Methods and compositions for treating Barth syndrome

Abstract

Disclosed herein, in one aspect, is a method for reducing immunogenicity comprising administering to a patient undergoing or having undergone BTHS gene therapy an effective amount of a non-depleting B cell inhibitor. Related compositions are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 293,514, filed December 23, 2021, the disclosure of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This specification includes a sequence listing submitted herewith, including a file named 178833-01101.xml, created on December 23, 2022, having the following size: 19,720 bytes, the contents of which are incorporated herein by reference.

[0003] Technical Field The present disclosure relates generally to compositions and methods for reducing the immunogenicity of biological therapeutics, and more specifically to gene therapy for treating Barth Syndrome. [Background technology]

[0004] First discovered in 1983 by Dr. Peter Barth, Barth Syndrome (OMIM#302060; BTHS) is a rare X-linked genetic disorder caused by pathological mutations in the TAFAZZIN gene, which encodes an acyltransferase required for cardiolipin remodeling. Thus, mutations in the TAFAZZIN gene, located at Xq28, cause pathological features by leading to the accumulation of intermediates in cardiolipin metabolism and mitochondrial dysfunction. Effects vary in presentation and severity, due in part to differences in tissue-specific gene expression. Clinical pathology typically includes cardiovascular dysfunction, myopathy, and immunodeficiency predisposing to infections. Mutations resulting in BTHS are typically familial, although it is estimated that 13% are de novo (Gonzalez 2012). It is estimated that there are approximately 150-200 known individuals with BTHS currently alive. However, it is increasingly recognized that the number of individuals living with this undiagnosed condition may be much higher, with 1 in 140,000 births affected.

[0005] Cardiomyopathy (CM) is estimated to occur in up to 70% of individuals with BTHS, developing within the first year of life. Cardiovascular pathology includes dilated CM, left ventricular noncompaction, hypertrophied CM, and rarely QTc prolongation, arrhythmias, and sudden cardiac death. Delayed diagnosis of CM in BTHS is common, and individuals require careful monitoring by a cardiologist for heart failure. Fortunately, most patients with CM respond well to standard medical treatment. However, a proportion of BTHS patients progress to require heart transplantation within the first 5 years of life. Additionally, larger cohort studies have noted that ventricular arrhythmias are common, with >10% of patients requiring implantable cardioverter defibrillator placement due to the risk of lethal arrhythmias. Arrhythmias can occur in individuals with BTHS during periods of otherwise seemingly healthy individuals.

[0006] Immunodeficiency, which predisposes to severe infections or sepsis, is common in BTHS. The most common is neutropenia, which occurs in up to 90% of patients. Neutropenia can range from mild and episodic to severe and chronic. In addition, pancytopenia has been noted in individuals with BTHS and is often mistaken for viral-induced bone marrow suppression (Rigaud 2013). It should be noted that half of the patients described as early dead in Barth's original paper succumbed to infectious complications (Barth 1983). The leading hypothesis for the cause of neutropenia is the presence of dysfunctional neutrophil precursors in the bone marrow. The use of granulocyte colony-stimulating factor (G-CSF) is often used to improve low neutrophil counts, but despite this, infections remain common.

[0007] Neuromuscular effects are common. Skeletal myopathies include proximal non-progressive muscle weakness. Hypotonia in childhood leads to global motor developmental delay, and hypotonia in adulthood leads to easy fatigability and reduced quality of life. Clinical trials aimed at supporting aerobic training appeared to improve quality of life, but provided little measurable physiological benefit.

[0008] Measurable metabolic abnormalities are useful in making the diagnosis of BTHS with a 5- to 20-fold increase in urinary 3-methylglutaconic acid. True metabolic sequelae are rare, but lactic acidosis and hypoglycemia are more common in infancy and can be fatal. Current treatment is aimed at supportive care during acute illness.

[0009] Advances in standard medical care have made BTHS a viable condition into adulthood. Current recommended treatments for BTHS are supportive but not curative. Supplements such as coenzyme Q, carnitine, pantothenic acid, and other B vitamins typically used to treat other mitochondrial diseases have not proven effective in BTHS (Rugolotto 2003).

[0010] However, there is significant variability in presentation, and once an individual has the severe spectrum of disease, complications can become life-threatening, requiring heart transplantation or extracorporeal membrane oxygenation (ECMO) to survive. Even milder or moderate cases of BTHS are at risk for acute metabolic crisis or severe infections that can manifest with little or no warning (barthsyndrome.org). This is a source of great anxiety for many patients who survive into adulthood.

[0011] Patients with BTHS endure lifelong limitations and reduced quality of life from both muscle weakness and chronic fatigue. Clinical trials utilizing formal exercise programs to build strength and endurance have proven ineffective, as have a myriad of supplements. Thus, there is an unmet need in the care of patients with BTHS. A panel of patients first met with the FDA in 2018 to explain their concerns and help advocate for treatment that goes beyond mere symptom management.

[0012] The advances offered by the rapidly evolving therapeutic field of gene therapy are highly attractive for the treatment of Barth syndrome. Gene therapy would allow for the restoration of normal production of acyltransferases, thereby reducing clinical symptoms.

[0013] In gene therapy, genetically modified cells are generated with the aim of incorporating defective copies of genes. However, there is a risk that parts of the "machinery" utilized for the genetic modification of the cells may be "presented" by the modified cells and recognized as a "foreign" agent by the host. Such recognition may induce a rejection reaction, which could potentially render such treatment ineffective or, in severe cases, potentially trigger an autoimmune reaction.

[0014] More recently, the advent of gene therapy has seen substantial obstacles in the immunogenicity of the viral vectors utilized to administer transgenes, as well as the immunogenicity of the transgene protein itself after expression by the recipient's cells. Immunogenicity of vectors and transgenes results in: 1) reduced efficacy when the vector and transgene are bound and eliminated by antibodies produced by the recipient; 2) the need for increased doses, which increases safety risks and costs; 3) difficult or impossible re-administration if the subject develops antibodies against the vector or transgene after previous administration. Recipients may have pre-existing antibodies against the vector even before the first administration due to cross-reaction with naturally occurring viruses. Summary of the Invention [Problem to be solved by the invention]

[0015] Thus, there is a need for methods and compositions for reducing the immunogenicity induced by Barth syndrome gene therapy. [Means for solving the problem]

[0016] Disclosed herein, in one aspect, is a method of reducing immunogenicity associated with gene therapy for Barth Syndrome (BTHS), comprising administering to a patient undergoing or who has undergone BTHS gene therapy an effective amount of a non-depleting B cell inhibitor.

[0017] In some embodiments, BTHS gene therapy can include a recombinant adeno-associated virus (rAAV) vector (such as an AAV9 vector) encoding a TAFAZZIN transgene.

[0018] In some embodiments, the B cell inhibitor is a CD32BxCD79B bispecific antibody capable of immunospecifically binding to an epitope of CD32B and an epitope of CD79B. In some embodiments, the CD32BxCD79B bispecific antibody is: (A) VL comprising the amino acid sequence of SEQ ID NO: 1 CD32B domain; (B) VH comprising the amino acid sequence of SEQ ID NO:2 CD32B domain; (C) VL comprising the amino acid sequence of SEQ ID NO:3 CD79B Domain; and (D) VH comprising the amino acid sequence of SEQ ID NO: 4 CD79B Includes the domain.

[0019] In some embodiments, the CD32B×CD79B bispecific antibody comprises: (A) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:5; (B) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:6; and (C) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:7. It is an Fc diabody comprising:

[0020] In some embodiments, the method may further comprise administering the Fc diabody at a dose of about 5 mg / kg to about 100 mg / kg, or about 5 mg / kg to about 50 mg / kg, or about 5 mg / kg to about 40 mg / kg, in a dosing regimen between a dose once per week and a dose once every six weeks. In some embodiments, the method may comprise administering the Fc diabody at a dose of about 10 mg / kg, and in a dosing regimen of a dose once every one to four weeks. In some embodiments, the method may comprise administering three doses of the Fc diabody at a dose of about 10 mg / kg, spaced two to six weeks apart.

[0021] One exemplary dosing regimen includes a first dose one week prior to the first BTHS gene therapy delivery, a second dose two weeks after the first BTHS gene therapy delivery, and a third dose three to four weeks after the second BTHS gene therapy delivery.

[0022] In some embodiments, the method may include administering a first dose about 2 days to about 6 weeks (e.g., 2 days, 6 days, 1 week, 2 weeks, 4 weeks) prior to administration of BTHS gene therapy, administering a second dose about simultaneously with administration of BTHS gene therapy, and administering a third dose about 2 days to about 6 weeks (e.g., 2 days, 6 days, 1 week, 2 weeks, 4 weeks) after administration of BTHS gene therapy.

[0023] In some embodiments, the Fc diabody results in inhibition of its own immunogenicity upon administration, resulting in lower prevalence and / or titers of anti-drug antibodies (ADA) at increased doses, hi some embodiments, ADA does not neutralize the Fc diabody.

[0024] In some embodiments, the Fc diabody binds in a dose-dependent manner to at least 80% of the B cells upon administration and remains bound to at least 50% of the B cells for at least 4 weeks after the last administration.

[0025] In some embodiments, the Fc diabody provides sustained inhibition of immunoglobulin production without depleting circulating B cells, hi some embodiments, the immunoglobulins comprise one or more of IgM, IgA, IgG, and IgE.

[0026] In some embodiments, the method may further include monitoring the patient by examining the presence of specific antibodies to the BTHS gene therapy. In some embodiments, the method may further include administering one or more doses of a B cell inhibitor to further modulate immunogenicity.

[0027] In some embodiments, the method may further comprise co-administering one or more immunomodulatory agents, such as sirolimus, rapamycin, abatacept, teplizumab, and Streptococcus pyogenes immunoglobulin G-degrading enzyme, hi some embodiments, the method may further comprise co-administering sirolimus.

[0028] Also provided herein is a pharmaceutical composition comprising a non-depleting B cell inhibitor as disclosed herein, provided (e.g., packaged) in a therapeutically effective unit dose. Instructions for administration regimens as disclosed herein may also be provided.

[0029] A further aspect relates to the use of a non-depleting B cell inhibitor disclosed herein in the manufacture of a medicament for reducing immunogenicity associated with Barth Syndrome (BTHS) gene therapy, optionally wherein the BTHS gene therapy comprises a genetically engineered cell harboring a recombinant adeno-associated virus (rAAV) vector encoding a TAFAZZIN transgene.

[0030] Also provided herein is a pharmaceutical composition for reducing immunogenicity associated with gene therapy for Barth Syndrome (BTHS), comprising an effective amount of a CD32B×CD79B bispecific antibody capable of immunospecifically binding to an epitope of CD32B and an epitope of CD79B prior to, concomitantly with, and / or after BTHS gene therapy, and optionally, the BTHS gene therapy comprises genetically engineered cells harboring a recombinant adeno-associated virus (rAAV) vector encoding a TAFAZZIN transgene.

[0031] In some embodiments, the CD32B×CD79B bispecific antibody comprises: (A) VL comprising the amino acid sequence of SEQ ID NO: 1 CD32B domain; (B) VH comprising the amino acid sequence of SEQ ID NO:2 CD32B domain; (C) VL comprising the amino acid sequence of SEQ ID NO:3 CD79B Domain; and (D) VH comprising the amino acid sequence of SEQ ID NO: 4 CD79B It may include a domain.

[0032] CD32B×CD79B bispecific antibodies: (A) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:5; (B) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:6; and (C) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:7. The Fc diabody may comprise:

[0033] The pharmaceutical compositions, in some embodiments, can include the Fc diabody at a dose of about 5 mg / kg to about 100 mg / kg, or about 5 mg / kg to about 50 mg / kg, or about 5 mg / kg to about 40 mg / kg, and in a dosing regimen of once a week to once every six weeks.

[0034] The pharmaceutical composition may, in some embodiments, comprise the Fc diabody at a dose of about 10 mg / kg, administered at 2-6 week intervals or once every 1-4 weeks. In certain embodiments, the administration regimen may comprise a first dose 1 week prior to the first BTHS gene therapy delivery, a second dose 2 weeks after the first BTHS gene therapy delivery, and a third dose 3-4 weeks after the second BTHS gene therapy delivery.

[0035] In some embodiments, the pharmaceutical composition may include a first dose administered about 2 days to about 6 weeks prior to administration of BTHS gene therapy, a second dose administered approximately simultaneously with administration of BTHS gene therapy, and a third dose administered about 2 days to about 6 weeks after administration of BTHS gene therapy.

[0036] The pharmaceutical composition, in some embodiments, may further comprise one or more immunomodulatory agents selected from sirolimus, rapamycin, abatacept, teplizumab, and Streptococcus pyogenes immunoglobulin G-degrading enzyme. [Brief description of the drawings]

[0037] [Figure 1] FIG. 2 is a schematic diagram of an exemplary vector. [Diagram 2] An exemplary construct is B951 (pds2TR-Des-coTAZ_Dual4). [Diagram 3]An exemplary construct is C064 (pds2TR-Des-coTAZiso2_Dual4). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Disclosed herein in one aspect is a method of reducing immunogenicity associated with gene therapy for Barth Syndrome (BTHS), comprising administering to a patient undergoing or who has undergone BTHS gene therapy an effective, non-depleting amount of a B cell inhibitory agent. In some embodiments, the B cell inhibitory agent is a CD32BxCD79B bispecific antibody, such as those disclosed in U.S. Patent Publication No. 2016 / 0194396, WO 2015 / 021089, and WO 2017 / 214096, each of which is incorporated by reference in its entirety.

[0039] definition For convenience, certain terms used in the specification, examples, and appended claims are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0040] The use of the terms "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0041] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device being used to determine the value or the variation that exists among test subjects. Typically, the term is meant to encompass a variation of less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the context.

[0042] The term "substantially" means more than 50%, preferably more than 80%, and most preferably more than 90% or more than 95%.

[0043] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports a definition that refers only to alternatives and "and / or."

[0044] As used in the specification and claims, the terms "comprising" (and including any form such as "comprise" and "comprises"), "having" (and can have any form such as "have" and "has"), "including" (and including any form such as "includes" and "include") or "containing" (and any form of inclusion such as "contains" and "contain") are inclusive or open ended and do not exclude additional unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method, system, host cell, expression vector, and / or composition of the invention. Additionally, the compositions, systems, host cells and / or vectors of the invention can be used to achieve the methods and proteins of the invention.

[0045] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the present disclosure.

[0046] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in that description of an embodiment.

[0047] Use of the term "for example" and its corresponding abbreviation "eg" means that the particular term recited (whether italicized or not) is representative of and is an embodiment of the invention that is not intended to be limited to the particular example referenced or cited, unless specifically stated otherwise.

[0048] "Gene" refers to a collection of nucleotides that encodes a polypeptide, and includes cDNA and genomic DNA nucleic acid molecules. "Gene" also refers to a nucleic acid fragment that can act as regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.

[0049] As used herein, "TAFAZZIN" refers to a phospholipid-lysophospholipid transacylase that can be responsible for the modification of cardiolipin (a membrane phospholipid) to its tetralinoleoyl form. In some embodiments, TAFAZZIN may refer to full-length human TAFAZZIN or human TAFAZZIN lacking exon 5, both of which may exhibit transacylase activity. In certain embodiments, tafaccin may refer to full-length mouse TAFAZZIN, which is homologous to human TAFAZZIN lacking exon 5.

[0050] As used herein, an "antibody" or "antibody molecule" refers to a protein that includes at least one immunoglobulin variable domain sequence, such as an immunoglobulin chain or a fragment thereof. Antibody molecules include antibodies (e.g., full-length antibodies) and antibody fragments. In some embodiments, an antibody molecule includes an antigen-binding or functional fragment of a full-length antibody, or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., IgG) that occurs naturally or is formed by recombinant processes of normal immunoglobulin gene fragments. In some embodiments, an antibody molecule refers to an immunologically active antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. An antibody fragment, e.g., a functional fragment, is a portion of an antibody, such as a Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). A functional antibody fragment binds to the same antigen that is recognized by an intact (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of the variable regions, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, or a recombinant single chain polypeptide molecule in which the light and heavy chain variable regions are linked by a peptide linker ("scFv protein"). In some embodiments, an antibody fragment does not include a portion of an antibody without antigen binding activity, such as an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments, such as dAb (domain antibodies), single chain, Fab, Fab' and F(ab')2 fragments, and single chain variable fragments (scFv). The terms "Fab" and "Fab fragment" are used interchangeably and refer to one constant domain and one variable domain from each heavy and light chain of an antibody, i.e., V L , C L , V H and C H Refers to the area containing 1.

[0051] Throughout this specification, the numbering of residues in the constant region of the IgG heavy chain is based on Kabat et al., Sequences of Proteins of Immunological Interest, 5 thThe numbering of the EU index as in Ed. Public Health Service, NH1, MD (1991) ("Kabat") is expressly incorporated herein by reference. The term "EU index as in Kabat" refers to the numbering of human IgG1 EU antibodies. Amino acids from the mature heavy and light chain variable domains of immunoglobulins are designated by the position of the amino acid in the chain. Kabat describes a number of amino acid sequences for antibodies, identifies amino acid consensus sequences for each subgroup, assigns a residue number to each amino acid, and CDRs are identified as defined by Kabat (CDR H It will be appreciated that 1 begins 5 residues earlier according to the definition by Chothia, C. & Lesk, A.M. ((1987) "Canonical structures for the hypervariable regions of immunoglobulins". J. Mol. Biol. 196:901-917). Kabat's numbering scheme can be extended to antibodies not included in his Summary of Invention by aligning the antibody in question with one of Kabat's consensus sequences with reference to the conserved amino acids. This method for assigning residue numbers has become standard in the art and readily identifies amino acids at equivalent positions in various antibodies, including chimeric or humanized variants. For example, the amino acid at position 50 of a human antibody light chain occupies the equivalent position as the amino acid at position 50 of a murine antibody light chain.

[0052] In some embodiments, the antibody molecule is monospecific, e.g., comprises binding specificity for a single epitope. In some embodiments, the antibody molecule is multispecific, e.g., comprises multiple immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule.

[0053] The terms "bispecific antibody molecule," "diabody," and "dual affinity retargeting (DART®)" antibody are used interchangeably herein to refer to an antibody molecule having specificity for two or more (e.g., 2, 3, 4, or more) epitopes and / or antigens. In some embodiments, the antibody may be a diabody or scaffold capable of antigen binding, such as those disclosed in U.S. Patent Application Publication No. 2016 / 0194396, WO 2015 / 021089, and WO 2017 / 214096, each of which is incorporated by reference in its entirety. In some embodiments, the antibody may be a CD32BxCD79B bispecific diabody (i.e., a "CD32BxCD79B diabody" and such diabodies further comprising an Fc domain (i.e., a "CD32BxCD79B Fc diabody"). In some embodiments, the antibody may be a humanized CD32BxCD79B DART® antibody produced in Chinese hamster ovary cells having a molecular weight of 111.5 kDa.

[0054] As used herein, "antigen" (Ag) refers to a macromolecule, including any protein or peptide. In some embodiments, an antigen is a molecule that can trigger an immune response, including, for example, activation of specific immune cells and / or antibody production. Antigens are not only involved in antibody production. T cell receptors also recognize antigens (albeit those in which peptides or peptide fragments form complexes with MHC molecules). Any macromolecule, including almost any protein or peptide, can be an antigen. Antigens can also be derived from genome recombinants or DNA. For example, any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that can elicit an immune response encodes an "antigen." In some embodiments, an antigen need not be encoded only by the full-length nucleotide sequence of a gene, nor need an antigen be encoded by a gene at all. In some embodiments, an antigen can be synthesized or derived from a biological sample, such as a tissue sample, a tumor sample, a cell, or a fluid that contains other biological components. As used herein, "tumor antigen" or, interchangeably, "cancer antigen" includes any molecule present on or associated with a cancer, such as a cancer cell or tumor microenvironment, that can trigger an immune response. As used herein, an "immune cell antigen" includes any molecule present on or associated with an immune cell that is capable of eliciting an immune response.

[0055] An "antigen-binding site" or "antigen-binding fragment" or "antigen-binding portion" of an antibody molecule (used interchangeably herein) refers to a portion of an antibody molecule, e.g., an immunoglobulin (Ig) molecule such as IgG, that is involved in antigen binding. In some embodiments, the antigen-binding site is formed by amino acid residues of the variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the variable regions of the heavy and light chains, called hypervariable regions, are arranged between more conserved adjacent stretches called "framework regions" (FRs). FRs are amino acid sequences naturally found between and adjacent to hypervariable regions in immunoglobulins. In some embodiments, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface that is complementary to the three-dimensional surface of a bound antigen. The three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs". Framework regions and CDRs are defined and described, for example, in Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy and variable light chains) is typically composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable light chain (VL) CDRs are generally defined to include residues 27-32 (CDR1), 50-56 (CDR2), and 91-97 (CDR3). The variable heavy chain (VH) CDRs are generally defined to include residues 27-33 (CDR1), 52-56 (CDR2), and 95-102 (CDR3).One of skill in the art will appreciate that loops may be of different lengths across antibodies and numbering systems, such as Kabat or Chothia controls, so that the frameworks have consistent numbering across antibodies.

[0056] In some embodiments, an antigen-binding fragment of an antibody (e.g., when included as part of a fusion molecule) may lack or not include a complete Fc domain. In some embodiments, an antibody-binding fragment may not include a complete IgG or a complete Fc, but may include one or more constant regions (or fragments thereof) from a light chain and / or a heavy chain. In some embodiments, an antigen-binding fragment may be completely free of any Fc domain. In some embodiments, an antigen-binding fragment may be substantially free of a complete Fc domain. In some embodiments, an antigen-binding fragment may include a portion of a complete Fc domain (e.g., a CH2 or CH3 domain or portion thereof). In some embodiments, an antigen-binding fragment may include a complete Fc domain. In some embodiments, the Fc domain is an IgG domain, such as an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the Fc domain includes a CH2 domain and a CH3 domain.

[0057] As used herein, "administering" and similar terms refer to delivering a composition to the individual being treated. Preferably, the compositions of the present disclosure are administered by parenteral routes, including, for example, subcutaneous, intramuscular, or preferably intravenous routes.

[0058] As used herein, "effective amount" means an amount of a bioactive agent or diagnostic agent sufficient to provide a desired local or systemic effect with a reasonable risk / benefit ratio corresponding to any medical treatment or diagnostic test. This will vary depending on the patient, the disease, the procedure being performed, and the nature of the drug. The therapeutically effective amount will vary depending on the patient and disease state being treated, the patient's weight and age, the severity of the disease state, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art. Dosage amounts are, for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, Approximately 500ng to approximately 4,000mg, approximately 1μg to approximately 3,500mg, approximately 5μg to approximately 3,000mg, approximately 10μg to approximately 2,600mg, approximately 20μg to approximately 2,575mg, approximately 30μg to approximately 2,550mg , about 40μg to about 2,500mg, about 50μg to about 2,475mg, about 100μg to about 2,450mg, about 200μg to about 2,425mg, about 300μg to about 2,000, about 400μg to about 1,17 5mg, about 500μg to about 1,150mg, about 0.5mg to about 1,125mg, about 1mg to about 1,100mg, about 1.25mg to about 1,075mg, about 1.5mg to about 1,050mg, about 2.0m g ~ about 1,025mg, about 2.5mg - about 1,000mg, about 3.0mg - about 975mg, about 3.5mg - about 950mg, about 4.0mg - about 925mg, about 4.5mg - about 900mg, about 5mg - about The antibody or antigen-binding portion thereof may be administered in an amount of about 875 mg, about 10 mg to about 850 mg, about 20 mg to about 825 mg, about 30 mg to about 800 mg, about 40 mg to about 775 mg, about 50 mg to about 750 mg, about 100 mg to about 725 mg, about 200 mg to about 700 mg, about 300 mg to about 675 mg, about 400 mg to about 650 mg, about 500 mg, or about 525 mg to about 625 mg of the antibody or antigen-binding portion thereof provided herein. Administration may be, for example, weekly, every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks. Dosing regimens may be adjusted to provide an optimal therapeutic response.An effective amount is also one in which any toxic or adverse effects (side effects) of the drug are minimized and / or outweighed by the beneficial effects. Dosing can be intravenously at or about 6 mg / kg or 12 mg / kg weekly, or 12 mg / kg or 24 mg / kg every other week. Further dosing regimens are described below.

[0059] As used herein, "pharmaceutical acceptable" refers to those useful for preparing pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise undesirable, including those that are acceptable for veterinary and human pharmaceutical use. Examples of "pharmaceutical acceptable liquid carriers" include water and organic solvents. Preferred pharmaceutical acceptable aqueous liquids include PBS, saline, dextrose solution, and the like.

[0060] The term "immunogenicity" refers to the ability of a particular substance, such as an antigen or epitope, to elicit an immune response, which may be humoral and / or cell-mediated, in humans and other animals. In some embodiments, administration of a composition of the present disclosure reduces the immunogenicity of a biological substance, such as a therapeutic agent, and / or increases immune tolerance to the biological substance. As used herein, "tolerance" or "immune tolerance" refers to the absence of an immune response to a particular antigen (e.g., a therapeutic biologic) in the context of an otherwise substantially normal immune system.

[0061] As used herein, "major histocompatibility complex" or "MHC" proteins refer to a set of cell surface molecules encoded by a large gene family that play a key role in the immune system of vertebrates. The key function of these proteins is to bind peptide fragments derived from endogenous or exogenous (foreign) proteins and present them on the cell surface for recognition by appropriate T cells of the host organism. The MHC gene family is divided into three subgroups: class I, class II, and class III. Human MHC class I and class II genes are also called human leukocyte antigen (HLA)-HLA class I and HLA class II, respectively. Some of the most studied HLA genes in humans are the nine MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, and HLA-DRB345.

[0062] Various aspects of the disclosure are described in further detail below. Additional definitions are set forth throughout the specification.

[0063] Non-depleting B cell inhibitors and pharmaceutical compositions In some embodiments, B cell inhibitors can be used to reduce or modulate immunogenicity. In some embodiments, such B cell inhibitors are non-depleting immunomodulators. As used herein, "non-depleting" or "non-depleting" means that the inhibitor or immunomodulator does not completely deplete B cell activity. In contrast, "depleting" B cells means that the agent acts to eliminate or destroy B cells, such as anti-CD20 antibodies, e.g., rituximab. Thus, in some embodiments, the non-depleting B cell inhibitor or immunomodulator disclosed herein is not rituximab. In some embodiments, the non-depleting B cell inhibitor or immunomodulator is not an anti-CD20 antibody or other CD20 inhibitor.

[0064] Exemplary non-depleting B cell inhibitors include, but are not limited to, CD32B×CD79B dual specific inhibitors; CD32B modulators; B cell receptor (BCR) blockers, such as anti-CD22 molecules; B cell survival and activation inhibitors, such as B cell activating factor (BAFF) or A proliferation-inducing ligand (APRIL) inhibitors, such as belimumab; anti-CD40 and anti-CD40L molecules; and Bruton's tyrosine kinase (BTK) inhibitors, such as ibrutinib (PCI-32765) and acalabrutinib.

[0065] In some embodiments, the B cell inhibitor may be a CD32BxCD79B bispecific antibody, such as those disclosed in US Patent Publication No. 2016 / 0194396, WO 2015 / 021089, and WO 2017 / 214096 (all incorporated by reference in their entireties), or an antigen-binding fragment thereof.

[0066] Exemplary CD32B×CD79B bispecific diabodies can comprise two or more polypeptide chains, (1) CD32B(VL CD32B ), e.g., a VL domain having the sequence (SEQ ID NO: 1) CD32B domain: [ka] (2) CD32B(VH CD32B ), e.g., a VH domain having the sequence (SEQ ID NO:2) of an antibody that binds to CD32B : [ka] (3) CD79B(VL CD79B ), e.g., a VL domain having the sequence (SEQ ID NO:3) CD79B domain: [ka] (4) CD79B (VH CD79B ), e.g., a VH domain having the sequence (SEQ ID NO: 4) of an antibody that binds to CD79B : [ka] may include.

[0067] In some embodiments, the B cell inhibitor can be PRV-3279, a humanized CD32B×CD79B Dual Affinity Re-Targeting (DART®) protein produced in Chinese hamster ovary cells with a molecular weight of 111.5 kDa. DART® proteins are bispecific antibody-based molecules that can simultaneously bind to two different antigens. PRV-3279 is designed to target CD32B (Fc gamma receptor IIb) and CD79B (immunoglobulin-associated beta subunit of the B cell receptor (BCR) complex) on B lymphocytes. Co-ligation of CD32B and CD79B in a preferential cis-binding mode on B lymphocytes triggers signaling of CD32B-binding immunoreceptor tyrosine-based inhibitory motifs, reducing antigen-mediated naive and memory B cell activation without extensive depletion. To extend in vivo half-life, PRV-3279 also contains a human immunoglobulin G (IgG)1 Fc region that has been mutated to significantly reduce or eliminate undesirable binding to FcγR and complement, while retaining affinity for neonatal FcR binding and utilizing the IgG salvage pathway mediated by this receptor.

[0068] The CD32B molecule is a transmembrane inhibitory receptor that is widely expressed on B cells and other immune effector cells such as macrophages, neutrophils, and mast cells. The anti-CD32B component of PRV-3279 is based on a humanized version of the mouse monoclonal antibody (mAb) 8B5 from MacroGenics. CD79B is an essential signaling component of the BCR that is expressed only on B cells. The anti-CD79B component of PRV-3279 is based on a humanized version of the mouse mAb CB3.

[0069] In some embodiments, PRV-3279 comprises the following sequence (CDRs are underlined and the coil domain is in bold): Chain 1 (Fc-CD32BVL-CD79bVH-E coil): (SEQ ID NO: 5) [ka] Strand 2 (CD79bVL-CD32BVH-K coil): (SEQ ID NO: 6) [ka] Chain 3 (Fc): (SEQ ID NO: 7) [ka]

[0070] PRV-3279 can bind to the majority of B cells (e.g., >80-90%) upon dosing, including both naive and memory phenotypes, in a dose-dependent manner and remain bound to at least 50% of B cells for >4 weeks, demonstrating sustained durability of the potential pharmacodynamic (PD) effects of PRV-3279 and supporting monthly (or longer) dosing frequencies.

[0071] PRV-3279 has previously been shown to reduce immunity to viral vaccines (hepatitis A vaccines) in healthy subjects, suggesting that it can inhibit immunogenicity to viral vectors. Additionally, a pilot study was conducted to evaluate the effect of PRV-3279 surrogates for gene therapy using adeno-associated viruses (AAV) to evaluate efficacy and immunogenicity in huCD32b transgenic mice (see PCT / US2020 / 044312 and US Patent Publication No. 2021 / 0130464, both of which are incorporated herein by reference in their entireties). This was a dose-response study with nine arms including a negative (no AAV9) control, followed by AAV9 in combination with placebo, PRV-3279 alone at three dose levels, co-administration of sirolimus with PRV-3279 at three dose levels, and sirolimus alone (Table 1). Longitudinal sampling included levels of IgM, IgG, AAV9 vector copies and tissue transgene activity at the end of the study.

[0072] Specific test objectives: 1. The amount of AAV9 vector detectable in the blood (vector clearance rate) 2. The amount of GAA transgene detectable in tissues (efficiency of gene therapy transfection) 3. Levels of anti-AAV9 antibodies in the blood (immunogenicity of AAV vectors) 4. Total IgM (PD marker of PRV-3279 activity)

[0073] [Table 1]

[0074] Dose-dependent and sustained B cell binding by PRV-3279 results in durable inhibition of immunoglobulin production without depleting circulating immune cell subsets, including B cells. Immunoglobulins reduced in peripheral blood include IgM, IgA, IgG, and IgE. Inhibition can be observed in the absence or presence of antigenic stimulation (e.g., vaccination). This is an advantageous safety feature of PRV-3279 as a non-depleting agent, allowing patients to retain circulating immune cells such as B cells as part of a functioning immune system. In contrast, patients receiving cell depleting agents (e.g., rituximab, ocrelizumab, imbilizumab) require significant time (e.g., up to 1 year) to restore depleted cell types. This is particularly important considering the treatment of BTHS, where immunodeficiency exists and the risk of serious infections remains throughout life. Immunocompetence is also clinically important for primary vaccination series in children, as BTHS is present from birth.

[0075] In another aspect, pharmaceutical compositions are provided that can be used in the methods disclosed herein, i.e., to reduce or suppress immunogenicity in a subject in need of such reduction or suppression during or after undergoing BTHS gene therapy that causes significant immunogenicity, or because the subject has pre-existing immunogenicity to a biotherapeutic agent (e.g., in the case of pre-existing anti-AAV antibodies due to a previous wild-type adenovirus infection or due to previous exposure to rAAV therapy). In some embodiments, the compositions disclosed herein can be administered to a patient prior to undergoing BTHS gene therapy to prevent immunogenicity and / or reduce pre-existing antibodies.

[0076] In some embodiments, the pharmaceutical composition comprises a B cell inhibitor disclosed herein and a pharma- ceutically acceptable carrier. The B cell inhibitor may be formulated into a pharmaceutical composition together with a pharma- ceutically acceptable carrier. Additionally, the pharmaceutical composition may include instructions for use of the composition to treat a patient to reduce or suppress immunogenicity in a subject in need thereof, for example, during or after administration of a biologic agent that causes significant immunogenicity.

[0077] As used herein, "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, and other excipients that are physiologically compatible. Preferably, the carrier is suitable for parenteral, oral, or topical administration. Depending on the route of administration, the active compound, e.g., small molecule or biological agent, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0078] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, as well as conventional excipients for the preparation of tablets, pills, capsules, etc. The use of such media and agents for the formulation of pharma-ceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions provided herein is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0079] The pharma- ceutically acceptable carrier may contain a pharma- ceutically acceptable antioxidant. Examples of pharma-ceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0080] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, as well as injectable organic esters such as ethyl oleate. If necessary, proper fluidity can be maintained, for example, by using coating materials such as lecithin, maintaining the required particle size in the case of dispersions, and by using surfactants. In many cases, it can be useful to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of an injectable composition can be achieved by including an agent that delays absorption, such as monostearate salts and gelatin, in the composition.

[0081] These compositions may also contain functional excipients such as preservatives, wetting agents, emulsifying agents and dispersing agents.

[0082] Therapeutic compositions typically must be sterile, non-phylogenic, and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0083] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the above-listed components as necessary, followed by sterilization, for example, by microfiltration.Generally, dispersions are prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other components required from those listed above.For the preparation of sterile powders for sterile injectable solutions, preparation methods include vacuum drying and freeze-drying (lyophilization), which allows the powder of active ingredient and any additional desired ingredients to be obtained from its solution that has been previously sterile-filtered.The active agent can be mixed under sterile conditions with additional pharmaceutically acceptable carriers, and any preservatives, buffers or propellants that may be required.

[0084] Prevention of presence of microorganisms can be ensured by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like, in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

[0085] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response) For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0086] Exemplary dosage ranges for administering antibodies include: 10-1000 mg (antibody) / kg (patient weight), 10-800 mg / kg, 10-600 mg / kg, 10-400 mg / kg, 10-200 mg / kg, 30-1000 mg / kg, 30-800 mg / kg, 30-600 mg / kg, 30-400 mg / kg, 30-200 mg / kg, 50-1000 mg / kg, 50-800mg / kg, 50-600mg / kg, 50-400mg / kg, 50-200mg / kg, 100-1000mg / kg, 100-900mg / kg, 100-800mg / kg, 100-700mg / kg, 100-600mg / kg, 100-500mg / kg, 100-400mg / kg, 100-300mg / kg, and 100-200mg / kg. Exemplary administration schedules include once every 3 days, once every 5 days, once every 7 days (i.e., once a week), once every 10 days, once every 14 days (i.e., once every 2 weeks), once every 21 days (i.e., once every 3 weeks), once every 28 days (i.e., once every 4 weeks), once a month, once every 5 weeks, and once every 6 weeks.

[0087] In some embodiments, about 5-40 mg / kg, about 5-20 mg / kg, or about 10 mg / kg per dose of PRV-3279 can be administered once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, or once every 6 weeks. One or more doses can be administered, such as one dose, two doses, or three doses. Administration can be by IV infusion. Any combination of the foregoing (e.g., three doses of 10 mg / kg / dose, once every 4 weeks) can be used for reducing the immunogenicity of biotherapeutics, including gene therapy products. In some embodiments, a first dose can be administered 2-6 weeks (e.g., 4 weeks) prior to gene therapy, a second dose about contemporaneous with gene therapy, and a third dose 2-6 weeks (e.g., 4 weeks) after gene therapy. The patient can then be monitored by examining the amount of specific antibodies against the gene therapy vector (e.g., rAAV) and / or transgene. If no or barely detectable antibodies, no additional PRV-3279 is necessary. If significant amounts of antibodies are present, one or more doses of PRV-3279 can be administered to further modulate immunogenicity.

[0088] For ease of administration and uniformity of dosage, it may be advantageous to formulate parenteral compositions in unit dosage form. Unit dosage form as used herein refers to a physically separate unit suitable as a unit dosage form for the patient to be treated. Each unit contains a predetermined amount of active agent calculated to produce a desired therapeutic effect in combination with any necessary pharmaceutical carrier. The specifications of unit dosage form are determined by and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the formulation technology, such as the active compound for treating a susceptibility in an individual.

[0089] The actual dosage level of the active ingredient in the pharmaceutical compositions disclosed herein may be varied to obtain an amount of the active ingredient that is not toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. As used herein in the context of administration, "parenteral" refers to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection, and infusion.

[0090] The phrases "parenteral administration" and "administered parenterally" as used herein refer to modes of administration other than enteral (i.e., via the digestive tract) and topical administration, usually by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Intravenous injection and infusion are frequently (although not exclusively) used for antibody administration.

[0091] When agents provided herein are administered to humans or animals as pharmaceuticals, they can be administered alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (e.g., 0.005 to 70%, e.g., 0.01 to 30%) of the active ingredient in combination with a pharma- ceutically acceptable carrier.

[0092] Therapeutic Uses and Methods The compositions disclosed herein can be used to reduce or suppress immunogenicity caused by BHTS gene therapy delivered by various means (e.g., AAV and other wild-type and recombinant vectors, lentivirus-modified human stem cells). In some embodiments, the B cell immunomodulators disclosed herein can be used to improve rAAV (recombinant adeno-associated virus) vector-based viral delivery of transgenes for inherited enzyme deficiencies, such as, for example, TAFAZZIN deficiency associated with BHTS.

[0093] In some embodiments, the B cell immunomodulators disclosed herein can be used to modulate limited immune responses elicited by multiple delivery routes (even in recognized immune privileged sites), such as systemic, intramuscular, ocular (requiring high local doses resulting in local immune responses), and central nervous system (CNS) (where leakage of viral capsid from the CNS induces a systemic response that reduces AAV uptake in the CNS).

[0094] In some embodiments, the B cell immunomodulators disclosed herein can be used to modulate multiple restricted immunological pathways that are B cell dependent, including: ·Development of neutralizing antibodies (nAbs) ·Antibody-dependent cell-mediated cytotoxicity ·Antibody-dependent complement-mediated cytotoxicity Autonomous B cell activation, e.g., via Toll-like receptors (TLRs) ·B cell-mediated T cell co-stimulation ·B cell-mediated antigen presentation

[0095] In some embodiments, the B cell immunomodulators disclosed herein can be used to improve multiple AAV clinical applications through B cell modulation, such as repeated administration and / or increased AAV dose.

[0096] In some embodiments, administration of PRV-3279 resulted in peak plasma concentrations at the end of infusion of the bispecific molecule with minimal accumulation upon multiple dosing, indicating that PRV-3279 has favorable pharmacokinetic properties.

[0097] In some embodiments, administration of PRV-3279 bispecific agent may result in inhibition of its own immunogenicity, i.e. lower prevalence and / or titer of anti-drug antibodies (ADA) with increasing dose of drug. This is in contrast to other immunomodulatory agents. Moreover, this suggests that increasing doses of PRV-3279, such as 20mg / kg, 30mg / kg or 40mg / kg, may be well tolerated without additional immunogenicity.

[0098] In some embodiments, it has been observed that PRV-3279 ADA does not affect pharmacokinetics (PK), pharmacodynamics (PD), safety or efficacy. This is surprising, since ADA usually affects at least PK and PD. Without being bound by theory, it is hypothesized that ADA does not neutralize PRV-3279.

[0099] In some embodiments, the PRV-3279 bispecific binds to most (e.g., >80-90%) of B cells, including both naive and memory phenotypes, upon administration in a dose-dependent manner and remains bound to at least 50% of B cells for at least 4 weeks after the last administration of a particular higher dose of the drug, demonstrating continued durability of the PD effect of PRV-3279 and supporting dosing once (or more) per month.

[0100] In some embodiments, dose-dependent and sustained B cell binding by PRV-3279 bispecific drug results in sustained inhibition of immunoglobulin production in the absence of depletion of any circulating cell subset, including B cells. Immunoglobulins reduced in peripheral blood include IgM, IgA, IgG and IgE. Inhibition can be observed in the absence or presence of antigenic stimulation (e.g., vaccination). This is an advantageous safety feature of PRV-3279 as a non-depleting agent, so that patients can retain circulating cells such as B cells to function as part of the immune system. In contrast, patients receiving depleting agents (e.g., rituximab, ocrelizumab, imbilizumab) take a long time (e.g., 1 year) to recover.

[0101] In some embodiments, BHTS gene therapy can be delivered via AAV-Des-TAZ, a recombinant AAV vector serotype 9 (AAV9) containing the full-length codon-optimized human TAFAZZIN gene. The selected serotype is less common in human viruses, reducing the likelihood that individuals will have circulating pre-treatment antibodies from a previous viral infection. Preclinical studies were performed to validate clinical efficacy in a knockdown (KD) mouse model of BTHS (Suzuki-Hatano 2019a). In mice, the vector penetrates recipient tissues, resulting in transgene expression and production of the TAFFAZIN gene product in skeletal and cardiomyocytes, leading to reversal of symptomology.

[0102] Testing with gene vectors in the KD BTHS mouse model demonstrated that promoter changes resulted in variation in transcription and therefore tissue expression based on the age at which the injection was performed, an important concept when considering optimal timing of administration or treatment to allow for application in humans and re-administration at later time points.

[0103] Figure 1 is a schematic diagram of the AAV-Des-TAZ vector design. The desmin promoter drives expression of the full-length human codon-optimized TAZ cDNA plus polyA tail. This sequence is flanked by the AAV inverted terminal repeats (ITRs), with the left flanking ITR containing the changes necessary to obtain dsAAV.

[0104] Figure 2 is an exemplary construct B951 (pds2TR-Des-coTAZ_Dual4). B951 is a self-complementary construct of codon-optimized full-length tafazzin controlled by the desmin promoter. The sequence is shown in Table 2.

[0105] [Table 2]

[0106] [Table 3]

[0107] [Table 4]

[0108] Figure 3 is an exemplary construct C064 (pds2TR-Des-coTAZiso2_Dual4). C064 replaces delta 5 or exon 5 deleted tafazzin cDNA. The sequence is shown in Table 3.

[0109] [Table 5]

[0110] [Table 6]

[0111] [Table 7]

[0112] In some embodiments, a combination therapy of PRV-3279 and AAV-Des-TAZ gene therapy is provided. As disclosed herein, the effect of PRV-3279 on B cell function can be transient. This allows gene therapy to be delivered and immunogenicity to remain low without depleting the B cell population and thus increasing the risk of infection in the long term. PRV-3279 is an ideal candidate for use in combination with gene therapy by reducing immunogenicity, which may increase the immediate efficacy of the gene vector while also allowing for re-administration of gene therapy at a later time point. The reduction in the formation of anti-viral vector antibodies is particularly important for pediatric patients who continue to undergo expected tissue and organ growth that requires repeated delivery of gene therapy. Furthermore, there is data supporting that PRV-3279 may help inhibit naive B cells, thereby reducing the immune response upon initial exposure to the gene vector.

[0113] Based on knowledge of the mechanism of PRV-3279 and understanding of the immunogenicity of gene therapy, one exemplary regimen can involve administering PRV-3279 three times during a course of AAV-Des-TAZ treatment as follows: 1 week prior to vector delivery, 2 weeks after delivery, and 3-4 weeks after the second administration. EXAMPLES

[0114] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the disclosure.

[0115] Example 1: Clinical trial design in adults 2 systemic doses - 3 × 10 13 μg / kg and 1×10 14Provided herein is a 96-week, 4 × 4 double-crossover Phase I / II clinical trial to test the safety and efficacy of gene therapy (AAV-Des-TAZ) at 1 μg / kg- in four cohorts (n = 8 total) of adolescents and young adults (ages 15-40 years) with BTHS, administered sirolimus + PRV-3279 at weeks -1, +2, and +4 with standard prophylaxis.

[0116] Subjects with standard prophylaxis may receive rituximab (Genentech, dose 375 mg / m2) weekly starting -21 days prior to dosing. Premedication is administered 30-60 minutes prior to rituximab and may include acetaminophen (Tylenol) and diphenhydramine (Benadryl). Additionally, subjects may receive sirolimus (Wyeth, dose 0.6-1 mg / m2 / day, adjusted to maintain trough serum sirolimus levels of 3-7 ng / mL) daily starting -7 days prior to dosing until week 12 after dosing.

[0117] Subjects with experimental prophylaxis may receive PRV-3279 starting -7, +14, and +28 days prior to dosing. Premedication may be administered 30-60 minutes prior to PRV-3279 and may include acetaminophen (Tylenol) and diphenhydramine (Benadryl). Additionally, subjects may receive sirolimus (Wyeth at a dose of 0.6-1 mg / m2 / day, adjusted to maintain a trough serum sirolimus level of 3-7 ng / mL) daily starting -7 days prior to dosing through 12 weeks post-dosing.

[0118] Subjects can be monitored for infection by physical examination and regular laboratory evaluations and can be treated appropriately if deemed clinically necessary.

[0119] The primary safety objective of this study is to investigate the safety and tolerability of systemic IV delivery of rAAV9-Des-TAZ containing PRV-3279 during BTHS. Safety outcomes may include immune system response, resting cardiac function, cardiac arrhythmias, and major adverse cardiac events.

[0120] Clinical objectives specific to AAV-Des-TAZ include: (1) to study the safety and tolerability of systemic delivery of AAV-Des-TAZ, and (2) to study the efficacy (VO2peak) of systemic delivery of AAV-Des-TAZ.

[0121] Clinical objectives specific to PRV-3279 include: (1) levels of anti-AAV9 antibodies in the blood (immunogenicity of the AAV vector), and (2) total IgM (a PD marker of PRV-3279 activity).

[0122] In cohort A (n=4), 3 subjects received 3 × 10 13 One subject was randomized to receive a single low dose of AAV-Des-TAZ at 1 × 10 14 Patients can be randomized to receive a single high dose of 100 μg / kg. This can be done using standard prophylaxis to anticipate rechallenge (rituximab and sirolimus).

[0123] After 16 weeks, the three subjects who received the low dose on day 1 can cross over and receive either the reverse dose or the high dose on week 16.

[0124] One subject who receives the high dose on day 1 can receive a placebo at week 16.

[0125] In cohort B (n=4), 3 subjects received 1×10 14 A single high dose of 3 × 10 μg / kg AAV9-Des-TAZ was administered to one subject. 13 They can be randomized to receive one low dose of rAAV9-Des-TAZ at 1 μg / kg. After 16 weeks, one subject who received the low dose at week 16 can cross over and receive the high dose at week 32. Three subjects who received the high dose at week 16 can receive placebo at week 32. This can be done using standard prophylaxis to anticipate rechallenge with rituximab and sirolimus.

[0126] In cohort C (n = 4), 3 subjects received 3 × 10 13 One subject was randomized to receive a single low dose of 1 × 10 14 Patients can be randomized to receive a single high dose of 100 μg / kg. This can be done with experimental prophylaxis to anticipate rechallenge with PRV-3279 and sirolimus.

[0127] In cohort D (n = 4), 3 subjects received 1 × 10 14 A single high dose of 3 × 10 μg / kg AAV9-Des-TAZ was administered to one subject. 13 They can be randomized to receive one low dose of μg / kg rAAV9-Des-TAZ. After 16 weeks, one subject who received the low dose at week 16 can cross over and receive the high dose at week 32. Three subjects who received the high dose at week 16 can receive placebo at week 32. This can be done using experimental prophylaxis to predict rechallenge with PRV-3279 and sirolimus.

[0128] Example 2: Toxicity study design for combination treatment of PRV-3279 and AAV-Des-TAZ gene therapy 120 age-matched huCD32B mice can be used in this study.

[0129] PRV-3279 Surrogate Requirements: 6 groups x 28 days x 10 mice x 25g mice x 50mg / kg = 2100mg 6 groups x 84 days x 10 mice x 25g mice x 50mg / kg = 6300mg +30% overdose = approximately 11 g of PRV-3279 surrogate.

[0130] Toxicity studies were performed at low doses (e.g., 3 × 10 13 μg / kg) or high doses (e.g., 1 × 10 14Gene therapy (AAV-Des-TAZ) at 50 mg / kg / day (µg / kg) can be performed in mice receiving 50 mg / kg / day PRV-3279 for 28 or 84 days.

[0131] [Table 8]

[0132] Modifications Modifications and variations of the described methods and compositions of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the present disclosure has been described in connection with specific embodiments, it should be understood that the present disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the present disclosure are contemplated and understood by those skilled in the relevant arts to which the present disclosure resides, and are within the scope of the present disclosure as expressed by the following claims.

[0133] Incorporation by Reference All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

[0134] References: Barth, PG et al. “An Barth Syndrome Foundation,2018.The Voice of the Patient:Barth Syndrome[WWW Document].www.fda.gov / media / 130562 / download Corti,M.et al.,2014.B-Cell Depletion is Protective Against Anti-AAV Capsid Immune Response:A Human Subject Case Study.Mol.Ther.Methods Clin.Dev.1.doi.org / 10.1038 / mtm.2014.33 Corti,Manuela et al.,“Safety of Intradiaphragmatic Delivery of Adeno-Associated Virus-Mediated Alpha-Glucosidase(rAAV1-CMV-hGAA)Gene Therapy in Children Affected by Pompe Disease.”Human gene therapy.Clinical development vol.28,4(2017):208-218.doi:10.1089 / humc.2017.146 Gonzalez IL,Human tafazzin(TAZ)gene mutation and variation database.2012.Science and Research section of www.barthsyndrome.org. Rigaud,C.,Lebre,AS.,Touraine,R.et al.Natural history of Barth syndrome:a national cohort study of 22 patients.Orphanet J Rare Dis 8,70(2013).doi.org / 10.1186 / 1750-1172-8-70 Roberts AE et al.,2012.The Barth Syndrome Registry:Distinguishing disease characteristics and growth data from a longitudinal study.Am J Med Genet Part A 158A:2726-2732. Rugolotto,Simone et al.“Long-term treatment of Barth syndrome with pantothenic acid:a retrospective study.”Molecular genetics and metabolism vol.80,4(2003):408-11.doi:10.1016 / j.ymgme.2003.07.002 Nathwani A.C.et al.(2007).Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates.Blood 109,1414-1421 Steward,C.Barth Syndrome,Orphanet Review,June 2011. McKusick,V.,OMIM entry #302060,September 2018.

Claims

1. A method for reducing immunogenicity associated with gene therapy for Barth Syndrome (BTHS), comprising administering to a patient undergoing or who has undergone BTHS gene therapy an effective amount of a non-depleting B cell inhibitor.

2. 2. The method of claim 1, wherein the BTHS gene therapy comprises administering to the patient genetically engineered cells harboring a recombinant adeno-associated virus (rAAV) vector encoding a TAFAZZIN transgene.

3. 2. The method of claim 1, wherein the B cell inhibitor is a CD32BxCD79B bispecific antibody capable of immunospecifically binding an epitope of CD32B and an epitope of CD79B.

4. the CD32B×CD79B bispecific antibody (A) a VL comprising the amino acid sequence of SEQ ID NO: 1 ＣＤ３２Ｂ domain; (B) VH comprising the amino acid sequence of SEQ ID NO:2 ＣＤ３２Ｂ domain; (C) VL comprising the amino acid sequence of SEQ ID NO:3 ＣＤ７９Ｂ Domain; and (D) VH comprising the amino acid sequence of SEQ ID NO: 4 ＣＤ７９Ｂ domain The method of claim 3 , comprising:

5. the CD32B×CD79B bispecific antibody (A) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:5; (B) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:6; and (C) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:

7. The method of claim 4, wherein the Fc diabody comprises:

6. 6. The method of claim 5, comprising administering said Fc diabody at a dose of about 5 mg / kg to about 100 mg / kg, or about 5 mg / kg to about 50 mg / kg, or about 5 mg / kg to about 40 mg / kg, in a dosing regimen of a dose once a week to once every 6 weeks.

7. 6. The method of claim 5, comprising administering said Fc diabody at a dose of about 10 mg / kg and on a dosing regimen of one dose every 1-4 weeks.

8. 6. The method of claim 5, comprising administering three doses of the Fc diabody at a dose of about 10 mg / kg, one week prior to the first BTHS gene therapy delivery, two weeks after the first BTHS gene therapy delivery, and three to four weeks after the second BTHS gene therapy delivery.

9. 10. The method of claim 8, comprising administering a first dose about 2 days to about 6 weeks prior to administration of the BTHS gene therapy, administering a second dose about simultaneously with administration of the BTHS gene therapy, and administering a third dose about 2 days to about 6 weeks after administration of the BTHS gene therapy.

10. 6. The method of claim 5, wherein the Fc diabody, upon administration, results in inhibition of its own immunogenicity and has a lower prevalence and / or potency of anti-drug antibodies (ADA) at increased doses.

11. The method of claim 10, wherein the ADA does not neutralize the Fc diabody.

12. 6. The method of claim 5, wherein said Fc diabody binds to at least 80% of B cells upon administration in a dose-dependent manner and remains bound to at least 50% of said B cells for at least 4 weeks after the final administration.

13. 6. The method of claim 5, wherein the Fc diabody provides sustained inhibition of immunoglobulin production without depleting circulating B cells.

14. 14. The method of claim 13, wherein the immunoglobulins comprise one or more of IgM, IgA, IgG and IgE.

15. 10. The method of claim 1, further comprising monitoring the patient by testing for the presence of specific antibodies to the BTHS gene therapy.

16. 16. The method of claim 15, further comprising administering one or more doses of said B cell inhibitor to further modulate immunogenicity.

17. 6. The method of claim 5, further comprising co-administering one or more immunomodulatory agents.

18. 18. The method of claim 17, wherein the one or more immunomodulatory agents are selected from sirolimus, rapamycin, abatacept, teplizumab, and Streptococcus pyogenes immunoglobulin G-degrading enzyme.

19. 6. The method of claim 5, further comprising co-administering sirolimus.

20. 1. Use of a non-depleting B cell inhibitor in the manufacture of a medicament for reducing immunogenicity associated with Barth Syndrome (BTHS) gene therapy, optionally wherein the BTHS gene therapy comprises a genetically engineered cell harboring a recombinant adeno-associated virus (rAAV) vector encoding a TAFAZZIN transgene.

21. A pharmaceutical composition for reducing immunogenicity associated with gene therapy for Barth Syndrome (BTHS), comprising an effective amount of a CD32BxCD79B bispecific antibody capable of immunospecifically binding to an epitope of CD32B and an epitope of CD79B prior to, concomitantly with, and / or after BTHS gene therapy, and optionally, the BTHS gene therapy comprises genetically engineered cells harboring a recombinant adeno-associated virus (rAAV) vector encoding a TAFAZZIN transgene.

22. the CD32B×CD79B bispecific antibody (A) a VL comprising the amino acid sequence of SEQ ID NO: 1 ＣＤ３２Ｂ domain; (B) VH comprising the amino acid sequence of SEQ ID NO:2 ＣＤ３２Ｂ domain; (C) VL comprising the amino acid sequence of SEQ ID NO:3 ＣＤ７９Ｂ Domain; and (D) VH comprising the amino acid sequence of SEQ ID NO: 4 ＣＤ７９Ｂ domain 22. The pharmaceutical composition of claim 21 , comprising:

23. the CD32B×CD79B bispecific antibody (A) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:5; (B) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:6; and (C) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:

7. The pharmaceutical composition of claim 22, wherein the Fc diabody comprises

24. 24. The pharmaceutical composition of claim 23, comprising said Fc diabody at a dose of about 5 mg / kg to about 100 mg / kg, or about 5 mg / kg to about 50 mg / kg, or about 5 mg / kg to about 40 mg / kg, in a dosing regimen of a once weekly dose to once every 6 weeks.

25. 24. The pharmaceutical composition of claim 23, comprising said Fc diabody at a dose of about 10 mg / kg, dosed once every 1-4 weeks, preferably in a regimen of 1 week prior to a first BTHS gene therapy delivery, 2 weeks after said first BTHS gene therapy delivery, and 3-4 weeks after a second BTHS gene therapy delivery.

26. 26. The pharmaceutical composition of claim 25, comprising a first dose about 2 days to about 6 weeks prior to administration of the BTHS gene therapy, a second dose about simultaneously with administration of the BTHS gene therapy, and a third dose about 2 days to about 6 weeks after administration of the BTHS gene therapy.

27. 22. The pharmaceutical composition of claim 21, further comprising one or more immunomodulatory agents selected from sirolimus, rapamycin, abatacept, teplizumab, and Streptococcus pyogenes immunoglobulin G-degrading enzyme.

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