WO2019152343A1

WO2019152343A1 - Methods of desensitizing against infusion associated reactions in lysosomal acid lipase deficiency (lal-d) patients treated with exogenous lysosomal acid lipase (lal

Info

Publication number: WO2019152343A1
Application number: PCT/US2019/015524
Authority: WO
Inventors: Mark Friedman; Suresh VIJAY; Julian RAIMAN; Srividya SREEKANTAM
Original assignee: Alexion Pharmaceuticals, Inc.
Priority date: 2018-02-01
Filing date: 2019-01-29
Publication date: 2019-08-08
Also published as: WO2019152343A8

Abstract

The disclosure features methods for safely and effectively administering exogenous LAL (e.g., sebelipase alfa) to pediatric LAL-D patients, as well as methods to prevent and ameliorate infusion reactions in LAL-D pediatric patients receiving exogenous LAL. In particular, disclosed herein are specific dosages and regimens for administering exogenous LAL to a pediatric patient having LAL-D, such dosages and regimens being designed to increase a patient's tolerance of the therapeutic, decrease the likelihood of (e.g., desensitize against) an infusion-related reaction, and maximize the amount of dose that may be administered to the patient safely, so as to safely achieve maximum therapeutic benefits.

Description

METHODS OF DESENSITIZING AGAINST INFUSION ASSOCIATED REACTIONS IN LYSOSOMAL ACID LIPASE DEFICIENCY (LAL-D) PATIENTS TREATED WITH

EXOGENOUS LYSOSOMAL ACID LIPASE (LAL)

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No.

62/624,890, filed on February 1, 2018 and U.S. Provisional Application Serial No. 62/768,524, filed on November 16, 2018. The entire contents of the above-referenced provisional patent applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted

electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 11, 2019, is named AXJ-249PC_SL.txt and is 3,717 bytes in size.

BACKGROUND

Lysosomal Acid Lipase Deficiency (LAL-D) is a rare lysosomal storage disease (LSD) characterized by a failure to breakdown cholesteryl esters (CE) and triglycerides (TAG) in lysosomes due to a deficiency of the enzyme. LAL-D resembles other lysosomal storage disorders with the accumulation of substrate in a number of tissues and cell types. In LAL-D, substrate accumulation is most marked in cells of the reticuloendothelial system including

Kupffer cells in the liver, histiocytes in the spleen and in the lamina propria of the small intestine. Reticuloendothelial cells expressing the macrophage mannose/N-acetyl glucosamine receptor (also known as macrophage mannose receptor, MMR, or CD206) and other cells which express the mannose-6-phosphate (MP6) receptor, mediate binding, cell uptake and lysosomal internalization of proteins with GlcNAc or mannose terminated N-glycans, and provide a pathway for the potential correction of the enzyme deficiency in these key cell types. LAL-D is a multi-system disease that most commonly manifests with gastrointestinal, liver and cardiovascular complications. The clinical effects of LAL deficiency are due to a massive accumulation of lipid material in the lysosomes in a number of tissues and a profound disturbance in cholesterol and lipid homeostatic mechanisms, including substantial increases in hepatic cholesterol synthesis. LAL deficiency presents as at least two phenotypes: Wolman Disease (WD) and Cholesteryl Ester Storage Disease (CESD).

Appropriate dosing of medications is a critical factor in their administration to patients, as infusion reactions can be associated with the administration of a medication to a patient. Such infusion reactions include chills, pyrexia and dizziness, which are often associated with hypersensitivity and allergic symptoms, such as urticaria. Severe infusion reactions can be life- threatening and include anaphylactoid symptoms, such as dyspnea, bronchospasm, hypotension, loss of consciousness and shock, or even myocardial infarction or cardiac arrest in some patients.

Patients with LAL-D are at risk of substantial morbidity and mortality. Accordingly, it is an object of the present invention to provide improved methods for treating patients with LAL- D, including treatment regimens to prevent or ameliorate infusion reactions in patients receiving exogenous LA, such as sebelipase alfa (KANUMA^®).

SUMMARY

Provided herein are methods to safely and effectively administer exogenous LAL to treat LAL-D, as well as methods to prevent and ameliorate infusion reactions in pediatric LAL-D patients receiving exogenous LAL. An exemplary exogenous LAL is sebelipase alfa

(KANUMA^®). In one embodiment, the exogenous LAL comprises the amino acid sequence set forth in SEQ ID NO:l.

In one aspect, methods of desensitizing a human pediatric patient having LAL-D against an infusion reaction associated with administration of sebelipase alfa are provided, wherein the method comprises intravenously administering sebelipase alfa once every other week to the patient, wherein:

a first infusion of sebelipase alfa is administered at a dose of 0.35 mg/kg,

a second infusion of sebelipase alfa is administered at a dose of 1 mg/kg at a dose, a third infusion of sebelipase alfa is administered at a dose of 2 mg/kg, and

a fourth and further infusions of sebelipase alfa are administered at a dose of 3 mg/kg; and wherein the dose is reduced if the patient exhibits an infusion reaction ( e.g ., pruritis, rash, headache, chills, anthralgia, myalgia, pyrexia, dizziness, urticaria, anaphylaxis, dyspnea, bronchospasm, hypotension, emesis, loss of consciousness, shock, tachycardia, myocardial infarction, and/or cardiac arrest).

Absent an infusion reaction associated with administration of sebelipase alfa, the initial infusions (e.g., first, second, third, fourth, fifth, sixth, seventh, and eighth infusions) are each administered over a course of at least 20 hours (e.g., about 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hour, 48 hours, 49 hours, or 50 hours). In one embodiment, the initial infusions (e.g., first, second, third, fourth, fifth, sixth, seventh, and eighth infusions) are each administered over a course of between 20 hours and 48 hours. In another embodiment, the first, second, third, and fourth infusions are each administered over at least 21 hours. In another embodiment, the first, second, third, and fourth infusions are each

administered over a course of between 24 hours and 48 hours. In another embodiment, the first, second, third, and fourth infusions are each administered over a course of between 21 hours and 43 hours.

If the patient exhibits an infusion reaction during or after administration of sebelipase alfa, the dose is reduced and sebelipase alfa is administered in incrementally increasing doses to the patient after the patient exhibits an adverse reaction. In one embodiment, the incrementally increasing doses are approximately double the amount of the preceding incremental dose. In another embodiment, sebelipase alfa is administered at a dose of 0.02 mg, followed by 0.04 mg ± 0.02, followed by 0.08 mg ± 0.04, followed by 0.16 mg ± 0.5, followed by 0.32 mg ± 0.5, followed by 0.62 mg ± 0.5, followed by 1.3 mg ± 0.5, followed by 2.5 mg ± 0.5, followed by 5.6 mg ± 2, followed by 6.5 mg ± 2. In another embodiment, sebelipase alfa is administered at a dose of 0.02 mg, followed by 0.04 mg ± 0.02, followed by 0.08 mg ± 0.04, followed by 0.16 mg ± 0.5, followed by 0.32 mg ± 0.5, followed by 0.62 mg ± 0.5, followed by 1.3 mg ± 0.5, followed by 2.5 mg ± 0.5, followed by 6.6 mg ± 2, followed by 7.6 mg ± 2.

In another embodiment, the pediatric patient weighs between 5 kg - 6 kg and the infusion dose is reduced after the patient exhibits an infusion reaction and sebelipase alfa is administered in incrementally increasing doses according to the following schedule:

In another embodiment, the patient weighs between 5 kg - 6 kg and the infusion dose is reduced after the patient exhibits an infusion reaction and sebelipase alfa is administered in incrementally increasing doses according to the following schedule:

In another aspect, methods of desensitizing a human pediatric patient having LAL-D against an infusion reaction associated with administration of sebelipase alfa are provided, wherein the method comprises intravenously administering sebelipase alfa once every other week to the patient, wherein the infusion dose is reduced after the patient exhibits an infusion reaction and administered in incrementally increasing doses according to the following schedule:

In one embodiment, Solution 1 is 100 ml volume; 0.0024 mg/ml concentration. In another embodiment, Solution 2 is 100 ml volume; 0.024 mg/ml concentration. In another embodiment, Solution 3 is 100 ml volume; 0.23529 mg/ml concentration.

In another aspect, methods of desensitizing a human pediatric patient having LAL-D against an infusion reaction associated with administration of sebelipase alfa are provided, wherein the method comprises intravenously administering sebelipase alfa once every other week to the patient, wherein the infusion dose is reduced after the patient exhibits an

infusion reaction and administered in incrementally increasing doses according to the following schedule:

n one embodiment, Solution 1 is 100 ml volume; 0.024 mg/ml concentration. In another embodiment, Solution 2 is 100 ml volume; 0.2355 mg/ml concentration.

In one embodiment, solution 1 is a 250 ml volume, 0.272 mg/ml concentration, and 68 mg total dose in solution.

Upon completion of the reduced dosing schedule and recovery from the infusion reaction, the dose resumes at the same dose or a lower dose prior to the infusion reaction. For example, in one embodiment, the patient is administered an infusion of sebelipase alfa at a dose of 1 mg/kg and exhibits an infusion reaction during or shortly thereafter after ( e.g ., within 1 hour, 59 minutes, 58 minutes, 57 minutes, 56 minutes, 55 minutes, 54 minutes, 53 minutes, 52 minutes, 51 minutes, 50 minutes, 49 minutes, 48 minutes, 47 minutes, 46 minutes, 45 minutes, 44 minutes, 43 minutes, 42 minutes, 41 minutes, 40 minutes, 39 minutes, 38 minutes, 37 minutes,

36 minutes, 35 minutes, 34 minutes, 33 minutes, 32 minutes, 31 minutes, 30 minutes, 29 minutes, 28 minutes, 27 minutes, 26 minutes, 25 minutes, 24 minutes, 23 minute, 22 minutes, 21 minutes, 20 minutes, 19 minutes, 18 minute, 17 minutes, 16 minutes, 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute). Upon completion of the reduced dosing schedule and recovery from the infusion reaction, the patient is then administered a subsequent infusion of sebelipase alfa at a dose of 0.35 mg/kg or 1 mg/kg. In another embodiment, the patient is administered an infusion of sebelipase alfa at a dose of 2 mg/kg, exhibits an infusion reaction during or shortly thereafter, completes the reduced dosing schedule, recovers from the infusion reaction, and is then administered a subsequent infusion of sebelipase alfa at a dose of 1 mg/kg or 2 mg/kg. In another embodiment, the patient is administered an infusion of sebelipase alfa at a dose of 3 mg/kg, exhibits an infusion reaction during or shortly thereafter, completes the reduced dosing schedule, recovers from the infusion reaction, and is then administered a subsequent infusion of sebelipase alfa at a dose of 2 mg/kg or 3 mg/kg.

Absent an infusion reaction associated with administration of sebelipase alfa or upon completion of the reduced dosing schedule and recovers from the infusion reaction, the infusion rate and/or volume of subsequent infusions can be increased upon confirmation that the dose is tolerated by the patient, thereby reducing the overall duration of a single infusion (e.g., to about 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, or 3 hours). In one embodiment, the infusion volume and infusion rates are increased commensurately upon confirmation that the dose is tolerated by the patient (e.g., in the absence of an infusion reaction).

DETAILED DESCRIPTION

Infusion reactions to therapeutic agents can be quite significant, resulting in discomfort and distress, hospitalization, treatment discontinuation, and even death. Unlike most adverse reactions, which often can be predicted, infusion reactions are unexpected and variable.

Moreover, infusion-related reactions make up a small percentage of adverse drug reactions, they still carry a significant economic impact. Infusion-related reactions can lead to prolonged infusion times, dose reductions, dose delays, and/or discontinuation of the drug.

The disclosure features methods for safely and effectively administering exogenous LAL ( e.g ., sebelipase alfa) and to prevent and ameliorate infusion reactions in pediatric patients receiving exogenous LAL. In particular, disclosed herein are specific dosages and regimens for administering exogenous LAL (e.g., sebelipase alfa) to a human pediatric patient having LAL-D, such dosages and regimens being designed to increase a patient’s tolerance of the therapeutic, decrease the likelihood of (e.g., desensitize against) an infusion-related reaction, and maximize the amount of dose that may be administered to the patient safely so as to safely achieve maximum therapeutic benefits.

While in no way intended to be limiting, exemplary solutions, formulations, therapeutic kits, and methods for making and using any of the foregoing are elaborated on below and exemplified in the working Examples. I. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the skilled artisan. Although any methods and

compositions similar or equivalent to those described herein can be used in practice or testing of the present invention, the preferred methods and compositions are described herein.

The singular form“a,”“an,” and“the” include plural reference unless the context clearly dictates otherwise.

The term“about”, particularly in reference to a given quantity or number, is meant to encompass deviations within plus or minus ten percent (± 10%), ( e.g ± 5%).

As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known

protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

As used herein, the percent homology between two amino acid sequences or two nucleotide sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology= # of identical positions/total # of positions X 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller ( Comput . Appl. Bioscl, 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

By an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment.

Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms "fragment," "variant,"

"derivative" and "analog" when referring to any of the polypeptides disclosed herein include any polypeptides which retain at least some of the activity of the corresponding native polypeptide ( e.g ., LAL polypeptide fragments, variants, derivatives, and analogs that retain the ability to hydrolyze cholesterol esters and/or triglycerides). Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments. Variants of a polypeptide include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can occur naturally or be non- naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions, or additions. Derivatives are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides can also be referred to herein as "polypeptide analogs." As used herein, a "derivative" of a subject polypeptide can contain one or more residues chemically derivatized by reaction of a functional side group. Also included as

"derivatives" are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and/or ornithine can be substituted for lysine.

II. LAL

"LAL" as used herein refers to "lysosomal acid lipase," and the two terms are used interchangeably throughout the specification. The LAL can be a human protein, i.e., human lysosomal acid lipase. LAL is also referred to in the literature as acid cholesteryl ester hydrolase, cholesteryl esterase, Lipase A, LIPA, and sterol esterase.

LAL catalyzes the hydrolysis of cholesterol esters and triglycerides to free cholesterol, glycerol, and free fatty acids. Thus, "LAL activity" can be measured, for example, by the cleavage of the fluorogenic substrate, 4-methylumbelliferyl oleate (4MUO). Cleavage of 4MUO can be detected, for example, by excitation at about 360 nm and emission at 460 nm of the released flurophore, 4-methylumbelliferone (4MU). Results can be reported in relative fluorescence units (RFU). For example, the amount of substrate cleaved in a 30 minute endpoint assay can be quantified relative to a 4MU standard curve, and one unit (U) of activity can be defined as the amount of enzyme required to cleave 1 micromole of 4MUO per minute at 37 °C. Accordingly, functional fragments or variants of LAL include fragments or variants that have LAL activity, e.g., the ability to hydrolyze cholesterol esters and/or triglycerides.

As used herein "exogenous LAL" refers to LAL that is not naturally produced by a patient. For example, exogenous LAL includes recombinant LAL protein that is administered to a patient, LAL protein that is isolated from a person or animal and administered to a patient, and LAL protein that is produced (i.e., expressed) in a patient as a result of administration of LAL- encoding RNA and/or DNA or another treatment that increases expression of endogenous LAL protein. In one embodiment, the exogenous LAL is sebelipase alfa.

An exemplary LAL is sebelipase alfa (KANUMA^®) (also known as“SBC- 102”).

Sebelipase alfa is a recombinant human lysosomal acid lipase (rhLAL). Lysosomal acid lipase is a lysosomal glycoprotein enzyme that catalyzes the hydrolysis of cholesteryl esters to free cholesterol and fatty acids and the hydrolysis of triglycerides to glycerol and free fatty acids. Sebelipase alfa is produced by recombinant DNA technology in the egg white of eggs laid by genetically engineered chickens. Purified sebelipase alfa is a monomeric glycoprotein containing 6 N-linked glycosylation sites and has a molecular mass of approximately 55,000 daltons. The amino acid sequence for sebelipase alfa is the same as the amino acid sequence for human LAL. The specific activity of sebelipase alfa is 195 to 345 units/mg. One unit is the amount of enzyme activity that catalyzes the hydrolysis of 1 micromole of the synthetic substrate 4-methylumbelliferyl oleate per minute at 37°C under specified assay conditions.

LAL deficiency is an autosomal recessive lysosomal storage disorder characterized by a genetic defect resulting in a marked decrease or loss in activity of the lysosomal acid lipase (LAL) enzyme. The primary site of action of the LAL enzyme is the lysosome, where the enzyme normally causes the breakdown of lipid particles including LDL-c. Deficient LAL enzyme activity results in progressive complications due to the lysosomal accumulation of cholesteryl esters and triglycerides in multiple organs, including the liver, spleen, intestine, and the walls of blood vessels. The resulting lipid accumulation in the liver may lead to increased liver fat content and progression of liver disease, including fibrosis and cirrhosis. Lipid accumulation in the intestinal wall leads to malabsorption and growth failure. In parallel, dyslipidemia due to impaired degradation of lysosomal lipid is common with elevated LDL-c and triglycerides and low HDL-cholesterol (HDL-c).

Sebelipase alfa binds to cell surface receptors (e.g., MMR receptors on

reticuloendothelial cells and/or MP6 receptors on other cells) via glycans expressed on the protein and is subsequently internalized into lysosomes. Sebelipase alfa catalyzes the lysosomal hydrolysis of cholesteryl esters and triglycerides to free cholesterol, glycerol and free fatty acids.

LAL (e.g., sebelipase alfa) includes any exogenous LAL which can be produced in any useful protein expression system including, without limitation, cell culture (e.g., CHO cells, COS cells), bacteria such as E. coli, transgenic animals such as mammals and avians (e.g., chickens, duck, and turkey) and in plant systems (e.g., duck weed and tobacco plants). One aspect of the invention relates to recombinant LAL produced in accordance with US Patent No. 7,524,626, issued October 3, 2006; US Patent Application Nos. 11/973,853, filed October 10, 2007;

11/978,360, filed October 29, 2007; and 12/319,396, filed January 7, 2009, the disclosures of which are incorporated in their entirety herein by reference. One aspect of the invention relates to recombinant LAL produced as described in Du et al., (2005) Am. J. Hum. Genet. 77: 1061- 1074, and Du et al., (2008) J. Lipid Res., 49: 1646-1657, the disclosures of which are incorporated in their entirety herein by reference. In one useful embodiment, the exogenous LAL is produced in the oviduct of a transgenic avian ( e.g ., a transgenic chicken), for example, according to a method described in WO 2011/133960 (PCT/US2011/033699), filed April 23, 2011, which is expressly incorporated by reference herein in its entirety. In some embodiments, the exogenous LAL is produced in an avian cell line. In some embodiments, the exogenous LAL is produced in a mammalian (e.g., a human) cell line.

The term "avian" as used herein refers to any species, subspecies or race of organism of the taxonomic class aves, such as, but not limited to chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows, and ratites including ostrich, emu and cassowary. The term includes the various known strains of Gallus gallus, or chickens (for example, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Australorp, Minorca, Amrox, California Gray), as well as strains of turkeys, pheasants, quails, duck, ostriches, and other poultry commonly bred in commercial quantities. It also includes an individual avian organism in all stages of development, including embryonic and fetal stages.

The term "poultry derived" or "avian derived" refers to a composition or substance produced by or obtained from poultry. "Poultry" refers to avians that can be kept as livestock, including but not limited to, chickens, duck, turkey, quail and ratites. For example, "poultry derived" may refer to chicken derived, turkey derived and/or quail derived.

In one embodiment, exogenous lysosomal acid lipase used in accordance with the invention contains glycans having substantial N-acetylglucosamine (GlcNAc) and mannose terminated N-linked structures. GlcNAc and mannose terminated glycans on exogenous LAL can be specifically recognized and internalized by macrophages and fibroblast. Mannose-6- phosphate (M6P), which can target proteins to the GlcNAc/mannose receptors which are expressed on cells implicated in conditions treatable by exogenous LAL administration, is also typically present on exogenous LAL used in accordance with the invention.

Typically, the LAL is human LAL. In one embodiment, the exogenous LAL has the amino acid sequence provided in Genbank RefSeq NM_000235.2). In another embodiment, the mature exogenous LAL has the following amino acid sequence: SGGKLTAVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKN

HSDKGPKPVVFLQHGLLADS SNWVTNLAN S S LGFILAD AGFD VWMGN S RGNTWSRKHKTLS VS QDEFW AFS YDEM AKYDLPAS INFILNKTGQEQVY Y V GHS QGTTIGFIAF S QIPELAKRIKMFF ALGP V AS V AFCT S PM AKLGRLP DHLIKDLF GD KEFLPQS AFLKWLGTH VCTH VILKELC GNLCFLLCGFNER NLNMSRVDVYTTHSPAGTSVQNMLHWSQAVKFQKFQAFDWGSSAKNY FHYN QS YPPTYNVKDMLVPT A VW S GGHDWLAD VYD VNILLTQITNLVF HES IPEWEHLDFIW GLD APWRLYNKIINLMRKY Q (SEQ ID NO:l)

In one embodiment, the LAL comprises amino acids 1-378 of SEQ ID NO:l, amino acids 3-378 of SEQ ID NO:l, amino acids 6-378 of SEQ ID NO:l, or amino acids 7-378 of SEQ ID NO:l. In another embodiment, the LAL comprises a mixture of at least two polypeptides selected from the group consisting of amino acids 1-378 of SEQ ID NO:l, amino acids 3-378 of SEQ ID NO:l, amino acids 6-378 of SEQ ID NO:l, and amino acids 7-378 of SEQ ID NO:l. In another embodiment, the LAL comprises a mixture of a polypeptide comprising amino acids 1- 378 of SEQ ID NO:l, a polypeptide comprising amino acids 3-378 of SEQ ID NO:l, and a polypeptide comprising amino acids 6-378 of SEQ ID NO:l.

In another embodiment, the LAL comprises a polypeptide that is identical to amino acids 1-378 of SEQ ID NO:l, amino acids 3-378 of SEQ ID NO:l, amino acids 6-378 of SEQ ID NO:l, or amino acids 7-378 of SEQ ID NO:l. In another embodiment, the LAL comprises a polypeptide that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to amino acids 1-378 of SEQ ID NO:l, amino acids 3-378 of SEQ ID NO:l, amino acids 6-378 of SEQ ID NO:l, or amino acids 7-378 of SEQ ID NO:l. In another embodiment, the LAL comprises a polypeptide that is a functional fragment of SEQ ID NO:l or is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical a functional fragment of SEQ ID NO:l.

In another embodiments the LAL is a recombinant LAL protein described in WO

2011/133960 (PCT/US2011/033699), filed April 23, 2011, which is expressly incorporated by reference herein in its entirety.

It is recognized that amino acid positions that are not identical often differ by

conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties ( e.g ., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the

conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. The scoring of conservative substitutions can be calculated according to, for example, the algorithm of Meyers & Millers, Computer Applic. Biol. Sci. 4:11-17 (1988).

A "comparison window" refers to a segment of contiguous positions, such as between about 25 and about 400 positions, or between about 50 to 200 positions, or between about 100 and 150 positions, over which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by a local homology algorithm (Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by a global alignment algorithm (Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by search for similarity methods (Pearson & Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444 (1988); Altschul et al., Nucl. Acids Res. 25:3389-402 (1997), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and BEAST in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), typically using the default settings, or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al. (eds.), 1994). For example, BEAST protein searches can be performed using the XBFAST program, score=50, wordlength=3 to obtain amino acid sequences that are more than 80% identical to the amino acid sequence of SEQ ID NO:l or a fragment thereof.

One example of a useful algorithm implementation is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignments. It can also plot a dendrogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng &

Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-3 (1989). The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster can then be aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. A series of such pairwise alignments that includes increasingly dissimilar sequences and clusters of sequences at each iteration produces the final alignment.

In one embodiment, the LAL polypeptides include variants of the wild-type sequences. These variants fall into one or more of three classes: substitutional, insertional, or deletional variants. These variants can be naturally occurring allelic or interspecies variants or they can be prepared by site-specific mutagenesis of nucleotides in the DNA encoding protein. Site-specific mutagenesis can be performed using cassette or PCR mutagenesis or other techniques well known in the art to produce DNA encoding the variant and, thereafter, expressing the DNA in recombinant cell culture. Variant target protein fragments having up to about 100-150 amino acid residues can be prepared by in vitro synthesis using established techniques. Conservative substitution tables providing functionally similar amino acids are well known in the art (Henikoff & Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 (1992)).

Amino acid substitutions are typically of single residues. Insertions usually will be on the order of from about 1 to about 20 amino acids, although considerably longer insertions can be tolerated. Deletions range from about 1 to about 20 residues, although in some cases, deletions can be much longer. Substitutions, deletions, and insertions or any combinations thereof can be used to arrive at a final derivative.

In one embodiment, the LAL ( e.g ., sebelipase alfa) has a specific activity of at least about 100 U/mg. In another embodiment, the exogenous LAL has a specific activity of at least about 200 U/mg. In another embodiment, the exogenous LAL has a specific activity of at least about 250 U/mg. In another embodiment, the exogenous LAL has a specific activity of about 100 to about 1,000 U/mg. In another embodiment, the exogenous LAL has a specific activity of about 100 to about 500 U/mg. In another embodiment, the exogenous LAL has a specific activity of about 100 to about 350 U/mg. In another embodiment, the exogenous LAL has a specific activity of about 200 to about 350 U/mg. In another embodiment, the exogenous LAL has a specific activity of about 250 to about 350 U/mg. In another embodiment, the exogenous LAL has a specific activity of about 250 U/mg. In another embodiment, the exogenous LAL has a specific activity of about 275 U/mg. In another embodiment, the exogenous LAL has a specific activity of about 300 U/mg. Human LAL has 6 potential sites in its amino acid sequence for N-linked glycosylation: Asn36, Asn72, AsnlOl, Asnl6l, Asn273, and Asn32l as set forth in SEQ ID NO:l. In one embodiment, at least 1, 2, 3, 4, or 5 of the N-linked glycosylation sites are glycosylated. In another embodiment all six glycosylation sites are glycosylated. In another embodiment, Asn36, AsnlOl, Asnl6l, Asn273, and Asn32l are glycosylated. In another embodiment, Asn36, AsnlOl, Asnl6l, Asn273, and Asn32l are glycosylated, and Asn72 is not glycosylated. In another embodiment, the N-glycan structures comprise bi, tri-, and tetraantennary structures with N-acetylglucosamine (GlcNAc), mannose, and/or mannose-6-phosphate (M6P). In another embodiment, the exogenous LAL comprises M6P-modified N-glycans at AsnlOl, Asnl6l, and Asn273. In another embodiment, the LAL does not comprise O-linked glycans. In another embodiment, the LAL does not comprise sialic acid. In another embodiment, the exogenous LAL has a glycosylation pattern as described in PCT/US2011/033699, filed April 23, 2011, which is incorporated by reference herein in its entirety.

In another embodiment, the molecular weight of the exogenous LAL is about 55 kD.

II. Infusion Reactions

Infusion reactions (also known as“infusion associated reactions” or“IARs”) are an adverse reaction associated with administration of a medication to a patient. Infusion reactions include, but are not limited to, pruritis, rash, headache, chills, anthralgia, myalgia, pyrexia, dizziness, urticaria, anaphylaxis, dyspnea, bronchospasm, hypotension, emesis, loss of consciousness, shock, tachycardia, myocardial infarction, and/or cardiac arrest.

Infusion related reactions are assigned grades according to the National Cancer Institute Common Terminology Criteria for Adverse Events (Version 4.0) as follows. Grade 1 includes a mild transient reaction, wherein interruption of infusion and/or interventions is not indicated. Grade 2 includes reactions that indicate the therapy or infusion should be interrupted, but the reaction responds promptly to symptomatic treatment ( e.g ., NSAIDS, narcotics, or intravenous (i.v.) fluids). In such cases, prophylactic medications are indicated for up to 24 hours at the discretion of the physician or staff. Grade 3 reactions include prolonged infusion related reactions (e.g., reactions not rapidly responsive to symptomatic medication and/or a brief interruption of infusion), a recurrence of symptoms following initial improvement, and hospitalization indicated for clinical sequelae. Grade 4 includes infusion related reactions with life-threatening consequences, and urgent intervention indicated as well as cessation of infusion. Grade 5 includes infusion related reactions causing the death of the patient.

III. Methods of Desensitization and Treatment

Provided herein are methods to safely and effectively administer exogenous LAL to treat LAL-D, as well as methods to prevent and ameliorate infusion reactions in pediatric LAL-D patients receiving exogenous LAL.

The term "patient" as used herein refers to any person receiving or who has received or is to receive medical care or treatment, e.g., as directed by a medical care provider.

The term“pediatric patient” refers to a patient under 18 years of age ( < 18 years).

The terms "treat," "treating," and "treatment" refer to methods of alleviating, abating, or ameliorating a disease or symptom, preventing an additional symptom, ameliorating or preventing an underlying cause of a symptom, inhibiting a disease or condition, arresting the development of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, relieving a condition caused by the disease or condition, or stopping a symptom of the disease or condition either prophylactically and/or after the symptom has occurred.

"Therapeutically effective dose" as used herein refers to the dose (e.g., amount and/or interval) of drug required to produce an intended therapeutic response. A therapeutically effective dose refers to a dose that, as compared to a corresponding subject who has not received such a dose, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of the occurrence or advancement of a disease or disorder. The term also includes within its scope, doses effective to enhance physiological functions.

"Intravenous infusion" refers to a route of administration in which medication is delivered over an extended period of time. To accomplish an intravenous infusion, an IV gravity drip or an IV pump can be used. IV infusion is typically used when a patient requires medications only at certain times and does not require additional intravenous fluids (e.g., water solutions which can contain sodium, chloride, glucose, or any combination thereof), such as those that restore electrolytes, blood sugar, and water loss. “Desensitizing” and“desensitization” refers to a process of reducing and/or eliminating a patient’s negative (adverse) reaction to a medication ( e.g ., sebelipase alfa). Desensitization involves building up a patient’s tolerance to a particular medication.

a first infusion of sebelipase alfa is administered at a dose of 0.35 mg/kg,

a fourth and further infusions of sebelipase alfa are administered at a dose of 3 mg/kg; and wherein the dose is reduced if the patient exhibits an infusion reaction.

In one embodiment, the LAL-D is Wolman Disease (WD). In another embodiment, the LAL-D is Cholesteryl Ester Storage Disease (CESD).

Exemplary infusion reactions include, but are not limited to, pruritis, rash, headache, chills, anthralgia, myalgia, pyrexia, dizziness, urticaria, anaphylaxis, dyspnea, bronchospasm, hypotension, emesis, loss of consciousness, shock, tachycardia, myocardial infarction, and/or cardiac arrest. In one embodiment, the patient exhibits one infusion reaction. In another embodiment, the patient exhibits multiple infusion reactions (e.g., one or more of pruritis, rash, headache, chills, anthralgia, myalgia, pyrexia, dizziness, urticaria, anaphylaxis, dyspnea, bronchospasm, hypotension, emesis, loss of consciousness, shock, tachycardia, myocardial infarction, and/or cardiac arrest).

Absent an infusion reaction associated with administration of sebelipase alfa, the initial infusions (e.g., first, second, third, fourth, fifth, sixth, seventh, and eighth infusions) are each administered over a course of at least 20 hours (e.g., about 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hour, 48 hours, 49 hours, or 50 hours). In one embodiment, the initial infusions (e.g., first, second, third, fourth, fifth, sixth, seventh, and eighth infusions) are each administered over a course of between 20 hours and 48 hours). In another embodiment, the first, second, third, and fourth infusions are each administered over at least 21 hours. In another embodiment, the first, second, third, and fourth infusions are each administered over a course of between 21 hours and 43 hours.

If the patient exhibits an infusion reaction during or after administration of sebelipase alfa, the dose is reduced and sebelipase alfa is administered in incrementally increasing doses to the patient after the patient exhibits an adverse reaction. In one embodiment, the incrementally increasing doses are about double the amount of the preceding dose. In another embodiment, sebelipase alfa is administered at a dose of 0.02 mg, followed by 0.04 mg ± 0.02, followed by 0.08 mg ± 0.04, followed by 0.16 mg ± 0.5, followed by 0.32 mg ± 0.5, followed by 0.62 mg ± 0.5, followed by 1.3 mg ± 0.5, followed by 2.5 mg ± 0.5, followed by 5.6 mg ± 2, followed by 6.5 mg ± 2. In another embodiment, sebelipase alfa is administered at a dose of 0.02 mg, followed by 0.04 mg ± 0.02, followed by 0.08 mg ± 0.04, followed by 0.16 mg ± 0.5, followed by 0.32 mg ± 0.5, followed by 0.62 mg ± 0.5, followed by 1.3 mg ± 0.5, followed by 2.5 mg ± 0.5, followed by 6.6 mg ± 2, followed by 7.6 mg ± 2.

In one embodiment, the pediatric patient weighs between 5 kg - 6 kg and the infusion dose is reduced after the patient exhibits an infusion reaction and sebelipase alfa is administered in incrementally increasing doses according to the following schedule:

In one embodiment, solution 1 is a 250 ml volume, 0.272 mg/ml concentration, and 68 mg total dose in solution. Upon completion of the reduced dosing schedule and recovery from the infusion reaction, the dose resumes at the same dose or a lower dose prior to the infusion reaction. For example, in one embodiment, the patient is administered an infusion of sebelipase alfa at a dose of 1 mg/kg and exhibits an infusion reaction during or shortly thereafter after (e.g., within 1 hour, 59 minutes, 58 minutes, 57 minutes, 56 minutes, 55 minutes, 54 minutes, 53 minutes, 52 minutes,

51 minutes, 50 minutes, 49 minutes, 48 minutes, 47 minutes, 46 minutes, 45 minutes, 44 minutes, 43 minutes, 42 minutes, 41 minutes, 40 minutes, 39 minutes, 38 minutes, 37 minutes,

Absent an infusion reaction associated with administration of sebelipase alfa or upon completion of the reduced dosing schedule and recovers from the infusion reaction, the infusion rate and/or volume of subsequent infusions can be increased upon confirmation that the dose is tolerated by the patient, thereby reducing the overall duration of a single infusion (e.g., to about 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, or 3 hours). In one embodiment, the infusion volume and infusion rates are increased commensurately upon confirmation that the dose is tolerated by the patient (e.g., in the absence of an infusion reaction). The following examples are merely illustrative and should not be construed as limiting the scope of this disclosure in any way as many variations and equivalents will become apparent to those skilled in the art upon reading the present disclosure.

The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

EXAMPLE

Example 1: Case Series of Sebelipase Alfa Hypersensitivity Reactions and Successful

Sebelipase Alfa Rapid Desensitization

Enzyme replacement therapy is life-changing for many with lysosomal storage diseases, but treatment can be hindered by immunologic reactions, including infusion and hypersensitivity reactions that interfere with medication administration, as well as development of neutralizing antibodies of unknown significance. Infusion reactions and allergic hypersensitivity reactions can have overlapping clinical features including wheezing, dyspnea, tachycardia, hypotension, and flushing (Demoly P., el al (2014) International Consensus on drug allergy. Allergy 69: 420- 437 and Aranda CS, et al (2016) J Allergy Clin Immunol Pract 4: 354-356). Infusion and hypersensitivity reactions typically occur during infusions, and management of both reaction types to enzyme replacement therapy has been previously described (Aranda et al. 2016 and

Karimian Z, et al. (2017) JIMD Rep 34: 63-70). Distinguishing a reaction as either an infusion reaction or a hypersensitivity reaction with evidence of mast cell degranulation is clinically important, as the latter can result in life-threatening anaphylaxis and is managed differently than the former, for which premedication and slowed infusion rate usually suffice (Demoly et al. 2014 and Aranda et al. 2016). Here we present three patients with lysosomal acid lipase deficiency with hypersensitivity reactions to sebelipase alfa, recombinant lysosomal acid lipase, who subsequently underwent successful rapid desensitization.

A. Case # 1

A 47-year-old Caucasian male with lysosomal acid lipase deficiency diagnosed at age 18, cerebral palsy with associated right-sided spasticity, was started on sebelipase alfa at 1 mg/kg every other week. He tolerated this dose without reaction until week 12. Ninety minutes into the infusion of sebelipase alfa, he developed diffuse urticaria, conjunctival injection, rhinorrhea, tachycardia, and tachypnea. The infusion was stopped and he was given IV dexchlorpheniramine and hydrocortisone with resolution of symptoms within an hour. Two weeks later, another infusion was attempted using pretreatment with dexchlorpheniramine and paracetamol. At 88 minutes into the infusion, he again developed diffuse rash, conjunctival injection, rhinorrhea, and tachypnea. The infusion was stopped and he was given IV

dexchlorpheniramine and hydrocortisone with resolution of symptoms within 50 minutes. Serum tryptase was measured and was normal at 4.7 ng/ml (normal value less than 13.0 ng/ml). Skin prick and intradermal testing to sebelipase alfa were performed per manufacturer’ s protocol, with prick testing at concentrations of 1:1000 (0.002 mg/ml) and 1:100 (0.02 mg/ml) and intradermal testing at 1:1000. Prick testing to 1:1000 and 1:100 was negative but intradermal testing was positive at 1:1000 with development of a 6-mm wheal. Drug-specific IgG antibodies were not detectable. Basophil activation test was performed as well and found to be positive. Based on a reaction consistent with immediate hypersensitivity and positive skin testing, the decision was made to proceed with desensitization to sebelipase alfa.

Five attempts were made to desensitize the patient to the full dose of 1 mg per kilogram of sebelipase alfa using a protocol ranging from 9 to 15 steps with initial concentrations as low as 100, 000^th of the target dose. Before each desensitization, the patient was given premedication of dexchlorpheniramine 5 mg IV one hour prior to the infusion. Within 3-6 hours of starting each desensitization, the patient developed a diffuse pruritic rash responsive to antihistamines. For the first three desensitizations, the desensitization protocol was able to be continued after administration of antihistamines and steroids without recurrence of symptoms. Upon the fourth attempt, the patient developed a diffuse pruritic rash and conjunctival injection 70 minutes after initial resolution and the infusion was stopped and methylprednisolone was administered.

Symptoms resolved within an hour. Upon the fifth attempt, however, he had multiple recurrences of diffuse pruritic rash despite antihistamines and the decision was made to stop the infusion and reduce his target dose. His target dose was reduced to 0.35 mg/kg every other week, which he tolerated through a desensitization protocol without recurrence of symptoms. Over the following weeks, his protocol was able to be shortened and simplified without recurrence of symptoms. Dose escalation was not attempted thereafter. The initial tolerated protocol and the final abbreviated protocol are shown in Table 1. Table 1: Sebelipase Alfa Desensitization Protocols for Case # 1

aInitial protocol:

Solution 1: 100 ml volume; 0.0024 mg/ml concentration.

Solution 2: 100 ml volume; 0.024 mg/ml concentration.

Solution 3: 100 ml volume; 0.23529 mg/ml concentration.

bFinal protocol:

Solution 1: 100 ml volume; 0.024 mg/ml concentration.

Solution 2: 100 ml volume; 0.2355 mg/ml concentration. B. Case # 2

A five- week old girl presented with enzymatically confirmed Lysosomal Acid Lipase deficiency (LAL-D). LAL-D was diagnosed before the onset of symptoms, as the parents had a previous child with the same condition. Infantile onset LAL-D is an autosomal recessive metabolic lysosomal storage disorder typically associated with a rapidly progressive, lethal course of disease.

The child was enrolled in a Phase 2, open label, single arm safety, efficacy and pharmacokinetics clinical study (“LAL-CL08”) in which she received weekly sebelipase alfa (SA) (KANUMA^®) infusions (l-3mg/kg) from 6 weeks of age. She was well when the first SA infusion was administered at the age of 6 weeks. The first SA infusion was administered at lmg/kg over 2 hours, followed by a flush of normal saline. The first three infusions were uneventful. The dose was increased at the fourth infusion. From the fourth infusion onwards, the child developed anaphylactic reactions as described below in Table 2. Table 2: Infusion Associated Reactions (I ARs)

IARs are known to be associated with recombinant protein based enzyme replacement therapies for other Lysosomal Storage Disorders (LSDs). SA is a recombinant human LAL isolated and purified from egg whites of transgenic chicken. Hypersensitivity reactions have been associated with infusions of SA. The conventional management of hypersensitivity reactions includes temporary interruption of infusion, lowering the infusion rate, and/or treatment with antihistamines, antipyretics, and/or corticosteroids or in severe case interruption / cessation of infusions. In such severe cases, cautious re-introduction of infusions, at a slower rate with increases as tolerated may be considered. Pre-treatment with antipyretics and/or antihistamines may prevent reactions subsequent infusions.

In the present case, the following treatment options were considered: increased duration of infusion at a slower rate, premedication with IV hydrocortisone and chlorphenamine, haematopoetic stem cell transplantation (HSCT), cessation of therapy and palliative care, and a desensitization protocol. Desensitization was elected as the best option and the desensitization plan set forth in Tables 3-5 was devised for the child, who weighed 5.3-6 kg.

Specifically, a modified“Boston protocol” was devised, which has been described for desensitization of hypersensitivity reactions to medications. Two stock solutions (0.02mg/ml and 0.2mg/ml) were prepared and administered according to the treatment protocol. The dose was increased from 0.35mg/kg, then 1 mg/kg to 2 mg/kg and then to 3 mg/kg. The volume of infusion (at the same dilution) was increased at the same infusion rate. Prolonged infusion times were administered, with doubling of doses as tolerated. Once the infusions at the initial fixed rate were deemed safe, the infusion rate was increased in steps to reduce infusion duration.

Small changes were made as required after every infusion. No premedication was used.

Table 3: Infusion 4 for Case # 2

Table 4: Infusion 8 for Case # 2

Table 5: Infusions for Case # 2

As a result, the child was successfully desensitized and did not experience any further IARs. Growth and development are normal for age, there are no gastrointestinal symptoms or organomegaly and biochemical markers have resolved. The significance of neutralizing antibodies remains unknown. There was no change in IFNy, TNFa, IL-2, IL-4, IL-6, or IL-10. Plasma tryptase measurements were normal. Anti-drug antibodies (ADA) were negative at the start of ERT. The ADA titre was 1/96 before the desensitization regime was commenced. ADA titre 1/500 - (neutralizing antibodies) to enzyme and cell uptake inhibition. Skin test 2 weeks after last IAR was negative for both 1 : 1000 and 1 : 100.

In conclusion, the desensitization protocol was effective in ameliorating hypersensitivity reactions associated with SA infusions.

C. Case # 3

A l3-year old Caucasian male with lysosomal acid lipase deficiency was started on sebelipase alfa at a dose of 1 mg/kg. He developed a pruritic truncal rash 5-7.5 hours after completion of his first 2-hour infusion, which was done without pretreatment. The rash resolved with diphenhydramine. He received pretreatment with prednisone and cetirizine prior to his second infusion, but 8.5 hours post-infusion he developed oropharyngeal angioedema, dyspnea, difficulty speaking, and a diffuse pruritic rash. Symptoms resolved within 1 hour of treatment with oral diphenhydramine. Tryptase was not drawn. He was subsequently referred to an Allergy Clinic. Skin prick and intradermal testing to sebelipase alfa was performed per manufacturer’s protocol, with prick testing at concentrations of 0.002 mg/ml (1:1000) and 0.02 mg/ml (1:100) and intradermal testing at 1:1000, 1:100, and 0.2 mg/ml (1:10). All testing was negative upon review at the standard reading time of 15 minutes after placement of the test. He then developed a local skin reaction at the site of testing several hours later. He had no detectable anti-drug antibodies. As his reactions and skin testing change occurred several hours after leaving the hospital, he did not have a clinician-observed reaction, but he and his parents were known to be reliable historians. His history of late-onset reaction with features consistent with anaphylaxis, worsening upon re-exposure despite premedication, and possible delayed positive skin testing suggested an immunologically mediated hypersensitivity, so desensitization was recommended. Initial desensitization took place in the Intensive Care Unit, given the severity of his reaction and the unusual delay in symptoms. Pre-medications, which could mask symptoms of a reaction, were avoided. His initial desensitization to a goal dose of 0.35 mg/kg was done very slowly with multiple dilutions of increasing concentration delivered at an increasing infusion rate such that each step of the lO-step protocol lasted 2 hours (Table 6). Because of concerns about drug stability at low concentrations and the duration of each step, each step used a separately mixed dilution. He tolerated the desensitization without reaction. Given that desensitization is a temporary state, desensitization was repeated for each subsequent infusion. The goal dose was increased to 1 mg/kg starting with the third desensitization. For each subsequent infusion, the duration of each step was serially shortened to a final protocol with one dilution delivered at increasing infusion rates and a duration of 12 minutes per step for the first 4 steps of a 6-step protocol (Table 6). The final desensitization protocol required 138 minutes, still longer than the standard infusion time for his dose, 68 mg, of 93.75 minutes. After multiple successful desensitization procedures, he transitioned his site of care to an outpatient infusion center.

Table 6: Sebelipase Alfa Desensitization Protocol for Case # 3.

aInitial desensitization protocol: Each step is a new syringe.

bSolution 1: 250 ml volume; 0.272 mg/ml concentration; 68 mg total dose in solution.

D. Discussion

A summary table of significant data from the three cases is provided below in Table 7.

Table 7: Summary of Cases

BAT: basophil activation test.

There are several clinically important lessons from these cases. The first is that clinicians must differentiate typical infusion reactions from hypersensitivity reactions that may require desensitization. Historical features are helpful in differentiating infusions reactions from hypersensitivity reactions, specifically the presence of symptoms suggestive of mast cell degranulation. Features seen in these cases suggestive of an antigen-specific, mast cell-mediated hypersensitivity reaction, include history of urticaria, angioedema, sneezing, and pruritus, and positive skin testing. Fever and rigors are more suggestive of typical infusion reactions, while wheezing, dyspnea, tachycardia, hypotension, and flushing are common to both. When available, skin testing can be helpful in determining the diagnosis and appropriate management (Demoly et al. 2014; Aranda el al. 2016). Studies have shown that those with infusion reactions with negative skin testing can often tolerate slower infusions without requiring desensitization (Aranda et al. 2016). Positive skin testing is consistent with allergic immediate hypersensitivity, and may indicate that the patient will not tolerate infusions with usual dosing and a slower rate or pre-medication (Aranda et al. 2016). The negative predictive value of skin testing to sebelipase alfa is not well-established, and a negative skin test is not sufficient to rule out immediate hypersensitivity as demonstrated in the second case. In addition, although features of mast cell degranulation better define hypersensitivity reactions, tryptase elevation does not appear to be sensitive or required. Furthermore, hypersensitivity reactions may occur either after multiple uneventful infusions as in Cases 1 and 2, or upon first exposure as in Case 3.

Clinicians must additionally be aware of the possibility of delayed reactions that may require slow dose escalation or desensitization. The phenomenon of delayed anaphylaxis suggests either biphasic anaphylaxis with a subtle immediate reaction and more severe late phase reaction, or anaphylaxis to a metabolite or by-product of the substance (Golden DB (2004) Novartis Found Symp 257: 101-110; discussion 110-105, 157-160, 276-185). In the third case presented here, the patient had reactions suggestive of either delayed-onset anaphylaxis or severe delayed infusion reactions. A missed immediate reaction is unlikely, as his first infusion was without premedication and was closely monitored, making it less plausible that an early phase reaction was missed. Additionally, his skin testing was negative upon initial read and

subsequently may have turned positive. Late-phase cutaneous reactions have been described for immediate hypersensitivity reactions, although they are described as being seen with positive immediate phase cutaneous reactions and their clinical importance is debated ( Bernstein /L, el al, Ann Allergy Asthma Immunol 100: S 1-148.).

Delayed-onset anaphylaxis to an infused medication is not well-described, and the diagnosis should be approached with caution and if suspected should prompt consultation and skin testing by an allergist. The best described cases of delayed anaphylaxis to any substance are those with alpha-gal (galactose- 1, 3-alpha-galactose) sensitization and anaphylaxis to non primate mammalian meat (Iweala OI, Burks AW (2016), Curr Allergy Asthma Rep 16:37). Alpha-gal hypersensitivity is also unique in its carbohydrate target, compared to protein antigens accounting for most food allergies (Iweala and Burks 2016). The pathophysiology of alpha-gal anaphylaxis is currently under investigation (Iweala and Burks 2016). Sebelipase alfa has a short serum half-life of only several minutes, making the delay in anaphylaxis even more puzzling in this case (Kanuma [package insert] (December 2015). New Haven, CT: Alexion Pharmaceuticals Inc). It remains unclear whether the metabolic pathway of enzyme replacement therapy with uptake into lysosomes plays a role in the delayed nature of his symptoms or if his reactions were not truly IgE-mediated anaphylactic reactions but rather delayed infusion reactions. Reports of delayed infusion reactions to enzyme replacement therapy have demonstrated that use of pre- medications, including IV steroids, prevent delayed reactions in only 50% of subjects, which raises the possibility that perhaps some of these patients had delayed-anaphylactic or other hypersensitivity reactions (Karimian Z, el al. (2017) Delayed infusion reactions to enzyme replacement therapies. JIMD Rep 34: 63-70). Perhaps a slow dose escalation protocol akin to a desensitization protocol would be a solution for those that do not respond to pre-medication and slowed infusion rates alone.

The delay in skin test results in Case 3 without an immediate phase response, in conjunction with the delayed anaphylactic clinical reaction, could be consistent with immune activation by a metabolite of the drug or presentation of the drug only after it is taken up into the lysosome. There are multiple mechanisms for anaphylaxis, the predominant being IgE-mediated and direct mast cell activation, but IgG and complement mediated mechanisms for anaphylaxis have also been described in animals (Finkelman FD, et al, (2016) J Allergy Clin Immunol 137: 1674-1680). IgG mediated anaphylaxis should be distinguished from IgG-mediated drug neutralization, which rather than causing significant clinical reaction reduces drug efficacy. Successful treatment of IgG neutralizing antibodies to enzyme replacement therapy has been described elsewhere (Messinger YH, et al. (2012) Genet Med 14: 135-142). General binding anti-sebelipase alfa antibodies as well as specific neutralizing anti-sebelipase alfa antibodies can be assayed, although in the phase 3 trial of sebelipase alfa, there was no evidence that these antibodies had any clinical significance (Messinger et al 2012 and Burton B, et al (2015), N Engl J Med 373: 1010-1020). Additionally, delayed positive skin testing as seen in Case 3 suggests activation of tissue resident mast cells in the skin by IgE or direct mast cell activation, although this does not exclude involvement of an IgG or complement-mediated process (Finkelman et al. 2016).

This is the first publication of desensitization protocols for sebelipase alfa. The biological mechanism behind desensitization remains unknown, and thus new desensitization protocols are developed with careful consideration to what has worked historically as well as the novel aspects of the desired drug and the nature of the patient’s reaction (Morales AR, et al., Ann Allergy Asthma Immunol 94: 575-580; Castells MC, et al., J Allergy Clin Immunol 122: 574- 580.; and Demoly et al. 2014). Each desensitization protocol in this case series was initially developed with prolonged steps, given the severity of reaction and concern for recurrence of severe anaphylaxis. The patient in the first case, unfortunately, suffered from relatively refractory symptoms despite these measures, and decision was made to reduce the target dose. Ultimately, each patient was able tolerate relatively short time intervals on subsequent desensitizations. It remains unknown whether the initial prolonged steps were necessary to be able to tolerate the final rapid desensitization. Furthermore, the final tolerated protocols for each patient were determined over time based on the success or reactions produced by previous protocols. In Cases 2 and 3, desensitization at lower doses preceded continued successful desensitizations at original or higher doses, given more rapidly. It is unknown whether the patient in Case 1 would have tolerated a dose increase after successful desensitization at the 0.35 mg/kg dose. Thus, development and tailoring of desensitization protocols should be done in conjunction with an allergist proficient in recognizing and managing anaphylaxis and differentiating it from infusion reactions.

This is the first reported case series of successful rapid desensitization to sebelipase alfa. In this case series, desensitization was a successful treatment option for patients with both immediate and delayed-onset reactions suggestive of anaphylaxis and allowed these patients to continue therapeutic enzyme replacement therapy.

Claims

CLAIMS: We claim:

1. A method of desensitizing a human pediatric patient having Lysosomal Acid Lipase Deficiency (LAL-D) against an infusion reaction associated with administration of sebelipase alfa, wherein the method comprises intravenously administering sebelipase alfa once every other week to the patient, wherein:

a first infusion of sebelipase alfa is administered at a dose of 0.35 mg/kg,

2. The method of claim 1, wherein the first, second, third, and fourth infusions are each administered over at least 21 hours.

3. The method of claim 1, wherein the first, second, third, and fourth infusions are each administered over a course of between 21 hours and 43 hours.

4. The method of any one of the preceding claims, wherein the dose is reduced and administered in incrementally increasing doses to the patient after the patient exhibits an infusion reaction.

5. The method of claim 4, wherein the patient weighs between 5 kg - 6 kg and wherein the infusion dose is reduced after the patient exhibits an infusion reaction and administered in incrementally increasing doses according to the following schedule:

6. The method of claim 4, wherein the patient weighs between 5 kg - 6 kg and wherein the infusion dose is reduced after the patient exhibits an infusion reaction and administered in incrementally increasing doses according to the following schedule:

7. The method of claim 4, wherein the infusion dose is reduced after the patient exhibits an infusion reaction and administered in incrementally increasing doses according to the following schedule:

aInitial protocol:

Solution 1: 100 ml volume; 0.0024 mg/ml concentration.

Solution 2: 100 ml volume; 0.024 mg/ml concentration.

Solution 3: 100 ml volume; 0.23529 mg/ml concentration.

8. The method of claim 4, wherein the infusion dose is reduced after the patient exhibits an infusion reaction and administered in incrementally increasing doses according to the following schedule:

aFinal protocol:

Solution 1: 100 ml volume; 0.024 mg/ml concentration.

Solution 2: 100 ml volume; 0.2355 mg/ml concentration.

9. The method of claim 4, wherein the infusion dose is reduced after the patient exhibits an infusion reaction and administered in incrementally increasing doses according to the following schedule:

10. The method of claim 4, wherein the infusion dose is reduced after the patient exhibits an infusion reaction and administered in incrementally increasing doses according to the following schedule:

aSolution 1: 250 ml volume; 0.272 mg/ml concentration; 68 mg total dose in solution.

11. The method of any one of claims 5-10, wherein the dose resumes at the same dose prior to the adverse event upon completion of the schedule and recovery from the infusion reaction

12. The method of any one of the preceding claims, wherein the infusion rate is increased upon confirmation that the dose is tolerated by the patient.

13. The method of any one of the preceding claims, wherein the infusion reaction is selected from the group consisting of pruritis, rash, headache, chills, anthralgia, myalgia, pyrexia, dizziness, urticaria, anaphylaxis, dyspnea, bronchospasm, hypotension, emesis, loss of consciousness, shock, tachycardia, myocardial infarction, and cardiac arrest.