WO2023196593A1 - Antigen-independent targeted delivery of therapeutic agents - Google Patents

Antigen-independent targeted delivery of therapeutic agents Download PDF

Info

Publication number
WO2023196593A1
WO2023196593A1 PCT/US2023/017884 US2023017884W WO2023196593A1 WO 2023196593 A1 WO2023196593 A1 WO 2023196593A1 US 2023017884 W US2023017884 W US 2023017884W WO 2023196593 A1 WO2023196593 A1 WO 2023196593A1
Authority
WO
WIPO (PCT)
Prior art keywords
modified
cancer
cyclooctyne
azide
analogues
Prior art date
Application number
PCT/US2023/017884
Other languages
French (fr)
Inventor
Andrew Wang
Hyesun HYUN
Original Assignee
The University Of North Carolina At Chapel Hill
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 The University Of North Carolina At Chapel Hill filed Critical The University Of North Carolina At Chapel Hill
Publication of WO2023196593A1 publication Critical patent/WO2023196593A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/54—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 compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic 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
    • A61K47/54—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 compound
    • A61K47/555—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 compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Cancer immunotherapy has improved clinical outcomes for patients with many different types of solid tumors, [1] including melanoma, non-small cell lung cancer, renal cell carcinoma, and urothelial cancers. [2] However, these approaches do not provide long-term antitumor immunity in all patients, for example, some reports show that long-term antitumor immunity occurs in less than 30% of patients with cancer. [3]
  • Cytotoxic agents are also a frontline therapy against cancers. However, owing to their cytotoxicity, undesirable and often serious toxicity is associated with this type of therapy. Targeted delivery of these types of agents to minimize side effects remains an ongoing challenge.
  • the subject matter described herein is directed to a method of delivering a 4- IBB agonist to a cancer cell, comprising: contacting a cancer cell with a particle comprising an azide- or tetrazinecontaining molecule to generate a surface-modified cancer cell; contacting the surface-modified cancer cell with a cyclooctyne-modified 4- 1BB agonist, wherein, the cyclooctyne-modified 4-1BB agonist binds to the surface modified cancer cell.
  • the subject matter described herein is directed to a method of delivering a 4-1BB agonist to a cancer cell, comprising: contacting a cancer cell with a particle comprising a cyclooctyne-containing molecule to generate a surface-modified cancer cell; contacting the surface-modified cancer cell with an azide- or tetrazinemodified 4- IBB agonist, wherein, the azide- or tetrazine-modified 4-1BB agonist binds to the surface modified cancer cell.
  • the subject matter described herein is directed to a method of delivering a therapeutic radionuclide to a cancer cell, comprising: contacting a cancer cell with a particle comprising an azide- or tetrazinecontaining molecule to prepare a surface-modified cancer cell; contacting the surface modified cancer cell with a cyclooctyne-modified therapeutic radionuclide, wherein, the cyclooctyne-modified therapeutic radionuclide binds to the surface modified cancer cell.
  • the subject matter described herein is directed to a method of delivering a therapeutic radionuclide to a cancer cell, comprising: contacting a cancer cell with a particle comprising a cyclooctyne-containing molecule to prepare a surface-modified cancer cell; contacting the surface modified cancer cell with an azide- or tetrazinemodified therapeutic radionuclide, wherein, the azide- or tetrazine-modified therapeutic radionuclide binds to the surface modified cancer cell.
  • the subject matter described herein is directed to a method of treating cancer in a subject by antigen-independent immunotherapy, comprising: administering to the subject a particle comprising an azide- or tetrazinecontaining molecule, wherein a cancer cell in the subject is modified to a surface- modified cancer cell; administering to the subject a cyclooctyne-modified 4-1 BB agonist, wherein the cyclooctyne-modified 4- IBB agonist binds to the surface modified cancer cell, wherein, the cancer is treated.
  • the subject matter described herein is directed to a method of treating cancer in a subject by antigen-independent immunotherapy, comprising: administering to the subject a particle comprising a cyclooctyne-containing molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; admimstenng to the subject an azide- or tetrazme-modified 4-1BB agonist, wherein the azide- or tetrazine-modified 4-1BB agonist binds to the surface modified cancer cell, wherein, the cancer is treated.
  • the subject matter described herein is directed to a method of treating cancer in a subject by antigen-independent therapy, comprising: administering to the subject a particle comprising an azide- or tetrazinecontaining molecule, wherein a cancer cell in the subject is modified to a surface- modified cancer cell; admimstenng to the subject a cyclooctyne-modified therapeutic radionuclide, wherein the cyclooctyne-modified therapeutic radionuclide binds to the surface modified cancer cell, wherein, the cancer is treated.
  • the subject matter described herein is directed to a method of treating cancer in a subject by antigen-independent immunotherapy, comprising: administering to the subject a particle comprising a cyclooctyne-containing molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; administering to the subject an azide- or tetrazine-modified therapeutic radionuclide, wherein the azide- or tetrazine-modified therapeutic radionuclide binds to the surface modified cancer cell, wherein, the cancer is treated.
  • the subject matter described herein is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising: an azide-containing molecule, a tetrazine-containing molecule, a cyclooctyne-modified 4- IBB agonist or a cyclooctyne-modified therapeutic radionuclide, an azide- or tetrazine-modified 4-1 BB agonist or an azide- or tetrazine-modified therapeutic radionuclide; and a pharmaceutically acceptable excipient.
  • Figures 1A-1G illustrate the proposed engineering and characterization of antibodies and nanoparticles for anti-gen independent targeting delivery.
  • Figure 1A is a schematic depicting MazNP and DBCO-a4-lBB improved cancer immunotherapy and reduced hepatotoxicity.
  • Figure IB displays Transmission Electron Microscopy (TEM) images of naked or Ac4ManNAz-loaded NP (MazNP), negatively stained with 2% uranyl acetate.
  • Figure ID depicts UV spectra of unmodified a4-lBB and DBCO-functionalized a4-lBB with target molar ratios of conjugation of DBCO to a4-lBB (20, 35, and 50: 1)
  • Figure IF depicts MALDI-TOF mass spectra of unmodified a4-lBB and DBCO-functionalized a4- 1 BB with different molar ratios of DBCO to a4-lBB.
  • Figure 1G displays the plot of the DOF (degree of functionalization) of different DBCO-functionalized a4-lBB determined by UV spectroscopic method and MALDI-TOF MS versus the molar equivalent of DBCO ligand used in the functionalization of a4-lBB (target DOF).
  • Figures 2A-2D illustrate the in vivo antitumor efficacy study conducted in Bl 6F 10 tumor-bearing mice.
  • Figure 2A displays the dosing schedule of antibodies and NPs.
  • Figure 2C demonstrates Kaplan-Meier survival curves of B16F10-tumor bearing mice. MST - median survival time.
  • Figure 2D presents the survival curves of cured animals following tumor rechallenge.
  • Figures 3A-3D illustrate the in vivo antitumor efficacy study conducted in orthotopic 4T1 breast tumor-bearing mice.
  • Figure 3A depicts the dosing schedule of treatments.
  • Figure 3C presents average tumor growth curves of animals shown in Figure 3B. Tumor growth over time was compared by Sidak’s multiple comparisons test following two-way ANOVA.
  • Figure 3D depicts the Kaplan-Meier survival curves of 4T1 tumor-bearing mice. MST - median survival time. P values were calculated by the log-rank test.
  • Figures 4A-4D illustrate CD8 + T cell expansion and NK cell activation in tumors stimulated by antigen-independent tumor targeted antibody with nanoparticle.
  • Figure 4C displays average tumor growth curves for each treatment shown in Figure 4B. **: p ⁇ 0.01 by Sidak’s multiple comparisons test following two-way ANOVA.
  • Figure 4D presents the differences in survival as determined for each group by Kaplan-Meier method. MST - median survival time. P values were calculated by the log-rank test. *: p ⁇ 0.05 and ***: p ⁇ 0.001.
  • Figures 5A-5E illustrate that the mD-az/NP did not induce a4-lBB-induced liver toxicity in B16F 10 tumor-bearing mice.
  • % Area of Figure 7A CD3 + CD8 + or Figure 7B CD3 + CD4 + T cells was estimated as the area of CD8 (red) or CD4 (cyan) fluorescence overlapped with CD3 (yellow)-positive area (orange or green, respectively) divided by the area of the tissue in the field of view (outlined by blue Hoechst 33258 staining).
  • % Area of Figure 7C CD3 + CD8 + T cells or Figure 7D CD3 + CD4 + T cells in total CD3 + T cells was estimated as the area of CD3 + CD8 + (green) or CD3 + CD4 + (orange) divided by the area of the total CD3 + T cell fluorescence (yellow), respectively.
  • Figure 8 illustrates the dosing schedule of treatments with aPD + DBCO-a4-lBB with MazNP with or without CD8 + T cell or NK cell depletion.
  • FIG 12 illustrates the quantification of TNF-a release from F4/80+ macrophages stimulated with a4-lBB or DBCO-a4-lBB in the absence and presence of 4- IBB ligand.
  • Figure 14 illustrates azide group generation on the B16F10 cell surface after 6 h incubation with PBS, Ac4ManNAz or non-PEGylated MazNP.
  • the cells were imaged with confocal microscopy (Green: streptavidin-FITC; Red: rhodamine-labeled MazNP; Blue: nuclei stained with Hoechst 33342). Scale bars: 10 pm.
  • Figure 15 illustrates azide group generation on the J774A.1 macrophage treated with chloroquine prior to 6 h incubation with PBS, Ac4ManNAz or non-PEGylated MazNP.
  • the cells were imaged with confocal microscopy (Green: streptavidin-FITC; Red: rhodamme- labeled MazNP; Blue: nuclei stained with Hoechst 33342). Scale bars: 10 pm.
  • Described herein are methods for treating cancer that can result in fewer off-target side effects using an antigen-independent approach to target delivery of a therapeutic agent to cancer cells.
  • the subject matter described herein is directed to a tumor-targeting approach based on unnatural sugar-mediated metabolic glycoengineering and bio-orthogonal click chemistry to selectively deliver a cancer therapeutic agent, such as a therapeutic radionuclide or a 4-1BB agonist to tumors.
  • a cancer therapeutic agent such as a therapeutic radionuclide or a 4-1BB agonist
  • an anti-4-lBB antibody a4-lBB
  • a4-lBB is delivered in an antigen-dependent manner, which eliminates the need for tumor-associated antigens.
  • antigen-independent targeted delivery of 4-1BB agonists improves anti-tumor immune responses while reducing hepatotoxicity. Described herein is the inhibition of tumor growth with prolonged survival, accompanied by inducing CD8 + T cells expansion in the tumor, along with significant reduction of CD8 + T cell accumulation in the liver and, thus, hepatotoxicity.
  • Tn certain embodiments, described herein are methods of treating cancer comprising administering a metabolic precursor, that is an azide-containing molecule, encapsulated in nanoparticles (NP) that enhance in vivo tumor-targeting of a cyclooctyne-modified therapeutic agent.
  • the methods described herein comprise contacting a cell wi th a particle comprising a type of unnatural sugar that can metabolically generate azide functional groups onto glycans on the surface of the tumor cell. In this way, the surface of the tumor cell is selectively decorated.
  • the cell surface azide groups can then be coupled with cyclooctyne-conjugated therapeutic agents via in vivo biorthogonal click chemistry reaction.
  • cancer immunotherapy has improved clinical outcomes for patients with many different types of solid tumors.
  • these approaches do not provide long-term antitumor immunity in all patients.
  • the methods described herein are antigen-independent immunotherapies that provide antitumor immune response and, advantageously, do not produce hepatotoxicity.
  • 4-1BB (CD137, TNFRSF9) is an inducible costimulatory receptor and expressed on the surface of macrophages/ 4 ’ 51 activated T
  • TCR T cell receptor
  • TIL tumor infiltrating lymphocytes
  • a4-lBB stimulation promotes CD8 + tumor infiltrating lymphocytes (TIL) with a memory phenotype, resulting in retardation of tumor growth and long-term protective immunological response in colon and breast cancer models/ 14, 151 Moreover, 4-1BB stimulation on NK cells induces interferon-gamma (IF
  • the surface of cancer cells can be covalently modified using the particles described herein, wherein the modified cell surface comprises at least one covalently atached azide-, tetrazine-, or cyclooctyne-containing molecule.
  • the term “surface modified cancer cell” refers to a cancer cell that comprises at least one covalent modification on the cell surface, whereby the modification is in the form of a metabolic glycoprotein labeling reagent.
  • This labeling reagent is a reactive motif for subsequent covalent binding to a modified 4- IBB agonist, such as an anti -4- IBB antibody, or a modified therapeutic radionuclide through the chemical linking strategies described herein.
  • a “metabolic glycoprotein labeling reagent” refers to the azide-, tetrazine-, or cyclooctyne-modified unnatural sugars that are metabolically incorporated into cell-surface glycoproteins to generate a biorthogonal click chemistry reagent that is accessible on the surface of the cell.
  • Metabolic glycoengineering 151 52 ' and biorthogonal click chemistry ⁇ 53 ' 55 ! are available tools. As described herein, these can be used to facilitate unique chemical decoration of cancer cells to enable targeted delivery of a modified 4-1BB agonist and/or modified therapeutic radionuclides onto the targeted cells
  • the methods described can result in a modified surface of a cancer cell comprising a glycoengineered moiety having the structure: (a transmembrane glycoprotein) — (a residue of an azide- containing molecule) — (a residue of a cyclooctyne) — (an optional linker 1) — (a residue of an antibody, small molecule, or a therapeutic radionuclide), wherein the dash represents a covalent bond; or, a cancer cell comprising a glycoengineered moiety having the structure: (a transmembrane glycoprotein) — (a residue of a cyclooctyne-containing molecule) — (a
  • the term “residue” or “residue of’ a chemical moiety refers to a chemical moiety that is bound to a molecule, whereby through the binding, at least one covalent bond has replaced at least one atom of the original chemical moiety, resulting in a residue of the chemical moiety in the molecule.
  • the term “small molecule” refers to a compound with a low molecular weight ( ⁇ 900 Daltons) that may regulate biological processes by engaging with biological targets. The binding of the small molecule to the azide, tetrazine or cyclooctyne can accomplished in any suitable way, including conjugation and chelation, using known chemistries.
  • the term “therapeutic radionuclide” refers to the source of therapeutic radiation. The radionuclide can be in the form of a radioisotope or a compound, such as a radiopharmaceutical, comprising the radionuclide.
  • the radioisotope and radiopharmaceutical can be any known radionuclide suitable for medical use.
  • the binding of the radionuclide to the azide, tetrazine or cyclooctyne can accomplished in any suitable way, including conj ugation and chelation, using known chemistries.
  • thiol-maleimide click chemistry can be used to modify the surface of a cell.
  • free thiol groups on the surface can be made to react with maleimide- functionalized biomolecule through stable thioester bond to form stable functionalized cells.
  • Maleimide-functionalized biomolecules can be prepared by amine-NHS reaction between desired biomolecule and NHS-maleimide crosslinker (e.g, sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane- 1 -carboxylate (sulfo-SMCC)).
  • the subject matter described herein is directed to a glycoengineered moiety comprising an azide moiety, a cyclooctyne moiety, or a tetrazine moiety.
  • the subject matter described herein is directed to the delivery of an azide-, cyclooctyne-, or a tetrazine-contaming molecule that is a metabolic labeling reagent.
  • the subject matter described herein is directed to the delivery of an azide-, cyclooctyne, or a tetrazine-containing metabolic labeling reagent using a particle.
  • the particle used to for the delivery of an azide-, cyclooctyne, or a tetrazine-containing metabolic labeling reagent is a nanoparticle comprising a polymer.
  • the particle used to for the delivery of an azide-, cyclooctyne, or a tetrazine-containing metabolic labeling reagent is a lipid-based nanoparticle.
  • the nanoparticle is a dendrimer, a liposome, an inorganic nanoparticle, or a polymeric nanoparticle.
  • the nanoparticle is about 2nm to about lOnm, about lOnm to about lOOnm, or about lOOnm to about lOOOnm.
  • the nanoparticle is about 2nm to about lOOOnm, about 2nm to about 750nm, about 2nm to about 500nm, about 2nm to about 250nm, about 2nm to about 200nm, about 2nm to about lOOnm, or 2nm to about 50nm.
  • the nanoparticle is about lOnm to about lOOOnm, about 25nm to about lOOOnm, about 50nm to about lOOOnm, about lOOnm to about lOOOnm, about 200 to about lOOOnm, about 500nm to about lOOOnm, or 750nm to about lOOOnm.
  • the nanoparticle is about 2nm, about 5nm, about lOnm, about 50nm, about lOOnm, about 200nm, about 300nm, about 400nm, about 500nm, about 600nm, about 700nm, about 800nm, about 900nm, or about lOOOnm.
  • the dendrimer is a multivalent dendrimer.
  • the multivalent dendrimer is a polyamidoamine dendrimer.
  • the nanoparticle is a pegylated nanoparticle (e.g., DBCO-functionalized PEG-PLGA nanoparticle). In embodiments, the pegylated nanoparticle is less than 200nm in diameter.
  • the at least one covalently attached antibody, therapeutic radionuclide, and/or small molecule is attached through a glycoengineered moiety.
  • the covalent attachment is via conjugating to thiol groups on cells.
  • the glycoengineered moiety comprises a residue of an amide of mannosamine, galactosamine, xylosamine, fucosamine, or other sugars and their analogues.
  • the glycoengineered moiety further comprises a residue of an azide, a cyclooctyne, or a tetrazine covalently attached to the residue of an amide of mannosamine, galactosamine, xylosamine, fucosamine, or other sugars and their analogues.
  • the cyclooctyne is DBCO.
  • analogues refer to compounds having a core structure of the parent compound but differing from it through chemical modifications that add functional moieties.
  • degree of functionalization refers to number of residues of azide, tetrazine or cyclooctyne that are covalently attached to the antibody, small molecule, or therapeutic radionuclide.
  • a degree of functionalization value refers to the number of residues per antibody, small molecule, or therapeutic radionuclide
  • Immune checkpoint refers to a molecule on the cell surface of a CD4 and CD8 T cell that down-modulates or inhibits an anti-tumor immune response.
  • Immune checkpoint molecules include, but are not limited to, Programmed Death 1 (PD1), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), PDL-1 (B7H1), PDL-2 (B7-DC), B7H3, B7H4, OX-40, CD137, CD40, CD27, LAG3, TIM3, ICOS, or BTLA, which directly inhibit immune cells.
  • Immunotherapeutic agents which can act as immune checkpoint inhibitors useful in the methods of the present invention include, but are not limited to, anti-PDl; anti -CTLA-4; anti- PDL-1; anti-B7-Hl; anti-PDL-2; anti-B7-H3; anti-B7-H4; anti-CD137; anti-CD40; anti- CD27; anti-LAG3; anti-TIM3; anti-ICOS, and anti-BTLA.
  • physiological conditions refers to the range of conditions of temperature, pH, and tonicity (or osmolality) normally encountered within tissues in the body of a living human.
  • zri vitro refers to artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube).
  • zri vzvo refers to natural environments (e.g., a cell or organism or body) and to processes or reactions that occur within a natural environment.
  • Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.
  • the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value or variations ⁇ 0.5%, 1%, 5%, or 10% from a specified value.
  • compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited.
  • a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients.
  • an antigen or “at least one antigen” can include a plurality of antigens, including mixtures thereof.
  • described herein is a pharmaceutical composition
  • a pharmaceutical composition comprising a functionalized antibody, therapeutic radionuclide, or an immune checkpoint molecule as described herein, and a pharmaceutically acceptable excipient.
  • a “pharmaceutically acceptable excipient” refers to a vehicle for containing a functionalized cell or an acellular extracellular matrix that can be introduced into a subject without significant adverse effects and without having deleterious effects on the functionalized cell or acellular extracellular matrix. That is, “pharmaceutically acceptable” refers to any formulation which is safe and provides the appropriate delivery for the desired route of administration of an effective amount of at least one functionalized cell or acellular extracellular matnx for use in the methods disclosed herein. Pharmaceutically acceptable carriers or vehicles or excipients are well known.
  • Such carriers can be suitable for any route of administration (e.g., parenteral, enteral (e.g., oral), or topical application).
  • Such pharmaceutical compositions can be buffered, for example, wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the functionalized cell or acellular extracellular matrix and route of administration.
  • Suitable pharmaceutically acceptable carriers include, for example, sterile water, salt solutions such as saline, glucose, buffered solutions such as phosphate buffered solutions or bicarbonate buffered solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates (e.g., lactose, amylose or starch), magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, and the like.
  • compositions or vaccines may also include auxiliary agents including, for example, diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffering agents, viscosity enhancing additives, lubricants, salts for influencing osmotic pressure, buffers, vitamins, coloring, flavoring, aromatic substances, and the like which do not deleteriously react with a functionalized cell or an acellular extracellular matrix.
  • auxiliary agents including, for example, diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffering agents, viscosity enhancing additives, lubricants, salts for influencing osmotic pressure, buffers, vitamins, coloring, flavoring, aromatic substances, and the like which do not deleteriously react with a functionalized cell or an acellular extracellular matrix.
  • auxiliary agents including, for example, d
  • pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions, or oils.
  • Non-aqueous solvents include, for example, propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include, for example, water, alcohohc/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • oils include those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.
  • Solid carriers/diluents include, for example, a gum, a starch (e.g., com starch, pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, or dextrose), a cellulosic matenal (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • a starch e.g., com starch, pregelatinized starch
  • a sugar e.g., lactose, mannitol, sucrose, or dextrose
  • a cellulosic matenal e.g., microcrystalline cellulose
  • an acrylate e.g., polymethylacrylate
  • sustained or directed release pharmaceutical compositions or vaccines can be formulated. This can be accomplished, for example, through use of liposomes or compositions wherein the active compound is protected with differentially degradable coatings (e.g., by microencapsulation, multiple coatings, and so forth). Such compositions may be formulated for immediate or slow release. It is also possible to freeze-dry the compositions and use the lyophilisates obtained (e.g., for the preparation of products for injection).
  • the subject matter described herein is directed to a method of delivering a 4- IBB agonist to a cancer cell comprising contacting a cancer cell with a particle comprising an azide- or tetrazine-containing molecule to prepare a surface-modified cancer cell, and further contacting the surface modified cancer cell with a cyclooctyne-modified 4- 1BB agonist, wherein, the cyclooctyne-modified 4-1BB agonist binds to the surface modified cancer cell.
  • the further contacting is after a period of time sufficient for formation of the surface modification on target cancer cells.
  • the azide- or tetrazine-containing molecule is an azide- or tetrazine-containing metabolic glycoprotein labeling reagent.
  • the azide- or tetrazine-containing metabolic glycoprotein labeling reagent is selected from among sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
  • the azide-containing metabolic glycoprotein labeling reagent is N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz).
  • the metabolic glycoprotein labeling reagent contains a cyclooctyne moiety or another click chemistry reactive group including but not limited to terminal alkynes, heterocyclic electrophiles, and reactive ketones, and the like.
  • the metabolic glycoprotein label reagent is DBCO (dibenzocyclooctyne).
  • the azide-containing metabolic glycoprotein labeling reagent is a bifunctional molecule, wherein it contains one or more additional reactive groups used to conjugate to other molecules on the cell surface.
  • the additional reactive groups are selected from among maleimides, thiols, amines, hydroxides, imidazoles, NHS esters, and other bioreactive functional groups.
  • the 4-1BB agonist of the cyclooctyne-modified 4-1BB agonist is an antibody or small molecule.
  • the 4-1BB agonist of the cyclooctyne-modified 4-1BB agonist is an anti-4-lBB antibody.
  • the anti-4-lBB antibody is functionalized with one or more residues of a cyclooctyne moiety.
  • the degree of functionalization of the antibody is 35 or less.
  • the cyclooctyne moiety is a dibenzocyclooctyne (DBCO).
  • the subject matter described herein is directed to a method of delivering a 4- IBB agonist to a cancer cell comprising contacting a cancer cell with a particle comprising a cy cl ooctyne-con taming molecule to prepare a surface-modified cancer cell, and further contacting the surface modified cancer cell with an azide-or tetrazine-modified 4-1BB agonist, wherein, the azide- or tetrazine-modified 4-1BB agonist binds to the surface modified cancer cell.
  • the cyclooctyne-containing molecule is an azide- or tetrazine-containing metabolic glycoprotein labeling reagent.
  • the azide containing molecule is a bifunctional molecule where it contains another reactive group that can be conjugated to molecules on the cell surface, including but not limited to maleimide, thiol, and amine.
  • the cyclooctyne-containing metabolic glycoprotein labeling reagent is selected from among sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
  • the cyclooctyne-containing metabolic glycoprotein labeling reagent is an unnatural sugar functionalized with dibenzocyclooctyne (DBCO).
  • DBCO dibenzocyclooctyne
  • the 4-1BB agonist of the azide- or tetrazine-modified 4-1BB agonist is an antibody or small molecule.
  • the 4-1BB agonist of the azide- or tetrazine-modified 4-1BB agonist is an anti-4-lBB antibody.
  • a therapeutic agent can be modified as described herein for click chemistry conjugation to a surface-modified cancer cell, wherein the therapeutic agent is anti-OX-40, IL-2 or IL-12.
  • the anti-4-lBB antibody is functionalized with one or more residues of an azide or tetrazine moiety.
  • the degree of functionalization of the antibody is 35 or less.
  • the subject matter described herein is directed to a method of delivering a therapeutic radionuclide to a cancer cell comprising contacting a cancer cell with a particle comprising an azide- or tetrazine-contammg molecule to prepare a surface- modified cancer cell, and further contacting the surface modified cancer cell with a cyclooctyne-modi fied therapeutic radionuclide, wherein, the cyclooctyne-modified therapeutic radionuclide binds to the surface modified cancer cell.
  • the subject matter described herein is directed to a method of delivering a therapeutic radionuclide to a cancer cell comprising contacting a cancer cell with a particle comprising an cyclooctyne-containing molecule to prepare a surface-modified cancer cell, and further contacting the surface modified cancer cell with a azide- or tetrazine- modified therapeutic radionuclide, wherein, the azide- or tetrazine-modified therapeutic radionuclide binds to the surface modified cancer cell.
  • the therapeutic radionuclide of the cyclooctyne-modified therapeutic radionuclide is a beta-emitter.
  • the therapeutic radionuclide of the cyclooctyne-modified therapeutic radionuclide is an alpha-emitter.
  • the therapeutic radionuclide of the azide- or tetrazine-modified therapeutic radionuclide is a beta-emitter.
  • the therapeutic radionuclide of the azide- or tetrazine- modified therapeutic radionuclide is an alpha-emitter.
  • the subject matter described herein is directed to a method of treating cancer in subject by antigen-independent immunotherapy comprising administering to the subject a particle comprising an azide- or tetrazine-containing molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; and subsequently, administering to the subject a cyclooctyne-modified 4-1BB agonist, wherein the cyclooctyne- modified 4- IBB agonist binds to the surface modified cancer cell, wherein, the cancer is treated.
  • the subject matter described herein is directed to a method of treating cancer in subject by antigen-independent immunotherapy comprising administering to the subject a particle comprising an cyclooctyne-containing molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; and subsequently, administering to the subject an azide or tetrazine-modified 4-1BB agonist, wherein the azide- or tetrazine-modified 4- IBB agonist binds to the surface modified cancer cell, wherein, the cancer is treated.
  • the subject matter described herein is directed to a method of treating cancer in subject by antigen-independent therapy comprising administering to the subject a particle comprising an azide- or tetrazine-contammg molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; and subsequently, administering to the subject a cyclooctyne-modified therapeutic radionuclide, wherein the cyclooctyne-modified therapeutic radionuclide binds to the surface modified cancer cell, wherein, the cancer is treated.
  • the subject matter described herein is directed to a method of treating cancer in subject by antigen-independent therapy comprising administering to the subject a particle comprising an cyclooctyne-containing molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; and subsequently, administering to the subject an azide or tetrazine-modified therapeutic radionuclide, wherein the azide- or tetrazine-modified radionuclide binds to the surface modified cancer cell, wherein, the cancer is treated.
  • the particle encapsulating the metabolic glycoprotein labeling reagent is a nanoparticle comprising a polymer.
  • the polymer used for the nanoparticle is selected from among the group consisting of mPEG-PLA, PLA, PLGA, and dextran.
  • the nanoparticle can be a lipid-based nanoparticle.
  • the particle has a loading efficiency from
  • the cancer cell lacks surface 4-1BB.
  • the cancer cell is selected from among melanoma, non-small cell lung cancer, small cell lung cancer, gastric cancer, esophageal cancer, GBM, head and neck cancer, pancreatic cancer, colorectal cancer, breast cancer, prostate cancer, bladder cancer, kidney cancer, lymphoma, and leukemia.
  • appropriate doses of the particles and agents depend upon its potency and can optionally be tailored to the particular recipient, for example, through administration of increasing doses until a preselected desired response is achieved. It is understood that the specific dose level for any particular animal subject can depend on a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • an effective amount is an amount sufficient to effect beneficial or desired clinical or biochemical results.
  • An effective amount can be administered one or more times.
  • the effective amount of the particle or agent will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount can include, but are not limited to, the severity of the subject's condition, the disorder being treated.
  • Described herein are methods that result in targeted modification of the surface of cancer cells comprising glycoengineering the cell to express a glycoengineered moiety, which can comprise a residue of an amide of mannosamine, galactosamine, xylosamine, fucosamine, or other sugars and their analogues, and can further comprise an azide moiety, a cyclooctyne moiety, or tetrazine moiety; and covalently linking an antibody, a therapeutic radioisotope, or a small molecule through the glycoengineered moiety, to target delivery to the cancer cell.
  • a glycoengineered moiety which can comprise a residue of an amide of mannosamine, galactosamine, xylosamine, fucosamine, or other sugars and their analogues, and can further comprise an azide moiety, a cyclooctyne moiety, or tetrazine moiety; and covalently linking an antibody, a therapeutic radioisotope
  • Glycoengineering a cell compnses contacting the cell with a compound, such as N-azidoacetylmannosaminetetraacelate, N-azidoacetylmannosamine, acetylated, N- azidoacetylgalactosamine-tetraacylated, or N-azidoacetylglucosamine, acetylated, to prepare a cell having an azide moiety, a cyclooctyne moiety, or tetrazine moiety, or mixtures thereof (referred to in each instance as a glycoengineered moiety) on the cell surface.
  • a compound such as N-azidoacetylmannosaminetetraacelate, N-azidoacetylmannosamine, acetylated, N- azidoacetylgalactosamine-tetraacylated, or N-azidoacetylglucosamine,
  • Covalently linking the moiety on the cell to an antibody, a therapeutic radionuclide, or a small molecule comprises attaching the antibody, therapeutic radionuclide, or a small molecule through the glycoengineered moiety on the cell surface by one of the strategies described herein.
  • nanoparticle-delivered antigen independent immunotherapy as described herein is in the absence of clinically relevant macrophage infiltration and hepatotoxicity associated with the use of an antibody for 4- IBB; or reduces, minimizes or prevents macrophage infiltration and hepatotoxicity associated with the use of an antibody for 4- IBB.
  • the antigen-independent immunotherapy as described herein is in the absence of clinically relevant liver toxicity through nanoparticle delivery of a glycoengineered moiety, wherein the nanoparticle delivery enables degradation of the glycoengineered moiety in lysosomes of macrophages; or reduces, minimizes or prevents liver toxicity through nanoparticle delivery of a glycoengineered moiety, wherein the nanoparticle delivery enables degradation of the glycoengineered moiety in lysosomes of macrophages.
  • SpectraPro® Float-A-Lyzer® G2 dialysis device from REPLIGEN (Waltham, MA).
  • Recombinant Mouse 4-1BB/TNFRSF9 Fc Chimera Protein, Recombinant Mouse 4-1BB Ligand/TNFSF9 Protein, and Enzyme-linked immunosorbent assay (ELISA) kits for mouse TNF-a, IL-6, and IFN-y were purchased from R&D Systems (Minneapolis, MN).
  • HRP horseradish peroxidase
  • 1-step ultra TMB-ELISA substrate solution stop solution for TMB Substrates
  • MaxiSorp flat-bottom plates stop solution for TMB Substrates
  • BCA protein assay BCA protein assay
  • MagniSortTM Mouse F4/80 Positive Selection Kit were from Thermo Fisher Scientific. All other chemicals were obtained from Sigma- Aldrich unless otherwise noted.
  • B16F10 and 4T1 cell lines were acquired from ATCC, where these lines were authenticated using morphology, karyotyping, and polymerase chain reaction (PCR)- based approaches and tested for mycoplasma.
  • B16F10 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Mediatech) and antibiotic-antimycotic (Anti -Anti: 100 U of penicillin, 1 0 pg ml’ 1 of streptomycin and 0.25 pg ml’ 1 of amphotericin B; Gibco).
  • DMEM Dulbecco’s modified Eagle’s medium
  • Mediatech fetal bovine serum
  • Anti -Anti 100 U of penicillin, 1 0 pg ml’ 1 of streptomycin and 0.25 pg ml’ 1 of amphotericin B; Gibco).
  • 4T1 cells were cultured in RPMI Medium 1640 (Gibco) supplemented with 10% fetal bovine serum (Mediatech) and Anti-Anti (1 x). The cell cultures were maintained below 50% confluence and early -passage cultures (between 5 and 8) were utilized for experiments.
  • NPs were prepared by a nanoprecipitation method. First, mPEG-PLGA was dissolved into acetonitrile (ACN) with a final polymer concentration of 10 mg ml’ 1 . Then the Ac4ManNAz was loaded and incorporated in the organic phase along with PLGA (1 :4 weight ratio) with a final polymer concentration of 10 mgml’ 1 . The organic phase was added dropwise into the aqueous phase (endotoxin free FLO) through a syringe under the oil to water ratio of 1:3. The solution was stirred at room temperature under a vacuum until the acetone completely evaporated. The NPs were collected and washed three times with endotoxin free H2O using ultrafiltration (Ami con Ultra Centrifugal Filter Units, 100,000 MWCO).
  • DBCO-functionalized a4-lBB (DBCO-a41BB) was synthesized via a primary amine A-hydroxysuccinimide (NHS) coupling reaction between NHS ester activated DBCO ligand and the primary amines in the antibody.
  • NHS A-hydroxysuccinimide
  • One milligram of a4-lBB in PBS was mixed with 0.14, 0.24, and 0.33 pmol of DBCO-PEGB-NHS (47.79 mM in DMSO) for target a4- 1BB:DBCO molar ratios of 1 :20, 1:35, and 1 :50, respectively.
  • the mixture was diluted to a final antibody concentration of 5 mg ml' 1 and incubated under horizontal shaking at 100 rpm for 2 h at room temperature (in the dark).
  • the DBCO-a41BB solution was then purified via dialysis using a Float-a Lyzer dialysis device (8-10 kD cutoff) against PBS at 4 °C for 3 days
  • the concentration of the purified DBCO-a41BB was determined by BCA protein assay and stored at 4 °C for further studies.
  • NPs were prepared as 1 mg ml 1 suspension in 10 mM NaCl.
  • the size, poly dispersity index (PI) and zeta potential of the produced NPs were measured using a Malvern Zetasizer Nano ZS instrument (Malvern, Inc.).
  • NPs were stained with 2 % uranyl acetate solution and imaged by transmission electron microscopy (TEM) using Thermofisher Talos F200X at an accelerating voltage of 200kV.
  • TEM transmission electron microscopy
  • the Ac4ManNAz loading efficiency, defined as loaded MazNP mass was determined by High Performance Liquid Chromatography (HPLC). NPs with a known mass were dissolved in 0.5 ml of ACN.
  • MWmconj, MW a 4-iBB, and MWoBCO-iinker are the MWs of the final DBCO-a41BB, a4-lBB, and DBCO-PEGB-NHS ester, respectively, and 115 is the MW of the departing NHS after DBCO-antibody coupling.
  • DBCO conjugation affects the binding properties of a4-lBB
  • enzyme-linked immunosorbent assay was performed.
  • Recombinant mouse 4- 1BB/TNFRSF9 Fc chimera proteins were immobilized (2 ug ml’ 1 ) on Maxisorp plates (NUNC Brand Products) overnight at 4 °C.
  • DBCO- functionalized antibodies with different concentrations were added and incubated for 1 h at room temperature.
  • 200 ng ml’ 1 of HRP-conjugated goat anti-rat IgG was then added as the detection antibody, followed by an HRP-sensitive colorimetric substrate.
  • the absorbance of ELISA test results was read at 450 nm.
  • DBCO-a41BB and MazNP Efficacy of DBCO-a41BB and MazNP in improving tumor immunotherapy
  • 75,000 B16F10 cells were suspended in DMEM, mixed with an equal volume of Matrigel (BD Biosciences), and subcutaneously inoculated on the right flank of C57BL/6 mice on day 0.
  • aPD-1 was intraperitoneally (IP) and 100 pg a4-lBB or DBCO-a4-lBB was intravenously (IV) injected into animals on day 8 and 13.
  • IV intravenously
  • a secondary challenge of 200,000 B16F10 cells was inoculated into the left flank and monitored without additional therapy.
  • 100,000 4T1 cells were suspended in RPMI Medium 1640, mixed with an equal volume of Matrigel, and injected on the left fourth mammary fat pad of BALB/c mice (8- week-old female) on day 0. 4T1 tumor-bearing mice were treated with antibodies in the same manner as Bl 6F10 tumor-bearing mice.
  • mice were treated with aPDl plus DBCO-a4-lBB with MazNP with the same procedure. 400 pg per dose of anti-CD8a or anti-NKl.l were injected intraperitoneally (IP) on day 14 (one day after the last treatment).
  • IP intraperitoneally
  • B16F10 tumor-bearing mice were randomized into 4 groups and received IV injections of PBS, AcrManNAz (17.5 mg kg' 1 ) or MazNP (eq. to Ac4ManNAz 17.5 mg kg' 1 ) on day 5, 6, 10, and 11.
  • aPD-1 was intraperitoneally (IP) and 100 pg a4-lBB or DBCO-a4-lBB was intravenously (IV) injected into animals on day 8 and 13.
  • IP intraperitoneally
  • IV intravenously
  • mice On day 18 after tumor inoculation (5 days after the last treatment of antibodies), mice were sacrificed, and tissues were fixed in 10% neutral buffered formalin for 48 h and then transferred to 70% ethanol.
  • Sequential IF staining was carried out on the Bond fully-automated slide staining system (Leica Microsystems Inc., Norwell, MA) using the Bond Research Detection System kit (DS9455, Leica). Five-micrometer sections of each tissue were deparaffinized in Leica Bond Dewax solution (AR9222, Leica), hydrated in Bond Wash solution (AR9590, Leica) and sequentially stained with the antibodies.
  • the samples were treated with Bond-epitope retrieval solution 1 pH 6.0 for 10 min at 1000C followed by blocking in Background Sniper. Then the samples were incubated in CD4 solution (1: 100) for 30 min followed by ImmPRESS goat anti-rat IgG treatment for 20 min. The antibody was stained with Cyanine 3 Tyramide Reagent. In the third round of antibody staining, the samples were treated once again with Bond-epitope retrieval solution 1 pH 6.0 for 10 min at 100 °C and incubated in CD3 antibody (1:400) for 1 h. Then the Novolink Polymer was used for 8 min as the secondary. The antibody was stained with Alexa Fluor 488 tyramide reagent (B40953, Invitrogen). For dual IF staining of liver sections with F4/80 and CDl lb, the slides were pretreated with 1 :140 diluted Enzyme 1 (Leica, AR9551) at 37 °C for 5 min.
  • Nonspecific binding was blocked by incubation in Background Sniper (BS966M, Biocare Medical, Pacheco, CA) at room temperature for 10 min. After pretreatment, the slides were incubated at RT in F4/80 antibody solution (1: 100) for 30 min followed by incubation in ImmPRESS goat anti-rat IgG (Vector Laboratories, Burlingame, CA) for 30 min and then stained with Cyanine 3 Tyramide Reagent (FP1046, Akoya Biosciences, Marlborough, MA). After completion of F4/80 staining, the second round of antigen retrieval was performed in Bond-epitope retrieval solution 1 pH 6.0 (AR9961, Leica) for 30 min at 100 °C followed by blocking in Background Sniper.
  • Bond-epitope retrieval solution 1 pH 6.0 (AR9961, Leica) for 30 min at 100 °C followed by blocking in Background Sniper.
  • the samples were incubated in CD1 lb solution (1 :5000) for 30 min followed by the Novolink Polymer (RE7161, Leica) and stained with Cyanine 5 Tyramide Reagent (FP1117, Akoya Biosciences).
  • the nuclei were counterstained with Hoechst 33258 (Invitrogen, Carlsbad, CA).
  • the slides were mounted with ProLong Gold antifade reagent (P36930, Life Technologies, Carlsbad, CA), and images were taken with a Zeiss LSM 700 laser scanning confocal microscope.
  • B16F10 tumor-bearing mice were treated the same as in immunofluorescence staining. Chromogenic immunohistochemistry (IHC) was performed on paraffin-embedded tissues that were sectioned at 5 pm. The IHC was carried out using the Leica Bond III Autostainer system. Slides were deparaffinized in Bond Dewax solution (Leica Biosystems Newcastle Ltd, United Kingdom) and hydrated in Bond Wash solution (Leica Biosystems Newcastle Ltd.). Heat induced antigen retrieval was performed in Bond-epitope retrieval solution 1 pH 6.0 (Leica Biosystems Newcastle Ltd ), and nonspecific binding was blocked by incubation in Background Sniper.
  • IHC Chromogenic immunohistochemistry
  • mice were incubated with mouse anti-CD8 (14-0808-80, ebioscience) solution at 1 : 1,000 for Ih followed by ImmPRESS goat anti-rat TgG (Vector Laboratories) for 30 min as the secondary.
  • Antibody detection with 3,3'- diaminobenzidine (DAB) was performed using the Bond Intense R detection system (Leica Biosystems Newcastle Ltd.). Stained slides were dehydrated and coverslipped with Cytoseal 60 (8310-4, Thermo Fisher Scientific). Images were taken with an Olympus BX61 microscope. The CD8-stained area in the liver was quantified by the Fiji image analysis software (National Institute of Health, Bethesda, MD) in 3-8 random fields per tissue.
  • mice 16F 10 tumor-beanng mice were treated the same as in immunofluorescence staining. Five days after the last dose of antibodies, mice were euthanized and the livers and spleens were surgically removed, weighted, and fixed in 10% neutral buffered formalin for 48 h and then transferred to 70% ethanol. Then fixed tissues were embedded in paraffin, sectioned (4 pm), and stained with hematoxylin and eosin for histological evaluation.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • Serum levels of cytokines were measured by ELISA in accordance with the manufacturer’s protocol.
  • the isolated F4/80 + cells were seeded in a flat-bottom 48 well plate at a density of 3x 10 4 cells per well with 0.4 ml serum-supplemented medium (DMEM). After 48 h, the F4/80 + cells were washed and incubated in a complete medium containing 10 ug ml’ 1 of a4-lBB in the absence and presence of 10 ug ml’ 1 of recombinant mouse 4-1BB ligand/TNFSF9 protein (4-1BBL) for 24 h at 37 °C. FormazNP and DBCO- a4-lBB group, the cells were incubated with MazNP (eq.
  • Fluorescently labeled/non-PEGylated MazNPs were prepared with PLGA- rhodamine B (50:50, 10 - 30k; AV011, Akina, IN). J774A. 1 mouse macrophages or B16F10 cells were plated in a 35 mm glass-bottomed dish at a density of 30,000 cells per well. After 24 h, the old medium was discarded, and cells were incubated with Ac4ManNAz (50 pM) or MazNP (eq. 50 uM Ac4ManNAz) in the serum-supplemented medium at 37 °C for 6 h.
  • Ac4ManNAz 50 pM
  • MazNP eq. 50 uM Ac4ManNAz
  • Nuclei were counterstained with NucBlue Live ReadyProbes (Hoechst 33342, Invitrogen) according to the manufacturer’s protocol.
  • Cells were washed with PBS, fixed with 4% paraformaldehyde (PF A) (Alfa Aesar, Tewksbury, MA) in PBS for 10 mm at room temperature, and then washed with DPBS (pH 7.4).
  • PF A paraformaldehyde
  • J774A.1 macrophages were pretreated with chloroquine (50 pM) for 1 h, washed twice, and incubated with rhodamine-labeled/non-PEGylated MazNPs for 6 h.
  • Asterisks represent different levels of significance; * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001, ##p ⁇ 0.01, ### p ⁇ 0.005, and #### p ⁇ 0.0001. All of image analyses were performed using the Fiji image analysis software (National Institute of Health, Bethesda, MD).
  • the Ac4ManNAz was encapsulated in methoxy polyethylene glycol)-b-poly(D,L-lactic-co-gly colic) acid (mPEG-PLGA) NPs to facilitate tumor accumulation and uptake of Ac4ManNAz through the enhanced permeation and retention (EPR) effect.
  • EPR enhanced permeation and retention
  • the particle size, PI, and zeta potential of NPs are summarized in the Table 1 below.
  • the PI values of NPs ranged from 0.11 to 0.21, which indicates homogeneous NPs.
  • the average size was 98 ⁇ 8 for naked NP and 119 ⁇ 4 for AcrManNAz-loaded PLGA NP (MazNP) with negative zeta potentials, as measured by dynamic light scattering (DLS) ( Figure 1C; Table 1).
  • the particle sizes measured by DLS were larger than those estimated by TEM (range 50 to 80 nm) ( Figure IB).
  • the thickness of the hydration layer on the NP is less than a nanometer, 1271 suggesting NP aggregation in the medium (10 mM NaCl).
  • the aggregation resolved in 50% serum; 74 nm for naked NP and 81 nm for MazNP (Table 2), which is unlikely to affect in vivo behavior of NPs.
  • the Ac4ManNAz loading efficiency of MazNP was 6.3 ⁇ 0.8%.
  • NPs were suspended in 50% FBS. The size and PI of NPs were measured by the DLS.
  • DBCO-a4-lBB DBCO-functionalized a4-lBB
  • DBCO-a4-lBB DBCO-functionalized a4-lBB
  • DBCO-a4-lBB was synthesized by coupling the NHS-ester modified DBCO ligand and the primary amines on the a4-lBB antibody.
  • the target molar ratios of conjugation of DBCO to a4-lBB were 20: 1, 35: 1, and 50:1.
  • the actual target degrees of functionalization of a4-lBB with DBCO were 8, 16, and 23, respectively, as determined by UV spectroscopy ( Figure ID; Figure 1G).
  • the conjugation was further confirmed using matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy (MALDI-TOF MS).
  • MALDI-TOF MS matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy
  • An increase in the mass of a4-lBB post reaction was observed, indicating the addition of DBCO reactive groups (Figure IF).
  • the degrees of functionalization (DOFs) determined using the MALDI-TOF MS method were higher than those determined by the UV spectroscopic method, because DBCO-PEGB-NHS contains only 90 mol% of DBCO moiety ( Figure 1G).
  • Example 3 Target DOF of 35: 1 or lower allows retention of antibody binding properties [00138]
  • Target DOF of 35: 1 or lower allows retention of antibody binding properties [00138]
  • DBCO conjugation affects binding properties of the antibody
  • the binding ability of murine 4-1BB ligand was evaluated with different concentrations of unmodified or functionalized a4-lBB using ELISA ( Figure IE).
  • Target DOF of 50:1 reduced the binding affinity of DBCO-a4-lBB was observed, indicating that a high degree of antibody modification with DBCO can compromise antibody binding to the DBCO ligand due to steric hindrances caused by the bulky DBCO ligand.
  • DBCO- a4-lBB with the ratio of 35: 1 or lower retained their binding properties relatively well.
  • Example 4 Antigen-independent delivery of a4-lBB improves combination immunotherapy of aPDl plus DBCO-O.4-1BB with MazNP in vivo
  • Example 5 Antitumor efficacy of combination immunotherapy of aPDl plus DBCO-a4-lBB with MazNP evaluated in 4T1 breast cancer model
  • Example 6 Augmentation of CD3 + CD8 + T cell expansion increases antitumor immune response of combination immunotherapy of aPDl plus DBCO-O.4-1BB with MazNP
  • aPDl plus DBC0-a4-lBB with MazNP increases the antitumor immune response was investigated.
  • costimulation of 4-1BB enhances activation and expansion of effective CD8 + tumor infiltrating lymphocytes (TIL) with a memory phenotype
  • TIL tumor infiltrating lymphocytes
  • CD3 + CD8 + /CD3 + T cells The notable difference in proportion of CD3 + CD8 + /CD3 + T cells between the Ac 4 ManNAz and MazNP is attributable to enhanced labeling of tumor cells with the azide group using NP, thereby increasing tumor accumulation of DBCO-a4-lBB.
  • CD3 + CD4 + T cells were less affected by treatments, as there appeared to be a slightly higher percentage in tumors of mice treated with aPDl plus DBCO-a4-lBB with MazNP (1.8 ⁇ 1.0%) than in those of mice treated with PBS (0.4 ⁇ 0.2%), aPDl plus a4-lBB (0.8 ⁇ 1.2%), or aPDl plus DBCO-a4-lBB with Ac 4 ManNAz (0.5 ⁇ 0.5%) (Figure 7B), while there was not much difference among the treatments irrespective of the a4-lBB delivery in the percentage of CD3 + CD4 + T cells in the total CD3 + T cells (Figure 7D).
  • the MazNP primarily augments the expansion of CD3 + CD8 + T cells that can eliminate tumors.
  • CD8 + T cells were depleted by intraperitoneally administering 400 pg per mouse of anti-CD8ato B16F10 tumor-bearing mice treated with aPDl plus DBCO-a4-lBB with MazNP on the day 14 (next day after the last treatment) ( Figure 8) and compared tumor growth.
  • CD8 + T cell depletion abolished tumor regression and eliminated the antitumor effects of aPDl plus DBCO-a4-lBB with MazNP (p ⁇ 0.01 on day 27).
  • NK cell depletion in B16F10 tumor-bearing mice was conducted to determine if the aPDl plus DBCO-a4-lBB with MazNP can expand its efficacy in innate immune cells. Mice were given anti-NKl.l to deplete NK cells on day 14 in the same manner as CD8 + T cell depletion study. NK depletion facilitated tumor development compared to aPDl plus DBCO-a4-lBB with MazNP treatment alone (Figure 4B).
  • Example 7 Antigen-independent delivery of O.4-1BB using MazNB does not cause liver toxicity
  • spleen and livers were observed in both aPDl plus a4-lBB and aPDl plus DBCO-a4-lBB with Ac4ManNAz groups, as indicated by w eights of spleens and livers, whereas not in the aPDl plus DBCO-O.4-1BB with MazNP group.
  • Serum liver enzyme analysis confirmed that alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were substantially elevated by aPDl plus a4-lBB (p ⁇ 0.001 for ALT,/? ⁇ 0.0001 for AST vs.
  • livers Histologic and morphologic analysis of livers revealed aPDl plus DBCO-a4-lBB and aPDl plus DBCO-a4-lBB with Ac4ManNAz increased immune cells, seen as small clusters in the liver parenchyma (arrowhead in Figure 5D) often surrounding portal triads, and in sinusoids (arrow in Figure 5D), supportive of CD8 + T cells expansion in blood circulation.
  • aPDl plus DBCO-a4-lBB with MazNP lessened immune cells, with livers appearing more similar to the PBS control (Figure 5B). This was consistent with findings in IHC analysis (Figure 5C) that the MazNP did not induce infiltration of immune cells in the liver, unlike free Ac4ManNAz.
  • Example 8 MazNP does not affect macrophage expansion in the liver [00146] Nonspecific hepatic CD8 + T cells triggered by O.4-1BB induce macrophage infiltration in the liver, causing pro-inflammatory cytokine production and initiating liver pathology. [5> 29] Having demonstrated that MazNP did not result in accumulation of CD8 + T cells and a4-lBB-associated hepatotoxicity, whether aPDl plus DBCO-a4-lBB with MazNP affects macrophage expansion in the liver was posited.
  • liver tissues were harvested 5 days after the last treatment of PBS, aPDl plus a4-lBB, aPDl plus DBC0-a4-lBB with Ac4ManNAz, or aPDl plus DBC0-a4-lBB with MazNP, and then stained for CDl lb (red) and F4/80 (green), which are markers of macrophages in the liver, known as Kupffer cells.
  • the isolated macrophages were then stimulated by a4-lBB or preincubated with MazNP prior to DBC0-a41BB, in the absence or presence of 4-1BBL. TNF-a released from macrophages was detected as a measure of macrophage stimulation by a4-lBB binding.
  • Anti- 4-1BB significantly induced TNF-a production by 3.2-fold relative to PBS control, whereas a slight increase was observed in MazNP plus DBC0-a4-lBB (1.6-fold) ( Figure 12).
  • the differential effect may be explained by redirecting DBC0-a4-lBB binding to the cell-surface azide groups on macrophages and subsequently prevents a4-lBB-mediated macrophage activation.
  • the a4-lBB treatment causes liver inflammation due to infiltration of monocytes/macrophages through interactions between its Fc domain and Fey receptor (FcyR) expressed on myeloid cells and sinusoidal endothelial cells.
  • Example 9 MazNP did not generate azide groups on the macrophage surfaces [00148] To further understand the lack of labeling of macrophages that take up
  • Kwon BS Weissman SM. cDNA sequences of two inducible T-cell genes. Proc Natl Acad Sci USA 1989, 86(6): 1963-1967.
  • ERK-dependent Bim modulation downstream of the 4-1BB-TRAF1 signaling axis is a critical mediator of CD8 T cell survival in vivo. Jlmmunol 2008, 180(12): 8093-8101. Shuford WW, Klu ssman K, Tritchler DD, Loo DT, Chalupny J, Siadak AW, et al. 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. JExp Med 1997, 186(1): 47-55. Zhu Y, Zhu G, Luo L, Flies AS, Chen L.
  • CD137 stimulation delivers an antigenindependent growth signal for T lymphocytes with memory phenotype.

Abstract

Described herein are engineered nanoparticle formulations of Ac4ManNAz and DBCO-functionalized α4-1BB for antigen-independent delivery of α4-1BB to tumors via biorthogonal click reaction. This approach overcomes the lack of targetable biomarkers/antigens in many cancers. Targeted delivery of α4-1BB not only significantly improved the efficacy of α4-1BB, but also reduced the dose-limiting hepatotoxicity. This antigen-independent delivery approach can be applied broadly for cancer treatment, for both immunotherapy and cytotoxic therapy.

Description

ANTIGEN-INDEPENDENT TARGETED DELIVERY OF THERAPEUTIC AGENTS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/328,873, filed on April 8, 2022.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant No. EB025651 awarded by the National Institute of Health (NIH). The government has certain rights in the invention.
BACKGROUND
[0003] Cancer immunotherapy has improved clinical outcomes for patients with many different types of solid tumors, [1] including melanoma, non-small cell lung cancer, renal cell carcinoma, and urothelial cancers. [2] However, these approaches do not provide long-term antitumor immunity in all patients, for example, some reports show that long-term antitumor immunity occurs in less than 30% of patients with cancer. [3]
[0004] The activation of 4-1BB costimulatory pathways has shown great promise for cancer immunotherapy. Despite encouraging efficacy results in preclinical studies, clinical progress of a4-lBB has been hampered by poor efficacy and severe dose-limiting toxicity, including hepatoxicity and systemic cytokine release syndrome. [18] Subsequent clinical safety' data revealed that a clinical candidate non-ligand-blocking IgG4 antibody caused hepatic polyclonal CD8+ T cell activation and IFN-y secretion in response to 4-1BB activation, limiting its therapeutic window, 1 18 whereas a clinical candidate ligand-blocking IgG2 antibody had a better safety profile but lower antitumor efficacy. [19]
[0005] Cytotoxic agents are also a frontline therapy against cancers. However, owing to their cytotoxicity, undesirable and often serious toxicity is associated with this type of therapy. Targeted delivery of these types of agents to minimize side effects remains an ongoing challenge.
[0006] At present, clinical development of 4-1BB agonists is hampered by off-target liver toxicity, and lack of tumor biomarkers and effector T cells within tumors. Similarly, many useful cytotoxic agents are plagued by off-target and dose-limiting side effects. What is therefore needed is improved and targeted delivery of cancer therapy agents to reduce side effects so that these therapies can reach full potential to better the lives of patients. These shortcomings and others are addressed herein.
BRIEF SUMMARY
[0007] In certain embodiments, the subject matter described herein is directed to a method of delivering a 4- IBB agonist to a cancer cell, comprising: contacting a cancer cell with a particle comprising an azide- or tetrazinecontaining molecule to generate a surface-modified cancer cell; contacting the surface-modified cancer cell with a cyclooctyne-modified 4- 1BB agonist, wherein, the cyclooctyne-modified 4-1BB agonist binds to the surface modified cancer cell. [0008] In certain embodiments, the subject matter described herein is directed to a method of delivering a 4-1BB agonist to a cancer cell, comprising: contacting a cancer cell with a particle comprising a cyclooctyne-containing molecule to generate a surface-modified cancer cell; contacting the surface-modified cancer cell with an azide- or tetrazinemodified 4- IBB agonist, wherein, the azide- or tetrazine-modified 4-1BB agonist binds to the surface modified cancer cell.
[0009] In certain embodiments, the subject matter described herein is directed to a method of delivering a therapeutic radionuclide to a cancer cell, comprising: contacting a cancer cell with a particle comprising an azide- or tetrazinecontaining molecule to prepare a surface-modified cancer cell; contacting the surface modified cancer cell with a cyclooctyne-modified therapeutic radionuclide, wherein, the cyclooctyne-modified therapeutic radionuclide binds to the surface modified cancer cell.
[0010] In certain embodiments, the subject matter described herein is directed to a method of delivering a therapeutic radionuclide to a cancer cell, comprising: contacting a cancer cell with a particle comprising a cyclooctyne-containing molecule to prepare a surface-modified cancer cell; contacting the surface modified cancer cell with an azide- or tetrazinemodified therapeutic radionuclide, wherein, the azide- or tetrazine-modified therapeutic radionuclide binds to the surface modified cancer cell.
[0011] In certain embodiments, the subject matter described herein is directed to a method of treating cancer in a subject by antigen-independent immunotherapy, comprising: administering to the subject a particle comprising an azide- or tetrazinecontaining molecule, wherein a cancer cell in the subject is modified to a surface- modified cancer cell; administering to the subject a cyclooctyne-modified 4-1 BB agonist, wherein the cyclooctyne-modified 4- IBB agonist binds to the surface modified cancer cell, wherein, the cancer is treated.
[0012] In certain embodiments, the subject matter described herein is directed to a method of treating cancer in a subject by antigen-independent immunotherapy, comprising: administering to the subject a particle comprising a cyclooctyne-containing molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; admimstenng to the subject an azide- or tetrazme-modified 4-1BB agonist, wherein the azide- or tetrazine-modified 4-1BB agonist binds to the surface modified cancer cell, wherein, the cancer is treated.
[0013] In certain embodiments, the subject matter described herein is directed to a method of treating cancer in a subject by antigen-independent therapy, comprising: administering to the subject a particle comprising an azide- or tetrazinecontaining molecule, wherein a cancer cell in the subject is modified to a surface- modified cancer cell; admimstenng to the subject a cyclooctyne-modified therapeutic radionuclide, wherein the cyclooctyne-modified therapeutic radionuclide binds to the surface modified cancer cell, wherein, the cancer is treated.
[0014] In certain embodiments, the subject matter described herein is directed to a method of treating cancer in a subject by antigen-independent immunotherapy, comprising: administering to the subject a particle comprising a cyclooctyne-containing molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; administering to the subject an azide- or tetrazine-modified therapeutic radionuclide, wherein the azide- or tetrazine-modified therapeutic radionuclide binds to the surface modified cancer cell, wherein, the cancer is treated.
[0015] In certain embodiments, the subject matter described herein is directed to a pharmaceutical composition comprising: an azide-containing molecule, a tetrazine-containing molecule, a cyclooctyne-modified 4- IBB agonist or a cyclooctyne-modified therapeutic radionuclide, an azide- or tetrazine-modified 4-1 BB agonist or an azide- or tetrazine-modified therapeutic radionuclide; and a pharmaceutically acceptable excipient.
[0016] Additional aspects are also described herein.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) [0017] Figures 1A-1G illustrate the proposed engineering and characterization of antibodies and nanoparticles for anti-gen independent targeting delivery. Figure 1A is a schematic depicting MazNP and DBCO-a4-lBB improved cancer immunotherapy and reduced hepatotoxicity. Figure IB displays Transmission Electron Microscopy (TEM) images of naked or Ac4ManNAz-loaded NP (MazNP), negatively stained with 2% uranyl acetate. Figure 1C displays the size and zeta potential measured by DLS. n = 3 identically and independently prepared samples (mean ± s.d.). Figure ID depicts UV spectra of unmodified a4-lBB and DBCO-functionalized a4-lBB with target molar ratios of conjugation of DBCO to a4-lBB (20, 35, and 50: 1), and Figure IE displays DBCO-a4-lBB binding affinity to 4- 1BB protein, as determined by ELISA, n = 3 identically and independently prepared samples (mean ± s.d.). ****: p < 0.0001 vs. a4-lBB at 200 ng/ml by Sidak’s multiple comparisons test following two-way ANOVA. Figure IF depicts MALDI-TOF mass spectra of unmodified a4-lBB and DBCO-functionalized a4- 1 BB with different molar ratios of DBCO to a4-lBB. Figure 1G displays the plot of the DOF (degree of functionalization) of different DBCO-functionalized a4-lBB determined by UV spectroscopic method and MALDI-TOF MS versus the molar equivalent of DBCO ligand used in the functionalization of a4-lBB (target DOF).
[0018] Figures 2A-2D illustrate the in vivo antitumor efficacy study conducted in Bl 6F 10 tumor-bearing mice. Figure 2A displays the dosing schedule of antibodies and NPs. Figure 2B depicts individual tumor growth curves of B16F10 tumors in C57BL/6 mice treated with PBS, aPDl, aPDl+a4-lBB, aPDl+DBCO-a4-lBB, aPDl+DBCO-a4-!BB+Ac4ManNAz, or aPDl+DBCO-a41BB+MazNP (n = 8-11) Figure 2C demonstrates Kaplan-Meier survival curves of B16F10-tumor bearing mice. MST - median survival time. Figure 2D presents the survival curves of cured animals following tumor rechallenge.
[0019] Figures 3A-3D illustrate the in vivo antitumor efficacy study conducted in orthotopic 4T1 breast tumor-bearing mice. Figure 3A depicts the dosing schedule of treatments. Figure 3B displays individual tumor grow th curves of 4T1 breast tumors in BALB/c mice treated with PBS, aPDl+a4-lBB, aPDl+DBCO-a4-lBB+free Ac4ManNAz or aPDl+DBCO-a4- IBB+MazNP (n = 8 per group). Figure 3C presents average tumor growth curves of animals shown in Figure 3B. Tumor growth over time was compared by Sidak’s multiple comparisons test following two-way ANOVA. ***: p < 0.001 and ****: p < 0.0001. Figure 3D depicts the Kaplan-Meier survival curves of 4T1 tumor-bearing mice. MST - median survival time. P values were calculated by the log-rank test.
[0020] Figures 4A-4D illustrate CD8+ T cell expansion and NK cell activation in tumors stimulated by antigen-independent tumor targeted antibody with nanoparticle. Figure 4A provides representative immunofluorescent images of B16F10 tumor sections. Scale bar in first row = 50 pm; scale bar in second row = 25 pm. Figure 4B depicts individual growth curves of B16F10 tumors in animals treated with aPDl+DBCO-a4-lBB+MazNP with or without CD8+ T cell or NK cell depletion, (n = 8 per group) Figure 4C displays average tumor growth curves for each treatment shown in Figure 4B. **: p < 0.01 by Sidak’s multiple comparisons test following two-way ANOVA. Figure 4D presents the differences in survival as determined for each group by Kaplan-Meier method. MST - median survival time. P values were calculated by the log-rank test. *: p < 0.05 and ***: p < 0.001.
[0021] Figures 5A-5E illustrate that the mD-az/NP did not induce a4-lBB-induced liver toxicity in B16F 10 tumor-bearing mice. Figure 5A depicts the serum levels of ALT and AST measured as units of enzyme liter (U/L) (n = 8 per group). *: p < 0.05; ***: p < 0.001; ****: p < 0.0001 vs. PBS by Dunnett’s multiple compansons test following one-way ANOVA.
Figure 5B displays immunohistochemistry staining for CD8+ on sectioned liver tissues. Scale bars = 50 pm. Figure 5C presents the quantification of CD8+ T cells that infiltrated surface in the liver of mice, shown in Figure 5B (n = 3). % CD8+ T cells area in livers was estimated as the area of CD8+ (brown) in the field of view (outlined by hematoxylin counterstaining). Randomly selected fields (11 images for PBS; 17 images for oPDl+a4-lBB; 20 images for aPDl+DBCO-a4-lBB+Ac4ManNAz; and 21 images for aPDl+DBCO-a4-lBB+MazNP were analyzed with Fiji image analysis software. ****: p < 0.0001 by Tukey’s multiple comparisons test following one-way ANOVA. Figure 5D displays hematoxylin and eosin (H&E) staining of representative tissue slides from livers. Scale bars = 200 pm. Figure 5E depicts the serum levels of TNF-a, IL-6, and IL-27 measured by ELISA (n = 8 per group). ##: p < 0.01; ###: p < 0.005; ####: p < 0.0001; *: p < 0.05; ***: p < 0.001; ****: p < 0.0001 vs. PBS by Tukey’s multiple comparisons test following one-way ANOVA.
[0022] Figures 6A-6D illustrate all images of immunofluorescently stained tumors treated with PBS Figure 6A; aPDl+a4-lBB Figure 6B; aPDl+DBCO-a4-lBB+free Ac4ManNAz Figure 6C, and aPDl+DBCO-a4-lBB+MazNP Figure 6D. Images were collected from three randomly selected fields per slide. Mice (n=3 per group) were given treatment the same as antitumor efficacy study and sacrificed 5 days after the last treatment. Scale bars = 25 pm [0023] Figures 7A-7D illustrate the quantitative analysis of immunofluorescent staining shown (Figure 4A). % Area of Figure 7A CD3+CD8+ or Figure 7B CD3+CD4+ T cells was estimated as the area of CD8 (red) or CD4 (cyan) fluorescence overlapped with CD3 (yellow)-positive area (orange or green, respectively) divided by the area of the tissue in the field of view (outlined by blue Hoechst 33258 staining). % Area of Figure 7C CD3+CD8+ T cells or Figure 7D CD3+CD4+ T cells in total CD3+ T cells was estimated as the area of CD3+CD8+ (green) or CD3+CD4+ (orange) divided by the area of the total CD3+ T cell fluorescence (yellow), respectively. Randomly selected fields (9 images per group) were analyzed with Fiji image analysis software. *: p < 0.05, **: p < 0.01, ***: p < 0.005, and ****: p < 0.0001 vs. MazNP by Dunnett’s multiple comparisons test following one-way ANOVA
[0024] Figure 8 illustrates the dosing schedule of treatments with aPD + DBCO-a4-lBB with MazNP with or without CD8+ T cell or NK cell depletion.
[0025] Figures 9A-9B illustrate the liver and spleen weights, respectively, from Bl 6F 10 tumor-bearing mice (n = 8 per group) treated with PBS, aPDl+a4-lBB, aPDl+DBCO-a4- lBB+Ac4ManNAz, or aPDl+DBCO-a4-lBB+MazNP. **: p < 0.01; ***: p < 0.001; ****: p < 0.0001 by Dunnett’s multiple comparisons test following one-way ANOVA.
[0026] Figures 10A-10D illustrate all images of liver sections stained against CD8 treated with Figure 10A PBS (11 images), Figure 10B aPDl+a4-lBB (17 images), Figure 10C aPDl+DBCO-a4-lBB+Ac4ManNAz (20 images), and Figure 10D aPDl+DBCO-a4- IBB+MazNP (21 images). Images were collected from randomly selected fields in the slide. Mice (n=3 per group) were given treatment the same as antitumor efficacy study and sacrificed 5 days after the last treatment. Scale bars = 50 gm.
[0027] Figures 11A-11D illustrates representative immunofluorescent images of liverresident CDl lb+ (red) and/or F4/80+ cells (green) treated with Figure 11A PBS, Figure 11B aPDl+a4-lBB, Figure 11C aPDl+DBCO-a4-lBB+Ac4ManNAz, and Figure 11D aPDl+DBC0-a4-lBB+MazNP. Scale bars = 50 pm.
[0028] Figure 12 illustrates the quantification of TNF-a release from F4/80+ macrophages stimulated with a4-lBB or DBCO-a4-lBB in the absence and presence of 4- IBB ligand. F4/80+ macrophages were isolated from livers in Bl 6F 10 tumor-bearing mice. The macrophages were stimulated by a4-lBB for 24 h or preincubated with MazNP for 3 days prior to stimulation by DBCO-a4-lBB for additional 24 hr, in the absence and presence of 4- 1BB ligand, n = 3 identically and independently prepared samples (mean ± s.d ). ***: p < 0.001; ****: p < 0.0001 vs. a4-lBB; #: p < 0.05; ###: p < 0.001 by Sidak’s multiple comparisons test following one-way ANOVA.
[0029] Figure 13A-13C illustrates that MazNP did not generate azide groups on macrophage surfaces or stimulate macrophage activation. Azide group generation on the surface of J774A.1 macrophages incubated with PBS, Ac4ManNAz, non-PEGylated MazNP for 6 h in Figure 13A or an additional 18 hr in Figure 13B. The cells were imaged with confocal microscopy (White: Lysotracker; Green: streptavidin-FITC; Red: rhodamine-labeled MazNP; Blue: nuclei stained with Hoechst 33258). Scale bars: 10 pm. Figure 13C illustrates immunofluorescent images of activated liver-resident CD163+ (Green) and/or CD206+ (Red) cells. Scale bars = 50 pm.
[0030] Figure 14 illustrates azide group generation on the B16F10 cell surface after 6 h incubation with PBS, Ac4ManNAz or non-PEGylated MazNP. The cells were imaged with confocal microscopy (Green: streptavidin-FITC; Red: rhodamine-labeled MazNP; Blue: nuclei stained with Hoechst 33342). Scale bars: 10 pm.
[0031] Figure 15 illustrates azide group generation on the J774A.1 macrophage treated with chloroquine prior to 6 h incubation with PBS, Ac4ManNAz or non-PEGylated MazNP. The cells were imaged with confocal microscopy (Green: streptavidin-FITC; Red: rhodamme- labeled MazNP; Blue: nuclei stained with Hoechst 33342). Scale bars: 10 pm.
DETAILED DESCRIPTION
[0032] Described herein are methods for treating cancer that can result in fewer off-target side effects using an antigen-independent approach to target delivery of a therapeutic agent to cancer cells. In certain embodiments, the subject matter described herein is directed to a tumor-targeting approach based on unnatural sugar-mediated metabolic glycoengineering and bio-orthogonal click chemistry to selectively deliver a cancer therapeutic agent, such as a therapeutic radionuclide or a 4-1BB agonist to tumors. In the case of the 4-1BB agonist, in certain embodiments, an anti-4-lBB antibody (a4-lBB) is delivered in an antigen-dependent manner, which eliminates the need for tumor-associated antigens. In certain embodiments, antigen-independent targeted delivery of 4-1BB agonists improves anti-tumor immune responses while reducing hepatotoxicity. Described herein is the inhibition of tumor growth with prolonged survival, accompanied by inducing CD8+ T cells expansion in the tumor, along with significant reduction of CD8+ T cell accumulation in the liver and, thus, hepatotoxicity.
[0033] Tn certain embodiments, described herein are methods of treating cancer comprising administering a metabolic precursor, that is an azide-containing molecule, encapsulated in nanoparticles (NP) that enhance in vivo tumor-targeting of a cyclooctyne-modified therapeutic agent. In certain embodiments, the methods described herein comprise contacting a cell wi th a particle comprising a type of unnatural sugar that can metabolically generate azide functional groups onto glycans on the surface of the tumor cell. In this way, the surface of the tumor cell is selectively decorated. The cell surface azide groups can then be coupled with cyclooctyne-conjugated therapeutic agents via in vivo biorthogonal click chemistry reaction. [34,351
[0034] The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented herein.
Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
I. Overview
[0035] As mentioned above, cancer immunotherapy has improved clinical outcomes for patients with many different types of solid tumors. However, these approaches do not provide long-term antitumor immunity in all patients. The methods described herein are antigen-independent immunotherapies that provide antitumor immune response and, advantageously, do not produce hepatotoxicity.
[0036] 4-1BB (CD137, TNFRSF9) is an inducible costimulatory receptor and expressed on the surface of macrophages/4’ 51 activated T|61 and B cells J4, 71 Tregs/81 dendritic cells (DCs)/91 natural killer (NK) cells/10, 111 and cells of myeloid origin/5, 121 Engagement of 4- 1BB by its ligand or an agonistic antibody increases signaling through the T cell receptor (TCR) and enhances T cell expansion, effector functions, resistance to apoptosis in CD8+ T cells, and cytokine production/13, 141 Preclinical studies have shown that anti-4-lBB agonist (a4-lBB) treatment promotes CD8+ tumor infiltrating lymphocytes (TIL) with a memory phenotype, resulting in retardation of tumor growth and long-term protective immunological response in colon and breast cancer models/14, 151 Moreover, 4-1BB stimulation on NK cells induces interferon-gamma (IFN-y) release1 1 11 and antibody-dependent cellular cytotoxicity (ADCC) on NK cells to kill cancer cells 161 Previous studies have demonstrated synergistic antitumor effects that the combination of a4-lBB (1 mg kg'1) with aPDl (10 mg kg'1) attenuated MC38 colon carcinoma growth over a4-lBB alone when administered twice intraperitoneally 5 days apart.1171 Several approaches have been used to reduce off-tumor toxicities while retaining therapeutic efficacy of a4-lBB, including fragment crystallizable (Fc) region-free tumor-targeted a4-lBB/201 a T cell bispecific (TCB) antibody co-targeting tumor antigens/21, 221 and antibody immobilization onto the nanoparticle (NP) surface/231 Although these methods could prevent toxicities associated with FcyR interactions by modifying the Fc region of the antibody, clinical responses in patients might be limited due to difficulty identifying reliable tumor biomarkers/241 Besides, most patients with cancer do not have sufficient effector T cell infiltration. The lack of effector T cells within the tumors can be a limiting factor in developing a4-lBB-based therapy. Previous studies reported that a4- 1BB treatment showed an ineffective anticancer immune response against poorly immunogenic B16F10 melanoma models compared to more immunogenic MC38 and CT26 models/21, 251
[0037] To further improve antitumor immunity and provide durable response, there is growing interest in combination immunotherapeutic treatments and combining immune checkpoint inhibitors with agonistic monoclonal antibodies (mAb) targeting costimulatory receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF). Again, however, the problem of off-target side effects and hepatotoxicity remains. [0038] The surface of cancer cells can be covalently modified using the particles described herein, wherein the modified cell surface comprises at least one covalently atached azide-, tetrazine-, or cyclooctyne-containing molecule. As used herein, the term “surface modified cancer cell” refers to a cancer cell that comprises at least one covalent modification on the cell surface, whereby the modification is in the form of a metabolic glycoprotein labeling reagent. This labeling reagent is a reactive motif for subsequent covalent binding to a modified 4- IBB agonist, such as an anti -4- IBB antibody, or a modified therapeutic radionuclide through the chemical linking strategies described herein.
[0039] As used herein, a “metabolic glycoprotein labeling reagent” refers to the azide-, tetrazine-, or cyclooctyne-modified unnatural sugars that are metabolically incorporated into cell-surface glycoproteins to generate a biorthogonal click chemistry reagent that is accessible on the surface of the cell.
[0040] Metabolic glycoengineering151 52' and biorthogonal click chemistry^53'55! are available tools. As described herein, these can be used to facilitate unique chemical decoration of cancer cells to enable targeted delivery of a modified 4-1BB agonist and/or modified therapeutic radionuclides onto the targeted cells Thus, in embodiments, the methods described can result in a modified surface of a cancer cell comprising a glycoengineered moiety having the structure: (a transmembrane glycoprotein) — (a residue of an azide- containing molecule) — (a residue of a cyclooctyne) — (an optional linker 1) — (a residue of an antibody, small molecule, or a therapeutic radionuclide), wherein the dash represents a covalent bond; or, a cancer cell comprising a glycoengineered moiety having the structure: (a transmembrane glycoprotein) — (a residue of a cyclooctyne-containing molecule) — (a residue of a azide) — (an optional linker 1) — (a residue of an antibody, small molecule, or a therapeutic radionuclide), wherein the dash represents a covalent bond.
[0041] As used herein, the term “residue” or “residue of’ a chemical moiety refers to a chemical moiety that is bound to a molecule, whereby through the binding, at least one covalent bond has replaced at least one atom of the original chemical moiety, resulting in a residue of the chemical moiety in the molecule.
[0042] As used herein, the term “small molecule” refers to a compound with a low molecular weight (< 900 Daltons) that may regulate biological processes by engaging with biological targets. The binding of the small molecule to the azide, tetrazine or cyclooctyne can accomplished in any suitable way, including conjugation and chelation, using known chemistries. [0043] As used herein, the term “therapeutic radionuclide” refers to the source of therapeutic radiation. The radionuclide can be in the form of a radioisotope or a compound, such as a radiopharmaceutical, comprising the radionuclide. The radioisotope and radiopharmaceutical can be any known radionuclide suitable for medical use. The binding of the radionuclide to the azide, tetrazine or cyclooctyne can accomplished in any suitable way, including conj ugation and chelation, using known chemistries.
[0044] In embodiments, thiol-maleimide click chemistry can be used to modify the surface of a cell. Generally, free thiol groups on the surface can be made to react with maleimide- functionalized biomolecule through stable thioester bond to form stable functionalized cells. Maleimide-functionalized biomolecules can be prepared by amine-NHS reaction between desired biomolecule and NHS-maleimide crosslinker (e.g, sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane- 1 -carboxylate (sulfo-SMCC)).
[0045] In embodiments, the subject matter described herein is directed to a glycoengineered moiety comprising an azide moiety, a cyclooctyne moiety, or a tetrazine moiety.
[0046] In embodiments, the subject matter described herein is directed to the delivery of an azide-, cyclooctyne-, or a tetrazine-contaming molecule that is a metabolic labeling reagent. [0047] In embodiments, the subject matter described herein is directed to the delivery of an azide-, cyclooctyne, or a tetrazine-containing metabolic labeling reagent using a particle. [0048] In certain embodiments, the particle used to for the delivery of an azide-, cyclooctyne, or a tetrazine-containing metabolic labeling reagent is a nanoparticle comprising a polymer. [0049] In certain embodiments, the particle used to for the delivery of an azide-, cyclooctyne, or a tetrazine-containing metabolic labeling reagent is a lipid-based nanoparticle.
[0050] In one embodiment, the nanoparticle is a dendrimer, a liposome, an inorganic nanoparticle, or a polymeric nanoparticle. In one embodiment, the nanoparticle is about 2nm to about lOnm, about lOnm to about lOOnm, or about lOOnm to about lOOOnm. In embodiments, the nanoparticle is about 2nm to about lOOOnm, about 2nm to about 750nm, about 2nm to about 500nm, about 2nm to about 250nm, about 2nm to about 200nm, about 2nm to about lOOnm, or 2nm to about 50nm. In embodiments, the nanoparticle is about lOnm to about lOOOnm, about 25nm to about lOOOnm, about 50nm to about lOOOnm, about lOOnm to about lOOOnm, about 200 to about lOOOnm, about 500nm to about lOOOnm, or 750nm to about lOOOnm. In embodiments, the nanoparticle is about 2nm, about 5nm, about lOnm, about 50nm, about lOOnm, about 200nm, about 300nm, about 400nm, about 500nm, about 600nm, about 700nm, about 800nm, about 900nm, or about lOOOnm. In one embodiment, the dendrimer is a multivalent dendrimer. In one embodiment, the multivalent dendrimer is a polyamidoamine dendrimer. In embodiments, the nanoparticle is a pegylated nanoparticle (e.g., DBCO-functionalized PEG-PLGA nanoparticle). In embodiments, the pegylated nanoparticle is less than 200nm in diameter.
[0051] In embodiments, the at least one covalently attached antibody, therapeutic radionuclide, and/or small molecule is attached through a glycoengineered moiety. In embodiments, the covalent attachment is via conjugating to thiol groups on cells.
[0052] In embodiments, the glycoengineered moiety comprises a residue of an amide of mannosamine, galactosamine, xylosamine, fucosamine, or other sugars and their analogues. In embodiments, the glycoengineered moiety further comprises a residue of an azide, a cyclooctyne, or a tetrazine covalently attached to the residue of an amide of mannosamine, galactosamine, xylosamine, fucosamine, or other sugars and their analogues. In embodiments, the cyclooctyne is DBCO.
[0053] As used herein, “analogues” refer to compounds having a core structure of the parent compound but differing from it through chemical modifications that add functional moieties. [0054] As used herein “degree of functionalization” refers to number of residues of azide, tetrazine or cyclooctyne that are covalently attached to the antibody, small molecule, or therapeutic radionuclide. A degree of functionalization value refers to the number of residues per antibody, small molecule, or therapeutic radionuclide
[0055] “Immune checkpoint” as used herein refers to a molecule on the cell surface of a CD4 and CD8 T cell that down-modulates or inhibits an anti-tumor immune response. Immune checkpoint molecules include, but are not limited to, Programmed Death 1 (PD1), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), PDL-1 (B7H1), PDL-2 (B7-DC), B7H3, B7H4, OX-40, CD137, CD40, CD27, LAG3, TIM3, ICOS, or BTLA, which directly inhibit immune cells. Immunotherapeutic agents which can act as immune checkpoint inhibitors useful in the methods of the present invention include, but are not limited to, anti-PDl; anti -CTLA-4; anti- PDL-1; anti-B7-Hl; anti-PDL-2; anti-B7-H3; anti-B7-H4; anti-CD137; anti-CD40; anti- CD27; anti-LAG3; anti-TIM3; anti-ICOS, and anti-BTLA.
[0056] As used herein, the term “physiological conditions” refers to the range of conditions of temperature, pH, and tonicity (or osmolality) normally encountered within tissues in the body of a living human.
[0057] The term “zri vitro" refers to artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube).
[0058] The term “zri vzvo” refers to natural environments (e.g., a cell or organism or body) and to processes or reactions that occur within a natural environment. [0059] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.
[0060] Unless otherwise apparent from the context, the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value or variations ± 0.5%, 1%, 5%, or 10% from a specified value.
[0061] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients.
[0062] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an antigen” or “at least one antigen” can include a plurality of antigens, including mixtures thereof.
[0063] Statistically significant means p <0.05.
IL Pharmaceutical compositions
[0064] In one embodiment, described herein is a pharmaceutical composition comprising a functionalized antibody, therapeutic radionuclide, or an immune checkpoint molecule as described herein, and a pharmaceutically acceptable excipient.
[0065] A “pharmaceutically acceptable excipient” refers to a vehicle for containing a functionalized cell or an acellular extracellular matrix that can be introduced into a subject without significant adverse effects and without having deleterious effects on the functionalized cell or acellular extracellular matrix. That is, “pharmaceutically acceptable” refers to any formulation which is safe and provides the appropriate delivery for the desired route of administration of an effective amount of at least one functionalized cell or acellular extracellular matnx for use in the methods disclosed herein. Pharmaceutically acceptable carriers or vehicles or excipients are well known. Descriptions of suitable pharmaceutically acceptable carriers, and factors involved in their selection, are found in a variety of readily available sources such as, for example, Remington ’s Pharmaceutical Sciences, 18th ed., 1990, herein incorporated by reference in its entirety for all purposes. Such carriers can be suitable for any route of administration (e.g., parenteral, enteral (e.g., oral), or topical application). Such pharmaceutical compositions can be buffered, for example, wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the functionalized cell or acellular extracellular matrix and route of administration. [0066] Suitable pharmaceutically acceptable carriers include, for example, sterile water, salt solutions such as saline, glucose, buffered solutions such as phosphate buffered solutions or bicarbonate buffered solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates (e.g., lactose, amylose or starch), magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, and the like. Pharmaceutical compositions or vaccines may also include auxiliary agents including, for example, diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffering agents, viscosity enhancing additives, lubricants, salts for influencing osmotic pressure, buffers, vitamins, coloring, flavoring, aromatic substances, and the like which do not deleteriously react with a functionalized cell or an acellular extracellular matrix.
[0067] For liquid formulations, for example, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Non-aqueous solvents include, for example, propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcohohc/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils include those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. Solid carriers/diluents include, for example, a gum, a starch (e.g., com starch, pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, or dextrose), a cellulosic matenal (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
[0068] Optionally, sustained or directed release pharmaceutical compositions or vaccines can be formulated. This can be accomplished, for example, through use of liposomes or compositions wherein the active compound is protected with differentially degradable coatings (e.g., by microencapsulation, multiple coatings, and so forth). Such compositions may be formulated for immediate or slow release. It is also possible to freeze-dry the compositions and use the lyophilisates obtained (e.g., for the preparation of products for injection).
III. Therapeutic methods
[0069] In embodiments, the subject matter described herein is directed to a method of delivering a 4- IBB agonist to a cancer cell comprising contacting a cancer cell with a particle comprising an azide- or tetrazine-containing molecule to prepare a surface-modified cancer cell, and further contacting the surface modified cancer cell with a cyclooctyne-modified 4- 1BB agonist, wherein, the cyclooctyne-modified 4-1BB agonist binds to the surface modified cancer cell. In certain embodiments, the further contacting is after a period of time sufficient for formation of the surface modification on target cancer cells.
[0070] In certain embodiments, the azide- or tetrazine-containing molecule is an azide- or tetrazine-containing metabolic glycoprotein labeling reagent.
[0071] In embodiments, the azide- or tetrazine-containing metabolic glycoprotein labeling reagent is selected from among sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
[0072] In certain embodiments, the azide-containing metabolic glycoprotein labeling reagent is N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz).
[0073] In embodiments, the metabolic glycoprotein labeling reagent contains a cyclooctyne moiety or another click chemistry reactive group including but not limited to terminal alkynes, heterocyclic electrophiles, and reactive ketones, and the like.
[0074] In certain embodiments, the metabolic glycoprotein label reagent is DBCO (dibenzocyclooctyne).
[0075] In certain embodiments, the azide-containing metabolic glycoprotein labeling reagent is a bifunctional molecule, wherein it contains one or more additional reactive groups used to conjugate to other molecules on the cell surface.
[0076] In embodiments, the additional reactive groups are selected from among maleimides, thiols, amines, hydroxides, imidazoles, NHS esters, and other bioreactive functional groups. [0077] In embodiments, the 4-1BB agonist of the cyclooctyne-modified 4-1BB agonist is an antibody or small molecule.
[0078] In certain embodiments, the 4-1BB agonist of the cyclooctyne-modified 4-1BB agonist is an anti-4-lBB antibody.
[0079] In embodiments described herein, the anti-4-lBB antibody is functionalized with one or more residues of a cyclooctyne moiety.
[0080] In certain embodiments, the degree of functionalization of the antibody is 35 or less. [0081] In certain embodiments, the cyclooctyne moiety is a dibenzocyclooctyne (DBCO). [0082] In embodiments, the subject matter described herein is directed to a method of delivering a 4- IBB agonist to a cancer cell comprising contacting a cancer cell with a particle comprising a cy cl ooctyne-con taming molecule to prepare a surface-modified cancer cell, and further contacting the surface modified cancer cell with an azide-or tetrazine-modified 4-1BB agonist, wherein, the azide- or tetrazine-modified 4-1BB agonist binds to the surface modified cancer cell.
[0083] In certain embodiments, the cyclooctyne-containing molecule is an azide- or tetrazine-containing metabolic glycoprotein labeling reagent.
[0084] In certain embodiments, the azide containing molecule is a bifunctional molecule where it contains another reactive group that can be conjugated to molecules on the cell surface, including but not limited to maleimide, thiol, and amine.
[0085] In embodiments, the cyclooctyne-containing metabolic glycoprotein labeling reagent is selected from among sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
[0086] In certain embodiments, the cyclooctyne-containing metabolic glycoprotein labeling reagent is an unnatural sugar functionalized with dibenzocyclooctyne (DBCO).
[0087] In embodiments, the 4-1BB agonist of the azide- or tetrazine-modified 4-1BB agonist is an antibody or small molecule.
[0088] In certain embodiments, the 4-1BB agonist of the azide- or tetrazine-modified 4-1BB agonist is an anti-4-lBB antibody.
[0089] In certain embodiments, a therapeutic agent can be modified as described herein for click chemistry conjugation to a surface-modified cancer cell, wherein the therapeutic agent is anti-OX-40, IL-2 or IL-12.
[0090] In embodiments described herein, the anti-4-lBB antibody is functionalized with one or more residues of an azide or tetrazine moiety.
[0091] In certain embodiments, the degree of functionalization of the antibody is 35 or less. [0092] In embodiments, the subject matter described herein is directed to a method of delivering a therapeutic radionuclide to a cancer cell comprising contacting a cancer cell with a particle comprising an azide- or tetrazine-contammg molecule to prepare a surface- modified cancer cell, and further contacting the surface modified cancer cell with a cyclooctyne-modi fied therapeutic radionuclide, wherein, the cyclooctyne-modified therapeutic radionuclide binds to the surface modified cancer cell.
[0093] In embodiments, the subject matter described herein is directed to a method of delivering a therapeutic radionuclide to a cancer cell comprising contacting a cancer cell with a particle comprising an cyclooctyne-containing molecule to prepare a surface-modified cancer cell, and further contacting the surface modified cancer cell with a azide- or tetrazine- modified therapeutic radionuclide, wherein, the azide- or tetrazine-modified therapeutic radionuclide binds to the surface modified cancer cell. [0094] In embodiments, the therapeutic radionuclide of the cyclooctyne-modified therapeutic radionuclide is a beta-emitter.
[0095] In certain embodiments, the therapeutic radionuclide of the cyclooctyne-modified therapeutic radionuclide is an alpha-emitter.
[0096] In embodiments, the therapeutic radionuclide of the azide- or tetrazine-modified therapeutic radionuclide is a beta-emitter.
[0097] In certain embodiments, the therapeutic radionuclide of the azide- or tetrazine- modified therapeutic radionuclide is an alpha-emitter.
[0098] In embodiments, the subject matter described herein is directed to a method of treating cancer in subject by antigen-independent immunotherapy comprising administering to the subject a particle comprising an azide- or tetrazine-containing molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; and subsequently, administering to the subject a cyclooctyne-modified 4-1BB agonist, wherein the cyclooctyne- modified 4- IBB agonist binds to the surface modified cancer cell, wherein, the cancer is treated.
[0099] In embodiments, the subject matter described herein is directed to a method of treating cancer in subject by antigen-independent immunotherapy comprising administering to the subject a particle comprising an cyclooctyne-containing molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; and subsequently, administering to the subject an azide or tetrazine-modified 4-1BB agonist, wherein the azide- or tetrazine-modified 4- IBB agonist binds to the surface modified cancer cell, wherein, the cancer is treated.
[00100] In embodiments, the subject matter described herein is directed to a method of treating cancer in subject by antigen-independent therapy comprising administering to the subject a particle comprising an azide- or tetrazine-contammg molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; and subsequently, administering to the subject a cyclooctyne-modified therapeutic radionuclide, wherein the cyclooctyne-modified therapeutic radionuclide binds to the surface modified cancer cell, wherein, the cancer is treated.
[00101] In embodiments, the subject matter described herein is directed to a method of treating cancer in subject by antigen-independent therapy comprising administering to the subject a particle comprising an cyclooctyne-containing molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; and subsequently, administering to the subject an azide or tetrazine-modified therapeutic radionuclide, wherein the azide- or tetrazine-modified radionuclide binds to the surface modified cancer cell, wherein, the cancer is treated.
[00102] In certain embodiments, the particle encapsulating the metabolic glycoprotein labeling reagent is a nanoparticle comprising a polymer.
[00103] In certain embodiments, the polymer used for the nanoparticle is selected from among the group consisting of mPEG-PLA, PLA, PLGA, and dextran.
[00104] In certain embodiments, the nanoparticle can be a lipid-based nanoparticle.
[00105] In embodiments disclosed herein, the particle has a loading efficiency from
0. 1 % to about 20%.
[00106] In embodiments described herein, the cancer cell lacks surface 4-1BB.
[00107] In certain embodiments, the cancer cell is selected from among melanoma, non-small cell lung cancer, small cell lung cancer, gastric cancer, esophageal cancer, GBM, head and neck cancer, pancreatic cancer, colorectal cancer, breast cancer, prostate cancer, bladder cancer, kidney cancer, lymphoma, and leukemia.
[00108] It is understood that appropriate doses of the particles and agents depend upon its potency and can optionally be tailored to the particular recipient, for example, through administration of increasing doses until a preselected desired response is achieved. It is understood that the specific dose level for any particular animal subject can depend on a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
[00109] As used herein, "effective amount" is an amount sufficient to effect beneficial or desired clinical or biochemical results. An effective amount can be administered one or more times.
[00110] The effective amount of the particle or agent will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount can include, but are not limited to, the severity of the subject's condition, the disorder being treated.
[00111] Described herein are methods that result in targeted modification of the surface of cancer cells comprising glycoengineering the cell to express a glycoengineered moiety, which can comprise a residue of an amide of mannosamine, galactosamine, xylosamine, fucosamine, or other sugars and their analogues, and can further comprise an azide moiety, a cyclooctyne moiety, or tetrazine moiety; and covalently linking an antibody, a therapeutic radioisotope, or a small molecule through the glycoengineered moiety, to target delivery to the cancer cell.
[00112] Glycoengineering a cell compnses contacting the cell with a compound, such as N-azidoacetylmannosaminetetraacelate, N-azidoacetylmannosamine, acetylated, N- azidoacetylgalactosamine-tetraacylated, or N-azidoacetylglucosamine, acetylated, to prepare a cell having an azide moiety, a cyclooctyne moiety, or tetrazine moiety, or mixtures thereof (referred to in each instance as a glycoengineered moiety) on the cell surface.
[001 13] Covalently linking the moiety on the cell to an antibody, a therapeutic radionuclide, or a small molecule comprises attaching the antibody, therapeutic radionuclide, or a small molecule through the glycoengineered moiety on the cell surface by one of the strategies described herein.
[00114] In some embodiments, nanoparticle-delivered antigen independent immunotherapy as described herein is in the absence of clinically relevant macrophage infiltration and hepatotoxicity associated with the use of an antibody for 4- IBB; or reduces, minimizes or prevents macrophage infiltration and hepatotoxicity associated with the use of an antibody for 4- IBB.
[00115] In some embodiments, the antigen-independent immunotherapy as described herein is in the absence of clinically relevant liver toxicity through nanoparticle delivery of a glycoengineered moiety, wherein the nanoparticle delivery enables degradation of the glycoengineered moiety in lysosomes of macrophages; or reduces, minimizes or prevents liver toxicity through nanoparticle delivery of a glycoengineered moiety, wherein the nanoparticle delivery enables degradation of the glycoengineered moiety in lysosomes of macrophages.
[00116] The disclosed subject matter is further described in the following non-limiting Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only.
EXAMPLES
Materials
[00117] mPEG-PLGA (AK102; LA:GA = 50:50 (w:w);Mw: =5000:30,000 Da) was obtained from Akina Inc. (West Lafayette, IN). N-azidoacetylmannosamine-tretraacylated (Ac4ManNAz) and DBCO-PEGB-NHS ester (Mw = 1046.2 g mol 1) were purchased from BroadPharm (San Diego, CA). Anti-PD-1 (clone: RMP1-14), anti-4-lBB (CD137) (clone: 3H3), anti-CD8a (clone: 2.43), anti-NKl. l (clone: PK136) were purchased from BioXcell (West Lebanon, NH). SpectraPro® Float-A-Lyzer® G2 dialysis device from REPLIGEN (Waltham, MA). Recombinant Mouse 4-1BB/TNFRSF9 Fc Chimera Protein, Recombinant Mouse 4-1BB Ligand/TNFSF9 Protein, and Enzyme-linked immunosorbent assay (ELISA) kits for mouse TNF-a, IL-6, and IFN-y were purchased from R&D Systems (Minneapolis, MN). Goat anti-rat IgG (H+L) secondary antibody horseradish peroxidase (HRP), 1-step ultra TMB-ELISA substrate solution, stop solution for TMB Substrates, MaxiSorp flat-bottom plates (NUNC brand products), BCA protein assay, and MagniSort™ Mouse F4/80 Positive Selection Kit were from Thermo Fisher Scientific. All other chemicals were obtained from Sigma- Aldrich unless otherwise noted.
Cell lines
[00118] The B16F10 and 4T1 cell lines were acquired from ATCC, where these lines were authenticated using morphology, karyotyping, and polymerase chain reaction (PCR)- based approaches and tested for mycoplasma. B16F10 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Mediatech) and antibiotic-antimycotic (Anti -Anti: 100 U of penicillin, 1 0 pg ml’1 of streptomycin and 0.25 pg ml’1 of amphotericin B; Gibco). 4T1 cells were cultured in RPMI Medium 1640 (Gibco) supplemented with 10% fetal bovine serum (Mediatech) and Anti-Anti (1 x). The cell cultures were maintained below 50% confluence and early -passage cultures (between 5 and 8) were utilized for experiments.
Preparation of Ac4ManNAz loaded NP
[00119] NPs were prepared by a nanoprecipitation method. First, mPEG-PLGA was dissolved into acetonitrile (ACN) with a final polymer concentration of 10 mg ml’1. Then the Ac4ManNAz was loaded and incorporated in the organic phase along with PLGA (1 :4 weight ratio) with a final polymer concentration of 10 mgml’1. The organic phase was added dropwise into the aqueous phase (endotoxin free FLO) through a syringe under the oil to water ratio of 1:3. The solution was stirred at room temperature under a vacuum until the acetone completely evaporated. The NPs were collected and washed three times with endotoxin free H2O using ultrafiltration (Ami con Ultra Centrifugal Filter Units, 100,000 MWCO).
Preparation of DBCO-functionalized a4-lBB [00120] DBCO-functionalized a4-lBB (DBCO-a41BB) was synthesized via a primary amine A-hydroxysuccinimide (NHS) coupling reaction between NHS ester activated DBCO ligand and the primary amines in the antibody. One milligram of a4-lBB in PBS was mixed with 0.14, 0.24, and 0.33 pmol of DBCO-PEGB-NHS (47.79 mM in DMSO) for target a4- 1BB:DBCO molar ratios of 1 :20, 1:35, and 1 :50, respectively. The mixture was diluted to a final antibody concentration of 5 mg ml'1 and incubated under horizontal shaking at 100 rpm for 2 h at room temperature (in the dark). The DBCO-a41BB solution was then purified via dialysis using a Float-a Lyzer dialysis device (8-10 kD cutoff) against PBS at 4 °C for 3 days The concentration of the purified DBCO-a41BB was determined by BCA protein assay and stored at 4 °C for further studies.
Characterization of MazNP
[00121] NPs were prepared as 1 mg ml 1 suspension in 10 mM NaCl. The size, poly dispersity index (PI) and zeta potential of the produced NPs were measured using a Malvern Zetasizer Nano ZS instrument (Malvern, Inc.). NPs were stained with 2 % uranyl acetate solution and imaged by transmission electron microscopy (TEM) using Thermofisher Talos F200X at an accelerating voltage of 200kV. The Ac4ManNAz loading efficiency, defined as loaded MazNP mass, was determined by High Performance Liquid Chromatography (HPLC). NPs with a known mass were dissolved in 0.5 ml of ACN. After 30 min, polymers were precipitated with the addition of 0.5 ml of DI water, centrifuged, and the supernatant was filtered with 0.45 mm syringe filter. The samples were analyzed via a Shimadzu SPD-M20A HPLC, equipped with C8 column. A linear gradient of 20% to 50% mobile phase B (ACN with 0.1% TFA) for 30 min was performed at a flow' rate of 0.5 ml min'1. Sample injection volume was 50 pL and UV absorbance was monitored at 280 nm.
Characterization of DBCO-functionalized a4-lBB
[00122] The degrees of functionalization (DOF, the number of DBCO conjugated to a4-lBB) of different DBCO-functionalized a4-lBB were determined via UV-vis absorption spectroscopy method. [34] Briefly, the concentrations and degrees of the DBCO incorporation of different DBCO-conjugated a4-lBB were determined spectroscopically and calculated using the molar extinction coefficient of DBCO at 310 nm = 12,000 M'1 cm'1, the correction factor of DBCO at 280 = 1.089, the extinction coefficient of a4-lBB at 280 nm = 1.33, and the molar extinction coefficient of a4-lBB at 280 nm = 210,000 M'1 cm'1 according to the manufacturer’s instructions. The DOF of different DBCO-functionalized a4-lBB were further confirmed with AB Sciex 5800 matrix-assisted laser desorption ionization-time-of- flight mass spectrometer (MALDI-TOF MS) at the UNC Michael Hooker Proteomics Center as previously published.1351 Briefly, antibody samples were dialyzed against deionized water at 4 °C for 36 h (6 cycles) using 8-10 kDa cutoff Float- A-Lyzer G2 dialysis devices. Desalted samples (1 mg ml’1) were loaded on a Teflon-coated plate with sinapinic acid as the matrix after five-fold dilution with 50% methanol containing 0.1% FA. Samples were laser- irradiated with 500 shots, and the molecular weight was determined by following standard
MALDI-TOF MS method using peptide calibration mixture 4700 (AB Sciex) and BSA as the standards. Using the MALDI molecular weights (MWs) of DBCO-a41BB, the number of DBCO conjugated to a4-lBB was estimated through the following equation.1361
[00123] DBCO linker
Figure imgf000023_0001
[00124] Where MWmconj, MWa4-iBB, and MWoBCO-iinker are the MWs of the final DBCO-a41BB, a4-lBB, and DBCO-PEGB-NHS ester, respectively, and 115 is the MW of the departing NHS after DBCO-antibody coupling.
[00125] To examine if DBCO conjugation affects the binding properties of a4-lBB, enzyme-linked immunosorbent assay (ELISA) was performed. [20] Recombinant mouse 4- 1BB/TNFRSF9 Fc chimera proteins were immobilized (2 ug ml’1) on Maxisorp plates (NUNC Brand Products) overnight at 4 °C. After washing and blocking, DBCO- functionalized antibodies with different concentrations were added and incubated for 1 h at room temperature. After washing, 200 ng ml’1 of HRP-conjugated goat anti-rat IgG was then added as the detection antibody, followed by an HRP-sensitive colorimetric substrate. The absorbance of ELISA test results was read at 450 nm.
Animal Study:
[00126] For all animal studies, eight-week-old female C57BL/6 mice (The Jackson Laboratory) were used. All animal work according to the protocol (protocol 19-001.0) was approved and monitored by the University of North Carolina Animal Care and Use Committee.
Efficacy of DBCO-a41BB and MazNP in improving tumor immunotherapy [00127] In the melanoma tumor model, 75,000 B16F10 cells were suspended in DMEM, mixed with an equal volume of Matrigel (BD Biosciences), and subcutaneously inoculated on the right flank of C57BL/6 mice on day 0. B16F10 tumor-bearing mice were randomized into 6 groups (n = 8 per group) and treated with intravenous (IV) inj ections of PBS, Ac4ManNAz (17.5 mg kg 1), or MazNP (eq. to Ac4ManNAz 17.5 mg kg 1) on day 5, 6, 10, and 11. 200 pg aPD-1 was intraperitoneally (IP) and 100 pg a4-lBB or DBCO-a4-lBB was intravenously (IV) injected into animals on day 8 and 13. For the surviving mice, at two months after primary inoculation, a secondary challenge of 200,000 B16F10 cells was inoculated into the left flank and monitored without additional therapy. In the breast tumor model, 100,000 4T1 cells were suspended in RPMI Medium 1640, mixed with an equal volume of Matrigel, and injected on the left fourth mammary fat pad of BALB/c mice (8- week-old female) on day 0. 4T1 tumor-bearing mice were treated with antibodies in the same manner as Bl 6F10 tumor-bearing mice. Tumor growth was monitored daily until the end point was reached. The length (L) and width (W) of each tumor were measured by a digital caliper, and the volume (V) was calculated by the modified ellipsoid formula: V = (L x W2)/2.[37] In the depletion study, mice were treated with aPDl plus DBCO-a4-lBB with MazNP with the same procedure. 400 pg per dose of anti-CD8a or anti-NKl.l were injected intraperitoneally (IP) on day 14 (one day after the last treatment). [38]
Immunofluorescence staining
[00128] To examine CD8+ T cells in tumors, either PBS, aPDl plus a4-lBB, aPDl plus DBCO-a4-lBB with AcrManNAz, or aPDl plus DBCO-a4-lBB with MazNP were injected to Bl 6F 10 tumor-bearing mice, and then tumors were harvested 5 days after receiving the last treatment (18 days after tumor inoculation) for fluorescent immunohistochemistry (IF). Briefly, seventy-five thousand B16F10 cells were suspended in DMEM, mixed with an equal volume of Matrigel, and subcutaneously inoculated on the right flank of C57BL/6 mice on day 0. B16F10 tumor-bearing mice were randomized into 4 groups and received IV injections of PBS, AcrManNAz (17.5 mg kg'1) or MazNP (eq. to Ac4ManNAz 17.5 mg kg'1) on day 5, 6, 10, and 11. Two hundred micrograms aPD-1 was intraperitoneally (IP) and 100 pg a4-lBB or DBCO-a4-lBB was intravenously (IV) injected into animals on day 8 and 13. On day 18 after tumor inoculation (5 days after the last treatment of antibodies), mice were sacrificed, and tissues were fixed in 10% neutral buffered formalin for 48 h and then transferred to 70% ethanol. Sequential IF staining was carried out on the Bond fully-automated slide staining system (Leica Microsystems Inc., Norwell, MA) using the Bond Research Detection System kit (DS9455, Leica). Five-micrometer sections of each tissue were deparaffinized in Leica Bond Dewax solution (AR9222, Leica), hydrated in Bond Wash solution (AR9590, Leica) and sequentially stained with the antibodies. For triple IF staining of tumor sections with CD3 (85061S, Cell Signaling, Danvers, MA), CD4 (14- 9766-82, Invitrogen, Eugene, OR), and CD8a (14-0808-80, Invitrogen), heat induced antigen retrieval was performed at 100 °C in Bond-epitope retrieval solution 1 pH 6.0 for 20 min. Nonspecific binding was blocked by incubation in Background Sniper at RT for 10 min. After pretreatment the slides were incubated at RT in CD8a antibody solution (1 :400) for 1 h followed by incubation in ImmPRESS goat anti -rat IgG for 30 min and stained with Cyanine 5 Tyramide Reagent. The next step of staining was performed with CD4. The samples were treated with Bond-epitope retrieval solution 1 pH 6.0 for 10 min at 1000C followed by blocking in Background Sniper. Then the samples were incubated in CD4 solution (1: 100) for 30 min followed by ImmPRESS goat anti-rat IgG treatment for 20 min. The antibody was stained with Cyanine 3 Tyramide Reagent. In the third round of antibody staining, the samples were treated once again with Bond-epitope retrieval solution 1 pH 6.0 for 10 min at 100 °C and incubated in CD3 antibody (1:400) for 1 h. Then the Novolink Polymer was used for 8 min as the secondary. The antibody was stained with Alexa Fluor 488 tyramide reagent (B40953, Invitrogen). For dual IF staining of liver sections with F4/80 and CDl lb, the slides were pretreated with 1 :140 diluted Enzyme 1 (Leica, AR9551) at 37 °C for 5 min.
Nonspecific binding was blocked by incubation in Background Sniper (BS966M, Biocare Medical, Pacheco, CA) at room temperature for 10 min. After pretreatment, the slides were incubated at RT in F4/80 antibody solution (1: 100) for 30 min followed by incubation in ImmPRESS goat anti-rat IgG (Vector Laboratories, Burlingame, CA) for 30 min and then stained with Cyanine 3 Tyramide Reagent (FP1046, Akoya Biosciences, Marlborough, MA). After completion of F4/80 staining, the second round of antigen retrieval was performed in Bond-epitope retrieval solution 1 pH 6.0 (AR9961, Leica) for 30 min at 100 °C followed by blocking in Background Sniper. Then the samples were incubated in CD1 lb solution (1 :5000) for 30 min followed by the Novolink Polymer (RE7161, Leica) and stained with Cyanine 5 Tyramide Reagent (FP1117, Akoya Biosciences). In each IF staining, the nuclei were counterstained with Hoechst 33258 (Invitrogen, Carlsbad, CA). The slides were mounted with ProLong Gold antifade reagent (P36930, Life Technologies, Carlsbad, CA), and images were taken with a Zeiss LSM 700 laser scanning confocal microscope.
Quantitative analysis of immunofluorescent staining was carried out with the Fiji image analysis software (National Institute of Health, Bethesda, MD) in 3-8 random fields per tissue.
Immunohistochemical staining
[00129] B16F10 tumor-bearing mice were treated the same as in immunofluorescence staining. Chromogenic immunohistochemistry (IHC) was performed on paraffin-embedded tissues that were sectioned at 5 pm. The IHC was carried out using the Leica Bond III Autostainer system. Slides were deparaffinized in Bond Dewax solution (Leica Biosystems Newcastle Ltd, United Kingdom) and hydrated in Bond Wash solution (Leica Biosystems Newcastle Ltd.). Heat induced antigen retrieval was performed in Bond-epitope retrieval solution 1 pH 6.0 (Leica Biosystems Newcastle Ltd ), and nonspecific binding was blocked by incubation in Background Sniper. After pretreatment, slides were incubated with mouse anti-CD8 (14-0808-80, ebioscience) solution at 1 : 1,000 for Ih followed by ImmPRESS goat anti-rat TgG (Vector Laboratories) for 30 min as the secondary. Antibody detection with 3,3'- diaminobenzidine (DAB) was performed using the Bond Intense R detection system (Leica Biosystems Newcastle Ltd.). Stained slides were dehydrated and coverslipped with Cytoseal 60 (8310-4, Thermo Fisher Scientific). Images were taken with an Olympus BX61 microscope. The CD8-stained area in the liver was quantified by the Fiji image analysis software (National Institute of Health, Bethesda, MD) in 3-8 random fields per tissue.
Toxicity studies
[00130] B 16F 10 tumor-beanng mice were treated the same as in immunofluorescence staining. Five days after the last dose of antibodies, mice were euthanized and the livers and spleens were surgically removed, weighted, and fixed in 10% neutral buffered formalin for 48 h and then transferred to 70% ethanol. Then fixed tissues were embedded in paraffin, sectioned (4 pm), and stained with hematoxylin and eosin for histological evaluation. Blood was collected via cardiac puncture, allowed to clot in a silica-coated tube, and submitted to Pathology Services Core at the University of North Carolina-Chapel Hill for analysis of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in serum.
Serum levels of cytokines (TNF-a, IL-6, and IFN-y) were measured by ELISA in accordance with the manufacturer’s protocol.
In vitro TNF-a release assay
[00131] Seventy-five thousand B16F10 cells were suspended in DMEM, mixed with an equal volume of Matrigel, and subcutaneously inoculated on the right flank of C57BL/6 mice on day 0. On day 18, livers were collected from the Bl 6F 10 tumor-bearing mice, crushed, and passed through a 70 pm cell strainer. Liver immune cells were isolated after cell fractionation on a 35% Percoll layer, and red blood cells were lysed with the ammonium- chloride-potassium (ACK) buffer.1391 Liver F4/80 positive cells were isolated by immunomagnetic selection method via the MagniSort Mouse F4/80 positive selection kit, following the manufacturer’s protocol. The isolated F4/80+ cells were seeded in a flat-bottom 48 well plate at a density of 3x 104 cells per well with 0.4 ml serum-supplemented medium (DMEM). After 48 h, the F4/80+ cells were washed and incubated in a complete medium containing 10 ug ml’1 of a4-lBB in the absence and presence of 10 ug ml’1 of recombinant mouse 4-1BB ligand/TNFSF9 protein (4-1BBL) for 24 h at 37 °C. For MazNP and DBCO- a4-lBB group, the cells were incubated with MazNP (eq. to 20 pM of Ac4ManNAz) for 3 days prior to 24 h incubation with 4-1BBL. The other steps were kept the same. After 24 h incubation, supernatants were collected and tested for secreted TNF-a using ELISA in accordance with the manufacturer’s protocol.
In vitro macrophage labeling of AciManNAz
[00132] Fluorescently labeled/non-PEGylated MazNPs were prepared with PLGA- rhodamine B (50:50, 10 - 30k; AV011, Akina, IN). J774A. 1 mouse macrophages or B16F10 cells were plated in a 35 mm glass-bottomed dish at a density of 30,000 cells per well. After 24 h, the old medium was discarded, and cells were incubated with Ac4ManNAz (50 pM) or MazNP (eq. 50 uM Ac4ManNAz) in the serum-supplemented medium at 37 °C for 6 h. After 6 h, cells were gently washed or incubated for an additional 18 h in the fresh serum- supplemented medium. After washing with PBS, cells were incubated with DBCO-PEG4- biotin (100 pM) in the serum-free medium for 1 h at 37 °C. The cells were gently rinsed again and incubated with 5 pl of FITC-streptavidin (1:200 dilution) in the serum-free medium for 40 min at 37 °C. For MazNP tracking, cells were rinsed and treated with 50 nM LysoTracker Deep Red (Invitrogen) at 37 °C for 25 min. Nuclei were counterstained with NucBlue Live ReadyProbes (Hoechst 33342, Invitrogen) according to the manufacturer’s protocol. Cells were washed with PBS, fixed with 4% paraformaldehyde (PF A) (Alfa Aesar, Tewksbury, MA) in PBS for 10 mm at room temperature, and then washed with DPBS (pH 7.4). To inhibit lysosomal activity, J774A.1 macrophages were pretreated with chloroquine (50 pM) for 1 h, washed twice, and incubated with rhodamine-labeled/non-PEGylated MazNPs for 6 h. After washing with PBS, cells were treated with DBCO-PEG4-biotin (100 pM) for 1 h, followed by 5 pl of FITC-streptavidin for 40 min at 37 °C. Cells were imaged with a Zeiss LSM 900 Spectral Confocal Laser Scanning Microscope in the Microscopy Services Laboratory Core Facility at the UNC School of Medicine.
Statistical analysis
[00133] All statistical analyses were performed with GraphPad Prism 9 (La Jolla, CA). The specifics of the statistical tests and number of replicates are stated in the figure legends. No collected experimental data was excluded for the quantitative analysis. All data are presented as mean ± s.d. One-way ANOVA with Tukey’s or Dunnett’s multiple comparisons test, Two-way ANOVA with Sidak’s multiple comparisons test were used to determine statistical significance. For the analysis of Kaplan-Meier survival curves, a Log-rank (Mantel-Cox) test was used. Asterisks represent different levels of significance; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ##p < 0.01, ### p < 0.005, and #### p < 0.0001. All of image analyses were performed using the Fiji image analysis software (National Institute of Health, Bethesda, MD).
Example 1: Functionalization ofMazNP
[00134] As a small-molecule sugar, the Ac4ManNAz was encapsulated in methoxy polyethylene glycol)-b-poly(D,L-lactic-co-gly colic) acid (mPEG-PLGA) NPs to facilitate tumor accumulation and uptake of Ac4ManNAz through the enhanced permeation and retention (EPR) effect. The particle size, PI, and zeta potential of NPs are summarized in the Table 1 below. The PI values of NPs ranged from 0.11 to 0.21, which indicates homogeneous NPs. The average size was 98 ± 8 for naked NP and 119 ± 4 for AcrManNAz-loaded PLGA NP (MazNP) with negative zeta potentials, as measured by dynamic light scattering (DLS) (Figure 1C; Table 1). The particle sizes measured by DLS were larger than those estimated by TEM (range 50 to 80 nm) (Figure IB). Typically, the thickness of the hydration layer on the NP is less than a nanometer, 1271 suggesting NP aggregation in the medium (10 mM NaCl). The aggregation resolved in 50% serum; 74 nm for naked NP and 81 nm for MazNP (Table 2), which is unlikely to affect in vivo behavior of NPs. The Ac4ManNAz loading efficiency of MazNP was 6.3 ± 0.8%.
[00135] Table 1. Size, zeta potential and Ac4ManNAz-loading efficiency ofMazNP.
Figure imgf000028_0001
a Poly dispersity index (PI), an estimate of the width of the particle size distribution, obtained from the cumulant analysis as described in the International Standard on DLS ISO 13321: 1996 and ISO 22412:2008 (Malvern DLS technical note MRK176401). PI < 0.1 is considered monodisperse, and >0.7 is very' broad. [1] b Zeta potential measured in 10 mM NaCl. n = 6 identically and independently prepared samples (mean ± s.d.). [00136] Table 2. Size of NPs in 50% FBS.
Figure imgf000029_0001
NPs were suspended in 50% FBS. The size and PI of NPs were measured by the DLS.
Example 2: Functionalization of a4-lBB with DBCO
[00137] To enable tumor-targeted delivery of a4-lBB, DBCO-functionalized a4-lBB (DBCO-a4-lBB) were formulated, which can conjugate to the azide reactive groups present on tumor cells via a bioorthogonal click reaction between the azide group and DBCO. DBCO-a4-lBB was synthesized by coupling the NHS-ester modified DBCO ligand and the primary amines on the a4-lBB antibody. The target molar ratios of conjugation of DBCO to a4-lBB were 20: 1, 35: 1, and 50:1. The actual target degrees of functionalization of a4-lBB with DBCO were 8, 16, and 23, respectively, as determined by UV spectroscopy (Figure ID; Figure 1G). The conjugation was further confirmed using matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy (MALDI-TOF MS). An increase in the mass of a4-lBB post reaction was observed, indicating the addition of DBCO reactive groups (Figure IF). The degrees of functionalization (DOFs) determined using the MALDI-TOF MS method were higher than those determined by the UV spectroscopic method, because DBCO-PEGB-NHS contains only 90 mol% of DBCO moiety (Figure 1G).
Example 3: Target DOF of 35: 1 or lower allows retention of antibody binding properties [00138] To determine whether DBCO conjugation affects binding properties of the antibody, the binding ability of murine 4-1BB ligand was evaluated with different concentrations of unmodified or functionalized a4-lBB using ELISA (Figure IE). At target DOF of 50:1 reduced the binding affinity of DBCO-a4-lBB was observed, indicating that a high degree of antibody modification with DBCO can compromise antibody binding to the DBCO ligand due to steric hindrances caused by the bulky DBCO ligand. In contrast, DBCO- a4-lBB with the ratio of 35: 1 or lower retained their binding properties relatively well. To take advantage of DBCO conjugation to the antibody without compromising binding, the target DOF of 35: 1 was selected for further studies. Example 4: Antigen-independent delivery of a4-lBB improves combination immunotherapy of aPDl plus DBCO-O.4-1BB with MazNP in vivo
[00139] The therapeutic effect of aPDl and DBCO-a4-lBB antibodies plus MazNP was evaluated in the C57BL/6 mice bearing subcutaneous B16F10 tumors. As controls, animals were given aPDl, a4-lBB or DBCO-a4-lBB along with aPDl which prevents T cell exhaustion (Figure 2A). To determine the effectiveness of NP in enhancing labeling of tumor cells with the azide group, animals given free Ac4ManNAz prior to DBCO-a4-lBB plus aPDl were also compared. The treatment of aPDl plus DBCO-a4-l BB with MazNP significantly attenuated the growth of Bl 6F 10 tumors (Figure 2B). The greater therapeutic efficacy also translated into an increased median survival as compared to aPDl plus a4-lBB (MST = 40 days,/? = 0.0071), aPDl plus DBCO-a4-lBB (MST = 36.5 days,/? = 0.0071). Interestingly, the MazNP extended the mouse survival as compared to free Ac.iManN Az (MST = 53 days,/? = 0.0697 vs aPDl plus DBCO-a4-lBB with MazNP) (Figure 2C; Table 3), likely due to the high accumulation and sustained intratumoral release of Ac4ManNAz from the NP. This is partly because circulating NPs can retain their cargo and extravasate at tumors via hyperpermeable vasculature surrounding tumors but not at normal tissues, whereas free Ac4ManNAz does not. |2S| These results suggest that the antigen-independent tumor targeting improved antitumor efficacy of a4-lBB, attributable to NP-mediated delivery of Ac4ManNAz to the tumors. In addition, the MazNP treatment cured 36.4% of tumors in mice, and 2/4 of cured mice survived without any further treatment after tumor rechallenge (Figure 2D), suggesting that the antigen-independent targeting using NP has the potential to produce persistence of antitumoral immunity.
[00140] Table 3. Statistics comparing the Kaplan-Meier survival curves of B16F10 bearing mice.
Figure imgf000030_0001
Figure imgf000031_0001
P values between groups were calculated by the log-rank test.
Example 5: Antitumor efficacy of combination immunotherapy of aPDl plus DBCO-a4-lBB with MazNP evaluated in 4T1 breast cancer model
[00141] To validate the in vivo results, the antitumor efficacy of the aPDl plus DBCO- a4-lBB with MazNP was evaluated in an orthotopic 4T1 breast cancer model. 4T1 cancer cells were injected into mouse mammary fat pads and treatments were administered in the same manner as B16F10 tumor-bearing mice (Figure 3A). The treatment of aPDl plus DBCO-a4-lBB with MazNP significantly delayed tumor growth compared to aPDl plus a4- 1BB or aPDl plus DBCO-a4-lBB with free AcrManNAz (Figures 3B, 3C), whereas there was not much difference between aPDl plus a4-lBB and aPDl plus DBCO-a4-lBB with free Ac4ManNAz. Survival data analyzed using the log-rank test showed that the median survival of aPDl plus DBCO-a4-lBB with MazNP (MST = 48 days) was increased by approximately 71%, compared to the PBS (MST = 28 days), and 26% to aPDl plus a4-lBB (MST = 38 days) or aPDl plus DBCO-a4-lBB with free AcrManNAz (MST = 38 days) (Figure 3d), indicating significant survival benefit of the aPDl plus DBCO-a4-lBB with MazNP treatment. These results, taken together with its improved therapeutic efficacy in B16F10 tumor-bearing mice, consistently support that the MazNP was able to increase duration of AcrManNAz action and facilitate targeted delivery to the tumor, resulting in a greater antitumor efficacy in companson with free AcrManN Az.
Example 6: Augmentation of CD3+CD8+ T cell expansion increases antitumor immune response of combination immunotherapy of aPDl plus DBCO-O.4-1BB with MazNP [00142] How aPDl plus DBC0-a4-lBB with MazNP increases the antitumor immune response was investigated. Given that costimulation of 4-1BB enhances activation and expansion of effective CD8+ tumor infiltrating lymphocytes (TIL) with a memory phenotype,114, 15] whether aPDl plus DBCO-a4-lBB with MazNP would increase frequency of CD8+ T cells in the tumors was investigated. Confocal microscopy showed highest fluorescent signals of T lymphocytes in the tumors from mice treated with aPDl plus DBCO- a4-lBB with MazNP across all treatment groups (Figure 4A; Figures 6A-6D). The immunofluorescence quantitative analysis found that the percentage of CD3+CD8+ T cells was three to four times higher in tumors from mice treated with aPDl plus DBCO-a4-lBB and MazNP (18.5 ± 10.3% of tissue area in the field of view) than in tumors from mice treated with PBS, aPDl plus a4-lBB, or aPDl plus DBCO-a4-lBB with AciManNAz (1.8 ± 1.4%, 5.7 ± 5.3%, or 3.9 ± 2.4%, respectively) (Figure 7A). In contrast, neither aPDl plus a4-lBB nor aPDl plus DBCO-a4-lBB with Ac4ManNAz induced tumor infiltration of CD3+CD8+ T cells when compared with the PBS control group. Subsequently, aPDl plus DBCO-a4-lBB with MazNP treatment increased the percentage of CD3+CD8+ T cells in the total CD3+ T cells (82.2% ± 8.3%) as compared to the PBS control (48.3 ± 46.2%), aPDl plus a4-lBB (56.2 ± 29.9%), or aPDl plus DBCO-a4-lBB with Ac4ManNAz (44.9± 20.7%) groups (Figure 7C). The notable difference in proportion of CD3+CD8+/CD3+ T cells between the Ac4ManNAz and MazNP is attributable to enhanced labeling of tumor cells with the azide group using NP, thereby increasing tumor accumulation of DBCO-a4-lBB. On the other hand, CD3+CD4+ T cells were less affected by treatments, as there appeared to be a slightly higher percentage in tumors of mice treated with aPDl plus DBCO-a4-lBB with MazNP (1.8 ± 1.0%) than in those of mice treated with PBS (0.4 ± 0.2%), aPDl plus a4-lBB (0.8 ± 1.2%), or aPDl plus DBCO-a4-lBB with Ac4ManNAz (0.5 ± 0.5%) (Figure 7B), while there was not much difference among the treatments irrespective of the a4-lBB delivery in the percentage of CD3+CD4+ T cells in the total CD3+ T cells (Figure 7D). These data suggest that the MazNP primarily augments the expansion of CD3+CD8+ T cells that can eliminate tumors.
[00143] To confirm that increased CD8+ T cells are responsible for the robust antitumor efficacy of aPDl plus DBCO-O.4-1BB with MazNP, CD8+ T cells were depleted by intraperitoneally administering 400 pg per mouse of anti-CD8ato B16F10 tumor-bearing mice treated with aPDl plus DBCO-a4-lBB with MazNP on the day 14 (next day after the last treatment) (Figure 8) and compared tumor growth. CD8+ T cell depletion abolished tumor regression and eliminated the antitumor effects of aPDl plus DBCO-a4-lBB with MazNP (p < 0.01 on day 27). The median survival of aPDl plus DBCO-a4-lBB with MazNP with CD8+ T cell depletion (MST = 27 days) was significantly shorter than aPDl plus DBCO-a4-lBB with MazNP alone (MST = undefined, p < 0.0005 vs. CD8+ T cell depletion), nearly similar to PBS-treated animals (MST = 25 days) (Figure 4D), supporting that a4- IBB -induced CD8+ T cell responses are essential for the antitumor activity of aPDl plus DBCO-a4-lBB with MazNP. Overall, the MazNP increases abundance of CD8+ T cell in tumors, which enables a robust and durable antitumor immune response as shown in in vivo tumor rechallenge without additional treatment (Figure 2D). NK cell depletion in B16F10 tumor-bearing mice was conducted to determine if the aPDl plus DBCO-a4-lBB with MazNP can expand its efficacy in innate immune cells. Mice were given anti-NKl.l to deplete NK cells on day 14 in the same manner as CD8+ T cell depletion study. NK depletion facilitated tumor development compared to aPDl plus DBCO-a4-lBB with MazNP treatment alone (Figure 4B). One out of 8 mice survived in the group of NK depletion through the end of the study (50 days), whereas the group of aPDl plus DBCO-a4-lBB with MazNP alone had 12.5% survival rate (Figure 4D), showing in part NK-dependent antitumor activities of aPDl plus DBCO-a4-lBB with MazNP. Given that our experiments showed the involvement of NK and CD8+ T cells in the antitumor efficacy of aPDl plus DBCO-a4-lBB with MazNP, the antigen-independent tumor targeting method can be inferred to have the potential to eradicate tumors by modulating both innate and adaptive immunity.
Example 7: Antigen-independent delivery of O.4-1BB using MazNB does not cause liver toxicity
[00144] Next, whether the antigen-independent delivery of a4-lBB using MazNP could reduce off-target toxi cities of a4-lBB in livers was determined. B16F10 tumor-bearing mice were treated with PBS, aPDl plus a4-lBB, aPDl plus DBCO-a4-lBB with AciManNAz, or aPDl plus DBCO-a4-lBB with MazNP, and hepatotoxicity of treatments were compared on day 18 (10 days after the initiation of antibody treatment). As shown in the Figure 9, enlargement of spleen and livers were observed in both aPDl plus a4-lBB and aPDl plus DBCO-a4-lBB with Ac4ManNAz groups, as indicated by w eights of spleens and livers, whereas not in the aPDl plus DBCO-O.4-1BB with MazNP group. Serum liver enzyme analysis confirmed that alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were substantially elevated by aPDl plus a4-lBB (p < 0.001 for ALT,/? < 0.0001 for AST vs. PBS) or aPDl plus DBCO-a4-lBB with Ac4ManNAz (p < 0.001 for ALT,/? < 0.05 for AST vs. PBS) treatments, as compared to PBS control group; aPDl plus DBCO-a4-lBB with Ac4ManNAz), while the aPDl plus DBC0-a4-lBB with MazNP -treated mice had normal serum ALT and AST levels except for one (Figure 5A). This indicates that the aPDl plus DBCO-a4-lBB with MazNP did not cause liver damage.
[00145] To examine a4-lBB-induced infiltration of CD8+ T cell in the liver, immunohistochemistry (IHC) was carried out to quantify liver-infiltrating CD8+ T cells (Figure 5B; Figure 10). Notably, a massive CD8+ T cell infiltration in the liver was observed in both aPDl plus a4-lBB (29.8 ± 18.2%) and aPDl plus DBCO-a4-lBB with Ac4ManNAz (22. 1 ± 10.9), statistically significantly higher than PBS control (0.9 ± 0.6%), while the percentage of CD8+ T cells in aPDl plus DBCO-a4-lBB with MazNP (5.4 ± 5.8%) was five times lower (Figure 5C). Histologic and morphologic analysis of livers revealed aPDl plus DBCO-a4-lBB and aPDl plus DBCO-a4-lBB with Ac4ManNAz increased immune cells, seen as small clusters in the liver parenchyma (arrowhead in Figure 5D) often surrounding portal triads, and in sinusoids (arrow in Figure 5D), supportive of CD8+ T cells expansion in blood circulation. By contrast, aPDl plus DBCO-a4-lBB with MazNP lessened immune cells, with livers appearing more similar to the PBS control (Figure 5B). This was consistent with findings in IHC analysis (Figure 5C) that the MazNP did not induce infiltration of immune cells in the liver, unlike free Ac4ManNAz. Since engagement of 4- IBB by its agonistic antibody leads to pro-inflammatory cytokine production, serum cytokine levels were examined to evaluate systemic toxicity and pro-inflammatory effects of the tumor targeting using MazNP in response to a4-lBB-induced activation of immune cells such as T cells and macrophages. The aPDl plus DBCO-a4-lBB with MazNP showed marginal alteration in serum levels of TNF-a, IL-6, and IFN-y, as compared to the PBS control (Figure 5E), whereas the levels of all pro-inflammatory cytokines or TNF-a and IFN-y significantly increased in aPDl plus a4-lBB or aPDl plus DBCO-a4-lBB with Ac4ManNAz, respectively. These results indicated that the antigen-independent targeted delivery using MazNP could prevent potential systemic toxicities of a4-lBB by reducing infiltration of immune cells in the liver.
Example 8: MazNP does not affect macrophage expansion in the liver [00146] Nonspecific hepatic CD8+ T cells triggered by O.4-1BB induce macrophage infiltration in the liver, causing pro-inflammatory cytokine production and initiating liver pathology. [5> 29] Having demonstrated that MazNP did not result in accumulation of CD8+ T cells and a4-lBB-associated hepatotoxicity, whether aPDl plus DBCO-a4-lBB with MazNP affects macrophage expansion in the liver was posited. To visualize hepatic macrophages in B16F10-tumor bearing mice, liver tissues were harvested 5 days after the last treatment of PBS, aPDl plus a4-lBB, aPDl plus DBC0-a4-lBB with Ac4ManNAz, or aPDl plus DBC0-a4-lBB with MazNP, and then stained for CDl lb (red) and F4/80 (green), which are markers of macrophages in the liver, known as Kupffer cells. With IF, we were able to locate clear CD1 lb+F4/80‘ monocyte-derived macrophages (a transient inflammatory stage from blood monocyte to tissue macrophage1301) or CD1 lb'F4/80+ macrophages in the liver, likely due to a low frequency of CD1 lb+F4/80+ macrophages. [31, 32] Confocal microscopy showed strong fluorescent signals of CD1 lb+F4/80‘ and CD1 lb'F4/80+ cells in the liver treated with aPDl plus DBC0-a4-lBB with Ac4ManNAz, comparable to aPDl plus a4-lBB (Figures 11A-11D), whereas aPDl plus a4-lBB with MazNP had much lower signal, similar to the PBS control. It is noteworthy that MazNP did not lead to a4-lBB-induced macrophage expansion in the liver, unlike Ac4ManNAz, despite a higher macrophage uptake rate of NP than small molecules. To understand the mechanism by which MazNP bypasses macrophage activation in response to a4-lBB, the production of pro-inflammatory cytokine, TNF-a, from hepatic macrophages with a4-lBB stimulation was evaluated. F4/80+ macrophages expressing 4-1BB were isolated from livers of B16F10 tumor-bearing mice, which could include their subsets of CD68+ with phagocytic and cytokine-producing activity.15, 32J The 4- 1BB ligand (4-1BBL) was used to block a4-lBB or DBC0-a4-lBB binding to 4-1BB on F4/80+ cells. The isolated macrophages were then stimulated by a4-lBB or preincubated with MazNP prior to DBC0-a41BB, in the absence or presence of 4-1BBL. TNF-a released from macrophages was detected as a measure of macrophage stimulation by a4-lBB binding. Anti- 4-1BB significantly induced TNF-a production by 3.2-fold relative to PBS control, whereas a slight increase was observed in MazNP plus DBC0-a4-lBB (1.6-fold) (Figure 12). Coincubation with 4-1BBL decreased TNF-a levels in the a4-lBB group by 66% (2.1-fold relative to PBS control) and MazNP plus DBCO-a4-lBB by 53% (0.9-fold), indicating that a4-lBB triggered induction of TNF-a production to a greater extent than MazNP plus DBC0-a4-lBB irrespective of the presence of 4-1BBL. Given that earlier experiments showed less effects of DBC0-a4-lBB on macrophage expansion in the liver and pro- inflammatory cytokine production than a4-lBB, the differential effect may be explained by redirecting DBC0-a4-lBB binding to the cell-surface azide groups on macrophages and subsequently prevents a4-lBB-mediated macrophage activation. By contrast, the a4-lBB treatment causes liver inflammation due to infiltration of monocytes/macrophages through interactions between its Fc domain and Fey receptor (FcyR) expressed on myeloid cells and sinusoidal endothelial cells. [22> 331 A contribution of the bulky DBCO ligand covering Fc region of a4-lBB and directly blocking interactions with FcyR needs to be further studied. [00147] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which the inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Example 9: MazNP did not generate azide groups on the macrophage surfaces [00148] To further understand the lack of labeling of macrophages that take up
MazNP, the in vitro cell-labeling capability of MazNP was investigated and the macrophagelabeling of MazNP to free Ac4ManNAz was compared. J774A.1 macrophages were incubated with free Ac4ManNAz or rhodamme-labeled/non-PEGylated MazNP for 6 h, followed by DBCO-PEG4-biotin that reacts with azide groups potentially expressed on the macrophage surface via click chemistry. Subsequently, DBCO-PEG4-biotin was detected by fluorescently-labeled streptavidin (streptavidin-FITC). Confocal microscopy showed strong fluorescent intensity of streptavidin in the free AcrManNAz group, showing azide groups on the macrophage surface, whereas MazNP did not (Figure 13A). Additionally, the celllabeling activity of MazNP in Bl 6F 10 was analyzed (Figure 14). The B16F10 cells showed FITC fluorescence intensity in both free Ac4ManNAz and MazNP groups. This differential cell labeling behavior could be a result of different cellular processes of MazNP, as macrophages are responsible for degrading foreign bodies (including nanomatenals) by lysosomal activities.56'58 To further investigate MazNP trafficking and accumulation in lysosomes, the localization of MazNP in lysosomes was examined using Lysotracker at different time points. It was observed that MazNP overlapped with Lysotracker signals at 6 h incubation and further showed negligible fluorescence intensity of MazNP without cellsurface azide groups after an additional 18 h incubation, unlike free Ac4ManNAz that retained FITC fluorescence signals (Figure 13B). The results not only demonstrated MazNP degradation in lysosomes, but also indicated that Ac4ManNAz failed to label macrophages following MazNP uptake by macrophages. These findings were further corroborated by azide groups generated on the surfaces of macrophages with pre-treatment of chloroquine that can inhibit lysosomal activity (Figure 15). The J774A.1 macrophages were pretreated with chloroquine, incubated with free Ac4ManNAz or rhodamine-labeled/non-PEGylated MazNP, followed by DBCO-PEG4-biotin and streptavidm-FITC. The J774A.1 macrophages treated with MazNP showed fluorescence intensities of MazNP in the cells and streptavidin-FITC on the cell surfaces, indicating cell-surface azide groups after MazNP uptake by macrophages. Free Ac4ManNAz showed macrophage surface labeling with azide groups, irrespective of the presence of chloroquine. These experiments demonstrated lysosomal degradation of Ac4ManNAz and MazNP by macrophages.
[00149] To investigate whether MazNP can prevent a4-lBB-induced macrophage activation, immunofluorescent staining was performed for macrophage activation markers, CD163 and CD206 (Figure 13C). 59, 60 While no significant difference in fluorescence signals of CD163+ was found among groups, a notable increase of activated CD206+ macrophage accumulation in aPDl plus a4-lBB and aPDl plus DBCO-a4-lBB with Ac4ManNAz groups was observed, indicating liver injury with potential chronic inflammation. 45-47 These findings provide evidence that that MazNP could reduce a4-lBB- mduced macrophage infiltration and hepatotoxicity.
References
1. Segal NH, He AR, Doi T, Levy R, Bhatia S, Pishvaian MJ, et al. Phase I Study of Single- Agent Utomilumab (PF-05082566), a 4-1BB/CD137 Agonist, in Patients with Advanced Cancer. Clin Cancer Res 2018, 24(8): 1816-1823.
2. Segal NH, Logan TF, Hodi FS, McDermott D, Melero I, Hamid O, et al. Results from an Integrated Safety Analysis of Urelumab, an Agonist Anti-CD137 Monoclonal Antibody. Clinical Cancer Research 2017, 23(8): 1929.
3. Bedard PL, Hansen AR, Ratain MJ, Siu LL. Tumour heterogeneity in the clinic. Nature 2013, 501(7467): 355-364.
4. Dagogo-Jack I, Shaw AT. Tumour heterogeneity and resistance to cancer therapies. Nat Rev Clin Oncol 2018, 15(2): 81-94.
5. You G, Lee Y, Kang YW, Park HW, Park K, Kim H, et al. B7-H3x4-1BB bispecific antibody augments antitumor immunity by enhancing terminally differentiated CD8(+) tumor-infiltrating lymphocytes. Sci Adv 2021, 7(3). 6. Kim JA, Averbook BJ, Chambers K, Rothchild K, Kjaergaard J, Papay R, et al. Divergent effects of 4- IBB antibodies on antitumor immunity and on tumor- reactive T-cell generation. Cancer Res 2001, 61(5): 2031-2037.
7. Wilcox RA, Flies DB, Zhu G, Johnson AJ, Tamada K, Chapoval Al, et al. Provision of antigen and CD 137 signaling breaks immunological ignorance, promoting regression of poorly immunogenic tumors. J Clin Invest 2002, 109(5): 651-659.
8. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010, 363(8): 711-723.
9. Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy. Science 2013, 342(6165): 1432-1433.
10. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-Ll antibody in patients with advanced cancer. N Engl J Med 2012, 366(26): 2455-2465.
11. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012, 366(26): 2443-2454.
12. Twomey JD, Zhang B. Cancer Immunotherapy Update: FDA- Approved Checkpoint Inhibitors and Companion Diagnostics. Aapsj 2021, 23(2): 39.
13. Topalian SL, Taube JM, Anders RA, Pardoll DM. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer 2016, 16(5): 275-287.
14. Pollok KE, Kim YJ, Hurtado J, Zhou Z, Kim KK, Kwon BS. 4-1BB T-cell antigen binds to mature B cells and macrophages, and costimulates anti-mu- primed splenic B cells. Eur J. Immunol 1994, 24(2): 367-374.
15. Bartkowiak T, Jaiswal AR, Ager CR, Chin R, Chen CH, Budhani P, et al. Activation of 4-1BB on Liver Myeloid Cells Triggers Hepatitis via an Interleukin- 27-Dependent Pathway. Clin Cancer Res 2018, 24(5): 1138-1151.
16. Kwon BS, Weissman SM. cDNA sequences of two inducible T-cell genes. Proc Natl Acad Sci USA 1989, 86(6): 1963-1967.
17. Zhang X, Voskens CJ, Sallin M, Maniar A, Montes CL, Zhang Y, et al. CD137 promotes proliferation and survival of human B cells. Jlmmunol 2010, 184(2): 787-795. So T, Lee SW, Croft M. Immune regulation and control of regulatory T cells by 0X40 and 4-1BB. Cytokine Growth Factor Rev 2008, 19(3-4): 253-262. Wilcox RA, Chapoval Al, Gorski KS, Otsuji M, Shin T, Flies DB, et al. Cutting edge: Expression of functional CD137 receptor by dendritic cells. Jlmmunol 2002, 168(9): 4262-4267. Kim DH, Chang WS, Lee YS, Lee KA, Kim YK, Kwon BS, et al. 4-1BB engagement costimulates NKT cell activation and exacerbates NKT cell ligand- induced airway hyperresponsivene ss and inflammation. Jlmmunol 2008, 1 0(4): 2062-2068. Wilcox RA, Tamada K, Strome SE, Chen L. Signaling through NK cell-associated CD 137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity. Jlmmunol 2002, 169(8): 4230- 4236. Croft M. Co-stimulatory members of the TNFR family: keys to effective T-cell immunity. Nature Reviews Immunology 2003, 3(8): 609-620. Vinay DS, Kwon BS. 4-1BB signaling beyond T cells. Cell Mol Immunol 2011, 8(4): 281-284. Palazon A, Teijeira A, Martinez-Forero I, Hervas-Stubbs S, Roncal C, Pefluelas I, et al. Agonist Anti-CD137 mAb Act on Tumor Endothelial Cells to Enhance Recruitment of Activated T Lymphocytes. Cancer Research 2011, 71(3): 801-811. Sabbagh L, Pulle G, Liu Y, Tsitsikov EN, Watts TH. ERK-dependent Bim modulation downstream of the 4-1BB-TRAF1 signaling axis is a critical mediator of CD8 T cell survival in vivo. Jlmmunol 2008, 180(12): 8093-8101. Shuford WW, Klu ssman K, Tritchler DD, Loo DT, Chalupny J, Siadak AW, et al. 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. JExp Med 1997, 186(1): 47-55. Zhu Y, Zhu G, Luo L, Flies AS, Chen L. CD137 stimulation delivers an antigenindependent growth signal for T lymphocytes with memory phenotype. Blood 2007, 109(11): 4882-4889. Houot R, Kohrt H, Levy R. Boosting antibody-dependant cellular cytotoxicity against tumor cells with a CD137 stimulatory antibody. Oncoimmunology 2012, 1(6): 957-958. 29. Chen S, Lee LF, Fisher TS, Jessen B, Elliott M, Evering W, et al. Combination of 4-1BB agonist and PD-1 antagonist promotes antitumor effector/memory CD8 T cells in a poorly immunogenic tumor model. Cancer Immunol Res 2015, 3(2): 149-160.
30. Compte M, Harwood SL, Mufloz IG, Navarro R, Zonca M, Perez-Chacon G, et al. A tumor-targeted trimeric 4-lBB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity. Nature Communications 2018, 9(1): 4809.
31 . Claus C, Ferrara C, Xu W, Sam J, Lang S, Uhlenbrock F, et al. Tumor-targeted 4- 1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Science Translational Medicine 2019, 11(496): eaav5989.
32. Jiang CT, Chen KG, Liu A, Huang H, Fan YN, Zhao DK, et al. Immunomodulating nano-adaptors potentiate antibody-based cancer immunotherapy. Nat Commun 2021, 12(1): 1359.
33. Santra S, Kaittanis C, Santiesteban OJ, Perez JM. Cell-specific, activatable, and theranostic prodrug for dual-targeted cancer imaging and therapy. J Am Chem Soc 2011, 133(41): 16680-16688.
34. Wang H, Wang R, Cai K, He H, Liu Y, Y en J, et al. Selective in vivo metabolic cell-labeling-mediated cancer targeting. Nature Chemical Biology 2017, 13(4): 415-424.
35. Lee S, Koo H, Na JH, Han SJ, Min HS, Lee SJ, et al. Chemical tumor-targeting of nanoparticles based on metabolic glycoengineering and click chemistry. ACS Nano 2014, 8(3): 2048-2063.
36. Anand U, Lu J, Loh D, Aabdin Z, Mirsaidov U. Hydration Layer-Mediated Pairwise Interaction of Nanoparticles. Nano Letters 2016, 16(1): 786-790.
37. Dong X, Mumper RJ. Nanomedicmal strategies to treat multidrug-resistant tumors: current progress. Nanomedicine (Lond) 2010, 5(4): 597-615.
38. Gullotti E, Yeo Y. Extracellularly activated nanocarriers: a new paradigm of tumor targeted drug delivery. Mol Pharm 2009, 6(4): 1041-1051.
39. Wang J, Zhao W, Cheng L, Guo M, Li D, Li X, et al. CD137-mediated pathogenesis from chronic hepatitis to hepatocellular carcinoma in hepatitis B virus-transgenic mice. J. Immunol 2010, 185(12): 7654-7662.
40. Helk E, Bemin H, Ernst T, Ittrich H, Jacobs T, Heeren J, et al. TNFa-mediated liver destruction by Kupffer cells and Ly6Chi m onocytes during Entamoeba histolytica infection. PLoS Pathog 2013, 9(1): el003096. 41. Movita D, Kreefft K, Biesta P, van Oudenaren A, Leenen PJ, Janssen HL, et al. Kupffer cells express a unique combination of phenotypic and functional characteristics compared with splenic and peritoneal macrophages. J Leukoc Biol 2012, 92(4): 723-733.
42. Kinoshita M, Uchida T, Sato A, Nakashima M, Nakashima H, Shono S, et al. Characterization of two F4/80-positive Kupffer cell subsets by their function and phenotype in mice. J Hepatol 2010, 53(5): 903-910.
43. Xu Y, Szalai AJ, Zhou T, Zinn KR, Chaudhuri TR, Li X, et al. Fc gamma Rs modulate cytotoxicity of anti-Fas antibodies: implications for agonistic antibodybased therapeutics. J. Immunol 2003, 171(2): 562-568.
44. Li F, Ravetch JV. Antitumor activities of agonistic anti-TNFR antibodies require differential FcyRIIB coengagement in vivo. Proc Natl Acad Sci USA 2013, 110(48): 19501-19506.
45. Au KM, Park SI, Wang AZ. Trispecific natural killer cell nanoengagers for targeted chem oimmunotherapy. Sci Adv 2020, 6(27): eaba8564.
46. Au KM, Tnpathy A, Lin CP-1, Wagner K, Hong S, Wang AZ, et al. Bespoke Pretargeted Nanoradioimmunotherapy for the Treatment of Non-Hodgkin Lymphoma. ACS Nano 2018, 12(2): 1544-1563.
47. Quiles S, Raisch KP, Sanford LL, Bonner JA, Safavy A. Synthesis and Preliminary Biological Evaluation of High-Drug-Load Paclitaxel -Antibody Conjugates for Tumor-Targeted Chemotherapy. Journal of Medicinal Chemistry 2010, 53(2): 586-594.
48. Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 1989, 24(3): 148-154.
49. Kwong B, Gai SA, Elkhader J, Wittrup KD, Irvine DJ. Localized immunotherapy via liposome-anchored Anti-CD137 + IL-2 prevents lethal toxicity and elicits local and systemic antitumor immunity. Cancer Res 2013, 73(5): 1547-1558.
50. Filliol A, Piquet-Pellorce C, Raguenes-Nicol C, Dion S, Farooq M, Lucas-Clerc C, et al. RIPK1 protects hepatocytes from Kupffer cells-mediated TNF -induced apoptosis in mouse models of PAMP-induced hepatitis. J Hepatol 2017, 66(6): 1205-1213.
51. Agatemor, C., et al., Exploiting metabolic glycoengineering to advance healthcare. Nat Rev Chem, 2019. 3(10): p. 605-620. Du, J., et al., Metabolic glycoengineering: sialic acid and beyond. Glycobiology, 2009. 19(12): p. 1382-401. Kim, E. and H. Koo, Biomedical applications of copper-free click chemistry: in vitro, in vivo, and ex vivo. Chemical Science, 2019. 10(34): p. 7835-7851. Chang, P.V., et al., Copper-free click chemistry in living animals. Proc Natl Acad Sci U S A, 2010. 107(5): p. 1821-6. Baskin, J.M., et al., Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci U S A, 2007. 104(43): p. 16793-7. Stem, S.T., Adiseshaiah, P.P. & Crist, R M. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Part Fibre Toxicol 9, 20 (2012). Bonam, S.R., Wang, F. & Muller, S. Lysosomes as a therapeutic target. Nature Reviews Drug Discovery 18, 923-948 (2019). Reichel, D., Tripathi, M. & Perez, J.M. Biological Effects of Nanoparticles on Macrophage Polarization in the Tumor Microenvironment. Nanotheranostics 3, 66-88 (2019). Nielsen, M.C. et al. Macrophage Activation Markers, CD163 and CD206, in Acute-on-Chronic Liver Failure. Cells 9 (2020). Singanayagam, A. & Triantafyllou, E. Macrophages in Chronic Liver Failure: Diversity, Plasticity' and Therapeutic Targeting. Front Immunol 12, 661182 (2021).

Claims

WHAT IS CLAIMED IS:
1. A method of delivering a 4- IBB agonist to a cancer cell, comprising: contacting a cancer cell with a particle comprising an azide- or tetrazinecontaining molecule to generate a surface-modified cancer cell; contacting the surface-modified cancer cell with a cyclooctyne-modified 4- 1BB agonist, wherein, the cyclooctyne-modified 4- IBB agonist binds to the surface modified cancer cell.
2. The method of claim 1, wherein the azide- or tetrazine-containing molecule is an azide- or tetrazine-containing glycoprotein labeling reagent.
3. The method of claim 2, wherein the azide- or tetrazine-containing metabolic glycoprotein labeling reagent is selected from the group consisting of modified sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
4. The method of claim 2, wherein the azide-containing metabolic glycoprotein labeling reagent is N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz).
5. The method of any one of claims 1-4, wherein the particle is a nanoparticle comprising a polymer.
6. The method of claim 5, wherein the polymer is selected from the group consisting of mPEG-PLA, PLA, PLGA, and dextran.
7. The method of claim 1, wherein in the particle is a lipid-based nanoparticle.
8. The method of any one of claims 1-7, wherein the particle has a loading efficiency from about 0.1% to about 20%.
9. The method of any one of claims 1-8, wherein the 4- IBB agonist of the cyclooctyne- modified 4- IBB agonist is an antibody or a small molecule.
10. The method of claim 9, wherein the 4-1BB agonist of the cyclooctyne-modified 4- 1BB agonist is an anti-4-lBB antibody.
11. The method of any one of claims 1-10, wherein the antibody is functionalized with more than one residue of a cyclooctyne moiety.
12. The method of claim 11, wherein the degree of functionalization of the antibody is 35 or less.
13. The method of claim 11 or 12, wherein the cyclooctyne is a dibenzocyclooctyne.
14. A method of delivering a 4-1BB agonist to a cancer cell, comprising: contacting a cancer cell with a particle comprising a cyclooctyne-containing molecule to generate a surface-modified cancer cell; contacting the surface-modified cancer cell with an azide- or tetrazine- modified 4- IBB agonist, wherein, the azide- or tetrazine-modified 4-1BB agonist binds to the surface modified cancer cell.
15. The method of claim 14, wherein the cyclooctyne-containing molecule is a cyclooctyne-containing glycoprotein labeling reagent.
16. The method of claim 15, cyclooctyne-containing metabolic glycoprotein labeling reagent is selected from among modified sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
17. The method of claim 15, wherein the cyclooctyne-containing metabolic glycoprotein labeling reagent is an unnatural sugar functionalized with dibenzocyclooctyne (DBCO).
18. The method of any one of claims 14-17, wherein the particle is a nanoparticle comprising a polymer.
19. The method of claim 18, wherein the polymer is selected from the group consisting of mPEG-PLA, PLA, PLGA, and dextran.
20. The method of claim 14, wherein in the particle is a lipid-based nanoparticle.
21. The method of any one of claims 14-20, wherein the particle comprising the cyclooctyne-containing molecule has a loading efficiency from about 0. 1% to about 20%.
22. The method of any one of claims 14-21, wherein the 4-1BB agonist of the azide- or tetrazine-modified 4- IBB agonist is an antibody or a small molecule.
23. The method of claim 22, wherein the 4- IBB agonist of the azide- or tetrazine- modified 4- IBB agonist is an anti -4- IBB antibody.
24. The method of any one of claims 14-23, wherein the antibody is functionalized with more than one residue of an azide or tetrazine moiety.
25. The method of claim 24, wherein the degree of functionalization of the antibody is 35 or less.
26. The method of claim 24 or 25, wherein the azide is N-azidoacetylmannosamine- tetraacylated (Ac4ManNAz).
27. The method of any one of claims 1-26, wherein the cancer cells lack surface 4-1BB.
28. The method of any one of claims 1-27, wherein the cancer cell is selected from among melanoma, non-small cell lung cancer, small cell lung cancer, gastric cancer, esophageal cancer, GBM, head and neck cancer, pancreatic cancer, colorectal cancer, breast cancer, prostate cancer, bladder cancer, kidney cancer, lymphoma, and leukemia.
29. A method of delivering a therapeutic radionuclide to a cancer cell, comprising: contacting a cancer cell with a particle comprising an azide- or tetrazinecontaining molecule to generate a surface-modified cancer cell; contacting the surface-modified cancer cell with a cyclooctyne-modified therapeutic radionuclide, wherein, the cyclooctyne-modified therapeutic isotope binds to the surface modified cancer cell.
30. The method of claim 29, wherein the azide- or tetrazine-containing molecule is an azide- or tetrazine-containing glycoprotein labeling reagent.
31. The method of claim 30, wherein the azide- or tetrazine-containing metabolic glycoprotein labeling reagent is selected from the group consisting of modified sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
32. The method of claim 30, wherein the azide-containing metabolic glycoprotein labeling reagent is N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz).
33. The method of any one of claims 29-32, wherein the particle is a nanoparticle comprising a polymer.
34. The method of claim 33, wherein the polymer is selected from the group consisting of mPEG-PLA, PLA, PLGA, and dextran.
35. The method of claim 29, wherein in the particle is a lipid-based nanoparticle.
36. The method of any one of claims 29-35, wherein the particle comprising the azide- or tetrazine-containing molecule has a loading efficiency from about 0. 1% to about 20%.
37. The method of any one of claims 29-36, wherein the therapeutic radionuclide of the cyclooctyne-modified therapeutic radionuclide is a beta emitter.
38. The method of any one of claims 29-36, wherein the therapeutic radionuclide of the cyclooctyne-modified therapeutic radionuclide is an alpha emitter.
39. The method of claim 37 or 38, wherein the cyclooctyne is a dibenzocyclooctyne.
40. A method of delivering a therapeutic radionuclide to a cancer cell, comprising: contacting a cancer cell with a particle comprising a cyclooctyne-containing molecule to generate a surface-modified cancer cell; contacting the surface-modified cancer cell with an azide- or tetrazine- modified therapeutic radionuclide, wherein, the azide- or tetrazine-modified therapeutic isotope binds to the surface modified cancer cell.
41. The method of claim 40, wherein the cyclooctyne-containing molecule is a cyclooctyne-containing glycoprotein labeling reagent.
42. The method of claim 41, cyclooctyne-containing metabolic glycoprotein labeling reagent is selected from the group consisting of modified sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
43. The method of claim 41, wherein the cyclooctyne-containing metabolic glycoprotein labeling reagent is an unnatural sugar functionalized with dibenzocyclooctyne (DBCO).
44. The method of any one of claims 40-43, wherein the particle is a nanoparticle comprising a polymer.
45. The method of claim 44, wherein the polymer is selected from the group consisting of mPEG-PLA, PLA, PLGA, and dextran.
46. The method of claim 40, wherein in the particle is a lipid-based nanoparticle.
47. The method of any one of claims 40-46, wherein the particle comprising the cyclooctyne-containing molecule has a loading efficiency from about 0. 1 % to about 20%.
48. The method of any one of claims 40-47, wherein the therapeutic radionuclide of the azide- or tetrazine-modified therapeutic radionuclide is a beta emitter.
49. The method of claim 48, wherein the therapeutic radionuclide of the azide- or tetrazine-modified therapeutic radionuclide is an alpha emitter.
50. The method of claim 48 or 49, wherein the azide is N-azidoacetylmannosamine- tetraacylated (Ac4ManNAz).
51. The method of any one of claims 29-50, wherein the cancer cell is selected from among melanoma, non-small cell lung cancer, small cell lung cancer, gastric cancer, esophageal cancer, GBM, head and neck cancer, pancreatic cancer, colorectal cancer, breast cancer, prostate cancer, bladder cancer, kidney cancer, lymphoma, and leukemia.
52. A method of treating cancer in a subject by antigen-independent immunotherapy, comprising: administering to the subject a particle comprising an azide- or tetrazinecontaining molecule, wherein a cancer cell in the subject is modified to a surface- modified cancer cell; and administering to the subject a cyclooctyne-modified 4-1BB agonist, wherein the cyclooctyne-modified 4- IBB agonist binds to the surface modified cancer cell, wherein, the cancer is treated.
53. The method of claim 52, wherein the azide- or tetrazine-containing molecule is an azide- or tetrazine-containing glycoprotein labeling reagent.
54. The method of claim 53, wherein the azide- or tetrazine-containing metabolic glycoprotein labeling reagent is selected from among modified sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
55. The method of claim 53, wherein the azide-containing metabolic glycoprotein labeling reagent is N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz).
56. The method of any one of claims 52-55, wherein the particle is a nanoparticle comprising a polymer.
57. The method of claim 56, wherein the polymer is selected from the group consisting of mPEG-PLA, PLA, PLGA, and dextran.
58. The method of claim 52, wherein in the particle is a lipid-based nanoparticle.
59. The method of any one of claims 52-58, wherein the particle comprising the azide- or tetrazine-containing molecule has a loading efficiency from about 0. 1% to about 20%.
60. The method of any one of claims 52-59, wherein the 4-1BB agonist of the cyclooctyne-modified 4- IBB agonist is an antibody or small molecule.
61. The method of claim 60, wherein the 4-1BB agonist of the cyclooctyne-modified 4- 1BB agonist is an anti-4-lBB antibody.
62. The method of any one of claims 52-61, wherein the antibody is functionalized with more than one residue of a cyclooctyne moiety.
63. The method of claim 61, wherein the degree of functionalization of the antibody is 35 or less.
64. The method of claim 61 or 62, wherein the cyclooctyne is a dibenzocyclooctyne.
65. A method of treating cancer in a subject by antigen-independent immunotherapy, comprising: administering to the subject a particle comprising a cyclooctyne-containing molecule, wherein a cancer cell in the subject is modified to a surface- modified cancer cell; and, administering to the subject an azide- or tetrazine-modified 4-1BB agonist, wherein the azide- or tetrazine-modified 4- IBB agonist binds to the surface modified cancer cell, wherein, the cancer is treated.
66. The method of claim 65, wherein the cyclooctyne-containing molecule is a cyclooctyne-containing glycoprotein labeling reagent.
67. The method of claim 66, wherein the cyclooctyne-containing metabolic glycoprotein labeling reagent is selected from the group consisting of modified sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
68. The method of claim 66, wherein the cyclooctyne-containing metabolic glycoprotein labeling reagent is an unnatural sugar functionalized with dibenzocyclooctyne (DBCO).
69. The method of any one of claims 65-68, wherein the particle is a nanoparticle comprising a polymer. The method of claim 69, wherein the polymer used for the nanoparticle is selected from the group consisting of mPEG-PLA, PLA, PLGA, and dextran. The method of claim 65, wherein in the particle is a lipid-based nanoparticle. The method of any one of claims 65-71, wherein the particle comprising the cyclooctyne-containing molecule has a loading efficiency from about 0. 1% to about 20%. The method of any one of claims 65-72, wherein the 4-1BB agonist of the azide- or tetrazine-modified 4- IBB agonist is an antibody or a small molecule. The method of claim 73, wherein the 4-1BB agonist of the azide- or tetrazine- modified 4- IBB agonist is an anti -4- IBB antibody. The method of any one of claims 65-74, wherein the antibody is functionalized with more than one residue of an azide or tetrazine moiety. The method of claim 75, wherein the degree of functionalization of the antibody is 35 or less. The method of claim 75 or 76, wherein the azide is N-azidoacetylmannosamine- tetraacylated (Ac4ManNAz). The method of any one of claims 52-77, wherein the cancer cells lack surface 4- IBB. The method of any one of claims 52-78, wherein the cancer cell is selected from among melanoma, non-small cell lung cancer, small cell lung cancer, gastric cancer, esophageal cancer, GBM, head and neck cancer, pancreatic cancer, colorectal cancer, breast cancer, prostate cancer, bladder cancer, kidney cancer, lymphoma, and leukemia. The method of claims 52 or 65, further comprising administering a checkpoint inhibitor to the subject. The method of claim 80, wherein the checkpoint inhibitor is administered prior to, concomitantly with, or after the administering of the azide- or tetrazine- or cyclooctyne- modified 4- IBB agonist. The method of claim 80, wherein the checkpoint inhibitor is an anti-PDl antibody. The method of any one of claims 52-82, wherein the administering does not cause hepatotoxicity. The method of any one of claims 52-83, wherein CD81 TIL is enhanced.
85. The method of claim 84, wherein said enhanced is at least two-fold to about five-fold.
86. A method of treating cancer in a subject by antigen-independent therapy, comprising: administering to the subject a particle comprising an azide- or tetrazinecontaining molecule, wherein a cancer cell in the subject is modified to a surface- modified cancer cell; and administering to the subject a cyclooctyne-modified therapeutic radionuclide, wherein the cyclooctyne-modified therapeutic radionuclide binds to the surface modified cancer cell, wherein, the cancer is treated.
87. The method of claim 86, wherein the azide- or tetrazine-containing molecule is an azide- or tetrazine-containing glycoprotein labeling reagent.
88. The method of claim 87, wherein the azide- or tetrazine-containing metabolic glycoprotein labeling reagent is selected from among modified sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
89. The method of claim 86, wherein the azide-containing metabolic glycoprotein labeling reagent is N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz).
90. The method of any one of claims 86-89, wherein the particle is a nanoparticle comprising a polymer.
91. The method of claim 90, wherein the polymer used for the nanoparticle is selected from among the group consisting of mPEG-PLA, PLA, PLGA, and dextran.
92. The method of claim 86, wherein in the particle is a lipid-based nanoparticle.
93. The method of any one of claims 86-92, wherein the particle comprising the azide- or tetrazine-containing molecule has a loading efficiency from about 0. 1% to about 20%.
94. The method of any one of claims 86-93, wherein the therapeutic radionuclide of the cyclooctyne-modified therapeutic radionuclide is a beta emitter.
95. The method of claim 94, wherein the therapeutic radionuclide of the cyclooctyne- modified therapeutic radionuclide is an alpha emitter.
96. The method of claim 94 or 95, wherein the cyclooctyne is a dibenzocyclooctyne.
97. A method of treating cancer in a subject by antigen-independent therapy, comprising: administering to the subject a particle comprising a cyclooctyne-containing molecule, wherein a cancer cell in the subject is modified to a surface-modified cancer cell; and, administering to the subject an azide- or tetrazine-modified therapeutic radionuclide, wherein the cyclooctyne-modified therapeutic radionuclide binds to the surface modified cancer cell, wherein, the cancer is treated.
98. The method of claim 97, wherein the cyclooctyne-containing molecule is a cyclooctyne-containing glycoprotein labeling reagent.
99. The method of claim 98, wherein the cyclooctyne-containing metabolic glycoprotein labeling reagent is selected from among modified sialic acid analogues, mannose analogues, xylose analogues, fucose analogues, galactose analogues, and other unnatural sugar analogues.
100. The method of claim 98, wherein the cyclooctyne-containing metabolic glycoprotein labeling reagent is an unnatural sugar functionalized with dibenzocyclooctyne (DBCO).
101. The method of any one of claims 97-100, wherein the particle is a nanoparticle comprising a polymer.
102. The method of claim 101, wherein the polymer is selected from the group consisting of mPEG-PLA, PLA, PLGA, and dextran.
103. The method of claim 97, wherein in the particle is a lipid-based nanoparticle.
104. The method of any one of claims 97-103, wherein the particle comprising the cyclooctyne-containing molecule has a loading efficiency from about 0. 1% to about 20%.
105. The method of any one of claims 97-104, wherein the therapeutic radionuclide of the azide- or tetrazine-modified therapeutic radionuclide is a beta emitter.
106. The method of claim 105, wherein the therapeutic radionuclide of the azide- or tetrazine-modified therapeutic radionuclide is an alpha emitter.
107. The method of claim 105 or 106, wherein the azide is N-azidoacetylmannosamine- tetraacylated (Ac4ManNAz).
108. The method of any one of claims 86-107, wherein the cancer cell is selected from among melanoma, non-small cell lung cancer, small cell lung cancer, gastric cancer, esophageal cancer, GBM, head and neck cancer, pancreatic cancer, colorectal cancer, breast cancer, prostate cancer, bladder cancer, kidney cancer, lymphoma, and leukemia.. The method of any one of claims 86-108, wherein the administering does not cause hepatotoxicity. . A pharmaceutical composition comprising: a particle comprising an azide- or tetrazme-containing molecule; and, a pharmaceutically acceptable excipient. . A pharmaceutical composition comprising: a particle comprising a cyclooctyne-containing molecule; and, a pharmaceutically acceptable excipient. . A pharmaceutical composition comprising: a cyclooctyne-modified 4-1BB agonist; and, a pharmaceutically acceptable excipient. . A pharmaceutical composition comprising: an azide- or tetrazine-modified 4- IBB agonist; and, a pharmaceutically acceptable excipient. . A pharmaceutical composition comprising: a cyclooctyne-modified therapeutic radionuclide; and, a pharmaceutically acceptable excipient. . A pharmaceutical composition comprising: an azide- or tetrazine-modified therapeutic radionuclide; and, a pharmaceutically acceptable excipient.
PCT/US2023/017884 2022-04-08 2023-04-07 Antigen-independent targeted delivery of therapeutic agents WO2023196593A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263328873P 2022-04-08 2022-04-08
US63/328,873 2022-04-08

Publications (1)

Publication Number Publication Date
WO2023196593A1 true WO2023196593A1 (en) 2023-10-12

Family

ID=88243549

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/017884 WO2023196593A1 (en) 2022-04-08 2023-04-07 Antigen-independent targeted delivery of therapeutic agents

Country Status (1)

Country Link
WO (1) WO2023196593A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180344770A1 (en) * 2017-02-17 2018-12-06 Rubius Therapeutics, Inc. Functionalized Erythroid Cells
US20200325225A1 (en) * 2016-12-19 2020-10-15 Hoffmann-La Roche Inc. Combination therapy with targeted 4-1bb (cd137) agonists
US20210346524A1 (en) * 2018-10-24 2021-11-11 Apa- Advanced Technologies Ltd. Fusogenic liposomes for selective imaging of tumor cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200325225A1 (en) * 2016-12-19 2020-10-15 Hoffmann-La Roche Inc. Combination therapy with targeted 4-1bb (cd137) agonists
US20180344770A1 (en) * 2017-02-17 2018-12-06 Rubius Therapeutics, Inc. Functionalized Erythroid Cells
US20210346524A1 (en) * 2018-10-24 2021-11-11 Apa- Advanced Technologies Ltd. Fusogenic liposomes for selective imaging of tumor cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HYESUN HYUN; BO SUN; STEPHANIE A. MONTGOMERY; TERESA GRIFFIN; JUANZHU YAN; ALBERT WIELGUS; YUE WANG; TIAN ZHANG; JIANJUN CHENG; AN: "Abstract 296: Antigen-independent delivery of 4-1BB agonist to the tumor microenvironment improves immune response while reducing hepatotoxicity", CANCER RESEARCH, vol. 82, no. 12_suppl., 15 June 2022 (2022-06-15), pages Abstract 296, XP009549680 *
TAKAYAMA YUKIYA, KUSAMORI KOSUKE, NISHIKAWA MAKIYA: "Click Chemistry as a Tool for Cell Engineering and Drug Delivery", MOLECULES, vol. 24, no. 1, pages 172, XP093101156, DOI: 10.3390/molecules24010172 *

Similar Documents

Publication Publication Date Title
JP7062298B2 (en) Compounds for targeted immunotherapy
EP3773737B1 (en) Saponin conjugated to epitope-binding proteins
Quiles et al. Synthesis and preliminary biological evaluation of high-drug-load paclitaxel-antibody conjugates for tumor-targeted chemotherapy
Andrikopoulou et al. Trastuzumab deruxtecan (DS-8201a): the latest research and advances in breast cancer
Hoang et al. Block copolymer micelles target Auger electron radiotherapy to the nucleus of HER2-positive breast cancer cells
WO2011130753A2 (en) Functionalized nano- and micro-materials for medical therapies
Zhu et al. Reversing activity of cancer associated fibroblast for staged glycolipid micelles against internal breast tumor cells
WO2017025889A1 (en) Polymeric nanoparticles with dec-205 ligand and co-encapsulating an antigen subject to an autoimmune response and a glucocorticoid receptor agonist
US20230416398A1 (en) Use of antibody against o-acetylated gd2 ganglioside to improve the therapeutic potential of drugs
Poudel et al. Combination chemotherapeutic and immune-therapeutic anticancer approach via anti-PD-L1 antibody conjugated albumin nanoparticles
Kumari et al. Antibody-conjugated nanoparticles for target-specific drug delivery of chemotherapeutics
CN113260653B (en) Stereocomplex for delivery of anticancer agents
WO2023196593A1 (en) Antigen-independent targeted delivery of therapeutic agents
Wittwer et al. Antibody drug conjugates: hitting the mark in pancreatic cancer?
Gunderson et al. Mirvetuximab soravtansine. FRalpha-targeting ADC, Treatment of epithelial ovarian cancer
JP2014504591A (en) Polymalic acid-based nanobiopolymer compositions and methods for treating cancer
KR102471267B1 (en) Methods of targeting tumor cells using chemotherapeutic agents conjugated to anti-Matriptase antibodies by in vivo cleavable linking moieties
Marques et al. Antibody-Functionalized Nanoparticles for Targeted Drug Delivery in Cancer Therapy
EP3328393A1 (en) Extracellular drug conjugates targeting cd20
US20220298225A1 (en) Methods and compositions for treating cancer with collagen binding drug carriers
WO2024034684A1 (en) New drug complex
Evans This chapter is a reprint of “Loujin Houdaihed, James C Evans, and Christine Allen. In Vivo Evaluation of Dual-Targeted Nanoparticles Encapsulating Paclitaxel and Everolimus. Cancers, 2019, 11 (6): 752.”
Lee et al. Photo-induced crosslinked and anti-PD-L1 peptide incorporated liposomes to promote PD-L1 multivalent binding for effective immune checkpoint blockade therapy
TW202412843A (en) new drug complex
IL305335A (en) Targeting conjugates comprising effector molecules and uses thereof

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: 23785443

Country of ref document: EP

Kind code of ref document: A1