Methods and compositions for reducing immunogenicity with non-depleting B cell inhibitors

Abstract

In one aspect, disclosed herein is a method for reducing immunogenicity, comprising administering an effective amount of a non-depleting B cell inhibitor to a patient receiving or who has received a biological therapeutic agent. Related compositions are also provided. In some embodiments, the biological therapeutic agent is selected from one or more of a gene therapy agent, a gene editing therapy agent, a messenger RNA (mRNA) therapy agent, an oncolytic virus, an enzyme replacement therapy agent, an antibody therapy agent, a protein therapy agent, and a cell therapy agent.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority and priority to U.S. Provisional Application No. 62 / 880,240, filed July 30, 2019, and U.S. Utility Application No. 16 / 943,849, filed July 30, 2020, which are incorporated herein by reference in their entirety.

[0002] [Sequence List] The 13,267-byte ASCII text file titled "010802seq.txt," created on July 30, 2020, and submitted via EFS-Web on July 30, 2020, is incorporated in its entirety within this specification for reference.

[0003] This disclosure relates in general to compositions and methods for reducing the immunogenicity of biological therapeutic agents, and more particularly to doing so with non-depleting B-cell inhibitors. [Background technology]

[0004] The use of biopharmaceuticals such as antibodies and polypeptides as therapeutic agents carries an associated risk of generating undesirable immune responses in patients, typically defined by the development of anti-drug antibody (ADA) responses. Such responses may be triggered by the presence of "exotic" epitopes in the molecule and may be exacerbated by exogenous factors, particularly the patient's genomic and disease background, the regimen and administration regimen used, the formulation, and the route of administration and impurities. These immune responses may have a range of consequences, from pharmacological alterations to increased drug clearance or neutralization and loss of therapeutic efficacy. In extreme cases, protein-based therapeutics may cause severe allergic and anaphylactic reactions that pose a significant risk to patients.

[0005] Another well-characterized immune response to “foreign” agents is so-called graft rejection (also known as host-versus-graft reaction), in which the endogenous immune system reacts to foreign tissue and causes its destruction. Tissue rejection can be mediated by humoral and cellular immune responses. In the case of genetically modified cells created for the purpose of incorporating a defective copy of a gene (gene therapy) or to help a patient eliminate cancer cells (e.g., CAR-T therapy), there is a risk that some of the “mechanisms” used to genetically modify the cells may be “presented” by the modified cells and recognized by the host as “foreign” agents. Such recognition may initiate a rejection reaction, which may potentially render such treatment ineffective or, in severe cases, potentially trigger an autoimmune reaction.

[0006] More recently, the emergence of gene therapy has encountered significant barriers, including the immunogenicity of the viral vectors used to deliver the transgene, and the immunogenicity of the transgene protein itself after it has been expressed by the recipient's cells. The immunogenicity of vectors and transgenes leads to: 1) reduced efficacy as vectors and transgenes due to binding to and elimination 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-administration if the subject develops antibodies against the vector or transgene after previous administration. Sometimes, recipients have pre-existing antibodies against the vector even before the first dose and die from cross-reactivity with naturally occurring viruses. Other therapies based on viruses (e.g., tumor regression viruses in cancer) and gene editing therapies (e.g., CRISPR-Cas9-based therapies) also suffer from immunogenicity.

[0007] Therefore, there is a need for methods and compositions to reduce immunogenicity induced by various biological therapeutic agents, including, but not limited to, antibodies, cell therapies, and gene therapies. [Overview of the project]

[0008] In one embodiment, a method for reducing immunogenicity is disclosed herein, which includes administering an effective amount of a non-depleting B-cell inhibitor to a patient who is receiving or has received a biological therapeutic agent.

[0009] In some embodiments, the biological therapeutic agent is selected from one or more of gene therapies, gene editing therapies, messenger RNA (mRNA) therapies, tumor regression viruses, enzyme replacement therapies, antibody therapies, protein therapies, and cell therapies. In some embodiments, the biological therapeutic agent is a gene therapy agent.

[0010] In some embodiments, the B cell inhibitor is a CD32B × CD79B bispecific antibody that can immunospecifically bind to the CD32B epitope and the CD79B epitope. In some embodiments, the CD32B × CD79B bispecific antibody is (A) VL containing the amino acid sequence of SEQ ID NO: 1 CD32B domain, (B) VH containing the amino acid sequence of SEQ ID NO: 2 CD32B domain, (C) VL containing the amino acid sequence of SEQ ID NO: 3 CD79B Domain, and (D) VH containing the amino acid sequence of SEQ ID NO: 4 CD79B domain Includes.

[0011] In some embodiments, the CD32B × CD79B bispecific antibody is (A) A first polypeptide chain containing the amino acid sequence of Sequence ID No. 5, (B) A second polypeptide chain containing the amino acid sequence of SEQ ID NO: 6, and (C) Third polypeptide chain containing the amino acid sequence of SEQ ID NO: 7 It is an Fc diamond body that includes this.

[0012] In some embodiments, the method can further include administering the Fc diabody at a dosage of about 5 mg / kg to about 40 mg / kg, with a dosing regimen of once-dosage / 2 weeks to once-dosage / 6 weeks. In some embodiments, the method can include administering the Fc diabody at a dosage of about 10 mg / kg, with a dosing regimen of once-dosage / 4 weeks. In some embodiments, the method can include administering three doses of the Fc diabody at a dosage of about 10 mg / kg, at an interval of 2 to 6 weeks.

[0013] In some embodiments, the method can include administering a first dose about 2 to 6 weeks (e.g., 4 weeks) before the administration of the biological therapeutic agent, a second dose substantially simultaneously with the administration of the biological therapeutic agent, and a third dose about 2 to 6 weeks (e.g., 4 weeks) after the administration of the biological therapeutic agent.

[0014] In some embodiments, the Fc diabody results in inhibition of its own immunogenicity upon administration and has a lower morbidity and / or titer of anti-drug antibodies (ADA) at increased dosages. In some embodiments, the ADA does not neutralize the Fc diabody.

[0015] In some embodiments, the 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 B cells for at least 4 weeks after the last administration.

[0016] In some embodiments, the Fc diabody results in a sustained inhibition of immunoglobulin production without depleting circulating B cells. In some embodiments, the immunoglobulin includes one or more of IgM, IgA, IgG, and IgE.

[0017] In some embodiments, the method can further include monitoring the patient by examining the presence of specific antibodies against the biological therapeutic agent. In some embodiments, the method can further include administering one or more doses of a B cell inhibitor to further modulate immunogenicity.

[0018] In some embodiments, the method can further include co-administering one or more immunomodulatory agents such as sirolimus, rapamycin, abatacept, teplyzumab, and the immunoglobulin G-degrading enzyme of Streptococcus pyogenes.

[0019] Also provided herein are pharmaceutical compositions comprising a non-depleting B cell inhibitor disclosed herein, provided in a therapeutically effective unit dose (e.g., packaged). Instructions for the dosing regimens disclosed herein can also be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] [Figure 1] A figure showing an overall schematic diagram of this study. [Figure 2A] A figure showing the daily average (±SD) PRV-3279 serum concentration (ng / mL) versus time on a linear scale (population for pharmacokinetic analysis) (Figure 2A: day 1). [Figure 2B] A figure showing the daily average (±SD) PRV-3279 serum concentration (ng / mL) versus time on a linear scale (population for pharmacokinetic analysis) (Figure 2B: day 15). [Figure 2C] A figure showing the daily average (±SD) PRV-3279 serum concentration (ng / mL) versus time on a linear scale (population for pharmacokinetic analysis) (Figure 2C: day 29). [Figure 3A] A figure showing the daily average PRV-3279 serum concentration (ng / mL) versus time on a semi-logarithmic scale (population for pharmacokinetic analysis) (Figure 3A: day 1).​​​​​​​ [Figure 4A] This figure shows the mean (±SD) versus time (days) of PRV-3279 serum concentration (ng / mL) for each dose and ADA result (pharmacokinetic analysis population) (Figure 4A: 3 mg / kg). [Figure 4B] This figure shows the mean (±SD) versus time (days) of PRV-3279 serum concentration (ng / mL) for each dose and ADA result (pharmacokinetic analysis population) (Figure 4B: 10 mg / kg). [Figure 5] This figure shows box plots of PRV-3279 serum pharmacokinetic parameters for each dose and ADA result (pharmacokinetic analysis population). [Figure 6] This figure shows the % maximum combined arithmetic mean (±SEM) of % anti-E / K+(CD3- / CD19+) per time and treatment (pharmacodynamic analysis population). [Figure 7] This figure shows the arithmetic mean (±SEM) of the number of B cells (CD19+) minus (returning to cell number / μL) (for the population targeted for pharmacodynamic analysis). [Figure 8] This figure shows the arithmetic mean (±SEM) of the decrease in circulating serum IgM levels (safety analysis population). [Figure 9] This figure shows the arithmetic mean (±SEM) of the decrease in circulating serum IgE levels (safety analysis population). [Figure 10] This figure shows the arithmetic mean (±SEM) of the decrease in circulating serum IgG levels (safety analysis population). [Modes for carrying out the invention]

[0021] In one embodiment, a method for reducing immunogenicity is disclosed herein, which includes administering an effective amount of a non-depleting B-cell inhibitor to a patient who is receiving or has received a biological therapy. In some embodiments, the B-cell inhibitor is a CD32B × CD79B bispecific antibody, such as those disclosed in U.S. Patent Application Publication 2016 / 0194396, International Publication 2015 / 021089, and International Publication 2017 / 214096, which are all part of this specification by reference.

[0022] [Definition] For convenience, the specific terms used in the specification, examples, and appended claims are set forth herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs.

[0023] The use of the words "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 meanings of "one or more," "at least one," and "one or more."

[0024] Throughout this application, the term “about” is used to indicate that a value includes the inherent variability of error in the methods / apparatus used to determine the value, or the variability present among the subjects of study. Typically, this term means, depending on the context, that it includes variability of approximately 1% or less, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0025] The term "substantially" means more than 50%, preferably more than 80%, and most preferably more than 90% or 95%.

[0026] The use of the term “or” in the claims is used to mean “and / or” unless it is explicitly indicated that it refers only to substitutes or that the substitutes are mutually exclusive, however, this disclosure supports the definitions of “and / or” as referring only to substitutes.

[0027] 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 comprehensive or open-ended and do not exclude any additional unlisted elements or method steps. Any embodiment described herein is intended to be implementable with respect to any method, system, host cell, expression vector, and / or composition of the present invention. Furthermore, the methods and proteins of the present invention can be achieved using the compositions, systems, host cells, and / or vectors of the present invention.

[0028] As used herein, the term “essentially consisting of” means elements necessary for a given embodiment. This term allows for the presence of additional elements that do not substantially affect the basic, novel, or functional features of that embodiment of the disclosure.

[0029] The term "consisting of" means the compositions, methods, and corresponding components thereof described herein, excluding any elements not listed in the description of embodiments.

[0030] The use of the term “for example” and its corresponding abbreviation “eg” (whether italicized or not) means that the specific terms listed are representative examples, and unless otherwise explicitly stated, embodiments of the present invention are not intended to be limited to the specific examples referenced or cited.

[0031] "Nucleic acid," "nucleic acid molecule," "oligonucleotide," or "polynucleotide" refers to a polymer compound containing covalently linked nucleotides. Examples of the term "nucleic acid" include polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. Examples of DNA, but not limited to, include complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA. In some embodiments, the present invention is directed towards polynucleotides encoding any one of the polypeptides disclosed herein, for example, polynucleotides encoding the Cas protein or its variants. In some embodiments, the present invention is directed towards polynucleotides encoding Cas3, Cas9, CaslO, or their variants.

[0032] A "gene" is an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acid molecules. A "gene" can also refer to nucleic acid fragments that can function as regulatory sequences before (5' non-coding sequence) and after (3' non-coding sequence) a coding sequence.

[0033] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to polymeric forms of amino acids of any length, including coding and non-coding amino acids, chemically modified or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone.

[0034] As used herein, “antibody” or “antibody molecule” means a protein, such as an immunoglobulin chain or fragment thereof, that contains at least one immunoglobulin variable domain sequence. Antibody molecules include antibodies (e.g., full-length antibodies) and antibody fragments. In one embodiment, an antibody molecule includes an antigen-binding fragment 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 is naturally occurring or formed by a normal immunoglobulin gene fragment recombination process. In embodiments, an antibody molecule means an immunologically active antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. An antibody fragment, such as a functional fragment, is a portion of an antibody, such as Fab, Fab', F(ab')2, F(ab)2, a variable fragment (Fv), a domain antibody (dAb), or a single-chain variable fragment (scFv). A functional antibody fragment binds to the same antigen recognized by an intact (e.g., full-length) antibody. Furthermore, the terms “antibody fragment” or “functional fragment” may also refer to isolated fragments consisting of variable regions, such as “Fv” fragments consisting of variable regions of the heavy and light chains, or recombinant single-chain polypeptide molecules (“scFv proteins”) in which the variable regions of the light and heavy chains are linked by a peptide linker. In some embodiments, antibody fragments do not include portions of the antibody that do not have antigen-binding activity, such as Fc fragments or single amino acid residues. Exemplary antibody molecules include full-length antibodies and antibody fragments, e.g., dAb (domain antibodies), single-chain, Fab, Fab', and F(ab')2 fragments, and single-chain variable fragments (scFvs). The terms “Fab” and “Fab fragment” are used interchangeably, referring to one constant domain and one variable domain from the respective heavy and light chains of the antibody, i.e., V L , C L , V H , and C H This refers to the region that includes the number 1.

[0035] Throughout this specification, the numbering of residues in the constant regions of IgG heavy chains is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th th Edition, Public Health Service, NIH, MD (1991) ("Kabat"). The term "EU index as in Kabat" refers to the numbering of human IgG1 EU antibodies. Amino acids from the variable domains of mature heavy and light chains of immunoglobulins are named by their position in the chain. Kabat has described the amino acid sequences of numerous antibodies, identified amino acid consensus sequences for each subgroup, assigned residue numbers to each amino acid, and the CDRs are identified as defined by Kabat (CDR1 starts 5 residues before the 5 residues that are understood to be the start of CDR2). The Kabat numbering scheme can be extended to antibodies not covered in the review by aligning the antibody in question with one of the consensus sequences in Kabat, with reference to conserved amino acids. This method of assigning residue numbers is standard in the art and facilitates the identification of amino acids at corresponding positions in different antibodies, including chimeric or humanized variants. For example, the amino acid at position 50 of the human antibody light chain occupies the position corresponding to the amino acid at position 50 of the mouse antibody light chain. H It should be understood that CDR1 starts 5 residues before the 5 residues that are understood to be the start of CDR2). The Kabat numbering scheme can be extended to antibodies not covered in the review by aligning the antibody in question with one of the consensus sequences in Kabat, with reference to conserved amino acids. This method of assigning residue numbers is standard in the art and facilitates the identification of amino acids at corresponding positions in different antibodies, including chimeric or humanized variants. For example, the amino acid at position 50 of the human antibody light chain occupies the position corresponding to the amino acid at position 50 of the mouse antibody light chain.

[0036] In some embodiments, the antibody molecule is monospecific, for example, it has binding specificity to a single epitope. In some embodiments, the antibody molecule is polyspecific, for example, it comprises multiple immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence has binding specificity to a first epitope and a second immunoglobulin variable domain sequence has binding specificity to a second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule.

[0037] The terms “bispecific antibody molecule,” “diabody,” and “biaffinity retargeting (DART®)” antibody are used interchangeably herein and refer to antibody molecules having specificity to multiple (e.g., two, three, four, or more) epitopes and / or antigens. In some embodiments, the antibody may be an antigen-binding diabody or scaffold, such as those disclosed in U.S. Patent Application Publication 2016 / 0194396, International Publication 2015 / 021089, and International Publication 2017 / 214096, each of which is incorporated in whole for reference. In some embodiments, the antibody may be a CD32B×CD79B bispecificity diabody (i.e., a "CD32B×CD79B diabody") and such a diabody further comprising an Fc domain (i.e., a "CD32B×CD79B Fc diabody"). In one embodiment, the antibody may be a humanized CD32B×CD79B DART® antibody with a molecular weight of 111.5 kDa produced in Chinese hamster ovary cells.

[0038] As used herein, “antigen” (Ag) means a macromolecule containing all proteins or peptides. In some embodiments, an antigen is a molecule that can induce an immune response, for example, by being involved in the activation of certain immune cells and / or the production of antibodies. Antigens are not only involved in antibody production; T cell receptors also recognize antigens (except for antigens whose peptides or peptide fragments form a complex with MHC molecules). Almost any macromolecule containing proteins or peptides can be an antigen. Antigens can also be derived from genomically recombinant DNA or DNA. For example, any DNA containing a nucleotide sequence or partial nucleotide sequence that codes for a protein capable of inducing an immune response codes for an “antigen”. In embodiments, an antigen does not have to be coded by a full-length nucleotide sequence of a gene, nor does an antigen have to be coded by a gene at all. In embodiments, an antigen can be synthetic or derived from a biological sample, such as a tissue sample, tumor sample, cell, or fluid of other biological components. As used herein, “tumor antigen,” or interchangeable “cancer antigen,” refers to cancer, for example, any molecule present on or associated with cancer cells or the tumor microenvironment that can induce an immune response. As used herein, "immune cell antigen" refers to any molecule present on or associated with immune cells that can induce an immune response.

[0039] The “antigen-binding site,” “antigen-binding fragment,” or “antigen-binding portion” of an antibody molecule (as used interchangeably herein) refers to a portion of an antibody molecule, such as an immunoglobulin (Ig) molecule like IgG, that participates in antigen binding. In some embodiments, the antigen-binding site is formed by amino acid residues in the variable (V) regions of the heavy (H) and light (L) chains. Three highly distinct stretches within the variable regions of the heavy and light chains are called hypervariable regions and are located between more conserved adjacent stretches called “framework regions” (FRs). FRs are amino acid sequences found naturally between and adjacent to hypervariable regions in immunoglobulins. In embodiments, within the 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 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, 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 in the amino-to-carboxyl amino acid sequence FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Variable light chain (VL) CDRs are generally defined as containing residues at positions 27-32 (CDR1), 50-56 (CDR2), and 91-97 (CDR3). Variable heavy chain (VH) CDRs are generally defined as containing residues at positions 27–33 (CDR1), 52–56 (CDR2), and 95–102 (CDR3).Those skilled in the art will understand that loops can be of varying lengths among antibodies, and that a numbering system such as Kabat or Chotia is in control to ensure the framework has consistent numbering across antibodies.

[0040] In some embodiments, the antigen-binding fragment of an antibody (for example, if included as part of a fusion molecule) may lack or have no complete Fc domain. In certain embodiments, the antibody-binding fragment may not contain complete IgG or complete Fc, but may contain one or more constant regions (or fragments thereof) from the light and / or heavy chains. In some embodiments, the antigen-binding fragment may not contain any Fc domain at all. In some embodiments, the antigen-binding fragment may substantially not contain a complete Fc domain. In some embodiments, the antigen-binding fragment may contain a portion of a complete Fc domain (for example, a CH2 or CH3 domain or a portion thereof). In some embodiments, the antigen-binding fragment may contain a complete Fc domain. In some embodiments, the Fc domain is an IgG domain, for example, an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the Fc domain includes a CH2 domain and a CH3 domain.

[0041] As used herein, “administer” and similar terms mean to deliver the composition to the individual being treated. Preferably, the compositions of this disclosure are administered parenterally, for example, by subcutaneous, intramuscular, or preferably intravenous routes.

[0042] As used herein, “effective dose” means the amount of a bioactive agent or diagnostic agent sufficient to provide the desired local or systemic effect with a reasonable risk-benefit ratio that may be associated with any medical or diagnostic test. This will vary depending on the patient, the disease, the procedure being performed, and the nature of the agent. The therapeutically effective dose will vary depending on the patient and condition being treated, the patient’s weight and age, the severity of the condition, the mode of administration, etc., which can be readily determined by those skilled in the art. The dosages are, for example, approximately 1 ng to 10,000 mg, approximately 5 ng to 9,500 mg, approximately 10 ng to 9,000 mg, approximately 20 ng to 8,500 mg, approximately 30 ng to 7,500 mg, approximately 40 ng to 7,000 mg, approximately 50 ng to 6,500 mg, approximately 100 ng to 6,000 mg, approximately 200 ng to 5,500 mg, approximately 300 ng to 5,000 mg, and approximately 400 ng to 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, approximately 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 1,175mg, Approximately 500μg to approximately 1,150mg, approximately 0.5mg to approximately 1,125mg, approximately 1mg to approximately 1,100mg, approximately 1.25mg to approximately 1,075mg, approximately 1.5mg to approximately 1,050mg, approximately 2.0mg to approximately 1,0 25mg, about 2.5mg to about 1,000mg, about 3.0mg to about 975mg, about 3.5mg to about 950mg, about 4.0mg to about 925mg, about 4.5mg to about 900mg, about 5mg to about 875mg, about The antibodies or their antigen-binding portions provided herein may range from 10 mg to approximately 850 mg, approximately 20 mg to approximately 825 mg, approximately 30 mg to approximately 800 mg, approximately 40 mg to approximately 775 mg, approximately 50 mg to approximately 750 mg, approximately 100 mg to approximately 725 mg, approximately 200 mg to approximately 700 mg, approximately 300 mg to approximately 675 mg, approximately 400 mg to approximately 650 mg, approximately 500 mg, or approximately 525 mg to approximately 625 mg. Dosage may be, for example, every week, every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks.The medication regimen may be adjusted to provide the optimal therapeutic response. An effective dose is one in which any toxic or adverse effects (side effects) of the drug are minimized and / or outweigh the beneficial effects. Administration may be intravenously, exactly or approximately 6 mg / kg or 12 mg / kg once weekly, or 12 mg / kg or 24 mg / kg every other week. Additional medication regimens are described below.

[0043] As used herein, “pharmaceutically acceptable” means a substance that is generally safe, non-toxic, and useful in the preparation of pharmaceutical compositions that are not biologically or otherwise undesirable, and is acceptable for veterinary and human pharmaceutical use. Examples of “pharmaceutically acceptable liquid carriers” include water and organic solvents. Preferred pharmaceutically acceptable aqueous liquids include PBS, physiological saline, and dextrose solutions.

[0044] The term “immunogenicity” means the ability of a particular substance, such as an antigen or epitope, to induce an immune response in the body of humans and other animals, which may be humoral and / or cell-mediated. In some embodiments, administration of the compositions of this disclosure reduces the immunogenicity of a biological substance, such as a therapeutic agent, and / or increases immune tolerance to it. As used herein, “tolerance” or “immune tolerance” means the absence of an immune response to a particular antigen (e.g., a therapeutic bioagent) in a setting of otherwise substantially normal immune systems.

[0045] As used herein, “Major Histocompatibility Complex” or “MHC” proteins refer to a set of cell surface molecules encoded by a large gene family that plays a significant role in the vertebrate immune system. The primary function of these proteins is to bind peptide fragments derived from endogenous or exogenous (foreign) proteins and display them on the cell surface for appropriate recognition by the host organism’s T cells. The MHC gene family is classified into three subgroups: Class I, Class II, and Class III. Human MHC Class I and Class II genes are also called 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-DPAl, HLA-DPBl, HLA-DQAl, HLA-DQBl, HLA-DRA, HLA-DRBl, and HLA-DRB345.

[0046] Various aspects of this disclosure are described in further detail below. Additional definitions are provided throughout this specification.

[0047] <Non-depletion B-cell inhibitors and pharmaceutical compositions> In various embodiments, B cell inhibitors can be used to reduce or modulate immunogenicity. In some embodiments, such B cell inhibitors are non-depletional immunomodulators. As used herein, “non-depletional” 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 they work to eliminate or destroy B cells, such as anti-CD20 antibodies, e.g., rituximab. Therefore, in one embodiment, the non-depletional B cell inhibitor or immunomodulator disclosed herein is not rituximab. In some embodiments, the non-depletional B cell inhibitor or immunomodulator is not an anti-CD20 antibody or other CD20 inhibitor.

[0048] Examples of non-depletion B cell inhibitors include, but are not limited to, CD32B × CD79B bispecificity inhibitors, CD32B modulators, B cell receptor (BCR) blockers such as anti-CD22 molecules, B cell survival and activation inhibitors such as B cell activator (BAFF) or A proliferation-inducing ligand (APRIL) inhibitors such as belimumanb, anti-CD40 and anti-CD40L molecules, and Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib (PCI-32765) and acalabrutinib.

[0049] In some embodiments, the B cell inhibitor may be a CD32B × CD79B bispecific antibody or its antigen-binding fragment, such as those disclosed in U.S. Patent Application Publication 2016 / 0194396, International Publication 2015 / 021089, and International Publication 2017 / 214096, all of which are incorporated in their entirety for reference.

[0050] An exemplary CD32B×CD79B bispecific diabody can contain two or more polypeptide chains. (1) CD32B(VL CD32B The VL domain of an antibody that binds to ) DIQMTQSPSS LSASVGDRVT ITCRASQEIS GYLSWLQQKP GKAPRRLIYA ASTLDSGVPS RFSGSESGTE FTLTISSLQP EDFATYYCLQ YFSYPLTFGG GTKVEIK VL having the sequence (sequence number 1) CD32B domain (2) CD32B(VH CD32B The VH domain of an antibody that binds to ) EVQLVESGGG LVQPGGSLRL SCAASGFTFS DAWMDWVRQA PGKGLEWVAE IRNKAKNHAT YYAESVIGRF TISRDDAKNS LYLQMNSLRA EDTAVYYCGA LGLDYWGQGT LVTVSS VH has the sequence (sequence number 2) CD32B domain (3) CD79B(VL CD79B The VL domain of an antibody that binds to ) DVVMTQSPLS LPVTLGQPAS ISCKSSQSLL DSDGKTYLNW FQQRPGQSPN RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP LTFGGGTKLE IK VL having the sequence (sequence number 3) CD79B domain (4) CD79B(VH CD79B The VH domain of an antibody that binds to ) QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMNWVRQA PGQGLEWIGM IDPSDSETHY NQKFKDRVTM TTDTSTSTAY MELRSLRSDD TAVYYCARAM GYWGQGTTVT VSS VH has the sequence (sequence number 4) CD79B domain It can include...

[0051] In one embodiment, the B cell inhibitor may be PRV-3279, a 111.5 kDa humanized CD32B × CD79B biaffinity retargeting (DART®) protein produced in Chinese hamster ovary cells. DART® proteins are bispecific, antibody-based molecules capable of simultaneously binding to two distinctly 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 manner on B lymphocytes initiates inhibitory motif signaling based on immunoreceptor tyrosine coupled with CD32B, which reduces antigen-mediated activation of naive and memory B cells without widespread depletion. To extend the in vivo half-life, PRV-3279 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 receptor-mediated IgG salvage pathway.

[0052] 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 signaling component of the BCR, exclusively expressed on B cells. The anti-CD79B component of PRV-3279 is based on a humanized version of mouse mAb CB3.

[0053] In one embodiment, PRV-3279 includes the following sequence (CDR is underlined and the coil domain is shown in bold): [ka] [ka] [ka]

[0054] In another embodiment, the present invention provides pharmaceutical compositions that can be used in the methods disclosed herein, i.e., pharmaceutical compositions for reducing or suppressing immunogenicity in a subject in need, for example, during or after administration of a biological product that elicits significant immunogenicity, or pharmaceutical compositions that can be used because the subject had pre-existing immunogenicity to a biotherapy agent (for example, in the case of pre-existing anti-AAV antibodies due to a previous wild-type adenovirus infection or previous exposure to rAAV treatment). In some embodiments, the compositions disclosed herein can be administered to a patient before administration of a biological product such as an antibody or gene therapy agent to prevent immunogenicity and / or reduce pre-existing antibodies.

[0055] In some embodiments, the pharmaceutical composition comprises a B-cell inhibitor and a pharmaceutically acceptable carrier as disclosed herein. The B-cell inhibitor can be incorporated into the pharmaceutical composition using a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical composition may include instructions for use, for example, for a composition used to treat a patient to reduce or suppress immunogenicity in a subject that requires it, for example, during or after administration of a biological agent that elicits significant immunogenicity.

[0056] As used herein, “pharmaceutically acceptable carriers” include any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and absorption retarders, buffers, and other physiologically compatible excipients. Preferably, the carrier is suitable for parenteral, oral, or topical administration. Depending on the route of administration, the active compound, for example, a small molecule or bioagent, may be coated with a material that protects the compound from the action of acids and other natural conditions that may deactivate the compound.

[0057] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, sterile powders for the immediate preparation of sterile injectable solutions or dispersions, and conventional excipients for the preparation of tablets, pills, capsules, and the like. The use of such media and agents for formulating pharmaceutically active substances is known in the art. Any conventional media or agent is intended for use in the pharmaceutical compositions provided herein, unless it is incompatible with the active compound. Auxiliary active compounds may also be incorporated into the compositions.

[0058] Examples of pharmaceutically acceptable carriers include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium bisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0059] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, as well as injectable organic esters such as ethyl oleate. Where necessary, adequate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it may be useful to include isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride in the composition. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition.

[0060] These compositions may also contain functional excipients such as preservatives, humectants, emulsifiers, and dispersants.

[0061] Therapeutic compositions typically must be sterile, non-phylogenic, and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0062] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into a suitable solvent, along with one or a combination of the components listed above as needed, and then sterilizing, for example, by microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for preparing sterile injectable solutions, preparation methods include vacuum drying and lyophilization, which yield a powder of the active ingredient and any additional desired components from the solution, which has been previously sterile filtered. Active agents may be mixed under sterile conditions with additional pharmaceutically acceptable carriers and any preservatives, buffers, or sprays as needed.

[0063] The prevention of microbial presence can be ensured by both the sterilization procedures described above and the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, sustained absorption of the injectable pharmaceutical form can be achieved by the inclusion of absorption-delaying agents such as aluminum monostearate and gelatin.

[0064] The medication regimen is adjusted to provide the optimal desired response (e.g., 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 urgency of the treatment situation.

[0065] Examples of dosage ranges for antibody administration include 10-1000 mg (antibody) / kg (patient's 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, and 50- Examples of dosages include 800 mg / kg, 50-600 mg / kg, 50-400 mg / kg, 50-200 mg / kg, 100-1000 mg / kg, 100-900 mg / kg, 100-800 mg / kg, 100-700 mg / kg, 100-600 mg / kg, 100-500 mg / kg, 100-400 mg / kg, 100-300 mg / kg, and 100-200 mg / kg. Exemplary dosing 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.

[0066] In some embodiments, doses of approximately 5–40 mg / kg, approximately 5–20 mg / kg, or approximately 10 mg / kg / PRV-3279 may be administered once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks. Single or multiple doses, such as single, double, or triple doses, may be administered. Administration may be via IV infusion. Any combination of the above (e.g., 10 mb / kg / dose in triple doses, once every four weeks) can be used to reduce the immunogenicity of biotherapy agents, including gene therapy products. In some embodiments, a first dose may be administered 2–6 weeks (e.g., four weeks) before gene therapy, a second dose approximately simultaneously with gene therapy, and a third dose 2–6 weeks (e.g., four weeks) after gene therapy. Thereafter, the patient can be monitored by testing the amount of specific antibodies against the gene therapy vector (e.g., rAAV) and / or the transgene. If no antibodies are detected or only small amounts are detected, additional PRV-3279 is not necessary. If a significant amount of antibodies is present, one or more doses of PRV-3279 may be administered to further modulate immunogenicity.

[0067] For ease of administration and uniformity of drug delivery, it may be advantageous to formulate parenteral compositions in unit dosage forms. As used herein, a unit dosage form means a physically distinct unit suitable for unit dosing of a patient being treated. Each unit contains a predetermined amount of the active agent, calculated to produce the desired therapeutic effect in association with any required pharmaceutical carrier. The specifications of a unit dosage form are indicated and directly depend on (a) the unique characteristics of the active compound and the specifics to be achieved, and (b) the limitations inherent in the field in which such an active compound is formulated for treating sensitivity in an individual.

[0068] The actual dosage levels of the active ingredients in the pharmaceutical compositions disclosed herein may be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response in a particular patient, composition, and mode of administration without causing toxicity to the patient. As used herein in the context of administration, “parenteral” means, but is not limited to, modes of administration other than enteral and topical administration, usually by injection, including intravenous, intramuscular, intra-arterial, subarachnoid, intra-articular, intra-orbital, intra-cardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections, as well as intravenous infusions.

[0069] As used herein, the terms "parenteral administration" and "administered parenterally" refer to, but are not limited to, methods of administration other than enteral (i.e., via the gastrointestinal tract) and topical administration, usually by injection or intravenous fluid administration, including, but not limited to, intravenous, intramuscular, intra-arterial, subarachnoid, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections, as well as intravenous fluid administration. Intravenous injection and intravenous fluid administration are frequently (but not exclusively) used for antibody administration.

[0070] When the agents provided herein are administered to humans or animals as pharmaceuticals, they can be given alone or as pharmaceutical compositions 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.

[0071] <Therapeutic Use and Methods> The compositions disclosed herein can be used to reduce or suppress immunogenicity caused by various biologic products, such as gene therapies delivered by various means (e.g., AAV and other wild-type and recombinant vectors, lentiviral-modified human stem cells), including encoded transgene proteins, gene editing therapies (e.g., CRISPR / Cas9), messenger RNA (mRNA) therapies (e.g., mRNA vaccines), tumor regression viruses (e.g., VSV, HSV-1), enzyme replacement therapy (e.g., factor VIII / IX exchange), antibody and fusion protein-based therapies (e.g., anti-TNF biologics), and cell therapies (e.g., CAR-T therapy).

[0072] In some embodiments, the B cell immunomodulators disclosed herein are used. 1. Therapies based on rAAV (recombinant adenovirus-associated virus) vectors, including the following: • "Conventional" viral delivery of transgenes, e.g., rAAV for hereditary enzyme deficiencies • Gene editing technologies (e.g., rAAV for in vivo delivery of clustered regular short-interval palindromic repeat structure (CRISPR)-associated nuclease Cas9 ("CRISPR / Cas9")) rAAV for the delivery of vaccine antibodies (e.g., influenza) 2. Human stem cell (HSC) therapy using lentivirus-modified HSCs, 3. Cas9 protein delivery (Cas9 is bacterial in origin and immunogenic), and 4. Tumor regression viruses such as vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1) This can improve multiple existing or emerging platforms for gene and cell-based therapies.

[0073] In some embodiments, B-cell immunomodulators disclosed herein can be used to modulate limiting immune responses induced by multiple delivery pathways (including known immune privileged sites) such as systemic, intramuscular, ocular (requiring high local doses and resulting in a local immune response), and central nervous system (CNS) (leaking of viral capsids from the CNS induces a systemic response that attenuates AAV uptake in the CNS).

[0074] In some embodiments, the B cell immunomodulators disclosed herein are used. Development of neutralizing antibodies (nAbs) ·Antibody-dependent cell-mediated cytotoxicity ·Antibody-dependent complement-mediated cytotoxicity • Autonomous B cell activation, for example, mediated by Toll-like receptors (TLRs) It can modulate multiple B cell-dependent restrictive immune pathways, including those mentioned above.

[0075] In some embodiments, the B-cell immunomodulators disclosed herein can be used to improve the clinical application of multiple AAVs through B-cell modulation, such as repeated dosing and / or increased AAV doses.

[0076] In some embodiments, after administration of PRV-3279, peak plasma concentrations occurred at the end of the infusion of the bispecific molecule, and accumulation was minimal during multiple doses. This indicates that PRV-3279 has good pharmacokinetic properties.

[0077] In some embodiments, administration of the PRV-3279 bispecific agent can result in inhibition of its own immunogenicity, i.e., lower morbidity and / or anti-drug antibody (ADA) titers with increasing drug doses. This is in contrast to other immunomodulators. Furthermore, this suggests that increased doses of PRV-3279, such as 20 mg / kg, 30 mg / kg, or 40 mg / kg, may be well-tolerated without additional immunogenicity.

[0078] In some embodiments, it has been observed that PRV-3279 ADA does not affect pharmacokinetics (PK), pharmacodynamics (PD), safety, or efficacy. This is surprising, as ADA typically affects at least PK and PD. While not bound by theory, it is assumed that ADA does not neutralize PRV-3279.

[0079] In some embodiments, the PRV-3279 bispecific agent binds to most B cells (e.g., >80–90%), including both native and memory phenotypes, in a dose-dependent manner upon administration, and remains bound to at least 50% of B cells for at least four weeks after the last dose at a particular higher dose of the drug. This demonstrates the sustained durability of the PD effect of PRV-3279, supporting once-monthly (or longer-term) administration.

[0080] In some embodiments, the dose-dependent and bispecific B-cell binding by the PRV-3279 drug results in a durable inhibition of immunoglobulin production when no circulating cell subset, including B cells, is depleted. Immunoglobulins reduced in peripheral blood include IgM, IgA, IgG, and IgE. Inhibition can be observed in the absence or presence of antigen stimulation (e.g., vaccination). This is a favorable safety feature of PRV-3279 as a non-depletion agent, allowing patients to retain circulating cells, such as B cells, to function as part of the immune system. In contrast, patients receiving depletion agents (e.g., rituximab, ocrelizumab, inebilizumab) take longer to recover (e.g., one year). [Examples]

[0081] The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and should not be construed as limiting the disclosure.

[0082] [Example 1] <Reduced immunogenicity against recombinant adeno-associated virus (rAAV)> In certain experiments, to maintain pharmacological coverage, CD32B × CD79B bispecific antibodies can be administered to mice before and at subsequent time points before administering an rAAV vector encoding a potentially therapeutic transgene, either as monotherapy or in combination with other immunomodulators such as sirolimus, rapamycin, abatacept, teprizumab, and Streptococcus pyogenes immunoglobulin G-degrading enzymes. At specific time points (e.g., 15–45 days), mice can be euthanized for immunological evaluation and assessment of adeno-associated virus gene transfer efficiency. Immunological endpoints include total antibody (IgM, IgG) against the rAAV vector and transgene, complement activation, B cell and T cell functional assays against the vector and transgene, and phenotypic characterization, respectively. Measures of the efficiency of adeno-associated virus gene transfer include the blood vector genome copy number determined by PCR, and, but not limited to, the activity of the transgene in tissues including the heart, skeletal muscle, liver, and spleen.

[0083] Compared to placebo controls, the results achieved by administering CD32B×CD79B bispecific antibodies to rAAV recipient animals include attenuation of the response to anti-rAAV and transgene-specific antibodies, decreased complement activation, and reduced anti-rAAV-specific T cell activity. Vector genome copy number and transgene activity were increased with CD32B×CD79B bispecific antibody administration compared to placebo animals, supporting the hypothesis that CD32B×CD79B bispecific antibody administration reduces the immunogenicity of recombinant AAV.

[0084] [Example 2] <Decreased immunogenicity of recombinant adeno-associated virus (rAAV) in response to repeated medication> In certain experiments, to maintain pharmacological coverage, CD32B×CD79B bispecific antibodies may be administered to mice as monotherapy or in combination with other immunomodulators, such as sirolimus, before and at subsequent time points before administering an rAAV vector encoding a potentially therapeutic transgene. At specific time points (e.g., 45, 90, 135 days), mice may receive additional doses (may be multiple) of the same rAAV vector / transgene. Mice may be euthanized at specific time points (e.g., 90, 135, 180 days) and continue to receive pharmacologically relevant doses of CD32B×CD79B bispecific antibodies before evaluating immunological endpoints and the efficiency of adeno-associated virus gene transfer. Measured immunological endpoints include total antibody against the rAAV vector and transgene, complement activation, B-cell and T-cell function assays against the vector and transgene, and phenotypic characterization, respectively. Measures of the efficiency of adeno-associated virus gene transfer include the vector genome copy number determined by PCR, as well as, but are not limited to, the activity of the transgene in various tissues, including the heart, skeletal muscle, liver, and spleen.

[0085] Compared to placebo controls, the results achieved by administering CD32B×CD79B bispecific antibodies to rAAV recipient animals include attenuation of the response to anti-rAAV and transgene-specific antibodies, decreased complement activation, and reduced anti-rAAV-specific T cell activity. Vector genome copy number and transgene activity can be increased with CD32B×CD79B bispecific antibody administration compared to placebo animals. These effects can be observed after a single dose of the rAAV vector and after subsequent doses of the rAAV vector, supporting the hypothesis that administration of CD32B×CD79B bispecific antibodies may enable repeated administration and increased efficacy of immunogenic recombinant AAV.

[0086] [Example 3] <Decreased pre-existing immune response to AAV or rAAV prior to administration of recombinant adeno-associated virus> In certain experiments, pre-existing immunity to wild-type AAV or rAAV can be induced in mice by administering each AAV or rAAV of the same AAV serotype that encodes a potentially therapeutic transgene. Subsequently, at a specific point in time, e.g., day 15, the same mice can be administered a CD32B × CD79B bispecific antibody, either as monotherapy or in combination with other immunomodulators, e.g., sirolimus, for a specific period, e.g., 14 days before and thereafter at subsequent points in time to maintain pharmacological coverage. At specific point in time (e.g., days 45, 90, 135), some mice may receive additional doses (multiple doses) of the same rAAV vector / transgene. These mice are euthanized at specific time points (e.g., 90, 135, 180 days) and can continue to receive pharmacologically relevant doses of CD32B × CD79B bispecific antibody before evaluating immunological endpoints and the efficiency of adeno-associated virus gene transfer. Measured immunological endpoints include total antibodies against wild-type AAV and / or rAAV vectors and transgenes, complement activation, B-cell and T-cell function assays against AAV and / or vectors and transgenes, and phenotypic characterization. Measures of adeno-associated virus gene transfer efficiency include vector genome copy number by PCR, and, but not limited to, transgene activity in tissues including the heart, skeletal muscle, liver, and spleen.

[0087] Compared to placebo controls, the results achieved by administering CD32B×CD79B bispecific antibodies to pre-immunized animals with AAV and / or rAAV include attenuation of the response to pre-existing anti-AAV and / or rAAV and transgene-specific antibodies, decreased complement activation, and reduced anti-rAAV-specific T cell activity. Following subsequent administration of rAAV, further attenuation of the response to anti-rAAV and transgene-specific antibodies, decreased complement activation, and reduced anti-rAAV-specific T cell activity may be observed. Vector genome copy number and transgene activity can be increased with CD32B×CD79B bispecific antibodies compared to placebo animals. These effects can be observed after a single administration of rAAV vectors to previously immunized animals and after subsequent administrations of rAAV vectors to previously immunized animals, supporting the hypothesis that administration of CD32B×CD79B bispecific antibodies may enable the administration of immunogenic recombinant AAV in the presence of a pre-existing immune response to AAV or rAAV.

[0088] [Example 4] <Decreased immunogenicity with repeated administration of enzyme replacement therapy (ERT)> In certain experiments, to maintain pharmacological coverage, CD32B×CD79B bispecific antibodies may be administered as monotherapy or in combination with other immunomodulators, such as sirolimus, to mice with a specific enzyme deficiency (such as knockout mice disclosed in Front Immunol. 2019 Mar 13;10:416, incorporated herein by reference) before and at subsequent time points after enzyme replacement therapy. At specific time points (e.g., days 7, 14, 21, 28, etc.), mice may receive additional doses (multiple doses) of the same ERT. Mice may be euthanized at specific time points (e.g., days 14, 21, 28, 35, etc.) and may continue to receive pharmacologically relevant doses of CD32B×CD79B bispecific antibodies before evaluating immunological endpoints and the efficiency of enzyme replacement therapy. Immunological endpoints include 1) characterization of total antibodies (IgM, IgG), B cell function assays, and phenotypic characteristics against the enzyme, and 2) measures of enzyme transduction efficiency, including reversal of the physiological consequences of enzyme deficiency and biochemical analysis of enzyme and substrate activity throughout the experiment.

[0089] Compared to placebo controls, the results achieved by administering CD32B×CD79B bispecific antibodies to enzyme replacement recipient animals include a reduced response to anti-enzyme-specific antibodies, improved enzyme-dependent physiological outcomes, increased duration of enzyme activity, and reduced substrate accumulation observed with CD32B×CD79B bispecific antibody administration, supporting the hypothesis that CD32B×CD79B bispecific antibody administration may reduce immunogenicity to enzyme replacement therapy, enabling repeated administration and increased efficacy of enzyme replacement therapy.

[0090] [Example 5] <Reduced immunogenicity to repeated administration of antibody and fusion protein-based therapies> In certain experiments, to maintain pharmacological coverage, CD32B×CD79B bispecific antibodies may be administered to mice as monotherapy or in combination with other immunomodulators, such as sirolimus, before and at subsequent time points, similar to human antibody and fusion protein-based therapies. At specific time points (e.g., days 7, 14, 21, 28, etc.), mice may receive additional doses (may be multiple) of the same antibody or fusion protein. Mice may be euthanized at specific time points (e.g., days 14, 21, 28, 35, etc.) and may continue to receive pharmacologically relevant doses of CD32B×CD79B bispecific antibodies before immunological endpoints and evaluation of antibody or fusion protein activity. Immunological endpoints include 1) characterization of total antibodies (IgM, IgG), B cell function assays, and phenotypic characteristics against the enzyme, and 2) measures of antibody or fusion protein efficiency, including pharmacokinetic, immunological, and / or pharmacodynamic analyses of the antibody or fusion protein activity throughout the experiment, such as the ability of the antibody or fusion protein to inhibit its target protein.

[0091] Compared to placebo control, the results achieved by administering CD32B×CD79B bispecific antibodies to antibody or fusion protein recipient animals include attenuation of the anti-antibody or fusion protein antibody response, reduced clearance, and half-life (t). 1 / 2 An increase in ) can be cited. Compared to placebo animals, improved and extended pharmacodynamic measures of efficacy can also be observed, supporting the hypothesis that administration of CD32B×CD79B bispecific antibodies may enable repeated administration and increased efficacy of immunogenic antibodies or fusion proteins.

[0092] [Example 6] <Phase 1b, double-blind, placebo-controlled, multi-dose-escalation study to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of PRV-3279 in healthy subjects> This study evaluated the safety, tolerability, and immunogenicity of multiple doses of PRV-3279 in healthy subjects at dose levels expected to provide sustained, high levels of receptor coverage. To avoid background drug characteristics that may interfere with the development of ADA, and signs and symptoms that may interfere with the assessment of tolerability, this study selected healthy subjects, thus allowing for a more thorough and safer examination of the immunogenicity and tolerability of repeated dosing of PRV-3279.

[0093] Sequential enrollment was planned for two cohorts. Cohort A was evaluated with PRV-3279 3 mg / kg every two weeks for a total of three doses. Cohort B was evaluated with PRV-3279 10 mg / kg every two weeks for three doses. Each cohort consisted of eight subjects randomly assigned in a 3:1 ratio to either PRV-3279 or placebo (i.e., n=6 for PRV-3279 and n=2 for placebo). The three doses of the study drug (PRV-3279 or placebo) were administered as a 2-hour IV infusion on days 1, 15, and 29 in each cohort.

[0094] Participants underwent screening assessments to determine eligibility within 28 days prior to randomization on day 1 and administration of the first dose. On day 1, participants were admitted to a clinical research unit (CRU) and underwent a baseline study to confirm their eligibility. On day 1, each participant was randomly assigned to receive either PRV-3279 or placebo for 2 hours via IV infusion in a double-blind manner and monitored for 4 hours post-administration. On day 2, participants underwent clinical laboratory safety assessments, pharmacokinetic (PK) assessments, and adverse event (AE) assessments and were discharged from the CRU. Participants returned to the CRU to receive the second (day 15) and third (day 29) doses of their assigned treatment. Similar to the first dose, participants were admitted to the CRU the day before administration and discharged the day after administration.

[0095] Each cohort included two sentinel subjects, one receiving PRV-3279 and the other a placebo in a double-blind manner. Sentinel subjects were evaluated for adverse events (e.g., fluid responses, delayed hypersensitivity) for at least 7 days from the start of the first infusion, before the rest of the cohort received their first infusion. The use of sentinel subjects and staggered dosing schedules ensures that any potential and high-frequency responses (e.g., ADA-related fluid responses) are identified before repeated dosing in the entire cohort.

[0096] Safety assessments included reported adverse events (AEs), including hypersensitivity or infusion reactions, vital signs measurements, physical examination, ECG, and laboratory tests. Physical examinations were performed to establish baseline and identify physical signs associated with AEs. Adverse events were collected at each visit and assessed for severity and association with the study drug. On days 1, 15, and 29, vital signs (temperature, pulse rate, blood pressure, and respiratory rate) were recorded immediately at time 0 (before dose, up to 5 minutes before infusion), 0.5 hours, 1 hour (midpoint of infusion), 2 hours (end of infusion), and 6 hours after the start of infusion (4 hours after the end of infusion). The start of IV infusion was designated as time "0". Vital signs were obtained within ±5 minutes of the scheduled time. Height was recorded only at the screening visit. Weight was obtained on days -1, 14, and 28.

[0097] Serum samples for PK, immunogenicity, and PD were obtained at the selected time. A schematic diagram of the study design is provided in Figure 1.

[0098] <Summary of Adverse Events> There were no AESIs, serious TEAEs, SAEs, or TEAEs resulting in death during the study. Four mild but recurrent TEAEs led to one patient (16.7%) withdrawing from PRV-3279 10 mg / kg. No other TEAEs resulted in patient withdrawal from the study. (Table error! Text of specified style is missing in the document.)

[0099] Overall, 34 TEAEs were reported by 9 subjects (56.3%). 18 TEAEs were reported in 5 subjects (83.3%) receiving PRV-3279 10 mg / kg, 12 TEAEs were reported in 3 subjects (50.0%) receiving PRV-3279 3 mg / kg, and 4 TEAEs were reported in 1 subject (25.0%) receiving placebo (Table error! Text of specified style is missing in the document.). 12 TEAEs in 4 subjects (66.7%) receiving PRV-3279 10 mg / kg and 4 TEAEs in 1 subject (16.7%) receiving PRV-3279 3 mg / kg were considered by investigators to be related to the study drug, while all other reported TEAEs were considered unrelated (Table error! Text of specified style is missing in the document.).

[0100] [Table 1]

[0101] During the study, two adverse events (AEs) that were deemed non-TEAEs by the investigators were reported in two subjects (9.2%). Both were considered unrelated to the study drug (Table 2).

[0102] [Table 2]

[0103] The table presents an overview of TEAEs by procedure and overall, and by SOC and PT. The table also presents an overview of TEAEs by SOC and PT, by procedure, and by severity, and an overview of related TEAEs. Finally, the table presents an overview of TEAEs that caused interruptions, by SOC and PT, by procedure, and overall.

[0104] [Table 3-1] [Table 3-2]

[0105] [Table 4-1] [Table 4-2] [Table 4-3]

[0106] [Table 5]

[0107] [Table 6]

[0108] <Pharmacokinetic concentration data> PRV-3279 is quantitatively measured from human serum using ECL. In this assay, an uncoated MSD Multi-Array® standard-conjugated plate is coated with rabbit anti-h8B5 antibody as a capture reagent for PRV-3279. A sample containing PRV-3279 is incubated on the coated plate. The conjugated PRV-3279 is detected with biotin-labeled 2A5 antibody. A streptavidin sulfo-tag conjugate is added and conjugated to the primary detection antibody. Tripropylamine (TPA, MSD Gold Read Buffer) is added to the plate, and an electrochemiluminescence signal is generated when a charge is applied, which is detected with an MSD SECTOR S 600 plate reader.

[0109] The arithmetic mean (±SD) PRV-3279 serum concentration-time data are shown in Figures 2A-2C. BLQ = less than the limit of quantification, LLOQ = lower limit of quantification, SD = standard deviation. Error bars: SD. Values ​​that were BLQ before dose and values ​​that were in the absorption phase before the first quantifiable concentration were replaced with zero. Thereafter, BLQ values ​​between evaluable concentrations were replaced with LLOQ / 2. LLOQ = 1.5 ng / mL

[0110] The arithmetic mean PRV-3279 serum concentration-time data are shown in Figures 3A-3C. BLQ = less than the limit of quantification, LLOQ = lower limit of quantification, SD = standard deviation. The BLQ value before dose and the value in the absorption phase before the first quantifiable concentration were replaced with zero. Thereafter, BLQ values ​​between evaluable concentrations were replaced with LLOQ / 2. LLOQ = 1.5 ng / mL.

[0111] Following 2-hour infusions of 3 mg / kg and 10 mg / kg of PRV-3279, mean peak concentrations occurred at the end of the infusion (2 hours) for days 1, 15, and 29. Mean concentrations remained above the lower limit of quantification (LLOQ, 1.5 ng / mL) up to 1344 hours after administration on day 29 for both dose levels. Mean pre-dose concentrations on days 15, 29, and 43 were 6145 ng / mL, 7590 ng / mL, and 12440 ng / mL for the 3 mg / kg dose, and 48383 ng / mL, 60460 ng / mL, and 77140 ng / mL for the 10 mg / kg dose, respectively. Pre-dose concentrations continued to increase on these days, and steady state was not achieved on either day 15 or day 29, as fewer than 5 half-lives had elapsed.

[0112] Arithmetic mean PRV-3279 concentration-time data are shown in Figures 4A-4B for each treatment and ADA result. ADA results in this plot are defined based on the immunogenicity of the sample at each specific time point. ADA = anti-drug antibody, BLQ = <limit of quantification, LLOQ = lower limit of quantification, SD = standard deviation. Values ​​that were BLQ before dose and values ​​that were in the absorption phase before the first quantifiable concentration were replaced with zero. Thereafter, BLQ values ​​between evaluable concentrations were replaced with LLOQ / 2. LLOQ = 1.5 ng / mL. At 3 mg / kg, the daily ADA negative / positive results were: days 1 and 8 = 6 / 0 (N=6), days 15, 22, and 29 = 5 / 1 (N=6), day 36 = 4 / 2 (N=6), day 43 = 3 / 3 (N=6), day 57 = 2 / 4 (N=6), day 71 = 1 / 5 (N=6), and day 85 = 0 / 6 (N=6). At 10 mg / kg, the daily ADA negative / positive results were: Days 1, 8, 15, and 22 = 6 / 0 (N=6), Day 29 = 5 / 0 (N=5), Day 36 = 5 / 0 (N=5), Day 43 = 5 / 0 (N=5), Day 57 = 5 / 0 (N=5), Day 71 = 3 / 2 (N=5), and Day 85 = 2 / 3 (N=5).

[0113] <Evaluation of immunogenicity data> Anti-PRV-3279 antibodies in human serum are detected and confirmed in human serum using a multi-step method in the MSD-ECL assay. In this assay, the sample, positive control (PC), and negative control (NC) are subjected to a minimum required dilution (MRD) of 1:10 in 300 mM acetic acid. The acidified samples are then neutralized and pre-incubated overnight with biotin-PRV-3279 coated on a NeutrAvidin high-volume plate. All anti-drug antibodies (ADAs) present in human serum bind to biotin-PRV-3279. After overnight incubation, the biotin-PRV-3279:ADA complex is subjected to a second acid treatment to break the complex. Subsequently, the acidified ADA sample is coated on a bare MSD high-binding plate. After blocking, the ADA sample is detected by the chemiluminescent signal generated when a potential is applied using sulfotag-PRV-3279. The resulting electrochemiluminescence (ECL) signal or relative luminescence (RLU) is directly proportional to the amount of ADA present in human serum.

[0114] Overall, ADA increased over time. Only 4 out of 6 subjects had ADA at day 85. Compared to subjects receiving a 10 mg / kg dose, subjects receiving a 3 mg / kg dose developed ADA earlier (day 15 vs. day 36), and all subjects developed ADA by day 85. Table 7 shows the titers of ADA against PRV-3279 over time from baseline, ranging from <10 to 270 and <10 to 2430. This indicates that PRV-3279 inhibits its own immunogenicity.

[0115] [Table 7]

[0116] <Evaluation of pharmacokinetic / immunogenicity data> Pharmacokinetic parameters of PRV-3279 (C 最大 and AUC 0~336 Table 8 provides a descriptive summary of the ADA results, treatments, and daily outcomes. PRV-3279 C最大 and AUC 0~336 A box plot of the parameter is shown in Figure 5, which indicates that ADA does not affect PK. ADA = anti-drug antibody, N = number of subjects in the pharmacokinetic analysis population within the corresponding ADA classification. The symbol inside the box represents the mean. The top (bottom) edge of the box represents the 75th (25th) percentile. Whiskers are drawn from the top (bottom) edge of the box to the maximum (minimum) value within 1.5 × interquartile range above (below) the edge of the box. Values ​​outside the whiskers are identified by symbols.

[0117] [Table 8-1] [Table 8-2]

[0118] <Evaluation of pharmacodynamic data> The maximum binding of PRV-3279 to B cells (CD19+), memory B cells (CD19+ / CD27+), and naive B cells (CD19+ / CD27-) was examined by staining with anti-PRV-3279 (anti-EK), including the maximum binding obtained in a PRV-3279 saturated sample, to determine PRV-3279 binding (percent bound B cells) and absolute and percentage receptor occupancy (MESF). For the calculation of % bound B cells and % receptor occupancy, the maximum binding of PRV-3279 to B cells in each individual sample was calculated by comparing the values ​​of % bound cells and the equivalent absolute receptor occupancy molecules of the soluble fluorescent dye (MESF) at each time point with the corresponding values ​​for the PRV-3279 saturated sample (total).

[0119] As shown in Figure 6, after doses of 3 and 10 mg / kg of PRV-3279, >85% of all available B cells (CD19+) were bound to the drug one day after administration in both dose groups. In both dose groups, binding decreased slightly to about 80% before the second dose and increased again to about 90% after the second dose. In the 10 mg / kg dose group, % bound B cells were maintained at this high level until approximately day 57, after which they dropped to less than 50% on day 85 (see Figure 6). At the 3 mg / kg dose, % bound B cells were comparable to the higher dose for the first 22 days and maintained around 70% until day 43, although binding was generally more volatile between doses at this dose level. Percent bound B cells were <20% on day 85, which was within the same range as placebo. The variability of the data was low to moderate.

[0120] Next, the percentages and absolute numbers of lymphocytes, monocytes (CD14+), T cells (CD3+), T helper cells (CD3+ / CD4+), cytotoxic T cells (CD3+ / CD8+), natural killer cells (CD3- / CD16+), natural killer T cells (CD3+ / CD16+ / CD56+), and B cells (CD19+) were investigated. The time course of the absolute number of B cells for each treatment is shown in Figure 7. Other cell types showed a similar pattern (data not shown). The number of B cells decreased by an average of -39% and -47% within 1 day after administration of 3 and 10 mg / kg of PRV-3279, respectively, but returned to baseline levels after 1 week. No comparable decrease was observed in subjects treated with placebo. The decline in cell numbers was slightly less pronounced after the second and third doses of PRV-3279. After the third dose, cell counts remained lower at 10 mg / kg than at 3 mg / kg, but on day 85, both counts were similar and again comparable to baseline.

[0121] In summary, the study demonstrated an initial transient decrease in peripheral B cell count, which became less pronounced after the second and third doses of PRV-3279 and recovered rapidly after each dose. Persistent B cell depletion did not occur in this study. Furthermore, none of the other immune cell types investigated showed clinically relevant depletion.

[0122] Next, circulating levels of immunoglobulin M (IgM), IgE, and IgG were measured using known methods. As shown in Figure 8, immunoglobulin M levels after administration of 3 and 10 mg / kg of PRV-3279 steadily decreased until approximately day 36 and remained at that level until day 85. However, the decrease was not clearly dose-dependent, and there was a tendency for less apparent decrease at 3 mg / kg than at 10 mg / kg at days 36 and 85. Immunoglobulin E levels after administration of 10 mg / kg of PRV-3279 were similar to those observed in placebo throughout the study, except at the final point on day 85, when the mean % change from baseline was -28.2% at 10 mg / kg compared to -5.0% at placebo (see Figure 9). Immunoglobulin G levels after administration of 3 and 10 mg / kg of PRV-3279 were highly variable and generally appeared indistinguishable from placebo throughout the study. The percentage change in IgG levels from baseline was mostly within ±5% for all treatments (see Figure 10).

[0123] <Conclusion> The primary objective of this Phase 1b, double-blind, placebo-controlled MAD study was to evaluate the safety and tolerability of multiple (3) IV infusions of two dose levels (3 and 10 mg / kg) of PRV-3279 in healthy subjects. A secondary objective was to characterize the multiple-dose PK and immunogenicity of PRV-3279. A preliminary objective was to explore the effects of PRV-3279 on potential biomarkers related to target binding and B cell function.

[0124] A total of 16 participants were enrolled, randomized, and administered medication. Two cohorts received either PRV-3279 or placebo, administered a total of three doses every two weeks. The three doses of the study drug (PRV-3279 3 mg / kg and 10 mg / kg or placebo) were administered intravenously to each cohort on days 1, 15, and 29. Fourteen participants completed the study, receiving all planned procedures as per the protocol. One placebo participant withdrew consent after receiving placebo doses on days 1 and 15, and one PRV-3279 10 mg / kg participant withdrew due to an adverse event after receiving the PRV-3279 10 mg / kg dose for 3 minutes on day 29.

[0125] All 16 subjects (100.0%) were included in the safety, PD, and immunogenicity populations. All 12 subjects (75.5%) who received the study drug were included in the PK population; however, data from day 29 onward were excluded for one subject who withdrew due to adverse events (AE) from PRV-3279 10 mg / kg in all PK summary plots and summary statistics.

[0126] This Phase 1b study builds upon the tolerability and PD information obtained in the first-in-human study and addresses the feasibility of readministration of PRV-3279. The results of the study confirm PRV-3279's ability to functionally suppress B cell function without depleting it, in a deep and sustained manner unaffected by ADA.

[0127] PRV-3279 was well tolerated and there were no SAEs. The PK characteristics support bi-weekly or, in some cases, lower frequency dosing. Anti-drug antibodies were lower in the higher dose group, which is consistent with PRV-3279's ability to inhibit its own immunogenicity.

[0128] The receptor occupancy PD effect of PRV-3279 persists well beyond drug discontinuation, is more sustained at a 10 mg / kg dose, and >50% binding was observed at least 28 days after the final dose, which is considered the minimum level of binding required for optimal B cell regulation.

[0129] A clear and sustained decrease in IgM levels was observed throughout the follow-up period, suggesting an extended PD effect. Importantly, and as expected, there was no B-cell depletion, and no observable adverse effects on immune cells or cytokines.

[0130] In conclusion, based on its excellent safety profile and superior PD effect at 10 mg / kg with lower immunogenicity, doses of 10 mg / kg or higher can be used to reduce the immunogenicity of biotherapy agents, including gene therapy products.

[0131] [Modified] Modifications and alterations of the methods and compositions described herein will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure is described in relation to specific embodiments, it will be understood that the claimed disclosure should not be unduly limited to such specific embodiments. In fact, various modifications of the embodiments for carrying out the described invention are intended and will be understood to be within the scope of the disclosure as expressed by the following claims.

[0132] [Integration by reference] All patents and publications referenced herein are incorporated herein by reference to the same extent as each individual patent and publication is specifically and individually indicated as being incorporated by reference.

Claims

A pharmaceutical composition for reducing immunogenicity in a patient who has received or is receiving gene therapy, wherein the immunogenicity is caused by the gene therapy agent, the gene therapy agent comprises a recombinant viral vector, the pharmaceutical composition comprises an effective amount of a non-depleting B cell inhibitor, 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, the CD32B×CD79B bispecific antibody is (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 and is an Fc diabody, wherein a first dose of the pharmaceutical composition is administered prior to the administration of the gene therapy agent and a second dose, and a third dose is administered after the administration of the second dose and the gene therapy agent. **Claim 2** The first dose of the pharmaceutical composition comprises an Fc diabody at a dose of about 5 mg / kg to about 40 mg / kg, wherein the first dose is administered 2 to 6 weeks prior to the administration of the second dose and the gene therapy agent, the dose of the Fc diabody in the second dose of the pharmaceutical composition is about 5 mg / kg to about 40 mg / kg, the dose of the Fc diabody in the third dose of the pharmaceutical composition is about 5 mg / kg to about 40 mg / kg, and the third dose is administered 2 to 6 weeks after the administration of the second dose and the gene therapy agent. The pharmaceutical composition according to claim 1. **Claim 3** The first dose of the pharmaceutical composition comprises an Fc diabody at a dose of about 10 mg / kg, wherein the first dose is administered 4 weeks prior to the administration of the second dose and the gene therapy agent, the dose of the Fc diabody in the second dose of the pharmaceutical composition is about 10 mg / kg, the dose of the Fc diabody in the third dose of the pharmaceutical composition is about 10 mg / kg, and the third dose is administered 4 weeks after the administration of the second dose and the gene therapy agent. The pharmaceutical composition according to claim 1. **Claim 4** The first dose of the pharmaceutical composition comprises an Fc diabody at a dose of about 10 mg / kg, wherein the first dose is administered 2 to 6 weeks prior to the administration of the second dose and the gene therapy agent, the dose of the Fc diabody in the second dose of the pharmaceutical composition is about 10 mg / kg, The dosage of the Fc diabody in the pharmaceutical composition of the third dosage is about 10 mg / kg, and the third dosage is administered 2 to 6 weeks after the administration of the second dosage and the gene therapeutic agent. The pharmaceutical composition according to claim 1.

5. The pharmaceutical composition according to claim 4, wherein the second dosage is administered substantially simultaneously with the administration of the gene therapeutic agent.

6. The pharmaceutical composition according to claim 1, wherein the Fc diabody brings about inhibition of its own immunogenicity upon administration and has a lower morbidity rate and / or titer of anti-drug antibodies (ADA) at increased dosages.

7. The pharmaceutical composition according to claim 6, wherein the ADA does not neutralize the Fc diabody.

8. The pharmaceutical composition according to claim 1, wherein the Fc diabody binds to at least 80% of B cells upon administration in a dosage-dependent manner and remains bound to at least 50% of the B cells for at least 4 weeks from the last administration.

9. The pharmaceutical composition according to claim 1, wherein the Fc diabody brings about a sustained inhibition of immunoglobulin production without depleting circulating B cells.

10. The pharmaceutical composition according to claim 9, wherein the immunoglobulin includes IgM, IgA, IgG, and IgE.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein one or more dosages are further administered to regulate the immunogenicity of specific antibodies detected against the gene therapeutic agent.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein one or more immunomodulatory agents are administered simultaneously.

13. The pharmaceutical composition according to claim 12, wherein the one or more immunomodulatory agents are selected from sirolimus, rapamycin, abatacept, teprotumumab, and the immunoglobulin G-degrading enzyme of Streptococcus pyogenes.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the gene therapeutic agent includes a recombinant adeno-associated virus vector.

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