CD40 antagonist or CD154 antagonist for use in mitigating immune responses in protein and gene therapies

Abstract

Featured are the use of pharmaceutical compositions containing a CD40 antagonist or a CD154 antagonist for the treatment, reduction, and / or inhibition of an immune response, such as an anti-drug antibody response, following, e.g., protein replacement therapy or gene therapy.

Description

[0001] CD40 ANTAGONIST OR CD154 ANTAGONIST FOR USE IN MITIGATING IMMUNE RESPONSES IN PROTEIN AND GENE THERAPIES

[0002] Sequence Listing

[0003] This application contains a Sequence Listing which has been filed electronically in Extensible Markup Language (XML) format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 6, 2025, is named 009WO2_Sequence_Listing_2_6_25.XML and is 20,979 bytes in size.

[0004] Background

[0005] Protein replacement therapy (PRT) and gene therapy are common therapeutic methods in which a functional protein (e.g., an enzyme) is provided for the treatment of a disease associated with a defective or insufficient amount of an endogenous protein. PRT methods include administration of the protein, such as an enzyme in enzyme replacement therapy (ERT), e.g., as a purified therapeutic protein or as a polynucleotide (e.g., an mRNA), or in a method of gene therapy, such as by administration of a delivery vehicle containing a polynucleotide encoding the protein (e.g., a cell, virus, or virus particle that includes a transgene). Gene therapy methods also provide for the delivery to a patient specific genetic material to modify the encoding of a gene product or to change the biological properties of tissues for the management of various disorders. Gene therapies act by different mechanisms including, replacing malfunctioning genes with the therapeutic genes, gene knockdown, or deactivating deleterious genes, and inserting a new gene (e.g., for PRT) to treat a disease or disorder. Subjects receiving PRT or gene therapy often develop an immune response that reduces the efficacy of the PRT or gene therapy. One such response is a T cell dependent antibody response (TDAR), where a subject may develop neutralizing antibodies (NAbs) to the protein (e.g., for PRT) or delivery vehicle (e.g., a cell, virus, or virus like particle), that either directly bind to their target (e.g., the protein or the delivery vehicle) and reduce its efficacy or leads to a downstream mechanism that results in increased clearance of the therapeutic agent or the vehicle delivering a therapeutic agent. This can include the development of anti-drug antibodies (ADA), which bind to and neutralize the protein (e.g., an enzyme in ERT) or the delivery vehicle. Another such response is T-cell-mediated cytotoxicity, which can result in T-cells lysing cells that have been transduced with viral delivery vehicle, which can reduce the efficiency of the virus to successfully transduce target cells with a transgene and express the therapeutic product of the transgene(s) to treat the subject.

[0006] Accordingly, methods are needed to reduce or suppress an immune response to improve PRT and gene therapy for the treatment of a wide variety of diseases and disorders.

[0007] Summary

[0008] A first aspect features a method of treating, reducing, or inhibiting a CD40-mediated immune activity (e.g., T cell-dependent antibody response or T cell-mediated cytotoxicity) in a human subject receiving or having received a therapeutic agent (e.g., a protein as part of a protein replacement therapy (PRT) or polynucleotide encoding a protein as part of a PRT or other gene therapy; or a delivery vehicle (e.g., a viral vectors, such as an AAV) containing a polynucleotide) by administering to the subject a pharmaceutical composition that includes a CD40 antagonist or a CD154 antagonist. In some embodiments, the pharmaceutical composition comprises the CD40 antagonist. In some embodiments, the CD40 antagonist is an anti-CD40 antibody or antigen binding fragment thereof. In some embodiments, the anti-CD40 antibody is iscalimab, bleselumab, BMS-986325, ravagalimab, lucatumumab or KPL-404, or, in each case, an antigen-binding fragment thereof. In some embodiments, the CD40 antagonist is a humanized anti-CD40 antibody or antigen-binding fragment thereof that includes a heavy chain variable region having an amino acid sequence with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region having an amino acid sequence with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the humanized anti-CD40 antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region with an amino acid sequence set forth in SEQ ID NO: 10 (e.g., the antibody is KPL-404). In some embodiments, the CD154 antagonist is an anti- CD154 antibody or antigen binding fragment thereof. In some embodiments, the anti-CD154 antibody is frexalimab, letolizumab, tegoprubart, toralizumab, dapirolizumab pegol, Lu AG22515, IBI-355, or ruplizumab, or, in each case, an antigen-binding fragment thereof. In one embodiment the CD154 antagonist is dazodalibep, or a CD154 binding fragment thereof. The protein replacement therapy may involve administration of the protein (e.g., an enzyme) as the therapeutic agent (e.g., as enzyme replacement therapy (ERT)) or administration of a polynucleotide encoding the protein as the therapeutic agent (e.g., as gene therapy). Gene therapy may involve administration of the polynucleotide encoding the protein (e.g., as mRNA or a similar mode of therapy) or administration of a vector (e.g., a viral vector) containing the polynucleotide. Gene therapy may also include delivery of a cell, virus, or virus like particle that includes a polynucleotide encoding a therapeutic protein or therapeutic nucleic acid. Gene therapy may include delivery of a cell, virus, or virus like particle that includes one or more polynucleotides for use in replacing a malfunctioning gene with a functional gene, modify the functionality or expression of an endogenous gene (e.g., via gene editing or gene silencing), insert a therapeutic gene, or to infect and lyse a target cell associated with a disease or disorder (e.g., oncolytic virotherapy). The vector may be used to deliver the polynucleotide (e.g., as a transgene (as DNA encoding the therapeutic agent)). The polynucleotide may encode a therapeutic protein of interest (e.g., an enzyme or other therapeutic protein) that is missing, defective, or deficient in the subject. The polynucleotide may encode one or more proteins and / or nucleic acids that edit a gene in the subject that is missing, defective or deficient in the subject.

[0009] In some embodiments, the therapeutic agent is one of the proteins set forth in Table 1 . In some embodiments, the subject has one of the diseases set forth in Table 1 .

[0010] In some embodiments, the pharmaceutical composition comprising a CD40 antagonist or a CD154 antagonist is administered at least once per day, every two weeks, every three weeks, per month, per quarter, per six months, or per year. In some embodiments, the pharmaceutical composition containing the CD40 antagonist or the CD154 antagonist is administered at the same dosing frequency as the therapeutic agent (e.g., 5 days, 4 days, 3 days, 2 days, or 1 day prior to administration of the therapeutic agent, or on the same day as the therapeutic agent). In some embodiments, the frequency of administration of a pharmaceutical composition containing the CD40 antagonist or the CD154 antagonist, or the dose administered, may be varied over time depending upon whether the subject exhibits a CD40-mediated immune activity (e.g., development of neutralizing antibodies against the therapeutic agent or a delivery vehicle (e.g., a viral vector) used to deliver the therapeutic agent). For example, the subject may be administered an initial dose of the pharmaceutical composition containing the CD40 antagonist or the CD154 antagonist once every month for six months while the subject is administered, or (in the case of gene therapy) expressing, the therapeutic agent. After six months, the frequency of administration of a pharmaceutical composition containing the CD40 antagonist or the CD154 antagonist may be reduced to once every other month (or less frequently or as needed) in order to reduce or inhibit development of a CD40- mediated immune activity.

[0011] In some embodiments, the pharmaceutical composition is administered as a prophylactic therapy (i.e., prior to development of a CD40-mediated immune activity against a therapeutic agent), as an acute therapy (e.g., upon detection of a CD40-mediated immune activity against a therapeutic agent (such as the development of an ADA against the therapeutic agent)), or as a chronic therapy (e.g., for the life of the subject, either before or after development of a CD40-mediated immune activity against a therapeutic agent).

[0012] In some embodiments, the pharmaceutical composition is administered prior to administration of a therapeutic agent (either as PRT or gene therapy). For example, the pharmaceutical composition may be administered at least one hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, or 1 month or more prior to administration of the therapeutic agent (e.g., PRT or gene therapy). The pharmaceutical composition may, for example, be administered prior to each administration of the therapeutic agent (e.g., PRT or gene therapy).

[0013] In some embodiments, the pharmaceutical composition is administered following administration of a therapeutic agent (e.g., as PRT or gene therapy). For example, the pharmaceutical composition may be administered at least one hour, 6 hours, 12 hours, 1 day, 1 week, or 1 month or more following administration of the therapeutic agent (e.g., as PRT or gene therapy). The pharmaceutical composition may, for example, be administered following each administration of the therapeutic agent (e.g., as PRT or gene therapy).

[0014] In some embodiments, the pharmaceutical composition may be administered prior to and following each administration of the therapeutic agent (e.g., as PRT or gene therapy).

[0015] In some embodiments, the pharmaceutical composition is administered substantially at the same time as the PRT or gene therapy (e.g., concurrently with administration of the PRT or gene therapy).

[0016] In some embodiments, the pharmaceutical composition is administered to the subject when or as needed (e.g., by detecting the presence of a CD-40 mediated immune response in the subject). For example, the pharmaceutical composition is administered to the subject following detection in a blood sample from the subject of a CD40-mediated immune response, such as a neutralizing antibody or an anti-drug antibody (ADA) that specifically binds to a therapeutic agent (e.g., a protein therapeutic agent (e.g., an enzyme), a polynucleotide therapeutic agent, a delivery vehicle that is used to deliver the therapeutic agent (e.g., a viral vector containing a transgene encoding a protein therapeutic agent (e.g., an enzyme)) or a cell containing, or transduced by, a delivery vehicle (e.g., a viral vector encoding the therapeutic agent). The assay may determine that a neutralization or ADA response includes the activation of plasma cells that produce the neutralizing antibody or ADA. In this case, the CD40 antagonist or the CD154 antagonist may be administered to the subject to attenuate the plasma cell-mediated neutralizing antibody or ADA response.

[0017] In some embodiments, the pharmaceutical composition is administered to the subject following detection of a reduction in a titer of a delivery vehicle (e.g., a viral vector), after administration of the delivery vehicle, e.g., during gene therapy, such as due to neutralization of the delivery vehicle as a result of a CD40-mediated immune activity. In other embodiments, the pharmaceutical composition is administered to the subject following detection of a reduction in levels of a transgene or therapeutic product encoded by a transgene, after administration of the delivery vehicle. For example, the expression of a transgene of a delivery vehicle may be reduced overtime from 30 days or more to less than 30 days once the subject receives one or more doses of the delivery vehicle. The reduction in transgene expression may be the result of (i) CD40-mediated immune activity resulting in a neutralizing antibody that clears the delivery vehicle from the subject, prior to delivering the transgene to the target tissue, (ii) CD40 and CD154 mediated T-cell cytotoxicity directed against a cell transduced by a delivery vehicle, (iii) a neutralizing antibody that clears the transgene expression product due to an increase overtime of a CD40-mediated immune activity or (iv) in the case of gene therapy based on episomal expression, dilution effects as cells divide and tissues grow. Accordingly, the CD40 antagonist or the CD154 antagonist may be administered to the subject to attenuate the CD40-mediated immune activity, e.g., by (a) reducing the development of neutralizing antibodies against the delivery vehicle or the transgene expression product; (b) reducing T-cell cytotoxicity against cells containing the delivery vehicle (e.g., a viral vector) or the transgene expression product that is a neoantigen.

[0018] In some embodiments, the pharmaceutical composition is administered to the subject following detection of a reduction in levels of a therapeutic product expressed from a transgene in the subject, after administration of the delivery vehicle (e.g., a viral vector) that includes the transgene. For example, during gene therapy, a reduction in levels of a therapeutic product expressed from a transgene in a subject may occur due to neutralization of the therapeutic product as a result of a CD40-mediated immune activity. The reduction in transgene expression may be the result of antibodies (e.g., neutralizing antibodies) that clear the transgene expression product more quickly as a result of an increase of a CD40-mediated immune activity overtime. In this case, the CD40 antagonist or the CD154 antagonist may be administered to the subject to attenuate the CD40- mediated immune activity, e.g., by reducing the development of antibodies (e.g., neutralizing antibodies) against the delivery vehicle. In addition, the reduction in transgene expression may be the result of a loss of transgene expression, a loss of vector retention in the subject’s tissue transduced with the delivery vehicle (e.g., a viral vector), or, in the case of gene therapy based on episomal expression, dilution effects as cells divide and tissues grow. For example, the expression level of a transgene of a delivery vehicle may be reduced after 1 , 2, 3, 4 or more years after the subject is administered the initial dose of the delivery vehicle, requiring the subject to receive one or more administrations of the delivery vehicle containing the transgene. In this case, the CD40 antagonist or the CD154 antagonist may be administered to the subject to attenuate the CD40-mediated immune activity, e.g., by reducing the development of neutralizing antibodies against the virus. In one embodiment, the subject is administered the CD40 antagonist or the CD154 antagonist, prior to, during or following administration of the initial dose of the delivery vehicle to attenuate CD40- mediated immune activity (e.g., TDAR or T-cell cytotoxicity) in response to the initial dose. This is desirable, as any subsequent dose of the delivery vehicle containing a transgene would not trigger a more potent (and potentially harmful) secondary immune response against, e.g., the virus containing the delivery vehicle with the transgene. In some embodiments, the subject is administered the CD40 antagonist or the CD154 antagonist, prior to or during any subsequent dose of the delivery vehicle to attenuate CD40-mediated immune activity (e.g., TDAR or T-cell cytotoxicity) in response to the subsequent dose.

[0019] In one aspect, the subject is administered (i) a first delivery vehicle (e.g., a viral vector) including a nucleic acid capable of integrating a transgene into the genome of the subject by homologous recombination for expressing a therapeutic protein in the subject and (ii) a second delivery vehicle (e.g., a viral vector) including a nucleic acid for episomal expression of a therapeutic protein in the subject. In certain embodiments, the therapeutic protein expressed from the transgene integrated into the genome and therapeutic protein expressed from the episome are identical. In one embodiment, the subject is administered one or more doses of the first delivery vehicle. For example, in some embodiments, the method includes administering to the subject 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of the first delivery vehicle. In another embodiment, the subject is administered one or more doses of the second delivery vehicle. For example, in some embodiments, the method includes administering to the subject 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of the second delivery vehicle. In a certain embodiment, the subject is administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of the first delivery vehicle and a single dose of the second delivery vehicle. In other embodiment, the first delivery vehicle and second delivery vehicle are administered to the subject at the same time, or within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 1 month of each other. In one embodiment, the subject is administered theCD40 antagonist or the CD154 antagonist, prior to, during or following administration each dose of the first or second delivery vehicle to attenuate CD40-mediated immune activity (e.g., TDAR or T-cell cytotoxicity) in response to administration of the first or second delivery vehicle.

[0020] In some embodiments, the subject is naive (seronegative) to the therapeutic agent or a delivery vehicle (e.g., a viral vector) used to deliver the therapeutic agent. In other embodiments, the subject has previously been exposed to the therapeutic agent or the same type of virus from which the delivery vehicle is derived (e.g., the seropositive subject may have been previously exposed to an adeno-associated virus (AAV) and may already have antisera that would recognize an AAV viral vector used as the vehicle to deliver a therapeutic agent). The pharmaceutical composition described herein can be used to treat, reduce, or inhibit a CD40-mediated immune response in a subject by administering the pharmaceutical composition: 1) prior to, with, or after administration of the therapeutic agent or the delivery vehicle containing a polynucleotide encoding the therapeutic agent in a subject seropositive for the therapeutic agent or delivery vehicle, or 2) prior to, with, or after administration of the therapeutic agent or the delivery vehicle containing a polynucleotide encoding the therapeutic agent in a subject in a subject seronegative for the therapeutic agent or delivery vehicle (e.g., the subject is naive to the therapeutic agent or the delivery vehicle).

[0021] In the absence of administration of a CD40 antagonist or a CD154 antagonist to a subject that has been determined to be seropositive for antibodies that bind a therapeutic agent or a delivery vehicle (e.g., the capsid of a viral vector), re-administration of the therapeutic agent or the delivery vehicle may result in a heightened immune response against the therapeutic agent or the delivery vehicle (e.g., an anamnestic response). Administration of the CD40 antagonist or the CD154 antagonist described herein to such a subject can reduce or inhibit the development of an anamnestic response.

[0022] In some embodiments, the method may include reducing the frequency or amount of the CD40 antagonist or the CD154 antagonist over time in a subject that exhibits a reduction over time of a CD40 mediated immune activity (e.g., the subject may become tolerized to the therapeutic agent or the delivery vehicle used to deliver the therapeutic agent)

[0023] In some embodiments, the pharmaceutical composition includes about 0.01 mg / mL to about 300 mg / mL of the CD40 antagonist or the CD154 antagonist (e.g., about 200 mg / mL of the CD40 antagonist or the CD154 antagonist). In some embodiments, the method includes administering about 0.1 mL to about 10.0 mL, e.g., about 1 .0 mL to about 5.0 mL, e.g., about 1 .0 mL or about 2.0 mL of the pharmaceutical composition to the subject, such as about 0.1 mL to about 2.0 mL (e.g., about 1 .0 mL) of a pharmaceutical composition containing about 0.01 mg / mL to about 300 mg / mL (e.g., about 200 mg / mL) of the CD40 antagonist or the CD154 antagonist.

[0024] In some embodiments, the method includes administering about 0.01 mg / kg to about 20 mg / kg (e.g., from about 0.03 mg / kg to about 10 mg / kg, e.g., about 0.03 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 1 .5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, or 10 mg / kg) of the CD40 antagonist or the CD154 antagonist.

[0025] In some embodiments, the pharmaceutical composition is administered intravenously, subcutaneously, or by infusion. For example, the method may include intravenously administering the pharmaceutical composition to the subject. Alternatively, the method may include subcutaneously administering the pharmaceutical composition to the subject. In some embodiments, the method may include intravenously administering the pharmaceutical composition followed by subcutaneously administering the pharmaceutical composition.

[0026] Definitions

[0027] The term “about,” as used herein, refers to a value that is ±10% of a reference value. The terms “CD40-mediated immune activity” and “CD40-mediated immune response,” as used herein, refer to an immune response that is activated upon CD40 binding to CD154 (e.g., upon binding of CD40 expressed on an immune cell, such as a B cell, to CD154 expressed on another immune cell, such as a T-cell). CD40 binding to CD154 can promote downstream signaling leading to an immune response, such as onset of a T cell dependent antibody response (TDAR), including the development of anti-drug antibodies (ADAs), as well as the activation of immune cells, including macrophages, antigen presenting cells, dendritic cells, mast cells, NK cells, and granulocytes. CD40 binding can also mediate a stromal response in epithelial / endothelial cells, fibroblasts, and osteoclasts. CD40, which is present on B cells, can also present antigens to helper T cells. If an activated T cell recognizes the peptide presented on the B cell, CD154 on the T cell binds to the B cell CD40 receptor, thus activating the B cell. Some exemplary CD40 related immune activities include, e.g., T cell activation, T cell proliferation, cytokine secretion, inflammation, tissue destruction, B cell activation, and B cell proliferation. A CD40 antagonist or a CD154 antagonist may reduce or abrogate a CD40-mediated immune activity by weakening, reducing, or preventing the interaction of CD40 with its CD154 ligand and / or by reducing or inhibiting downstream effects resulting from CD40 / CD154- mediated activity.

[0028] The term “gene therapy” as used herein refers to the administration of foreign genomic material into a subject’s tissue to modify the expression of a gene product or to change the biological properties of cells, for treating a disease or disorder. Gene therapy includes providing a polynucleotide (e.g., an mRNA encoding a therapeutic protein, or a vector, e.g., a viral vector, containing a polynucleotide encoding a therapeutic protein), or a host cell, virus, or virus like particle that includes a polynucleotide encoding a therapeutic protein (e.g., an enzyme); a therapeutic polynucleotide (e.g., an RNAi); components for carrying out genome-editing in the subject (e.g., transgenes for homologous recombination-based gene editing, nucleases for nuclease-based gene editing (e.g., zinc finger nucleases, transcription activator-like effector nucleases, meganucleases, or the CRISPR / Cas9 system); or a replication-competent virus, that can proliferate selectively in cells associated with a disease or disorder (e.g., an oncolytic virus). Examples of gene therapy methods are described in the international application PCT / US2018 / 058301 , filed on October 30, 2018, and published as W02020 / 032986, the disclosure of which, and the sequences therein, are incorporated herein by reference in their entirety. Thus, gene therapy may involve administration of the polynucleotide encoding the protein (e.g., as mRNA or similar therapy) or administration of a vector (e.g., a viral vector) containing the polynucleotide. Gene therapy may also include delivery of a cell, virus, or virus like particle that includes a vector (e.g., viral vector) encoding the protein. The polynucleotide may be formulated in a lipid-based delivery system.

[0029] The term “neutralizing antibody” refers to an antibody that reduces efficacy of a PRT or gene therapy. A neutralizing antibody may be an anti-drug antibody (ADA) that specifically binds to the protein, polynucleotide, vector, virus, or cell that is administered to a subject. A neutralizing antibody may be an antibody that results in increased clearance or decreased half-life of the protein, polynucleotide, vector, virus, or cell that is administered to a subject. The term “prevent,” as used herein, refers to reducing the risk of onset of a disease, e.g., as a prophylactic therapy for a subject who is at risk of developing a disorder, e.g., the development of NAbs or an immune response (e.g., TDAR) to a PRT or gene therapy. A subject can be characterized as “at risk” of developing a disorder by identifying a mutation associated with the disorder, according to any suitable method known in the art. Additionally, or alternatively, a subject can be characterized as “at risk” of developing a disorder if the subject has a family history of the disorder or is receiving a PRT.

[0030] The terms ‘protein replacement therapy” and “PRT, as used herein, refer to a therapeutic treatment in which an exogenous protein is provided to a subject (e.g., a human subject). The exogenous protein may be an enzyme having catalytic activity (e.g., enzyme replacement therapy (ERT)) or a non-catalytic protein. The protein may be formulated in a lipid-based delivery system.

[0031] The terms “treating,” “reducing,” and “inhibiting,” as used herein, refers to reducing the progression of a disease, reducing the severity of a disease symptom, reducing progression of a disease symptom, removing a disease symptom, or delaying onset of a disease (e.g., the development of a neutralizing antibody, such as an ADA, or a symptom associated with an immune response, such as a TDAR, or a symptom resulting from the presence of a neutralizing antibody, e.g., ADA). These terms encompass reducing a level of neutralizing antibodies, e.g., anti-drug antibodies (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) relative to a control subject (e.g., a subject that does not receive an anti-CD40 antibody or antigen-binding fragment thereof) or the subject prior to receiving a treatment as described herein (e.g., a naive subject).

[0032] The term “viral vector” as used herein refers to a virus or virus-like particle that is capable of transducing a subject’s cell with a polynucleotide or other cargo (e.g., proteins or other therapeutic agents) for directly or indirectly exerting a therapeutic effect.

[0033] Brief Description of the Drawings

[0034] FIGS. 1A and 1B are histograms showing CD80 (FIG. 1A) and CD86 (FIG. 1 B) expression in monocytes after exposure of the cell cultures to AAV2 and treatment with KPL-404.

[0035] FIGS. 2A and 2B are graphs showing CD80 (FIG. 2A) and CD86 (FIG. 2B) expression in monocytes after exposure of the cell cultures to AAV2 and treatment with iscalimab (CFZ533) or KPL- 404.

[0036] FIG. 3 is a set of graphs showing intracellular staining for TNF-a and IFN-y. KPL-404 treatment suppressed TNF-a and IFN-y production in AAV2-capsid-specific T cells.

[0037] FIG. 4. is a schematic diagram showing KPL-404 or placebo dosing and recombinant AAV8 infection in three arms. AAV8 contained neoantigens Gaussia luciferase (Glue) or human secreted alkaline phosphatase (hSEAP) cargos.

[0038] FIG. 5 is a graph showing KPL-404 plasma concentrations in cynomolgous serum from arm 1 in FIG. 4.

[0039] FIG. 6 is a graph showing enhanced Glue expression in AAV8 mediated gene therapy following treatment with KPL-404 versus placebo. FIG. 7 is a graph showing hSEAP expression in AAV8 mediated gene therapy following treatment with KPL-404 versus placebo.

[0040] FIGS. 8A-8C are graphs showing anti-AAV8 IgG and anti-AAV8 IgM serum concentrations in each of the three dosing arms of FIG. 4. FIG. 8A shows AAV redosing with KPL-404. FIG. 8B shows AAV redosing with placebo. FIG. 8C shows AAV single dose with placebo.

[0041] FIG. 9 is a graph showing AAV8 neutralizing antibody titer in plasma following treatment with KPL-404 or placebo. KPL-404 suppressed neutralizing antibodies induced by AAV8.

[0042] FIG. 10 is a graph showing anti-Gluc neutralizing antibody titer in plasma following treatment with KPL-404 or placebo. KPL-404 enhanced expression of neoantigen cargo Glue.

[0043] Detailed Description

[0044] Featured are methods of treating, reducing, or inhibiting a CD-40 mediated immune response, such as a T cell dependent antibody response (TDAR), in a subject (e.g., a human subject) receiving a therapeutic agent, such as, e.g., a protein (e.g., as a protein replacement therapy (PRT), such as, e.g., an enzyme replacement (ERT)) or a gene therapy (e.g., administration of a transgene encoding a therapeutic protein (e.g., using a vector, such as a viral vector, or a cell, virus, or virus like particle that includes the vector), such as an enzyme or other therapeutic protein).

[0045] The method includes administering to the subject a pharmaceutical composition that includes a CD40 antagonist or a CD154 antagonist. In some embodiments, the pharmaceutical composition contains anti-CD40 antibodies or antigen-binding fragments thereof (e.g., KPL-404) that can block CD40-mediated immune activity (e.g., by blocking the CD40-CD154 co-stimulatory pathway or by blocking the activation of a downstream effect that would otherwise follow activation of the CD40- CD154 co-stimulatory pathway). The pharmaceutical composition can also be used to prevent CD40 mediated signaling. In some embodiments, the pharmaceutical composition contains anti-CD154 antibodies, or antigen-binding fragments thereof, that can block CD40-mediated immune activity (e.g., by blocking the CD40-CD154 co-stimulatory pathway or by blocking the activation of a downstream effect that would otherwise follow activation of the CD40-CD154 co-stimulatory pathway). The pharmaceutical composition can also be used to prevent CD154 mediated signaling.

[0046] The CD40-CD154 costimulatory pathway is involved in the development of T cell dependent immune responses, development of humoral memory, and antigen presenting cell function.

[0047] The methods described herein may be used for subjects administered a therapeutic protein, such as an enzyme (e.g., administered as a purified protein), or for subjects administered a gene therapy (e.g., administration of a polynucleotide encoding a therapeutic protein, such as an enzyme, e.g., delivered using a vector, such as a viral vector). By blocking the activity of CD40, e.g., expressed by a B cell, or blocking the activity of CD154, or reducing or inhibiting CD40-mediated immune activity, the CD40 antagonist or the CD154 antagonist is able to reduce or inhibit the development of neutralizing immune response, including, e.g., a T cell dependent antibody response, against a therapeutic agent, such as a therapeutic protein (e.g., an enzyme), a polynucleotide encoding the therapeutic protein, or a vector (e.g., an AAV vector) used to deliver the therapeutic cargo (e.g. a nucleic acid encoding a therapeutic protein). The CD40 antagonist or the CD154 antagonist should enhance the efficacy of PRT, gene therapy or cellular therapy by inhibiting adverse immunogenic reaction to the therapeutic protein, vectors and cells (or proteins directly or indirectly expressed therefrom), used therein, respectively. For example, the CD40 antagonist or the CD154 antagonist can be used to inhibit humoral and cellular immune responses against delivery vehicles administered to a subject, such as cells or viral vectors (e.g., retroviral vectors, adenoviral vectors). Also, the use of such antagonists can enable a delivery vehicle (e.g., cell or viral vector) to be administered repeatedly, which will facilitate treatment of chronic diseases such as cancers, autoimmune diseases or diseases associated with a defective endogenous protein or insufficient amount of an endogenous protein. The methods described herein allow a delivery vehicle (e.g., cells or viral vectors) to be administered repeatedly, or at higher dosages without an adverse immunogenic response by administering the CD40 antagonist or the CD154 antagonist, prior to or during a first or any subsequent dose of the delivery vehicle to attenuate CD40-mediated immune activity (e.g., TDAR or T-cell cytotoxicity) in response to the first or subsequent dose.

[0048] The methods and the pharmaceutical compositions used therein are described in more detail below.

[0049] Indications

[0050] The methods described herein include administration of a pharmaceutical composition containing a CD40 antagonist or a CD154 antagonist as described herein. The CD40 antagonist or the CD154 antagonist can be administered to a subject as part of an in vivo (e.g., therapeutic or prophylactic) protocol for the treatment, reduction, or inhibition of a CD40-mediated immune activity, including, e.g., a neutralizing antibody response, T-cell cytotoxicity or a TDAR. For in vivo embodiments, the contacting step includes administration of the CD40 antagonist or the CD154 antagonist to the subject under conditions effective to permit binding of the CD40 antagonist to CD40 in the subject, or to permit the binding of the CD154 antagonist to CD154 in the subject. The CD40 antagonist or the CD154 antagonist can be administered to treat, reduce, or inhibit a CD40-mediated immune response, such as the development of neutralizing antibodies, T-cell cytotoxicity or a TDAR, or to treat or reduce the likelihood of developing one or more symptoms associated with the development of neutralizing antibodies, T-cell cytotoxicity or TDAR, e.g., relative to a subject receiving PRT or gene therapy without administration of the CD40 antagonist or the CD154 antagonist.

[0051] A subject to be treated according to the methods described herein has received and / or is undergoing treatment with a therapeutic agent (e.g., PRT or gene therapy). The method may be used for any PRT in which a functional protein (e.g., enzyme) or a bioequivalent thereof (e.g., an active protein or enzyme fragment or an engineered variant of the protein or enzyme) is provided for the treatment of a disease associated with a defective endogenous protein or insufficient amount of an endogenous protein. The PRT may be used for providing a protein (e.g., administration a purified protein), while a gene therapy may be used to provide a polynucleotide encoding a therapeutic protein (e.g., in the context of a delivery vehicle, e.g., viral vector, or a cell or virus like particle containing the vector, that contains the polynucleotide) for the treatment of a disease or disorder. The therapeutic protein can be any protein, including the proteins described in Table 1 below. The disease can be any disease, such as those described in Table 1 below.

[0052] Table 1. Examples of proteins and their associated diseases that can be treated by PRT or gene therapy

[0053]

[0054] The subject to be treated according to the methods described herein may have received and / or is undergoing treatment with a therapeutic agent (e.g., PRT or a gene therapy that provides a nucleic acid encoding a therapeutic protein (e.g., an enzyme or other therapeutic protein)). The method may be used for any gene therapy in which a nucleic acid encoding a functional therapeutic protein or a bioequivalent thereof is provided for the treatment of a disease or disorder associated with expression of a defective endogenous protein or the lack of a sufficient amount of an endogenous protein. Several types of delivery vehicles that may be used to deliver a therapeutic protein by gene therapy are known in the art, including, e.g., mammalian, bacterial, and viral vectors. The vector may also be an expression vector. Examples of viral vectors include vectors developed from a virus selected from the group consisting of a retrovirus, adenovirus, adeno-associated virus, parvovirus, coronavirus, negative strand RNA viruses, orthomyxovirus, rhabdovirus, paramyxovirus, positive strand RNA viruses, picornavirus, alphavirus, double stranded DNA viruses, herpesvirus, Epstein-Barr virus, cytomegalovirus, fowlpox, and canarypox. In some embodiments, the vector is an adenovirus, such as, e.g., Ad2, Ad5, Ad11 , Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52, Ad59, and Pan9. In some embodiments, the vector is an adeno-associated virus (AAV) vector, such as an AAV serotype selected from, but not limited to, AAV1 , AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. Unless otherwise specified, the AAV inverted terminal repeats (ITRs), or other AAV components, may be readily selected from among any AAV serotype, including, without limitation, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or other known and unknown AAV serotypes.

[0055] A subject to be treated with the CD40 antagonist or the CD154 antagonist, as described herein, may be one that is administered a therapeutic agent, e.g., as part of a gene therapy. The gene therapy may include administration of a virus that results in the development in the subject of neutralizing antibodies to the virus, such as to the capsid protein of the virus or antibodies to the therapeutic protein expressed as a result of the gene therapy, or the development of an immune response in the subject against cells transduced with the virus, or combinations thereof.

[0056] A subject to be treated according to the methods described herein may also be a subject who has not received a therapeutic agent (e.g., a PRT or a gene therapy, e.g., the subject is a naive subject). Alternatively, the subject may be one who has already received a first administration of a therapeutic agent (e.g., a PRT or gene therapy). In either case, administration of a pharmaceutical composition comprising a CD40 antagonist or a CD154 antagonist, as described herein, may be particularly useful for reducing or inhibiting the development of a secondary recall response (e.g., an anamnestic response), in which an initial administration of a therapeutic agent primes the immune system, and the second administration of the therapeutic agent produces a substantially larger immune response.

[0057] The subject may also be one who has been exposed to a particular antigen but is otherwise naive to the PRT or gene therapy. For example, a subject may have not previously received an AAV (e.g., AAV9) mediated gene therapy, but the subject may have been previously infected with a naturally occurring AAV (e.g., AAV9) in an unrelated context, and this prior infection may have primed the subject’s immune system against the AAV gene therapy vehicle. Such a subject may also exhibit an elevated recall response in response to a gene therapy upon the second administration. Such a subject would benefit from administration of a pharmaceutical composition containing the CD40 antagonist or the CD154 antagonist, as described herein.

[0058] CD40 Antagonists and CD154 Antagonists

[0059] In certain embodiments, the pharmaceutical composition includes a CD40 antagonist. In some embodiments, the CD40 antagonist is an anti-CD40 antibody, or antigen-binding fragment thereof. In other embodiments, the anti-CD40 antibody is iscalimab, bleselumab, BMS-986325, ravagalimab, lucatumumab, or, in each case, an antigen-binding fragment thereof. In one embodiment, the anti-CD40 antibody is KPL-404 (abiprubart), or an antigen binding fragment thereof, e.g., as described in PCT Pub. Nos. WO 2012 / 125569 and WO 2017 / 040932, including the sequences disclosed therein, which are herein incorporated by reference in their entirety.

[0060] The methods described herein include administration of anti-CD40 antibodies derived from the murine 2C10 antibody, or antigen-binding fragment thereof. An anti-CD40 antibody derived from the murine 2C10 antibody includes KPL-404 (abiprubart). The heavy chain variable regions, light chain variable regions, and CDRs of certain humanized anti-CD40 antibodies developed from the murine 2C10 antibody are shown in Table 2.

[0061] Table 2: Anti-CD40 antibody sequences

[0062] In certain embodiments, the anti-CD40 antibody or antigen-binding fragment thereof includes a heavy chain variable region including an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 9.

[0063] In certain embodiments, the anti-CD40 antibody or antigen-binding fragment thereof includes a light chain variable region including an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to a light chain variable region amino acid sequence as set forth in SEQ ID NO: 10.

[0064] In certain embodiments, the anti-CD40 antibodies or antigen-binding fragments thereof include both a heavy chain variable region including an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to a heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 9, and a light chain variable region including an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to a variable light chain amino acid sequence as set forth in SEQ ID NO: 10.

[0065] In certain embodiments, a heavy chain variable region of the anti-CD40 antibody or antigenbinding fragment thereof includes complementarity determining regions (CDRs) that are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the CDRs of a heavy chain variable region of the KPL-404 antibody (CDR1 , CDR2 and CDR3 as set forth in SEQ ID NOs: 3, 4, 5, respectively).

[0066] In certain embodiments, the light chain variable region of the anti-CD40 antibody or antigenbinding fragment thereof includes CDRs that are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the CDRs of a light chain variable region of the KPL-404 antibody (CDR1 , CDR2 and CDR3 as set forth in SEQ ID NOs: 6, 7, 8, respectively).

[0067] In certain embodiments, the heavy chain includes the CDRs as set forth in SEQ ID NOs: 3-5, respectively, and the light chain includes the CDRs as set forth in SEQ ID NOs: 6-8, respectively.

[0068] In certain embodiments, the heavy chain has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 9 and the CDRs set forth in SEQ ID NOs: 3-5, respectively, and the light chain has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 10 and the CDRs set forth in SEQ ID NOs: 6-8, respectively. For example, the antibody is KPL-404.

[0069] In other embodiments, the pharmaceutical composition includes a CD154 antagonist. In some embodiments, the CD154 antagonist is an anti-CD154 antibody or antigen binding fragment thereof. In some embodiments, the anti-CD154 antibody is frexalimab, letolizumab, tegoprubart, toralizumab, Lu AG22515, IBI-355, or ruplizumab, or, in each case, an antigen-binding fragment thereof. In one embodiment the CD154 antagonist is dazodalibep, or a CD154 binding fragment thereof. Also within the scope of the disclosure are antibodies or antigen-binding fragments thereof in which specific amino acids have been substituted, deleted, or added. These alternations do not have a substantial effect on the peptide’s biological properties such as binding activity. For example, antibodies may have amino acid substitutions in the framework region, such as to improve binding to the antigen. In another example, a selected, small number of acceptor framework residues can be replaced by the corresponding donor amino acids. The donor framework can be a mature or germline human antibody framework sequence or a consensus sequence. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in, e.g., Bowie et al. (Science, 247: 1306- 1310, 1990), Cunningham et al. (Science, 244: 1081-1085, 1989), Ausubel (ed.) (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., 1994), T. Maniatis, E. F. Fritsch and J. Sambrook (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y., 1989), Pearson (Methods Mol. Biol. 243:307-31 , 1994), and Gonnet et al. (Science 256:1443-45, 1992); each of which is incorporated herein by reference.

[0070] The polypeptides described herein may be a functionally active variant of the antibodies or antigen-binding fragments thereof disclosed herein, e.g., with less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 1 % amino acid residues substituted or deleted but that retain essentially the same immunological properties including, but not limited to, binding to CD40 or CD154.

[0071] In some embodiments, the dissociation constant (KD) of the antibody or antigen-binding fragment thereof is less than about 1 x 108, e.g., less than about 1 x 109.

[0072] The antibodies or antigen-binding fragments thereof may also include variants, analogs, orthologs, homologs and derivatives of polypeptides, that exhibit a biological activity, e.g., binding of an antigen such as CD40. The polypeptides may contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art.

[0073] The antibody or antigen-binding fragment thereof can be derivatized or linked to another functional molecule. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent interaction, etc.) to one or more other molecular entities, such as another antibody, a detectable agent, an immunosuppressant, or a pharmaceutical agent.

[0074] Pharmaceutical compositions

[0075] A pharmaceutical composition containing a CD40 antagonist can be used in the methods of treating, reducing, or inhibiting a CD40-mediated immune activity (e.g., development of a neutralizing antibody against a therapeutic agent (e.g., a therapeutic protein or a delivery vehicle (e.g., a viral vector) used to administer a therapeutic agent), a T cell dependent antibody response, or an ADA) in a human subject receiving or having received the therapeutic agent (e.g., in PRT or gene therapy), as described herein. In some embodiments, the CD40 antagonist is an anti-CD40 antibody, or antigenbinding fragment thereof. In other embodiments, the anti-CD40 antibody is iscalimab, bleselumab, BMS-986325, ravagalimab, lucatumumab, or, in each case, an antigen-binding fragment thereof. In other embodiments, the anti-CD40 antibody is KPL-404, or an antibody or antigen-binding fragment thereof containing a heavy chain variable region having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 9 and a light chain variable region having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 10), formulated together with a pharmaceutically acceptable carrier. A pharmaceutical composition containing a CD154 antagonist can be used in the methods of treating, reducing, or inhibiting a CD40-mediated immune activity (e.g., development of a neutralizing antibody against a therapeutic agent (e.g., a therapeutic protein or a delivery vehicle (e.g., a viral vector) used to administer a therapeutic agent), a T cell dependent antibody response, or an ADA) in a human subject receiving or having received the therapeutic agent (e.g., in PRT or gene therapy), as described herein. In some embodiments, the CD154 antagonist is an anti-CD154 antibody, or antigen-binding fragment thereof. In some embodiments, the anti-CD154 antibody is frexalimab, letolizumab, tegoprubart, toralizumab, dapirolizumab pegol, Lu AG22515, IBI- 355, or ruplizumab, or, in each case, an antigen-binding fragment thereof. In one embodiment the CD154 antagonist is dazodalibep, or a CD154 binding fragment thereof.

[0076] Pharmaceutically acceptable carriers that can be included in the pharmaceutical composition include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Depending on the route of administration, the CD40 antagonist or the CD154 antagonist (or antigen-binding portion(s) thereof) may be coated in a material to protect the antibodies (or antigen-binding portion(s) thereof) from the action of acids and other natural conditions that may inactivate the CD40 anagonist or the CD154 antagonist (or antigen-binding portion(s) thereof). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In certain embodiments, the present composition may include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0077] Pharmaceutical compositions of the invention may contain the CD40 antagonist or the CD154 antagonist, and the second therapeutic agent as described herein (e.g., one or more immunosuppressants).

[0078] The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a solid form (e.g., a dry powder) to be reconstituted with water or another suitable vehicle before use. The compositions may be in the form of an oil emulsion, water-in-oil emulsion, water-in-oil-in-water emulsion, site-specific emulsion, long-residence emulsion, sticky emulsion, microemulsion, nanoemulsion, liposome, microparticle, microsphere, nanosphere, nanoparticle and various natural or synthetic polymers, such as nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures, that allow for sustained release of the vaccine.

[0079] The composition can be in the form of a pill, tablet, capsule, liquid, or sustained release tablet for oral administration; or a liquid for intravenous, intrathecal, subcutaneous or parenteral administration; or a polymer or other sustained release vehicle for local administration.

[0080] The composition can be in the form of a solution containing the CD40 antagonist or the CD154 antagonist and a pharmaceutically acceptable carrier, e.g., an aqueous carrier if the composition is water-soluble. Examples of aqueous solutions include, e.g., water, saline, phosphate buffered saline, Hank’s solution, Ringer’s solution, dextrose / saline, glucose solutions and the like. The formulations can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. Additives can also include additional active ingredients such as bactericidal agents, or stabilizers. For example, the solution can contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate or triethanolamine oleate.

[0081] The composition can also be prepared as a solid formulation. For example, the composition can be formulated as, e.g., pills, tablets, powders, or capsules. For solid compositions, conventional solid carriers can be used which include, e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. Suitable pharmaceutical excipients include e.g., starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol.

[0082] Methods well known in the art for making formulations are found, for example, in “Remington: The Science and Practice of Pharmacy” (20th ed., ed. A.R. Gennaro AR., 2000, Lippincott Williams & Wilkins, Philadelphia, PA).

[0083] The pharmaceutical composition may also be provided as or incorporated into lipid monolayers or bilayers, e.g., as liposomes (see, e.g., U.S. Patent Nos. 6,110,490; 6,096,716; 5,283,185 and 5,279,833, which are incorporated herein by reference).

[0084] The composition can be prepared with a carrier that protects the CD40 antagonist or the CD154 antagonist against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. U.S. Patent No. 4,522,811.

[0085] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0086] A pharmaceutical composition containing the CD40 antagonist or the CD154 antagonist thereof can be formulated for administration by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. Administration may be parenteral, intravenous, intrathecal, subcutaneous, oral, topical, local, intramuscular, intradermal, transdermal, subdermal, rectal, spinal, or epidermal. Intravenous delivery by continuous infusion is one exemplary method of administration.

[0087] Methods for preparing parenterally administrable compositions are known in the art (see, e.g., Bai, J. Neuroimmunol. 80: 65-75, 1997; Warren, J. Neurol. Sci. 152: 31-38, 1997; and Tonegawa, J. Exp. Med. 186: 507-515, 1997, which are incorporated herein by reference).

[0088] Formulations for parenteral administration may, for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide / glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present agent. Nanoparticulate formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the present agent. Other potentially useful delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, intrathecal pumps, implantable infusion systems, and liposomes. The concentration of the agent in the formulation varies depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.

[0089] Sterile injectable solutions can be prepared by incorporating the present agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the present agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered overtime, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For example, the CD40 antagonist or the CD154 antagonist described herein may be administered once or twice weekly by subcutaneous injection or once or twice monthly by subcutaneous injection, for use as described herein.

[0090] Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. When administered orally, the present compositions may be protected from digestion. This can be accomplished either by complexing the CD40 antagonist or the CD154 antagonist with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the CD40 antagonist or the CD154 antagonist in an appropriately resistant carrier such as a liposome. Means of protecting agents from digestion are well known in the art (see, e.g., Fix, Pharm Res. 13: 1760-1764, 1996; Samanen, J. Pharm. Pharmacol. 48: 119-135, 1996; and U.S. Patent No. 5,391 ,377, which are incorporated herein by reference).

[0091] Routes of Administration and Dosing

[0092] A pharmaceutical composition containing a CD40 antagonist or a CD154 antagonist as described herein can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. Administration may be parenteral, intravenous, intrathecal, subcutaneous, oral, topical, local, intramuscular, intradermal, transdermal, subdermal, rectal, spinal, or epidermal. Intravenous delivery by continuous infusion is one exemplary method for administering theCD40 antagonist or the CD154 antagonist.

[0093] The composition described herein may include a pharmaceutically acceptable diluent. Pharmaceutically acceptable diluents include, for example, saline and aqueous buffer solutions.

[0094] Parenteral administration can include modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.

[0095] The methods described herein may include administration of the pharmaceutical composition by infusion, intravenously, or subcutaneously.

[0096] A therapeutically effective dose of a CD40 antagonist (e.g., an anti-CD40 antibody or an antigen binding fragment thereof) or a CD154 antagonist (e.g., an anti-CD154 antibody or an antigen binding fragment thereof) for treating a CD40-mediated immune activity in accordance with the invention can occur at various dosages. In some embodiments of the invention, a therapeutically effective dose is equal to or greater than 40 mg, 60 mg, 80 mg, 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, 200 mg, 220 mg, 240 mg, 280 mg, 300 mg, 320 mg, 340 mg, 360 mg, 380 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, or 500 mg, 520 mg, 540 mg, 580 mg, 600 mg, 620 mg, 640 mg, 660 mg, 680 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg or 1000 mg.

[0097] In some embodiments, the therapeutically effective dose is about 50 mg. In some embodiments, the therapeutically effective dose is about 60 mg. In some embodiments, the therapeutically effective dose is about 70 mg. In some embodiments, the therapeutically effective dose is about 80 mg. In some embodiments, the therapeutically effective dose is about 90 mg. In some embodiments, the therapeutically effective dose is about 100 mg. In some embodiments, the therapeutically effective dose is about 120 mg. In some embodiments, the therapeutically effective dose is about 140 mg. In some embodiments, the therapeutically effective dose is about 150 mg. In some embodiments, the therapeutically effective dose is about 160 mg. In some embodiments, the therapeutically effective dose is about 170 mg. In some embodiments, the therapeutically effective dose is about 180 mg. In some embodiments, the therapeutically effective dose is about 190 mg. In some embodiments, the therapeutically effective dose is about 200 mg. In some embodiments, the therapeutically effective dose is about 210 mg. In some embodiments, the therapeutically effective dose is about 220 mg. In some embodiments, the therapeutically effective dose is about 240 mg. In some embodiments, the therapeutically effective dose is about 250 mg. In some embodiments, the therapeutically effective dose is about 260 mg. In some embodiments, the therapeutically effective dose is about 270 mg. In some embodiments, the therapeutically effective dose is about 280 mg. In some embodiments, the therapeutically effective dose is about 290 mg. In some embodiments, the therapeutically effective dose is about 300 mg. In some embodiments, the therapeutically effective dose is about 310 mg. In some embodiments, the therapeutically effective dose is about 320 mg. In some embodiments, the therapeutically effective dose is about 330 mg. In some embodiments, the therapeutically effective dose is about 340 mg. In some embodiments, the therapeutically effective dose is about 350 mg. In some embodiments, the therapeutically effective dose is about 360 mg. In some embodiments, the therapeutically effective dose is about 370 mg. In some embodiments, the therapeutically effective dose is about 380 mg. In some embodiments, the therapeutically effective dose is about 390 mg. In some embodiments, the therapeutically effective dose is about 400 mg. In some embodiments, the therapeutically effective dose is about 420 mg. In some embodiments, the therapeutically effective dose is about 440 mg. In some embodiments, the therapeutically effective dose is about 460 mg. In some embodiments, the therapeutically effective dose is about 480 mg. In some embodiments, the therapeutically effective dose is about 500 mg. In some embodiments, the therapeutically effective dose is about 520 mg. In some embodiments, the therapeutically effective dose is about 540 mg. In some embodiments, the therapeutically effective dose is about 560 mg. In some embodiments, the therapeutically effective dose is about 580 mg. In some embodiments, the therapeutically effective dose is about 600 mg. In some embodiments, the therapeutically effective dose is about 620 mg. In some embodiments, the therapeutically effective dose is about 640 mg. In some embodiments, the therapeutically effective dose is about 660 mg. In some embodiments, the therapeutically effective dose is about 680 mg. In some embodiments, the therapeutically effective dose is about 700 mg. In some embodiments, the therapeutically effective dose is about 720 mg. In some embodiments, the therapeutically effective dose is about 740 mg. In some embodiments, the therapeutically effective dose is about 760 mg. In some embodiments, the therapeutically effective dose is about 800 mg. In some embodiments, the therapeutically effective dose is about 820 mg. In some embodiments, the therapeutically effective dose is about 840 mg. In some embodiments, the therapeutically effective dose is about 860 mg. In some embodiments, the therapeutically effective dose is about 880 mg. In some embodiments, the therapeutically effective dose is about 900 mg. In some embodiments, the therapeutically effective dose is about 920 mg. In some embodiments, the therapeutically effective dose is about 940 mg. In some embodiments, the therapeutically effective dose is about 960 mg. In some embodiments, the therapeutically effective dose is about 980 mg. In some embodiments, the therapeutically effective dose is about 1000 mg.

[0098] In some embodiments, a therapeutically effective dose is about 100-800 mg, about 150-500 mg, about 200-500 mg, about 250-500 mg, about 300-500 mg, about 350-500 mg, about 400-500 mg, about 450-500 mg, about 100-750 mg, about 100-700 mg, about 100-650 mg, about 100-600 mg, about 100-550 mg, about 100-500 mg, about 100-450 mg, about 100-400 mg, about 100-350 mg, about 100-300 mg, about 100-250 mg, about 100-200 mg, about 350-650 mg, about 400-600 mg or about 450-550 mg.

[0099] In some embodiments, the therapeutically effective dose is between 0.5 mg / kg and 100 mg / kg. In some embodiments, the therapeutically effective dose is about 2 mg / kg. In some embodiments, the therapeutically effective dose is about 2.5 mg / kg. In some embodiments, the therapeutically effective dose is about 3 mg / kg. In some embodiments, the therapeutically effective dose is about 4 mg / kg. In some embodiments, the therapeutically effective dose is about 4.5 mg / kg. In some embodiments, the therapeutically effective dose is about 5 mg / kg. In some embodiments, the therapeutically effective dose is about 5.5 mg / kg. In some embodiments, the therapeutically effective dose is about 6 mg / kg. In some embodiments, the therapeutically effective dose is about 6.5 mg / kg. In some embodiments, the therapeutically effective dose is about 7 mg / kg. In some embodiments, the therapeutically effective dose is about 8 mg / kg. In some embodiments, the therapeutically effective dose is about 9 mg / kg. In some embodiments, the therapeutically effective dose is about 10 mg / kg. In some embodiments, the therapeutically effective dose is about 15 mg / kg. In some embodiments, the therapeutically effective dose is about 18 mg / kg. In some embodiments, the therapeutically effective dose is about 20 mg / kg.

[0100] In some embodiments, the therapeutically effective dose is between 1 mg / kg and 10 mg / kg. In some embodiments, the therapeutically effective dose is between 5 mg / kg and 10 mg / kg. In some embodiments, the therapeutically effective dose is between 6 mg / kg and 10 mg / kg. In some embodiments, the therapeutically effective dose is between 7 mg / kg and 10 mg / kg. In some embodiments, the therapeutically effective dose is between 8 mg / kg and 10 mg / kg. In some embodiments, the therapeutically effective dose is between 9 mg / kg and 10 mg / kg. In some embodiments, the therapeutically effective dose is between 5 mg / kg and 9 mg / kg. In some embodiments, the therapeutically effective dose is between 5 mg / kg and 8 mg / kg. In some embodiments, the therapeutically effective dose is between 5 mg / kg and 7 mg / kg. In some embodiments, the therapeutically effective dose is between 5 mg / kg and 6 mg / kg. In some embodiments, the therapeutically effective dose is between 2 mg / kg and 10 mg / kg. In some embodiments, the therapeutically effective dose is between 3 mg / kg and 9 mg / kg. In some embodiments, the therapeutically effective dose is between 4 mg / kg and 8 mg / kg. In some embodiments, the therapeutically effective dose is between 5 mg / kg and 7 mg / kg.

[0101] In some embodiments, the step of administering the pharmaceutical composition including a CD40 antagonist (e.g., an anti-CD40 antibody or an antigen binding fragment thereof) or a CD154 antagonist (e.g., an anti-CD154 antibody or an antigen binding fragment thereof) for treating a CD40- mediated immune activity in accordance with the invention comprises at least one loading dose. In some embodiments the at least one loading dose is followed by at least one maintenance dose.

[0102] In some embodiments, the at least one loading dose is greater than the at least one maintenance dose. In some embodiments, the at least one loading dose is two-fold greater in dosage than the dosage of the at least one maintenance dose. In some embodiments, the at least one loading dose is three-fold greater in dosage than the dosage of the at least one maintenance dose. In some embodiments, the at least one loading dose is four-fold greater in dosage than the dosage of the at least one maintenance dose. In some embodiments, the at least one loading dose is five-fold greater in dosage than the dosage of the at least one maintenance dose. In some embodiments, the at least one loading dose is seven-fold greater in dosage than the dosage of the at least one maintenance dose. In some embodiments, the at least one loading dose is ten-fold greater in dosage than the dosage of the at least one maintenance dose.

[0103] In some embodiments, the loading dose is greater than the maintenance doses. In some embodiments, the loading dose and the maintenance doses are administered subcutaneously. In some embodiments, the step of administering the pharmaceutical composition comprises subcutaneous administration of at least one loading dose, followed by subcutaneous administration of at least one maintenance dose at a dosage of from about 100-800 mg, about 150-500 mg, about 200- 500 mg, about 250-500 mg, about 300-500 mg, about 350-500 mg, about 400-500 mg, about 450-500 mg, about 100-750 mg, about 100-700 mg, about 100-650 mg, about 100-600 mg, about 100-550 mg, about 100-500 mg, about 100-450 mg, about 100-400 mg, about 100-350 mg, about 100-300 mg, about 100-250 mg, about 100-200 mg, about 350-650 mg, about 400-600 mg or about 450-550 mg. In some embodiments, at least one maintenance dose is about 50 mg, 100 mg, 120 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 220 mg, 240 mg, 250 mg, 260 mg, 280 mg, 300 mg, 320 mg, 340 mg, 350 mg, 360 mg, 380 mg, 390 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520mg, 540mg, 580 mg, 600 mg, 620 mg, 640 mg, 660 mg, 680 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg or 1000 mg.

[0104] In some embodiments, the step of administering the pharmaceutical composition comprises subcutaneous administration of at least one loading dose, followed by subcutaneous administration of at least one maintenance dose, where the loading dose is at a dosage of from about 100-800 mg, about 150-500 mg, about 200-500 mg, about 250-500 mg, about 300-500 mg, about 350-500 mg, about 400-500 mg, about 450-500 mg, about 100-750 mg, about 100-700 mg, about 100-650 mg, about 100-600 mg, about 100-550 mg, about 100-500 mg, about 100-450 mg, about 100-400 mg, about 100-350 mg, about 100-300 mg, about 100-250 mg, about 100-200 mg, about 350-650 mg, about 400-600 mg or about 450-550 mg. In some embodiments, the loading dose is about 100 mg, 120 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 220 mg, 240 mg, 250 mg, 260 mg, 280 mg, 300 mg, 320 mg, 340 mg, 350 mg, 360 mg, 380 mg, 390 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520mg, 540mg, 580 mg, 600 mg, 620 mg, 640 mg, 660 mg, 680 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg or 1000 mg, 1 ,5g , or 2 g.

[0105] The composition may be administered, e.g., once per day, once per week, once every two weeks, once every three weeks, once per month, once every three months, or once per year. In some embodiments, the composition is administered during a treatment regimen of at least one day, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, or longer. In some embodiments, the pharmaceutical composition is administered at least once per month, per quarter, per six months, or per year.

[0106] In some embodiments, the pharmaceutical composition is administered prior to administration of the therapeutic agent (e.g., by PRT or gene therapy). For example, the pharmaceutical composition may be administered at least one hour, 6 hours, 12 hours, 1 day, 1 week, or 1 month prior to administration of the therapeutic agent. The pharmaceutical composition may be administered prior to each administration of the therapeutic agent.

[0107] In some embodiments, the pharmaceutical composition is administered following administration of the therapeutic agent. For example, the pharmaceutical composition may be administered at least one hour, 6 hours, 12 hours, 1 day, 1 week, or 1 month following administration of the therapeutic agent. The pharmaceutical composition may be administered following each administration of the therapeutic agent (e.g., by PRT or gene therapy).

[0108] In some embodiments, the pharmaceutical composition is administered substantially at the same time as the therapeutic agent. For example, the pharmaceutical composition may be administered concurrently with administration of the therapeutic agent.

[0109] In some embodiments, the pharmaceutical composition is administered to the subject following detection in a blood sample from the subject of a neutralizing antibody (e.g., ADA) that specifically binds the therapeutic agent (e.g., the therapeutic protein (e.g., an enzyme), a polynucleotide encoding a therapeutic protein, a delivery vehicle containing the polynucleotide (e.g., a viral vector), or a cell).

[0110] The pharmaceutical composition may be administered to the subject as a chronic therapy, e.g., for the life of the subject. Alternatively, the pharmaceutical composition may be administered to the subject only as needed (e.g., following detection of neutralizing antibodies in the subject). The dose of the pharmaceutical composition may also be varied overtime, as needed. For example, the subject may be administered an initial low dose (e.g., between 1-100 mg per dose) of the CD40 antagonist or the CD154 antagonist, or a lower frequency of dosing of the CD40 antagonist or the CD154 antagonist (e.g., no more than once per month), prior to development of neutralizing antibodies. The subject may be administered a higher dose of the CD40 antagonist or the CD154 antagonist (e.g., between 100-300 mg per dose), or a higher frequency of dosing of the CD40 antagonist or the CD154 antagonist (e.g., greater than once per month, or administration with each administration of the therapeutic agent (e.g., by PRT or gene therapy)), after development of neutralizing antibodies.

[0111] The likelihood that a gene therapy strategy will be successful depends in part on the ability of the vector to deliver the therapeutic product within the target organ or cells, with acceptable kinetics and minimal side effects. Circumventing an immune response against a viral vector is a challenge with all vector types. As mentioned above, viral vectors are detected by the immune system and illicit an immune response against them that becomes effective before the virus infects its target cells. This immune response can interfere with efficient gene transfer. An immune response against a gene therapy vector may eliminate the vector and the cells transduced by the viral vector, decreasing both the intensity and the duration of transgenic protein expression. Furthermore, the immune response to viral vectors involves the production of proinflammatory cytokines and chemokines that have harmful effects to the subject. In many cases, achieving sufficient expression of the therapeutic protein to treat a disease is a challenge in gene therapy. Administering higher doses of viral vector to increase gene transfer is challenging, as PBMC infection by adenoviruses induces cytokine production in a dosedependent manner; higher doses of viral vector induce a stronger inflammatory response responsible for increased elimination of the infected cells expressing the transgene. The immune response generated by administration of the viral vector and expression of the therapeutic product as well as the waning expression of the therapeutic product overtime often encountered with episomal-based gene therapy are key barriers to successfully treating subjects with gene therapy. Homologous recombination-based methods provide a more durable expression of a therapeutic product and can increase expression levels overtime due to a selective advantage conferred to cells containing the transgene, leading to an increased proportion of transduced cells (see, e.g., W02020032986). Notwithstanding the many advantages of gene transfer by homologous recombination, this method faces similar challenges associated with the activation of an immune response upon administration (and re-administration) of the viral vector in addition to challenges stemming from homologous recombination being a relatively rare event, resulting in a low transduction efficiency, which can impact the ability to achieve therapeutic levels of the product promptly after the first administration of the viral vector to the subject.

[0112] In order to overcome the above challenges, the present invention includes methods of expressing a therapeutic product in a subject by providing two types of gene transfer to the subject; a first viral vector for integrating a transgene in the subject’s genome by homologous recombination to achieve durable expression of a therapeutic product; and a second viral vector for episomal-based expression of a therapeutic product to rapidly achieve therapeutic levels of the therapeutic product. The subject is further administeredthe CD40 antagonist or the CD154 antagonist, prior to, during or following administration of the initial dose of the first and / or second viral vectors to attenuate CD40- mediated immune activity (e.g., TDAR or T-cell cytotoxicity) in response to the initial dose(s). Attenuation of the CD40-mediated immune activity allows for re-administrations of first viral vector and / or second viral vector for increasing the levels of transgene integrations in the genome of the subject and / or episomal-based expression of the therapeutic product, respectively, as appropriate.

[0113] Accordingly, in one aspect of the invention, the subject is administered (i) a first delivery vehicle (e.g., a viral vector) including a nucleic acid capable of integrating a transgene into the genome of the subject by homologous recombination for expressing a therapeutic protein in the subject and (ii) a second delivery vehicle (e.g., a viral vector) including a nucleic acid for episomal expression of a therapeutic protein in the subject. In certain embodiments, the therapeutic protein expressed from the transgene integrated into the genome and therapeutic protein expressed from the episome are identical. In one embodiment, the subject is administered one or more doses of the first delivery vehicle. For example, in some embodiments, the method includes administering to the subject 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of the first delivery vehicle. In another embodiment, the subject is administered one or more doses of the second delivery vehicle. For example, in some embodiments, the method includes administering to the subject 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of the second delivery vehicle. In a certain embodiment, the subject is administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of the first delivery vehicle and a single dose of the second delivery vehicle. In other embodiments, the first delivery vehicle and second delivery vehicle are administered to the subject at the same time, or within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 1 month of each other. In one embodiment, the subject is administered theCD40 antagonist or the CD154 antagonist, prior to, during or following administration of each dose of the first and / or second delivery vehicle, and any subsequent dose(s) of the first or second delivery vehicles, to attenuate CD40-mediated immune activity (e.g., TDAR or T-cell cytotoxicity) in response to administration of the first and / or second delivery vehicle.

[0114] Evaluation of treatment efficacy

[0115] Assays for assessing the health state of a subject receiving the therapeutic agent (e.g., by PRT or gene therapy) are described below. If the assays(s) show a reduction in the health state of the subject, the subject can be further tested for the presence of a CD40-mediated immune activity (e.g., the development of neutralizing antibodies against the therapeutic agent). Signs that the subject is beginning to show a decline in responsiveness to the therapeutic agent may signal a need to test the subject for the presence of neutralizing antibodies. If a reduction in efficacy of the PRT or gene therapy is suspected, the subject can be tested for the presence of neutralizing antibodies. This test can be performed especially in a subject that has been undergoing treatment with a PRT or gene therapy and has otherwise been observed to exhibit an improvement in symptoms of the disorder over a period of, e.g., about 1 year or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more years). The subject can be tested to determine whether the neutralizing antibodies, if present, are responsible for the reduction of efficacy of the PRT or gene therapy using assays described herein. If it is determined that the subject has, e.g., neutralizing antibodies that reduce the efficacy of the PRT or gene therapy, CD40 antagonist or a CD154 antagonist treatment can be initiated.

[0116] Several metrics can be used to evaluate the treatment efficacy of a PRT or gene therapy that is administered to a subject. These metrics may also be used as a benchmark to assess whether a subject that has developed CD40-mediated immune activities, such as the development of neutralizing antibodies, is experiencing a loss of PRT or gene therapy efficacy caused by the CD40- mediated immune activities. Furthermore, these metrics can be used in combination with CD40 antagonist or CD154 antagonist therapy to monitor a change in PRT or gene therapy efficacy (e.g., a restoration of PRT or gene therapy efficacy). In particular, these metrics may be used in combination with, e.g., neutralizing antibody assays, as described herein, both before CD40 antagonist or CD154 antagonist therapy, to signal the need for CD40 antagonist or CD154 antagonist therapy, and after CD40 antagonist or CD154 antagonist therapy, to signal successful CD40 antagonist or CD154 antagonist therapy (e.g., a reduction or inhibition of one or more CD40-mediated immune activities).

[0117] For example, the metrics may be used to measure a reduction in efficacy of a therapeutic agent administered by PRT or gene therapy (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in efficacy), thereby signaling a need for CD40 antagonist or CD154 antagonist therapy. The reduction in efficacy may be measured with respect to a baseline (e.g., a subject’s value(s) in one or more of the metrics before any reduction in efficacy, e.g., within a predetermined time period after initiation of PRT or gene therapy, e.g., 1 month, 3 months, 6 months, 1 year, 2 years, or more after the initiation of PRT or gene therapy PRT or gene therapy), a reference subject with the same disease undergoing PRT or gene therapy, or an average of reference subjects who have received PRT or gene therapy. A reduction in efficacy of PRT or gene therapy that surpasses a predetermined threshold (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in efficacy, e.g., relative to a time prior to reduction in efficacy), according to one or more of the metrics described herein, may signal a need for CD40 antagonist or CD154 antagonist therapy. When it is determined that CD40 antagonist or CD154 antagonist therapy is needed to treat a reduction in PRT or gene therapy efficacy due to the presence of neutralizing antibodies against a therapeutic agent, one or more of these metrics may also be used to track a restoration of, or increase in, PRT or gene therapy efficacy (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase in efficacy, e.g., relative to a time prior to CD40 antagonist or CD154 antagonist therapy). An increase in efficacy may be measured with respect to a baseline (e.g., the subject’s values before increase or restoration of efficacy), a reference subject with the same disease or disorder, or an average of reference subjects who have experienced a reduction in efficacy of PRT or gene therapy during PRT or gene therapy or who have received CD40 antagonist or CD154 antagonist therapy. When a subject experiences an improvement in efficacy following CD40 antagonist or CD154 antagonist therapy that surpasses a predetermined threshold (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase in efficacy, e.g., relative to a time prior to CD40 antagonist or CD154 antagonist therapy) or the subject exhibits a recovery in a level of efficacy of the PRT or gene therapy relative to a time prior to the reduction in efficacy, CD40 antagonist or CD154 antagonist therapy may be deemed successful and may be halted or administered at a reduced frequency or dose.

[0118] In some embodiments, the titer of neutralizing antibodies against a therapeutic agent in the subject may be used to guide administration of a pharmaceutical composition containing a CD40 antagonist or a CD154 antagonist. For example, if the titer of neutralization antibodies decreases below a predetermined threshold, the subject may discontinue CD40 antagonist or CD154 antagonist treatment. For example, a subject may receive a first CD40 antagonist or CD154 antagonist treatment prior to, at the same time as, or following, the first administration of the PRT or gene therapy. The CD40 antagonist or CD154 antagonist treatment may be discontinued after this administration. In another example, the subject may receive a first CD40 antagonist or CD154 antagonist treatment prior to, at the same time as, or following, the second administration of the PRT or gene therapy. The CD40 antagonist or the CD154 antagonist treatment may be discontinued after this administration. In some embodiments, the subject may receive CD40 antagonist or CD154 antagonist treatment prior to, at the same time as, or following, the first and second administrations of the PRT or gene therapy. The CD40 antagonist or CD154 antagonist treatment may be discontinued after the second administration. In some embodiments, the subject may receive CD40 antagonist or CD154 antagonist treatment prior to, at the same time as, or following, for example, the first, second, and third administrations of the PRT or gene therapy. The CD40 antagonist or CD154 antagonist treatment may be discontinued after the third administration.

[0119] Neutralizing antibody assays

[0120] An assay may be used to determine the presence of neutralizing antibodies in a subject (e.g., a human subject) against a therapeutic agent. Accordingly, the pharmaceutical composition comprising a CD40 antagonist or a CD154 antagonist may be administered to the subject following detection in a blood sample from the subject of neutralizing antibodies (e.g., an ADA) that specifically bind to a therapeutic protein (e.g., an enzyme), a polynucleotide encoding a therapeutic protein, or a delivery vehicle containing the polynucleotide (e.g., a viral vector).

[0121] The presence of neutralizing antibodies in a subject can be assessed by using an assay as described herein. Not all antibodies that bind to the therapeutic agent or polynucleotide reduce the efficacy of the PRT or gene therapy in a treated subject. For example, the antibodies that arise following PRT or gene therapy may bind to a domain of the therapeutic protein (e.g., an enzyme) that does not affect its therapeutic activity or function. Therefore, one or more additional assays (e.g., functional assays) may be used to determine if the antibodies are both neutralizing and reduce the efficacy of the therapeutic protein.

[0122] Following the identification of a reduction in efficacy of a therapeutic protein (e.g., an enzyme) during PRT or gene therapy, a subject can undergo further tests to detect and characterize the presence of neutralizing antibodies. For example, a subject’s sample (e.g., blood, serum) can be tested for the presence and / or titer of neutralizing antibodies against the enzyme administered as the PRT. The overall strategy for detecting an antibody response to PRT is similar to that for other protein therapeutics. A sensitive screening assay for binding antibodies can be used as a first step. Furthermore, the level or titer of antibody can be measured relative to a control or a baseline value, such as the amount of the antibody prior to the observed reduction in efficacy or of a reference subject with a disease not experiencing a reduction in efficacy of the enzyme. This step can be followed by an assay that confirms that a positive response is antibody-mediated and a competition assay using antigen to confirm that a positive response is specific. Positive samples can then be tested for the neutralizing activity of the antibody in both enzyme activity and functional assays, and the antibody may be further assessed for titer and isotype.

[0123] In particular, an evaluation of an immune response to an enzyme can involve one or more immunoassays that detect sensitivity, specificity, and / or robustness of the antibody / therapeutic interaction. Neutralization of catalytic activity can be assessed by mixing serially diluted subject sera into a validated potency assay that measures the activity of the enzyme toward its specific substrate. This assay can be validated according to recommended standards.

[0124] Assays that can be used with the methods described herein may include an ADA assay, which includes, for example, a screening assay, confirmatory assay, titration assay, characterization assay, and / or validation assay. The ADA assay may detect and characterize the immunogenicity of a therapeutic agent using one or more assays or a suite of validated assays. The ADA assay may include an electrochemiluminescent (ECL) bridging assay, which may include a sample acidification step to dissociate drug-antibody complexes and extend the drug tolerance by allowing unbound antidrug antibodies to compete freely in the assay. A positive control may be used, such as a polyclonal antibody purified from a rabbit hyperimmunized with the therapeutic agent. An ADA assay may use an anti-therapeutic agent antibody bridge of immobilizing antigen and reporting antigen. The immobilizing antigen may be a biotin-labeled enzyme (B-enzyme) and the reporting antigen may be ruthenium- labeled therapeutic agent (Ru-agent). The measurement output then may be produced by a chemiluminescent signal produced by electrical stimulation of the ruthenium-labelled agent when captured by a streptavidin plate. Additionally, following the ADA assay, a neutralizing antibody (NAb) assay may be performed, e.g., for samples confirmed positive in a confirmatory assay.

[0125] Following confirmation of the presence of an ADA, a subject can subsequently undergo treatment with a CD40 antagonist or a CD154 antagonist as described herein to treat, reduce, or inhibit the development of or production of the ADA or to treat, reduce, or inhibit one or more symptoms associated with a CD40-mediated activity (e.g., a TDAR or ADA developed against a therapeutic agent (including against a delivery vehicle (e.g., a viral vector) used to deliver the therapeutic protein)).

[0126] Examples

[0127] The following are examples of aspects for carrying out the methods of the present disclosure and are offered for illustrative purposes only. The examples are not intended to limit the scope of the present disclosure in any way.

[0128] Example 1. Co-cultures with adeno-associated virus serotype 2 (AAV2)-activated primary human peripheral blood mononuclear cells (PBMCs)

[0129] The objective of this study was to evaluate the role of KPL-404 on antigen specific immune responses to AAV2 capsids in a primary human PBMC cell culture setting. Antigen-presenting cells (APCs) and AAV2 capsids-specific T cells from PBMCs of 5 healthy donors were activated and expanded in vitro. For each donor, a separate experiment as performed on an independent occasion. CD80 and CD86 expression on APCs as well as TNF-a and IFN-y production on T cells were evaluated by flow cytometry. The following methods were used.

[0130] Methods:

[0131] 1 . Thaw cryopreserved PBMCs (Day 0). a. Remove a cryovial containing cryopreserved PBMCs from liquid nitrogen storage. b. Immediately place cryovial into 37°C water bath. c. Remove vial from the water bath as soon as cells are almost thawed (typically <1 min) with a small pellet remaining. d. Spray the outside of the vial with 70% ethanol. e. Transfer the vials into a biological safety cabinet.

[0132] 2. Treat thawed cells with DNase I (Day 0). a. Add 25 pL of Dnase I stock solution (1 mg / mL, in DPBS with Ca++ and Mg++) into 24 mL X-VIVO 15 media to obtain a final 1 pg / mL concentration (approximately 2 U / mL). b. Dilute 1 mL thawed cells by adding drop by drop into 24 mL X-VIVO 15 media containing Dnase I.

[0133] 3. Resuspend PBMCs at 5x105cells / mL in X-VIVO 15 (Day 0). a. Centrifuge diluted cells at 1700rpm for 5 min. b. Aspirate supernatant and resuspend cells in 5 mL X-VIVO 15 media. c. Pass the resuspended cells through a 70 mm cell strainer. d. Take an aliquot and count the total number of cells. e. Adjust the volume of X-VIVO 15 media to obtain a cell concentration of 5x105cells / mL.

[0134] 4. Seed cells (Day 0). a. Transfer 200 pL of the resuspended cells per well into 96-well U bottom plates to seed 105cells / well. b. Rest the seeded cells 12-24 h in a humidified incubator at 37°C with 5% CO2.

[0135] 5. Treat the cells with KPL-404 (Day 1). a. Transfer the plates into a biological safety cabinet. b. Add 2 pL KPL-404 (1 mg / mL) or lgG4 (1 mg / mL) into the designed wells, yielding the final concentrations: 10 pg / mL. c. Culture cells for 1 h in a humidified incubator at 37°C with 5% CO2.

[0136] 6. Add cytokines (Day 1). a. Prepare 2x cytokine solution from stock solution: GM-CSF (2x106lU / mL), IL-4 (2x106lU / mL), and Flt3-L (100 pg / mL) as shown in Table 3 below containing 10 pg / mL KPL-404 or lgG4.

[0137] Table 3. 2X cytokine solution (for 60 wells including 10% extra volume) b. Transfer the plates into a biological safety cabinet. c. Remove 100 pL of solution from each well. d. Add 100 pL of the 2x cytokine solution into the wells containing 100 mL of cells into the wells, yielding the following final concentrations: 1000 ILI / mL GM-CSF, 500 lU / mL IL-4, and 50 ng / mL Flt3-L. e. Mix gently by pipetting. f. Culture cells for 24 h in a humidified incubator at 37°C with 5% CO2.

[0138] 7. Stimulate cells (Day 2). a. Prepare 2x adjuvant solution from stock solution: R848 (20 mM), LPS (1 mg / mL) and IL-1 b (5 pg / mL) as shown in Table 4 below containing 10 pg / mL KPL-404 or lgG4. Table 4. 2x adjuvant solution (for 60 wells including 10% extra volume) b. Transfer the plates into a biological safety cabinet. c. Remove 100 pL of solution from each well. d. Add 100 pL of 2x adjuvant solution into each well to obtain a final concentration of 10 mM R848,

[0139] 100 ng / mL LPS, 10 ng / mL IL-1 p. e. Add 1 pL AAV2 empty particles (1 .27x1012VP / mL) into each well to a final concentration of about 1x 104particles / cell. f. Mix gently by pipetting. g. Culture cells for 24 h in a humidified incubator at 37°C with 5% CO2.

[0140] 8. Feed cells (Day 3). a. Prepare 2x feeding solution from stock solution: IL-2 (2x105 lU / mL), IL-7 (20 pg / mL), and IL-15 (20 pg / mL) as shown in Tables 5 and 6 below containing 10 pg / mL KPL-404 or lgG4.

[0141] Table 5. 2X feeding solution (for 60 wells including 10% extra volume)

[0142] Table 6. complete RPMI media (R10) b. Transfer the plates into a biological safety cabinet. c. Remove 100 pL of solution from each well. d. Add 100 pL of 2x feeding solution to obtain a final concentration of 10 lU / mL IL-2, 10 ng / mL IL-7, 10 ng / mL IL-15. e. Mix gently by pipetting. f. Culture cells for 48-72 h in a humidified incubator at 37°C with 5% CO2. g. Feed the cells on day 5, day 7, and day 9 repeatedly.

[0143] 9. Harvest cells (Day 10). a. Label tubes with the names of each test condition. b. Transfer the plates into a biological safety cabinet. c. Mix the contents of each well vigorously by pipetting. d. Collect and transfer the contents of wells into appropriate tubes (Combine all replicate wells for each test condition). e. Centrifuge cells at 1700rpm for 5 min. f. Aspirate supernatant and resuspend the cells in R10 media. g. Take an aliquot and count the number of cells for each test condition. h. Adjust the volume of R10 media to obtain a cell concentration of 2x106 cells / mL.

[0144] 10. Re-Seed cells (Day 10). a. Transfer 100 pL of cells per well into a new 96-well U bottom plate in triplicates for each test group to obtain 2x105 cells / well.

[0145] 11 . Re-Treat the cells with KPL-404 (Day 10). a. Add 1 pL KPL-404 (1 mg / mL) or lgG4(1 mg / mL) into the designed wells, yielding the final concentrations: 10 pg / mL. b. Culture cells for 1 h in a humidified incubator at 37°C with 5% CO2.

[0146] 12. Re-stimulate cells (Day 10). a. Calculate the total volume of 2x re-stimulation solution needed based on plate design with an extra amount to account for volume loss during transfer. b. Prepare 2x re-stimulation solution as shown in Table 7 below containing 10 pg / mL KPL-404 or !gG4.

[0147] Table 7. 2X re-stimulation solution (for 60 wells including 10% extra volume) c. Add 2x re-stimulation solution into designed wells. d. Add 2 pL AAV2 empty particles (1 .27x1012VP / mL) into designed wells to a final concentration of about 1x 104particles / cell. e. Mix gently by pipetting. f. Culture cells for 1 h in a humidified incubator at 37°C with 5% CO2.

[0148] 13. Add protein transport inhibitors (Day 10). a. Prepare 11x protein transport inhibition solution as shown in Table 8 below and add 20 pL into each well.

[0149] Table 8. 11x protein transport inhibition solution (for 60 wells including 10% extra volume) b. Mix by pipetting. c. Culture cells for 5 h in a humidified incubator at 37°C with 5% CO2.

[0150] 14. Harvest cells (Day 10). a. Label tubes with the names of each test condition. b. Transfer the plates into a biological safety cabinet. c. Mix the contents of each well vigorously by pipetting. d. Collect and transfer the contents of wells into FACS tubes (Combine all replicate wells for each test condition). e. Centrifuge cells at 1700rpm for 5 min. f. Aspirate supernatant and resuspend the cells in 1x PBS. g. Wash the cells with 1x PBS two times.

[0151] 15. Stain cells for viability and extracellular markers (Day 10). a. Prepare the viability and extracellular staining reagents (as below) in 1x PBS:

[0152] Panel for T cells response:

[0153] Live / Dead - Aqua

[0154] CD45-Pacific blue

[0155] CD3-Brilliant Violet 786

[0156] CD4-Brilliant Violet 750

[0157] CD8-Brilliant Violet 570

[0158] CD69-PE / Cy5 b. Resuspend cells in 100 pL extracellular staining solution. c. Mix well by pipetting. d. Incubate at 4°C for 20 min protected from light.

[0159] 16. Fix and permeabilize the cells (Day 10). a. Add 2 mL of 1x PBS into each tube. b. Centrifuge plates at 1700rpm for 5 min. c. Remove the supernatant. d. Add 100 pL of Fixation / Permeabilization solution (BD) into each tube. e. Mix well by pipetting. f. Incubate at 4°C for 30 min protected from light. g. Add 2 mL of 1x Perm / Wash Buffer (BD) into each tube. h. Centrifuge tubes at 1700rpm for 5 min. i. Remove the supernatant. j. Add 100 pL of 1x Perm / Wash Buffer (BD) into each tube. k. Mix well by pipetting. l. store the cells at 4°C overnight.

[0160] 17. Stain cells for intracellular proteins (Day 11). a. Add anti-IFN-g and anti-TNF-a at 1 :200 dilution to obtain a final concentration of 0.25 mg / mL, 1 mg / mL, respectively. c. Mix well by pipetting. d. Incubate at 4°C for 30 min protected from light. c. Add 2 mL of 1x Perm / Wash Buffer (BD) into each tube. d. Centrifuge at 1700rpm for 5 min. c. Remove the supernatant. d. Resuspend cells in 200 mL PBS. e. Store cells at 4°C protected from light until ready to acquire by flow cytometer.

[0161] 18. Prepare single color compensation controls (Day 11). a. Label separate test tubes for each antibody used in staining for flow cytometry. b. Vigorously vortex the beads. c. Combine 1 drop of beads, 200 mL PBS and 1 mL antibody in a test tube. d. Mix and incubate at 4°C protected from light for 30 min. e. Add 2 mL of 1x PBS and centrifuge tubes at 1700rpm for 5 min. f. Decant supernatant and resuspend pellet in 250 mL 1x PBS. g. Store cells at 4°C protected from light until ready to acquire by flow cytometer.

[0162] 19. Acquire and record data from experimental samples using a multi-color flow cytometer following fluorescence detector voltage and compensation setup (Day 11).

[0163] 20. Use compatible software to analyze flow data, such as FlowJo (Day 11).

[0164] Results:

[0165] As shown FIGS. 1A and 1 B, exposure of the cell cultures to AAV2 without addition of a blocking antibody was characterized by notable expression levels of either CD80 or CD86 (compare histograms depicting the positive control to histograms depicting the negative control in FIGS. 1 A and 1 B). Treatment of these cell cultures with KPL-404 suppressed CD80 and CD86 expression in monocytes of human PBMCs stimulated with empty AAV2 particles. A different CD40-blockng antibody, CFZ533 (Iscalimab) similarly inhibited the expression of CD80 and CD86. Mean fluorescence intensity (MFI) of CD80 and CD86, respectively, on monocytes treated with KPL-404, control cells, and monocytes treated with Iscalimab are shown in FIGS. 2A and 2B. The average MFI of CD80 on monocytes treated with KPL-404 was 2548 compared with 4206 in positive control cells. The average MFI of CD86 on monocytes treated with KPL-404 was 4002 compared with 7581 in positive control cells. These data demonstrate that KPL-404 attenuates an antigen presenting cell (APC) response.

[0166] As shown in FIG. 3, intracellular staining for TNF-a and IFN-y also revealed that KPL-404 treatment suppressed TNF-a and IFN-y production in AAV2-capsid-specific T cells. The average frequency of IFN-y+CD8+, TNF-a+CD8+, IFN-y+CD4+, and TNF-a+CD4+T cells was 0.77%, 2.83%, 0.83%, and 3.88% in KPL-404 treated PBMCs, respectively, compared with 1.85%, 6.57%, 2.41 %, 8.97% in non-treated PBMC, respectively. These data demonstrate that KPL-404 attenuates T cell response to AAV2.

[0167] Example 2. Cynomolgus Monkey Study

[0168] The objective of this study was to evaluate the effect of KPL-404 on inhibition of adeno- associated virus serotype 8 (AAV8) vector-mediated induction of immune responses in cynomolgus (cyno) monkeys.

[0169] Prior to the start of the study, animals were acclimated for 2 weeks. Animal health (including physical examination, blood chemistry and hematology and PT / APTT) was monitored by a veterinarian. During the 2-week acclimation period, 59 animals were screened for anti-AAV8 neutralizing antibodies (NAb) and total anti-AAV8 antibody (TAb) IgG and IgM levels before the initiation of the in vivo experiment. The inclusion criteria for the cyno monkeys in this study are listed below. a. NAb titer: <4 b. IgM TAb titer: <300 c. IgG TAb titer: <1 ,000 d. Monkeys with IgG TAb titers at low (<260), medium (260 - 550) and high levels (550 - 1 ,000) were equally distributed among the study arms.

[0170] Selected monkeys were assigned into three groups. KPL-404 dosing and recombinant AAV8 infection are summarized in FIG. 4.

[0171] The following methods were used:

[0172] Quantification of KPL-404 in serum by ELISA

[0173] Monkey serum KPL-404 concentration were quantified with an Elisa assay.

[0174] (1) Diluted capture protein to 0.5 pg / mL in 1 x PBS to prepare the capture protein solution.

[0175] (2) Added 100 pL of the capture protein solution to wells, sealed the plates and incubated at 4°C for 18 to 24 h.

[0176] (3) On the second day, kept the plate at RT for at least 30 min.

[0177] (4) Washed the plate 4x 300 pL per well with wash buffer, then tapped on an absorbent paper.

[0178] (5) Added 300 pL nonfat milk blocking buffer to each well and sealed the plates at RT for 2 h. (6) Prepared the calibration standards (Table 9), QCs (Table 10) and study samples. Vortexed the mixed solution from each step before preparing the next solution.

[0179] -STD1 to STD9 were used as calibration standard samples and STD1 to STD3 were used as anchor point. -All the samples including calibration standards, QC samples and study samples were diluted with dilution buffer (2% BSA in PBS) at MRD (25-fold).

[0180] -Study samples that had KPL-404 concentrations greaterthan the ULOQ of the standard were further diluted with 4% serum after dilution with MRD. Table 9. Preparation of calibration standards samples

[0181] Table 10. Preparation of QC samples LLQC 100

[0182] (7) After incubation with the blocking buffer, washed the plate as described in step (4).

[0183] (8) Added 100 pL of sample to wells, and sealed the plates at RT for 1 h.

[0184] (9) Prepared the detection antibody solution (1 :2000 dilute with dilution buffer).

[0185] (10) After incubation with samples, washed the plate as described in step (4).

[0186] (11) Added 100 pL detection antibody solution to wells, and sealed and incubated the plates at RT for 1 h.

[0187] (12) Kept aliquot of TMB substrate to RT for at least 30 min.

[0188] (13) After the incubation with the detection antibody, washed the plate as described in step (4).

[0189] (14) Added 100 pL TMB substrate to wells.

[0190] (15) Incubated the reaction at RT for 10 min.

[0191] (16) Add 100 pL / well of hydrochloric acid.

[0192] (17) Read absorbance at OD45onm immediately with Spectramax M3.

[0193] Titration of anti-AAV8 IgG and IgM

[0194] Monkey plasma IgG and IgM anti-AAV8 TAb were assessed with an Elisa assay.

[0195] (1) Coated corning 96-half-area well plates with 50 pL of AAV8 antigens (1.1 e+9 cp / mL AAV8 empty capsid) per well at 4°C overnight.

[0196] (2) Washed the plates with 150 pL of PBST per well, 5 times.

[0197] (3) Blocked the plates with 150 pL of PBST-5% milk per well at RT for 2 h.

[0198] (4) Washed the plates as described in step (2).

[0199] (5) Added 50 pL of plasma diluted in PBST-1 % BSA to each well and incubated at RT for 2 h.

[0200] For IgG test, made 100x dilution of plasma (3 pL plasma + 297 pL dilution buffer), followed with further 3-fold dilution (100 pL sample + 200 pL dilution buffer).

[0201] For IgM test, made 200x dilution of plasma (3 pL plasma + 597 pL dilution buffer), followed with further 2-fold dilution (150 pL sample + 150 pL dilution buffer).

[0202] (6) Washed the plates as described in step (2).

[0203] (7) Added 50 pL of secondary antibody per well and incubated at RT for 1 h.

[0204] For IgG test, 1 :15,000 diluted peroxidase conjugated Anti-Monkey IgG.

[0205] For IgM test, 1 :80,000 diluted peroxidase conjugated Anti-Monkey IgM Antibody.

[0206] (8) Washed the plates as described in step (2).

[0207] (9) Added 50 pL of TMB substrate per well and incubated at RT for 20 min.

[0208] (10) Added 50 pL of stop solution per well and read OD450nm values with Spectramax M3.

[0209] Assessment of AAV8 neutralizing antibody

[0210] Monkey plasma anti-AAV8 NAb was assessed with a cell-based assay conducted in 293T cells.

[0211] (1) The neutralization assay was conducted in 293T cells. 40,000 Cells per well were seeded into 96- well plates and cultured at 37 °C and 5% CO2 overnight.

[0212] (2) Next day, serially diluted the reference antibody and the monkey plasma samples with the cell culture medium.

[0213] (3) Diluted the AAV8-CMV-GFP to 50,000 MOI / 50 pL using the cell culture medium.

[0214] (4) Mixed antibody or plasma samples prepared in step (2) with diluted virus in equal volume in 96-well plate and incubated at 37 °C for 2.5 h.

[0215] (5) Added 100 pL of the Ab / virus mix to wells.

[0216] (6) Cultured the cells at 37 °C, 5% CO2 for 2 days.

[0217] (7) Measured fluorescence signal using an Acumen Cellista.

[0218] (8) Data analysis

[0219] Inhibition rate (%) = (Raw data sample - Average VC) I (Average CC - Average VC) *100. Neutralizing antibody titers were analyzed using the 4-parameter nonlinear logistic regression of the inhibition rate (%) with the Prism Graphpad software.

[0220] Measurement of plasma hSEAP

[0221] Monkey plasma hSEAP were measured with a chemiluminescence assay.

[0222] (1) Equilibrated all the reagents to RT prior to use.

[0223] (2) Diluted the plasma samples, and incubated at 65°C for 30 min, then kept at 4°C. The standard did not need to be heated.

[0224] (3) Added 50 pL of the standard (S1-S7 plus Blank) to the plates in duplicate wells.

[0225] (4) Standard: PAP was tested at 0.3 ng / pL. The highest concentration of the Standard curve was 1 ,875 pg / mL, with a serial 2-fold dilutions in total 7 concentrations (S1-S7).

[0226] (5) Added 50 pL of the test samples. To avoid interference, wells adjacent to the highest concentration of the standard were kept in empty.

[0227] (6) Added 50 pL of Assay Buffer to all wells, then incubated for 5 min.

[0228] (7) Added 50 pL of Reaction Buffer to all wells, then incubated for 15 min.

[0229] Reaction Buffer: CSPD was made a 20x dilution with Reaction Buffer Diluent within 5 min prior to use.

[0230] (8) Read chemiluminescence signal with 500 ms / well using Spectramax M3.

[0231] Measurement of serum Glue

[0232] Monkey serum Glue were measured with a chemiluminescence assay.

[0233] (1) Equilibrate all reagents to room temperature before use;

[0234] (2) Add 10 pL standard (S1-S7+Blank) to the experimental plate, duplicate wells;

[0235] (3) Add 10 pL serum sample to the assay plate;

[0236] (4) Add 50 pL Working Solution to all wells and incubate for 10 minutes;

[0237] (5) The microplate reader detects chemiluminescence, and the detection time is 500 ms / well.

[0238] (6) Read chemiluminescence signal with 500 ms / well using Spectramax M3.

[0239] Measurement of anti-Gluc antibody

[0240] Anti-Gluc antibody was measured with an ELISA assay. All of the reagents were prepared at time of use: a) Wash buffer: mixed 100mL 1Ox PBST and 900 mL deionized water b) Coating buffer: mixed 1 mL 20x coating buffer and 19 mL deionized water c) Dilution buffer: added 1g BSA to 100mL PBST d) Blocking buffer: added 5g nonfat powdered milk to 100mL PBST e) Antigen solution: diluted the Gaussia Luciferase Protein protein (0.1 mg / mL) to 0.5pg / mL with coating buffer f) Secondary antibody solution: diluted Gaussia Luciferase Protein -biotin to 1pg / mL with dilution buffer.

[0241] (1) Added 50pL / well of the antigen solution to the plate, sealed the plate and incubated at 4°C, overnight.

[0242] (2) On the second day, removed the plate from refrigerator and set on bench top at room temperature for a minimum of 30 minutes.

[0243] (3) Washed the plate 3 x 150 pL per well with wash buffer then tapped dry on absorbent paper.

[0244] (4) Added 150 pL / well blocking buffer to the plate and sealed the plate. Incubated the plate at room temperature for 2 hours.

[0245] (5) Prepared the controls and study samples. Samples were diluted four-fold serially in duplicate across the plate with a starting dilution of 1 :10. Vortexed the mixed solution from each step before preparing the next solution.

[0246] (6) Washed the plate 3 x 150 pL per well with wash buffer then tapped dry on absorbent paper.

[0247] (7) Added 50 pL of plasma samples to each well and sealed the plate. Incubated the plate at room temperature for 2 hours.

[0248] (8) Washed the plate 5 x 150 pL per well with wash buffer then tapped dry on absorbent paper.

[0249] (9) Added 50 pL / well of secondary antibody dilution to the plate and sealed the plate. Incubated the plate at room temperature for 1 hours.

[0250] (10) Washed the plate 5 x 150 pL per well with wash buffer then tapped dry on absorbent paper.

[0251] (11) Diluted Streptavidin-HRP at a ratio of 1 :200 with dilution buffer and added 50 pL to each well. Incubated the plate at room temperature for 30 mins.

[0252] (12) Washed the plate 5 x 150 pL per well with wash buffer then tapped dry on absorbent paper.

[0253] (13) Added 50 pL / well TMB substrate to the plate and put the plate into a drawer for 20 minutes.

[0254] (14) Added 50 pL / well 2N hydrochloric acid to all assigned wells.

[0255] (15) Read absorbance at 450 nm immediately.

[0256] (16) Data analysis: Acquired data was analyzed using Microsoft Excel. Plotted a graph with the absorbance value as the dependent variable (y-axis) and the reciprocal of dilution factor as the independent variable (x-axis). End point titers were expressed as the reciprocal of the sample dilution factor giving a y=OD450nm of blank+3xSD.

[0257] %CV of absorbance at 450nm in duplicate was calculated. Results:

[0258] Serum concentration of KPL-404 of monkeys in Arm 1

[0259] Serum KPL-404 concentration of monkeys in Arm 1 was assessed biweekly by ELISA. The results of serum KPL-404 concentration in individual monkeys in Arm 1 are shown in FIG. 5. As shown in FIG. 5, serum concentrations of KPL-404 in the monkeys in Arm 1 maintained plateau at high levels between day 14 and day 84, the time points tested.

[0260] Serum Glue protein of monkeys in Arms 1 and 2

[0261] The monkeys in Arm 1 and Arm 2 were IV injected with AAV8-Gluc recombinant virus on day 1 . Serum Glue protein of the monkeys was quantified biweekly by chemiluminescence. The results are shown in FIG. 6 .

[0262] Serum hSEAP of monkeys in all 3 arms

[0263] Monkeys in all three arms were IV injected with AAV8-hSEAP recombinant virus on day 43. Plasma hSEAP protein of the monkeys was determined biweekly by chemiluminescence. The results are shown in FIG. 7.

[0264] Plasma AAV8 IgG and IgM of monkeys in all 3 arms

[0265] Plasma anti-AAV8 IgG and IgM of the monkeys were determined biweekly by ELISA. The results of anti-AAV8 IgG and IgM titers in monkey plasma are shown in FIGS. 8A-8C.

[0266] Plasma AAV8 neutralizing antibody of monkeys in all 3 arms

[0267] Plasma AAV8 NAb of monkeys in all 3 arms was assessed biweekly by bioluminescence. The results of AAV8 NAb in the monkey plasma are shown in FIG. 9.

[0268] Serum anti-Gluc neutralizing antibodies

[0269] The monkeys in Arm 1 and Arm 2 were IV injected with AAV8-Gluc recombinant virus on day 1 . Serum anti-Gluc neutralizing antibodies of the monkeys was quantified biweekly by ELISA. The results are shown in FIG. 10.

[0270] Conclusions

[0271] KPL-404 was well absorbed and maintained stable in monkeys. Monkeys treated with KPL- 404 (Arm 1) exhibited lower levels of the specific immune responses to AAV8 than monkeys without KPL-404 treatment (Arm 2). Monkeys treated with KPL-404 exhibited higher-level expression of recombinant AAV8 proteins than non-KPL-404 treated monkeys. These results demonstrate that KPL- 404 can inhibit the immune responses to AAV8 vectors in monkeys.

[0272] Example 3. Antibody treatment of a subject with Hemophilia B.

[0273] A subject with Hemophilia B is undergoing gene therapy with an AAV vector encoding Factor IX. Following receipt of a first administration of the AAV gene therapy vector, the serum of the subject can be tested for the presence of neutralizing antibodies to, e.g., the AAV capsid. Prior to the administration of a second dose of the AAV gene therapy vector, such as one month after the first administration of the AAV gene therapy vector, the subject may be administered 2.0 mL of a pharmaceutical composition containing the KPL-404 antibody formulated at 200 mg / mL by subcutaneous administration. One day following administration of the anti-CD40 antibody, the subject may receive the second dose of the AAV gene therapy vector. Any subsequent redosing of the AAV to the subject will be carried out one day after another administration of the anti-CD40 antibody to prevent the generation of neutralizing antibodies for each redosing of the AAV.

[0274] Example 4. Suppression of T cell dependent antibody response to viral vectors

[0275] A subject with Hemophilia B is undergoing gene therapy with two different viral vectors encoding Factor IX. The subject receives a first delivery vehicle that includes a viral vector including a nucleic acid capable of integrating the Factor IX transgene into the genome of the subject by homologous recombination. The subject receives a second delivery vehicle that includes a viral vector including a nucleic acid capable of expressing Factor IX by episomal expression. Following receipt of a first administration of each gene therapy vector, the serum of the subject can be tested for the presence of neutralizing antibodies to, e.g., a capsid of the viral vector. Priorto the administration of a second dose of the first and second gene therapy vectors, such as one month after the first administration of the gene therapy vectors, the subject may be administered 2.0 mL of a pharmaceutical composition containing the KPL-404 antibody formulated at 150 mg / mL by subcutaneous administration. One day following administration of the anti-CD40 antibody, the subject may receive the second dose of each of the gene therapy vectors. Following the second dose of the gene therapy vectors, the serum of the subject can be assayed for the development of neutralizing antibodies against the capsid. One month later, the subject may receive a second anti-CD40 antibody treatment, and one day later, the subject may receive a third dose of each of the gene therapy vectors. Following administration of the third dose of each of the gene therapy vectors, the serum of the subject can be assayed for neutralizing antibodies. The subject may, if necessary, continue anti- CD40 antibody therapy while they continue to receive treatment with the two gene therapy vectors.

[0276] Other Embodiments

[0277] While specific aspects of the invention have been described and illustrated, such aspects should be considered illustrative of the invention only and not as limiting the invention as construed in accordance with the accompanying claims.

[0278] All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes.

[0279] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

CLAIMS1. A method of treating, reducing, or inhibiting a CD40-mediated immune response in a human subject receiving or having received a therapeutic agent, the method comprising administering to the subject a pharmaceutical composition comprising a CD40 antagonist or a CD154 antagonist.

2. The method of claim 1 , wherein the therapeutic agent comprises gene therapy comprising administration of a polynucleotide encoding the protein.

3. The method of claim 2, wherein gene therapy comprises administration of a vector comprising the polynucleotide encoding the protein.

4. The method of claim 3, wherein the vector is a viral vector.

5. The method of any one of claims 1 -4, wherein the therapeutic agent a protein is selected from those set forth in Table 1 administered to the subject in protein replacement therapy.

6. The method of any one of claims 1-5, wherein the subject has a disease selected from those set forth in Table 1 .

7. The method of any one of claims 1-6, wherein the pharmaceutical composition is administered at least once per month, per quarter, per six months, or per year.

8. The method of any one of claims 1-7, wherein the pharmaceutical composition is administered prior to administration of the therapeutic agent.

9. The method of claim 8, wherein the pharmaceutical composition is administered at least one hour, 6 hours, 12 hours, 1 day, 1 week, or 1 month prior to administration of the therapeutic agent.

10. The method of claim 8 or 9, wherein the pharmaceutical composition is administered prior to each administration of the therapeutic agent.

11. The method of any one of claims 1-10, wherein the pharmaceutical composition is administered following administration of the therapeutic agent.

12. The method of claim 11 , wherein the pharmaceutical composition is administered at least one hour, 6 hours, 12 hours, 1 day, 1 week, or 1 month following administration of the therapeutic agent.

13. The method of claim 11 or 12, wherein the pharmaceutical composition is administered following each administration of the therapeutic agent.

14. The method of any one of claims 1-13, wherein the pharmaceutical composition is administered substantially concurrently with the therapeutic agent.

15. The method of claim 14, wherein the pharmaceutical composition is administered at each administration of the therapeutic agent.

16. The method of any one of claims 1-15, wherein the pharmaceutical composition is administered to the subject following detection in a blood sample from the subject of a CD40-mediated immune activity, wherein the CD40-mediated immune activity comprises development of an anti-drug antibody (ADA) that specifically binds the therapeutic agent, wherein, optionally, the therapeutic agent is a protein, a polynucleotide encoding the protein, or a vector comprising the polynucleotide.

17. The method of any one of claims 1-16, wherein the therapeutic agent is an enzyme administered to the subject as an enzyme replacement therapy (ERT).

18. The method of any one of claims 1-17, wherein the CD40-mediated immune response comprises activation of a cellular immune response, such as activation of antigen-presenting cells against the therapeutic agent, or activation of a humoral immune response, such as development of neutralizing antibodies against the therapeutic agent.

19. The method of any one of claims 1-18, wherein the pharmaceutical composition comprises the CD40 antagonist.

20. The method of claim 19, wherein the CD40 antagonist is an anti-CD40 antibody or antigen binding fragment thereof.21 . The method of claim 20, wherein the CD40 antagonist comprises a humanized anti-CD40 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 10.

22. The method of claim 21 , wherein the CD40 antagonist is KPL-404.

23. The method of claim 20, wherein the CD40 antagonist is iscalimab.

24. The method of claim 20, wherein the CD40 antagonist is BMS-986325.

25. The method of claim 20, wherein the CD40 antagonist is bleselumab.

26. The method of claim 20, wherein the CD40 antagonist is ravagalimab.

27. The method of claim 20, wherein the CD40 antagonist is lucatumumab.

28. The method of any one of claims 1-18, wherein the pharmaceutical composition comprises the CD154 antagonist.

29. The method of claim 28, wherein the CD154 antagonist is an anti-CD154 antibody or antigen binding fragment thereof.

30. The method of claim 29, wherein the CD154 antagonist is frexalimab.

31. The method of claim 29, wherein the CD154 antagonist is dapirolizumab pegol.

32. The method of claim 29, wherein the CD154 antagonist is tegoprubart.

33. The method of claim 28, wherein the CD154 antagonist is dazodalibep.

34. The method of claim 29, wherein the CD154 antagonist is Lu AG22515.

35. The method of claim 29, wherein the CD154 antagonist is letolizumab.

36. The method of claim 29, wherein the CD154 antagonist is toralizumab.

37. The method of claim 29, wherein the CD154 antagonist is IBI-355.

38. The method of claim 24, wherein the CD154 antagonist is ruplizumab.

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