Methods and compositions for treating abis syndrome

Abstract

Disclosed herein in one aspect is a method of reducing immunogenicity comprising administering an effective amount of a non-depleting B-cell inhibitor to a patient who is receiving or has received BTHS gene therapy. Related compositions are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0002] This specification includes a sequence listing filed herewith, which includes a file entitled 178833-011401.xml created on December 23, 2022, having the following size: 19,720 bytes, the contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates generally to compositions and methods for reducing the immunogenicity of biotherapeutics, and more particularly to gene therapy for treating Barth Syndrome. Background Art

[0004] Barth syndrome (OMIM#302060; BTHS), first discovered in 1983 by Dr. Peter Barth, is a rare X-linked genetic disorder caused by pathological variants in the TAFAZZIN gene, which encodes an acyltransferase essential for cardiolipin remodeling. Thus, mutations in the TAFAZZIN gene, located at Xq28, cause pathological features by leading to accumulation of intermediates of cardiolipin metabolism and mitochondrial dysfunction. The manifestation and severity of the effects vary, in part due to differences in tissue-specific gene expression. Clinical pathology typically includes cardiovascular dysfunction, myopathy, and immune deficiency that predisposes to infection. Mutations that cause BTHS are typically familial, but it is estimated that 13% of mutations are de novo (Gonzalez 2012). It is estimated that there are currently approximately 150-200 individuals known to have BTHS. However, there is a growing recognition that the number of undiagnosed individuals with this condition may be much larger, affecting 1 in 140,000 births.

[0005] It is estimated that cardiomyopathy (CM) occurs in up to 70% of individuals with BTHS and presents within the first year of life. Cardiovascular pathology includes dilated CM, noncompaction of the left ventricular myocardium, hypertrophic CM, and rarely, QTc prolongation, arrhythmias, and sudden cardiac death. Delays in the diagnosis of CM in BTHS are common, and individuals require close monitoring by a cardiologist for heart failure. Fortunately, most patients with CM respond well to standard medical therapy. However, a subset of BTHS patients progress to require a heart transplant within the first 5 years of life. In addition, ventricular arrhythmias have been noted to be common in larger cohort studies, and >10% of patients require implantable defibrillator placement due to the risk of fatal arrhythmias. Arrhythmias may occur in individuals with BTHS when they otherwise appear to be in good health.

[0006] Immunologic deficiencies that predispose to severe infection or sepsis are 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. Additionally, pancytopenia has also occurred 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 who died early in the original paper described by Barth died from infectious complications (Barth 1983). The leading hypothesis for the etiology of neutropenia is the presence of dysfunctional neutrophil precursors in the bone marrow. Administration of granulocyte colony-stimulating factor (G-CSF) is often used to improve low neutrophil counts, however, despite this, infections remain common.

[0007] Neuromuscular effects are common. Skeletal myopathy includes proximal, nonprogressive muscle weakness. Hypotonia leads to delayed gross motor development in childhood and fatigability and reduced quality of life in adulthood. Clinical trials designed to support aerobic training appear to improve quality of life but with few measurable physiological benefits.

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

[0009] Advances in standard medical care have made BTHS a condition that can be survived into adulthood. Currently recommended treatments for BTHS are supportive rather than curative. Supplements typically used to treat other mitochondrial diseases (e.g., coenzyme Q, carnitine, pantothenic acid, and other B vitamins) have not proven effective in BTHS (Rugolotto 2003).

[0010] However, presentation varies widely, and when individuals fall into the severe end of the spectrum, complications can be life-threatening, requiring a heart transplant or extracorporeal membrane oxygenation (ECMO) for survival. Even in milder or moderate cases of BTHS, there is a risk of acute metabolic crisis or severe infection that can occur with little or no warning (barthsyndrome.org). This is a significant source of anxiety for many patients who survive into adulthood.

[0011] Patients with BTHS suffer lifelong limitations and reduced quality of life due to muscle weakness and chronic fatigue. Clinical trials utilizing formal exercise programs to increase strength and endurance have proven ineffective, as have multiple supplements. Therefore, there is an unmet need in caring for patients with BTHS. A group of patients first met with the FDA in 2018 to help explain their concerns and advocate for therapies that go beyond symptom management.

[0012] Advances in the emerging therapeutic field of gene therapy are very attractive for the treatment of Barth syndrome. Gene therapy allows the restoration of normal production of acyltransferases, thereby reducing clinical symptoms.

[0013] In gene therapy, genetically modified cells are produced in order to incorporate a missing copy of a gene. However, there is a risk that some of the "machinery" used for genetic modification of cells may be "presented" by the modified cells and recognized by the host as "foreign" factors. Such recognition will trigger a rejection reaction, which could potentially render such treatment ineffective, or in severe cases potentially cause an autoimmune reaction.

[0014] Recently, the advent of gene therapy has encountered substantial obstacles: the immunogenicity of the viral vectors used to administer the transgene and the immunogenicity of the transgene protein itself after expression in the recipient cells. Immunogenicity of the vector and transgene results in: 1) reduced efficacy when the vector and transgene are bound and cleared by antibodies produced by the recipient; 2) the need for increased doses, which increases safety risks and costs; and 3) difficulty or impossibility of re-dosing if the subject develops antibodies to the vector or transgene after a previous dose. Sometimes, the recipient has pre-existing antibodies to the vector even before the first administration due to cross-reactivity with naturally occurring viruses.

[0015] Therefore, there is a need for methods and compositions for reducing immunogenicity induced by Barth syndrome gene therapy. Summary of the invention

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

[0017] In some embodiments, BTHS gene therapy may comprise a recombinant adeno-associated virus (rAAV) vector (eg, AAV9 vector) encoding a TAFAZZIN transgene.

[0018] In some embodiments, the B cell inhibitor is a CD32B×CD79B bispecific antibody that can immunospecifically bind to an epitope of CD32B and an epitope of CD79B. In some embodiments, the CD32B×CD79B bispecific antibody comprises: (A) VL comprising the amino acid sequence of SEQ ID NO: 1 CD32B Structural domain; (B) VH comprising the amino acid sequence of SEQ ID NO: 2 CD32B Structural 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 Structural domain.

[0019] In some embodiments, the CD32B×CD79B bispecific antibody is an Fc diabody comprising: (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.

[0020] In some embodiments, the method may further include administering the Fc diabody at a dosage of between about 5 mg / kg and about 100 mg / kg, or between about 5 mg / kg and about 50 mg / kg, or between about 5 mg / kg and about 40 mg / kg and at a dosage regimen between one dose per week and one dose per 6 weeks. In some embodiments, the method may include administering the Fc diabody at a dosage of about 10 mg / kg and at a dosage regimen of one dose per 1-4 weeks. In some embodiments, the method may include administering 3 doses of Fc diabody at a dosage of about 10 mg / kg at intervals of 2-6 weeks.

[0021] One exemplary dosing regimen includes a first dose 1 week before 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.

[0022] In some embodiments, the method can include administering a first dose about 2 days to about 6 weeks (e.g., 2 days, 6 days, 1 week, 2 weeks, 4 weeks) before administering the BTHS gene therapy, administering a second dose at about the same time as administering the 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 administering the BTHS gene therapy.

[0023] In some embodiments, the Fc diabody achieves inhibition of its own immunogenicity after administration, wherein the incidence and / or titer of anti-drug antibodies (ADA) is reduced at increased doses. In some embodiments, ADA does not neutralize the Fc diabody.

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

[0025] In some embodiments, the Fc diabody promotes sustained suppression of immunoglobulin production without depleting circulating B cells. In some embodiments, the immunoglobulin comprises one or more of IgM, IgA, IgG, and IgE.

[0026] In some embodiments, the method may further include monitoring the patient by checking for 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 immunomodulators, such as sirolimus, rapamycin, abatacept, teligrumumab, and an immunoglobulin G degrading enzyme of Streptococcus pyogenes. In some embodiments, the method may further comprise co-administering sirolimus.

[0028] Also provided herein are pharmaceutical compositions comprising a non-depleting B cell inhibitor disclosed herein provided (eg, packaged) in therapeutically effective unit doses. Instructions for a dosage regimen as disclosed herein may also be provided.

[0029] Additional aspects relate to the use of a non-depleting B cell inhibitor as disclosed herein in the manufacture of a medicament for reducing immunogenicity associated with Barth syndrome (BTHS) gene therapy, wherein optionally the BTHS gene therapy comprises genetically modified cells with a recombinant adeno-associated virus (rAAV) vector encoding a TAFAZZIN transgene.

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

[0031] In some embodiments, the CD32B×CD79B bispecific antibody may comprise: (A) VL comprising the amino acid sequence of SEQ ID NO: 1 CD32B Structural domain; (B) VH comprising the amino acid sequence of SEQ ID NO: 2 CD32B Structural 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 Structural domain.

[0032] The CD32B×CD79B bispecific antibody may be an Fc diabody comprising: (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.

[0033] In some embodiments, the pharmaceutical composition may comprise an Fc diabody at a dose of between about 5 mg / kg to about 100 mg / kg, or between about 5 mg / kg to about 50 mg / kg, or between about 5 mg / kg to about 40 mg / kg and a dosage regimen of between once a week and every 6 weeks.

[0034] In some embodiments, the pharmaceutical composition may comprise an Fc diabody at a dose of about 10 mg / kg and at intervals of 2-6 weeks, or at doses every 1-4 weeks. In certain embodiments, the dosing regimen may include a first dose 1 week before 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 comprise a first dose about 2 days to about 6 weeks before administration of the BTHS gene therapy, a second dose at about the same time as administration of the BTHS gene therapy, and a third dose about 2 days to about 6 weeks after administration of the BTHS gene therapy.

[0036] In some embodiments, the pharmaceutical composition may further comprise one or more immunomodulators selected from the group consisting of sirolimus, rapamycin, abatacept, teligrumumab, and immunoglobulin G-degrading enzyme of Streptococcus pyogenes. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic overview of an exemplary vector.

[0038] Figure 2 The exemplary construct is B951 (pds2TR-Des-coTAZ_Dual4).

[0039] Figure 3 is an exemplary construct C064 (pds2TR-Des-coTAZiso2_Dual4). DETAILED DESCRIPTION

[0040] Disclosed herein in one aspect is a method of reducing immunogenicity associated with Barth syndrome (BTHS) gene therapy, the method comprising administering an effective amount of a non-depleting B cell inhibitor to a patient who is receiving or has received BTHS gene therapy. In some embodiments, the B cell inhibitor is a CD32B×CD79B bispecific antibody, such as those disclosed in U.S. Publication No. 2016 / 0194396, WIPO Publication No. WO 2015 / 021089, and WO 2017 / 214096 (each incorporated by reference in its entirety). definition

[0041] For convenience, certain terms employed 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.

[0042] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" or "an" can mean "one", but it is also consistent with the meaning of "one or more", "at least one" and "more than one".

[0043] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error of the method / device employed to determine the value, or the variation between study subjects. Typically, the term is meant to cover approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% variation, depending on the specific circumstances.

[0044] The term "substantially" means more than 50%, preferably more than 80%, most preferably more than 90% or 95%.

[0045] The term "or" used in the claims is used to mean "and / or" unless explicitly indicated to refer to only alternatives or the alternatives are mutually exclusive, although the present disclosure supports a definition referring to only alternatives and "and / or."

[0046] As used in this specification and claims, the terms "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, 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 in this specification can be implemented with respect to any method, system, host cell, expression vector and / or composition of the invention. In addition, the compositions, systems, host cells and / or vectors of the invention can be used to implement the methods and proteins of the invention.

[0047] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiment of the disclosure.

[0048] The term "consisting of" refers to the compositions, methods, and respective components described herein, which do not include any elements not enumerated in the description of that embodiment.

[0049] Unless expressly stated otherwise, use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) means that the specific terms recited are representative examples and embodiments of the invention and they are not intended to be limited to the specific examples referenced or cited.

[0050] "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 may serve as regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.

[0051] As used herein, "TAFAZZIN" refers to a phospholipid-lysophospholipid transacylase that can be responsible for modifying cardiolipin (a membrane phospholipid) into its tetralinoleyl form. In certain embodiments, TAFAZZIN can refer to full-length human TAFAZZIN or human TAFAZZIN lacking exon 5, both of which can exhibit transacylase activity. In certain embodiments, tafazzin can refer to full-length mouse TAFAZZIN homologous to human TAFAZZIN lacking exon 5.

[0052] As used herein, "antibody" or "antibody molecule" refers to a protein comprising at least one immunoglobulin variable domain sequence, such as an immunoglobulin chain or a fragment thereof. Antibody molecules encompass antibodies (e.g., full-length antibodies) and antibody fragments. In some embodiments, antibody molecules comprise antigen-binding fragments or functional fragments of full-length antibodies or full-length immunoglobulin chains. For example, full-length antibodies are naturally occurring immunoglobulin (Ig) molecules (e.g., IgG) formed by normal immunoglobulin gene fragment recombination processes. In some embodiments, antibody molecules refer to immunologically active antigen-binding portions of immunoglobulin molecules, such as antibody fragments. Antibody fragments (e.g., functional fragments) are a part of an antibody, such as Fab, Fab', F(ab')2, F(ab)2, variable fragments (Fv), domain antibodies (dAb), or single-chain variable fragments (scFv). Functional antibody fragments bind to the same antigen as the antigen recognized by a complete (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of variable regions, such as an "Fv" fragment consisting of the variable regions of the heavy chain and the light chain, or a recombinant single-chain polypeptide molecule ("scFv protein") in which the light chain and the heavy chain variable regions are connected by a peptide linker. In some embodiments, the antibody fragment does not include an antibody portion that has no antigen binding activity, such as an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments (e.g., dAb (domain antibodies), single chains, 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 a region that includes one constant domain and one variable domain from each heavy chain and light chain of an antibody, i.e., V L , C L 、V H and C H 1.

[0053] Throughout this specification, the numbering of residues in the IgG heavy chain constant region is that of the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1, Maryland (1991) ("Kabat"), which is expressly incorporated herein by reference. The term "EU index as in Kabat" refers to the numbering of the human IgG1 EU antibody. Amino acids from the variable domains of mature heavy and light chains of immunoglobulins are designated by the position of the amino acid in the chain. Kabat described many amino acid sequences of antibodies, identified the amino acid consensus sequence for each subgroup, and assigned residue numbers to each amino acid, and the CDRs as defined by Kabat (it should be understood as Chothia, C. and Lesk, AM ((1987) "Canonical structures for the hypervariable regions of immunoglobulins", J. Mol. Biol. [Journal of Molecular Biology] 196: 901-917) H 1) is defined for identification. Kabat's numbering scheme can be extended to antibodies not included in its summary by comparing the antibody in question with one of the consensus sequences in Kabat by reference to the conserved amino acids. This method for assigning residue numbers has become a standard in the art and easily identifies amino acids at equivalent positions in different antibodies (including chimeric or humanized variants). For example, the amino acid at position 50 of a human antibody light chain occupies a position equivalent to the amino acid at position 50 of a mouse antibody light chain.

[0054] In some embodiments, the antibody molecule is monospecific, e.g., it comprises a binding specificity for a single epitope. In some embodiments, the antibody molecule is multispecific, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence has a binding specificity for a first epitope, and a second immunoglobulin variable domain sequence has a binding specificity for a second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule.

[0055] The terms "bispecific antibody molecule", "diabody" and "dual affinity retargeting" are "Antibody" is used interchangeably herein and refers to an antibody molecule that is specific for more than one (e.g., two, three, four or more) epitopes and / or antigens. In some embodiments, the antibody can be a diabody or scaffold capable of binding an antigen, such as those disclosed in U.S. Publication No. 2016 / 0194396, WIPO Publication No. WO 2015 / 021089, and WO 2017 / 214096 (each of which is incorporated by reference in its entirety). In some embodiments, the antibody can be a CD32B×CD79B bispecific diabody (i.e., a "CD32B×CD79B diabody," as well as such diabodies that additionally comprise an Fc domain (i.e., a "CD32B×CD79B Fc diabody"). In some embodiments, the antibody can be a humanized CD32B×CD79B produced in Chinese hamster ovary cells. Antibody, its molecular weight is 111.5kDa.

[0056] As used herein, "antigen" (Ag) refers to macromolecules, including all proteins or peptides. In some embodiments, antigens are molecules that can cause an immune response (e.g., involving the activation of certain immune cells and / or antibody production). Antigens are not only related to antibody production. T cell receptors also recognize antigens (although it is an antigen in which its peptide or peptide fragment is complexed with an MHC molecule). Any macromolecule including almost all proteins or peptides can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA containing a nucleotide sequence or a partial nucleotide sequence encoding a protein that can cause an immune response encodes an "antigen". In some embodiments, antigens do not need to be encoded only by the full-length nucleotide sequence of a gene, and antigens do not need to be encoded by a gene. In some embodiments, antigens can be synthetic or can be derived from biological samples, such as tissue samples, tumor samples, cells, or fluids with other biological components. As used herein, "tumor antigens" or interchangeably "cancer antigens" include any molecules present on or associated with cancer, such as cancer cells or tumor microenvironments that can cause an immune response. As used herein, "immune cell antigens" include any molecules present on or associated with immune cells that can cause an immune response.

[0057] The "antigen binding site" or "antigen binding fragment" or "antigen binding portion" of an antibody molecule (used interchangeably herein) refers to the portion of an antibody molecule (e.g., an immunoglobulin (Ig) molecule, such as IgG) that participates 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 distinct segments within the variable regions of the heavy and light chains (called hypervariable regions) are arranged between more conservative flanking segments called "framework regions" (FRs). FRs are amino acid sequences that naturally occur between and adjacent to the 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 the bound antigen. The three hypervariable regions of each heavy and light chain are called "complementarity determining regions" or "CDRs." Framework regions and CDRs have been defined and described, for example, in Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department 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 chain and variable light chain) typically consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following amino acid sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Variable light chain (VL) CDRs are generally defined as including residues at positions 27-32 (CDR1), positions 50-56 (CDR2), and positions 91-97 (CDR3). The variable heavy chain (VH) CDRs are generally defined as comprising residues at positions 27-33 (CDR1), positions 52-56 (CDR2), and positions 95-102 (CDR3). One of ordinary skill in the art will appreciate that loops can have different lengths among antibodies and numbering systems (e.g., Kabat or Chothia controls) such that the framework has consistent numbering among antibodies.

[0058] In some embodiments, the antigen binding fragment of an antibody (e.g., when included as part of a fusion molecule) may lack or contain a complete Fc domain. In some embodiments, the antibody binding fragment does 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, the antigen binding fragment may be completely free of any Fc domain. In some embodiments, the antigen binding fragment may be substantially free of a complete Fc domain. In some embodiments, the antigen binding fragment may include a portion of a complete Fc domain (e.g., a CH2 or CH3 domain or a portion thereof). In some embodiments, the 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.

[0059] As used herein, "administering" and similar terms mean delivering the composition to the individual being treated. Preferably, the compositions of the present disclosure are administered by, for example, parenteral (including subcutaneous, intramuscular), or preferably intravenous routes.

[0060] As used herein, "effective amount" means an amount of a bioactive agent or diagnostic agent sufficient to provide a local or systemic effect desired as would be required for any medical treatment or diagnostic test at a reasonable risk / benefit ratio. This will vary depending on the patient, the disease, the treatment being administered, and the nature of the agent. The therapeutically effective amount will vary depending on the patient and the disease condition being treated, the patient's weight and age, the severity of the disease condition, the mode of administration, etc., which can be readily determined by one of ordinary skill in the art. The range of administered doses can be, for example, from about 1 ng to about 10,000 mg, from about 5 ng to about 9,500 mg, from about 10 ng to about 9,000 mg, from about 20 ng to about 8,500 mg, from about 30 ng to about 7,500 mg, from about 40 ng to about 7,000 mg, from about 50 ng to about 6,500 mg, from about 100 ng to about 6,000 mg, from about 200 ng to about 5,500 mg, from about 300 ng to about 5,000 mg, from about 400 ng to about 4,5 00mg, about 500ng to about 4,000mg, about 1μg to about 3,500mg, about 5μg to about 3,000mg, about 10μg to about 2,600mg, about 20μg to about 2,575mg, about 30μg to about 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 2,000mg, about 400μg to about 2 g to about 1,175 mg, about 500 μg to about 1,150 mg, about 0.5 mg to about 1,125 mg, about 1 mg to about 1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5 mg to about 1,050 mg, about 2.0 mg to about 1,025 mg, about 2.5 mg to about 1,000 mg, about 3.0 mg to about 975 mg, about 3.5 mg to about 950 mg, about 4.0 mg to about 925 mg, about 4.5 mg to about 900 mg. The present invention provides the antibody or its antigen-binding portion thereof as provided herein, for example, about 5mg to about 875mg, about 10mg to about 850mg, about 20mg to about 825mg, about 30mg to about 800mg, about 40mg to about 775mg, about 50mg to about 750mg, about 100mg to about 725mg, about 200mg to about 700mg, about 300mg to about 675mg, about 400mg to about 650mg, about 500mg or about 525mg to about 625mg of antibody or its antigen-binding portion thereof as provided herein.Administration can be, for example, once a week, once every 2 weeks, once every three weeks, once every 4 weeks, once every 5 weeks or once every 6 weeks.Dosage regimen can be adjusted to provide best therapeutic response.Effective dose is also the amount that any toxicity or harmful effect (side effect) of medicament is minimized and / or exceeded by beneficial effect.It can be administered intravenously at exactly or about 6 mg / kg or 12 mg / kg per week, or 12 mg / kg or 24 mg / kg every two weeks. Additional dosing regimens are described below.

[0061] As used herein, "pharmaceutically acceptable liquid carrier" shall mean a liquid carrier that can be used to prepare a pharmaceutical composition that is generally safe, non-toxic, and biologically or otherwise undesirable, and includes liquid carriers that are acceptable for veterinary use as well as human pharmaceutical use. Examples of "pharmaceutically acceptable liquid carriers" include water and organic solvents. Preferred pharmaceutically acceptable aqueous liquids include PBS, saline, and dextrose solutions, etc.

[0062] The term "immunogenicity" refers to the ability of a particular substance (e.g., an antigen or epitope) to stimulate an immune response that may be humoral and / or cell-mediated in humans and other animals. In some embodiments, administration of the compositions disclosed herein reduces the immunogenicity of a biological substance (e.g., 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 an otherwise substantially normal immune system.

[0063] As used herein, "major histocompatibility complex" or "MHC" proteins refer to a group of cell surface molecules encoded by a large gene family that plays an important 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 display 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 referred to as human leukocyte antigens (HLA) - HLA Class I and HLA Class II, respectively. Some of the most studied HLA genes in humans are nine MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, and HLA-DRB345.

[0064] Various aspects of the disclosure are described in further detail below. Additional definitions are listed throughout the specification. Non-depleting B cell inhibitors and pharmaceutical compositions

[0065] 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. On the other hand, "depletion" of B cells means that the agent acts to eliminate or destroy B cells, such as anti-CD20 antibodies, such as rituximab. Therefore, 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.

[0066] Exemplary non-depleting B cell inhibitors include, but are not limited to, CD32B×CD79B bispecific 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 proliferation-inducing ligand (APRIL) inhibitors, such as belimumanb; anti-CD40 and anti-CD40L molecules; and Bruton's tyrosine kinase (BTK) inhibitors, such as ibrutinib (PCI-32765) and acalabrutinib.

[0067] In some embodiments, the B cell inhibitor may be a CD32B×CD79B bispecific antibody, such as those disclosed in U.S. Publication No. 2016 / 0194396, WIPO Publication Nos. WO 2015 / 021089 and WO 2017 / 214096 (all of which are incorporated by reference in their entirety), or an antigen-binding fragment thereof.

[0068] Exemplary CD32B×CD79B bispecific diabodies may comprise two or more polypeptide chains, and may comprise: (1) VL domain of an antibody that binds to CD32B (VL CD32B ), such VL CD32B The domain has the sequence (SEQ ID NO: 1): DIQMTQSPSS LSASμGDRVT ITCRASQEIS GYLSWLQQKP GKAPRRLIYA ASTLDSGVPS RFSGSESGTE FTLTISSLQP EDFATYYCLQ YFSYPLTFGG GTKVEIK (2) VH domain of an antibody that binds to CD32B (VH CD32B ), such VH CD32B The domain has the sequence (SEQ ID NO: 2): EVQLVESGGG LVQPGGSLRL SCAASGFTFSDAWMDWVRQA PGKGLEWVAE IRNKAKNHAT YYAESVIGRF TISRDDAKNS LYLQMNSLRA EDTAVYYCGA LGLDYWGQGT LVTVSS (3) VL domain of an antibody that binds to CD79B (VL CD79B ), such VL CD79B The domain has the sequence (SEQ ID NO: 3): DVVMTQSPLS LPVTLGQPAS ISCKSSQSLL DSDGKTYLNW FQQRPGQSPN RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDμGV YYCWQGTHFP LTFGGGTKLE IK (4) VH domain of an antibody that binds to CD79B (VH CD79B ), such VH CD79B The domain has the sequence (SEQ ID NO:4): QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMNWVRQA PGQGLEWIGM IDPSDSETHY NQKFKDRVTM TTTDSTSTAY MELRSLRSDD TAVYYCARAM GYWGQGTTVT VSS

[0069] In some embodiments, the B cell inhibitor can be PRV-3279, a humanized CD32B×CD79B dual-affinity retargeted Protein with a molecular weight of 111.5 kDa. The protein is a bispecific antibody-based molecule that can bind to 2 different antigens simultaneously. PRV-3279 is designed to target CD32B (Fcγ receptor IIb) and CD79B (immunoglobulin-related β subunit of the B cell receptor (BCR) complex) on B lymphocytes. The co-ligation of CD32B and CD79B on B lymphocytes in a preferential cis-binding mode triggers CD32B-coupled immunoreceptor tyrosine-based inhibitory motif signaling, which reduces antigen-mediated activation of naive B cells and memory B cells without extensive depletion. In order to extend the half-life in vivo, PRV-3279 also contains a human immunoglobulin G (IgG) 1 Fc region that has been mutated to greatly reduce or eliminate undesirable binding to FcγR and complement, but retains affinity for binding to neonatal FcR to utilize the IgG salvage pathway mediated by this receptor.

[0070] The CD32B molecule is a transmembrane inhibitory receptor 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 MacroGenics' proprietary mouse monoclonal antibody (mAb) 8B5. CD79B is an essential signal transduction component of the BCR expressed only on B cells. The anti-CD79B component of PRV-3279 is based on a humanized version of the mouse mAb CB3.

[0071] In some embodiments, PRV-3279 comprises the following sequence (CDRs are underlined and helical domains are in bold): Chain 1 (Fc-CD32BVL-CD79bVH-E helix): (SEQ ID NO.: 5) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGKAPSSSPMEDIQMTQSPSSLS ASμGDRVTITC RASQEISGYLS WLQQKPGKAPRRLIYAASTLDSGVPSRFS GSESGTEFTLTISSLQPEDFATYYC LQFYFSYPLT FGGGTKVEIKGGGSGGG GQVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMNW VRQAPGQGLE WIG MIDPSDSETHYNQKFKD RVTMTTDTSTSTAYMELRSLRSDDTAVYY CAR AMGY WGQGTTVTVSSGGCGGGEVAALEKEVAALEKEVAALEKE VAALEKGGGNS Chain 2 (CD79b VL - CD32B VH - K helix): (SEQ ID NO.:6) DVVMTQSPLSLPVTLGQPASISC KSSQSLLDSDGKTYLN WFQQRPGQSPN RLIY LVSKLDS GVPDRFSGSGSGTDFTLKISRVEAEDμGVYYC QTfP LT FGGGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFS DAWMDW VRQAPGKGLEWVA EIRNKAKNHATYYAESVIG RFTISRDDAK NSLYLQMNSLRAEDTAVYYC GALGLDY WGQGTLVTVSSGGCGGGKVA ALKEKVAALKEKVAALKEKVAALKE Chain 3 (Fc): (SEQ ID NO.:7) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCA VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRW QQGNVFSCSVMHEALHNRYTQKSLSLSPGK

[0072] PRV-3279 binds to most (e.g., >80%-90%) B cells (including naive and memory phenotypes) in a dose-dependent manner after administration, and can remain bound to at least 50% of B cells for more than 4 weeks. This demonstrates the sustained persistence of the potential pharmacodynamic (PD) effect of PRV-3279 and supports a dosing frequency of once a month (or longer).

[0073] PRV-3279 has previously been shown to reduce immunity to viral vaccines (hepatitis A vaccine) in healthy subjects, indicating that immunogenicity to viral vectors can be suppressed. In addition, a pilot study was conducted to evaluate the effects of PRV-3279 alternatives on gene therapy using adeno-associated virus (AAV) to assess its efficacy and immunogenicity in huCD32b transgenic mice (see PCT / US2020 / 044312 and US2021 / 0130464, both of which are incorporated herein by reference in their entirety). This is a dose-response study with nine groups (including negative (no AAV9) controls, then AAV9 combined with placebo, 3 dose levels of PRV-3279 alone, co-administration of sirolimus with three dose levels of PRV-3279, and sirolimus alone) (Table 1). Longitudinal sampling included the levels of IgM, IgG, AAV9 vector copies, and tissue transgenic activity at the end of the study.

[0074] Specific research objectives: 1. Amount of AAV9 vector detectable in the blood (vector clearance rate) 2. The amount of GAA transgene detectable in tissues (gene therapy transfection efficiency) 3. Levels of anti-AAV9 antibodies in the blood (immunogenicity of AAV vectors) 4. Total IgM (PD marker of PRV-3279 activity) Table 1: Design of the PRV-3279 dose-response study

[0075] The dose-dependent and sustained B-cell binding of PRV-3279 promotes the persistent suppression of immunoglobulin production without depleting any circulating immune cell subsets (including B cells). The immunoglobulins reduced in peripheral blood include IgM, IgA, IgG and IgE. Suppression can be observed in the absence or presence of antigenic stimulation (e.g., vaccination). This is a favorable safety feature of PRV-3279 as a non-depleting agent, allowing patients to retain circulating immune cells (e.g., B cells) as part of a functional immune system. In contrast, patients receiving cell depleting agents (e.g., rituximab, ocrelizumab, inebilizumab) need a considerable amount of time (e.g., up to one year) to restore depleted cell types. This is particularly important when considering the therapy of BTHS, in which there is immune deficiency and there is still a risk of severe infection throughout the life cycle. Immune competence is also clinically significant for the primary vaccination series of children because BTHS exists from birth.

[0076] In another aspect, pharmaceutical compositions useful in the methods disclosed herein are provided, i.e., pharmaceutical compositions for reducing or inhibiting immunogenicity in a subject in need thereof, e.g., during or after receiving BTHS gene therapy that causes significant immunogenicity, or because the subject has pre-existing immunogenicity to the biotherapeutic (e.g., in the case of pre-existing anti-AAV antibodies due to prior wild-type adenovirus infection or due to prior exposure to rAAV therapy). In some embodiments, the compositions disclosed herein can be administered to a patient prior to receiving BTHS gene therapy to prevent immunogenicity and / or reduce pre-existing antibodies.

[0077] In some embodiments, the pharmaceutical composition comprises a B cell inhibitor as disclosed herein and a pharmaceutically acceptable carrier. The B cell inhibitor can be formulated into a pharmaceutical composition together with a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition can include instructions for use of the composition, for example, for treating a patient to reduce or inhibit the immunogenicity of a subject in need thereof, such as when or after receiving a biological agent that causes significant immunogenicity.

[0078] As used herein, "pharmaceutically acceptable carriers" include 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 biologic) can be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0079] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, and conventional excipients for the preparation of tablets, pills, capsules, etc. The use of such media and medicaments for the preparation of pharmaceutically active substances is known in the art. Unless any conventional media or medicaments are incompatible with the active compound, it is contemplated to use them in the pharmaceutical compositions provided herein. Supplementary active compounds may also be incorporated into the composition.

[0080] The pharmaceutically acceptable carrier may include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically 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, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0081] The example of suitable aqueous and non-aqueous carrier that can be used in the pharmaceutical composition provided herein includes water, ethanol, polyol (such as glycerol, propylene glycol, polyethylene glycol etc.) and suitable mixture thereof, and injectable organic ester (such as ethyl oleate).When needed, suitable fluidity can be maintained, for example, in the following manner: by using coating material (such as lecithin), by maintaining required particle size in the case of dispersion, and by using surfactant.In many cases, it is useful to include isotonic agent in the composition, for example sugar, polyol (such as mannitol, sorbitol) or sodium chloride.The absorption of injectable composition can be extended by including the medicament (such as, monostearate and gelatin) of delayed absorption in the composition.

[0082] These compositions may also contain functional excipients such as preservatives, wetting agents, emulsifiers and dispersants.

[0083] The therapeutic composition typically must be sterile, non-sterile 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.

[0084] Sterile injectable solutions can be prepared in the following manner: the active compound of the desired amount is mixed with a suitable solvent with one or more composition combinations (if necessary) listed above, followed by sterilization, such as by microfiltration. Typically, dispersions are prepared by mixing the active compound into a sterile vehicle containing a basic dispersion medium and other components required from the above listing. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method includes vacuum drying and freeze drying (freeze drying) to produce active ingredients plus powders of any other required ingredients from its previous sterile filtered solution. Active agent can be mixed with other pharmaceutically acceptable carriers under aseptic conditions, and mixed with any preservatives, buffers or propellants that may be required.

[0085] Prevention of the presence of microorganisms can be ensured by the above sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the composition. In addition, prolonged absorption of the injectable pharmaceutical form may be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0086] The dosage regimen is adjusted to provide the optimum desired response (eg, 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.

[0087] Exemplary dosage ranges for administering the antibody include: 10-1000 mg (antibody) / kg (patient body 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 00-800mg / 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 dosage regimens include once every three days, once every five days, once every seven days (i.e., once a week), once every 10 days, once every 14 days (i.e., once every two weeks), once every 21 days (i.e., once every three weeks), once every 28 days (i.e., once every four weeks), once a month, once every 5 weeks and once every 6 weeks.

[0088] In some embodiments, each dose of about 5-40mg / kg, about 5-20mg / kg or about 10mg / kg 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, such as 1 dose, 2 doses or 3 doses can be administered. Administration can be via IV infusion. Any combination of the foregoing (e.g., 3 doses of 10mg / kg / dose, once every 4 weeks) can be used to reduce the immunogenicity of biotherapeutics (including gene therapy products). In some embodiments, the first dose can be administered 2-6 weeks (e.g., 4 weeks) before gene therapy, the second dose can be administered at about the same time of gene therapy, and the third dose can be administered 2-6 weeks (e.g., 4 weeks) after gene therapy. Thereafter, the patient can be monitored by checking the amount of specific antibodies against gene therapy vectors (e.g., rAAV) and / or transgenics. If no antibodies are detected or very few antibodies are detected, no additional PRV-3279 will be needed. If significant amounts of antibodies are present, one or more doses of PRV-3279 may be administered to further modulate immunogenicity.

[0089] It may be advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suited as unitary dosages for the patient to be treated; each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic effect in association with any required pharmaceutical carriers. The specifications for the unit dosage form are dictated by and directly dependent on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the art in compounding an active compound, for example, to treat the sensitivity of an individual.

[0090] Actual dosage levels of the active ingredients in the pharmaceutical compositions disclosed herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, mode of administration without being toxic to the patient. As used herein, "parenteral" in the context of administration means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0091] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral (i.e., via the digestive tract) and topical administration, typically by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Intravenous injection and infusion are typically (but not exclusively) used for antibody administration.

[0092] When the agents provided herein are administered to humans or animals as medicines, 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 pharmaceutically acceptable carrier. Therapeutic uses and methods

[0093] The compositions disclosed herein can be used to reduce or inhibit immunogenicity caused by BHTS gene therapy delivered by various means (e.g., AAV and other wild-type and recombinant vectors, lentiviral modified human stem cells). In some embodiments, the B cell immunomodulators disclosed herein can be used to improve viral delivery of transgenes based on rAAV (recombinant adeno-associated virus) vectors, for example, for inherited enzyme deficiencies, such as TAFAZZIN deficiency associated with BHTS.

[0094] In some embodiments, the B cell immunomodulators disclosed herein can be used to modulate restricted immune responses elicited by a variety of delivery routes (even in sites of perceived immune privilege), such as systemic, intramuscular, ocular (requiring local high doses to result in local immune responses), and central nervous system (CNS) (where leakage of viral capsids from the CNS induces a systemic response that reduces AAV uptake in the CNS).

[0095] In some embodiments, the B cell immune modulators disclosed herein can be used to modulate various restricted immune pathways that are B cell dependent, including: Development of neutralizing antibodies (nAb) 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

[0096] In some embodiments, the B cell immunomodulators disclosed herein can be used to improve various AAV clinical applications through B cell modulation (e.g., repeated dosing and / or increased AAV doses).

[0097] In some embodiments, after administration of PRV-3279, peak plasma concentrations occurred at the end of the bispecific molecule infusion, and there was minimal accumulation upon multiple dosing, demonstrating that PRV-3279 has favorable pharmacokinetic properties.

[0098] In some embodiments, administration of the PRV-3279 bispecific can achieve suppression of its own immunogenicity, i.e., the incidence and / or titer of anti-drug antibodies (ADA) decreases with increasing drug doses. This is in contrast to other immunomodulators. Additionally, this suggests that increasing doses of PRV-3279 (e.g., 20 mg / kg, 30 mg / kg, or 40 mg / kg) can be well tolerated without added immunogenicity.

[0099] In some embodiments, it has been observed that PRV-3279 ADA does not affect pharmacokinetics (PK), pharmacodynamics (PD), safety or efficacy. This is unexpected because ADA usually affects at least PK and PD. Without being bound by theory, it has been assumed that ADA does not neutralize PRV-3279.

[0100] In some embodiments, the PRV-3279 bispecific binds to the majority (e.g., >80%-90%) of B cells (including naive and memory phenotypes) in a dose-dependent manner after administration, and remains bound to at least 50% of B cells for at least 4 weeks after the last administration of certain higher doses of the drug. This demonstrates the sustained durability of the PD effect of PRV-3279 and supports monthly (or longer) administration.

[0101] In some embodiments, the dose-dependency and sustained B cell binding of PRV-3279 bispecific drugs promotes the persistent inhibition of immunoglobulin production in the absence of any circulating cell subsets (including B cells). The immunoglobulins reduced in peripheral blood include IgM, IgA, IgG and IgE. Inhibition can be observed in the absence or presence of (e.g., vaccination) antigen stimulation. This is a favorable safety feature of PRV-3279 as a non-depleting agent, so that patients can retain circulating cells (e.g., B cells) as part of the immune system. In contrast, patients receiving depleting agents (e.g., rituximab, omezumab, inelizumab) need a long time (e.g., one year) to recover.

[0102] In some embodiments, BHTS gene therapy can be delivered via AAV-Des-TAZ, a recombinant AAV vector serotype 9 (AAV9) containing a full-length codon-optimized human TAFFAZIN gene. The selected serotype is less prevalent in the human virome, thereby reducing the chance that an individual has pre-treatment antibodies circulating from a previous viral infection. Preclinical trials were conducted to validate the clinical efficacy of a knockdown (KD) mouse model of BTHS (Suzuki-Hatano 2019a). In mice, the vector penetrated the recipient's tissues and resulted in transgene expression and production of the TAFFAZIN gene product in skeletal and cardiomyocytes, leading to reversal of symptoms.

[0103] Studies with gene vectors in the KD BTHS mouse model demonstrated that changes in the promoter lead to changes in transcription and therefore tissue expression depending on age at injection. This is an important concept when considering applications in humans and the optimal timing of administration or therapy to allow re-administration at a later time point.

[0104] Figure 1 Schematic diagram of the AAV-Des-TAZ vector design. The desmin promoter drives the expression of the full-length human codon-optimized TAZ cDNA + poly A tail. This sequence is flanked by the AAV inverted terminal repeats (ITRs), where the left flanking ITR contains the necessary changes to generate dsAAV.

[0105] Figure 2 The exemplary construct is B951 (pds2TR-Des-coTAZ_Dual4). B951 is a self-complementary construct of codon-optimized full-length tafazin controlled by the desmin promoter. The sequence is shown in Table 2. Table 2. B951 (pds2TR-Des-coTAZ Dual4)

[0106] Figure 3 The exemplary construct is C064 (pds2TR-Des-coTAZiso2_Dual4). C064 replaces δ5 or exon 5 deleted tafazin cDNA. The sequence is shown in Table 3. Table XX C064 (pds2TR-Des-coTAZiso2 Dual4)

[0107] 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 instantaneous. This allows gene therapy to be delivered and maintains low immunogenicity without depleting B cell populations, and therefore does not increase the risk of long-term infection. PRV-3279 is an ideal candidate for use in combination with gene therapy by reducing immunogenicity, thereby enhancing the immediate effectiveness of gene vectors, while also potentially allowing gene therapy to be re-administered at a later time point. The reduction in the formation of antiviral vector antibodies is particularly important for pediatric patients, who will continue to have expected tissue and organ growth, thereby requiring repeated delivery of gene therapy. In addition, there are data to support that PRV-3279 can help suppress naive B cells, thereby reducing immune responses when initially exposed to gene vectors.

[0108] Based on the knowledge of the mechanism of PRV-3279 and the understanding of the immunogenicity of gene therapy, an exemplary schedule may include three administrations of PRV-3279 during the course of AAV-Des-TAZ treatment as follows: 1 week before vector delivery, 2 weeks after delivery, and 3-4 weeks after the second dose. Examples

[0109] The following examples, including experiments performed and results achieved, are provided for illustrative purposes only and should not be construed as limiting the present disclosure. Example 1: Clinical trial design in adults

[0110] This paper presents a 96-week, 4×4 double-crossover Phase I / II clinical trial to test two systemic doses (3x10 13 μg / kg and 1x 10 14 μg / kg) gene therapy (AAV-Des-TAZ) compared with standard prevention and sirolimus + PRV-3279.

[0111] Subjects receiving standard prophylaxis may receive rituximab (Genentech, dose 375 mg / m2) weekly starting -21 days prior to dosing. Premedication will be administered 30-60 minutes prior to rituximab and may include acetaminophen (Tylenol) and diphenhydramine (Benadryl). In addition, 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 post-dosing.

[0112] Subjects receiving experimental prophylaxis may receive PRV-3279 starting at -7 days, +14 days, and +28 days prior to dosing. Premedication may be administered 30-60 minutes prior to PRV-3279 and may include acetaminophen (Tylenol) and diphenhydramine (Diphenhydramine). In addition, 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 at -7 days prior to dosing until week 12 post-dosing.

[0113] Subjects may be monitored for infection through physical examinations and periodic laboratory evaluations, and may be treated appropriately as clinically necessary.

[0114] The primary safety objective of this study is to investigate the safety and tolerability of systemic IV delivery of rAAV9-Des-TAZ and PRV-3279 in BTHS. Safety outcomes may include immune system response, resting cardiac function, arrhythmias, and major adverse cardiac events.

[0115] Clinical objectives specific to AAV-Des-TAZ include: (1) testing the safety and tolerability of systemically delivered AAV-Des-TAZ, and (2) testing the efficacy (VO2 peak) of systemically delivered AAV-Des-TAZ.

[0116] 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).

[0117] In cohort A (n=4), 3 subjects were randomized to receive a single low dose of 3 x 10 13 μg / kg of AAV-Des-TAZ, and one subject could be randomized to receive a single high dose of 1x 10 14μg / kg. This can be done along with standard prophylaxis to anticipate re-administration of rituximab and sirolimus.

[0118] After 16 weeks, the 3 subjects who received the low dose on Day 1 could cross over and receive either the reverse dose or the high dose on Week 16.

[0119] One subject who received the high dose on Day 1 could receive placebo at Week 16.

[0120] In cohort B (n=4), 3 subjects were randomized to receive a single high-dose of 1 x 10 14 μg / kg of AAV9-Des-TAZ, and one subject could be randomized to receive a single low-dose of 3x 10 13 After 16 weeks, one subject who received the low dose at week 16 can cross over and receive the high dose at week 32. The three subjects who received the high dose at week 16 can receive placebo at week 32. This can be done along with standard prophylaxis to anticipate re-dosing of rituximab and sirolimus.

[0121] In cohort C (n=4), 3 subjects were randomized to receive a single low-dose of 3 x 10 13 μg / kg of AAV-Des-TAZ, and one subject could be randomized to receive a single high dose of 1x 10 14 μg / kg. This can be done along with experimental prophylaxis to predict re-administration of PRV-3279 and sirolimus.

[0122] In cohort D (n=4), 3 subjects were randomized to receive a single high dose of 1 x 10 14 μg / kg of AAV9-Des-TAZ, and one subject could be randomized to receive a single low-dose of 3x 10 13 After 16 weeks, one subject who received the low dose at week 16 can cross over and receive the high dose at week 32. The three subjects who received the high dose at week 16 can receive placebo at week 32. This can be done in conjunction with experimental prophylaxis to anticipate re-dosing of PRV-3279 and sirolimus. Example 2: Toxicology study design for the combination therapy of PRV-3279 and AAV-Des-TAZ gene therapy

[0123] One hundred and twenty age-matched huCD32B mice were available for this study.

[0124] PRV-3279 Replacement Requirements: 6 groups × 28 days × 10 mice × 25g mice × 50 mg / kg = 2100 mg 6 groups × 84 days × 10 mice × 25g mice × 50 mg / kg = 6300 mg + 30% overage = about 11 g of PRV-3279 replacement.

[0125] Toxicology studies can be performed on a schedule of low doses (e.g., 3 x 10 13 μg / kg) or high doses (e.g., 1x 10 14 In this study, we performed gene therapy (AAV-Des-TAZ) in mice treated with 50 mg / kg / day PRV-3279 for 28 or 84 days. Modification

[0126] Without departing from the scope and spirit of the present disclosure, modifications and variations of the methods and compositions described in the present disclosure will be apparent to those skilled in the art. Although the present disclosure has been described in conjunction with specific embodiments, it should be understood that the disclosure claimed for protection should not be unduly limited to such specific embodiments. In fact, various modifications of the modes for implementing the present disclosure are intended and understood by those skilled in the relevant art to be within the scope of the present disclosure as represented by the following claims. Incorporated by Reference

[0127] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. References: Barth, PG et al. "An Barth Syndrome Foundation,2018.The Voice of the Patient:BarthSyndrome[WWW Document].www.fda.gov / media / 130562 / download Corti,M.et al.,2014.B-Cell Depletion is Protective Against Anti-AAVCapsid Immune Response:A Human Subject Case Study.Mol.Ther.MethodsClin.Dev.1. doi.org / 10.1038 / mtm.2014.33 Corti,Manuela et al.,“Safety ofIntradiaphragmatic Delivery ofAdeno-Associated Virus-Mediated Alpha-Glucosidase(rAAV1-CMV-hGAA)Gene Therapy inChildren Affected by Pompe Disease.”Human gene therapy.Clinical developmentvol.28,4(2017):208-218.doi:10.1089 / humc.2017.146 Gonzalez IL,Human tafazzin(TAZ)gene mutation andvariationdatabase.2012.Science and Research section of www.barthsyndrome.org . Rigaud,C.,Lebre,AS.,Touraine,R.et al.Natural history ofBarthsyndrome: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:Distinguishingdisease characteristics and growth data from a longitudinalstudy.Am J MedGenet Part A 158A:2726-2732. Rugolotto,Simone et al.“Long-term treatment of Barth syndromewithpantothenic acid:a retrospective study.”Molecular genetics and metabolismvol.80,4(2003):408-11.doi:10.1016 / j.ymgme.2003.07.002 Nathwani A.C.et al.(2007).Safe and efficient transduction of theliver afterperipheral vein infusion of self-complementary AAV vector resultsin stabletherapeutic 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), the method comprising administering an effective amount of a non-depleting B cell inhibitor to a patient who is receiving or has received gene therapy for BTHS.

2. The method of claim 1, wherein the BTHS gene therapy comprises administering to the patient genetically modified cells with a recombinant adeno-associated virus (rAAV) vector encoding a TAFAZZIN transgene. 3 . The method of claim 1 , wherein the B cell inhibitor is a CD32B×CD79B bispecific antibody capable of immunospecifically binding to an epitope of CD32B and an epitope of CD79B.

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

5. The method of claim 4, wherein the CD32B×CD79B bispecific antibody is an Fc diabody comprising: (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.

6. The method of claim 5, comprising administering the Fc diabody at a dose of between about 5 mg / kg and about 100 mg / kg, or between about 5 mg / kg and about 50 mg / kg, or between about 5 mg / kg and about 40 mg / kg and at a dose schedule of between once a week and every 6 weeks.

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

8. The method of claim 5, comprising administering three doses of the Fc diabody at a dose of about 10 mg / kg, 1 week before the first BTHS gene therapy delivery, 2 weeks after the first BTHS gene therapy delivery, and 3-4 weeks after the second BTHS gene therapy delivery.

9. The method of claim 8, comprising administering a first dose about 2 days to about 6 weeks before administering the BTHS gene therapy, administering a second dose at about the same time as administering the BTHS gene therapy, and administering a third dose about 2 days to about 6 weeks after administering the BTHS gene therapy.

10. The method of claim 5, wherein the Fc diabody achieves inhibition of its own immunogenicity upon administration, wherein the incidence and / or titer of anti-drug antibodies (ADA) is reduced at increased doses.

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

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

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

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

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

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

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

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

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

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

21. A pharmaceutical composition for reducing immunogenicity associated with Barth syndrome (BTHS) gene therapy for administration before, simultaneously with and / or after BTHS gene therapy, the pharmaceutical composition comprising an effective amount of a CD32B×CD79B bispecific antibody capable of immunospecifically binding to an epitope of CD32B and an epitope of CD79B, wherein optionally the BTHS gene therapy comprises genetically modified cells containing a recombinant adeno-associated virus (rAAV) vector encoding a TAFAZZIN transgene.

22. The pharmaceutical composition of claim 21, wherein the CD32B×CD79B bispecific antibody comprises: (A) VL comprising the amino acid sequence of SEQ ID NO: 1 CD32B Structural domain; (B) VH comprising the amino acid sequence of SEQ ID NO: 2 CD32B Structural 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 Structural domain.

23. The pharmaceutical composition of claim 22, wherein the CD32B×CD79B bispecific antibody is an Fc diabody comprising: (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.

24. The pharmaceutical composition of claim 23, comprising the Fc diabody, at a dosage of between about 5 mg / kg and about 100 mg / kg, or between about 5 mg / kg and about 50 mg / kg, or between about 5 mg / kg and about 40 mg / kg, and at a dosage regimen of between once a week and once every 6 weeks.

25. The pharmaceutical composition of claim 23, comprising the Fc diabody at a dose of about 10 mg / kg, administered every 1-4 weeks, preferably 1 week before the first BTHS gene therapy delivery, 2 weeks after the first BTHS gene therapy delivery, and 3-4 weeks after the second BTHS gene therapy delivery.

26. The pharmaceutical composition of claim 25, comprising a first dose about 2 days to about 6 weeks before administration of the BTHS gene therapy, a second dose at about the same time as 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. The pharmaceutical composition of claim 21, further comprising one or more immunomodulators selected from the group consisting of sirolimus, rapamycin, abatacept, teligrumumab, and immunoglobulin G degrading enzyme of Streptococcus pyogenes.

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