugate and use thereof - Google Patents

A therapeutic gpiib/iiia binding-protein drug conjugate and use thereof Download PDF

Info

Publication number
WO2019241847A1
WO2019241847A1 PCT/AU2019/050638 AU2019050638W WO2019241847A1 WO 2019241847 A1 WO2019241847 A1 WO 2019241847A1 AU 2019050638 W AU2019050638 W AU 2019050638W WO 2019241847 A1 WO2019241847 A1 WO 2019241847A1
Authority
WO
WIPO (PCT)
Prior art keywords
scfv
binding protein
gpiib
cancer
antibody
Prior art date
Application number
PCT/AU2019/050638
Other languages
French (fr)
Inventor
Karlheinz Peter
Xiaowei Wang
Geoffrey Allan Pietersz
May Lin YAP
James David MCFADYEN
Original Assignee
Baker Heart and Diabetes Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2018902247A external-priority patent/AU2018902247A0/en
Application filed by Baker Heart and Diabetes Institute filed Critical Baker Heart and Diabetes Institute
Publication of WO2019241847A1 publication Critical patent/WO2019241847A1/en

Links

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
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2848—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • 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
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • 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

Definitions

  • the specification is in the field of platelet and cancer biology, haematology and medicine.
  • the specification describes activated platelet binding proteins including antigen-binding fragments of antibodies and antibody drug conjugates (ADCs) comprising same including compositions and methods of using said binding proteins and ADCs.
  • ADCs antibody drug conjugates
  • Antibody drug conjugates represent an emerging class of therapeutics comprising an antibody conjugated to a drug such as a cytotoxic or immunomodulatory drug via a chemical linker.
  • the therapeutic concept of ADCs is to combine binding capabilities of an antibody with a drug, where the antibody is used to deliver the drug to a tumor cell by means of binding to a target tumor cell antigen.
  • the expression level of traditional tumor antigens within a tumor can be inadequate for effective tumor reduction and there is a need for new approaches to make ADC more effective. Even if the expression level is adequate, the ability of the ADC to access tumor cells in the solid tumor microenvironment about which little is understood, may lead to treatment failures. Nanoparticle based approaches have been used to try to trap ADC within solid tumors. SUMMARY OF THE DISCLOSURE
  • composition includes a single composition, as well as two or more compositions; reference to “an agent” includes one agent, as well as two or more agents; reference to “the disclosure” includes single and multiple aspects of the disclosure and so forth.
  • the present disclosure describes and enables a binding protein drug conjugate (also referred to as an ADC or immunoconjugate) for delivering a therapeutic drug to a tissue comprising activated platelets comprising (i) a binding protein comprising an antibody variable domain that binds specifically to the activated form of platelet-specific glycoprotein (GP)IIb/IIIa conjugated to (ii) a therapeutic drug, wherein the binding protein concentrates the therapeutic drug to a target tissue comprising activated platelets.
  • a binding protein drug conjugate also referred to as an ADC or immunoconjugate
  • the present disclosure describes and enables a binding protein drug conjugate for delivering a therapeutic drug to a tumor comprising (i) a binding protein comprising an antibody variable domain that binds specifically to the activated form of platelet- specific GPIIb/IIIa conjugated to (ii) a therapeutic drug, wherein the binding protein concentrates the therapeutic drug in the tumor microenvironment and provides targeted delivery of the therapeutic drug to the tumor cells.
  • the therapeutic drug is chemotherapeutic prodrug and the binding protein drug conjugate comprises a cleavable linker and optionally a spacer between the prodrug and the binding protein which upon cleavage of the linker releases the active chemotherapeutic drug from the binding protein.
  • the cleavable linker is a pH sensitive linker, such as a hydrazone or cis-aconityl, providing a non-specific pH sensing mechanism to provide activation or release of the therapeutic drug in a mildly acidic environment.
  • a pH sensitive linker such as a hydrazone or cis-aconityl
  • a disulfide linker is employed to release the therapeutic drug based upon the higher reducing potential found within the tumor microenvironment.
  • hindrance groups can be introduced near the cleavage site.
  • the cleavable linker is selectively cleaved by a molecule present in the tumor stroma.
  • cleavable linker comprises a dipeptide, such as Val-Cit, Phe-Lys and Val-Ala.
  • the binding protein is an Fv, scFv, di-scFv, diabody, triabody, tetrabody, Fab, F(ab')2, bispecific antibodies, full length antibody, chimeric, human etc antibody.
  • Non-Ig binding proteins include monobodies, anticalins, and Darpins, LoopDarbins affibodies (Jost et al. Current opinion in Structural Biology 2014 27:102-112). Synthetic antibody mimetic s are also contemplated, as are ibodies (Adalta).
  • the binding protein and/or the therapeutic drug are modified for conjugation (coupling). In one embodiment, the modifications are for site- specific conjugation. In one embodiment, the binding protein and the therapeutic agent are conjugated by sortase A mediated conjugation.
  • the therapeutic drug is selected from the group consisting of a mitotic inhibitor, a plant alkaloid, and an anti-tumor antibiotic.
  • the antibody variable domain binds an epitope of GPIIb/IIIa recognised by a scFv comprising of an amino acid sequence set forth in SEQ ID NO: 1 or 5.
  • the antibody variable domain comprises a complementary determining region (CDR) of the heavy chain variable region (V H ) having the sequence of SEQ ID NO:2 or 6 and/or a CDR of the light chain variable region (V L ) having the amino acid sequence of SEQ ID NO: 3 or 7.
  • CDR complementary determining region
  • Variant sequences are also contemplated which have a small number of modified amino acids as known in the art while retaining or displaying enhanced binding and stability features.
  • Reference to a small number includes for example 1 amino acid difference, or 2, 3, 4, 5, 6 amino acid substitutions, deletions such as conservative substitutions or use of unnatural amino acids.
  • the antibody variable domain comprises a heavy chain variable region (VH) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 2 or 6.
  • VH heavy chain variable region
  • the antibody variable domain comprises a light chain variable region (VL) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 3 or 7.
  • the antibody variable domain comprises a heavy chain variable region (V H ) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 2 or 6 and a light chain variable region (VL) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 3 or 7.
  • the antibody variable domain comprises an amino acid sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 1 or 5.
  • the antibody variable domain is derived from a human scFv library.
  • Reference to a spacer includes a self immolative spacer, such as a para- aminobenzylalcohol (PABA) spacer or a non-self immolative spacer.
  • a self immolative spacer such as a para- aminobenzylalcohol (PABA) spacer or a non-self immolative spacer.
  • PABA para- aminobenzylalcohol
  • the cytotoxic agent is cytotoxic to tumour cells at nanomolar concentrations, picomolar concentrations or less.
  • the binding protein drug conjugate comprising a detectable label or molecule suitable for imaging tumors, diagnostic or monitoring.
  • the ratio of antibody variable domain to drug is 1:1 or 1:2.
  • the ratio of antibody variable domain to drug is 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8.
  • the binding protein drug conjugate as described herein is in the form of a composition including a pharmaceutical composition.
  • Pharmaceutical compositions include pharmaceutically acceptable salts and isoforms.
  • the composition comprises a pharmacologically or physiologically acceptable diluent and/or carrier.
  • composition as described herein is for use in medical therapy or imaging.
  • methods for imaging cancer are provide using the herein described binding proteins.
  • composition comprising the binding protein drug conjugate as described herein in, or in the preparation of a medicament for, the treatment of cancer including reduction of solid tumors and reduced metastasis in a subject with cancer.
  • the cancer is selected from the group consisting of breast cancer, lung cancer, a glioblastoma, prostate cancer, pancreatic cancer, colon cancer, colorectal cancer, head and neck cancer, mesothelioma, kidney cancer, ovarian, oesophageal, squamous cell carcinoma, triple negative breast cancer, and non-small cell lung cancer.
  • a fusion protein or a kit is contemplated comprising a binding protein comprising the antibody variable domain as described herein labelled with a detectable label suitable for imaging, diagnostics, theranostics or monitoring.
  • a fusion protein or a kit comprising a binding protein comprising the antibody variable domain as described herein conjugated via a suitable linker or spacer to a therapeutic agent suitable for therapy, theranostics or monitoring.
  • the present disclosure provides a vector or host cell comprising a polynucleotide sequence capable of expressing the binding protein comprising the antibody variable domain as described herein.
  • Illustrative polynucleotide sequences are provided in SEQ ID NOs 4 (encoding the SCE5 scFv- nucleotides 1 to 138 encode the vector leader sequence) and SEQ ID NO:8 (encoding the anti-LIBs scFv).
  • the present disclosure provides a method of treating cancer and reducing metastasis comprising administering an effective amount of a binding protein drug conjugate as described herein to a subject with cancer.
  • the present application provides a method for treating cancer in a subject, comprising (i) administering a binding protein comprising an antibody variable region that binds specifically to activated platelets such as to the activated form of platelet specific GPIIb/IIIa wherein the binding protein is coupled to an imaging contrast agent to determine the presence of activated platelets within a tumor (ii) administering a binding protein comprising an antibody variable region that binds specifically to activated platelets such as to the activated form of platelet specific GPIIb/IIIa wherein the binding protein is coupled to a drug such as a therapeutic or cytotoxic agent contingent upon a determination from (i) of the presence of activated platelets within a tumor.
  • the binding protein comprises an imaging contrast agent and one or more anti-cancer drugs such as a therapeutic or cytotoxic agent.
  • Therpeutic drugs include immune or inflammatory agonists.
  • the binding protein drug conjugate is administered in combination with an additional drug selected from the group consisting of an anti- apoptotic agent, a mitotic inhibitor, an anti-tumor antibiotic, an immunomodulating agent, a nucleic acid for gene therapy, an alkylating agent, an anti-angiogenic agent, an anti-metabolite, a boron-containing agent, a chemoprotective agent, a hormone agent, an anti-hormone agent, a corticosteroid, a photoactive therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor, and a tyrosine kinase inhibitor.
  • the agent is attached or otherwise part of the ADC.
  • a binding protein comprising an antibody variable domain that binds specifically to the activated form of platelet- specific GPIIb/IIIa in the preparation of a cancer imaging agent.
  • FIG. 1 Structure of GGG-Val-Cit-PAB-MMAE and labeling strategy for the generation of scFv GpIIb/IIIa -Cy7-MMAE and Cathepsin B drug release.
  • scFv GpiIb/IIIa was conjugated to GGG-Val-Cit-PAB-MMAE using an enzymatic sortase A reaction (I), followed by Cy7 conjugation, via NHS labelling to produce scFv GpiIb/IIIa - Cy7-MMAE (II).
  • cathepsin B cleaves the scFv Gpnb/IIIa -Cy7-MMAE at the MMAE Val-Cit linker, releasing the potent MMAE for tumor killing (III).
  • FIG. 1 scFv GpIIb/IIIa -Cy7-MMAE characterization and specific binding to activated platelets.
  • A Coomassie (red) and near infrared (green) imaging of scFv GPiib/iiia - C y 7 - MMAE 0) > unmodified scFv GpiIb/IIIa (II), scFv mut -Cy7-MMAE (III) and unmodified scFv mut (IV). Yellow color indicates the overlap between Coomassie in red and near infrared in green.
  • the scFv GPiib/iiia contains a V5 tag, which allows using an anti-V5-FITC secondary antibody for detection.
  • FIG. 4 Tumor cells induce platelet activation.
  • A, B Washed human platelets were added to MDA-MB-231 tumor cells and stained with a PE-conjugated anti-CD4l antibody (red) and an activation- specific anti-GPIIb/IIIa antibody (scFv GPiib/ ni a -GFP or FITC-conjugated PAC-l).
  • Fluorescence imaging (20x) demonstrates that MDA-MB-231 cells induces platelet binding and activation as shown by scFv GPiib/ ni a -GFP and PAC-l binding (green)
  • C Washed platelet rich plasma was added to MDA-MB-231, HT29, HT1080 and PC3 tumor cells and stained with a PE- conjugated anti-CD4l antibody and scFv GPiib/iiia or scFv mut , detected with an anti-V5- FITC antibody.
  • Flow cytometry dot plots represent the gating strategy differentiating regions of cancer cells and platelets.
  • the cancer cell region was further gated to select for CD4l-positive cancer cells (CD4l+ve).
  • D Analysis of the CD4l-positive region demonstrated that 100% of platelets were activated upon incubation with cancer cells for 6 hours, determined by positive scFv GPiib/ ni a binding (red), which was equivalent to scFv GPiib/iiia binding to ADP-activated platelets (green).
  • scFv GPiib/ ni a binding red
  • scFv GPiib/iiia binding to ADP-activated platelets green
  • Activated platelets are present in the tumor microenvironment and can be detected and imaged using the scFvGPiib/nia.
  • A In vivo bioluminescence imaging of a representative mouse with an orthotopic mammary tumor.
  • B In vivo bioluminescence imaging of metastatic lung and lymph node lesions, by shielding the area of the primary tumor.
  • C In vivo fluorescence imaging of MDA-MB-231 tumor bearing mice injected with scFv GpIIb/IIIa -Cy7-MMAE.
  • Activated platelets are present in the tumor microenvironment and can be detected and imaged using the scFvGPiib/iiia.
  • A In vivo fluorescence imaging of MDA-MB-231 tumor-bearing mice, injected with scFv GPiib/iiia -Cy7-MMAE and scFv mut -Cy7-MMAE.
  • B In vivo bioluminescence imaging of MDA-MB-231 tumor-bearing mice confirming primary tumor localization in the mammary gland.
  • FIG. 7 Activated platelets are present in the tumor microenvironment but absent in the spleen and bone marrow.
  • A, B Immunofluorescence imaging of tumor sections of MDA-MB-231 tumor-bearing mice injected with DyLight 649 anti- GPlbp (red) and scFv GPiib/iiia -GFP or scFv mut -GFP (green), counterstained with Hoechst® (blue).
  • A Immunofluorescence imaging (20x) demonstrated the abundance of platelets within the tumor microenvironment (red), and the specificity of the platelet binding of scFv GPiib/iiia to tumor-associated platelets in vivo (green).
  • C, D Flow cytometry of the spleen and bone marrow of BALB/C nude mice injected with PBS (control) or DyLight 649 anti-GPlbp and scFv GPiib/iiia -GFP or scFv mut -GFP.
  • Black dotted lines represent guideline for normal range within the 95% interval published by Charles River for female BALB/C athymic nude mice. Data expressed as mean ⁇ SEM. WBC - white blood cells, ALT - alanine aminotransferase and ALP - alkaline phosphatase.
  • FIG. 9 scFvGPiib/nia-MMAE binds to activated platelets and induces cancer cell killing.
  • A Cytotoxicity assay of MDA-MB-231, HT29, HT1080 and PC3 tumor cells, cultured in GGG-Val-Cit PAB-MMAE and scFv GPiib/ ni a -MMAE, in the presence of cathepsin B (+C). Data expressed as mean ⁇ SEM.
  • Figure 10 provides illustrative nucleotide and amino acids sequence of antibody variable domains and CDR and linker sequences of an anti-LIBs antibody clone.
  • Figure 11 provides illustrative nucleotide and amino acids sequence of antibody variable domains and CDR and linker sequences of an SCE5 antibody clone described in the examples.
  • SEQ ID NO: 4 Illustrative nucleotide sequence encoding amino acid sequence of single-chain (scFv) antibody against activated GPIIb/IIIa) (SCE5) including a leader sequence at nucleotides 1 to 138
  • SEQ ID NO: 8 Illustrative nucleotide sequence encoding amino acid sequence of single-chain (scFv) antibody against activated GPIIb/IIIa) (anti- LIBs)
  • antibody variable domains/regions, antigen binding fragments and parts thereof may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991.
  • antigen binding site or domain of naturally occurring antibodies is formed by the precise juxtaposition of typically six hypervariable loops (also regarded as complementary determining regions or CDR) provided by the light chain variable region and the heavy chain variable region and aided by more conserved framework regions of the variable domains.
  • antibody fragments or derivatives comprising an antibody variable region able to bind to the antigen include without limitation Fab, Fab', Fd, Fd', Fv, dAb, isolated CDR region, F(ab') 2 bivalent fragments, diabodies and liner antibodies.
  • EU numbering system of Kabat will be understood to mean the numbering of an antibody heavy chain is according to the EU index as taught in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda.
  • the EU index is based on the residue numbering of the human IgGl EU antibody.
  • references herein to a range of, e.g., residues, will be understood to be inclusive.
  • reference to "a region comprising amino acids 56 to 65 of SEQ ID NO: 1" will be understood to mean that the region comprises a sequence of amino acids as numbered 56, 57, 58, 59, 60, 61, 62, 63, 64 and 65 in SEQ ID NO: 1.
  • Antibody variable domain refers to a binding protein that is capable of interacting with or specifically binding to an antigen (e.g., a platelet receptor or molecule, such as a protein, e.g., a glycoprotein).
  • an antigen e.g., a platelet receptor or molecule, such as a protein, e.g., a glycoprotein.
  • the antibody variable domain can be an antigen binding fragment of an antibody (e.g., a Fv or a scFv, etc.).
  • Glycoprotein Ilb/IIIa Glycoprotein Ilb/IIIa (GPIIb/IIIa, also known as integrin al Ibp3 ) is an integrin complex found on platelets. It is a receptor for fibrinogen and von Willebrand factor and aids platelet activation.
  • the GPIIb/IIIa complex is formed via calcium-dependent association of gpllb and gpllla, a required step in normal platelet aggregation and endothelial adherence.
  • Platelet activation by ADP leads to the aforementioned conformational change in platelet gpIIb/IIIa receptors that induces binding to fibrinogen.
  • GPIIb For the purposes of nomenclature only and not limitation an exemplary sequence of human GPIIb is set out in NCBI Gene ID: 3674, NCBI Reference Sequence: NG_00833l.l.For the purposes of nomenclature only and not limitation an exemplary sequence of human GPIIIa is set out in NCBI Gene ID: 3690, NCBI Reference Sequence: NG_008332.2. Additional sequences of GPIIb and/or Ilia from other species can be determined using sequences provided herein and/or in publically available databases and/or determined using standard techniques (e.g., as described in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
  • the term "binds" in reference to the interaction of antibody variable domain or fusion protein comprising same with GPIIb/IIIa means that the interaction is dependent upon the presence of a particular structure (e.g., epitope) on the component.
  • a particular structure e.g., epitope
  • an antibody or antibody variable domain recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope "A”, the presence of a molecule containing epitope "A" (or free, unlabeled "A"), in a reaction containing labeled "A" and the protein, will reduce the amount of labeled "A" bound to the antibody or antibody variable domain.
  • the term "specifically binds" shall be taken to mean that the binding interaction between the antibody variable domain and GPIIb/IIIa is dependent on the presence of the antigenic determinant or epitope.
  • the binding region preferentially binds or recognizes a specific antigenic determinant or epitope even when present in a mixture of other molecules or organisms.
  • the antibody variable domain reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with the specific component or cell expressing same than it does with alternative antigens or cells. It is also understood by reading this definition that, for example, a binding region the specifically binds to a particular component may or may not specifically bind to a second antigen.
  • binding does not necessarily require exclusive binding or non-detectable binding of another antigen.
  • the term “specifically binds” can be used interchangeably with “selectively binds” herein.
  • reference herein to binding means specific binding, and each term shall be understood to provide explicit support for the other term. Methods for determining specific binding will be apparent to the skilled person. For example, a binding protein comprising the binding region of the disclosure is contacted with the component or a cell expressing same or a mutant form thereof or an alternative antigen. The binding to the component or mutant form or alternative antigen is then determined and a binding region that binds as set out above is considered to specifically bind to the component.
  • epitope (syn. "antigenic determinant”) shall be understood to mean a region of GPIIb/IIIa to which a protein comprising a antibody variable domain of an antibody binds. This term is not necessarily limited to the specific residues or structure to which the protein makes contact. For example, this term includes the region spanning amino acids contacted by the protein and/or at least 5 to 10 or 2 to 5 or 1 to 3 amino acids outside of this region.
  • the epitope is a linear series amino acids.
  • An epitope may also comprise a series of discontinuous amino acids that are positioned close to one another when GPIIb/IIIa is folded, that is, a "conformational epitope".
  • epitope is not limited to peptides or polypeptides.
  • the term “epitope” includes chemically active surface groupings of molecules such as sugar side chains, phosphoryl side chains, or sulfonyl side chains, and, in certain examples, may have specific three dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope or peptide or polypeptide comprising same can be administered to an animal to generate antibodies against the epitope.
  • a fusion protein comprising the antibody variable domain of the disclosure reduces or prevents binding of a recited antibody to GPIIb/IIIa, for example, a scFv consisting of a sequence set forth in SEQ ID NO: 1. This may be due to antibody variable domain binding to the same or an overlapping epitope as the scFv. It will be apparent from the foregoing that the protein need not completely inhibit binding of the antibody, rather it need only reduce binding by a statistically significant amount, for example, by at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Methods for determining competitive inhibition of binding are known in the art and/or described herein.
  • the antibody is exposed to GPIIb/IIIa either in the presence or absence of the protein. If less antibody binds in the presence of the protein than in the absence of the protein, the protein is considered to competitively inhibit binding of the antibody.
  • the competitive inhibition of binding is caused by the antigen binding domain of the protein on GPIIb/IIIa overlapping with the antigen binding domain of the antibody.
  • the ability of antibody variable domains and ADC comprising same as described herein to bind to their cognate receptor on activated platelets in vivo in an appropriate subject or model is determined in accordance with the present invention and examples.
  • “Overlapping" in the context of two epitopes means that two epitopes share a sufficient number of amino acid residues to permit a binding protein of the disclosure that binds to one epitope to competitively inhibit the binding of a recited antibody to GPIIb/IIIa that binds to the other epitope.
  • the "overlapping" epitopes share at least 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 amino acids.
  • recombinant shall be understood to mean the product of artificial genetic manipulation. Accordingly, in the context of an antibody or antigen binding fragment thereof, this term does not encompass an antibody naturally occurring within a subject’s body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, in it is to be considered an isolated protein comprising an antibody variable region. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein. A recombinant protein also encompasses a protein expressed by means of artificial genetic manipulation when it is within a cell.
  • protein shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex).
  • the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond.
  • non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.
  • polypeptide or "polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.
  • an "antibody” is generally considered to be a protein that comprises a variable region made up of a plurality of polypeptide chains, e.g., a polypeptide comprising a light chain variable region (VL) and a polypeptide comprising a heavy chain variable region (V H ).
  • An antibody also generally comprises constant domains, some of which can be arranged into a constant region, which includes a constant fragment or fragment crystallizable (Fc), in the case of a heavy chain.
  • a V H and a V L interact to form an Fv comprising an antigen binding region that is capable of specifically binding to one or a few closely related antigens.
  • a light chain from mammals is either a k light chain or a l light chain and a heavy chain from mammals is a, d, e, g, or m.
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, dlgA and IgY), class (e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , dlgAi and dIgA 2, IgAi and IgA 2 ) or subclass.
  • antibody and antibody variable domain also encompasses humanized antibodies and humanized antibody variable domain, primatized antibodies and monized antibody variable domain, human antibodies and human antibody variable domains , synhumanized antibodies and chimeric antibodies and synhumanized and chimeric antibody variable domain.
  • variable region refers to the portions of the light and/or heavy chains of an antibody as defined herein that specifically binds to an antigen and, for example, includes amino acid sequences of CDRs; i.e., CDR1, CDR2, and CDR3, and framework regions (FRs).
  • the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs.
  • V H refers to the variable region of the heavy chain.
  • V L refers to the variable region of the light chain.
  • CDRs complementarity determining regions
  • CDR1, CDR2, and CDR3 refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding.
  • Each variable region typically has three CDR regions identified as CDR1, CDR2 and CDR3.
  • the amino acid positions assigned to CDRs and FRs are defined according to Rabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as "the Rabat numbering system”.
  • V H FRS and CDRs are positioned as follows: residues 1-30 (FR1), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and 103- 113 (FR4).
  • V L FRS and CDRs are positioned as follows: residues 1- 23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-107 (FR4).
  • "Framework regions" (hereinafter FR) are those variable domain residues other than the CDR residues.
  • the term "Fv” shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a V L and a V H associate and form a complex having an antigen binding site, i.e., capable of specifically binding to an antigen.
  • the V H and the V L which form the antigen binding site can be in a single polypeptide chain or in different polypeptide chains.
  • an Fv of the disclosure (as well as any protein of the disclosure) may have multiple antigen binding sites which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means.
  • the V H is not linked to a heavy chain constant domain (C H ) 1 and/or the V L is not linked to a light chain constant domain (C L ).
  • exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab’ fragment, a F(ab’) fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., C H 2 or C H 3 domain, e.g., a minibody.
  • a “Fab fragment” consists of a monovalent antigen-binding fragment of an antibody, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means.
  • a "Fab' fragment” of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a V H and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner.
  • a Fab’ fragment can also be produced by recombinant means.
  • a “F(ab')2 fragment” of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • a "Fab 2 " fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a C H 3 domain.
  • a "single chain Fv” or “scFv” is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.
  • an "antigen binding fragment" of an antibody comprises one or more antibody variable domains of an intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, half antibodies and multispecific antibodies formed from antibody fragments.
  • mutant refers to a scFv (e.g., scFv mut ) which has undergone modification (e.g., deletion or truncation) of one or more amino acids using well known techniques to inactivate the receptor binding and/or functional activity of the scFv.
  • modification e.g., deletion or truncation
  • identity refers to the percentage number of amino acids that are identical or constitute conservative substitutions.
  • identity may be determined using sequence comparison programs such as GAP (Deveraux et al, 1984, Nucleic Acids Research 12, 387-395), which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
  • the terms “treating”, “treat” or “treatment” include administering a ADC described herein to thereby reduce or eliminate at least one symptom of a cancer or to slow progression of cancer. This term also encompasses treatment of a subject in remission to prevent or ameliorate a relapse or metastasis.
  • Cancers that may be treated include those selected from the group consisting of breast cancer, lung cancer, a glioblastoma, prostate cancer, pancreatic cancer, colon cancer, colorectal cancer, head and neck cancer, mesothelioma, kidney cancer, ovarian, oesophageal, squamous cell carcinoma, triple negative breast cancer, and non-small cell lung cancer.
  • an “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result.
  • the desired result may be a therapeutic or prophylactic or imaging result or an imaging and therapeutic or prophylactic result.
  • An effective amount can be provided in one or more administrations.
  • the term "effective amount” is meant an amount necessary to effect treatment of a cancer as hereinbefore described.
  • the term "effective amount” is meant an amount necessary to effect a change in a factor associated with a disease or condition as hereinbefore described.
  • the effective amount may be sufficient to effect a beneficial change in the size of a solid tumour with for example, minimal systemic side effects.
  • the effective amount may vary according to the disease or condition to be treated or factor to be altered and also according to the weight, age, racial background, sex, health and/or physical condition and other factors relevant to the mammal being treated. Typically, the effective amount will fall within a relatively broad range (e.g. a "dosage" range) that can be determined through routine trial and experimentation by a medical practitioner. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., weight or number of binding proteins.
  • the effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.
  • a “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease or condition.
  • a therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antigen binding protein -drug conjugate to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the ADC are outweighed by the therapeutically beneficial effects.
  • a therapeutically effective amount shall be taken to mean a sufficient quantity of ADC to treat a cancer such as a cancer associated with a solid tumor and metastasis.
  • prophylactically effective amount shall be taken to mean a sufficient quantity of ADC to prevent or inhibit or delay the onset of cancer or the side effects of the cancer in a subject.
  • subject shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.
  • ADC Antibody Drug Conjugates
  • the present inventors have developed an scFv-drug conjugate directed at activated platelets such as those found within the tumor microenvironment. They have determined its ability in vivo to concentrate potent cytotoxic agent within solid primary tumors and metastasis of the MDA-MB-231 murine metastatis model for triple negative breast cancer, and provide significant regression of primary tumors and prevention of metastases without systemic side effects.
  • the present disclosure provides an Antibody-Fragment- Drug-Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and (ii) a drug which mediates its effect in the vicinity of the activated platelet conjugated in releasable from the (i).
  • ADC Antibody-Fragment- Drug-Conjugate
  • an Antibody-Fragment- Drug-Conjugate comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and (ii) a drug construct comprising a prodrug of a highly potent cytotoxic agent such as an auristatin or equivalent cytotoxic agent conjugated to (i).
  • a site-directed enzymatic conjugation method using sortase A to catalyse the condensation reaction is used to conjugate an antibody variable domain with a C-terminal FPETG tag to a triglycine bridge or other spacer such as glycine at the N-terminus of a cytotoxic drug construct.
  • the scFvGPIIb/IIIA- MMAE ADC displayed a band at 34kDa after western blotting ( Figure 2B) and the specific binding of the antibody variable domain (scFV) to activated platelets was retained after conjugation to MMAE by the present method (Figure 2C).
  • the drug construct comprises a cleavable linker which, upon cleavage in the tumor microenvironment, releases the active cytotoxic drug which is able to induce tumor regression.
  • the drug construct comprises a cleavable linker which, upon cleavage in the tumor microenvironment, releases the drug which is able to mediates its effect in the vicinity of the activated platelet.
  • the drug construct comprises a cleavable linker which, upon cleavage in the tumor microenvironment, releases the drug which is able to induce tumor regression.
  • the present application illustrates the use of a cathepsin cleavable contruct to activate the pro-drug.
  • a pro-drug approach may not be required.
  • Other illustrative microenvironment enzymes include MMPs and FAP.
  • MMPs Since proteinases and peptidases are abundant in the tumor interstitial space, therapeutic nanosystems fused with peptides that are specific substrates of these enzymes can be designed to control the release of therapeutic agents within the tumor microenvironment.
  • MMPs with an elevated expression in tumor ECM, are crucial in tumor progression. Therefore, making use of MMP-cleavable sequences achieves drug release or active site exposure.
  • the substrate peptide for MMP-2 is GPLGIAGQ; this sequence is cleaved into GPLG and IAGO by MMP-2.
  • Fibroblast activation protein-a is another accessible protease that is specifically expressed on the surface of CAFs, a major cellular component in the tumor microenvironment.
  • FAP-a selectively cleaves the sequence GPAX (X designates any amino acid) between proline, and alanine (Ji et ah, Angew Chem Int Ed Engl 55:1050-1055, 2016; Qin et al Molecular Pharmacology 92(3): 219- 231).
  • an Antibody-Fragment- Drug-Conjugate comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet-specific GPIIb/IIIa and part of a C-terminal sortase A sequence conjugated to the N-terminus of (ii) a drug construct comprising an N-terminal nucleophilic group (e.g.
  • glycine or polyglycine e.g., GGG sequence linked via a peptidase cleavable linker and optionally a spacer to a cytotoxic agent such as an auristatin or equivalent cytotoxic agent.
  • an Antibody-Fragment- Drug-Conjugate comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet-specific GPIIb/IIIa and part of a C-terminal sortase A conjugated to the N-terminus of (ii) a drug construct comprising an N-terminal polyglycine (e.g. GGG) sequence linked via a cleavable linker and a spacer to a cytotoxic agent such as an auristatin or equivalent cytotoxic agent.
  • ADC Antibody-Fragment- Drug-Conjugate
  • the present disclosure is of an ADC comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet-specific GPIIb/IIIa and a C-terminal sortase A recognition sequence and (ii) a cathepsin B dependent cytotoxic prodrug comprising an N-terminal polyglycine (e.g. GGG) sequence linked via a cathepsin B cleavable peptide linker to an auristatin or equivalent cytotoxic agent.
  • a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet-specific GPIIb/IIIa and a C-terminal sortase A recognition sequence
  • a cathepsin B dependent cytotoxic prodrug comprising an N-terminal polyglycine (e.g. GGG) sequence linked via a cathepsin B cleavable peptide linker to an auristatin or equivalent cytotoxic agent
  • the antibody variable domain binds an epitope of GPIIb/IIIa recognised by a scFv consisting of a sequence set forth in SEQ ID NO: 1.
  • the antibody variable domain comprises a complementary determining region (CDR) of the heavy chain variable region (V H ) having the sequence of SEQ ID NO:2 and/or a CDR of the light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 3.
  • CDR complementary determining region
  • the antibody variable domain comprises a heavy chain variable region (VH) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 2 and a light chain variable region (VL) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 3.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody variable domain comprises an amino acid sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 1.
  • antibody variable domain variants encompassed comprise one to four or two to eight amino acid substitutions and retain high affinity specific binding to activated GPIIb/IIIa.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys,
  • sequence identity are understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Conservative substitutions known in the art may also be assessed and included in the percent identity calculation. As known in the art, even non-conservative substitutions within CDR regions are tolerated and can be routinely tested.
  • the antibody variable domain is derived from a human scFv library.
  • the antibody variable domain is scFVGPiib/iiia that binds only to the activated form of platelet- specific GPIIb/IIIa having the amino acid sequence set out in SEQ ID NO:l.
  • the ADC comprises the Val-Citrulline cleavable linker.
  • the ADC comprises a spacer wherein the spacer is a self immolative spacer, such as a para-aminobenzylalcohol (PABA) spacer.
  • PABA para-aminobenzylalcohol
  • the ADC comprises (i) a fusion protein comprising scFV GPiib/iiia and a C-terminal sortase A recognition sequence and (ii) an N-terminal polyglycine sequence linked via a cleavable linker and immolative spacer to MMAE.
  • cytotoxic agent includes cytotoxic agents that are active at picomolar concentrations.
  • the cytotoxic agent is a mitotic inhibitor or an anti-tumor antibiotic or a plant alkaloid.
  • Illustrative mitotic inhibitors include a dolastatin, an auristatin, a maytansinoid, and a plant alkaloid.
  • auristatin refers to a family of antimitotic agents. Auristatin derivatives are also included within the definition of the term "auristatin”.
  • auristatins include, but are not limited to, auristatin E (AE), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), and synthetic analogs of auristatin.
  • Maytansinoids include, for example, DM1, DM2, DM3, and DM4.
  • anti-tumor antibiotics are selected from the group consisting of an actinomycine (e.g., PBD), an anthracycline, a calicheamicin, and a duocarmycin.
  • the chemotherapeutic agent is selected from a chemical compound useful in the treatment of cancer, regardless of mechanism of action.
  • Classes of chemotherapeutic agents include, but are not limited to: alkyating agents, antimetabolites, spindle poison plant alkaloids, cytoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors.
  • Chemotherapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy.
  • chemotherapeutic agents include: erlotinib docetaxel), 5-FU, gemcitabine, cisplatin, carboplatin, paclitaxel, trastuzumab temozolomide, tamoxifen, doxorubicin and rapamycin.
  • chemotherapeutic agents include: oxaliplatin imatinib mesylate, Mek inhibitor, PI3K inhibitor, fulvestrant, leucovorin), rapamycin, lapatinib, lonafarnib sorafenib gefitinib irinotecan tipifarnib, albumin-engineered nanoparticle formulations of paclitaxel vandetanib chloranmbucil, AG1478, AG1571, temsirolimus, pazopanib canfosfamide, thiotepa and cyclosphosphamide alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophospho
  • chemotherapeutic agent or “drug” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands; (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a l,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras,
  • SERMs selective
  • Immune modulators are an important class of compounds that are directed to activated platelets in concentrated form by the present invention. Examples include check point inhibitors, TLR agonists.
  • chemotherapeutic agent therapeutic antibodies such as alemtuzumab, bevacizumab, cetuximab, panitumumab rituximab pertuzumab trastuzumab tositumomab and the antibody drug conjugate, gemtuzumab.
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies.
  • the antibody variable domain, fusion protein or ADC as described herein comprises a detectable label.
  • detectable labels are suitable for imaging and tracing tumors.
  • Detectable labels include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin and the like.
  • Detectable labels include detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. As well understood, detectable enzyme are detected by adding enzyme substrates to produce a detectable reaction product.
  • An antibody or ADC may also be labelled with biotin, and detected through indirect measurement of avidin or streptavidin binding.
  • the antibody variable domain is conjugated to an imaging agent/label/dye known in the art.
  • imaging agents include, but are not limited to, MRI contrast agents such as Gd, Mn and F etc, a radiolabel, radiotracer, an enzyme, a fluorescent label or dye a luminescent label, a bioluminescent label, a magnetic label, and biotin.
  • Contrast agents for molecular imaging of cancer using a range of distinct molecular imaging modalities are provided.
  • Illustrative modalities include without limitation, MRI, fluorescence imaging (e.g. Cy7 and FLECT imaging) and molecular imaging via for example PET/CT (e.g. 64 Cu) and ultrasound (e.g., ultrasound enhancing microbubbles such as streptavidin-coated microbubbles, and photoacoustic imaging).
  • the ADC as described herein comprises a 1 : 1 ratio of antibody variable domain to pro-drug.
  • the ratio in the ADC or composition is about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
  • vectors comprising the polynucleotide sequences encoding the binding proteins of the present invention are contemplated.
  • Illustrative polynucleotide sequences are described in Figure 8 and 9 and in the sequence listing, or sequences having at least 90%, 95% or 99% sequence identity.
  • Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, BHK, VERO, HT1080, 293, 293T, 293F, RD, COS-7, CHO, Jurkat, HUT, SUPT, C8166, HepG2, MOLT4/clone8, MT-2, MT-4, H9, PM1, CEM, myeloma cells (e.g., SB20 cells) and CEMX174, HEK293T, NSO, SP2 cells, HeLa cells, A549 cells, 3T3 cells, and a number of other cell lines.
  • ATCC American Type Culture Collection
  • Other cells that may be used include insect cell lines, such as Sf9cells, avian, amphibian cells, plant cells, yeast and fungal yeast cells.
  • a non mammalian cell may be employed such as a yeast cell, such as Hansenula polymorpha. Such cells are useful for providing controlled levels of expression.
  • Vectors available for cloning and expression in host cell lines are well known in the art, and include but are not limited to vectors for cloning and expression in mammalian or yeast cell lines, vectors for cloning and expression in bacterial cell lines, vectors for cloning and expression in phage and vectors for cloning and expression insect cell lines.
  • the fusion proteins can be recovered using standard protein purification methods.
  • the composition comprises an Antibody-Fragment-Drug-Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and (ii) a drug construct comprising a prodrug of a highly potent cytotoxic agent such as an auristatin or equivalent cytotoxic agent conjugated to
  • ADC Antibody-Fragment-Drug-Conjugate
  • the composition comprises an Antibody-Fragment-Drug- Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and part of a C-terminal sortase A sequence conjugated to the N-terminus of (ii) a drug construct comprising an N-terminal glycine sequence linked via a cleavable linker and optionally a spacer to a cytotoxic agent such as an auristatin or equivalent cytotoxic agent.
  • ADC Antibody-Fragment-Drug- Conjugate
  • the composition comprises a pharmacologically or physiologically acceptable diluent and/or carrier.
  • pharmaceutically acceptable carrier or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction.
  • Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emusifying agents, pH buffering agents, preservatives, and the like.
  • a variety of acceptable carriers known in the art may be used, as for example described in Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991).
  • the composition comprises ADC wherein the ratio of antibody variable domain to pro-drug is substantially 1:1.
  • each antibody or each antibody variable domain is conjugated to 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug molecules.
  • sortase A mediated conjugation of the drug construct to the fusion protein comprising the antibody variable domain produces an essentially homogeneous ADC with substantially no undesired species having heterogenous numbers of drug to antibody ratios. This is useful for both medical and imaging applications.
  • composition comprising the ADC as described herein is for use in (or used for) medical therapy or medical imaging.
  • the ADC is preferably administered in a therapeutically effective amount.
  • the actual amount administered and the rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington’s Pharmaceutical Sciences, supra.
  • the binding protein is administered parenterally, such as subcutaneously or intravenously.
  • the binding protein administered intravenously.
  • Formulation of a ADC to be administered will vary according to the route of administration and formulation (e.g., solution, emulsion, capsule) selected.
  • An appropriate pharmaceutical composition comprising an ADC to be administered can be prepared in a physiologically acceptable carrier as discussed herein.
  • suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • aqueous carriers include water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, l6th Edition, Mack, Ed. 1980).
  • the compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate.
  • the ADC can be stored in the liquid stage or can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.
  • the present disclosure also provides for the use of a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet-specific GPIIb/IIIa and a C-terminal sortase A recognition sequence in the preparation of a cancer imaging agent.
  • the disclosure provides an fusion protein comprising the antibody variable domain as described herein and a detectable label suitable for imaging.
  • the disclosure provides a vector or host cell comprising a polynucleotide sequence capable of expressing the fusion protein comprising the fusion proteins comprising the antibody variable domain as described herein.
  • the antibody variable domain is conjugated to a bifunctional sarcophagine chelator, MeCOSar and labelled with copper- 64 .
  • cancer cells induce activation and can be imaged using ScFv GPiib/ ni a -Cy7-MMAE in vitro and in vivo.
  • imaging can be carried out using the antibody variable domain linked to a detectable agent.
  • the antibody variable domain is conjugated to a detectable label via sortase A mediated conjugation. Fusion proteins comprising the antibody variable domain and a sortase A recognition sequences can conveniently be produce recombinantly.
  • One non-limiting illustrative cancer imaging agent is ScFv GPiib/ ni a -Cy7.
  • Detectable labels include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin and the like.
  • Detectable labels include detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. As well understood, detectable enzyme are detected by adding enzyme substrates to produce a detectable reaction product.
  • An antibody or ADC may also be labelled with biotin, and detected through indirect measurement of avidin or streptavidin binding.
  • the antibody variable domain is conjugated to an imaging agent/label/dye known in the art.
  • imaging agents include, but are not limited to, a radiolabel, radiotracer, an enzyme, a fluorescent label or dye a luminescent label, a bioluminescent label, a magnetic label, and biotin.
  • Contrast agents for molecular imaging of cancer using a range of distinct molecular imaging modalities include, fluorescence imaging (e.g. Cy7) and molecular imaging via for example PET/CT (e.g. 64 Cu) and ultrasound (e.g., ultrasound enhancing microbubbles such as streptavidin-coated microbubbles)
  • negative control imaging agents include mutated antibody variable domains.
  • Illustrative controls include scFvmut-detectable label formats.
  • Cancer imaging agents and controls as described herein may be provided in the form of kits for cancer imaging. Kits are optionally provided with instructions for use in cancer imaging.
  • Kits comprising vectors or host cells expressing a fusion protein comprising the antigen variable domain as described herein and a sortase A recognition sequence are contemplated.
  • Vectors or host cells expressing a negative control imaging agents include mutated antibody variable domains and a sortase A recognition sequence.
  • the present disclosure enables the use of a composition comprising the ADC as described herein in, or in the preparation of a medicament for, the treatment of cancer including reduction of solid tumors and reduced metastasis in a subject diagnosed with cancer.
  • the composition comprises an Antibody- Fragment-Drug-Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and (ii) a drug construct comprising a prodrug of a highly potent cytotoxic agent such as an auristatin or equivalent cytotoxic agent conjugated to (i).
  • ADC Antibody- Fragment-Drug-Conjugate
  • the composition comprises an Antibody-Fragment-Drug- Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and part of a C-terminal sortase A sequence conjugated to the N-terminus of (ii) a drug construct comprising an N-terminal glycine sequence linked via a cleavable linker and optionally a spacer to a cytotoxic agent such as an auristatin or equivalent cytotoxic agent.
  • ADC Antibody-Fragment-Drug- Conjugate
  • FIG. 1 An illustrative ADC is shown in Figure 1 being produced by sortase A mediated site specific biological conjugation and then cleaved by cathepsin B to release the active cytotoxic agent MMAE.
  • tumor microenvironment is pre-identified as comprising abundant activated platelets.
  • the cancer is selected from the group consisting of breast cancer, lung cancer, a glioblastoma, prostate cancer, pancreatic cancer, colon cancer, colorectal cancer, head and neck cancer, mesothelioma, kidney cancer, ovarian, oesophageal, squamous cell carcinoma, triple negative breast cancer, and non- small cell lung cancer.
  • the disclosure enables a method of treating cancer and of reducing metastasis comprising administering an effective amount of a composition comprising an ADC as described herein to a subject with cancer.
  • the composition comprises an Antibody-Fragment-Drug-Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and (ii) a drug construct comprising a prodrug of a highly potent cytotoxic agent such as an auristatin or equivalent cytotoxic agent conjugated to (i).
  • ADC Antibody-Fragment-Drug-Conjugate
  • the composition comprises an Antibody-Fragment-Drug- Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and part of a C-terminal sortase A sequence conjugated to the N-terminus of (ii) a drug construct comprising an N-terminal polyglycine (e.g. GGG) sequence linked via a cleavable linker and optionally a spacer to a cytotoxic agent such as an auristatin or equivalent cytotoxic agent.
  • ADC Antibody-Fragment-Drug- Conjugate
  • the effective amount concentrates therapeutic doses of cytotoxic agent within the tumor and at sites of metastasis.
  • the same amount of ADC comprising a non-binding (mutant) antibody provides substantially no therapeutic/cytotoxic/tumor/metastasis reduction activity.
  • the effective amount also has substantially no or minimal off-target side effects on haematological parameters, liver function and renal function comparable to vehicle control.
  • the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.
  • the composition comprises an effective amount of the binding protein.
  • the composition comprises a therapeutically effective amount of the binding protein.
  • the composition comprises a prophylactically effective amount of the binding protein.
  • the dosage should not be so large as to cause adverse side effects.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any complication.
  • Dosages can vary from about 0.01 mg/kg to about 5 mg/kg, or 0.01 -5 mg/kg or 0.1 - 2 mg/kg or 0.03 - 2 mg/kg, or 0.05 mg/kg to about 100 mg/kg, e.g., from about 0.1 mg/kg to about 50 mg/kg, such as, from about 0.5 mg/kg to about 20 mg/kg or 2 mg/kg to about 5 mg/kg, in one or more dose administrations daily for one or several days or weekly or every four days for three to six weeks or more. Please advise if a different dose is preferred.
  • the ADC is administered at an initial (or loading) dose which is higher than subsequent (maintenance doses).
  • the ADC is administered at an initial dose of between about 1 mg/kg to about 50mg/kg.
  • the binding protein is then administered at a maintenance dose of between about O.OOOlmg/kg to about lOmg/kg.
  • the maintenance doses may be administered every 7- 35 days, such as, every 7 or 14 or 28 days.
  • a dose escalation regime is used, in which a ADC is initially administered at a lower dose than used in subsequent doses.
  • multiple doses in a week may be administered.
  • increasing doses may be administered.
  • a subject may be retreated with ADC, by being given more than one exposure or set of doses, such as at least about two exposures of the binding protein, for example, from about 2 to 60 exposures, and more particularly about 2 to 40 exposures, most particularly, about 2 to 20 exposures.
  • any retreatment may be given after imaging with the labelled antibody variable domain protein to confirm the presence of activated platelets in the tumor.
  • any retreatment may be given at defined intervals.
  • subsequent exposures may be administered at various intervals, such as, for example, about 24-28 weeks or 48-56 weeks or longer.
  • such exposures are administered at intervals each of about 24-26 weeks or about 38-42 weeks, or about 50-54 weeks.
  • given the ability to administer an effective dose without substantial systemic side effects means that the patient may tolerate long term treatment, if required.
  • the ADC is administered systemically and displays minimal systemic side effects.
  • the ADC is concentrated in the tumor microenvironment by binding of the antibody variable domain to activated platelets within the tumor microenvironment and wherein the cytotoxic agent is activated in the tumor microenvironment by endogenous cleavage of the ADC within the tumor microenvironment.
  • Subjects contemplated in the present description include patients including humans or animals including laboratory animals or art accepted test animals. Patients include human subjects in need of treatment or prophylaxis.
  • the ADC is administered in combination with an additional cytotoxic or therapeutic agent.
  • the additional agent is selected from the group consisting of an anti-apoptotic agent, a mitotic inhibitor, an anti-tumor antibiotic, an immunomodulating agent, a nucleic acid for gene therapy, an alkylating agent, an anti-angiogenic agent, an anti-metabolite, a boron-containing agent, a chemoprotective agent, a hormone agent, an anti-hormone agent, a corticosteroid, a photoactive therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor, and a tyrosine kinase inhibitor.
  • scFv single-chain antibody
  • the scFv was conjugated to three different contrast agents; Cy7 for fluorescence imaging, 64 Cu for PET imaging and microbubbles for ultrasound imaging. This novel approach provides holds promise as a universal and flexible diagnostic method for the detection and imaging of cancer.
  • a subject is screened using the binding protein-imaging agent and, if positive, is treated with the binding protein-therapeutic agent.
  • Theranostic methods are enabled comprising (i) screening for cancer by detection with the binding agent-imaging agent conjugate, followed by (ii) treatment with the binding agent-therapeutic drug conjugate as described and enabled herein.
  • scFv -Biotin The generation of biotinylated scFv GPiib/iiia and scFv mut has been described previously. Both scFvs were sub-cloned into the AviTagTM containing pAC6 vector system, the DNA was then transformed into electrocompetent cell E. coli EVB101 (Avidity LLC) by electroporation. In vivo biotinylation was performed according to the manufacturer’s instruction producing the attachment of one biotin molecule on every scFv.
  • Alternative tags are known in the art, such as a spycatcher/spyTag system.
  • the resultant scFv-BCN was further conjugated to an azide-NIR dye (azide-Cyanine 7 dye) via copper-free click reaction to generate the FFECT tracer.
  • azide-NIR dye azide-Cyanine 7 dye
  • Excess free dye was dialysed in PBS and the purified scFv- Cy7 was analyzed on a SDS-PAGE gel and NIR signal from the band of interest was confirmed using the Odyssey Imager.
  • 64 Cu Production and scFv Radiolabeling - 64 CuCl 2 produced by the 64 Ni(p,n) 64 Cu reaction was obtained from the Austin Health Centre for PET. The solution was provided with a radionuclidic purity, tested using gamma ray spectrometry of >99% and a radiochemical purity, tested using HPFC of >95%.
  • the scFv used for PET/CT imaging were conjugated to a sarcophagine chelator, MeCOSar (Clarity Pharmaceuticals) 21,22 .
  • MeCOSar MeCOSarity Pharmaceuticals
  • diethylenetriaminepentaacetic acid (10 pF, 10 mM) was added to the mixture and incubated for a further 5 min at room temperature. Samples were washed twice with PBS in spin columns (Millipore, cutoff 10,000 MWCO) and resuspended in PBS at a final concentration of 0.2 mg/mL to produce scFv GPiib/ ni a - 64 Cu or scFv mut - 64 Cu.
  • Cancer Cell Lines -Human cancer cell lines SKBr3, MDA-MB-231, Ramos and HT- 1080 were cultured in RPMI media (GIBCO® #21870) supplemented with 10% (v/v) FBS (Invitrogen), 100 ET/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in a 5% C0 2 humidified atmosphere.
  • Preparation of Human Washed Platelets -Fresh blood was drawn from informed healthy volunteers who had not taken anti-platelet drugs at least two weeks prior to venesection. Blood was collected into acid citrate dextrose (ACD; 85 mM sodium citrate, 72.9 mM citric acid, 110 mM D-glucose, 70 mM theophylline) (at a ratio of ACD:blood of 1:6) and supplemented with apyrase 0.005 U/mF and enoxaparin 20 U/mF. Whole blood was centrifuged at 200 g for 10 min. Platelet rich plasma (PRP) was obtained and then centrifuged at l700g for 7 min.
  • ACD acid citrate dextrose
  • PRP Platelet rich plasma
  • PPP platelet poor plasma
  • platelet washing buffer pH 6.5; 4.3 mM K 2 HP0 4 ; 4.3 mM Na 2 HP0 4 ; 24.3 mM NaH 2 P0 4 ; NaCl 0.113 M; 5.5 mM D-glucose; 10 mM theophylline
  • enoxaparin 20 U/mL
  • apyrase O.OlU/mL
  • washed platelets were resuspended in Tyrode’s buffer (pH 7.2- 7.4; 12 mM NaHCOs; Hepes 10 mM; NaCl 0.137 M; KCL 27 mM; D-glucose 55 mM) containing 1 mM CaCl and apyrase 0.02 U/mL.
  • Tumour Xenograft Model - 5-6 weeks old female BALB/c nude mice were purchased from the Animal Resources Centre, Canningvale. To establish tumour xenografts, mice were injected subcutaneously with exponentially growing SKBr3, MDA-MB-231, Ramos or HT-1080 cells (2.0 x 10 6 cells per mouse) in 0.3 ml of matrigel (BD Biosciences #356234) into the left flank region. Tumours were left to grow and measured daily until reaching a diameter of approximately 4 mm, which usually appears 2-3 weeks post xenograft cell injection.
  • ROI region-of-interest
  • MFI mean fluorescence intensity
  • PET Imaging In vivo PET Imaging -Animals were injected intravenously with 20 pg scFv GPiib/iiia - 64 Cu or scFv mut - 64 Cu. Tracer was allowed to circulate for two hours. Animals were anesthetized with ketamine (50 mg/kg; Parnell Laboratories) and xylazine (10 mg/kg; Troy Laboratories) and placed in the PET/CT scanner supplied with continuous 0 2 and 2% isofluorane. PET/CT imaging was performed using a NanoPET/CT in vivo Preclinical Imager (Mediso) with a 30 min PET acquisition time, and coincidence mode of 1:3.
  • Tumour sections were stained overnight with a polyclonal rabbit anti-CD4l antibody (Abeam #ab63983), and detected with an Alexa Fluor 647 labeled anti-rabbit antibody (Life Technologies #A-2l245), counterstained with Hoechst® (Thermo Fisher Scientific #33342) and visualized using the Nikon Alr Plus Confocal Microscope, 60x oil objective.
  • the central element of the imaging study is the ability to target and image activated platelets as a general component of the tumour microenvironment, thereby defining a novel non-invasive technique to diagnose and localize tumours in vivo.
  • the results demonstrate the feasibility of Cy7, 64 Cu and microbubbles as conjugates to the scFv GPiib/ ni a for imaging tumours in vivo.
  • the ability to use the recombinant single-chain antibody scFv GPiib/ ni a which specifically binds to activated platelets have been characterized extensively in previous in vivo studies, and thus provides a solid foundation to conclude based on the studies described herein that activated platelets can serve as a marker of the tumour microenvironment.
  • the specificity and sensitivity of the scFv GPiib/ ni a allowed strong targeting to activated platelets and thus represents a unique foundation of this molecular target as a unique approach for tumour imaging.
  • imaging of activated platelets in cancer can be used as an auxiliary imaging strategy to allow the accurate delineation of the anatomic distribution of tumours..
  • the scFvG Piib/ ni a illustrates the important role for targeting activated platelets in tumor therapy and the use of activated platelet- specific antibody variable domain targeted drug delivery as means to enhance the efficacy, whilst minimizing the systemic toxicity of chemotherapy.
  • the protein comprising an antibody variable domain of the present disclosure comprises a binding region that is an inhibitor of GPIIb/IIIa receptor function and/or activity.
  • the binding region specifically binds an epitope on GPIIb/IIIa recognised by a scFv consisting of a sequence set forth in SEQ ID NO: 1.
  • the binding region competitively inhibits binding of a scFv consisting of a sequence set forth in SEQ ID NO: 1 to an epitope on GPIIb/IIIa.
  • the binding region comprises an antibody variable region, e.g., is an antibody or an antibody fragment that binds to GPIIb/IIIa.
  • the antibody variable region binds specifically to activated GPIIb/IIIa.
  • Suitable antibodies and proteins comprising variable regions thereof are known in the art and/or described herein.
  • the binding protein comprises a binding region, wherein the binding region is a protein comprising a Fv.
  • the protein comprises a single chain Fv fragment (scFv).
  • scFv Single Chain Fv
  • Fragments comprise VH and VL regions in a single polypeptide chain and a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv).
  • the linker comprises the sequence SSGS.
  • the present disclosure also contemplates a disulfide stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of VH and a FR of VL and the cysteine residues linked by a disulfide bond to yield a stable Fv.
  • a dimeric scFv i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun).
  • two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367.
  • a humanized antibody or fragment thereof e.g., a protein comprising a human like variable region, which includes CDRs from an antibody from a non-human species (e.g., mouse or rat or non-human primate) grafted onto or inserted into FRs from a human antibody (e.g., produced by methods described in US5225539, US6054297, US7566771 or US5585089) • a human antibody or fragment thereof, e.g., antibodies having variable and, optionally, constant antibody regions found in humans, e.g. in the human germline or somatic cells or from libraries produced using such regions.
  • the "human” antibodies can include amino acid residues not encoded by human sequences, e.g. mutations introduced by random or site directed mutations in vitro (e.g., produced by methods described in US5565332) and affinity matured forms of such antibodies.
  • a synhumanized antibody or fragment thereof e.g., an antibody that includes a variable region comprising FRs from a New World primate antibody variable region and CDRs from a non-New World primate antibody variable region (e.g., produced by methods described in W02007019620).
  • a primatized antibody or fragment thereof e.g., an antibody comprising variable region(s) from an antibody generated following immunization of a non-human primate (e.g., a cynomolgus macaque) (e.g., produced by methods described in US6113898).
  • a non-human primate e.g., a cynomolgus macaque
  • a chimeric antibody or chimeric antigen binding fragment e.g., an antibody or fragment in which one or more of the variable domains is from a particular species (e.g., murine, such as mouse or rat) or belonging to a particular antibody class or subclass, while the remainder of the antibody or fragment is from another species (such as, for example, human or non-human primate) or belonging to another antibody class or subclass (e.g., produced by methods described in US6331415; US5807715; US4816567 and US4816397).
  • a chimeric antibody or chimeric antigen binding fragment e.g., an antibody or fragment in which one or more of the variable domains is from a particular species (e.g., murine, such as mouse or rat) or belonging to a particular antibody class or subclass, while the remainder of the antibody or fragment is from another species (such as, for example, human or non-human primate) or belonging to another antibody class or subclass (e.g., produced by methods described
  • a deimmunized antibody or antigen binding fragment thereof e.g., antibodies and fragments that have one or more epitopes, e.g., B cell epitopes or T cell epitopes removed (i.e., mutated) to thereby reduce the likelihood that a subject will raise an immune response against the antibody or protein (e.g., as described in W02000034317 and W02004108158).
  • epitopes e.g., B cell epitopes or T cell epitopes removed (i.e., mutated) to thereby reduce the likelihood that a subject will raise an immune response against the antibody or protein (e.g., as described in W02000034317 and W02004108158).
  • a bispecific antibody or fragment thereof e.g., an antibody comprising two types of antibodies or antibody fragments (e.g., two half antibodies) having specificities for different antigens or epitopes (e.g., as described in US5731168).
  • Additional exemplary antibody fragments for use in the present disclosure are described herein or known in the art and include:
  • single-domain antibodies domain antibody or dAb
  • a single polypeptide chain comprising all or a portion of the heavy chain variable domain of an antibody.
  • a diabody, triabody, tetrabody or higher order protein complex e.g., as described in W098/044001, W094/007921 see also Kim et al Mol Cancer Ther 2008; 7(8) 2008).
  • a half-antibody or a half-molecule e.g., a protein comprising a single heavy chain and a single light chain.
  • the present disclosure also contemplates other antibodies and antibody fragments, such as:
  • heteroconjugate proteins e.g., as described in US4676980;
  • heteroconjugate proteins produced using a chemical cross-linker, e.g., as described in US4676980;
  • the present disclosure provides an ADC comprising one or more linkers.
  • the present disclosure contemplates various forms of covalent and non- covalent linkages.
  • the regions can be linked by a chemical or flexible or peptide linker.
  • Peptide linkers may comprise between 2 and 30 amino acids in length. Linkers may comprise non-naturally occurring amino acids.
  • the linker sequence is at least about 3 amino acids in length.
  • a linker comprises the sequence (Ala) 3 .
  • a "flexible" linker is an amino acid sequence which does not have a fixed structure (secondary or tertiary structure) in solution. Such a flexible linker is therefore free to adopt a variety of conformations.
  • Flexible linkers suitable for use in the present disclosure are known in the art. Flexible linkers are also disclosed in WO1999045132.
  • the linker may comprise any amino acid sequence that does not substantially hinder interaction of the binding region with its target.
  • Preferred amino acid residues for flexible linker sequences include, but are not limited to, glycine, alanine, serine, threonine proline, lysine, arginine, glutamine and glutamic acid.
  • the linker is a rigid linker.
  • a "rigid linker” refers to a linker having limited flexibility.
  • the relatively rigid linker comprises the sequence (EAAAK) n , where n is between 1 and 3.
  • the value of n can be between 1 and about 10 or between about 1 and 100.
  • n is at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10.
  • n is less than 100.
  • n is less than 90, or less than about 80, or less than about 60, or less than about 50, or less than about 40, or less than about 30, or less than about 20, or less than about 10.
  • a rigid linker need not completely lack flexibility.
  • the linker is a cleavable linker.
  • the linker comprises a cleavage site for a peptidase.
  • the linker comprises a cleavage site for urokinase, pro-urokinase, plasmin, plasminogen, TGFP, staphylokinase, Thrombin, a coagulation factor (e.g., Factor IXa, Factor Xa) or a metalloproteinase, such as an interstitial collagenase, a gelatinase or a stromelysin.
  • Exemplary cleavable linkers are described in US6,004,555, US5,877,289, US6,093,399 and US5,877,289.
  • linkers include hydrazone linkers, disulphide linkers, peptide linkers and beta-glucuronide linkers.
  • Stable conjugation of antibody or smaller antibody fragments known in the art to drugs can be achieved using various different approaches and methods.
  • Chemical strategies include amide linkages, Schiff base linkages, hydrazone formation (unstable in acidic pHs), thiols and click reaction technologies resulting in for example stable l,2,3-triaazole linkages.
  • specific conjugation strategies have been developed with recombinant antibody fragments, these include streptavidin-biotin links, peptide bonds formed by sortase A peptidase between LPXTG and an N-terminal glycine, other engineer able site specific modifications include sulfhydryl groups His6- tags, N-terminal serine/threonine, and the above mentioned enzyme tags/SNAP tags.
  • SNAP tag technology may be used to conjugate (covalently couple) a single chain fragment variable drugs such as auristatin F via benzylguanine (BG).
  • AURIF is a BG-modified version of MMAF suitable for SNAP-tag coupling.
  • the SNAP-tag is an engineered version of the human DNA repair enzyme 06-alky lguaninc-DNA- alkyltransferase, which allows the covalent coupling of BG-modified components with a defined 1:1 stoichiometry.
  • ADCs may employ chemical linkage to cysteine or lysine residues in mAbs, resulting in a heterogeneous mixture of products with a undefined drug-to-antibody ratio (DAR) and varying conjugation sites.
  • DAR drug-to-antibody ratio
  • SNAP- tag and similar strategies can overcome these limitations.
  • the binding protein of the disclosure is conjugated to a detectable label, for example, a fluorescent label or a radioactive label.
  • the labeled protein and the test binding protein are then mixed and contacted with GPIIb/IIIa or a peptide comprising an epitope thereof.
  • the level of labeled protein is then determined and compared to the level determined when the labeled protein is contacted with the GPIIb/IIIa or the peptide comprising an epitope thereof in the absence of the binding protein. If the level of labeled protein is reduced in the presence of the binding protein compared to the absence of the binding protein, the binding protein competitively inhibits binding of the scFv.
  • the epitope bound by a protein described herein is mapped.
  • Epitope mapping methods will be apparent to the skilled artisan. For example, a series of overlapping peptides spanning the GPIIb/IIIa sequence or a region thereof comprising an epitope of interest, for example, peptides comprising 10 to 15 amino acids are produced. The binding protein is then contacted to each peptide or a combination thereof and the peptide(s) to which it binds determined. This permits determination of peptide(s) comprising the epitope to which the binding protein binds. If multiple non-contiguous peptides are bound by the protein, the protein may bind a conformational epitope.
  • amino acid residues within GPIIb/IIIa are mutated, for example, by alanine scanning mutagenesis, and mutations that reduce or prevent protein binding are determined. Any mutation that reduces or prevents binding of the binding protein is likely to be within the epitope bound by the protein.
  • the present disclosure includes the following non-limiting Examples.
  • scFv was then subcloned into a pSectag 2A vector (Invitrogen) for expression in human embryonic kidney (HEK) cells (Invitrogen), see Hohmann et al. Blood 2013: 12l (6):3067-3075.
  • Sortase A is used to induce an enzymatic reaction used for the conjugation of the scFv, carrying an LPETG sequence to MMAE which was produced carrying a triple glycine sequence.
  • Sortase A a transpeptidase cloned from Staphylococcus aureus was produced and purified as previously described, see Wang et al. Theranostics 20l6;6(5):726-738. All proteins (scFvs and sortase A) contains a 6x His-tag, which was used for purification with nickel-based affinity chromatography (Invitrogen).
  • the purified scFv-Cy7- MMAE was then analyzed by SDS-PAGE gel and the protein and near-infrared signal from the band of interest was confirmed using the Odyssey Imager. Additionally, western blot was performed with rabbit anti-MMAE antibody (Levena Biopharma), detected with an anti-rabbit HRP antibody (Cell Signalling) to confirm conjugation of MMAE to the scFv.
  • Flow cytometry was performed using a FACS Fortessa scanner (BD Biosciences, Franklin Lakes, NJ, United States). Results were analyzed using the Flowlogic software.
  • platelet rich plasma was incubated with the cancer cell lines MDA-MB-231, HT29, HT1080 and PC3 for 6 hours at 37°C.
  • ADP-activated platelet rich plasma was incubated with the cancer cells for 6 hours at 37°C.
  • the cancer cell and platelet (resting/ ADP-activated platelets) mixtures were then stained with an anti-CD4l-PE monoclonal antibody (BD Biosciences, Franklin Lakes, NJ, United States) and scFv GPiib/iiia or scFv mut binding was detected by an anti-V5-FITC monoclonal antibody (Abeam, Cambridge, United Kingdom). Flow cytometry and analysis was performed as described above.
  • Cancer Cell Lines A metastatic variant of the MDA-MB-231 triple-negative breast adenocarcinoma cell line (a kind gift from Dr Zhou Ou, Fudan University Shanghai Cancer Center, China) was transduced with a lentiviral vector containing codon- optimized firefly luciferase-mCherry under the control of the ubiquitin-C promoterF 23,24 Li et al. European Journal of Cancer 2006;42(18):3274-3286 and Le et al. Nature Communications 2016;7: 10634) and was cultured in DMEM medium + Glutamax (GIBCO®), supplemented with 10% (v/v) FBS (Invitrogen), at 37°C in a 5% C0 2 humidified atmosphere. Cell identity was confirmed by karyotyping.
  • Cytotoxicity Assay The cytotoxic activity of GGG-Val-Cit-PAB-MMAE and the scFv-MMAE conjugates were assessed in a cytotoxicity assay in the presence of cathepsin B. To induce cathepsin B cleavage, MMAE, GGG-Val-Cit-PAB-MMAE or scFv-MMAE conjugate was incubated with 0.01 units of pre- activated cathepsin B (Sigma Aldrich) for 4 hours at 37°C in 25 mM Acetate Buffer pH 4.8. MDA-MB-231 cells were seeded on a 96 well plate at 6000 cells/well overnight.
  • the platelet-scFv-MMAE mixture was centrifuged at 2000 g for 2 minutes, the supernatant was removed, and platelets were resuspended in PBS.
  • unwashed platelet-scFv-MMAE mixture was used. Either washed or unwashed (control) platelet-scFv-MMAE mixture was added to MDA-MB-231, HT29, HT1080 and PC3 cells in the absence of exogenous cathepsin B and incubated for 72 hours before determination of XTT metabolism as described above.
  • Tumor sections were stained overnight with an anti-cathepsin B antibody (Abeam) and detected with an Alexa Fluor 488 labeled anti-mouse antibody (Life Technologies) and cell surface membrane-reactive anti-sodium/potassium ATPase antibody (Abeam), counterstained with Hoechst® (Thermo Fisher Scientific) and visualized using the Nikon Alr Plus Confocal Microscope, 20x water objective.
  • Abeam anti-cathepsin B antibody
  • Abeam Alexa Fluor 488 labeled anti-mouse antibody
  • Abeam cell surface membrane-reactive anti-sodium/potassium ATPase antibody
  • Metastasis development was monitored via bioluminescence using IVIS Lumina II by measuring luciferase activity in the chest region, distant from the primary tumor, for a longer time point (60 sec) as previously described Le CP et al. Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination. Nature Communications. 2016; 7: 10634. Metastasis development was quantified using the Living Image software v4.5.1 (Perkin Elmer, Waltham, MA, United States) by quantifying photon/s in a region of interest around the chest bioluminescence signal.
  • mice underwent bioluminescence imaging on day 3 and day 7 post tumor inoculation to confirm tumor growth. Mice with tumors were then randomly assigned to three groups and treated with either scFvGPn b/ nia-M AE, scFv mut -MMAE or left untreated. Treatment was initiated 7 days following tumor inoculation, via intravenous injection of 6 mg/kg body weight of either scFv Gpiib/ m a - MMAE or scFv mut -MMAE followed by three additional treatments every fourth day. Primary tumor size and metastasis development was monitored twice a week as described above.
  • Tumor samples were fixed in formalin (Sigma Aldrich, St Louis, MO, United States) for 24 hours, paraffin embedded, and microtome sectioned (Leica, Wetzlar, Germany) to 20 pm onto a glass slide. Sections were deparaffinized, stained with Hoechst® and visualized using the Alr Plus Confocal Microscope using a 60x oil objective.
  • Mouse blood collection and toxicity measurements Mouse blood was collected via submandibular bleeds into EDTA-coated microtainer collection tubes (BD Biosciences, Franklin Lakes, NJ, United States) and blood counts were performed using the XS- 1000 ⁇ hematologic analyzer (Sysmex Corporation, Kobe, Hyogo, Japan) to determine white blood cells (WBC) and platelets counts. For liver and kidney function tests, 500 pL of mouse blood was collected in citrate.
  • ALP alkaline phosphatase
  • ALT alanine aminotransferase
  • urea levels were measured using the Synchron LX20PRO System (Beckman Coulter Diagnostics, High Wycombe, United Kingdom) by Monash Pathology.
  • ADC antibody-drug conjugate
  • ADP adenosine diphosphate
  • AF Alexa Fluor
  • ALP alkaline phosphatase
  • ALT alanine aminotransferase
  • BLI Bioluminescence
  • Cy7 Cyanine 7
  • GPIIb/IIIa Glycoprotein Ilb/IIIa
  • IC50 half maximal inhibitory concentration
  • IVIS In vivo imaging system
  • MMAE monomethyl auristatin E
  • Mut Mutant
  • NHS N-hydroxysuccinimide
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • scFv single-chain antibodies
  • Val-Cit Valine-citrulline
  • HRP horseradish peroxidase
  • WBC white blood cells
  • XTT tetrazolium salt.
  • a novel antibody-drug-conjugate was developed.
  • the unique scFv GPiib/iiia was employed which specifically targets activated platelets, combined with a sortase A based site-directed biological conjugation method to produce a novel ADC, scFv GPiib/iiia -MMAE incorporating the highly potent anti-mitotic agent (MMAE).
  • a MMAE linker was designed with a triglycine (GGG) sequence, a cathepsin B cleavable peptide (Val-Cit) and a para-aminobenzylalcohol (PABA) self-immolative spacer ( Figure 1).
  • GGG triglycine
  • Val-Cit cathepsin B cleavable peptide
  • PABA para-aminobenzylalcohol
  • the scFv-MMAE was labeled with a Cy7 dye by NHS labeling to yield, scFv GPiib/ ni a -Cy7-MMAE ( Figure 1).
  • SDS page electrophoresis and visualized using the Odyssey reader confirms successful Cy7 labeling of the scFv which has a molecular weight of 34 kDa ( Figure 2A).
  • the scFv GPiib/iiia carries a hexahistidine tag downstream of the LPETG sequence, which is cleaved following sortase A reaction, resulting in a final scFv GPiib/ ni a -Cy7- MMAE product with reduced molecular weight compared to the uncleaved scFv GPiib/iiia .
  • MMAE conjugation to the scFv was analyzed via Western blotting using an anti-MMAE antibody, confirmed by the presence of a band at 34 kDa ( Figure 2B).
  • cytotoxic activity of MMAE on tumor cells was assessed in culture after release from the conjugate by cathepsin B, and the results confirmed equal dose- dependent killing of the triple-negative breast adenocarcinoma cell line MDA-MB- 231 by scFvopii b/iiia -MMAE and scFv mut- MMAE.
  • the IC 50 of scFv GPiib/ ni a- MMAE was l.98xlO 10 M while the IC 50 of the scFv mut- MMAE was l.78xlO 10 M ( Figure 3A).
  • cathepsin B cleavable linker enables the activation of an inactive prodrug to an active cytotoxic form by utilizing the abundance of cathepsin B in the tumor microenvironment.
  • Schmid et al Bioconjug. Chem. 2007:18(3):702-716 disclose the activity of cathepsin B over expressed in solid tumors to cleave a prodrug.
  • the IC50 killing efficacy of GGG-Val-Cit-PAB-MMAE on MDA-MB-231 was l.86xl0 8 M and the addition of cathepsin B increased the cellular killing efficacy by approximately 40-fold to 4.70xl0 10 M ( Figure 3B), which is similar to the IC50 of the unmodified MMAE.
  • the IC50 killing efficacy of scFv GPiib/ ni a -MMAE was 2.2lxl0 8 M and the addition of cathepsin B enhanced the killing efficacy to l.96xlO 10 M ( Figure 3B).
  • MDA-MB-231 cells were incubated with washed human platelets and stained with a CD41 (GPIIb)-specific, non-activation- dependent antibody and two antibodies that are specific for activated GPIIb/IIIa on activated platelets; scFv GPiib/iiia- GFP and PAC-l, the latter antibody binds selectively to activated human GPIIb/IIIa and thus provides a positive control for our scFv GPiib/iiia .
  • GPIIb CD41
  • MDA-MB-231 cells were found to bind to and directly activated platelets, as shown by increased scFv GPiib/iiia- GFP ( Figure 4A) and PAC-l binding ( Figure 4B). Furthermore, for in vivo characterization, the scFv GPiib/iiia -MMAE was conjugated to Cy7 and the construct injected intravenously to MDA-MB-231 metastatic tumor-bearing mice. Bioluminescence imaging was used to detect areas of primary tumor as shown in Figure 5 A.
  • MDA-MB-231, HT29, HT1080 and PC3 cells were incubated with washed human platelets and stained with anti-CD4l antibody as a platelet marker and scFv GPiib/iiia, as a platelet activation marker.
  • Bioluminescence imaging of lung and lymph node metastasis was performed by covering the area of the primary tumor site and imaging with a longer exposure time (60 sec) as shown in Figure 5B. Fluorescence imaging of mice was performed 24 hours post injection of scFv GPiib/ ni a -Cy7-MMAE and this demonstrated the enrichment of scFv GPiib/ ni a -Cy7-MMAE at the primary tumor region as well as some nonspecific uptake by the liver (Figure 5C). Following imaging, mice were killed, organs perfused to remove circulating blood and reimaged.
  • Example 5 scFvGPiib/nia-Cy7-MMAE localizes to activated platelets in the tumor microenvironment and sites of metastasis but not to resting platelets in the spleen and bone marrow
  • MDA-MB-231 tumor-bearing mice were injected with scFv GPiib/ ni a -GFP or scFv mut -GFP and a Dylight 649 anti-GPl b antibody.
  • This in vivo approach for immunofluorescence was used as the activated form of GPIIb/IIIa undergoes a conformational change and antigen masking upon tissue fixation and is not recognized by the scFv GPiib/ ni a.
  • Example 6 Treatment with scFvGPiib / nia-Cy7-MMAE reduces tumor growth and metastasis
  • mice were imaged for bioluminescence signal at day 3 post MDA-MB-231 inoculation, and again at day 7 to confirm the presence of growing tumors (data not shown).
  • mice were treated with scFv GPiib/ ni a -MMAE or scFv mut -MMAE and the effect of therapy on tumor growth and metastasis formation was assessed.
  • the specification discloses the targeting of activated platelets within the tumor microenvironment as novel strategy for the treatment of cancers including primary tumors and metastatic disease.
  • This approach is based on the development of a unique ADC, which targets tumor-associated platelets in the tumor microenvironment using the activated GPIIb/IIIa as an epitope to deliver a therapeutic agent such as a highly potent synthetic anti-mitotic agent.
  • a therapeutic agent such as a highly potent synthetic anti-mitotic agent.
  • this novel approach allows the specific targeting and release of MMAE, via tumor-derived cathepsin B, whilst sparing untoward systemic side effects.
  • ADC efficacy of ADC is highly contingent upon the presence and abundance of the antibody target within the tumor, representing a major limitation for the use of ADCs in several cancer types, which do not express a clinically validated specific antigenic molecular target.
  • a particular advantage of the present approach is the possible ubiquitous nature of activated platelets in a broad range of human tumors, including breast (Lal et al., Breast Cancer Research. 20l3;l5:207), colorectal (Li et al., Scientific Reports. 20l7;7(l): 10261), lung (Ji et al., Platelets. 20l5;26(2): 138-142), ovarian (Stone et al., N Engl J Med.
  • the inventors initially established the ability of activated platelet-specific scFv for tumor imaging (Yap et al., Theranostics. 20l7;7(l0):2565-2574), thus providing a theranostic approach, allowing for the molecular imaging of tumors as a means to detect tumor-associated platelets and therefore predict those likely to respond to platelet-targeting therapy and treatment of patients with the same antibody conjugated to a cytotoxic agent
  • the Examples demonstrate the ability to target the activated platelet as a novel approach to deliver a pro-drug for killing of tumor cells.
  • the Valine-citrulline (Val-Cit) linker is used as a cathepsin B cleavable linker requiring the presence of cathepsin B for cleavage and release of free MMAE, the active anti-cancer agent.
  • Therapeutic approaches using ADCs, such as MMAE are commonly used to target antigens on the cancer cell surface followed by internalization.
  • Activation of the prodrug in the tumor microvasculature might not have taken place (due to lack of Cathepsin B access to the microvasculature) or the active drug might have been flushed from the tumor vessels without any therapeutic effect.
  • the inventors proposed that the drug would achieve a high enough local concentration to inhibit tumour growth and surprisingly this is what they found raising the prospect that sufficiently abundent activated platelets are found within the tumour microenvironment for effective delivery of drug to tumor and validating the novel and inventive concept of platelet targeting for the treatment of solid tumors and also for reducing metastases.
  • activated platelets Prior to the present disclosure, it was not expected that activated platelets would be abundant in the stroma. Similarly Cathepsin B is not expected to be within the microvasculature.
  • an scFv is used as an antibody format, and it offers several advantages.
  • the small size allows quick and efficient penetration of the tumor environment.
  • the recombinant nature of design offers flexibility with respect to conjugation with drugs to generate a homogenous ADC, as described using a sortase A conjugation method.
  • scFvs can be produced in various expression systems, adaptable to clinically applicable purity and scale.
  • Our specific scFv GPiib/iiia also possesses two additional advantages for future work aiming for clinical translation: Firstly, the scFv GPiib/ ni a was developed from a human scFv library, reducing the risk of antigenicity. Secondly, scFv GPiib/ ni a is cross reactive between activated platelets from mice and humans (Schwarz et al., Circulation Research. 2006;99(l):25-33).
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

Abstract

A binding protein drug conjugate for delivering a therapeutic drug to a target tissue such as a tumor comprising (i) a binding protein comprising an antibody variable domain that specifically binds specifically to the activated form of platelet-specific GPIIb/IIIa conjugated to (ii) a therapeutic drug, wherein the binding protein 5 concentrates the therapeutic drug in the tissue and provides targeted delivery of the therapeutic drug to the tissue. Compositions comprising the conjugate is used in the treatment of tumors. Theranostic methods employ administration of the binding protein conjugated to an imaging agent to diagnose a tumor and of the binding protein conjugated to a therapeutic agent to effect treatment.

Description

A THERAPEUTIC GPIIB/IIIA BINDING-PROTEIN DRUG CONJUGATE
AND USE THEREOF
FIELD OF THE DISCLOSURE
The specification is in the field of platelet and cancer biology, haematology and medicine. The specification describes activated platelet binding proteins including antigen-binding fragments of antibodies and antibody drug conjugates (ADCs) comprising same including compositions and methods of using said binding proteins and ADCs.
BACKGROUND ART
Bibliographic details of references are listed at the end of the specification. Reference to any disclosure in this specification is not, and should not be taken as, acknowledgement or any form of suggestion that this disclosure forms part of the prior art or common general knowledge in any country.
The use of antibodies in medical therapy and imaging appears to be gaining momentum although their manufacture can be costly and there are problems associated with expression levels, delivery and stability in vivo. Particularly useful is the recombinant production of antibodies using a variety of expression hosts. There remains a need for improved methods of producing and delivering biological molecules.
Antibody drug conjugates (ADC) represent an emerging class of therapeutics comprising an antibody conjugated to a drug such as a cytotoxic or immunomodulatory drug via a chemical linker. The therapeutic concept of ADCs is to combine binding capabilities of an antibody with a drug, where the antibody is used to deliver the drug to a tumor cell by means of binding to a target tumor cell antigen. However, the expression level of traditional tumor antigens within a tumor can be inadequate for effective tumor reduction and there is a need for new approaches to make ADC more effective. Even if the expression level is adequate, the ability of the ADC to access tumor cells in the solid tumor microenvironment about which little is understood, may lead to treatment failures. Nanoparticle based approaches have been used to try to trap ADC within solid tumors. SUMMARY OF THE DISCLOSURE
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a single composition, as well as two or more compositions; reference to "an agent" includes one agent, as well as two or more agents; reference to "the disclosure" includes single and multiple aspects of the disclosure and so forth.
In one embodiment the present disclosure describes and enables a binding protein drug conjugate (also referred to as an ADC or immunoconjugate) for delivering a therapeutic drug to a tissue comprising activated platelets comprising (i) a binding protein comprising an antibody variable domain that binds specifically to the activated form of platelet- specific glycoprotein (GP)IIb/IIIa conjugated to (ii) a therapeutic drug, wherein the binding protein concentrates the therapeutic drug to a target tissue comprising activated platelets.
In one embodiment the present disclosure describes and enables a binding protein drug conjugate for delivering a therapeutic drug to a tumor comprising (i) a binding protein comprising an antibody variable domain that binds specifically to the activated form of platelet- specific GPIIb/IIIa conjugated to (ii) a therapeutic drug, wherein the binding protein concentrates the therapeutic drug in the tumor microenvironment and provides targeted delivery of the therapeutic drug to the tumor cells.
In one embodiment, the therapeutic drug is chemotherapeutic prodrug and the binding protein drug conjugate comprises a cleavable linker and optionally a spacer between the prodrug and the binding protein which upon cleavage of the linker releases the active chemotherapeutic drug from the binding protein.
In one embodiment, the cleavable linker is a pH sensitive linker, such as a hydrazone or cis-aconityl, providing a non-specific pH sensing mechanism to provide activation or release of the therapeutic drug in a mildly acidic environment. See, for example, McCombs et al. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015, 17(2): 339-351.
In one embodiment, a disulfide linker is employed to release the therapeutic drug based upon the higher reducing potential found within the tumor microenvironment. As known in the art, hindrance groups can be introduced near the cleavage site.
In one embodiment, the cleavable linker is selectively cleaved by a molecule present in the tumor stroma.
In one embodiment, cleavable linker comprises a dipeptide, such as Val-Cit, Phe-Lys and Val-Ala.
In one embodiment, the binding protein is an Fv, scFv, di-scFv, diabody, triabody, tetrabody, Fab, F(ab')2, bispecific antibodies, full length antibody, chimeric, human etc antibody. Non-Ig binding proteins include monobodies, anticalins, and Darpins, LoopDarbins affibodies (Jost et al. Current opinion in Structural Biology 2014 27:102-112). Synthetic antibody mimetic s are also contemplated, as are ibodies (Adalta).
In one embodiment, the binding protein and/or the therapeutic drug are modified for conjugation (coupling). In one embodiment, the modifications are for site- specific conjugation. In one embodiment, the binding protein and the therapeutic agent are conjugated by sortase A mediated conjugation.
In one embodiment, the therapeutic drug is selected from the group consisting of a mitotic inhibitor, a plant alkaloid, and an anti-tumor antibiotic.
In one embodiment, the antibody variable domain binds an epitope of GPIIb/IIIa recognised by a scFv comprising of an amino acid sequence set forth in SEQ ID NO: 1 or 5.
In one embodiment, the antibody variable domain comprises a complementary determining region (CDR) of the heavy chain variable region (VH) having the sequence of SEQ ID NO:2 or 6 and/or a CDR of the light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 3 or 7.
Variant sequences are also contemplated which have a small number of modified amino acids as known in the art while retaining or displaying enhanced binding and stability features. Reference to a small number includes for example 1 amino acid difference, or 2, 3, 4, 5, 6 amino acid substitutions, deletions such as conservative substitutions or use of unnatural amino acids.
In one embodiment, the antibody variable domain comprises a heavy chain variable region (VH) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 2 or 6.
In one embodiment, the antibody variable domain comprises a light chain variable region (VL) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 3 or 7. In one embodiment, the antibody variable domain comprises a heavy chain variable region (VH) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 2 or 6 and a light chain variable region (VL) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 3 or 7.
In one embodiment, the antibody variable domain comprises an amino acid sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 1 or 5.
In one embodiment, the antibody variable domain is derived from a human scFv library.
Reference to a spacer includes a self immolative spacer, such as a para- aminobenzylalcohol (PABA) spacer or a non-self immolative spacer.
In one embodiment the cytotoxic agent is cytotoxic to tumour cells at nanomolar concentrations, picomolar concentrations or less.
In one embodiment, the binding protein drug conjugate comprising a detectable label or molecule suitable for imaging tumors, diagnostic or monitoring.
In one embodiment, the ratio of antibody variable domain to drug is 1:1 or 1:2.
In one embodiment, the ratio of antibody variable domain to drug is 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8.
In one embodiment, the binding protein drug conjugate as described herein is in the form of a composition including a pharmaceutical composition. Pharmaceutical compositions include pharmaceutically acceptable salts and isoforms. In one embodiment, the composition comprises a pharmacologically or physiologically acceptable diluent and/or carrier.
In one embodiment, the composition as described herein is for use in medical therapy or imaging. In one embodiment, methods for imaging cancer are provide using the herein described binding proteins.
The specification also provides for the use of a composition comprising the binding protein drug conjugate as described herein in, or in the preparation of a medicament for, the treatment of cancer including reduction of solid tumors and reduced metastasis in a subject with cancer.
In one embodiment, the cancer is selected from the group consisting of breast cancer, lung cancer, a glioblastoma, prostate cancer, pancreatic cancer, colon cancer, colorectal cancer, head and neck cancer, mesothelioma, kidney cancer, ovarian, oesophageal, squamous cell carcinoma, triple negative breast cancer, and non-small cell lung cancer. In another embodiment, a fusion protein or a kit is contemplated comprising a binding protein comprising the antibody variable domain as described herein labelled with a detectable label suitable for imaging, diagnostics, theranostics or monitoring.
In another embodiment, a fusion protein or a kit is contemplated comprising a binding protein comprising the antibody variable domain as described herein conjugated via a suitable linker or spacer to a therapeutic agent suitable for therapy, theranostics or monitoring.
The present disclosure provides a vector or host cell comprising a polynucleotide sequence capable of expressing the binding protein comprising the antibody variable domain as described herein. Illustrative polynucleotide sequences are provided in SEQ ID NOs 4 (encoding the SCE5 scFv- nucleotides 1 to 138 encode the vector leader sequence) and SEQ ID NO:8 (encoding the anti-LIBs scFv).
In one embodiment the present disclosure provides a method of treating cancer and reducing metastasis comprising administering an effective amount of a binding protein drug conjugate as described herein to a subject with cancer.
In one embodiment the present application provides a method for treating cancer in a subject, comprising (i) administering a binding protein comprising an antibody variable region that binds specifically to activated platelets such as to the activated form of platelet specific GPIIb/IIIa wherein the binding protein is coupled to an imaging contrast agent to determine the presence of activated platelets within a tumor (ii) administering a binding protein comprising an antibody variable region that binds specifically to activated platelets such as to the activated form of platelet specific GPIIb/IIIa wherein the binding protein is coupled to a drug such as a therapeutic or cytotoxic agent contingent upon a determination from (i) of the presence of activated platelets within a tumor.
In one embodiment, the binding protein comprises an imaging contrast agent and one or more anti-cancer drugs such as a therapeutic or cytotoxic agent. Therpeutic drugs include immune or inflammatory agonists.
In one embodiment, the binding protein drug conjugate is administered in combination with an additional drug selected from the group consisting of an anti- apoptotic agent, a mitotic inhibitor, an anti-tumor antibiotic, an immunomodulating agent, a nucleic acid for gene therapy, an alkylating agent, an anti-angiogenic agent, an anti-metabolite, a boron-containing agent, a chemoprotective agent, a hormone agent, an anti-hormone agent, a corticosteroid, a photoactive therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor, and a tyrosine kinase inhibitor. In one embodiment, the agent is attached or otherwise part of the ADC.
In one embodiment there is provided for the use of a binding protein comprising an antibody variable domain that binds specifically to the activated form of platelet- specific GPIIb/IIIa in the preparation of a cancer imaging agent.
This summary is not an exhaustive recitation of all embodiments of the invention.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Structure of GGG-Val-Cit-PAB-MMAE and labeling strategy for the generation of scFvGpIIb/IIIa-Cy7-MMAE and Cathepsin B drug release. scFvGpiIb/IIIa was conjugated to GGG-Val-Cit-PAB-MMAE using an enzymatic sortase A reaction (I), followed by Cy7 conjugation, via NHS labelling to produce scFvGpiIb/IIIa- Cy7-MMAE (II). In vivo , cathepsin B cleaves the scFvGpnb/IIIa-Cy7-MMAE at the MMAE Val-Cit linker, releasing the potent MMAE for tumor killing (III).
Figure 2. scFvGpIIb/IIIa-Cy7-MMAE characterization and specific binding to activated platelets. (A) Coomassie (red) and near infrared (green) imaging of scFv GPiib/iiia-Cy7-MMAE 0)> unmodified scFvGpiIb/IIIa (II), scFvmut-Cy7-MMAE (III) and unmodified scFvmut (IV). Yellow color indicates the overlap between Coomassie in red and near infrared in green. (B) Western blot of scFvGpnb/ma-Cy7-MMAE (I), unmodified scFvGpiIb/IIIa (II), scFvmut-Cy7-MMAE (III) and unmodified scFvmut (IV). (C) Flow cytometry profile showing comparable binding of scFvGPiib/iiia (red) and scFvGPiib/iiia-Cy7-MMAE (blue) to activated platelets. The scFvGPiib/iiia contains a V5 tag, which allows using an anti-V5-FITC secondary antibody for detection. The anti- V5-FITC antibody alone was used as a negative control (grey) (all n=3). (D) Flow cytometry profile of scFvGPIIb/IIIa-Cy7-MMAE and scFvmut-Cy7-MMAE binding to ADP activated platelets (red), resting platelets (black), and secondary anti-V5 antibody (grey) only.
Figure 3. Conjugation of MMAE to scFvGpIIb/IIIa and scFvmut produces an active and highly potent MMAE in the presence of cathepsin B present in tumor cells and the tumor microenvironment. (A) Cytotoxicity assay of triple negative breast adenocarcinoma MDA-MB-231 cells, cultured in scFvGPIIb/IIIa-MMAE versus scFVmut-MMAE in the presence of cathepsin B. (B) Cytotoxicity assay of MDA-MB- 231 cells, cultured in MMAE, GGG-ValCit PAB-MMAE and scFvGPIIb/IIIa-MMAE, with (+C) or without (-C) cathepsin B. (C) Tumor sections were stained with an anti- cathepsin B antibody (green) and anti-sodium/potassium ATPase antibody (red) and counterstained with Hoechst® nucleic acid stain (blue). Imaging 20x) demonstrates the abundance of cathepsin B (green) within the tumor microenvironment, which was localized in both intra- and pericellular locations (left panel). Secondary antibody staining of the sections shows no fluorescence signal (right panel). . (D) Cytotoxicity assay of MDA-MB-231 cells, which were cultured with activated platelets that had been pre-incubated with scFvGPiib/iiia-MMAE (■) or scFvmut-MMAE (■), and undergone washing to remove unbound scFv (wash) in the absence of exogenous cathepsin B. As a control, MDA-MB-231 cells were cultured in activated platelets that had been pre- incubated with scFvGPiib/iiia-MMAE (▼) or scFvmut-MMAE (▼), which did not undergo a washing step to remove unbound scFv (all n=3). Data expressed as mean ± SEM.
Figure 4. Tumor cells induce platelet activation. (A, B) Washed human platelets were added to MDA-MB-231 tumor cells and stained with a PE-conjugated anti-CD4l antibody (red) and an activation- specific anti-GPIIb/IIIa antibody (scFvGPiib/nia-GFP or FITC-conjugated PAC-l). Fluorescence imaging (20x) demonstrates that MDA-MB-231 cells induces platelet binding and activation as shown by scFvGPiib/nia-GFP and PAC-l binding (green) (C) Washed platelet rich plasma was added to MDA-MB-231, HT29, HT1080 and PC3 tumor cells and stained with a PE- conjugated anti-CD4l antibody and scFvGPiib/iiia or scFvmut, detected with an anti-V5- FITC antibody. Flow cytometry dot plots represent the gating strategy differentiating regions of cancer cells and platelets. The cancer cell region was further gated to select for CD4l-positive cancer cells (CD4l+ve). (D) Analysis of the CD4l-positive region demonstrated that 100% of platelets were activated upon incubation with cancer cells for 6 hours, determined by positive scFvGPiib/nia binding (red), which was equivalent to scFvGPiib/iiia binding to ADP-activated platelets (green). As a negative control, platelets incubated with cancer cells were also stained with scFvmut (blue - peak on far left) and displayed negative binding (all n=3). (E) Analysis of the region of cancer cells positive for CD41 (red), confirming that platelets can bind directly to cancer cells (all n=3). (F) Fluorescence imaging (20x) demonstrated that MDA-MB-231 cells bind directly to platelets (red) and induces platelet activation as shown by positive scFvGPiib/iiia staining (green) and negative scFvmut staining.
Figure 5. Activated platelets are present in the tumor microenvironment and can be detected and imaged using the scFvGPiib/nia. (A) In vivo bioluminescence imaging of a representative mouse with an orthotopic mammary tumor. (B) In vivo bioluminescence imaging of metastatic lung and lymph node lesions, by shielding the area of the primary tumor. (C) In vivo fluorescence imaging of MDA-MB-231 tumor bearing mice injected with scFvGpIIb/IIIa-Cy7-MMAE. (D) Ex vivo biodistribution of tumor, lung, muscle, heart and skin of MDA-MB-231 mice injected with scFvGPIIb/IIIa- Cy7-MMAE. (E, F) Immunofluorescence imaging of MDA-MB-231 tumor sections of mice injected with DyLight 649 anti-GPlbp (red) and scFvGPIIb/IIIa-GFP or scFvmut-GFP (green), counterstained with Hoechst® (blue). (E) Immunofluorescence imaging (20x) demonstrates the abundance of platelets within the tumor microenvironment (red), and the specificity of the platelet binding of scFvGpiIb/IIIa t0 tumor-associated platelets in vivo (green). (F) In contrast the scFvmut does not bind platelets in vivo.
Figure 6. Activated platelets are present in the tumor microenvironment and can be detected and imaged using the scFvGPiib/iiia. (A) In vivo fluorescence imaging of MDA-MB-231 tumor-bearing mice, injected with scFvGPiib/iiia-Cy7-MMAE and scFvmut-Cy7-MMAE. (B) In vivo bioluminescence imaging of MDA-MB-231 tumor-bearing mice confirming primary tumor localization in the mammary gland. (C) Ex vivo biodistribution of the primary tumor of MDA-MB-231 tumor-bearing mice injected with scFvGPiib/nia-Cy7-MMAE and scFvmut- Cy 7 -MM AE. (D) Quantification of fluorescence signal of MDA-MB-231 tumor-bearing mice, injected with scFvGPiib/iiia- Cy7-MMAE and scFvmut-Cy7-MMAE (all n=3). Data expressed as mean ± SEM. *P< 0.05 analyzed by unpaired Student’s T tests. (E) Representative bioluminescence in vivo lung imaging of MDA-MB-231 tumor-bearing mice demonstrated the presence of lung metastases (left panel) and representative ex vivo fluorescence imaging of the lung (right panel) demonstrating positive scFvGPiib/iiia-Cy7-MMAE signal but negative scFvmut-Cy7-MMAE. (F) Representative in vivo lung imaging of MDA-MB-231 tumor-bearing mice with no lung metastasis (left panel) and representative ex vivo fluorescence imaging, demonstrating negative scFvGPiib/iiia-Cy7-MMAE and scFvmut- Cy7-MMAE signal (right panel). BLI-Bioluminescence.
Figure 7. Activated platelets are present in the tumor microenvironment but absent in the spleen and bone marrow. (A, B) Immunofluorescence imaging of tumor sections of MDA-MB-231 tumor-bearing mice injected with DyLight 649 anti- GPlbp (red) and scFvGPiib/iiia-GFP or scFvmut-GFP (green), counterstained with Hoechst® (blue). (A) Immunofluorescence imaging (20x) demonstrated the abundance of platelets within the tumor microenvironment (red), and the specificity of the platelet binding of scFvGPiib/iiia to tumor-associated platelets in vivo (green). (B) In contrast, the scFvmut does not bind platelets in vivo. (C, D) Flow cytometry of the spleen and bone marrow of BALB/C nude mice injected with PBS (control) or DyLight 649 anti-GPlbp and scFvGPiib/iiia-GFP or scFvmut-GFP. (C) Flow cytometry of spleen cells, which were co-stained for CD41 and gated on the CD41 -positive region, demonstrated the presence of platelets in the spleen via DyLight 649 anti-GPlbp staining, but the absence of activated platelets, demonstrated by the absence of scFvGPiib/iiia-GFP staining (n=3). (D) Flow cytometry of bone marrow cells from the femur, co-stained for CD41 and gated on the CD41 -positive region, demonstrates the presence of platelets in the bone marrow via DyLight 649 anti-GPlbp staining, but the absence of activated platelets, demonstrated by the absence of scFvGPiib/iiia-GFP staining (n=3).
Figure 8. scFvGpIIb/IIIaMMAE treatment inhibits tumor growth and metastasis development in a murine model of triple negative breast cancer. (A)
Primary tumor volume of MDA-MB-231 tumor bearing mice treated with 4 mg/kg of scFv GPiib/ma-MMAE (■) (n=7 scFvmut-MMAE (·) (n=6) or PBS (A) (n=6). (B) Metastasis burden of MDA-MB-231 tumor-bearing mice treated with 4 mg/kg of scFvGpiIb/IIIa-MMAE (■) (n=7), scFvmut-MMAE (·) (n=6) or PBS (A) (n=6), measured bioluminescence imaging, 60 sec exposure, and representative images of lung and lymph node metastasis from each group. (C) Body weight of MDA-MB-231 tumor bearing mice treated with 4 mg/kg of scFvGpiIb/IIIa-MMAE (■) (n=7), scFvmut-MMAE (·) (n=6) or PBS (A) (n=6). ****F<0.0001 between untreated and scFvGPTTh/TTTa- MMAE and ###iTP< 0.000! between scFvmut-MMAE and scFvGPTTh/TTTa-MMAE analyzed by two-way ANOVA with Tukey’s multiple comparison’s test. *P<0 05 analyzed by one-way ANOVA with Dunnetts’s multiple comparison’s test. (D) Blood cell counts of tumor-bearing mice after four treatment rounds of 6 mg/kg of seFvGPiib/ma- AE (s) (n=5), scFvmut-MMAE (®) (n=5) or untreated (A) (n=5). (B) Liver and renal function tests of tumor-bearing mice after four treatment rounds of 6 mg/kg of scFvGPiib/nia-MMAE (■) ( u 4/5 }, scFv -MMAE (·) (n 5 ) or untreated ( A ) {u 5). Black dotted lines represent guideline for normal range within the 95% interval published by Charles River for female BALB/C athymic nude mice. Data expressed as mean ± SEM. WBC - white blood cells, ALT - alanine aminotransferase and ALP - alkaline phosphatase.
Figure 9. scFvGPiib/nia-MMAE binds to activated platelets and induces cancer cell killing. (A) Cytotoxicity assay of MDA-MB-231, HT29, HT1080 and PC3 tumor cells, cultured in GGG-Val-Cit PAB-MMAE and scFvGPiib/nia-MMAE, in the presence of cathepsin B (+C). Data expressed as mean ± SEM. (B) Cytotoxicity assay of HT29, HT1080 and PC3 tumor cells, cultured in activated platelets which have been pre-incubated with scFvGPiib/nia-MMAE (■) or scFvmut-MMAE (■) and washed to remove unbound scFv (wash) in the absence of exogenous cathepsin B. As a control, HT29, HT1080 and PC3 tumor cells were cultured in activated platelets that had been pre-incubated with scFvGPiib/nia-MMAE (▼) or scFvmut-MMAE (▼), which did not undergo a washing step (all n=3). Data expressed as mean ± SEM. Figure 10 provides illustrative nucleotide and amino acids sequence of antibody variable domains and CDR and linker sequences of an anti-LIBs antibody clone.
Figure 11 provides illustrative nucleotide and amino acids sequence of antibody variable domains and CDR and linker sequences of an SCE5 antibody clone described in the examples.
KEY TO SEQUENCE LISTING
SEQ ID NO: 1 amino acid sequence of single-chain (scFv) antibody against activated GPIIb/IIIa) (SCE5)
SEQ ID NO: 2 heavy chain VH amino acid sequence of scFv of SCE5
SEQ ID NO: 3 light chain VL amino acid sequence of scFv of SCE5
SEQ ID NO: 4 Illustrative nucleotide sequence encoding amino acid sequence of single-chain (scFv) antibody against activated GPIIb/IIIa) (SCE5) including a leader sequence at nucleotides 1 to 138
SEQ ID NO: 5 amino acid sequence of single-chain (scFv) antibody against activated GPIIb/IIIa) (anti-LIBs)
SEQ ID NO: 6 heavy chain VH amino acid sequence of scFv of anti-LIBs
SEQ ID NO: 7 light chain VL amino acid sequence of scFv of anti-LIBs
SEQ ID NO: 8 Illustrative nucleotide sequence encoding amino acid sequence of single-chain (scFv) antibody against activated GPIIb/IIIa) (anti- LIBs)
DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, histochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present), Colowick and Kaplan, eds., Methods In Enzymology, Academic Press, Inc.; Weir and Blackwell.
The description and definitions of antibody variable domains/regions, antigen binding fragments and parts thereof, may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991. As known to those of average skill in the art the antigen binding site or domain of naturally occurring antibodies is formed by the precise juxtaposition of typically six hypervariable loops (also regarded as complementary determining regions or CDR) provided by the light chain variable region and the heavy chain variable region and aided by more conserved framework regions of the variable domains. However a multitude of antibody fragments or derivatives comprising an antibody variable region able to bind to the antigen are also known to the skilled addressee include without limitation Fab, Fab', Fd, Fd', Fv, dAb, isolated CDR region, F(ab')2 bivalent fragments, diabodies and liner antibodies.
The term "EU numbering system of Kabat" will be understood to mean the numbering of an antibody heavy chain is according to the EU index as taught in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on the residue numbering of the human IgGl EU antibody.
The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.
Reference herein to a range of, e.g., residues, will be understood to be inclusive. For example, reference to "a region comprising amino acids 56 to 65 of SEQ ID NO: 1" will be understood to mean that the region comprises a sequence of amino acids as numbered 56, 57, 58, 59, 60, 61, 62, 63, 64 and 65 in SEQ ID NO: 1.
"Antibody variable domain" refers to a binding protein that is capable of interacting with or specifically binding to an antigen (e.g., a platelet receptor or molecule, such as a protein, e.g., a glycoprotein). For example, the antibody variable domain can be an antigen binding fragment of an antibody (e.g., a Fv or a scFv, etc.). Glycoprotein Ilb/IIIa (GPIIb/IIIa, also known as integrin al Ibp3 ) is an integrin complex found on platelets. It is a receptor for fibrinogen and von Willebrand factor and aids platelet activation. The GPIIb/IIIa complex is formed via calcium-dependent association of gpllb and gpllla, a required step in normal platelet aggregation and endothelial adherence. Platelet activation by ADP (blocked by clopidogrel) leads to the aforementioned conformational change in platelet gpIIb/IIIa receptors that induces binding to fibrinogen. For the purposes of nomenclature only and not limitation an exemplary sequence of human GPIIb is set out in NCBI Gene ID: 3674, NCBI Reference Sequence: NG_00833l.l.For the purposes of nomenclature only and not limitation an exemplary sequence of human GPIIIa is set out in NCBI Gene ID: 3690, NCBI Reference Sequence: NG_008332.2. Additional sequences of GPIIb and/or Ilia from other species can be determined using sequences provided herein and/or in publically available databases and/or determined using standard techniques (e.g., as described in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
As used herein, the term "binds" in reference to the interaction of antibody variable domain or fusion protein comprising same with GPIIb/IIIa means that the interaction is dependent upon the presence of a particular structure (e.g., epitope) on the component. For example, an antibody or antibody variable domain recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope "A", the presence of a molecule containing epitope "A" (or free, unlabeled "A"), in a reaction containing labeled "A" and the protein, will reduce the amount of labeled "A" bound to the antibody or antibody variable domain.
As used herein, the term "specifically binds" shall be taken to mean that the binding interaction between the antibody variable domain and GPIIb/IIIa is dependent on the presence of the antigenic determinant or epitope. The binding region preferentially binds or recognizes a specific antigenic determinant or epitope even when present in a mixture of other molecules or organisms. In one example, the antibody variable domain reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with the specific component or cell expressing same than it does with alternative antigens or cells. It is also understood by reading this definition that, for example, a binding region the specifically binds to a particular component may or may not specifically bind to a second antigen. As such, "specific binding" does not necessarily require exclusive binding or non-detectable binding of another antigen. The term "specifically binds" can be used interchangeably with "selectively binds" herein. Generally, reference herein to binding means specific binding, and each term shall be understood to provide explicit support for the other term. Methods for determining specific binding will be apparent to the skilled person. For example, a binding protein comprising the binding region of the disclosure is contacted with the component or a cell expressing same or a mutant form thereof or an alternative antigen. The binding to the component or mutant form or alternative antigen is then determined and a binding region that binds as set out above is considered to specifically bind to the component.
As used herein, the term "epitope" (syn. "antigenic determinant") shall be understood to mean a region of GPIIb/IIIa to which a protein comprising a antibody variable domain of an antibody binds. This term is not necessarily limited to the specific residues or structure to which the protein makes contact. For example, this term includes the region spanning amino acids contacted by the protein and/or at least 5 to 10 or 2 to 5 or 1 to 3 amino acids outside of this region. In some examples, the epitope is a linear series amino acids. An epitope may also comprise a series of discontinuous amino acids that are positioned close to one another when GPIIb/IIIa is folded, that is, a "conformational epitope". The skilled artisan will also be aware that the term "epitope" is not limited to peptides or polypeptides. For example, the term "epitope" includes chemically active surface groupings of molecules such as sugar side chains, phosphoryl side chains, or sulfonyl side chains, and, in certain examples, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope or peptide or polypeptide comprising same can be administered to an animal to generate antibodies against the epitope.
The term "competitively inhibits" shall be understood to mean that a fusion protein comprising the antibody variable domain of the disclosure reduces or prevents binding of a recited antibody to GPIIb/IIIa, for example, a scFv consisting of a sequence set forth in SEQ ID NO: 1. This may be due to antibody variable domain binding to the same or an overlapping epitope as the scFv. It will be apparent from the foregoing that the protein need not completely inhibit binding of the antibody, rather it need only reduce binding by a statistically significant amount, for example, by at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Methods for determining competitive inhibition of binding are known in the art and/or described herein. For example, the antibody is exposed to GPIIb/IIIa either in the presence or absence of the protein. If less antibody binds in the presence of the protein than in the absence of the protein, the protein is considered to competitively inhibit binding of the antibody. In one example, the competitive inhibition of binding is caused by the antigen binding domain of the protein on GPIIb/IIIa overlapping with the antigen binding domain of the antibody. In a further particular, the ability of antibody variable domains and ADC comprising same as described herein to bind to their cognate receptor on activated platelets in vivo in an appropriate subject or model is determined in accordance with the present invention and examples.
"Overlapping" in the context of two epitopes means that two epitopes share a sufficient number of amino acid residues to permit a binding protein of the disclosure that binds to one epitope to competitively inhibit the binding of a recited antibody to GPIIb/IIIa that binds to the other epitope. For example, the "overlapping" epitopes share at least 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 amino acids.
The term "recombinant" shall be understood to mean the product of artificial genetic manipulation. Accordingly, in the context of an antibody or antigen binding fragment thereof, this term does not encompass an antibody naturally occurring within a subject’s body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, in it is to be considered an isolated protein comprising an antibody variable region. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein. A recombinant protein also encompasses a protein expressed by means of artificial genetic manipulation when it is within a cell.
The term "protein" shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.
The term "polypeptide" or "polypeptide chain" will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.
The skilled artisan will be aware that an "antibody" is generally considered to be a protein that comprises a variable region made up of a plurality of polypeptide chains, e.g., a polypeptide comprising a light chain variable region (VL) and a polypeptide comprising a heavy chain variable region (VH). An antibody also generally comprises constant domains, some of which can be arranged into a constant region, which includes a constant fragment or fragment crystallizable (Fc), in the case of a heavy chain. A VH and a VL interact to form an Fv comprising an antigen binding region that is capable of specifically binding to one or a few closely related antigens. Generally, a light chain from mammals is either a k light chain or a l light chain and a heavy chain from mammals is a, d, e, g, or m. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, dlgA and IgY), class (e.g., IgGi, IgG2, IgG3, IgG4, dlgAi and dIgA2, IgAi and IgA2) or subclass. The term "antibody" and "antibody variable domain" also encompasses humanized antibodies and humanized antibody variable domain, primatized antibodies and primitized antibody variable domain, human antibodies and human antibody variable domains , synhumanized antibodies and chimeric antibodies and synhumanized and chimeric antibody variable domain.
As used herein, "variable region" refers to the portions of the light and/or heavy chains of an antibody as defined herein that specifically binds to an antigen and, for example, includes amino acid sequences of CDRs; i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.
As used herein, the term "complementarity determining regions" (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2 and CDR3. In one example, the amino acid positions assigned to CDRs and FRs are defined according to Rabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as "the Rabat numbering system". According to the numbering system of Rabat, VH FRS and CDRs are positioned as follows: residues 1-30 (FR1), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and 103- 113 (FR4). According to the numbering system of Rabat, VL FRS and CDRs are positioned as follows: residues 1- 23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-107 (FR4). "Framework regions" (hereinafter FR) are those variable domain residues other than the CDR residues.
As used herein, the term "Fv" shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex having an antigen binding site, i.e., capable of specifically binding to an antigen. The VH and the VL which form the antigen binding site can be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the disclosure (as well as any protein of the disclosure) may have multiple antigen binding sites which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means. In some examples, the VH is not linked to a heavy chain constant domain (CH) 1 and/or the VL is not linked to a light chain constant domain (CL). Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab’ fragment, a F(ab’) fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., CH2 or CH3 domain, e.g., a minibody. A "Fab fragment" consists of a monovalent antigen-binding fragment of an antibody, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means. A "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab’ fragment can also be produced by recombinant means. A "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A "Fab2" fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a CH3 domain. A "single chain Fv" or "scFv" is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.
An "antigen binding fragment" of an antibody comprises one or more antibody variable domains of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, half antibodies and multispecific antibodies formed from antibody fragments.
As used herein, the term "mutant" or "mutated" refers to a scFv (e.g., scFvmut) which has undergone modification (e.g., deletion or truncation) of one or more amino acids using well known techniques to inactivate the receptor binding and/or functional activity of the scFv.
The term "identity" or "identical" as used herein refers to the percentage number of amino acids that are identical or constitute conservative substitutions. In one embodiment, identity may be determined using sequence comparison programs such as GAP (Deveraux et al, 1984, Nucleic Acids Research 12, 387-395), which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
As used herein, the terms "treating", "treat" or "treatment" include administering a ADC described herein to thereby reduce or eliminate at least one symptom of a cancer or to slow progression of cancer. This term also encompasses treatment of a subject in remission to prevent or ameliorate a relapse or metastasis. Cancers that may be treated include those selected from the group consisting of breast cancer, lung cancer, a glioblastoma, prostate cancer, pancreatic cancer, colon cancer, colorectal cancer, head and neck cancer, mesothelioma, kidney cancer, ovarian, oesophageal, squamous cell carcinoma, triple negative breast cancer, and non-small cell lung cancer.
An "effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, the desired result may be a therapeutic or prophylactic or imaging result or an imaging and therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" is meant an amount necessary to effect treatment of a cancer as hereinbefore described. In some examples of the present disclosure, the term "effective amount" is meant an amount necessary to effect a change in a factor associated with a disease or condition as hereinbefore described. For example, the effective amount may be sufficient to effect a beneficial change in the size of a solid tumour with for example, minimal systemic side effects. The effective amount may vary according to the disease or condition to be treated or factor to be altered and also according to the weight, age, racial background, sex, health and/or physical condition and other factors relevant to the mammal being treated. Typically, the effective amount will fall within a relatively broad range (e.g. a "dosage" range) that can be determined through routine trial and experimentation by a medical practitioner. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., weight or number of binding proteins. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.
A "therapeutically effective amount" is at least the minimum concentration required to effect a measurable improvement of a particular disease or condition. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antigen binding protein -drug conjugate to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the ADC are outweighed by the therapeutically beneficial effects. In one example, a therapeutically effective amount shall be taken to mean a sufficient quantity of ADC to treat a cancer such as a cancer associated with a solid tumor and metastasis. As used herein, the term "prophylactically effective amount" shall be taken to mean a sufficient quantity of ADC to prevent or inhibit or delay the onset of cancer or the side effects of the cancer in a subject. As used herein, the term "subject" shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.
Antibody Drug Conjugates (ADC )
The present inventors have developed an scFv-drug conjugate directed at activated platelets such as those found within the tumor microenvironment. They have determined its ability in vivo to concentrate potent cytotoxic agent within solid primary tumors and metastasis of the MDA-MB-231 murine metastatis model for triple negative breast cancer, and provide significant regression of primary tumors and prevention of metastases without systemic side effects.
In one embodiment, the present disclosure provides an Antibody-Fragment- Drug-Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and (ii) a drug which mediates its effect in the vicinity of the activated platelet conjugated in releasable from the (i).
In one embodiment, the present disclosure provides an Antibody-Fragment- Drug-Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and (ii) a drug construct comprising a prodrug of a highly potent cytotoxic agent such as an auristatin or equivalent cytotoxic agent conjugated to (i).
In one embodiment, a site-directed enzymatic conjugation method using sortase A to catalyse the condensation reaction is used to conjugate an antibody variable domain with a C-terminal FPETG tag to a triglycine bridge or other spacer such as glycine at the N-terminus of a cytotoxic drug construct. The scFvGPIIb/IIIA- MMAE ADC displayed a band at 34kDa after western blotting (Figure 2B) and the specific binding of the antibody variable domain (scFV) to activated platelets was retained after conjugation to MMAE by the present method (Figure 2C).
In one embodiment, the drug construct comprises a cleavable linker which, upon cleavage in the tumor microenvironment, releases the active cytotoxic drug which is able to induce tumor regression.
In one embodiment, the drug construct comprises a cleavable linker which, upon cleavage in the tumor microenvironment, releases the drug which is able to mediates its effect in the vicinity of the activated platelet.
In one embodiment, the drug construct comprises a cleavable linker which, upon cleavage in the tumor microenvironment, releases the drug which is able to induce tumor regression. The present application illustrates the use of a cathepsin cleavable contruct to activate the pro-drug. When a non-cytotoxic drug is employed or where adequate agent concentration is achieved a pro-drug approach may not be required. Other illustrative microenvironment enzymes include MMPs and FAP.
Since proteinases and peptidases are abundant in the tumor interstitial space, therapeutic nanosystems fused with peptides that are specific substrates of these enzymes can be designed to control the release of therapeutic agents within the tumor microenvironment. For example, MMPs, with an elevated expression in tumor ECM, are crucial in tumor progression. Therefore, making use of MMP-cleavable sequences achieves drug release or active site exposure. The substrate peptide for MMP-2 is GPLGIAGQ; this sequence is cleaved into GPLG and IAGO by MMP-2. Others have developed a liposome modified with cell-penetrating peptide (CPP) and a poly(ethylene glycol) (PEG)-conjugated antibody PEG was linked to the liposome via a MMP-2 responsive sequence. In the presence of MMP-2, long PEG chains were removed from the liposomes. As a result, the exposed CPP mediated the internalization of the liposomes. In another example, the MMP-2 and MMP-9 sensitive sequence, PVGLIG, can be cleaved between glycine and leucine (Gao et ah, Biomaterials 34:4137-4149, 2013). This sequence can also be used for construction of tumor niche-responsive nanoformulations. Fibroblast activation protein-a (FAP-a) is another accessible protease that is specifically expressed on the surface of CAFs, a major cellular component in the tumor microenvironment. FAP-a selectively cleaves the sequence GPAX (X designates any amino acid) between proline, and alanine (Ji et ah, Angew Chem Int Ed Engl 55:1050-1055, 2016; Qin et al Molecular Pharmacology 92(3): 219- 231).
In vitro and in vivo studies were undertaken which disclose that the conjugated MMAE is released and maintains cytotoxicity upon exposure to cathepsin B. In one embodiment, the present disclosure provides an Antibody-Fragment- Drug-Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet-specific GPIIb/IIIa and part of a C-terminal sortase A sequence conjugated to the N-terminus of (ii) a drug construct comprising an N-terminal nucleophilic group (e.g. glycine or polyglycine e.g., GGG) sequence linked via a peptidase cleavable linker and optionally a spacer to a cytotoxic agent such as an auristatin or equivalent cytotoxic agent.
In one embodiment, the present disclosure provides an Antibody-Fragment- Drug-Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet-specific GPIIb/IIIa and part of a C-terminal sortase A conjugated to the N-terminus of (ii) a drug construct comprising an N-terminal polyglycine (e.g. GGG) sequence linked via a cleavable linker and a spacer to a cytotoxic agent such as an auristatin or equivalent cytotoxic agent.
In one embodiment, the present disclosure is of an ADC comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet-specific GPIIb/IIIa and a C-terminal sortase A recognition sequence and (ii) a cathepsin B dependent cytotoxic prodrug comprising an N-terminal polyglycine (e.g. GGG) sequence linked via a cathepsin B cleavable peptide linker to an auristatin or equivalent cytotoxic agent.
In one embodiment, the antibody variable domain binds an epitope of GPIIb/IIIa recognised by a scFv consisting of a sequence set forth in SEQ ID NO: 1.
In one embodiment, the antibody variable domain comprises a complementary determining region (CDR) of the heavy chain variable region (VH) having the sequence of SEQ ID NO:2 and/or a CDR of the light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 3.
In another embodiment, the antibody variable domain comprises a heavy chain variable region (VH) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 2 and a light chain variable region (VL) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 3.
In one embodiment, the antibody variable domain comprises an amino acid sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 1.
At least 90% identity includes 91, 92, 93, 94, 95, 96, 97, 98, 99% or 100% sequence identity to the reference amino acid sequence. In another embodiment, antibody variable domain variants encompassed comprise one to four or two to eight amino acid substitutions and retain high affinity specific binding to activated GPIIb/IIIa.
In one embodiment, the term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" are understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Conservative substitutions known in the art may also be assessed and included in the percent identity calculation. As known in the art, even non-conservative substitutions within CDR regions are tolerated and can be routinely tested.
In one embodiment, the antibody variable domain is derived from a human scFv library.
In one embodiment, the antibody variable domain is scFVGPiib/iiia that binds only to the activated form of platelet- specific GPIIb/IIIa having the amino acid sequence set out in SEQ ID NO:l.
In one embodiment, the ADC comprises the Val-Citrulline cleavable linker.
In one embodiment, the ADC comprises a spacer wherein the spacer is a self immolative spacer, such as a para-aminobenzylalcohol (PABA) spacer.
In one embodiment, the ADC comprises (i) a fusion protein comprising scFV GPiib/iiia and a C-terminal sortase A recognition sequence and (ii) an N-terminal polyglycine sequence linked via a cleavable linker and immolative spacer to MMAE.
Reference herein to an "equivalent cytotoxic agent" includes cytotoxic agents that are active at picomolar concentrations. In one embodiment the cytotoxic agent is a mitotic inhibitor or an anti-tumor antibiotic or a plant alkaloid. Illustrative mitotic inhibitors include a dolastatin, an auristatin, a maytansinoid, and a plant alkaloid. The term "auristatin", as used herein, refers to a family of antimitotic agents. Auristatin derivatives are also included within the definition of the term "auristatin". Examples of auristatins include, but are not limited to, auristatin E (AE), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), and synthetic analogs of auristatin. Maytansinoids include, for example, DM1, DM2, DM3, and DM4. In one embodiment, anti-tumor antibiotics are selected from the group consisting of an actinomycine (e.g., PBD), an anthracycline, a calicheamicin, and a duocarmycin.
In one embodiment, the chemotherapeutic agent is selected from a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkyating agents, antimetabolites, spindle poison plant alkaloids, cytoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy. Examples of chemotherapeutic agents include: erlotinib docetaxel), 5-FU, gemcitabine, cisplatin, carboplatin, paclitaxel, trastuzumab temozolomide, tamoxifen, doxorubicin and rapamycin.
Further examples of chemotherapeutic agents include: oxaliplatin imatinib mesylate, Mek inhibitor, PI3K inhibitor, fulvestrant, leucovorin), rapamycin, lapatinib, lonafarnib sorafenib gefitinib irinotecan tipifarnib, albumin-engineered nanoparticle formulations of paclitaxel vandetanib chloranmbucil, AG1478, AG1571, temsirolimus, pazopanib canfosfamide, thiotepa and cyclosphosphamide alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, calicheamicin gammall, calicheamicin dynemicin, dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine, novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine, ibandronate; CPT-l l; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.
Also included in the definition of "chemotherapeutic agent" or "drug" are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands; (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a l,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, such as oblimersen (vii) ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines (ix) anti- angiogenic agents such as bevacizumab; and pharmaceutically acceptable salts, acids and derivatives of any of the above.
Immune modulators (immune agonists) are an important class of compounds that are directed to activated platelets in concentrated form by the present invention. Examples include check point inhibitors, TLR agonists.
Also included in the definition of "chemotherapeutic agent" are therapeutic antibodies such as alemtuzumab, bevacizumab, cetuximab, panitumumab rituximab pertuzumab trastuzumab tositumomab and the antibody drug conjugate, gemtuzumab.
Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies.
In one embodiment, the antibody variable domain, fusion protein or ADC as described herein comprises a detectable label. In one embodiment detectable labels are suitable for imaging and tracing tumors.
Detectable labels include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin and the like. Detectable labels include detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. As well understood, detectable enzyme are detected by adding enzyme substrates to produce a detectable reaction product. An antibody or ADC may also be labelled with biotin, and detected through indirect measurement of avidin or streptavidin binding.
In one embodiment, the antibody variable domain is conjugated to an imaging agent/label/dye known in the art. Examples of imaging agents include, but are not limited to, MRI contrast agents such as Gd, Mn and F etc, a radiolabel, radiotracer, an enzyme, a fluorescent label or dye a luminescent label, a bioluminescent label, a magnetic label, and biotin. Contrast agents for molecular imaging of cancer using a range of distinct molecular imaging modalities are provided. Illustrative modalities include without limitation, MRI, fluorescence imaging (e.g. Cy7 and FLECT imaging) and molecular imaging via for example PET/CT (e.g. 64Cu) and ultrasound (e.g., ultrasound enhancing microbubbles such as streptavidin-coated microbubbles, and photoacoustic imaging).
In another embodiment, the ADC as described herein comprises a 1 : 1 ratio of antibody variable domain to pro-drug.
In one embodiment the ratio in the ADC or composition is about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
Vectors and polynucleotide sequences
In another embodiment, vectors comprising the polynucleotide sequences encoding the binding proteins of the present invention are contemplated. Illustrative polynucleotide sequences are described in Figure 8 and 9 and in the sequence listing, or sequences having at least 90%, 95% or 99% sequence identity.
Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, BHK, VERO, HT1080, 293, 293T, 293F, RD, COS-7, CHO, Jurkat, HUT, SUPT, C8166, HepG2, MOLT4/clone8, MT-2, MT-4, H9, PM1, CEM, myeloma cells (e.g., SB20 cells) and CEMX174, HEK293T, NSO, SP2 cells, HeLa cells, A549 cells, 3T3 cells, and a number of other cell lines. Other cells that may be used include insect cell lines, such as Sf9cells, avian, amphibian cells, plant cells, yeast and fungal yeast cells. In one embodiment, a non mammalian cell may be employed such as a yeast cell, such as Hansenula polymorpha. Such cells are useful for providing controlled levels of expression. Vectors available for cloning and expression in host cell lines are well known in the art, and include but are not limited to vectors for cloning and expression in mammalian or yeast cell lines, vectors for cloning and expression in bacterial cell lines, vectors for cloning and expression in phage and vectors for cloning and expression insect cell lines. The fusion proteins can be recovered using standard protein purification methods.
Compositions
The present disclosure provides a composition comprising the ADC as described herein. Accordingly, in one embodiment the composition comprises an Antibody-Fragment-Drug-Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and (ii) a drug construct comprising a prodrug of a highly potent cytotoxic agent such as an auristatin or equivalent cytotoxic agent conjugated to
CD-
In one embodiment, the composition comprises an Antibody-Fragment-Drug- Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and part of a C-terminal sortase A sequence conjugated to the N-terminus of (ii) a drug construct comprising an N-terminal glycine sequence linked via a cleavable linker and optionally a spacer to a cytotoxic agent such as an auristatin or equivalent cytotoxic agent.
In one embodiment, the composition comprises a pharmacologically or physiologically acceptable diluent and/or carrier. By "pharmaceutically acceptable" carrier, or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emusifying agents, pH buffering agents, preservatives, and the like. Depending upon the particular route of administration, a variety of acceptable carriers, known in the art may be used, as for example described in Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991).
In one embodiment, the composition comprises ADC wherein the ratio of antibody variable domain to pro-drug is substantially 1:1.
In one embodiment each antibody or each antibody variable domain (antigen binding fragment of a full length antibody) is conjugated to 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug molecules.
As determined herein sortase A mediated conjugation of the drug construct to the fusion protein comprising the antibody variable domain produces an essentially homogeneous ADC with substantially no undesired species having heterogenous numbers of drug to antibody ratios. This is useful for both medical and imaging applications.
In one embodiment, the composition comprising the ADC as described herein is for use in (or used for) medical therapy or medical imaging.
Pharmaceutical compositions
The ADC is preferably administered in a therapeutically effective amount. The actual amount administered and the rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington’s Pharmaceutical Sciences, supra.
In one example, the binding protein is administered parenterally, such as subcutaneously or intravenously. For example, the binding protein administered intravenously.
Formulation of a ADC to be administered will vary according to the route of administration and formulation (e.g., solution, emulsion, capsule) selected. An appropriate pharmaceutical composition comprising an ADC to be administered can be prepared in a physiologically acceptable carrier as discussed herein. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, l6th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The ADC can be stored in the liquid stage or can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.
Cancer Imaging reagents
The present disclosure also provides for the use of a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet-specific GPIIb/IIIa and a C-terminal sortase A recognition sequence in the preparation of a cancer imaging agent.
The disclosure provides an fusion protein comprising the antibody variable domain as described herein and a detectable label suitable for imaging. The disclosure provides a vector or host cell comprising a polynucleotide sequence capable of expressing the fusion protein comprising the fusion proteins comprising the antibody variable domain as described herein.
In one illustrative embodiment, the antibody variable domain is conjugated to a bifunctional sarcophagine chelator, MeCOSar and labelled with copper-64.
As determined herein cancer cells induce activation and can be imaged using ScFvGPiib/nia-Cy7-MMAE in vitro and in vivo.
In one embodiment, imaging can be carried out using the antibody variable domain linked to a detectable agent. In one embodiment the antibody variable domain is conjugated to a detectable label via sortase A mediated conjugation. Fusion proteins comprising the antibody variable domain and a sortase A recognition sequences can conveniently be produce recombinantly. One non-limiting illustrative cancer imaging agent is ScFvGPiib/nia-Cy7.
Detectable labels include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin and the like. Detectable labels include detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. As well understood, detectable enzyme are detected by adding enzyme substrates to produce a detectable reaction product. An antibody or ADC may also be labelled with biotin, and detected through indirect measurement of avidin or streptavidin binding.
In one embodiment, the antibody variable domain is conjugated to an imaging agent/label/dye known in the art. Examples of imaging agents include, but are not limited to, a radiolabel, radiotracer, an enzyme, a fluorescent label or dye a luminescent label, a bioluminescent label, a magnetic label, and biotin.
Contrast agents for molecular imaging of cancer using a range of distinct molecular imaging modalities. Illustrative modalities include, fluorescence imaging (e.g. Cy7) and molecular imaging via for example PET/CT (e.g. 64Cu) and ultrasound (e.g., ultrasound enhancing microbubbles such as streptavidin-coated microbubbles)
In another embodiment, negative control imaging agents include mutated antibody variable domains. Illustrative controls include scFvmut-detectable label formats.
Cancer imaging agents and controls as described herein may be provided in the form of kits for cancer imaging. Kits are optionally provided with instructions for use in cancer imaging.
Kits comprising vectors or host cells expressing a fusion protein comprising the antigen variable domain as described herein and a sortase A recognition sequence are contemplated. Vectors or host cells expressing a negative control imaging agents include mutated antibody variable domains and a sortase A recognition sequence.
Methods and uses
In one embodiment, the present disclosure enables the use of a composition comprising the ADC as described herein in, or in the preparation of a medicament for, the treatment of cancer including reduction of solid tumors and reduced metastasis in a subject diagnosed with cancer.
Accordingly, in one embodiment the composition comprises an Antibody- Fragment-Drug-Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and (ii) a drug construct comprising a prodrug of a highly potent cytotoxic agent such as an auristatin or equivalent cytotoxic agent conjugated to (i).
In one embodiment, the composition comprises an Antibody-Fragment-Drug- Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and part of a C-terminal sortase A sequence conjugated to the N-terminus of (ii) a drug construct comprising an N-terminal glycine sequence linked via a cleavable linker and optionally a spacer to a cytotoxic agent such as an auristatin or equivalent cytotoxic agent.
An illustrative ADC is shown in Figure 1 being produced by sortase A mediated site specific biological conjugation and then cleaved by cathepsin B to release the active cytotoxic agent MMAE.
Usefully the tumor microenvironment is pre-identified as comprising abundant activated platelets.
As determined herein platelets are abundant in a broad range of primary human tumors including breast, bowel and lung adenocarcinoma. In one embodiment, the cancer is selected from the group consisting of breast cancer, lung cancer, a glioblastoma, prostate cancer, pancreatic cancer, colon cancer, colorectal cancer, head and neck cancer, mesothelioma, kidney cancer, ovarian, oesophageal, squamous cell carcinoma, triple negative breast cancer, and non- small cell lung cancer.
In one embodiment, the disclosure enables a method of treating cancer and of reducing metastasis comprising administering an effective amount of a composition comprising an ADC as described herein to a subject with cancer. Accordingly, in one embodiment the composition comprises an Antibody-Fragment-Drug-Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and (ii) a drug construct comprising a prodrug of a highly potent cytotoxic agent such as an auristatin or equivalent cytotoxic agent conjugated to (i).
In one embodiment, the composition comprises an Antibody-Fragment-Drug- Conjugate (ADC) comprising: (i) a fusion protein comprising an antibody variable domain that binds only to the activated form of platelet- specific GPIIb/IIIa and part of a C-terminal sortase A sequence conjugated to the N-terminus of (ii) a drug construct comprising an N-terminal polyglycine (e.g. GGG) sequence linked via a cleavable linker and optionally a spacer to a cytotoxic agent such as an auristatin or equivalent cytotoxic agent.
As described and enabled herein, the effective amount concentrates therapeutic doses of cytotoxic agent within the tumor and at sites of metastasis. As a control, the same amount of ADC comprising a non-binding (mutant) antibody, provides substantially no therapeutic/cytotoxic/tumor/metastasis reduction activity.
In one embodiment, the effective amount also has substantially no or minimal off-target side effects on haematological parameters, liver function and renal function comparable to vehicle control.
The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.
The dosage ranges for the administration of the ADC of the disclosure are those large enough to produce the desired effect. For example, the composition comprises an effective amount of the binding protein. In one example, the composition comprises a therapeutically effective amount of the binding protein. In another example, the composition comprises a prophylactically effective amount of the binding protein.
The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.
Dosages can vary from about 0.01 mg/kg to about 5 mg/kg, or 0.01 -5 mg/kg or 0.1 - 2 mg/kg or 0.03 - 2 mg/kg, or 0.05 mg/kg to about 100 mg/kg, e.g., from about 0.1 mg/kg to about 50 mg/kg, such as, from about 0.5 mg/kg to about 20 mg/kg or 2 mg/kg to about 5 mg/kg, in one or more dose administrations daily for one or several days or weekly or every four days for three to six weeks or more. Please advise if a different dose is preferred. In some examples, the ADC is administered at an initial (or loading) dose which is higher than subsequent (maintenance doses). For example, the ADC is administered at an initial dose of between about 1 mg/kg to about 50mg/kg. The binding protein is then administered at a maintenance dose of between about O.OOOlmg/kg to about lOmg/kg. The maintenance doses may be administered every 7- 35 days, such as, every 7 or 14 or 28 days.
In some examples, a dose escalation regime is used, in which a ADC is initially administered at a lower dose than used in subsequent doses.
In the case of a subject that is not adequately responding to treatment, multiple doses in a week may be administered. Alternatively, or in addition, increasing doses may be administered.
A subject may be retreated with ADC, by being given more than one exposure or set of doses, such as at least about two exposures of the binding protein, for example, from about 2 to 60 exposures, and more particularly about 2 to 40 exposures, most particularly, about 2 to 20 exposures. In one example, any retreatment may be given after imaging with the labelled antibody variable domain protein to confirm the presence of activated platelets in the tumor.
In another example, any retreatment may be given at defined intervals. For example, subsequent exposures may be administered at various intervals, such as, for example, about 24-28 weeks or 48-56 weeks or longer. For example, such exposures are administered at intervals each of about 24-26 weeks or about 38-42 weeks, or about 50-54 weeks. Ideally, given the ability to administer an effective dose without substantial systemic side effects means that the patient may tolerate long term treatment, if required.
In one embodiment the ADC is administered systemically and displays minimal systemic side effects. As disclosed herein, the ADC is concentrated in the tumor microenvironment by binding of the antibody variable domain to activated platelets within the tumor microenvironment and wherein the cytotoxic agent is activated in the tumor microenvironment by endogenous cleavage of the ADC within the tumor microenvironment.
"Subjects" contemplated in the present description include patients including humans or animals including laboratory animals or art accepted test animals. Patients include human subjects in need of treatment or prophylaxis.
In another embodiment, the ADC is administered in combination with an additional cytotoxic or therapeutic agent. In one embodiment, the additional agent is selected from the group consisting of an anti-apoptotic agent, a mitotic inhibitor, an anti-tumor antibiotic, an immunomodulating agent, a nucleic acid for gene therapy, an alkylating agent, an anti-angiogenic agent, an anti-metabolite, a boron-containing agent, a chemoprotective agent, a hormone agent, an anti-hormone agent, a corticosteroid, a photoactive therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor, and a tyrosine kinase inhibitor.
The following reagents and methods may be employed in the production and practise of the present invention.
Imaging agents
Using a single-chain antibody (scFv) which specifically targets the active conformation of GPIIb/IIIa of both human and mouse activated platelets we demonstrate for the first time the ability to image tumours in vivo by targeting activated platelets using a GPIIb/IIIa scFv. The scFv was conjugated to three different contrast agents; Cy7 for fluorescence imaging, 64Cu for PET imaging and microbubbles for ultrasound imaging. This novel approach provides holds promise as a universal and flexible diagnostic method for the detection and imaging of cancer. In one embodiment, a subject is screened using the binding protein-imaging agent and, if positive, is treated with the binding protein-therapeutic agent.
Theranostic methods are enabled comprising (i) screening for cancer by detection with the binding agent-imaging agent conjugate, followed by (ii) treatment with the binding agent-therapeutic drug conjugate as described and enabled herein.
Generation of scFv -Biotin. -The generation of biotinylated scFvGPiib/iiia and scFvmut has been described previously. Both scFvs were sub-cloned into the AviTag™ containing pAC6 vector system, the DNA was then transformed into electrocompetent cell E. coli EVB101 (Avidity LLC) by electroporation. In vivo biotinylation was performed according to the manufacturer’s instruction producing the attachment of one biotin molecule on every scFv. Alternative tags are known in the art, such as a spycatcher/spyTag system.
Generation of scFv-LPETG- Using polymerase chain reaction (PCR) reaction, the LPETG tag as a sortase recognition sequence was introduced to the C-terminal end of the scFv. The entire scFv was then subcloned into a pSectag 2A vector (Invitrogen) for expression in human embryonic kidney cells (Invitrogen)29.
Conjugation of scFvs to Cy7 - The scFvGPiib/iiia and scFvmut constructed with a LPETG tag on the C-terminus were labelled with Cy7 using a Sortase A enzyme-based protocol31. Briefly, the Sortase A enzymatic reaction enables the conjugation of the LPETG with a glycine residue on the N-terminal of a cyclooctyne compound, bicyclo- [6.l.0]-non-4-yne (BCN) to form scFv-BCN. The resultant scFv-BCN was further conjugated to an azide-NIR dye (azide-Cyanine 7 dye) via copper-free click reaction to generate the FFECT tracer. Excess free dye was dialysed in PBS and the purified scFv- Cy7 was analyzed on a SDS-PAGE gel and NIR signal from the band of interest was confirmed using the Odyssey Imager.
64Cu Production and scFv Radiolabeling -64CuCl2 produced by the 64Ni(p,n)64Cu reaction was obtained from the Austin Health Centre for PET. The solution was provided with a radionuclidic purity, tested using gamma ray spectrometry of >99% and a radiochemical purity, tested using HPFC of >95%. The scFv used for PET/CT imaging were conjugated to a sarcophagine chelator, MeCOSar (Clarity Pharmaceuticals)21,22. For radiolabeling: to a mixture of scFv-MeCOSar (100 pg, in PBS), was 64CuCl2 (50 MBq) added at room temperature. After 30 min, diethylenetriaminepentaacetic acid (10 pF, 10 mM) was added to the mixture and incubated for a further 5 min at room temperature. Samples were washed twice with PBS in spin columns (Millipore, cutoff 10,000 MWCO) and resuspended in PBS at a final concentration of 0.2 mg/mL to produce scFvGPiib/nia-64Cu or scFvmut-64Cu.
Conjugation of scFv -Biotin to Microbubbles - Biotinlyated scFvs (4 pg) were directly added to a vial of target-ready microbubbles MBs (VisualSonics, Inc) and incubated at room temperature for 20 min23. The scFv-MBs were placed on ice and used within 6 hours of conjugation.
Cancer Cell Lines -Human cancer cell lines SKBr3, MDA-MB-231, Ramos and HT- 1080 were cultured in RPMI media (GIBCO® #21870) supplemented with 10% (v/v) FBS (Invitrogen), 100 ET/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in a 5% C02 humidified atmosphere.
Preparation of Human Washed Platelets -Fresh blood was drawn from informed healthy volunteers who had not taken anti-platelet drugs at least two weeks prior to venesection. Blood was collected into acid citrate dextrose (ACD; 85 mM sodium citrate, 72.9 mM citric acid, 110 mM D-glucose, 70 mM theophylline) (at a ratio of ACD:blood of 1:6) and supplemented with apyrase 0.005 U/mF and enoxaparin 20 U/mF. Whole blood was centrifuged at 200 g for 10 min. Platelet rich plasma (PRP) was obtained and then centrifuged at l700g for 7 min. The platelet poor plasma (PPP) was removed and platelets resuspended in platelet washing buffer (pH 6.5; 4.3 mM K2HP04; 4.3 mM Na2HP04; 24.3 mM NaH2P04; NaCl 0.113 M; 5.5 mM D-glucose; 10 mM theophylline) supplemented with enoxaparin (20 U/mL), apyrase (O.OlU/mL) and 0.5% BSA. Finally, washed platelets were resuspended in Tyrode’s buffer (pH 7.2- 7.4; 12 mM NaHCOs; Hepes 10 mM; NaCl 0.137 M; KCL 27 mM; D-glucose 55 mM) containing 1 mM CaCl and apyrase 0.02 U/mL.
Tumour Xenograft Model - 5-6 weeks old female BALB/c nude mice were purchased from the Animal Resources Centre, Canningvale. To establish tumour xenografts, mice were injected subcutaneously with exponentially growing SKBr3, MDA-MB-231, Ramos or HT-1080 cells (2.0 x 106 cells per mouse) in 0.3 ml of matrigel (BD Biosciences #356234) into the left flank region. Tumours were left to grow and measured daily until reaching a diameter of approximately 4 mm, which usually appears 2-3 weeks post xenograft cell injection.
In vivo Fluorescence Tomography Imaging of Tumour -Animals were injected intravenously with 20 pg scFvGPiib/nia-Cy7 or scFvmut-Cy7 and tracer was allowed to circulate for 20 hours. Animals were anesthetized with ketamine (50 mg/kg; Parnell Laboratories) and xylazine (10 mg/kg; Troy Laboratories) and placed in the FLECT scanner supplied with continuous 02 and 2% isofluorane. Fluorescence imaging was performed using the Trifoil InSyTe FLECT® imager using the following filters (Excitation - 730 and emission - 803) at 500ms at each frame. A CT scan was then performed using a Trifoil microCT in vivo Preclinical Imager using the following settings (X-ray voltage = 55 kVp, Exposure time = 1100 ms and Pitch= 1). A total projection of 180 projects over 360° of rotation was acquired. Projected data were rebinned by 1:4 and reconstructed using Butterworth filter. The FLECT and CT scan images were then fused and co-registered using the InVivoScope version 2.00-analysis software. For fluorescence quantification, a region-of-interest (ROI) was drawn around the area of tumour and mean fluorescence intensity (MFI) was quantified using the InVivoScope version 2.00 analysis software.
IVIS Imaging -Following FLECT imaging, 2D fluorescence imaging of mice was performed using the IVIS Lumina Series II Imaging System (Perkin Elmer) using the following settings (Filter Passband = Excitation 710-760 nm, Emission 810-875 nm). Fluorescence intensity of tumours and other organs was calculated using the Living Image software v4.5.l (Perkin Elmer) and presented as Average Radiant Efficiency [p/s/cm2/sr] / [pW/cm2].
In vivo PET Imaging -Animals were injected intravenously with 20 pg scFvGPiib/iiia- 64Cu or scFvmut-64Cu. Tracer was allowed to circulate for two hours. Animals were anesthetized with ketamine (50 mg/kg; Parnell Laboratories) and xylazine (10 mg/kg; Troy Laboratories) and placed in the PET/CT scanner supplied with continuous 02 and 2% isofluorane. PET/CT imaging was performed using a NanoPET/CT in vivo Preclinical Imager (Mediso) with a 30 min PET acquisition time, and coincidence mode of 1:3. This was followed by a CT scan with the following parameters (X-ray voltage = 55 kVp, Exposure time = 1100 ms and Pitch= 0.5). A total projection of 240 projects over 360° of rotation was acquired. Projected data were rebinned by 1:4 and reconstructed using a Ramlak filter. Following the CT scans, animals were killed, perfused with PBS and tumours and muscle sections were removed and weighed. The radioactivities of each organ including tumours were measured using a gamma counter (Perkin Elmer). Tumour uptake was then calculated as percentage injected dose/gram (% ID/g) of tumour over muscle signals.
In vivo Ultrasound Imaging -Ultrasound of animals was performed with a Vevo2l00 small animal high frequency ultrasound scanner (VisualSonics Inc) using the MS250 non-linear contrast transducer. Animals were placed under light sedation (range of 1% to 2% isofluorane), on the VisualSonics imaging station. The imaging station was heated to prevent hypothermia. Imaging was performed on the flank of the animal where the tumour was induced. Animals were injected intravenously with either scFvGPiib/iiia-MBs or scFvmut-MBs. Videos and images were acquired before, and at defined time points after injecting L5xl07 microbubbles in a total volume of 150 pL. Analysis was performed using VisualSonics imaging software (VisualSonics Inc).
Immunofluorescence of Cancer Cells and Platelets -Washed platelets were incubated with 5xl05 SkBr3 cells at 37°C for 2 hours. The platelets and cancer cell culture were then stained with CD41 (Beckman Coulter #A0778l or #IM0649U) and either PAC-l- FITC (BD Biosciences #340507), scFvGPiib/iiia-GFP or CD62P (P-selectin) (BD Biosciences #550561) antibody for 30 min. Cells were fixed with BD Cytofix solution (BD Biosciences) and was imaged using the Nikon Alr Plus Confocal Microscope, 40x objective.
Immunohistochemistry of Human and Mouse Tumour Sections- Human tumour sections were obtained from biopsy specimens from patients with histologically confirmed breast, bowel or lung adenocarcinoma. Mouse of human tumour sections were fixed in formalin solution (Sigma Aldrich #HT50l l28) for 24 hours, paraffin embedded and microtome sectioned (Leica) to 5 mM - 30 pM onto a glass slide. Sections were deparaffinized, and underwent antigen retrieval with 0.01 M Citric Acid in 90°C for 20 min. Tumour sections were stained overnight with a polyclonal rabbit anti-CD4l antibody (Abeam #ab63983), and detected with an Alexa Fluor 647 labeled anti-rabbit antibody (Life Technologies #A-2l245), counterstained with Hoechst® (Thermo Fisher Scientific #33342) and visualized using the Nikon Alr Plus Confocal Microscope, 60x oil objective.
Flow Cytometry of Tumour Cells- Tumours were excised from mice and placed into RPMI media. Ramos tumours and spleen sections were teased apart and filtered through a 100 pm filter into single cell suspensions. MDA-MB-231 tumours and mouse muscle sections were smashed in between two frosted slide glasses, digested with Liberase (Sigma #5401020001) for 2 hours and filtered through a 100 pm filter into single cell suspensions. Cells were then stained with mouse CD41 APC (eBioscience #17-0411) and flow cytometry was performed using a FACSCantoII scanner (BD Biosciences). Results were analyzed using the Flowlogic software.
Statistical Analysis -All data are reported as mean ± SEM of at least 3 independent assays unless otherwise noted. Statistical analyses were performed using unpaired Student’s T tests for comparison of two groups and one way ANOVA for comparisons of more than two groups. A P value of less than 0.05 was considered significant.
The central element of the imaging study is the ability to target and image activated platelets as a general component of the tumour microenvironment, thereby defining a novel non-invasive technique to diagnose and localize tumours in vivo. In addition, the results demonstrate the feasibility of Cy7, 64Cu and microbubbles as conjugates to the scFvGPiib/nia for imaging tumours in vivo. The ability to use the recombinant single-chain antibody scFvGPiib/nia, which specifically binds to activated platelets have been characterized extensively in previous in vivo studies, and thus provides a solid foundation to conclude based on the studies described herein that activated platelets can serve as a marker of the tumour microenvironment. Irrespective of the coupling approach, the specificity and sensitivity of the scFvGPiib/nia allowed strong targeting to activated platelets and thus represents a unique foundation of this molecular target as a unique approach for tumour imaging. Clinically, imaging of activated platelets in cancer can be used as an auxiliary imaging strategy to allow the accurate delineation of the anatomic distribution of tumours.. Secondly, as determined herein the scFvGPiib/nia illustrates the important role for targeting activated platelets in tumor therapy and the use of activated platelet- specific antibody variable domain targeted drug delivery as means to enhance the efficacy, whilst minimizing the systemic toxicity of chemotherapy. GPIIb/IIIa binding region
The protein comprising an antibody variable domain of the present disclosure comprises a binding region that is an inhibitor of GPIIb/IIIa receptor function and/or activity. In one example, the binding region specifically binds an epitope on GPIIb/IIIa recognised by a scFv consisting of a sequence set forth in SEQ ID NO: 1. In one example, the binding region competitively inhibits binding of a scFv consisting of a sequence set forth in SEQ ID NO: 1 to an epitope on GPIIb/IIIa. In one example, the binding region comprises an antibody variable region, e.g., is an antibody or an antibody fragment that binds to GPIIb/IIIa. For example, the antibody variable region binds specifically to activated GPIIb/IIIa. Suitable antibodies and proteins comprising variable regions thereof are known in the art and/or described herein. In one embodiment, the binding protein comprises a binding region, wherein the binding region is a protein comprising a Fv. For example, the protein comprises a single chain Fv fragment (scFv).
Single Chain Fv (scFv) Fragments -The skilled artisan will be aware that scFvs comprise VH and VL regions in a single polypeptide chain and a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv). In one example, the linker comprises the sequence SSGS. The present disclosure also contemplates a disulfide stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of VH and a FR of VL and the cysteine residues linked by a disulfide bond to yield a stable Fv. Alternatively, or in addition, the present disclosure encompasses a dimeric scFv, i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367.
Other Antibodies or Antigen Binding Fragments
Exemplary antibodies or antigen binding fragments thereof for use in the present disclosure are described herein or known in the art and include:
• a humanized antibody or fragment thereof, e.g., a protein comprising a human like variable region, which includes CDRs from an antibody from a non-human species (e.g., mouse or rat or non-human primate) grafted onto or inserted into FRs from a human antibody (e.g., produced by methods described in US5225539, US6054297, US7566771 or US5585089) • a human antibody or fragment thereof, e.g., antibodies having variable and, optionally, constant antibody regions found in humans, e.g. in the human germline or somatic cells or from libraries produced using such regions. The "human" antibodies can include amino acid residues not encoded by human sequences, e.g. mutations introduced by random or site directed mutations in vitro (e.g., produced by methods described in US5565332) and affinity matured forms of such antibodies.
• a synhumanized antibody or fragment thereof, e.g., an antibody that includes a variable region comprising FRs from a New World primate antibody variable region and CDRs from a non-New World primate antibody variable region (e.g., produced by methods described in W02007019620).
• a primatized antibody or fragment thereof, e.g., an antibody comprising variable region(s) from an antibody generated following immunization of a non-human primate (e.g., a cynomolgus macaque) (e.g., produced by methods described in US6113898).
• a chimeric antibody or chimeric antigen binding fragment, e.g., an antibody or fragment in which one or more of the variable domains is from a particular species (e.g., murine, such as mouse or rat) or belonging to a particular antibody class or subclass, while the remainder of the antibody or fragment is from another species (such as, for example, human or non-human primate) or belonging to another antibody class or subclass (e.g., produced by methods described in US6331415; US5807715; US4816567 and US4816397).
• a deimmunized antibody or antigen binding fragment thereof, e.g., antibodies and fragments that have one or more epitopes, e.g., B cell epitopes or T cell epitopes removed (i.e., mutated) to thereby reduce the likelihood that a subject will raise an immune response against the antibody or protein (e.g., as described in W02000034317 and W02004108158).
• a bispecific antibody or fragment thereof, e.g., an antibody comprising two types of antibodies or antibody fragments (e.g., two half antibodies) having specificities for different antigens or epitopes (e.g., as described in US5731168).
Additional exemplary antibody fragments for use in the present disclosure are described herein or known in the art and include:
• single-domain antibodies (domain antibody or dAb), e.g., a single polypeptide chain comprising all or a portion of the heavy chain variable domain of an antibody. • a diabody, triabody, tetrabody or higher order protein complex (e.g., as described in W098/044001, W094/007921 see also Kim et al Mol Cancer Ther 2008; 7(8) 2008).
• a half-antibody or a half-molecule, e.g., a protein comprising a single heavy chain and a single light chain.
The present disclosure also contemplates other antibodies and antibody fragments, such as:
• minibodies, e.g., as described in US5837821;
• heteroconjugate proteins, e.g., as described in US4676980;
• heteroconjugate proteins produced using a chemical cross-linker, e.g., as described in US4676980; and
• Fab3 (e.g., as described in EP19930302894).
Linkers
The present disclosure provides an ADC comprising one or more linkers.
The present disclosure contemplates various forms of covalent and non- covalent linkages. For example, the regions can be linked by a chemical or flexible or peptide linker. Peptide linkers may comprise between 2 and 30 amino acids in length. Linkers may comprise non-naturally occurring amino acids. In one embodiment, the linker sequence is at least about 3 amino acids in length. In one example, a linker comprises the sequence (Ala)3. A "flexible" linker is an amino acid sequence which does not have a fixed structure (secondary or tertiary structure) in solution. Such a flexible linker is therefore free to adopt a variety of conformations. Flexible linkers suitable for use in the present disclosure are known in the art. Flexible linkers are also disclosed in WO1999045132. The linker may comprise any amino acid sequence that does not substantially hinder interaction of the binding region with its target. Preferred amino acid residues for flexible linker sequences include, but are not limited to, glycine, alanine, serine, threonine proline, lysine, arginine, glutamine and glutamic acid.
In one example, the linker is a rigid linker. A "rigid linker" (including a "semi-rigid linker") refers to a linker having limited flexibility. For example, the relatively rigid linker comprises the sequence (EAAAK)n, where n is between 1 and 3. The value of n can be between 1 and about 10 or between about 1 and 100. For example, n is at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10. In one example, n is less than 100. For example, n is less than 90, or less than about 80, or less than about 60, or less than about 50, or less than about 40, or less than about 30, or less than about 20, or less than about 10. A rigid linker need not completely lack flexibility.
In one embodiment, the linker is a cleavable linker. For example, the linker comprises a cleavage site for a peptidase. For example, the linker comprises a cleavage site for urokinase, pro-urokinase, plasmin, plasminogen, TGFP, staphylokinase, Thrombin, a coagulation factor (e.g., Factor IXa, Factor Xa) or a metalloproteinase, such as an interstitial collagenase, a gelatinase or a stromelysin. Exemplary cleavable linkers are described in US6,004,555, US5,877,289, US6,093,399 and US5,877,289.
Further illustrative linkers include hydrazone linkers, disulphide linkers, peptide linkers and beta-glucuronide linkers.
Stable conjugation of antibody or smaller antibody fragments known in the art to drugs can be achieved using various different approaches and methods. Chemical strategies include amide linkages, Schiff base linkages, hydrazone formation (unstable in acidic pHs), thiols and click reaction technologies resulting in for example stable l,2,3-triaazole linkages. More recently, specific conjugation strategies have been developed with recombinant antibody fragments, these include streptavidin-biotin links, peptide bonds formed by sortase A peptidase between LPXTG and an N-terminal glycine, other engineer able site specific modifications include sulfhydryl groups His6- tags, N-terminal serine/threonine, and the above mentioned enzyme tags/SNAP tags.
SNAP tag technology may be used to conjugate (covalently couple) a single chain fragment variable drugs such as auristatin F via benzylguanine (BG). AURIF is a BG-modified version of MMAF suitable for SNAP-tag coupling. The SNAP-tag is an engineered version of the human DNA repair enzyme 06-alky lguaninc-DNA- alkyltransferase, which allows the covalent coupling of BG-modified components with a defined 1:1 stoichiometry. Conventional ADCs may employ chemical linkage to cysteine or lysine residues in mAbs, resulting in a heterogeneous mixture of products with a undefined drug-to-antibody ratio (DAR) and varying conjugation sites. SNAP- tag and similar strategies can overcome these limitations.
Suitable linker technologies are described in US 2018/0154011, US 20180133315 and US 20180106809.
Competitive Binding Assays
Assays for determining a binding protein that competitively inhibits binding of a scFv of the disclosure will be apparent to the skilled artisan. For example, the binding protein of the disclosure is conjugated to a detectable label, for example, a fluorescent label or a radioactive label. The labeled protein and the test binding protein are then mixed and contacted with GPIIb/IIIa or a peptide comprising an epitope thereof. The level of labeled protein is then determined and compared to the level determined when the labeled protein is contacted with the GPIIb/IIIa or the peptide comprising an epitope thereof in the absence of the binding protein. If the level of labeled protein is reduced in the presence of the binding protein compared to the absence of the binding protein, the binding protein competitively inhibits binding of the scFv.
Epitope Mapping Assays
In another example, the epitope bound by a protein described herein is mapped. Epitope mapping methods will be apparent to the skilled artisan. For example, a series of overlapping peptides spanning the GPIIb/IIIa sequence or a region thereof comprising an epitope of interest, for example, peptides comprising 10 to 15 amino acids are produced. The binding protein is then contacted to each peptide or a combination thereof and the peptide(s) to which it binds determined. This permits determination of peptide(s) comprising the epitope to which the binding protein binds. If multiple non-contiguous peptides are bound by the protein, the protein may bind a conformational epitope. Alternatively, or in addition, amino acid residues within GPIIb/IIIa are mutated, for example, by alanine scanning mutagenesis, and mutations that reduce or prevent protein binding are determined. Any mutation that reduces or prevents binding of the binding protein is likely to be within the epitope bound by the protein.
The present disclosure includes the following non-limiting Examples.
EXAMPLES
Example 1: Materials and Methods
Generation of targeting antibody scFv-LPETG and coupling enzyme sortase A- The generation of the single-chain antibody directed against GPIIb/IIIa (scFvGPiib/nia) and a control, non-binding, single-chain antibody (scFvmut) has been described previously in Wang et al. Circulation 2012:725(25):3117-3126. Using polymerase chain reaction, an LPETG-tag (sortase A recognition sequence), a V5-tag and a His-tag was introduced to the C-terminal end of the scFv. Ta et al. Circ. Res. 201 1;109(4):365-373. The entire scFv was then subcloned into a pSectag 2A vector (Invitrogen) for expression in human embryonic kidney (HEK) cells (Invitrogen), see Hohmann et al. Blood 2013: 12l (6):3067-3075. Sortase A is used to induce an enzymatic reaction used for the conjugation of the scFv, carrying an LPETG sequence to MMAE which was produced carrying a triple glycine sequence. Sortase A, a transpeptidase cloned from Staphylococcus aureus was produced and purified as previously described, see Wang et al. Theranostics 20l6;6(5):726-738. All proteins (scFvs and sortase A) contains a 6x His-tag, which was used for purification with nickel-based affinity chromatography (Invitrogen).
Conjugation of scFv with MMAE and Cy7 -The potent anti-mitotic drug, MMAE, carrying a Val-Cit linker and a triple glycine sequence (GGG-Val-Cit-PAB-MMAE) was synthesized by Levena Biopharma (Levena Biopharma). The scFvGPiib/nia and scFvmut constructed with a LPETG-tag were linked to GGG-Val-Cit-PAB-MMAE using a sortase A enzyme -based protocol described previously (Ta et al. Circ. Res. 20l l;l09(4):365-373) was used to produce scFvGPiib/m-MMAE and scFvmut-MMAE . Excess scFv which contains a His-tag was removed using metal affinity chromatography (Invitrogen) and excess MMAE was removed using a 10 kDa spin column. For imaging studies, Cy7 was incorporated into the conjugate by incubating scFvGPiib/m-MMAE and scFvmut-MMAE with 2x excess Cy7 via amine labeling (AAT Bioquest). Excess free dye was removed by dialysis in PBS. The purified scFv-Cy7- MMAE was then analyzed by SDS-PAGE gel and the protein and near-infrared signal from the band of interest was confirmed using the Odyssey Imager. Additionally, western blot was performed with rabbit anti-MMAE antibody (Levena Biopharma), detected with an anti-rabbit HRP antibody (Cell Signalling) to confirm conjugation of MMAE to the scFv.
Preparation of platelet rich plasma and flow cytometry - Blood was collected from healthy volunteers in citrate and centrifuged at 180 g for 10 minutes. The platelet rich plasma was then collected, stored at 37°C and used within two hours. For flow cytometry, platelet rich plasma was diluted 1 :20 in Tyrode’s buffer. To induce platelet activation, ADP was added at a final concentration of 20 mM and incubated for 5 minutes before adding the scFv. Binding was determined by anti-V5-FITC (Thermofisher Scientific) or rabbit anti-MMAE antibody (Levena Biopharma) and detected with anti -rabbit mAb coupled with AF647 (Invitrogen). Flow cytometry was performed using a FACS Fortessa scanner (BD Biosciences, Franklin Lakes, NJ, United States). Results were analyzed using the Flowlogic software. To determine the ability of cancer cells to activate platelets, platelet rich plasma was incubated with the cancer cell lines MDA-MB-231, HT29, HT1080 and PC3 for 6 hours at 37°C. As a positive control with the same experimental setting, ADP-activated platelet rich plasma was incubated with the cancer cells for 6 hours at 37°C. The cancer cell and platelet (resting/ ADP-activated platelets) mixtures were then stained with an anti-CD4l-PE monoclonal antibody (BD Biosciences, Franklin Lakes, NJ, United States) and scFvGPiib/iiia or scFvmut binding was detected by an anti-V5-FITC monoclonal antibody (Abeam, Cambridge, United Kingdom). Flow cytometry and analysis was performed as described above.
Cancer Cell Lines - A metastatic variant of the MDA-MB-231 triple-negative breast adenocarcinoma cell line (a kind gift from Dr Zhou Ou, Fudan University Shanghai Cancer Center, China) was transduced with a lentiviral vector containing codon- optimized firefly luciferase-mCherry under the control of the ubiquitin-C promoterF23,24 Li et al. European Journal of Cancer 2006;42(18):3274-3286 and Le et al. Nature Communications 2016;7: 10634) and was cultured in DMEM medium + Glutamax (GIBCO®), supplemented with 10% (v/v) FBS (Invitrogen), at 37°C in a 5% C02 humidified atmosphere. Cell identity was confirmed by karyotyping.
Cytotoxicity Assay - The cytotoxic activity of GGG-Val-Cit-PAB-MMAE and the scFv-MMAE conjugates were assessed in a cytotoxicity assay in the presence of cathepsin B. To induce cathepsin B cleavage, MMAE, GGG-Val-Cit-PAB-MMAE or scFv-MMAE conjugate was incubated with 0.01 units of pre- activated cathepsin B (Sigma Aldrich) for 4 hours at 37°C in 25 mM Acetate Buffer pH 4.8. MDA-MB-231 cells were seeded on a 96 well plate at 6000 cells/well overnight. The next day, cells were treated with MMAE, GGG-Val-Cit-PAB-MMAE or scFv-MMAE in the presence or absence of cathepsin B and incubated for 72 hours. Metabolism of the yellow tetrazolium salt (XTT) was determined according to the manufacturer’s instructions (Sigma Aldrich). Percentage killing relative to untreated control cultures was calculated using the following formula: 100 - (test value/untreated value xlOO). Each assay was repeated in triplicate. For platelet assays, 100 pL of platelet rich plasma (containing approximately 2xl07 platelets) was activated with 20 pM ADP. Activated platelets were incubated with scFv-MMAE for 30 minutes. To remove unbound scFv-MMAE, the platelet-scFv-MMAE mixture was centrifuged at 2000 g for 2 minutes, the supernatant was removed, and platelets were resuspended in PBS. As a control, unwashed platelet-scFv-MMAE mixture was used. Either washed or unwashed (control) platelet-scFv-MMAE mixture was added to MDA-MB-231, HT29, HT1080 and PC3 cells in the absence of exogenous cathepsin B and incubated for 72 hours before determination of XTT metabolism as described above.
Immunohistochemical analysis of cathepsin B in tumors - Tumors were fixed in formalin solution (Sigma Aldrich) for 24 hours, paraffin embedded, and microtome sectioned (Leica) to 5 pm - 30 pm onto a glass slide. Sections were deparaffinized and underwent antigen retrieval with 0.01 M Citric Acid in 90°C for 20 minutes. Tumor sections were stained overnight with an anti-cathepsin B antibody (Abeam) and detected with an Alexa Fluor 488 labeled anti-mouse antibody (Life Technologies) and cell surface membrane-reactive anti-sodium/potassium ATPase antibody (Abeam), counterstained with Hoechst® (Thermo Fisher Scientific) and visualized using the Nikon Alr Plus Confocal Microscope, 20x water objective.
Breast cancer metastasis model - To establish metastasis, 2 x 103 MDA-MB-231 cells in 20 pL PBS were injected into the fourth left mammary fat pad of 5 -6 weeks old anaesthetized BALB/C athymic nude mice. Primary tumor growth was measured by caliper and tumor volume was determined using the formula (length x width2)/2. Additionally, primary tumor growth was measured via biolumineseence using the !YlS Lumina II (Perkin Elmer, Waltham, MA, United States) imaging system by measuring luciferase activity after a tail-vein injection of 150 mg/kg D-luciferin (ThermoFisher Scientific, Waltham, MA, United States). Metastasis development was monitored via bioluminescence using IVIS Lumina II by measuring luciferase activity in the chest region, distant from the primary tumor, for a longer time point (60 sec) as previously described Le CP et al. Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination. Nature Communications. 2016; 7: 10634. Metastasis development was quantified using the Living Image software v4.5.1 (Perkin Elmer, Waltham, MA, United States) by quantifying photon/s in a region of interest around the chest bioluminescence signal.
For therapy studies, mice underwent bioluminescence imaging on day 3 and day 7 post tumor inoculation to confirm tumor growth. Mice with tumors were then randomly assigned to three groups and treated with either scFvGPnb/nia-M AE, scFvmut-MMAE or left untreated. Treatment was initiated 7 days following tumor inoculation, via intravenous injection of 6 mg/kg body weight of either scFvGpiib/ma- MMAE or scFvmut-MMAE followed by three additional treatments every fourth day. Primary tumor size and metastasis development was monitored twice a week as described above.
In vivo Fluorescence Imaging of scFvGPm/iiia-Cy 7 -MM AE - Animals were injected intravenously with 20 pg of scFvGPiib/nia-Cy7-MMAE or scFvmut-Cy7-MMAE. Fluorescence imaging of the ADC in mice was performed 24 hours later using the IVIS Lumina using the following settings (Filter Passband = Excitation 710-760 nm, Emission 810-875 nm). Following imaging, mice were sacrificed, organs perfused to remove circulating blood and reimaged. Flow cytometry and ex vivo fluorescence imaging of scFvGPm/iiia-GFP and Plbfl B ALB/C nude mice with MDA-MB-231 mammary tumor, or non-tumor bearing mice were injected intravenously with 20 mg of scFvGPiib/iiia-GFP or scFvmut-GFP and 20 pL of DyLight 649 anti-GPlbp (Emfret Analytics, Eibelstadt, Bayern, Germany). After 24 hours, animals were sacrificed, and tumor, spleen and femurs were extracted.
Tumor samples were fixed in formalin (Sigma Aldrich, St Louis, MO, United States) for 24 hours, paraffin embedded, and microtome sectioned (Leica, Wetzlar, Germany) to 20 pm onto a glass slide. Sections were deparaffinized, stained with Hoechst® and visualized using the Alr Plus Confocal Microscope using a 60x oil objective.
For flow cytometry, spleen sections were teased apart, disaggregated and filtered through a 100 pm filter into single cell suspensions. Bone marrow cells were extracted from the femurs and filtered through a 100 pm filter into single cell suspensions. Spleen and bone marrow cells were then suspended in 1 mL red cell lysis buffer (0.155 M NH4Cl, 0.01 M KHCO3, 0.01 mM EDTA) for 10 min and neutralized with wash buffer (PBS, 2mM EDTA, 0.1% BSA). Cells were stained with mouse CD41-PE (eBioscience, Waltham, MA, United States) and flow cytometry was performed using the Fortessa scanner. Results were analyzed using the Flowlogic software.
Mouse blood collection and toxicity measurements Mouse blood was collected via submandibular bleeds into EDTA-coated microtainer collection tubes (BD Biosciences, Franklin Lakes, NJ, United States) and blood counts were performed using the XS- 1000Ϊ hematologic analyzer (Sysmex Corporation, Kobe, Hyogo, Japan) to determine white blood cells (WBC) and platelets counts. For liver and kidney function tests, 500 pL of mouse blood was collected in citrate. Plasma was collected following centrifugation of blood at 1000 g for 10 minutes and alkaline phosphatase (ALP), alanine aminotransferase (ALT) and urea levels were measured using the Synchron LX20PRO System (Beckman Coulter Diagnostics, High Wycombe, United Kingdom) by Monash Pathology.
Statistical Analysis All data are reported as mean ± SEM of at least 3 independent assays unless otherwise noted. Statistical analyses were performed using one-way ANOVA, with Dunnett’s multiple comparisons test for analysis of metastasis volume and two-way ANOVA, with Tukey’s multiple comparisons test for analysis of primary tumor growth volume. A P value of < 0.05 was considered significant. Abbreviations ADC: antibody-drug conjugate; ADP: adenosine diphosphate; AF: Alexa Fluor; ALP: alkaline phosphatase; ALT: alanine aminotransferase; BLI: Bioluminescence; Cy7: Cyanine 7; GPIIb/IIIa: Glycoprotein Ilb/IIIa; IC50: half maximal inhibitory concentration; IVIS: In vivo imaging system; MMAE: monomethyl auristatin E; Mut: Mutant; NHS: N-hydroxysuccinimide; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; scFv: single-chain antibodies; Val-Cit: Valine-citrulline; HRP: horseradish peroxidase; WBC: white blood cells and XTT: tetrazolium salt.
Example 2: scFvGPiib/nia-Cy7-MMAE binds to activated platelets
To allow the unique targeting of a potent anti-mitotic agent to activated platelets, a novel antibody-drug-conjugate was developed. The unique scFvGPiib/iiia was employed which specifically targets activated platelets, combined with a sortase A based site-directed biological conjugation method to produce a novel ADC, scFvGPiib/iiia-MMAE incorporating the highly potent anti-mitotic agent (MMAE). In order to facilitate its conjugation to scFv and enzymatic release, a MMAE linker was designed with a triglycine (GGG) sequence, a cathepsin B cleavable peptide (Val-Cit) and a para-aminobenzylalcohol (PABA) self-immolative spacer (Figure 1). Each scFv has a sortase A recognition sequence, LPETG, at the C-terminus used for a site- directed, enzymatic coupling to the glycine sequence of GGG-Val-Cit-PAB-MMAE. To enable in vivo detection, the scFv-MMAE was labeled with a Cy7 dye by NHS labeling to yield, scFvGPiib/nia-Cy7-MMAE (Figure 1). Analysis of this final construct using SDS page electrophoresis and visualized using the Odyssey reader confirms successful Cy7 labeling of the scFv which has a molecular weight of 34 kDa (Figure 2A). The scFvGPiib/iiia carries a hexahistidine tag downstream of the LPETG sequence, which is cleaved following sortase A reaction, resulting in a final scFvGPiib/nia-Cy7- MMAE product with reduced molecular weight compared to the uncleaved scFvGPiib/iiia. MMAE conjugation to the scFv was analyzed via Western blotting using an anti-MMAE antibody, confirmed by the presence of a band at 34 kDa (Figure 2B). Additionally, flow cytometry experiments demonstrated that the specific binding of the scFvGPiib/nia-Cy7-MMAE construct to activated platelets was maintained post conjugation (Figure 2C). The scFvGPiib/iiia used in this study binds to only the activated form of platelet- specific GPIIb/IIIa, importantly both in human, as well as in mouse platelets. As a negative control, scFvmut was used comprising a point mutation, such that it acts as a non -binding control (Figure 2D)15Schwarz el al. Circulation Research 2006:99(l):22-33. Example 3: Cathepsin B releases scFvGPiib/nia-MMAE and scFvmut-MMAE to its potent and cytotoxic form
The cytotoxic activity of MMAE on tumor cells was assessed in culture after release from the conjugate by cathepsin B, and the results confirmed equal dose- dependent killing of the triple-negative breast adenocarcinoma cell line MDA-MB- 231 by scFvopiib/iiia-MMAE and scFvmut-MMAE. On MDA-MB-231 cells, the IC50 of scFvGPiib/nia-MMAE was l.98xlO 10 M while the IC50 of the scFvmut-MMAE was l.78xlO 10 M (Figure 3A). These studies confirmed that the present approach of utilizing a sortase A conjugation of MMAE to the scFv allowed for a controlled and equal coupling of MMAE to the scFv and the equivalent cathepsin B -mediated release of cytotoxic MMAE by scFvGPiib/nia and scFvmut.
The incorporation of a cathepsin B cleavable linker enables the activation of an inactive prodrug to an active cytotoxic form by utilizing the abundance of cathepsin B in the tumor microenvironment. In the prior art Schmid et al Bioconjug. Chem. 2007:18(3):702-716 disclose the activity of cathepsin B over expressed in solid tumors to cleave a prodrug.
Prior to animal studies, in vitro studies were performed to demonstrate that the conjugated MMAE is released and maintains cytotoxicity upon exposure to cathepsin B. MDA-MB-231 cells were incubated with either MMAE, GGG-Val-Cit-PAB- MMAE or scFv-MMAE in the absence and presence of cathepsin B. Increased cell killing by the GGG-Val-Cit-PAB-MMAE and scFv-MMAE was observed in the presence of cathepsin B, indicating release of MMAE. Upon cleavage, an exposed self immolative spacer can self-hydrolyse upon deacylation and free cytotoxic molecules are released. The IC50 killing efficacy of GGG-Val-Cit-PAB-MMAE on MDA-MB-231 was l.86xl0 8 M and the addition of cathepsin B increased the cellular killing efficacy by approximately 40-fold to 4.70xl0 10 M (Figure 3B), which is similar to the IC50 of the unmodified MMAE. The IC50 killing efficacy of scFvGPiib/nia-MMAE was 2.2lxl0 8 M and the addition of cathepsin B enhanced the killing efficacy to l.96xlO 10 M (Figure 3B). The results confirmed an increased cell killing by the GGG-Val-Cit-PAB-MMAE and scFv-MMAE in the presence of cathepsin B, indicating release of MMAE. A lower level (~lO 8 M) of non-specific killing of cancer cells was also observed with scFv- MMAE and GGG-Val-Cit-PAB-MMAE in the absence of exogenous Cathepsin B.
This is proposed herein to be due to the secretion of small amounts of cathepsin B from the cancer cells, or release of other cathepsins and proteases that can also cleave the
Val-Cit linker, as previously reported (Tan et al World J Biol Chem 4(4):9l- 101, 2013; Sudhan DR et al. Pharmacol Ther 775: 105-15, 2015; and Caculitan et al. Cancer Res 77(24):7027-37, 2017. These findings confirm the cytotoxic potency and the efficacy of the cathepsin B dependent release and thereby activation of the potent MMAE from the inactive form present in the newly created scFvGPiib/nia-MMAE-conjugate.
In this study, a novel approach of utilizing Val-Cit-PAB-MMAE, a cathepsin B dependent prodrug, conjugated to an antibody that targets activated platelets as a component of the tumor microenvironment.
Previous studies have indicated the abundance of secreted or cell surface- associated cathepsin B within tumor cells. To confirm the suitability of our targeting approach for a therapeutic application, the presence and localization, of cathepsin B within the tumor was investigated via immunofluorescence imaging of tumor xenograft sections stained with a cathepsin B antibody. This confirmed the abundance of cathepsin B within the MDA-MB-231 tumor cells and tumor microenvironment (Figure 3C). To ascertain the cellular localization of cathepsin B in tumor cells, a magnified image was acquired (data not shown). This confirmed the intracellular and pericellular location (indicated by white arrows) of cathepsin B within tumor cells.
To investigate if the therapeutic efficacy of scFvGPiib/iiia-MMAE is based on its specificity for activated platelets, the scFvGPiib/iiia-MMAE or scFvmut-MMAE was incubated with ADP-stimulated platelets in vitro. Platelets were subsequently washed to remove unbound scFvGPiib/iiia-MMAE or scFvmut-MMAE and then added to MDA- MB-231 cells (Figure 3D). Consistent with the specific binding of the targeted ADC to activated GPIIb/IIIa, incubation of scFvGPiib/iiia-MMAE with activated platelets resulted in increased MDA-MB-231 cell killing, with an IC50 of 5.9xl0 9 M. In contrast, activated platelets incubated with scFvmut-MMAE demonstrated no cellular killing. As a control to confirm the equal coupling of MMAE to the scFvGPiib/iiia and scFvmut, a mixture of activated platelets and either scFvGPiib/iiia-MMAE or scFvmut- MMAE, which did not undergo washing, were added to MDA-MB-231 tumor cells (Figure 3D). No significant differences in killing were observed between the scFvGPiib/iiia-MMAE and scFvmut-MMAE. The IC50 killing efficacy of scFvGPiib/nia- MMAE on MDA-MB-231 was l.27xl0 8 M and the IC50 killing efficacy of scFvmut- MMAE on MDA-MB-231 was l.38xl0 8 M, thereby confirming equal coupling efficacy of MMAE to both, scFvGPiib/iiia and scFvmut. Example 4: Cancer cells induces platelet activation and can be imaged using scF VGPiib/nia-Cy7-MMAE
The ability of cancer cells to activate platelets and the functionality of the scFvopiib/iiia-MMAE to target activated platelets in the tumor microenvironment was first analyzed in vitro via fluorescence imaging. MDA-MB-231 cells were incubated with washed human platelets and stained with a CD41 (GPIIb)-specific, non-activation- dependent antibody and two antibodies that are specific for activated GPIIb/IIIa on activated platelets; scFvGPiib/iiia-GFP and PAC-l, the latter antibody binds selectively to activated human GPIIb/IIIa and thus provides a positive control for our scFvGPiib/iiia. MDA-MB-231 cells were found to bind to and directly activated platelets, as shown by increased scFvGPiib/iiia-GFP (Figure 4A) and PAC-l binding (Figure 4B). Furthermore, for in vivo characterization, the scFvGPiib/iiia-MMAE was conjugated to Cy7 and the construct injected intravenously to MDA-MB-231 metastatic tumor-bearing mice. Bioluminescence imaging was used to detect areas of primary tumor as shown in Figure 5 A.
In further studies, the ability of cancer cells to activate platelets and the functionality of the scFvGPiib/iiia-MMAE to target activated platelets in the tumor microenvironment was first analyzed in vitro via fluorescence imaging. MDA-MB-231, HT29, HT1080 and PC3 cells were incubated with washed human platelets and stained with anti-CD4l antibody as a platelet marker and scFvGPiib/iiia, as a platelet activation marker. Strikingly, flow cytometry analysis of the platelet population (as defined by CD41 expression) revealed that incubation of platelets with MDA-MB-231, HT29, HT1080 or PC3 tumor cells resulted in activation of the entire platelet population, to a similar extent as ADP-activated platelets, thus confirming the ability of scFvGPiib/iiia to detect tumor cell-induced platelet activation (Figure 4C and 4D, red line). In contrast, scFvmut, as negative control, showed no binding to platelets incubated with cancer cells (Figure 4D, blue line- left most line). Furthermore, gating on the cancer cell population confirmed that platelets bind directly to cancer cells, to varying degrees, depending on the cell line (Figure 4C and 4E). Indeed, the percentage of MDA-MB-231, HT29, HT1080 and PC3 cells with adherent platelets were 11%, 18%, 54% and 12%, respectively (Figure 4E). To further illustrate the platelet-cancer cell interactions, we performed immunofluorescence imaging, demonstrating that platelets can bind directly to cancer cells and that cancer cells induce platelet activation, which can be detected by binding of the activation- specific scFvGPiib/iiia (Figure 4F).
Bioluminescence imaging of lung and lymph node metastasis was performed by covering the area of the primary tumor site and imaging with a longer exposure time (60 sec) as shown in Figure 5B. Fluorescence imaging of mice was performed 24 hours post injection of scFvGPiib/nia-Cy7-MMAE and this demonstrated the enrichment of scFvGPiib/nia-Cy7-MMAE at the primary tumor region as well as some nonspecific uptake by the liver (Figure 5C). Following imaging, mice were killed, organs perfused to remove circulating blood and reimaged. A highly significant enrichment of the scFvGPiib/nia-Cy7-MMAE occurred in the primary tumors as well as the lung metastases (Figure 5D). Additionally, to confirm the selectivity of the construct for platelets in the tumor microenvironment in vivo , MDA-MB-231 tumor-bearing mice were injected with scFvGPiib/nia-GFP or scFvmut-GFP and a Dylight 649 anti-GPIb antibody. This in vivo approach for immunofluorescence was used as the activated form of GPIIb/IIIa undergoes a conformational change and antigen masking upon tissue fixation and is not recognized by the scFvGPiib/iiia. The antibodies were allowed to circulate for 24 hours and tumor sections were extracted, sectioned and imaged using fluorescence microscopy to demonstrate the localization of platelets, detected by GPlb staining co localizing with activated platelets, detected by scFvGPiib/nia-GFP staining in the tumor microenvironment (Figure 5E). In contrast, no GFP signal or GPlb co-localization was observed in mice injected with scFvmut-GFP (Figure 5F), indicating the specificity of the scFvGPiib/iiia targeting to activated platelets.
Example 5 scFvGPiib/nia-Cy7-MMAE localizes to activated platelets in the tumor microenvironment and sites of metastasis but not to resting platelets in the spleen and bone marrow
In further studies, for in vivo characterization, scFvGPiib/nia-MMAE and scFvmut- MMAE were conjugated to Cy7 and injected intravenously to mice with MDA-MB-231 primary tumor that had already metastasized. Fluorescence imaging of mice performed 24 hours post injection of scFv-Cy7-MMAE demonstrated enrichment of scFvGPiib/nia- Cy7-MMAE at the primary tumor region, with no tumor localization of the scFvmut- Cy7-MMAE (Figure 6A). As the MDA-MB-231 cells had been previously transduced with a lentiviral vector to express luciferase, bioluminescence imaging was used to detect areas of primary tumor as shown in Figure 6B. Concordant with in vivo imaging, ex vivo analyses showed significant enrichment of scFvGPiib/nia-Cy7-MMAE but not scFvmut-Cy7-MMAE in the primary tumors (Figure 6C and 6D). Additionally, uptake of the ADC was observed most strongly in the center of the tumor area in mice injected with scFvGPiib/nia-Cy7-MMAE and minimal uptake of the control ADC was also observed in mice injected with scFvmut-Cy7-MMAE. The specific mechanism of the scFv based ADC uptake will be investigated in future studies. Bioluminescence imaging of lung and lymph node metastases was performed by covering the primary tumor site and imaging with a longer exposure time (60 sec) (Figure 6E and 6F). We further demonstrated that the scFvGPiib/nia-Cy7-MMAE localized in the lungs of MDA- MB-231 tumor-bearing mice with lung metastases (Figure 6E), but not in mice without lung metastases (Figure 6F), confirming that scFvGPiib/nia-Cy7-MMAE localization was specific to activated platelets within tumors. Again, no tumor localization of the scFvmut-Cy7-MMAE was observed in the lungs in the presence or absence of metastases (Figure 6E and 6F).
To further confirm the selectivity of the targeted ADC for platelets in the tumor microenvironment in vivo , MDA-MB-231 tumor-bearing mice were injected with scFvGPiib/nia-GFP or scFvmut-GFP and a Dylight 649 anti-GPl b antibody. This in vivo approach for immunofluorescence was used as the activated form of GPIIb/IIIa undergoes a conformational change and antigen masking upon tissue fixation and is not recognized by the scFvGPiib/nia. After 24 hours, tumors were extracted, sectioned and imaged using fluorescence microscopy to demonstrate the localization of platelets, detected by GP 16b staining co-localizing with activated platelets, as indicated by scFvGPiib/nia-GFP in the tumor microenvironment (Figure 7A). In contrast, we saw no GFP signal in mice injected with scFvmut-GFP (Figure 7B), indicating the specificity of the scFvGPiib/nia targeting to activated platelets.
To demonstrate the specificity of the scFvGPiib/nia to activated platelets in the tumor microenvironment, it was next investigated whether the scFv localized to the spleen and bone marrow, which are two abundant sources of non-activated platelets. BALB/C nude mice were injected with scFvGPiib/nia-GFP or scFvmut-GFP and the Dylight 649 anti-GPll^ antibody and the femurs and spleen were extracted after 24 hours and stained for CD41. Single cell analysis via flow cytometry of the spleen and bone marrow, gated on CD41 -positive cells displayed binding of the GPlt^ antibody in the spleen and bone marrow but no scFvGPiib/nia binding to these regions (Figure 7C and 7D), thus confirming the specificity of scFvGPiib/nia for activated platelets in vivo.
Example 6: Treatment with scFvGPiib/nia-Cy7-MMAE reduces tumor growth and metastasis
To investigate the in vivo effect of the scFvGPiib/nia-MMAE therapeutic, MDA- MB-231 metastatic tumor-bearing mice were imaged for bioluminescence signal at day 3 post MDA-MB-231 inoculation, and again at day 7 to confirm the presence of growing tumors (data not shown). To investigate the in vivo effect of the scFvGPiib/iiia- MMAE therapeutic, using the MDA-MB-231 murine metastasis model, mice were treated with scFvGPiib/nia-MMAE or scFvmut-MMAE and the effect of therapy on tumor growth and metastasis formation was assessed. Strikingly, treatment of tumor bearing mice with scFvGPiib/nia-MMAE at a dose of 6 mg/kg (administered every 4 days) resulted in a marked reduction in the primary tumor size by over 4-fold (70%) as compared to mice treated with scFvmut-MMAE or PBS (Figure 8A). Accordingly, a significant reduction in the development of lung and lymph node metastases on day 23, as measured by total bioluminescence, was seen in scFvGPiib/nia-MMAE treated mice compared to non-targeted and vehicle controls (Figure 8B). Significantly, treatment with scFvGPiib/nia-MMAE prevented the development of metastasis in 40% of mice (3 out of 7 mice), as indicated by bioluminescence imaging, while all mice treated with scFvmut-MMAE or PBS developed metastasis. Importantly, no therapeutic effects were observed with scFvmut-MMAE, thus highlighting the impressive ability of the instant targeting approach to concentrate therapeutic doses of MMAE specifically within the tumor and at sites of metastasis. Consistent with the data showing that the subject targeted MMAE does not cause off-target side effects, treated mice were healthy during the course of the treatment, did not lose weight and hematological parameters, liver function and renal function were comparable to vehicle control (Figure 8D) Taken together, these findings demonstrate the potential of using activated platelets as a therapeutic targeting strategy for the delivery of chemotherapy to maximize potency whilst sparing systemic side effects.
To examine whether the novel platelet targeted ADC disclosed herein displays efficacy against different tumor types, these experiments were repeated using a human colorectal cell line (HT29), a human fibrosarcoma cell line (HT1080) and a human prostate cancer cell line (PC3). It was first demonstrated that MDA-MB-231, HT29, HT1080 and PC3 tumor cells cultured in GGG-Val-Cit PAB-MMAE and scFvGPiib/iiia- MMAE, in the presence of cathepsin B were able to induce cellular killing, albeit at slightly varying concentrations (Figure 9A). Next, HT29, HT1080 and PC3 cell lines were cultured in ADP-activated platelets mixed with scFvGPiib/nia-MMAE and scFvmut- MMAE (Figure 9B). These studies confirmed that the scFvGPiib/nia-MMAE bound to activated platelets and induced cell killing of the HT29, HT1080 and PC3 cell lines, thus supporting the notion that this platelet-targeted ADC is active across a broad range of tumor cell types.
The specification discloses the targeting of activated platelets within the tumor microenvironment as novel strategy for the treatment of cancers including primary tumors and metastatic disease. This approach is based on the development of a unique ADC, which targets tumor-associated platelets in the tumor microenvironment using the activated GPIIb/IIIa as an epitope to deliver a therapeutic agent such as a highly potent synthetic anti-mitotic agent. Importantly, this novel approach allows the specific targeting and release of MMAE, via tumor-derived cathepsin B, whilst sparing untoward systemic side effects. The efficacy of ADC is highly contingent upon the presence and abundance of the antibody target within the tumor, representing a major limitation for the use of ADCs in several cancer types, which do not express a clinically validated specific antigenic molecular target. In this regard, a particular advantage of the present approach is the possible ubiquitous nature of activated platelets in a broad range of human tumors, including breast (Lal et al., Breast Cancer Research. 20l3;l5:207), colorectal (Li et al., Scientific Reports. 20l7;7(l): 10261), lung (Ji et al., Platelets. 20l5;26(2): 138-142), ovarian (Stone et al., N Engl J Med. 2012;366(7):610- 618) and esophageal (Shimada et al., J. Am. Coll. Surg. 2004;l98(5):737-74l) tumors, indicating that the platelet- targeting strategy holds promise for a large range of tumors. The inventors initially established the ability of activated platelet-specific scFv for tumor imaging (Yap et al., Theranostics. 20l7;7(l0):2565-2574), thus providing a theranostic approach, allowing for the molecular imaging of tumors as a means to detect tumor-associated platelets and therefore predict those likely to respond to platelet-targeting therapy and treatment of patients with the same antibody conjugated to a cytotoxic agent
Additionally, the Examples demonstrate the ability to target the activated platelet as a novel approach to deliver a pro-drug for killing of tumor cells. In one example, the Valine-citrulline (Val-Cit) linker is used as a cathepsin B cleavable linker requiring the presence of cathepsin B for cleavage and release of free MMAE, the active anti-cancer agent. Therapeutic approaches using ADCs, such as MMAE are commonly used to target antigens on the cancer cell surface followed by internalization. In this study, the tumor- specific cytotoxic potency of scFvGPiib/iiia- MMAE, conjugated via a Val-Cit linker targeting activated platelets within the tumor microenvironment is demonstrated. Whilst cathepsin B is well characterized to be highly expressed in tumor cells, the inventors have established surprisingly that cathepsin B is also released by tumor cells and able to contact activated platelets labelled with the subject ADC . In initial work showed the ability of activated platelets to serve as useful markers for cancer imaging. However, imaging does not require contact between the activated platelet (anticipated to reside in the tumor microvasculature) and the tumor cell, nor between the imaging agent and the tumor cell. Activation of the prodrug in the tumor microvasculature might not have taken place (due to lack of Cathepsin B access to the microvasculature) or the active drug might have been flushed from the tumor vessels without any therapeutic effect. The inventors proposed that the drug would achieve a high enough local concentration to inhibit tumour growth and surprisingly this is what they found raising the prospect that sufficiently abundent activated platelets are found within the tumour microenvironment for effective delivery of drug to tumor and validating the novel and inventive concept of platelet targeting for the treatment of solid tumors and also for reducing metastases. Prior to the present disclosure, it was not expected that activated platelets would be abundant in the stroma. Similarly Cathepsin B is not expected to be within the microvasculature.
In one embodiment an scFv is used as an antibody format, and it offers several advantages. The small size allows quick and efficient penetration of the tumor environment. The recombinant nature of design offers flexibility with respect to conjugation with drugs to generate a homogenous ADC, as described using a sortase A conjugation method. Furthermore, scFvs can be produced in various expression systems, adaptable to clinically applicable purity and scale. Our specific scFvGPiib/iiia also possesses two additional advantages for future work aiming for clinical translation: Firstly, the scFvGPiib/nia was developed from a human scFv library, reducing the risk of antigenicity. Secondly, scFvGPiib/nia is cross reactive between activated platelets from mice and humans (Schwarz et al., Circulation Research. 2006;99(l):25-33).
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.
The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.
Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).
Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps. Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention.
The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.
BIBLIOGRAPHY
Armstrong PC, Peter K. GPIIb/IIIa inhibitors: from bench to bedside and back to bench again. Thromb. Haemost. 2012;107(5):808-814.
Castiglioni T, Merino MJ, Elsner B, et al. Immunohistochemical analysis of cathepsins D, B, and L in human breast cancer. Human Pathology. l994;25(9):857- 862.
Chubak J, Kamineni A, Buist DS, Anderson ML, Whitlock EP. Aspirin Use for the Prevention of Colorectal Cancer: An Updated Systematic Evidence Review for the U.S. Preventive Services Task Force. Rockville (MD): Agency for Healthcare Research and Quality (US); 2015.
Doronina SO, Toki BE, Torgov MY, et al. Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nature Biotechnology. 2003;2l(7):778-784.
Gondi CS, Rao JS. Cathepsin B as a cancer target. Expert Opin. Ther. Targets. 2013;17(3):281-291.
Hohmann JD, Wang X, Krajewski S, et al. Delayed targeting of CD39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding? Blood. 2013;121(16):3067— 3075.
Ji Y, Sheng L, Du X, Qiu G, Su D. Elevated platelet count is a strong predictor of poor prognosis in stage I non-small cell lung cancer patients. Platelets. 20l5;26(2): 138—142.
Lal I, Dittus K, Holmes CE. Platelets, coagulation and fibrinolysis in breast cancer progression. Breast Cancer Research. 20l3;l5:207.
Le CP, Nowell CJ, Kim-Fuchs C, et al. Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination. Nature Communications. 20l6;7:l0634.
Li D-Q, Hou Y-F, Wu J, et al. Gene expression profile analysis of an isogenic tumour metastasis model reveals a functional role for oncogene AF1Q in breast cancer metastasis. European Journal of Cancer. 2006;42(l8):3274-3286.
Li N, Yu Z, Zhang X, et al. Elevated mean platelet volume predicts poor prognosis in colorectal cancer. Scientific Reports. 20l7;7(l): 10261.
Pietersz GA, Wang X, Yap ML, Lim B, Peter K. Therapeutic targeting in nanomedicine: the future lies in recombinant antibodies. Nanomedicine. 2017; 12(15): 1873-1889.
Roshy S, Sloane BF, Moin K. Pericellular cathepsin B and malignant progression. Cancer Metastasis Rev. 2003;22(2-3):27l-286. Ruan H, Hao S, Young P, Zhang H. Targeting Cathepsin B for Cancer Therapies. Horiz Cancer Res. 2015;56:23-40.
Sanderson RJ, Hering MA, James SF, et al. In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin. Cancer Res. 2005;l l(2 Pt l):843-852.
Schmid B, Chung D-E, Warnecke A, Fichtner I, Kratz F. Albumin-binding prodrugs of camptothecin and doxorubicin with an Ala-Leu-Ala-Leu-linker that are cleaved by cathepsin B: synthesis and antitumor efficacy. Bioconjug. Chem. 2007;18(3):702-716.
Schwarz et al. JPET 2004 308:1002.
Schwarz M, Meade G, Stoll P, et al. Conformation-Specific Blockade of the Integrin GPIIb/IIIa A Novel Antiplatelet Strategy That Selectively Targets Activated Platelets. Circulation Research. 2006;99(l):25-33.
Schwarz M, Rottgen P, Takada Y, et al. Single-chain antibodies for the conformation-specific blockade of activated platelet integrin allbp3 designed by subtractive selection from naive human phage libraries. FASEB J. 2004;
Shimada H, Oohira G, Okazumi S, et al. Thrombocytosis associated with poor prognosis in patients with esophageal carcinoma. J. Am. Coll. Surg. 2004;l98(5):737- 741.
Sloane BF, Yan S, Podgorski I, et al. Cathepsin B and tumor proteolysis: contribution of the tumor microenvironment. Semin. Cancer Biol. 2005;15(2):149-157.
Stone RL, Nick AM, McNeish IA, et al. Paraneoplastic Thrombocytosis in Ovarian Cancer. N Engl J Med. 2012;366(7):610-618.
Ta HT, Prabhu S, Leitner E, et al. Enzymatic single-chain antibody tagging: a universal approach to targeted molecular imaging and cell homing in cardiovascular disease. Circ. Res. 20l l;l09(4):365-373.
Wang X, Gkanatsas Y, Palasubramaniam J, et al., Theranostics. 20l6;6(5):726-
738.
Wang X, Hagemeyer CE, Hohmann JD, et al., Circulation. 2012;125(25):3117-
3126.
Yap ML, McFadyen JD, Wang X, et al. Targeting Activated Platelets: A Unique and Potentially Universal Approach for Cancer Imaging. Theranostics. 20l7;7(l0):2565-2574.

Claims

CLAIMS:
1. A binding protein drug conjugate for delivering a therapeutic drug to a tumor comprising (i) a binding protein comprising an antibody variable domain that specifically binds specifically to the activated form of platelet- specific GPIIb/IIIa conjugated to (ii) a therapeutic drug, wherein the binding protein concentrates the therapeutic drug in the tumor microenvironment and provides targeted delivery of the therapeutic drug to the tumor cells.
2 The binding protein drug conjugate of claim 1 wherein the therapeutic drug is chemotherapeutic prodrug and the binding protein drug conjugate comprises a cleavable linker and optionally a spacer between the prodrug and the binding protein which upon cleavage of the linker releases the active chemotherapeutic drug from the binding protein.
3. The binding protein drug conjugate of claim 2 wherein the cleavable linker is selectively cleaved by a molecule present in the tumor stroma.
4. The binding protein drug conjugate of claim 3 wherein the cleavable linker is a peptide or comprises a dipeptide, such as Val-Cit, Phe-Lys and Val-Ala.
5. The binding protein drug conjugate of any one of claim 1 to 3 wherein the binding protein is an antibody, a Fv, scFv, di-scFv, diabody, triabody, tetrabody, Fab, F(ab')2, an ibody or darpin.
6. The binding protein drug conjugate of any one of claims 1 to 5, wherein the binding protein and/or the therapeutic drug are modified for conjugation (coupling).
7. The binding protein drug conjugate of any one of claim 6 wherein the modifications are for site- specific conjugation.
8. The binding protein drug conjugate of claim 7 wherein binding protein and the therapeutic agent are conjugated by sortase A mediated conjugation.
9. The binding agent of any one of claim 1 to 8 wherein the therapeutic drug is selected from the group consisting of a mitotic inhibitor, a plant alkaloid, and an anti tumor antibiotic.
10. The binding protein drug conjugate of any one of claims 1 to 9 wherein the antibody variable domain binds an epitope of GPIIb/IIIa recognised by a scFv comprising of an amino acid sequence set forth in SEQ ID NO: 1 or 5.
11. The binding protein drug conjugate of any one of claims 1 to 10, wherein the antibody variable domain comprises a complementary determining region (CDR) of the heavy chain variable region (VH) having the sequence of SEQ ID NO:2 or 6 and/or a CDR of the light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 3 or 7.
12. The binding protein drug conjugate of any one of claims 1 to 10, wherein the antibody variable domain comprises a heavy chain variable region (VH) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 2 or 6 and a light chain variable region (VL) comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 3 or 7.
13. The binding protein drug conjugate of any one of claims 1 to 10, wherein the antibody variable domain comprises an amino acid sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 1 or 5.
14. The binding protein drug conjugate of any one of claims 1 to 10, wherein the antibody variable domain is derived from a human, such as a human scFv, library.
15. The binding protein drug conjugate of claim 2, comprising a spacer wherein the spacer is a self immolative spacer, such as a para-aminobenzylalcohol (PABA) spacer or a non- self immolative spacer.
16. The binding protein drug conjugate of any one of claims 1 to 15 wherein cytotoxic agent is cytotoxic to tumour cells at nanomolar or picomolar concentrations or less.
17. The binding protein drug conjugate of any one of claims 1 to 16 comprising a detectable label suitable for imaging tumors, diagnostic or monitoring.
18. The binding protein drug conjugate of any one of claims 1 to 17 wherein the ratio of antibody variable domain to drug is between about 1:1 and 1:10, or 1:1 and 1:2, inclusive.
19. A composition comprising the binding protein drug conjugate of any one of claims 1 to 18.
20. A composition comprising the binding protein drug conjugate of any one of claims 1 to 18 and a pharmacologically or physiologically acceptable diluent and/or carrier.
21. A composition comprising the binding protein drug conjugate of any one of claim 1 to 18 for use in medical therapy or imaging.
22. Use of a composition comprising the binding protein drug conjugate of any one of claims 1 to 16 in, or in the preparation of a medicament for, the treatment of cancer including reduction of solid tumors and reduced metastasis in a subject with cancer.
23. Use of claim 22 or method of claim 26 wherein the cancer is selected from the group consisting of breast cancer, lung cancer, a glioblastoma, prostate cancer, pancreatic cancer, colon cancer, colorectal cancer, head and neck cancer, mesothelioma, kidney cancer, ovarian, oesophageal, squamous cell carcinoma, triple negative breast cancer, and non-small cell lung cancer.
24. A fusion protein or a kit comprising a binding protein comprising the antibody variable domain of any one of claims 1 to 13 labelled with a detectable label suitable for imaging, diagnostics or monitoring.
25. A vector or host cell comprising a polynucleotide sequence capable of expressing the binding protein comprising the antibody variable domain of any one of claims 1 to 13.
26. A method of treating cancer and reducing metastasis comprising administering an effective amount of an binding protein drug conjugate according to any one of claims 1 to 13 to a subject with cancer.
27. The method of claim 21 wherein the binding protein drug conjugate is administered in combination with an additional drug selected from the group consisting of an anti-apoptotic agent, a mitotic inhibitor, an anti-tumor antibiotic, an immunomodulating agent, a nucleic acid for gene therapy, an alkylating agent, an anti- angiogenic agent, an anti-metabolite, a boron-containing agent, a chemoprotective agent, a hormone agent, an anti-hormone agent, a corticosteroid, a photoactive therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor, and a tyrosine kinase inhibitor.
28. Use of a binding protein comprising an antibody variable domain that binds specifically to the activated form of platelet- specific GPIIb/IIIa in the preparation of a cancer imaging agent.
PCT/AU2019/050638 2018-06-22 2019-06-21 A therapeutic gpiib/iiia binding-protein drug conjugate and use thereof WO2019241847A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2018902247 2018-06-22
AU2018902247A AU2018902247A0 (en) 2018-06-22 Therapeutic agent

Publications (1)

Publication Number Publication Date
WO2019241847A1 true WO2019241847A1 (en) 2019-12-26

Family

ID=68982510

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2019/050638 WO2019241847A1 (en) 2018-06-22 2019-06-21 A therapeutic gpiib/iiia binding-protein drug conjugate and use thereof

Country Status (1)

Country Link
WO (1) WO2019241847A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005091805A2 (en) * 2004-02-13 2005-10-06 The Scripps Research Institute Methods and compositions for inhibition of metastasis
US20090180960A1 (en) * 2007-12-21 2009-07-16 Baker Medical Research Institute Diagnosis and treatment of diseases involving platelet activation
WO2012006633A1 (en) * 2010-07-09 2012-01-12 Biogen Idec Hemophilia Inc. Chimeric clotting factors
WO2015070014A1 (en) * 2013-11-08 2015-05-14 Biogen Idec Ma Inc. Procoagulant fusion compound

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005091805A2 (en) * 2004-02-13 2005-10-06 The Scripps Research Institute Methods and compositions for inhibition of metastasis
US20090180960A1 (en) * 2007-12-21 2009-07-16 Baker Medical Research Institute Diagnosis and treatment of diseases involving platelet activation
WO2012006633A1 (en) * 2010-07-09 2012-01-12 Biogen Idec Hemophilia Inc. Chimeric clotting factors
WO2015070014A1 (en) * 2013-11-08 2015-05-14 Biogen Idec Ma Inc. Procoagulant fusion compound

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BONNARD T. ET AL.: "Novel Thrombolytic Drug Based on Thrombin Cleavable Microplasminogen Coupled to a Single-Chain Antibody Specific for Activated GPIIb/IIIa", J AM HEART ASSOC., vol. 6, no. 2, 2017, pages 1 - 18, XP055664231 *
COHEN S. ET AL.: "Potential Future Clinical Applications for the GPIIb/IIIa Antagonist, Abciximab in Thrombosis, Vascular and Oncological Indications", PATHOLOGY ONCOLOGY RESEARCH, vol. 6, no. 3, 2000, pages 163 - 174, XP055664258 *
HOHMANN J. ET AL.: "Delayed targeting of CD 39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding?", BLOOD, vol. 121, no. 16, 2013, pages 3067 - 3075, XP055664257 *
NAKADA M. ET AL.: "c7E3 Fab inhibits human tumor angiogenesis in a SCID mouse human skin xenograft model", ANGIOGENESIS, vol. 9, 2006, pages 171 - 176, XP019446557, DOI: 10.1007/s10456-006-9053-x *
YAP M. ET AL.: "Targeting Activated Platelets: A Unique and Potentially Universal Approach for Cancer Imaging", THERANOSTICS, vol. 7, no. 10, 2017, pages 2565 - 2574, XP055664228 *

Similar Documents

Publication Publication Date Title
AU2020200324B2 (en) Anti-Axl antibodies, antibody fragments and their immunoconjugates and uses thereof
ES2788457T3 (en) Anti-GCC Antibody Molecules and Related Compositions and Methods
CN101573384B (en) Antibodies and immunoconjugates and uses therefor
TW202330036A (en) Manufacturing method of antibody-drug conjugates
ES2749181T3 (en) Anti-GCC antibody molecules and use thereof to test for susceptibility to GCC-targeted therapy
ES2549362T3 (en) Antigens associated with endometriosis
TW201831514A (en) Anti-met antibodies, bispecific antigen binding molecules that bind met, and methods of use thereof
JP2010528056A (en) Humanized anti-TROP-2 antibody and chimeric anti-TROP-2 antibody that mediate cell killing effect of cancer cells
EP2668209B1 (en) Antibodies selective for cells presenting egfr at high density
CN103119442A (en) Immuno-PEG imaging of antibodies and immunoconjugates and uses therefor
ES2930255T3 (en) Anti-Ror2 antibodies, antibody fragments, their immunoconjugates and uses thereof
US11198738B2 (en) Therapeutic antibodies and uses thereof
SG188795A1 (en) Chimeric and humanized anti-cd44 antibodies that mediate cancer cell cytotoxicity
JP7383682B2 (en) Anti-EDB antibodies and antibody-drug conjugates
CA3005469A1 (en) Novel anti-emr2 antibodies and methods of use
JP2009528994A (en) Cancer-like disease-modifying antibody
JP2013144700A (en) Cytotoxicity mediation of cell evidencing surface expression of cd44
CA3118542A1 (en) Cdcp1-targeted therapies
US9931416B2 (en) Anti-claudin 1 antibodies for use in the treatment of colorectal cancer
JP2010530361A (en) Cell cytotoxicity mediating surface expression of CD59
JP2020523414A (en) Antibody drug conjugates that bind to LGR5
WO2021228044A1 (en) Drug conjugates containing alpha-enolase antibodies and uses thereof
WO2019241847A1 (en) A therapeutic gpiib/iiia binding-protein drug conjugate and use thereof
AU2017261369A1 (en) Novel anti-TNFRSF21 antibodies and methods of use
CA3009488A1 (en) Novel anti-upk1b antibodies and methods of use

Legal Events

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

Ref document number: 19821752

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19821752

Country of ref document: EP

Kind code of ref document: A1