WO2025015005A2

WO2025015005A2 - Polymer-protein conjugate, pharmaceutical composition comprising said conjugate, therapeutic use thereof

Info

Publication number: WO2025015005A2
Application number: PCT/US2024/037314
Authority: WO
Inventors: Masakazu Kamata; Jie Ren
Original assignee: The Uab Research Foundation
Priority date: 2023-07-11
Filing date: 2024-07-10
Publication date: 2025-01-16
Also published as: WO2025015005A3

Abstract

The present disclosure provides for compounds including bioactive proteins conjugated with biocompatible polymers to form polymer-protein conjugates. The disclosure also provides for pharmaceutical compositions including the polymer-protein conjugates, methods of use of the polymer-protein conjugates and their pharmaceutical compositions, methods of making the polymer-protein conjugates, and the like.

Description

POLYMER-PROTEIN CONJUGATE, PHARMACEUTICAL COMPOSITION COMPRISING SAID CONJUGATE, THERAPEUTIC USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to each of the following: U.S. Provisional Application Serial No. 63/513,033, having the title “SITE-ORIENTED POLYMER MODIFICATION OF ANTIBODY FOR ENHANCED BRAIN DELIVERY,” filed on July 11 , 2023; U.S. Provisional Application Serial No. 63/579,586, having the title “POLYMER- PROTEIN CONJUGATE, PHARMACEUTICAL COMPOSITION COMPRISING SAID CONJUGATE, THERAPEUTIC USE THEREOF,” filed on August 30, 2023; and U.S. Provisional Application Serial No. 63/542,916, having the title “POLYMER-PROTEIN CONJUGATE, PHARMACEUTICAL COMPOSITION COMPRISING SAID CONJUGATE, THERAPEUTIC USE THEREOF,” filed on October 6, 2023; each of which is incorporated by reference.

STATEMENT ON FUNDING PROVIDED BY THE U.S. GOVERNMENT

This invention was made with Government support under contract R01CA232015 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

Antibodies have been widely employed as therapeutics and diagnostics. To date, more than 100 antibody-based therapeutics have been approved by the Food and Drug Administration (FDA) for treatments of various diseases. However, the merit of antibody therapeutics is negated in treating some diseases. For example, there are limitations using antibodies for treating brain diseases due to the blood-brain barrier (BBB) that separates the peripheral circulating system from the brain via tight junctions and restricted transcytosis. Therefore, an effective and biocompatible brain delivery system would boost antibody applications and change the landscape of treatment regimens for brain diseases.

SUMMARY

Embodiments of the present disclosure provides for compounds including bioactive proteins (e.g., antibodies) conjugated with biocompatible polymers (e.g., zwitterionic polymers) to form polymer-protein conjugates (e.g., zwitterionic polymer-antibody conjugates). The disclosure also provides for pharmaceutical compositions including the polymer-protein conjugates, methods of use of the polymer-protein conjugates and their pharmaceutical compositions, methods of making the polymer-protein conjugates, and the like.

In an aspect, the present disclosure provides for a composition comprising: a bioactive protein and a at least one zwitterionic polymer, wherein the zwitterionic polymer is conjugated to at least one site of the protein. In an aspect, the zwitterionic polymer has the following structure:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen and a C1-C3 alkyl group and n is an integer from 25 to 400.

In an aspect, the present disclosure provides for a pharmaceutical composition comprising a therapeutically effective amount of the composition as described above or herein and a pharmaceutically-acceptable carrier, formulated for administering to a subject.

In an aspect, the present disclosure provides for a method of making a zwitterionic polymer-protein conjugate comprising: synthesizing a at least one zwitterionic polymer and covalently bonding the at least one zwitterionic polymer to a bioactive protein.

In an aspect, the present disclosure provides for a method of making a zwitterionic polymer-antibody conjugate comprising: synthesizing a at least one zwitterionic polymer, cleaving at least one interchain disulfide bond of an antibody, and mixing the cleaved antibody with the zwitterionic polymers to form an antibody covalently bonded to the at least one zwitterionic polymer.

In an aspect, the present disclosure provides for a method of treating a condition of the central nervous system by penetrating a blood brain barrier of a subject comprising: administering to the subject in need thereof, a pharmaceutical composition, wherein the pharmaceutical composition includes a therapeutically effective amount of the composition or pharmaceutical composition as described above or herein. BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

Figures 1A-1 B illustrate the synthetic process of PMPC polymers and Tmab^PMPCs (Tmab: Trastuzumab, PMPC: Poly (2-Methacryloyloxyethyl phosphorylcholine)). Figure 1A illustrate the synthetic process of thiol-reactive maleimide-modified PMPC. MPC was first polymerized following a reversible addition-fragmentation chain transfe (RAFT) polymerization to obtain Carboxyl-terminated PMPC. Then the end group was converted to an amino group with Ethylenediamine modification, followed by converting the amino group to maleimide group by Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1- carboxylate) to obtain maleimide-modified PMPC. Figure 1 B illustrates the synthetic process of Tmab^PMPC production. The interchain disulfide bonds of Tmab were first cleaved by tris(2- carboxyethyl)phosphine (TCEP) to create thiol groups, followed by reaction with maleimide- modified PMPC to obtain Tmab^PMPC.

Figures 2A-2E illustrate structural and biofunctional characterizations of Tmab^PMPC Figure 2A illustrates SDS page images of Tmab, TCEP-reduced Tmab, Tmab^PMPC50, Tmab^PMPC10°, and Tmab^PMPC10° (lanes 1-5). Figure 2B illustrates the size distributions and the average diameters of Tmab, Tmab^PMPC50, Tmab^PMPC10°, Tmab^PMPC200, and nTmab determined by DLS measurement in PBS at 25 °C. Figure 2C illustrates CLSM images of alexa fluor 647 (AF 647) labeled Tmab, Tmab^PMPC50, Tmab^PMPC10°, and Tmab^PMPC200 following incubation with HER2+ human ovarian cancer cell line, SKOV-3 cells. The cell nuclei were labeled by Hoechst 33342 (blue) after 2 h incubation with each sample at 37 °C. The scale bar represents 50 pm. Figure 2D illustrates the saturation binding curves of Tmab, Tmab^PMPC5°, Tmab^PMPC10°, Tmab^PMPC10°, and nanoencapsulated trastuzumab (nTmab) in SKOV-3 cells. Figure 2E illustrates the confocal laser scanning microscope (CLSM) images of AF647 labeled Tmab, Tmab^PMPC5°, Tmab^PMPC10°, and Tmab^PMPC200 binding to SKOV-3 cells. The cell nuclei were stained by Hoechst 33342 (blue) while surface bound Tmab was stained by alexa fluor488 (AF488) labeled anti-human IgG antibody after 24 h incubation with each sample at 37 °C. The scale bar represents 50 pm. Figure 2F illustrates the pearson correlation coefficient of AF488 and AF647 signals in (E) figures. Three different areas of each sample were measured. Figure 2G illustrates phagocytic percentage of THP-1 cells incubated with HER2+ Jurkat cells pre-treated with Tmab samples for 2 h. Error bars represent the s.e.m. of triplicate samples (n = 3), ****P < 0.0001 . ns: not significant. Figures 3A-3G illustrate the internalization and penetration of Tmab^PMPC in mouse brain endothelial cells (bEND.3). Figure 3A illustrates the cellular internalization of Tmab, Tmab^PMPC50, Tmab^PMPC10°, Tmab^PMPC200, and nTmab in bEND.3 cells. Each sample was labeled with 5-(and-6)-carboxytetrarnethylrhodamine, succinimidyl ester (TAMRA )dye and incubated with bEND.3 cells for 2 h at 37 °C. The cells were then harvested and subjected to flow cytometry to measure the MFI of TAMRA. Figure 3B illustrates CLSM images showing intracellular trafficking of TAMRA (green) labeled Tmab, Tmab^PMPC10°, and nTmab in bEND.3 cells. The cells were incubated with each sample for 4 h at 37 °C, followed by nuclear staining with Hoechst 33342 (blue) and lysosome/late endosomal staining with lysotracker deep red (red). The scale bar represents 50 pm. Figure 3C illustrates the pearson correlation coefficient of lysotracker and TAMRA signals in Figure 3B figures. 3 different areas of each sample were analyzed. Figure 3D illustrates the scheme to show the experimental procedure of the transwell assay. Figure 3E illustrates the accumulative penetration efficiency of TAMRA labeled Tmab, Tmab^PMPC5°, Tmab^PMPC10°, Tmab^PMPC20°, and nTmab through a monolayer of bEND.3 cells in transwells. The fluorescence intensity in the basolateral compartment was measured at designated time points with a plate reader. Figure 3F illustrates the penetration efficiency of Tmab^PMPC200 through the bEND.3 layers with or without PMPC200 competition. PMPC200 was mixed with Tmab^PMPC200 at a molar ratio of 20: 1 or 200: 1 and added to the apical surface of the transwell. Figure 3G illustrates the binding affinity of penetrated T mab, T mab^PMPC50, T mab^PMPC10°, T mab^PMPC20°, and nT mab to SKOV-3 cells. The medium in the basolateral compartment was collected 10 h post sample addition and incubated with SKOV-3 cells. The mean fluorescent intensity (MFI) was measured by flow cytometry. Error bars represent the s.e.m. of triplicate samples (n = 3), ****P < 0.0001.

Figures 4A-4E illustrate the biodistribution and brain delivery of Tmab^PMPC NSG mice were treated with AF647-labeled Tmab, Tmab^PMPC5°, Tmab^PMPC10°, Tmab^PMPC200, and nTmab via intravenous (i.v.) injection (10 mg/kg). Figure 4A illustrates the whole-body imaging of AF647 fluorescence by IVIS imaging. Figure 4B illustrates AF647 visualization by IVIS imaging, including the heart, liver, spleen, lung, and kidneys from the mice shown in Figure 4A. Figure 4C illustrates the total flux (photons/s) of the dissected organs. Figure 4D illustrates the total flux (photons/s) of the dissected brains. Figure 4E illustrates the Tmab concentrations in the brains following PBS perfusion. The brains were weighed and homogenized after IVIS imaging. The amount of T mab in the brains was quantified by sandwich ELSA with anti-human IgG antibodies. Error bars represent the s.e.m. of triplicate samples (n = 3), ****P < 0.0001 .

Figures 5A-5F illustrate the in vitro and in vivo toxicity assays of Tmab^PMPC. In vitro cytotoxicity assay on Figure 5A bEND.3 and Figure 5B JX14P cells (human glioblastoma cell line). Cells were incubated with PMPC200 or Tmab^PMPC200 at the PMPC concentrations ranging from 5 to 1000 pg/mL for 72 h. The cellular viability was measured by Cell counting kit-8 (CCK8)( CCK-8) assay. Figure 5C illustrates fold changes of AST and ALT in the plasma of the mice treated with Tmab, PMPC200, or Tmab^PMPC200. The Tmab dosage was 50 mg/kg while the PMPC dosage was 90 mg/kg, and the plasma was collected before and 72 h post-treatment. Figure 5D illustrates mice were treated with Tmab, PMPC200, and Tmab^PMPC200. Evans blue dye solution (4 mg per mouse) was intravenously injected to mice 24 h post-treatment, and the brains were obtained 2 h post-Evans blue dye injection. The brain images were taken by MS imaging. Figure 5E illustrates immunofluorescent images of the brain sections from the mice treated with T mab, PMPC200, or T mab^PMPC20°. The brains were collected 72 h post-treatment and processed to sections with a vibratome, followed by staining with anti-l ba1 and anti-GFAP primary antibodies and the corresponding dye-labeled secondary antibodies. The nuclei were visualized by Hoechst 33342 staining. Figure 5F illustrates the quantification of the ionized calcium-binding adapter molecule 1 ( Iba1) or glial fibrillary acidic protein (GFAP) positive area fractions from the images in Figure 5E.

Figure 6 illustrates fourier-transform infrared spectroscopy (FT-IR) spectra of PMPC- COOH, deprotected PMPC, and PMPC-Mal.

Figure 7 illustrates the ¹H nuclear magnetic resonance (NMR) spectra of a) PMPC100-COOH, b) deprotected PMPC100-COOH, and c) PMPC100-Mal.

Figure 8 illustrates whole body imaging of NSG mice treated with AF647 or AFTmab. Figure 9 illustrates enzyme-linked immunosorbent assay (ELISA) standard curves of Tmab and AFTmab.

Figure 10 illustrates CLSM images of SKOV-3 cells treated with PBS, AF647, AFIgG, or AFTmab.

Figure 1 1 illustrates Tmab-dependent binding of AFTmab to SKOV-3 cells. Figure 12 illustrates the assay for Tmab-saturation binding.

DETAILED DESCRIPTION

Embodiments of the present disclosure provides for compounds including bioactive proteins (e.g., antibodies) conjugated with biocompatible polymers (e.g., zwitterionic polymers) to form polymer-protein conjugates (e.g., zwitterionic polymer-antibody conjugates). In addition, the present disclosure provides for pharmaceutical compositions including the polymer-protein conjugates, methods of use of the polymer-protein conjugates and their pharmaceutical compositions, methods of making the polymer-protein conjugates, and the like.

This disclosure is not limited to particular embodiments described, and as such may, of course, vary. The terminology used herein serves the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, dimensions, frequency ranges, applications, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence, where this is logically possible. It is also possible that the embodiments of the present disclosure can be applied to additional embodiments involving measurements beyond the examples described herein, which are not intended to be limiting. It is furthermore possible that the embodiments of the present disclosure can be combined or integrated with other measurement techniques beyond the examples described herein, which are not intended to be limiting.

It should be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; “application cited documents’’), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference.

Further, documents or references cited in this text, in a Reference List before the claims, or in the text itself; and each of these documents or references (“herein cited references’’), as well as each document or reference cited in each of the herein-cited references (including any manufacturer’s specifications, instructions, etc.) are hereby expressly incorporated herein by reference.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

Definitions

It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

It will be understood by those skilled in the art that the moieties substituted can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like. Cycloalkyls can be substituted in the same manner.

The terms “administering” and “administration” as used herein refer to introducing a composition (e.g., a vaccine, adjuvant, or immunogenic composition) of the present disclosure into a subject. As used herein, “administering” can refer to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernous, intrathecal, intravireal, intracerebral, and intracerebroventricular, intratympanic, intracochlear, rectal, vaginal, by inhalation, by catheters, stents or via an implanted reservoir or other device that administers, either actively or passively (e.g. by diffusion) a composition the perivascular space and adventitia. A preferred route of administration of the vaccine composition is intravenous.

The term "alkyl", either alone or within other terms such as "thioalkyl" and "arylalkyl", as used herein, means a monovalent, saturated hydrocarbon radical which may be a straight chain (i.e. linear) or a branched chain. An alkyl radical for use in the present disclosure generally comprises from about 1 to 20 carbon atoms, particularly from about 1 to 10, 1 to 8 or 1 to 7, more particularly about 1 to 6 carbon atoms, or 3 to 6. Illustrative alkyl radicals include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, isopentyl, amyl, sec-butyl, tert-butyl, tert-pentyl, n-heptyl, n-actyl, n-nonyl, n-decyl, undecyl, n-dodecyl, n- tetradecyl, pentadecyl, n-hexadecyl, heptadecyl, n-octadecyl, nonadecyl, eicosyl, dosyl, n- tetracosyl, and the like, along with branched variations thereof. In certain aspects of the disclosure an alkyl radical is a Ci-C₆ lower alkyl comprising or selected from the group consisting of methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, isopentyl, amyl, tributyl, sec-butyl, tert-butyl, tert-pentyl, and n-hexyl. An alkyl radical may be optionally substituted with substituents as defined herein at positions that do not significantly interfere with the preparation of compounds of the disclosure and do not significantly reduce the efficacy of the compounds. In certain aspects of the disclosure, an alkyl radical is substituted with one to five substituents including halo, lower alkoxy, lower aliphatic, a substituted lower aliphatic, hydroxy, cyano, nitro, thio, amino, keto, aldehyde, ester, amide, substituted amino, carboxyl, sulfonyl, sulfuryl, sulfenyl, sulfate, sulfoxide, substituted carboxyl, halogenated lower alkyl (e.g. CF₃), halogenated lower alkoxy, hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy, lower alkylcarbonylamino, cycloaliphatic, substituted cycloaliphatic, or aryl (e.g., phenylmethyl benzyl)), heteroaryl (e.g., pyridyl), and heterocyclic (e.g., piperidinyl, morpholinyl). Substituents on an alkyl group may themselves be substituted.

The terms "alkoxyl" or "alkoxyalkyl" as used herein refer to an alkyl-O- group wherein alkyl is as previously described. The term "alkoxyl" as used herein can refer to C1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, t-butoxyl, and pentoxyl.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms or 2 to 8 carbon atoms or 2 to 6 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (R¹R²)C=C(R³R⁴) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

As used herein, "alkynyl" or “alkynyl group” refers to straight or branched chain hydrocarbon groups having 2 to 40, 2 to 20, 2 to 10, or 2 to 5 carbon atoms and at least one triple carbon to carbon bond, such as ethynyl. Reference to "alkynyl" or “alkynyl group” includes unsubstituted and substituted forms of the hydrocarbon moiety.

The Ar (e.g., A , Ar₂, etc) group is an aromatic system or group such as an aryl group. “Aryl”, as used herein, refers to C₅-C2o-membered aromatic, heterocyclic, fused aromatic, fused heterocyclic, biaromatic, or bihetereocyclic ring systems. In an aspect, “aryl”, can include 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, functional groups that correspond to benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics”. The aromatic ring can be substituted at one or more ring positions with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF₃, -CN; and combinations thereof.

The term “aryl” also includes polycyclic ring systems (C₅-C₃o) having two or more cyclic rings in which two or more carbons are common to two adjoining rings (i.e., “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples of heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H- 1 ,5,2-dithiazinyl, dihydrofuro[2,3 bjtetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1 H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1 ,2,5-thiadiazinyl, 1 ,2,3- thiadiazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,5-thiadiazolyl, 1 ,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or more of the rings can be substituted as defined above for “aryl”.

The term "antibody" as used herein refers to polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, F(ab')₂ fragments, F(ab) fragments, Fv fragments, single domain antibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule.

The term "antibody" as used herein further refers to an immunoglobulin which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of another molecule. The antibody can be monoclonal, polyclonal, or a recombinant antibody, and can be prepared by techniques that are well known in the art such as immunization of a host and collection of sera (polyclonal) or by preparing continuous hybrid cell lines and collecting the secreted protein (monoclonal), or by cloning and expressing nucleotide sequences, or mutagenized versions thereof, coding at least for the amino acid sequences required for specific binding of natural antibodies. Antibodies may include a complete immunoglobulin or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, IgG 1 , lgG2a, lgG2b and lgG3, IgM, IgY, etc. Fragments thereof may include Fab, Fv and F(ab')₂, Fab', scFv, and the like. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for a particular molecule is maintained.

The term "composition" as used herein refers to a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such a term in relation to a pharmaceutical composition is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation, or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present disclosure encompass any composition made by administering a compound of the present disclosure and a pharmaceutically acceptable carrier. When a compound of the present disclosure is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound of the present disclosure is contemplated. Accordingly, the pharmaceutical compositions of the present disclosure include those that also contain one or more other active ingredients, in addition to a compound of the present disclosure. The weight ratio of the compound of the present disclosure to the second active ingredient may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, but not intended to be limiting, when a compound of the present disclosure is combined with another agent, the weight ratio of the compound of the present disclosure to the other agent will generally range from about 1000: 1 to about 1 : 1000, preferably about 200: 1 to about 1 :200. Combinations of a compound of the present disclosure and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used. In such combinations the compound of the present disclosure and other active agents may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent to, or subsequent to the administration of other agent(s).

A composition of the disclosure can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Various delivery systems are known and can be used to administer a composition of the disclosure, e.g. encapsulation in liposomes, microparticles, microcapsules, and the like.

A therapeutic composition of the disclosure may comprise a carrier, such as one or more of a polymer, carbohydrate, peptide or derivative thereof, which may be directly or indirectly covalently attached to the compound. A carrier may be substituted with substituents described herein including without limitation one or more alkyl, amino, nitro, halogen, thiol, thioalkyl, sulfate, sulfonyl, sulfinyl, sulfoxide, hydroxyl groups. In aspects of the disclosure the carrier is an amino acid including alanine, glycine, praline, methionine, serine, threonine, asparagine, alanyl-alanyl, prolyl-methionyl, or glycyl-glycyl. A carrier can also include a molecule that targets a compound of the disclosure to a particular tissue or organ.

Compounds of the disclosure can be prepared using reactions and methods generally known to the person of ordinary skill in the art, having regard to that knowledge and the disclosure of this application including the Examples. The reactions are performed in solvent appropriate to the reagents and materials used and suitable for the reactions being affected. It will be understood by those skilled in the art of organic synthesis that the functionality present on the compounds should be consistent with the proposed reaction steps. This will sometimes require modification of the order of the synthetic steps or selection of one particular process scheme over another in order to obtain a desired compound of the disclosure. It will also be recognized that another major consideration in the development of a synthetic route is the selection of the protecting group used for protection of the reactive functional groups present in the compounds described in this disclosure. An authoritative account describing the many alternatives to the skilled artisan is Greene and Wuts (Protective Groups In Organic Synthesis, Wiley and Sons, 1991).

A compound of the disclosure of the disclosure may be formulated into a pharmaceutical composition for administration to a subject by appropriate methods known in the art. Pharmaceutical compositions of the present disclosure or fractions thereof comprise suitable pharmaceutically acceptable carriers, excipients, and vehicles selected based on the intended form of administration, and consistent with conventional pharmaceutical practices. Suitable pharmaceutical carriers, excipients, and vehicles are described in the standard text, Remington: The Science and Practice of Pharmacy (21 .sup. st Edition. 2005, University of the Sciences in Philadelphia (Editor), Mack Publishing Company), and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. By way of example for oral administration in the form of a capsule or tablet, the active components can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, methyl cellulose, magnesium stearate, glucose, calcium sulfate, dicalcium phosphate, mannitol, sorbitol, and the like. For oral administration in a liquid form, the chug components may be combined with any oral, non-toxic, pharmaceutically, acceptable inert carrier such as ethanol, glycerol, water, and the like. Suitable binders (e.g., gelatin, starch, corn sweeteners, natural sugars including glucose; natural and synthetic gums, and waxes), lubricants (e.g. sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride), disintegrating agents (e.g. starch, methyl cellulose, agar, bentonite, and xanthan gum), flavoring agents, and coloring agents may also be combined in the compositions or components thereof. Compositions as described herein can further comprise wetting or emulsifying agents, or pH buffering agents.

The terms "subject", "individual", or "patient" as used herein are used interchangeably and refer to an animal preferably a warm-blooded animal such as a mammal. Mammal includes without limitation any members of the Mammalia. A mammal, as a subject or patient in the present disclosure, can be from the family of Primates, Carnivora, Proboscidea, Perissodactyla, Artiodactyla, Rodentia, and Lagomorpha. In a particular embodiment, the mammal is a human. In other embodiments, animals can be treated; the animals can be vertebrates, including both birds and mammals. In aspects of the disclosure, the terms include domestic animals bred for food or as pets, including equines, bovines, sheep, poultry, fish, porcines, canines, felines, and zoo animals, goats, apes (e.g. gorilla or chimpanzee), and rodents such as rats and mice.

The term "pharmaceutically acceptable carrier" as used herein refers to a diluent, adjuvant, excipient, or vehicle with which a probe of the disclosure is administered and which is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. When administered to a patient, the probe and pharmaceutically acceptable carriers can be sterile. Water is a useful carrier when the probe is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The present compositions advantageously may take the form of solutions, emulsion, sustained-release formulations, or any other form suitable for use.

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition or prevention of a disease or condition or enhance and/or tune the immune system of the subject to the desirable responses for certain pathogens (e.g., virus). For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms or prevention of a disease or condition and/or tune the immune system of the subject to the desirable responses for certain pathogens, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

As used herein, the terms “treating” and “treatment” can refer generally to obtaining a desired pharmacological and/or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, such as infections and consequences thereof and/or tuning the immune system of the subject to the desirable responses for certain pathogens. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of infections in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or infection but has not yet been diagnosed as having it; (b) inhibiting the disease or infection, i.e., arresting its development; and (c) relieving the disease or infection i.e., mitigating or ameliorating the disease and/or its symptoms or conditions, (d) and/or tune the immune system of the subject to the desirable responses for certain pathogens. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and/or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition, and/or tuning the immune system of the subject to the desirable responses for certain pathogens. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

As used herein, “therapeutic” can refer to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect and/or tuning the immune system of the subject to the desirable responses for certain pathogens.

The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

The term “pharmaceutically acceptable prodrug” or “prodrug” represents those prodrugs of the compounds of the present disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use. Prodrugs of the present disclosure can be rapidly transformed in vivo to a parent compound having a structure of a disclosed compound, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).

Discussion

The present disclosure provides for compounds including bioactive proteins (e.g., antibodies) conjugated with biocompatible polymers (e.g., zwitterionic polymers) to form polymer-protein conjugates (e.g., zwitterionic polymer-antibody conjugates). The disclosure also provides for pharmaceutical compositions including the polymer-protein conjugates, methods of use of the polymer-protein conjugates and their pharmaceutical compositions, methods of making the polymer-protein conjugates, and the like. Compounds and pharmaceutical compositions of the present disclosure can be used in combination with one or more other therapeutic agents for treating cancers, autoimmune diseases, and other diseases. For example, compounds and pharmaceutical compositions of the present disclosure can be employed in combination with other therapeutics, such as antibody therapeutics, to treat brain diseases.

Antibody therapeutics in treating brain diseases are limited due to poor blood-brain barrier (BBB) penetration. One method for brain delivery of therapeutic antibodies is in a polymer-shell-based platform, termed nanocapsule. However, the platform can result in functional loss of antibodies due to epitope masking by the polymer network, necessitating the incorporation of a targeting moiety and degradable crosslinkers to enable on-site antibody release. Alternatively, site-specific conjugation of biocompatible polymers (e.g. zwitterionic polymers) to the antibody can result in the antibody maintaining some functionalities, while the polymer-antibody conjugate enables brain delivery and maintains epitope recognition, cellular internalization, and antibody-dependent cellular phagocytic activity. The polymer-antibody conjugate can work without additional components, thereby addressing the issues of the nanocapsule delivery system. Additionally, the conjugation of the zwitterionic polymer enables decreasing systemic toxicity of native antibodies or antibodies conjugated with harmful compounds such as antibody-drug conjugates.

In one aspect, the polymer of the polymer-protein conjugates can be a zwitterionic polymer. The zwitterionic polymer can be modified via the addition of at least one maleimide end group. In another aspect, the zwitterionic polymer can be based on phosphorylcholine. In general, the phosphorylcholine-based polymer can have the following structure:

In one aspect, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ can each independently be selected from hydrogen or a C1-C3 alkyl group (e.g., -CH₃). In one aspect, n can be about 25 to 400. In another aspect, n can be about 50 to 200, or about 75 to 125, or about 100.

In one aspect, the polymer can be conjugated to the bioactive protein via a covalent bond. In some aspects, the bioactive protein can have at least one disulfide bond. At least one of the disulfide bonds present in the bioactive protein can be cleaved to form thiol residues. A polymer, in some aspects a zwitterionic polymer, can be conjugated to the protein via a covalent bond with at least one thiol residue. Optionally, multiple polymers (e.g., 2 to 160, 10 to 140, 20 to 120, 30 to 100, or 40 to 80) can be conjugated to the cleaved antibody in this manner by covalently bonding with different thiol residues.

In one aspect, the bioactive protein of the polymer-protein conjugate can be an antibody. The antibody can be a member of the immunoglobulin G (IgG) isotype. The antibody can be a member of the subclass lgG1 , for example trastuzumab, cetuximab, panitumumab, denosumab, pertuzumab, or avelumab. In one aspect, at least one of the interchain disulfide bonds of the IgG antibody can be cleaved, resulting in thiol residues. A polymer, in some aspects a zwitterionic polymer, can be conjugated to the antibody via a covalent bond with at least one thiol residue of the cleaved antibody. Optionally, multiple polymers (e.g., 2 to 160, 10 to 140, 20 to 120, 30 to 100, or 40 to 80) can be conjugated to the cleaved antibody in this manner by covalently bonding with different thiol residues.

In one aspect, the polymer-protein conjugates can be formulated to penetrate the BBB in a subject to treat a condition of the central nervous system. The condition to be treated in a subject (e.g., mammal) in need of treatment can include those for which the antibody is directed towards. The condition can be a brain disease (e.g., brain tumors, Alzheimer’s disease, stroke, Parkinson’s disease), a disease such as cancer, or the like. The polymer-protein conjugates can further comprise a pharmaceutically-acceptable carrier to form a pharmaceutical composition that can be formulated for administering to a subject.

A method of making a polymer-protein conjugate is disclosed. The method can include synthesizing at least one biocompatible polymer, such as a zwitterionic polymer. In one aspect, the synthesizing step can include adding at least one maleimide end group to the polymer. The protein and the polymer can be mixed together to allow the polymer to form at least one covalent bond with the protein.

In another aspect, a method of making a polymer-antibody conjugate is disclosed. The method can include synthesizing at least one biocompatible polymer, such as a zwitterionic polymer. In one aspect, the synthesizing step can include adding at least one maleimide end group to the polymer. The method of making can further include cleaving at least one interchain disulfide bond of the antibody to create thiol residues. The cleaved antibody and the polymers can be mixed together to allow the polymers to covalently bond to the antibody. In one aspect, the zwitterionic polymers can covalently bond to the thiol residues of the cleaved antibody. Additional features are provided in Example 1.

Pharmaceutical Formulations and Routes of Administration

Embodiments of the present disclosure include the agent (e.g., the polymer-protein conjugates) as identified herein and can be formulated with one or more pharmaceutically acceptable excipients, diluents, carriers and/or adjuvants. In addition, embodiments of the present disclosure include the agent formulated with one or more pharmaceutically acceptable auxiliary substances. In particular the agent can be formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and/or adjuvants to provide an embodiment of a composition of the present disclosure.

A wide variety of pharmaceutically acceptable excipients are known in the art. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7^th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3^rd ed. Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

In an embodiment of the present disclosure, the agent can be administered to the subject using any means capable of resulting in the desired effect. Thus, the agent can be incorporated into a variety of formulations for therapeutic administration. For example, the agent can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, the agent may be administered in the form of its pharmaceutically acceptable salts, or a subject active composition may be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the agent can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

Embodiments of the agent can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

Embodiments of the agent can be utilized in aerosol formulation to be administered via inhalation. Embodiments of the agent can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, embodiments of the agent can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Embodiments of the agent can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compositions. Similarly, unit dosage forms for injection or intravenous administration may comprise the agent in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

Embodiments of the agent can be formulated in an injectable composition in accordance with the disclosure. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient (triamino-pyridine derivative and/or the labeled triamino-pyridine derivative) encapsulated in liposome vehicles in accordance with the present disclosure.

In an embodiment, the agent can be formulated for delivery by a continuous delivery system. The term “continuous delivery system” is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.

Mechanical or electromechanical infusion pumps can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; and the like. In general, delivery of the agent can be accomplished using any of a variety of refillable, pump systems. Pumps provide consistent, controlled release over time. In some embodiments, the agent can be in a liquid formulation in a drug-impermeable reservoir, and is delivered in a continuous fashion to the individual.

In one embodiment, the drug delivery system is an at least partially implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices well known in the art. An implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body. Subcutaneous implantation sites are used in some embodiments because of convenience in implantation and removal of the drug delivery device.

Drug release devices suitable for use in the disclosure may be based on any of a variety of modes of operation. For example, the drug release device can be based upon a diffusive system, a convective system, or an erodible system (e.g., an erosion-based system). For example, the drug release device can be an electrochemical pump, osmotic pump, an electroosmotic pump, a vapor pressure pump, or osmotic bursting matrix, e.g., where the drug is incorporated into a polymer and the polymer provides for release of drug formulation concomitant with degradation of a drug-impregnated polymeric material (e.g., a biodegradable, drug-impregnated polymeric material). In other embodiments, the drug release device is based upon an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, etc.

Drug release devices based upon a mechanical or electromechanical infusion pump can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like. In general, a subject treatment method can be accomplished using any of a variety of refillable, non-exchangeable pump systems. Pumps and other convective systems are generally preferred due to their generally more consistent, controlled release over time. Osmotic pumps are used in some embodiments due to their combined advantages of more consistent controlled release and relatively small size (see, e.g., PCT published application no. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and 5,728,396). Exemplary osmotically-driven devices suitable for use in the disclosure include, but are not necessarily limited to, those described in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631 ; 3,916,899; 4,016,880; 4,036,228; 4,111 ,202; 4,111 ,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396; and the like.

In some embodiments, the drug delivery device is an implantable device. The drug delivery device can be implanted at any suitable implantation site using methods and devices well known in the art. As noted herein, an implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body.

In some embodiments, the agent can be delivered using an implantable drug delivery system, e.g., a system that is programmable to provide for administration of the agent. Exemplary programmable, implantable systems include implantable infusion pumps. Exemplary implantable infusion pumps, or devices useful in connection with such pumps, are described in, for example, U.S. Pat. Nos. 4,350,155; 5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171 ,276; 6,241 ,704; 6,464,687; 6,475,180; and 6,512,954. A further exemplary device that can be adapted for the present disclosure is the Synchromed infusion pump (Medtronic).

Suitable excipient vehicles for the agent are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Methods of preparing such dosage forms are known, or will be apparent upon consideration of this disclosure, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.

Compositions of the present disclosure can include those that comprise a sustained- release or controlled release matrix. In addition, embodiments of the present disclosure can be used in conjunction with other treatments that use sustained-release formulations. As used herein, a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-based hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. A sustained- release matrix desirably is chosen from biocompatible materials, but not limited, such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxcylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. Illustrative biodegradable matrices include a polylactide matrix, a polyglycolide matrix, and a polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) matrix.

In another embodiment, the pharmaceutical composition of the present disclosure (as well as combination compositions) can be delivered in a controlled release system. For example, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (Sefton (1987). CRC Crit. Ref. Biomed. Eng. 14:201 ; Buchwald et al. (1980). Surgery 88:507; Saudek et al. (1989). N. Engl. J. Med. 321 :574). In another embodiment, polymeric materials are used. In yet another embodiment a controlled release system is placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose. In yet another embodiment, a controlled release system is placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic. Other controlled release systems are discussed in the review by Langer (1990). Science 249:1527- 1533.

In another embodiment, the compositions of the present disclosure (as well as combination compositions separately or together) include those formed by impregnation of the agent described herein into absorptive materials, such as sutures, bandages, and gauze, or coated onto the surface of solid phase materials, such as surgical staples, zippers and catheters to deliver the compositions. Other delivery systems of this type will be readily apparent to those skilled in the art in view of the instant disclosure.

Dosages

Embodiments of the agent (e.g., the polymer-protein conjugates) can be administered to a subject in one or more doses. Those of skill will readily appreciate that dose levels can vary as a function of the specific the agent administered, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

In an embodiment, multiple doses of the agent are administered. The frequency of administration of the agent can vary depending on any of a variety of factors, e.g., severity of the symptoms, and the like. For example, in an embodiment, the agent can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid). As discussed above, in an embodiment, the agent is administered continuously.

The duration of administration of the agent, e.g., the period of time over the agent is administered, can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, the agent in combination or separately, can be administered over a period of time of about one day to one week, about two weeks to four weeks, about one month to two months, about two months to four months, about four months to six months, about six months to eight months, about eight months to 1 year, about 1 year to 2 years, or about 2 years to 4 years, or more.

Dosage at concentrations as high as 60 micrograms/kilograms that are non-toxic can be used. Also, lower concentrations, such as 1-4 micrograms/kilogram, show biological activity in in vivo systems depending on the disease types. The concentration in in vitro established at 10⁹-10⁶ M are active and this concentration is expected to be achieved in the cell environment. (See Slominski AT, Janjetovic Z, Fuller BE, Zmijewski MA, Tuckey RC, et al. (2010) Products of vitamin D3 or 7-dehydrocholesterol metabolism by cytochrome P450scc show anti-leukemia effects, having low or absent calcemic activity. PLoS ONE 5(3): e990; Slominski AT, Kim T-K., Janjetovic Z, Tuckey RC, Bieniek, R, Yue Y, Li W, Chen J, Miller D, Chen T, Holick M (2011) 20-hydroxyvitamin D2 is a non-calcemic analog of vitamin D with potent antiproliferative and prodifferentiation activities in normal and malignant cells. Am J Physiol: Cell Physiol 300:C526-C541 ; Wang J, Slominski AT, T uckey RC, Janjetovic Z, Kulkarni A, Chen J, Postlethwaite A, Miller D, Li W (2012) 20-Hydroxylvitamin D₃ possesses high efficacy against proliferation of cancer cells while being non-toxic. Anticancer Res 32: 739-746; Slominski A, Janjetovic Z, Tuckey RC, Nguyen MN, Bhattacharya KG, Wang J, Li W, Jiao Y, Gu W, Brown M, Postlethwaite AE (2013) 20-hydroxyvitamin D3, noncalcemic product of CYP11 A1 action on vitamin D3, exhibits potent antifibrogenic activity in vivo. J Clin Endocrinol Metab 98, E298-E30; Chen, J., J. Wang, T. Kim, E. Tieu, E. Tamg, Lin Z, D. Kovacic, D. Miller, A. Postlethwaite, R. Tuckey, A. Slominski and W. Li (2014). Novel Vitamin D Analogs as Potential Therapeutics: The Metabolism, Toxicity Profiling, and Antiproliferative Activity. Anticancer Res 34: 2153-2163.)

In an aspect, the dosage for administering to a subject (e.g., a mammal such as a human) having a condition (e.g., COVID-19) of any single agent the present disclosure is about 2 to 60 micrograms/kilogram or a combination of agents, each agent can be about 2 to 60 micrograms/kilogram.

Routes of Administration

Embodiments of the present disclosure provide methods and compositions for the administration of the agent (e.g., the polymer-protein conjugates) to a subject (e.g., a human) using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.

Routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. An agent can be administered in a single dose or in multiple doses.

Embodiments of the agent can be administered to a subject using available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the disclosure include, but are not limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administration include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration can be conducted to effect systemic or local delivery of the agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.

In an embodiment, the agent can also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not limited to, oral and rectal (e.g., using a suppository) delivery. Methods of administration of the agent through the skin or mucosa include, but are not limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration. For transdermal transmission, absorption promoters or iontophoresis are suitable methods, lontophoretic transmission may be accomplished using commercially available "patches" that deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.

While embodiments of the present disclosure are described in connection with the Examples and the corresponding text and figures, there is no intent to limit the disclosure to the embodiments in these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

EXAMPLE 1

Antibodies have been widely employed as therapeutics and diagnostics due to their high binding affinity, specificity, and biocompatibility compared with other molecules (1). To date, more than 100 antibody-based therapeutics have been approved by the Food and Drug Administration (FDA) for treatments of various diseases, including cancers (2), autoimmune diseases (3), infectious diseases (4), and metabolic diseases (5). However, the merit of antibody therapeutics is negated in treating brain diseases, even though many antibodies have been proven to target the pathological antigens of brain diseases effectively ex vivo (6, 7, 8). Such a limitation mainly results from the blood-brain barrier (BBB) that separates the peripheral circulating system from the brain via tight junctions and restricted transcytosis (9, 10). Generally, only 0.1 -0.2% of antibodies infused to the blood can penetrate the BBB and be deposited in the brain (11). Therefore, an effective and biocompatible brain delivery system would boost antibody applications and change the landscape of treatment regimens for brain diseases.

The current strategies mainly utilize receptor-mediated transcytosis through the endothelial cells in the BBB (12). BBB-penetrating ligands have been developed to target the receptors on the BBB, including glucose transporter-1 (13), low-density lipoprotein receptor- related protein-1 (14), and transferrin receptor (15). Antibodies are fused or conjugated with these ligands to facilitate their entry into the brain (16). Yet the ligands are either derived from microbes/toxins such as rabies virus (17) that are highly immunogenic or endogenous proteins like lipoproteins (18) that are highly hydrophobic or charged. Thus, antibodies with those ligands suffer from undesirable changes in surface properties, which may compromise antibody blood circulation and functions and expose the patients to high risks.

Poly 2-Methacryloyloxyethyl phosphorylcholine (PMPC) is a biocompatible, non- immunogenic polymer approved by the FDA as a coating material in transplantable devices (19,20). Our group has discovered that PMPC polymers can interact with nicotinic acetylcholine receptors and choline transporters similarly to acetylcholine and choline (21). Such interactions facilitate receptor-mediated transcytosis by endothelial cells of the BBB, enhancing PMPC delivery to the brain. Utilizing PMPC and designated crosslinkers, we have encapsulated various macromolecular cargos within PMPC shells, termed MPC nanocapsules, and demonstrated prolonged blood circulation, reduced immunogenicity, and enhanced brain delivery in mice and non-human primates (22, 23, 24, 25). Moreover, the nanocapsule surface can be modified with target-specific ligands, which further guide it to disease sites after brain entry. For instance, we encapsulated the therapeutic antibody rituximab (RTX) that targets CD20 of B-cell lymphoma into MPC nanocapsules with degradable crosslinkers, followed by conjugation of CXCL13 as a ligand (nRTX^CXCL13) on the surface to target B-cell lymphomas (24). We demonstrated superior brain delivery via MPC nanoencapsulation, tumor targeting through CXCL13, and complete elimination of brain- metastasized B-cell lymphoma by releasing RTX in the brain. This could not be achieved by native RTX, suggesting that PMPC engineering is a powerful strategy for brain delivery of therapeutic antibodies.

The current methodology of MPC nanoencapsulation fabricates an MPC network surrounding the surface of antibodies to form an MPC shell (26). The polymer network protects the antibody from immune surveillance and minimizes on-target/off-tumor toxicity but also conceals the epitope recognition and biological activities, necessitating the addition of targeting ligands, such as CXCL13, and on-site antibody release following the destruction of the shell via degradable crosslinkers. However, the disease-associated microenvironment often lacks or differs in stimuli, such as acidity and overexpression of certain enzymes that can trigger the degradation of crosslinkers (27). Moreover, some types of cancer, like glioblastoma, differ in the microenvironment, leading to difficulty in selecting targeting ligands (28). In those cases, it is more favorable to endow antibodies with enhanced BBB penetrability while maintaining their biofunctionality. Inspired by ligand antibody fusion methodology, we hypothesize that direct conjugation of PMPC to antibodies can preserve epitope recognition while enhancing brain entry.

The majority of FDA-approved therapeutic antibodies are IgG 1 subtype (29). In the IgG 1 structure, four interchain disulfide bonds are located in the hinge and near-hinge area distant from the functional binding epitopes in the Fab and Fc domains (30). Those disulfide bonds can be cleaved into thiol groups by reductive reagents without disturbing the integrity or binding affinity of the antibody, which can be further coupled to thiol-reactive species (31). It has been reported that site-specific conjugation of polyethylene glycol to the thiol groups of the Fab doesn't interfere with epitope recognition (32). We reason that PMPC can be conjugated to the thiol groups cleaved from the interchain disulfide bridges of IgG 1 antibodies to avoid masking the binding epitopes. We synthesized a series of PMPC polymers with three lengths by reversible addition-fragmentation chain-transfer (RAFT) polymerization. Then, we conjugated these polymers to trastuzumab (Tmab), which targets human epidermal growth factor receptor 2 (HER2) (Figure 1). We investigated the impact of PMPC conjugation length on target recognition, cellular internalization, antibody-dependent cellular phagocytosis (ADCP), and brain delivery efficiency. With an optimal length of PMPC, we achieved effective brain delivery of conjugated antibody over 4.5 times higher than the native counterpart with its biofunctionality maintained. This simple strategy paves the way for novel approaches to brain delivery of antibody therapeutics toward clinical practice.

Materials and methods Materials

Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Trastuzumab (Tmab) was purchased from Bio X Cell (Lebanon, NH). Human IgG, Corning transwell with permeable polyester membrane inserts, and Pierce bicinchoninic acid (BCA) colorimetric protein assay kit were purchased from Thermo Fisher Scientific (Waltham, MA). 5-Carboxytetramethylrhodamine (TAMRA) N-hydroxysuccinimide (NHS) Ester and Alexa fluor 647 (AF647) NHS ester were purchased from Click Chemistry Tools (Scottsdale, AZ). PD-10 desalting column, zeba desalting column, dialysis tubing (Molecular weight (MW) cutoff 3 KD), 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), NHS, 2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044), Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1 -carboxylate (Sulfo-SMCC), and Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Fisher scientific. SKOV-3, THP-1 , Jurkat, and bEND.3 cells were purchased from American Type Culture Collection (ATCC). The parental Jurkat cells were transduced with a lentiviral vector encoding human HER2 or mCherry to obtain HER2+ mCherry+ Jurkat cells, as reported . Human glioblastoma cell line, JX14P cells were derived from a glioblastoma biopsy from the patient (33). Iscove's Modified Dulbecco's Medium (IMDM), GlutaMax, Antibiotic-Antimycotic solution, Trypsin-EDTA, and heat-inactivated fetal bovine serum (FBS) were obtained from Corning (Corning, NY). Cell Counting Kit-8 (CCK-8) was purchased from ApexBio Technology LLC (Houston, TX). Corning transwell with permeable polyester membrane, CellTrace™ Far Red Cell Proliferation Kit, Pierce BCA protein assay kit, Lysotracker deep red, and 96 ELISA well-plates were purchased from Thermo Fisher Scientific. The capture (goat anti-human IgG Fab) and detection (HRP-conjugated goat anti-human IgG (H+L)) antibodies were purchased from BioRad Laboratories (Hercules, CA). NOD.Cg-Prkdc scid H2rg tm1 Wjl /SzJ (NSG) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) colorimetric assay kits were purchased from Cayman Chemicals (Ann Arbor, Ml). Mouse anti-glial fibrillary acidic protein (GFAP) antibody was purchased from Biolegend (San Diego, CA). Rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1) antibody was purchased from FujiFILM Pure Chemical Corporation (Richmond, VA). AF647 labeled donkey anti-mouse IgG and Alexa fluor 488 (AF488) labeled goat anti-rabbit IgG antibodies were purchased from Jackson immunoresearch laboratories, Inc (West Grove, PA).

Synthesis of PMPC polymers

PMPC polymers with carboxyl end groups were synthesized following a fast RAFT polymerization procedure (34) with MPC as the monomer, 4-Cyano-4- (phenylcarbonothioylthio)pentanoic acid (CPPA) as the chain transfer agent, and VA-044 as the initiator. Briefly, 2 mM MPC and VA-044 were mixed and dissolved in 500 L cell culture grate water (Corning), followed by CPPA (10% w/v in dimethyl sulfoxide, (DMSO). The molar ratios of VA-044 to MPC were set to 1000:1 , 500:1 , and 250:1 , while those of CPPA to MPC were set to 200:1 , 100:1 , 50:1 to synthesize PMPC200, PMPC100, and PMPC50. The polymerization was allowed to stir at 95 °C for 3 min, followed by cooling down in liquid nitrogen to stop the reaction. Then the solutions were dialyzed against water for 24 h to remove the unreacted materials. The obtained PMPC polymers were stored in an aqueous solution at -80 °C for the next step.

Synthesis of Maleimide-modified PMPC polymers

The terminal groups of PMPC polymers were first converted into primary amine groups. The carboxyl groups were activated by EDC and NHS at molar ratios to PMPC of 10:1 and 4:1 , respectively, in 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 5.0, 100 mM). The activation was allowed to stir at 4 °C for 1 h, adding ethylenediamine (molar ratio to PMPC at 20:1). The pH value was quickly adjusted to 8.0, followed by stirring at room temperature for 2 h. The reaction mixture was dialyzed against water (MW cutoff: 3 KD) for 24 h to remove the unreacted species. The resultant polymer solutions were reacted with Sulfo-SMCC (molar ratio to PMPC at 2:1) at 4 °C for 2 h, then passed through Zeba desalting columns to obtain maleimide-modified PMPC polymers. The polymer solution was lyophilized and stored at -80 °C.

Fluorescent labeling of Tmab Tmab was modified by TAMRA or AF647 to the surface lysine groups, respectively. For TAMRA conjugation, TAMRA NHS ester (1% w/v in DMSO) was added to Tmab (2 mg/mL) solution in PBS at a molar ratio of 10:1 and incubated at 4 °C for 2 h. The Tmab solution was then passed through PD-10 disalting column to remove the unconjugated TAMRA and stored at 4 °C. The conjugation of AF647 followed the protocol above with an AF647 NHS ester to Tmab molar ratio of 2:1.

Conjugation of PMPC polymers to Tmab

The interchain disulfide bridges of Tmab were converted into thiol groups by TCEP following a published protocol (35). 2 mL of Tmab (2 mg/mL) in PBS was added by 0.16 mL of TCEP (1 mg/mL) (molar ratio: 40:1), and 20 pL of EDTA (0.5 M), followed by incubation at 37 °C for 2 h. The T mab solution was then purified by passing through PD-10 column to remove extra TCEP. The concentration of Tmab in the solution was determined by BCA assay, as described below. The thiol group number per antibody was measured by the Ellman assay as published (36). Then maleimide-modified PMPC polymers were added to the Tmab solution (molar ratio of PMPC to Tmab at 100:1) and incubated at 4 °C for 12 h for PMPC conjugation. The unconjugated polymers were removed by passing through an ultrafilter (MW cutoff: 100 KD) 5 times. The resultant Tmab^PMPCs were stored at 4 °C for further experiments. The synthesis of fluorescence labeled Tmab^PMPCs followed the same protocol as described above with AF647 or TAMRA labeled Tmab.

Synthesis of Tmab encapsulated MPC nanocapsule (nTmab)

Tmab was encapsulated into nanocapsules via in situ polymerization as reported previously (21) using MPC (50% w/v in water) as the monomer, glycerol dimethacrylate (GDMA, 5% w/v in DMSO) as the crosslinker, ammonium persulfate (APS, 10% w/v in PBS) as the initiator, tetramethylethylenediamine (TEMED, 10% w/v in PBS) as the catalyst. Tmab in PBS (1 mg/mL) was mixed with MPC, GDMA, and APS at the molar ratios of 1 : 12000: 1200: 2000, after which the polymerization was initiated by adding TEMED solution (molar ratio 1 :1 to APS). The mixture was incubated at 4 °C for 2 h, followed by dialysis against PBS to remove unreacted reagents. The resultant nTmab was stored at 4 °C for further experiments.

Protein concentration assay

Antibody content in the form of Tmab^PMPC and nTmab was determined by BCA colorimetric protein assay. Briefly, a tartrate buffer (pH 11 .25) containing BCA (25 mM), CuSO₄ (3.2 mM), and Tmab samples were incubated at 37 °C for 2 h. After the reaction was cooled to room temperature, the absorbance reading at 562 nm was determined with a UV/Vis spectrometer. Native Tmab was used to generate a standard curve to calculate the Tmab concentration in samples.

SDS-PAGE and dynamic light scattering (DLS) measurement The conjugation of PMPC was validated through SDS-PAGE gel electrophoresis (4- 12% Precast gel, Nakalai USA, San Diego, CA). 5 pg of Tmab, Tmab after disulfide- cleavage, or Tmab^PMPCs were mixed with 5x protein loading dye. Gel electrophoresis was run in 1x SDS running buffer (Nakalai USA) at 160 mV for 2 h. Gels were stained overnight in Coomassie blue staining solution, followed by destaining in a methanol/acetic acid mixture to visualize the bands. Gel images were taken with an iBright 1500 gel imaging system (Thermo Fisher Scientific, Waltham, MA). The size distributions of Tmab^PMPCs and nTmab were determined by DLS at the Tmab concentration of 1 mg/mL in PBS solutions at 25 °C with a DynaPro NanoStar II (Wyatt, Santa Barbara, CA). DLS measurements were performed 3 times to determine the average diameter.

Cell culture and Cellular viability measurement

THP-1 and bEND.3 cells were cultured in IMDM supplemented with 10% FBS, 1% GlutaMax, and 1 % Antibiotic-Antimycotic. JX14P cells were cultured in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (37). HER2+ mCherry+ Jurkat cells were cultured in IMDM supplemented with 10% FBS, 1% GlutaMax, 1% Antibiotic-Antimycotic, Doxycycline (1 pg/mL), and neomycin (250 pg/mL).

To measure the cytotoxicity of Tmab^PMPC, bEND.3 cells and JX14P cells were selected as the model BBB endothelial cells and glial cells, respectively. bEND.3 cells and JX14P cells were seeded to 96-well plates (10,000 cells per well). After 24 h, 100 pL PMPC200 or Tmab^PMPC200 in cell culture medium (concentration ranging from 5 to 1000 pg/mL) was added to each well and incubated at 37 °C for 72 h. The cell viability was then measured with Cell Counting Kit-8 (UV adsorption at 450 nm).

Binding affinity assay

To investigate the binding affinity of Tmab^PMPCs to HER2, SKOV-3 was used as the HER2+ cell line. SKOV-3 cells were seeded on glass coverslips. After 24 h, cells were incubated with AF647 labeled Tmab^PMPCs, Tmab, or nTmab at the final Tmab concentration of 1 pg/mL at 37 °C for 2 h, followed by 4% paraformaldehyde for 15 min at room temperature for fixation after 3 washes with PBS. Nuclei were stained by 10 pg/mL Hoechst 33342 at room temperature for 20 min. Fluorescent images were taken with a Nikon A1 R/SIM confocal microscope (Nikon, Melville, NY).

The half maximal effective concentration (EC50) of each Tmab sample was determined as follows: 0.2 million SKOV-3 cells in FACS buffer (5% FBS in PBS, 2 mM EDTA, 0.02% NaN₃) were incubated with AF647 labeled Tmab, Tmab^PMPCs, and nTmab with a Tmab concentration gradient ranging from 0.002 to 400 nM at 4 °C for 1 h. The cells were then washed and fixed. The mean fluorescent intensity (MFI) of AF647 was measured with a FACSymphony flow cytometer (BD Biosciences, San Jose, CA). The EC50 was determined by analyzing the profiles curved from the mean fluorescent intensity at each concentration. ADCP assay

The ADCP ability of Tmab^PMPC was determined as follows: THP-1 cells were used as the phagocytic cell line, while HER2+ mCherry+ Jurkat cells were used as the target cell line. THP-1 cells were labeled with CellTrace™ Far Red Cell Proliferation Kit at room temperature for 30 min. 0.5 million Jurkat cells were incubated with Tmab^PMPCs, Tmab, or nTmab at room temperature for 30 min and then added to Far-red labeled THP-1 cells (1 x 10⁵). The cells were incubated at 37 °C for 2 h and then fixed with 4% formaldehyde in PBS. The phagocytic levels were determined by flow cytometry.

Tmab internalization

The levels of Tmab internalization were determined in SKOV-3 cells and bEND.3 cells. For the assay in SKOV-3 cells, TAMRA-labeled Tmab^PMPCs, Tmab, or nTmab (50 pg/mL each) were incubated with SKOV-3 cells at 37 °C for 24 h in a 12-well plate, followed by 3 PBS washes. The cells were then incubated with AF488-labeled anti-human IgG antibody (10 pg/mL) at 4 °C for 30 min to stain the Tmab bound on the surface of cells. Following fixation by 4% formaldehyde, the nuclei were stained by Hoechst 33342. The fluorescent images were taken with a Nikon A1 R/SIM confocal microscope. Pearson correlation coefficients were obtained by analyzing 3 different areas with the colocalizationfinder plugin in ImageJ.

For the assay in bEND.3 cells, cells were incubated with TAMRA-labeled T mab^PMPCs, Tmab, or nTmab (50 pg/mL each) at 37 °C for 2 h, followed by 3 PBS washes. The cells were then harvested by trypsin-EDTA digestion and fixed with 4% formaldehyde in PBS solution. The MFI was measured with a FACSymphony flow cytometer as above. Intracellular trafficking

The intracellular trafficking of Tmab^PMPCs was investigated by measuring the colocalization of internalized Tmab and the late endosome/lysosome. bEND.3 cells were seeded onto glass coverslips, followed by adding TAMRA-labeled Tmab samples to each well at the final Tmab concentration of 50 pg/mL. The cells were incubated at 37 °C for 4 h, followed by 3 PBS washes. Lysotracker deep red in the fresh medium was added to each well at the final concentration of 50 nM and incubated at 37 °C for 30 min. The cells were then washed with PBS and fixed. Nuclei were stained with Hoechst 33342. The fluorescent images of the cells were taken with a Nikon A1 R/SIM confocal microscope. Pearson correlation coefficients were obtained by analyzing 3 different areas with the colocalizationfinder plugin in ImageJ.

Transwell assay

Transwell assay to determine the levels of transcytosis was performed as follows: bEND.3 cells were seeded on the polyester membrane inserts (pore size, 0.4 pm) of transwells at 10⁵ cells/well. The cell culture media in the apical and basolateral compartments were replaced every three days for 3 weeks. TAMRA-labeled Tmab samples in 150 pL of culture medium were added to each insert of transwells at T mab concentration of 0.05 mg/mL while the medium volume in the basolateral compartment was set to 600 pL.

200 pL of the medium in the basolateral chamber was collected at 0, 2, 4, 6, 8, and 10 h after adding samples and replenished with 200 pL fresh medium. The fluorescent intensity of the medium collected from the basolateral compartment was analyzed by a Varioskan LUX plate reader. The profile of the penetration rate as a function of time was plotted. For the transwell assay with PMPC competition, PMPC200 and Tmab^PMPC20° in cell culture medium were mixed at the PMPC molar ratio of 20:1 or 200:1 and then added to the apical chamber.

The profile of the penetration efficiency (%) vs time was plotted and fitted with the equation:

FLb X 600 PE(n - 1)(%) x 200

PEn (%)

FLa X 150 ^{X 100% +} 600

Where PEn (%) represents the penetration efficiency at a certain time point; FLb represents the fluorescent intensity of the medium taken from the basolateral chamber at that time point; PE(n-1) (%) represents the penetration efficiency (%) at the previous time point; PE0 (%) equals to 0.

To validate the epitope recognition ability of Tmab after BBB penetration, the medium in the basolateral chamber was collected 10 h post the sample addition, diluted 10 times with FACS buffer, and then incubated with SKOV-3 cells (2 x 10⁵) in FACS buffer at 4 °C for 1 h. The mean fluorescent intensity was measured by FACSymphony flow cytometer. Biodistribution

The biodistribution was investigated by optical imaging. NSG mice were intravenously administrated with 100 pL of AF647 labeled Tmab, Tmab^PMPCs, or nTmab (10 mg/kg). In vivo fluorescent imaging was performed with an MS Lumina II (Perkin Elmer, Waltham, MA) 24 h post-injection (Ex.=640 nm, Em. =710 nm). Organ imaging of the heart, liver, spleen, lungs, kidneys, and brain was performed following PBS perfusion. The quantitative measurement of the fluorescence in average radiant efficiency (brains) or total radiant efficiency (other organs) was analyzed by the region of interest (ROI) tool in Living Image® software.

Enzyme-linked immunosorbent assay (ELISA)

The delivery of Tmab to the brain was quantified by ELISA. Briefly, NSG mice were intravenously administrated with 100 pL Tmab, Tmab^PMPCs, or nTmab (10 mg/kg). The brains were harvested 24 h post-administration following PBS perfusion, homogenized, and centrifuged to collect the brain extract. A 96 ELISA well-plate was coated with anti-human IgG antibody (1 pg/mL in carbonate buffer, pH 9.5) at 4 °C overnight, followed by 3 washes with PBST (0.1% Tween in PBS). Non-specific binding was blocked with blocking buffer (2% BSA in PBST) at room temperature for 2 h, followed by 3 washes with PBST. The brain extract was then diluted 1000-fold with blocking buffer and incubated in the well at room temperature for 2 h, followed by 5 washes with PBST. Tmab and Tmab^PMPCs in blocking buffer (concentration ranging from 0 to 20 ng/mL) were used as a standard. HRP-coupled anti-human IgG antibody (10 pg/mL) was added to each well and incubated at room temperature for 1 h, followed by 5 washes with PBST. Finally, 3,3',5,5'-Tetramethylbenzidine and H2O2 solutions were added and incubated at room temperature for 15 min. The UV adsorption at 450 nm was measured with a Varioskan LUX plate reader.

Evaluation of organ damages

Liver toxicity was determined by measuring the levels of AST and ALT in plasma samples. Mice were intravenously administered 150 pL of Tmab, PMPC200, or Tmab^PMPC20° at Tmab dosage of 50 mg/kg or PMPC dosage of 90 mg/kg. PMPC dosage in PMPC200 is the same as that in Tmab^PMPC20°, as calculated by the molecular weight, average conjugation number of PMPC200 per T mab, and the molecular weight of T mab. PBS was injected as a negative control. The blood was collected from the retroorbital vein before or 72 h posttreatment into heparin-coated tubes and centrifuged to obtain the plasma as the supernatant. The levels of AST and ALT in plasma samples were measured with Cayman AST and ALT colorimetric activity assay kits.

The levels of I ba1 and GFAP were used to determine neurotoxicity in the brain. The brains were fixed in 4% paraformaldehyde overnight, followed by incubation with 15% and 30% sucrose in PBS for 6 h, respectively. The brains were processed to sections using a vibratome (VT1000S, Leica Biosystems, Dear Park, IL) in PBS with a thickness of 100 pm. Anti-lba1 and anti-GFAP antibodies were used as primary antibodies. The sections were permeablized with 1 % Triton-100 in PBS for 5 min and blocked with blocking buffer (1 % BSA, 0.2% Triton-100 in PBS) at room temperature for 2 h. The sections were incubated with anti-GFAP (1 :200) and anti- 1 ba1 (1 :500) overnight in a blocking buffer at 4 °C. Following a wash with 0.2% Triton-100 in PBS, the sections were incubated with secondary antibodies (AF488 labeled goat anti-rabbit IgG for anti-GFAP, AF647 labeled donkey anti-mouse IgG for anti-lba1) in blocking buffer for 1 h at room temperature. After washing with 0.2% Triton- 100 in PBS, the sections were analyzed on A1 R/SIM confocal microscope. The area fractions to quantify the expression levels of I ba 1 and GFAP were determined with Imaged, respectively.

Results:

Site-oriented conjugation of PMPC to Tmab retains Tmab functionalities.

As illustrated in Figure 1 A, we synthesized PMPC polymers with three different chain lengths via RAFT polymerization. The feed ratio of the monomer MPC to the chain transfer agent was set to 50:1 , 100:1 , and 200:1 , respectively, to obtain PMPC with the degrees of polymerization 50, 100, and 200, denoted as PMPC50, PMPC100, and PMPC200. The end group of PMPC polymers was modified with Sulfo-SMCC to produce maleimide-modified PMPC polymers that are thiol-reactive. To produce thiol residues on the antibody, we incubated Tmab with excess TCEP (Figure 1 B) and confirmed the disulfide-cleavage with SDS-PAGE since SDS can break down the Van der waals force and hydrogen bonds that hold the light chain and heavy chain together after disulfide-cleavage (38). As shown in the SDS-PAGE image, Tmab was mainly broken down into two distinct fragments with molecular weights of 50 KD and 25 KD, representing heavy and light chains, respectively (lane 2, Figure 2A). The average number of free thiol residues per Tmab following TCEP cleavage was calculated from the Ellman assay to be 7.1 per Tmab.

To prepare Tmab PMPC conjugates (Tmab^PMPCs), we mixed the disulfide bonds- cleaved Tmab with maleimide-modified PMPC polymers (Figure 1 B). The conjugates with corresponding PMPC lengths were denoted as Tmab^PMPC5°, Tmab^PMPC10°, and Tmab^PMPC20°, respectively. The PMPC conjugation shifts up bands depending on the feed ratios of the monomer MPC (lanes 3-5, Figure 2A), validating the successful PMPC conjugation. The average conjugation numbers of the polymer chains per antibody are 5.8, 4.7, and 4.2, which were determined by the average consumed thiol number after the conjugation through the Ellman assay. The size of each Tmab^PMPCs was determined by DLS measurements (Figure 2B). MPC nanoencapsulated Tmab (nTmab) was synthesized as reported to serve as a control (21). Compared to the average diameter of native Tmab (8 nm), that of nTmab was 22 nm. Whereas the average diameters of Tmab^PMPCs were 8-12 nm, the same or slightly larger compared to native Tmab. PMPC conjugation did not change the size intensely compared to MPC nanoencapsulation, implying a partial surface coverage by conjugated PMPC chains rather than a full coverage in nTmab.

We next tested the impact of PMPC chain length on the biofunctionalities of Tmab. The mechanisms of action of T mab in HER2+ tumor elimination mainly falls into 3 categories: 1) blockade of the surface HER2 receptor on tumor cells (39); 2) degradation of the HER2 receptor after endocytosis (40); 3) antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP) (41). To investigate the HER2-binding affinity after PMPC conjugation, we fluorescently labeled Tmab^PMPCs with AF647 and incubated them with the HER2+ human ovarian cancer cell line SKOV-3. Antibody binding was assessed by CLSM imaging and flow cytometry. CLSM images indicated that the affinity of Tmab^PMPCs decreased as the length of PMPC increased, which was more apparent in Tmab^PMPC200 than in the other two Tmab^PMPCs (Figure 2C). The loss of binding to SKOV-3 cells was also validated by flow cytometry (Figure 2D). There was a modest change in EC50 of Tmab^PMPCs depending on the PMPC chain length; EC50 of Tmab^PMPC5°, Tmab^PMPC10°, Tmab^PMPC20° was 1.2, 1.6, and 4.9 nM, respectively, which were comparable to that of Tmab (0.9 nM). Whereas nTmab did not show HER2-dependent binding due to the strong cytophobicity (20). We next tested the impact of PMPC conjugation on antibody internalization (Figure 2E). AF647-labeled Tmab^PMPCs were incubated with SKOV-3 cells for 24 h, followed by staining the Tmab on the cell surface with AF488-labeled anti-human IgG antibody. Neglectable AF488 fluorescence was observed on the cell surface incubated with Tmab, Tmab^PMPC5°, and Tmab^PMPC10°. In contrast, a definitive AF488 signal was seen in the case of Tmab^PMPC20°, which was colocalized with AF647 signals (Figure 2E). Pearson analysis revealed the highest degree of co-localization in the case of T mab^PMPC200, indicating that the conjugation of PMPC50 or PMPC100 did not interfere with the internalization of Tmab (Figure 2F). Lastly, we measured the capability of Tmab to induce ADCP. The assay was performed by using THP-1 cells as the phagocytic cell line and HER2+ mCherry+ Jurkat cells as the target cell line. The Jurkat cells were pre-incubated with each Tmab sample (5 pg/mL), followed by the incubation with Far-Red labeled THP-1 cells. Human IgG was used as a negative control. The levels of ADCP were determined by the percentage of THP-1 with phagocytosed Jurkat cells. Tmab, Tmab^PMPC50, and Tmab^PMPC10° exhibited similar levels of phagocytosis activity, which were higher than those of IgG control, nTmab, or Tmab^PMPC200. These results indicate that PMPC conjugation with shorter chains (<100) does not interfere with the induction of ADCP.

PMPC conjugation enhances BBB penetration of Tmab through the transcytosis pathway.

PMPC facilitates BBB penetration through receptor-mediated transcytosis in BBB endothelial cells (21). To study the in vitro BBB penetration efficiency of Tmab^PMPC, we incubated bEND.3 cells with TAMRA-labeled Tmab^PMPCs. Tmab and nTmab were also included as controls. The levels of internalization in bEND.3 cells were determined by measuring the MFI of TAMRA signals on flow cytometry. The MFI of Tmab^PMPC100 or Tmab^PMPC200was significantly higher than Tmab or Tmab^PMPC5°, indicating that enhanced internalization of Tmab was achieved through conjugation of PMPC with longer length (>100) (Figure 3A). To validate that the internalized Tmab^PMPC proceeded to the transcytosis pathway, we stained the late endosome and lysosome of bEND.3 cells with lysotracker. More of the TAMRA signals were localized outside of the late endosome or lysosome in bEND.3 cells treated with Tmab^PMPC10° or nTmab. In contrast, the signals of Tmab were mostly overlapped with the lysotracker signals in CLSM images (Figure 3B). Quantitative Pearson analysis revealed that there was no detectable level of differences in co-localization between the late endosome or lysosome and Tmab^PMPC10° or nTmab, which was significantly lower than that of Tmab (Figure 3C). This suggests that the internalized Tmab^PMPC is proceeded to the transcytosis pathway but not trapped in endosomal compartments. We next quantified levels of transcytosis using bEND.3 transwell assay as reported (21) (Figure 3D). Tmab samples labeled with TAMRA were added to the apical inserts of the transwell. The medium in the basolateral chamber was collected at designated time points, and the TAMRA fluorescent intensity was measured (Figure 3E). The penetration of Tmab^PMPC100 and Tmab^PMPC200 reached approximately 10% after 10 h incubation, which was lower than that of nTmab (20%) but remarkably higher than those of Tmab and Tmab^PMPC5° (2% and 2.3%). Presence of the free polymer PMPC200 interfered with transcytosis, suggesting the binding of PMPC to bEND.3 cell layers mediated the transcytosis (Figure 3F). Those findings collectively prove that the conjugation of PMPC to Tmab enhances BBB penetration through PMPC-mediated transcytosis in vitro. To confirm that Tmab maintains the epitope affinity following the transcytosis, we incubated SKOV-3 cells with the medium in the basolateral chamber 10 h post sample addition to the apical chamber and measured the MFI by flow cytometry (Figure 3G). Both Tmab^PMPC100 and Tmab^PMPC20° showed a higher MFI than that of Tmab or nTmab, suggesting that the HER2 binding of Tmab was maintained after transcytosis. Although the penetration efficiency of Tmab^PMPC200 is slightly higher than that of Tmab^PMPC10° in terms of absolute antibody amount, the penetrated Tmab^PMPC10° exhibits the highest MFI in SKOV-3 cells due to its better retainment of the binding compared to that of Tmab^PMPC200, indicating that PMPC100 is the optimal chain length to achieve the highest BBB penetration while retaining the epitope recognition.

PMPC conjugation enhances the brain delivery of Tmab.

The above results indicated that PMPC conjugation facilitated transcytosis of Tmab in vitro. We next tested the delivery of Tmab^PMPCs to the brain in vivo using NSG mice. AF647-labeled Tmab^PMPCs, Tmab, or nTmab were administered to mice via the retroorbital vein, and the fluorescent signals were imaged by IVIS imaging (Figure 4A). Native Tmab, as well as Tmab^PMPC50 were evenly distributed in mice. In contrast, Tmab^PMPC10° and Tmab^PMPC20° were enriched around the head area similar to nTmab 24 h post-injection. After a standard perfusion process, we collected the organs from these mice, including the heart, liver, spleen, lung, and kidney, and validated the biodistribution of Tmab in each organ by IVIS imaging. The organ images (Figure 4B) and the quantitative ROI data in total flux (p/s) (Figure 4C) indicated that the liver was the primary organ for Tmab accumulation, while PMPC conjugation induced no significant change in the level of liver accumulation (P value>0.65). These results indicated the PMPC conjugation didn't increase the non-specific clearance of Tmab by the reticuloendothelial system (RES) in the liver. We also collected the brains from the same mice and measured AF647 intensity by IVIS imaging (Figure 4D). The brain images and the quantitative ROI data in average radiance (p/s/cm²/sr) (Figure 4E) showed that Tmab^PMPC10°, Tmab^PMPC20°, and nTmab had similar average radiances that were over 5.5-fold of Tmab, indicating that PMPC conjugation with longer chain lengths significantly enhanced brain penetration of Tmab. To quantify the deposited Tmab level in the brain, we homogenized brain tissues after imaging and titrated the Tmab concentration by sandwich ELISA assay with anti-human IgG antibodies. Samples obtained from mice treated with Tmab^PMPC100 and Tmab^PMPC20° contained over 4-fold higher Tmab than that of native Tmab or Tmab^PMPC5°, which was consistent with the quantitative ROI data (Figure 4F). Those data collectively indicated that Tmab^PMPCs with longer chain lengths (>100 units per chain) enhanced brain delivery of T mab, which was comparable to that of the nanocapsule. PMPC conjugation doesn't induce neurotoxicity.

We next assessed the toxicities of Tmab, PMPC polymer (PMPC200), and Tmab PMPC conjugate (Tmab^PMPC20°) in in vitro and in vivo assays. The in vitro cytotoxicity assay was performed using two different cell lines; bEND.3 cells as the BBB endothelial cell model cell and JX14P cells derived from a glioblastoma patient brain biopsy as the glial cell model. Cells were incubated with Tmab, PMPC200, or Tmab^PMPC20° for 72 h. The cytotoxicity was determined by cellular viability assay with a CCK-8 assay kit (Figure 5A, B). Neither PMPC200 nor Tmab^PMPC200 exhibited detectable toxicity on both cells at the PMPC concentrations ranging from 5 to 1000 pg/mL. The toxicity was also evaluated in vivo using NSG mice. Mice were injected with Tmab, PMPC200, or Tmab^PMPC200 at the Tmab dosage of 50 mg/kg (corresponding to a dosage 4 mg/kg in humans), or PMPC dosage of 90 mg/kg (estimated by the calculation described in the Materials and Methods section) or 72 h postinjection. The plasma levels of two liver enzymes, AST and ALT, were measured to evaluate liver toxicity. There was no change in the level of AST or ALT 72 h after the treatment in all three groups compared with the plasma prior to the treatment (baseline), suggesting that PMPC or its conjugate did not induce liver toxicity (Figure 5C). To rule out the possibility of BBB disruption by PMPC, we injected Evans blue dye intravenously 24 h post-treatment, which can deposit in the brain only upon disruption of the BBB. There was no sign of Evans- blue dye in the brain of mice treated with Tmab, PMPC200, or Tmab^PMPC200, indicating that the enhanced brain delivery of Tmab via PMPC conjugation is not a result of BBB disruption (Figure 5D). At last, we tested whether PMPC conjugation induces neurotoxicity by measuring the expression of Iba1 and GFAP, as reported previously (21 , 24). Iba1 and GFAP were used as biomarkers for microglia and astrocytes in the brain, respectively, whose expressions increase upon brain damage (42). The brains were obtained from mice treated with PBS (control), Tmab, PMPC200, or Tmab^PMPC20° at the PMPC dosage of 90 mg/kg or Tmab dosage of 50 mg/kg 72 h post-treatment and processed for immunofluorescent imaging of Iba1 and GFAP. No significant difference in levels of either I ba 1 or GFAP signal density was seen in the immunofluorescent figures of the brains (Figure 5E) treated by Tmab, PMPC200, or Tmab^PMPC20°. The expression levels of Iba1 and GFAP quantified by area fraction analysis also supported this observation (Figure 5F). Those findings collectively indicated that PMPC conjugation achieved effective brain delivery of Tmab without induction of adverse effects, at least in the liver, the BBB, or the brain.

Discussion

Antibodies have emerged as the major player in precision medicine. Although an extensive antibody library has been established to target pathological antigens, their applications in treating brain diseases are limited mainly due to poor brain entry. To this end, we developed a simple methodology that directly conjugates the brain-penetrable polymer, PMPC, to specific sites of Tmab as a representative human lgG1 antibody. PMPC length of 100 units allowed effective delivery of Tmab to the brain via enhanced BBB penetration while preserving its essential functionalities, such as epitope recognition, receptor-mediated internalization, and effector function. Moreover, we did not observe detectable levels of toxicities in mice treated with T mab-conjugated with PMPC, indicating that this simple methodology for PMPC engineering of human IgG 1 confers a brain-penetrable moiety more safely and should be highly beneficial for future clinical translation.

Low reticuloendothelial system (RES) clearance is the prerequisite for targeting reagents to guarantee their targeting efficiency, which is one of the advantages of antibodies over other types of targeting reagents. A group of BBB-penetrating strategies has been reported to enhance brain delivery of therapeutic antibodies. However, many of them are peptide-based ligands that are either hydrophobic or highly charged. Such modifications induce non-specific accumulation and disrupt their long-circulating profiles, resulting in poor delivery to the target sites. PMPC is a super-hydrophilic polymer that is neutrally charged at physiological pH values. The conjugation does not change the biodistribution of Tmab in the liver, indicating that PMPC modification does not induce the RES clearance of the antibody and, thereby, could achieve a higher antigen-specific targeting efficiency than other brain targeting ligands.

A typical lgG1 antibody contains two identical light and heavy chains. It includes 16 disulfide bonds, including 4 interchain disulfide bonds in the hinge region and 12 intrachain disulfide bonds associated with 12 individual domains (43). Among those disulfide bonds, the interchain bonds are preferably cleaved by TCEP, providing 8 free thiol residues. The results from Ellman assays to determine the thiol number indicated that an average of 7.1 thiol residues were produced from one Tmab molecule while approximately 4-5 residues were consumed by PMPC conjugation. T mab functionality results indicated that the thiol-residue- specific PMPC conjugation minimized the function loss of the antibody compared with MPC nanocapsulation. Among the PMPC polymers with different degrees of polymerization, Tmab^PMPC50’ and Tmab^PMPC10° exhibited almost comparable antibody functions as Tmab, suggesting that PMPC conjugation with a degree of polymerization equal to or lower than 100 can retain the antibody functions. These studies would genuinely allow site-specific PMPC conjugation on therapeutic antibodies, beneficial for further tuning of PMPC modification.

The MPC polymer network enhanced brain delivery of therapeutic antibodies through receptor-mediated transcytosis. The surface ligand density was the most dominant viable determining the binding strength. One key concern is that the conjugated PMPC, with a linear polymer topology and partial coverage of the antibody surface, could have a lower MPC density and be insufficient to enhance brain delivery effectively. The results from both in vitro BBB penetration using bEND.3 cell layer and in vivo brain delivery experiments showed that Tmab^PMPC10° and Tmab^PMPC20° exhibited comparable levels of BBB penetration and brain deposition of T mab, which were nearly identical to those of nT mab. These results indicated that the MPC density in Tmab^PMPC100 as comparable to that in nTmab, which was required to achieve effective brain delivery. Due to the retainment of epitope recognition following transcytosis, PMPC100 is the optimal chain length to enable enhanced antibody brain delivery with preserved functionality.

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Supplemental Information for Example 1

As shown in Figure 6, the functional chemical species presenting in PMPC polymer were characterized by FT-IR analysis using a Bruker Alpha ATR-FTIR spectrometer. The ATR accessory was used for signal enhancement of chemical species by internal total reflectance. The number of scans was set to 32 with a resolution on four for all samples analyzed.

As shown in Figure 7, the number of repeat units in the PMPC backbone was characterized by proton nuclear magnetic resonance spectroscopy (¹H NMR) analysis using a Bruker 400 MHz NMR spectrometer. The PMPCs were prepared in D2O at a concentration of 1 mg/mL and analyzed at room temperature with 64 scans for determination of degree of polymerization. Additionally, the percent conversion from PMPC-COOH to PMPC-Mal was calculated by ¹H-NMR analysis, and the PMPC-Mal samples were prepared for ¹H-NMR and with 256 number of scans to resolve the end group proton signals appearing at S- 6.8 ppm. PMPC100-COOH ¹H NMR (400 MHz, D₂O, 0) from one side: 2.4-2.8 (a and b, 4H, HOOCCH2CH2-), 1.7 (c, 3H, -CH3), 1.6-2.2 (d, 2H, -CH2C-), 0.7-1.1 (e, 3H, -CCH3), 7.4-8.0 (f, g, and h, 5H, -CCHCHCHCHCH), 3.8-4.2 (i, j, and k, 6H, -COOCH2CH2OPOCH2-), 3.6 (I, 2H, -CH2N-), 3.2 (-N(CH3)3).

As shown in Figure 8, mice (n=3) were retro-orbitally injected with 100 pL AFTmab (10 mg/kg) or AF647 in PBS. AF647 was used as equivalent to the total fluorescent intensity of AFTmab. The mouse on the far left was treated with PBS as a negative control (n=1). The images were taken on an IVIS imaging system 12 hours post-injection.

As shown in Figure 9, a 96 ELISA well-plate was coated with anti-human IgG antibody (1 pg/mL in carbonate buffer, pH 9.5) at 4 °C overnight, followed by three washes with PBST (0.1% Tween in PBS). Non-specific binding was blocked with blocking buffer (2% BSA in PBST) at room temperature for two hours, followed by three washes with PBST. Tmab and AFTmab were diluted with blocking buffer (concentration ranging from 0 to 10 ng/mL) and incubated in the well at room temperature for two hours, followed by five washes with PBST. HRP-coupled anti-human IgG antibody (10 pg/mL) was added to each well and incubated at room temperature for one hour, followed by five washes with PBST. Finally, 3,3',5,5'-Tetramethylbenzidine and H₂O₂ solutions were added and incubated at room temperature for 15 minutes. The UV adsorption at 450 nm was measured with a Varioskan LUX plate reader.

As shown in Figure 10, SKOV-3 cells (2 x 10⁵) were seeded on glass coverslips in 12-well plate. After 24 hours, cells were incubated with AFTmab (2 pg/mL), AF647, AFIgG (equivalent to the fluorescent intensity of AFTmab), and PBS (10 pL) at 37 °C for two hours, followed by 4% paraformaldehyde for 15 minutes at room temperature for fixation after three washes with PBS. Nuclei were stained by 10 pg/mL Hoechst 33342 at room temperature for 20 minutes. Fluorescent images were taken with a Nikon A1 R/SIM confocal microscope.

As shown in Figure 11 SKOV-3 cells (1 x 10⁵) were incubated with AFTmab (2 pg/mL) and AF647 (equivalent to the fluorescent intensity of AFTmab) for two hours at 37 °C. The cells were then washed by PBS, harvested, and fixed. The mean-fluorescent intensity of the cell surface was measured by flow cytometry.

As shown in Figure 12, SKOV-3 cells (2 x 10⁵) in FACS buffer were included with AFTmab and Tmab (concentration ranging from 0.001 to 300 nM) for two hours at 4 oC, followed by three washes with PBS. The mean-fluorescent intensity of the cell surface was measured by flow cytometry.

Supplemental Table 1 . The summary of DP, yield, and modification ratio of PMPC-COOHs, and PMPC-Mals with different designated feed ratios.

It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1 percent to about 5 percent” should be interpreted to include not only the explicitly recited concentration of about 0.1 weight percent to about 5 weight percent but also include individual concentrations (e.g., 1 percent, 2 percent, 3 percent, and 4 percent) and the sub-ranges (e.g., 0.5 percent, 1.1 percent, 2.2 percent, 3.3 percent, and 4.4 percent) within the indicated range. The term “about” can include traditional rounding according to significant figures of the numerical value. In addition, the phrase “about ‘x’ to ‘y’” includes “about x’ to about ‘y

Many variations and modifications may be made to the above-described aspects. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

Claims

CLAIMS What is claimed:

1. A composition comprising: a bioactive protein and a at least one zwitterionic polymer, wherein the zwitterionic polymer is conjugated to at least one site of the protein.

2. The composition of claim 1 , wherein the protein comprises a at least one disulfide bond.

3. The composition of either claim 1 and 2, wherein the protein is an antibody.

4. The composition of claim 3, wherein the antibody is a member of the IgG isotype.

5. The composition of claim 3, wherein the antibody is a member of the IgG 1 subclass.

6. The composition of claim 3, wherein the antibody is trastuzumab.

7. The composition of any one of claims 1-6, wherein the zwitterionic polymer is conjugated to the protein via a at least one covalent bond.

8. The composition of any one of claims 1-6, wherein the at least one disulfide bond of the protein is cleaved.

9. The composition of claim 8, wherein the zwitterionic polymer is bonded to a at least one thiol group of the at least one cleaved disulfide bond.

10. The composition of any one of claims 1-9, wherein the zwitterionic polymer comprises a at least one maleimide end group.

11 . The composition of any one of claims 1-9, wherein the zwitterionic polymer is a phosphorylcholine-based polymer.

12. The composition of any one of claims 1-9, wherein the zwitterionic polymer has the following structure:

13. The composition of claim 12, wherein n is an integer from 50 to 200.

14. The composition of claim 12, wherein n is 100.

15. A pharmaceutical composition comprising a therapeutically effective amount of the composition of any one of claims 1 to 14 and a pharmaceutically-acceptable carrier, formulated for administering to a subject.

16. A method of making a zwitterionic polymer- protein conjugate comprising: synthesizing a at least one zwitterionic polymer and covalently bonding the at least one zwitterionic polymer to a bioactive protein.

17. The method of claim 16, wherein the protein comprises a at least one disulfide bond.

18. The method of claim 17, wherein a at least one disulfide bond of the protein is cleaved before bonding the at least one zwitterionic polymer to the protein.

19. The method of claim 18, wherein the at least one zwitterionic polymer is bonded to a at least one thiol group of the at least one cleaved disulfide bond.

20. A method of making a zwitterionic polymer-antibody conjugate comprising: synthesizing a at least one zwitterionic polymer, cleaving at least one interchain disulfide bond of an antibody, and mixing the cleaved antibody with the zwitterionic polymers to form an antibody covalently bonded to the at least one zwitterionic polymer.

21 . The method of claim 20, wherein the zwitterionic polymer is conjugated to a at least one thiol group of the cleaved antibody.

22. The method of claim 20, wherein the antibody is a member of the IgG isotype.

23. The method of claim 20, wherein the antibody is selected from the IgG 1 subclass.

24. The method of claim 20, wherein the IgG antibody is trastuzumab.

25. The method of any one of claims 16-24, wherein the zwitterionic polymer comprises a at least one maleimide end group.

26. The method of any one of claims 16-24, wherein the zwitterionic polymer is a phosphorylcholine-based polymer.

27. The method of any one of claims 16-24, wherein the zwitterionic polymer has the following structure:

28. The method of claim 27, wherein n is an integer from 50 to 200.

29. The method of claim 27, wherein n is 100.

30. A method of treating a condition of the central nervous system by penetrating a blood brain barrier of a subject comprising: administering to the subject in need thereof, a pharmaceutical composition, wherein the pharmaceutical composition includes a therapeutically effective amount of the composition or pharmaceutical composition of any one of claims 1 to 15.