WO2019136118A2 - Cell-associating immunologic adjuvants for treatment enhancement - Google Patents

Cell-associating immunologic adjuvants for treatment enhancement Download PDF

Info

Publication number
WO2019136118A2
WO2019136118A2 PCT/US2019/012138 US2019012138W WO2019136118A2 WO 2019136118 A2 WO2019136118 A2 WO 2019136118A2 US 2019012138 W US2019012138 W US 2019012138W WO 2019136118 A2 WO2019136118 A2 WO 2019136118A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
cell
treatment kit
nanoparticles
adjuvant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2019/012138
Other languages
French (fr)
Other versions
WO2019136118A3 (en
Inventor
Che-Ming Jack HU
Saborni CHATTOPADHYAY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Academia Sinica
Original Assignee
Academia Sinica
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 Academia Sinica filed Critical Academia Sinica
Priority to JP2020537204A priority Critical patent/JP7230035B2/en
Priority to US16/959,758 priority patent/US11813326B2/en
Priority to CN201980007309.4A priority patent/CN111727054A/en
Publication of WO2019136118A2 publication Critical patent/WO2019136118A2/en
Publication of WO2019136118A3 publication Critical patent/WO2019136118A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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)
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A61K31/175—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine having the group, >N—C(O)—N=N— or, e.g. carbonohydrazides, carbazones, semicarbazides, semicarbazones; Thioanalogues thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/538—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with carbocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2884—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44

Definitions

  • This present disclosure relates to a treatment kit and methods for using the same.
  • tumor cell derivatives are formulated with adjuvants exogenously prior to administration.
  • the adjuvant mixtures may then increase the immunogenicity of tumor-associated antigens in the tumor cell derivatives.
  • dead cancer cells were functionali zed with adjuvant-nanoparticles ex vivo to vaccinate against subcutaneous tumorsp].
  • the platform in itself has a 30% efficiency that can be increased up to 78% when combined with an immune check-point blocker therapy.
  • allogenic cancer cells were formulated with a STING agonist ex vivo as a vaccine formulation[4].
  • the formulation, known as STING-VAX was able to elicit anti-tumor immunity upon administration in animal models.
  • immunologic adjuvants that can be associated with cells and demonstrate its use to enhance the immunogenicity of released antigens from ceils during treatment.
  • FIG. 1 by associating exogenous immunologic adjuvants with functionalities that can associate with cancer cells—such as through the means of cellular internationalization, membrane association, or surface ligand binding— the adjuvants can flag the cellular targets for immune potentiation.
  • Subsequent treatment that induces release of cellular debris, vesicles, and antigens would thus trigger immune responses regardless of whether the treatment itself triggers endogenous danger signals.
  • This application thus concerns about both functionalization of immunologic adjuvants and treatment modalities associated with the functionalized adjuvants.
  • an immunologic adjuvants are designed to associate with malignant cells, which can be through either intracellular (cellular internationalization) or extracellular means (membrane-tethering or surface ligand binding). Malignant cells treated with the functionalized adjuvants are then induced to release cellular debris, vesicles, and antigens. Induction of antigen release can be triggered by any stimuli know in the art.
  • the stimuli may at least comprise chemical agents, biological agents, light, or mechanical stimuli.
  • a treatment modality for pathogenic cell where the diseased cells are first administered with exogenous adjuvants.
  • the exogenous adjuvants can be associated with the cells as follows; (1 ) an adjuvant formulation that allows intracellular delivery of the desired adjuvants, e.g. nanoparticles, exosomes, engineered cell penetrating peptides etc.; (2) an adjuvant formulation that can be partitioned into the cell membrane, e.g. adjuvant with lipophilic linkers, amphiphilic adjuvants etc.; and (3) an adjuvant formulation that has binding affinity to cell surface moieties and capable of physically associating with cell membrane moieties, e.g. adjuvant coupled with antibody, adjuvant coupled with aptamers, adjuvant-sugar formulations etc
  • the cell is then treated with a vesiculating, antigen-releasing stimuli that include but not limited to chemotherapy drugs, biologic agents, irradiation, photolytic agents, mechanical disruption etc.
  • a vesiculating, antigen-releasing stimuli that include but not limited to chemotherapy drugs, biologic agents, irradiation, photolytic agents, mechanical disruption etc.
  • the regimen facilitates the production of co-localized antigen with exogenous adjuvants in vivo.
  • the cell -associating immunogenic adjuvants can be applied to complement any existing or emerging treatment modalities aimed at eradicating pathogenic ceils.
  • a treatment kit comprising: an immunologic adjuvant formulation capable of physically associating with ceils; and a means for inducing antigen release from a cell.
  • a method of treating a disease comprising; administering to a subject in need thereof an immunologic adjuvant formulation capable of physically associating with cells, and administering to the subject in need thereof a means for inducing antigen release from a cell.
  • the immunologic adjuvant formulation may comprise a. polymeric nanoparticle encapsulating an adjuvant, and the polymeric nanoparticle may further comprise: a polymeric shell impermeable to water, and one or more aqueous cores enclosed by the polymeric shell
  • the polymeric shell may have a thickness less than 25 nm and the polymeric nanoparticle has an outer diameter of 30-600 nm.
  • the means for inducing antigen release from a cell may be encapsulated in the polymeric nanoparticle.
  • the immunologic adjuvant formulation may comprise MPLA, CpG, poly(I:C), or variants of cyclic-dinucleotides.
  • the immunologic adjuvant formulation may comprise an agonist to a cytoplasmic pattern recognition receptor or a stimulator of interferon genes (STING).
  • STING stimulator of interferon genes
  • the agonist may be cyclic di-GMP or cyclic GAMP
  • the immunologic adjuvant formulation may comprises an adjuvant conjugated to a small molecule, a peptide, an antibody, an aptamer, a sugar moiety, a polymer or a combination thereof for recognizing and binding to a moiety on a cell surface.
  • the antibody may be an immunoglobulin molecule, an Pv, a disulfide linked Fv, a monoclonal antibody, a scFv, a chimeric antibody, a single domain antibody, a CDR- grafted antibody, a diabody, a humanized antibody, a multispecific antibody, an Fab, a dual specific antibody, an Fab’ fragment, a bispecific antibody, an F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementarity determining region (CDR), or a single chain antibody.
  • CDR complementarity determining region
  • the immunologic adjuvant formulation may comprises an adjuvant conjugated to a molecule capable of tethering to a cell membrane.
  • the molecule may be lipophilic or amphiphilic.
  • the lipophilic molecule may comprise fatty acid chains, cholesterol, or phospholipids.
  • the amphiphilic molecule may comprises lipid-PEG conjugates.
  • the immunologic adjuvant formulation may comprises an adjuvant formulated with nanoparticulates or cell penetrating peptides for intracellular delivery.
  • the cell penetrating peptides may comprise HIV-1 TAT peptide (GRKKRRQRRRPPQ), penetratin (RQDQWFQNRRMKWKK), poly arginines (Rn), or transportan (GWTLNSAGYLLGINLKALAALAKKIL).
  • HIV-1 TAT peptide GRKKRRQRRRPPQ
  • penetratin RQDQWFQNRRMKWKK
  • poly arginines Rn
  • transportan GWTLNSAGYLLGINLKALAALAKKIL
  • the means for inducing antigen release may comprise chemical agents, biological agents, irradiation, photolytic agents, mechanical disruptions, or a combination thereof.
  • the chemical agents may comprise a cytotoxic agent.
  • the cytotoxic agent may comprise Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (DM1), anthracycline, pyrrolobenzodiazepine, a-amanitin, tubulysin, benzodiazepine, erlotinib, bortezomib, fulvestrant, sunitinib, !etrozoie, imatimb mesylate, PTK787/ZK 222584, oxalipiatin, leucovorin, rapamycin, lapatinib, lonafamib (SARASAR®, SCH 66336), sorafenib, gefitinib, AG1478, AG1571, alkylating agent; alkyl sulfonate; aziridines; ethylenimme; methylamelamine; acetogenins; camptothecin; br
  • the photolytic agents comprises Photofrin, Laserphyrin, Aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m -tetrahy droxy phenyl chi orin (mTHPC), chlorin e6 (Ce6), Allumera, Levulan, Foscan, Metvix, Hexvix, Photochlor, Photosens, Photrex, Lumacan, Visonac, Amphinex, Verteporfin, Purlytin, ATMPn, Zinc phthalocyanine (ZnPc), Protoporphyrin IX (PpIX), Pyropheophorbidea (PPa), Pheophorbide a (PhA), or a combination thereof.
  • the method may further comprise: encapsulating an adjuvant in a polymeric nanoparticle.
  • the polymeric nanoparticle may comprise: a polymeric shell impermeable to water, and one or more aqueous cores enclosed by the polymeric shell.
  • the means for inducing antigen release may be administered to the subject in need before or after administration of the immunologic adjuvant formulation.
  • the means for inducing antigen release may be administered to the subject in need at the same time as the immunologic adjuvant formulation.
  • the method may further comprise: encapsulating the means for inducing antigen release from a cell in the immunologic adjuvant formulation.
  • the method may further comprise: conjugating an adjuvant with a small molecule, a peptide, an antibody, an aptamer, a sugar moiety, a polymer or a combination thereof recognizing and binding to a moiety on a cell surface to form the immunologic adjuvant formulation.
  • the method may further comprise: conjugating an adjuvant with a molecule capable of tethering to a cell membrane.
  • the method may further comprise: formulating an adjuvant with nanoparticulates or cell penetrating peptides for intracellular delivery.
  • the step of administering to the subject may be by at least one mode selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intra- articular, intrabronchiai, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelia!, intracerebe!lar, intracerebroventricuiar, intracolic, intracervicai, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasai, and transdermal.
  • parenteral subcutaneous, intramuscular, intravenous, intra- articular, intrabronchiai, intraabdominal, intracapsular
  • the disease may be cardiovascular disease, cancer, autoimmune disease, or infection.
  • FIG. 1 illustrates pathogenic cell-associating immunologic adjuvants and their function in enhancing subsequent treatments.
  • Immunologic adjuvants are functionalized to associate with pathogenic cells through various means such as intracellular delivery, membrane tethering, and surface binding.
  • the resulting cells are then subjected to treatments that induce generation of adjuvant-associated cellular debris, which can promote adaptive immunity via coordinated antigen and adjuvant transport.
  • FIG. 2 depicts encapsulation efficiency of polymeric nanoparticles for a nucleic acid and a protein.
  • A Observation of empty nanoparticles (left), nanoparticles loaded with dye- labelled DNA (middle), and nanoparticles loaded with dye-labelled bovine serum albumin (right) following pelleting by ultracentrifugation. Distinctive colored pellets indicate successful encapsulation of DNA and BSA proteins.
  • B Empty nanoparticles visualized by cryoEM.
  • C DNA-loaded nanoparticles visualized by cryoEM.
  • D BSA protein-loaded nanoparticles visualized by cryoEM. Effective DNA and protein loading could be observed through the highly grainy textures inside nanoparticles.
  • FIG. 3 depicts the effect of polymeric nanoparticles on encapsulation and controlled release of STING agonists for immune stimulation.
  • A Preparation of adjuvant-loaded thin- shell hollow nanoparticles.
  • B Size of nanoparticles as measured by nanoparticle tracking analysis.
  • C Encapsulation of cyclic-di-GMP as verified by gradient HPLC.
  • D CryoEM visualization of thin-shell hollow nanoparticles.
  • E Cargo release study revealed a pH-sensitive triggered release profile for the nanoparticles.
  • FIG. 4 depicts STING agonist-loaded nanoparticles on enhancing lymphatic cytokines while minimizing systemic cytokines.
  • A Induction of TNF-a in a mouse dendritic cell line following incubation with cd-GMP nanoparticles and free cd-GMP.
  • B Induction of IL-6 in the same mouse dendritic cell line.
  • C Induction of IFN-b in the mouse dendritic cell line.
  • D Activation of JAWSII upon incubation with equivalent doses of cd-GMP in free molecule form and in nanoparticle formulation.
  • E Quantification of lymph node IFN-b 48 hours following footpad injection of free cd-GMP or nanoparticle cd-GMP.
  • F Level of systemic TNF-a following footpad injection of free cd-GMP and nanoparticle cd-GMP.
  • FIG. 5 depicts a treatment modality based on a tumor cell-associating immunologic adjuvant (cGAMP-loaded nanoparticle). Following nanoparticJe uptake, the cells containing exogenous adjuvants are then treated to generate a dead cellular debris. The cellular debris containing the exogenous adjuvants subsequently triggers adaptive immunity to improve treatment outcome.
  • FIG. 6 depicts (a) CryoEM visualization of a fluorescently labelled cGAMP -loaded nanoparticle (b) Confocal analysis and (c) flow-cytometric analysis show association of cGAMP with dead cells and debris following treatment with cGAMP-loaded nanoparticles.
  • FIG. 7 depicts cellular debris generated by HeLa cells incubated with cGAMP nanoparticles were more immunogenic than those incubated with free cGAMP.
  • the HeLa cell associating cGAMP nanoparticles resulted in increased DC expression of CD80 (a), CD86 (b), and elevated DC secretion of TNF-a(c) and IFN-p(d)
  • FIG. 8 depicts adjuvant retention with tumors and cancer cells were examined upon intratumoral injection of a dye-labelled STING agonist (red) in its free (Free Adjuvant) and nanoparticle form (Adjuvant Nanoparticle). 24 hours following the adjuvant administration the tumors and extracted cancer cells were examine by (a) whole-body imaging, (b) flow- cytometric analysis, and (c) fluorescence microscopy. The examinations demonstrate significant adjuvant retention inside the tumor and within cancer cells upon nanoparticle administration.
  • FIG. 9 depicts tumor treatment efficacy with different adjuvant and chemodrug combinations, including placebo, cisplatin only (CTTP only), cGAMP-loaded nanoparticle only (NP only), free cGAMP followed by cisplatin (cGAMP+CTTP), and cGAMP-loaded nanoparticle followed by ci splatin (NP +CTTP).
  • CTTP cisplatin only
  • NP cGAMP+CTTP
  • cGAMP-loaded nanoparticle followed by ci splatin ci splatin
  • FIG. 10 depicts the preparation of antibody-conjugated CpG-ODN.
  • FIG. 1 1 depicts (A ) Fluorescence microscopy observation of HeLa cellular monolayer following 3 h of incubation with free CpG-F M (middle) and antibody-conjugated CpG-F M (aCD44-CpG-FAM; right). Enhanced CpG association by the antibody-conjugated CpG-FAM was observed with a higher number of fluorescence punctates. (B) Flow' cytometric analysis of cells with CpG-FAM. (C) Quantification of cells associated with CpG-FAM. Anti-CD44 conjugates (aCD44) yielded higher CpG-FAM association on HeLa cells than free CpG-FAM (free).
  • FIG. 12 depicts expression of maturation markers (A) CD80 and (B) CD86 by JA W SII ceils following incubation with HeLa ceil debris treated with free CpG (free) or anti-CD44 conjugated CpG (aCD44).
  • FIG. 13 depicts (A) Schematic presentation of nanoparticle-encapsulated CpG. (B) CryoEM visualization of nanoparticle-encapsulated CpG.
  • FIG. 14 depicts (A) Fluorescence microscopy observation of HeLa cellular monolayer following 24 h of incubation with free CpG-FAM (middle) and nanoparticle-encapsulated CpG (CpG-FAM NP; right). Enhanced CpG association was observed with a high number of fluorescence punctates. (B) Flow cytometric analysis of cells with CpG-FAM. (C) Quantification of cells associated with CpG-FAM Anti-CD44 conjugates yielded higher CpG- FAM association on HeLa cells than free CpG-FAM.
  • FIG. 15 depicts expression of maturation markers (A) CD80 and (B) CD86 by JAWSII ceils following incubation with HeLa cell debris treated with free CpG (free) or nanoparticle- encapsulated CpG (NP).
  • FIG. 16 depicts (A) Schematic presentation of a nanoparticle co-encapsulating a chemotherapeutic drug (daunorubicin) and an adjuvant (cd-GMP). (B) CryoEM visualization of nanoparticles co-encapsulating daunorubicin and cd-GMP
  • FIG. 17 depicts expression of maturation markers (A,B) CD86 and (C,D) CD80 by JAWSII cells following incubation with CT26 cell debris treated with nanoparticles coencapsulating daunorubicin and cd-GMP ((cdGMP+DR)NP), free cd-GMP and daunorubicin nanoparticles (cdGMP + DR NP) or daunorubicin nanoparticle only (DR NP)
  • FIG. 18 depicts secretion of IFN-b from JAWSII cells following incubation with CT26 cell debris treated with nanoparticles coencapsulating daunorubicin and cd-GMP ((cdGMP+DR)NP), free cd-GMP and daunorubicin nanoparticles or daunorubicin nanoparticle only (DR NP).
  • compositions “comprising,”“includes,”“including,”“has,” “having,”“contains”,“containing,”“characterized by” or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any limitation explicitly indicated.
  • a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, or method.
  • transitional phrase“consisting essentially of’ is used to define a composition, method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel character! stic(s) of the claimed invention.
  • the term“consisting essentially of’ occupies a middle ground between“comprising” and “consisting of’.
  • the term“about” is used to indicate that a value includes for example, the inherent variation of error for a measuring device, the method being employed to determine the value, or the variation that exists among the study subjects. Typically the term is meant to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%0, 15%, 16%, 17%, 18%, 19% or 20% variability depending on the situation.
  • “treating,” or“treatment” is referred to herein as administration of a therapeutic composition to a subject with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate a disorder, symptoms of the disorder, a disease state secondary to the disorder, or predisposition toward the disorder.
  • Subject refers to a mammalian subject diagnosed with or suspected of having or developing diseases such as cardiovascular disease, cancer, autoimmune disease, or infection.
  • exemplary' patients may be humans, apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents and other mammalians with the diseases that can benefit from the treatment.
  • administration may be performed via parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intraeerebe!lar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transderma!.
  • injection may be performed by intravenous (i.v.) injection, sub- cutaneous (s.c.) injection, intraderm al (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection.
  • i.v. intravenous
  • s.c. sub- cutaneous
  • i.d. intraderm al
  • i.p. intraperitoneal
  • i.m. intramuscular injection.
  • Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time.
  • administration may be by the oral route.
  • an "immunologic adjuvant formulation” means a formulation that when administered to a subject is capable of inducing an immune response in the subject. When administered in combination with an antigen, the“immunologic adjuvant formulation” are capable of eliciting an anti gen -specific immune response.
  • The“immunologic adjuvant formulation” will be associated with cells via cellular internationalization, membrane association, and/or surface ligand binding after being administered by a subject.
  • cellular internationalization means an adjuvant formulation that allows intracellular delivery' of the desired adjuvants.
  • cellular internationalization of the desired adjuvants may be achieved at least by nanoparti cies, exosomes, engineered cell penetrating peptides etc
  • “membrane association” means an adjuvant formulation that can be partitioned into the cell membrane.
  • “membrane association” of the desired adjuvants may be achieved at least by lipophilic linkers, amphiphilic adjuvants etc.
  • “surface ligand binding” means an adjuvant formulation that has binding affinity to cell surface moieties and capable of physically associating with cell membrane moieties.
  • “surface ligand binding” of the desired adjuvants may be achieved at least by antibody, aptamers, sugar formulations etc.
  • a "means for inducing antigen release from a cell” means using a chemical or physical approaches to induce the treated cells to release cellular debris, vesicles, and/or antigens that trigger immune response.
  • the means may comprise chemical agents, biological agents, irradiation, photolytic agents, mechanical disruptions, or a combination thereof.
  • a treatment kit for enhancing an immunogenicity of released antigens from cells comprises an immunologic adjuvant formulation capable of physically associating with cells; and a means for inducing antigen release from a cell.
  • the immunologic adjuvant formulation is a polymeric nanoparticle encapsulating an adjuvant, which comprises: a polymeric shell impermeable to water, and one or more aqueous cores enclosed by the polymeric shell.
  • the polymeric shell may have a thickness less than 25 nm and the polymeric nanoparticle has an outer diameter of 30-600 nm.
  • the thickness is less than 24 nm, 23 nm, 22 nm, 21 nm, 20 n n, 18 nm, 16 nm, 14 nm, 12 nm, or 10 nm.
  • the outer diameter is preferably in a range from 30-550 nm, 30-500 nm, 30-450 nm, 30-400 nm, 30-350 nm, 30-300 nm, 30-250 nm, 30-200 nm, 30-150 nm, 30-100 nm, 35-550 nm, 35-500 nm, 35-450 nm, 35- 400 nm, 35-350 nm, 35-300 nm, 35-250 nm, 35-200 nm, 35-150 nm, 35-100 nm, 40-550 nm, 40-500 nm, 40-450 nm, 40-400 nm, 40-350 nm, 40-300 nm, 40-250 nm, 40-200 nm, 40-150 nm, 40-100 nm, 45-550 nm, 45-500 nm, 45-450 nm, 45-400 nm, 45-350 nm, 45-300 nm, 45
  • the polymeric shell is formed of an amphiphilic polymer that contains a non -polar segment and a polar terminal group.
  • the non-polar segment include, but are not limited to, poly(lactie acid), poJy(lactic-co-glycolic acid) (PLGA), polycaprolactone, and polyurethane.
  • the lactic acid to glycolic acid molar ratio of the PLGA varies from 1 :99 to 99: 1 , from 10:90 to 90: 10, from 15:85 to 85:15, from 20:80 to 80:20, from 25:75 to 75:25, from 30:70 to 70:30.
  • the lactic acid to glycolic acid molar ratio of the PLGA is 50:50 or 75:25.
  • the polar terminal group can be a negatively charged group, a positively charged group, a zwitterionic group, or a neutral group.
  • the negatively charged group include a carboxylic acid, a succinic acid, and a sulfonic acid.
  • Examples of the positively charged group include an amine and an ami dine.
  • the zwiterionic group include a carboxybetaine and a sulfobetaine.
  • An example of the neutral group is a saccharide.
  • An exemplary polymeric shell is formed of a polymer containing poly(lactic-co- giycolic acid) as the non-polar segment and a carboxylic acid as the polar terminal group.
  • the polymeric nanoparticle described herein can be a polymeric hollow nanoparticle platform with a defect-free polymeric shell having a thickness of 25 nm or less.
  • the hollow polymeric nanoparticle typically has an outer diameter between 30 and 600 nm.
  • the polymeric nanoparticle can be formed with a large i nterior aqueous space capable of maximizing the cargo loading.
  • the interior aqueous space can possess a diameter at least 80% of the particle’s outer diameter or can be of multiple compartments with a large collective volume.
  • High efficiency encapsulation of hydrophilic dyes and nucleic acids are demonstrated with the thin-shell hollow nanoparticles in the absence of complementary' binding molecules.
  • the thin- sheil nanoparticles are demonstrated to be resistant to osmotic stress, a feature attributable to complete, defect-free polymeric shell that is impermeable to water.
  • the polymeric nanoparticle of this application can be used for delivering bioactive agents in various fields, including drug delivery' and vaccine development.
  • Polymeric nanoparticles particularly those consisting of biodegradable and biocompatible polymers such as poly(lactic-co-giycolic acid)(PLGA), have received considerable attention in nanomedicine research because of the polymer’s numerous features including biocompatibility, biodegradability, and synthetic flexibility.
  • PLGA-based nanoparticles have been limited to the delivery of water-insoluble compounds in clinical. Encapsulation of hydrophilic and macromolecular cargoes in polymeric nanoparticles remains a challenge as polymers tend to form solid nanospheres with little or no aqueous core space to carry' hydrophilic and macromolecular cargoes, e.g., siRNA.
  • a method of treating a disease comprises administering to a subject in need thereof an immunologic adjuvant formulation capable of physically associating with cells; and administering to the subject in need thereof a means for inducing antigen release from a cell.
  • the disease includes, but is not limited to, cardiovascular disease, cancer, autoimmune disease, or infection.
  • the thin-shell hollow nanoparticle is prepared based on a double emulsion process using amphiphilic polymers with high contrast of polarity at their terminus. More specifically, a solution of carboxyl-terminated PLGA in dichloromethane (DCM) is first used to emulsify an aqueous phase containing a cargo under sonic dispersion to form an emulsion. The emulsion thus formed is subsequently emulsified in an outer aqueous phase using fluidic dispersion.
  • DCM dichloromethane
  • the preparation method described above can provide hollow polymeric nanoparticles with outer diameters between 30 and 600 nm, e.g., 30-40 nm and 100-600 nm.
  • the nanoparticles are prepared based on a water-oil-water double emulsion process in which polymers dissolved in a solvent system is first used to emulsify an aqueous phase.
  • the emulsion is subsequently emulsified by a secondary aqueous phase.
  • the inner and outer aqueous phases can be of any polar solution, e.g., water, acetic acid, and ethanol.
  • the aqueous phase contains solubilized molecules to modulate the solution’s acidity and viscosity, which include sodium phosphate and sodium bicarbonate.
  • water is used as an anti-solvent for the nanoparticle preparation.
  • the water-oil-water double emulsion method described above for preparing the polymeric nanoparticle of in the present application has two key features: namely, (i) emulsion between different phases is achieved through polymers with inherently high contrast in polarity (PLGA with a carboxyl-terminal group) rather than using an surfactant, e.g., vitamin E-D-a- tocopherol polyethylene glycol succinate and polyfvinyl alcohol), which enhances the emulsifying capability to minimize polymer shell thickness and has a higher commercial value without using surfactant materials, and (ii) controlled fluidic dispersion using either a microfluidizer or sonication for the second emulsion process to balance homogenization of the oil phase and retention of encapsulated cargo in the inner aqueous phase.
  • PLGA polymers with inherently high contrast in polarity
  • an surfactant e.g., vitamin E-D-a- tocopherol polyethylene glycol succinate and polyfvinyl
  • the polymeric nanoparticle prepared by the above-described method serves as a platform technology for drug delivery, theranostics, and vaccine development applications. It can facilitate delivery of a large class of bioactive agents, including small molecules, peptides, nucleic acids, and proteins, to enhance their therapeutic potency.
  • the thin-shell polymeric hollow nanoparticles can be used to encapsulate bioactive agents, including but not limited to small molecules, peptides, proteins, nucleic acids, imaging agents, inorganic nanoparticles, organic nanoparticles, and any combination of the above.
  • the surface of the platform can be optionally decorated with functional moieties, including small molecules, peptides, proteins, nucleic acids, imaging agents, nanoparticles, for different applications such as long-circulating drug delivery, targeted drug delivery, and antigen delivery.
  • Thin-shell polymeric nanoparticles were produced according to a protocol including the following steps:
  • the thin-shell hollow nanoparticles were found to be osmoticaily resistant resulting from the water impermeable polymeric shells.
  • 100 nm hollow nanoparticles encapsulating a hydrophilic red food coloring were suspended in solutions ranging from water to 3X PBS, the difference in osmolarity (between 0 to 850 Osmo/kg) did not cause the hollow nanoparticles to release their cargoes.
  • nanoparticles were pelleted under centrifugation at 30,000 g for 5 min, and the resulting pellets showed similar, reddish color indicating retention of hydrophilic dye in the particles.
  • a distinguishing feature of the thin-shell polymeric nanoparticle platform is its capacity to encapsulate a large amount of hydrophilic cargoes with its large interior aqueous space.
  • the thin-shell hollow nanoparticles were subjected to encapsulate several bioactive agents, including siRNA and an immunological adjuvant cyclic di-GMP.
  • sulfo-cy5, cyclic di-GMP, and cyclic cGAMP small molecules
  • peptides e.g., ovalbumin peptide OTI (SIINFEKL) or OTII (AAHAEINEA)
  • nucleic acids e.g, CpG- oligodeoxynucleotides, 20-mer single standed DNA, and 20-mer siRNA
  • proteins e.g, bovine serum albumin (BSA) and CRISPR-Cas9 nuclease
  • siRNA was encapsulated at an efficiency of 50% with a final loading yield of about 1 nmol per mg of nanoparticles and cyclic di-GMP was encapsulated at a 37% loading efficiency.
  • Silencing of a green fluorescent protein (GFP) gene in GFP-expressing HeLa cells was observed using siRNA loaded thin-shell hollow nanoparticles.
  • GFP green fluorescent protein
  • FIG. 2 Please refer to FIG. 2 for a depiction of encapsulation efficiency of polymeric nanoparticles for a nucleic acid and a protein.
  • effective encapsulation of a nucleic acid and a protein was demonstrated using dye-labelled 20-mer single stranded DNA (A, middle) and dye-labelled BSA (A, right).
  • the particle pellets were found to be yellow colored (indicative of the yellowish FAM dye label) as opposed to the white pellet of the empty nanoparticles (A; left). Fluorescence quantification indicates that the nanoparticles unexpectedly exhibited encapsulation efficiencies of 42% and 35% for the nucleic acid and protein, respectively.
  • FIG. 3 depicts the effect of polymeric nanoparticles on encapsulation and controlled release of STING agonists for immune stimulation.
  • the present platform was applied for vaccine development.
  • a major technical challenge in preparing nanoparticle vaccines lies in reliably associating antigens and adjuvants on a nanoscale substrate. For example, see Brannon-Peppas et a/., Adv Drug Deliv Rev, 2004, 56(11): 1649-59; and Lima- Tenorio et al, Ini J Pharm, 2015, 493(1-2): 313-27.
  • thin-shell polymeric hollow nanoparticles were used to package a high density of functional cargoes using a biodegradable polymer, i.e., PLGA.
  • hollow particles between 100 to 200 nm in diameter were prepared using a double emulsion process (FIG. 3A and 3B). They efficiently encapsulated a STING agonist adjuvant, cyclic di-GMP (cd-GMP), at about 40% efficiency (FIG. 3C), which is notable as nucleic acids and hydrophilic cargoes are notoriously difficult to encapsulate in nanoparticle platforms.
  • cd-GMP cyclic di-GMP
  • FIG. 3C 40% efficiency
  • the high encapsulation efficiency yielded approximately 2,000 STING agonist molecules per nanoparticle. No nanoformulations of STING agonists based on polymeric nanoparticles have been reported.
  • FIG. 4 depicts STING agonist-loaded nanoparticles on enhancing lymphatic cytokines while minimizing systemic cytokines.
  • Immune activation by the cd-GMP -loaded nanoparticles was first examined in vitro using a mouse dendritic cell line, JAWSII. Following 24 hours of incubation with either free cd-GMP or cd-GMP nanoparticles, the culture supernatants were collected for ELISA analysis to quantify the levels of TNF-a, IL-6, and IFN-b. As compared to free cd-GMP formulation, the nanoparticle formulation more effectively triggered the production of TNF-a, IL-6, and IFN-b (FIG.
  • CD80 expression on the ceils was also enhanced when incubated with the nanoparticle formulation as compared to an equivalent dose of free cd-GMP (FIG. 4D). It was observed that the nanoparticles enhanced the adjuvanticity of the STING agonist by about 30 folds, attributable to increased intracellular delivery by the nanocarrier. It can be inferred that the free cyclic-di -nucleotide is not readily membrane permeable and may not easily access its cytosolic target. Upon nanoparticle encapsulation, cellular uptake is enhanced via particle endocytosis and the subsequent intracellular release facilitates cytosolic entry of cd-GMP, thereby enhancing its immune potentiating effect.
  • the treatment kit and the method described herein can be used to treat diseases, for example, cardiovascular disease, cancer, autoimmune disease, or infection.
  • the treatment kit and the method described herein can be used to cancers, such as cancer of the oral cavity and pharynx (lip, tongue, salivary gland, floor of mouth, gu and other mouth, nasopharynx, tonsil, oropharynx, hypopharynx, other oral/pharynx); cancers of the digestive system (esophagus; stomach; small intestine; colon and rectum; anus, anal canal, and anorectum; liver; intrahepatic bile duct; gallbladder; other biliary ; pancreas; retroperitoneum; peritoneum, omentum, and mesentery; other digestive); cancers of the respiratory system (nasal cavity, middle ear, and sinuses; larynx, lung and bronchus; pleura, trache
  • cancers such as cancer of the
  • the immunologic adjuvant formulation comprises a polymeric nanoparticle encapsulating an adjuvant
  • the polymeric nanoparticle may further comprise: a polymeric shell impermeable to water, and one or more aqueous cores enclosed by the polymeric shell.
  • the adjuvant comprises MPLA, CpG, poly(LC), variants of cyclic-dinucleotides, an agonist to a cytoplasmic pattern recognition receptor such as cyclic di-GMP or cyclic GAMP, or a stimulator of interferon genes (STING).
  • FIG. 5 Please refer to FIG. 5 for depiction of a treatment modality based on a tumor cell- associating immunologic adjuvant (cGAMP -loaded nanoparticle).
  • cGAMP is an agonist to cytoplasmic pattern recognition receptor, a stimulator of interferon genes (STING) that activate the TBK I/interferon regulatory factor 3 (IRF3)/type 1 interferon (IFN) signaling.
  • STING stimulator of interferon genes
  • IRF3 interferon regulatory factor 3
  • IFN type 1 interferon
  • the means for inducing antigen release may comprise chemical agents, biological agents, irradiation, photolytic agents, mechanical disruptions, or a combination thereof.
  • the chemical agents comprise any cytotoxic agents known in the art.
  • the cytotoxic agent comprises Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (DM1), anthracycline, pyrrolobenzodiazepine, a-amanitin, tubulysin, benzodiazepine, erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787/ZK 222584, oxaliplatin, leucovorin, rapamycin, lapatinib, lonafamib (SARASAR ® , SCH 66336), sorafenib, gefitinib, AG1478, AG1571, alkylating agent; alkyl sulfonate, aziridines; ethylenimine; methylamelamine
  • the photo! ytic agents which is generally a photosensitizing agent used in the photodynamic therapy, comprises Photofrin, Laserphyrin, Aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m -tetrahy droxy phenyl chi orin (mTHPC), chlorin e6 (Ce6), Allumera, Levulan, Foscan, Metvix, Hexvix, Photochlor, Photosens, Photrex, Lutnacan, Visonac, Amphinex, Verteporfin, Purlytin, ATMPn, Zinc phthaJocyanine (ZnPc), Protoporphyrin IX (PpIX), Pyropheophorbidea (PPa), Pheophorbide a (PhA), or a combination thereof.
  • ALA Aminolevulinic acid
  • mTHPC Silicon Phthalocyanine Pc 4
  • mTHPC m
  • the means for inducing antigen release may be administered to the subject in need before or after administration of the immunologic adjuvant formulation.
  • the means for inducing antigen release and the immunologic adjuvant formulation are formulated as two distinct doses for being administered at different times.
  • the means for inducing antigen release may be administered to the subject in need at the same time as the immunologic adjuvant formulation.
  • the means for inducing antigen release from a cell can be formulated with or encapsulated in the immunologic adjuvant formulation as a single dose or two distinct doses for being administered simultaneously.
  • the adjuvants and the cytotoxic agents may be formulated in one nanoparticle or conjugated to one antibody.
  • the cancer cell debris were then collected and incubated with a JAWSII mouse dendritic cell (DC) line to examine their immunogenicity.
  • DC JAWSII mouse dendritic cell
  • HeLa cells incubated with either free cGAMP or cGAMP -loaded nanoparticles for 24 hr were treated with cisplatin, and the resulting cellular debris w'ere collected to stimulate DCs.
  • the DCs were collected after 24 hr of incubation and studied for DC activation markers CD86 and CD80.
  • In vitro activation of DCs by immunogenic cellular debris was demonstrated by FACS analysis.
  • the cytokine profile of the activated DCs was estimated by ELISA analysis for TNF-a and EFN-b.
  • Nanoparticle-mediated adjuvant retention with the B16 ceils was validated using whole body imaging, flow cytometric analysis, and fluorescence microscopy.
  • Dye-labelled STING agonist (2'-[D Y -547]-AHC-c-di GMP) was administered in its free form and nanoparticle form intra-turaoraily and examined after 24 hours. It was observed that the nanoparticle formulation of the STING agonist w3 ⁇ 4s well retained inside the tumor (FIG. 8(a)).
  • flow cytometry and confocal microscopy Upon cancer cell extraction from the dissected tumors, significant STING agonist association within the cancer cells was also confirmed from flow cytometry and confocal microscopy (FIG. 8(a)).
  • flow cytometry and confocal microscopy 2'-[D Y -547]-AHC-c-di GMP
  • mice were inoculated subcutaneously with B 16-OVA tumors. When the tumors appeared palpable, they w ? ere pre treated with free cGAMP or cGAMP-loaded nanoparticles prior to chemotherapy by cisplatin (CTTP). 24 hours following intra-tumoral adjuvant administration, the mice were given an intra-tumoral cisplatin injection to induce cell death (FIG. 9(a)). These two groups are designated cGAMP+CTTP and NP+CTTP to differentiate the use of free cGAMP and cGAMP-loaded nanoparticles, respectively.
  • CTTP cisplatin
  • mice receiving no treatment (placebo), cistplatin only (CTTP only) and cGAMP-loaded nanoparticles only (NP only) w ? ere also prepared.
  • CTP only cistplatin only
  • NP only cGAMP-loaded nanoparticles only
  • cGAMP administration 2.5 pg of the adjuvant was administered in 50 pL of solution.
  • cisplatin administration 235 pg of the ehemodrug was administered in 100 pL of solution.
  • the tumor volumes were monitored over time.
  • 50% of the animals (5 out of 10) showed complete tumor regression. In contrast, no other group had any tumor with complete regression (FIG. 9(b), (c)).
  • the immunologic adjuvant formulation comprises an adjuvant conjugated with a small molecule, a peptide, an antibody, an aptamer, a sugar moiety, a polymer or a combination thereof for recognizing and binding to a moiety on a cell surface.
  • the antibody comprises an immunoglobulin molecule, an Fv, a disulfide linked Fv, a monoclonal antibody, a scFv, a chimeric antibody, a single domain antibody, a CDR-grafted antibody, a diabody, a humanized antibody, a multispecific antibody, an Fab, a dual specific antibody, an Fab’ fragment, a bispecific antibody, an F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementarity determining region (CDR), or a single chain antibody.
  • an immunoglobulin molecule an Fv, a disulfide linked Fv, a monoclonal antibody, a scFv, a chimeric antibody,
  • CpG-FAM was conjugated to antibodies for pathogenic cell association.
  • Anti-CD44 which targets CD44 that are highly expressed on HeLa cells was adopted for adjuvant conjugation.
  • 1 mg of the antibodies was first mixed with sulfo-SMCC at a molar ratio of 1 :20 in IX PBS for 24 h.
  • the functionalized antibodies were purified using a centrifugal filter with a molecular weight cut off (MWCO) of 30 kDa.
  • the antibodies were then mixed with CpG-ODN containing a terminal cysteine group.
  • a FAM dye was attached to the CpG-ODN for visualization (FIG. 10).
  • the CpG-conjugated antibodies were collected using a centrifugal filter with a MWCO of 30 kDa
  • CpG-FAM was encapsulated into polymeric nanoparticles for enhancing tumor cell uptake.
  • a double emulsion process was employed to prepare the CpG-FAM loaded nanoparticles.
  • 20 mg/mL of CpG was first dissolved in 50 pL of 100 mM of phosphate buffer, which was then emulsified in 500 _uL of ethyl acetate containing 50 mg/mL of PLGA (10,000 Da, carboxyl -terminated, 50:50 iactide:glycolide).
  • the resulting water-in-oil emulsion was then emulsified in 5 mL of 10 mM phosphate buffer.
  • the water-in-oil-in-water emulsion was then poured into 10 mL of 10 mM of phosphate buffer and the ethyl acetate was evaporated under nitrogen bombardment. After 1 h of solvent evaporation, the nanoparticle-encapsulated CpG was collected using a centrifugal filter with a MWCO of 30 kDa. Successful preparation of sub- 100 nm nanoparticles containing CpG was verified using CryoEM (FIG. 13).
  • the HeLa cells were then treated with a cytotoxic agent, doxorubicin, to induce cell death and generate cellular debris. Following 20 h of doxorubicin treatment, the cellular debris were collected and fed to JAWSII mouse dendritic cells. The JAWSII cells were then assessed for activation markers such as CD80 and CD86 after 20 hr of incubation with the cellular debris. It was observed that the debris of HeLa cells treated with nanoparticle-encapsulated CpG triggered the highest levels maturation markers (FIG. 15).
  • doxorubicin cytotoxic agent
  • the means for inducing antigen release may be administered to the subject in need at the same time as the immunologic adjuvant formulation.
  • the means for inducing antigen release may be administered to the subject in need before or after administration of the immunologic adjuvant formulation.
  • daunorubicin was encapsulated into nanoparticles along with ed ⁇ GMP STING agonist to simultaneously enable intracellular adjuvant delivery while triggering ceil death.
  • a double emulsion process was employed to prepare the nanoparticles co- encapsulating daunorubicin and cd-GMP.
  • Solution containing 10 mg/mL of daunorubicin and 10 mg/mL of cd-GMP was first prepared with 100 mM of phosphate buffer, and 50 pL of the solution was emulsified in 500 pL of ethyl acetate containing 50 mg/mL of PLGA (10,000 Da, carboxyl -terminated, 50:50 lactide:gfycolide). The resulting water-in-oil emulsion was then emulsified in 5 rnL of 10 mM phosphate buffer.
  • the water-in-oil-in- water emulsion was then poured into 10 rnL of 10 mM of phosphate buffer and the ethyl acetate was evaporated under nitrogen bombardment.
  • the daunorubicin/cd-GMP nanoparticles were collected using a centrifugal filter with a MWCO of 30 kDa.
  • nanoparticles encapsulating daunorubicin in the absence of cd-GMP was prepared with 20 mg/mL of daunorubi cin as the inner aqueous phase.
  • Successful preparation of sub- 100 nm nanoparticles containing daunorubicin and cd-GMP was verified by cryoEM (FIG. 16).
  • CT26 colon cancer cells with three distinctive formulations: 1) daunorubicin nanoparticles only (DRNP); 2) free cd-GMP and daunorubicin nanoparticles (cd- GMP+DR NP); and 3) Nanoparticles co-eneapsuiating daunorubicin and cd-GMP ((cd- GMP+DR) NP.
  • DRNP daunorubicin nanoparticles only
  • cd- GMP+DR NP free cd-GMP and daunorubicin nanoparticles
  • Nanoparticles co-eneapsuiating daunorubicin and cd-GMP (cd- GMP+DR) NP.
  • To 2 mL of CT26 culture were given 10 itg of daunorubicin and 10 pg of cd- GMP. Following 3 h of treatment, cellular debris was collected and fed to JAWSII mouse dendritic cells.
  • the JAWSII cells were then assessed for activation markers such as CD80 and CD86 after 24 hr of incubation with the cellular debris. It was observed that the debris of CT26 ceils treated with nanoparticles co-encapsuiating daunorubicin and cd-GMP triggered the highest levels maturation markers such as CD80 and CD86 (FIG. 17).
  • the description above demonstrates that the treatment kit comprising immunologic adjuvant formulation is capable of facilitating immune response target specific cells.
  • a novel treatment modality was also demonstrated based on the cell-associating immunologic adjuvant, which is capable of enhancing adaptive immunity to the diseased cell.
  • the method and the treatment kit are applicable to any existing treatments that induce release of cellular debris and antigens, including but not limited to chemotherapy, radiation therapy, photothermal therapy, immunotherapy, and surgical interventions.
  • Other approaches for associating immunologic adjuvants with pathogenic cells including but not limited to methods that facilitate intracellular entiy, membrane tethering, and surface binding of immunologic adjuvants, are also contemplated.
  • the immunologic adjuvants can also include but not limited to MPLA, CpG, poly(LC), variants of cyclic-dinucleotides, and other immune-stimulatory molecules.
  • Treatment kit that contain both immunologic adjuvant formulation and means for inducing antigen release, such as nanoparticles containing both cGAMP and chemodrugs, for ceil association are also contemplated.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Optics & Photonics (AREA)
  • Nanotechnology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)

Abstract

This disclosure provides treatment kit that are capable of modulating the immune response. The treatment kit may also be used enhance the immunogenicity of antigens released from cell debris. Also provided are methods of using the treatment kit.

Description

CELL-ASSOCIATING IMMUNOLOGIC ADJU VANTS FOR TREATMENT
ENHANCEMENT
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provi sional Application No 62/613,574, filed January 4, 2018.
FIELD OF INVENTION
[0002] This present disclosure relates to a treatment kit and methods for using the same.
BACKGROUND OF THE INVENTION:
[0003] Effective immune potentiation against antigenic targets hinges on the coordinated presentation of antigens and immune-stimulatory signals. Activation of innate immune signals via agonists of the pathogen recognition receptors trigger the secretion of specific cytokine milieu, which in turn shape the adaptive immunity against the antigen target. This mechanism is frequently exploited in vaccine designs as vaccine antigens are often formulated with immunologic adjuvants to enhance immune potentiation. Once the immune system is primed against a foreign antigen, the adaptive immune cells work to recognize and eliminate similarly suspicious cells, while retaining a memory of the same antigen, allowing a quicker, more potent assault upon recurrence of infectionf l ]
[0004] The role of adaptive immunity is increasingly recognized in the battle against malignant cells, i.e. cancer, as immune cells such as cytotoxic T lymphocytes can facilitate eradication of these cellular targets. Tumor cells frequently express tumor-associated antigens, which serve as targets for vaccine development. Recent studies have shown that by formulating tumor- associated antigens with immunologic adjuvants, the resulting vaccine formulations can enhance tumor-specific immunity and facilitate better cancer treatment outcomes [2] These cancer vaccine formulations require genomic analysis of tumor samples for the identification of specific peptide sequences, which can then be synthesized for vaccine development.
[0005] Alternatively, whole cell-based tumor vaccines have also been developed by extracting patient cancer cell samples or using allogeneic cancer cells. In these approaches, tumor cell derivatives are formulated with adjuvants exogenously prior to administration. The adjuvant mixtures may then increase the immunogenicity of tumor-associated antigens in the tumor cell derivatives. In one example, dead cancer cells were functionali zed with adjuvant-nanoparticles ex vivo to vaccinate against subcutaneous tumorsp]. The platform in itself has a 30% efficiency that can be increased up to 78% when combined with an immune check-point blocker therapy. In another example, allogenic cancer cells were formulated with a STING agonist ex vivo as a vaccine formulation[4]. The formulation, known as STING-VAX, was able to elicit anti-tumor immunity upon administration in animal models.
[0006] The induction of endogenous immune-stimulatory molecules has also been identified as an important factor behind successful cancer treatment. Several cancer treatments, including several types of chemotherapy, radiation, and oncolytic viruses, have been demonstrated to induce cellular stress in cancer cell and trigger immunogenic cell death (ICD). This type of cell death is associated with endogenous release of danger associated molecular patterns (DAMPs), which consist of a combination of peptides, proteins, or nucleic acids originated from the dying cells[5]. Together with the antigenic materials from the dying cells, these endogenous danger signals promote antigen-specific adaptive immunity. Mounting evidence suggests that this innate mechanism of co-displaying antigens and immune-stimulatory molecules is critical to containing disease progression in patients affected by a plethora of neoplasms[6]. It is thus conceivable that such mechanism may be enhanced with the aid of exogenous adjuvants, which may further amplify the immune response to released tumor antigens following cancer cell death.
SUMMARY OF THE INVENTION
[0007] In the present application, we develop immunologic adjuvants that can be associated with cells and demonstrate its use to enhance the immunogenicity of released antigens from ceils during treatment. As shown in FIG. 1, by associating exogenous immunologic adjuvants with functionalities that can associate with cancer cells— such as through the means of cellular internationalization, membrane association, or surface ligand binding— the adjuvants can flag the cellular targets for immune potentiation. Subsequent treatment that induces release of cellular debris, vesicles, and antigens would thus trigger immune responses regardless of whether the treatment itself triggers endogenous danger signals. This application thus concerns about both functionalization of immunologic adjuvants and treatment modalities associated with the functionalized adjuvants. The claimed invention may be applied to any pathogenic cells beyond cancers. Specifically, an immunologic adjuvants are designed to associate with malignant cells, which can be through either intracellular (cellular internationalization) or extracellular means (membrane-tethering or surface ligand binding). Malignant cells treated with the functionalized adjuvants are then induced to release cellular debris, vesicles, and antigens. Induction of antigen release can be triggered by any stimuli know in the art. For instance, the stimuli may at least comprise chemical agents, biological agents, light, or mechanical stimuli.
[0008] In the scope of present invention, a treatment modality for pathogenic cell is provided where the diseased cells are first administered with exogenous adjuvants. The exogenous adjuvants can be associated with the cells as follows; (1 ) an adjuvant formulation that allows intracellular delivery of the desired adjuvants, e.g. nanoparticles, exosomes, engineered cell penetrating peptides etc.; (2) an adjuvant formulation that can be partitioned into the cell membrane, e.g. adjuvant with lipophilic linkers, amphiphilic adjuvants etc.; and (3) an adjuvant formulation that has binding affinity to cell surface moieties and capable of physically associating with cell membrane moieties, e.g. adjuvant coupled with antibody, adjuvant coupled with aptamers, adjuvant-sugar formulations etc
[0009] Once the adjuvant is associated with the infected cell, the cell is then treated with a vesiculating, antigen-releasing stimuli that include but not limited to chemotherapy drugs, biologic agents, irradiation, photolytic agents, mechanical disruption etc. The regimen facilitates the production of co-localized antigen with exogenous adjuvants in vivo. In principle, the cell -associating immunogenic adjuvants can be applied to complement any existing or emerging treatment modalities aimed at eradicating pathogenic ceils.
[0010] In one aspect, provided herein is a treatment kit, comprising: an immunologic adjuvant formulation capable of physically associating with ceils; and a means for inducing antigen release from a cell.
[0011] In another aspect, provided herein is a method of treating a disease, comprising; administering to a subject in need thereof an immunologic adjuvant formulation capable of physically associating with cells, and administering to the subject in need thereof a means for inducing antigen release from a cell.
[0012] Preferably, the immunologic adjuvant formulation may comprise a. polymeric nanoparticle encapsulating an adjuvant, and the polymeric nanoparticle may further comprise: a polymeric shell impermeable to water, and one or more aqueous cores enclosed by the polymeric shell
[0013] Preferably, the polymeric shell may have a thickness less than 25 nm and the polymeric nanoparticle has an outer diameter of 30-600 nm.
[0014] Preferably, the means for inducing antigen release from a cell may be encapsulated in the polymeric nanoparticle. [0015] Preferably, the immunologic adjuvant formulation may comprise MPLA, CpG, poly(I:C), or variants of cyclic-dinucleotides.
[0016] Preferably, the immunologic adjuvant formulation may comprise an agonist to a cytoplasmic pattern recognition receptor or a stimulator of interferon genes (STING).
[0017] Preferably, the agonist may be cyclic di-GMP or cyclic GAMP
[0018] Preferably, the immunologic adjuvant formulation may comprises an adjuvant conjugated to a small molecule, a peptide, an antibody, an aptamer, a sugar moiety, a polymer or a combination thereof for recognizing and binding to a moiety on a cell surface.
[0019] Preferably, the antibody may be an immunoglobulin molecule, an Pv, a disulfide linked Fv, a monoclonal antibody, a scFv, a chimeric antibody, a single domain antibody, a CDR- grafted antibody, a diabody, a humanized antibody, a multispecific antibody, an Fab, a dual specific antibody, an Fab’ fragment, a bispecific antibody, an F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementarity determining region (CDR), or a single chain antibody.
[0020] Preferably, the immunologic adjuvant formulation may comprises an adjuvant conjugated to a molecule capable of tethering to a cell membrane.
[0021] Preferably, the molecule may be lipophilic or amphiphilic.
[0022] Preferably, the lipophilic molecule may comprise fatty acid chains, cholesterol, or phospholipids.
[0023] Preferably, the amphiphilic molecule may comprises lipid-PEG conjugates.
[0024] Preferably, the immunologic adjuvant formulation may comprises an adjuvant formulated with nanoparticulates or cell penetrating peptides for intracellular delivery.
[0025] Preferably, the cell penetrating peptides may comprise HIV-1 TAT peptide (GRKKRRQRRRPPQ), penetratin (RQDQWFQNRRMKWKK), poly arginines (Rn), or transportan (GWTLNSAGYLLGINLKALAALAKKIL).
[0026] Preferably, the means for inducing antigen release may comprise chemical agents, biological agents, irradiation, photolytic agents, mechanical disruptions, or a combination thereof.
[0027] Preferably, the chemical agents may comprise a cytotoxic agent.
[0028] Preferably, the cytotoxic agent may comprise Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (DM1), anthracycline, pyrrolobenzodiazepine, a-amanitin, tubulysin, benzodiazepine, erlotinib, bortezomib, fulvestrant, sunitinib, !etrozoie, imatimb mesylate, PTK787/ZK 222584, oxalipiatin, leucovorin, rapamycin, lapatinib, lonafamib (SARASAR®, SCH 66336), sorafenib, gefitinib, AG1478, AG1571, alkylating agent; alkyl sulfonate; aziridines; ethylenimme; methylamelamine; acetogenins; camptothecin; bryostatin; callystatin; CC-1065; cryptophycins; dolastatin; duocarmycin; eleutherobin; pancrati statin, sarcodictyin; spongistatin; chlorambucil, chlomaphazine; cholophosphamide; estramustine; ifosfamide; mechlorethamine; mechlorethamine oxide hydrochloride; melphalan; novembichin; phenesterine; prednimustine; trofosfamide; uracil mustard; carmustine; chlorozotocin; fotemustine; lomustine; nimustine; ranimustine; calicheamicin; dynemicin; clodronate; esperamiein, neocarzinostatin chromophore; aclacinomysins; actinomycin; authramycin; azaserine; bleomycins; cactinomycin; carabicin; caminomycin; carzinophilin; chromomycinis; dactinomycin; daunorubicin; detorubicin; 6-diazo-5-oxo-L-norleucine; doxorubicin; epirubicin; esorubicin; idarubicin; marcellomycin,;mitomycin; mycophenolic acid; nogalamycin, olivomycins; peplomycin; potfiromycin; puromycin; quelamycin; rodorubicin; streptonigrin; streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate; 5-fluorouracil (5-FU); denopterin; pteropterin; trimetrexate; fludarabine; 6- mercaptopurine; thiamiprine; thioguanine; ancitabine; azacitidine; 6-azauridine; carmofur; cytarabine; dideoxyuridine; doxifluridine; enocitabine; floxuridine; calusterone; dromostanolone propionate; epitiostanol; mepitiostane; testolactone; aminoglutethimide; mitotane; trilostane; frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea, lentinan; lonidainine; maytansine, ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecene; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoid; paclitaxel; doxetaxel; chloranbucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; cisplatin; carboplatin; vinblastine; platinum; etoposide; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor; difiuorom ethyl ornithine (DMFO), retinoid; capecitabine; or a combination thereof.
[0029] Preferably, the photolytic agents comprises Photofrin, Laserphyrin, Aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m -tetrahy droxy phenyl chi orin (mTHPC), chlorin e6 (Ce6), Allumera, Levulan, Foscan, Metvix, Hexvix, Photochlor, Photosens, Photrex, Lumacan, Visonac, Amphinex, Verteporfin, Purlytin, ATMPn, Zinc phthalocyanine (ZnPc), Protoporphyrin IX (PpIX), Pyropheophorbidea (PPa), Pheophorbide a (PhA), or a combination thereof.
[0030] Preferably, the method may further comprise: encapsulating an adjuvant in a polymeric nanoparticle. The polymeric nanoparticle may comprise: a polymeric shell impermeable to water, and one or more aqueous cores enclosed by the polymeric shell.
[0031] Preferably, the means for inducing antigen release may be administered to the subject in need before or after administration of the immunologic adjuvant formulation.
[0032] Preferably, the means for inducing antigen release may be administered to the subject in need at the same time as the immunologic adjuvant formulation.
[0033] Preferably, the method may further comprise: encapsulating the means for inducing antigen release from a cell in the immunologic adjuvant formulation.
[0034] Preferably, the method may further comprise: conjugating an adjuvant with a small molecule, a peptide, an antibody, an aptamer, a sugar moiety, a polymer or a combination thereof recognizing and binding to a moiety on a cell surface to form the immunologic adjuvant formulation.
[0035] Preferably, the method may further comprise: conjugating an adjuvant with a molecule capable of tethering to a cell membrane.
[0036] Preferably, the method may further comprise: formulating an adjuvant with nanoparticulates or cell penetrating peptides for intracellular delivery.
[0037] Preferably, the step of administering to the subject may be by at least one mode selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intra- articular, intrabronchiai, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelia!, intracerebe!lar, intracerebroventricuiar, intracolic, intracervicai, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasai, and transdermal.
[0038] Preferably, the disease may be cardiovascular disease, cancer, autoimmune disease, or infection.
BRIEF DESCRIPTION OF THE DRAWINGS [0039] FIG. 1 illustrates pathogenic cell-associating immunologic adjuvants and their function in enhancing subsequent treatments. Immunologic adjuvants are functionalized to associate with pathogenic cells through various means such as intracellular delivery, membrane tethering, and surface binding. The resulting cells are then subjected to treatments that induce generation of adjuvant-associated cellular debris, which can promote adaptive immunity via coordinated antigen and adjuvant transport.
[0040] FIG. 2 depicts encapsulation efficiency of polymeric nanoparticles for a nucleic acid and a protein. A: Observation of empty nanoparticles (left), nanoparticles loaded with dye- labelled DNA (middle), and nanoparticles loaded with dye-labelled bovine serum albumin (right) following pelleting by ultracentrifugation. Distinctive colored pellets indicate successful encapsulation of DNA and BSA proteins. B: Empty nanoparticles visualized by cryoEM. C: DNA-loaded nanoparticles visualized by cryoEM. D: BSA protein-loaded nanoparticles visualized by cryoEM. Effective DNA and protein loading could be observed through the highly grainy textures inside nanoparticles.
[0041] FIG. 3 depicts the effect of polymeric nanoparticles on encapsulation and controlled release of STING agonists for immune stimulation. A: Preparation of adjuvant-loaded thin- shell hollow nanoparticles. B: Size of nanoparticles as measured by nanoparticle tracking analysis. C: Encapsulation of cyclic-di-GMP as verified by gradient HPLC. D: CryoEM visualization of thin-shell hollow nanoparticles. E: Cargo release study revealed a pH-sensitive triggered release profile for the nanoparticles.
[0042] FIG. 4 depicts STING agonist-loaded nanoparticles on enhancing lymphatic cytokines while minimizing systemic cytokines. A: Induction of TNF-a in a mouse dendritic cell line following incubation with cd-GMP nanoparticles and free cd-GMP. B: Induction of IL-6 in the same mouse dendritic cell line. C: Induction of IFN-b in the mouse dendritic cell line. D: Activation of JAWSII upon incubation with equivalent doses of cd-GMP in free molecule form and in nanoparticle formulation. E: Quantification of lymph node IFN-b 48 hours following footpad injection of free cd-GMP or nanoparticle cd-GMP. F; Level of systemic TNF-a following footpad injection of free cd-GMP and nanoparticle cd-GMP.
[0043] FIG. 5 depicts a treatment modality based on a tumor cell-associating immunologic adjuvant (cGAMP-loaded nanoparticle). Following nanoparticJe uptake, the cells containing exogenous adjuvants are then treated to generate a dead cellular debris. The cellular debris containing the exogenous adjuvants subsequently triggers adaptive immunity to improve treatment outcome. [0044] FIG. 6 depicts (a) CryoEM visualization of a fluorescently labelled cGAMP -loaded nanoparticle (b) Confocal analysis and (c) flow-cytometric analysis show association of cGAMP with dead cells and debris following treatment with cGAMP-loaded nanoparticles.
[0045] FIG. 7 depicts cellular debris generated by HeLa cells incubated with cGAMP nanoparticles were more immunogenic than those incubated with free cGAMP. The HeLa cell associating cGAMP nanoparticles resulted in increased DC expression of CD80 (a), CD86 (b), and elevated DC secretion of TNF-a(c) and IFN-p(d)
[0046] FIG. 8 depicts adjuvant retention with tumors and cancer cells were examined upon intratumoral injection of a dye-labelled STING agonist (red) in its free (Free Adjuvant) and nanoparticle form (Adjuvant Nanoparticle). 24 hours following the adjuvant administration the tumors and extracted cancer cells were examine by (a) whole-body imaging, (b) flow- cytometric analysis, and (c) fluorescence microscopy. The examinations demonstrate significant adjuvant retention inside the tumor and within cancer cells upon nanoparticle administration.
[0047] FIG. 9 depicts tumor treatment efficacy with different adjuvant and chemodrug combinations, including placebo, cisplatin only (CTTP only), cGAMP-loaded nanoparticle only (NP only), free cGAMP followed by cisplatin (cGAMP+CTTP), and cGAMP-loaded nanoparticle followed by ci splatin (NP +CTTP). (a) Tumor implantation and treatment regimen for B 16-OVA murine melanoma subcutaneous model (b) Tumor progression of all treatment groups over 60 days (c) Survival analysis curve of the different treatment groups (n=9-l 1). (c) Comparison of mean tumor volume in a tumor rechallenge experiment with tumor-free surviving animals from the NP+CTTP group (n i). The tumor growth was compared to a naive control animal that was inoculated the same number of tumor cells (n=:l).
[0048] FIG. 10 depicts the preparation of antibody-conjugated CpG-ODN.
[0049] FIG. 1 1 depicts (A ) Fluorescence microscopy observation of HeLa cellular monolayer following 3 h of incubation with free CpG-F M (middle) and antibody-conjugated CpG-F M (aCD44-CpG-FAM; right). Enhanced CpG association by the antibody-conjugated CpG-FAM was observed with a higher number of fluorescence punctates. (B) Flow' cytometric analysis of cells with CpG-FAM. (C) Quantification of cells associated with CpG-FAM. Anti-CD44 conjugates (aCD44) yielded higher CpG-FAM association on HeLa cells than free CpG-FAM (free).
[0050] FIG. 12 depicts expression of maturation markers (A) CD80 and (B) CD86 by JA W SII ceils following incubation with HeLa ceil debris treated with free CpG (free) or anti-CD44 conjugated CpG (aCD44). [0051] FIG. 13 depicts (A) Schematic presentation of nanoparticle-encapsulated CpG. (B) CryoEM visualization of nanoparticle-encapsulated CpG.
[0052] FIG. 14 depicts (A) Fluorescence microscopy observation of HeLa cellular monolayer following 24 h of incubation with free CpG-FAM (middle) and nanoparticle-encapsulated CpG (CpG-FAM NP; right). Enhanced CpG association was observed with a high number of fluorescence punctates. (B) Flow cytometric analysis of cells with CpG-FAM. (C) Quantification of cells associated with CpG-FAM Anti-CD44 conjugates yielded higher CpG- FAM association on HeLa cells than free CpG-FAM.
[0053] FIG. 15 depicts expression of maturation markers (A) CD80 and (B) CD86 by JAWSII ceils following incubation with HeLa cell debris treated with free CpG (free) or nanoparticle- encapsulated CpG (NP).
[0054] FIG. 16 depicts (A) Schematic presentation of a nanoparticle co-encapsulating a chemotherapeutic drug (daunorubicin) and an adjuvant (cd-GMP). (B) CryoEM visualization of nanoparticles co-encapsulating daunorubicin and cd-GMP
[0055] FIG. 17 depicts expression of maturation markers (A,B) CD86 and (C,D) CD80 by JAWSII cells following incubation with CT26 cell debris treated with nanoparticles coencapsulating daunorubicin and cd-GMP ((cdGMP+DR)NP), free cd-GMP and daunorubicin nanoparticles (cdGMP + DR NP) or daunorubicin nanoparticle only (DR NP)
[0056] FIG. 18 depicts secretion of IFN-b from JAWSII cells following incubation with CT26 cell debris treated with nanoparticles coencapsulating daunorubicin and cd-GMP ((cdGMP+DR)NP), free cd-GMP and daunorubicin nanoparticles or daunorubicin nanoparticle only (DR NP).
DETAILED DESCRIPTION
[0057] The foregoing and other aspects of the present disclosure will now be described in more detail with respect to other embodiments described herein. It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
[0058] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
[0059] As used herein, the terms“comprises,”“comprising,”“includes,”“including,”“has,” “having,”“contains”,“containing,”“characterized by” or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any limitation explicitly indicated. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, or method.
[0060] The transitional phrase“consisting of’ excludes any elements, steps, or ingredients not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
[0061] The transitional phrase“consisting essentially of’ is used to define a composition, method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel character! stic(s) of the claimed invention. The term“consisting essentially of’ occupies a middle ground between“comprising” and “consisting of’.
[0062] Where applicants have defined an invention or a portion thereof with an open-ended term such as“comprising,” it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms“consisting essentially of’ or“consisting of.”
[0063] As used herein, the term“about” is used to indicate that a value includes for example, the inherent variation of error for a measuring device, the method being employed to determine the value, or the variation that exists among the study subjects. Typically the term is meant to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%0, 15%, 16%, 17%, 18%, 19% or 20% variability depending on the situation.
[0064] The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.”
[0065]“treating,” or“treatment” is referred to herein as administration of a therapeutic composition to a subject with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate a disorder, symptoms of the disorder, a disease state secondary to the disorder, or predisposition toward the disorder.
[0066]“Subject” as used herein refers to a mammalian subject diagnosed with or suspected of having or developing diseases such as cardiovascular disease, cancer, autoimmune disease, or infection. Exemplary' patients may be humans, apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents and other mammalians with the diseases that can benefit from the treatment.
[0067]“Administering” or“Administration” is referred to herein as providing a treatment kit of the present application to a subject. By way of example and not limitation, administration may be performed via parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intraeerebe!lar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transderma!. For example, injection may be performed by intravenous (i.v.) injection, sub- cutaneous (s.c.) injection, intraderm al (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes may be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route.
[0068] As used herein, an "immunologic adjuvant formulation" means a formulation that when administered to a subject is capable of inducing an immune response in the subject. When administered in combination with an antigen, the“immunologic adjuvant formulation” are capable of eliciting an anti gen -specific immune response. The“immunologic adjuvant formulation” will be associated with cells via cellular internationalization, membrane association, and/or surface ligand binding after being administered by a subject.
[0069] As used herein,“cellular internationalization” means an adjuvant formulation that allows intracellular delivery' of the desired adjuvants. For instance, “cellular internationalization” of the desired adjuvants may be achieved at least by nanoparti cies, exosomes, engineered cell penetrating peptides etc
[0070] As used herein,“membrane association” means an adjuvant formulation that can be partitioned into the cell membrane. For instance,“membrane association” of the desired adjuvants may be achieved at least by lipophilic linkers, amphiphilic adjuvants etc.
[0071] As used herein,“surface ligand binding” means an adjuvant formulation that has binding affinity to cell surface moieties and capable of physically associating with cell membrane moieties. For instance,“surface ligand binding” of the desired adjuvants may be achieved at least by antibody, aptamers, sugar formulations etc.
[0072] As used herein, a "means for inducing antigen release from a cell" means using a chemical or physical approaches to induce the treated cells to release cellular debris, vesicles, and/or antigens that trigger immune response. For example, the means may comprise chemical agents, biological agents, irradiation, photolytic agents, mechanical disruptions, or a combination thereof.
[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.
[0074] In an embodiment, a treatment kit for enhancing an immunogenicity of released antigens from cells is disclosed. The treatment kit comprises an immunologic adjuvant formulation capable of physically associating with cells; and a means for inducing antigen release from a cell.
[0075] In a preferred embodiment, the immunologic adjuvant formulation is a polymeric nanoparticle encapsulating an adjuvant, which comprises: a polymeric shell impermeable to water, and one or more aqueous cores enclosed by the polymeric shell.
[0076] The polymeric shell may have a thickness less than 25 nm and the polymeric nanoparticle has an outer diameter of 30-600 nm. Preferably, the thickness is less than 24 nm, 23 nm, 22 nm, 21 nm, 20 n n, 18 nm, 16 nm, 14 nm, 12 nm, or 10 nm. The outer diameter is preferably in a range from 30-550 nm, 30-500 nm, 30-450 nm, 30-400 nm, 30-350 nm, 30-300 nm, 30-250 nm, 30-200 nm, 30-150 nm, 30-100 nm, 35-550 nm, 35-500 nm, 35-450 nm, 35- 400 nm, 35-350 nm, 35-300 nm, 35-250 nm, 35-200 nm, 35-150 nm, 35-100 nm, 40-550 nm, 40-500 nm, 40-450 nm, 40-400 nm, 40-350 nm, 40-300 nm, 40-250 nm, 40-200 nm, 40-150 nm, 40-100 nm, 45-550 nm, 45-500 nm, 45-450 nm, 45-400 nm, 45-350 nm, 45-300 nm, 45- 250 nm, 45-200 nm, 45-150 nm, 45-100 nm, 50-550 nm, 50-500 nm, 50-450 nm, 50-400 nm, 50-350 nm, 50-300 nm, 50-250 n n, 50-200 nm, 50-150 nm, or 50-100 nrn.
[0077] In one embodiment, the polymeric shell is formed of an amphiphilic polymer that contains a non -polar segment and a polar terminal group. Examples of the non-polar segment include, but are not limited to, poly(lactie acid), poJy(lactic-co-glycolic acid) (PLGA), polycaprolactone, and polyurethane. The lactic acid to glycolic acid molar ratio of the PLGA varies from 1 :99 to 99: 1 , from 10:90 to 90: 10, from 15:85 to 85:15, from 20:80 to 80:20, from 25:75 to 75:25, from 30:70 to 70:30. Preferably, the lactic acid to glycolic acid molar ratio of the PLGA is 50:50 or 75:25. The polar terminal group can be a negatively charged group, a positively charged group, a zwitterionic group, or a neutral group. Examples of the negatively charged group include a carboxylic acid, a succinic acid, and a sulfonic acid. Examples of the positively charged group include an amine and an ami dine. Examples of the zwiterionic group include a carboxybetaine and a sulfobetaine. An example of the neutral group is a saccharide.
[0078] An exemplary polymeric shell is formed of a polymer containing poly(lactic-co- giycolic acid) as the non-polar segment and a carboxylic acid as the polar terminal group.
[0079] The polymeric nanoparticle described herein can be a polymeric hollow nanoparticle platform with a defect-free polymeric shell having a thickness of 25 nm or less. The hollow polymeric nanoparticle typically has an outer diameter between 30 and 600 nm.
[0080] By minimizing the shell thickness, the polymeric nanoparticle can be formed with a large i nterior aqueous space capable of maximizing the cargo loading. For particles above 100 nm in outer diameter, the interior aqueous space can possess a diameter at least 80% of the particle’s outer diameter or can be of multiple compartments with a large collective volume. High efficiency encapsulation of hydrophilic dyes and nucleic acids are demonstrated with the thin-shell hollow nanoparticles in the absence of complementary' binding molecules. The thin- sheil nanoparticles are demonstrated to be resistant to osmotic stress, a feature attributable to complete, defect-free polymeric shell that is impermeable to water. The polymeric nanoparticle of this application can be used for delivering bioactive agents in various fields, including drug delivery' and vaccine development.
[0081] Polymeric nanoparticles, particularly those consisting of biodegradable and biocompatible polymers such as poly(lactic-co-giycolic acid)(PLGA), have received considerable attention in nanomedicine research because of the polymer’s numerous features including biocompatibility, biodegradability, and synthetic flexibility. However, due to the polymer’s inherent hydrophobicity, PLGA-based nanoparticles have been limited to the delivery of water-insoluble compounds in clinical. Encapsulation of hydrophilic and macromolecular cargoes in polymeric nanoparticles remains a challenge as polymers tend to form solid nanospheres with little or no aqueous core space to carry' hydrophilic and macromolecular cargoes, e.g., siRNA.
[0082] Given that macromolecular encapsulation is common in natural nanoparticulates in the form of viruses, an ideal nanocarrier should possess a thin shell enclosing a large aqueous volume for the packaging of bioactive molecules. The thin shell is also preferably defect free and water impermeable to allow reliable cargo encapsulation. [0083] In an embodiment, a method of treating a disease is provided. The method comprises administering to a subject in need thereof an immunologic adjuvant formulation capable of physically associating with cells; and administering to the subject in need thereof a means for inducing antigen release from a cell. Examples of the disease includes, but is not limited to, cardiovascular disease, cancer, autoimmune disease, or infection.
[0084] Further disclosed in detail herein is a method of preparing the above-described polymeric nanoparticle
[0085] In general, the thin-shell hollow nanoparticle is prepared based on a double emulsion process using amphiphilic polymers with high contrast of polarity at their terminus. More specifically, a solution of carboxyl-terminated PLGA in dichloromethane (DCM) is first used to emulsify an aqueous phase containing a cargo under sonic dispersion to form an emulsion. The emulsion thus formed is subsequently emulsified in an outer aqueous phase using fluidic dispersion.
[0086] By adjusting the polymer concentration and dispersion force or using polymers with defined length and sharp polarity in the double emulsion process, the preparation method described above can provide hollow polymeric nanoparticles with outer diameters between 30 and 600 nm, e.g., 30-40 nm and 100-600 nm.
[0087] The nanoparticles are prepared based on a water-oil-water double emulsion process in which polymers dissolved in a solvent system is first used to emulsify an aqueous phase. The emulsion is subsequently emulsified by a secondary aqueous phase. The inner and outer aqueous phases can be of any polar solution, e.g., water, acetic acid, and ethanol. The aqueous phase contains solubilized molecules to modulate the solution’s acidity and viscosity, which include sodium phosphate and sodium bicarbonate. In one embodiment, water is used as an anti-solvent for the nanoparticle preparation.
[0088] The water-oil-water double emulsion method described above for preparing the polymeric nanoparticle of in the present application has two key features: namely, (i) emulsion between different phases is achieved through polymers with inherently high contrast in polarity (PLGA with a carboxyl-terminal group) rather than using an surfactant, e.g., vitamin E-D-a- tocopherol polyethylene glycol succinate and polyfvinyl alcohol), which enhances the emulsifying capability to minimize polymer shell thickness and has a higher commercial value without using surfactant materials, and (ii) controlled fluidic dispersion using either a microfluidizer or sonication for the second emulsion process to balance homogenization of the oil phase and retention of encapsulated cargo in the inner aqueous phase. [0089] The polymeric nanoparticle prepared by the above-described method serves as a platform technology for drug delivery, theranostics, and vaccine development applications. It can facilitate delivery of a large class of bioactive agents, including small molecules, peptides, nucleic acids, and proteins, to enhance their therapeutic potency. The thin-shell polymeric hollow nanoparticles can be used to encapsulate bioactive agents, including but not limited to small molecules, peptides, proteins, nucleic acids, imaging agents, inorganic nanoparticles, organic nanoparticles, and any combination of the above. The surface of the platform can be optionally decorated with functional moieties, including small molecules, peptides, proteins, nucleic acids, imaging agents, nanoparticles, for different applications such as long-circulating drug delivery, targeted drug delivery, and antigen delivery.
[0090] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference.
10091 f Examples
[0092] Preparation of polymeric nanoparticles
[0093] Thin-shell polymeric nanoparticles were produced according to a protocol including the following steps:
1. preparing 10 rng/mL carboxy-terminated PLGA polymers in DCM
2. emulsifying 50 pL of inner aqueous phase in 500 pL of PLGA/DCM solution to form a first emulsion. Probe sonicate continuously at 50% sonication amplitude for 30 seconds.
3. emulsifying the first emulsion in 5 ml of aqueous solution and disperse the mixture under controlled fluidic shear using a microfluidizer to form a second emulsion.
4. adding an additional 30 mL of aqueous solution to the second emulsion and evaporate the solvent at 35 °C.
5. evaporating the DCM in a fume hood for 3 hours to afford a solution.
6. isolating particles from the solution by an ultracentrifuge at 22 kG for 35 min.
7. re-dispersing the particles in a desired solution.
0094] Characterisation and encapsulation of polymeric nanoparticles [0095] By employing the steps described above, hollow polymeric nanoparticles with an average diameter of 110.9 nm were prepared. Statistical average of the particles’ shell thickness was derived based on parameters obtained by nanoparticle tracking analysis. Based on the total polymer weight, PLGA density, and the number of resulting nanoparticles, it was calculated that the nanoparticles have a statistical average of 16.5 nm in shell thickness. Unexpectedly, certain polymeric nanoparticles had diameters less than 40 nm.
[0096] The thin-shell hollow nanoparticles were found to be osmoticaily resistant resulting from the water impermeable polymeric shells. In a test, 100 nm hollow nanoparticles encapsulating a hydrophilic red food coloring were suspended in solutions ranging from water to 3X PBS, the difference in osmolarity (between 0 to 850 Osmo/kg) did not cause the hollow nanoparticles to release their cargoes. Following 10 min of incubation in their respective solutions, nanoparticles were pelleted under centrifugation at 30,000 g for 5 min, and the resulting pellets showed similar, reddish color indicating retention of hydrophilic dye in the particles. Detection of the supernatant for the released dye based on an absorbance method showed no detectable signals. The study shows that despite having a thin polymeric shell below 20 nm, the hollow nanoparticles had defect-free shells that made them resistant to osmotic stress.
[0097] To further demonstrate that the shell of the thin-shell hollow nanoparticles were solid rather than fluid, hollow nanoparticles were subjected to mechanical stress to break the shell. In a cryo-EM visualization, a broken hollow nanoparticle was observed. The observed image of the broken hollow7 nanoparticles was indicative of a hollow sphere with a solid shell, in contrast to the polymeric vesicles that undergo vesicular reorganization upon mechanical perturbation. The solid polymeric shell led to the water impermeability and osmotic resistance that were not observed in known hollow nanostructures.
[0098] A distinguishing feature of the thin-shell polymeric nanoparticle platform is its capacity to encapsulate a large amount of hydrophilic cargoes with its large interior aqueous space. The thin-shell hollow nanoparticles were subjected to encapsulate several bioactive agents, including siRNA and an immunological adjuvant cyclic di-GMP. Unexpectedly, high encapsulation efficiency was achieved for hydrophilic contents of various length scales, including small molecules (e.g, sulfo-cy5, cyclic di-GMP, and cyclic cGAMP), peptides (e.g., ovalbumin peptide OTI (SIINFEKL) or OTII (AAHAEINEA)), nucleic acids (e.g, CpG- oligodeoxynucleotides, 20-mer single standed DNA, and 20-mer siRNA), and proteins (e.g, bovine serum albumin (BSA) and CRISPR-Cas9 nuclease), which were successfully encapsulated with an efficiency above 30% within the compounds’ solubility limits. For example, siRNA was encapsulated at an efficiency of 50% with a final loading yield of about 1 nmol per mg of nanoparticles and cyclic di-GMP was encapsulated at a 37% loading efficiency. Silencing of a green fluorescent protein (GFP) gene in GFP-expressing HeLa cells was observed using siRNA loaded thin-shell hollow nanoparticles.
[0099] Encapsulation of polymeric nanoparticles with hydrophilic macromolecules
[0100] An assay was performed to evaluate the encapsulation efficiency of polymeric nanoparticles for two hydrophilic macromolecules, i.e., a nucleic acid (dye-labelled 20-mer single stranded DNA) and a protein (dye-labelled BSA).
[0101] Please refer to FIG. 2 for a depiction of encapsulation efficiency of polymeric nanoparticles for a nucleic acid and a protein. In this example, effective encapsulation of a nucleic acid and a protein was demonstrated using dye-labelled 20-mer single stranded DNA (A, middle) and dye-labelled BSA (A, right). Upon pelleting the nanoparticles, the particle pellets were found to be yellow colored (indicative of the yellowish FAM dye label) as opposed to the white pellet of the empty nanoparticles (A; left). Fluorescence quantification indicates that the nanoparticles unexpectedly exhibited encapsulation efficiencies of 42% and 35% for the nucleic acid and protein, respectively.
[0102] Effective encapsulation of the nucleic acid and protein was also visualized using cryoEM. While empty particles showed a plain, even texture in its aqueous core (FIG. 2B), DNA-loaded particles (FIG. 2C) and BSA-loaded particles (FIG. 2D) showed highly grainy textures that are characteristics of concentrated biological contents under cryoEM. Based on the loading concentration of 8 rnM DNA and 50 mg/mL BSA for the present embodiments, the DNA-loaded nanoparticle contained about 3500 DNA molecules and the BSA-loaded nanoparticle contains about 260 proteins. Importantly, such high loading of a nucleic acid and a protein have not been previously achieved. Indeed, known PLGA -based nanoformulations consistently showed very poor nucleic acids encapsulation, and poly cationic polymers had to be employed to neutralize the negative charges on nucleic acids to enhance loading. For example, see Shi et al. , Angew Chem Int Ed Engl, 201 1, 50(31): 7027-31; and Woodrow et ah, Nature Materials, 2009, 8(6): 526-533.
[0103] These results described above indicate that the thin-shell polymeric nanoparticles of the present application unexpectedly exhibited high encapsulation efficiency, i.e., high loading efficiency, for hydrophilic macromolecules in their native, soluble state, highlighting the advantage and uniqueness of the thin-shell hollow nanoparticles. [0104] Effect of polymeric nanoparticles on encapsulation and controlled release of stimulator of interferon gene (STING) agonists for immune stimulation in lymph nodes
[0105] An assay was performed to evaluate the effect of polymeric nanoparticles on encapsulation and controlled release of STING agonists for immune stimulation in lymph nodes.
[0106] FIG. 3 depicts the effect of polymeric nanoparticles on encapsulation and controlled release of STING agonists for immune stimulation. In this example, the present platform was applied for vaccine development. A major technical challenge in preparing nanoparticle vaccines lies in reliably associating antigens and adjuvants on a nanoscale substrate. For example, see Brannon-Peppas et a/., Adv Drug Deliv Rev, 2004, 56(11): 1649-59; and Lima- Tenorio et al, Ini J Pharm, 2015, 493(1-2): 313-27. To overcome this technical challenge, thin-shell polymeric hollow nanoparticles were used to package a high density of functional cargoes using a biodegradable polymer, i.e., PLGA.
[0107] More specifically, hollow particles between 100 to 200 nm in diameter were prepared using a double emulsion process (FIG. 3A and 3B). They efficiently encapsulated a STING agonist adjuvant, cyclic di-GMP (cd-GMP), at about 40% efficiency (FIG. 3C), which is notable as nucleic acids and hydrophilic cargoes are notoriously difficult to encapsulate in nanoparticle platforms. The high encapsulation efficiency yielded approximately 2,000 STING agonist molecules per nanoparticle. No nanoformulations of STING agonists based on polymeric nanoparticles have been reported. Examination by cryoEM revealed large interior cores in these hollow nanoparticles, which w'ere responsible for the high loading efficiency of cd-GMP (FIG. 3D). Upon intracellular delivery, the thin polymeric shell of the hollow' nanoparticles was triggered by acidic pH to rapidly release the interior content (FIG. 3E).
[0108] These results indicate that the thin-shell polymeric nanoparticles enabled effective preparation of synthetic vaccines.
[0109] Evaluation of STING agonist-loaded nanoparticles on enhancing lymphatic cytokines while minimizing systemic cytokines
[0110] An assay was performed to evaluate STING agonist-loaded nanoparticles on enhancing lymphatic cytokines while minimizing systemic cytokines.
[0111] FIG. 4depicts STING agonist-loaded nanoparticles on enhancing lymphatic cytokines while minimizing systemic cytokines. Immune activation by the cd-GMP -loaded nanoparticles was first examined in vitro using a mouse dendritic cell line, JAWSII. Following 24 hours of incubation with either free cd-GMP or cd-GMP nanoparticles, the culture supernatants were collected for ELISA analysis to quantify the levels of TNF-a, IL-6, and IFN-b. As compared to free cd-GMP formulation, the nanoparticle formulation more effectively triggered the production of TNF-a, IL-6, and IFN-b (FIG. 4A, 4B, and 4C). CD80 expression on the ceils was also enhanced when incubated with the nanoparticle formulation as compared to an equivalent dose of free cd-GMP (FIG. 4D). It was observed that the nanoparticles enhanced the adjuvanticity of the STING agonist by about 30 folds, attributable to increased intracellular delivery by the nanocarrier. It can be inferred that the free cyclic-di -nucleotide is not readily membrane permeable and may not easily access its cytosolic target. Upon nanoparticle encapsulation, cellular uptake is enhanced via particle endocytosis and the subsequent intracellular release facilitates cytosolic entry of cd-GMP, thereby enhancing its immune potentiating effect.
[0112] Immune potentiation by the cd-GMP nanoparticles was further compared to free cd- GMP in vivo in mice. 48 hours following footpad injections, the draining popliteal lymph nodes were collected for IFN-b quantification. It was observed that the nanoparticle formulation induced a significantly higher level of IFN-b in the lymph node (FIG. 4E), which is important for proper T cell maturation. In addition, the systemic level of TNF-a, an indicator of reactogenicity, was also monitored following the footpad administration of free cd-GMP and cd-GMP nanoparticles. It was observed that the nanoparticles resulted in a significantly lower serum level of TNF-a, which reflects the nanoparticles’ distribution in the lymphatic system. The free cd-GMP, on the other hand, induced an elevated level of serum TNF-a and these small molecules could freely diffuse into the blood stream (FIG. 4F).
[0113] These results indicate that STING agonist-loaded nanoparticles effectively enhanced lymphatic cytokines for lymph node-targeted immune potentiation
[0114] Treatment modality based on a cell-associating immunologic adjuvant
[0115] In one embodiment, the treatment kit and the method described herein can be used to treat diseases, for example, cardiovascular disease, cancer, autoimmune disease, or infection. In one embodiment, the treatment kit and the method described herein can be used to cancers, such as cancer of the oral cavity and pharynx (lip, tongue, salivary gland, floor of mouth, gu and other mouth, nasopharynx, tonsil, oropharynx, hypopharynx, other oral/pharynx); cancers of the digestive system (esophagus; stomach; small intestine; colon and rectum; anus, anal canal, and anorectum; liver; intrahepatic bile duct; gallbladder; other biliary ; pancreas; retroperitoneum; peritoneum, omentum, and mesentery; other digestive); cancers of the respiratory system (nasal cavity, middle ear, and sinuses; larynx, lung and bronchus; pleura, trachea, mediastinum, and other respirator}-); cancers of the mesothelioma; bones and joints; and soft tissue, including heart; skin cancers, including melanomas and other non-epithelial skin cancers; Kaposi’s sarcoma and breast cancer; cancer of the female genital system (cervix uteri; corpus uteri; uterus, ovary; vagina; vulva; and other female genital); cancers of the male genital system (prostate gland; testis, penis; and other male genital), cancers of the urinary- system (urinary bladder; kidney and renal pelvis; ureter; and other urinary'); cancers of the eye and orbit; cancers of the brain and nervous system (brain, and other nervous system); cancers of the endocrine system (thyroid gland and other endocrine, including thymus); lymphomas (Hodgkin’s disease and non-Hodgkin’s lymphoma), multiple myeloma, and leukemias (lymphocytic leukemia; myeloid leukemia; monocytic leukemia; and other leukemias).
[0116] In an embodiment, the immunologic adjuvant formulation comprises a polymeric nanoparticle encapsulating an adjuvant, and the polymeric nanoparticle may further comprise: a polymeric shell impermeable to water, and one or more aqueous cores enclosed by the polymeric shell. In a preferred embodiment, the adjuvant comprises MPLA, CpG, poly(LC), variants of cyclic-dinucleotides, an agonist to a cytoplasmic pattern recognition receptor such as cyclic di-GMP or cyclic GAMP, or a stimulator of interferon genes (STING).
[0117] Please refer to FIG. 5 for depiction of a treatment modality based on a tumor cell- associating immunologic adjuvant (cGAMP -loaded nanoparticle). In one embodiment of the present application, 2’ 3’ -cGAMP is encapsulated inside a polymeric hollow nanoparticle to enhance cell internalization via endocytosis. cGAMP is an agonist to cytoplasmic pattern recognition receptor, a stimulator of interferon genes (STING) that activate the TBK I/interferon regulatory factor 3 (IRF3)/type 1 interferon (IFN) signaling. By facilitating cGAMP association with cells, the adjuvant formulation primes the antigens for immune processing. Upon treatment that induces cell death by a means of for inducing antigen release, cellular debris are then processed along with the exogenously delivered adjuvants from the adjuvant formulation, enabling enhanced adaptive immunity for cell killing (FIG. 5).
[0118] In an embodiment, the means for inducing antigen release may comprise chemical agents, biological agents, irradiation, photolytic agents, mechanical disruptions, or a combination thereof.
[0119] In a preferred embodiment, the chemical agents comprise any cytotoxic agents known in the art. For example, the cytotoxic agent comprises Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (DM1), anthracycline, pyrrolobenzodiazepine, a-amanitin, tubulysin, benzodiazepine, erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787/ZK 222584, oxaliplatin, leucovorin, rapamycin, lapatinib, lonafamib (SARASAR®, SCH 66336), sorafenib, gefitinib, AG1478, AG1571, alkylating agent; alkyl sulfonate, aziridines; ethylenimine; methylamelamine; acetogenins; camptothecin; bryostatin; callystatin; CC-1065; cryptophycins; dolastatin; duocarmycin; eleutherobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlomaphazine; eholophospharnide; estrarnustine, ifosfamide; mechlorethamine; mechlorethamine oxide hydrochloride; melphalan; novembichin; phenesterine; prednimustine; trofosfamide; uracil mustard; carmustine; chlorozotocin; fotemustine; lomustine; nimustine; ranimustine; calicheamicin; dynemicin; clodronate; esperamicin; neocarzinostatin chromophore; aclacinomysins; actinomycin; authramycin; azaserine; bleomycins; cactinornycin; carabicin; caminomycin; carzinophilin; chromomycinis; dactinomycin; daunorubicin; detorubicin; 6-diazo-5-oxo-L-norleucine; doxorubicin; epirubicin; esorubicin; idarubicin; marcellomycin,;mitomycin; mycophenolic acid; nogalamycin; olivomycins; peplomycin; potfiromycin; puromycin; quelamycin; rodorubicin; streptonigrin; streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate; 5-fluorouracil (5-FU); denopterin; pteropterin; trimetrexate; fludarabine; 6- mercaptopurine; thiamiprine; thioguanine; ancitabine; azacitidine; 6-azauridine; carmofur; cytarabine; dideoxyuridine; doxifluridine; enocitabine; floxuridine; calusterone; dromostanolone propionate; epitiostanol; mepitiostane; testolactone; aminoglutethimide; mitotane; trilostane; frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine; ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecene; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoid; paclitaxel; doxetaxel; chloranbucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; cisplatin; carboplatin; vinblastine, platinum; etoposide; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor, difluoromethylornithine (DMFO); retinoid; capecitabine; or a combination thereof.
[0120] In a preferred embodiment, the photo! ytic agents, which is generally a photosensitizing agent used in the photodynamic therapy, comprises Photofrin, Laserphyrin, Aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m -tetrahy droxy phenyl chi orin (mTHPC), chlorin e6 (Ce6), Allumera, Levulan, Foscan, Metvix, Hexvix, Photochlor, Photosens, Photrex, Lutnacan, Visonac, Amphinex, Verteporfin, Purlytin, ATMPn, Zinc phthaJocyanine (ZnPc), Protoporphyrin IX (PpIX), Pyropheophorbidea (PPa), Pheophorbide a (PhA), or a combination thereof.
[0121] The means for inducing antigen release may be administered to the subject in need before or after administration of the immunologic adjuvant formulation. In this course of treatments, the means for inducing antigen release and the immunologic adjuvant formulation are formulated as two distinct doses for being administered at different times.
[0122] Alternatively, the means for inducing antigen release may be administered to the subject in need at the same time as the immunologic adjuvant formulation. In this course of treatments, the means for inducing antigen release from a cell can be formulated with or encapsulated in the immunologic adjuvant formulation as a single dose or two distinct doses for being administered simultaneously. For example, the adjuvants and the cytotoxic agents may be formulated in one nanoparticle or conjugated to one antibody.
[0123] To demonstrate the treatment modality, HeLa cells were incubated with fluorescently labelled cGAMP-loaded nanoparticles (FIG. 6(a)). Upon intracellular uptake of the nanoparticles following 24 hr of incubation, the cells were allowed to vesiculate and generate cellular debris under the treatment of a chemotherapy drug, cisplatin as shown in FIG. 5. To demonstrate the association of the adjuvant nanoparticles with the cancer cell vesicles, the collected cellular debris were analyzed with confocal microscopy and quantified with fluorescence-activated cell sorting (FACS). The results show that the cellular debris contained a significant amount of adjuvant-loaded nanoparticles (FIG. 6(b), (c)>
[0124] The cancer cell debris were then collected and incubated with a JAWSII mouse dendritic cell (DC) line to examine their immunogenicity. HeLa cells incubated with either free cGAMP or cGAMP -loaded nanoparticles for 24 hr were treated with cisplatin, and the resulting cellular debris w'ere collected to stimulate DCs. The DCs were collected after 24 hr of incubation and studied for DC activation markers CD86 and CD80. In vitro activation of DCs by immunogenic cellular debris was demonstrated by FACS analysis. The cytokine profile of the activated DCs was estimated by ELISA analysis for TNF-a and EFN-b. The results show that incubation with the cGAMP-loaded nanoparticles significantly increased the level of CD80 and CD86 and elevated the secretion of TNF-a and IFN-b by the DCs as compared to the free cGAMP, which has little capacity to associate with the cells (FIG. 7) In particular, IFN-b, a type-I IFN that is recognized as a crucial factor for shaping adaptive cellular immunity[7], is boosted by more than 100-foid with the adjuvant formulation. [0125] To validate cell-associating effect of the nanoparticle adjuvant formulation, B6 mice were inoculated subcutaneously with B 16-OVA tumors. Nanoparticle-mediated adjuvant retention with the B16 ceils was validated using whole body imaging, flow cytometric analysis, and fluorescence microscopy. Dye-labelled STING agonist (2'-[D Y -547]-AHC-c-di GMP) was administered in its free form and nanoparticle form intra-turaoraily and examined after 24 hours. It was observed that the nanoparticle formulation of the STING agonist w¾s well retained inside the tumor (FIG. 8(a)). Upon cancer cell extraction from the dissected tumors, significant STING agonist association within the cancer cells was also confirmed from flow cytometry and confocal microscopy (FIG. 8(b), (c)). In contrast, little adjuvant retention either within the tumor or inside the cancer cells w¾s observed upon free adjuvant administration.
[0126] To study the in vivo efficiency of the treatment modality, B6 mice were inoculated subcutaneously with B 16-OVA tumors. When the tumors appeared palpable, they w?ere pre treated with free cGAMP or cGAMP-loaded nanoparticles prior to chemotherapy by cisplatin (CTTP). 24 hours following intra-tumoral adjuvant administration, the mice were given an intra-tumoral cisplatin injection to induce cell death (FIG. 9(a)). These two groups are designated cGAMP+CTTP and NP+CTTP to differentiate the use of free cGAMP and cGAMP-loaded nanoparticles, respectively. For controls, tumor-bearing mice receiving no treatment (placebo), cistplatin only (CTTP only) and cGAMP-loaded nanoparticles only (NP only) w?ere also prepared. For cGAMP administration, 2.5 pg of the adjuvant was administered in 50 pL of solution. For cisplatin administration, 235 pg of the ehemodrug was administered in 100 pL of solution. Following the different treatments, the tumor volumes were monitored over time. In the NP+CTTP group, 50% of the animals (5 out of 10) showed complete tumor regression. In contrast, no other group had any tumor with complete regression (FIG. 9(b), (c)). Notably, neither CTTP only nor NP only treatment induced tumor regression, indicating that a synergistic effect was induced with their combination. In addition, none of the cGAMP+CTTP animals showed complete tumor regression, indicating that the synergistic effect in the NP+CTTP group was attributable to the enhanced adjuvant retention within the cancer cells. With the tumor-free surviving mice from the NP+CTTP group, 4 of them were re-challenged with B 16-OVA cells on day 60 and compared to animals that had no prior tumor challenge and treatment. It w¾s observed that the tumor growth was significantly reduced among the surviving animals from the NP+CTTP group, which indicate that these animals had acquired adaptive anti-cancer immunity (FIG. 9(d)). These results validate that by applying a cancer cell-associating immunologic adjuvant, the efficacy of a subsequent anticancer treatment can be significantly enhanced owing to the promotion of adaptive immunity. [0127] In one embodiment of the present application, the immunologic adjuvant formulation comprises an adjuvant conjugated with a small molecule, a peptide, an antibody, an aptamer, a sugar moiety, a polymer or a combination thereof for recognizing and binding to a moiety on a cell surface. Preferably, the antibody comprises an immunoglobulin molecule, an Fv, a disulfide linked Fv, a monoclonal antibody, a scFv, a chimeric antibody, a single domain antibody, a CDR-grafted antibody, a diabody, a humanized antibody, a multispecific antibody, an Fab, a dual specific antibody, an Fab’ fragment, a bispecific antibody, an F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementarity determining region (CDR), or a single chain antibody.
[0128] In one preferred embodiment, CpG-FAM was conjugated to antibodies for pathogenic cell association. Anti-CD44, which targets CD44 that are highly expressed on HeLa cells was adopted for adjuvant conjugation. 1 mg of the antibodies was first mixed with sulfo-SMCC at a molar ratio of 1 :20 in IX PBS for 24 h. Following the incubation, the functionalized antibodies were purified using a centrifugal filter with a molecular weight cut off (MWCO) of 30 kDa. The antibodies were then mixed with CpG-ODN containing a terminal cysteine group. A FAM dye was attached to the CpG-ODN for visualization (FIG. 10). Following 24 h of incubation, the CpG-conjugated antibodies were collected using a centrifugal filter with a MWCO of 30 kDa
[0129] We then assessed the CpG association to HeLa cells by incubating free CpG-FAM and anti -CD44-conj ugated CpG-FAM with HeLa cells for 3 h. Following the incubation, the cells were washed and the CpG association was evaluated based on the FAM fluorescence. It was observed under fluorescence microscopy that the anti-CD44 CpG conjugates yielded higher fluorescence retention to the HeLa cell monolayer (FIG. 11 A). Upon detaching the cells for flow cytometric analysis, enhanced CpG/ cancer cell association was also observed with the anti-CD44 conjugates, demonstrating successful adjuvant targeting to the pathogenic cells (FIG. 1 IB, C).
[0130] Following adjuvant association, the HeLa cells were then treated with a cytotoxic agent, doxorubicin, to induce cell death and generate cellular debris. Following 20 h of doxorubicin treatment, the cellular debris were collected and fed to JAWSII mouse dendritic cells. The JAWSII cells were then assessed for activation markers such as CD80 and CD86 after 20 hr of incubation with the cellular debris. It was observed that the debris of HeLa cells treated with anti-CD44 conjugated CpG triggered the highest levels maturation markers (FIG. 12). [0131] In another embodiment of the present application, CpG-FAM was encapsulated into polymeric nanoparticles for enhancing tumor cell uptake. A double emulsion process was employed to prepare the CpG-FAM loaded nanoparticles. 20 mg/mL of CpG was first dissolved in 50 pL of 100 mM of phosphate buffer, which was then emulsified in 500 _uL of ethyl acetate containing 50 mg/mL of PLGA (10,000 Da, carboxyl -terminated, 50:50 iactide:glycolide). The resulting water-in-oil emulsion was then emulsified in 5 mL of 10 mM phosphate buffer. Following the incubation, the water-in-oil-in-water emulsion was then poured into 10 mL of 10 mM of phosphate buffer and the ethyl acetate was evaporated under nitrogen bombardment. After 1 h of solvent evaporation, the nanoparticle-encapsulated CpG was collected using a centrifugal filter with a MWCO of 30 kDa. Successful preparation of sub- 100 nm nanoparticles containing CpG was verified using CryoEM (FIG. 13).
[0132] We then assessed the CpG association to HeLa cells by incubating free CpG-FAM and nanoparticle-encapsulated CpG-FAM with HeLa cells for 24 h. Following the incubation, the ceils were washed and the CpG association was evaluated based on the FAM fluorescence. It was observed under fluorescence microscopy that the nanoparticle-encapsulated CpG yielded higher fluorescence retention to the HeLa cell monolayer (FIG. 14.A) Upon detaching the cells for flow' cytometric analysis, enhanced CpG/cancer cell association was also observed with the nanoparticle-encapsulated CpG, demonstrating successful adjuvant targeting to the cells (FIG. 14B, C).
[0133] Following adjuvant association, the HeLa cells were then treated with a cytotoxic agent, doxorubicin, to induce cell death and generate cellular debris. Following 20 h of doxorubicin treatment, the cellular debris were collected and fed to JAWSII mouse dendritic cells. The JAWSII cells were then assessed for activation markers such as CD80 and CD86 after 20 hr of incubation with the cellular debris. It was observed that the debris of HeLa cells treated with nanoparticle-encapsulated CpG triggered the highest levels maturation markers (FIG. 15).
[0134] In another embodiment of the present application, the means for inducing antigen release may be administered to the subject in need at the same time as the immunologic adjuvant formulation. Alternatively, the means for inducing antigen release may be administered to the subject in need before or after administration of the immunologic adjuvant formulation.
[0135] In one embodiment, daunorubicin was encapsulated into nanoparticles along with ed~ GMP STING agonist to simultaneously enable intracellular adjuvant delivery while triggering ceil death. A double emulsion process was employed to prepare the nanoparticles co- encapsulating daunorubicin and cd-GMP. Solution containing 10 mg/mL of daunorubicin and 10 mg/mL of cd-GMP was first prepared with 100 mM of phosphate buffer, and 50 pL of the solution was emulsified in 500 pL of ethyl acetate containing 50 mg/mL of PLGA (10,000 Da, carboxyl -terminated, 50:50 lactide:gfycolide). The resulting water-in-oil emulsion was then emulsified in 5 rnL of 10 mM phosphate buffer. Following the incubation, the water-in-oil-in- water emulsion was then poured into 10 rnL of 10 mM of phosphate buffer and the ethyl acetate was evaporated under nitrogen bombardment. After 1 h of solvent evaporation, the daunorubicin/cd-GMP nanoparticles were collected using a centrifugal filter with a MWCO of 30 kDa. As a control, nanoparticles encapsulating daunorubicin in the absence of cd-GMP was prepared with 20 mg/mL of daunorubi cin as the inner aqueous phase. Successful preparation of sub- 100 nm nanoparticles containing daunorubicin and cd-GMP was verified by cryoEM (FIG. 16).
[0136] We then treated CT26 colon cancer cells with three distinctive formulations: 1) daunorubicin nanoparticles only (DRNP); 2) free cd-GMP and daunorubicin nanoparticles (cd- GMP+DR NP); and 3) Nanoparticles co-eneapsuiating daunorubicin and cd-GMP ((cd- GMP+DR) NP. To 2 mL of CT26 culture were given 10 itg of daunorubicin and 10 pg of cd- GMP. Following 3 h of treatment, cellular debris was collected and fed to JAWSII mouse dendritic cells. The JAWSII cells were then assessed for activation markers such as CD80 and CD86 after 24 hr of incubation with the cellular debris. It was observed that the debris of CT26 ceils treated with nanoparticles co-encapsuiating daunorubicin and cd-GMP triggered the highest levels maturation markers such as CD80 and CD86 (FIG. 17).
[0137] The supernatants from the JAWSII cells were also assessed for IFN-b, which is a major lymphocyte activation signal for T cell activation and expansion. It was observed that the debris of CT26 cells treated with nanoparticles co-encapsulating daunorubicin and cd-GMP induced the highest level IFN-b production in JAWSII cell (FIG. 18). These results demonstrate the adjuvant formulation co-delivering cytotoxic agents and immunologic adjuvants to cells can induce cellular debris with significant immunogenicity.
[0138] In summary, the description above demonstrates that the treatment kit comprising immunologic adjuvant formulation is capable of facilitating immune response target specific cells. A novel treatment modality was also demonstrated based on the cell-associating immunologic adjuvant, which is capable of enhancing adaptive immunity to the diseased cell. The method and the treatment kit are applicable to any existing treatments that induce release of cellular debris and antigens, including but not limited to chemotherapy, radiation therapy, photothermal therapy, immunotherapy, and surgical interventions. Other approaches for associating immunologic adjuvants with pathogenic cells, including but not limited to methods that facilitate intracellular entiy, membrane tethering, and surface binding of immunologic adjuvants, are also contemplated. The immunologic adjuvants can also include but not limited to MPLA, CpG, poly(LC), variants of cyclic-dinucleotides, and other immune-stimulatory molecules. Treatment kit that contain both immunologic adjuvant formulation and means for inducing antigen release, such as nanoparticles containing both cGAMP and chemodrugs, for ceil association are also contemplated.
[0139] It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments. It is intended that the specification and examples be considered as exemplary only, with a true scope of the disclosure being indicated by the following claims and their equivalents.
REFERENCES
[0140] The references listed below and referred to herein are hereby incorporated into this specification by reference unless this specification expressly provides otherwise.
1. Bachmann, M.F. and G.T. Jennings, Vaccine delivery: a matter of size, geometry, kinetics and molecular paterns. Nat Rev Immunol, 2010. 10(11): p. 787-96.
2. Ott, P.A., et al, An immunogenic personal neoantigen vaccine for patients with melanoma. Nature, 2017. 547(7662): p. 217-+.
3. Fan, Y., et al., Immunogenic Cell Death Amplified by Co-localized Adjuvant Delivery for Cancer Immunotherapy. Nano Lett, 2017.
4. Fu, J., et al., STING agonist formulated cancer vaccines can cure established tumors resistant to PD- 1 blockade. Sci Transl Med, 2015. 7(283): p. 283ra52.
5. Galluzzi, L., et al., Immunogenic cell death in cancer and infectious disease. Nat Rev Immunol, 2017. 17(2): p. 97-111.
6. Fridman, W.H., et al., The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer, 2012. 12(4): p. 298-306.
7. Hertzog, P.J., Overview. Type I interferons as primers, activators and inhibitors of innate and adaptive immune responses. Immunology and Cell Biology, 2012. 90(5): p. 471- 473.

Claims

WHAT IS CLAIMED IS:
1. A treatment kit, comprising;
an immunologic adjuvant formulation capable of physically associating with cells; and a means for inducing antigen release from a cell.
2. The treatment kit of claim 1 , wherein the immunologic adjuvant formulation comprises a polymeric nanoparticle encapsulating an adjuvant, wherein the polymeric nanoparticle comprises:
a polymeric shell impermeable to water, and
one or more aqueous cores enclosed by the polymeric shell.
3. The treatment kit of claim 2, wherein the polymeric shell has a thickness less than 25 nm and the polymeric nanoparticle has an outer diameter of 30-600 nm.
4. The treatment kit of claim 2, wherein the means for inducing antigen release from a cell is encapsulated in the polymeric nanoparticle.
5. The treatment kit of claim 1 , the immunologic adjuvant formulation comprises MPLA, CpG, poly(LC), or variants of cyclic-dinucleotides.
6. The treatment kit of claim 1 , wherein the immunologic adjuvant formulation comprises an agonist to a cytoplasmic pattern recognition receptor or a stimulator of interferon genes (STING).
7. The treatment kit of claim 6, wherein the agonist is cyclic di-GMP or cyclic GAMP.
8. The treatment kit of claim 1, wherein the immunologic adjuvant formulation comprises an adjuvant conjugated to a small molecule, a peptide, an antibody, an aptamer, a sugar moiety, a polymer or a combination thereof for recognizing and binding to a moiety on a cell surface
9. The treatment kit of claim 8, wherein the antibody is an immunoglobulin molecule, an Fv, a disulfide linked Fv, a monoclonal antibody, a scFv, a chimeric antibody, a single domain antibody, a CDR-grafted antibody, a diabody, a humanized antibody, a multispecific antibody, an Fab, a dual specific antibody, an Fab’ fragment, a bispecific antibody, an F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementarity determining region (CDR), or a single chain antibody.
10. The treatment kit of claim 1, wherein the immunologic adjuvant formulation is conjugated with a molecule capable of tethering to a cell membrane.
11. The treatment kit of cl aim 10, wherein the molecule is lipophilic or amphiphilic.
12. The treatment kit of claim 11, wherein the lipophilic molecule comprises fatty acid chains, cholesterol, or phospholipids.
13. The treatment kit of claim 11, wherein the amphiphilic molecule comprises lipid-PEG conjugates.
14. The treatment kit of claim 1, wherein the immunologic adjuvant formulation comprises an adjuvant conjugated to nanoparticulates or cell penetrating peptides for intracellular delivery.
15. The treatment kit of claim 14, wherein the cell penetrating peptides comprises HIV-1 TAT peptide (GRKKRRQRRRPPQ), penetratin (RQIKIWFQNRRMKWKK), poiyarginines (Rn), or transportan (GWTLNSAGYLLGINLKALAALAKKIL).
16. The treatment kit of claim 1 , wherein the means for inducing antigen release comprises chemical agents, biological agents, irradiation, photoiytic agents, mechanical disruptions, or a combination thereof
17. The treatment kit of claim 16, wherein the chemical agents comprises a cytotoxic agent.
18. The treatment kit of claim 17, wherein the cytotoxic agent comprises Monomethyl auri statin E (MMAE), Monomethyl auri statin F (MMAF), mertansine (DM1), anthracycline, pyrrolobenzodiazepine, a-amanitin, tubulysin, benzodiazepine, eriotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787/ZK 222584, oxaliplatin, !eucovorin, rapamycin, lapatinib, lonafamib (SARAS AR®, SCH 66336), sorafenib, gefitinib, AG1478, AG1571, alkylating agent; alkyl sulfonate; aziridines, ethylenimine; methylamelamine; acetogenins; camptothecin; bryostatin; callystatin; CC-1065; cryptophycins; dolastatin; duocarmycin; eleutherobin, pancrati statin; sarcodictyin; spongistatin; chlorambucil; chlornaphazine; cholophosphamide; estramustine; ifosfamide; mechlorethamine; mechlorethamine oxide hydrochloride, melphalan; novembichin; phenesterine; prednimustine; trofosfamide; uracil mustard; carmustine; chlorozotocin; fotemustine; lomustine; nimustine; ranimustine; calicheamicin; dynemicin; clodronate; esperamicin; neocarzinostatin chromophore; aclacinomysins; actinomycin; authramycin; azaserine; bleomycins; cactinomycin; carabicin; caminomycin; carzinophilin; chromomycinis; dactinomycin; daunorubicin; detorubicin; 6-diazo-5-oxo-L-norleucine; doxorubicin; epirubicin; esorubicin; idarubicin; marcellomycin,;mitomycin; mycophenolic acid; nogalamycin; olivomycins; peplomycin; potfiromycin; puromycin; queiamycin; rodorubicin; streptonigrin; streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate; 5-fluorouracil (5-FU); denopterin; pteropterin; trimetrexate; fludarabine; 6-mercaptopurine; thiamiprine; thioguanine; ancitabine; azacitidine; 6-azauridine; carmofur; cytarabine; dideoxyuridine; doxifluridine; enocitabine; floxuridine; calusterone; dromostanolone propionate; epitiostanol; mepitiostane; testolactone; aminoglutethimide; mitotane; trilostane; frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracii; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate, hydroxyurea; lentinan; lonidainine; maytansine; ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecene; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoid; paclitaxel; doxetaxel; chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; cisplatin; earboplatin; vinblastine; platinum; etoposide; ifosfamide; mitoxantrone, vincristine; vinorelbine; novantrone; teniposide; edatrexate, daunomycin; aminopterin; xeloda; ibandronate; topoisom erase inhibitor; difluoromethylornithine (DMFO); retinoid, capecitabine; or a combination thereof
19 The treatment kit of claim 16, wherein the photolytic agents comprises Photofrin, Laserphyrin, Aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m- tetrahydroxyphenylchlorin (mTHPC), chlorin e6 (Ce6), Allumera, Levulan, Foscan, Metvix, Hexvix, Photochlor, Photosens, Photrex, Lumacan, Visonac, Amphinex, Verteporfm, Purlytin, ATMPn, Zinc phthalocyanine (ZnPc), Protoporphyrin IX (PpIX), Pyropheophorbidea (PPa), Pheophorbide a (PhA), or a combination thereof.
20 The treatment kit of claim I, wherein the immunologic adjuvant formulation and the means for inducing antigen release from a cell are formulated in single dose
21 A method of treating a disease, comprising:
administering to a subject in need thereof an immunologic adjuvant formulation capable of physically associating with ceils; and administering to the subject in need thereof a means for inducing antigen release from a cell.
22. The method of claim 21, further comprising: encapsulating an adjuvant in a polymeric nanoparticle, wherein the polymeric nanoparticle comprises: a polymeric shell impermeable to water, and one or more aqueous cores enclosed by the polymeric shell .
23. The method of claim 21, wherein the means for inducing antigen release is administered to the subject in need before or after administration of the immunologic adjuvant formulation.
24. The method of claim 21 , wherein the means for inducing antigen release is administered to the subject in need at the same time as the immunologic adjuvant formulation.
25. The method of claim 24, further comprising: encapsulating the means for inducing antigen release from a cell in the immunologic adjuvant formulation.
26. The method of claim 21, the immunologic adjuvant formulation comprises MPLA, CpG, poly(LC), or variants of cyclic-dinucleotides.
27. The method of claim 21, wherein the adjuvant comprises an agonist to a cytoplasmic pattern recognition receptor or a stimulator of interferon genes (STING).
28. The method of claim 21, wherein the agonist is cyclic di-GMP or cyclic GAMP.
29. The method of claim 21, further comprising: conjugating an adjuvant with a small molecule, a peptide, an antibody, an aptamer, a sugar moiety, a polymer or a combination thereof recognizing and binding to a moiety on a cell surface to form the immunologic adjuvant formulation.
30. The method of claim 29, wherein the antibody is an immunoglobulin molecule, an Fv, a disulfide linked Fv, a monoclonal antibody, a scFv, a chimeric antibody, a single domain antibody, a CDR-grafted antibody, a diabody, a humanized antibody, a multispecific antibody, an Fab, a dual specific antibody, an Fab’ fragment, a bispecific antibody, an F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementarity determining region (CDR), or a single chain antibody.
31. The method of claim 21, further comprising:
conjugating an adjuvant with a molecule capable of tethering to a cell membrane.
32. The method of claim 31, wherein the molecule is lipophilic or amphiphilic.
33. The method of claim 32, wherein the lipophilic molecule comprises fatty acid chains, cholesterol, or phospholipids.
34. The method of claim 32, wherein the amphiphilic molecule comprises lipid-PEG conjugates.
35. The method of claim 21 , further comprising:
formulating an adjuvant with nanoparticulates or cell penetrating peptides for intracellular delivery.
36. The method of claim 35, wherein the cell penetrating peptides comprises HIV-1 TAT peptide (GRKKRRQRRRPPQ), penetratin (RQIKIWF QNRRMK WKK), poiyarginines (Rn), or transportan (GWTLNSAGYLLGINLKALAALAKKIL).
37. The method of claim 21, wherein the means for inducing antigen release comprises chemical agents, biological agents, irradiation, photolytic agents, mechanical disruptions, or a combination thereof.
38. The method of claim 37, wherein the chemical agents comprises a cytotoxic agent.
39. The method of claim 38, wherein the cytotoxic agent comprises Monomethyl auri statin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (DM1), anthracycline, pyrrol obenzodiazepine, a-amanitin, tubulysin, benzodiazepine, erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787/ZK 222584, oxaliplatin, leucovorin, rapamycin, lapatinib, lonafarnib (SARASAR®, SCH 66336), sorafenib, gefitinib, AG1478, AG1571, alkylating agent; alkyl sulfonate, aziri dines, ethylenimine; methylamelamine; acetogenins; camptothecin; bryostatin; cailystatin; CC-1065; cryptophycins; dolastatin; duocarmycin; eleutherobin; pancrati statin; sarcodictyin, spongistatin; chlorambucil; chiomaphazine; cholophosphamide; estramustme; ifosfamide; mechlorethamine; mechloretharnine oxide hydrochloride; melphalan; novembichin; phenesterine; prednimustine; trofosfamide; uracil mustard; carmustine; chlorozotocin; fotemustine; lomustine; nimustine, ranimustine; calichearnicin, dynemicin; clodronate; esperamicin; neocarzinostatin chromophore; aclacinomysins; actinomycin; authramycin; azaserine; bleomycins; cactinomycin; carabicin; eaminornycin; carzinophilin; chromomycinis; dactinomycin; daunorubicin; detorubicin; 6-diazo-5-oxo-L-norleucine; doxorubicin; epirubicin; esorubicin; idarubicin; marcellomycin,;mitomycin; mycophenolic acid; nogalamycin; olivomycins; peplomycin; potfiromycin; puromycin; quelamycin; rodorubicin; streptonigrin; streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate; 5-fluorouracil (5-FU); denopterin; pteropterin; trimetrexate; fludarabine; 6-mercaptopurine; thiamiprine; thioguanine; ancitabine; azacitidine; 6-azauridine; carmofur; cytarabine; dideoxyuridine, doxifluridine; enocitabine; floxuridine; calusterone; dromostanolone propionate; epitiostanol; mepitiostane; testolactone; aminoglutethimide; mitotane; trilostane; frolinic acid, acegl atone; aldophosphamide glycoside; aminolevulinic acid, eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; e!formithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine; ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone, podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin, sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecene; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoid; paclitaxel; doxetaxel; chloranbucil; gemcitabine; 6-thioguanine; rnereaptopurine; methotrexate; cisplatin; carboplatin; vinblastine; platinum; etoposide; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide, edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor; difluoromethylomithine (DMFO); retinoid; capecitabine; or a combination thereof.
40. The method of claim 37, wherein the photolytic agents comprises Photofrin, Laserphyrin, Aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m- tetrahydroxyphenylchlorin (mTHPC), chlorin e6 (Ce6), Allumera, Levulan, Foscan, Metvix, Hexvix, Photochlor, Photosens, Photrex, Lumacan, Visonac, Amphinex, Verteporfm, Purlytin, ATMPn, Zinc phthalocyanine (ZnPc), Protoporphyrin IX (PpIX), Pyropheophorbidea (PPa), Pheophorbide a (PhA), or a combination thereof.
41. The method of claim 21, wherein administering to the subject is by at least one mode selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intra-articu!ar, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectai, intrarenal, intraretinal, intraspinai, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transderrnal
42. The method of claim 21, wherein the disease is cardiovascular disease, cancer, autoimmune disease, or infection.
PCT/US2019/012138 2018-01-04 2019-01-03 Cell-associating immunologic adjuvants for treatment enhancement Ceased WO2019136118A2 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2020537204A JP7230035B2 (en) 2018-01-04 2019-01-03 Cell-associated immune adjuvants and kits and formulations containing same for enhancing therapy
US16/959,758 US11813326B2 (en) 2018-01-04 2019-01-03 Cell-associating immunologic adjuvants for treatment enhancement
CN201980007309.4A CN111727054A (en) 2018-01-04 2019-01-03 Cell-Binding Adjuvants for Increased Efficacy

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201862613574P 2018-01-04 2018-01-04
US62/613,574 2018-01-04

Publications (2)

Publication Number Publication Date
WO2019136118A2 true WO2019136118A2 (en) 2019-07-11
WO2019136118A3 WO2019136118A3 (en) 2019-09-26

Family

ID=67143800

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/012138 Ceased WO2019136118A2 (en) 2018-01-04 2019-01-03 Cell-associating immunologic adjuvants for treatment enhancement

Country Status (5)

Country Link
US (1) US11813326B2 (en)
JP (1) JP7230035B2 (en)
CN (1) CN111727054A (en)
TW (1) TWI818943B (en)
WO (1) WO2019136118A2 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021183131A1 (en) * 2020-03-12 2021-09-16 Aphios Corporation Dosage forms for improving organ transplantation, graft versus host disease and stem cells transplants
WO2022223619A1 (en) 2021-04-20 2022-10-27 Institut Curie Compositions and methods for use in immunotherapy
EP4003906A4 (en) * 2019-07-30 2024-03-06 Academia Sinica PEPTIDE-LOADED CARRIER SYSTEMS AND USES THEREOF

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2023260654A1 (en) * 2022-04-26 2024-11-28 The University Of Sydney Bispecific nanoparticle bioconjugates
CN114748632A (en) * 2022-05-16 2022-07-15 清华大学 Application of microtubule inhibitor in preparation of antitumor and/or antiviral drugs

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2001298061A1 (en) * 2000-12-13 2003-07-09 Purdue Research Foundation Microencapsulation of drugs by solvent exchange
CA2586913A1 (en) * 2004-11-09 2006-05-18 University Of Southern California Targeted innate immunity
EP1850663A4 (en) * 2005-02-22 2008-03-19 Cedars Sinai Medical Center USE OF MINOXIDIL SULPHATE AS ANTI-TUMOR DRUG
DE102006007433A1 (en) * 2006-02-17 2007-08-23 Curevac Gmbh Adjuvant in the form of a lipid-modified nucleic acid
WO2007133728A1 (en) * 2006-05-12 2007-11-22 The Trustees Of The University Of Pennsylvania Photodynamic therapy-generated mesothelioma vaccine
US9549901B2 (en) * 2010-09-03 2017-01-24 The Brigham And Women's Hospital, Inc. Lipid-polymer hybrid particles
WO2012054425A2 (en) * 2010-10-18 2012-04-26 University Of Iowa Research Foundation Biodegradable particulate formulations
US8926994B2 (en) * 2011-12-07 2015-01-06 The Methodist Hospital Research Institute Mesoporous silicon particles for the presentation of tumor antigens and adjuvant for anti-cancer immunity
US20130236504A1 (en) * 2012-03-06 2013-09-12 Medical University Of South Carolina Delivery System for Enhancing Drug Efficacy
CN102688198B (en) * 2012-06-19 2015-04-15 广州帝奇医药技术有限公司 Polypeptide drug sustained-release microsphere preparation and preparation method thereof
EP2934598B1 (en) * 2012-12-19 2018-04-18 Board Of Regents, The University Of Texas System Pharmaceutical targeting of a mammalian cyclic di-nucleotide signaling pathway
PE20160080A1 (en) * 2013-05-18 2016-02-21 Aduro Biotech Inc COMPOSITIONS AND METHODS TO ACTIVATE THE SIGNALING THAT DEPENDS ON THE INTERFERON GENE STIMULATOR
KR20150034517A (en) * 2013-09-26 2015-04-03 삼성전자주식회사 Liposome comprising a complex of a hydrophobic active ingredient and a polypeptide, and use thereof
EP3546473B1 (en) * 2014-12-16 2025-12-10 Kayla Therapeutics Fluorinated cyclic [(2',5')p(3',5')p]-dinucleotides for cytokine induction
WO2017023749A1 (en) * 2015-07-31 2017-02-09 Tarveda Therapeutics, Inc. Compositions and methods for immunomodulation
KR102222186B1 (en) * 2015-08-13 2021-03-03 머크 샤프 앤드 돔 코포레이션 Cyclic di-nucleotide compounds as STING agonists
US10835604B2 (en) * 2015-08-13 2020-11-17 Northeastern University Biomaterials for combined radiotherapy and immunotherapy of cancer
CA3016457A1 (en) * 2016-03-02 2017-09-08 The Board Of Regents Of The University Of Texas System Sting activating nanovaccine for immunotherapy
US20190105282A1 (en) * 2016-03-23 2019-04-11 Che-Ming Jack Hu Thin-shell polymeric nanoparticles and uses thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4003906A4 (en) * 2019-07-30 2024-03-06 Academia Sinica PEPTIDE-LOADED CARRIER SYSTEMS AND USES THEREOF
WO2021183131A1 (en) * 2020-03-12 2021-09-16 Aphios Corporation Dosage forms for improving organ transplantation, graft versus host disease and stem cells transplants
WO2022223619A1 (en) 2021-04-20 2022-10-27 Institut Curie Compositions and methods for use in immunotherapy

Also Published As

Publication number Publication date
JP2021509674A (en) 2021-04-01
US11813326B2 (en) 2023-11-14
TW201938193A (en) 2019-10-01
TWI818943B (en) 2023-10-21
CN111727054A (en) 2020-09-29
US20210361765A1 (en) 2021-11-25
JP7230035B2 (en) 2023-02-28
WO2019136118A3 (en) 2019-09-26

Similar Documents

Publication Publication Date Title
US11813326B2 (en) Cell-associating immunologic adjuvants for treatment enhancement
Kuerban et al. Doxorubicin-loaded bacterial outer-membrane vesicles exert enhanced anti-tumor efficacy in non-small-cell lung cancer
Jang et al. Exosome-based photoacoustic imaging guided photodynamic and immunotherapy for the treatment of pancreatic cancer
Zhang et al. Doxorubicin‐loaded nanoparticle coated with endothelial cells‐derived exosomes for immunogenic chemotherapy of glioblastoma
Wang et al. Designer exosomes enabling tumor targeted efficient chemo/gene/photothermal therapy
Peng et al. Herceptin-conjugated paclitaxel loaded PCL-PEG worm-like nanocrystal micelles for the combinatorial treatment of HER2-positive breast cancer
Lee et al. Recent progress in tumor pH targeting nanotechnology
US10314854B2 (en) Methods for treating tumors in situ including intratumor injection of cytotoxic particles and immune checkpoint blockade therapy
Bostad et al. Light-controlled endosomal escape of the novel CD133-targeting immunotoxin AC133–saporin by photochemical internalization—a minimally invasive cancer stem cell-targeting strategy
Yang et al. PTN-PTPRZ1 signaling axis blocking mediates tumor microenvironment remodeling for enhanced glioblastoma treatment
JP2019524885A (en) Polyglutamine oxidized antifolate and use thereof
Qu et al. Drug-drug conjugates self-assembled nanomedicines triggered photo−/immuno-therapy for synergistic cancer treatments
Yu et al. Combined chemo–immuno–photothermal therapy for effective cancer treatment via an all-in-one and one-for-all nanoplatform
Zhang et al. Hyaluronate-based self-stabilized nanoparticles for immunosuppression reversion and immunochemotherapy in osteosarcoma treatment
Wang et al. Nanotechnology applications in breast cancer immunotherapy
Raikwar et al. Opportunities in combinational chemo-immunotherapy for breast cancer using nanotechnology: an emerging landscape
Yan et al. Highly triple-effective synergy based on tetrahedral DNA nanostructure-induced tumor vaccines for cancer therapy
Zhou et al. Alternative and injectable preformed albumin-bound anticancer drug delivery system for anticancer and antimetastasis treatment
Myat et al. Antibody-decorated chitosan-iodoacetamide-coated nanocarriers for the potential delivery of doxorubicin to breast cancer cells
Wu et al. Enhancing chemoimmunotherapy for colorectal cancer with paclitaxel and alantolactone via CD44-Targeted nanoparticles: A STAT3 signaling pathway modulation approach
Jiao et al. Intelligent porphyrin nano-delivery system for photostimulated and targeted inhibition of angiogenesis
Wang et al. Co-encapsulated Ce6+ CpG and biopeptide-modified liposomes for enhanced transdermal photo-immunotherapy of superficial tumors
Hunt Precision targeting of intraperitoneal tumors with peptideguided nanocarriers
CN113244175B (en) Immune vesicle maytansine conjugate as well as preparation method and application thereof
KR102708057B1 (en) A gas precursor-encapsulated nanoparticle for inducing necroptosis-mimicking cell death and a pharmaceutical composition for the prevention or treatment of cancer comprising the same as an active ingredient

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: 19736047

Country of ref document: EP

Kind code of ref document: A2

ENP Entry into the national phase

Ref document number: 2020537204

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19736047

Country of ref document: EP

Kind code of ref document: A2