WO2023196593A1 - Administration ciblée indépendante de l'antigène d'agents thérapeutiques - Google Patents

Administration ciblée indépendante de l'antigène d'agents thérapeutiques Download PDF

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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
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modified
cancer
cyclooctyne
azide
analogues
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PCT/US2023/017884
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English (en)
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Andrew Wang
Hyesun HYUN
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The University Of North Carolina At Chapel Hill
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    • 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.

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Abstract

L'invention concerne des formulations de nanoparticules modifiées de Ac4ManNAz et de α4-1BB à DBCO fonctionnalisée pour une administration indépendante de l'antigène d'α4-1BB à des tumeurs par l'intermédiaire d'une réaction « click » biorthogonale. Cette approche surmonte l'absence de biomarqueurs/antigènes pouvant être ciblés dans de nombreux cancers. L'administration ciblée d'α4-1BB non seulement améliore significativement l'efficacité d'α4-1BB, mais réduit également l'hépatotoxicité limitant les doses. Cette approche d'administration indépendante de l'antigène peut être appliquée largement pour le traitement du cancer, à la fois pour l'immunothérapie et la thérapie cytotoxique.
PCT/US2023/017884 2022-04-08 2023-04-07 Administration ciblée indépendante de l'antigène d'agents thérapeutiques WO2023196593A1 (fr)

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US20180344770A1 (en) * 2017-02-17 2018-12-06 Rubius Therapeutics, Inc. Functionalized Erythroid Cells
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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

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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 *

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