[go: up one dir, main page]

WO2024187116A2 - Thermophobic trehalose glycopolymers as smart c-type lectin receptor vaccine adjuvants - Google Patents

Thermophobic trehalose glycopolymers as smart c-type lectin receptor vaccine adjuvants Download PDF

Info

Publication number
WO2024187116A2
WO2024187116A2 PCT/US2024/019138 US2024019138W WO2024187116A2 WO 2024187116 A2 WO2024187116 A2 WO 2024187116A2 US 2024019138 W US2024019138 W US 2024019138W WO 2024187116 A2 WO2024187116 A2 WO 2024187116A2
Authority
WO
WIPO (PCT)
Prior art keywords
tcta
monomer
copolymer
kda
virus
Prior art date
Application number
PCT/US2024/019138
Other languages
French (fr)
Other versions
WO2024187116A3 (en
Inventor
Hector Aguilar-Carreno
Rock Joseph Mancini
Amy Esther Nielsen
Original Assignee
Cornell University
Washington State University
Astante Therapeutics Inc.
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 Cornell University, Washington State University, Astante Therapeutics Inc. filed Critical Cornell University
Publication of WO2024187116A2 publication Critical patent/WO2024187116A2/en
Publication of WO2024187116A3 publication Critical patent/WO2024187116A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
    • 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
    • A61K39/12—Viral antigens
    • 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
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • A61K2039/55583—Polysaccharides
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2438/00—Living radical polymerisation
    • C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]

Definitions

  • thermophobic trehalose glycopolymers as smart C-type lectin receptor vaccine adjuvants.
  • VAERS Vaccine Adverse Event Reporting System
  • Adjuvant activity within a vaccine formulation is currently limited to minimize adverse events, which simultaneously decreases efficacy (Herve et al., “The How’s and What’s of Vaccine Reactogenicity,” NPJ Vaccines, 4(39): 1-11 (2019); Burny et al., “Inflammatory Parameters Associated with Systemic Reactogenicity Following Vaccination with Adjuvanted Hepatitis B Vaccines in Humans,” Vaccine, 37(14):2004-2015 (2019)). Significant efforts are underway to design new adjuvants that enhance efficacy without inflammation (Moser et al., “Increased Vaccine Tolerability and Protection via NF-KB Modulation,” Sci.
  • One aspect of the present disclosure relates to a copolymer comprising at least one PA block and at least one PB block, where PA represents a polymer block comprising one or more units of monomer A and PB represents a polymer block comprising one or more units of monomer B, with the monomer A being an amide or ester, and the monomer B being a trehalose- based monomer, where at least one hydroxyl group on the trehalose is esterified.
  • Another aspect of the present disclosure relates to a bioactive agent delivery system comprising the copolymer according to the present disclosure and a bioactive agent.
  • Another aspect of the present disclosure relates to a composition comprising the copolymer according to the present disclosure and a vaccine.
  • Yet another aspect of the present disclosure relates to a method of vaccinating a subject against infection by an enveloped virus.
  • This method involves providing a composition according to the present disclosure and treating the subject with the composition to vaccinate the subject against the enveloped virus.
  • a further aspect of the present disclosure relates to a method of preparing a copolymer. This method involves providing a radically polymerizable amide monomer A, providing a trehalose-based monomer B comprising at least one esterified hydroxyl group or a polymer block PB comprising one or more units of the monomer B, and polymerizing the monomer A with the monomer B or the polymer block PB.
  • Thermophobic adjuvants were synthesized by combining a rationally designed trehalose glycolipid vaccine adjuvant with thermoresponsive poly-N-isoporpylacrylamide (NIP AM) via Reversible Addition-Fragmentation Chain-Transfer polymerization.
  • NIP AM thermoresponsive poly-N-isoporpylacrylamide
  • Thermophobic adjuvants exhibited LCSTs near 37 °C, and self-assembled into nanoparticles with temperaturedependent sizes.
  • Thermophobic adjuvants activated HEK-mMINCLE and other innate immune cell lines as well as primary mouse Bone Marrow-Derived Dendritic Cells and Macrophages.
  • thermophobic adjuvants enhanced efficacy of an inactivated Influenza A/Califomia/04/2009 virus vaccine, by increasing neutralizing antibody titers and CD4+/44+/62L+ lung and lymph node central memory T cells, as well as providing better protection from morbidity.
  • thermophobic trehalose glycolipid polymers as well as their in-vitro and in-vivo performance as the first synthetic vaccine adjuvants that modulate their potency across an LCST in physiological temperature ranges.
  • FIG. 1 A illustrates the multivalent thermophobic glycolipid vaccine adjuvants disclosed herein, which self-assemble into nanoparticles which decrease in size, and immunogenicity, in response to temperature.
  • FIG. IB shows 6,6’ -trehalose dibehenate, a minimally active adjuvant motif, Brartemicin, a high affinity C-type Lectin Receptor (CLR) ligand, and 4,6-vinylbenylidene trehalose, which is polymerizable.
  • FIG. 1C shows a polymerizable glycolipid adjuvant that activates the Macrophage-Inducible C-Type Lectin Receptor (MINCLE) class of CLR on innate immune cells and could be readily incorporated within a thermoresponsive polymer.
  • MINCLE Macrophage-Inducible C-Type Lectin Receptor
  • FIG. 2 shows a synthesis of glycolipid monomer 1, and thermophobic glycopolymer adjuvants (Pl and P2) along with unadjuvanted thermoresponsive glycopolymer P3 synthesized as a negative control.
  • the synthesis began from an established intermediate, 4,6- O-(4-vinylbenzylidene)-a,a-trehalose monomer. Preparation of the glycolipid monomer is then accomplished via a Steglich esterification of the 6’ hydroxyl group with behenic acid to yield the polymerizable adjuvant 1.
  • the copolymer is then synthesized via RAFT polymerization, where 1 and NIP AM are used to create a statistical copolymer. Subsequent re-initiation with AIBN using Pl as a macro-Chain Transfer Agent and additional NIP AM monomer afforded P2 which contained a congruent adjuvant degree of polymerization, with an additional thermoresponsive NIP AM block.
  • FIGs. 3A-3C show characterization of thermophobic glycopolymer adjuvant copolymers.
  • FIG. 3 A shows that polymers (1 mg/mL in PBS) were observed to have Lower Critical Solution Temperatures (LCSTs) near 37 °C by UV/Vis.
  • DLS Dynamic Light Scattering
  • FIG. 3C shows NOESY-NMR of Pl and P2 confirmed interactions between the behenic ester lipid and isopropyl amide on NIP AM indicating a possible mechanism for the attenuated potency observed at increased temperatures.
  • FIG. 4 shows the 'H NMR of l-(diethoxymethyl)-4-vinylbenzene.
  • FIG. 5 shows the 13 C NMR of l-(diethoxymethyl)-4-vinylbenzene.
  • FIG. 6 shows the 'H NMR of 4,6-O-(4-vinylbenzylidene)-a,a-trehalose.
  • FIG. 7 shows the 1 H NMR for 4,6-O-(4-vinylbenzylidene)-6’-O-behenoyl- a,a-trehalose.
  • FIG. 8 shows the 13 C NMR for 4,6-O-(4-vinylbenzylidene)-6’-0-behenoyl- a,a-trehalose.
  • FIG. 9 shows the NOESY-NMR spectrum for P2 at 35°C.
  • FIG. 10 shows the results of HEK293-mMINCLE Immunoassay. Activation of HEK293-mMINCLE cells with colorimetric readout proportional to Secreted Embryonic Alkaline Phosphatase production (SEAP). Pl, P2 were tested compared to (+)-control D-(+)- Trehalose-6, 6’ -dibehenate (TDB) by mass (pg/mL) and by mols (pM). i.e) lOpM Pl is 73 pg/mL and lOpM P2 is 253 pg/mL. In addition, Pl and P2 were tested by molarity of the subunit agonist, “ag” (pM trehalose glycolipid content). P3 was tested by mass, and did not contain any lipid.
  • SEAP Secreted Embryonic Alkaline Phosphatase production
  • FIG. 11 shows a representative Live-Dead Assay.
  • FIG. 12 shows a Bone Marrow Derived Dendritic Cell (BMDC) (CD1 lc + dendritic cells) full cytokine profile and purity assessment.
  • BMDC Bone Marrow Derived Dendritic Cell
  • FIG. 13 shows a Bone Marrow Derived Macrophage (BMDM) (Mo macrophages) full cytokine profile and purity assessment.
  • BMDM Bone Marrow Derived Macrophage
  • FIG. 14 shows the full range of Lower Critical Solution Temperature (LCST) data at Img / mL.
  • FIG. 15 shows the effect of concentration on polymer Lower Critical Solution
  • FIGs. 16A-16C show Dynamic Light Scattering (DLS) data for Pl (FIG.
  • FIG. 17 shows Gel Permeation Chromatography (GPC) chromatograms for co-polymers Pl, P2, and P3 (baseline corrected and normalized).
  • FIG. 18 shows the Nuclear Overhauser Enhancement Spectroscopy (NOESY-
  • FIG. 19 shows the NOESY-NMR spectrum for Pl at 35°C.
  • FIG. 20 shows the NOESY-NMR spectrum for P2 at 25°C.
  • FIG. 21 A shows a selected binding pose of truncated adjuvant glycolipid subunit 1 docked with the MINCLE crystal structure (PDB: 4ZRW) using Autodock Vina suggested preservation of key bridging interactions between Arg 182 and Phel98 in the binding groove.
  • FIG. 2 IB shows Pl and P2 activated HEK-mMINCLE cells comparable to equimolar amounts of D-(+)-Trehalose-6, 6’ -dibehenate (TDB) (polymer concentrations indicated are with respect to agonist subunit). Error bars are standard deviations of the mean for experiments performed in triplicate; *p ⁇ 0.05, **p ⁇ 0.01 for Pl or P2 compared to negative control (PBS).
  • FIG. 21C shows proinfl ammatory response of Pl and P2 in a model JAWS II innate immune cell line treated with 100 ug/mL of each adjuvant and incubated for 48 hours at the indicated temperatures.
  • Cytokines were assessed by cytometric bead array, and error bars are standard deviations of the mean for experiments performed in triplicate; *p ⁇ 0.05, **p ⁇ 0.01 for attenuated cytokine production at 39 °C compared to the same adjuvant at 35 °C.
  • FIGs. 22A-22C show characterization of thermophobic glycopolymer adjuvant in primary murine immune cells.
  • Polymers or TDB 100 pg/mL were incubated with F4/80+ Bone Marrow Derived Macrophages (BMDMs) (FIG. 22 A) or CD1 lc+ Bone Marrow Derived Dendritic Cells (BMDCs) (FIG. 22B) for 48 hours at the indicated temperatures (35-39 °C).
  • BMDMs Bone Marrow Derived Macrophages
  • BMDCs Bone Marrow Derived Dendritic Cells
  • FIG. 22C shows the observed relation between particle size and adjuvant activity. Linking these observations to the DLS and NOSEY data, it was concluded that the thermally induced change in activity could be due to either decreased particle size itself or enhanced lipid-NIPAM shielding interactions that result from this transition. [0035] FIGs. 23 A-23H show in-vivo performance of thermophobic vaccine adjuvants.
  • FIG. 23 A shows that mice exhibited normal weight gain post vaccination.
  • FIG. 23B shows IL-6 levels measured from mouse sera 24 hours after second vaccination. IL-6 production was increased for P2 and TDB compared to unvaccinated cohorts.
  • FIG. 23 C shows that levels of CD4+/44+/62L+ central memory T-cells found in lung and lymph node samples were increased in the P2 cohort, consistent with latent IL-6 levels measured 7 days after administering booster vaccination (FIG. 23D) and increases in central memory T-cell populations (FIG. 23E) following the full vaccination schedule as well.
  • FIG. 23F shows weight loss of mice post challenge with influenza indicated protective effects conferred by the vaccine adjuvanted with P2 was comparable to that of alum.
  • FIG. 24 shows the 1 H Correlation Spectroscopy (COSY) NMR of 4,6-O-(4- vinylbenzylidene)-a,a-trehalose.
  • FIG. 25 compares the diastereomers of 4,6-O-(4-vinylbenzylidene)-a,a- trehalose.
  • MM2 energy minimization predicts a difference of 0.40 kcal/mol between the (R) and (S) isomers, where the styrenyl substituent is favored in the equatorial position.
  • the NOESY spectra indicate interactions between the acetal hydrogen and carbohydrate ring protons P and O/S.
  • FIG. 26 shows an expansion of carbohydrate region of the COSY NMR for
  • FIG. 27 shows the COSY NMR for 4,6-O-(4-vinylbenzylidene)-6’-0- behenoyl-a,a-trehalose.
  • FIG. 28 shows the NOESY NMR for 4,6-O-(4-vinylbenzylidene)-6’-0- behenoyl-a,a-trehalose. E/T and E/P crosspeaks indicate retention of (R) stereochemistry at the benzylidene acetal.
  • FIG. 29 shows the UV-VIS spectrum of 4,6-O-(4-vinylbenzylidene)-6’-0- behenoyl-a,a-trehalose.
  • FIG. 30 shows the IR spectrum of 4,6-O-(4-vinylbenzylidene)-6’-0-behenoyl- a,a-trehalose.
  • FIG. 31 shows the 1 HNMR of poly(NIPAM)-co-poly(4,6-O-(4- vinylbenzylidene)-6’-O-behenoyl-a,a-trehalose).
  • FIG. 32 shows the 1 HNMR of poly(NIPAM)-co-poly(4,6-O-(4- vinylbenzylidene)-6’-0-behenoyl-a,a-trehalose)-Z>/oc -poly(NIPAM).
  • FIG. 33 shows temperature dependent aggregation of P2 (Bright Field 40x).
  • FIG. 34 shows the Neuraminidase Activity Assay data.
  • One aspect of the present disclosure relates to a copolymer comprising at least one PA block and at least one PB block, where PA represents a polymer block comprising one or more units of monomer A and PB represents a polymer block comprising one or more units of monomer B, with the monomer A being an amide or ester, and the monomer B being a trehalose- based monomer, where at least one hydroxyl group on the trehalose is esterified.
  • transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed subject matter. In some embodiments or claims where the term comprising is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of’ or “consisting essentially of.” [0052] Terms of degree such as “substantially,” “about,” and “approximately” and the symbol as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed.
  • alkyl means an aliphatic hydrocarbon group which may be straight or branched having 1 to about 30 carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule, or 1 to about 20 carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule, or 1 to about 15 carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule, or 1 to about 10 carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule, or 1 to about 2, 3, 4, or 5 carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule, or any number of carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule between 1 and about 30 carbon atoms.
  • Branched means that one or more alkyl groups, e.g., a lower alkyl group such as methyl, ethyl or propyl are attached to a linear alkyl chain.
  • alkyl groups include, without limitation, methyl, ethyl, w-propyl, i- propyl, //-butyl, /-butyl, w-pentyl, and 3 -pentyl.
  • Compounds described herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms.
  • Each chiral center may be defined, in terms of absolute stereochemistry, as (R)- or (S)-. This technology is meant to include all such possible isomers, as well as mixtures thereof, including racemic and optically pure forms.
  • Optically active (R)- and (S)-, (-)- and (+)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.
  • copolymer refers to a polymer derived from more than one species of monomer.
  • block copolymer or “block polymer” refers to a macromolecule comprising long sequences of different repeat units.
  • exemplary block polymers include, but are not limited to, A n B m , A n B m Am, A n B m Ck, or A n B m CkA n , where n, m and k are independently any integer.
  • LCST lower critical solution temperature
  • a mixture of polymer and solvent will form a single phase when mixed in any proportion at a temperature below the LCST.
  • a mixture of polymer and solvent will separate into different phases when mixed in certain proportions and heated to a temperature above the LCST thereby traversing through a temperature known as a cloud point.
  • a cloud point is defined as the temperature at which transmittance of the polymer solution reaches 50% of the maximum observed transmittance.
  • the LCST of a polymer solution means that the polymer of a given proportion or concentration is uniformly solvated in a solution below that temperature (i.e., LCST), but separates into 1 or more additional phases as the solution temperature is increased above the LCST.
  • number average molecular weight (Mn) refers to the total weight of the polymer divided by the number of molecules of the polymer.
  • the copolymer is a block copolymer.
  • the block copolymer comprises an architecture of PA-PB, PB-PA, PA-PB- TCTA, PA-TCTA-PB, TCTAkPA-PB-TCTA 2 , or TCTAkPB-PA-TCTA 2 , where TCTA, TCTA 1 , and TCTA 2 are moieties derived from a telechelic chain transfer agent.
  • the block copolymer comprises an architecture of PA-
  • PB-PA PA-PB-TCTA-PB
  • PA-PB-TCTA-PA PA-TCTA-PB
  • PA-TCTA-PB -PA PA-TCTA-PB
  • PA-PB-TCTA-PB-PA PA-PB-TCTA-PB-PA
  • TCTA'-PA-PB-PA-TCTA 2 TCTA ⁇ PB-PA-PB-TCTA 2
  • TCTA'-PA-PB-TCTA-PB-TCTA 2 TCTAkpA-PB-TCTA-PA-TCTA 2
  • TCTAkPA-TCTA-PB-PA-TCTA 2 or TCTA'-PA-PB- TCTA-PB-PA-TCTA 2
  • TCTA, TCTA 1 , and TCTA 2 are moieties derived from a telechelic chain transfer agent.
  • monomer A is a monomer that, if used to produce a homopolymer, produces a homopolymer having a lower critical solution temperature (LCST) that causes a cloud point between 30 °C and 45 °C at appropriate polymer concentrations known to exhibit an LCST.
  • LCST critical solution temperature
  • said monomer A is an acrylamide, methacrylamide, acrylate, or methacrylate.
  • monomer A is selected from the group consisting of N,N-diethyl acrylamide, N,N-dimethyl acrylamide, N-isopropyl acrylamide, N-ethyl acrylamide, N-tert-butyl acrylamide, N-n-propyl acrylamide, N-methyl-N-ethyl acrylamide, and methacrylamide derivates thereof.
  • said monomer A is N- isopropyl acrylamide.
  • the at least one PB block has a chemical structure of
  • L is a linker; and m is 1 to 100,000.
  • L is and each / is a point of attachment of L to the at least one hydroxyl group on the trehalose.
  • the trehalose-based monomer is a monomer comprising trehalose attached to a side chain of the monomer.
  • monomer B has a chemical structure of Formula (II):
  • R is Ci -30 alkyl
  • monomer B has a chemical structure of Formula (Ila): [0070] In some embodiments, monomer B has a chemical structure of Formula (lib):
  • the copolymer comprises a moiety selected from where is the terminal group of the polymer; a is 1 to 100,000; b is 1 to 100,000; x is 1 to 100,000; and y is 1 to 100,000.
  • the copolymer has a chemical structure selected from where a is 1 to 100,000, b is 1 to 100,000, x is 1 to 100,000, and y is 1 to 100,000.
  • the copolymer has a chemical structure selected from
  • the copolymer has a chemical structure selected from where a is 1 to 100,000, b is 1 to 100,000, x is 1 to 100,000, and y is 1 to 100,000.
  • a is about 1 to about 10, about 1 to about 20, about 1 to about 40, about 1 to about 60, about 1 to about 80, about 1 to about 100, about 1 to about 1,000, about 1 to about 10,000, about 1 to about 50,000, about 1 to about 100,000, about 5 to about 20, about 5 to about 40, about 5 to about 60, about 5 to about 80, about 5 to about 100, about 5 to about 1,000, about 5 to about 10,000, about 5 to about 50,000, about 5 to about 100,000, about 10 to about 20, about 10 to about 40, about 10 to about 60, about 10 to about 80, about 10 to about 100, about 10 to about 1,000, about 10 to about 10,000, about 10 to about 50,000, about 10 to about 100,000, about 100 to about 1,000, about 100 to about 10,000, about 100 to about 50,000, about 100 to about 100,000, about 1,000 to about 10,000, about 1,000 to about 50,000, about 1,000 to about 100,000, about 10,000 to about 50,000, about 10,000 to about 100,000, or about 50,000 to about 100,000. In some embodiments, a is 1, 2, 3,
  • b is about 1 to about 100, about 1 to about 500, about 1 to about 1,000, about 1 to about 5,000, about 1 to about 10,000, about 1 to about 20,000, about 1 to about 30,000, about 1 to about 40,000, about 1 to about 50,000, about 1 to about 60,000, about 1 to about 70,000, about 1 to about 80,000, about 1 to about 90,000, about 1 to about 100,000, about 500 to about 1,000, about 500 to about 5,000, about 500 to about 10,000, about 500 to about 20,000, about 500 to about 30,000, about 500 to about 40,000, about 500 to about 50,000, about 500 to about 60,000, about 500 to about 70,000, about 500 to about 80,000, about 500 to about 90,000, about 500 to about 100,000, about 1,000 to about 5,000, about 1,000 to about 10,000, about 1,000 to about 20,000, about 1,000 to about 30,000, about 1,000 to about 40,000, about 1,000 to about 50,000, about 1,000 to about 60,000, about 1,000 to about 70,000, about 1,000 to about 80,000, about 1,000 to about 90,000, about 500 to about 100,000
  • x is about 1 to about 100, about 1 to about 500, about 1 to about 1,000, about 1 to about 5,000, about 1 to about 10,000, about 1 to about 20,000, about 1 to about 30,000, about 1 to about 40,000, about 1 to about 50,000, about 1 to about 60,000, about 1 to about 70,000, about 1 to about 80,000, about 1 to about 90,000, about 1 to about 100,000, about 500 to about 1,000, about 500 to about 5,000, about 500 to about 10,000, about 500 to about 20,000, about 500 to about 30,000, about 500 to about 40,000, about 500 to about 50,000, about 500 to about 60,000, about 500 to about 70,000, about 500 to about 80,000, about 500 to about 90,000, about 500 to about 100,000, about 1,000 to about 5,000, about 1,000 to about 10,000, about 1,000 to about 20,000, about 1,000 to about 30,000, about 1,000 to about 40,000, about 1,000 to about 50,000, about 1,000 to about 60,000, about 1,000 to about 70,000, about 1,000 to about 80,000, about 1,000 to about 90,000, about 500 to about 100,000
  • y is about 1 to about 100, about 1 to about 500, about 1 to about 1,000, about 1 to about 5,000, about 1 to about 10,000, about 1 to about 20,000, about 1 to about 30,000, about 1 to about 40,000, about 1 to about 50,000, about 1 to about 60,000, about
  • the copolymer according to the present disclosure can have a number average molecular weight (Mn) above 1 kDa, above 2 kDa, above 3 kDa, above 4 kDa, above 5 kDa, above 6 kDa, above 7 kDa, above 8 kDa, above 9 kDa, above 10 kDa, above
  • Mn number average molecular weight
  • the copolymer can have a number average molecular weight (Mn) ranging from about 0.1 kDa to about 200 kDa.
  • Mn number average molecular weight
  • the copolymer can have a number average molecular weight (Mn) from about 0.1 kDa to about 100 kDa, from about 0.5 kDa to about 90 kDa, from about 1 kDa to about 80 kDa, from about 2 kDa to about 70 kDa, from about 3 kDa to about 60 kDa, from about 4 kDa to about 50 kDa, from about 5 kDa to about 40 kDa, from about 6 kDa to about 30 kDa, from about 7 kDa to about 30 kDa, from about
  • the copolymers according to the present disclosure can have a broad or sharp lower critical solution temperature (LCST).
  • the copolymers of the present disclosure have a LCST from about 30°C to about 50°C, from about 31°C to about 49°C, from about 32°C to about 48°C, from about 33°C to about 47°C, from about 34°C to about 46°C, from about 35°C to about 45°C, from about 36°C to about 44°C, or from about 37°C to about 43°C.
  • the copolymers of the present disclosure have a LCST of about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C.
  • Another aspect of present disclosure relates to a bioactive delivery system comprising the copolymer as disclosed herein and a bioactive agent.
  • the bioactive agent is selected from the group consisting of a protein, a (poly)peptide, a vaccine, a nucleic acid, a hormone, a cancer drug, an angiogenesis inhibitor, a growth factor, and an anti-microbial substance.
  • Another aspect of present disclosure relates to a composition comprising the copolymer as disclosed herein and a vaccine.
  • the vaccine is selected from an inactivated enveloped virus, whole cell, cell lysate, tumor-associated antigen, tumor-specific antigen, protein, nucleic acid, and (poly)peptide.
  • suitable inactivated enveloped viruses include, without limitation, herpesviruses, poxviruses, hepadnaviruses, asfarviridae, flavivirus, alphavirus, togavirus, coronavirus, hepatitis viruses, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, filovirus, and retroviruses.
  • the inactivated enveloped virus is selected from the group consisting of Ebola virus, human immunodeficiency virus, influenza virus, Lassa fever virus, Nipah virus, respiratory syncytial virus, Rift Valley fever virus, SARS virus, MERS virus, Marbury virus, swine pox virus, Cytomegalovirus, Crimean hemorrhagic fever virus, and COVID-19.
  • bioactive agent delivery system or the composition disclosed herein further comprise an adjuvant, antibiotic, antiviral, pharmaceutically acceptable carrier, stabilizer, and/or preservative.
  • suitable adjuvants include, without limitation, an aluminum salt, inulin, argamline, a combination of inulin and aluminum hydroxide, monophosphoryl lipid A (MPL), resiquimoid, muramyl dipeptide (MDP), N -Glycolyl dipeptide (GMDP, N-glycolyl dipeptide), poly IC, CpG oligonucleotide, anciclovir, resiquimod, aluminum hydroxide containing MPL, a water-in-oil emulsion, squalene or analogs thereof, any pharmaceutically acceptable oil, tween-80, sorbitan trioleate, alpha-tocopherol, cholecalciferol or any analogs thereof, derivatives thereof, calcium-modified forms thereof, phosphate-modified forms thereof, and combinations thereof.
  • suitable antibiotics include, without limitation,
  • suitable preservatives include, without limitation, chlorobutanol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal, phenylmercuric nitrate, and combinations thereof.
  • suitable pharmaceutically acceptable carriers include, without limitation, pyrogen-free water, isotonic saline, buffered aqueous solutions, including aqueous phosphate buffers, aqueous citrate buffers, and combinations thereof.
  • suitable stabilizers include, without limitation, sorbitol,
  • L- glycine L-glycine, mannitol, L-glutamic acid, human serum albumin, and combinations thereof.
  • suitable antivirals include, without limitation, zidovudine, acyclovir, anciclovir, ganciclovir, vidarabine, idoxuridine, trifluridine, ribavirin, foscamet, amantadine, peramivir, rimantadine, saquinavir, indinavir, ritonavir, alpha-interferons, AZT, t-705, zanamivir, oseltamivir, influenza virus vaccines, and combinations thereof.
  • the vaccine composition (composition comprising the copolymer as disclosed herein and a vaccine) can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraperitoneal, intranasal, or intramuscular means for prophylactic or therapeutic treatment.
  • the vaccine compositions of the present disclosure may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or it may be enclosed in hard or soft shell capsules, or it may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the vaccine compositions of the present disclosure may also be administered in a time release manner incorporated within such devices as time-release capsules or nanotubes. Such devices afford flexibility relative to time and dosage.
  • the vaccine compositions may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like.
  • compositions and preparations should contain at least 0.1% of the copolymer, although lower concentrations may be effective and indeed optimal.
  • the percentage of the copolymer in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit.
  • the amount of a copolymer of the present invention in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, com starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, sucrulose, or saccharin.
  • a liquid carrier such as a fatty oil.
  • oral dosage forms comprising the copolymers of the present disclosure.
  • tablets may be coated with shellac, sugar, or both.
  • a syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
  • the vaccine compositions may also be administered parenterally.
  • Solutions or suspensions of the vaccine compositions can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils.
  • Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
  • water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
  • compositions of the present disclosure When it is desirable to deliver the vaccine compositions of the present disclosure systemically, they may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi -dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • Intraperitoneal or intrathecal administration of the vaccine compositions of the present disclosure can also be achieved using infusion pump devices such as those described by Medtronic, Northridge, CA. Such devices allow continuous infusion of desired compositions avoiding multiple injections and multiple manipulations.
  • the vaccine compositions may also be formulated as a depot preparation.
  • Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the vaccine compositions may also be administered directly to the airways in the form of an aerosol.
  • the vaccine compositions in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • the vaccine compositions also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
  • Another aspect of the present disclosure relates to a method of vaccinating a subject against infection by an enveloped virus. This method comprises providing a composition according to the present disclosure and treating the subject with the composition to vaccinate the subject against the enveloped virus.
  • the subject is a human.
  • the subject is a non-human animal such as non-human primate, dog, cat, sheep, goat, cow, pig, horse, or rodent.
  • Factors to be accounted for when administering the vaccine of the present disclosure in order to produce a robust immune response include without limitation the concentrations of the vaccine, the presence of an adjuvant, the mode and frequency of administration, and the subject’s medical details, such as age, weight and overall health and immune condition.
  • a clinician may administer the vaccine composition until a dosage is reached that provides the desired or required prophylactic effect, e.g., the desired antibody titers. The progress of this therapy can be easily monitored by conventional assays.
  • the method further comprises selecting a subject in need of vaccination against infection by an enveloped virus.
  • the vaccine composition as descried herein is administered prophylactically to prevent, delay, or inhibit the development of the infection in a subject at risk of being infected with an enveloped virus.
  • prophylactic administration of the vaccine composition is effective to fully prevent infection in an individual of the enveloped virus of the vaccine composition.
  • prophylactic administration is effective to prevent the full extent of infection that would otherwise develop in the absence of such administration, i.e., substantially prevent or inhibit the enveloped virus infection in an individual.
  • Another aspect of the present disclosure relates to a method of preparing a copolymer.
  • This method comprises providing a radically polymerizable amide monomer A, providing a trehalose-based monomer B comprising at least one esterified hydroxyl group or a polymer block PB comprising one or more units of said monomer B, and polymerizing the monomer A with the monomer B or the polymer block PB.
  • RAFT reversible additionfragmentation chain transfer
  • Radical Addition-Fragmentation Chain Transfer polymerization limits the number of initiation sites and drastically reduces the rate of polymer-to-polymer chain transfer and termination reactions, and also introduces the capability of producing custom chain architectures such as block copolymers (BCPs) and statistical copolymers. This degree of control is superior to that offered by other controlled radical polymerization methods —that is, polymers of more narrow dispersity may be obtained over a shorter period of time with less rigorous purification and lower batch-to-batch variability.
  • BCPs block copolymers
  • statistical copolymers statistical copolymers
  • RAFT polymerization is a type of living polymerization or controlled polymerization, utilizing a chain transfer agent (CTA).
  • CTA chain transfer agent
  • the RAFT polymerization reaction starts with initiation.
  • Initiation is accomplished by adding an agent capable of decomposing to form free radicals; the decomposed free radical fragment of the initiator attacks a monomer yielding a propagating radical (P' n ), in which additional monomers are added producing a growing polymer chain.
  • CTA chain transfer agent
  • This radical (R') reacts with a new monomer molecule forming a new propagating radical (P'm).
  • P' n ) and P'm reach equilibrium and the dormant polymer chain (3) provides an equal probability to all polymers chains to grow at the same rate, allowing polymers to be synthesized with narrow polydispersity. Termination is limited in RAFT and, if occurring, is negligible. For traditional monomers which contain only one polymerizable species, targeting a specific molecular weight in RAFT can be calculated by multiplying the ratio of monomer consumed to the concentration of CTA used by the molecular weight of the monomer. When applied to monomers containing species with two or more polymerizable moieties, branched polymers may form that further increase the molecular weight. Initiation
  • initiators The initiating agents often are referred to as "initiators.” Suitable initiators depend greatly on the details of the polymerization, including the types of monomers being used, the type of catalyst system, the solvent system, and the reaction conditions.
  • a typical radical initiator can be azo compounds, which provide a two-carbon centered radical.
  • Radical initiators such as benzoyl peroxide, azobisisobutyronitrile (AIBN), 1,1' azobis(cyclohexanecarbonitrile) or (ABCN), or 4,4’ -azobis(4-cyanoval eric acid) (ACVA); redox initiator such as benzoyl peroxide/N,N-dimethylaniline; microwave heating initiator; photoinitiator such as (2,4,6-trimethylbenzoyl)-diphenylphosphine oxide; gamma radiation initiator; or Lewis Acids such as scandium(III) tritiate or yttrium (III) tritiate, are typically used in RAFT polymerization.
  • AIBN azobisisobutyronitrile
  • ABCN 1,1' azobis(cyclohexanecarbonitrile) or (ABCN), or 4,4’ -azobis(4-cyanoval eric acid)
  • redox initiator such as benzoyl
  • Z can also be sulfonyl, phosphonate, or phosphine.
  • a CTA agent’s effectiveness depends on the monomer being used and is determined by the properties of the free radical leaving group R and the Z group. These groups activate and deactivate the thiocarbonyl double bond of the RAFT agent and modify the stability of the intermediate radicals (Moad et al., “Living Radical Polymerization by the Raft Process- a Second Update,” Australian Journal of Chemistry 62(11): 1402-72 (2009), which is hereby incorporated by reference in its entirety).
  • Typical CTA agents used are 1 -phenylethyl benzodithioate or 1 -phenylethyl 2- phenylpropanedithioate.
  • the chain transfer agent used is a telechelic chain transfer agent, which typically is based on trithiocarb onate functionality.
  • Polymers produced from the chain transfer agent based on a trithiocarb onate functional group retain the CTA functionality in the statistical center of the chain, as opposed to polymers produced by a dithiocarbonate-based CTA, which retain the CTA functionality at the end of the polymeric chain.
  • the telechelic chain transfer agent is capable of adding polymer blocks symmetrically from the interior where the trithiocarbonate functionality is located, i.e., polymerizing monomers from both ends, forming symmetrical architecture or polymer blocks.
  • the RAFT process begins with the chain transfer of a growing A radical to a functional trithiocarb onate, as shown in Scheme 2.
  • the formed radical intermediate is stable against coupling or disproportion reactions with other free radicals.
  • one of the thioate groups reversibly fragments allowing propagation of one of the three arms.
  • Arr Scheme 3 Schematic showing of the basic propagation mechanism of RAFT polymerization using a trithiocarb onate CTA
  • RAFT Reversible Addition-Fragmentation Chain Transfer
  • the polymerizing step is carried out in a solvent at a temperature of 50 to 140 °C.
  • the polymerizing is carried out in a solvent at a temperature of about 50 to about 60 °C, about 50 to about 70 °C, about 50 to about 80 °C, about 50 to about 90 °C, about 50 to about 100 °C, about 50 to about 110 °C, about 50 to about 120 °C, about 50 to about 130 °C, about 50 to about 140 °C, about 60 to about 70 °C, about 60 to about 80 °C, about 60 to about 90 °C, about 60 to about 100 °C, about 60 to about 110 °C, about 60 to about 120 °C, about 60 to about 130 °C, about 60 to about 140 °C, about 70 to about 80 °C, about 70 to about 90 °C, about 70 to about 100 °C, about 70 to about 110 °C, about 70 to about 120 °C, about 70 to about 120 °C, about
  • Suitable solvents include, without limitation, dimethylformamide, dimethylacetamide, dimethylsulfoxide, acetonitrile, water, tetrahydrofuran, dioxane, saturated hydrocarbons such as heptane, halogenated hydrocarbons such as chloroform or di chloromethane, toluene, benzene, halogenated derivates of benzene such as chlorobenzene, or xylenes.
  • the polymerizing is carried out via reversible additionfragmentation chain-transfer polymerization (RAFT).
  • RAFT reversible additionfragmentation chain-transfer polymerization
  • said polymerizing is carried out in the presence of a free radical initiator and a chain transfer agent.
  • the free radical initiator is selected from the group consisting of benzoyl peroxide, azobisisobutyronitrile, 1,1' azobis(cyclohexanecarbonitrile), t- butylperoxide, and 4,4’ -azobis(4-cyanoval eric acid).
  • the chain transfer agent is a thiocarb onylthio compound, a dithioester compound, a trithiocarb onate compound, a dithiocarbamate compound, or a xanthate compound capable of reversible association with polymerizable free radicals.
  • the chain transfer agent is a telechelic chain transfer agent
  • said polymerizing comprises polymerizing said monomer A and said monomer B via RAFT to form a TCTA ⁇ PA-PB-TCTA 2 or PA-PB-TCTA-PB-PA, wherein TCTA 1 , TCTA 2 , and TCTA are moieties derived from a telechelic chain transfer agent.
  • the method further comprises providing a radically polymerizable amide, represented by A, providing a polymer TCTA ⁇ PA-PB-TCTA 2 or PA-PB- TCTA-PB-PA, and polymerizing said monomer A with the polymer TCTA ⁇ PA-PB-TCTA 2 or PA-PB-TCTA-PB-PA.
  • A a radically polymerizable amide
  • the step of providing a polymer block PB comprising one or more units of said monomer B comprises providing said monomer B, polymerizing said monomer B in a solvent suitable for dissolving at least one of said PB block, and functionalizing the at least one of said PB block with one or more chain transfer groups or one or more initiator groups to produce a functionalized PB block.
  • Polymer cloud point (Cp) was determined via UV-Vis transmittance at 600 nm and a 50% decrease in transmittance was noted as the LCST of the polymer.
  • LCST Polymer cloud point
  • Particle size distributions of the synthesized polymers were determined using a Malvern Zetasizer Pro Blue (Malvern Panalytical). Samples were dissolved in Endotoxin Free H2O (Millipore Sigma) at a concentration of 100 ug/mL. A representative trace is shown in FIG. 3B. Samples were measured in triplicate and averaged to provide the reported particle sizes.
  • the HEK-Blue mMincle cell line was purchased from Invivogen (San Diego,
  • HEK-Blue mMincle cells were grown in complete media composed of high glucose DMEM (Cytiva HyClon Dulbecco's Modified Eagles Medium) (Cytiva, Marlborough, USA, SH30243.01) with 4.5 g L' 1 glucose, 2mM L-glutamine, 100 U mL' 1 PenStrep (Caisson Labs, Smithfield, UT, USA, PSLOl-lOOmL) and 0.1 mg mL' 1 Zeocin (Invivogen, San Diego, CA, USA) supplemented with 10% premium grade Heat Inactivated (HI)-FBS (VWR, Radnor, PA, USA, 97068-091). The media was changed every 3-4 days. Cells were passaged upon 80% confluence. Passaging involved changing media, counting, and seeding 3xl0 5 cells in 35 mL of new complete media in a
  • BMDCs Bone Marrow-Derived Dendritic Cells
  • BMDMs Macrophages
  • the monocytes were seeded at a density of 2 x 10 6 cells/mL in 10 mL of complete media which comprised of RPMI (Fisher Scientific, Waltham, MA, USA, 11-875-093) with 2 mM glutamine, 50 pM P-mercaptoethanol and supplemented with 10% HI-FBS and 20 ng/mL recombinant murine GMCSF (Peprotech, Westlake Village, CA, USA, 315-03).
  • complete media comprised of RPMI (Fisher Scientific, Waltham, MA, USA, 11-875-093) with 2 mM glutamine, 50 pM P-mercaptoethanol and supplemented with 10% HI-FBS and 20 ng/mL recombinant murine GMCSF (Peprotech, Westlake Village, CA, USA, 315-03).
  • RPMI Fisher Scientific, Waltham, MA, USA, 11-875-093
  • mM glutamine 50 pM P-mercaptoethanol
  • the media was removed and centrifuged at 200 G for 10 min to isolate the non-adherent fraction containing BMDCs.
  • the adherent cells were released by adding 0.25% Trypsin (Sigma Aldrich, St. Louis, MO, USA, T- 4049-100mL) and incubated at 37 °C for 10 min before neutralizing with Trypsin neutralizing solution (10% HI-FBS in IX PBS) to isolate the BMDMs.
  • Trypsin neutralizing solution (10% HI-FBS in IX PBS
  • BMDCs and BMDMs isolated from mouse femurs were added to Eppendorf vials and blocked using Purified Anti-Mouse CD16/CD32 blocking antibody (Tonbo Biosciences, San Diego, CA, USA, 70- 0161-U500) following a 1 :50 dilution for 20 min on ice. The vials were then centrifuged at 200G for 5 min at 4 °C and the supernatant discarded. The antibodies were then added to the cells and incubated for 1 hour at 4 °C in the dark.
  • the BMDCs were stained with the following antibodies: FITC Anti-Mouse CD11c (Tonbo Biosciences, San Diego, CA, USA, 35-0114-U100) at 0.25 pg mL' 1 , APC Anti-Human/Mouse CD1 lb (Tonbo Biosciences, San Diego, CA, USA, 20-0112- U100) at 0.125 pg ml 1 , and PE Anti-Mouse F4/80 (Tonbo Biosciences, San Diego, CA, USA, 50-4801-U100) at 0.25 pg mL' 1 all within the range suggested by the manufacturers.
  • the BMDMs were stained with the following antibodies: FITC Anti-Mouse CD86 (Tonbo Biosciences, San Diego, CA, USA, 35-0862-U100) at 0.125 pg mL’ 1 , APC Anti-Mouse CD206 (Biolegend, San Diego, CA, USA, 141708) at 0.25 pg mL’ 1 , and PE Anti-Mouse F4/80 (Tonbo Biosciences, San Diego, CA, USA, 50-4801-U100) at 0.25 pg mL’ 1 .
  • FITC Anti-Mouse CD86 Teonbo Biosciences, San Diego, CA, USA, 35-0862-U100
  • APC Anti-Mouse CD206 Biolegend, San Diego, CA, USA, 141708
  • PE Anti-Mouse F4/80 Teonbo Biosciences, San Diego, CA, USA, 50-4801-U100
  • Influenza virus A/Ca/04/2009 was propagated using standard protocols
  • mice Twenty, eight-week-old female C57BL6 mice (Charles River Laboratories) were housed in a BSL-2 vivarium. There were six groups of three mice per treatment group and a group of two unchallenged unvaccinated mice. Three mice per group were immunized intramuscularly with 100 pl of a 1 : 1 mixture of inactivated influenza virus and adjuvant or control. The groups were divided as follows: alum, TDB, Pl, P2, no adjuvant (inactivated virus only), and the final group received only phosphate-buffered saline (PBS) as control. The mice were given a vaccine boost on day 15 post prime vaccination and body weights were recorded every two days for 28 days.
  • PBS phosphate-buffered saline
  • mice were bled on days 0, 7, 14, 16, 22, and 28 post vaccination and challenged intranasally with mouse-adapted live Influenza A/Califomia/04/2009 strain virus on day 29. Mice were observed for signs of disease and weight loss after infection. All mice groups were euthanized five days post infection followed by collection of blood and lung samples.
  • NI assays were adapted from Leang and Hurt (Leang and Hurt,
  • 2'-(4-Methy1umbellifery1)-a ⁇ D-N-acetylneuraminic acid (MUNANA) substrate was then added, and the sealed plates were incubated at 37 °C for 1 hour followed by addition of stop solution. Inhibition of enzymatic activity was assessed using a plate reader (Tecan Spark, Zurich, Switzerland) set to 355 nm excitation measuring absorbance at 460 nm. These results are shown in FIG. 34.
  • IL-6 levels were quantified from vaccinated mouse sera using the High
  • Lymphocytes were extracted from mouse lungs with mechanical and enzymatic digestion (Liberase TL and DNase I (Sigma-Aldrich, St. Louis)). Flow cytometry data was acquired using Attune NxT (Thermo Fisher) and analyzed using FlowJo (Tree Star, Ashland, OR), as described. Central memory T cells were identified using the following antibody (clone) combinations: anti-CD16/CD32 Fc Block (93), anti-CD45 (30-F11), anti-CD3 (145- 2C11), anti-CD4 (RM4-5), anti-CD62L (MEL-14), anti-CD44 (IM7), viability dye (Invitrogen 65-0866-14).
  • the Cloud point (Cp) of the copolymers was determined via UV-Vis transmittance at 600 nm and a decrease in transmittance to 50% of the initial was reported as the LCST of the polymer.
  • the polymers were tested at concentrations of 1 mg/mL, 100 ug/mL, 50 ug/mL 25 ug/mL, 10 ug/mL, and 1 ug/mL.
  • the LCST values were affected by both temperature and the varied weight fractions of polymer in solution. These results are shown in FIG. 14 and FIG. 15.
  • HEK-Blue mMincle cells (Invivogen, CA) were plated in 96 well plates in
  • SEAP detection media (1.17 mg mL' 1 5-bromo-4- chloro-3’-indolylphosphate p-toluidine salt in 1 M aqueous diethanolamine) was used to measure SEAP for all MINCLE assays.
  • 180 pL of SEAP detection media was added to each well.
  • 20 pL of supernatant from the MINCLE assays was added to each well and the plates were incubated for 1-4 hours to achieve significant positive control absorbance. Following incubation, the absorbance of each well was measured at 620 nm. Experiments were performed in hexaplet and normalized to positive and negative controls.
  • BMDC and BMDM primary cultures were isolated from mouse femurs and seeded at a density of 2.5 x 105 cells per well in 24 well plates. The plates were then incubated for approximately 6 hours at the temperatures tested (35 °C, 37 °C, and 39 °C) prior to adding the test compounds. Pl, P2, P3, as well as TDB were added to the wells at a concentration of 100 pg mL' 1 while the more potent LPS (Sigma Aldrich, St. Louis, MO, USA, L2018-5MG) was tested at 40 pg mL' 1 . The plates were then incubated at the given temperatures for 48 hours before the supernatant was collected.
  • methyltriphenylphosphonium bromide (5.4 g, 14.5 mmol) and 45 mL of tetrahydrofuran (THF) were added to a flame dried 250 mL round bottom flask.
  • THF tetrahydrofuran
  • the reaction mixture was cooled to -78 °C and stirred for 20 min.
  • 1.6M n-butyllithium in hexanes (9.94 mL, 15.9 mmols n-BuLi) was added drop wise over the course of 20 min.
  • the reaction was stirred for 30 min at -78 °C, warmed to 25 °C and stirred for 10 min.
  • the orange-red colored reaction solution was then cooled down to -78 °C.
  • Ethyl-(l-phenylethyl) Trithiocarbonate [0159] Ethyl-(l-phenylethyl) trithiocarb onate was synthesized according to a literature procedure (Wood et al., “Selective One-Pot Synthesis of Trithiocarbonates, Xanthates, and Dithiocarbamates for Use in Raft/Madix Living Radical Polymerizations,” Organic Letters, 8 (4): 553-556 (2006), which is hereby incorporated by reference in its entirety). First, a suspension of K3PO4 (31.94 mg, 0.52 mmol) was formed in dry acetone (15mL) and cooled to 0°C.
  • thermoresponsive adjuvant linear poly-N-isopropylacrylamide
  • NIP AM aqueous Lower Critical Solution Temperature
  • LCST Lower Critical Solution Temperature
  • NIP AM has been previously conjugated to established vaccine adjuvants (Hoshi et al., “Synthesis of a Hemin-Containing Copolymer as a Novel Immunostimulator that Induces IFN-gamma Production,” Ini. J.
  • thermoresponsive polymers including other acrylamides (Francica et al., “Thermoresponsive Polymer Nanoparticles Co-deliver RSV F Trimers with a TLR-7/8 Adjuvant,” Bioconjug.
  • polyesters/ethers Bobbala et al., “Novel Injectable Pentablock Copolymer Based Thermoresponsive Hydrogels for Sustained Release Vaccines,” AAPS J., 18(1):261-269 (2016), which is hereby incorporated by reference in its entirety
  • polyacetals Vinyl Herck et al., “Transiently Thermoresponsive Acetal Polymers for Safe and Effective Administration of Amphotericin B as a Vaccine Adjuvant,” Bioconjug.
  • CLRs C-Type Lectin Receptors
  • MINCLE Macrophage- Inducible C-type Lectin
  • TDB hydroxyls weakly coordinate with hydrophilic binding domains that share structural similarity among CLRs (Furukawa et al., “Structural Analysis for Glycolipid Recognition by the C-type Lectins Mincle and MCL,” Proc. Natl. Acad. Sci., 110(43): 17438— 17443 (2013), which is hereby incorporated by reference in its entirety), and in the case of MINCLE, bind with Arg 182.25 Although these polar binding interactions are somewhat weak, the hydrophobic portions of TDB can also interact with their own binding domains and facilitate coordination.
  • TDB and related analogues
  • multivalent physiochemical presentation which can enhance avidity and also induce clustering of receptors, cumulatively resulting in synergistic MINCLE activation
  • Retzinger et al. “The Role of Surface in the Biological Activities of Trehalose 6,6’-dimycolate. Surface Properties and Development of a Model System,” J. Biol. Chem., 256(15):8208-8216 (1981); Retzinger, “Dissemination of Beads Coated with Trehalose 6,6'-dimycolate: A Possible Role for Coagulation in the Dissemination Process,” Exp. Mol.
  • Molecular design of a polymerizable trehalose glycoplipid adjuvant required the construction of a polymerizable adjuvant subunit consisting of a trehalose carbohydrate core and a behenic ester lipid chain that can interact with the hydrophobic domains of MINCLE.
  • inspiration was drawn from the 4,6-vinylbenzylidene acetal monomer which can be accessed in a single step from trehalose with no protecting groups and moderate yields (Mancini et al., “Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors,” J. Am. Chem.
  • RAFT Reversible Addition-Fragmentation Chain-Transfer
  • NIP AM 1, a trithiocarb onate Chain Transfer Agent (CTA)
  • CTA trithiocarb onate Chain Transfer Agent
  • AIBN AIBN initiator
  • thermophobic trehalose glycolipid adjuvant P2 which had an identical degree of polymerization with respect to adjuvant, but higher molecular weight and sharper LCST.
  • Adjuvant M n was determined by GPC
  • lipidated glycomonomer 1, Pl, and P2 would each activate the Macrophage-Inducible C-type Lectin (MINGLE) receptor.
  • MINGLE Macrophage-Inducible C-type Lectin
  • Pl and P2 activated HEK-mMINCLE cells in a more potent and dosedependent manner, consistent with previous reports illustrating that glycolipid multivalency dramatically affects MINCLE binding and subsequent activation.
  • Pl, and particularly P2 were more potent relative to TDB positive control, although this could be due to the multivalent nature of the polymers.
  • responses were comparable to TDB (FIG. 2 IB).
  • only minimal activity was observed for P3, indicating that neither the NIP AM nor trehalose blocks (or the cumulative trehalose-NIPAM combination) were significant MINCLE agonists (FIG. 10).
  • thermophobic adjuvants would enhance the efficacy of a vaccine.
  • temperature-dependent potency confirmed in-vitro, the thermophobic glycolipid polymers were next examined to determine if they would perform as functional vaccine adjuvants in-vivo.
  • TDB, Pl, and P2 adjuvants were standardized according to the number of adjuvant subunits; 100 nmol of adjuvant subunit was used for each of these cohorts.
  • Mice were vaccinated on day 0 and boosted on day 15, followed by influenza challenge on day 30 post vaccination. Blood samples were drawn to assess antibody response (day 14 and 29) and inflammatory cytokines (24 hours and 7 days post second vaccination). Vaccines were well tolerated by all vaccinated cohorts (FIG. 23 A) and did not result in significant changes in weight-gain. Serum samples were also assayed for IL-6 as a measure for systemic inflammation (FIG. 23B).
  • Pl and P2 produced inflammation comparable to alum adjuvanted control but with enhanced production of lung and lymph node central memory CD4+/44+/62L+ T cells (lung and lymph node central memory CD4+/44+/62L+ T cells (FIG. 23C). Further, at 7 days post-boost, this effect of lower IL-6 production and enhanced T cell proliferation was extended, particularly for the P2 cohort relative to control adjuvants (FIGs. 23D and 23E). Following vaccination, mice were challenged intranasally with mouse-adapted live Influenza A/California/04/2009 strain virus on day 30 and monitored for weight loss over 5 days followed by euthanasia.
  • thermophobic adjuvants were synthesized and characterized for their thermoresposive behavior and performance as thermophobic vaccine adjuvants. Consistent with other multivalent trehalose glycolipids, the thermophobic adjuvants exhibited promiscuous agonism among HEK-mMINCLE, and JAWSII innate immune cell lines indicating that, at a minimum, these polymers activate the MINCLE receptor, and likely activate other innate immune cell receptors as well.
  • thermophobic inflammatory immunological profiles that were modulated by small (1-2 °C) changes in temperature in an innate immune cell line (JAWS II) and primary Bone Marrow-Derived Dendritic Cells and Macrophages in vitro.
  • the molecular mechanism of action for thermophobic immune cell activation is suspected to arise from either inter- or intra- molecular changes in the polymer conformation; decreased particle size could result in attenuated activity, or this could arise from shielding interactions between the lipids, critical for activity, with hydrophobic portions of NIP AM subunits.
  • Further structural elucidation by variable-temperature DLS revealed a decrease in particle size for Pl and a slightly decreased particle size, but variable D for P2.
  • thermophobic adjuvants that modulate their potency in response to temperature in-vitro and are effective vaccine adjuvants in-vivo. It is envisioned that the thermophobic adjuvant concept will help address the highly heterogeneous immune responses vaccine adjuvants elicit at the population-level by coupling potency to inflammation-induced pyrexia thereby personalizing adjuvant activity to an individual’s own immune response.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pulmonology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Inorganic Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)

Abstract

The present disclosure relates to a copolymer comprising at least one PA block and at least one PB block, wherein PA represents a polymer block comprising one or more units of monomer A and PB represents a polymer block comprising one or more units of monomer B, with the monomer A being an amide or ester, and the monomer B being a trehalose-based monomer, wherein at least one hydroxyl group on the trehalose is esterified. The present disclosure also relates to a method of preparing the copolymer and a bioactive agent delivery system comprising the copolymer and a bioactive agent. The present disclosure further relates to a composition comprising the copolymer and a vaccine and a method of vaccinating a subject against infection by an enveloped virus.

Description

THERMOPHOBIC TREHALOSE GLYCOPOLYMERS AS SMART C-TYPE LECTIN RECEPTOR VACCINE ADJUVANTS
[0001] This application claims the priority benefit of U.S. Provisional Patent
Application Serial No. 63/450,875, filed March 8, 2023, which is hereby incorporated by reference in its entirety.
[0002] This invention was made with government support under DI 8AC00031 awarded by Department of Defense and CA234115 awarded by National Institutes of Health. The government has certain rights in the invention.
FIELD
[0003] The present disclosure relates to thermophobic trehalose glycopolymers as smart C-type lectin receptor vaccine adjuvants.
BACKGROUND
[0004] Pyrexia, a hallmark of the non-resolved inflammatory response, along with other symptoms of systemic reactogenicity, limits both the dosing and type of adjuvant that may be added to vaccine formulations to remain within their strict safety margins (Pasquale et al., “Vaccine Adjuvants: from 1920 to 2015 and Beyond,” Vaccines, 3(2):320-343 (2015); Bonam et al., “An Overview of Novel Adjuvants Designed for Improving Vaccine Efficacy,” Trends Pharmacol. Sci., 38(9):771— 793 (2017); Apostolico et al., “Adjuvants: Classification, Modus Operandi, and Licensing,” J. Immunol. Res., 2016(1459394): 1-16 (2016)). Even today, pyrexia is the most common adverse event reported to the Vaccine Adverse Event Reporting System (VAERS) for adjuvanted influenza vaccines (VAERS - Data, https://vaers.hhs.gov/data.html). Adjuvant activity within a vaccine formulation is currently limited to minimize adverse events, which simultaneously decreases efficacy (Herve et al., “The How’s and What’s of Vaccine Reactogenicity,” NPJ Vaccines, 4(39): 1-11 (2019); Burny et al., “Inflammatory Parameters Associated with Systemic Reactogenicity Following Vaccination with Adjuvanted Hepatitis B Vaccines in Humans,” Vaccine, 37(14):2004-2015 (2019)). Significant efforts are underway to design new adjuvants that enhance efficacy without inflammation (Moser et al., “Increased Vaccine Tolerability and Protection via NF-KB Modulation,” Sci. Adv., 6(37):eaaz8700 (2020)); however, from an epidemiological perspective, striking this balance in a vaccine formulation remains a challenge, because the immune response elicited by adjuvants is highly heterogeneous among individuals or cohorts (Beyer et al., “Cold-Adapted Live Influenza Vaccine Versus Inactivated Vaccine: Systemic Vaccine Reactions, Local and Systemic Antibody Response, and Vaccine Efficacy: A Meta-analysis,” Vaccine, 20(9-10): 1340-1353 (2002)).
[0005] The present disclosure is directed to overcoming these and other deficiencies in the art.
SUMMARY
[0006] One aspect of the present disclosure relates to a copolymer comprising at least one PA block and at least one PB block, where PA represents a polymer block comprising one or more units of monomer A and PB represents a polymer block comprising one or more units of monomer B, with the monomer A being an amide or ester, and the monomer B being a trehalose- based monomer, where at least one hydroxyl group on the trehalose is esterified.
[0007] Another aspect of the present disclosure relates to a bioactive agent delivery system comprising the copolymer according to the present disclosure and a bioactive agent.
[0008] Another aspect of the present disclosure relates to a composition comprising the copolymer according to the present disclosure and a vaccine.
[0009] Yet another aspect of the present disclosure relates to a method of vaccinating a subject against infection by an enveloped virus. This method involves providing a composition according to the present disclosure and treating the subject with the composition to vaccinate the subject against the enveloped virus.
[0010] A further aspect of the present disclosure relates to a method of preparing a copolymer. This method involves providing a radically polymerizable amide monomer A, providing a trehalose-based monomer B comprising at least one esterified hydroxyl group or a polymer block PB comprising one or more units of the monomer B, and polymerizing the monomer A with the monomer B or the polymer block PB.
[0011] It was envisioned that a synthetic adjuvant which attenuates activity in response to heating on the scale associated with pyrexia (AT = 1-2 °C) could present a safer, more personalized, approach to adjuvanting vaccines (FIG. 1 A). This disclosure reports the first synthetic vaccine adjuvants that attenuate potency in response to small, 1-2 °C, changes in temperature about their Lower Critical Solution Temperature (LCST). Adjuvant additives significantly increase vaccine efficacy. However, adjuvants also cause inflammatory side-effects, such as pyrexia, which currently limits their use. To address this, a thermophobic vaccine adjuvant engineered to attenuate potency at temperatures correlating to pyrexia was created. Thermophobic adjuvants were synthesized by combining a rationally designed trehalose glycolipid vaccine adjuvant with thermoresponsive poly-N-isoporpylacrylamide (NIP AM) via Reversible Addition-Fragmentation Chain-Transfer polymerization. The resulting thermophobic adjuvants exhibited LCSTs near 37 °C, and self-assembled into nanoparticles with temperaturedependent sizes. Thermophobic adjuvants activated HEK-mMINCLE and other innate immune cell lines as well as primary mouse Bone Marrow-Derived Dendritic Cells and Macrophages. Inflammatory cytokine production was attenuated under conditions mimicking pyrexia (above the LCST) relative to homeostasis (37 °C) or below the LCST. This thermophobic behavior correlated with decreased adjuvant Rg observed by DLS, as well as glycolipid-NIPAM shielding interactions observed by NOESY-NMR. In-vivo, thermophobic adjuvants enhanced efficacy of an inactivated Influenza A/Califomia/04/2009 virus vaccine, by increasing neutralizing antibody titers and CD4+/44+/62L+ lung and lymph node central memory T cells, as well as providing better protection from morbidity.
[0012] The present disclosure describes the synthesis of thermophobic trehalose glycolipid polymers, as well as their in-vitro and in-vivo performance as the first synthetic vaccine adjuvants that modulate their potency across an LCST in physiological temperature ranges.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 A illustrates the multivalent thermophobic glycolipid vaccine adjuvants disclosed herein, which self-assemble into nanoparticles which decrease in size, and immunogenicity, in response to temperature. FIG. IB shows 6,6’ -trehalose dibehenate, a minimally active adjuvant motif, Brartemicin, a high affinity C-type Lectin Receptor (CLR) ligand, and 4,6-vinylbenylidene trehalose, which is polymerizable. FIG. 1C shows a polymerizable glycolipid adjuvant that activates the Macrophage-Inducible C-Type Lectin Receptor (MINCLE) class of CLR on innate immune cells and could be readily incorporated within a thermoresponsive polymer.
[0014] FIG. 2 shows a synthesis of glycolipid monomer 1, and thermophobic glycopolymer adjuvants (Pl and P2) along with unadjuvanted thermoresponsive glycopolymer P3 synthesized as a negative control. The synthesis began from an established intermediate, 4,6- O-(4-vinylbenzylidene)-a,a-trehalose monomer. Preparation of the glycolipid monomer is then accomplished via a Steglich esterification of the 6’ hydroxyl group with behenic acid to yield the polymerizable adjuvant 1. The copolymer is then synthesized via RAFT polymerization, where 1 and NIP AM are used to create a statistical copolymer. Subsequent re-initiation with AIBN using Pl as a macro-Chain Transfer Agent and additional NIP AM monomer afforded P2 which contained a congruent adjuvant degree of polymerization, with an additional thermoresponsive NIP AM block.
[0015] FIGs. 3A-3C show characterization of thermophobic glycopolymer adjuvant copolymers. FIG. 3 A shows that polymers (1 mg/mL in PBS) were observed to have Lower Critical Solution Temperatures (LCSTs) near 37 °C by UV/Vis. FIG. 3B shows that further dilution diminished this effect and instead resulted in polymer nanoparticles (100 pg/mL in PBS, pH =7.4) observed by Dynamic Light Scattering (DLS) at 25, 35, 37, and 39 °C. For Pl and P2, particle size decreased as temperature increased. This was the opposite trend observed for the control polymer P3. FIG. 3C shows NOESY-NMR of Pl and P2 confirmed interactions between the behenic ester lipid and isopropyl amide on NIP AM indicating a possible mechanism for the attenuated potency observed at increased temperatures.
[0016] FIG. 4 shows the 'H NMR of l-(diethoxymethyl)-4-vinylbenzene.
[0017] FIG. 5 shows the 13C NMR of l-(diethoxymethyl)-4-vinylbenzene.
[0018] FIG. 6 shows the 'H NMR of 4,6-O-(4-vinylbenzylidene)-a,a-trehalose.
[0019] FIG. 7 shows the 1 H NMR for 4,6-O-(4-vinylbenzylidene)-6’-O-behenoyl- a,a-trehalose.
[0020] FIG. 8 shows the 13C NMR for 4,6-O-(4-vinylbenzylidene)-6’-0-behenoyl- a,a-trehalose.
[0021] FIG. 9 shows the NOESY-NMR spectrum for P2 at 35°C.
[0022] FIG. 10 shows the results of HEK293-mMINCLE Immunoassay. Activation of HEK293-mMINCLE cells with colorimetric readout proportional to Secreted Embryonic Alkaline Phosphatase production (SEAP). Pl, P2 were tested compared to (+)-control D-(+)- Trehalose-6, 6’ -dibehenate (TDB) by mass (pg/mL) and by mols (pM). i.e) lOpM Pl is 73 pg/mL and lOpM P2 is 253 pg/mL. In addition, Pl and P2 were tested by molarity of the subunit agonist, “ag” (pM trehalose glycolipid content). P3 was tested by mass, and did not contain any lipid.
[0023] FIG. 11 shows a representative Live-Dead Assay.
[0024] FIG. 12 shows a Bone Marrow Derived Dendritic Cell (BMDC) (CD1 lc+ dendritic cells) full cytokine profile and purity assessment.
[0025] FIG. 13 shows a Bone Marrow Derived Macrophage (BMDM) (Mo macrophages) full cytokine profile and purity assessment.
[0026] FIG. 14 shows the full range of Lower Critical Solution Temperature (LCST) data at Img / mL. [0027] FIG. 15 shows the effect of concentration on polymer Lower Critical Solution
Temperature (LCST).
[0028] FIGs. 16A-16C show Dynamic Light Scattering (DLS) data for Pl (FIG.
16A), P2 (FIG. 16B), and P3 (FIG. 16C).
[0029] FIG. 17 shows Gel Permeation Chromatography (GPC) chromatograms for co-polymers Pl, P2, and P3 (baseline corrected and normalized).
[0030] FIG. 18 shows the Nuclear Overhauser Enhancement Spectroscopy (NOESY-
NMR) for Pl at 25 °C.
[0031] FIG. 19 shows the NOESY-NMR spectrum for Pl at 35°C.
[0032] FIG. 20 shows the NOESY-NMR spectrum for P2 at 25°C.
[0033] FIG. 21 A shows a selected binding pose of truncated adjuvant glycolipid subunit 1 docked with the MINCLE crystal structure (PDB: 4ZRW) using Autodock Vina suggested preservation of key bridging interactions between Arg 182 and Phel98 in the binding groove. FIG. 2 IB shows Pl and P2 activated HEK-mMINCLE cells comparable to equimolar amounts of D-(+)-Trehalose-6, 6’ -dibehenate (TDB) (polymer concentrations indicated are with respect to agonist subunit). Error bars are standard deviations of the mean for experiments performed in triplicate; *p < 0.05, **p < 0.01 for Pl or P2 compared to negative control (PBS). FIG. 21C shows proinfl ammatory response of Pl and P2 in a model JAWS II innate immune cell line treated with 100 ug/mL of each adjuvant and incubated for 48 hours at the indicated temperatures. Cytokines were assessed by cytometric bead array, and error bars are standard deviations of the mean for experiments performed in triplicate; *p < 0.05, **p < 0.01 for attenuated cytokine production at 39 °C compared to the same adjuvant at 35 °C.
[0034] FIGs. 22A-22C show characterization of thermophobic glycopolymer adjuvant in primary murine immune cells. Polymers or TDB (100 pg/mL) were incubated with F4/80+ Bone Marrow Derived Macrophages (BMDMs) (FIG. 22 A) or CD1 lc+ Bone Marrow Derived Dendritic Cells (BMDCs) (FIG. 22B) for 48 hours at the indicated temperatures (35-39 °C). Secreted inflammatory cytokines were assessed by cytometric bead array. Error bars are standard deviation of the mean for experiments repeated in hexaplet, * p < 0.05, ** p < 0.01, *** p < 0.001 for attenuated cytokines produced at 39 °C relative to 37 or 35 °C. See FIGs. 12 and 13 for complete cytokine profiles and additional positive control (LPS). FIG. 22C shows the observed relation between particle size and adjuvant activity. Linking these observations to the DLS and NOSEY data, it was concluded that the thermally induced change in activity could be due to either decreased particle size itself or enhanced lipid-NIPAM shielding interactions that result from this transition. [0035] FIGs. 23 A-23H show in-vivo performance of thermophobic vaccine adjuvants.
FIG. 23 A shows that mice exhibited normal weight gain post vaccination. FIG. 23B shows IL-6 levels measured from mouse sera 24 hours after second vaccination. IL-6 production was increased for P2 and TDB compared to unvaccinated cohorts. FIG. 23 C shows that levels of CD4+/44+/62L+ central memory T-cells found in lung and lymph node samples were increased in the P2 cohort, consistent with latent IL-6 levels measured 7 days after administering booster vaccination (FIG. 23D) and increases in central memory T-cell populations (FIG. 23E) following the full vaccination schedule as well. FIG. 23F shows weight loss of mice post challenge with influenza indicated protective effects conferred by the vaccine adjuvanted with P2 was comparable to that of alum. This was consistent with neutralizing antibodies assessed by Hemagglutination (HA) inhibition assay at day 14 and 28 (FIG. 23 G) as well as neuraminidase (NA) inhibition assay at day 28 post vaccination (FIG. 23H), each of which indicated that HA and NA neutralization in the P2 adjuvanted cohort is comparable to, or better than, the alum adjuvanted cohort. According to the HA inhibition assay, all adjuvanted vaccines performed better than unadj uvanted control by day 28.
[0036] FIG. 24 shows the 1 H Correlation Spectroscopy (COSY) NMR of 4,6-O-(4- vinylbenzylidene)-a,a-trehalose.
[0037] FIG. 25 compares the diastereomers of 4,6-O-(4-vinylbenzylidene)-a,a- trehalose. MM2 energy minimization predicts a difference of 0.40 kcal/mol between the (R) and (S) isomers, where the styrenyl substituent is favored in the equatorial position. The NOESY spectra indicate interactions between the acetal hydrogen and carbohydrate ring protons P and O/S.
[0038] FIG. 26 shows an expansion of carbohydrate region of the COSY NMR for
4,6-O-(4-vinylbenzylidene)-a,a-trehalose.
[0039] FIG. 27 shows the COSY NMR for 4,6-O-(4-vinylbenzylidene)-6’-0- behenoyl-a,a-trehalose.
[0040] FIG. 28 shows the NOESY NMR for 4,6-O-(4-vinylbenzylidene)-6’-0- behenoyl-a,a-trehalose. E/T and E/P crosspeaks indicate retention of (R) stereochemistry at the benzylidene acetal.
[0041] FIG. 29 shows the UV-VIS spectrum of 4,6-O-(4-vinylbenzylidene)-6’-0- behenoyl-a,a-trehalose.
[0042] FIG. 30 shows the IR spectrum of 4,6-O-(4-vinylbenzylidene)-6’-0-behenoyl- a,a-trehalose. [0043] FIG. 31 shows the 1HNMR of poly(NIPAM)-co-poly(4,6-O-(4- vinylbenzylidene)-6’-O-behenoyl-a,a-trehalose).
[0044] FIG. 32 shows the 1HNMR of poly(NIPAM)-co-poly(4,6-O-(4- vinylbenzylidene)-6’-0-behenoyl-a,a-trehalose)-Z>/oc -poly(NIPAM).
[0045] FIG. 33 shows temperature dependent aggregation of P2 (Bright Field 40x).
[0046] FIG. 34 shows the Neuraminidase Activity Assay data.
DETAILED DESCRIPTION OF THE INVENTION
[0047] One aspect of the present disclosure relates to a copolymer comprising at least one PA block and at least one PB block, where PA represents a polymer block comprising one or more units of monomer A and PB represents a polymer block comprising one or more units of monomer B, with the monomer A being an amide or ester, and the monomer B being a trehalose- based monomer, where at least one hydroxyl group on the trehalose is esterified.
[0048] As used above, and throughout the description herein, the following terms, unless otherwise indicated, shall be understood to have the following meanings. If not defined otherwise herein, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this technology belongs. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.
[0049] In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
[0050] The terms “comprising,” “comprises,” and “comprised of’ as used herein are synonymous with “including,” “includes,” or “containing,” “contains,” and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps. [0051] The terms “comprising,” “comprises,” and “comprised of’ also encompass the term “consisting of.” The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, un-recited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed subject matter. In some embodiments or claims where the term comprising is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of’ or “consisting essentially of.” [0052] Terms of degree such as “substantially,” “about,” and “approximately” and the symbol as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±0.1% (and up to ±1%, ±5%, or ±10%) of the modified term if this deviation would not negate the meaning of the word it modifies. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. All numerical values provided herein that are modified by terms of degree set forth in this paragraph (e.g., “substantially,”
Figure imgf000010_0001
are also explicitly disclosed without the term of degree. For example, “about 1%” is also explicitly disclosed as “1%”.
[0053] The term “and/or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.
[0054] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.
[0055] The term “alkyl” means an aliphatic hydrocarbon group which may be straight or branched having 1 to about 30 carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule, or 1 to about 20 carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule, or 1 to about 15 carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule, or 1 to about 10 carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule, or 1 to about 2, 3, 4, or 5 carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule, or any number of carbon atoms in a hydrocarbon chain or branched hydrocarbon molecule between 1 and about 30 carbon atoms. Branched means that one or more alkyl groups, e.g., a lower alkyl group such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include, without limitation, methyl, ethyl, w-propyl, i- propyl, //-butyl, /-butyl, w-pentyl, and 3 -pentyl.
[0056] Compounds described herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. Each chiral center may be defined, in terms of absolute stereochemistry, as (R)- or (S)-. This technology is meant to include all such possible isomers, as well as mixtures thereof, including racemic and optically pure forms. Optically active (R)- and (S)-, (-)- and (+)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.
[0057] The term “copolymer” refers to a polymer derived from more than one species of monomer.
[0058] The term “block copolymer” or “block polymer” refers to a macromolecule comprising long sequences of different repeat units. In some embodiments, exemplary block polymers include, but are not limited to, AnBm, AnBmAm, AnBmCk, or AnBmCkAn, where n, m and k are independently any integer.
[0059] The term “lower critical solution temperature (LCST)” refers to a temperature, below which, a polymer and solvent are completely miscible. A mixture of polymer and solvent will form a single phase when mixed in any proportion at a temperature below the LCST. A mixture of polymer and solvent will separate into different phases when mixed in certain proportions and heated to a temperature above the LCST thereby traversing through a temperature known as a cloud point. A cloud point is defined as the temperature at which transmittance of the polymer solution reaches 50% of the maximum observed transmittance. For example, “the LCST of a polymer solution” means that the polymer of a given proportion or concentration is uniformly solvated in a solution below that temperature (i.e., LCST), but separates into 1 or more additional phases as the solution temperature is increased above the LCST.
[0060] The term “number average molecular weight (Mn)” refers to the total weight of the polymer divided by the number of molecules of the polymer.
[0061] In some embodiments, the copolymer is a block copolymer. For example, in some embodiments, the block copolymer comprises an architecture of PA-PB, PB-PA, PA-PB- TCTA, PA-TCTA-PB, TCTAkPA-PB-TCTA2, or TCTAkPB-PA-TCTA2, where TCTA, TCTA1, and TCTA2 are moieties derived from a telechelic chain transfer agent.
[0062] In some embodiments, the block copolymer comprises an architecture of PA-
PB-PA, PA-PB-TCTA-PB, PA-PB-TCTA-PA, PA-TCTA-PB -PA, PA-PB-TCTA-PB-PA, TCTA'-PA-PB-PA-TCTA2, TCTA^PB-PA-PB-TCTA2, TCTA'-PA-PB-TCTA-PB-TCTA2, TCTAkpA-PB-TCTA-PA-TCTA2, TCTAkPA-TCTA-PB-PA-TCTA2, or TCTA'-PA-PB- TCTA-PB-PA-TCTA2, where TCTA, TCTA1, and TCTA2 are moieties derived from a telechelic chain transfer agent.
[0063] In some embodiments, monomer A is a monomer that, if used to produce a homopolymer, produces a homopolymer having a lower critical solution temperature (LCST) that causes a cloud point between 30 °C and 45 °C at appropriate polymer concentrations known to exhibit an LCST.
[0064] In some embodiments, said monomer A is an acrylamide, methacrylamide, acrylate, or methacrylate. For example, in some embodiments, monomer A is selected from the group consisting of N,N-diethyl acrylamide, N,N-dimethyl acrylamide, N-isopropyl acrylamide, N-ethyl acrylamide, N-tert-butyl acrylamide, N-n-propyl acrylamide, N-methyl-N-ethyl acrylamide, and methacrylamide derivates thereof. In some embodiments, said monomer A is N- isopropyl acrylamide.
[0065] In some embodiments, the at least one PB block has a chemical structure of
Formula (I):
-(L-B)m- (I), where
L is a linker; and m is 1 to 100,000.
Figure imgf000012_0001
✓
[0066] In some embodiments, L is and each / is a point of attachment of L to the at least one hydroxyl group on the trehalose.
[0067] In some embodiments, the trehalose-based monomer is a monomer comprising trehalose attached to a side chain of the monomer.
[0068] In some embodiments, monomer B has a chemical structure of Formula (II):
Figure imgf000013_0001
where R is Ci -30 alkyl.
[0069] In some embodiments, monomer B has a chemical structure of Formula (Ila):
Figure imgf000013_0002
[0070] In some embodiments, monomer B has a chemical structure of Formula (lib):
Figure imgf000013_0003
[0071] In some embodiments, the copolymer comprises a moiety selected from
Figure imgf000014_0001
where is the terminal group of the polymer; a is 1 to 100,000; b is 1 to 100,000; x is 1 to 100,000; and y is 1 to 100,000.
[0072] In some embodiments, the copolymer has a chemical structure selected from
Figure imgf000014_0002
where a is 1 to 100,000, b is 1 to 100,000, x is 1 to 100,000, y is 1 to 100,000, and Ra and Rb are moi eties derived from a thiocarbonylthio compound, a dithioester compound, a trithiocarb onate compound, a dithiocarbamate compound, or a xanthate compound. [0073] In some embodiments, Ra and Rb are independently selected from the group
Figure imgf000015_0001
[0074] In some embodiments, the copolymer has a chemical structure selected from
Figure imgf000015_0002
where a is 1 to 100,000, b is 1 to 100,000, x is 1 to 100,000, and y is 1 to 100,000.
[0075] In some embodiments, the copolymer has a chemical structure selected from
Figure imgf000016_0001
wherein a is 1 to 100,000; b is 1 to 100,000; x is 1 to 100,000; and y is 1 to 100,000.
[0076] In some embodiments, the copolymer has a chemical structure selected from
Figure imgf000016_0002
where a is 1 to 100,000, b is 1 to 100,000, x is 1 to 100,000, and y is 1 to 100,000.
[0077] In some embodiments, a is about 1 to about 10, about 1 to about 20, about 1 to about 40, about 1 to about 60, about 1 to about 80, about 1 to about 100, about 1 to about 1,000, about 1 to about 10,000, about 1 to about 50,000, about 1 to about 100,000, about 5 to about 20, about 5 to about 40, about 5 to about 60, about 5 to about 80, about 5 to about 100, about 5 to about 1,000, about 5 to about 10,000, about 5 to about 50,000, about 5 to about 100,000, about 10 to about 20, about 10 to about 40, about 10 to about 60, about 10 to about 80, about 10 to about 100, about 10 to about 1,000, about 10 to about 10,000, about 10 to about 50,000, about 10 to about 100,000, about 100 to about 1,000, about 100 to about 10,000, about 100 to about 50,000, about 100 to about 100,000, about 1,000 to about 10,000, about 1,000 to about 50,000, about 1,000 to about 100,000, about 10,000 to about 50,000, about 10,000 to about 100,000, or about 50,000 to about 100,000. In some embodiments, a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a is 1.
[0078] In some embodiments, b is about 1 to about 100, about 1 to about 500, about 1 to about 1,000, about 1 to about 5,000, about 1 to about 10,000, about 1 to about 20,000, about 1 to about 30,000, about 1 to about 40,000, about 1 to about 50,000, about 1 to about 60,000, about 1 to about 70,000, about 1 to about 80,000, about 1 to about 90,000, about 1 to about 100,000, about 500 to about 1,000, about 500 to about 5,000, about 500 to about 10,000, about 500 to about 20,000, about 500 to about 30,000, about 500 to about 40,000, about 500 to about 50,000, about 500 to about 60,000, about 500 to about 70,000, about 500 to about 80,000, about 500 to about 90,000, about 500 to about 100,000, about 1,000 to about 5,000, about 1,000 to about 10,000, about 1,000 to about 20,000, about 1,000 to about 30,000, about 1,000 to about 40,000, about 1,000 to about 50,000, about 1,000 to about 60,000, about 1,000 to about 70,000, about 1,000 to about 80,000, about 1,000 to about 90,000, about 1,000 to about 100,000, about 5,000 to about 10,000, about 5,000 to about 20,000, about 5,000 to about 30,000, about 5,000 to about 40,000, about 5,000 to about 50,000, about 5,000 to about 60,000, about 5,000 to about 70,000, about 5,000 to about 80,000, about 5,000 to about 90,000, about 5,000 to about 100,000, about 10,000 to about 20,000, about 10,000 to about 30,000, about 10,000 to about 40,000, about
10,000 to about 50,000, about 10,000 to about 60,000, about 10,000 to about 70,000, about
10,000 to about 80,000, about 10,000 to about 90,000, about 10,000 to about 100,000, about 20,000 to about 30,000, about 20,000 to about 40,000, about 20,000 to about 50,000, about
20,000 to about 60,000, about 20,000 to about 70,000, about 20,000 to about 80,000, about
20,000 to about 90,000, about 20,000 to about 100,000, about 30,000 to about 40,000, about 30,000 to about 50,000, about 30,000 to about 60,000, about 30,000 to about 70,000, about
30,000 to about 80,000, about 30,000 to about 90,000, about 30,000 to about 100,000, about
40,000 to about 50,000, about 40,000 to about 60,000, about 40,000 to about 70,000, about
40,000 to about 80,000, about 40,000 to about 90,000, about 40,000 to about 100,000, about
50,000 to about 60,000, about 50,000 to about 70,000, about 50,000 to about 80,000, about 50,000 to about 90,000, about 50,000 to about 100,000, about 60,000 to about 70,000, about
60,000 to about 80,000, about 60,000 to about 90,000, about 60,000 to about 100,000, about
70,000 to about 80,000, about 70,000 to about 90,000, about 70,000 to about 100,000, about
80,000 to about 90,000, about 80,000 to about 100,000, or about 90,000 to about 100,000.
[0079] In some embodiments, x is about 1 to about 100, about 1 to about 500, about 1 to about 1,000, about 1 to about 5,000, about 1 to about 10,000, about 1 to about 20,000, about 1 to about 30,000, about 1 to about 40,000, about 1 to about 50,000, about 1 to about 60,000, about 1 to about 70,000, about 1 to about 80,000, about 1 to about 90,000, about 1 to about 100,000, about 500 to about 1,000, about 500 to about 5,000, about 500 to about 10,000, about 500 to about 20,000, about 500 to about 30,000, about 500 to about 40,000, about 500 to about 50,000, about 500 to about 60,000, about 500 to about 70,000, about 500 to about 80,000, about 500 to about 90,000, about 500 to about 100,000, about 1,000 to about 5,000, about 1,000 to about 10,000, about 1,000 to about 20,000, about 1,000 to about 30,000, about 1,000 to about 40,000, about 1,000 to about 50,000, about 1,000 to about 60,000, about 1,000 to about 70,000, about 1,000 to about 80,000, about 1,000 to about 90,000, about 1,000 to about 100,000, about 5,000 to about 10,000, about 5,000 to about 20,000, about 5,000 to about 30,000, about 5,000 to about 40,000, about 5,000 to about 50,000, about 5,000 to about 60,000, about 5,000 to about 70,000, about 5,000 to about 80,000, about 5,000 to about 90,000, about 5,000 to about 100,000, about 10,000 to about 20,000, about 10,000 to about 30,000, about 10,000 to about 40,000, about
10,000 to about 50,000, about 10,000 to about 60,000, about 10,000 to about 70,000, about
10,000 to about 80,000, about 10,000 to about 90,000, about 10,000 to about 100,000, about 20,000 to about 30,000, about 20,000 to about 40,000, about 20,000 to about 50,000, about
20,000 to about 60,000, about 20,000 to about 70,000, about 20,000 to about 80,000, about
20,000 to about 90,000, about 20,000 to about 100,000, about 30,000 to about 40,000, about 30,000 to about 50,000, about 30,000 to about 60,000, about 30,000 to about 70,000, about
30,000 to about 80,000, about 30,000 to about 90,000, about 30,000 to about 100,000, about
40,000 to about 50,000, about 40,000 to about 60,000, about 40,000 to about 70,000, about
40,000 to about 80,000, about 40,000 to about 90,000, about 40,000 to about 100,000, about
50,000 to about 60,000, about 50,000 to about 70,000, about 50,000 to about 80,000, about
50,000 to about 90,000, about 50,000 to about 100,000, about 60,000 to about 70,000, about 60,000 to about 80,000, about 60,000 to about 90,000, about 60,000 to about 100,000, about
70,000 to about 80,000, about 70,000 to about 90,000, about 70,000 to about 100,000, about
80,000 to about 90,000, about 80,000 to about 100,000, or about 90,000 to about 100,000. [0080] In some embodiments, y is about 1 to about 100, about 1 to about 500, about 1 to about 1,000, about 1 to about 5,000, about 1 to about 10,000, about 1 to about 20,000, about 1 to about 30,000, about 1 to about 40,000, about 1 to about 50,000, about 1 to about 60,000, about
I to about 70,000, about 1 to about 80,000, about 1 to about 90,000, about 1 to about 100,000, about 500 to about 1,000, about 500 to about 5,000, about 500 to about 10,000, about 500 to about 20,000, about 500 to about 30,000, about 500 to about 40,000, about 500 to about 50,000, about 500 to about 60,000, about 500 to about 70,000, about 500 to about 80,000, about 500 to about 90,000, about 500 to about 100,000, about 1,000 to about 5,000, about 1,000 to about 10,000, about 1,000 to about 20,000, about 1,000 to about 30,000, about 1,000 to about 40,000, about 1,000 to about 50,000, about 1,000 to about 60,000, about 1,000 to about 70,000, about 1,000 to about 80,000, about 1,000 to about 90,000, about 1,000 to about 100,000, about 5,000 to about 10,000, about 5,000 to about 20,000, about 5,000 to about 30,000, about 5,000 to about 40,000, about 5,000 to about 50,000, about 5,000 to about 60,000, about 5,000 to about 70,000, about 5,000 to about 80,000, about 5,000 to about 90,000, about 5,000 to about 100,000, about 10,000 to about 20,000, about 10,000 to about 30,000, about 10,000 to about 40,000, about
10,000 to about 50,000, about 10,000 to about 60,000, about 10,000 to about 70,000, about
10,000 to about 80,000, about 10,000 to about 90,000, about 10,000 to about 100,000, about 20,000 to about 30,000, about 20,000 to about 40,000, about 20,000 to about 50,000, about
20,000 to about 60,000, about 20,000 to about 70,000, about 20,000 to about 80,000, about
20,000 to about 90,000, about 20,000 to about 100,000, about 30,000 to about 40,000, about 30,000 to about 50,000, about 30,000 to about 60,000, about 30,000 to about 70,000, about 30,000 to about 80,000, about 30,000 to about 90,000, about 30,000 to about 100,000, about 40,000 to about 50,000, about 40,000 to about 60,000, about 40,000 to about 70,000, about 40,000 to about 80,000, about 40,000 to about 90,000, about 40,000 to about 100,000, about 50,000 to about 60,000, about 50,000 to about 70,000, about 50,000 to about 80,000, about 50,000 to about 90,000, about 50,000 to about 100,000, about 60,000 to about 70,000, about 60,000 to about 80,000, about 60,000 to about 90,000, about 60,000 to about 100,000, about 70,000 to about 80,000, about 70,000 to about 90,000, about 70,000 to about 100,000, about 80,000 to about 90,000, about 80,000 to about 100,000, or about 90,000 to about 100,000.
[0081] In some embodiments, the copolymer according to the present disclosure can have a number average molecular weight (Mn) above 1 kDa, above 2 kDa, above 3 kDa, above 4 kDa, above 5 kDa, above 6 kDa, above 7 kDa, above 8 kDa, above 9 kDa, above 10 kDa, above
I I kDa, above 12 kDa, above 13 kDa, above 14 kDa, above 15 kDa, above 16 kDa, above 17 kDa, above 18 kDa, above 19 kDa, above 20 kDa, above 21 kDa, above 22 kDa, above 23 kDa, above 24 kDa, above 25 kDa, above 26 kDa, above 27 kDa, above 28 kDa, above 29 kDa, above 30 kDa, above 31 kDa, above 32 kDa, above 33 kDa, above 34 kDa, above 35 kDa, above 36 kDa, above 37 kDa, above 38 kDa, above 39 kDa, above 40 kDa, above 41 kDa, above 42 kDa, above 43 kDa, above 44 kDa, above 45 kDa, above 46 kDa, above 47 kDa, above 48 kDa, above 49 kDa, above 50 kDa, above 51 kDa, above 52 kDa, above 53 kDa, above 54 kDa, above 55 kDa, above 56 kDa, above 57 kDa, above 58 kDa, above 59 kDa, or above 60 kDa.
[0082] In some embodiments, the copolymer can have a number average molecular weight (Mn) ranging from about 0.1 kDa to about 200 kDa. For example, the copolymer can have a number average molecular weight (Mn) from about 0.1 kDa to about 100 kDa, from about 0.5 kDa to about 90 kDa, from about 1 kDa to about 80 kDa, from about 2 kDa to about 70 kDa, from about 3 kDa to about 60 kDa, from about 4 kDa to about 50 kDa, from about 5 kDa to about 40 kDa, from about 6 kDa to about 30 kDa, from about 7 kDa to about 30 kDa, from about
8 kDa to about 30 kDa, from about 9 kDa to about 30 kDa, from about 10 kDa to about 30 kDa, from about 12 kDa to about 30 kDa, from about 14 kDa to about 30 kDa, from about 16 kDa to about 30 kDa, from about 18 kDa to about 30 kDa, from about 20 kDa to about 30 kDa, from about 2 kDa to about 20 kDa, from about 3 kDa to about 20 kDa, from about 4 kDa to about 20 kDa, from about 5 kDa to about 20 kDa, from about 6 kDa to about 20 kDa, from about 7 kDa to about 20 kDa, from about 8 kDa to about 20 kDa, from about 9 kDa to about 20 kDa, from about 10 kDa to about 20 kDa, from about 11 kDa to about 20 kDa, from about 12 kDa to about 20 kDa, from about 13 kDa to about 20 kDa, from about 14 kDa to about 20 kDa, from about 15 kDa to about 20 kDa, from about 2 kDa to about 15 kDa, from about 3 kDa to about 15 kDa, from about 4 kDa to about 15 kDa, from about 5 kDa to about 15 kDa, from about 6 kDa to about 15 kDa, from about 7 kDa to about 15 kDa, from about 8 kDa to about 15 kDa, from about
9 kDa to about 15 kDa, from about 10 kDa to about 15 kDa, from about 1 kDa to about 10 kDa, from about 2 kDa to about 10 kDa, from about 3 kDa to about 10 kDa, from about 4 kDa to about 10 kDa, or from about 5 kDa to about 10 kDa.
[0083] The copolymers according to the present disclosure can have a broad or sharp lower critical solution temperature (LCST). In some embodiments, the copolymers of the present disclosure have a LCST from about 30°C to about 50°C, from about 31°C to about 49°C, from about 32°C to about 48°C, from about 33°C to about 47°C, from about 34°C to about 46°C, from about 35°C to about 45°C, from about 36°C to about 44°C, or from about 37°C to about 43°C. In some embodiments, the copolymers of the present disclosure have a LCST of about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. [0084] Another aspect of present disclosure relates to a bioactive delivery system comprising the copolymer as disclosed herein and a bioactive agent.
[0085] Any of the copolymers disclosed herein may be used in accordance with this aspect of the present disclosure.
[0086] In some embodiments, the bioactive agent is selected from the group consisting of a protein, a (poly)peptide, a vaccine, a nucleic acid, a hormone, a cancer drug, an angiogenesis inhibitor, a growth factor, and an anti-microbial substance.
[0087] Another aspect of present disclosure relates to a composition comprising the copolymer as disclosed herein and a vaccine.
[0088] Any of the copolymers disclosed herein may be used in accordance with this aspect of the present disclosure.
[0089] In some embodiments of the composition, the vaccine is selected from an inactivated enveloped virus, whole cell, cell lysate, tumor-associated antigen, tumor-specific antigen, protein, nucleic acid, and (poly)peptide.
[0090] In some embodiments, suitable inactivated enveloped viruses include, without limitation, herpesviruses, poxviruses, hepadnaviruses, asfarviridae, flavivirus, alphavirus, togavirus, coronavirus, hepatitis viruses, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, filovirus, and retroviruses. In other embodiments, the inactivated enveloped virus is selected from the group consisting of Ebola virus, human immunodeficiency virus, influenza virus, Lassa fever virus, Nipah virus, respiratory syncytial virus, Rift Valley fever virus, SARS virus, MERS virus, Marbury virus, swine pox virus, Cytomegalovirus, Crimean hemorrhagic fever virus, and COVID-19.
[0091] In some embodiments, the bioactive agent delivery system or the composition disclosed herein further comprise an adjuvant, antibiotic, antiviral, pharmaceutically acceptable carrier, stabilizer, and/or preservative.
[0092] In some embodiments, suitable adjuvants include, without limitation, an aluminum salt, inulin, argamline, a combination of inulin and aluminum hydroxide, monophosphoryl lipid A (MPL), resiquimoid, muramyl dipeptide (MDP), N -Glycolyl dipeptide (GMDP, N-glycolyl dipeptide), poly IC, CpG oligonucleotide, anciclovir, resiquimod, aluminum hydroxide containing MPL, a water-in-oil emulsion, squalene or analogs thereof, any pharmaceutically acceptable oil, tween-80, sorbitan trioleate, alpha-tocopherol, cholecalciferol or any analogs thereof, derivatives thereof, calcium-modified forms thereof, phosphate-modified forms thereof, and combinations thereof. [0093] In some embodiments, suitable antibiotics include, without limitation,
Amikacin, Amoxicillin, Amoxicillin-clavulanic acid, Amphothericin-B, Ampicillin, Ampicllin- sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin, Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin, Cefalothin, Cefazolin, Cefdinir, Cefepime, Cefixime, Cefmenoxime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefoxitin, Cefbirome, Cefpodoxime, Cefpodoxime-clavulanic acid, Cefpodoxime-sulbactam, Cefbrozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole, Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin, Clarithromycin, Clinafloxacin, Clindamycin, Cioxacillin, Colistin, Cotrimoxazol (Trimthoprim/sulphamethoxazole), Dalbavancin, Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin, Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin, Flucl oxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid, Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem, Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin, Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem, Metronidazole, Meziocillin, Mezlocillin-sulbactam, Minocycline, Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin, Penicillin V, Piperacillin, Piperacillin-sulbactam, Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfloxacin, Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazole, Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyklin, Ticarcillin, Ticarcillin- clavulanic acid, Tigecycline, Tobramycin, Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin, Voriconazole, and combinations thereof.
[0094] In some embodiments, suitable preservatives include, without limitation, chlorobutanol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal, phenylmercuric nitrate, and combinations thereof.
[0095] In some embodiments, suitable pharmaceutically acceptable carriers include, without limitation, pyrogen-free water, isotonic saline, buffered aqueous solutions, including aqueous phosphate buffers, aqueous citrate buffers, and combinations thereof.
[0096] In some embodiments, suitable stabilizers include, without limitation, sorbitol,
L- glycine, mannitol, L-glutamic acid, human serum albumin, and combinations thereof.
[0097] In some embodiments, suitable antivirals include, without limitation, zidovudine, acyclovir, anciclovir, ganciclovir, vidarabine, idoxuridine, trifluridine, ribavirin, foscamet, amantadine, peramivir, rimantadine, saquinavir, indinavir, ritonavir, alpha-interferons, AZT, t-705, zanamivir, oseltamivir, influenza virus vaccines, and combinations thereof. [0098] In some embodiments, the vaccine composition (composition comprising the copolymer as disclosed herein and a vaccine) can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraperitoneal, intranasal, or intramuscular means for prophylactic or therapeutic treatment.
[0099] The vaccine compositions of the present disclosure may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or it may be enclosed in hard or soft shell capsules, or it may be compressed into tablets, or they may be incorporated directly with the food of the diet. The vaccine compositions of the present disclosure may also be administered in a time release manner incorporated within such devices as time-release capsules or nanotubes. Such devices afford flexibility relative to time and dosage. For oral therapeutic administration, the vaccine compositions may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% of the copolymer, although lower concentrations may be effective and indeed optimal. The percentage of the copolymer in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit. The amount of a copolymer of the present invention in such therapeutically useful compositions is such that a suitable dosage will be obtained.
[0100] The tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, com starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, sucrulose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.
[0101] Also specifically contemplated are oral dosage forms comprising the copolymers of the present disclosure.
[0102] Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar, or both. A syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
[0103] The vaccine compositions may also be administered parenterally. Solutions or suspensions of the vaccine compositions can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0104] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
[0105] When it is desirable to deliver the vaccine compositions of the present disclosure systemically, they may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi -dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
[0106] Intraperitoneal or intrathecal administration of the vaccine compositions of the present disclosure can also be achieved using infusion pump devices such as those described by Medtronic, Northridge, CA. Such devices allow continuous infusion of desired compositions avoiding multiple injections and multiple manipulations.
[0107] In addition to the formulations described previously, the vaccine compositions may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
[0108] The vaccine compositions may also be administered directly to the airways in the form of an aerosol. For use as aerosols, the vaccine compositions in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The vaccine compositions also may be administered in a non-pressurized form such as in a nebulizer or atomizer. [0109] Another aspect of the present disclosure relates to a method of vaccinating a subject against infection by an enveloped virus. This method comprises providing a composition according to the present disclosure and treating the subject with the composition to vaccinate the subject against the enveloped virus.
[0110] Any of the copolymers disclosed herein may be used in accordance with this aspect of the present disclosure.
[OHl] In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal such as non-human primate, dog, cat, sheep, goat, cow, pig, horse, or rodent.
[0112] Factors to be accounted for when administering the vaccine of the present disclosure in order to produce a robust immune response, include without limitation the concentrations of the vaccine, the presence of an adjuvant, the mode and frequency of administration, and the subject’s medical details, such as age, weight and overall health and immune condition. A clinician may administer the vaccine composition until a dosage is reached that provides the desired or required prophylactic effect, e.g., the desired antibody titers. The progress of this therapy can be easily monitored by conventional assays.
[0113] In some embodiments, the method further comprises selecting a subject in need of vaccination against infection by an enveloped virus.
[0114] In some embodiments, the vaccine composition as descried herein is administered prophylactically to prevent, delay, or inhibit the development of the infection in a subject at risk of being infected with an enveloped virus. In some embodiments, prophylactic administration of the vaccine composition is effective to fully prevent infection in an individual of the enveloped virus of the vaccine composition. In other embodiments, prophylactic administration is effective to prevent the full extent of infection that would otherwise develop in the absence of such administration, i.e., substantially prevent or inhibit the enveloped virus infection in an individual.
[0115] Another aspect of the present disclosure relates to a method of preparing a copolymer. This method comprises providing a radically polymerizable amide monomer A, providing a trehalose-based monomer B comprising at least one esterified hydroxyl group or a polymer block PB comprising one or more units of said monomer B, and polymerizing the monomer A with the monomer B or the polymer block PB.
[0116] One suitable polymerization technique that can be used to polymerize the monomer A with the monomer B or the polymer block PB is the reversible additionfragmentation chain transfer (RAFT) polymerization (Moad, “The Emergence of RAFT Polymerization,” Aust. J. Chem. 59(10):661-662 (2006); Moad et al., “Living Radical Polymerization by the RAFT Process-A First Update,” Aust. J. Chem. 59(10):669-692 (2006); Moad et al., “Living Radical Polymerization by the RAFT Process - A Second Update,” Aust. J. Chem. 62(11): 1402-1472 (2009); Moad et al., “Living Radical Polymerization by the RAFT Process - A Third Update,” Aust. J. Chem. 65(8):985-1076 (2012), which are hereby incorporated by reference in their entirety).
[0117] Radical Addition-Fragmentation Chain Transfer polymerization limits the number of initiation sites and drastically reduces the rate of polymer-to-polymer chain transfer and termination reactions, and also introduces the capability of producing custom chain architectures such as block copolymers (BCPs) and statistical copolymers. This degree of control is superior to that offered by other controlled radical polymerization methods — that is, polymers of more narrow dispersity may be obtained over a shorter period of time with less rigorous purification and lower batch-to-batch variability.
[0118] RAFT polymerization is a type of living polymerization or controlled polymerization, utilizing a chain transfer agent (CTA). A conventional RAFT polymerization mechanism, consisting of a sequence of addition-fragmentation equilibria, is shown in Scheme 1 (Moad et al., “Living Radical Polymerization by the Raft Process- a First Update,” Australian Journal of Chemistry 59: 669-92 (2006), which is incorporated herein by reference in its entirety). As depicted, the RAFT polymerization reaction starts with initiation. Initiation is accomplished by adding an agent capable of decomposing to form free radicals; the decomposed free radical fragment of the initiator attacks a monomer yielding a propagating radical (P'n ), in which additional monomers are added producing a growing polymer chain. In the propagation step, the propagating radical (P'n) adds to a chain transfer agent (CTA), such as a thiocarb onylthio compound (RSC(Z)=S, 1), followed by the fragmentation of the intermediate radical (2) forming a dormant polymer chain with a thiocarb onylthio ending (PnS(Z)C=S, 3) and a new radical (R'). This radical (R') reacts with a new monomer molecule forming a new propagating radical (P'm). In the chain propagation step, (P'n) and (P'm) reach equilibrium and the dormant polymer chain (3) provides an equal probability to all polymers chains to grow at the same rate, allowing polymers to be synthesized with narrow polydispersity. Termination is limited in RAFT and, if occurring, is negligible. For traditional monomers which contain only one polymerizable species, targeting a specific molecular weight in RAFT can be calculated by multiplying the ratio of monomer consumed to the concentration of CTA used by the molecular weight of the monomer. When applied to monomers containing species with two or more polymerizable moieties, branched polymers may form that further increase the molecular weight. Initiation
Figure imgf000027_0001
Termination p + u
Figure imgf000027_0002
dead polymer
Scheme 1. Schematic showing the Radical Addition-Fragmentation Chain Transfer (RAFT) polymerization mechanism, described in Moad et al., “Living Radical Polymerization by the Raft Process- a First Update,” Australian Journal of Chemistry 59: 669-92 (2006), which is incorporated herein by reference in its entirety.
[0119] The initiating agents often are referred to as "initiators." Suitable initiators depend greatly on the details of the polymerization, including the types of monomers being used, the type of catalyst system, the solvent system, and the reaction conditions. A typical radical initiator can be azo compounds, which provide a two-carbon centered radical. Radical initiators such as benzoyl peroxide, azobisisobutyronitrile (AIBN), 1,1' azobis(cyclohexanecarbonitrile) or (ABCN), or 4,4’ -azobis(4-cyanoval eric acid) (ACVA); redox initiator such as benzoyl peroxide/N,N-dimethylaniline; microwave heating initiator; photoinitiator such as (2,4,6-trimethylbenzoyl)-diphenylphosphine oxide; gamma radiation initiator; or Lewis Acids such as scandium(III) tritiate or yttrium (III) tritiate, are typically used in RAFT polymerization.
[0120] RAFT polymerization can use a wide variety of CTA agents. Suitable CTA agents should be capable of initiating the polymerization of the monomers and achieve a narrow poly dispersity in the process. For RAFT polymerization to be efficient, the initial CTA agents should have a reactive C=S double bond; the intermediate radical should fragment predictably without side reactions; the intermediate should partition in favor of products, and the expelled radicals (R’) should efficiently re-initiate polymerization. A suitable CTA agent is a s thiocarb onylthio compound (ZC(=S)SR:
Figure imgf000028_0001
, where R is free radical leaving group and Z is a group that modifies addition and fragmentation rates of RAFT polymerization. Exemplary CTA agents include, but are not limited to, a dithioester compound (where Z = aryl, heteraryl, or alkyl), a trithiocarb onate compound (where Z = alkylthio, arylthio, or heteroarylthio), a dithiocarbamate compound (where Z = arylamine or heterarylamine or alkylamine), and a xanthate compound (where Z = alkoxy, aryloxy, or heteroaryl oxy), that are capable or reversible association with polymerizable free radicals. Z can also be sulfonyl, phosphonate, or phosphine. A more extensive list of suitable CTA agents (or RAFT agents) can be found in Moad et al., “Living Radical Polymerization by the Raft Process- a First Update,” Australian Journal of Chemistry 59: 669-92 (2006); Moad et al., “Living Radical Polymerization by the Raft Process- a Second Update,” Australian Journal of Chemistry 62(11): 1402-72 (2009); Moad et al., “Living Radical Polymerization by the Raft Process- a Third Update,” Australian Journal of Chemistry 65: 985-1076 (2012); Skey et al., "Facile one pot synthesis of a range of reversible additionfragmentation chain transfer (RAFT) agents." Chemical Communications 35: 4183-85 (2008), which are hereby incorporated by reference in their entirety. A CTA agent’s effectiveness depends on the monomer being used and is determined by the properties of the free radical leaving group R and the Z group. These groups activate and deactivate the thiocarbonyl double bond of the RAFT agent and modify the stability of the intermediate radicals (Moad et al., “Living Radical Polymerization by the Raft Process- a Second Update,” Australian Journal of Chemistry 62(11): 1402-72 (2009), which is hereby incorporated by reference in its entirety). Typical CTA agents used are 1 -phenylethyl benzodithioate or 1 -phenylethyl 2- phenylpropanedithioate.
[0121] In some RAFT polymerization processes, the chain transfer agent used is a telechelic chain transfer agent, which typically is based on trithiocarb onate functionality. Polymers produced from the chain transfer agent based on a trithiocarb onate functional group retain the CTA functionality in the statistical center of the chain, as opposed to polymers produced by a dithiocarbonate-based CTA, which retain the CTA functionality at the end of the polymeric chain. The telechelic chain transfer agent is capable of adding polymer blocks symmetrically from the interior where the trithiocarbonate functionality is located, i.e., polymerizing monomers from both ends, forming symmetrical architecture or polymer blocks. For example, the RAFT process begins with the chain transfer of a growing A radical to a functional trithiocarb onate, as shown in Scheme 2.
Figure imgf000029_0001
Scheme 2. Schematic showing the transfer of the initial radical to the trithiocarb onate CTA, in the start of the RAFT polymerization process.
[0122] The formed radical intermediate is stable against coupling or disproportion reactions with other free radicals. As shown in Scheme 3 infra, one of the thioate groups reversibly fragments allowing propagation of one of the three arms. Arr
Figure imgf000029_0002
Scheme 3. Schematic showing of the basic propagation mechanism of RAFT polymerization using a trithiocarb onate CTA
[0123] See also Scheme 4 infra for the basic mechanism of RAFT polymerization using a telechelic chain transfer agent.
Figure imgf000029_0003
Scheme 4. Schematic showing of the basic mechanism of RAFT polymerization using a telechelic chain transfer agent. AIBN is an exemplary chain initiator, azobisisobutyronitrile; and
Figure imgf000029_0004
is an exemplary monomer unit, a vinyl monomer (Tasdelen et al., “Telechelic Polymers by Living and Controlled/Living Polymerization Methods,” Progress in Polymer Science 36 (4), 455-567 (2011), which is hereby incorporated by reference in its entirety). [0124] Suitable telechelic CTA agents include any trithiocarbonate compound (e.g., s 11 R z s/ , where Z = alkylthio, arylthio, or heteroarylthio and R is free radical leaving group). A more extensive list of suitable telechelic CTA agents (trithiocarb onate compounds) can be found in Skey et al., "Facile One Pot Synthesis of a Range of Reversible Addition-Fragmentation Chain Transfer (RAFT) Agents." Chemical Communications 35: 4183-85 (2008), which is hereby incorporated by reference in its entirety. A typical telechelic chain transfer agent is dibenzyl
> .....A... .. carb onotri thioate o c
[0125] In some embodiments, the polymerizing step is carried out in a solvent at a temperature of 50 to 140 °C. For example, in some embodiments, the polymerizing is carried out in a solvent at a temperature of about 50 to about 60 °C, about 50 to about 70 °C, about 50 to about 80 °C, about 50 to about 90 °C, about 50 to about 100 °C, about 50 to about 110 °C, about 50 to about 120 °C, about 50 to about 130 °C, about 50 to about 140 °C, about 60 to about 70 °C, about 60 to about 80 °C, about 60 to about 90 °C, about 60 to about 100 °C, about 60 to about 110 °C, about 60 to about 120 °C, about 60 to about 130 °C, about 60 to about 140 °C, about 70 to about 80 °C, about 70 to about 90 °C, about 70 to about 100 °C, about 70 to about 110 °C, about 70 to about 120 °C, about 70 to about 130 °C, about 70 to about 140 °C, about 80 to about 90 °C, about 80 to about 100 °C, about 80 to about 110 °C, about 80 to about 120 °C, about 80 to about 130 °C, about 80 to about 140 °C, about 90 to about 100 °C, about 90 to about 110 °C, about 90 to about 120 °C, about 90 to about 130 °C, about 90 to about 140 °C, about 100 to about 110 °C, about 100 to about 120 °C, about 100 to about 130 °C, about 100 to about 140 °C, about 110 to about 120 °C, about 110 to about 130 °C, about 110 to about 140 °C, about 120 to about 130 °C, about 120 to about 140 °C, or about 130 to about 140 °C.
[0126] Suitable solvents that can be used include, without limitation, dimethylformamide, dimethylacetamide, dimethylsulfoxide, acetonitrile, water, tetrahydrofuran, dioxane, saturated hydrocarbons such as heptane, halogenated hydrocarbons such as chloroform or di chloromethane, toluene, benzene, halogenated derivates of benzene such as chlorobenzene, or xylenes. [0127] In some embodiment, the polymerizing is carried out via reversible additionfragmentation chain-transfer polymerization (RAFT).
[0128] In some embodiments, said polymerizing is carried out in the presence of a free radical initiator and a chain transfer agent.
[0129] In some embodiments, the free radical initiator is selected from the group consisting of benzoyl peroxide, azobisisobutyronitrile, 1,1' azobis(cyclohexanecarbonitrile), t- butylperoxide, and 4,4’ -azobis(4-cyanoval eric acid).
[0130] In some embodiments, the chain transfer agent is a thiocarb onylthio compound, a dithioester compound, a trithiocarb onate compound, a dithiocarbamate compound, or a xanthate compound capable of reversible association with polymerizable free radicals.
[0131] In some embodiments, the chain transfer agent is a telechelic chain transfer agent, and said polymerizing comprises polymerizing said monomer A and said monomer B via RAFT to form a TCTA^PA-PB-TCTA2 or PA-PB-TCTA-PB-PA, wherein TCTA1, TCTA2, and TCTA are moieties derived from a telechelic chain transfer agent.
[0132] In some embodiments, the method further comprises providing a radically polymerizable amide, represented by A, providing a polymer TCTA^PA-PB-TCTA2 or PA-PB- TCTA-PB-PA, and polymerizing said monomer A with the polymer TCTA^PA-PB-TCTA2 or PA-PB-TCTA-PB-PA.
[0133] In some embodiments, the step of providing a polymer block PB comprising one or more units of said monomer B comprises providing said monomer B, polymerizing said monomer B in a solvent suitable for dissolving at least one of said PB block, and functionalizing the at least one of said PB block with one or more chain transfer groups or one or more initiator groups to produce a functionalized PB block.
[0134] The above disclosure is general. A more specific description is provided below in the following examples. The examples are described solely for the purpose of illustration and are not intended to limit the scope of the present disclosure. Changes in form and substitution of equivalents are contemplated as circumstances suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
EXAMPLES
[0135] The following Examples are presented to illustrate various aspects of the present disclosure, but are by no means intended to limit its scope. Example 1 - Materials and Methods
Analytical Techniques
[0136] All reactions were monitored by thin-layer chromatography using glass- backed Silica plates (SiliCycle, Quebec City, Quebec) and visualized by UV absorbance (254 nm) in addition to either Cerium Ammonium Molybdate or ethanolic H2SO4. All reactions were performed in flame-dried glassware under Argon atmosphere, unless otherwise noted. Column chromatography was performed using a Teledyne ISCO Combi Flash Rf+ Purion purification system using Silica Flash Cartridges purchased from Silicycle. Purification of the glycopolymers was performed via Dialysis in 1 mM NaHCCh H2O in 3.5 MWCO dialysis membrane (ThermoFischer Scientific). 1H, 13C, COSY, NOESY, and HSQCAD NMR spectra were taken on a Varian 400 MHz or Agilent DD2 600 MHz NMR spectrometers. CDCI3 and DMSO-de were purchased from Cambridge Isotope Laboratories (Tewksbury, MA). All NMR spectra were analyzed using MestreNova software (Version 12.0.1).
Gel Permeation Chromatography (GPC)
[0137] Molecular weights and distributions of the synthesized polymers were characterized using a Dionex UltiMate 3000 HPLC (Thermo Scientific) equipped with Chromeleon software V6.80 SR14 and 2x PIGel Mixed D columns PL1110-6500 (Aglient Technoligies Santa Clara, CA), at a flow rate of 1 mL/min with RI detection. Samples were dissolved in the mobile phase (0.05 M LiBr in DMF) at a concentration of 10 mg mL. Calibration was performed with known PMMA standards in the same mobile phase.
Lower Critical Solution Temperature (LCST) Measurements
[0138] Polymer cloud point (Cp) was determined via UV-Vis transmittance at 600 nm and a 50% decrease in transmittance was noted as the LCST of the polymer. To observe how the LCST values were affected by the varied weight fractions of polymer in solution, polymers were tested at concentrations of 1 mg/mL, 100 ug/mL, 50 ug/mL 25 ug/mL, 10 ug/mL, and 1 ug/mL.
Dynamic Light Scattering (DLS)
[0139] Particle size distributions of the synthesized polymers were determined using a Malvern Zetasizer Pro Blue (Malvern Panalytical). Samples were dissolved in Endotoxin Free H2O (Millipore Sigma) at a concentration of 100 ug/mL. A representative trace is shown in FIG. 3B. Samples were measured in triplicate and averaged to provide the reported particle sizes.
HEK-mMINCLE Cell Culture
[0140] The HEK-Blue mMincle cell line was purchased from Invivogen (San Diego,
CA, USA, hkb-mmcl) and passages 5-18 were used for all experiments. As per manufacturer instructions, HEK-Blue mMincle cells were grown in complete media composed of high glucose DMEM (Cytiva HyClon Dulbecco's Modified Eagles Medium) (Cytiva, Marlborough, USA, SH30243.01) with 4.5 g L'1 glucose, 2mM L-glutamine, 100 U mL'1 PenStrep (Caisson Labs, Smithfield, UT, USA, PSLOl-lOOmL) and 0.1 mg mL'1 Zeocin (Invivogen, San Diego, CA, USA) supplemented with 10% premium grade Heat Inactivated (HI)-FBS (VWR, Radnor, PA, USA, 97068-091). The media was changed every 3-4 days. Cells were passaged upon 80% confluence. Passaging involved changing media, counting, and seeding 3xl05 cells in 35 mL of new complete media in a new T-175 culture flask.
Harvest and Culture of Bone Marrow-Derived Dendritic Cells (BMDCs) and Macrophages (BMDMs)
[0141] 8 week old C57BL/6J mice purchased from The Jackson Laboratory (Bar
Harbor, ME, USA, 000664) and pluripotent monocytes were harvested from mouse femurs and cultured (Matheu et al., “Generation of Bone Marrow Derived Murine Dendritic Cells for use in 2 -Photon Imaging,” J Vis Exp., 17:773 (2008), which is hereby incorporated by reference in its entirety). Briefly, the monocytes were seeded at a density of 2 x 106 cells/mL in 10 mL of complete media which comprised of RPMI (Fisher Scientific, Waltham, MA, USA, 11-875-093) with 2 mM glutamine, 50 pM P-mercaptoethanol and supplemented with 10% HI-FBS and 20 ng/mL recombinant murine GMCSF (Peprotech, Westlake Village, CA, USA, 315-03). On the 3rd day the volume of the cell culture was doubled by addition of complete media and the cells were cultured for an additional 3 days before use. On the 6th day, the media was removed and centrifuged at 200 G for 10 min to isolate the non-adherent fraction containing BMDCs. The adherent cells were released by adding 0.25% Trypsin (Sigma Aldrich, St. Louis, MO, USA, T- 4049-100mL) and incubated at 37 °C for 10 min before neutralizing with Trypsin neutralizing solution (10% HI-FBS in IX PBS) to isolate the BMDMs. The BMDCs and BMDMs were centrifuged and transferred to RPMI complete media before seeding at a density of 2.5 x 105 cells per well in 24 well plates.
BMDC and BMDM Purity Assessment
[0142] A purity assessment was performed with BMDCs and BMDMs isolated from mouse femurs. Briefly, 1 x 106 cells were added to Eppendorf vials and blocked using Purified Anti-Mouse CD16/CD32 blocking antibody (Tonbo Biosciences, San Diego, CA, USA, 70- 0161-U500) following a 1 :50 dilution for 20 min on ice. The vials were then centrifuged at 200G for 5 min at 4 °C and the supernatant discarded. The antibodies were then added to the cells and incubated for 1 hour at 4 °C in the dark. The BMDCs were stained with the following antibodies: FITC Anti-Mouse CD11c (Tonbo Biosciences, San Diego, CA, USA, 35-0114-U100) at 0.25 pg mL'1, APC Anti-Human/Mouse CD1 lb (Tonbo Biosciences, San Diego, CA, USA, 20-0112- U100) at 0.125 pg ml 1, and PE Anti-Mouse F4/80 (Tonbo Biosciences, San Diego, CA, USA, 50-4801-U100) at 0.25 pg mL'1 all within the range suggested by the manufacturers. The BMDMs were stained with the following antibodies: FITC Anti-Mouse CD86 (Tonbo Biosciences, San Diego, CA, USA, 35-0862-U100) at 0.125 pg mL’1, APC Anti-Mouse CD206 (Biolegend, San Diego, CA, USA, 141708) at 0.25 pg mL’1, and PE Anti-Mouse F4/80 (Tonbo Biosciences, San Diego, CA, USA, 50-4801-U100) at 0.25 pg mL’1. Following incubation, the cells were washed 3x with ice cold blocking buffer (10% HI-FBS in lx PBS) and re-suspended in 600 pL ice cold FACS buffer (for analysis on the BD Accuri C6 plus Flow Cytometer. 10000 gated events were collected for all samples analyzed (see bottom of FIG. 12 and FIG. 13). The experiments were performed in hexaplet.
Virus Propagation and Inactivation for Vaccine Generation
[0143] Influenza virus A/Ca/04/2009 was propagated using standard protocols
(Szretter et al., “Influenza: Propagation, Quantification, and Storage,” Curr Protoc Microbiol, 3: 15G.1.1-15G.1.22 (2006), which is hereby incorporated by reference in its entirety). Briefly, in a biosafety cabinet the surface of fertilized specific pathogen-free eggs (10-12 days old) were decontaminated. 100 pL containing 100 PFU of Influenza A/Califomia/04/2009 were injected into the amniotic sac. The injection site was glue-sealed, and the eggs were placed in incubator for 72 hours, followed by another incubation at 4 °C for 24 hours. The amniotic fluid was collected and cleared at 1000 RCF for 10 minutes. Viral stocks were snap-frozen in liquid nitrogen and kept at -80 °C.
[0144] Large-scale cultivated Influenza A/California were inactivated with 0.02% paraformaldehyde (PF A) for 4 hours at room temperature. Vaccine inactivation was confirmed by passaging inactivated virus in cell culture and by injecting 100 pL inactivated virus in eggs as described above followed by confirmation via observance of CPE or plaque assays. Cell cultures were passaged at minimum 3 times to confirm absence of viable virus.
Immunization and Challenge of Mice
[0145] Twenty, eight-week-old female C57BL6 mice (Charles River Laboratories) were housed in a BSL-2 vivarium. There were six groups of three mice per treatment group and a group of two unchallenged unvaccinated mice. Three mice per group were immunized intramuscularly with 100 pl of a 1 : 1 mixture of inactivated influenza virus and adjuvant or control. The groups were divided as follows: alum, TDB, Pl, P2, no adjuvant (inactivated virus only), and the final group received only phosphate-buffered saline (PBS) as control. The mice were given a vaccine boost on day 15 post prime vaccination and body weights were recorded every two days for 28 days. [0146] The mice were bled on days 0, 7, 14, 16, 22, and 28 post vaccination and challenged intranasally with mouse-adapted live Influenza A/Califomia/04/2009 strain virus on day 29. Mice were observed for signs of disease and weight loss after infection. All mice groups were euthanized five days post infection followed by collection of blood and lung samples.
[0147] The parameters listed below were measured: serum cytokine analysis 24 hours and 7 days post boost, antibody titers via hemagglutinin inhibition assay on day 14 and 28 and neural India session inhibition assay on day 28. Flow cytometry analysis was performed using lung samples 5 days post challenge.
Hemagglutinin Inhibition (HI) Assay
[0148] Mouse sera obtained from the vaccinated and control groups were placed in a water bath at 56 °C for 30 min. Hemagglutinin Inhibition (HI) buffer was used to dilute 2-fold successive dilutions of sera (25 pL) starting at 1 : 10 to 1 : 1280 on U-bottom 96-well microtiter plates. HI buffer containing 4 hemagglutinin units/50 pL from Ca/04/2009 was added to the diluted sera followed by 1 hour incubation at room temperature. To eliminate the existence of hemagglutinins in rooster red blood cells (RBCs) and regulate spontaneous RBC agglutination, a control sample was set up for each serum (HI buffer). Following serum/virus incubation, a 0.5% RBC suspension of rooster blood was overlaid and plates were read 30-40 min after overlay. The antiserum's HAI titer is expressed by the fold change in RBC agglutination between all groups.
Neuraminidase Inhibition Assay (NI)
[0149] NI assays were adapted from Leang and Hurt (Leang and Hurt,
“Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals,” J Vis Exp, 122:e55570 (2017), which is hereby incorporated by reference in its entirety). Briefly, 1/10 dilution of sera were 2- fold serially diluted in assay buffer, then incubated with diluted A/Califomia/04/2009 virus in black flat-bottom 96-well plates. Plates were sealed and incubated for 30-45 min at 37 °C to allow for antibody binding. 2'-(4-Methy1umbellifery1)-a~D-N-acetylneuraminic acid (MUNANA) substrate was then added, and the sealed plates were incubated at 37 °C for 1 hour followed by addition of stop solution. Inhibition of enzymatic activity was assessed using a plate reader (Tecan Spark, Zurich, Switzerland) set to 355 nm excitation measuring absorbance at 460 nm. These results are shown in FIG. 34.
Serum Cytokine Analysis
[0150] IL-6 levels were quantified from vaccinated mouse sera using the High
Sensitivity 5-Plex Mouse ProcartaPlex™ Panel (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions, read on the MagPix Instrument (Luminex), and calculated with Luminex xPONENT 4.1 Software.
Flow Cytometry
[0151] Lymphocytes were extracted from mouse lungs with mechanical and enzymatic digestion (Liberase TL and DNase I (Sigma-Aldrich, St. Louis)). Flow cytometry data was acquired using Attune NxT (Thermo Fisher) and analyzed using FlowJo (Tree Star, Ashland, OR), as described. Central memory T cells were identified using the following antibody (clone) combinations: anti-CD16/CD32 Fc Block (93), anti-CD45 (30-F11), anti-CD3 (145- 2C11), anti-CD4 (RM4-5), anti-CD62L (MEL-14), anti-CD44 (IM7), viability dye (Invitrogen 65-0866-14).
[0152] Statistical analysis was performed using GraphPad Prism v6.0 and p<0.05 was deemed to be statistically significant at all the calculations. *<0.05 **<0.01
LCST Measurements
[0153] The Cloud point (Cp) of the copolymers was determined via UV-Vis transmittance at 600 nm and a decrease in transmittance to 50% of the initial was reported as the LCST of the polymer. The polymers were tested at concentrations of 1 mg/mL, 100 ug/mL, 50 ug/mL 25 ug/mL, 10 ug/mL, and 1 ug/mL. The LCST values were affected by both temperature and the varied weight fractions of polymer in solution. These results are shown in FIG. 14 and FIG. 15.
MINCLE Assay
[0154] HEK-Blue mMincle cells (Invivogen, CA) were plated in 96 well plates in
180 pL of complete media at a density of 3 x 105 cells per well. The plates were incubated for approximately 6 hours at the temperatures tested (35 °C, 37 °C, and 39 °C) prior to adding the test compounds. Pl, P2, P3, along with TDB (Avanti Polar Lipids, Alabaster, AL, USA, 809808), which served as the positive control, were tested at various concentrations (1 pg mL1, 10 pg mL1, 50 pg mL1, and 100 pg mL1) by adding 20 pL of each solution to the respective wells to achieve the indicated concentrations at a final volume of 200 pL/well. Plates were incubated for 16 hours before measuring NF -kB transcription via secreted embryonic alkaline phosphatase (SEAP) detection protocol. SEAP detection media (1.17 mg mL'1 5-bromo-4- chloro-3’-indolylphosphate p-toluidine salt in 1 M aqueous diethanolamine) was used to measure SEAP for all MINCLE assays. To an optically transparent-bottomed 96-well plate, 180 pL of SEAP detection media was added to each well. Next, 20 pL of supernatant from the MINCLE assays was added to each well and the plates were incubated for 1-4 hours to achieve significant positive control absorbance. Following incubation, the absorbance of each well was measured at 620 nm. Experiments were performed in hexaplet and normalized to positive and negative controls.
BMDC and BMDM Assay
[0155] BMDC and BMDM primary cultures were isolated from mouse femurs and seeded at a density of 2.5 x 105 cells per well in 24 well plates. The plates were then incubated for approximately 6 hours at the temperatures tested (35 °C, 37 °C, and 39 °C) prior to adding the test compounds. Pl, P2, P3, as well as TDB were added to the wells at a concentration of 100 pg mL'1 while the more potent LPS (Sigma Aldrich, St. Louis, MO, USA, L2018-5MG) was tested at 40 pg mL'1. The plates were then incubated at the given temperatures for 48 hours before the supernatant was collected. The supernatant samples were then analyzed by using Mouse TH1/ TH2/ TH17 Cytometric Bead Array Kit (BD Biosciences, San Jose, CA, USA 560485) according to the manufacturer’s instructions using a BD Accuri C6 plus Flow Cytometer. The experiments were performed in triplicate. These results are shown in the top portion of FIG. 12 and FIG. 13.
Example 2 - Synthetic Methods
Synthesis of l-(Diethoxymethyl)-4-vinylbenzene
[0156] l-(Diethoxymethyl)-4-vinylbenzene was synthesized as described in the literature (Mancini et al., “Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors,” J. Am. Chem. Soc., 134(20):8474-8479 (2012); Lee et al., “Trehalose Glycopolymers as Excipients for Protein Stabilization,” Biomacromolecules, 14 (8):2561-2569 (2013); which are hereby incorporated by reference in their entirety). Briefly, methyltriphenylphosphonium bromide (5.4 g, 14.5 mmol) and 45 mL of tetrahydrofuran (THF) were added to a flame dried 250 mL round bottom flask. The reaction mixture was cooled to -78 °C and stirred for 20 min. 1.6M n-butyllithium in hexanes (9.94 mL, 15.9 mmols n-BuLi) was added drop wise over the course of 20 min. The reaction was stirred for 30 min at -78 °C, warmed to 25 °C and stirred for 10 min. The orange-red colored reaction solution was then cooled down to -78 °C. Terephthaldehyde monodiethyl acetal (2.5 g, 12.0 mmol) dissolved in 7.5 mL of THF was added drop wise over the course of 1 hour. After stirring at -78 °C for 30 min, the reaction flask was warmed to 0 °C and stirred for 3 hours. Then the reaction warmed to 25 °C and stirred for 1 hour. The reaction was quenched by adding 10 mL saturated NaHCCh solution. The organic layer was collected after adding 50 mL of H2O, and the aqueous layer was extracted with 20 mL of ether three times. The combined organic layers were dried over MgSCh. After purification by silica gel column chromatography with Hex:EtOAc = 20: 1, 2.40 g of product in liquid form was obtained (89% yield). 'HNMR (600 MHz, in CDCh) 8: 7.55- 7.45 (m, 2H), 7.45-7.36 (m, 2H), 6.78-6.68 (m, 1H), 5.81-5.74 (d, J = 17 Hz, 1H), 5.57- 5.51 (s, 1H), 5.29-5.22 (d, J = 11 Hz, 1H), 3.70-3.50 (m, 4H), 1.40-1.10 (t, J = 7 Hz, 6H) (FIG. 4). 13C NMR (600 MHz in CDCh) 6: 138.80, 137.43, 136.59, 126.84, 125.93, 113.77, 101.00, 60.65, 15.10 (FIG. 5).
Synthesis of 4,6-0-(4-Vinylbenzylidene)-a,a-trehalose
[0157] 4,6-O-(4-Vinylbenzylidene)-a,a-trehalose was synthesized according to a literature procedure (Mancini et al., “Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors,” J. Am. Chem. Soc., 134(20):8474-8479 (2012); Lee et al., “Trehalose Glycopolymers as Excipients for Protein Stabilization,” Biomacromolecules, 14 (8):2561-2569 (2013); which are hereby incorporated by reference in their entirety). To a flame dried round bottom flask a,a-trehalose (4.15 g, 12.1 mmol, 5.00 eq) and 150 mL DMF were added, and the resulting mixture was warmed to 100 °C. Next, l-(diethoxymethyl)-4- vinylbenzene (0.500 g, 2.42 mmol, 1.00 eq) and camphorsulphonic acid (CSA, 46.5 mg, 0.20 mmol, 0.12 eq) were added, and the solution was stirred for 3 hours at 100 °C, with venting. The reaction vessel was vented to allow for the gradual removal of EtOH byproduct. Solvent was removed from the resulting crude reaction product in vacuo to give a white solid that was further purified by column chromatography (40:60 to 10:90 watermethanol linear gradient over 30 min at lOmL/min). After removal of solvent, the resulting product (456 mg, 0.99 mmol, 40% yield) was obtained as a white solid. 'H NMR (600 MHz, d6DMSO) 6: 7.510 (d, 2H, J=8.4Hz), 7.458 (d, 2H, 8.4Hz), 6.775 (dd, 1H, 10.8, 17.4Hz), 5.887 (d, 1H, 17.4Hz), 5.590 (s, 1H), 5.320, (d, 1H, 11.4Hz), 5.259 (d, 1H, 4.8Hz), 5.033-5.016 (m, 2H), 4.990 (d, 1H, 3.6Hz), 4.921 (d, 1H, 3.6Hz), 4.868-4.853 (m, 2H), 4.452 (dd, 1H, 6, 6Hz), 4.137 (dd, 1H, 4.8, 9.6Hz), 4.036 (ddd, 1H, 4.8, 9.6, 10.2Hz), 3.795 (ddd, 1H, 4.8, 5.4, 9.3Hz), 3.746 (ddd, 1H, 2.1, 4.5, 9.9Hz), 3.691 (dd, 1H, 10.2, 10.2Hz), 3.644-3.595 (m, 2H), 3.530 (ddd, 1H, 5.4, 5.7, 11.7Hz), 3.454-3.402 (m, 2H), 3.316 (ddd, 1H, 3.8, 5.6, 9.4Hz), 3.203 (ddd, 1H, 4.8, 5.4, 9.3Hz) (FIG. 6). COSY-NMR characterization is shown in FIG. 24 with predicted acetal stereochemistry (FIG. 25) and expanded carbohydrate region of the COSY-NMR shown in FIG. 26. 13C NMR (600 MHz d6DMSO) 6: 138.4, 138.3, 137.2, 127.6, 126.7, 115.8, 101.6, 101.5, 95.3, 94.8, 82.4, 73.6, 73.1, 72.4, 70.9, 70.5, 69.2, 63.3, 61.6. MS (ESI-MS) calc, for C2iH280iiNa+ : 479 observed: 479.
Synthesis of 4,6-O-(4-Vinylbenzylidene)-6’-0-behenoyl-a,a-trehalose (1)
[0158] To a flame dried round bottom flask, a solution of HBTU (0.378 g, 0.99 mmols, 1.10 eq) and behenic acid (0.340g, 0.99 mmols, 1.10 eq) in 5 mL anhydrous pyridine was added and the mixture was allowed to stir for 30 min at room temperature. Next, 4,6-O-(4- vinylbenzylidene)-a,a-trehalose (0.414 g, 0.98 mmol, 1.00 eq) was added, and the solution stirred for 3 days at room temperature while the reaction was monitored by TLC. Solvent was removed azeotropically with toluene from the resulting crude reaction product in vacuo to give a pale brown solid that was further purified by silica chromatography (5%-25% MeOH- DCM:EtOAc 1 : 1) linear gradient over 20 min at 40 mL/min). After removal of solvent the resulting product (176 mg, 25% yield) was obtained as a beige solid. 1 H NMR (600 MHz, d6DMSO) 5: 7.510 (d, 2H, J=8.4Hz), 7.458 (d, 2H, 8.4Hz), 6.775 (dd, 1H, 10.8, 17.4Hz), 5.887 (d, 1H, 17.4Hz), 5.590 (s, 1H), 5.320, (d, 1H, 11.4Hz), 5.259 (d, 1H, 4.8Hz), 5.033-5.016 (m, 2H), 4.990 (d, 1H, 3.6Hz), 4.921 (d, 1H, 3.6Hz), 4.868-4.853 (m, 2H), 4.452 (dd, 1H, 6, 6Hz), 4.137 (dd, 1H, 4.8, 9.6Hz), 4.036 (ddd, 1H, 4.8, 9.6, 10.2Hz), 3.795 (ddd, 1H, 4.8, 5.4, 9.3Hz), 3.746 (ddd, 1H, 2.1, 4.5, 9.9Hz), 3.691 (dd, 1H, 10.2, 10.2Hz), 3.644-3.595 (m, 2H), 3.530 (ddd, 1H, 5.4, 5.7, 11.7Hz), 3.454-3.402 (m, 2H), 3.316 (ddd, 1H, 3.8, 5.6, 9.4Hz), 3.203 (ddd, 1H, 4.8, 5.4, 9.3Hz) (FIG. 7). This was further characterized by COSY-NMR (FIG. 27) with the predicted stereochemistry of the acetal position confirmed by NOESY-NMR (FIG. 28). 13C NMR (600 MHz d6DMSO) 5: 138.4, 138.3, 137.2, 127.6, 126.7, 115.8, 101.6, 101.5, 95.3, 94.8, 82.4, 73.6, 73.1, 72.4, 70.9, 70.5, 69.2, 63.3, 61.6 (FIG. 8). MS (ESI-MS) calc, for C21H28O1 lNa+: 479 observed: 479. The product was also characterized by UV/Vis spectroscopy (FIG. 29) and IR spectroscopy (FIG. 30).
Synthesis of Chain Transfer Agent Ethyl-(l-phenylethyl) Trithiocarbonate [0159] Ethyl-(l-phenylethyl) trithiocarb onate was synthesized according to a literature procedure (Wood et al., “Selective One-Pot Synthesis of Trithiocarbonates, Xanthates, and Dithiocarbamates for Use in Raft/Madix Living Radical Polymerizations,” Organic Letters, 8 (4): 553-556 (2006), which is hereby incorporated by reference in its entirety). First, a suspension of K3PO4 (31.94 mg, 0.52 mmol) was formed in dry acetone (15mL) and cooled to 0°C. Next, ethanethiol (4.2mL, 0.48 mmol) and carbon disulfide (3.67mL, 0.48 mmol) were added and allowed to stir for 10 min (a dramatic dark yellow color appears due to the formation of the trithioate anion intermediate). 1 -Bromo- 1 -phenylethane (2.56 mL, 0.50mmols) was added and allowed to stir for 30 min. The reaction was allowed to warm to room temperature and the solution was concentrated in vacuo. The solution was washed with 30 mL of H2O three times and the organic layer was dried over MgSCU. After silica gel column chromatography with Hex : EtOAc (1 :4) the resulting product (1.38mL, 83% yield) was characterized. 'HNMR (600MHz CDCI3) 8: LOO (t, 3H, CH3CH2-), 1.50, 1.73 (m, 2H, CH2-S), 1.81 (d, 3H, CH3(CH-)), 3.41 (t, 2H, -S-CH2), 5.41 (q, CH), 7.42 (m, 5H, ArH). 13C NMR 5: 21.4 (CH3CH), 30.1, 36.5 (-CH2-S), 50.1 (CH), 127.7 (p-Ph, CH), 127.8 (m-Ph, CH), 128.7 (o-Ph, CH) 141.2 (Ph, C), 223.1(CS3). Preparation of Poly(NIPAM)-co-poly(4,6-O-(4-vinylbenzylidene)-6’-0-behenoyl-a,a- trehalose) (Pl)
[0160] In a 25 mL Schleck flask, 1 (0.185 g, 0.240 mmol) was dissolved in dimethylformamide (DMF, 3.04 mL). NIP AM (0.241 g, 2.13 mmol) was added as a comonomer. 6.4 pL CTA (0.0073 g, 0.030 mmol) was added ([Trehalose:NIPAM:CTA:I] = [20: 180:2.5: 1]). AIBN (2.00 mg, 12.2 x 10 3 mmol) was added to the solution. Three cycles of freeze-pump-thaw were undertaken, and the reaction was initiated by immersion in an oil bath at 90°C. The polymerization was stopped by cooling with liquid nitrogen after 16 hours. As determined by JH NMR spectroscopy, 35% monomer conversion was achieved. The product was purified by dialysis against H2O with 1 mM NaHCCL for 3 days (3.5 kDa) and dried using a lyophilizer resulting in Pl. Number-average molecular weight (Mn) of the polymer was 7.3 kDa, and D was 1.27 by GPC). 'H NMR (600MHz d6DMSO) 5 7.56, 7.25, 5.45, 5.06, 5.01, 4.20, 4.23, 3.93, 3.81, 3.71, 3.60, 3.54, 3.11, 2.25, 1.94, 1.48, 1.20, 1.01, 0.82 (FIG. 31).
Preparation of Poly(NIPAM)-co-poly(4,6-O-(4-vinylbenzylidene)-6’-0-behenoyl-a,a- trehalose)-b-pNIPAM (P2)
[0161] In a 25 mL Schleck flask Pl macroCTA (0.07 g, 9.13 mmol) was dissolved in dimethylformamide (DMF, 2.00 mL). NIP AM (0.138 g, 1.22 mmol) was added as a comonomer. AIBN (1.00 mg, 6.09 x I O 3 mmol) was added to the solution ([M:CTA:I] = [200: 1.5: 1], Three cycles of freeze-pump-thaw were undertaken, and the reaction was initiated by immersion in an oil bath at 90°C. The polymerization was stopped by cooling with liquid nitrogen after 16 hours. As determined by 'H NMR spectroscopy, 56% NIP AM monomer conversion was achieved. The product was purified by dialysis against H2O with ImM NaHCCh for 3 days (3.5 kDa) and dried using a lyophilizer resulting in P2. Number-average molecular weight (Mn) of the polymer was 25.3 kDa, and D was 1.85 by GPC. *HNMR (600MHz d6DMSO) 5 7.58, 7.30-7.17, 5.45, 5.06, 4.89, 4.87, 4.83, 4.20, 4.03, 3.81, 3.11, 2.90, 2.82, 2.74, 2.70, 2.65, 2.58, 2.25, 1.94, 1.42, 1.20, 1.01, 0.82 (FIG. 32).
Preparation of Poly(4,6-0-(4-vinylbenzylidene)a,a-trehalose)-b-poly(NIPAM) (P3) [0162] 4,6 -O-(4-vinylbenzylidene)-a,a-trehalose was prepared according to literature procedure, to be used as a MacroCTA (Mancini et al., “Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors,” J. Am. Chem. Soc., 134(20): 8474-8479 (2012); which is hereby incorporated by reference in its entirety). In a 25 mL Schleck flask 4,6-O-(4-vinylbenzylidene)-a,a-trehalose (39.6 kda) macroCTA (0.603 g, 15.2 mmol) was dissolved in dimethylformamide (DMF, 3.04 mL). NIP AM (0.206 g, 1.82 mmol) was added as a comonomer. ([M:CTA:I] = [300:2.5: 1], AIBN (1.00 mg, 6.09 x 10-3 mmol) was added to the solution. Three cycles of freeze-pump-thaw were undertaken, and the reaction was initiated by immersion in an oil bath at 90°C. The polymerization was stopped by cooling with liquid nitrogen after 16 hours. As determined by 'H NMR spectroscopy, 72% NIP AM monomer conversion was achieved. The product was purified by dialysis against H2O with ImM NaHCCh for 3 days (3.5kDa) and dried via lyophilization resulting in P3. Number-average molecular weight (Mn) of the polymer was 52.0 kDa, and D was 1.18 by GPC. JH NMR (600MHz d6DMSO) 5 7.13, 6.40, 5.40, 5.19, 4.92, 4.37, 4.02, 3.82, 3.56, 2.82, 2.52 2.48, 1.96, 1.90, 1.44, 1.02.
Example 3 - Results and Discussion
[0163] To create a thermoresponsive adjuvant, linear poly-N-isopropylacrylamide
(NIP AM) was considered, as it exhibits an aqueous Lower Critical Solution Temperature (LCST) (Heskins et al., “Solution Properties of Poly(N-isopropylacrylamide),” J. Macromol. Sci. Part A — Chem., 2(8): 1441-1455 (1968), which is hereby incorporated by reference in its entirety) that can be engineered to occur sharply within physiological ranges (Pei et al., “Synthesis and Properties of Poly (N-isopropyl acrylamide) and Poly (N-isopropylacrylamide-co- acrylamide) Hydrogels,” J. Shanghai Univ. Engl. Ed., 9:466-470 (2005), which is hereby incorporated by reference in its entirety), and has been used to thermally attenuate activity when conjugated to enzymes (Shimoboji et al., “Temperature-Induced Switching of Enzyme Activity with Smart Polymer-Enzyme Conjugates,” Bioconjug. Chem., 14(3):517— 525 (2003), which is hereby incorporated by reference in its entirety). NIP AM has been previously conjugated to established vaccine adjuvants (Hoshi et al., “Synthesis of a Hemin-Containing Copolymer as a Novel Immunostimulator that Induces IFN-gamma Production,” Ini. J. Nanomedicine , 13:4461- 4472 (2018), which is hereby incorporated by reference in its entirety), and likely acts as an adjuvant itself (Shakya et al., “Adjuvant Properties of a Biocompatible Thermo-Responsive Polymer of N-isopropyl acrylamide in Autoimmunity and Arthritis,” J. R. Soc. Interface, 8(65): 1748-1759 (2011), which is hereby incorporated by reference in its entirety), although the exact mechanism, such as direct cell activation relative to enhancement of depot effects, remains conjectural (Shakya and Nandakumar, “Applications of Polymeric Adjuvants in Studying Autoimmune Responses and Vaccination Against Infectious Diseases,” J. R. Soc. Interface, 10(79):20120536 (2013); Shakya et al., “Macrophage-Derived Reactive Oxygen Species Protects Against Autoimmune Priming with a Defined Polymeric Adjuvant,” Immunology, 147(1): 125— 132 (2016); Monaco et al., “Multi-Arm Star-Shaped Glycopolymers with Precisely Controlled Core Size and Arm Length,” Biomacromolecules 21(9):3736-3744 (2020); which are hereby incorporated by reference in their entirety). Apart from NIP AM, a wide-range of additional thermoresponsive polymers including other acrylamides (Francica et al., “Thermoresponsive Polymer Nanoparticles Co-deliver RSV F Trimers with a TLR-7/8 Adjuvant,” Bioconjug. Chem., 27(10):2372-2385 (2016), which is hereby incorporated by reference in its entirety), polyesters/ethers (Bobbala et al., “Novel Injectable Pentablock Copolymer Based Thermoresponsive Hydrogels for Sustained Release Vaccines,” AAPS J., 18(1):261-269 (2016), which is hereby incorporated by reference in its entirety), polyacetals (Van Herck et al., “Transiently Thermoresponsive Acetal Polymers for Safe and Effective Administration of Amphotericin B as a Vaccine Adjuvant,” Bioconjug. Chem., 29(3):748-760 (2018), which is hereby incorporated by reference in its entirety), and poloxomer-chitosan sol-gels (Kojarunchitt et al., “Modified Thermoresponsive Poloxamer 407 and Chitosan Sol-Gels as Potential Sustained-Release Vaccine Delivery Systems,” Eur. J. Pharm. Biopharm., 89:74-81 (2015); Clausse et al., “Polymeric Antigen BLSOmp31 in Aluminium Hydroxide Induces Serum Bactericidal and Opsonic Antibodies against Brucella Canis in Dogs,” Vet. Immunol.
ImmunopathoL , 184:36-41 (2017); which are hereby incorporated by reference in their entirety) have also been tested in therapeutic vaccine formulations for diseases ranging from HIV to cancer. However, in all cases, the thermoresponsive nature of the polymers has been exploited for their rheological properties or ability to impart controlled-release mechanisms to the antigen/adjuvant depot, rather than thermoregulation of adjuvant potency itself. To-date, no examples exist where temperature is used to modulate adjuvant activity about an LCST. As such, in this disclosure copolymers of thermoresponsive NIP AM and a polymerizable adjuvant were investigated to determine whether they will attenuate potency above the LCST.
[0164] Selecting a suitable adjuvant to polymerize with NIP AM, synthetically accessible adjuvants with large hydrophobic regions critical for activity were considered, as it was envisioned that access to these regions would be occluded as the polymer traversed the hydrophilic/hydrophobic transition characteristic of the LCST. This motif is exemplified by the highly lipophilic adjuvant trehalose-6, 6’ -dibehenate (TDB) (FIG. IB) (Khan et al., “Long-Chain Lipids Are Required for the Innate Immune Recognition of Trehalose Diesters by Macrophages,” ChemBioChem, 12(17):2572-2576 (2011), which is hereby incorporated by reference in its entirety), which itself is the minimally active motif of trehalose 6,6’-dimycolate (Al Dulayymi et al., “The First Unique Synthetic Mycobacterial Cord Factors,” Tetrahedron Lett., 50(26):3702- 3705 (2009), which is hereby incorporated by reference in its entirety), the primary immunostimulatory component of mycobacterium tuberculosis (Geisel et al., “In Vivo Activity of Released Cell Wall Lipids of Mycobacterium bovis Bacillus Calmette-Guerin Is Due Principally to Trehalose Mycolates,” J. Immunol., 174(8):5007-5015 (2005), which is hereby incorporated by reference in its entirety). Both the mycolic ester and synthetic TDB activate innate immune cells through C-Type Lectin Receptors (CLRs), including the Macrophage- Inducible C-type Lectin (MINCLE) receptor (Feinberg et al., “Mechanism for Recognition of an Unusual Mycobacterial Glycolipid by the Macrophage Receptor Mincle,” J. Biol. Chem., 288(40):28457-28465 (2013), which is hereby incorporated by reference in its entirety), among other CLR pathways (Hansen et al., “Macrophage Phosphoproteome Analysis Reveals MINCLE-dependent and -independent Mycobacterial Cord Factor Signaling,” Mol. Cell. Proteomics, 18(4):669-685 (2019); Kodar et al., “The Mincle Ligand Trehalose Dibehenate Differentially Modulates Ml -Like and M2 -Like Macrophage Phenotype and Function via Syk Signaling,” Immun. Inflamm. Dis., 5(4):503-514 (2017); Miyake et al., “C-type Lectin MCL Is an FcRy — Coupled Receptor that Mediates the Adjuvanticity of Mycobacterial Cord Factor,” Immunity, 38(5): 1050-1062 (2013); which are hereby incorporated by reference in their entirety), as well as inflammasomes (Schweneker et al., “The Mycobacterial Cord Factor Adjuvant Analogue Trehalose-6,6'-dibehenate (TDB) Activates the Nlrp3 Inflammasome,” Immunobiology, 218(4):664-673 (2013), which is hereby incorporated by reference in its entirety), to elicit a TH1/TH17 polarized immune response (Desel et al., “The Mincle-Activating Adjuvant TDB Induces MyD88-Dependent Thl and Thl7 Responses through IL-1R Signaling,” PloS One, 8(l):e53531 (2013), which is hereby incorporated by reference in its entirety). Overall, this polarization can be detrimental in some viral vaccines (Maroof et al., “Intranasal Vaccination Promotes Detrimental Thl7-Mediated Immunity against Influenza Infection,” PLoS Pathog., 10(l):el 003875 (2014), which is hereby incorporated by reference in its entirety), but alternatively could be beneficial, if adequately regulated, making TDB (and related derivatives) a promising target for further control with thermoresponsive polymers.
[0165] TDB hydroxyls weakly coordinate with hydrophilic binding domains that share structural similarity among CLRs (Furukawa et al., “Structural Analysis for Glycolipid Recognition by the C-type Lectins Mincle and MCL,” Proc. Natl. Acad. Sci., 110(43): 17438— 17443 (2013), which is hereby incorporated by reference in its entirety), and in the case of MINCLE, bind with Arg 182.25 Although these polar binding interactions are somewhat weak, the hydrophobic portions of TDB can also interact with their own binding domains and facilitate coordination. As a result, one approach to enhance the potency of TDB (and related analogues) is through multivalent physiochemical presentation, which can enhance avidity and also induce clustering of receptors, cumulatively resulting in synergistic MINCLE activation (Retzinger et al., “The Role of Surface in the Biological Activities of Trehalose 6,6’-dimycolate. Surface Properties and Development of a Model System,” J. Biol. Chem., 256(15):8208-8216 (1981); Retzinger, “Dissemination of Beads Coated with Trehalose 6,6'-dimycolate: A Possible Role for Coagulation in the Dissemination Process,” Exp. Mol. Pathol., 46(2): 190-198 (1987); which are hereby incorporated by reference in their entirety). Together, these observations imply that 1) the multivalency of polymeric glycolipids should enhance activation of MINCLE and 2) facilitating MINCLE receptor access to the hydrophobic portion of the glycolipid is critical for activity. Based upon this observation, it was hypothesized that combining thermoresponsive NIP AM with a polymerizable TDB derivative would allow NIP AM to participate in temperature-dependent shielding of the hydrophobic portion of the glycolipid adjuvant, thereby inversely linking adjuvant potency to temperature.
[0166] Molecular design of a polymerizable trehalose glycoplipid adjuvant required the construction of a polymerizable adjuvant subunit consisting of a trehalose carbohydrate core and a behenic ester lipid chain that can interact with the hydrophobic domains of MINCLE. To create a motif that was polymerizable, inspiration was drawn from the 4,6-vinylbenzylidene acetal monomer which can be accessed in a single step from trehalose with no protecting groups and moderate yields (Mancini et al., “Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors,” J. Am. Chem. Soc., 134(20):8474-8479 (2012), which is hereby incorporated by reference in its entirety). It was assumed that adding the behenic ester found in TDB to the 6’ position of the monomer would create a glycolipid immunostimulant owing to previous reports demonstrating a broad range of alkyl or aryl (Rasheed et al., “Design of Trehalose-based Amide/Sulfonamide C-type Lectin Receptor Ligands,” ChemMedChem, 16(8): 1246-1251 (2021), which is hereby incorporated by reference in its entirety) substituents (as in Brartemicin) (Jacobsen et al., “The Natural Product Brartemicin is a High Affinity Ligand for the Carbohydrate-Recognition Domain of the Macrophage Receptor Mincle,”
MedChemComm, 6(4):647-652 (2015), which is hereby incorporated by reference in its entirety) are tolerated at the 6/6’ positions and only a single lipid chain is required for adjuvant activity (FIGs. IB and 1C) (Stocker et al., “On One Leg: Trehalose Monoesters Activate Macrophages in a Mincle-Dependent Manner,” ChemBioChem, 15(3):382— 388 (2014), which is hereby incorporated by reference in its entirety).
[0167] Briefly, the synthesis commenced with a transacetalization linking 1-
(diethoxymethyl)-4-vinylbenzene with anhydrous a,a-trehalose to form a 4,6-O-(4- vinylbenzylidene)-a,a-trehalose glycomonomer according to established literature procedure (Mancini et al., “Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors,” J. Am. Chem. Soc., 134(20):8474-8479 (2012), which is hereby incorporated by reference in its entirety). Next, to synthesize the immunostimulant glycomonomer, an established protocol for the Steglich esterification of trehalose glycolipids was employed (Paul et al., “Direct Synthesis of Maradolipids and Other Trehalose 6-Monoesters and 6,6'-Diesters,” J. Org. Chem., 78(2):363-369 (2013), which is hereby incorporated by reference in its entirety), resulting in 4,6-O-(4-vinylbenzylidene)-6’-O-behenoyl-a,a-trehalose monoester 1 as the polymerizable glycolipid adjuvant following pyridine/HBTU coupling of monomer with behenic acid (FIG. 2). Next, Reversible Addition-Fragmentation Chain-Transfer (RAFT) polymerization was conducted with NIP AM, 1, a trithiocarb onate Chain Transfer Agent (CTA), and AIBN initiator to arrive at the random copolymer Pl following standard dialysis work-up (MWCO: 3.5KDa, 0. ImM NaHCCh H2O, 2 days). This resulted in a low molecular weight polymer (Mn = 7 kDa) with a broad LCST range (35-45 °C). To sharpen the LCST, Pl was used as a macro-CTA in a second RAFT polymerization which extended the NIP AM block. Following dialysis as above, this provided a multivalent thermophobic trehalose glycolipid adjuvant P2 which had an identical degree of polymerization with respect to adjuvant, but higher molecular weight and sharper LCST. A third polymer, P3, containing NIP AM and 4- vinylbenzylidene-a,a-trehalose without the 6’ behenic ester modification, was also synthesized and evaluated as a control for both long-range NIPAM-trehalose interactions as well as any immunogenicity that might arise from the NIPAM-trehalose polymer system itself. Each polymer exhibited well-defined glycomonomer degree of polymerization and modest overall dispersity (D) (Table 1).
Table 1. Polymer Data
Figure imgf000045_0001
[a] Adjuvant Mn was determined by GPC
[b] ratios of starting materials used in each polymerization, T = Glycolipid adjuvant 1, N = N- isopropyl acrylamide, CTA = Ethyl -(1 -phenylethyl) trithiocarb onate, I = Azobisisobutyronitrile with [c] synthesized as a NIP AM block from glycopolymer macro CTA
[d] Determined by NMR
[e] Determined by GPC
[f] Determined by NMR [0168] The physicochemical behavior of Pl, P2, and P3 was characterized in solution. Examining the LCST for each polymer revealed that, while Pl exhibited a broad LCST, P2 and P3 had sharp LCSTs near 37 °C at a concentration of 1 mg/mL (FIGs 3A and 14). Decreasing polymer concentration broadened and shifted LCST values to higher temperatures (39-43 °C) before becoming undetectable in the lower pg/mL range (FIG. 15). Next, variations in average particle size as a function of temperature were examined by DLS. All 3 polymers formed nanoparticles at 100 pg/mL, the concentration ultimately used in-vitro (FIGs. 3B and 16A-16C). Increasing temperature caused particles formed by Pl and P2 to decrease in size, while the opposite trend was observed for P3. This suggested that lipid chains within the Pl and P2 nanoparticles play an important role in the formation of secondary structure in aqueous media, perhaps by enhancing the exclusion of water from the nanoparticles at higher temperatures. The apparent broadening in dispersity for P2 from 37°C to 39°C may also be attributed to competing interactions from the increased NIP AM content which strengthens selfassociation due to hydrophobic interactions that predominate above the LCST. Conversely, P3, which did not contain lipid esters, exhibited the expected increase in particle size as aggregates formed with increased temperature. Characterizing the polymers by GPC provided multimodal peaks; however, this could be due to poor behavior of trehalose glycopolymers with the mixed-D packing material, as has been previously reported (FIG. 17) (Das et al., “Aqueous RAFT Synthesis of Glycopolymers for Determination of Saccharide Structure and Concentration Effects on Amyloid P Aggregation,” Biomacromolecules, 18(10):3359- 3366 (2017); Liang et al., “Self-Association of Poly[2-(P-D-glucosyloxy)ethyl Acrylate] in Water,” J. Colloid Interface Sci., 224(l):84-90 (2000); which are hereby incorporated by reference in their entirety). As such, the reported molecular weights were those calculated from NMR. NOESY-NMR also revealed significant through-space interactions between the behenic ester methylene regions in Pl and P2 with the isopropyl substituents on the NIP AM subunits of each polymer. Interactions for the well-established trehalose clam-shell conformation (Messina et al., “Effect of Trehalose Polymer Regioisomers on Protein Stabilization,” Polym. Chem., 8(33):4781-4788 (2017), which is hereby incorporated by reference in its entirety) were also present (FIG. 3C). All of these interactions were possibly enhanced at higher temperatures, although this also significantly increased noise in the spectra, making this conclusion less definitive (FIGs. 9 and 18-20).
[0169] Following physicochemical characterization of P1-P3, performance of the glycolipid polymers as adjuvants in-vitro was determined. Due to both the established glycolipid SAR, as well as own docking experiments which indicated preservation of a critical bridging between Argl82 and Phel98 (Ryter et al., “Aryl Trehalose Derivatives as Vaccine Adjuvants for Mycobacterium tuberculosis," J. Med. Chem., 63(l):309-320 (2020), which is hereby incorporated by reference in its entirety) (FIG. 21 A), it was first hypothesized that lipidated glycomonomer 1, Pl, and P2 would each activate the Macrophage-Inducible C-type Lectin (MINGLE) receptor. To test this HEK-mMINCLE cells were used which couple downstream signaling from the activation of mMINCLE to an alkaline phosphatase output that may be quantified calorimetrically. Testing 1 on HEK-mMINCLE cells revealed statistically significant, but weak, activity at high (>100 pg/mL) concentrations indicating it was not a potent mMINCLE agonist. In contrast, Pl and P2 activated HEK-mMINCLE cells in a more potent and dosedependent manner, consistent with previous reports illustrating that glycolipid multivalency dramatically affects MINCLE binding and subsequent activation. On an equimolar basis, Pl, and particularly P2, were more potent relative to TDB positive control, although this could be due to the multivalent nature of the polymers. Indeed, when adjuvants were compared equally with respect to molar equivalents or wt% of adjuvant subunit, responses were comparable to TDB (FIG. 2 IB). In contrast, only minimal activity was observed for P3, indicating that neither the NIP AM nor trehalose blocks (or the cumulative trehalose-NIPAM combination) were significant MINCLE agonists (FIG. 10). Having confirmed that Pl and P2 were MINCLE agonists, it was next determined if temperature would affect inflammatory cytokine production. This was first tested by incubating Pl and P2 with JAWSII immune cells, a model for the inflammatory innate immune response. Here attenuated inflammatory cytokine production at elevated temperatures (FIG. 21C) was observed for both TNF-a and IL-6 indicating that Pl and P2 are thermophobic adjuvants.
[0170] Although functional production of cytokines in the positive control likely indicates that attenuated cytokine production at elevated temperatures is not from cell death, a live/dead assay across this same temperature range was also performed (FIG. 11). For a more detailed characterization of temperature-dependent cytokine production, Pl and P2 were tested in primary Bone Marrow-Derived Dendritic Cells (BMDCs) and Macrophages (BMDMs). As with JAWSII cells, cultures were dosed with adjuvant (100 pg /mL) and incubated at temperatures of 35, 37, or 39 °C, effectively traversing the previously observed LCST for P2 at this concentration (FIGs. 22A and 22B). Here decreases in inflammatory cytokine production (IL-6 and TNF-a) were again observed at increased temperatures for the thermophobic adjuvants (Pl and P2) relative to TDB, which did not activate the cells as strongly, but did exhibit a modest temperature-dependent effect with regards to TNF-a. This reduction in cytokine production was attributed to attenuated adjuvant potency, as positive LPS control demonstrated each of the primary cell types remained capable of producing inflammatory cytokines at 39°C consistent with historical reports of cell viability over 40°C (Watanabe and Okada, “Effects of Temperature on Growth Rate of Cultured Mammalian Cells (L5178Y),” J. Cell Biol., 32(2):309- 323 (1967), which is hereby incorporated by reference in its entirety). This, along with the polymer characterization data, led to the hypothesis that two possible temperature-driven mechanisms were in operation: 1) adjuvant activity could be decreased simply from the decrease in particle size that occurs with temperature as particle size is known to affect glycolipid activity (Stocker et al., “The Effects of Trehalose Glycolipid Presentation on Cytokine Production by GM-CSF Macrophages,” Glycoconj. J., 36(1):69— 78 (2019), which is hereby incorporated by reference in its entirety) or 2) activity could be attenuated due to lipid-NIPAM shielding, as observed by NOESY-NMR (FIG. 22C). While there is support for both mechanistic hypotheses, it seemed important to also know if these thermophobic adjuvants would enhance the efficacy of a vaccine. As such, with temperature-dependent potency confirmed in-vitro, the thermophobic glycolipid polymers were next examined to determine if they would perform as functional vaccine adjuvants in-vivo.
[0171] To do this, the performance of the glycolipid polymer adjuvants were evaluated in a whole inactivated Influenza A/Califomia/04/2009 virus (IIV) vaccine model (FIGs. 23 A-23H). Both Pl and P2 were tested as adjuvants alongside conventional alum and the known MINGLE agonist trehalose 6, 6‘ -dibehenate as positive controls. Seven study groups of C57BL/6 mice (n=3) were used, consisting of Pl, P2; TDB and alum (positive controls); unadjuvanted vaccine, unvaccinated, and unchallenged unvaccinated mice (negative controls). TDB, Pl, and P2 adjuvants were standardized according to the number of adjuvant subunits; 100 nmol of adjuvant subunit was used for each of these cohorts. Mice were vaccinated on day 0 and boosted on day 15, followed by influenza challenge on day 30 post vaccination. Blood samples were drawn to assess antibody response (day 14 and 29) and inflammatory cytokines (24 hours and 7 days post second vaccination). Vaccines were well tolerated by all vaccinated cohorts (FIG. 23 A) and did not result in significant changes in weight-gain. Serum samples were also assayed for IL-6 as a measure for systemic inflammation (FIG. 23B). Pl and P2 produced inflammation comparable to alum adjuvanted control but with enhanced production of lung and lymph node central memory CD4+/44+/62L+ T cells (lung and lymph node central memory CD4+/44+/62L+ T cells (FIG. 23C). Further, at 7 days post-boost, this effect of lower IL-6 production and enhanced T cell proliferation was extended, particularly for the P2 cohort relative to control adjuvants (FIGs. 23D and 23E). Following vaccination, mice were challenged intranasally with mouse-adapted live Influenza A/California/04/2009 strain virus on day 30 and monitored for weight loss over 5 days followed by euthanasia. Interestingly, weight-loss post- challenge demonstrated that Pl was not particularly protective, resulting in more than 20% weight loss which was comparable to unvaccinated and/or unadjuvanted control groups. However, adding a NIP AM block, as in P2, enhanced protection despite an apparent decrease in potency relative to Pl in-vitro. Mice vaccinated with P2 adjuvant experienced only modest (-10%) weight-loss which was comparable to TDB or alum adjuvanted controls (FIG. 23F). This also correlated with observed increases in lung and lymph node central memory CD4+/44+/62L+ T cells for the P2 vaccinated cohort, an effect consistent with vaccines adjuvanted by trehalose glycolipids (Lindenstrom et al., “Tuberculosis Subunit Vaccination Provides Long-Term Protective Immunity Characterized by Multifunctional CD4 Memory T Cells,” J. Immunol., 182(12):8047-8055 (2009), which is hereby incorporated by reference in its entirety). In a hemagglutination and neuraminidase inhibition assays, both Pl and P2 adjuvants displayed 2-3 fold increases in HA and NA inhibition relative to unadjuvanted vaccine, suggesting the production of neutralizing antibodies as one mechanism of protection that is promoted by the adjuvants, particularly P2 (FIGs. 23G and 23H).
[0172] NIPAM-trehalose glycolipid copolymers Pl and P2 were synthesized and characterized for their thermoresposive behavior and performance as thermophobic vaccine adjuvants. Consistent with other multivalent trehalose glycolipids, the thermophobic adjuvants exhibited promiscuous agonism among HEK-mMINCLE, and JAWSII innate immune cell lines indicating that, at a minimum, these polymers activate the MINCLE receptor, and likely activate other innate immune cell receptors as well. Both Pl and P2 displayed thermophobic inflammatory immunological profiles that were modulated by small (1-2 °C) changes in temperature in an innate immune cell line (JAWS II) and primary Bone Marrow-Derived Dendritic Cells and Macrophages in vitro. The molecular mechanism of action for thermophobic immune cell activation is suspected to arise from either inter- or intra- molecular changes in the polymer conformation; decreased particle size could result in attenuated activity, or this could arise from shielding interactions between the lipids, critical for activity, with hydrophobic portions of NIP AM subunits. Further structural elucidation by variable-temperature DLS revealed a decrease in particle size for Pl and a slightly decreased particle size, but variable D for P2. This coupled with NOESY-NMR spectra suggested that lipid interactions with the polymer backbone or isopropyl side-chain plays a primary role in driving the thermophobic phenomenon, however, changes in particle size likely also contribute. Interestingly, the P2 outperformed Pl as a vaccine adjuvant in-vivo even though P2 was less potent than Pl, across all cell types tested in-vitro. The superior in-vivo performance of P2 could be explained by increased particle size seen from DLS characterization or alternatively, an enhanced depoting effect, from temperature-dependent aggregation induced by the additional NIP AM block observed by bright-field microscopy (FIG. 33). Regardless, both copolymers induced less inflammation than the parent glycolipid (TDB) and comparable (in the case of Pl) or superior (in the case of P2) protective immunity. Overall, these copolymers establish the first thermophobic adjuvants that modulate their potency in response to temperature in-vitro and are effective vaccine adjuvants in-vivo. It is envisioned that the thermophobic adjuvant concept will help address the highly heterogeneous immune responses vaccine adjuvants elicit at the population-level by coupling potency to inflammation-induced pyrexia thereby personalizing adjuvant activity to an individual’s own immune response. [0173] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED IS:
1. A copolymer comprising at least one PA block and at least one PB block, wherein PA represents a polymer block comprising one or more units of monomer A and PB represents a polymer block comprising one or more units of monomer B, with said monomer A being an amide or ester, and said monomer B being a trehalose-based monomer, wherein at least one hydroxyl group on the trehalose is esterified.
2. The copolymer of claim 1, wherein the copolymer is a block copolymer.
3. The copolymer of claim 2, wherein the block copolymer comprises an architecture of PA-PB, PB-PA, PA-PB-TCTA, PA-TCTA-PB, TCTA'-PA-PB-TCTA2, or TCTAkPB-PA-TCTA2, wherein TCTA, TCTA1, and TCTA2 are moieties derived from a telechelic chain transfer agent.
4. The copolymer of claim 2, wherein the block copolymer comprises an architecture of PA-PB-PA, PA-PB-TCTA-PB, PA-PB-TCTA-PA, PA-TCTA-PB-PA, PA-PB- TCTA-PB-PA, TCTA'-PA-PB-PA-TCTA2, TCTAkPB-PA-PB-TCTA2, TCTAkPA-PB-TCTA- PB-TCTA2, TCTAkPA-PB-TCTA-PA-TCTA2, TCTA1 -PA-TCTA-PB -PA-TCT A2, or TCTA1- PA-PB-TCTA-PB-PA-TCTA2 wherein TCTA, TCTA1 and TCTA2 are moieties derived from a telechelic chain transfer agent.
5. The copolymer of any one of claims 1-4, wherein said monomer A is an acrylamide, methacrylamide, acrylate, or methacrylate.
6. The copolymer of claim 5, wherein said monomer A is selected from the group consisting of N,N-diethyl acrylamide, N,N-dimethyl acrylamide, N-isopropyl acrylamide, N- ethyl acrylamide, N-tert-butyl acrylamide, N-n-propyl acrylamide, N-methyl-N-ethyl acrylamide, and methacrylamide derivates thereof.
7. The copolymer of claim 6, wherein said monomer A is N-isopropyl acrylamide.
8. The copolymer of any one of claims 1-7, wherein the at least one PB block has a chemical structure of Formula (I):
-(L-B)m- (I), wherein
L is a linker; and m is 1 to 100,000.
Figure imgf000052_0001
9. The copolymer of claim 8, wherein L is and each / is a point of attachment of L to the at least one hydroxyl group on the trehalose.
10. The copolymer of any one of claims 1-8, wherein the trehalose-based monomer is a monomer comprising trehalose attached to a side chain of the monomer.
11. The copolymer of any one of claims 1-10, wherein said monomer B has a chemical structure of Formula (II):
Figure imgf000052_0002
wherein
R is Ci -30 alkyl.
12. The copolymer of any one of claims 1-11, wherein said monomer B has a chemical structure of Formula (Ila):
Figure imgf000053_0001
13. The copolymer of any one of claims 1-12, wherein said monomer B has a chemical structure of Formula (lib):
Figure imgf000053_0002
14. The copolymer of any one of claims 1-13, wherein the copolymer comprises a moiety selected from
Figure imgf000053_0003
wherein is the terminal group of the polymer; a is 1 to 100,000 ; b is 1 to 100,000; x is 1 to 100,000; and y is 1 to 100,000.
15. The copolymer of any one of claims 1-14, wherein the copolymer has a chemical structure selected from
Figure imgf000054_0001
a is 1 to 100,000; b is 1 to 100,000; x is 1 to 100,000; and y is 1 to 100,000.
16. The copolymer of any one of claims 1-15, wherein the copolymer has a chemical structure selected from
Figure imgf000055_0001
wherein a is 1 to 100,000; b is 1 to 100,000; x is 1 to 100,000; and y is 1 to 100,000.
17. The copolymer of any one of claims 1-16, wherein the copolymer has a chemical structure selected from
Figure imgf000055_0002
wherein a is 1 to 100,000; b is 1 to 100,000; x is 1 to 100,000; and y is 1 to 100,000.
18. A bioactive agent delivery system comprising: the copolymer according to any one of claims 1-17; and a bioactive agent.
19. The bioactive agent delivery system of claim 18, wherein the bioactive agent is selected from the group consisting of a protein, a (poly)peptide, a vaccine, a nucleic acid, a hormone, a cancer drug, an angiogenesis inhibitor, a growth factor, and an anti-microbial substance.
20. A composition comprising: the copolymer according to any one of claims 1-17; and a vaccine.
21. The composition of claim 20, wherein the vaccine is selected from an inactivated enveloped virus, whole cell, cell lysate, tumor-associated antigen, tumor-specific antigen, protein, nucleic acid, and (poly)peptide.
22. The composition of claim 21, wherein the inactivated enveloped virus is selected from the group consisting of herpesviruses, poxviruses, hepadnaviruses, asfarviridae, flavi virus, alphavirus, togavirus, coronavirus, hepatitis viruses, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, filovirus, and retroviruses.
23. The composition of claim 21, wherein the inactivated enveloped virus is selected from the group consisting of Ebola virus, human immunodeficiency virus, influenza virus, Lassa fever virus, Nipah virus, respiratory syncytial virus, Rift Valley fever virus, SARS virus, MERS virus, Marbury virus, swine pox virus, Cytomegalovirus, Crimean hemorrhagic fever virus, and COVID-19.
24. The bioactive agent delivery system of claims 18 or 19 or the composition of any one of claims 20-23 further comprising: an adjuvant, antibiotic, antiviral, pharmaceutically acceptable carrier, stabilizer, and/or preservative.
25. The bioactive agent delivery system of claims 18 or 19 or the composition of any one of claims 20-23, further comprising: an adjuvant selected from the group consisting of an aluminum salt, inulin, argamline, a combination of inulin and aluminum hydroxide, monophosphoryl lipid A (MPL), resiquimoid, muramyl dipeptide (MDP), N -Glycolyl dipeptide (GMDP, N- glycolyl dipeptide), poly IC, CpG oligonucleotide, resiquimod, aluminum hydroxide containing MPL, a water-in-oil emulsion, squalene or analogs thereof, any pharmaceutically acceptable oil, tween- 80, sorbitan trioleate, alpha-tocopherol, cholecalciferol or any analogs thereof, derivatives thereof, calcium-modified forms thereof, phosphate-modified forms thereof, and combinations thereof.
26. The bioactive agent delivery system of any one of claims 18, 19, or 25 or the composition of any one of claims 20-23 or 25, further comprising: an antibiotic selected from the group consisting of Amikacin, Amoxicillin, Amoxicillin- clavulanic acid, Amphothericin-B, Ampicillin, Ampicllin-sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin, Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin, Cefalothin, Cefazolin, Cefdinir, Cefepime, Cefixime, Cefmenoxime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefoxitin, Cefbirome, Cefpodoxime, Cefpodoxime- clavulanic acid, Cefpodoxime-sulbactam, Cefbrozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole, Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin, Clarithromycin, Clinafloxacin, Clindamycin, Cioxacillin, Colistin, Cotrimoxazol (Trimthoprim/sulphamethoxazole), Dalbavancin, Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin, Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin, Flucl oxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid, Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem, Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin, Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem, Metronidazole, Meziocillin, Mezlocillin-sulbactam, Minocycline, Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin, Penicillin V, Piperacillin, Piperacillin-sulbactam, Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfloxacin, Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazole, Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyklin, Ticarcillin, Ticarcillin- clavulanic acid, Tigecycline, Tobramycin, Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin, Voriconazole, and combinations thereof.
27. The bioactive agent delivery system of any one of claims 18, 19, 25, or 26 or the composition of any one of claims 20-23, 25, or 26, further comprising: a preservative selected from the group consisting of chlorobutanol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal, phenylmercuric nitrate, and combinations thereof.
28. The bioactive agent delivery system of any one of claims 18, 19, or 25-27 or the composition of any one of claims 20-23 or 25-27, further comprising: a pharmaceutically acceptable carrier selected from the group consisting of pyrogen-free water, isotonic saline, buffered aqueous solutions, including aqueous phosphate buffers, aqueous citrate buffers, and combinations thereof.
29. The bioactive agent delivery system of any one of claims 18, 19, or 25-28 or the composition of any one of claims 20-23 or 25-28, further comprising: a stabilizer selected from the group consisting of sorbitol, L- glycine, mannitol, L- glutamic acid, human serum albumin, and combinations thereof.
30. The bioactive agent delivery system of any one of claims 18, 19, or 25-29 or the composition of any one of claims 20-23 or 25-29, further comprising: an antiviral selected from the group consisting of zidovudine, acyclovir, gancyclovir, vidarabine, idoxuridine, trifluridine, ribavirin, foscarnet, amantadine, peramivir, rimantadine, saquinavir, indinavir, ritonavir, alpha-interferons, AZT, t-705, zanamivir, oseltamivir, influenza virus vaccines, and combinations thereof.
31. A method of vaccinating a subject against infection by an enveloped virus, said method comprising: providing a composition according to any one of claims 20-30; and treating the subject with said composition to vaccinate the subject against the enveloped virus.
32. The method of claim 31 further comprising: selecting a subject in need of vaccination against infection by an enveloped virus.
33. A method of preparing a copolymer, said method comprising: providing a radically polymerizable amide monomer A; providing a trehalose-based monomer B comprising at least one esterified hydroxyl group or a polymer block PB comprising one or more units of said monomer B; and polymerizing said monomer A with said monomer B or said polymer block PB.
34. The method of claim 33, wherein said polymerizing is carried out in a solvent at a temperature of 50 to 140 °C.
35. The method of claim 33, wherein said polymerizing is carried out via reversible addition-fragmentation chain-transfer polymerization (RAFT).
36. The method of claim 35, wherein said polymerizing is carried out in the presence of a free radical initiator and a chain transfer agent.
37. The method of claim 36, wherein the free radical initiator is selected from the group consisting of benzoyl peroxide, azobisisobutyronitrile, 1,1' azobis(cyclohexanecarbonitrile), t-butylperoxide, and 4,4’ -azobis(4-cyanoval eric acid).
38. The method of claim 36, wherein the chain transfer agent is a thiocarb onylthio compound, a dithioester compound, a trithiocarb onate compound, a dithiocarbamate compound, or a xanthate compound capable of reversible association with polymerizable free radicals.
39. The method of claim 33, wherein said providing a polymer block PB comprising one or more units of said monomer B comprises: providing said monomer B; polymerizing said monomer B in a solvent suitable for dissolving at least one of said PB block; and functionalizing the at least one of said PB block with one or more chain transfer groups or one or more initiator groups to produce a functionalized PB block.
40. The method of claim 36, wherein said chain transfer agent is a telechelic chain transfer agent, and wherein said polymerizing comprises: polymerizing said monomer A and said monomer B via RAFT to form a TCTA1- PA-PB-TCTA2 or PA-PB-TCTA-PB-PA, wherein TCTA1, TCTA2’ and TCTA are moieties derived from a telechelic chain transfer agent.
41. The method of claim 40, further comprising: providing a radically polymerizable amide, represented by A; providing a polymer TCTA^PA-PB-TCTA2 or PA-PB-TCTA-PB-PA; and polymerizing said monomer A with the polymer TCTA^PA-PB-TCTA2 or PA- PB-TCTA-PB-PA.
PCT/US2024/019138 2023-03-08 2024-03-08 Thermophobic trehalose glycopolymers as smart c-type lectin receptor vaccine adjuvants WO2024187116A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363450875P 2023-03-08 2023-03-08
US63/450,875 2023-03-08

Publications (2)

Publication Number Publication Date
WO2024187116A2 true WO2024187116A2 (en) 2024-09-12
WO2024187116A3 WO2024187116A3 (en) 2024-10-24

Family

ID=92675738

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/019138 WO2024187116A2 (en) 2023-03-08 2024-03-08 Thermophobic trehalose glycopolymers as smart c-type lectin receptor vaccine adjuvants

Country Status (1)

Country Link
WO (1) WO2024187116A2 (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2798289B1 (en) * 1999-09-15 2004-12-31 Cll Pharma QUICKLY DELITING MOUTH GALENIC FORMS AND THEIR PREPARATION METHOD
EP2807177B1 (en) * 2012-01-27 2019-11-13 The Regents of The University of California Stabilization of biomolecules using sugar polymers
ES2758985T3 (en) * 2014-08-12 2020-05-07 Univ California Trehalose hydrogels for protein stabilization and administration

Also Published As

Publication number Publication date
WO2024187116A3 (en) 2024-10-24

Similar Documents

Publication Publication Date Title
JP6704012B2 (en) Compositions containing cyclic purine dinucleotides with defined stereochemistry and methods for their preparation and use
TWI745275B (en) HUMAN iNKT CELL ACTIVATION USING GLYCOLIPIDS
US20180296663A1 (en) Vaccine composition
EP2680880B2 (en) Vaccination in newborns and infants
JP6175451B2 (en) Pharmaceutical composition comprising a polymer carrier cargo complex and at least one protein or peptide antigen
EA020962B1 (en) Compounds and compositions as tlr activity modulators
Andrianov et al. Supramolecular assembly of Toll-like receptor 7/8 agonist into multimeric water-soluble constructs enables superior immune stimulation in vitro and in vivo
JP6646056B2 (en) Branched and linear chimeric compounds, polynucleotides, uses and methods for their preparation
JP5933106B2 (en) Carbohydrate-glycolipid conjugate vaccine
JP2021512963A (en) Toll-like receptor ligand
WO2022192438A1 (en) Subunit vaccines with dinucleotide-loaded hydrogel adjuvant
WO2024187116A2 (en) Thermophobic trehalose glycopolymers as smart c-type lectin receptor vaccine adjuvants
Li et al. Enhancing COVID-19 vaccine efficacy: dual adjuvant strategies with TLR7/8 agonists and glycolipids
Hendricksen Synthesis and Performance of Stimuli-Responsive Polymeric Biomaterials for Immunological and Therapeutic Application
WO2024006915A2 (en) Methods and compositions for modulation of immune responses
WO2024064391A2 (en) Use of protein kinase c (pkc) activating compounds as an adjuvant
Van Zanten Rational design of combination influenza nanovaccines to enhance immunity in aged subjects
Keller A Modular pH-Responsive Polymer Platform for Protein-Based Vaccines
US20210401977A1 (en) Biodegradable Cationic Polycarbonates as Adjuvants for Vaccines
HK40013049A (en) Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use
Lynn Polymer carriers of toll-like receptor-7/8 agonists as vaccine adjuvants
HK1212707B (en) Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use
NZ707561B2 (en) Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use