[go: up one dir, main page]

WO2014138186A1 - Bioorthogonal two-component delivery systems for enhanced internalization of nanotherapeutics - Google Patents

Bioorthogonal two-component delivery systems for enhanced internalization of nanotherapeutics Download PDF

Info

Publication number
WO2014138186A1
WO2014138186A1 PCT/US2014/020620 US2014020620W WO2014138186A1 WO 2014138186 A1 WO2014138186 A1 WO 2014138186A1 US 2014020620 W US2014020620 W US 2014020620W WO 2014138186 A1 WO2014138186 A1 WO 2014138186A1
Authority
WO
WIPO (PCT)
Prior art keywords
alb
composition
peg
range
integer corresponding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2014/020620
Other languages
French (fr)
Inventor
Sudath HAPUARACHCHIGE
Dmitri Artemov
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Johns Hopkins University
Original Assignee
Johns Hopkins University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Johns Hopkins University filed Critical Johns Hopkins University
Publication of WO2014138186A1 publication Critical patent/WO2014138186A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61K49/0056—Peptides, proteins, polyamino acids
    • 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
    • A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • 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
    • A61K47/6851—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 determinant of a tumour cell
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • 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
    • 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
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • 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
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers

Definitions

  • Targeted delivery of nanocarriers offers a great promise to significantly improve the efficacy of traditional cytotoxic cancer therapy while reducing its systemic side effects.
  • Important features of targeted nanocarriers include chemical stability, high affinity to target sites, low non-specific interactions, optimal pharmacokinetics, and high drug loading capacity.
  • a traditional strategy to develop targeted nanocarriers utilizes cytotoxic antibody- drug conjugates (ADCs) where chemotherapeutics are directly attached to the target-specific antibody. This emerging new class of nanoscale therapeutic agents is currently used clinically for targeted therapy of cancer cells that overexpress a suitable surface receptor.
  • ADCs cytotoxic antibody- drug conjugates
  • the HER2 receptor regulates multiple physiological pathways including cell proliferation and differentiation.
  • the HER2 receptor is also known for its poor internalization capability, even after trastuzumab binding and subsequent heterodimerization, possibly due to localization of the receptor in membrane protrusion or/and in lipid raft areas where it has poor contact with the lipid bilayer.
  • the humanized anti-HER2 monoclonal antibody (mAb), trastuzumab (Herceptin ® ) is used as a first line treatment for HER2/wew positive breast cancers.
  • the cytotoxic mechanism of trastuzumab includes the inhibition of P13K/Akt and Ras/MAPK signaling pathways leading to cell cycle arrest.
  • approximately 50% of patients with HER2 -positive disease do not benefit from trastuzumab or become refractory to it even though the HER2 level remains high.
  • T-DM1 trastuzumab-emtansine conjugate
  • T-DM1 trastuzumab-emtansine conjugate
  • the direct conjugation of chemotherapeutics to mAb can reduce the in vivo therapeutic index, by reducing the binding affinity of the antibody with targeted receptors.
  • antibody conjugation does not enhance the ADC internalization and results in the systemic toxicity due to long systemic circulation.
  • thrombocytopenia in which the blood has a lower than normal number of platelets, has been observed in a small number of patients treated with ADCs.
  • the present invention provides a composition comprising a molecule of formula I:
  • Ab is a monoclonal antibody to a target of interest
  • (Peg a -Az) is a polyethylene glycol chain containing a terminal azide (Az) functional group linked to the Ab by the other end
  • a is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24
  • b is an integer corresponding to the number of (Peg a -Az) groups covalently and directly linked to the Ab in a range from 1-30.
  • the present invention provides a composition comprising a molecule of formula II:
  • Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers);
  • CTX is a chemotherapeutic agent covalently and directly linked to Alb and x is an integer corresponding to the number of C7 molecules attached per Alb in a range from 1-6;
  • (Peg favor-DBCO) is a polyethylene glycol chain containing a terminal dibenzylcyclooctyne (DBCO) functional group linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and y is an integer corresponding to the number of (Peg perennial-DBCO) groups covalently and directly linked to the Alb in a range from 5- 20.
  • albumin bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers
  • CTX is a chemotherapeutic agent covalently and directly linked to Alb and x is an integer corresponding
  • the present invention provides a composition comprising a molecule of formula III: Ab(Peg a -Az) b (Fl) c (Ill);
  • Ab is a monoclonal antibody to a target of interest
  • (Peg a -Az) is a polyethylene glycol chain containing a terminal azide (Az) functional group linked to the Ab by the other end
  • a is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24
  • b is an integer corresponding to the number of (Peg a -Az) groups covalently and directly linked to the Ab in a range from 1-30
  • Fl is a first fluorophore covalently and directly linked to the Ab
  • c is an integer corresponding to the number of Fl fluorophores attached per Ab in a range from 1-5.
  • the present invention provides a composition comprising a molecule of formula IV:
  • Alb denotes albumin (bovine serum albumin, human serum albumin, or other similar and biocompatible proteins and polymers);
  • CTX is a chemotherapeutic agent covalently and directly linked to Alb and x is an integer corresponding to the number of CT molecules attached per Alb in a range from 1-6;
  • (Peg favor-DBCO) is a polyethylene glycol chain containing a terminal DBCO functional group, wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; y is an integer corresponding to the number of (Peg perennial-DBCO) groups covalently and directly linked to the Alb in a range from 5-20; and
  • F2 is a second fluorophore covalently and directly linked to the Alb, and z is an integer corresponding to the number of fluorophores attached per Alb in a range from 1-5.
  • the present invention provides a composition comprising a molecule of formula V:
  • Ab is a monoclonal antibody to a target of interest
  • TCO is a iraws-cyclooctene functional group linked to the Ab by the other end
  • d is an integer corresponding to the number TCO groups covalently and directly linked to the Ab in a range from 5-6.
  • the present invention provides a composition comprising a molecule of formula VI:
  • Alb(CTX) y (Peg n -Tt) z (VI); wherein Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers); CTX is a chemotherapeutic agent covalently and directly linked to Alb and y is an integer corresponding to the number of C7 molecules attached per Alb in a range from 1-6;
  • (Peg perennial-Tt) is a polyethylene glycol chain containing terminal tetrazine (Tt) functional groups linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and z is an integer corresponding to the number of (Peg perennial-Tt) groups covalently and directly linked to the Alb in a range from 5-20.
  • the present invention provides a composition comprising a molecule of formula VII:
  • Ab is a monoclonal antibody to a target of interest
  • TCO is a iraws-cyclooctene functional group linked to the Ab by the other end; wherein d is an integer corresponding to the number TCO; and Fl is the first fluorophore covalently and directly linked to the Ab, and e is an integer corresponding to the number of F 1 fluorophores attached per Ab in a range from 1-5.
  • the present invention provides a composition comprising a molecule of formula VIII:
  • Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers);
  • CTX is a chemotherapeutic agent covalently and directly linked to Alb and y is an integer corresponding to the number of CT molecules attached per Alb in a range from 1-6;
  • (Pegschreib-Tt) is a polyethylene glycol chain containing terminal tetrazine (Tt) functional groups linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24;
  • z is an integer corresponding to the degree of functionalization of (Peg-Tt) groups covalently and directly linked to the Alb in a range from 5-20;
  • F2 is the second fluorophore covalently and directly linked to the Alb, and
  • v is an integer corresponding to the number of F2 fluorophores attached per Alb in a range from 1-5.
  • the present invention provides a composition for the diagnosis and/or treatment of a disease comprising: a composition of formula I and a composition of formula II.
  • the present invention provides a composition for the diagnosis or treatment of a disease comprising: a composition of formula III and a composition of formula IV; wherein F 1 and F2 cannot be the same.
  • the present invention provides a composition for the diagnosis and/or treatment of a disease comprising: a composition of formula V and a composition of formula VI.
  • the present invention provides a composition for the diagnosis or treatment of a disease comprising: a composition of formula VII and a composition of formula VIII; wherein F 1 and F2 cannot be the same.
  • the present invention provides a method of treatment of breast cancer in a subject comprising: a) administering to the subject a therapeutically effective amount of a composition of formula I or III or V, or VII, wherein Ab is trastuzumab (or oxher d specified above) and optionally, Fl is a first fluorescent dye (eg.
  • Figure 1 illustrates the internalization strategy and formulation of components for in vitro study
  • (la) Two-component delivery strategy via immuno-conjugation between trastuzumab on HER2 overexpressing cell surface, bioorthogonal multiple "click” reactions between azide functionality in trastuzumab and strained dibenzylcyclooctyne in albumin on cell surface followed by cluster formation and internalization
  • (lb) Modification of trastuzumab.
  • Antibody was first substituted by PEGylated azide using NHS-PEG 4 -Azide followed by fluorescent labeling to obtain Tz(Peg 4 -Az) x (Rhod) y .
  • Albumin was first functionalized with PEGylated strained dibenzylcyclooctyne using NHS- PEG4-DBCO followed by fluorescent labeling to obtain Alb(Peg 4 -Az) x (Alexa488) y .
  • Figure 2 illustrates the cell images of two-component delivery systems.
  • FIG. 3 illustrates the formulation, imaging, and intracellular delivery of drug conjugated two-component delivery system.
  • (3a) Synthesis of sulfo-NHS-succinate paclitaxel. Following the literature procedure, paclitaxel was first, functionalized with a carboxylate linker and derivatized to sulfo-NHS functionality increasing the solubility in aqueous media.
  • (3b) Formulation of paclitaxel conjugated BSA. (i) Substitution of paclitaxel on albumin, (ii) DBCO functionalization, (iii) Fluorescent dye labeling. (3c) Image-guided in vitro therapy, Control, two-component delivery with unmodified trastuzumab on BT-474 cells. Treated, in vitro click therapy on BT-474 cells.
  • FIG. 4 is a series of graphs depicting the toxicological study.
  • (4a) Cells were treated with each component alone and cell viability was given as a percentage with respect to cells with no treatment. Cells were treated with unmodified trastuzumab, Tz(Peg 4 - Az) 2 o(Rhod)2, or Tz(Peg4-Az) 2 9(Rhod)2. Unmodified Tz only shows a significant therapeutic effect even after incubation for 30 minutes. Cells were also treated with modified BSA, Alb(CTX) 2 .
  • Figure 5 shows schematics of the in vivo click therapy based on iraws-cyclooctene (TCO) and Tetrazine (Tt) based bioorthogonal click chemistry.
  • Figure 6 depicts the formulation of components for in vivo study. (6a)
  • TCO iraws-cyclooctene
  • Tt Tetrazine
  • FIG. 7 depicts the in vivo click therapy.
  • 7a Mice were first injected with one of first components, saline (untreated control), Tz(Fl) 1 (treated control), and Tz(TCO) 6 (Fl) 1 (Treated). After 6-7 hours of blood clearance of first component, saline (untreated control) and Alb(CTX) 2.6 (Peg 5 -Tt)i 5 (F2)i (treatment control and treatment) was injected as the second component.
  • 7d Change of body weights of mice during the treatment.
  • the present invention provides a new strategy for an improved target-specific drug delivery that utilizes enhanced
  • This novel two-step/two- component system for intracellular delivery of therapeutics is based on the induced internalization of cross-linked and clustered mAb to target receptors of interest, including, for example, HER2 receptors.
  • the system provides target-specific, and optionally, image-guided drug delivery, and highly efficient internalization and accumulation of chemotherapeutics in the target cells of interest.
  • the present invention provides in situ complexation of two or more delivery components by the bioorthogonal click reactions between mAb functionalized with multiple azide groups and nano/macromolecular carriers such as bovine serum albumin functionalized with multiple dibenzylcyclooctyne groups and substituted with chemotherapeutics, such as paclitaxel.
  • the present invention provides copper- free, strained-promoted, bioorthogonal click chemistry for therapy.
  • the present invention provides in situ complexation of two or more delivery components by the bioorthogonal click reactions between mAb functionalized with /raws-cyclooctene, and nano/macromolecular carriers such as bovine serum albumin functionalized with multiple tetrazine groups and substituted with chemotherapeutics, such as paclitaxel.
  • these novel drug compositions and the accompanying delivery systems of the present invention were evaluated in HER2 -positive and negative breast cancer cell lines. It was demonstrated that the inventive click therapy provided herein delivers highly efficient intracellular drug accumulation and substantially increased cytotoxicity for HER2 overexpressing breast cancer cells in comparison to conventional single component or combination therapy, both in vitro and in vivo.
  • the present invention provides a composition comprising a molecule of formula I:
  • Ab is a monoclonal antibody to a target of interest
  • (Peg a -Az) is a polyethylene glycol chain containing a terminal azide (Az) functional group linked to the Ab by the other end
  • a is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24
  • b is an integer corresponding to the number of (Peg a -Az) groups covalently and directly linked to the Ab in a range from 1-30.
  • the term “Ab” means a monoclonal antibody having antigenic specificity for a target antigen of interest.
  • the phrase "having antigenic specificity" as used herein means that the monoclonal antibody can specifically bind to and immunologically recognize the antigen.
  • the antigen is a cancer antigen.
  • the terminal azide functional group is covalently linked to the Fc portion of the Ab.
  • the term "other end" when used in describing the linkage of the molecule to the Ab is synonymous with the Fc portion of the antibody molecule, or C-terminal end.
  • cancer antigen refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) solely or predominantly expressed or over-expressed by a tumor cell or cancer cell, such that the antigen is associated with the tumor or cancer.
  • the cancer antigen can additionally be expressed by normal, non-tumor, or non-cancerous cells.
  • normal, non-tumor, or noncancerous cells are normally expressed by normal, non-tumor, or noncancerous cells.
  • the tumor or cancer cells can over-express the antigen or express the antigen at a significantly higher level, as compared to the expression of the antigen by normal, non-tumor, or noncancerous cells.
  • the cancer antigen can additionally be expressed by cells of a different state of development or maturation.
  • the cancer antigen can be additionally expressed by cells of the embryonic or fetal stage, which cells are not normally found in an adult host.
  • the cancer antigen can be additionally expressed by stem cells or precursor cells, which cells are not normally found in an adult host.
  • the cancer antigens used in the compositions of the present invention can be an antigen expressed by any cell of any cancer or tumor, including the cancers and tumors described herein.
  • the cancer antigen may be a cancer antigen of only one type of cancer or tumor, such that the cancer antigen is associated with or characteristic of only one type of cancer or tumor.
  • the cancer antigen may be a cancer antigen (e.g., may be characteristic) of more than one type of cancer or tumor.
  • the cancer antigen may be expressed by both breast and prostate cancer cells and not expressed at all by normal, non-tumor, or non-cancer cells.
  • the monoclonal antibody of the invention can be a recombinant antibody.
  • "recombinant antibody” refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the invention and a polypeptide chain of an antibody, or a portion thereof.
  • the polypeptide of an antibody, or portion thereof can be a heavy chain, a light chain, a variable or constant region of a heavy or light chain, a single chain variable fragment (scFv), or an Fc, Fab, or F(ab)2 ! fragment of an antibody, etc.
  • polypeptide chain of an antibody, or portion thereof can exist as a separate polypeptide of the recombinant antibody.
  • the polypeptide chain of an antibody, or portion thereof can exist as a polypeptide, which is expressed in frame (in tandem) with the polypeptide of the invention.
  • the polypeptide of an antibody, or portion thereof can be a polypeptide of any antibody or any antibody fragment, including any of the antibodies and antibody fragments described herein.
  • Suitable methods of making antibodies are known in the art. For instance, standard hybridoma methods are described in, e.g., K5hler and Milstein, Eur. J. Immunol, 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and C.A. Janeway et al. (eds.), Immunobiology, 5 th Ed., Garland Publishing, New York, NY (2001)). Alternatively, other methods, such as EBV-hybridoma methods (Haskard and Archer, J. Immunol.
  • Antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and described in, for example U.S. Patents 5,545,806 and 5,569,825, and Janeway et al, supra.
  • the invention also provides antigen binding portions of any of the antibodies described herein.
  • the antigen binding portion can be any portion that has at least one antigen binding site, such as Fab, F(ab')2, dsFv, sFv, diabodies, and triabodies.
  • a single-chain variable region fragment (sFv) antibody fragment which consists of a truncated Fab fragment comprising the variable (V) domain of an antibody heavy chain linked to a V domain of a light antibody chain via a synthetic peptide, can be generated using routine recombinant DNA technology techniques (see, e.g., Janeway et al, supra).
  • dsFv disulfide-stabilized variable region fragments
  • Antibody fragments of the invention are not limited to these exemplary types of antibody fragments.
  • the Ab used in the compositions and methods of the present invention include, for example, trastuzumab or another monoclonal antibody such as anti- HER2 mAb (eg. Pertuzumab), anti-PSMA mAb (eg. J591, J415, Hybritech PEQ226.5 and PM2J004.5), and anti-CD30 mAb (eg. Brentuximab).
  • trastuzumab or another monoclonal antibody such as anti- HER2 mAb (eg. Pertuzumab), anti-PSMA mAb (eg. J591, J415, Hybritech PEQ226.5 and PM2J004.5), and anti-CD30 mAb (eg. Brentuximab).
  • a number of other therapeutic monoclonal antibodies have been approved for use in humans, and can be used in the present invention, including, but not limited to, alemtuzumab, bevacizumab, vedotin, cetuximab, gemtuzumabozogamicin, ibritumomabtiuxetan, ofatumumab, panitumumab, rituximab, and tositumomab.
  • (Peg a -Az) refers to a polyethylene glycol molecule chain containing a terminal azide (Az) functional group.
  • the Ab is linked to the Peg moiety on a first end through an amide linkage and it is linked on its second end to the azide moiety covalently to the terminal carbon of the Peg molecule chain.
  • a is an integer corresponding to the number of covalently linked ethylene glycol units in a polyethylene glycol chain in a range from 0-24. In some embodiments about 4 to 10 ethylene glycol units are used.
  • b is an integer corresponding to the number of molar equivalents of (Peg a -Az) groups covalently and directly linked to a molar equivalent of Ab in a range from 1-30. In some embodiments between 10 and 20 (Peg a -Az) groups are used.
  • the present invention provides a composition comprising a molecule of formula II: Alb(CTX) x (Pegn-DBCO) y (II);
  • Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers);
  • CTX is a chemotherapeutic agent covalently and directly linked to Alb and x is an integer corresponding to the number of molar equivalents of CTX molecules attached per molar equivalent oiAlb in a range from 1-6;
  • (Peg cont-DBCO) is a polyethylene glycol chain containing a terminal dibenzylcyclooctyne(D5COj functional group linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and y is an integer corresponding to the number of molar equivalents of (Peg n -DBCO) groups covalently and directly linked to a molar equivalent oiAlb in a range from 5-20.
  • Alb in the compositions and methods of the present invention denotes the protein albumin, including, for example, bovine serum albumin and human serum albumin. It will be understood by those of skill in the art that other similar and biocompatible proteins and polymers can be used with the inventive compositions such as dextran, chitosan, polylactic-co-glycolic acid (PLGA), etc. In some embodiments, the albumin used is human.
  • DBCO means a strained cyclooctyne molecule dibenzylcyclooctyne.
  • DIBO dibenzocyclooctyne
  • CTX means a chemotherapeutic agent covalently and directly linked to the albumin or biocompatible protein.
  • the term can be synonymous with the term “biologically active agent.”
  • the biologically active agent may vary widely with the intended purpose for the composition.
  • active is art-recognized and refers to any moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject.
  • Examples of biologically active agents that may be referred to as "drugs", are described in well-known literature references such as the Merck Index, the Physicians' Desk Reference, and The Pharmacological Basis of Therapeutics, and they include, without limitation, medicaments; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.
  • the term "CTX" means an anti-cancer or anti-neoplastic agent.
  • antineoplastic agents include alkylating agents, nitrogen mustard alkylating agents, nitrosourea alkylating agents, antimetabolites, purine analog antimetabolites, pyrimidine analog antimetabolites, hormonal antineoplastics, natural antineoplastics, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • chemotherapeutic agent generally includes pharmaceutically or therapeutically active compounds that work by interfering with DNA synthesis or function in cancer cells. Based on their chemical action at a cellular level, chemotherapeutic agents can be classified as cell- cycle specific agents (effective during certain phases of cell cycle) and cell-cycle nonspecific agents (effective during all phases of cell cycle). Without being limited to any particular example, examples of chemotherapeutic agents can include alkylating agents, angiogenesis inhibitors, aromatase inhibitors, antimetabolites, anthracyclines, antitumor antibiotics, monoclonal antibodies, platinums, topoisomerase inhibitors, and plant alkaloids. In some embodiments CTX is selected from the group consisting of taxanes (e.g. paclitaxel, docetaxel), antimetabolites (e.g. fluorouracil), intercalation and inhibitory drugs (e.g.
  • taxanes e.g. paclitaxel, docetaxel
  • antimetabolites e.g. fluorour
  • the number of molar equivalents of CTX per unit oiAlb is in the range of 1 to 6. In some embodiments, the number of molar equivalents of CTX per unit of Alb is between 2 to 3.
  • n is an integer corresponding to the number of covalently linked ethylene glycol units in a polyethylene glycol chain in a range from 0-24. In some embodiments about 4 to 10 ethylene glycol units are used.
  • y is an integer corresponding to the number of molar equivalents of (Peg Berry-DBCO) groups covalently and directly linked to a molar equivalent oiAlb in a range from 5-20. In some embodiments, y is in the range of about 14 to 15.
  • the present invention provides a composition comprising a molecule of formula III: Ab(Peg a -Az) b (Fl) c (Ill);
  • molecule is defined the same as formula I, with the addition of a fluorophore (Fl), which is covalently and directly linked to the Ab, and wherein c is an integer corresponding to the number of molar equivalents of fluorophores attached per molar equivalent of Ab in a range from 1-5.
  • Fl fluorophore
  • the present invention provides a composition comprising a molecule of formula IV:
  • molecule is defined the same as formula II, with the addition of a fluorophore (F2), a second fluorophore covalently and directly linked to the Alb, and z is an integer corresponding to the number of molar equivalents of fluorophores attached per molar equivalent oiAlb in a range from 1-5.
  • F2 fluorophore
  • z is an integer corresponding to the number of molar equivalents of fluorophores attached per molar equivalent oiAlb in a range from 1-5.
  • the term "fluorophore” is synonymous with "imaging agent.”
  • the fluorophore is a fluorescent dye.
  • the dyes may be emitters in the visible or near-infrared (MR) spectrum.
  • MR near-infrared
  • Known dyes useful in the present invention include carbocyanine, indocarbocyanine, oxacarbocyanine, thuicarbocyanine and merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, boron-dipyrromethane
  • the fluorophore is selected from the group consisting of: rhodamine, Alexa488 or clinically approved dyes such as ICG, Cy5.5, Cy7, Irdye800 CW, ProSense 750).
  • NIR dyes which are active in the NIR region are known in biomedical applications. However, there are only a few NIR dyes that are readily available due to the limitations of conventional dyes, such as poor hydrophilicity and photostability, low quantum yield, insufficient stability and low detection sensitivity in biological system, etc. Significant progress has been made on the recent development of NIR dyes (including cyanine dyes, squaraine, phthalocyanines, porphyrin derivatives and BODIPY (borondipyrromethane) analogues) with much improved chemical and photostability, high fluorescence intensity and long fluorescent life. Examples of R dyes include cyanine dyes (also called as
  • polymethine cyanine dyes are small organic molecules with two aromatic nitrogen- containing heterocycles linked by a polymethine bridge and include Cy5, Cy5.5, Cy7 and their derivatives.
  • Squaraines (often called Squarylium dyes) consist of an oxocyclobutenolate core with aromatic or heterocyclic components at both ends of the molecules, an example is KSQ-4-H.
  • Phthalocyanines are two-dimensional 187t-electron aromatic porphyrin derivatives, consisting of four bridged pyrrole subunits linked together through nitrogen atoms.
  • BODIPY (borondipyrromethane) dyes have a general structure of 4,4'-difluoro- 4- bora-3a, 4a-diaza-s-indacene) and sharp fluorescence with high quantum yield and excellent thermal and photochemical stability.
  • Imaging agents which are attached to compositions of the present invention can include PET, SPECT, and MRI imaging agents.
  • the most widely used agents include branched chelating agents such as di-ethylene tri-amine penta-acetic acid (DTPA), 1,4,7, 10- tetra-azacyclododecane-l,4,7, 10-tetraacetic acid (DOTA) and their analogs for complexation with metals such as Gd and Cu.
  • Chelating agents such as di-amine dithiols, activated mercaptoacetyl-glycyl-glycyl-gylcine (MAG3), and hydrazidonicotinamide (HY IC), are able to chelate metals like 99m Tc and 186 Re.
  • the present invention provides compositions wherein the fluorophore comprises a metal isotope suitable for imaging.
  • isotopes useful in the present invention include Tc-94m, Tc-99m, In-11 1, Ga-67, Ga-68, Y-86, Y-90, Lu-177, Re-186, Re-188, Cu-64, Cu-67, Co-55, Co-57, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, or Dy-166, 1-125, 1-124, 1-123, F-18.
  • the present invention provides a composition comprising a molecule of formula V:
  • TCO iraws-cyclooctene functional group linked to the Ab by the other end
  • d is an integer corresponding to the number degrees of substitution of TCO. It is understood by those of ordinary skill that iraws-cyclooctene is another click-chemistry reagent.
  • the present invention provides a composition comprising a molecule of formula VI:
  • Alb(CTX) y (Peg n -Tt) z (VI); wherein Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers); CTX is a chemotherapeutic agent covalently and directly linked to Alb and y is an integer corresponding to the number of C7 molecules attached per Alb in a range from 1-6;
  • (Peg staggering-Tt) is a polyethylene glycol chain containing a terminal tetrazine (Tt) functional group linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and z is an integer corresponding to the degree of functionalization of Peg-Tt groups covalently and directly linked to the Alb in a range from 5-20.
  • the present invention provides a composition comprising a molecule of formula VII:
  • Ab is a monoclonal antibody to a target of interest
  • TCO is a iraws-cyclooctene functional group linked to the Ab by the other end; wherein d is an integer corresponding to the number degrees of substitution of TCO; and Fl is the first fluorophore covalently and directly linked to the Ab, and e is an integer corresponding to the number of fluorophores attached per Ab in a range from 1-5.
  • the present invention provides acomposition comprising a molecule of formula VIII:
  • Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers);
  • CTX is a chemotherapeutic agent covalently and directly linked to Alb and y is an integer corresponding to the number of CT molecules attached per Alb in a range from 1-6;
  • Peg contun-Tt is a polyethylene glycol chain containing a terminal tetrazine (Tt) functional group linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24;
  • z is an integer corresponding to the degree of functionalization of Peg-Tt groups covalently and directly linked to the Alb in a range from 5-20;
  • F2 is the second fluorophore covalently and directly linked to the Alb, and
  • v is an integer corresponding to the number of fluorophores attached per Alb in a range from 1-5.
  • the present invention provides compositions for diagnosis or treatment of a disease comprising the compositions of formula V and VI.
  • the present invention provides a composition for the diagnosis or treatment of a disease comprising the composition of formula III and the composition of formula IV; wherein F 1 and F2 cannot be the same.
  • the present invention provides a composition for the diagnosis or treatment of a disease comprising the composition of formula VII and the composition of formula VIII; wherein F 1 and F2 cannot be the same.
  • the present invention provides a
  • compositions comprising one or compositions described herein and a pharmaceutically acceptable carrier.
  • the present invention provides a method of imaging and/or inducing cytotoxicity in a host cell or population of cells comprising administering to the cell or population of cells the compositions described herein, or the pharmaceutical composition described herein, in an amount sufficient to image and/or kill the host cell or population of cells.
  • the present invention provides a use of the compositions described herein, in an effective amount, to prepare a medicament, preferably for use as a medicament for treating a disease in a subject.
  • the medicament further comprises a pharmaceutically acceptable carrier.
  • the medicament further comprises a second therapeutic agent.
  • the disease is cancer, and in a preferred embodiment, the disease is breast cancer.
  • the present invention provides a method of treatment of breast cancer in a subject comprising: a) administering to the subject a therapeutically effective amount of a composition of formulas I and/or III, and/or V, and/or VII, wherein Ab is trastuzumab (or other Ab specified above) and optionally, F 1 is a first fluorescent dye (e.g., ICG, Cy5.5, Cy7, Ir dye 800 CW, ProSense 750); b) administering to the subject a therapeutically effective amount of a composition of formula II and/or IV and/or VI and/or VIII, wherein Alb is serum albumin (or similar proteins), CTX is paclitaxel (or other chemotherapeutics specified above), and optionally, F2 is a second fluorescent dye specified above; wherein a) and b) are administered consecutively or concurrently.
  • F 1 is a first fluorescent dye (e.g., ICG, Cy5.5, Cy7, Ir dye 800 CW, ProSense 750)
  • F2 is a
  • the first compositions of formulas I, III, V and VII are administered to the cells or the subject first.
  • the second compositions of formulas II, IV, VI and VIII are administered to the cells or the subject and the compositions conjugate at the Ab target receptors.
  • the amount of time between administration of the first component or composition and the second component or composition can vary.
  • the time sufficient for the first component or composition to bind the target can be within 30 minutes up to 5, 6, 7, 8, 10 hours. This is followed by administration of the second component or composition.
  • compositions and methods of the present invention described above can also encompass a pharmaceutical composition comprising the compositions and a pharmaceutically acceptable carrier.
  • the carriers or diluents used herein may be solid carriers or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.
  • Solid carriers or diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, or suspensions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include, for example, water, alcoholic/aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles for subcutaneous, intravenous, intraarterial, or intramuscular injection
  • parenteral vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Formulations suitable for parenteral administration include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
  • water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • compositions of the invention are exemplary, and are in no way limiting. More than one route can be used to administer the compositions of the present invention, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
  • injectable formulations are in accordance with the invention.
  • the requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ⁇ SHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)).
  • pharmaceutically active compound or “therapeutically active compound” means a compound useful for the treatment or modulation of a disease or condition in a subject suffering therefrom.
  • pharmaceutically active compounds can include any drugs known in the art for treatment of disease indications.
  • a particular example of a pharmaceutically active compound is a chemotherapeutic agent.
  • the amount or dose of the compositions of the present invention that is administered should be sufficient to effectively target the cell, or population of cells in vivo, such that the imaging of the target cell or population of cells, as well as the cytotoxicity of the compositions can be detected, in the subject over a reasonable time frame.
  • the dose will be determined by the efficacy of the particular composition formulation and the location of the target population of cells in the subject, as well as the body weight of the subject to be treated.
  • the dose of the compositions of the present invention also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of the inventive compositions. Typically, an attending physician will decide the dosage of the compositions with which to treat each individual subject, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound to be administered, route of administration, and the severity of the condition being treated.
  • the dose of the compositions of the present invention can be about 0.001 to about 1000 mg/kg body weight of the subject being treated, from about 0.01 to about 100 mg/kg body weight, from about 0.1 mg/kg to about 10 mg/kg, and from about 0.5 mg to about 5 mg/kg body weight.
  • the dose of the compositions of the present invention can be at a concentration from about 1 nM to about 10,000 nM, preferably from about 10 nM to about 5,000 nM, more preferably from about 100 nM to about 500 nM.
  • inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.
  • the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • the invention further provides a host cell comprising any of the compositions described herein.
  • the term "host cell” refers to any type of cell that can contain the inventive compositions.
  • the host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae.
  • the host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human.
  • the host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension.
  • the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell.
  • suitable human host cells can include, but are not limited to, cells of the major organs of the body, including, for example, cells of the lung, including hepatocytes and hepatic stellate cells, cells of the breast, cells of the prostate, cells of the cornea, including corneal epithelial cells, cells of the lung, including lung epithelial cells, and cells of the brain, such as neurons.
  • the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a cancer cell, specifically a breast cancer cell.
  • the population of cells can be a heterogeneous population comprising the host cell comprising any of the compositions described, in addition to at least one other cell, e.g., a host cell (e.g., a epithelial cell), which does not comprise any of the compositions, or a cell other than a epithelial cell, e.g., a macrophage, a neutrophil, an erythrocyte, a hepatocyte, a hepatic stellate cell, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc.
  • the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) comprising the compositions.
  • the disease can include cancer.
  • Cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor.
  • the cancer is breast cancer.
  • administering means that the one or more compositions of the present invention are introduced into a sample having at least one cell, or population of cells, and appropriate enzymes or reagents, in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit uptake of the at least one compositions of the present invention into the cytosol through the click-chemical reactions occurring on the cell membrane.
  • administering means that at least one or more compositions of the present invention are introduced into a subject, preferably a subject receiving treatment for a disease, and the at least one or more compositions are allowed to come in contact with the one or more disease related cells or population of cells of interest in vivo.
  • the term “subject” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
  • ATCC American Type Culture Collection
  • ATCC American Type Culture Collection
  • ATCC 46-X media
  • FBS FBS
  • Third or fourth passages of cells with 70-80% confluency were used for each experiment unless otherwise mentioned.
  • paclitaxel and Taxol were purchased from LKT Laboratories, Inc. and Sagent Pharmaceuticals, Inc. respectively.
  • Trastuzumab Hereceptin® was purchased from Genentech Inc., or kindly gifted by Dr. Robert Ivkov (The Johns Hopkins School of Medicine) and used after purification.
  • Bioconjugation reagents NHS-PEG 4 -Azide and NHS- PEG 4 -DBCO ester were purchased from Thermo Fisher and Click Chemistry Tools respectively. Dry or HPLC grade solvents were purchased from Sigma-Aldrich and used without further purification.
  • BT-474 cells were placed in 4-well chamber slides (5x 10 5 cells in 0.5 mL of 46-X medium in each well) and grown for 24-48 hours. Two slides were maintained at 4 °C or 37 °C throughout the experiment. First, cells were incubated with modified trastuzumab (20 /g/mL in DPBS-FBS) for 20 minutes and washed twice with DPBS-FBS (Dulbecco's Phosphate Buffered Saline Solution with 1% Fetal Bovine Serum) to remove the unbound trastuzumab.
  • modified trastuzumab (20 /g/mL in DPBS-FBS
  • DPBS-FBS Dulbecco's Phosphate Buffered Saline Solution with 1% Fetal Bovine Serum
  • modified albumin C-2, 100 /g/mL in DPBS-FBS
  • DPBS-FBS modified albumin
  • Alexa Fluor 488 labeled albumin without DBCO alkyne was used in controls as the second delivery component.
  • Cytotoxic study Her2/wew overexpressing BT-474 (study cell line) and Her2/wew basal level MDA-MB 231 (control cell line) cells were seeded in a 96-well plate at a density of 5x l0 3 cells and incubated at 37 °C in a humidified atmosphere containing 5% C0 2 . After 24 hours, cultured cells treated with or without modified or unmodified trastuzumab (100 pL, 20 /g/mL) and incubated for 30 minutes at 4°C. Cells were washed once with 0.05%
  • BSA/DPBS BSA/DPBS and incubated with or without second components, DBCO modified albumin substituted with paclitaxel, or equivalent amount of paclitaxel as Taxol (100 //L, 40 /g/mL).
  • Cells were incubated at 37°C for 2 hours, washed once using 0.05% BSA/DPBS and refill with fresh media (200 /L). After the incubation at 37 °C for 72 hours in a humidified atmosphere containing 5% C0 2 , 100 //L from total media was removed and viability of cells was determined using WST-8 assay following the manufactures protocol (10 //L of WST-8 reagent per well with 100 //L of media, incubation at 37° for 4hours, and measuring the absorbance at 450 nm). Each test was quadruplicated per plate and triplicate independent experiments were done for the statistical analysis. The -value ⁇ 0.05 calculated by independent two-sample t-test, is considered as a significant difference between the study groups.
  • trastuzumab a pre-targeting, delivery component was functionalized with PEGylated azides.
  • the degree of functionalization plays a major role in antibody-receptor binding affinity and the efficiency of the click conjugation.
  • the degree of functionalization was determined based on the change of molecular weight measured by MALDI-TOF/MS.
  • Functional groups of azides were determined using TAMRA-DIBO via click labeling.
  • trastuzumab was used with 20 functional groups attached which gave an optimum cell surface labeling pattern and the click reaction with DBCO
  • BSA was first functionalized with approximately 15 units of NHS-PEG 4 -DBCO and labeled with Alexa Fluor 488 (Invitrogen) and obtained and used for the imaging experiments. Imaging experiments were performed at 4 °C and 37 °C to observe the surface labeling and the internalization ( Figure 2).
  • HER2 overexpressing BT-474 cells shows a typical cell surface labeling pattern with labeled trastuzumab (control without azide functionalization) at 4 °C and doesn't show significant internalization at 37 °C alone.
  • the live cells were incubated at 4 °C and 37 °C and the internalization was observed after incubation of BT-474 cells for 2 hours.
  • the control MDA-MB-231 cells show no surface labeling by trastuzumab and negligible level of internalization of second delivery components.
  • Tz(Peg4-Az) 2 o(Rhod)2, Tz(Peg4-Az) 2 8(Rhod)2, were used as the pre-targeting first delivery component.
  • the second delivery component was formulated by the substitution of paclitaxel (Figure 3a), followed by the DBCO
  • the optimized bioorthogonal two-component nanocarrier delivery system can be used for in vivo experiments in preclinical stage.
  • Tz Trastuzumab
  • TCO iraws-cyclooctene
  • NHS-TCO ester reagent No significant decrease of binding affinity of functionalized Tz was detected at the substitution of six groups of TCO.
  • Resulted Tz conjugate was labeled by NIR CF-680 fluorophores (Fl) ( Figure 6a).
  • the common formula for first components is Tz(TCO) d (Fl) e , where d and e are degree of substitutions of TCO and Fl respectively.
  • Alb(CTX) x was substituted with (Pegs-Tt) using NHS-Pegs-Tetrazine reagent followed by labeling with NIR dye CF-750 ( Figure 6b).
  • the common formula for the second component is
  • mice were injected with first components, saline (group M-l for no treatment control), Tz(Fl)i (group M-2 control treatment, 0.2 mg in 200 ⁇ ⁇ of saline), Tz(TCO) 6 (Fl)i (group M-3 click therapy, 0.2 mg in 200 ⁇ ., of saline).
  • mice in group M-l were injected with saline and mice in group M-2 and M-3 were treated with second delivery components Alb(CTX) 2.6 (Peg 5 -Tt)i 5 (F2)i (2.0 mg in 200 ⁇ ., of saline).
  • the second and third doses were given on the 14 th and 28 th days. Tumor measurements were taken every 6 th day. The relative growth of tumors was analyzed based on the ratio of tumor size to the initial tumor size ( Figure 7c). The change of body weights of mice was analyzed during the click therapy treatment.
  • Tumors of mice in group M-3 treated with TCO/Tt based click therapy exhibited slowest growth rates (Figure 7c). After 48 days, tumors were extracted and cross sections were stained with H&E, the tumor sections in M-3 exhibited the significant area of necrosis (Data not shown). Tumors of control mice showed higher growth rate than the treated group. However, tumors of untreated mice show highest growth rate. During the treatment period, control and treated mice show a slight loss of body weight, but it was not significant when compared to untreated mice (Figure 7d).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

In accordance with one or more embodiments, the present invention provides a new strategy for an improved target-specific drug delivery that utilizes enhanced internalization of therapeutic conjugates by in situ complexation driven by bioorthogonal click chemistry, which is defined herein as "click therapy". This novel two-step/two-component system for intracellular delivery of therapeutics is based on the induced internalization of cross-linked and clustered mAb to target receptors of interest, including, for example, HER2 receptors. The system provides target-specific, and optionally, image-guided drug delivery, and highly efficient internalization and accumulation of chemotherapeutics in the target cells of interest. The present invention provides in situ complexation of two or more delivery components by the bioorthogonal click reactions between multiple azido-functionalized or tetrazine functionalized mAb and multiple cyclooctyne-functionalized nanocarriers, or trans- cyclooctene functionalization, and bovine serum albumin (BSA) substituted with chemotherapeutics, such as paclitaxel. The present invention provides copper-free, strained- promoted, bioorthogonal click chemistry for therapy.

Description

BIOORTHOGONAL TWO-COMPONENT DELIVERY SYSTEMS FOR ENHANCED INTERNALIZATION OF NANOTHERAPEUTICS
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/772,782, filed on March 5, 2013, which is hereby incorporated by reference for all purposes as if fully set forth herein.
BACKGROUND OF THE INVENTION
[0002] Targeted delivery of nanocarriers offers a great promise to significantly improve the efficacy of traditional cytotoxic cancer therapy while reducing its systemic side effects. Important features of targeted nanocarriers include chemical stability, high affinity to target sites, low non-specific interactions, optimal pharmacokinetics, and high drug loading capacity. A traditional strategy to develop targeted nanocarriers utilizes cytotoxic antibody- drug conjugates (ADCs) where chemotherapeutics are directly attached to the target-specific antibody. This emerging new class of nanoscale therapeutic agents is currently used clinically for targeted therapy of cancer cells that overexpress a suitable surface receptor.
[0003] For example, approximately 20-30% of breast cancers exhibit HER2
overexpression due to the gene amplification leading to the high aggressiveness with generally poor prognosis. The HER2 receptor regulates multiple physiological pathways including cell proliferation and differentiation. The HER2 receptor is also known for its poor internalization capability, even after trastuzumab binding and subsequent heterodimerization, possibly due to localization of the receptor in membrane protrusion or/and in lipid raft areas where it has poor contact with the lipid bilayer. The humanized anti-HER2 monoclonal antibody (mAb), trastuzumab (Herceptin®) is used as a first line treatment for HER2/wew positive breast cancers. The cytotoxic mechanism of trastuzumab includes the inhibition of P13K/Akt and Ras/MAPK signaling pathways leading to cell cycle arrest. Unfortunately, approximately 50% of patients with HER2 -positive disease do not benefit from trastuzumab or become refractory to it even though the HER2 level remains high.
[0004] The novel ADC, trastuzumab-emtansine conjugate (T-DM1), was recently reported to have significantly improved efficacy compared to standard monotherapeutics for trastuzumab-refractory disease. However, the direct conjugation of chemotherapeutics to mAb can reduce the in vivo therapeutic index, by reducing the binding affinity of the antibody with targeted receptors. In addition, antibody conjugation does not enhance the ADC internalization and results in the systemic toxicity due to long systemic circulation. In fact, thrombocytopenia, in which the blood has a lower than normal number of platelets, has been observed in a small number of patients treated with ADCs.
[0005] Therefore, there still exists an unmet need for improved delivery systems associated with antibody chemotherapeutics and their use in the diagnosis and treatment of cancer and other diseases.
SUMMARY OF THE INVENTION
[0006] In accordance with an embodiment, the present invention provides a composition comprising a molecule of formula I:
Ab(Pega-Az)b (I);
wherein Ab is a monoclonal antibody to a target of interest; (Pega-Az) is a polyethylene glycol chain containing a terminal azide (Az) functional group linked to the Ab by the other end; wherein a is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and b is an integer corresponding to the number of (Pega-Az) groups covalently and directly linked to the Ab in a range from 1-30.
[0007] In accordance with another embodiment, the present invention provides a composition comprising a molecule of formula II:
Alb(CTX)x(Pegn-DBCO)y (II);
wherein Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers); CTX is a chemotherapeutic agent covalently and directly linked to Alb and x is an integer corresponding to the number of C7 molecules attached per Alb in a range from 1-6; (Peg„-DBCO) is a polyethylene glycol chain containing a terminal dibenzylcyclooctyne (DBCO) functional group linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and y is an integer corresponding to the number of (Peg„-DBCO) groups covalently and directly linked to the Alb in a range from 5- 20.
[0008] In accordance with a further embodiment, the present invention provides a composition comprising a molecule of formula III: Ab(Pega-Az)b(Fl)c (Ill);
wherein Ab is a monoclonal antibody to a target of interest; (Pega-Az) is a polyethylene glycol chain containing a terminal azide (Az) functional group linked to the Ab by the other end; wherein a is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; b is an integer corresponding to the number of (Pega-Az) groups covalently and directly linked to the Ab in a range from 1-30; and Fl is a first fluorophore covalently and directly linked to the Ab, and c is an integer corresponding to the number of Fl fluorophores attached per Ab in a range from 1-5.
[0009] In accordance with still another embodiment, the present invention provides a composition comprising a molecule of formula IV:
Alb(CTX)x(Pegn-DBCO)y(F2)z (IV);
wherein Alb denotes albumin (bovine serum albumin, human serum albumin, or other similar and biocompatible proteins and polymers); CTX is a chemotherapeutic agent covalently and directly linked to Alb and x is an integer corresponding to the number of CT molecules attached per Alb in a range from 1-6; (Peg„-DBCO) is a polyethylene glycol chain containing a terminal DBCO functional group, wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; y is an integer corresponding to the number of (Peg„-DBCO) groups covalently and directly linked to the Alb in a range from 5-20; and F2 is a second fluorophore covalently and directly linked to the Alb, and z is an integer corresponding to the number of fluorophores attached per Alb in a range from 1-5.
[0010] In accordance with an embodiment, the present invention provides a composition comprising a molecule of formula V:
Ab(TCO)d (V);
wherein Ab is a monoclonal antibody to a target of interest; (TCO) is a iraws-cyclooctene functional group linked to the Ab by the other end; and wherein d is an integer corresponding to the number TCO groups covalently and directly linked to the Ab in a range from 5-6.
[0011] In accordance with another embodiment, the present invention provides a composition comprising a molecule of formula VI:
Alb(CTX)y(Pegn-Tt)z (VI); wherein Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers); CTX is a chemotherapeutic agent covalently and directly linked to Alb and y is an integer corresponding to the number of C7 molecules attached per Alb in a range from 1-6; (Peg„-Tt) is a polyethylene glycol chain containing terminal tetrazine (Tt) functional groups linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and z is an integer corresponding to the number of (Peg„-Tt) groups covalently and directly linked to the Alb in a range from 5-20.
[0012] In accordance with a further embodiment, the present invention provides a composition comprising a molecule of formula VII:
Ab(TCO)d(Fl)e (VII);
wherein Ab is a monoclonal antibody to a target of interest; (TCO) is a iraws-cyclooctene functional group linked to the Ab by the other end; wherein d is an integer corresponding to the number TCO; and Fl is the first fluorophore covalently and directly linked to the Ab, and e is an integer corresponding to the number of F 1 fluorophores attached per Ab in a range from 1-5.
[0013] In accordance with yet another embodiment, the present invention provides a composition comprising a molecule of formula VIII:
Alb(CTX)y(Pegn-Tt)z(F2)v (VIII);
wherein Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers); CTX is a chemotherapeutic agent covalently and directly linked to Alb and y is an integer corresponding to the number of CT molecules attached per Alb in a range from 1-6; (Peg„-Tt) is a polyethylene glycol chain containing terminal tetrazine (Tt) functional groups linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; z is an integer corresponding to the degree of functionalization of (Peg-Tt) groups covalently and directly linked to the Alb in a range from 5-20; F2 is the second fluorophore covalently and directly linked to the Alb, and v is an integer corresponding to the number of F2 fluorophores attached per Alb in a range from 1-5.
[0014] In accordance with an embodiment, the present invention provides a composition for the diagnosis and/or treatment of a disease comprising: a composition of formula I and a composition of formula II. [0015] In accordance with an embodiment, the present invention provides a composition for the diagnosis or treatment of a disease comprising: a composition of formula III and a composition of formula IV; wherein F 1 and F2 cannot be the same.
[0016] In accordance with an embodiment, the present invention provides a composition for the diagnosis and/or treatment of a disease comprising: a composition of formula V and a composition of formula VI.
[0017] In accordance with an embodiment, the present invention provides a composition for the diagnosis or treatment of a disease comprising: a composition of formula VII and a composition of formula VIII; wherein F 1 and F2 cannot be the same.
[0018] In accordance with an embodiment, the present invention provides a method of treatment of breast cancer in a subject comprising: a) administering to the subject a therapeutically effective amount of a composition of formula I or III or V, or VII, wherein Ab is trastuzumab (or oxher d specified above) and optionally, Fl is a first fluorescent dye (eg. ICG, Cy5.5, Cy7, Irdye800 CW, ProSense 750); b) administering to the subject a therapeutically effective amount of a composition of formula II or IV, or VI, or VIII, wherein Alb is serum albumin (or similar proteins), CTX is paclitaxel (or other chemotherapeutics specified above), and optionally, F2 is a second fluorescent dye; wherein a) and b) are administered consecutively or concurrently.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 illustrates the internalization strategy and formulation of components for in vitro study, (la) Two-component delivery strategy via immuno-conjugation between trastuzumab on HER2 overexpressing cell surface, bioorthogonal multiple "click" reactions between azide functionality in trastuzumab and strained dibenzylcyclooctyne in albumin on cell surface followed by cluster formation and internalization, (lb) Modification of trastuzumab. Antibody was first substituted by PEGylated azide using NHS-PEG4-Azide followed by fluorescent labeling to obtain Tz(Peg4-Az)x(Rhod)y. (lc) Modification of BSA. Albumin was first functionalized with PEGylated strained dibenzylcyclooctyne using NHS- PEG4-DBCO followed by fluorescent labeling to obtain Alb(Peg4-Az)x(Alexa488)y.
[0020] Figure 2 illustrates the cell images of two-component delivery systems. (2a) Control imaging experiment; MDA-MB-231 cells treated with Tz(Peg4-Az)2o(Rhod)2 for 20 min at 37 °C, followed by Alb(Peg4-DBCO)i5(Alexa 488)i for 2 hours at 37°C. (2b) Control imaging experiment; BT-474 cells treated with Tz(Rhod)2 for 20 min at 37 °C followed by Alb(Peg4-DBCO)i5(Alexa 488)i for 2 h at 37 °C. (2c) Treated imaging experiment; BT-474 cells were treated with Tz(Peg4-Az)2o(Rhod)2 for 20 minutes at 37 °C followed by Alb(Peg4- DBCO)i5(Alexa 488)i for 2 hours at 37 °C.
[0021] Figure 3 illustrates the formulation, imaging, and intracellular delivery of drug conjugated two-component delivery system. (3a) Synthesis of sulfo-NHS-succinate paclitaxel. Following the literature procedure, paclitaxel was first, functionalized with a carboxylate linker and derivatized to sulfo-NHS functionality increasing the solubility in aqueous media. (3b) Formulation of paclitaxel conjugated BSA. (i) Substitution of paclitaxel on albumin, (ii) DBCO functionalization, (iii) Fluorescent dye labeling. (3c) Image-guided in vitro therapy, Control, two-component delivery with unmodified trastuzumab on BT-474 cells. Treated, in vitro click therapy on BT-474 cells.
[0022] Figure 4 is a series of graphs depicting the toxicological study. (4a) Cells were treated with each component alone and cell viability was given as a percentage with respect to cells with no treatment. Cells were treated with unmodified trastuzumab, Tz(Peg4- Az)2o(Rhod)2, or Tz(Peg4-Az)29(Rhod)2. Unmodified Tz only shows a significant therapeutic effect even after incubation for 30 minutes. Cells were also treated with modified BSA, Alb(CTX)2.2(Peg4-DBCO)i5(Alexa488)i, Alb(CTX)3.3(Peg4-DBCO)15(Alexa488)1, or Taxol equivalent to the Paclitaxel loaded on BSA and determined the cell viability in each component alone. (4b) The therapeutic effect of modified Alb with 2.2 or 3.3 of paclitaxel or equivalent amount of Taxol (control) as two-component delivery system using unmodified trastuzumab, modified trastuzumab, Tz(Peg4-Az)2o(Rhod)2, Tz(Peg4-Az)29(Rhod)2, or modified trastuzumab after blocking receptors with unmodified trastuzumab.
[0023] Figure 5 shows schematics of the in vivo click therapy based on iraws-cyclooctene (TCO) and Tetrazine (Tt) based bioorthogonal click chemistry.
[0024] Figure 6 depicts the formulation of components for in vivo study. (6a)
Formulation of iraws-cyclooctene (TCO) based first component. (6b) Formulation of Tetrazine (Tt) based second component.
[0025] Figure 7 depicts the in vivo click therapy. (7a) Mice were first injected with one of first components, saline (untreated control), Tz(Fl)1 (treated control), and Tz(TCO)6(Fl)1 (Treated). After 6-7 hours of blood clearance of first component, saline (untreated control) and Alb(CTX)2.6(Peg5-Tt)i5(F2)i (treatment control and treatment) was injected as the second component. (7b) Tumor uptake of first component after 6 hours of injection. M-l; group of untreated mice, M-2; group of control mice, M-3; group of click therapy treated mice. (7c) Relative tumor growth during the treatment (Tumor size at t= every 6th day/Initial tumor size t=0 day). (7d) Change of body weights of mice during the treatment.
DETAILED DESCRIPTION OF THE INVENTION
[0026] In accordance with one or more embodiments, the present invention provides a new strategy for an improved target-specific drug delivery that utilizes enhanced
internalization of therapeutic conjugates by in situ complexation driven by bioorthogonal click chemistry, which is defined herein as "click therapy." This novel two-step/two- component system for intracellular delivery of therapeutics is based on the induced internalization of cross-linked and clustered mAb to target receptors of interest, including, for example, HER2 receptors. The system provides target-specific, and optionally, image-guided drug delivery, and highly efficient internalization and accumulation of chemotherapeutics in the target cells of interest.
[0027] In accordance with some embodiments, the present invention provides in situ complexation of two or more delivery components by the bioorthogonal click reactions between mAb functionalized with multiple azide groups and nano/macromolecular carriers such as bovine serum albumin functionalized with multiple dibenzylcyclooctyne groups and substituted with chemotherapeutics, such as paclitaxel. The present invention provides copper- free, strained-promoted, bioorthogonal click chemistry for therapy.
[0028] In accordance with some alternative embodiments, the present invention provides in situ complexation of two or more delivery components by the bioorthogonal click reactions between mAb functionalized with /raws-cyclooctene, and nano/macromolecular carriers such as bovine serum albumin functionalized with multiple tetrazine groups and substituted with chemotherapeutics, such as paclitaxel.
[0029] In some embodiments, these novel drug compositions and the accompanying delivery systems of the present invention were evaluated in HER2 -positive and negative breast cancer cell lines. It was demonstrated that the inventive click therapy provided herein delivers highly efficient intracellular drug accumulation and substantially increased cytotoxicity for HER2 overexpressing breast cancer cells in comparison to conventional single component or combination therapy, both in vitro and in vivo. [0030] In accordance with an embodiment, the present invention provides a composition comprising a molecule of formula I:
Ab(Pega-Az)b (I);
[0031] wherein Ab is a monoclonal antibody to a target of interest; (Pega-Az) is a polyethylene glycol chain containing a terminal azide (Az) functional group linked to the Ab by the other end; wherein a is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and b is an integer corresponding to the number of (Pega-Az) groups covalently and directly linked to the Ab in a range from 1-30.
[0032] As used herein, the term "Ab" means a monoclonal antibody having antigenic specificity for a target antigen of interest. The phrase "having antigenic specificity" as used herein means that the monoclonal antibody can specifically bind to and immunologically recognize the antigen. In some embodiments, the antigen is a cancer antigen.
[0033] With regard to the linkage of the (Pega-Az) or TCO moiety to the Ab, the terminal azide functional group is covalently linked to the Fc portion of the Ab. As such, the term "other end" when used in describing the linkage of the molecule to the Ab, is synonymous with the Fc portion of the antibody molecule, or C-terminal end.
[0034] The term "cancer antigen" as used herein refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) solely or predominantly expressed or over-expressed by a tumor cell or cancer cell, such that the antigen is associated with the tumor or cancer. The cancer antigen can additionally be expressed by normal, non-tumor, or non-cancerous cells. However, in such cases, the expression of the cancer antigen by normal, non-tumor, or noncancerous cells is not as robust as the expression by tumor or cancer cells. In this regard, the tumor or cancer cells can over-express the antigen or express the antigen at a significantly higher level, as compared to the expression of the antigen by normal, non-tumor, or noncancerous cells. Also, the cancer antigen can additionally be expressed by cells of a different state of development or maturation. For instance, the cancer antigen can be additionally expressed by cells of the embryonic or fetal stage, which cells are not normally found in an adult host. Alternatively, the cancer antigen can be additionally expressed by stem cells or precursor cells, which cells are not normally found in an adult host.
[0035] The cancer antigens used in the compositions of the present invention can be an antigen expressed by any cell of any cancer or tumor, including the cancers and tumors described herein. The cancer antigen may be a cancer antigen of only one type of cancer or tumor, such that the cancer antigen is associated with or characteristic of only one type of cancer or tumor. Alternatively, the cancer antigen may be a cancer antigen (e.g., may be characteristic) of more than one type of cancer or tumor. For example, the cancer antigen may be expressed by both breast and prostate cancer cells and not expressed at all by normal, non-tumor, or non-cancer cells.
[0036] The monoclonal antibody of the invention can be a recombinant antibody. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the invention and a polypeptide chain of an antibody, or a portion thereof. The polypeptide of an antibody, or portion thereof, can be a heavy chain, a light chain, a variable or constant region of a heavy or light chain, a single chain variable fragment (scFv), or an Fc, Fab, or F(ab)2! fragment of an antibody, etc. The polypeptide chain of an antibody, or portion thereof, can exist as a separate polypeptide of the recombinant antibody. Alternatively, the polypeptide chain of an antibody, or portion thereof, can exist as a polypeptide, which is expressed in frame (in tandem) with the polypeptide of the invention. The polypeptide of an antibody, or portion thereof, can be a polypeptide of any antibody or any antibody fragment, including any of the antibodies and antibody fragments described herein.
[0037] Suitable methods of making antibodies are known in the art. For instance, standard hybridoma methods are described in, e.g., K5hler and Milstein, Eur. J. Immunol, 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and C.A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). Alternatively, other methods, such as EBV-hybridoma methods (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al, Methods Enzymol, 121, 140-67 (1986)), and bacteriophage vector expression systems (see, e.g., Huse et al, Science, 246, 1275-81 (1989)) are known in the art. Further, methods of producing antibodies in non-human animals are described in, e.g., U.S. Patents 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002/0197266 Al).
[0038] Antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and described in, for example U.S. Patents 5,545,806 and 5,569,825, and Janeway et al, supra.
[0039] Methods for generating humanized antibodies are well known in the art and are described in detail in, for example, Janeway et al, supra, U.S. Patents 5,225,539, 5,585,089 and 5,693,761, European Patent No. 0239400 Bl, and United Kingdom Patent No. 2188638. Humanized antibodies can also be generated using the antibody resurfacing technology described in U.S. Patent 5,639,641 and Pedersen et al, J. Mol. Biol, 235, 959-973 (1994).
[0040] The invention also provides antigen binding portions of any of the antibodies described herein. The antigen binding portion can be any portion that has at least one antigen binding site, such as Fab, F(ab')2, dsFv, sFv, diabodies, and triabodies.
[0041] A single-chain variable region fragment (sFv) antibody fragment, which consists of a truncated Fab fragment comprising the variable (V) domain of an antibody heavy chain linked to a V domain of a light antibody chain via a synthetic peptide, can be generated using routine recombinant DNA technology techniques (see, e.g., Janeway et al, supra). Similarly, disulfide-stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology (see, e.g., Reiter et al, Protein Engineering, 7, 697-704 (1994)). Antibody fragments of the invention, however, are not limited to these exemplary types of antibody fragments.
[0042] In some embodiments the Ab used in the compositions and methods of the present invention include, for example, trastuzumab or another monoclonal antibody such as anti- HER2 mAb (eg. Pertuzumab), anti-PSMA mAb (eg. J591, J415, Hybritech PEQ226.5 and PM2J004.5), and anti-CD30 mAb (eg. Brentuximab). A number of other therapeutic monoclonal antibodies have been approved for use in humans, and can be used in the present invention, including, but not limited to, alemtuzumab, bevacizumab, vedotin, cetuximab, gemtuzumabozogamicin, ibritumomabtiuxetan, ofatumumab, panitumumab, rituximab, and tositumomab.
[0043] As used herein, (Pega-Az) refers to a polyethylene glycol molecule chain containing a terminal azide (Az) functional group. The Ab is linked to the Peg moiety on a first end through an amide linkage and it is linked on its second end to the azide moiety covalently to the terminal carbon of the Peg molecule chain. As used herein, a is an integer corresponding to the number of covalently linked ethylene glycol units in a polyethylene glycol chain in a range from 0-24. In some embodiments about 4 to 10 ethylene glycol units are used.
[0044] As used herein, b is an integer corresponding to the number of molar equivalents of (Pega-Az) groups covalently and directly linked to a molar equivalent of Ab in a range from 1-30. In some embodiments between 10 and 20 (Pega-Az) groups are used.
[0045] In accordance with another embodiment, the present invention provides a composition comprising a molecule of formula II: Alb(CTX)x(Pegn-DBCO)y (II);
wherein Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers); CTX is a chemotherapeutic agent covalently and directly linked to Alb and x is an integer corresponding to the number of molar equivalents of CTX molecules attached per molar equivalent oiAlb in a range from 1-6; (Peg„-DBCO) is a polyethylene glycol chain containing a terminal dibenzylcyclooctyne(D5COj functional group linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and y is an integer corresponding to the number of molar equivalents of (Pegn-DBCO) groups covalently and directly linked to a molar equivalent oiAlb in a range from 5-20.
[0046] As used herein, Alb in the compositions and methods of the present invention denotes the protein albumin, including, for example, bovine serum albumin and human serum albumin. It will be understood by those of skill in the art that other similar and biocompatible proteins and polymers can be used with the inventive compositions such as dextran, chitosan, polylactic-co-glycolic acid (PLGA), etc. In some embodiments, the albumin used is human.
[0047] As used herein, the term "DBCO" means a strained cyclooctyne molecule dibenzylcyclooctyne. In other embodiments, another strained cyclooctyne molecule, dibenzocyclooctyne (DIBO) can be used. It will be understood by those of ordinary skill that other equivalent molecules are included within the scope of the inventive compositions.
[0048] In accordance with one or more embodiments, the term "CTX" means a chemotherapeutic agent covalently and directly linked to the albumin or biocompatible protein. As used herein the term can be synonymous with the term "biologically active agent." The biologically active agent may vary widely with the intended purpose for the composition. The term active is art-recognized and refers to any moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of biologically active agents, that may be referred to as "drugs", are described in well-known literature references such as the Merck Index, the Physicians' Desk Reference, and The Pharmacological Basis of Therapeutics, and they include, without limitation, medicaments; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. In some embodiments, the term "CTX" means an anti-cancer or anti-neoplastic agent. Examples of antineoplastic agents include alkylating agents, nitrogen mustard alkylating agents, nitrosourea alkylating agents, antimetabolites, purine analog antimetabolites, pyrimidine analog antimetabolites, hormonal antineoplastics, natural antineoplastics, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
[0049] Moreover, the term "chemotherapeutic agent" as well as words stemming therefrom, as used herein, generally includes pharmaceutically or therapeutically active compounds that work by interfering with DNA synthesis or function in cancer cells. Based on their chemical action at a cellular level, chemotherapeutic agents can be classified as cell- cycle specific agents (effective during certain phases of cell cycle) and cell-cycle nonspecific agents (effective during all phases of cell cycle). Without being limited to any particular example, examples of chemotherapeutic agents can include alkylating agents, angiogenesis inhibitors, aromatase inhibitors, antimetabolites, anthracyclines, antitumor antibiotics, monoclonal antibodies, platinums, topoisomerase inhibitors, and plant alkaloids. In some embodiments CTX is selected from the group consisting of taxanes (e.g. paclitaxel, docetaxel), antimetabolites (e.g. fluorouracil), intercalation and inhibitory drugs (e.g.
doxorubicin).
[0050] In accordance with some embodiments, the number of molar equivalents of CTX per unit oiAlb is in the range of 1 to 6. In some embodiments, the number of molar equivalents of CTX per unit of Alb is between 2 to 3.
[0051] As used herein, the term "(Pegn-DBCO)" means a polyethylene glycol chain as described above, wherein the first end of the polyethylene glycol chain is covalently linked via an amide linkage to the Alb, and wherein the second end of the polyethylene glycol chain is covalently linked to a dibenzylcyclooctyne moiety. As used herein, n is an integer corresponding to the number of covalently linked ethylene glycol units in a polyethylene glycol chain in a range from 0-24. In some embodiments about 4 to 10 ethylene glycol units are used.
[0052] In accordance with some embodiments, y is an integer corresponding to the number of molar equivalents of (Peg„-DBCO) groups covalently and directly linked to a molar equivalent oiAlb in a range from 5-20. In some embodiments, y is in the range of about 14 to 15.
[0053] In accordance with a further embodiment, the present invention provides a composition comprising a molecule of formula III: Ab(Pega-Az)b(Fl)c (Ill);
wherein the molecule is defined the same as formula I, with the addition of a fluorophore (Fl), which is covalently and directly linked to the Ab, and wherein c is an integer corresponding to the number of molar equivalents of fluorophores attached per molar equivalent of Ab in a range from 1-5.
[0054] In accordance with still another embodiment, the present invention provides a composition comprising a molecule of formula IV:
Alb(CTX)x(Pegn-DBCO)y(F2)z (IV);
wherein the molecule is defined the same as formula II, with the addition of a fluorophore (F2), a second fluorophore covalently and directly linked to the Alb, and z is an integer corresponding to the number of molar equivalents of fluorophores attached per molar equivalent oiAlb in a range from 1-5.
[0055] As used herein the term "fluorophore" is synonymous with "imaging agent." In some embodiments, the fluorophore is a fluorescent dye. The dyes may be emitters in the visible or near-infrared (MR) spectrum. Known dyes useful in the present invention include carbocyanine, indocarbocyanine, oxacarbocyanine, thuicarbocyanine and merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, boron-dipyrromethane
(BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and
ADS832WS. In some embodiments the fluorophore is selected from the group consisting of: rhodamine, Alexa488 or clinically approved dyes such as ICG, Cy5.5, Cy7, Irdye800 CW, ProSense 750).
[0056] Organic dyes which are active in the NIR region are known in biomedical applications. However, there are only a few NIR dyes that are readily available due to the limitations of conventional dyes, such as poor hydrophilicity and photostability, low quantum yield, insufficient stability and low detection sensitivity in biological system, etc. Significant progress has been made on the recent development of NIR dyes (including cyanine dyes, squaraine, phthalocyanines, porphyrin derivatives and BODIPY (borondipyrromethane) analogues) with much improved chemical and photostability, high fluorescence intensity and long fluorescent life. Examples of R dyes include cyanine dyes (also called as
polymethine cyanine dyes) are small organic molecules with two aromatic nitrogen- containing heterocycles linked by a polymethine bridge and include Cy5, Cy5.5, Cy7 and their derivatives. Squaraines (often called Squarylium dyes) consist of an oxocyclobutenolate core with aromatic or heterocyclic components at both ends of the molecules, an example is KSQ-4-H. Phthalocyanines, are two-dimensional 187t-electron aromatic porphyrin derivatives, consisting of four bridged pyrrole subunits linked together through nitrogen atoms. BODIPY (borondipyrromethane) dyes have a general structure of 4,4'-difluoro- 4- bora-3a, 4a-diaza-s-indacene) and sharp fluorescence with high quantum yield and excellent thermal and photochemical stability.
[0057] Other imaging agents which are attached to compositions of the present invention can include PET, SPECT, and MRI imaging agents. The most widely used agents include branched chelating agents such as di-ethylene tri-amine penta-acetic acid (DTPA), 1,4,7, 10- tetra-azacyclododecane-l,4,7, 10-tetraacetic acid (DOTA) and their analogs for complexation with metals such as Gd and Cu. Chelating agents, such as di-amine dithiols, activated mercaptoacetyl-glycyl-glycyl-gylcine (MAG3), and hydrazidonicotinamide (HY IC), are able to chelate metals like 99mTc and 186Re.
[0058] In accordance with an alternate embodiment, the present invention provides compositions wherein the fluorophore comprises a metal isotope suitable for imaging.
Examples of isotopes useful in the present invention include Tc-94m, Tc-99m, In-11 1, Ga-67, Ga-68, Y-86, Y-90, Lu-177, Re-186, Re-188, Cu-64, Cu-67, Co-55, Co-57, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, or Dy-166, 1-125, 1-124, 1-123, F-18.
[0059] In accordance with an embodiment, the present invention provides a composition comprising a molecule of formula V:
Ab(TCO)d (V);
wherein Ab is a monoclonal antibody to a target of interest; (TCO) is a iraws-cyclooctene functional group linked to the Ab by the other end; and wherein d is an integer corresponding to the number degrees of substitution of TCO. It is understood by those of ordinary skill that iraws-cyclooctene is another click-chemistry reagent.
[0060] In accordance with another embodiment, the present invention provides a composition comprising a molecule of formula VI:
Alb(CTX)y(Pegn-Tt)z (VI); wherein Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers); CTX is a chemotherapeutic agent covalently and directly linked to Alb and y is an integer corresponding to the number of C7 molecules attached per Alb in a range from 1-6; (Peg„-Tt) is a polyethylene glycol chain containing a terminal tetrazine (Tt) functional group linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and z is an integer corresponding to the degree of functionalization of Peg-Tt groups covalently and directly linked to the Alb in a range from 5-20.
[0061] In accordance with a further embodiment, the present invention provides a composition comprising a molecule of formula VII:
Ab(TCO)d(Fl)e (VII);
wherein Ab is a monoclonal antibody to a target of interest; (TCO) is a iraws-cyclooctene functional group linked to the Ab by the other end; wherein d is an integer corresponding to the number degrees of substitution of TCO; and Fl is the first fluorophore covalently and directly linked to the Ab, and e is an integer corresponding to the number of fluorophores attached per Ab in a range from 1-5.
[0062] In accordance with yet another embodiment, the present invention provides acomposition comprising a molecule of formula VIII:
Alb(CTX)y(Pegn-Tt)z(F2)v (VIII);
wherein Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers); CTX is a chemotherapeutic agent covalently and directly linked to Alb and y is an integer corresponding to the number of CT molecules attached per Alb in a range from 1-6; (Peg„-Tt) is a polyethylene glycol chain containing a terminal tetrazine (Tt) functional group linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; z is an integer corresponding to the degree of functionalization of Peg-Tt groups covalently and directly linked to the Alb in a range from 5-20; F2 is the second fluorophore covalently and directly linked to the Alb, and v is an integer corresponding to the number of fluorophores attached per Alb in a range from 1-5.
[0063] In accordance with an embodiment, the present invention provides compositions for diagnosis or treatment of a disease comprising the compositions of formula V and VI. [0064] In accordance with an embodiment, the present invention provides a composition for the diagnosis or treatment of a disease comprising the composition of formula III and the composition of formula IV; wherein F 1 and F2 cannot be the same.
[0065] In accordance with an embodiment, the present invention provides a composition for the diagnosis or treatment of a disease comprising the composition of formula VII and the composition of formula VIII; wherein F 1 and F2 cannot be the same.
[0066] In accordance with an embodiment, the present invention provides a
pharmaceutical composition comprising one or compositions described herein and a pharmaceutically acceptable carrier.
[0067] In accordance with an embodiment, the present invention provides a method of imaging and/or inducing cytotoxicity in a host cell or population of cells comprising administering to the cell or population of cells the compositions described herein, or the pharmaceutical composition described herein, in an amount sufficient to image and/or kill the host cell or population of cells.
[0068] In accordance with an embodiment, the present invention provides a use of the compositions described herein, in an effective amount, to prepare a medicament, preferably for use as a medicament for treating a disease in a subject.
[0069] In another embodiment, the medicament further comprises a pharmaceutically acceptable carrier.
[0070] In a further embodiment, the medicament further comprises a second therapeutic agent. In still another embodiment, the disease is cancer, and in a preferred embodiment, the disease is breast cancer.
[0071] In accordance with an embodiment, the present invention provides a method of treatment of breast cancer in a subject comprising: a) administering to the subject a therapeutically effective amount of a composition of formulas I and/or III, and/or V, and/or VII, wherein Ab is trastuzumab (or other Ab specified above) and optionally, F 1 is a first fluorescent dye (e.g., ICG, Cy5.5, Cy7, Ir dye 800 CW, ProSense 750); b) administering to the subject a therapeutically effective amount of a composition of formula II and/or IV and/or VI and/or VIII, wherein Alb is serum albumin (or similar proteins), CTX is paclitaxel (or other chemotherapeutics specified above), and optionally, F2 is a second fluorescent dye specified above; wherein a) and b) are administered consecutively or concurrently.
[0072] In some embodiments, the first compositions of formulas I, III, V and VII are administered to the cells or the subject first. After a sufficient period of time, the second compositions of formulas II, IV, VI and VIII are administered to the cells or the subject and the compositions conjugate at the Ab target receptors. The amount of time between administration of the first component or composition and the second component or composition can vary. In some embodiments, the time sufficient for the first component or composition to bind the target can be within 30 minutes up to 5, 6, 7, 8, 10 hours. This is followed by administration of the second component or composition.
[0073] It is contemplated that any of the compositions and methods of the present invention described above can also encompass a pharmaceutical composition comprising the compositions and a pharmaceutically acceptable carrier.
[0074] The carriers or diluents used herein may be solid carriers or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.
[0075] Solid carriers or diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
[0076] For liquid formulations, pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, or suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic/aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.
[0077] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Formulations suitable for parenteral administration include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
[0078] Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
[0079] The choice of carrier will be determined, in part, by the particular composition, as well as by the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and interperitoneal administration are exemplary, and are in no way limiting. More than one route can be used to administer the compositions of the present invention, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
[0080] Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ^SHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)).
[0081] As used herein the term "pharmaceutically active compound" or "therapeutically active compound" means a compound useful for the treatment or modulation of a disease or condition in a subject suffering therefrom. Examples of pharmaceutically active compounds can include any drugs known in the art for treatment of disease indications. A particular example of a pharmaceutically active compound is a chemotherapeutic agent.
[0082] For purposes of the invention, the amount or dose of the compositions of the present invention that is administered should be sufficient to effectively target the cell, or population of cells in vivo, such that the imaging of the target cell or population of cells, as well as the cytotoxicity of the compositions can be detected, in the subject over a reasonable time frame. The dose will be determined by the efficacy of the particular composition formulation and the location of the target population of cells in the subject, as well as the body weight of the subject to be treated.
[0083] The dose of the compositions of the present invention also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of the inventive compositions. Typically, an attending physician will decide the dosage of the compositions with which to treat each individual subject, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound to be administered, route of administration, and the severity of the condition being treated. By way of example, and not intending to limit the invention, the dose of the compositions of the present invention can be about 0.001 to about 1000 mg/kg body weight of the subject being treated, from about 0.01 to about 100 mg/kg body weight, from about 0.1 mg/kg to about 10 mg/kg, and from about 0.5 mg to about 5 mg/kg body weight. In another embodiment, the dose of the compositions of the present invention can be at a concentration from about 1 nM to about 10,000 nM, preferably from about 10 nM to about 5,000 nM, more preferably from about 100 nM to about 500 nM.
[0084] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.
Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.
[0085] The invention further provides a host cell comprising any of the compositions described herein. As used herein, the term "host cell" refers to any type of cell that can contain the inventive compositions. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. For purposes of imaging or killing a target cell, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. Examples of suitable human host cells can include, but are not limited to, cells of the major organs of the body, including, for example, cells of the lung, including hepatocytes and hepatic stellate cells, cells of the breast, cells of the prostate, cells of the cornea, including corneal epithelial cells, cells of the lung, including lung epithelial cells, and cells of the brain, such as neurons. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a cancer cell, specifically a breast cancer cell.
[0086] The population of cells can be a heterogeneous population comprising the host cell comprising any of the compositions described, in addition to at least one other cell, e.g., a host cell (e.g., a epithelial cell), which does not comprise any of the compositions, or a cell other than a epithelial cell, e.g., a macrophage, a neutrophil, an erythrocyte, a hepatocyte, a hepatic stellate cell, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) comprising the compositions.
[0087] With respect to the inventive methods, the disease can include cancer. Cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor. Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non- Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer. Preferably, the cancer is breast cancer.
[0088] As defined herein, in one or more embodiments, "administering" means that the one or more compositions of the present invention are introduced into a sample having at least one cell, or population of cells, and appropriate enzymes or reagents, in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit uptake of the at least one compositions of the present invention into the cytosol through the click-chemical reactions occurring on the cell membrane.
[0089] In another embodiment, the term "administering" means that at least one or more compositions of the present invention are introduced into a subject, preferably a subject receiving treatment for a disease, and the at least one or more compositions are allowed to come in contact with the one or more disease related cells or population of cells of interest in vivo.
[0090] As used herein, the term "treat," as well as words stemming therefrom, includes diagnostic and preventative as well as disorder remitative treatment. [0091] As used herein, the term "subject" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
EXAMPLES
[0092] Cell lines. BT-474 and MDA-MB-231 cell lines were purchased from the
American Type Culture Collection (ATCC) and cultures according to the ATCC direction using 46-X media (ATCC) with antibiotics, excess L-glutamine and 10% FBS. Third or fourth passages of cells with 70-80% confluency were used for each experiment unless otherwise mentioned.
[0093] Therapeutics. Pure paclitaxel and Taxol were purchased from LKT Laboratories, Inc. and Sagent Pharmaceuticals, Inc. respectively. Trastuzumab (Hereceptin®) was purchased from Genentech Inc., or kindly gifted by Dr. Robert Ivkov (The Johns Hopkins School of Medicine) and used after purification.
[0094] Chemicals and solvents. Bioconjugation reagents, NHS-PEG4-Azide and NHS- PEG4-DBCO ester were purchased from Thermo Fisher and Click Chemistry Tools respectively. Dry or HPLC grade solvents were purchased from Sigma-Aldrich and used without further purification.
[0095] HPLC purification. Waters binary pump/dual absorbance HPLC system was used with an YMC-Pack Diol-300 (300 x 8.0 mm I.D., S-5 μιη, 3.0 nm) size exclusion column was used for the purification of modified trastuzumab and BSA eluting with 0.2M NaCl in 0.1M PBS (pH 7.2).
[0096] Formulation of first-component series. For the optimization of Peg4-Az substitution, five fractions (500 μΐ^ of 2.5 mg/mL, ~8.5x l0~9 moles each) of pure monoclonal antibody trastuzumab (Genentech, Inc. South San Francisco, CA) were treated with different equivalence (20, 50, 100, 200, 500eq) of NHS-PEG4-Azidein 5% DMSO/PBS(lX). The mixture was vortexed and allowed for conjugation for 2 hours at ambient temperature. Excess small molecular reactants and reagents were removed by Amicon Ultra (0.5 mL, 3 KDa) centrifugal filtration and further purified by HPLC size exclusion chromatography. Pure fractions were concentrated using Amicon Ultra (0.5 mL, 3 KDa) centrifugal filtration. Degree of substitution and functional modification were determined by MALDI-TOF and TAMRA-DIBO (Click-iT®, Invitrogen) methods respectively. Two methods of the determination show a good correlation (data not shown).
[0097] Formulation of second-component series. For the optimization of Peg4-DBCO substitution, five samples (500 of 10 mg/mL) of BSA (Sigma-Aldrich, Inc.) were treated with NHS-PEG4-DBCO (50, 100, 200, 500, 1000 equiv, in 10 of DMSO), vortexed and allowed for conjugations at ambient temperature for 2 hours. Excess small molecular reactants and reagents were removed by Amicon Ultra (0.5 mL, 3 KDa) centrifugal filtration and further purified by HPLC size exclusion chromatography. Pure fractions were concentrated using Amicon Ultra (0.5 mL, 3 KDa) centrifugal filtration. The degree of modification was calculated based on the change of molecular weight determined by
MALDI-TOF (data not shown). Albumin with ten DBCO valencies were labeled with Alexa Fluor 488 and used for the in vitro imaging experiments.
[0098] For the therapeutic study, BSA was first conjugated with paclitaxel following a modified literature protocol. Briefly, a vacuum dried mixture of paclitaxel (LKT
Laboratories, Inc. 20 mg, 0.025 mmol), two molar equivalents, of succinic anhydride (5 mg, 0.05 mmol), and 10 mole% of 4-dimethylaminopyridine (0.3 mg, 0.0025) was dissolved in dry pyridine (0.5 mL) and stirred for 3 hours at room temperature under an argon atmosphere. Solvents were evaporated by a rotavap and the solid residue was dissolved in
dichloromethane (10 mL). The organic layer was washed with 0.1 M HC1 twice (10 mLx3) and dried with anhydrous Na2S04. The solvent was evaporated, and the crude material was further purified by silica gel column chromatography using 2-3% methanol in chloroform to obtain the pure product (22 mg, 95% yield) as a white solid. Then 2'- succinate paclitaxel (4.5 mg, 4.5 μιηο^) dissolved in dry DMSO (100 μί) was added to a solution of dry DMSO:DMF (70:30, 100 μΐ containing N-hydroxy-3-sulfo-succinimide (1.8 mg, 9 μπιοΐεβ). The mixture was stirred for 5 hours and the completion of the reaction was determined by TLC. Resulting 2'-sulfo-NHS-succinyl-paclitaxel containing solution was directly used for the conjugation with BSA.
[0099] Three samples of BSA (4 mg, 0.06 μιηο^ each in PBS) were treated with N- hydroxy-3-sulfo-succinimide from original reaction solution (25, 50, 100 μΐ^, 0.6, 1.2, 2.4 μηιοΐε) and stirred for lhour at room temperature. The reaction mixtures were centrifuged to remove any precipitates and the excess reactants were removed by Amicon Ultra (0.5 mL, 3 KDa) centrifugal filtration. Products were further purified by HPLC size exclusion chromatography. Pure fractions were concentrated using Amicon Ultra (0.5 mL, 3 KDa) centrifugal filtration units. The paclitaxel conjugated BSA was substituted with DBCO following the general procedure given above and labeled with Alexa Fluor 488 following the manufacturer's protocol.
[0100] MALDI analysis. A fraction of pure sample was exchange to water and concentrated (~5 mg/mL) using Amicon Ultra (0.5 mL, 3 KDa) centrifugal filter. Samples (1 μί) were loaded on voyager Teflon coated plate following "dried drop" method using sinapinic acid (10 mg/mL in 50% acetonitrile/0.3% trifluoroacetic acid) as the matrix.
Samples were laser-irradiated by 500 shots and obtained the corresponding molecular mass following the standard method for protein analysis, γ-globulin or pure albumin was used to standardize and calibrate the instrument. Degree of substitutions was calculated based on the change of molecular weight with respect to unmodified trastuzumab and albumin.
[0101] Click-iT® TAMRA DIBO assay. Degree of functional azide groups substituted on trastuzumab was determined by click reaction using Click-iT® TAMRA DIBO fluorescent dye (Invitrogen). Approximately 100 //L of 0.2 mg/mL modified trastuzumab was reacted with excess (300-500 eq) Click-iT® TAMRA DIBO alkyne, and incubated for 2 hours at room temperature in 5% DMSO/DPBS. Samples were purified by HPCL size-exclusion chromatography. The number of functional azides per antibody was determined by the standard absorption method used to calculate degree of labeling.
[0102] Confocal microscopic imaging. The BT-474 cells were placed in 4-well chamber slides (5x 105 cells in 0.5 mL of 46-X medium in each well) and grown for 24-48 hours. Two slides were maintained at 4 °C or 37 °C throughout the experiment. First, cells were incubated with modified trastuzumab (20 /g/mL in DPBS-FBS) for 20 minutes and washed twice with DPBS-FBS (Dulbecco's Phosphate Buffered Saline Solution with 1% Fetal Bovine Serum) to remove the unbound trastuzumab. As the second delivery component, modified albumin (C-2, 100 /g/mL in DPBS-FBS) was added and incubated for 2hours. Cells were washed with DPBS-FBS and fixed by 4% PFA. Cells were counterstained by Hoechst 33258 (1 /g/mL in PBS) to visualize nuclei and wet-mounted for confocal microscopic imaging. Alexa Fluor 488 labeled albumin without DBCO alkyne was used in controls as the second delivery component. [0103] In vitro cytotoxic study of two component drug delivery system.
[0104] Formulation: Four different components, Tz(Peg4-Az)2o( hod)2, Tz(Peg4- Az)28(Rhod)2, Alb(Px)2.2(Peg4-DBCO)i5(Alexa488)i, Alb(Px)3.3(Peg4-DBCO)i5(Alexa488)i, were synthesized for the cytotoxic effect of two component delivery systems following the general procedure. Degree of substitution and labeling were determined and optimized by MALDI-TOF and absorbance methods.
[0105] Cytotoxic study: Her2/wew overexpressing BT-474 (study cell line) and Her2/wew basal level MDA-MB 231 (control cell line) cells were seeded in a 96-well plate at a density of 5x l03 cells and incubated at 37 °C in a humidified atmosphere containing 5% C02. After 24 hours, cultured cells treated with or without modified or unmodified trastuzumab (100 pL, 20 /g/mL) and incubated for 30 minutes at 4°C. Cells were washed once with 0.05%
BSA/DPBS and incubated with or without second components, DBCO modified albumin substituted with paclitaxel, or equivalent amount of paclitaxel as Taxol (100 //L, 40 /g/mL). Cells were incubated at 37°C for 2 hours, washed once using 0.05% BSA/DPBS and refill with fresh media (200 /L). After the incubation at 37 °C for 72 hours in a humidified atmosphere containing 5% C02, 100 //L from total media was removed and viability of cells was determined using WST-8 assay following the manufactures protocol (10 //L of WST-8 reagent per well with 100 //L of media, incubation at 37° for 4hours, and measuring the absorbance at 450 nm). Each test was quadruplicated per plate and triplicate independent experiments were done for the statistical analysis. The -value <0.05 calculated by independent two-sample t-test, is considered as a significant difference between the study groups.
EXAMPLE 1
[0106] Trastuzumab, a pre-targeting, delivery component was functionalized with PEGylated azides. The degree of functionalization plays a major role in antibody-receptor binding affinity and the efficiency of the click conjugation. The degree of functionalization was determined based on the change of molecular weight measured by MALDI-TOF/MS. Functional groups of azides were determined using TAMRA-DIBO via click labeling. For the imaging experiments trastuzumab was used with 20 functional groups attached which gave an optimum cell surface labeling pattern and the click reaction with DBCO
functionalized BSA. There were approximately 20 functional azide groups in this modified trastuzumab and it was labeled with rhodamine and obtained Tz(Peg4-Az)2o(Rhod)2 for the imaging experiments.
[0107] For the second delivery component, BSA was first functionalized with approximately 15 units of NHS-PEG4-DBCO and labeled with Alexa Fluor 488 (Invitrogen) and obtained and used for the imaging experiments. Imaging experiments were performed at 4 °C and 37 °C to observe the surface labeling and the internalization (Figure 2). HER2 overexpressing BT-474 cells shows a typical cell surface labeling pattern with labeled trastuzumab (control without azide functionalization) at 4 °C and doesn't show significant internalization at 37 °C alone. After treating surface labeled cells with second delivery component the live cells were incubated at 4 °C and 37 °C and the internalization was observed after incubation of BT-474 cells for 2 hours. The control MDA-MB-231 cells show no surface labeling by trastuzumab and negligible level of internalization of second delivery components.
EXAMPLE 2
[0108] For the cytotoxic study, Tz(Peg4-Az)2o(Rhod)2, Tz(Peg4-Az)28(Rhod)2, were used as the pre-targeting first delivery component. The second delivery component was formulated by the substitution of paclitaxel (Figure 3a), followed by the DBCO
functionalization and labeling of Alexa Fluor 488 to obtain Alb(Px)2.2(Peg4- DBCO)i5(Alexa488)i, Alb(Px)3.3(Peg4-DBCO)15(Alexa488)1. Internalization of therapeutic components were tracked by the confocal imaging (Figure 3b). The control experiment with trastuzumab without azide functionalization exhibit the cell surface labeling but not the significant loading of second delivery components. The treated experiment using Tz(Peg4- Az)2o(Rhod)2 and Alb(Px)2.2(Peg4-DBCO)i5(Alexa488)ishows the internalization of delivery components and colocalization the cytoplasm.
[0109] The cytotoxic study was performed using BT-474 and MDA-MB-231 cell lines (Figure 4 and Table 1). After the incubation of trastuzumab for 30 minutes pure trastuzumab shows significant therapeutic effect on BT-474 cells (Figure 4a). After the modification of trastuzumab with azides and fluorophores, the trastuzumab did not change the cytotoxicity of unmodified trastuzumab. Cells were treated with Taxol as a control with a concentration equivalent to paclitaxel substituted in BSA (BSA2.2 and BSA3.3), and show a significant cytotoxic effects (cell viability 61% for Taxol equivalent to BSA2.2, and 50% for Taxol equivalent to BSA3.3). The same equivalent amount of paclitaxel was substituted on BSA and treated the cells as second component alone. BSA bound paclitaxel has low cytotoxic effect compared to the corresponding Taxol treated cells (73% for BSA2.2 and 71% for BSA3.3). The same one-component study was carried out for the HER2 basal level cell line MDA-MB-231 (Figure 4b). Here, neither pure trastuzumab, nor any of its modifications, show cytotoxic effects on MDA-MB-231. Effect of click therapy was performed in both BT- 474 and MDA-MB-231 cell lines. The two-component delivery systems with unmodified trastuzumab have low cytotoxic effect in both cell lines. The highest cytotoxic effect was observed in Tz(Peg4-Az)2o(Rhod)2 and Alb(Px)2.2(Peg4-DBCO)i5(Alexa488)i delivery combination (cell viability 41%). BSA with 2.2 equiv. of drug loading was more effective than 3.3 equiv. of drug loading. This can have a significant effect on cell surface click conjugation or cellular internalization. The degree of trastuzumab modification is 20 for optimal cytotoxicity. Results were proven by the negative control cell line MDA-MB-231.
[0110] Modification of trastuzumab with 20 PEGylated azide groups did not affect binding affinity and therapeutic effect of the antibody. Paclitaxel attached directly to the BSA did not show cytotoxicity comparing with equivalent amount of paclitaxel in Taxol. Therefore, paclitaxel loaded BSA does not have an effect on non-specific sites as a single delivery component. After the first component formed a receptor-antibody complexation, the second-delivery component forms multiple bioorthogonal click reactions on cell surface, forming clusters and enhanced internalization by endocytosis. Compared to 2.2 and 3.3 paclitaxel loaded second delivery components, the BSA2.2 provides a better cytotoxic effect. The cytotoxic effect is even greater than the equivalent amount of Taxol. Without being constrained to any particular mechanism of action, this observation may be due to the better click reactions without steric hindrance or suitable hydrophilicity of the component. The optimized bioorthogonal two-component nanocarrier delivery system can be used for in vivo experiments in preclinical stage.
[0111] Table 1 : In Vitro Toxicity Study
Figure imgf000027_0001
Figure imgf000028_0001
EXAMPLE 3
[0112] Click therapy was applied in vivo using HER2(+) BT-474 tumor mouse models of human breast cancer. For in vivo study, iraws-cyclooctene (TCO) and Tetrazine (Tt) based bioorthogonal click chemistry was used. This novel bioorthogonal click reaction is 1000 times faster than the azide (Az) and dibenzylcyclooctyne (DBCO) click chemistry. The delivery strategy and hypothesis of the internalization of TCO-Tt click therapy is similar to the strategy we observed in Az-DBCO click therapy (Figure 5).
[0113] The formulation of components was similar to the bioconjugation techniques used in in vitro study (Figure 6). Trastuzumab (Tz) was functionalized with iraws-cyclooctene (TCO) using NHS-TCO ester reagent. No significant decrease of binding affinity of functionalized Tz was detected at the substitution of six groups of TCO. Resulted Tz conjugate was labeled by NIR CF-680 fluorophores (Fl) (Figure 6a). The common formula for first components is Tz(TCO)d(Fl)e, where d and e are degree of substitutions of TCO and Fl respectively. For the drug-loaded nanocarrier second-component, Alb(CTX)x was substituted with (Pegs-Tt) using NHS-Pegs-Tetrazine reagent followed by labeling with NIR dye CF-750 (Figure 6b). The common formula for the second component is
Alb(CTX)y(Peg5-Tt)z(F2)v where y is the degree of substitution paclitaxel (CTX), z is the degree of functionalization of pegylated tetrazine and v is the degree of labeling of F2 fluorophore. [0114] As shown in Figure 7a, mice were injected with first components, saline (group M-l for no treatment control), Tz(Fl)i (group M-2 control treatment, 0.2 mg in 200 μϊ^ of saline), Tz(TCO)6(Fl)i (group M-3 click therapy, 0.2 mg in 200 μΐ., of saline). After blood clearance of the first component (6-7 hours post injection, Figure 7b), mice in group M-l were injected with saline and mice in group M-2 and M-3 were treated with second delivery components Alb(CTX)2.6(Peg5-Tt)i5(F2)i (2.0 mg in 200 μΐ., of saline). The second and third doses were given on the 14th and 28th days. Tumor measurements were taken every 6th day. The relative growth of tumors was analyzed based on the ratio of tumor size to the initial tumor size (Figure 7c). The change of body weights of mice was analyzed during the click therapy treatment.
[0115] Tumors of mice in group M-3 treated with TCO/Tt based click therapy exhibited slowest growth rates (Figure 7c). After 48 days, tumors were extracted and cross sections were stained with H&E, the tumor sections in M-3 exhibited the significant area of necrosis (Data not shown). Tumors of control mice showed higher growth rate than the treated group. However, tumors of untreated mice show highest growth rate. During the treatment period, control and treated mice show a slight loss of body weight, but it was not significant when compared to untreated mice (Figure 7d).
[0116] These in vivo results show that, TCO/Tt based click therapy is effective in vivo and the system reduced the tumor growth rate significantly.
[0117] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[0118] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0119] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A composition comprising a molecule of formula I:
Ab(Pega-Az)b (I);
wherein Ab is a monoclonal antibody to a target of interest;
(Pega-Az) is a polyethylene glycol chain containing a terminal azidQ(Az) functional group linked to the Ab by the other end;
wherein a is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and
b is an integer corresponding to the number of (Pega-Az) groups covalently and directly linked to the Ab in a range from 1-30.
2. A composition comprising a molecule of formula II:
Alb(CTX)x(Pegn-DBCO)y(II);
wherein Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers);
CTX is a chemotherapeutic agent covalently and directly linked to Alb and x is an integer corresponding to the number of CT molecules attached per
Alb in a range from 1-6;
(Pegn-DBCO) is a polyethylene glycol chain containing a terminal
dibenzylcyclooctyne (DBCO) functional group linked to the Alb by the other end;
wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and y is an integer corresponding to the number of (Pegn-DBCO) groups
covalently and directly linked to the Alb in a range from 5-20.
3. A composition comprising a molecule of formula III:
Ab(Pega-Az)b(Fl)c(III);
wherein Ab is a monoclonal antibody to a target of interest;
(Pega-Az) is a polyethylene glycol chain containing a terminal azidQ(Az) functional group linked to the Ab by the other end; wherein a is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24:
b is an integer corresponding to the number of (Pega-Az) groups covalently and directly linked to the Ab in a range from 1-30; and
Fl is the first fluorophore covalently and directly linked to the Ab, and c is an integer corresponding to the number of fluorophores attached per Ab in a range from 1-5.
4. A composition comprising a molecule of formula IV:
Alb(CTX)x(Pegn-DBCO)y(F2)z(IV);
wherein Alb denotes albumin (bovine serum albumin, human serum albumin, or other similar and biocompatible proteins and polymers);
CTX is a chemotherapeutic agent covalently and directly linked to Alb and x is an integer corresponding to the number of CT molecules attached per Alb in a range from 1-6;
(Pegn-DBCO) is a polyethylene glycol chain containing a terminal DBCO functional group, wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24;
y is an integer corresponding to the number of (Peg„-DBCO) groups
covalently and directly linked to the Alb in a range from 5-20; and
F2 is the second fluorophore covalently and directly linked to the Alb, and z is an integer corresponding to the number of fluorophores attached per Alb in a range from 1-5.
5. A composition comprising a molecule of formula V:
Ab(TCO)d (V);
wherein Ab is a monoclonal antibody to a target of interest;
(TCO) is trans-cyclooctene functional group linked to the Ab by the other end; and
wherein d is an integer corresponding to the number degrees of
substitution of TCO.
6. A composition comprising a molecule of formula VI: Alb(CTX)y(Pegn-Tt)z (VI);
wherein Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers);
CTX is a chemotherapeutic agent covalently and directly linked to Alb and y is an integer corresponding to the number of CT molecules attached per
Alb in a range from 1-6;
(Pegn-Tt) is a polyethylene glycol chain containing a terminal tetrazine (Tt) functional group linked to the Alb by the other end;
wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; and z is an integer corresponding to the degree of functionalization oiPeg-Tt groups covalently and directly linked to the Alb in a range from 5-20.
7. A composition comprising a molecule of formula VII:
Ab(TCO)d(Fl)e (VII);
wherein Ab is a monoclonal antibody to a target of interest;
(TCO) is trans-cyclooctene functional group linked to the Ab by the other end; wherein d is an integer corresponding to the number degrees of
substitution of TCO; and
Fl is the first fluorophore covalently and directly linked to the Ab, and e is an integer corresponding to the number of fluorophores attached per Ab in a range from 1-5.
8. A composition comprising a molecule of formula VIII:
Alb(CTX)y(Pegn-Tt)z(F2)v (VIII);
wherein Alb denotes albumin (bovine serum albumin, human serum albumin or other similar and biocompatible proteins and polymers);
CTX is a chemotherapeutic agent covalently and directly linked to Alb and y is an integer corresponding to the number of CT molecules attached per Alb in a range from 1-6;
(Peg„-Tt) is a polyethylene glycol chain containing a terminal tetrazine (Tt) functional group linked to the Alb by the other end; wherein n is an integer corresponding to the number of ethylene glycol units in a polyethylene glycol chain in a range from 0-24; z is an integer corresponding to the degree of functionalization of (Peg„-Tt) groups covalently and directly linked to the Alb in a range from 5-20; and
F2 is the second fluorophore covalently and directly linked to the Alb, and v is an integer corresponding to the number of fluorophores attached per Alb in a range from 1-5.
9. A composition for the diagnosis or treatment of a disease comprising:
the composition of claim 1 and the composition of claim 2.
10. A composition for the diagnosis or treatment of a disease comprising,
the composition of claim 3 and the composition of claim 4;
wherein F 1 and F2 cannot be the same.
1 1. A composition for the diagnosis or treatment of a disease comprising:
the composition of claim 5 and the composition of claim 6.
12 A composition for the diagnosis or treatment of a disease comprising:
the composition of claim 7 and the composition of claim 8;
wherein Fl and F2 cannot be the same.
13. The composition of any of claims 1 to 12, wherein Ab is trastuzumab or another monoclonal antibody such as anti-HER2 mAb (eg. Pertuzumab), anti-PSMA mAb (eg. J591, J415, Hybritech PEQ226.5 and PM2J004.5), and anti-CD30 mAb (eg. Brentuximab).
14. The composition of any of claims 1 to 12, wherein is a chemotherapeutic agent selected from the group consisting of: Taxanes (eg.Paclitaxel, Docetaxel), antimetabolites (e.g., Fluorouracil), intercalation and inhibitory drugs (eg. Doxorubicin).
15. The composition of any of claims 3 to 4, or 7 to 8,wherein the fluorophore is selected from the group consisting of: rhodamine, Alexa488 or approved dyes such as ICG, Cy5.5, Cy7, Irdye800 CW, ProSense 750, BODIPY dyes, Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700,
AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.).
16. Use of the compositions of any of claims 1 to 8, as a medicament for the treatment of breast cancer in a subject comprising:
a) administering a therapeutically effective amount of the composition of any of claims 1, 3, 5, or 7, is administered to the subject, and
b) administration of a therapeutically effective amount of the composition of any of claims 2, 4, 6, or 8;
wherein Ab is trastuzumab and optionally,
Fland F2 are different fluorescents dyes; comprising
wherein a) and b) are administered consecutively or concurrently.
17. The use of claim 16, wherein the a) and b) are consecutively administered between 3 to 10 hours apart, preferably about 6 to 7 hours apart.
18. A method of treatment of breast cancer in a subject comprising:
a) administering to the subject a therapeutically effective amount of a
composition of any of claims 1, 3, 5, or 7 wherein .4/? is trastuzumab (or other Ab specified above) and optionally,
Fl is a fluorescent dye;
b) administering to the subject a therapeutically effective amount of a
composition of any of claims 2, 4, 6, or 8, wherein Alb is serum albumin (or similar proteins), CTX is paclitaxel (or other
chemotherapeutics specified above), and optionally,
F2 is a fluorescent dye that is not the same as F l; and
19. The method of claim 18, wherein the a) and b) are consecutively administered between 3 to 10 hours apart, preferably about 6 to 7 hours apart.
PCT/US2014/020620 2013-03-05 2014-03-05 Bioorthogonal two-component delivery systems for enhanced internalization of nanotherapeutics Ceased WO2014138186A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361772782P 2013-03-05 2013-03-05
US61/772,782 2013-03-05

Publications (1)

Publication Number Publication Date
WO2014138186A1 true WO2014138186A1 (en) 2014-09-12

Family

ID=51491898

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/020620 Ceased WO2014138186A1 (en) 2013-03-05 2014-03-05 Bioorthogonal two-component delivery systems for enhanced internalization of nanotherapeutics

Country Status (1)

Country Link
WO (1) WO2014138186A1 (en)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108473574A (en) * 2015-10-20 2018-08-31 索伦托治疗有限公司 Intracellular Delivery of Compounds
US10130711B2 (en) 2013-06-19 2018-11-20 The Regents Of The University Of California Chemical structures for localized delivery of therapeutic agents
US10130723B2 (en) 2014-03-14 2018-11-20 The Regents Of The University Of California TCO conjugates and methods for delivery of therapeutic agents
WO2019084323A1 (en) * 2017-10-25 2019-05-02 Georgia State University Research Foundation, Inc. Enrichment-triggered chemical delivery system
US10828373B2 (en) 2015-09-10 2020-11-10 Tambo, Inc. Bioorthogonal compositions
US11253600B2 (en) 2017-04-07 2022-02-22 Tambo, Inc. Bioorthogonal compositions
US20230001009A1 (en) * 2019-12-04 2023-01-05 The Trustees Of The University Of Pennsylvania Nanoparticle compositions and methods of use
US11560384B2 (en) 2017-05-04 2023-01-24 University Of Utah Research Foundation Benzonorbornadiene derivatives and reactions thereof
CN117599001A (en) * 2023-11-29 2024-02-27 南京鼓楼医院 Biological orthogonal Janus microsphere and preparation method and application thereof
EP4425179A3 (en) * 2016-09-01 2024-12-11 Life Technologies Corporation Compositions and methods for enhanced fluorescence
WO2025059685A1 (en) * 2023-09-15 2025-03-20 Tambo, Inc. Methods for in vivo targeted delivery of a payload

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120076727A1 (en) * 2007-01-11 2012-03-29 Immunomedics, Inc. In Vivo Copper-Free Click Chemistry for Delivery of Therapeutic and/or Diagnostic Agents
US20120107302A1 (en) * 2008-03-18 2012-05-03 Leanne Berry Combinations of an anti-her2 antibody-drug conjugate and chemotherapeutic agents, and methods of use
WO2012075361A2 (en) * 2010-12-02 2012-06-07 Immunomedics, Inc. In vivo copper-free click chemistry for delivery of therapeutic and/or diagnostic agents

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120076727A1 (en) * 2007-01-11 2012-03-29 Immunomedics, Inc. In Vivo Copper-Free Click Chemistry for Delivery of Therapeutic and/or Diagnostic Agents
US20120107302A1 (en) * 2008-03-18 2012-05-03 Leanne Berry Combinations of an anti-her2 antibody-drug conjugate and chemotherapeutic agents, and methods of use
WO2012075361A2 (en) * 2010-12-02 2012-06-07 Immunomedics, Inc. In vivo copper-free click chemistry for delivery of therapeutic and/or diagnostic agents

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BOEGGEMAN, E. ET AL.: "Site Specific Conjugation of Fluoroprobes to the Remodeled Fc N-Glycans of Monoclonal Antibodies Using Mutant Glycosyltransferases: Application for Cell Surface Antigen Detection", BIOCONJUGATE CHEMISTRY, vol. 20, 2009, pages 1228 - 1236 *
CHAN, D. P. Y. ET AL.: "Double Click: Dual Functionalized Polymeric Micelles with Antibodies and Peptides", BIOCONJUGATE CHEMISTRY, vol. 24, 4 January 2013 (2013-01-04), pages 105 - 113 *
HAPUARACHCHIGE, S. ET AL.: "Bioorthogonal, Two-Component Delivery Systems Based on Antibody and Drug-Loaded Nanocarriers for Enhanced Internalization of Nanotherapeutics", BIOMATERIALS, vol. 35, 15 December 2013 (2013-12-15), pages 2346 - 2354 *
KOO, H. ET AL.: "Bioorthogonal Copper-Free Click Chemistry In Vivo for Tumor-Targeted Delivery of Nanoparticles", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 51, 2012, pages 11836 - 11840 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10130711B2 (en) 2013-06-19 2018-11-20 The Regents Of The University Of California Chemical structures for localized delivery of therapeutic agents
US10130723B2 (en) 2014-03-14 2018-11-20 The Regents Of The University Of California TCO conjugates and methods for delivery of therapeutic agents
US12257326B2 (en) 2014-03-14 2025-03-25 The Research Foundation For The State University Of New York TCO conjugates and methods for delivery of therapeutic agents
US10342882B2 (en) 2014-03-14 2019-07-09 The Regents Of The University Of California TCO conjugates and methods for delivery of therapeutic agents
US10806807B2 (en) 2014-03-14 2020-10-20 The Regents Of The University Of California TCO conjugates and methods for delivery of therapeutic agents
US10828373B2 (en) 2015-09-10 2020-11-10 Tambo, Inc. Bioorthogonal compositions
EP3365375A4 (en) * 2015-10-20 2019-09-04 Sorrento Therapeutics, Inc. INTRACELLULAR ADMINISTRATION COMPOUNDS
CN108473574A (en) * 2015-10-20 2018-08-31 索伦托治疗有限公司 Intracellular Delivery of Compounds
EP4425179A3 (en) * 2016-09-01 2024-12-11 Life Technologies Corporation Compositions and methods for enhanced fluorescence
US11253600B2 (en) 2017-04-07 2022-02-22 Tambo, Inc. Bioorthogonal compositions
US12296017B2 (en) 2017-04-07 2025-05-13 Tambo, Inc. Bioorthogonal compositions
US11560384B2 (en) 2017-05-04 2023-01-24 University Of Utah Research Foundation Benzonorbornadiene derivatives and reactions thereof
US11607458B2 (en) 2017-10-25 2023-03-21 Georgia State University Research Foundation, Inc. Enrichment-triggered chemical delivery system
WO2019084323A1 (en) * 2017-10-25 2019-05-02 Georgia State University Research Foundation, Inc. Enrichment-triggered chemical delivery system
US20230001009A1 (en) * 2019-12-04 2023-01-05 The Trustees Of The University Of Pennsylvania Nanoparticle compositions and methods of use
WO2025059685A1 (en) * 2023-09-15 2025-03-20 Tambo, Inc. Methods for in vivo targeted delivery of a payload
CN117599001A (en) * 2023-11-29 2024-02-27 南京鼓楼医院 Biological orthogonal Janus microsphere and preparation method and application thereof

Similar Documents

Publication Publication Date Title
WO2014138186A1 (en) Bioorthogonal two-component delivery systems for enhanced internalization of nanotherapeutics
Jain et al. Penetratin improves tumor retention of single-chain antibodies: a novel step toward optimization of radioimmunotherapy of solid tumors
CN106995493B (en) Chlorotoxin polypeptides and conjugates and uses thereof
Nabi et al. Mucin-1 conjugated polyamidoamine-based nanoparticles for image-guided delivery of gefitinib to breast cancer
US10780179B2 (en) Conjugates for the treatment of cancer targeted at intracellular tumor-associated antigens
JP7126956B2 (en) Conjugates that enhance whole-cell accumulation
US20100284921A1 (en) Targeted nanoparticles for intracellular cancer therapy
Chen et al. Melanocortin-1 receptor-targeting ultrasmall silica nanoparticles for dual-modality human melanoma imaging
US20240299599A1 (en) Dimerization strategies and compounds for molecular imaging and/or radioimmunotherapy
KR20190003630A (en) Compositions and methods for targeted particle penetration, distribution, and response in malignant brain tumors
AU2025200954A1 (en) Molecules with solubility tag and related methods
Chen et al. MUC1 aptamer-based near-infrared fluorescence probes for tumor imaging
WO2018217528A1 (en) Ultrasmall nanoparticles labeled with zirconium-89 and methods thereof
CA2955438A1 (en) Dextran conjugates for targeting macrophages and other mannose binding c-type lectin receptor expressing cells
Prakash et al. A novel approach to deliver anticancer drugs to key cell types in tumors using a PDGF receptor-binding cyclic peptide containing carrier
Châtre et al. In vivo synthesis of triple-loaded albumin conjugate for efficient targeted cancer chemotherapy
US20250049963A1 (en) Glycosylated dendrimers for targeted intracellular delivery
US20180250360A1 (en) Method for detecting cancer cells, reagent for introducing substance into cancer cells, and composition for treating cancer
US12281161B2 (en) Polypeptides and uses thereof
WO2013003507A1 (en) Multifunctional agents
Popova et al. Biotin-decorated anti-cancer nucleotide theranostic conjugate of human serum albumin: Where the seed meets the soil?
US20020119095A1 (en) Chemotherapeutic agents conjugated to p97 and their methods of use in treating neurological tumours
CN121079294A (en) Compound nuclei for multifunctionalization of surfactants and their applications
Kang et al. Catabolism of 64Cu and Cy5. 5-labeled human serum albumin in a tumor xenograft model
WO2024047172A1 (en) An integrin alpha10 antibody drug conjugate

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: 14759766

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: 14759766

Country of ref document: EP

Kind code of ref document: A1