Peg suppression of anaphylaxis

Abstract

Described herein are compositions and methods for reducing or preventing a hypersensitivity reaction in a subject. In particular, described are compositions and methods for reducing or prophylactically preventing hypersensitivity reactions in a subject otherwise due to a response to the PEG portion of PEGylated compounds and substrates, including PEGylated drugs and vaccines.

Description

PEG SUPPRESSION OF ANAPHYLAXISCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to 63 / 610,090, titled “PEG SUPRESSION OF ANAPHYLAXIS,” filed on December 14, 2023 and herein incorporated by reference in its entirety.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01 HL141934, awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE

[0003] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.BACKGROUND

[0004] Polyethylene glycol (PEG) is frequently covalently grafted to a variety of nanoparticle and protein therapeutics. The highly flexible and hydrophilic nature of PEG creates a non-fouling coating that reduces the adsorption of opsonins on nanoparticles and liposomes, and markedly increases the hydrodynamics diameter of various biomacromolecules. Both effects lead to slower hepatic and renal clearance and consequently prolonged circulation. PEG can also reduce the immunogenicity of the underlying drug, enabling repeated dosing. There are now over 30 PEGylated protein and liposome products on the market. Some, such as Krystexxa (PEG- uricase) for the treatment of chronic refractory tophaceous gout, represent the last-line therapy. Others, such as Doxil / Caelyx (PEGylated liposomal doxorubicin), are part of the most effective treatment available for a number of cancers. PEGylated drugs have been dosed into billions of individuals worldwide, as both Pfizer / BioNTech and Modema COVID mRNA vaccines are formulated with lipid-PEG conjugates. PEG can be expected to continue to be a main fixture in medicine for the considerable future.

[0005] Despite the advantages of PEGylation, repeated dosing of some PEGylated drugs can induce elevated titers of anti-PEG antibodies (APA) within days. Unlike other anti-drug antibodies (ADA), there is a naturally high prevalence of pre-existing APA, with detectible APA in at least about 70% of the general population. This high pre-existing prevalence is consistent with T-independent mechanism of APA induction, which bypasses the conventional process of somatic hypermutation to evolve high affinity antibodies specific to an antigen. Thus, the high prevalence of pre-existing APA likely reflects both high homology between APA and select germline sequence of B-cells in most individuals, and also the almost ubiquitous presence of PEG in many hygiene, skincare, and food products. Pre-existing APA may mean that a subject will develop pseudoallergic or other hypersensitivity reactions (HSRs), an adverse event commonly called infusion reaction. Symptoms of hypersensitivity reactions include shortness of breath, facial redness, chest pain, flashing and rash. While most HSRs spontaneously resolve or can be managed, some cases can escalate into anaphylactic shock. PEGylated nanomedicines that have been reported to cause such reactions include PEGylated liposomal doxorubicin (Doxil / Caelyx), PEGylated G-CSF (pegfilgrastim, Neulasta), PEGylated erythropoietin (mono- mPEG-epoetin-p, Mircera), PEGylated recombinant human factor VIII (Adynovate), and PEGylated phenylalanine ammonia lyase (pegvaliase-pqpz, Palynziq). At least three PEGylated drugs have been withdrawn from clinical use partly due to severe HSRs. This underscores an urgent need for methods that can alleviate allergic reactions to PEGylated drugs.SUMMARY

[0006] Covalent conjugation of polyethylene glycol (PEG) is frequently employed to enhance the pharmacokinetics and biodistribution of various protein and nanoparticle therapeutics. Unfortunately, some PEGylated drugs can induce elevated levels of antibodies that can bind PEG, i.e. anti-PEG antibodies (APA) in some patients, which in turn can reduce the efficacy and increase the risks of allergic reactions, including anaphylaxis. There is currently no intervention available in the clinic that specifically mitigates allergic reactions to PEGylated drugs without the use of broad immunosuppression. The methods and compositions described herein may surprisingly use free PEG as a prophylaxis against anaphylaxis induced by PEG- specific allergic reactions in swine. As shown here, although injection of PEG-liposomes (PL) resulted in anaphylactoid shock (pseudo-anaphylaxis) within 1-3 mins in both naive and PL-sensitized swine, repeated injection of free PEG alone did not result in allergic reactions, and injection of free PEG effectively suppressed allergic reactions to PL, including in previously PL- sensitized swine. The methods and compositions described herein include the use of free PEG, in tandem with one or more PEGylated therapeutic agents, to reduce APA and allergic response to PEGylated therapeutic agents. Specifically, described herein are compositions and methods for suppressing hypersensitivity reactions that may be especially useful for suppressing drug and vaccine hypersensitivity associated with carrier.

[0007] Thus, the methods and composition described herein may reduce or eliminate the APA and allergic responses, e.g., infusion reactions or CARP A (Complement activation-related pseudoallergy), that may be present after administration of a therapeutic PEGylated agent. For instance, previous studies have shown that approximately 45% of cancer patients who received the PEGylated therapeutic agent Doxil® developed hypersensitivity reactions that included shortness of breath, flushing, and dizziness. The methods and composition described, herein, including administering one or more doses of free PEG (e.g., free PEG having a molecular weight between about 1 kDa and about 100 kDa, between about 5 kDa and about 100 kDa, between about 10 kDa and about 100 kDa, between about 20 kDa and about 100 kDa, etc.) concurrently with or before (e.g., one week before, two weeks before, three weeks before, four weeks before, four weeks before, between 1 hour and 30 days before, between 1 day and 30 days before, between 2 days and 30 days before, between 3 days and 30 days before, between 4 days and 30 days before, etc.) may prevent or reduce the hypersensitivity reaction.

[0008] For the recently approved Pfizer / BioNTech BNT162b2 and Modema mRNA-1273 vaccines, several cases with PEG-related allergic reactions have been reported, with and without anaphylaxis, the symptoms of which include hives, diarrhea, dizziness, shortness of breath and irregular heart rate. The methods and apparatuses described herein may reduce or prevent the APA and allergic response to PEGylated therapeutic agents including PEGylated vaccines.

[0009] For example, described herein are pharmaceutical composition for use in preventing a hypersensitivity reaction, the composition comprising: free PEG at a concentration of from about 0.10 mg / ml to about 100 mg / ml, wherein the free PEG is not conjugated to a PEGylated therapeutic agent, further wherein the free PEG has a molecular weight between about 1 kDa and about 100 kDa (e.g., between about 5 kDa and about 100 kDa, between about 10 kDa and about100 kDa, between about 20 kDa and about 100 kDa, etc.), and a pharmaceutically acceptable carrier.

[0010] A pharmaceutical composition for use in preventing a hypersensitivity reaction may include: a PEGylated therapeutic agent; free PEG at a concentration of from about 0.10 mg / ml to about 100 mg / ml, wherein the free PEG is not conjugated to the PEGylated therapeutic agent, further wherein the free PEG has a molecular weight between about 1 kDa and about 100 kDa (e.g., between about 5 kDa and about 100 kDa, between about 10 kDa and about 100 kDa, between about 20 kDa and about 100 kDa, etc.); and a pharmaceutically acceptable carrier.

[0011] Any of these pharmaceutical compositions may include free PEG having an average molecular weight of about 80 kDa or less, e.g., about 50 kDa or less (e.g., free high molecular weight PEG having an average weight of between about 1 kDa and about 100 kDa, about 50 kDa, etc.).

[0012] In general, the pharmaceutical composition may be a PEGylated therapeutic agent, including one or more of: a PEGylated protein, a PEGylated small molecule and / or a PEGylated nanoparticle. The composition may be configured as an injectable composition.

[0013] Also described herein are methods for reducing a likelihood of a hypersensitivity reaction and / or reducing the hypersensitivity reaction, the comprising: delivering to a subject a therapeutically effective dose, fa, of a free PEG in a pharmaceutically acceptable carrier; and delivering to the subject a therapeutically effective dose, da, of a PEGylated drug in a pharmaceutically acceptable carrier at a time represented by t(d).

[0014] The therapeutically effective dose, fa, of the free PEG and the therapeutically effective dose, da, of the PEGylated drug may be in a single formulation. In some examples the therapeutically effective dose, fa, of the free PEG is delivered before the therapeutically effective dose, da, of a PEGylated drug is delivered.

[0015] Any of these methods may include delivering to the subject a therapeutically effective dose, fb, of a free PEG in a pharmaceutically acceptable carrier, wherein the therapeutically effective dose fb is delivered to the subject before the therapeutic dose, fa, is delivered to the subject. The therapeutically effective dose, fb, may be delivered to the subject at least 7 days before the therapeutic dose, fa, is delivered to the subject. In some examples delivering to the subject a therapeutically effective dose, fc, of free PEG in a pharmaceutically acceptable carrier,wherein the therapeutically effective dose, fc, is delivered to the subject before the therapeutic dose, fb, is delivered to the subject.

[0016] Any of these methods may include delivering to the subject a therapeutically effective dose, fd, of free PEG in a pharmaceutically acceptable carrier, wherein the therapeutically effective dose, fd, is delivered to the subject before the therapeutic dose, fc, is delivered to the subject.

[0017] In any of these methods, the free PEG may have an average molecular weight of between about 1 kDa and about 80 kDa or less (e.g., about 50 kDa or less, etc.).

[0018] In any of these methods delivering may comprise injection.

[0019] The therapeutically effective amount of free PEG may comprise from between 0.1 mg / kg (free PEG / subject’s bodyweight) to 100 mg / kg (free PEG / subject bodyweight).

[0020] The hypersensitivity reaction may include one or more of: shortness of breath, dizziness, skin redness, chest pain, flashing and rash.

[0021] Also described herein are methods of determining a therapy schedule. These methods may include: delivering to a non-human subject therapeutically effective dose, fa, of a free PEG in a pharmaceutically acceptable carrier at a time represented by t(f); delivering to the non- human subject a therapeutically effective dose, da, of a PEGylated drug at a time represented by t(d), wherein the free PEG is not conjugated to the PEGylated therapeutic; measuring an extent of one or more than one hypersensitivity reaction(s) in the non-human subject to thereby obtain one or more than one hypersensitivity reaction measurement(s), wherein the one or more than one hypersensitivity reaction measurements are indicative of an effectiveness of the therapy schedule for treating non-human subject; determining a time represented by t(s), wherein t(s) is the time difference between t(f) and t(d); and determining, based on the hypersensitivity reaction measurement, a therapy schedule, wherein the therapy schedule comprises determining a free PEG dose and a time t(s).

[0022] For example, described herein are compositions and methods for reducing a likelihood of a hypersensitivity reaction and / or reducing the hypersensitivity reaction to a PEGylated drug in a subject. These compositions may include free PEG between 0.1 mg / kg (free PEG / subject’s bodyweight) to 100 mg / kg (free PEG / subject bodyweight), wherein the free PEG is not conjugated to a PEGylated therapeutic agent, further wherein the free PEG has a molecular weight between 1 kDa and 100 kDa, and may be used to reduce the likelihood and / or severity ofa hypersensitivity reaction in a subject, and in particular a human subject, by: administering to a subject free PEG between 0.1 mg / kg (free PEG / subject’s bodyweight) to 100 mg / kg (free PEG / subject bodyweight), wherein the free PEG is not conjugated to a PEGylated therapeutic agent, further wherein the free PEG has a molecular weight between 1 kDa and 100 kDa, wherein the free PEG is delivered simultaneously with or prior to delivery of the PEGylated drug.

[0023] The free PEG may be in a single formulation with the PEGylated drug. In some cases, the free PEG may be delivered before the PEGylated drug is delivered to the subject.

[0024] In particular, the compositions described herein may match the linear PEG polymer in the composition to the form used in the PEGylated drug. In particular, the free PEG composition may be just linear methoxy PEG (mPEG) (e.g., pure mPEG, 99% or more linear methoxy PEG (mPEG), 95% or more linear methoxy PEG (mPEG), 90% or more linear methoxy PEG (mPEG), than 85% or more linear methoxy PEG (mPEG), 80% or more linear methoxy PEG (mPEG), etc.), may be just linear hydroxy PEG (OH-PEG) (e.g., pure OH-PEG, 99% or more OH-PEG, 95% or more OH-PEG, 90% or more OH-PEG, 85% or more OH-PEG, 80% or more OH-PEG, etc.), or may be a mixture of mPEG and OH-PEG (e.g., 50% mPEG / 50% OH-PEG, 55% mPEG / 45% OH-PEG, 60% mPEG / 40% OH-PEG, 65% mPEG / 35% OH-PEG, between about 50%-about 75% mPEG and about 25%-about 50% OH-PEG, 45% mPEG / 55% OH-PEG, 40% mPEG / 60% OH-PEG, 35% mPEG / 65% OH-PEG, between about 25%-about 50% mPEG and about 75%-about 50% OH-PEG, etc.). For example, the free PEG comprises a mixture of linear methoxy PEG (mPEG) and hydroxy PEG (OH-PEG). In use, the free PEG may be delivered to the subject at least 7 days before the PEGylated drug is delivered to the subject.

[0025] The free PEG may have an average molecular weight of between 1 kDa and 80 kDa. The free PEG may have an average molecular weight of between 1 kDa and 50 kDa.

[0026] The PEG may be administered by injection. The therapeutically effective amount of free high molecular weight PEG may comprise from between about 0.1 mg / kg (free PEG / subject’s bodyweight) to about 100 mg / kg (free PEG / subject bodyweight).

[0027] As mentioned, in general, the hypersensitivity reaction may include one or more of: pulmonary arterial pressure, shortness of breath, dizziness, skin redness, chest pain, flashing and rash.

[0028] Thus, described herein are products (e.g., the free PEG compositions described herein) for the use in treating a hypersensitivity reaction to a PEGylated drug in a subject.

[0029] All of the methods and apparatuses described herein, in any combination, are herein contemplated and can be used to achieve the benefits as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] A better understanding of the features and advantages of the methods and apparatuses described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, and the accompanying drawings of which:

[0031] FIGS. 1 A-l J illustrate free PEG effectively prevents PL-induced pseudoallergy in naive swine. FIGS. 1 A-l J show results assessing PEG intervention against allergic responses to PL in naive pigs via invasive monitoring of hemodynamic changes. FIG. 1 A illustrates a schematic of study design.

[0032] FIGS. 1B-1C show representative PAP response over time in pigs (FIG. IB) first treated with PL before PEG, vs. (FIG. 1C) first treated with PEG prior to PL. Saline and Zymosan were included in all animals as controls.

[0033] FIGS. ID- IE show normalized PAP response over time in pigs (FIG. ID) first treated with PL before PEG (n=3), vs. (FIG. IE) first treated with PEG prior to PL (n=4).

[0034] FIGS. 1F-1H show a comparison of FIG. IF averaged PAP values normalized to saline, FIG. 1G peak PAP values normalized to saline, and FIG. 1H increase in PAP AUC normalized to saline. Peak PAP values are calculated as the average of top 3 recorded PAP readings for each treatment condition in each pig.

[0035] FIGS 11-1 J show abdominal region of the skin 3 mins after infusion of saline, followed by either free PEG or PL. FIG. II shows example photographs of pigs before and after infusion with PL and shows PL induces confluent skin flushing and rash. FIG. 1 J shows example photographs of pigs after infusion of PEG but before PL, and after infusion of PL, and shows pre-infusion of PEG eliminates PL-induced confluent skin flush and rash to either very light skin flush or no discernible skin flush.

[0036] FIG. 2A-i 21 show effects of repeated free PEG dosing on PEG-liposomes-induced allergic response. FIG. 2A-i - 21 show results of assessing repeated dosing of free PEG on allergic responses to PEG-liposomes via invasive monitoring of hemodynamic changes.

[0037] FIG. 2Ai-2Aii shows schematic of study design. FIG. 2B shows anti-PEG antibodies levels before each round of PEG dosing (n=6).

[0038] FIGS. 2C-2D shows normalized PAP response over time in pigs previously repeated dosed with free PEG, then FIG. 2C first treated with PL before PEG, vs. FIG. 2D first treated with free PEG prior to PL.

[0039] FIGS. 2E-2G show comparison of (FIG. 2E) averaged PAP values normalized to saline, (FIG. 2F) peak PAP values normalized to saline, and (FIG. 2G) increase in PAP AUC normalized to saline. Peak PAP values are calculated as the average of top 3 recorded PAP readings for each treatment condition in each pig.

[0040] FIGS. 2H-2I show abdominal region of the skin 3 mins after infusion of saline, followed by either free PEG or PL. FIG. 2H shows example photographs of pigs that had received 5 rounds of PEG, taken before and after infusion of PL and shows PL induces only light skin flush. FIG. 21 shows example photographs of pigs that had received 5 rounds of PEG, taken after infusion of PEG but before PL.

[0041] FIGS. 3A-3T show safety and immunogenicity of repeated free PEG dosing. FIGS. 3A-3T show safety assessment of repeated IV dosing of 40 kDa free PEG (50 mg / kg) on Days 0, 7, 14, 21, 28, and 35 into swine (n=4). (FIGS. 3A-3H) No effect on CBC before and after free PEG injection. FIGS. 3L3P show no observable toxicity to the liver and kidney function was observed.

[0042] FIGS. 3Q-3T show that body weight, body temperature, heart rate and hemostasis are all in normal range across the duration of study.

[0043] FIG. 4 shows normal shape and morphologies of cells in the tissue after PEG dosing. FIG. 4 shows histology assessment on safety of repeated dosing of free PEG. Pigs, after receiving at least 5 weekly rounds of PEG injection and hypersensitivity assay on Day 35, were sacrificed at the end of the experiment. Various organs were then harvested and prepared for immunohistology HE staining.

[0044] FIG. 5A-5I show free PEG reduces allergic response to PL in swine previously exposed to PEG-liposomes. FIG. 5A-5I show results assessing free PEG intervention on allergic response to PL via invasive monitoring of hemodynamic changes, in swine previously exposed to PL. FIG. 5 A shows a schematic of experiment design. FIG. 5B shows changes of IgG APA levels 7 days after injection of PL, and 5mins after infusion of free PEG during hypersensitivityassay on Day 7. FIGS. 5C-5D show representative PAP response over time in pigs (FIG. 5C) first treated with PL before PEG, vs. (FIG. 5D) first treated with free PEG prior to PL. Saline and Zymosan were included as controls. FIGS. 5E-5F show normalized PAP response over time in pigs previously dosed with PL, then (FIG. 5E) first treated with PL before PEG, vs. (FIG. 5F) first treated with free PEG prior to PL. FIGS. 5G-5H show a Comparison of (FIG. 5G) averaged PAP values normalized to saline, (FIG. 5H) peak PAP values normalized to saline, and (FIG. 51) increase in PAP AUC normalized to saline. Peak PAP values represent the average of top 3 recorded PAP readings for each treatment condition in each pig.

[0045] FIGS. 6A-6D illustrate level of ArnPA vs. AbPA IgG and IgM levels in human serum prior to (FIGS. 6A and 6C) a first dose of CO VID mRNA vaccine and (FIGS. 6B and 6D) before a second dose of the CO VID mRNA vaccines. Serum from 40 distinct donors were included in this pilot study.

[0046] FIG. 7A is a graph showing induction of AmPA IgM in mice receiving mPEG-uricase together with either saline or with different doses of OH-PEG. FIG. 7B shows a graph of the induction of AmPA IgM. FIG. 7C shows a graph of the induction of AbPA IgG and FIG. 7D shows a graph of the induction of AbPA IgM in mice receiving mPEG-uricase together with either saline or different doses of mPEG. Note that AmPA IgG is not shown as it was not detected in any of the mice in the study.DETAILED DESCRIPTION

[0047] Described herein are compositions and methods for reducing or preventing a hypersensitivity reaction in a subject. In particular, described are compositions and methods for reducing or prophylactically preventing hypersensitivity reactions in a subject otherwise due to a response to the PEG portion of PEGylated compounds and substrates, including PEGylated drugs and vaccines. Applicant has surprisingly found that treatment of a subject with free PEG can reduce or prevent hypersensitivity reactions due to PEGylation.Definitions

[0048] The terms administering or introducing can be used interchangeably in the context of the placement of a PEG (or a PEGylated substance) into a subject. A PEG composition (or PEGylated substance) either separately or together can be administered by any appropriate route that results in delivery to a desired location in the subject. A PEG composition (or PEGylatedsubstance) either separately or together can be administered via a route such as, but not limited to, enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroven- tricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavemous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intraci sternal (within the cistema magna cerebellomedu- laris), intracorneal (within the cornea), dental intracomal, intracoronary (within the coronary arteries), intracorporus cavemosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intrat- esticular (within thetesticle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratym- panic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration, which is then covered by a dressing that occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis and spinal.

[0049] The term adverse drug reaction (ADR) (also referred to as side effects) refers to unwanted, and harmful effects attributed to the use of a drug. Examples of adverse drug reactions are pseudoallergic reactions, including shortness of breath, facial redness, chest pain, flashing, and / or rash.

[0050] The term anti-PEG antibodies (APA) refers to antibodies (such as IgA, IgD, IgE, IgG, IgM types of antibodies) that specifically recognize PEG. In some examples, anti-PEG antibodies can be anti-PEG IgM or anti-PEG IgG antibodies.

[0051] The terms delivering or providing refers to carrying and turning over a substance to a subject or other intended recipient.

[0052] The term dosage refers to how to take or administer a therapeutic substance. Dosage can refer to a specific amount of a therapeutic substance taken at one specific time and the frequency of doses over a period of time.

[0053] The term dose refers to a specified amount of a therapeutic substance taken at one specific time.

[0054] The term drug refers to a substance, other than food, used in the prevention, diagnosis, alleviation, treatment of cure of a disorder, disease, or syndrome in a subject.

[0055] The term effective treatment refers to a treatment that has a desired effect. Generally, a treatment is considered an “effective treatment,” if any one or more than one or all the signs or symptoms of a condition, disorder, disease, or syndrome are altered in a beneficial matter. In one example, an effective treatment alters levels of hypersensitivity in a beneficial manner (e.g., decreased by at least 10%). Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of hypersensitivity is halted or at least slowed. An effective treatment can refer to providing a formulation to an individual and can include one or more of: (1) inhibiting the reaction, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the reaction, e.g., causing regression of symptoms; and (3) preventing the development of symptoms or reducing the risk (likelihood) risk of the development of symptoms. Methods of measuring these indicators can be qualitative or quantitative and are known to those of skill in the art and / or described herein.

[0056] The term free PEG refers to PEG which is not conjugated to a drug or another PEGylated compound, drug, or substrate. Free PEG can be in the form of a solid, power, liquid, or gas.

[0057] The terms individual, subject, and patient refer to any subject for whom diagnosis, treatment, prevention, or therapy is desired. A “subject,” as used herein, includes any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated with the compositions and methods described herein. Suitable subjects include animals, such as mammals, including non-human primates and human patients. Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Subjects may be transgenic or non-transgenic.

[0058] The term liposome refers to lipid vesicles composed of one or more than one lipid bilayer. In addition to lipids, liposomes can include other components such as drugs. Liposomes can be manufactured by emulsifying natural or synthetic lipids in an aqueous medium. Some liposomes are PEGylated liposomes. Some liposomes are mRNA vaccines, such as liposomebased mRNA anti-COVID-19 vaccines) and can contain liposome-entrapped mRNA. These andother liposomes may include PEGylated lipid that provides an advantage. Liposomes can be a type of nanoparticle.

[0059] The term nanoparticle(s) refers to a very small particle with a dimension on the scale of nanometers. Nanoparticles are typically less than about 100 nm in diameter or in a longest dimension, and more than 1 nm in diameter or in a longest dimension. In some variations, nanoparticles can be less than 1000 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, or less than 100 nm in diameter or in a longest dimension, and including all sizes between these. Nanoparticles can also or instead be at least 1 nm, at least 10 nm, at least 50 nm, or at least 100 nm in diameter or in a shortest or longest dimension, including all sizes between these (such as at least 80 nm and not more than 300 nm, etc.). Nanoparticles can be spherical or non-spherical (e.g., caged, oval-shaped, rod-shaped, tube-shaped, etc.). A nanoparticle can be composed of one or more than one natural or non-natural (artificial) polymer. Nanoparticles can include one or more other components, such as antibodies, drugs (including prodrugs that need to be converted into a pharmacologically active agent in the body by the action of enzymes or other chemicals), nucleic acids, peptides, and vaccines.Nanoparticles can be liposomes, dendrimers, solid, carbon nanoparticles, etc.

[0060] The term pharmaceutical composition refers to a mixture or composition suitable for administering to a subject that includes a pharmaceutical or therapeutic agent.

[0061] The term polyethylene glycol (frequently abbreviated as “PEG”) refers to polymers composed of repeating units of ethylene glycol (CH2 CH2O) monomers. PEG can include an n number of (CH2 CH2O) monomers (i.e., CH2 CJLOjn), where n is an integer greater than 1 and typically greater than 4. Polyethylene glycol can be categorized into different types based on various factors such as synthesis geometry, molecular weight, and specific functional groups. In some examples, molecules of PEG in a composition can be all about the same size (e.g., monodispersed or discrete) and have defined molecular weights and chain size. In some examples, molecules of PEG in a composition can vary somewhat in size. These molecule sizes typically center around one size. These so-called polydispersed polymers have a dispersion of size / weight (MW), with the help of Gaussian distribution of molecular weights and chain lengths. A number following PEG (e.g., PEG-800) refers to the molecular weight of the PEG. Molecular weights of the PEG are typically in units of Daltons (Da) unless otherwise specified or apparent based on context. As PEG compositions typically contain a range of PEG sizes, themolecular weight typically refers to the average molecular weight of the PEG. Polyethylene glycol can have one or more than one PEG chain. Polyethylene glycol can be categorized as a linear polymer and a non-linear polymer. Linear PEG polymers have a long chain. Non-linear PEG polymers have more than one PEG chain. Examples of non-linear polymers include branched polymers, comb-like polymers, and star polymers (with at least three arms radiating from one center (e.g., atom, small molecule, branched macromolecule, etc.)). Examples of branched PEG polymers include three-arm PEGs, four-arm PEGs, five-arm PEGs, six -arm PEGs, seven-arm PEGs, eight-arm PEGs, etc. PEG may have one or more PEG polymer ends. A polymer end can be represented by “Rm”, where R indicates a chemical group and m represents an integer to identify a different R group (e.g., Ri, R2, R3, etc.). The Rmgroup on two or more PEG ends can be the same or different. For example, a linear PEG polymer can be a homobifunctional PEG and have the same functional group (e.g., an Ri group) at both ends (e.g., at a first end and a second end). A linear PEG polymer can be a heterobifunctional PEG and have different functional groups (e.g., Ri, R2) at each polymer chain end (first and second ends). A non-linear PEG polymer can have the same Rmfunctional group at all ends or have different functional groups (e.g., Ri, Ri, Ri, or Ri, R2, R3) at different ends (first, second, and third ends). Linear methoxy PEG (mPEG) derivatives are a PEG chain with a reactive group at one end, while the other end is capped with a methoxy group. Any of the PEG compositions described herein may be linear PEG that is terminated with any appropriate end group, including (but not limited to) a hydroxy group or a methoxy group.

[0062] PEGs can be produced in a wide range of molecular weights, such as from molecular weight (MW) of around 200 to several million. PEGS are sometimes classified by their weight, such as low molecular weight PEG (less than 600 Da; e.g., PEG-200, PEG-400), medium molecular weight PEG (from 600 Da up to 2000 Da; e.g., PEG-600, PEG- 1000), and high molecular weight PEG (e.g., PEG-2000 and above). The PEGs of these different weight classifications may roughly share characteristics, such as somewhat similar solubility in water and organic solvents, form (waxy solid vs. gel) etc. A PEG with 80 PEG monomers (e.g., n=80) could have an average molecular weight of approximately 3500 Da and be labeled PEG-3500. PEG molecules, particularly very high molecule weight PEG molecules, may sometimes be referred to as polyethylene oxide (PEG), such as, for example, PEG molecules having molecular weight values above (about) 20,000 g / mol may be referred to as PEO.

[0063] The term PEGylated refers to a compound, drug, or substrate to which polyethylene glycol has been added. Examples of PEGylated compounds include PEGylated antibodies or antibody fragments, PEGylated drugs, PEGylated lipids, PEGylated oligonucleotides, PEGylated protein, PEGylated peptide, PEGylated nanoparticles, etc. Examples of PEGylated biological drugs include enzymes, mRNA, oligonucleotides, peptides, and proteins (recombinant or non-recombinant). Any of these can be recombinant or nonrecombinant. Examples of PEGylated non-biological drugs include PEGylated small organic molecules, synthetic peptides, and aptamers. Examples of PEGylated vaccines include liposomebased conformation-specific amyloid peptide antigen vaccines and liposome-based mRNA anti-COVTD-19 vaccines, Examples of PEGylated drugs include those that are FDA-approved PEGylated drugs. PEGylated drugs include those in Table 1.

[0064] Table 1

[0065] Thus, as used herein, therapeutic PEGylated agents (also referred to as PEGylated drugs) may include therapeutic PEGylated proteins, small molecules and / or nanoparticles. Specific examples of PEGylated therapeutic proteins that may be used as described herein include, but are not limited to: Elfabrio® (Pegunigalsidase alfa-iwx), Fylnetra™ (Pegfilgrastim- pbbk), Stimufend® (Pegfilgrastim-fpgk), Rolvedon™ (Eflapegrastim-xnst), Skytrofa™ (Lonapegsomatropin-tcgd), Besremi™ (Ropeginterferon alfa-2b-njft), Nyvepria™ (Pegfilgrastim-apgf), Esperoct® (Turoctocog alfa pegol), Ziextenzo™ (Pegfilgrastim-bmez), Jivi™ (Damoctocog alfa pegol), Palynziq™ (Pegvaliase-pqpz), Revcovi™ (Elapegademase- Ivlr), Asparlas™ (Calaspargase pegol-mknl), Fulphila™ (Pegfilgrastim-jmdb), Udenyca™ (Pegfilgrastim-cbqv), Rebinyn® (Nonacog beta pegol), Adynovate® (Rurioctocog alfa pegol), Plegridy™ (Peginterferon beta- la), Sylatron™ (Peginterferon alfa-2b), Krystexxa® (Pegloticase), Cimzia™ (Certolizumab pegol), Mircera™ (Methoxy polyethylene glycol-epoetin beta), Somavert™ (Pegvisomant), Neulasta® (Pegfilgrastim), Pegasys™ (Peginterferon alfa-2a), Pegintron™ (Peginterferon alfa-2b), Oncaspar™ (Pegaspargase), and Adagen™ (Pegademase bovine). Specific examples of pegylated therapeutic small molecules that may be used as described herein include, but are not limited to: Syfovre™ (Pegcetacoplan), Empaveli™ (Pegcetacoplan), Movantik® (Naloxegol), Omontys™ (Peginesatide), and Macugen™ (Pegaptanib sodium). Specific examples of PEGylated therapeutic nanoparticles that maybe used as described herein include, but are not limited to: Spikevax® (COVID-19 Vaccine, mRNA), Comimaty™ (COVID-19 Vaccine, mRNA), Onpattro® (Patisiran), Onivyde™ (Irinotecan liposome), and Doxil® (Doxorubicin HC1 liposome).

[0066] Although any size of PEG (e.g., high molecular weight PEG) can be added to a compound, drug, or substrate, many drugs have attached PEGs in the 2-40 kDa range.

[0067] The term PEGylation method (or PEGylation) refers to techniques that modify compounds or supports by adding polymeric chains of ethylene glycol (PEG) to the compound or support. The PEGylation method is typically covalent conjugation in which the PEG and compound or support are covalently linked. In some variations, the PEGylation method involves generation of intermolecular forces between the PEG and compounds or supports. PEG and the compounds or supports can be conjugated without covalent linkage. The PEGylation method can include self-assembly. PEGylation can lend desirable properties to compounds, such as improved drug solubility, decreased drug immunogenicity, protection of drug destruction by the immune system enhanced aqueous biocompatibility, longer drug bloodstream half-life, improved solubility, and less frequent dosing requirement.

[0068] Drug PEGylation can provide a significant advantage for patients. Unless indicated otherwise, or by context, PEGylation includes direct PEGylation as well as PEG containing vehicles such as dendrimers, liposomes, micelles, and nanoparticles.

[0069] Typically, a branched PEG has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core. PEG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol. The central branch moiety can also be derived from several amino acids, such as lysine. The branched PEG can be represented in general form as R(-PEG-OX)qin which R represents the core moiety, such as glycerol or pentaerythritol, X represents a capping group or an end group, and q represents the number of arms.

[0070] The term pseudoallergy (also referred to hypersensitivity reaction(s), HSR(s), or infusion reaction(s) refers to exaggerated or inappropriate responses occurring in response to a substance, such as to a pseudoallergen. Symptoms of hypersensitivity reactions can include one or more of shortness of breath, facial redness, chest pain, flashing and rash. In some instances, a hypersensitivity reaction can escalate into anaphylactic shock, which can occasionally be fatal. A hypersensitivity reaction can include one or more of the following stages: prodrome, early reaction, or late reaction. Prodrome refers to the first stage of a pseudoallergic reaction and usually occurs within minutes of exposure to a pseudoallergen. Early reaction occurs within minutes to hours after exposure to a pseudoallergen. Late reaction occurs several hours to days after exposure to a pseudoallergen.

[0071] The term reduce may relate generally to the ability of one or more formulations to “decrease” a relevant physiological or cellular response, such as a symptom of pseudoallergy or hypersensitivity reaction described herein, or to reduce a risk of having a relevant physiological or cellular response. A reduced response or reduced risk can be measured according to routine techniques in the diagnostic art. Examples include decreased shortness of breath, decreased facial redness, decreased chest pain, decreased flashing and decreased rash. A measurable physiological response may include decreased inflammation, as measured, for example, by body temperature, redness, swelling, or other clinical marker of inflammation. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to persons skilled in the art. A “decrease” in a response may be statistically significant as compared to the response produced by no PEG or a control composition, and may include, for example, a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers in between.

[0072] The term risk refers to the chance that an activity or action could happen and harm a subject.

[0073] The term systemically refers to the administration of a formulation other than directly into a target site, tissue, or organ, such that it enters, instead, the subject’s circulatory system. A PEG formulation can be administered systemically or non-systemically.

[0074] The term treatment refers to use of a substance or process to preserve to give particular properties to a subject. A treatment can be a prophylactic treatment designed and / or used to prevent or minimize a response. In some examples, administration of PEG to a subject is a treatment to prevent or minimize a pseudoallergy response in the subject.

[0075] The term vaccine refers to a preparation useful for stimulating the body's immune response against diseases. Vaccines can be injections (shots), liquids, pills, or nasal sprays formulated to teach the subject’s immune system to recognize and defend against a harmful stimulus, such as a bacteria, a virus, cancer cells, etc. Vaccines include one or more than one active ingredient(s). In some examples, an active ingredient can be PEGylated. Vaccines can also include one or more than one excipient such as stabilizers, adjuvants, and preservatives that serve a specific purpose. A stabilizer, such as sugar or gelatin, can keep a vaccine potent during transportation and storage. An adjuvant, such as an aluminum salt, can help stimulate a strongerimmune response. A preservative, such as thimerosal, can prevent contamination. Vaccines can also include residual amounts of materials that were used during the manufacturing process and removed.

[0076] The term vaccination refers to administering a vaccine to a subject, such as administering injections (shots), liquids, pills, or nasal sprays.

[0077] Described herein are methods for reducing a risk of or a likelihood of a hypersensitivity reaction. This and other methods can include a step of delivering to a subject a therapeutically effective dose, fa, of a free PEG in a pharmaceutically acceptable carrier. This and other methods can include a step of delivering to a subject a therapeutically effective dose, da, of a therapeutic PEGylated agent (e.g., PEGylated drug) in a pharmaceutically acceptable carrier at a time represented by t(d). The therapeutically effective dose, fa, of a free PEG and the therapeutically effective dose, da, of the PEGylated drug can be a single formulation. The therapeutically effective dose, fa, of a free PEG and the therapeutically effective dose, da, of the PEGylated drug can be delivered to the subject at the same time. In some variations, one or more therapeutically effective doses of free PEG can be delivered to the subject before delivering the PEGylated drug to the subject. One or more therapeutically effective doses of free PEG can include one or more than one dose, two or more than two doses, three or more than three doses, four or more than four doses, five or more than five doses, etc. Each dose may be represented here by an f, with a lowercase letter to indicate different doses (e.g., fa, fb, fc, etc.) The doses delivered to the subject can be delivered one day or more than one day, one week or more than one week, two weeks or more than two weeks, three weeks or more than three weeks, or four weeks or more than four week before delivering the PEGylated drug to the subject. When more than two doses are delivered, the time between the delivery of the doses can be about the same (e.g., every 7 days, every 14 days) or the time between the delivery of the doses can be variable (e.g., 5 days between delivering the first and second does, 9 days between delivering the second and third doses, etc.).

[0078] A therapeutically effective dose of free PEG can have an average molecule weight of from about 1 kDa to about 50000 kDa, and more typically from about 1 kDa to about 200 kDa, such as between about 1 kDa and less than about 200kDa, less than about 150 kDa, less than about 100 kDa, less than about 50 kDa. Also described herein is wherein the subject has a body weight and the therapeutically effective amount of free PEG is from about 5 mg / kg (freePEG / subject bodyweight) to about 200 mg / kg (free PEG / subject bodyweight). In some embodiments, the pH value of the composition containing the free PEG can range between about 7.0 and about 9.0. Typically, the pH value of the composition can be between about 7.5 and about 8.5, e.g., apH value of about 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.Pharmaceutically acceptable carriers include liquid carriers that are sterile aqueous solutions that contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.

[0079] Generally, a subject is a mammal (an organism) which are within the class Mammalia, including the order carnivore (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the subject is a human. In some embodiments, the subject is a laboratory animal, such as a mouse or rat. Modes of administration include injection, infusion, instillation, and / or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion. In some examples, the route is intravenous.

[0080] Also disclosed herein are methods of determining a therapy schedule. This and other methods can include one or both of the steps of: delivering to a non-human subject therapeutically effective dose, fa, of a free PEG in a pharmaceutically acceptable carrier at a time represented by t(f); delivering to the non-human subject a therapeutically effective dose, da, of a PEGylated drug at a time represented by t(d), wherein the free PEG is not conjugated to the PEGylated therapeutic. As mentioned, t(f may be relative to the delivery of the first and / or subsequent doses of the PEGylated drug. For example, t(f) may be approximately . This and other methods can include measuring an extent of one or more than one hypersensitivity reaction(s) in the non-human subject to thereby obtain one or more than one hypersensitivity reaction measurement(s). Measuring an extent of one or more hypersensitivity reaction can beperformed quantitatively or qualitatively, such as observing hypersensitivity reaction (e.g., one or more of shortness of breath, facial redness, chest pain, flashing, and rash, etc.). This and other methods can include one or more of wherein the one or more than one hypersensitivity reaction measurements are indicative of an effectiveness of the therapy schedule for treating non-human subject; determining a time represented by t(s), wherein t(s) is the time difference between t(f) and t(d); and determining, based on the hypersensitivity reaction measurement, a therapy schedule, wherein the therapy schedule comprises determining a free PEG dose and a time t(s).

[0081] Also disclosed herein are compositions that may be useful for practicing the methods. A pharmaceutical composition may include a PEGylated therapeutic and free PEG, wherein the free PEG is not conjugated to the PEGylated therapeutic; and a pharmaceutically acceptable carrier. Also disclosed herein are pharmaceutical composition that may include a PEGylated therapeutic and a free PEG, for use in preventing a hypersensitivity reaction. Also disclosed herein are compositions free PEG at a concentration of from about 500 mg / ml to about 40,000 mg / ml, wherein the free PEG is not conjugated to a PEGylated therapeutic, further wherein the free PEG has a molecular weight between about 1 kDa and about 100 kDa (e.g., between about 5 kDa and about 100 kDa, between about 10 kDa and about 100 kDa, between about 20 kDa and about 100 kDa, between about 20kDa and about 80 kDa, etc.); and a pharmaceutically acceptable carrier. In these and other pharmaceutical compositions herein, the free PEG has an average molecular weight of between about 1 kDa and less than about 1000 kDa, such as less than about 500 kDa, less than about 100 kDa, or less than about 50 kDa. In these and other compositions, wherein when a porcine hypersensitivity animal model appropriately receives the pharmaceutical composition, the porcine hypersensitivity animal model exhibits less hypersensitivity reaction compared with a comparable porcine hypersensitivity animal model that appropriately receives a control pharmaceutical composition with the PEGylated therapeutic and without the free PEG. In some cases it may be beneficial to use high molecular weight PEG having a molecular weight of between about 15 kDa and about 100 kDa (e.g., between about 20 kDa and about 100 kDa, between about 15 kDa and about 80 kDa, etc.).

[0082] Preliminary work on the delivery of free PEG in conjunction with a PEGylated drug (e.g., therapeutic PEGylated agent) may be given in a dose or amount that is particularly effective in reducing or eliminating an APA and / or allergic response. In some cases the methodsand compositions described herein may be configured to provide a dose of between about 0.1 mg / kg (free PEG / subject’s bodyweight) to 100 mg / kg (free PEG / subject bodyweight). Thus, the composition may be configured to allow for this dosage to be delivered as part of a treatment, depending on the route of treatment (e.g., IV, etc.). For example, a vial of drug to be given by IV infusion may first be added into an IV bag (typically 100-250mL) before being infused into a patient over, e.g., over the course of 1 -2 hrs. At an intended dose of 0.1 mg / kg to 100 mg / kg, assuming a patient is 80 kg in weight, the total amount of free PEG included in the composition may be between about 80-400 mg (or about 45-225 mg for someone 45 kg in weight, etc.). The cone in the vial or in the IV bag may be different. In a first use scenario, the cone of free PEG may be kept below a threshold to prevent it from becoming too viscous to be effectively injected. For example, the concentration of the free PEG composition may be maintained at less than about 15-20 mg / mL in solution to prevent it from being too viscous to be infused. In some examples a separate solution of free PEG that may be configured to be injected into an IV bag (e.g., with drug added either before or after), which may then be infused into a patient. In some examples the composition may be configured to be ready to be directly injected (e.g., IV / IM / SC) without a separate free PEG preparation. For an IV, for example, the composition may be instilled as-is or may be again diluted. For SC / IM, the composition may be configured for use without further dilution.

[0083] Thus, in some examples the lower end of the cone range of free PEG in the composition may be as low as about 0.18 mg / mL in the IV bag (e.g., 250mL vol for 45 kg weight individual at 1 mg / kg free PEG dose); at the higher dose (e.g., lOOmL vol for 80 kg weight individual at 5 mg / kg free PEG dose) the concentration may be about 4 mg / mL in the IV bag. Thus in some examples, the concentration of the composition may be between about 0.1 mg / mL and about 15 mg / mL of free PEG. For PEG within a concentrated vial, approximately 100 mg / mL may be used without viscosity issues. In some cases the method and composition may be configured to delivery both drug and PEG at the same time. Alternatively in some cases, the free PEG may be infused without the therapeutic PEGylated drug, e.g., for a first period of time (e.g., about 1 hr.) and then drug may be infused immediately after (or concurrently therewith) for a second period of time (e.g., for another hour).

[0084] The use of high molecular weight free PEG may help overcome pre-existing APA as well as induction of APA by PEG-liposomes (PL) and PEGylated proteins. This may be due, inpart, because grafting PEGs to a scaffold (protein, nanoparticle) increases the probability of crosslinking of B -cell receptors and thus markedly enhances the hapten effect, leading to much stronger immunogenicity than free PEG alone. In turn, the presence of the less immunogenic free PEG would not only competitively inhibit APA from binding PEGylated drugs, but also reduce immuno-stimulation. Free PEG can markedly reduce induction of APA to a number of PEGylated drugs, as well as effectively saturate preexisting APA in the circulation, thereby restoring prolonged circulation of PEGylated drugs. Repeated weekly administration of free PEG in mice did not increase the preexisting APA, and no organ toxicity was detected. However, these results did not address allergic reactions to PEGylated drugs. Described herein are results showing free PEG can serve as an intervention against PEG-specific pseudoallergy. By using the complement activation-related pseudoallergy (CARP A) pioneered by Szebani and colleagues, which is 1000-times more sensitive to allergic reactions than rodent models, the results described below illustrate, for the first time, the surprising result that free PEG can serve as an intervention against PEG-specific pseudoallergy.

[0085] The results described herein are particularly surprising given the current belief in the art that PEG having a molecular weight of greater than about 1 kDa (e.g., so-called “high molecular weight PEG”) trigger hypersensitivity. See, e.g., Liu et al., “An injectable excipient prevents anti-polyethylene glycol antibody mediated hypersensitivity” (Allergy. 2021 Sep; 76(9): 2902-2904). It is widely believed that reactions to PEG are molecular weight dependent, and that high molecular weight (HMW > lkDa) PEGs are likely to trigger reactions hypersensitivity mediated by anti-PEG antibodies.EXAMPLESResults

[0086] Free PEG effectively prevents PL-induced pseudoallergy in naive swine

[0087] In the porcine CARPA model, the change in pulmonary arterial pressure (PAP) over baseline provides an objective and quantitative readout for allergic responses, these results were examined to assess changes in PAP in swine receiving different interventions, with PL serving as a model PEGylated drug, saline as negative control, and Zymosan as a positive control. The findings from Szebani and colleagues, who first reported CARPA against PEG-liposomes in naive animals (FIG. 1 A) were first reproduced. Consistent with previous observations, in each of the 3 pigs that received saline followed by PL (Group 1), a rapid increase in PAP within a fewminutes of the PL infusion (FIG. IB, FIG. ID) was seen. Consistent with the jump in PAP, the animals displayed increased respiratory rate as well as confluent skin flushing and rash emerged shortly after PL injection (FIG. II). The anaphylactic response was so strong that 2 of the animals experienced cardiac arrest and had to be resuscitated, including closed chest compression and administration of one or more (as many as seven) shots of epinephrine. In these animals, subsequent dosing of Zymosan induced no appreciable increase in PAP, likely due to the epinephrine present as well as the fact that the PL-induced CARPA response likely substantially depleted complement factors. These results underscore both the dynamic nature of the swine CARPA model, and its suitability for assessing allergic response to PEGylated drugs like PL.

[0088] In sharp contrast to the swine that received PL immediately after saline, a completely different response was seen when the animals were injected with free PEG first after saline (Group 2), prior to the animals receiving PL. The pigs that received free PEG after saline displayed no discernible allergic response to the free PEG, with PAP readings over time that were indifferentiable from saline (FIG. 1C, FIG. 1 E). More importantly, the free PEG intervention greatly alleviated any CARPA response to PL injected shortly after (FIG. 1C, FIG. IE). No appreciable increase in PAP was seen over time relative to free PEG and relative to even saline. Compared to when PL was injected first, injection of free PEG before PL reduced the change in PAP after PL dosing, including the averaged PAP normalized to saline over time (6% with dosing free PEG first vs. 138% when PL is dosed first; FIG. IF), the maximum increase in PAP relative to saline (9% vs. 158%; FIG. 1G), and overall magnitude of the CARPA response (reflected by increase in AUC relative to saline, 0.73 vs. 18.7; FIG. 1H). The difference in PL- induced change in PAP in pigs receiving PL prior to PEG and in pigs receiving PL after PEG is statistically significant. PL-induced change in PAP in animals following free PEG dosing was not statistically different than PAP following free PEG dosing. Consistent with the lack of CARPA, free PEG dosing prior to PL virtually eliminated the confluent skin flushing and rash that was observed in animals receiving PL first; Instead, either no to very light skin flush, and no rash (FIG. 1 J) was seen. In the Group 2 animals, injection of Zymosan (positive control) induced an appreciable change in PAP. This indicates that the lack of increase in PAP is indeed due to free PEG mitigating the PL-induced CARPA effect, and not an experimental artifact from an inability to measure changes in PAP in select animals. It suggests the free PEG interventionmitigates only PL-induced CARPA and not all CARP A, underscoring the molecular specificity of the free PEG intervention. Altogether, these results suggest free PEG can effectively mitigate PL-induced allergic response in naive swine.Effects of repeated free PEG dosing on PL-induced allergic response

[0089] Based on the promising results in naive swine, and prior findings that repeatedly injection of free PEG did not appreciably increase APA titers in mice, the impact of repeated weekly dosing of free PEG on mitigating PL-induced pseudoallergy (FIG. 2A) was examined. Each of the animals was given either 3 or 5 doses of free PEG on a weekly basis. While PAP measurements were not performed following each free PEG dosing, it was noted that none of the animals developed any discernible skin flush or rash, and none of them had appreciable differences in heart rate, suggesting that repeated injection of free PEG alone is likely well tolerated and does not induce allergic reaction. Consistent with findings in mice, these experiments detected no induction of APA following 5 weekly doses of PEG: the levels of APA detected in the animals were consistently lower than the corresponding baseline levels (FIG. 2B).

[0090] On the PAP assessment day, the animals were again divided into two groups. In the group that received PL first (Group 1), an appreciable increase in the PAP was seen within minutes following PL-dosing, which typically lasted ~10 minutes (FIG. 2C). Compared to the response in naive swine, the average PAP increase (~87% vs. ~140% increase), peak PAP values (135% vs. 160% above baseline) and PAP-AUC (13.8 vs. 18.7) were all modestly to marginally lower in swine with prior exposure to free PEG (FIGS. 2E-2G). Interestingly, consistent with a reduced change in PAP, only one of four swine actually developed visible skin flush, which was mild (FIG. 2H). None of the animals experienced anaphylaxis that required resuscitation or epinephrine intervention. Altogether, these results suggest that free PEG dosed well in advance of PL may modestly reduce PL-induced allergic response.

[0091] The results were substantially more promising in the swine group that received free PEG dosed shortly prior to PL (Group 2), similar to findings in naive swine. Specifically, swine that received free PEG first after saline displayed no discernible allergic response to the free PEG, with PAP readings over time that were again indifferentiable from saline (FIG. 2D). More importantly, injection of PL shortly after free PEG again saw few signs of PL-induced CARP A, with the change in PAP once again indifferentiable relative to free PEG and relative to saline. Compared to PEG-exposed swine that first received PL, injecting free PEG prior to PL greatlyreduced the averaged PAP normalized to saline over time (15% vs. 87% when PL is dosed first), the maximum increase in PAP relative to saline (9% vs. 135%), and overall magnitude of the CARPA response (reflected by increase in AUC relative to saline, 3.2 vs. 13.8) (FIGS. 2E-2G). The difference in changes to PAP induced by PL, dosed before or after PEG, is statistically significant. Consistent with the PAP measurements, none of the swine receiving free PEG prior to PL exhibited any discernible skin flush or rashes (FIG. 21). These data once again underscored the effectiveness of the free PEG intervention, even in animals that had prior repeated exposure to PEG.Safety and immunogenicity of repeated free PEG dosing

[0092] Despite the long track record of safety of various PEGylated drugs, including those modified with comparably high MW PEG, the free PEG dose substantially exceeds the typical amount of PEG included in most clinical formulations. The safety and immunogenicity of repeated free PEG dosing was then evaluated by analyzing the blood (plasma) and urine collected at different time points in the above study. As shown in FIGS. 3A, 3B, 3C, and 3D, the enzymes ALT, ALP, AST and BUN levels in blood are all comparable to the baseline values before any PEG dosing, and within normal range, indicating that free PEG dosing does not interfere with liver function and health. With the complete blood count (CBC) panel, significant decrease in MBC and RBC were detected on Day 28. Other than this, no concerning changes were seen over time relative to the baseline level, and all readings fluctuated within the normal expected range (FIGS. 3E-3H). An increase in MONO count on Day 28 was detected. Other than this, no changes over time regarding PLT, RET-He, and NEUT (FIGS. 3I-3L) were seen. Likewise, tests for MALB, MTP, UCREAT and BUN levels in urine were similar over time compared to the baseline prior to PEG dosing, indicating that there was no damage in kidneys with repeated PEG dosing (FIG. 3M-3P). Finally, the body weight, body temperature, heart rate, and hemostasis were all within normal range (FIG, 3Q). These results are consistent with histology from H&E stained tissues from heart, liver, spleen and kidney, all of which show normal shape and morphologies of cells in the tissue (FIG. 4). There were prior reports of increased vacuolation in tissues from animals administered proteins modified with high MW PEG. To obtain an independent assessment of potential vacuolation after exposure to free PEG, organs were harvested at the end point of the study and prepared for the paraffin section, then had the tissues prepared and assessed by the pathology core facility in University of NorthCarolina, at Chapel Hill. The core did not find any vacuolation in any tissues evaluated (lung, liver, spleen, kidney, heart, brain). Overall, the lack of toxicological findings is consistent with the lack of increase in APA.Free PEG reduces allergic response to PL in swine previously exposed to PL

[0093] Finally, the effectiveness of free PEG intervention was evaluated in animals that were previously sensitized to PL, and thus can more strongly induce allergic response. All swine were injected with PL one week in advance (FIG. 5A). After a week, a significant increase of APA was detected in pigs receiving PL from ~1 ug / mL to ~6 ug / mL (FIG. 5B). On the study day, the animals were split into two groups: one that received PL first, and the other that received free PEG prior to PL dosing. In each of the 4 swine that received PL first, a rapid increase in PAP was seen within a few minutes of the infusion (FIG. 5C, FIG. 5E). Consistent with the jump in PAP, the animals displayed increased respiratory rate, as well as confluent skin flushing and rash. The anaphylactic response was so strong that 2 of 4 animals experienced cardiac arrest and had to be resuscitated, including closed chest compressions and administration of one or more (as many as four) shots of epinephrine.

[0094] In contrast to dosing PL first in PL-sensitized animals, dosing of free PEG first into PL-sensitized swine did not result in discernible increase in the average PAP, peak PAP or PAP- AUC in 3 of 4 swine, which were no different than saline (FIG. 5G). In one swine, a modest increase in PAP was detected. Nevertheless, even in that one swine, the average PAP (53% increase over baseline), peak PAP (53% higher) and PAP-AUC were only modestly higher, and the animal did not exhibit any other outward symptoms of allergic response (labored breathing, skin flush or rash), similar to the other 3 PL-sensitized swine that received free PEG first and displayed no change in PAP. Averaged over all 4 animals, the responses to free PEG in PL- sensitized swine were only marginally greater than those observed in naive animals or animals sensitized with free PEG alone.

[0095] Most importantly, free PEG once again effectively suppressed allergic reaction to PL even in swine sensitized to PL. While the PAP was noticeably elevated soon after PL dosing relative to free PEG, they were greatly attenuated compared to dosing PL directly. The averaged PAP normalized to saline over time (68% vs. 204% when PL is dosed first), the maximum increase in PAP relative to saline (97% vs. 259%), and overall magnitude of the CARPA response (reflected by increase in AUC relative to saline, 14.4 vs. 48.3) were all statisticallysignificantly lower in animals infused with free PEG first than in animals infused with saline control (FIGS. 5G-5I). The PAP measurements were consistent with visual observations of labored breathing, skin flush and rashes; none of the swine given PL following free PEG exhibited any severe symptoms (only one pig had light skin flush.). Free PEG indeed lowered the APA levels in pigs within minutes (FIG. 5B). These data once again underscored the effectiveness of the free PEG intervention, even in animals that had prior exposure to PL.

[0096] Cardiac anaphylaxis is one of the most severe, potentially lethal manifestations of immediate allergy to a variety of allergens and drugs, including PEGylated drugs. As such, the swine CARPA model pioneered by Szebeni and colleagues to assess allergic reactions to different PEGylated drugs, which displays typical symptoms of cardiac anaphylaxis, offers a highly rigorous approach to evaluate the effectiveness of any intervention designed to alleviate PEG-specific anaphylaxis. Indeed, findings from the same swine CARPA model contributed to FDA approval of a transthyretin-directed small inhibitory RNA-containing nanoparticle (Patisiran, Onpattro). Here, free PEG is shown to be surprisingly effective at mitigating allergic reactions directed against PL in both PL-naive and PL-sensitized swine. Likewise, despite the apparent PEG-sensitivity in PL-naive animals (confirmed here against PL, in good agreement with earlier studies), repeated dosing of free PEG was found to be very safe with no discernible renal, hepatic or cardiac toxicities, to not induce increase in APA, to not induce any detectable allergic reactions, and to effectively block PL-induced allergy. These findings, combined with our earlier work demonstrating free PEG can also reduce dosing of free PEG to be very safe with no discernible renal, hepatic or cardiac toxicities, to not induce increase in APA, to not induce any detectable allergic reactions, and to effectively block PL-induced allergy. These findings, combined with our earlier work demonstrating free PEG can also reduce APA-induction and restore prolonged circulation of PEGylated drugs in animals with drug-induced APA, strongly underscore the translational potential and support fiirther investigation of free PEG as an intervention to restore the safe and efficacious use of select PEGylated drugs impacted by PEG- specific immunity / hypersensitivity.

[0097] The approach of using free PEG to suppress APA induction or overcome pre-existing APA has never previously been explored. This is likely due to the longstanding dogma that introducing a large quantity of antigens to already sensitized patients would greatly stimulate antigen-specific B-cell expansion and trigger massive hypersensitive reactions. Nevertheless, thepresumption that free PEG stimulates APA+ B-cells to the same extent as PEGylated drugs with multiple covalently linked PEGs such as PL does not account for the size or highly flexible nature of PEG chains, or the hapten effect upon conjugating PEG to lipids / proteins. Indeed, PEG-drugs are relatively large: for instance, Krystexxa is a tetramer with MW -540 kDa, with 36 chains of 10 kDa PEG, Palynziq has a MW -1,000 kDa with 9 chains of 20 kDa PEG, and most PL are -80-120 nm in size with hundreds of PEG chains on the surface of each PL. Thus, multiple APA (IgG -150 kDa) can readily accumulate on the same PEGylated drug, and the close spatial proximity of multiple bound APA-Fc can trigger complement activation through the classical pathway. In contrast, when dosed at molar excess relative to circulating APA, PEG is unlikely to form such immune complexes. Furthermore, stimulation of APA, a T-independent response, requires cross-linking of multiple B-cell receptors (BCR) on the surface of APA+ B- cells. While free PEG can engage BCRs, it is unlikely to crosslink BCRs to the same extent as the much larger PL and select PEGylated protein drugs. Thus, free PEG should not strongly stimulate APA+ B-cell expansion in the same way PEG-drugs would. Limited BCR stimulation is consistent with earlier work that showed infusion of free PEG effectively reduced induction of elevated APA titers by a number of different PEGylated drugs.

[0098] Hence, one strategy herein of using free PEG to prevent hypersensitivity reactions to PEGylated drugs can be explained as that the spatial presentation of free PEG contrasts sharply to when PEG is linked to proteins or lipids. Described herein, where sharply distinct allergic response in swine to PL vs. free PEG was shown, indicates that free PEG should not be viewed synonymous as PEGylated drugs, and that free PEG interacts very differently with the immune system than many PEGylated drugs.

[0099] Like other medical interventions, the effectiveness and safety of the free PEG intervention will likely vary among individuals. Indeed, there are case reports of anaphylactic response to free PEG incorporated as formulation excipient in a dose- and MW- dependent manner. It is interesting to contrast the incidence rate of hypersensitivity to PEG (37 cases reported between 1977 and 2016) to the corresponding exposure to drugs that contain free PEG. While difficult to precisely estimate, free PEG is likely used as part of the formulation in at least hundreds of drugs, which in turn has likely been dosed into at least tens, if not hundreds, of millions of individuals over the 4 decades time span. Even if likely substantial underreporting of hypersensitivity is accounted for, the use of free PEG is still likely to be safe relative to theobserved incidence rates of allergic reactions to a number of PEGylated drugs, and also relative to other medical interventions such as broad immunosuppression. For perspective, infusion reaction is common in patients treated with Krystexxa, with -24-30% of patients experiencing IR and 6.5% at risk of anaphylaxis. Likewise, >70% of patients treated with Palynziq experienced hypersensitivity reactions, with -10% at risk for anaphylaxis, leading to its black box warning in the FDA-approved product insert. For such PEGylated drugs, reducing the incidence rate of allergic reaction to those observed with free PEG in many other drug products would provide a meaningful improvement in the safety and quality of the treatments.[000100] Moghimi and colleagues explored the use of poloxamines to reduce the clearance of the poloxamine-coated particles. Szebeni and colleagues also investigated the use of placebo (empty) PL to prevent infusion reactions to PEGylated liposomal doxorubicin, based on the tachyphylaxis phenomenon. These experiments intentionally sought to saturate pre-existing APA and mitigate APA induction via the use of high MW free PEG, rather than empty PL or block copolymers that can form micelles, for a number of reasons. First, it was initially feared that multiple APA can bind the same PL or PEG-containing micelles, and thus create immune complexes that can similarly trigger CARPA or otherwise stimulate additional APA secretion. While tachyphylaxis is clearly a viable strategy, doing so likely requires careful monitoring in clinical setting, and may need to be optimized for each patient. In contrast, a vast molar excess of free-PEG:APA compared to PL:APA may be more readily achieved, thus limiting generation of immune complexes or stimulation of APA secretion. Second, no PEG-based intervention can be more cost-effective than free-PEG itself, and USP-grade PEG is readily available.[000101] Many alternatives to PEG are under active investigation. However, replacing PEG in FDA-approved PEGylated drugs with alternative polymers necessitates many years of development for each drug, which is particularly impractical when coupled with the enormous costs of clinical development. It is also possible that these PEG alternatives may prove no better at mitigating formation of ADA or preventing hypersensitivity. Given the large number of PEGylated drugs already on the market and in clinical development, the most cost-effective, impactful, and readily translatable approach to overcoming possible APA-associated loss of efficacy and side effects is to specifically limit the induction of high-titer APA and PEG-specific allergy in the first place. The “off-the-shelf’ interventions described herein may be likely effective for multiple PEGylated drugs, can be quite inexpensive in the long run, and can quicklyadvance into clinical studies given the very low costs to establish GMP manufacturing or produce clinical trial materials. These attributes are important, as there are already dozens of PEG-protein and PL products on the market, including the CO VID mRNA vaccines from Pfizer and Modema, with more in clinical development. With the near ubiquitous exposure to PEGylated drugs and associated risks of drug-induced APA, it is likely that patients will be increasingly dosed with multiple PEGylated drugs, leading to higher incidence of APA-mediated allergic reactions or loss of efficacy in the years ahead. This underscores the urgency to develop methods that can specifically reduce APA induction and hypersensitivity without broad immunosuppression for essential disease management in many patients.Methoxy-terminated PEG[000102] The chemical nature of free PEG may impact the type of APA responses in vivo. Specifically, PEG can be terminated with different end groups, for instance hydroxy (OH-PEG, often abbreviated as simply PEG) or methoxy (H3CO-PEG, or mPEG). Different types of APAs may bind to different types of PEG. For instance, APAs that bind the PEG backbone (i.e. anti- backbone-PEG antibody, or AbPA), and APSs that bind the methoxy-terminal portion of PEG (i.e. anti-methoxy-PEG antibody, or AmPA). Note that AbPA can bind both OH-PEG and mPEG with comparable affinities, as they share the same repeated units of CH2-CH2-O in the polymer backbone, whereas AmPA can only bind mPEG with high affinity. Nevertheless, the field rarely distinguishes the two types of APAs, leaving many questions unanswered.[000103] For example, prior to the experiments described herein, it was not clear if anti-mPEG antibodies (AmPA) are present in humans, and it is not understood in the art that preventing AmPA in humans is of any value. Described herein for the first time is evidence that AmPA exists in humans (and not just animals) and that the induction of AmPA can be reduced to a greater extent than when using OH-PEG. For example, the type of APA present in humans was determined as an accurate quantitation of AmPA vs. AbPA allows better prediction and / or modeling of the extent to which APA may accumulate on PEGylated drugs over time, and consequently better prediction of safety and efficacy. To do so, we analyzed ~40 serum samples selected at random from the total repository for a Phase 2 study of individuals receiving COVID mRNA vaccines (>700 individuals). Notably, mRNA vaccines are formulated with lipid-PEG conjugates containing mPEG.[000104] Interestingly, as described herein, AmPA IgG either pre-existied or potentially boosted after the first dose of either Pfizer or Modema COVID mRNA vaccines (FIGS. 6A and 6C). In contrast, AmPA IgM was detected at levels that are ~10-fold lower than AbPA IgM (FIGS. 6B and 6D). While both AbPA IgM and AmPA IgM appears to be boosted following COVID mRNA vaccination, the magnitude of the increase appears modestly greater for AbPA than AmPA. These results establish for the first time that AmPA exists in humans, and can be induced following exposure to a PEGylated entity (in this instance, CO VID mRNA vaccine). Thus, to adequately reduce APA as well as potential allergic response mediated by APA, it would be desirable to eliminate both AbPA and AmPA.[000105] To investigate this, we first confirmed that AmPA can be induced in mice, which would allow us to assess whether we can reduce induction of AmPA. We first reanalyzed serum samples in which PEG-uricase was co-administered with OH-PEG. We found no detectable levels of anti-methoxy-PEG (AmPA) IgG, but we did detect AmPA IgM induced at the highest OH-PEG dose in mice (FIG. 7A). The lack of AmPA IgG but detectable levels of AmPA IgM is consistent with our observations above, suggesting that mice may be a suitable animal model for our investigation. It also suggests that while OH-PEG may fully saturate all existing AbPA+ B cell receptor (BCR) at that dose, it was not able to do so against AmPA+ B cells, thus resulting in clear induction of AmPA IgM.[000106] We next investigated whether the exact nature of the free PEG could impact the type of APA induced by assessing dosing PEG-uricase with mPEG instead of OH-PEG. Similar to an earlier study with OH-PEG, mPEG afforded clear suppression of AbPA IgG response (FIG. 7A), with no detectable AmPA IgG (not shown). mPEG was more effective at suppressing AmPA IgM induction than OH-PEG (comparing FIGS. 7B to 7A). mPEG was also able to reduce induction of AbPA (FIGS. 7C and 7D). These results directly underscore the specific chemical nature of PEG can impact its effectiveness in suppressing different APAs.[000107] Thus, described herein are methods of using methoxy-PEG (mPEG), and / or methoxy- PEG (mPEG) and OH-PEG to reduce anti -mPEG antibodies (e.g., AmPA and AbPA), or specifically AmPA. In some cases it may be particularly beneficial to inhibit AmPA, e.g., by administering mPEG and / or to preferentially reduce the induction of AmPA. In some cases it may be particularly beneficial to inhibit both AmPA and AbPA by administering both mPEG and OH-PEG, and / or to reduce the induction of AmPA and AbPA.[000108] This data, in combination with the results described above, support compositions for treating (e.g., reducing the likelihood and / or reducing the severity of) a hypersensitivity reaction to a PEGylated drug in a subject. In particular, these compositions may comprise (or in some cases, consist of) free PEG, e.g., between 0.1 mg / kg (free PEG / subject’s bodyweight) to 100 mg / kg (free PEG / subject bodyweight), wherein the free PEG is not conjugated to a PEGylated therapeutic agent, further wherein the free PEG has a molecular weight between 1 kDa and 100 kDa. The free PEG is delivered simultaneously with and / or prior to delivery of the PEGylated drug. In cases where the free PEG composition is administered with the PEGylated drug (e.g., such as a therapeutic mRNA vaccine, etc.), the free PEG may be in a single formulation with the PEGylated drug. In cases where the free PEG composition is administered before the PEGylated drug, the free PEG composition may be present with any pharmaceutically acceptable carrier. In particular, these compositions may be compositions in which the free PEG comprises linear methoxy PEG (mPEG) (e.g., pure mPEG, 99% or greater mPEG, 95% or greater mPEG, 90% or greater mPEG, 98% or greater mPEG, 80% or greater mPEG, etc.). The us of mPEG alone or in combination with OH-PEG may be particularly helpful where the PEGylated drug is PEGylated with a mPEG. Thus, the composition of the PEG may be matched to the composition used for PEGylation.Materials and MethodsPreparation of PL and PEG[000109] LPD NPs were synthesized according to published procedures from Dr. Leaf Huang. Briefly, DOTAP and cholesterol (1:1, mol / mol) liposomes were prepared by a hydration-extrusion method. DSPE-PEG 2K was then introduced at 60 °C for 15 min. Finally, 20 .L of 20% glucose solution was added to adjust the osmotic pressure. NP size and NP surface charge were measured by a Malvern ZetaSizer Nano series (Westborough, MA). Free PEG solution was prepared by dissolving 40k Da PEG (SERVA, 33139) in saline and filter sterilized, prepared right before animal study.Pig study[000110] The detail of porcine hypersensitivity model was described in some earlier studies. Yorkshire cross market swine were used in this study, with average weights of 30-40 kg at study entry and 40-50 kg at study end.[000111] Surgical setup: Swine were anesthetized with isoflurane and were placed in dorsal recumbency on the surgical table with the head and neck exposed. After final surgical prep, the left or right jugular furrow was draped off with sterile drapes and a 3-5 incision was made directly over the jugular furrow. Blunt dissection was used to isolate the jugular vein and carotid artery individually. Both vessels were isolated and carefully cleared of adventitia. Two ties (2-0 to 3-0 sterile suture) were placed around the jugular vein, the tie closest to the heart was occluded to distend the vessel and a large bore over the needle IV catheter (14-18 gauge) was directly introduced into the vessel. The catheter was secured to the vessel with at least two ties and the catheter was connected to an IV bag. The isolated carotid artery is cannulated as described with the jugular vein. The tie closest to the heart was lifted to occlude the vessel and a small (1-3 mm) arteriotomy was performed with a scalpel. A Millar catheter (to monitor aortic pressure) was placed via this arteriotomy and temporarily secured to prevent slipping, and hemorrhage from the artery. An identical procedure was performed in the right neck to isolate and cannulate the right jugular vein for placement of a pulmonary artery pressure catheter. Alternately, the Millar catheter introduction was placed in the right or left inguinal region via the femoral artery using the same cutdown, dissect, and cannulate procedure. All catheters were attached to the data collection unit which read and recorded physiologic data and pressure waveforms. Pressure waveform readings were used to determine when catheter sensors were advanced into their respective locations for desired physiologic monitoring / data collection (the Millar catheter into the aortic root and the PA pressure catheter in the pulmonary artery beyond the right ventricle. After confirmed placement all catheters were secured to their respective vessels with at least two sutures (2-0 to 4-0 sterile suture).[000112] Three swine studies were designed and performed. In Study 1, saline was injected into swine on Day 0. On Day 7, swine were divided into two groups for CARPA assessment. Group One received four IV injections spaced -15-20 mins apart in the following order: saline, PL, PEG, and Zym. Group Two received four IV injections, again spaced -15-20 mins apart, in the following order: saline, PEG, PL, and Zym. PL were administrated at 0.1 mg / kg via a bolus injection. PEG (40 kDa) were dosed at 50 mg / kg, with PEG dissolved in 20 mL saline and infused at 1 mL / min over 20 mins. Finally, Zymosan at 0. Img / kg was administrated via a bolus injection as the positive control. Blood and urine were collected both before and after the four injections. After each injection, hypersensitivity studies were performed. Specifically, thepulmonary arterial pressure (PAP) and heart rate (HR) starting immediately before each injection was measured, and continued for -15-20 mins. For animals that displayed an appreciable change, at least 15 mins was allowed to pass, in order to ensure the pigs recovered to normal before the next injection. Respiratory rate (RR) was controlled by mechanical ventilation for the whole study. After the study, the animals were sacrificed by overdose of pentobarbital (> 100 mg / kg IV) under general anesthesia. Euthanasia was confirmed by the veterinarian or veterinary technician by lack of heartbeat, lack of blood pressure waveforms and flat lining of the end-tidal carbon dioxide (EtCO2) monitor curve. Liver, kidney, spleen, heart, brain, and lung were collected for histology tests of toxicity and PEG vacuolation / accumulation.[000113] In the first part of Study 2 (Study 2a), pigs were injected with PEG a total of 5 times on Days 0, 7, 14, 21, and 28. Blood was collected immediately before and after each round of injection. Hypersensitivity studies with the same protocol as Study 1 were then performed on Day 35. In the second part of Study 2 (Study 2b), pigs were dosed with saline on Day 0, and with PEG 3 times on Days 7, 14, and 21. CARPA assessments following the same aforementioned protocols were performed on both Day 28 and Day 35. Finally, in Study 3, pigs were immunized with PL on Day 0, and CARPA assessments were performed on Day 7.Blood collection[000114] Blood samples were taken from the ear vein before the start of treatment, as well as 5 mins after the intervention / treatment. After collection of the whole blood, the blood was allowed to clot by leaving it undisturbed at room temperature for 30 mins. The clot was removed by centrifuging at 2,000 x g for 10 minutes in a refrigerated centrifuge at 4 °C. The resulting plasma samples were aliquoted into 0.5 ml aliquots, and stored at -80°C until use.Anti-PEG Antibodies quantification via competition ELISA[000115] APA were quantified using a competitive ELISA, as previously described . ELISA plates (Coming Costar 3695) were coated overnight with 50 pg / mL DSPE-mPEG 5k Da at 4 °C. Plates were then washed with PBS and blocked with 5% (w / v) non-fat milk (Lab scientific bioKEMIX, M0841) in PBS. Diluted pig plasma samples as well as the pig anti-PEG IgG standard were incubated in 1% milk in PBS overnight at 4 °C, after adding to the blocked and washed ELISA plate. Pig anti-PEG IgG standard was designed by grafting PEG-binding domain of human 15 -2b antibody onto constant region of pig IgG. Each plasma sample was measured intriplicate. To verity specificity of the detected signal (i.e., verity PEG-specificity), PEG competition samples were in included where 10 kDa free mPEG were added to the plasma samples; subtraction of the signal from these PEG-competition wells allows us to determine and eliminate non-PEG-specific background signal (e.g., from lipid-binding antibodies). To detect binding, a secondary anti-pig IgG HRP-conjugate (ThermoFisher Supraclonal, SA5- 10226) were diluted 1 : 10,000 in 1% milk and added to all wells. Following overnight incubation at 4 °C, plates were washed before addition of TMB (Thermo Fisher, 34029), and quenched with IN HCL. Absorbance was read at 450 nm (signal) and 570 nm (background); the standard curve allows for conversion of absorbance values to mass concentrations of APA.Clinical blood chemistry[000116] Blood were taken from the ear vein, before and after treatment. The blood was kept in hirudin- or K3-EDTA tubes to achieve anticoagulation. Complete blood counts (CBCs) were performed on the IDEXX Procyte Dx hematology analyzer utilizing pig species-specific parameters by core facility at University of North Carolina, at Chapel Hill (UNC-CH). Blood samples from day 0 before treatment were considered as negative controls (n = 16). CBC data for treatment groups was from 2 separate studies (n = 16).Clinical plasma and urine toxicity analysis[000117] Liver and Kidney function assays were performed by core facility at University of North Carolina at Chapel Hill (UNC-CH). Plasma samples and urine samples were collected before and after treatment. Samples from day 0 before treatment were used as controls (n = 16).[000118] Statistical analysis[000119] One-way ANOVA was used to compare the difference among more than three groups and student’s t-test was used to compare between two groups. Statistical analysis was performed with GraphPad Prism software (GraphPad Software Inc., USA). Significance was determined as p value less than 0.05 (p < 0.05). * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001. [000120] Any of the methods (including user interfaces) described herein may be implemented as software, hardware or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions capable of being executed by a processor (e.g., computer, tablet, smartphone, etc.), that when executed by the processor causes the processor tocontrol perform any of the steps, including but not limited to: displaying, communicating with the user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, alerting, or the like.[000121] When a feature or element is herein referred to as being "on" another feature or element, it can be directly on the other feature or element or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being "connected", "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected", "directly attached" or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature.[000122] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".[000123] Spatially relative terms, such as "under", "below", "lower", "over", "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as "under" or "beneath" other elements or features wouldthen be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise. [000124] Although the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element without departing from the teachings of the present invention.[000125] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.[000126] In general, any of the apparatuses and methods described herein should be understood to be inclusive, but all or a sub-set of the components and / or steps may alternatively be exclusive, and may be expressed as “consisting of’ or alternatively “consisting essentially of’ the various components, steps, sub-components or sub-steps.[000127] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about orapproximately that value, unless the context indicates otherwise. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "X" is disclosed the "less than or equal to X" as well as "greater than or equal to X" (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.[000128] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.[000129] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for thespecific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

CLAIMSWhat is claimed is:

1. A pharmaceutical composition for use in preventing a hypersensitivity reaction, the composition comprising: free PEG at a concentration of from 0.10 mg / ml to 100 mg / ml, wherein the free PEG is not conjugated to a PEGylated therapeutic agent, further wherein the free PEG has a molecular weight between 1 kDa and 100 kDa; and a pharmaceutically acceptable carrier.

2. A pharmaceutical composition for use in preventing a hypersensitivity reaction, the composition comprising: a PEGylated therapeutic agent; free PEG at a concentration of from 0.10 mg / ml to 100 mg / ml, wherein the free PEG is not conjugated to the PEGylated therapeutic agent, further wherein the free PEG has a molecular weight between 1 kDa and 100 kDa; and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition of any of claims 1-2, wherein the free PEG has an average molecular weight of 80 kDa or less.

4. The pharmaceutical composition of any of claims 1 -2, wherein the free PEG has an average molecular weight of 50 kDa or less.

5. The pharmaceutical composition of any of claims 1 -2, further comprising the PEGylated therapeutic agent.

6. The pharmaceutical composition of claim 5, configured as an injectable composition.

7. The pharmaceutical composition of any of claims 1 -6, wherein the PEGylated therapeutic agent comprises one or more of: a PEGylated protein, a PEGylated small molecule, a PEGylated liposome, and / or a PEGylated nanoparticle.

8. The pharmaceutical composition of any of claims 1 -7, wherein the free PEG comprises linear methoxy PEG (mPEG).

9. The pharmaceutical composition of any of claims 1 -7, wherein the free PEG comprises a mixture of linear methoxy PEG (rnPEG) and hydroxy PEG (OH-PEG).

10. A method for reducing a likelihood of a hypersensitivity reaction and / or reducing the hypersensitivity reaction, the comprising: delivering to a subject a therapeutically effective dose, fa, of a free high molecular weight PEG in a pharmaceutically acceptable carrier; and delivering to the subject a therapeutically effective dose, da, of a PEGylated drug in a pharmaceutically acceptable carrier at a time represented by t(d).

11. The method of claim 10, wherein the therapeutically effective dose, fa, of the free high molecular weight PEG and the therapeutically effective dose, da, of the PEGylated drug are in a single formulation.

12. The method of claim 10, wherein the therapeutically effective dose, fa, of the free high molecular weight PEG is delivered before the therapeutically effective dose, da, of a PEGylated drug is delivered.

13. The method of claim 10, further comprising delivering to the subject a therapeutically effective dose, ft, of a free high molecular weight PEG in a pharmaceutically acceptable carrier, wherein the therapeutically effective dose ft is delivered to the subject before the therapeutic dose, fa, is delivered to the subject.

14. The method of claim 13, wherein the therapeutically effective dose, ft, is delivered to the subject at least 7 days before the therapeutic dose, fa, is delivered to the subject.

15. The method of claim 13, further comprising delivering to the subject a therapeutically effective dose, ft, of free high molecular weight PEG in a pharmaceutically acceptable carrier, wherein the therapeutically effective dose, ft, is delivered to the subject before the therapeutic dose, ft, is delivered to the subject.

16. The method of claim 15, further comprising delivering to the subject a therapeutically effective dose, fa, of free high molecular weight PEG in a pharmaceutically acceptable carrier, wherein the therapeutically effective dose, fa, is delivered to the subject before the therapeutic dose, fc, is delivered to the subject.

17. The method of claim 10, wherein the free high molecular weight PEG has an average molecular weight of between 1 kDa and 80 kDa.

18. The method of claim 10, wherein the free high molecular weight PEG has an average molecular weight of between 1 kDa and 50 kDa.

19. The method of claim 10, wherein delivering comprises injection.

20. The method of claim 10, wherein the therapeutically effective amount of free high molecular weight PEG comprises from between 0.1 mg / kg (free PEG / subject’s bodyweight) to 100 mg / kg (free PEG / subject bodyweight).

21. The method of claim 10, wherein the hypersensitivity reaction includes one or more of: pulmonary arterial pressure, shortness of breath, dizziness, skin redness, chest pain, flashing and rash.

22. A method of determining a therapy schedule, comprising delivering to a non-human subject therapeutically effective dose, fa, of a free high molecular weight PEG in a pharmaceutically acceptable carrier at a time represented by t(f); delivering to the non-human subject a therapeutically effective dose, da, of a PEGylated drug at a time represented by t(d), wherein the free high molecular weight PEG is not conjugated to the PEGylated therapeutic; measuring an extent of one or more than one hypersensitivity reaction(s) in the non-human subject to thereby obtain one or more than one hypersensitivity reaction measurement(s), wherein the one or more than one hypersensitivity reaction measurements are indicative of an effectiveness of the therapy schedule for treating non-human subject;determining a time represented by t(s), wherein t(s) is the time difference between t(f) and t(d); and determining, based on the hypersensitivity reaction measurement, a therapy schedule, wherein the therapy schedule comprises determining a free high molecular weight PEG dose and a time t(s).

23. A method for reducing a likelihood of a hypersensitivity reaction and / or reducing the hypersensitivity reaction to a PEGylated drug in a subject, the method comprising: administering to a subject free PEG between 0.1 mg / kg (free PEG / subject’s bodyweight) to 100 mg / kg (free PEG / subject body weight), wherein the free PEG is not conjugated to a PEGylated therapeutic agent, fiirther wherein the free PEG has a molecular weight between 1 kDa and 100 kDa, wherein the free PEG is delivered simultaneously with or prior to delivery of the PEGylated drug.

24. The method of claim 23, wherein the free PEG is in a single formulation with the PEGylated drug.

25. The method of claim 23, wherein the free PEG is delivered before the PEGylated drug is delivered to the subject.

26. The method of claim 23, wherein the free PEG comprises greater than 95% linear methoxy PEG (mPEG).

27. The method of claim 23, wherein the free PEG comprises a mixture of linear methoxy PEG (mPEG) and hydroxy PEG (OH-PEG).

28. The method of claim 23, further comprising delivering to the subject the free PEG at least 7 days before the PEGylated drug is delivered to the subject.

29. The method of claim 23, wherein the free PEG has an average molecular weight of between 1 kDa and 80 kDa.

30. The method of claim 23, wherein the free PEG has an average molecular weight of between 1 kDa and 50 kDa.

31. The method of claim 23, wherein administering comprises injection.

32. The method of claim 23, wherein the therapeutically effective amount of free high molecular weight PEG comprises from between 0.1 mg / kg (free PEG / subject’s bodyweight) to 100 mg / kg (free PEG / subject body weight).

33. The method of claim 10, wherein the hypersensitivity reaction includes one or more of: pulmonary arterial pressure, shortness of breath, dizziness, skin redness, chest pain, flashing and rash.

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