Poly-nitrophenyl boronic acid-containing polymers

Abstract

Provided herein are polymers comprising polyethylene glycol and two or more terminal nitrophenylboronic acid groups. The polymers can additionally comprise one or more targeting molecules and / or one or more therapeutic molecules. In addition, the polymers disclosed herein can form part of a conjugate with a second polymer, and these polymer conjugates can form nanoparticles useful for various therapeutic purposes.

Description

POLY-NITROPHENYL BORONIC ACID-CONTAINING POLYMERSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The current application claims priority to U.S. Prov. Appl. No. 63 / 588,185, filed Oct. 5, 2023, which is incorporated herein by reference in its entirety.FIELD

[0002] The present disclosure is in the field of polymers, and nanoparticles comprising said polymers, for use in delivery of biological agents; for example, therapeutic molecules.BACKGROUND

[0003] Nanoparticles formed from various types of polymer have enjoyed success as delivery vehicles for therapeutics, improving the pharmacokinetics of the therapeutic and enabling delivery of therapeutic molecules across the blood-brain barrier. See, for example, US Patent Application Publication No. 2021 / 0220483 and US Patent No. 10,182,986.

[0004] In addition to containing therapeutic agents, such as small molecule chemotherapeutics, such nanoparticles also often contain targeting agents (or targeting molecules) to direct the nanoparticle, and its therapeutic payload, to a target cell. Such nanoparticles also can contain large-molecule therapeutic agents, that work in concert with the small molecule for more effective treatment.SUMMARY

[0005] The present disclosure provides polyethylene glycol (PEG)-containing polymers, useful, e.g., in the preparation of therapeutic nanoparticles, that also contain two or more nitrophenyl boronic acid (NPBA) moieties. The NPBA-PEG polymers disclosed herein can also optionally contain a targeting agent, that is covalently conjugated to the PEG-containing end of the NPBA-PEG polymer. The NPBA-PEG polymers disclosed herein can also optionally contain a therapeutic agent {e.g., an antibody), that is covalently conjugated to the PEG-containing end of the NPBA-PEG polymer. Additionally, the NPBA-PEG polymers disclosed herein, with or without a targeting agent and / or a therapeutic agent, can form conjugates with a second polymercontaining vicinal diol residues (c. ., a mucic acid-containing polymer) through a reversible (pH- sensitive) covalent complexation between NPBA group(s) of the NPBA-PEG polymer and the vicinal diols present in the second polymer.

[0006] In certain aspects, the invention provides polyethylene glycol (PEG) polymers containing one or more nitrophenylboronic acid (NPBA) moieties. For example, a NPBA-PEG polymer can have a structure as shown in Formula III, in which the NPBA portion of the polymer is to the left of the spacer in the formula, and the PEG portion of the polymer is to the right of the spacer:wherein:L3 is a linking group, or linking groups, between the one or more boronic acid moieties and the spacer;L4 is a linking group, or linking groups, between the spacer and the one or more polyethylene oxide linkages;Spacer is a moiety containing at least one terminal amine and / or at least one terminal carboxylic acid; q is independently at each occurrence 0, 1, 2, 3, or 4; r is 1, 2, or 3; s is independently at each occurrence 20-1200; t is 1, 2, or 3; andXi is a terminal functional group.

[0007] In certain embodiments, Xi is either of:(a) -C1-6 alkyl, optionally substituted with -OH, -COOH, -C(=O)O(alkyl), - C(=O)O(aryl), -NH2, -NH(alkyl), or N(alkyl)2; or(b) a salt or protected analog of any of the groups of (a); orXi comprises a targeting molecule or a therapeutic molecule. In certain embodiments, a targeting molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) above, such that Xi has the structure -C-C(=O)-NH-targeting molecule. In additionalembodiments, a therapeutic molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) above, such that Xi has the structure -C-C(=O)-NH-therapeutic molecule.

[0008] In certain embodiments, the spacer is an amino acid. In a preferred embodiment, q is 1, r is 2, s is 50-350, t is 1, L3 is -C(=O)-NH-, Lj is -C(=O)-NH-, Xi is -CH3, and the spacer is a lysyl group. In an additional embodiment, q is 1, r is 2, s is 50-350, t is 1, L3 is -C(=O)-NH-, L4 is -C(=O)-NH-, Xi is -C-C(=O)-OH, and the spacer is a lysyl group.

[0009] In certain embodiments, L3 and L4 linkers shown in Formula III can be any organic moiety suitable for the purpose. For example, L3 and L4 can be independently, at each occurrence, one or more of the following groups:-(Co-6alkylene)-NH-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-NH-(Co-6alkylene)-, -(Co-6alkylene)-0-C(=0)-(Co-6alkylene)-, -(Co-6alkylene)-C(=0)-0-(Co-6alkylene)-, -(Co-6alkylene)-NH-C(=0)-0-(Co-6alkylene)-, or -(Co-6alkylene)-0-C(=0)-NH-(Co-6alkylene)-.

[0010] Additional linking groups include, for example, amides, esters, carbamates, thioesters, disulfides, anhydrides and / or ethers.

[0011] In certain embodiments, the spacer is an organic group bearing at least one terminal amine and at least one terminal carboxylic acid.

[0012] In certain embodiments, the spacer has the formula -NH-CH-Ri-C(=O)-, wherein Ri is an amino acid functional group.

[0013] In certain embodiments,(a) Ri is the functional group of lysine;(b) L3 is -C(=O)-NH-; and(c) L4is -C(=O)-NH-.

[0014] In certain embodiments: the spacer is a lysyl group; q is 1;s is 50-300; t is 1; for the first instance of r, L3 is -C(=0)-NH-; for the second instance of r, L3 is -C(=0)-NH-;L4is -C(=0)-NH-; andXi is -CH3or -C-C(=O)-OH.

[0015] In certain embodiments, the polymer further comprises one or more targeting molecules.

[0016] In certain embodiments, the targeting molecule is selected from the group consisting of a protein, a peptide, an aptamer, and a ligand for a cellular receptor. In certain embodiments, the protein is an antibody or an antibody fragment. In certain embodiments, the antibody or antibody fragment is directed to CD3, CD4, CD8, the epidermal growth factor receptor (EGFR) or the transferrin receptor (TfR). In certain embodiments, the cellular receptor is CD3, CD4, CD8, the epidermal growth factor receptor (EGFR) or the transferrin receptor (TfR). In certain embodiments, the ligand for a cellular receptor is transferrin.

[0017] In certain embodiments, the targeting molecule is a covalent conjugate of Xi, such that Xi has the structure -C-C(=O)-NH-targeting molecule. In certain embodiments, the composition further comprises a therapeutic molecule. In certain embodiments, the therapeutic molecule is a covalent conjugate of Xi, such that Xi has the structure -C-C(=O)-NH-therapeutic molecule.

[0018] In certain embodiments, the therapeutic molecule is an antibody. In certain embodiments, the antibody is herceptin. In certain embodiments, the invention provides a polymer conjugate comprising the polymer of Formula (III), and a second polymer containing at least one set of vicinal diols.

[0019] In certain embodiments, the polymer conjugate comprises a plurality of the polymers of Formula (III). In certain embodiments, the conjugate comprises a plurality of molecules of Formula III, wherein:(a) in at least one of the polymers of claim 1, Xi is -CH3; and(b) in at least one of the polymers of claim 1, Xi comprises a targeting agent.

[0020] In certain embodiments, the targeting agent is an anti-CD8 antibody.

[0021] In certain embodiments, the invention provides a nanoparticle comprising(a) the polymer of any of claims 1, 10, 15, 18 or 21; or(b) the polymer conjugate of any of claims 22-25.

[0022] In certain embodiments, the invention provides a pharmaceutical composition comprising the nanoparticles provided above and a pharmaceutically acceptable carrier or excipient.

[0023] In certain embodiments, the invention provides a method for the treatment of a disease or disorder in a subject, the method comprising administering the pharmaceutical composition provided above to the subject. In certain embodiments, the disease or disorder is a malignancy. In certain embodiments, the malignancy is selected from the group consisting of a hematologic malignancy and a solid tumor. In certain embodiments, the hematologic malignancy is a B-cell malignancy. In certain embodiments, the B-cell malignancy is a leukemia or a lymphoma. In certain embodiments, the solid tumor is a breast tumor, a lung tumor, an ovarian tumor, a gastric tumor, a colorectal tumor, a pancreatic tumor, a primary brain tumor or a brain metastasis.DETAILED DESCRIPTION

[0024] The present disclosure may be understood more readily by reference to the following description taken in connection with the accompanying Examples, all of which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific products, methods, conditions or parameters described or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of any claimed invention. Similarly, unless specifically otherwise stated, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosure herein is not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement. Throughout this text, it is recognized that the descriptions refer to compositions and methods of making and using said compositions. That is, where the disclosure describes or claims a feature or embodiment associated with a composition or a method of making or using a composition, it isappreciated that such a description or claim is intended to extend these features or embodiment to embodiments in each of these contexts (i.e., compositions, methods of making, and methods of using).

[0025] In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a material” is a reference to at least one of such materials and equivalents thereof known to those skilled in the art, and so forth.

[0026] When a value is expressed as an approximation by use of the descriptor “about,” it will be understood that the particular value forms another embodiment. In general, use of the term “about” indicates approximations that can vary depending on the desired properties sought to be obtained by the disclosed subject matter and is to be interpreted in the specific context in which it is used, based on its function. The person skilled in the art will be able to interpret this as a matter of routine. In some cases, the number of significant figures used for a particular value may be one non-limiting method of determining the extent of the word “about.” In other cases, the gradations used in a series of values may be used to determine the intended range available to the term “about” for each value. Where present, all ranges are inclusive and combinable. That is, references to values stated in ranges include every value within that range.

[0027] It is to be appreciated that certain features of the disclosure which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. That is, unless obviously incompatible or specifically excluded, each individual embodiment is deemed to be combinable with any other embodiment(s) and such a combination is considered to be another embodiment. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. Finally, while an embodiment may be described as part of a series of steps or part of a more general structure, each said step may also be considered an independent embodiment in itself, combinable with others.

[0028] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.” Likewise, a termsuch as Cl -3 alkyl also includes, as separate embodiments, Cl alkyl, C2 alkyl, C3 alkyl, Cl -2 alkyl, and C2-3 alkyl.

[0029] Reference to alcohols, aldehydes, amines, carboxylic acids, ketones, or other similarly reactive functional groups also includes their protected analogs. For example, reference to hydroxy or alcohol also includes those substituents wherein the hydroxy is protected by acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), P-Methoxyethoxymethyl ether (MEM), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT), methoxymethyl ether (MOM), methoxytrityl [(4- methoxyphenyl)diphenylmethyl, MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ether (most popular ones include trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso- propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers), ethoxyethyl ethers (EE). Reference to amines also includes those substituents wherein the amine is protected by a BOC glycine, carbobenzyloxy (Cbz), p-methoxybenzyl carbonyl (Moz or MeOZ), tertbutyloxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p- methoxyphenyl (PMP), tosyl (Ts) group, or sulfonamide (Nosyl & Nps) group. Reference to substituent containing a carbonyl group also includes those substituents wherein the carbonyl is protected by an acetal or ketal, acylal, or diathane group. Reference to substituent containing a carboxylic acid or carboxylate group also includes those substituents wherein the carboxylic acid or carboxylate group is protected by its methyl ester, benzyl ester, tert-butyl ester, an ester of 2,6- disubstituted phenol (e g. 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol), a silyl ester, an orthoester, or an oxazoline.Abbreviations

[0030] The following abbreviations and acronyms may be used in this disclosure:Ab: antibodyACN: acetonitrileARS: alizarin red SAsp: aspartate, aspartic acidBBB: blood-brain barrierBoc: tert-butyloxycarbonylBoc-Lys(Boc)-OSu: bis-Boc-L-lysine-N-hydroxysuccinimide esterBom: benzyloxymethylBTB: blood-tumor barrierCAB: cabazitaxelCAR: chimeric antigen receptorCbz: carboxybenzylCPME: cyclopentyl methyl etherCPT: camptothecinC V : column volume cMAP: cationic mucic acid polymer, cationic mucic acid-containing polymer cP: cationic polymerDa: daltonDCM: dichloromethane, methylene chlorideDF: diafiltrationDIC: N,NEdiisopropylcarbodiimideDIPEA: N,NEdiisopropylethy amine diSPA-PEG: di(succinimidyl propionate)-PEGDLS: dynamic light scatteringDMA: dimethylacetamideDMAP: 4-dimethylaminopyridineDMF: N,N-dimethyl -formamideDMSO: dimethyl sulfoxideDnp: 2,4-dinitrophenylEDC: l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlorideEEDQ: N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinolineEGFR: epidermal growth factor receptorESI: electrospray ionizationEtOAc: ethyl acetateFPLC: fast protein liquid chromatography g: gramGABA: gamma-amino butyric acidGly: glycine, glycylGPC: gel permeation chromatography h: hourHBSS: Hank’s balanced salt solutionHC1: hydrochloric acidHEPES: N-2-hydroxyethylpiperazine-NBZ-ethanesulfbnic acidHex: hexanoic acid, w-caproic acidHIC: hydrophobic interaction chromatographyHOPO: hydroxypyridine-N-oxideHPLC: high-performance liquid chromatographyHPLC-UV: HPLC with ultraviolet absorbance detectionIm: imidazole, imidazoliumIm-cMAP-Im: cationic mucic acid flanked by imidazole moietiesIm.Na: 4-imidazoleacetic acid, sodium salt hydrateIPA: isopropyl alcohol kDa: kilodaltonL: literLAR: linker-to-antibody ratioMAM: polymerizable mucic acid monomerMAP: mucic acid polymerMeOH: methanolMES: 2-(N-morpholino)ethanesulfonic acid, 2-ethanesulfonic acid mg: milligram mL: milliliter mmol: millimoleMmt: monomethoxytrityl mol: mole mPEG: methoxy polyethylene glycol mRNA: messenger ribonucleic acidMTBE: methyl tert-butyl etherMtt: 4-methyltritylMWCO: molecular weight cut-offN-Boc diamine: N-Boc-ethylenediamineNHS: N-hydroxy succinimideNMM: N-methylmorpholineNP: nanoparticleNPBA: 3-carboxy-5-nitrophenylboronic acid, nitrophenylboronic acidPAA: poly(amino acid)PAMAM: poly(amidoamine)PBAE: poly(p-amino ester)PBS: phosphate-buffered salinePd / C: palladium on carbon catalystPEG: polyethylene glycolPEGm: methoxy polyethylene glycolPent: pentanoic acid; w-valeric acidPEG: polyethylene oxidePES: polyethersulfonePEI: polyethyleneiminePFP: pentaflurorphenyl(PFPO)2O: bis(pentafluorophenyl) carbonatePrOAc: isopropyl acetatePVDF: poly vinylidene fluoride, polyvinylidene difluoridePyAOP: (7-azabenzotriazol- 1 -yloxy)tripyrrolidinophosphonium hexafluorophosphateRNA: ribonucleic acid siRNA: short interfering ribonucleic acid, silencing ribonucleic acidSN38: 7-ethyl-10-hydroxy-camptothecinSpP: strands per particleTBDPS: tert-butyl diphenylsilane; tert-butyldiphenylsilylTBDP SCI: tert-butly 1 (chi oro)di phenyl sil aneTEA: triethylamineTf: holo-transferrinTfR: transferrin receptorTFA: trifluoroacetate, trifluoroacetic acidTHF : tetrahydrofuranTIPS: triisopropyl silaneTras: trastuzumab (Herceptin)Trt: tritylUF / DF: ultrafiltration & diafiltration pg: microgram uL: microliter pmol: micromoleUV: ultravioletVai: valine, valylReaction components

[0031] The procedures described herein include standard solvents and catalysts as are known in the art. Although particular compounds (e.g., acids, bases, coupling agents) are recited in the disclosure, it is clear to one of skill in the art that different reagents can be used.

[0032] To that end, exemplary solvents include but are not limited to chlorinated solvents (e.g., dichloromethane, chloroform, 1,2-di chloroethane), ethers (e.g., diethyl ether, tert-butyl methyl ether, cyclopentyl methyl ether, diglyme, 1,4-dioxane, 2-m ethyltetrahydrofuran), alcohols (e g., methanol, ethanol, isopropanol, tert-butanol), alkanes (e.g., pentane, hexanes, heptanes), glycols (e.g., ethylene glycol, polyethylene glycol), polar aprotic solvents (e.g., dimethylacetamide, acetonitrile, dimethyl sulfoxide, dimethyl formamide, acetone, N-methyl-2- pyrrolidone, ), and polar protic solvents (e.g., water, ethanol, acetic acid, propionic acid).

[0033] Exemplary acids include but are not limited to mineral acids (e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid) and organic acids (e.g., acetic acid, malonic acid, methanesulfonic acid, propionic acid, thioacetic acid, p-toluenesulfonic acid, tribromoacetic acid, trichloroacetic acid, trifluoroacetic acid).

[0034] Exemplary bases include but are not limited to amino bases (e.g. 1,4- diazabicyclo[2.2.2]octane, diethylamine, triethylamine, N,N-diisopropylethylamine, lithium amide, lithium bis(trimethylsilyl)amide, morpholine, piperidine), alkoxides (e.g., barium tert- butoxide, lithium tert-butoxide, sodium methoxide), hydroxides (e.g., tetrabutylammoniumhydroxide, sodium hydroxide, potassium hydroxide), organometallic bases (e.g., n-butyllithium, tert-butyllithium, butyl magnesium chloride), pyridines (e g., 4-dimethylaminopyridine, 2,6- lutidine, pyridine), carbonates (e.g., lithium carbonate, sodium carbonate, magnesium carbonate, potassium carbonate), and hydrides (e.g., sodium hydride, calcium hydride, potassium hydride).

[0035] Exemplary coupling agents include but are not limited to carbodiimide reagents (e.g., N,NEdiisopropylcarbodiimide, 1 -(3 -dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride, N,Ntdicyclopentylcarbodiimide), additives for carbodiimide reagents (e.g., 1- hydroxy-7-azabenzotriazole, 6-chl oro-1 -hydroxy benzotri azole, N-hydroxysuccinimide, 1- hydroxy-2-pyridinone, 6-chloro-N-hydroxy-2-phenylbenzimidazole, ethyl 2-cyano-2- (hydroxyimino)acetate), anhydride based or forming reagents (e.g., ditertbutyl carbonate, acetic anhydride, 2-ethoxy-l -ethoxy carbonyl- 1,2-dihydroquinoline, ethyl chloroformate), acylazoles (e.g., carbonyl diimidazole), acid halide generating reagents (e.g., thionyl chloride, phosgene, cyanuric chloride, benzyltriphenylphosphonium dihydrogen trifluoride), phosphonium salt coupling reagents (e.g., benzotriazol- l-yloxytris(diemthylamino)phosphonium hexafluorophosphate), tetramethyl aminium reagents (e.g., 2-(2-oxo-l(2H)-pyridyl-l,l,3,3- tetramethyluronium tetrafluoroborate), aminium reagents (e.g., 2-chloro-l,3- dimethylimidazolidinium hexafluorophosphate), oxyma uranium salts (e.g., l-((l-cyano-2- ethoxy-2-oxoethylideneaminooxy)(morpholino)methylene)pyrrolidinium hexafluorophosphate), antimonate uranium salts (e.g., benzotriazol-l-yloxuy-N,N-dimethyl-methaniminium hexachloroantimonate), organophosphorus reagents (e.g., diethylcyanophosphonate, 1 -oxochlorophospholane, 2-propanephosphonic acid anhydride), triazine based reagents (e.g., 2-chloro- 4,6-dimethoxy-l,3,5-triazine), organosulfur reagents (e.g., pentafluorophenyl-4- nitrobenzenesulfonate), pyridinium reagents (e.g., 2-chloro-l -methylpyridinium iodide), polymer bound reagents (e.g., polymer supported N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline).Nitrophenylboronic acid (NPBA)-containing polyethylene glycol (PEG) polymers

[0036] Disclosed herein are polyethylene glycol (PEG) polymers containing one or more nitrophenylboronic acid (NPBA) moieties. For example, a NPBA-PEG polymer can have a structure as shown in Formula III, in which the NPBA portion of the polymer is to the left of the spacer in the formula, and the PEG portion of the polymer is to the right of the spacer:wherein:L3 is a linking group, or linking groups, between the one or more boronic acid moieties and the spacer;L4 is a linking group, or linking groups, between the spacer and the one or more polyethylene oxide linkages;Spacer is a moiety containing at least one terminal amine and / or at least one terminal carboxylic acid; q is independently at each occurrence 0, 1, 2, 3, or 4; r is 1, 2, or 3; s is independently at each occurrence 20-1200; t is 1, 2, or 3; andXi is a terminal functional group.

[0037] In certain embodiments, Xi is either of:(a) -C1-6 alkyl, optionally substituted with -OH, -COOH, -C(=O)O(alkyl), - C(=O)O(aryl), -NH2, -NH(alkyl), or N(alkyl)2; or(b) a salt or protected analog of any of the groups of (a); orXi comprises a targeting molecule or a therapeutic molecule. In certain embodiments, a targeting molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) above, such thatXi has the structure -C-C(=O)-NH-targeting molecule. In additional embodiments, a therapeutic molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) above, such that Xi has the structure -C-C(=O)-NH-therapeutic molecule.

[0038] In certain embodiments, the spacer is an amino acid. In a preferred embodiment, q is 1, r is 2, s is 50-350, t is 1, L3 is -C(=O)-NH-, L4 is -C(=O)-NH-, Xi is -CH3, and the spacer is a lysyl group. In an additional embodiment, q is 1, r is 2, s is 50-350, t is 1, L3 is -C(=O)-NH-, L4 is -C(=O)-NH-, Xi is -C-C(=O)-OH, and the spacer is a lysyl group.

[0039] A NPBA-PEG polymer can be synthesized, for example, by combining an amine- functionalized PEG (e.g., PEG2k, PEG3.5k, PEG5k, PEGlOk) with a carboxy NPBA to form a NPBA-PEG acetic acid (AA) conjugate joined by an amide linkage. In certain embodiments, the PEG derivative contains an amino group at one terminus (for reaction with NPBA) and a carboxyl group at the opposite terminus (which can, in a subsequent reaction, be activated to react with a macromolecule such a polypeptide).3-carboxy-5-nitrophenylboronic acid(NPBA)

[0040] In certain embodiments, n is between 2 and 2,000 or any integral values therebetween; e.g., between 100 and 300, between 20 and 300, between 120 and 180, or between 140 and 160.

[0041] In certain embodiments, the value of n is such that the PEG (i.e. -CH2-CH2-O-) portion of the compound has a weight average molecular weight of between about 2 and about 15 kDa; e.g., about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.

[0042] The NPBA-PEG-AA conjugate is activated, for reaction with a polypeptide (such as, for example, transferrin or an antibody), by forming a pentafluorophenyl (PFP) derivative. In these embodiments, bis(pentafluorophenyl) carbonate is combined with a NPBA-PEG-AA conjugate to form a NPBA-PEG-pentafluorophenyl ester (e g., NPBA-PEG5k-AA-PFP).

[0043] In certain embodiments, n is between 2 and 2,000 or any integral values therebetween; e.g., between 100 and 300, between 20 and 300, between 120 and 180, and / or between 140 and 160.

[0044] In certain embodiments, the value of n is such that the PEG (i.e. -CH2-CH2-O-) portion of the compound has a weight average molecular weight of between about 2 and about 1 kDa; e.g., about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.

[0045] The NPBA-PEG-pentafluorophenyl ester is then reacted with a targeting molecule or with a therapeutic molecule (or any other polypeptide) to form a NPBA-PEG polymer with a covalently bound-targeting molecule or a NPBA-PEG polymer with a covalently bound therapeutic molecule.Linking groups in NPBA-PEG Polymers

[0046] The J and L4 linkers shown in Formula III can be any organic moiety suitable for the purpose. For example, L3 and L4 can be independently, at each occurrence, one or more of the following groups:-(Co-6alkylene)-NH-C(=0)-(Co-6alkylene)-, -(Co-6alkylene)-C(=0)-NH-(Co-6alkylene)-, -(Co-6alkylene)-0-C(=0)-(Co-6alkylene)-, -(Co-6alkylene)-C(=0)-0-(Co-6alkylene)-, -(Co-6alkylene)-NH-C(=0)-0-(Co-6alkylene)-, or -(Co-6alkylene)-0-C(=0)-NH-(Co-6alkylene)-.Additional linking groups include, for example, amides, esters, carbamates, thioesters, disulfides, anhydrides and / or ethers.Spacers in NPBA-PEG Polymers

[0047] The spacer moiety shown in Formula III can be any organic moiety suitable for the purpose. In certain embodiments, the spacer is an organic group bearing at least one terminal amine and at least one terminal carboxylic acid. In additional embodiments, an amino acid, orportion thereof, is used as a spacer molecule in the NPBA-PEG polymers disclosed herein. The twenty essential amino acids have the general structure:in which Ri is a functional group that is specific to each particular amino acid. Functional groups for the twenty essential amino acids are given in Table 1. Chemical structures for a number of non-essential amino acids are given in Table 2.Table 1: Essential amino acid functional groupsLegend: The left column provides the name of the amino acid and its three-letter and one-letter abbreviations. The right column shows the chemical structure of the functional group (Ri) for each ammo acid, except for proline* For proline, the structure of the entire amino acid is shown, since Ri cannot be represented independently.Table 2: Non-essential amino acids and their chemical structures

[0048] In certain embodiments, lysine is used as a spacer for attaching two NPB A moi eties to a PEG-containing polymer, as shown in Formula IV below:wherein: s is 20-1200; andXi is(a) -Ci-6 alkyl, optionally substituted with -OH, -COOH, -C(=O)O(alkyl), - C(=O)O(aryl), -NH2, -NH(alkyl), or N(alkyl)2; or(b) a salt or protected analog of any of the groups of (a); orXi comprises a targeting molecule or a therapeutic molecule. In certain embodiments, a targeting molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) above, such that Xi has the structure -C-C(=O)-NH-targeting molecule. In additional embodiments, a therapeutic molecule (as described elsewhere herein) is a covalent conjugate ofone of the functional groups of (a) above, such that Xi has the structure -C-C(=O)-NH-therapeutic molecule.

[0049] Additional moieties that can be used as spacers include y-shaped polyethylene glycol groups, branched polyethylene glycol groups, and branched polyethyleneimine groups.Terminal functional groups in NPBA-PEG Polymers

[0050] The terminal functional group of a NPBA-PEG polymer (denoted Xi in Formulas III and IV) can be any moiety suitable for the purpose. In certain embodiments, Xi comprises a reactive group, such as an amine, an alcohol or a carboxylic acid. In additional embodiments, Xi is -CH3. Additional terminal groups include, for example, -C(i-6) alkyl; optionally substituted with -OH, -COOH, -C(=O)-O-(alkyl), -C(=O)-O-(aryl), -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl) or - N(aryl2).

[0051] In additional embodiments, Xi comprises a targeting agent or targeting molecule, as described elsewhere in this disclosure A targeting molecule is generally attached to a NPBA- PEG polymer, in which Xi contains a reactive group, by conversion of the reactive group (e.g, - COOH) to an activated derivative (e.g, pentafluorophenyl (PFP) ester), and reaction of the activated derivative with the targeting molecule. The exemplary PFP ester moiety is known to react with amino groups in proteins, particularly antibodies. Pham, G.H et al. (2018) ChemBioChem 19:790-804.

[0052] In further embodiments, Xi comprises a therapeutic agent, as described elsewhere in this disclosure. A therapeutic agent is generally attached to a NPBA-PEG polymer, in which Xi contains a reactive group, by conversion of the reactive group (e.g, -COOH) to an activated derivative (e.g, pentafluorophenyl (PFP) ester), and reaction of the activated derivative with the targeting agent. The exemplary PFP ester moiety is known to react with amino groups in proteins, particularly antibodies. Pham, G.H e / a / . (2018) ChemBioChem 19:790-804.

[0053] A NPBA-PEG polymer comprising two NPBA moieties, a lysine spacer and a - COOH reactive group ((NPBA)2-Lys-PEG-COOH) is activated, for reaction with a targeting agent (such as, for example, an antibody) or with a therapeutic agent (such as, for example, an antibody), by forming a PFP ester derivative of the NPBA-PEG polymer. In these embodiments, bis(pentafluorophenyl) carbonate is combined with a (NPBA)2-Lys-PEG-COOH polymer to form a NPBA-PEG-pentafluorophenyl ester (c.g, (NPBA)2-Lys-PEG-COO-PFP).1 DCMIn certain embodiments, s is between 2 and 1,200 or any integral values therebetween; e.g., between 100 and 300, between 20 and 300, between 120 and 180, and / or between 140 and 160.

[0054] In certain embodiments, the value of s is such that the PEG (i.e. -CH2-CH2-O-) portion of the compound has a number average molecular weight of between about 2 kDa and about 15 kDa; e.g., about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.

[0055] The NPBA-PEG-pentafluorophenyl ester is then reacted with the targeting agent or with the therapeutic agent to form a NPBA-PEG polymer with a covalently bound-targeting agent or a NPBA-PEG polymer with a covalently bound therapeutic agent.Targeting molecules

[0056] In certain embodiments, a targeting molecule is covalently coupled to a NPBA- PEG polymer. For example, a targeting molecule can be part of the Xi moiety of the NPBA-PEG polymer, e.g., as a covalent conjugate of one of the functional groups that are specified for Xi elsewhere herein. In certain embodiments, a targeting molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) in the description of Formula III above, such that Xi has the structure -C-C(=O)-NH-targeting molecule.

[0057] Targeting molecules are known in the art and include, for example, small molecules (e.g., vitamins, e.g., folate), saccharides (e.g, mannose, allose, altrose, glucose, gulose, idose, galactose, talose, disaccharides, trisaccharides, oligosaccharides), peptides (e.g., RGD), polypeptides (e.g, antibodies, proteins that bind cell-surface receptors such as transferrin), nucleic acids (e.g., aptamers), peptoids and peptide nucleic acids (PNAs).

[0058] For example, NPBA-PEG polymers can comprise the targeting molecule transferrin, which targets the polymer or polymer conjugate (or a nanoparticle formed from the polymer or the polymer conjugate) to endothelial cells of the blood-brain barrier or the bloodtumor barrier. In these embodiments, the targeting molecule is attached to a NPBA-PEG polymer by way of reaction with a PFP-activated ester, as described elsewhere herein. Inasmuch as primary amine groups in proteins react with the PFP-activated ester, attachment of a polypeptide (such as, for example, holo-transferrin) to the NPBA-PEG-pentafluorophenyl ester is achieved by combining the ester with holo-transferrin. Progress of the reaction is monitored by high- performance liquid chromatography (HPLC), and the monoPEGylated transferrin conjugate is purified by hydrophobic interaction chromatography (HIC), based on the differential binding of monoPEGylated transferrin compared to species with higher degrees of PEGylation ( .g., diPEGylated and triPEGylated species). This method prevents loss of iron from transferrin during purification, as occurred using previous methods, thereby preserving the holo-transferrin structure.

[0059] In additional embodiments, a targeting molecule is a protein, a peptide, an aptamer, or a ligand for a cellular receptor. In certain embodiments, the cellular receptor is CD3, CD4, CD8, the epidermal growth factor receptor (EGFR) or the transferrin receptor (TfR).

[0060] A targeting molecule that is a protein can be, for example, an antibody or fragment thereof. In certain embodiments, a targeting molecule is an antibody directed to CD3, an antibody directed to CD4, an antibody directed to CD8, an antibody directed to the epidermal growth factor receptor (EGFR) or an antibody directed to the TfR.

[0061] In further embodiments, for targeting to a tumor cell, one or more small molecule targeting molecules can be used. These include, but are not limited to, carbohydrates (such as, for example, fructose, glucose, mannose, levan; or a derivative thereof), vitamins (such as, for example, riboflavin, biotin, thiamine, vitamin B12, and folic acid), and amino acids (such as, for example, glycine, alanine, arginine lysine and tryptophan). Additional small molecule targeting agents include adenosine, anisamide, anacardic acid, phenylboronic acid and methotrexate. See, for example, Kaur et al. (2023) J. Controlled Release 355:417-433, incorporated by reference in its entirety.

[0062] Additional targeting molecules are disclosed in co-owned US provisional patent application No. 63 / 588,162, entitled “Cationic Polymers and Polymer Conjugates for Delivery ofBiological Agents” the disclosure of which is incorporated by reference in its entirety for all purposes.Therapeutic Molecules

[0063] In certain embodiments, NPBA-PEG polymers, as described herein, comprise a therapeutic molecule (e.g., a large molecule therapeutic). For example, a therapeutic molecule can be part of the Xi moiety of the NPBA-PEG polymer, e.g., as a covalent conjugate of one of the functional groups that are specified for Xi elsewhere herein. In certain embodiments, a therapeutic molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) in the description of Formula III above, such that Xi has the structure -C-C(=O)-NH- targeting molecule.

[0064] Large molecule therapeutics are known in the art and include, for example, therapeutic polypeptides (e.g., antibodies, enzymes, and bioactive proteins) and nucleic acids.

[0065] Exemplary therapeutic polypeptides include, but are not limited to, antibodies such as trastuzumab (Herceptin, an anti-Her2 antibody), anti-Her3, anti-Trop2, anti-PSMA, anti-LIV- 1, anti-FOLRl, anti-DLL3, anti-PDGF, anti-FRalpha, anti-PTK7, anti-mesothelin, anti-c-MET, anti-MUCl, anti-CD70, anti-CD74, anti-CD30, anti-CD33, anti-FLT3, anti-CD22, anti-CD20, anti-CD19, anti-CD4, anti-CD8, and antibodies directed againstB7-H3, B7-H4, Nectin-4, Claudin 18.2, R0R1, ROR2, BCMA, EpCAM, Claudin 6, CDH6, CEACAM5, and Integrin beta-6.

[0066] Certain polypeptide therapeutics, such as antibodies directed to tumor markers like those exemplified in the previous paragraph, are also used at targeting molecules.

[0067] For example, NPBA-PEG polymers (and nanoparticles made therefrom) can comprise the anti-Her2 antibody trastuzumab (also known as Herceptin). In these embodiments, the antibody is attached to a NPBA-PEG polymer by way of reaction with a PFP -activated ester, as described elsewhere in this disclosure.

[0068] Inasmuch as the primary amine groups in proteins react with PFP- activated esters, attachment of trastuzumab to the NPBA-PEG-pentafluorophenyl ester is achieved by reacting the NPBA-PEG-pentafluorophenyl ester with trastuzumab. Progress of the reaction is monitored by HPLC and the monoPEGylated trastuzumab conjugate is purified by HIC, based on the differential binding of monoPEGylated trastuzumab compared to species containing higher degrees of PEGylation (e.g., diPEGylated and triPEGylated species).

[0069] A therapeutic molecule can be a nucleic acid such as, for example an antisense oligonucleotide, a mRNA, a siRNA, a miRNA, an antisense RNA; or a DNA or RNA molecule used for CRISPR gene editing, such as a guide RNA, a tracr RNA or a DNA or RNA encoding the Cas9 nuclease.

[0070] Additional therapeutic molecules are disclosed in co-owned US provisional patent application No. 63 / 588,162, entitled “Cationic Polymers and Polymer Conjugates for Delivery of Biological Agents”; the disclosure of which is incorporated by reference in its entirety for all purposes.Polymer conjugates

[0071] In certain embodiments, a NPBA-PEG polymer, with or without an attached targeting molecule or therapeutic molecule, forms a conjugate with a second polymer containing vicinal diol residues; with the NPBA-PEG polymer being bound to the second polymer through a reversible (pH-sensitive) covalent complexation between NPBA group(s) of the NPBA-PEG polymer and vicinal diols present in the second polymer.

[0072] In certain embodiments, conjugates of a NPBA-PEG polymer and any of the polymers described in US Patent No. 10,342,879 are provided. In certain of these embodiments, a plurality of NPBA-PEG polymers are conjugated to a single second polymer. In conjugates of one or more NPBA-PEG polymers and a second polymer, the NPBA-PEG polymer can contain a targeting molecule, a therapeutic molecule, neither or both.

[0073] In additional embodiments, conjugates of a NPBA-PEG polymer and any of the polymers described in US Patent No. 9,446,149 are provided. In certain of these embodiments, a plurality of NPBA-PEG polymers are conjugated to a single second polymer. In conjugates of one or more NPBA-PEG polymers and a second polymer, the NPBA-PEG polymer can contain a targeting molecule, a therapeutic molecule, neither or both.

[0074] In additional embodiments, conjugates of a NPBA-PEG polymer and any of the polymers described in US Patent No. 11,285,212 are provided. In certain of these embodiments, a plurality of NPBA-PEG polymers are conjugated to a single second polymer. In conjugates of one or more NPBA-PEG polymers and a second polymer, the NPBA-PEG polymer can contain a targeting molecule, a therapeutic molecule, neither or both.

[0075] In additional embodiments, conjugates of a NPBA-PEG polymer and any of the polymers described in US Patent No. 10,182,986 are provided. In certain of these embodiments, a plurality of NPBA-PEG polymers are conjugated to a single second polymer. In conjugates of one or more NPBA-PEG polymers and a second polymer, the NPBA-PEG polymer can contain a targeting molecule, a therapeutic molecule, neither or both.

[0076] In additional embodiments, conjugates of a NPBA-PEG polymer and any of the polymers described in US Patent No. 11,041,050 are provided. In certain of these embodiments, a plurality of NPBA-PEG polymers are conjugated to a single second polymer. In conjugates of one or more NPBA-PEG polymers and a second polymer, the NPBA-PEG polymer can contain a targeting molecule, a therapeutic molecule, neither or both.

[0077] In additional embodiments, conjugates of a NPBA-PEG polymer and any of the polymers described in US Patent Application Publication No. 2021 / 0220483 are provided. In certain of these embodiments, a plurality of NPBA-PEG polymers are conjugated to a single second polymer. In conjugates of one or more NPBA-PEG polymers and a second polymer, the NPBA-PEG polymer can contain a targeting molecule, a therapeutic molecule, neither or both.

[0078] In additional embodiments, conjugates of a NPBA-PEG polymer and any of the polymers described in US Patent No. 11,738,092 are provided. In certain of these embodiments, a plurality of NPBA-PEG polymers are conjugated to a single second polymer. In conjugates of one or more NPBA-PEG polymers and a second polymer, the NPBA-PEG polymer can contain a targeting molecule, a therapeutic molecule, neither or both.

[0079] The disclosures ofUS Patent No. 9,446,149; US Patent No. 10,182,986; US Patent No. 10,342,879; US Patent No. 11,041,050; US Patent No. 11,285,212; US Patent No. 11,738,092 and US Patent Application Publication No. 2021 / 0220483 are incorporated by reference in their entireties, for the purposes of describing polymers containing vicinal diols, their synthesis, their properties and their uses.Formation of nanoparticles

[0080] Conjugates of a NPBA-PEG polymer and a second polymer, as described above, can be converted to nanoparticles for therapeutic use. To convert a polymer conjugate (as described above) to nanoparticles, the lyophilized polymer conjugate is dissolved to aconcentration of 1-10 mg / mL (e.g., 4 mg / mL) in water or dilute saline at pH 4. The solution is then filtered and can be frozen until further use.Formulations, kits, and routes of administration

[0081] Therapeutic compositions comprising nanoparticles as disclosed herein are also provided. Such compositions typically comprise the nanoparticles and a pharmaceutically acceptable carrier. Supplementary active compounds can also be incorporated into nanoparticle compositions.

[0082] The therapeutic compositions disclosed herein are useful for, inter alia, treating cancer, cancer metastases and other disorders of the brain and central nervous system. Accordingly, a “therapeutically effective amount” of a composition comprising nanoparticles is any amount that reduces symptoms or, e.g., stimulates tumor regression. For example, dosage amounts of nanoparticles can vary from about 0.1 -1.0 mg / kg body weight, or from about 0.5 to 2.0 mg / kg body weight or from about 1-5 mg / kg body weight or from about 1 mg / kg body weight to about 10 mg / kg body weight or more (or any integral value therebetween); with a frequency of administration of, e.g., hourly, twice per day, once per day, twice per week, once per week, twice per month, once per month, depending upon, e.g., body weight, route of administration, severity of disease, etc. Thus, a therapeutically effective amount can comprise a plurality of administrations of the same amount, or different amounts, of nanoparticles. In certain embodiments, a single administration of nanoparticles is a therapeutically effective amount.

[0083] In certain embodiments, nanoparticles are administered at a dosage of 1-20 mg nanoparticle (or any integral or decimal value therebetween) per square meter of the surface area of the body of the subject, as is typical for dosages of chemotherapeutics.

[0084] Various pharmaceutical compositions and techniques for their preparation and use are known to those of skill in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and techniques for their administration one may refer to texts such as Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., “Goodman and Gilman’s The Pharmacological Basis of Therapeutics,” McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), “Remington: The Principles of Pharmacy Practice,” Lippincott Williams & Wilkins, 2008.

[0085] The nanoparticles described herein can be suspended in a physiologically compatible carrier for administration. As used herein, the term “physiologically compatible carrier” refers to a carrier that is compatible with the nanoparticles and with any other ingredients of the formulation, and is not deleterious to the recipient thereof. Those of skill in the art are familiar with physiologically compatible carriers. Examples of suitable carriers include water (e.g., pH 4 water), phosphate-buffered saline, Hank’s balanced salt solution+ / -glucose (HESS), and multiple electrolyte solutions such as, e.g., Plasma-LyteTM A (Baxter).

[0086] The volume of a nanoparticle suspension administered to a subject will vary depending on the site of administration, treatment goal and number of nanoparticles in solution. Typically the amount of nanoparticles administered will be a therapeutically effective amount. As used herein, a “therapeutically effective amount” or “effective amount” refers to the number of administered nanoparticles which are required to effect treatment of the particular disorder; i.e., to produce a reduction in the amount and / or severity of the symptoms associated with that disorder. For example, in the case of a cancer metastasis to the brain, administration of a therapeutically effective amount of nanoparticles results in reduction and / or reversal of the symptoms of metastasis; e.g., regression of the metastatic tumor. Therapeutically effective amounts vary with the type and extent of brain damage, and can also vary depending on the overall condition of the subject.

[0087] The disclosed therapeutic compositions can also include pharmaceutically acceptable materials, compositions or vehicle, such as a liquid or solid fdler, diluent, excipient, solvent or encapsulating material, i.e., carriers. These carriers can, for example, stabilize the nanoparticles and / or facilitate the retention of the nanoparticles in the body. Each carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically - acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol and polyethylene glycol; polyols, such as glycerin, sorbitol and mannitol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer® solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0088] Nanoparticles can be administered to a subject by any suitable route, including, but not limited to, inhalation, topically, nasally, orally, parenterally (e.g., intravenously, intraperitoneally, intravesically or intrathecally) or rectally in a vehicle comprising one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard practice. Administration of the compounds described herein can be carried out using any method known in the art. For example, administration may be transdermal, parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intraci sternal, intraperitoneal, intracerebroventricular, intrathecal, intranasal, aerosol, by suppositories, or by oral administration. A pharmaceutical composition of the nanoparticles described herein can be for administration by injection, or for oral, pulmonary, nasal, transdermal, or ocular administration.

[0089] Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intrapulmonary, intravenous, intra-arterial, intra-ocular, intra-cranial, sub- meningeal, or subcutaneous administration, including formulations encapsulated in micelles, liposomes or drug-release capsules (active agents incorporated within a biocompatible coating designed for slow-release); ingestible formulations; formulations for topical use, such as eye drops, creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays. The dosage of the compositions of the disclosure will vary according to the extent and severity of the need for treatment, the activity of the administered composition, the general health of the subject, and other considerations well known to the skilled artisan.

[0090] In additional embodiments, the compositions described herein are delivered intracranially at or near a site of brain injury or metastasis. Such localized delivery allows for the delivery of the composition non-systemically, thereby reducing the body burden of the composition as compared to systemic delivery. Local delivery can be achieved, for example, by intra-cranial injection, or through the use of various medically implanted devices including, butnot limited to, stents and catheters, or can be achieved by inhalation, phlebotomy, or surgery. Methods for coating, implanting, embedding, and otherwise attaching desired agents to medical devices such as stents and catheters are established in the art and contemplated herein.

[0091] Another aspect of the present disclosure relates to kits for carrying out the administration of nanoparticles, optionally in combination with another therapeutic agent, to a subject. In one embodiment, a kit comprises a composition of nanoparticles formulated in a pharmaceutical carrier, suitable for administration, e.g., by injection.Synthetic methods

[0092] Also provided are methods for the synthesis of NPBA-PEG polymers, as shown in the Examples provided elsewhere in this disclosure.EXAMPLESExample 1: Synthesis of (NPBA)2-Lys-PEGm

[0093] A reactor was charged with amine PEGm (NFE-PEGsooom, 300 mg, 0.06 mmol) and bis-Boc-L-lysine-N-hydroxysuccinimide ester (Boc-Lys(Boc)-OSu, 75 mg, 0.17 mmol). The reactor was purged with argon, and methylene chloride (DCM) was added to achieve an amine PEGm concentration of 0.2 g / mL. N,N-diisopropylethylamine (DIPEA, 35 pL, 0.18 mmol) was added, and the reaction was stirred at 19-25°C for 16-22 hours. The reaction mixture was dried under vacuum. The solids were dissolved in dimethyl sulfoxide (DMSO) to obtain a homogenous solution with a concentration of about 0.05 g amine PEGm / mL. The product was purified by dialysis using a pre-washed 2 kDa MWCO Spectra Por membrane. Dialysis was performed three times against DMSO and three times against Milli-Q water (each exchange is into about 125x of the product solution volume). The product solution was frozen and lyophilized for at least 72 hours to yield purified Boc-Lys(Boc)-PEGm (Boc-Lys(Boc)-PEGsooom, 291 mg, 91 % yield) as a solid. The solid was stored at < -20°C in glass vials under argon.

[0094] A reactor was charged with Boc-Lys(Boc)-PEGm (201 mg, 0.45 mmol), purged with argon, and vented with a needle. Triisopropyl silane (TIPS), DCM, and trifluoroacetic acid (TFA) were added at a volume ratio of 5:72:23, with TFA added dropwise, to achieve a Boc- Lys(Boc)-PEGm concentration of 0.08 g Boc-Lys(Boc)-PEGm / mL of liquid volume (TIPS :DCM: TFA). The reaction was stirred under vent at 19-25 °C for 16-22 hours. DCM andTFA were removed under vacuum. The solids were dissolved in a minimum amount of DCM and added dropwise to methyl tert-butyl ether (MTBE, about 60x the DCM volume), and the suspension was stirred at 19-25 °C for approximately 1 hour. The resulting suspension was centrifuged, and supernatant liquid was decanted. These steps (dissolving in DCM, adding MTBE, stirring, centrifuging and decanting) were repeated, and the isolated solid was dried under vacuum to remove excess solvent. Optionally, the solid was dissolved in a minimum amount of Milli-Q water and lyophilized for at least 72 hours to yield purified Lys-PEGm (Lys-PEGsooom, 124 mg, 64 % yield) as a solid. The solid was stored at < -20 °C in glass vials under argon.

[0095] A reactor was charged with Lys-PEGm (100 mg, 0.020 mmol), and was purged with argon. DCM was added to achieve a Lys-PEGm concentration of about 0.1 g Lys-PEGm / mL DCM. DIPEA (10 pL, 0.059 mmol) was added, and the mixture was stirred at 19-25 °C until a clear, homogenous solution was achieved (ca. 10 minutes). A separate reactor was charged with (7-azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP, 29 mg, 0.056 mmol) and 3-carboxy-5-nitrophenylboronic acid (NPBA, 59 mg, 0.28 mmol). The reactor was purged with argon, and DCM was added to achieve a NPBA concentration of 0.08 g NPBA / mL. To this suspension, N,N-dimethylformamide (DMF) was added dropwise, and the mixture was stirred at 19-25 °C until a clear, homogenous solution was achieved (ca. 0.18 volume of DCM volume and 15 minutes). DIPEA (97 pL, 0.56 mmol) was added dropwise resulting in color change of the solution to yellow. The Lys-PEGm was transferred dropwise to the NPBA solution, and the reaction mixture was stirred at 19-25 °C for 16-22 hours, then dried under vacuum. 0.5 N hydrochloric acid (HC1) was added to achieve a NPBA concentration of about 0.03 g NPBA / mL, and the milky white suspension was stirred at 19-25 °C for approximately 1 hour. The resulting suspension was centrifuged, and the product solution was decanted and filtered using a 0.2 pm Supor membrane syringe filter. The product was purified by dialysis against pH 3 water using a 3 kDa MWCO Amicon centrifugal filtration unit. The product solution was frozen and lyophilized for at least 72 hours to yield purified (NPBA)2-Lys-PEGm (2NPBA-Lys-PEGsooo- COOMe, 87 mg, 80 % yield) as a solid. The solid was stored at < -20 °C in glass vials under argon. H NMR (500 MHz, D2O): 8.41, 8.38, 8.29, 8.15, 8.06, 4.57, 3.6-3.3 (PEG), 3.22, 1.92, 1.85, 1.64, 1.57, 1.49.

[0096] For (NPBA)2-Lys-PEGm synthesized by this method, s is about 114. Accordingly, the PEG component (NFE-PEGsooom) has a number average molecular weight of about 5,000 Da. This value will change if a different starting material is used (e.g., s is about 80 for NFE-PEGssoom or about 227 for NEh-PEGioooom).Example 2: Synthesis of (NPBA)2-Lys-PEG-COOH

[0097] A reactor was charged with amine PEG acetic acid (NH2-PEG5000-COOH, 403 mg, 0.081 mmol) and bis-Boc-L-lysine-N-hydroxysuccinimide ester (Boc-Lys(Boc)-OSu, 100 mg, 0.23 mmol). The reactor was purged with argon, and DCM was added to achieve an amine PEG acetic acid concentration of 0.2 g / mL. DIPEA (47 pL, 0.24 mmol) was added, and the reaction mixture was stirred at 19-25 °C for 16-22 hours, then dried under vacuum. The solids were dissolved in DMSO to obtain a homogenous solution and concentration of about 0.05 g starting amine PEG acetic acid / mL. The product was purified by dialysis using a pre-washed 2 kDa MWCO Spectra Por membrane. Dialysis was performed three times against DMSO and three times against Milli-Q water (each exchange is into about 125x of the product solution volume). The product solution was frozen and lyophilized for at least 72 hours to yield purified Boc-Lys(Boc)-PEG-COOH (Boc-Lys(Boc)-PEGsooo-COOH, 377 mg, 88 % yield) as a solid. The solid was stored at < -20 °C in glass vials under argon.

[0098] A reactor was charged with Boc-Lys(Boc)-PEG-COOH (186 mg, 0.42 mmol), purged with argon, and vented with a needle. TIPS, DCM, and TFA were added at a volume ratio of 5:72:23, with TFA added dropwise, to achieve a Boc-Lys(Boc)-PEG-COOH concentration of 0.08 g / mL total volume TIPS:DCM:TFA. The reaction was stirred under vent at 19-25 °C for 16- 22 hours. DCM and TFA were removed under vacuum. The solids were dissolved in a minimum amount of DCM and added dropwise to methyl tert-butyl ether (MTBE, about 60x the DCM volume), and the suspension was stirred at 19-25 °C for approximately 1 hour. The resulting suspension was centrifuged, and supernatant liquid was decanted. These steps (dissolving in DCM, adding MTBE, stirring, centrifuging and decanting) were repeated, and the isolated solid was dried under vacuum to remove excess solvent. Optionally, the solid was dissolved in a minimum amount of Milli-Q water and lyophilized for at least 72 hours to yield purified Lys- PEG-COOH (Lys-PEGsooo-COOH, 102 mg, 57 % yield) as a solid. The solid was stored at < -20 °C in glass vials under argon.

[0099] A reactor was charged with Lys-PEG-COOH (300 mg, 0.059 mmol), and was purged with argon. DCM was added to achieve a Lys-PEG-COOH concentration of about 0.1 g / m . DIPEA (31 pL, 0.18 mmol) was added, and the mixture was stirred at 19-25 °C until a clear, homogenous solution was achieved (ca. 10 minutes). A separate reactor was charged with PyAOP (87 mg, 0.17 mmol) and NPBA (176 mg, 0.84 mmol). The reactor was purged with argon, and DCM was added to achieve an NPBA concentration of 0.08 g NPBA / mL. To this suspension, DMF was added dropwise, and the mixture was stirred at 19-25 °C until a clear, homogenous solution was achieved (ca. 0.18x DCM volume and 15 minutes). DIPEA (291 pL, 1.67 mmol) was added dropwise resulting in a change in color of the solution to yellow. The Lys-PEG-COOH was transferred dropwise to the NPBA solution, and the reaction was stirred at 19-25 °C for 16- 22 hours, then dried under vacuum. 0.5 N HC1 was added to achieve a NPBA concentration of about 0.03 g NPBA / mL, and the milky white suspension was stirred at 19-25 °C for approximately 1 hour. The resulting suspension was centrifuged, and the product solution was decanted and filtered using a 0.2 pm Supor membrane syringe filter. The product was purified by dialysis against pH 3 water using a 3 kDa MWCO Amicon centrifugal filtration unit. The product solution was frozen and lyophilized for at least 72 hours to yield purified (NPBA)i-Lys-PEG-COOH((NPBA)2-Lys-PEGsooo-COOH, 258 mg, 79 % yield) as a solid. The solid was stored at < -20 °C in glass vials under argon. 'H NMR (500 MHz, D2O): 8.41, 8.37, 8.28, 8.15, 8.06, 4.57, 4.09, 3.6-3.3 (PEG), 1.91, 1.85, 1.65, 1.58, 1.49.

[0100] For (NPBA)2-Lys-PEG-COOH synthesized by this method, s is about 114.Accordingly, the PEG component (NH2-PEG5000-COOH) has a number average molecular weight of about 5,000 Da. This value will change if a different starting material is used (e.g., s is about 80 for NH2-PEG3500-COOH or about 227 for NH2-PEG10000-COOH).Example 3: Synthesis of (NPBA)2-Lys-PEG-COO-PFP

[0101] A reactor was charged with (NPBA)2-Lys-PEG-COOH (102 mg, 0.018 mmol), and was purged with argon. DCM (2 m ) was added to achieve a 2NPBA-Lys-PEG-COOH concentration of 0.05 g / mL. N-methylmorpholine (NMM, 6 pL, 0.054 mmol) and bis(pentafluorophenyl) carbonate ((PFPO)2O, 30 mg, 0.076 mmol) were added, and the reaction mixture was stirred at 19-25 °C for 2-4 hours. Progress of the reaction was monitored by HPLC. After reaction completion, the reaction mixture was added dropwise into MTBE (about lOx the DCM volume), and the suspension was stirred at 19-25 °C for approximately 15 minutes under argon. The resulting suspension was centrifuged, and supernatant liquid was decanted. Heptane (about lOx the DCM volume) was added, and the suspension was stirred at 19-25 °C forapproximately 15 minutes under argon. The resulting suspension was centrifuged, and supernatant liquid was decanted. The isolated solid was dried under vacuum to remove excess solvent to yield purified (NPBA)2-Lys-PEG-COO-PFP ((NPBA)2-Lys-PEG5ooo-COO-PFP, 94 mg, 90 % yield).The solid was stored at < -20 °C in glass vials under argon.1. DCM

[0102] As stated in Example 6, s is about 114 for (NPBA)2-Lys-PEG-COO-PFP synthesized by this method. This value will change if a different starting material is used.Example 4: Preparation of (NPBA)2-Lys-PEG-AbSynthesis of Crude Mixture

[0103] A 15 mL 30 kDa MWCO centrifugal filtration unit was pre-washed with 50 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) pH 7.8 buffer and centrifuged at 3,200 x g for 5 minutes. 100 mg (0.0069 mmol) of anti-CD8 antibody at 25 mg / mL was added and centrifuged at 3,200 x g for 15 minutes to allow buffer exchange into 50 mM HEPES pH 7.8 buffer. The flow-through was then replaced with 50 mM HEPES buffer and centrifugation was repeated until < 0.01 % of the initial solution remained. The concentration of antibody was determined with an Unchained Labs Little Lunatic UV / Vis absorbance spectrometer. The antibody concentration was then adjusted to 10 mg / mL in 50 mM HEPES pH 7.8 buffer. Following solubilization, 10.21 mg (0.00187 mmol) of (NPBA)2-Lys-PEG-COO-PFP in 0.093 mL DMSO was added slowly to the antibody solution and mixed well. The reaction solution was stirred at 19-25 °C and sampled periodically to determine progress of the reaction. Reaction progress was analyzed on a Cytiva AKTA Go fast protein liquid chromatography (FPLC) system attached to a 1 mL HiTrap Phenyl HP HIC column with a mobile phase A of 1.5 M Ammonium Sulfate, 20 mM Sodium Phosphate, pH 7.4 and a mobile phase B of 20 % isopropyl alcohol (IP A),10 mM Sodium Phosphate, pH 7.4 at a flow rate of 1 mL / min. The gradient was run from 40 % B to 100 % B over 25 column volumes. The column was connected to an absorbance detector set to 280 nm. FPLC analysis indicated that the reaction was complete after two hours, at which time the (NPBA)i-Lys-PEG-Ab crude mixture was added to a pre-washed 30 kDa MWCO Amicon centrifugal filtration unit with 50 mM HEPES pH 7.8 buffer and centrifuged at 3,200 x g in 15-20 minutes intervals with intermediate mixing to achieve a concentration of 30-35 mg / mL of Ab in the crude mixture.Purification of (NPBAfi-Lys-PEG-Ab (Linker-to-Ab Ratio (LAR) 1)

[0104] 2 mL of (NPBA)i-Lys-PEG-Ab crude mixture, at approximately 30 mg / mL in 50 mM HEPES, pH 7.8, was added to a reaction vessel. 3 mL of mobile phase A (1.5 M Ammonium Sulfate, 20 mM Sodium Phosphate pH 7.4) was added to achieve a final solution of 0.9 M ammonium sulfate, 12 mM sodium phosphate, pH 7.4. After mixing, a 2.5 mL sample was loaded onto a 5 mL HiTrap Phenyl HP HIC column attached to an AKTA Go FPLC system using a 5 mL sample loop. Mobile phase B (20 % IP A, 10 mM Sodium Phosphate pH 7.4) was used as the eluent. The monoPEGylated (NPBA)2-Lys-PEG-Ab (LAR 1) was isolated using a continuous gradient of elution buffer 40 % to 100 % B over 25 column volumes at a flow rate of 5 mL / min. The fractions containing monoPEGylated 2NPBA-Lys-PEG-Ab (LAR 1) were combined and transferred to a pre-washed 15 mL 30 kDa MWCO Amicon centrifugal filtration unit with lx PBS, pH 7.4. The fractions were concentrated using the 15 mL 30 kDa MWCO Amicon centrifugal filtration unit and centrifuged at 3,200 x g for 15 minutes. The flow-through was replaced with lx PBS pH 7.4 buffer and centrifugation was repeated. This process was repeated until < 0.01 % of the initial solution remained. The concentration of the purified monoPEGylated (NPBA)2-Lys- PEG-Ab (LAR 1) was adjusted to 30-40 mg / mL and the purified (NPBA)2-Lys-PEG-Ab material was stored at -80 °C.

[0105] Substitution, in the method described above, of anti-CD8 antibody with any other protein-, peptide-, or aptamer- based targeting agent yields the respective targeting agentcontaining (NPBA)2-Lys-PEG polymer crude mixtures and purified components.Incorporation by Reference

[0106] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, publicly accessible databases, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.Equivalents

[0107] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

CLAIMSWhat is claimed is:

1. A boronic acid-containing polymer having the structure:wherein: the spacer is selected from any linear or branched organic group;L3 is a linking group, or linking groups, between the one or more boronic acid moi eties and the spacer;L4is a linking group, or linking groups, between the spacer and the one or more polyethylene oxide linkages; q is independently at each occurrence 0, 1, 2, 3, or 4; r is 1, 2, or 3; s is independently at each occurrence 20-1200; t is 1, 2, or 3; andXi is(a) -Ci-6 alkyl, optionally substituted with -OH, -COOH, -C(=O)O(alkyl), -C(=O)O(aryl), -NH2, -NH(alkyl), or N(alkyl)2; or(b) a salt or protected analog of any of the foregoing.

2. The polymer of claim 1, wherein q is 1.

3. The polymer of claim 1, wherein r is 2.

4. The polymer of claim 1, wherein s is 50-300.

5. The polymer of claim 1, wherein t is 1.

6. The polymer of claim 1, wherein L3 and L4are independently at each occurrence:-(Co-6alkylene)-NH-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-NH-(Co-6alkylene)-, -(Co-6alkylene)-0-C(=0)-(Co-6alkylene)-, -(Co-6alkylene)-C(=0)-0-(Co-6alkylene)-, -(Co-6alkylene)-NH-C(=0)-0-(Co-6alkylene)-, or -(Co-6alkylene)-0-C(=0)-NH-(Co-6alkylene)-.

7. The polymer of claim 1, wherein the spacer is an organic group bearing at least one terminal amine and at least one terminal carboxylic acid.

8. The polymer of claim 7, wherein the spacer has the formula -NH-CH-Ri-C(=O)-, wherein Ri is an amino acid functional group.

9. The polymer of claim 8, wherein:(a) Ri is the functional group of lysine;(b) L3is -C(=O)-NH-; and(c) L4is -C(=O)-NH-.

10. The polymer of claim 1, wherein: the spacer is a lysyl group; q is 1; r is 2; s is 50-300; t is 1; for the first instance of r, L3is -C(=O)-NH-; for the second instance of r, L3is -C(=O)-NH-;L4is -C(=O)-NH-; andXi is -CH3or -C-C(=O)-OH.

11. The polymer of claim 10, further comprising one or more targeting molecules.

12. The polymer of claim 11, wherein the targeting molecule is selected from the group consisting of a protein, a peptide, an aptamer, and a ligand for a cellular receptor.

13. The polymer of claim 12, wherein the protein is an antibody or an antibody fragment.

14. The polymer of claim 13, wherein the antibody or antibody fragment is directed to CD3, CD4, CD8, the epidermal growth factor receptor (EGFR) or the transferrin receptor (TfR).

15. The polymer of claim 12, wherein the cellular receptor is CD3, CD4, CD8, the epidermal growth factor receptor (EGFR) or the transferrin receptor (TfR).

16. The polymer of claim 12, wherein the ligand for a cellular receptor is transferrin.

17. The polymer of claim 11, wherein the targeting molecule is a covalent conjugate of Xi, such that Xi has the structure -C-C(=O)-NH-targeting molecule.

18. The polymer of claim 10, further comprising a therapeutic molecule.

19. The polymer of claim 18, wherein the therapeutic molecule is a covalent conjugate of Xi, such that Xi has the structure -C-C(=O)-NH-therapeutic molecule.

20. The polymer of claim 19, wherein the therapeutic molecule is an antibody.

21. The polymer of claim 20, wherein the antibody is Herceptin.

22. A polymer conjugate comprising the polymer of claim 1 and a second polymer containing at least one set of vicinal diols.

23. The polymer conjugate of claim 22, wherein the polymer conjugate comprises aplurality of the polymers of claim 1.

24. The polymer conjugate of claim 23, the conjugate comprising a plurality of molecules of Formula III, wherein:(a) in at least one of the polymers of claim 1, Xi is -CH3; and(b) in at least one of the polymers of claim 1, Xi comprises a targeting agent.

25. The polymer conjugate of claim 24, wherein the targeting agent is an anti-CD8 antibody.

26. A nanoparticle comprising:(a) the polymer of any of claims 1, 10, 15, 18 or 21; or(b) the polymer conjugate of any of claims 22-25.

27. A pharmaceutical composition comprising the nanoparticle of claim 26 and a pharmaceutically acceptable carrier or excipient.

28. A method for the treatment of a disease or disorder in a subject, the method comprising administering the pharmaceutical composition of claim 27 to the subject.

29. The method of claim 28, wherein the disease or disorder is a malignancy.

30. The method of claim 29, wherein the malignancy is selected from the group consisting of a hematologic malignancy and a solid tumor.

31. The method of claim 30, wherein the hematologic malignancy is a B-cell malignancy.

32. The method of claim 31, wherein the B-cell malignancy is a leukemia or a lymphoma.

33. The method of claim 30, wherein the solid tumor is a breast tumor, a lung tumor, an ovarian tumor, a gastric tumor, a colorectal tumor, a pancreatic tumor, a primary brain tumor or a brain metastasis.

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