A method for treating IgA nephropathy with APRIL-binding antibodies

Abstract

Formulations and uses of antibodies that bind to human APRIL for the treatment of conditions associated with the overproduction or deposition of IgA, such as IgA nephropathy.

Description

[Technical Field]

[0001] Related applications This application claims the benefit of U.S. Provisional Application No. 62 / 704,831, filed on 29 May 2020, with priority claimed, and the entire application, including all tables, figures, and claims, is incorporated herein by reference.

[0002] Technical field This invention relates to the use of isolated antibodies, fragments thereof, that bind to human APRIL for the treatment of IgA nephropathy. [Background technology]

[0003] APRIL is expressed as a type II transmembrane protein, but unlike most other TNF family members, it is primarily processed as a secreted protein, cleaved in the Golgi apparatus, where it is cleaved by furin convertase to release a soluble active form (Lopez-Fraga et al., 2001, EMBO Rep 2:945-51). APRIL assembles as a non-covalent homotrimer with similar structural homology in the protein fold to several other TNF family ligands (Wallweber et al., 2004, Mol Biol 343, 283-90). APRIL binds to two TNF receptors: B cell maturation antigen (BCMA) and transmembrane activator, as well as calcium regulator and cyclophylline ligand interacting factor (TACI) (Kimberley et al., 2009, J Cell Physiol. 218(1):1-8). Furthermore, APRIL has recently been shown to bind to heparan sulfate proteoglycan (HSPG) (Hendriks et al., 2005, Cell Death Differ 12, 637-48). APRIL plays a role in B cell signaling and has been shown to promote both the proliferation and survival of human and mouse B cells in vitro (reviewed in Kimberley et al., 2009, J Cell Physiol. 218(1):1-8).

[0004] APRIL is primarily expressed by a subset of immune cells, including monocytes, macrophages, dendritic cells, neutrophils, B cells, and T cells, many of which also express BAFF. Furthermore, APRIL can be expressed by non-immune cells such as osteoclasts, epithelial cells, and various tumor tissues (reviewed in Kimberley et al., 2009, J Cell Physiol. 218(1):1-8). In fact, APRIL was initially identified based on its expression in cancer cells (Hahne et al., 1998, J Exp Med 188, 1185-90). High levels of APRIL mRNA have been found in a panel of tumor cell lines as well as in primary human tumors such as colorectal and lymphoid carcinomas.

[0005] APRIL serum levels were found to be elevated in patients with IgA nephropathy (McCarthy et al., 2011, J.Clin.Invest. 121(10):3991-4002).

[0006] APRIL plays a crucial role in the survival and proliferation of several B-cell malignancies (and potentially some solid tumors). APRIL also plays a major role in inflammatory diseases or autoimmune disorders. Therefore, strategies to antagonize APRIL are therapeutic targets for these multiple diseases. In fact, clinical trials targeting APRIL with TACI-Fc (atacicept) are currently underway for the treatment of several autoimmune diseases. However, TACI-Fc also targets BAFF, a factor involved in the maintenance of normal B cells. Antibodies directed against APRIL are described in WO9614328, WO2001 / 60397, WO2002 / 94192, WO9912965, WO2001 / 196528, WO9900518, and WO2010 / 100056. WO2010 / 100056 describes an antibody that specifically targets APRIL. The antibody for WO2010 / 100056 completely blocks the binding of APRIL to TACI and at least partially blocks its binding to BCMA. The antibody hAPRIL.01A completely blocks its binding to both BCMA and TACI. The hAPRIL.01A antibody inhibited B cell proliferation, survival, and antigen-specific immunoglobulin secretion in vitro and in vivo (Guadagnoli et al., 2011, Blood 117(25):6856-65). Furthermore, hAPRIL.01A inhibited the proliferation and survival of malignant cells representative of human CLL and MM disease in vitro and in vivo (Guadagnoli et al., 2011, Blood 117(25):6856-65; Lascano et al., 2013, Blood 122(24):3960-3; Tai et al., 2014, ASH poster 2098). Finally, hAPRIL.01A inhibited the secretion of antigen-specific IgA (Guadagnoli et al., 2011, Blood 117(25):6856-65). Anti-APRIL antibodies, including those that completely block binding to both BCMA and TACI, may be useful in treating IgA nephropathy, and there is a need to provide more effective formulations and dosing regimens for treating this disease. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention relates to anti-APRIL antibody formulations suitable for delivery via parenteral routes, particularly intravenous and / or subcutaneous routes. The formulations described herein can provide a high-concentration drug solution while maintaining acceptable viscosity, antibody solubility, levels of protein degradation and aggregation (especially during long-term storage), and pain at the administration site resulting from certain inactive components of the formulation.

[0008] In a first aspect, the present invention provides an antibody preparation comprising: Anti-APRIL antibodies at concentrations of approximately 20 mg / mL to 190 mg / mL; Approximately 10 mM L-histidine; Approximately 75 mM L-arginine; Approximately 3% by weight of sorbitol; Approximately 0.01% by weight of polysorbate 20; and pH is approximately 6.0 to 6.6.

[0009] In various embodiments, the formulation exhibits one or more, preferably two, three, or all four of the following characteristics: Having a viscosity of 16 cP or less, Free from glutamic acid or its salts, Having a gravimetric osmolality of approximately 250 mOsm / kg to approximately 390 mOsm / kg, It has an optical density (OD330) of approximately 1.0 or less at 330 nm.

[0010] In certain embodiments, the formulation maintains at least 96% purity of anti-APRIL antibody after 9 months of storage at 2-8°C following the manufacturing of the formulation. Preferably, the formulation maintains at least 95% purity of anti-APRIL antibody after 6 months of storage at 25°C following the manufacturing of the formulation.

[0011] Protein aggregation is generally thought to occur as a result of two instability factors: conformation and colloidality. Conformational stability is the difference in free energy between the folded and unfolded states of a protein. Although it cannot be directly measured as an energy value, it is related to the melting temperature T. M or a coagulation temperature T to a different degree Agg This allows for the qualitative determination of an increase or decrease in conformational stability between formulations. Colloidal stability is the result of a balance between attractive and repulsive intermolecular interactions. That is, the less protein interaction there is, the less likely the sample is to aggregate. The second virial coefficient of osmotic interaction can provide a tool for predicting the tendency of proteins to aggregate in the formulation state. In certain embodiments, the formulations of the present invention, when measured at 25°C, are approximately 2.5 x 10⁻⁶ -5 mol·mL / g 2 The second virial coefficient is as described above. The second virial coefficient can be measured, for example, by static light scattering or membrane osmotic pressure measurement, as is known in the art.

[0012] High-concentration antibody preparations are often described as "milky white." This is a characteristic resulting from turbidity in the sample, which may be a precursor to antibody self-association and aggregation. Measurement of optical density at 330 nm reflects this turbidity. In preferred embodiments, the preparation has an OD330 of approximately 0.8 or less.

[0013] In various embodiments, the anti-APRIL antibody of the formulation has a calculated isoelectric point (pI) of approximately 7.4 or higher. The pI of the protein is calculated using the amino acid pK values ​​described in Bjellqvist et al., Electrophoresis 1993, 14, 1023-1031.

[0014] In preferred embodiments, the anti-APRIL antibody is present in the formulation at a concentration of approximately 150 mg / mL. In particularly preferred embodiments, such formulations have a weight osmolality of approximately 290 mOsm / kg to approximately 390 mOsm / kg and most preferably an OD330 of approximately 0.8 or less.

[0015] In other preferred embodiments, the anti-APRIL antibody is present in the formulation at a concentration of approximately 20 mg / mL. In particularly preferred embodiments, such formulations have a weight osmolality of approximately 293 mOsm / kg to approximately 333 mOsm / kg and most preferably an OD330 of approximately 0.8 or less.

[0016] In certain embodiments, the formulation does not contain one or more of glycine, carbonate, HEPES, phosphate, citrate, and acetate, and most preferably does not contain any of them.

[0017] Preferably, the anti-APRIL antibody in the formulation is a humanized antibody containing a heavy chain variable region / light chain variable region pair selected from the group consisting of VH11.VL15, VH12.VL15, VH13.VL15, VH14.VL15, VH14_1.VL15, VH14_1C.VL15, VH14_1D.VL15, VH14_1E.VL15, and VH14_1G.VL15. These sequences are defined below. VH14_1G.VL15 is the most preferred.

[0018] As used herein, the term “about” refers to any given value plus or minus 10%, preferably plus or minus 5%.

[0019] In a related embodiment, the claims relating to a method for administering an anti-APRIL antibody to an individual that requires administration of an anti-APRIL antibody, the method comprising administering the formulation described herein to the individual by subcutaneous injection.

[0020] In another related embodiment, the claims relating to a method for administering an anti-APRIL antibody to an individual that requires such administration, the method comprising administering the formulation described herein to the individual by intravenous injection.

[0021] Various dosing schedules can be employed, as described below. In certain embodiments, this method involves repeating infusion or subcutaneous administration on a weekly ("QW") schedule for multiple cycles (e.g., 4 weeks, 6 weeks, 8 weeks, etc.). In other embodiments, this method involves repeating at least every two weeks ("every other week" or "Q2W" as used herein) on a schedule for multiple cycles (e.g., 4 weeks, 6 weeks, 8 weeks, etc.). Alternatively, this method involves repeating at least every four weeks ("Q4W") or once a month ("QMT") on a schedule for multiple cycles (e.g., 8 weeks, 12 weeks, 16 weeks, etc.). In certain embodiments, a front-loading dosing schedule is used. For example, a loading dosing schedule is followed by administration by either intravenous infusion or subcutaneous administration, repeated at least every two weeks to at least every four weeks, followed by a maintenance dosing schedule, which includes administration by either intravenous infusion or subcutaneous administration. Here, the maintenance administration schedule involves administering fewer anti-APRIL antibodies with each dose containing fewer anti-APRIL antibodies, or by administering them at longer intervals than during the loading dose schedule. In another example, the loading dose schedule includes administration by either intravenous infusion or subcutaneous injection, repeated at least daily, more preferably twice daily, for at least four days, followed by a maintenance dose schedule, which includes administration by either intravenous infusion or subcutaneous injection in a schedule such as QW, Q2W, Q4W, QM. In one embodiment, the loading dose schedule includes administering the antibody by intravenous infusion, and the maintenance dose schedule includes administering the antibody by subcutaneous injection. In another embodiment, both the loading dose schedule and the maintenance dose schedule include administering the antibody by subcutaneous injection. In yet another embodiment, both the loading dose schedule and the maintenance dose schedule include administering the antibody by intravenous infusion. This is not intended to be an exhaustive list of administration schedules.

[0022] As an example, the subcutaneous injection of this method involves administering approximately 2 mL of the antibody preparation to a patient's preferred injection site (e.g., thigh, abdomen, upper arm). In a preferred embodiment, the anti-APRIL antibody in the preparation is at a concentration of approximately 150 mg / mL, resulting in the administration of approximately 300 mg of anti-APRIL antibody in a single injection. In a specific embodiment, the subcutaneous injection of this method involves administering approximately 4 mL of the antibody preparation with an anti-APRIL antibody concentration of approximately 150 mg / mL (as a single injection or as 2 x 2 mL injections), resulting in the administration of approximately 600 mg of anti-APRIL antibody. The dosage and the number of injections required as part of a single dose can be adjusted as needed to achieve a desired total dose of anti-APRIL antibody of approximately 10 mg to approximately 1350 mg.

[0023] In some other embodiments, intravenous infusion of this method comprises (a) diluting a formulation of the first aspect and embodiments of the present invention in 0.9% physiological saline to a concentration of about 0.1 mg / mL to about 10 mg / mL; and (b) administering a total dose of anti-APRIL antibody of about 10 mg to about 1350 mg to an individual over about 2 hours using a single intravenous dose of the diluted formulation. Here again, as just one example, about 15 mL of a formulation with an anti-APRIL antibody concentration of about 20 mg / mL is added to about 235 mL of 0.9% physiological saline to provide an intravenous dose with a concentration of about 1.2 mg / mL.

[0024] In certain embodiments, a method for administering anti-APRIL antibodies to an individual requiring administration of anti-APRIL antibodies includes administering the formulation described herein by a loading / maintenance dose protocol. Such a protocol may include a loading component of the protocol comprising one or more doses of anti-APRIL antibodies at a concentration higher than that of the maintenance component of the loading / maintenance dose protocol; one or more doses of anti-APRIL antibodies at a frequency higher than that of the maintenance component of the loading / maintenance dose protocol; and / or one or more doses of anti-APRIL antibodies via a route different from that of the maintenance component of the loading / maintenance dose protocol.

[0025] For example, the loading component of a loading / maintenance protocol may include one or more intravenous doses of anti-APRIL antibody, and the maintenance component of a loading / maintenance protocol may include one or more subcutaneous doses of anti-APRIL antibody. In such examples, the load dose(s) may be high in concentration and / or administered more frequently than the maintenance dose(s).

[0026] As another example, the loading component of a loading / maintenance protocol may include one or more subcutaneous doses of anti-APRIL antibody, and the maintenance component of a loading / maintenance protocol may include one or more intravenous doses of anti-APRIL antibody. In such examples, the load dose(s) may be high in concentration and / or administered more frequently than the maintenance dose(s).

[0027] As another example, the loading component of a loading / maintenance protocol may include one or more subcutaneous doses of anti-APRIL antibody, and the maintenance component of a loading / maintenance protocol may include one or more subcutaneous doses of anti-APRIL antibody. In such examples, the concentration of the loading dose(s) may be high and / or the frequency of administration may be greater than that used in the maintenance dose(s).

[0028] In one embodiment, the loading dose comprises an intravenous infusion of 150-1350 mg of anti-APRIL antibody, followed by at least one infusion of the same amount at a first time interval, and the maintenance dose comprises administering one of the following: i) after the last loading dose infusion, at a first time interval, administering a smaller amount of anti-APRIL antibody, and continuing to administer the smaller amount for at least 12 weeks, and at least one subsequent dose at the same time interval; ii) after the last loading dose infusion, administering the same amount of anti-APRIL antibody at a second time interval, and continuing to administer the same amount and at least one subsequent dose at a second time interval for at least 12 weeks, where the second time interval is longer than the first time interval; or iii) after the last loading dose infusion, administering a smaller amount of anti-APRIL antibody at a second time interval, and continuing to administer the same amount and at least one subsequent dose at a second time interval for at least 12 weeks, where the maintenance dose may be administered by intravenous infusion or subcutaneous injection, preferably subcutaneous injection. In one embodiment, the loading dose comprises a subcutaneous injection of 150-1350 mg of anti-APRIL antibody, followed by at least one subcutaneous injection of the same amount at a first time interval, and the maintenance dose comprises administering one of the following: i) after the last loading dose infusion, at a first time interval, administering a smaller amount of anti-APRIL antibody, and continuing to administer the smaller amount for at least 12 weeks, and at least one subsequent dose at the same time interval; ii) after the last loading dose infusion, at a second time interval, administering the same amount of anti-APRIL antibody, and continuing to administer the same amount and at least one subsequent dose at a second time interval for at least 12 weeks, wherein the second time interval is longer than the first time interval; or iii) after the last loading dose infusion, at a second time interval, administering a smaller amount of anti-APRIL antibody, and continuing to administer the same amount and at least one subsequent dose at a second time interval for at least 12 weeks, wherein the maintenance dose may be administered by intravenous infusion or subcutaneous injection.

[0029] In another embodiment of the present invention, a product is provided comprising a material useful for treating the above-mentioned disorder. The product comprises a container, a label, and an accompanying leaflet. Suitable containers include, for example, bottles, vials, syringes (pre-filled or filled from the container at the time of administration), auto-injectors, injector pens, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for treating the above-mentioned condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a subcutaneous injection needle). At least one activator in the product is a container containing the anti-APRIL antibody composition according to the present invention. In certain embodiments, a label on or accompanying the container indicates that the composition is used to treat a selected above-mentioned condition, such as IgA nephropathy. The product may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. This product may further contain other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, and syringes. Furthermore, this product may include a package insert with instructions for use. The formulations of the present invention may be provided in various forms, such as single-use or multi-use vials containing the antibody formulation, or pre-filled syringes, auto-injectors, or injector pens containing the antibody formulation. The concentration of the anti-APRIL antibody in such containers is about 20 mg / mL to about 190 mg / mL, most preferably about 150 mg / mL. The volume of the formulation in such containers may be 0.5 mL to 50 mL, preferably between 1 mL and 10 mL, most preferably 1 mL, 2 mL, 3 mL, 4 mL, or 5 mL.

[0030] A brief explanation of arrays The sequences shown in the sequence listing are V of the preferred antibody for the formulations and methods described herein. H and V LThis relates to the amino acid sequences and coding DNA sequences of the domains, heavy chains, and light chains, such as the preferred heavy chain and light chain amino acid sequences and coding DNA sequences of the preferred antibodies described herein. Furthermore, the amino acid sequences of both the heavy chain and light chain CDRs of the antibodies described herein are presented. Table 1 below associates the sequence ID with each sequence. [Table 1] [Brief explanation of the drawing]

[0031] [Figure 1] This figure shows, in tabular format, the turbidity, appearance, and pH results of various VH14_1G.VL15 antibody preparations after temperature stress, freeze / thaw, and shaking stress. [Figure 2] This figure shows, in tabular format, the results of the percentage purity of various VH14_1G.VL15 antibody preparations after temperature stress, freeze / thaw, and shaking stress, as measured by SE-UPLC. [Figure 3] This figure shows a graph of SE-UPLC percentage purity, measured by peak area, of various VH14_1G.VL15 antibody preparations after storage at -70°C for 12 weeks. [Figure 4] This figure shows a graph of SE-UPLC percentage purity, measured by peak area, of various VH14_1G.VL15 antibody preparations after storage at 2-8°C for 12 weeks. [Figure 5] This figure shows a graph of SE-UPLC percentage purity, measured by peak area, of various VH14_1G.VL15 antibody preparations after 12 weeks of storage at 25°C. [Figure 6] This figure shows a graph of SE-UPLC percentage purity, measured by peak area, of various VH14_1G.VL15 antibody preparations after 12 weeks of storage at 45°C. [Figure 7]This figure shows, in tabular form, the results of the percentage purity of various VH14_1G.VL15 antibody preparations after temperature stress, freeze / thaw, and shaking stress, as measured by CEX-UPLC. [Figure 8] This figure shows the ln purity (%) as a percentage of time (days) at 25°C for various VH14_1G.VL15 antibody preparations. [Figure 9] This figure shows Arrhenius relation plots (ln kobs vs. 1 / T (Kelvin)) for four VH14_1G.VL15 antibody preparations at 2-8°C, 25°C, and 45°C. [Figure 10] This figure shows the ln purity (%) over time (days) for four VH14_1G.VL15 antibody preparations at 25℃. [Figure 11] This figure shows Arrhenius relationship plots (ln kobs vs. 1 / T (Kelvin)) at 25°C and 45°C for various VH14_1G.VL15 antibody preparations. [Figure 12] This figure shows the hydrodynamic radius (nm) and % mass of species populations in various VH14_1G.VL15 antibody test samples. [Figure 13] This figure shows the turbidity, appearance, and pH results of various VH14_1G.VL15 antibody preparations after temperature stress in a table format. [Figure 14] This figure shows the HIAC particle counting results in a table format for various VH14_1G.VL15 antibody preparations after temperature stress. [Figure 15] This figure shows, in tabular format, the percentage purity results of various VH14_1G.VL15 antibody preparations after temperature stress, as measured by SE-HPLC. [Figure 16] This figure shows, in tabular format, the percentage purity results of various VH14_1G.VL15 antibody preparations after temperature stress, as measured by CE-HPLC. [Figure 17] This figure shows the ln purity (%) as a percentage of time (days) at 25°C for various VH14_1G.VL15 antibody preparations. [Figure 18]This figure shows the ln purity (%) as a percentage of time (days) at 45°C for various VH14_1G.VL15 antibody preparations. [Figure 19] This figure shows Arrhenius relationship plots (ln kobs vs. 1 / T (Kelvin)) at 25°C and 45°C for various VH14_1G.VL15 antibody preparations. [Figure 20] This figure shows the ln purity (%) as a percentage of time (days) at 5°C for various VH14_1G.VL15 antibody preparations. [Figure 21] This figure shows the ln purity (%) as a percentage of time (days) at 25°C for various VH14_1G.VL15 antibody preparations. [Figure 22] This figure shows the ln purity (%) as a percentage of time (days) at 45°C for various VH14_1G.VL15 antibody preparations. [Figure 23] This figure shows Arrhenius relation plots (ln kobs vs. 1 / T (Kelvin)) for various VH14_1G.VL15 antibody preparations at 5°C, 25°C, and 45°C. [Figure 24] This figure shows the clinical trial protocol for evaluating the safety, tolerability, pharmacokinetics (PK), and disease progression (PD) of intravenously administered VH14_1G.VL15. [Figure 25] This figure shows the mean serum BION-1301 concentration + / - SD versus nominal time after IV administration of various doses of BION-1301. [Figure 26] This figure shows the mean free APRIL concentration in serum as a percentage of the initial baseline concentration after intravenous administration of various doses of BION-1301. [Figure 27A] This figure shows the mean change in serum immunoglobulin IgA, IgG, and IgM concentrations in the serum after IV administration of various doses of BION-1301, expressed as a percentage of the initial baseline concentration. [Figure 27B] This figure shows the mean change in serum immunoglobulin IgA, IgG, and IgM concentrations in the serum after IV administration of various doses of BION-1301, expressed as a percentage of the initial baseline concentration. [Figure 27C]This figure shows the mean change in serum immunoglobulin IgA, IgG, and IgM concentrations in the serum after IV administration of various doses of BION-1301, expressed as a percentage of the initial baseline concentration. [Figure 27D] This figure shows the mean change in serum immunoglobulin IgA, IgG, and IgM concentrations in the serum after IV administration of various doses of BION-1301, expressed as a percentage of the initial baseline concentration. [Figure 27E] This figure shows the mean change in serum immunoglobulin IgA, IgG, and IgM concentrations in the serum after IV administration of various doses of BION-1301, expressed as a percentage of the initial baseline concentration. [Figure 27F] This figure shows the mean change in serum immunoglobulin IgA, IgG, and IgM concentrations in the serum after IV administration of various doses of BION-1301, expressed as a percentage of the initial baseline concentration. [Figure 28A] This figure shows the percentage change in serum immunoglobulin IgA, IgG, and IgM concentrations in the serum at 29 days after IV administration of various doses of BION-1301, expressed as a percentage of the initial baseline concentration. [Figure 28B] This figure shows the percentage change in serum immunoglobulin IgA, IgG, and IgM concentrations in the serum at 85 days after IV administration of various doses of BION-1301, expressed as a percentage of the initial baseline concentration. [Figure 29] This figure shows the clinical trial protocol for evaluating the safety, tolerability, pharmacokinetics (PK), and disease progression (PD) of BION-1301 administered intravenously versus via suppository administration. [Figure 30] This figure shows the mean (±SD) serum concentration of BION-1301 versus the time (semi-logarithmic scale) after a single IV or SC administration of 300 mg of BION-1301. [Figure 31A] This figure shows the mean (±SD) fAPRIL concentration after a single IV or SC administration of 300 mg of BION-1301. [Figure 31B]This figure shows the mean (±SD) percentage change in fAPRIL compared to baseline after a single IV or SC administration of 300 mg of BION-1301. [Figure 32A] This figure shows the mean percentage change in serum immunoglobulin levels over time relative to baseline (32A=IgA, 32B=IgG, 32C=IgM) after a single IV or SC administration of 300 mg of BION-1301. [Figure 32B] This figure shows the mean percentage change in serum immunoglobulin levels over time relative to baseline (32A=IgA, 32B=IgG, 32C=IgM) after a single IV or SC administration of 300 mg of BION-1301. [Figure 32C] This figure shows the mean percentage change in serum immunoglobulin levels over time relative to baseline (32A=IgA, 32B=IgG, 32C=IgM) after a single IV or SC administration of 300 mg of BION-1301. [Figure 33] This figure shows the reduction in serum IgA and Gd-IgA1 in single-dose escalation (SAD) and multiple-dose escalation (MAD) trials (ADU-CL-19; ClinicalTrials.gov Identifier:NCT03945318) of BION-1301 administered by intravenous (IV) infusion in healthy human volunteers. [Figure 34] This figure shows changes in free APRIL levels, Gd-IgA1 levels, mesangial cell proliferation, and proteinuria in IgAN patients after treatment with BION-1301. [Modes for carrying out the invention]

[0032] Accordingly, the present invention relates to an antibody, its use, and formulation effective for the treatment of IgA nephropathy as described herein. The antibody described herein is exemplified by an anti-hAPRIL antibody (VH14_1G.VL15, or also known as BION-1301 as used in clinical trials) having the amino acid sequence of SEQ ID NO: 28 for the heavy chain and SEQ ID NO: 30 for the light chain. This antibody has been shown in healthy volunteers to block the binding of human APRIL to human B cell maturation antigen (BCMA), transmembrane activator, calcium regulator, and cyclophylline ligand interactor (TACI), thereby significantly reducing IgA levels. Such a reduction in IgA levels is expected to be similar in subjects with IgA nephropathy, and therefore is expected to have a significant therapeutic effect. Further characteristics and discussion of the antibody useful in the formulation and method described herein can be found in International Publication No. 2016110587, the disclosure of which is incorporated herein by reference as it relates to an anti-APRIL antibody useful for the treatment of IgA nephropathy. The formulations and methods provided herein are expected to provide stable formulations of anti-hAPRIL antibodies for the treatment of IgA nephropathy.

[0033] In the description of the present invention, a sequence similarity of at least 90% is understood to mean, more preferably, at least 95%, for example, at least 99% sequence similarity.

[0034] Those skilled in the art will understand that "sequence similarity" refers to the degree to which individual nucleotide or peptide sequences are similar. The degree of similarity between two sequences is based on the degree of identity combined with the degree of conservative change. The percentage of "sequence similarity" is the percentage of identical or conservatively modified amino acids or nucleotides. That is, "sequence similarity" = (% sequence identity) + (% conservative change).

[0035] For the purposes of this invention, “conservative variation” and “identity” are considered to be subcategories of the broader term “similarity.” Therefore, whenever the term “sequence similarity” is used, it encompasses both sequence “identity” and “conservative variation.” According to certain embodiments, conservative variation is ignored, and % sequence similarity refers to % sequence identity.

[0036] The term “sequence identity” is known to those skilled in the art. To determine the degree of sequence identity shared by two amino acid sequences or two nucleic acid sequences, sequences are aligned for the purpose of optimal comparison (for example, gaps can be introduced in the sequence of the first amino acid sequence or nucleic acid sequence to achieve optimal alignment with the second amino acid sequence or nucleic acid sequence). Such alignment may be performed over the entire length of the sequences being compared. Alternatively, the alignment may be performed over a short comparison length, e.g., about 20, about 50, about 100 or more nucleic acids / bases or amino acids.

[0037] Next, amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The degree of identity shared between sequences is typically expressed as an identity percentage between the two sequences, which corresponds to the number of identical positions shared by identical residues in the sequences (i.e., % identity = number of identical residues at corresponding positions / total number of positions x 100). Preferably, the two sequences being compared are the same or substantially the same length.

[0038] The percentage of "conservative changes" can be determined in the same way as the percentage of sequence identity. However, in this case, changes at specific positions in the amino acid or nucleotide sequence that are likely to retain the functional properties of the original residue are recorded as if no change has occurred.

[0039] In the case of amino acid sequences, the relevant functional properties are the physicochemical properties of the amino acids. Conservative substitutions of amino acids in the polypeptide of the present invention can be selected from other members of the class to which the amino acid belongs. For example, in the field of protein biochemistry, it is well known that amino acids belonging to a group of amino acids having a particular size or property (such as charge, hydrophobicity, and hydrophilicity) can be substituted with other amino acids without substantially altering the activity of the protein, especially the activity in regions of the protein not directly related to biological activity (see, for example, Watson, et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Edition 1987)). For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Arg and its inverse for Lys to maintain a positive charge, Glu and its inverse for Asp to maintain a negative charge, Ser and its inverse for Thr to maintain a free -OH group, and Gln and its inverse for Asn to maintain a free -NH2 group.

[0040] In the case of nucleotide sequences, the relevant functional properties are primarily the biological information that specific nucleotides carry within the open reading frame of the sequence in relation to the transcription and / or translation mechanisms. It is a common understanding that the genetic code is degenerate (or redundant), and that multiple codons can carry the same information with respect to the amino acids they encode. For example, in certain species, the amino acid leucine is encoded by the UUA, UUG, CUU, CUC, CUA, CUG codons (or TTA, TTG, CTT, CTC, CTA, CTG in the case of DNA), and the amino acid serine is designated by the UCA, UCG, UCC, UCU, AGU, AGC (or TCA, TCG, TCC, TCT, AGT, AGC in the case of DNA). Nucleotide changes that do not alter the translated information are considered conservative changes.

[0041] In this invention, it is most preferable to use BLAST (Basic Local Alignment Tool) to determine the percentage identity and / or percentage similarity between nucleotide sequences or amino acid sequences.

[0042] Queries using the BLASTn, BLASTp, BLASTx, tBLASTn, and tBLASTx programs of Altschul et al. (1990) can be submitted from the online version of BLAST accessible via http: / / www.ncbi.nlm.nih.gov. Alternatively, a stand-alone version of BLAST downloadable via the NCBI Internet site (e.g., version 2.2.29 (released on January 3, 2014)) can be used. Preferably, BLAST queries are run with the following parameters. To determine percent identity and / or percent similarity between amino acid sequences: algorithm: blastp; word size: 3; scoring matrix: BLOSUM62; gap cost: existence: 11, extension: 1; composition adjustment: conditional compositional score matrix adjustment; filter: off; mask: off. To determine the percentage of identity and / or similarity between nucleotide sequences: algorithm: blastn; word size: 11; maximum number of matches within query: 0; match / mismatch scores: 2, -3; gap cost: existence: 5, extension: 2; filter: low complexity regions; mask: mask with only lookup table.

[0043] The percentage of "conservative changes" can be determined in the same manner as percent sequence identity using the indicated algorithms and computer programs. For example, some computer programs such as BLASTp present the number / percentage of positives (= similarities) and the number / percentage of identities. The percentage of conservative changes can be derived therefrom by subtracting the percentage of identities from the percentage of positives / similarities (percentage of conservative changes = percentage of similarity - percentage of identity).

[0044] According to a further aspect, the invention relates to an isolated polynucleotide encoding the V H domain and / or V L domain of an antibody according to the invention, or the heavy and / or light chains of an antibody. V HThe polynucleotide sequence encoding the domain is preferably a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, and 23, preferably SEQ ID NOs: 13, 15, or 23, and more preferably SEQ ID NO: 23, and having at least 90% sequence similarity. L The polynucleotide sequence encoding the domain is preferably a polynucleotide sequence having at least 90% sequence similarity to the polynucleotide sequence of SEQ ID NO: 25. The polynucleotide sequence encoding the heavy chain is preferably a polynucleotide sequence having at least 90% sequence similarity to the polynucleotide sequence of SEQ ID NO: 27. The polynucleotide sequence encoding the light chain is preferably a polynucleotide sequence having at least 90% sequence similarity to the polynucleotide sequence of SEQ ID NO: 29.

[0045] The present invention further relates to an expression unit comprising a plurality of expression vectors comprising a plurality of polynucleotides according to the present invention under the control of a preferred regulatory sequence, wherein the plurality of polynucleotides are the V of the antibody according to the present invention. H Domain or heavy chain and V L Encodes a domain or light chain. The expression unit is V H Polynucleotide sequences encoding a domain or heavy chain and V L The expression vector can be designed so that a polynucleotide sequence encoding a domain or light chain may be present on the same expression vector. Therefore, the expression unit may contain a single vector. Alternatively, V H Polynucleotide sequences encoding a domain or heavy chain and V L The polynucleotide sequences encoding the domain or light chain may be on different expression vectors. In such embodiments, the expression unit comprises multiple, for example, two, expression vectors.

[0046] Further aspects of the present invention relate to a host cell comprising a plurality of polynucleotides and / or expression units of the present invention. The expression units are preferably V HPolynucleotide sequences encoding a domain or heavy chain and V L This is an expression unit that includes an expression vector containing both a domain and a polynucleotide sequence encoding a light chain.

[0047] treatment The antibody formulations and methods of use of the present invention are suitable for the treatment of conditions known or expected to be improved by blocking the interaction between human april and BCMA and / or TACI. As already known in the art, blocking the interaction between human april and BCMA and / or TACI inhibits the proliferation and / or survival of immune cells, and therefore such blocking of the proliferation and / or survival of immune cells may be valuable in the treatment of conditions such as inflammatory diseases, diseases mediated by Ig secretion and / or autoimmune diseases, where such blocking is beneficial. Blocking the interaction between human april and BCMA and / or TACI may also be beneficial in the treatment of cancer.

[0048] Furthermore, the antibodies of the present invention may be beneficial in the treatment of other conditions in which a reduction in immunoglobulin levels, such as IgA1 or IgA2 levels, IgG, IgM, and Gd-IgA levels, is beneficial, such as conditions related to Ig secretion, particularly IgA secretion, Ig overproduction, such as IgA1 or IgA2 levels, IgG, IgM, and Gd-IgA overproduction, particularly IgA overproduction, or Ig deposition, particularly IgA deposition. Examples of such conditions, but not limited to, include IgA nephropathy and other forms of glomerulonephritis, celiac disease, bullous pemphigoid, Henoch-Schonlein purpura, and other autoimmune diseases associated with Ig deposition. The formulations and methods of use of the anti-hAPRIL antibodies described herein are particularly suitable for the treatment of IgA nephropathy.

[0049] IgA nephropathy (IgAN) is the leading cause of primary glomerulonephritis (Berthelot L, et al., 2015, Kidney Int, 88:815-22). The prognosis for IgAN patients varies and depends on several factors. In patients with mild to moderate proteinuria and normal renal function on biopsy, 2.8% developed end-stage renal disease (ESRD) at 25-year follow-up (Knoop T, et al., 2017, Nephrol Dial Transplant 32:1841-50), resulting in dialysis or kidney transplantation. In the general IgAN population, 14%–39% have been reported to develop ESRD within 20 years of diagnosis (Berthoux FC, et al., 2008, Semin Nephrol 28:4-9; Manno C, et al., 2007, Am J Kidney Dis, 49:763-75). A key early stage in the pathology of IgAN is the production of autoantibodies against galactose-deficient IgA1 (gd-IgA1), which leads to the formation of immune complexes that cause inflammation, mesangial cell proliferation, and complement activation resulting in kidney damage. APRIL binds to BCMA and TACI, promoting the proliferation and survival of human plasmablasts / plasma cells (O'Connor BP, et al., 2004, J Exp Med, 199:91-8; Moreaux J, et al., 2007, Haematologica, 92:803-11). APRIL contributes to IgAN by promoting B-cell class switching to IgA-producing plasma cells (He B, et al., 2010, NatImmunol 11:836-45). Importantly, anti-APRIL antibodies reduce renal injury, serum IgA, IgA deposition, and proteinuria in IgAN mouse models.

[0050] Serum Gd-IgA1 levels have been reported to be significantly higher in IgAN patients than in disease controls and healthy controls. In IgAN patients, serum Gd-IgA1 levels were significantly correlated with estimated glomerular filtration rate, serum IgA levels, and tubular atrophy / interstitial fibrosis. CKD progression was more frequent in IgAN patients with high serum Gd-IgA1 levels than in patients with low serum Gd-IgA1 levels. A Cox proportional hazards model, after adjusting for several confounding factors, showed that high Gd-IgA1 levels were an independent risk factor for CKD progression. Kim et al., J.Clin.Med. 2020 Nov 4;9(11):3549.doi:10.3390 / jcm9113549.

[0051] A humanized APRIL antagonist monoclonal antibody (described herein) is under development for the treatment of IgAN and is undergoing clinical trials in healthy volunteers (see clinicaltrials.gov NCT03945318). Blocking APRIL with an anti-hAPRIL antibody has been shown to significantly reduce IgA and IgM levels and decrease IgG levels in healthy cynomolgus monkeys, and similar results have been shown in healthy human volunteers. Furthermore, this blockade reduced Gd-IgA1 in healthy human volunteers. Consequently, blocking APRIL in IgAN patients is expected to lead to a decrease in IgA, IgG, and IgM levels, as well as a reduction in corresponding gd-IgA1, autoantibody and immune complex deposition against gd-IgA1, and renal damage.

[0052] Myette et al. (2019, Kidney International 96(1):104-116) demonstrated the efficacy of a mouse anti-APRIL antibody in a mouse model of IgA nephropathy, and the human antibody VIS649 is included in a Phase 2 clinical trial (clinicaltrials.gov NCT04287985).

[0053] General definition The term “antibody” refers to any form of antibody that exhibits a desired biological activity by inhibiting the binding of a ligand to a receptor or by inhibiting ligand-induced signaling of a receptor. In this specification, biological activity includes blocking the binding of APRIL to its receptor BCMA and / or TACI. Thus, “antibody” is used in its broadest sense and encompasses, in particular, monoclonal antibodies (including full-length monoclonal antibodies) and, for example, multispecific antibodies (e.g., bispecific antibodies) or antibody fragments based on Duobody® technology (Genmab) or Hexabody® technology (Genmab).

[0054] "Antibody fragment" and "antibody-binding fragment" refer to antigen-binding fragments and analogues of an antibody, typically comprising at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least a portion of the binding specificity of the parent antibody. Typically, the antibody fragment retains at least 10% of the parent binding activity, when its activity is expressed in molar terms. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody to the target. Examples of antibody fragments, but not limited to these, include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv, unibodies (Genmab technology); nanobodies (Ablynx technology); domain antibodies (Domantis technology); and multispecific antibodies formed from antibody fragments. The manipulated antibody variants are outlined in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.

[0055] A "Fab fragment" consists of one light chain and one heavy chain with a CH1 group and a variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0056] The "Fc" region is the C of the antibody. H 1 and CH It contains two heavy chain fragments, each containing two domains. The two heavy chain fragments have two or more disulfide bonds and C H The three domains are held together by hydrophobic interactions.

[0057] "Fab' fragment" consists of one light chain and V H Domain and C H 1 domain, and C H 1 domain and C H It includes a portion of one heavy chain containing the region between the two domains, which allows for the formation of an interchain disulfide bond between the two heavy chains of the two Fab' fragments, thereby forming an F(ab')2 molecule.

[0058] The "F(ab')2 fragment" consists of two light chains and C H 1 domain and C H It contains two heavy chains, each containing a portion of the constant region between the two domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0059] The "Fv region" includes the variable regions of both the heavy and light chains, but does not include the steady region.

[0060] A "single-chain Fv antibody" (or "scFv antibody") is an antibody with V H and V L This refers to antibody fragments containing domains, which are present within a single polypeptide chain. Generally, an Fv polypeptide is a V polypeptide that allows scFv to form the desired structure for antigen binding. H Domain and V LIt further includes a polypeptide linker between the domains. For a review of scFv, see Pluckthun, 1994, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also International Publication No. 88 / 01649 and U.S. Publications No. 4,946,778 and No. 5,260,203.

[0061] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragment is the same polypeptide chain (V H -V L or V L -V H ) Light chain variable domain (V L ) is linked to the heavy chain variable domain (V H ) includes. By using a short linker to allow pairing between two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. The diabody is fully described, for example, in European Patent Application Publication No. 404,097; International Publication No. 93 / 11161; and Holligeretal., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0062] "Duobody" is a bispecific antibody that possesses a normal IgG structure (Labrijn et al., 2013, Proc. Natl. Acad. Sci. USA 110(13):5145-5150).

[0063] "Hexabodies" are antibodies that possess high lethality while maintaining a regular structure and specificity (Diebolder et al., 2014, Science 343 (6176): 1260-3).

[0064] A "domain antibody fragment" is an immunologically functional immunoglobulin fragment that contains only the variable region of the heavy chain or the variable region of the light chain. In some cases, it may contain two or more V H The region is covalently linked to the peptide linker, creating a bivalent domain antibody fragment. The two Vs of the bivalent domain antibody fragment H The region may target the same or different antigens.

[0065] The antibody fragment of the present invention may include a substantial portion of a constant region that enables dimerization (or polymerization) of a heavy chain with reduced disulfide-binding capacity, where, for example, at least one of the hinge cysteines normally involved in inter-heavy-chain disulfide linkage is modified as described herein. In another embodiment, an antibody fragment, for example, one including an Fc region, may retain at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life regulation, ADCC (antibody-dependent cell-mediated cytotoxicity) function, and / or complement binding (for example, if the antibody has a glycosylation profile required for ADCC function or complement binding).

[0066] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence of an antibody originating from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence of an antibody originating from another species or belonging to another antibody class or subclass, as well as a fragment of such an antibody, insofar as it exhibits the desired biological activity (see, for example, U.S. Patent Application Publication No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855).

[0067] As used herein, the term “humanized antibody” refers to an antibody form that includes sequences derived from non-human (e.g., mouse) antibodies and human antibodies. Such antibodies include minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FR region are from a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically the region of a human immunoglobulin. Humanized forms of rodent antibodies essentially contain the same CDR sequence as the parent rodent antibody, but may include certain amino acid substitutions to increase affinity, to improve the stability of the humanized antibody, or for other reasons.

[0068] The antibodies of the present invention also include antibodies having modified (or blocked) Fc regions to provide modified effector functions. See, for example, U.S. Patent No. 5,624,821; International Publication No. 2003 / 086310; International Publication No. 2005 / 120571; International Publication No. 2006 / 0057702; Presta, 2006, Adv. Drug Delivery Rev. 58:640-656. Such modifications can be used to enhance or suppress various immune system responses and may have beneficial effects on diagnostics and therapeutics. Modifications to the Fc region include changes in amino acids (substitutions, deletions, and insertions), glycosylation or deglycosylation, and the addition of multiple Fc regions. Modifications to the Fc region may also alter the half-life of the antibody in therapeutic antibodies, and a longer half-life may lead to reduced administration frequency, improved convenience, and reduced material usage. See Presta, 2005, J. Allergy Clin. Immunol. 116:731 at 734-35.

[0069] The antibodies of the present invention also include antibodies having an intact Fc region that provides complete effector function, such as an isotype IgG1 antibody that induces complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells.

[0070] Antibodies can also be conjugated (e.g., covalently linked) to molecules that improve the stability of the antibody during storage or extend its half-life in vivo. Examples of molecules that extend the half-life include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-conjugated and PEGylated derivatives of antibodies can be prepared using techniques well known in the art. See, for example, Chapman, 2002, Adv. Drug Deliv. Rev. 54:531-545; Anderson and Tomasi, 1988, J. Immunol. Methods 109:37-42; Suzuki et al., 1984, Biochim. Biophys. Acta 788:248-255; and Brekke and Sandlie, 2003, Nature Rev. 2:52-62.

[0071] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable region is defined by sequence alignment as amino acid residues from the “complementarity-determining region” or “CDR,” for example, residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) within the light chain domain, and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) within the heavy chain variable domain (Kabat et al., 1991, Sequences of proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of This includes residues from the Health, Bethesda, Md. and / or structurally defined "hypervariable loops" (HVLs), such as residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) within the light chain variable domain, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) within the heavy chain variable domain (see Chothia and Leskl, 1987, J.Mol.Biol.196:901-917).

[0072] A "framework" or "FR" residue or sequence is a variable domain residue or sequence other than a CDR residue as defined herein.

[0073] In certain embodiments of the present invention, the antibodies may be isolated antibodies. “Isolated antibodies” are those identified, isolated, and / or recovered from components of their natural environment. The contaminants in the natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to (1) more than 95% by weight, most preferably more than 99% by weight, of the antibody as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) until homogenized by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Because at least one component of the antibody’s natural environment is absent, isolated antibodies include in situ antibodies from recombinant cells. However, typically, isolated antibodies are prepared by at least one purification step.

[0074] An "isolated" nucleic acid molecule is a nucleic acid molecule identified and separated from at least one contaminating nucleic acid molecule that normally associates with the natural source of antibody nucleic acids. An isolated nucleic acid molecule is a nucleic acid molecule in a form or setting other than those found in nature. Therefore, isolated nucleic acid molecules are distinct from nucleic acid molecules present in natural cells. However, isolated nucleic acid molecules include, for example, nucleic acid molecules found in cells that normally express antibodies, where the nucleic acid molecule is located at a different chromosomal position than in natural cells.

[0075] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies in the population are identical except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. The modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and is not to be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by the hybridoma method first described by Kohler et al., (1975, Nature 256:495) or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., 1991, Nature 352:624-628 and Marks et al., 1991, J.Mol.Biol.222:581-597. Monoclonal antibodies as used herein specifically include chimeric antibodies.

[0076] As used herein, the term "immune cells" includes cells of hematopoietic origin that play a role in the immune response. Examples of immune cells include lymphocytes such as B cells and T cells, myeloid cells such as natural killer cells and monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0077] As used herein, “immunoconjugate” refers to an anti-human APRIL antibody or fragment thereof conjugated with a therapeutic portion, such as a bacterial toxin, cytotoxic agent, or radiotoxin. The toxic portion can be conjugated to the antibody of the present invention using methods available in the art.

[0078] As used herein, a sequence "variant" or "variant sequence" refers to a sequence that differs from the disclosed sequence in one or more amino acid residues but retains the biological activity of the parent molecule. The present invention includes variants of antibodies clearly disclosed by various sequences. For the VH domain CDR1, CDR2, and CDR3 sequences, according to some embodiments, the variant sequence may include up to six amino acid substitutions for the CDR1, CDR2, and CDR3 sequences incorporated together, e.g., 1, 2, 3, 4, 5, or 6 amino acid substitutions. Similarly, for the VL domain CDR1, CDR2, and CDR3 sequences, according to some embodiments, the variant sequence may include up to six amino acid substitutions for the CDR1, CDR2, and CDR3 sequences incorporated together, e.g., 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0079] A “conservatively modified variant” or “conservative amino acid substitution” refers to an amino acid substitution that is known to those skilled in the art and can generally be performed without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson, et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p.224 (4th Edition 1987)).

[0080] As used herein, the term “about” means a value that is within an acceptable margin of error for a particular value, as determined by those skilled in the art, and is subject in part to the limitations of the method or method of measuring or determining the value, i.e., the measuring system. For example, “about” may, by convention in the art, mean within 1 or a standard deviation greater than 1. Alternatively, “about” or “essentially including” may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, these terms may mean a value of one order of magnitude or five times or less. Where a particular value is provided in the application and claims, unless otherwise specified, the meaning of “about” or “essentially including” shall be assumed to be within an acceptable margin of error for that particular value.

[0081] The term “multiple” shall be understood to mean one or more. Depending on the context of its use, “multiple” may refer to any preferred number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In certain embodiments, “a number of” may mean “a plurality.” Depending on the context of its use, “multiple” may refer to any preferred number selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0082] "Specifically" binding, when referring to ligand / receptor, antibody / antigen, or other binding pairs, indicates a binding reaction that determines the presence of a protein in a heterogeneous population and / or other biologic, such as APRIL. Therefore, under specified conditions, a particular ligand / antigen will bind to a particular receptor / antibody and will not bind in large quantities to other proteins present in the sample.

[0083] When “administration,” “treatment,” and “therapy” apply to animals, humans, experimental subjects, cells, tissues, organs, or body fluids, the term “administration,” “treatment,” and “therapy” refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or body fluid. “Administration,” “treatment,” and “therapy” may also refer to, for example, a therapeutic method, a pharmacokinetic method, a diagnostic method, a research method, and an experimental method. Treatment of cells includes the contact of a reagent with cells, as well as the contact of a reagent with a liquid in which the liquid is in contact with cells. “Administration,” “treatment,” and “therapy” also mean in vitro and ex vivo treatment of cells, for example, with a reagent, diagnostic, conjugate composition, or with another cell. In this specification of the present invention, the terms “in vitro” and “ex vivo” have similar meanings and may be used interchangeably. In the present invention, the treatment of a “condition” includes any therapeutic use, including the prophylactic and therapeutic use of anti-human APRIL antibodies. Thus, the term “condition” may refer not only to a disease state, but also to a physiological state in a prophylactic environment in which physiological functions are not altered to an adverse state.

[0084] Antibody DNA can also be modified, for example, by substituting the coding sequences of human heavy and light chain constant domains for homologous mouse sequences (U.S. Patent No. 4,816,567; Morrison, et al., 1984, Proc. Natl Acad. Sci. USA, 81:6851), or by covalently linking all or part of the coding sequence of a non-immunoglobulin material (e.g., a protein domain) to the immunoglobulin coding sequence. Typically, such non-immunoglobulin material can be used to substitute the constant domain of an antibody or to substitute the variable domain of one antigen-binding site of an antibody, creating a chimeric bivalent antibody containing one antigen-binding site with specificity for a particular antigen and another antigen-binding site with specificity for a different antigen.

[0085] The amino acid sequence variants of the anti-human APRIL antibody of the present invention are prepared by introducing appropriate nucleotide changes into the coding DNA or by peptide synthesis. Such variants include, for example, deletions from and / or insertions and / or substitutions of residues in the amino acid sequence shown for the anti-APRIL antibody. Any combination of deletions, insertions, and substitutions is performed to arrive at the final construct, provided that the final construct has the desired characteristics. Amino acid modifications may also alter the post-translational processes of the anti-APRIL antibody, such as changes in the number or location of glycosylation sites.

[0086] A useful method for identifying specific residues or regions of an anti-APRIL antibody polypeptide that are favorable sites for mutagenesis is called "alanine scanning mutagenesis" and was described by Cunningham and Wells, 1989, Science 244:1081-1085. Here, a target residue or group of residues is identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and substituted with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) that affects the interaction between the amino acid and the APRIL antigen. The amino acid residue that exhibits functional sensitivity to the substitution is then refined by introducing further or other variants at or to the substitution site. Thus, while the sites for introducing amino acid sequence diversity are predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a specific site, Ala scanning or random mutagenesis is performed at the target codon or region, and variants of the expressed anti-APRIL antibody are screened for the desired activity.

[0087] Typically, an amino acid sequence variant of an anti-APRIL antibody has an amino acid sequence that has at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, 98%, or 99% amino acid sequence similarity to the amino acid sequence of either the heavy chain or light chain of the original antibody. This sequence similarity or homology is as defined above.

[0088] Antibodies possessing the characteristics identified as desirable in this specification can be screened for increased biological activity in vitro or for suitable binding affinity. To screen for antibodies that bind to the same epitope on human APRIL as hAPRIL.01A, a standard cross-blocking assay described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) can be performed. Antibodies that bind to the same epitope may cross-block in such an assay, but not all cross-blocking antibodies necessarily bind to exactly the same epitope. This is because cross-blocking can result from steric hindrance of antibody binding due to the antibody binding to overlapping epitopes or even nearby non-overlapping epitopes.

[0089] Alternatively, for example, as described in Champe et al., 1995, J. Biol. Chem. 270:1388-1394, this can be done to determine whether the antibody binds to the target epitope. "Alanine scanning mutagenesis" as described in Cunningham and Wells, 1989, Science 244:1081-1085, or several other forms of point mutagenesis of amino acid residues in human APRIL, can also be used to determine the functional epitope of the anti-APRIL antibody of the present invention.

[0090] Another method for mapping antibody epitopes is to study the binding of antibodies to synthetic linear and CLIPS peptides, which can be screened using credit card-style mini PEPSCAN cards, as described by Slootstra et al. (Slootstra et al., 1996, Mol. Diversity 1:87-96) and Timmerman et al. (Timmerman et al., 2007, J. Mol. Recognit. 20:283-299). The binding of antibodies to each peptide is determined by a PEPSCAN-based enzyme-linked immunosorbent assay (ELISA).

[0091] Further antibodies that bind to the same epitope as hAPRIL.01A can be obtained, for example, by screening antibodies produced against APRIL for epitope binding, or by immunizing animals with peptides containing a human APRIL fragment that includes the epitope sequence. Antibodies that bind to the same functional epitope may be expected to exhibit similar APRIL binding as well as similar biological activities such as BCMA and TACI blocking activity, and such activities can be confirmed by functional assays of the antibodies.

[0092] The antibody can be selected from any class of immunoglobulin, such as IgM, IgG, IgD, IgA, and IgE. Preferably, the antibody is an IgG antibody. Any isotype of IgG, such as IgG1, IgG2, IgG3, and IgG4, can be used. Variants of IgG isotypes are also intended. The antibody may contain sequences derived from multiple classes or isotypes. Optimization of the constant domain sequence necessary to produce the desired biological activity is readily achieved by screening the antibody using biological assays known in the art or described herein.

[0093] Similarly, light chains of any class can be used in the compositions and methods described herein. Specifically, kappa, lambda, or their variants are useful in these compositions and methods.

[0094] The antibodies and antibody fragments of the present invention may also be used with cytotoxic agents or radioactive nucleotides, for example, 99 Tc, 90 Y, 111 In, 32 P, 14 C, 125 I, 3 H, 131 I, 11 C, 15 O, 13 N, 18 F, 35 S, 51 Cr, 57 To, 226 Ra, 60 Co, 59 Fe, 57 Se, 152 EU, 67 Cu, 217 Carbon, 211 At, 212 Pb, 47 Sc, 109 Pd, 234 Th, and 40 K, 157 Gd, 55 Mn, 52 Tr and 56 Such antibody conjugates can be conjugated to cytotoxic payloads such as Fe. In immunotherapy, these antibody conjugates may be used to selectively target and kill cells that express a target (the antigen of the antibody) on their surface. Exemplary cytotoxic agents include lysine, vinca alkaloids, methotrexate, Pseudomonas exotoxin, saporin, diphtheria toxin, cisplatin, doxorubicin, abrin toxin, geronin, and pokeweed antiviral protein.

[0095] The antibodies and antigen-binding fragments of this specification may also be conjugated with fluorescent or chemiluminescent labels, such as fluorophores, e.g., rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluoresamine, 152Examples include Eu, dansyl, umbelliferone, luciferin, luminal labeling, isoluminal labeling, aromatic acridinium ester labeling, imidazole labeling, acridimium salt labeling, oxalate ester labeling, aequorin labeling, 2,3-dihydrophthalazinedione, biotin / avidin, spin labeling, and stable free radicals.

[0096] Any method known in the art for conjugating the antibody molecule or protein molecule of the present invention to various parts, such as the methods described in Hunter et al., 1962, Nature 144:945; David et al., 1974, Biochemistry 13:1014; Pain et al., 1981, J.Immunol.Meth. 40:219; and Nygren, J., 1982, Histochem. and Cytochem. 30:407, may be used. Methods for conjugating antibodies and proteins are conventional and well known in the art.

[0097] Antibody purification When recombinant technology is used, antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If antibodies are produced intracellularly, the first step is to remove particulate debris, which is either host cells or lysed fragments, by, for example, centrifugation or ultrafiltration. Carter et al., 1992, Bio / Technology 10:163-167 describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If antibodies are secreted into the culture medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in one of the aforementioned steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of accidental contaminants.

[0098] Antibody compositions prepared from cells can be purified using, for example, hydroxyl apatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc region present in the antibody. Protein A can be used to purify antibodies based on human Ig.γ1, Ig.γ2, or Ig.γ4 heavy chains (Lindmark et al., 1983, J.Immunol.Meth. 62:1-13). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., 1986, EMBO J 5:1567-1575). The matrix to which the affinity ligand adheres is often agarose, but other matrices can also be used.

[0099] Mechanically stable matrices such as pore-controlled glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. H For antibodies containing three domains, Bakerbond ABX® resin (JTBaker, Phillipsburg, NJ) is useful for purification. Depending on the antibody to be recovered, fractionation by ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography with silica, chromatography with heparin, SEPHAROSE® chromatography with anion or cation exchange resin (e.g., polyaspartate column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available.

[0100] In one embodiment, glycoproteins can be purified by adsorption to a lectin substrate (e.g., a lectin affinity column), and fucose-containing glycoproteins can be removed from the preparation, thereby concentrating fucose-free glycoproteins.

[0101] Pharmaceutical preparations This invention comprises pharmaceutical formulations of anti-human APRIL antibodies. To prepare pharmaceutical or sterile compositions, antibodies, particularly antibodies or fragments thereof, are mixed with pharmaceutically acceptable carriers or excipients. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984). Therapeutic and diagnostic formulations can be prepared by mixing them with physiologically acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, or suspensions (e.g., Hardman, et al., 2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.), 1993, Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms See Forms:Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0102] The toxicity and therapeutic effects of antibody compositions administered alone or in combination with other drugs, such as conventional anticancer agents, can be determined, for example, by standard pharmaceutical procedures in cell cultures or experimental animals to determine the LD50 (the dose at which 50% of the population dies) and ED50 (the dose at which 50% of the population is therapeutically effective). The dose-to-toxicity ratio is the therapeutic index and can be expressed as the ratio between LD50 and ED50. Data obtained from these cell culture assays and animal studies can be used in constructing dose ranges for use in humans. Doses of such compounds are preferably within the range of circulating concentrations that include an ED50 with little to no toxicity. Doses may vary within this range depending on the form of administration used and the route of administration utilized.

[0103] Suitable routes of administration include parenteral administration, such as intramuscular, intravenous, or subcutaneous administration, and oral administration. The antibody used in the pharmaceutical composition or in carrying out the method of the present invention can be administered by various conventional methods, including oral ingestion, inhalation, topical application, or injection into the skin, subcutaneously, intraperitoneally, parenterally, intra-arterially, or intravenously. In one embodiment, the antibody of the present invention is administered intravenously. In another embodiment, the antibody of the present invention is administered subcutaneously.

[0104] Alternatively, antibodies can be administered locally rather than systemically by direct injection of the antibody into the site of action, often in the form of depot or sustained-release formulations. Furthermore, antibodies may be administered via targeted drug delivery systems.

[0105] A preferred administration protocol includes the maximum dose or frequency of administration that achieves the desired therapeutic effect (e.g., reduction in IgA levels) while avoiding serious undesirable side effects. The antibodies described herein may be administered by intravenous or subcutaneous injection (e.g., thigh, abdomen, upper arm) approximately weekly, approximately every two weeks, approximately every three weeks, approximately every four weeks, approximately every eight weeks, etc. The dose per injection or infusion may be approximately 10 to 1350 mg, for example, approximately 50 mg, approximately 150 mg, approximately 300 mg, approximately 450 mg, approximately 600 mg, approximately 750 mg, approximately 1000 mg, or approximately 1350 mg. In certain embodiments, administration of the anti-APRIL antibody is by subcutaneous injection at a dose of approximately 600 mg per administration event ("administration event" refers to one or more deliveries, such as injections, intended to provide a single dose to an individual, where the administration is performed at the same or different site on the individual), with a frequency of administration of once a week or once every two weeks. Preferred formulations for intravenous administration are aqueous buffer solutions at concentrations of approximately 15–25 mg / mL or approximately 20 mg, while preferred formulations for subcutaneous administration are approximately 125–175 mg or approximately 150 mg. These formulations preferably contain L-histidine, L-arginine, sorbitol, and polysorbate 20 at pH 6.3 ± 0.2. Preferably, the L-histidine is at a concentration of about 8–12 mM or about 10 mM, the L-arginine at a concentration of about 60–90 mM or about 75 mM, the sorbitol at about 2.4–3.6% or about 3% (w / w), and the polysorbate 20 at a concentration of about 0.008–0.012% or about 0.01% (w / w). More preferably, the aqueous buffer solution has a pH of 6.3 ± 0.2 and contains, essentially consists of, or comprises 10 mM L-histidine, 75 mM L-arginine, 3% (w / w) sorbitol, and 0.01% (w / w) polysorbate 20. The pH of the aqueous buffer solution can be adjusted to 6.3 ± 0.2 using a suitable sterile acid / base such as hydrochloric acid and sodium hydroxide. Preparations for intravenous infusion can be diluted with sterile saline (0.9%) before infusion.For example, the desired amount of anti-APRIL antibody can be diluted to a volume of approximately 250 mL. For instance, 15 mL of a 20 mg / mL antibody formulation can be diluted with 235 mL of sterile saline before injecting a dose of 300 mg. The formulation for subcutaneous injection can be used without further dilution.

[0106] The therapeutically effective dose and frequency of administration of the anti-APRIL antibodies disclosed herein for treating antibody-associated conditions, as well as the duration of treatment, may depend on a variety of factors, including the nature and severity of the condition, the potency of the antibody, the mode of administration, the subject's age, weight, general health status, sex and diet, and the subject's response to treatment, and may be determined by the treating physician. Anti-APRIL antibodies may be administered once daily, every two days, every three days, twice a week, once a week, every two weeks, every three weeks, once a month, every six weeks, every two months, or every three months, or as the treating physician deems appropriate.

[0107] Anti-APRIL antibodies may be administered for periods of at least approximately 1 week, 2 weeks, 1 month (4 weeks), 6 weeks, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, or longer, as the treating physician deems appropriate. APRIL-related illness can be a chronic condition. A chronic condition may exist for, for example, at least approximately 6 weeks, 2 months, 1 year, or longer. Antibodies may be administered for periods of at least approximately 6 weeks, 2 months, 3 months, or 6 months, 1 year, or even more years, as required for the individual's medical care.

[0108] Anti-APRIL antibodies can also be administered in an irregular manner to treat antibody-associated conditions. Early achievement of an effective concentration (therapeutic dose level) of the target antibody through a loading dose and subsequent maintenance dose (front-loading) may be more effective than conventional therapies in that it reduces the total antibody dose required and shortens the time to maximum target engagement. As used herein, such a dosing protocol is referred to as a “loading / maintenance dosing protocol.” An effective target antibody concentration can be achieved using a loading dose within four weeks, preferably within three weeks, more preferably within two weeks, and most preferably within one week, including within one day. The target serum concentration is then maintained by administering an equivalent or lesser (or less frequent) maintenance dose for the remainder of the treatment regimen or until suppression of disease symptoms is achieved.

[0109] When referring to drug administration, the term “front-loading” refers to an initial loading dose and subsequent maintenance doses. The initial loading dose (single or multiple doses) is intended to more rapidly increase the serum drug concentration in an animal or human patient to an effective target serum concentration. In various embodiments, front-loading is achieved by an initial dose delivered over a period of up to three weeks so that the antibody reaches the target serum concentration. Preferably, the loading dose or a series of doses are administered over a period of up to two weeks, more preferably within one week, for example, within one day. Most preferably, the loading dose is a single dose, followed by no maintenance dose for at least one week, and the loading dose is performed within one day. To avoid adverse immune responses to the antibody drug, it may be preferable to deliver the loading dose of the antibody by intravenous injection. The present invention includes loading doses and maintenance doses for front-loading drug delivery by intravenous or subcutaneous administration.

[0110] The loading dose may be administered as one or more doses at time intervals of at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks apart. In some embodiments, at least one loading dose is administered by one or more intravenous injections, followed by at least one maintenance dose administered by one or more intravenous or subcutaneous injections. In other embodiments, instructions for use may be for administering at least one loading dose and at least one maintenance dose by one or more intravenous or subcutaneous injections. In certain embodiments, both at least one loading dose and at least one maintenance dose are administered subcutaneously. In other embodiments, at least one loading dose is administered by intravenous infusion, followed by at least one maintenance dose administered subcutaneously. For example, a treatment method may include administering a loading dose of 150-1350 mg of anti-APRIL antibody by intravenous infusion or subcutaneous injection. After the loading dose (for example, one, two, three, or four weeks after the loading dose), a maintenance dose of 600 mg or less of anti-APRIL antibody may be administered by subcutaneous injection every four weeks or less, preferably every three weeks or less, more preferably every two weeks or less, and in embodiments, every week or less. The selection of the loading dose, maintenance dose, and interval can be made according to the ability of the animal or human patient to tolerate the administration of the antibody to the body, and according to the desired serum level of APRIL to be achieved.

[0111] The loading dose of a drug may be greater than the subsequent maintenance dose (e.g., about 1.5, 2, 3, 4, or 5 times). One or more therapeutically effective maintenance doses may be any therapeutically effective dose described herein. The loading dose may be about 2 or 3 times greater than the maintenance dose. The anti-APRIL antibody may be administered in two (or more) loading doses prior to the maintenance dose. The first loading dose of the antibody or a fragment thereof may be administered on day 1 during the treatment period, the second loading dose may be administered, for example, about 1 or 2 weeks later, and the maintenance dose may be administered, for example, once a week or once every two weeks. The first loading dose may be about 3 or 4 times greater than the maintenance dose, and the second loading dose may be about 2, 3, 4, 5, or more times greater than the maintenance dose.

[0112] As used herein, “inhibit,” “treat,” or “treat” includes delaying the onset of disease-related symptoms and / or reducing the severity of symptoms that develop or are expected to develop in the disease. These terms further include improving existing symptoms, preventing additional symptoms, and improving or preventing the underlying causes of such symptoms. Thus, these terms mean that beneficial results have been given to a vertebrate subject with the disease.

[0113] The antibodies of the present invention for therapeutic purposes are administered in a therapeutically effective dose. As used herein, the terms “therapeutically effective dose” or “effective dose” refer to the amount of anti-APRIL antibody or fragment thereof that, when administered alone or in combination with additional therapeutic agents to cells, tissues, or subjects, is effective to a patient in preventing or improving the disease or condition being treated. A therapeutically effective dose further refers to the amount of a compound sufficient to result in improvement of symptoms, e.g., treatment, cure, prevention or improvement of the associated medical condition, or an increase in the rate of treatment, cure, prevention or improvement of such condition. When applied to individual active ingredients administered alone, the therapeutically effective dose refers to that ingredient only. When applied to combinations, the therapeutically effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective dose of a therapeutic agent typically reduces symptoms by at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%.

[0114] Methods of co-administration or treatment with a second therapeutic agent are well known in the art. For example, see Hardman, et al. (eds.), 2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.), 2001, Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo (eds.), 2001, Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., PA.

[0115] The pharmaceutical compositions of the present invention may also contain other agents, for example, but not limited to, cytotoxic agents, chemotherapeutic agents, cell proliferation inhibitors, anti-angiogenic agents or antimetabolites, tumor targeting agents, immunostimulants or immunomodulators, or antibodies conjugated to cytotoxic agents, cell proliferation inhibitors, or other toxic agents. The pharmaceutical compositions may also be used in conjunction with other therapeutic modalities such as surgery, chemotherapy, and radiation.

[0116] Preferred Embodiment The following are preferred embodiments of the present invention: (1) An antibody preparation suitable for pharmaceutical infusion or subcutaneous injection, Anti-APRIL antibodies at concentrations of approximately 20 mg / mL to 190 mg / mL; Approximately 10 mM L-histidine; Approximately 75 mM L-arginine; Approximately 3% by weight of sorbitol; Approximately 0.01% by weight of polysorbate 20; and It contains a pH of approximately 6.0 to 6.6. An antibody preparation that (i) has a viscosity of approximately 16 cP or less, (ii) does not contain glutamic acid or its salts, (iii) has a weight osmolality of approximately 250 mOsm / kg to approximately 390 mOsm / kg, and (iv) has an OD330 of less than approximately 1.0. (2) The antibody preparation according to Embodiment 1, wherein, after manufacturing the preparation, it maintains an anti-APRIL antibody with a purity of at least 96% after storage at 2-8°C for 9 months. (3) The antibody preparation according to Embodiment 2, wherein, after being stored at 25°C for 6 months following the manufacture of the preparation, the preparation maintains an anti-APRIL antibody with a purity of at least 95%. (4) When measured at 25℃, 2.5 x 10 -5 mol·mL / g 2 An antibody preparation according to one of Embodiments 1 to 3, having the above-mentioned second virial coefficient. (5) An antibody preparation according to one of Embodiments 1 to 4, having a calculation isoelectric point of approximately 7.4 or higher. (6) The antibody preparation according to one of Embodiments 1 to 5, wherein the concentration of anti-APRIL antibody in the preparation is approximately 150 mg / mL. (7) The antibody preparation according to Embodiment 6, having a weight osmolality of approximately 290 mOsm / kg to approximately 390 mOsm / kg. (8) The antibody preparation according to Embodiment 6 or 7, having an OD330 of approximately 0.8 or less. (9) An antibody preparation according to one of Embodiments 1 to 5, wherein the anti-APRIL antibody is concentrated at a concentration of approximately 20 mg / mL. (10) The antibody preparation according to Embodiment 9, having a weight osmolality of approximately 293 mOsm / kg to approximately 333 mOsm / kg. (11) The antibody preparation according to one of Embodiments 1 to 10, wherein the anti-APRIL antibody is a humanized antibody containing a heavy chain variable region / light chain variable region pair selected from the group consisting of VH11.VL15, VH12.VL15, VH13.VL15, VH14.VL15, VH14_1.VL15, VH14_1D.VL15, VH14_1E.VL15, and VH14_1G.VL15. (12) An antibody preparation according to one of Embodiments 1 to 11, which does not contain glycine, carbonate, HEPES, phosphate, citrate, or acetate. (13) A single-use or multi-use vial containing the antibody preparation described in one of Embodiments 1 to 12. (14) A single-use or multi-use vial according to Embodiment 13, containing a formulation with a volume of 0.5 mL to 50 mL having an anti-APRIL antibody concentration of approximately 20 mg / mL to approximately 190 mg / mL. (15) A single-use or multi-use vial according to Embodiment 14, wherein the formulation has an anti-APRIL antibody concentration of approximately 20 mg / mL. (16) A single-use or multi-use vial according to Embodiment 14, wherein the formulation has an anti-APRIL antibody concentration of approximately 150 mg / mL. (17) A single-use or multi-use vial from one of embodiments 14 to 16, containing a formulation with a volume of 5 mL. (18) A pre-filled syringe, auto-injector, or injector pen containing one antibody preparation from Embodiments 1 to 12. (19) A pre-filled syringe, auto-injector, or injector pen according to Embodiment 18, containing a formulation in a volume of approximately 0.5 mL to approximately 10 mL having an anti-APRIL antibody concentration of approximately 20 mg / mL to approximately 190 mg / mL. (20) A pre-filled syringe, auto-injector, or injector pen according to Embodiment 19, wherein the formulation has an anti-APRIL antibody concentration of approximately 20 mg / mL. (21) A pre-filled syringe, auto-injector, or injector pen according to Embodiment 19, wherein the formulation has an anti-APRIL antibody concentration of approximately 150 mg / mL. (22) A pre-filled syringe, auto-injector, or injector pen according to any of Embodiments 19 to 21, containing a formulation in a volume of approximately 2 mL. (23) A method for administering an anti-APRIL antibody to an individual that requires administration of an anti-APRIL antibody, comprising administering the formulation described in one of Embodiments 1 to 12 to the individual by subcutaneous injection. (24) The method according to Embodiment 23, comprising repeating administration on a schedule at least weekly (QW) for at least two administration cycles. (25) The method according to Embodiment 23, comprising repeating administration at least every two weeks (Q2W) for at least two administration cycles. (26) The method according to Embodiment 23, comprising repeating administration at least every four weeks (Q4W) or monthly (QMT) for at least two administration cycles. (27) The method according to one of embodiments 23 to 26, wherein a total dose of approximately 10 mg to approximately 1350 mg of anti-APRIL antibody is administered at each administration event. (28) The method according to Embodiment 27, wherein an anti-APRIL antibody concentration of approximately 150 mg / mL is delivered in a single dose of approximately 2 mL of the formulation. (29) The method according to Embodiment 27, wherein an anti-APRIL antibody concentration of approximately 150 mg / mL and approximately 4 mL of the formulation are delivered in a single dose, and each administration event comprises one or more subcutaneous injections. (30) The method according to one of Embodiments 23 to 29, wherein the formulation is administered subcutaneously to a site in the thigh, abdomen, or upper arm of an individual. (31) A method for administering an anti-APRIL antibody to an individual that requires administration of an anti-APRIL antibody, comprising administering the formulation described in one of Embodiments 1 to 12 to the individual by intravenous infusion. (32) The method according to Embodiment 31, comprising repeating administration in at least a QW schedule for at least two administration cycles. (33) The method according to Embodiment 31, comprising repeating administration on at least a Q2W schedule for at least two administration cycles. (34) The method according to Embodiment 31, comprising repeating administration at least on a Q4W or monthly schedule for at least two administration cycles. (35) Intravenous infusion: Dilute the preparation described in any one of Embodiments 1 to 12 with 0.9% physiological saline to a concentration of approximately 0.1 mg / mL to approximately 10 mg / mL; The method according to any one of Embodiments 31 to 34, comprising administering a total dose of approximately 10 mg to approximately 1350 mg of anti-APRIL antibody to an individual as a single intravenous dose of a diluted formulation over approximately 2 hours. (36) The method according to Embodiment 35, wherein 15 mL of the preparation having a concentration of 20 mg / mL is added to 235 mL of 0.9% physiological saline to obtain an intravenous dose with a concentration of 1.2 mg / mL. (37) A method for administering an anti-APRIL antibody to an individual that requires administration of an anti-APRIL antibody, the method comprising administering anti-APRIL by a loading / maintenance administration protocol. (38) The method according to Embodiment 37, wherein the loading component of the loading / maintenance administration protocol includes an anti-APRIL antibody administered once or more at a concentration higher than the anti-APRIL antibody concentration in the maintenance component of the loading / maintenance administration protocol. (39) The method according to Embodiment 37, wherein the loading component of the loading / maintenance administration protocol includes an anti-APRIL antibody administered at a higher frequency than the administration frequency of the anti-APRIL antibody in the maintenance component of the loading / maintenance administration protocol, once or more times. (40) The method according to one of Embodiments 37 to 39, wherein the loading component of the loading / maintenance administration protocol includes an anti-APRIL antibody administered once or more times via a route different from the administration route of the anti-APRIL antibody in the maintenance component of the loading / maintenance administration protocol. (41) The method according to Embodiment 37, wherein the loading component of the loading / maintenance administration protocol comprises an anti-APRIL antibody administered intravenously once or more times, and the maintenance component of the loading / maintenance administration protocol comprises an anti-APRIL antibody administered subcutaneously once or more times. (42) The method according to one of Embodiments 37 to 41, wherein the anti-APRIL antibody is the antibody preparation described in one of Embodiments 1 to 12. (43) The method according to one of embodiments 23 to 41, wherein the individual requiring administration of anti-APRIL antibody has a serum IgA level greater than 4 g / L. (44) The method according to one of embodiments 23 to 41, wherein the individual requiring administration of an anti-APRIL antibody is a patient with IgA nephropathy. (45) The method according to one of embodiments 23 to 41, wherein the individual has hyperimmunoglobulinemia.

[0117] The present invention will be further explained and supported here by reference to the following non-limiting experiments.

[0118] Examples Example 1: The following examples describe the development of high-dose anti-APRIL formulations with a viscosity of less than 16 cP. The formulations were subjected to temperature stress (-70°C, 2–8°C, 25°C, and 45°C), freeze / thaw cycles, and gentle shaking, with the intention of selecting the formulation best suited to the bulk active pharmaceutical ingredient, and then filled with the pharmaceutical product at the desired concentration. The predicted stability of the formulations by SE- and CEX-UPLC at 5°C was approximately 2 and 6 years, respectively. The samples revealed the following characteristics: Appearance (C=Transparent; O=Opal white; NC=Colorless; Y=Pale yellow; NP=No visible particulate matter) concentration Turbidity at 330nm Size exclusion ultrafast chromatography (SE-UPLC) Cation exchange UPLC (CEX-UPLC) Dynamic light scattering / Static light scattering pH Osmolality viscosity reagent: Anti-APRIL antibody:VH14_1G.VL15 Histidine JTBaker Jun-2080 Arginine JTBaker Jun-2066 Glutamic acid JTBaker 2077.06 Sorbitol EMD Millipore 1.03583.2503 Sodium Chloride JTBaker 7647-145 Polysorbate-20 JTBaker 4116.04 Piperazine Sigma 80621 Imidazole Fluka 56749 Tris Sigma 252859 Disodium hydrogen phosphate anhydrous JTBaker Jan-8327 Potassium dihydrogen phosphate Millipore 7778-77-0

[0119] A total of 11 formulations were prepared (see Table 2), including six formulations with target concentrations exceeding 150 mg / mL and five formulations with a concentration of 200 mg / mL. All formulations were prepared in histidine buffer at pH 6.1 and 6.3. [Table 2]

[0120] Anti-APRIL antibodies were concentrated to 50 mg / mL at a maximum flow rate of 40 mL / min by tangential flow filtration (TFF). The concentrated material was then collected, centrifuged at 8500 rcf for 5 minutes, and filtered through a 0.22 μm PVDF membrane. The material was loaded into a pre-hydrated 10 kDa MWCO dialysis cassette and dialyzed against 200 mL of buffer at 2–8°C (3 changes of 200 mL). The total dialysis time was 2 days. Next, the recovered protein samples were spin-concentrated in a 10 kDa MWCO centrifuge filter unit at 3750 rcf for several hours to reach the target value. The concentrated formulations were analyzed as over-concentrated samples (e.g., greater than 150 mg / mL) or diluted to a specific target concentration (e.g., 200 mg / mL) using the corresponding buffer. Polysorbate-20 (PS20), prepared with the corresponding buffer solution, was added to each formulation to achieve a final concentration of 0.01% (w / v).

[0121] The recovery rate calculation and visual observation results are shown in Table 3 below. After the TFF step, a recovery rate of 98.2% was calculated from the protein content. A cloudy white solution was observed. Therefore, centrifugation and filtration of this protein solution were performed before the dialysis step to remove particles. The filtration step had little to no effect on the protein concentration. During the spin concentration step, all promising formulations evaluated in this screening reached a target concentration of over 150 mg / mL. [Table 3]

[0122] Table 4 below summarizes the analysis obtained for this study. In this step, the recovery rate % compared to the initial amount of material loaded into the cassette ranged from 70.2% to 87.1%. Viscosity ranged from 7.7% to 15.4 cP, with formulation 1 having the highest concentration and lowest viscosity. The comparison of formulations 3 and 11 suggests that the combination of arginine and glutamic acid, in the presence of sorbitol, results in lower viscosity than arginine alone. Overall, this initial screening experiment suggests that salts should be avoided to maintain low viscosity. This finding is reinforced by comparing formulations 3 and 5. Overall, colloidal stability was within acceptable limits for all formulations tested, with positive A2 values ​​and similar ranges. [Table 4]

[0123] Example 2 To assess the possibility of obtaining protein concentrations exceeding 200 mg / mL, fresh samples were prepared from the initial anti-APRIL antibody. Preparations containing sodium chloride were excluded because salt is detrimental to maintaining lower viscosity values. Samples were overconcentrated to 239–252 mg / mL during the spin concentration step. Notably, recovery data calculated from protein quantification by A280 were lower than previously observed values, ranging from 42.7% to 69.4%. The samples were then diluted to 200 mg / mL with the corresponding preparation buffer, while adjusting the PS20 content to a final concentration of 0.01%. Viscosity measurements were high, ranging from 26.2 cP to 47.0 cP. Notably, the viscosity measurements obtained in samples concentrated to 155–190 mg / mL were significantly lower than those observed in the 200 mg / mL samples. This increase in viscosity suggests that some harmful protein-protein interactions occur during extreme protein overconcentration. While centrifugation allows for the concentration of small sample volumes, the test sample experiences high transmembrane pressure due to gravity during the centrifugation process, which can further cause protein denaturation. The results are provided in Tables 5 and 6 below. [Table 5] [Table 6]

[0124] To closely monitor sample viscosity between 190 mg / mL and 200 mg / mL and to evaluate the effect of surfactants on viscosity, a third set of fresh samples was prepared from the initial anti-APRIL antibody. Formulations 1 and 6 were prepared with this composition. The protein was reduced to 28.8 mg / mL by TFF, and the material after 12 mL of TFF was then dialyzed. During the spin concentration process, a qualitative increase in viscosity was observed at a concentration of approximately 180 mg / mL, and the final concentration achieved was approximately 200 mg / mL. Half of the sample was maintained at the final concentration, and the other half was reduced to 190 mg / mL with the corresponding formulation buffer without PS20. Viscosity was measured at both concentrations, and the results are shown in Table 7 below. High viscosity was observed in samples at concentrations above 200 mg / mL, with values ​​of 46.9 cP and 33.0 cP for formulations 1 and 6, respectively. When the sample concentration was reduced to 190 mg / mL by dilution, the viscosity decreased to 34.2 cP and 22.5 cP. Preparation of a sample containing 0.01% PS20 resulted in a viscosity of formulation 6 of 20 cP. [Table 7]

[0125] Example 3 Four 150 mg / mL anti-APRIL (VH14_1G.VL15) antibody preparations (Table 8 below) containing histidine buffer at pH 6.1 and 6.3 were prepared. 0.6 mL of test sample was aseptically placed into a 2 mL glass vial and then stoppered. The stability test conditions and assays used are shown in Table 9. [Table 8] [Table 9]

[0126] The thawed anti-APRIL antibody was centrifuged at 3000 rcf for 20 minutes and filtered through a 0.22 μm membrane to remove any observed particles. The filtered material was concentrated by TFF over two days to a final concentration of 37.9 mg / mL. The concentrated material was then placed in a 70 mL volume dialysis cassette of prehydrate 10 kDa MWCO and dialyzed over two days in 2 L of buffer (2 L changed three times) at 2-8°C. Next, the recovered protein sample was spin-concentrated in Amicon Ultra 15 tubes (regenerated cellulose 10 kDa MWCO) at 2300 rcf and 15°C for several hours to a final concentration exceeding 150 mg / mL. The material to be concentrated was divided in half and concentrated in two spin concentrators for large-scale processing. Before analysis, half was pooled immediately after the concentration step once the target volume was reached. The excess concentrated protein solution was set aside for viscosity testing before adding surfactants. The concentrated formulation was diluted to the target 150 mg / mL using the corresponding buffer. PS20, prepared in the corresponding buffer, was added to the formulation to a target concentration of 150 mg / mL for VH14_1G.VL15 until a final concentration of 0.01% (w / v). Under sterile conditions, the VH14_1G.VL15 formulation was filtered through a 0.22 μm PVDF membrane, dispensed into depyrogenic borosilicate vials, stoppered, and crimped with aluminum seals. The vials were then subjected to stress conditions for stability testing according to Table 9. At each required time point, the sample was removed from the conditions and examined for appearance in the original vial. Osmolality, viscosity, A330, pH, and DLS assays were performed on undiluted samples, while A280, SE-UPLC, and CEX-UPLC were performed on diluted samples according to their respective method specifications. Osmolality and viscosity were performed only at time T=0. All remaining sample volumes were kept at -70°C for backup testing.

[0127] After applying various stress conditions, the appearance, turbidity using A330, pH, and concentration of the samples were measured. Sample concentration was determined by absorbance measurement at 280 nm using an extinction coefficient of 1.0 mg / mL = 1.4 AU. The reported final protein concentration was corrected for scattering ((A280 - A330) / ε280 = corrected protein concentration). Sample viscosity values ​​were measured for over-enriched samples (before PS20 addition) and T=0 samples during the process. Osmolality values ​​were determined only for T=0 samples.

[0128] During the sample preparation step, aliquots of over-concentrated samples were set aside for viscosity measurement. Viscosities ranged from 12.5 to 15.6 cP. Notably, differences in viscosity were observed in approximately 190 mg / mL samples during formulation screening and sample preparation in the 12-week formulation test. This difference may be due to differences in the amount of sample processed during the spin concentration step or differences in the starting materials used in this test. During the dilution step, a target antibody concentration of 150 mg / mL was achieved for all test formulations while introducing the surfactant to a final concentration of 0.01% PS20. The resulting 150 mg / mL formulations exhibited viscosities of 6.3 to 7.8 cP and gravimetric osmolality of 324 to 358 mOsm / kg. As an important note, the overview of the preparation information indicated that the buffer corresponding to formulation 11 was prepared at pH 6.1 instead of the target pH 6.3, thus explaining the discrepancy with the target pH of this formulation. The measured pH values ​​remained within 0.1 units of the starting value at all observed time points.

[0129] After stress incubation, the appearance, protein concentration, turbidity, and pH of the samples were recorded (Figure 1). No significant differences in pH were observed after different stress conditions. Except for the appearance of a few particles after 12 weeks of incubation at 25°C (formulation 6) and 3 weeks of incubation at 45°C (formulations 3 and 6), the appearance of all formulations remained clear and pale yellow after different stress conditions, with no visible particles. Turbidity values ​​were generally lower for formulations 1 and 3. After five freeze / thaw cycles or 3 days of shaking at 2–8°C, no discernible analytical changes were evident in any of the formulations.

[0130] The target antibody concentration of 150 mg / mL was estimated for all test formulations. The pH value of the samples was within 0.1 units of the dialysis buffer. Note that formulation 11 was prepared at pH 6.14, not the target pH 6.30. Visually, all formulations remained clear and pale yellow after various stress conditions, although some particles were observed after 12 weeks of incubation at 25°C (formulation 6) and 3 weeks of incubation at 45°C (formulations 3 and 6). Turbidity, when measured from A330, was generally lower for formulations 1 and 3. The results are shown in Tables 10 and 11 below. [Table 10] [Table 11]

[0131] Example 4 Size exclusion chromatography (also known as gel filtration chromatography) separates the molecular morphology of proteins based on size. In this method, larger molecular species (e.g., aggregates, IgG dimers, and oligomers) elute earlier than the desired (monomer) IgG species as pre-peaks. Smaller molecular species (e.g., degradation products and fragments) elute later as post-peaks. SE-UPLC was performed on antibody-stable samples. Briefly, the sample was diluted to 0.5 mL / min in the mobile phase, and 2.5 μg was injected into a Waters Acquity UPLC BEH 200 SEC, 1.7 μm, 4.6 x 300 mm column. Chromatographic separation was performed in a mobile phase of 10 mM phosphate, 0.4 M NaCl, pH 7.0 ± 0.1, flow rate 0.2 mL / min, at ambient temperature.

[0132] The tabular data obtained by SE-UPLC is shown in Figure 2, and the relative main peaks are shown graphically in Figures 3-6. After 12 weeks of incubation at 2-8°C, the main peaks decreased by 0.1-0.3% in all formulations. Since the obtained data were comparable to the T=0 results and within the range of assay variability, no distinguishable differences were observed between any of the test formulations after exposure to 5 cycles of freeze / thaw and shaking stress conditions at 2-8°C.

[0133] After 12 weeks of incubation at 2–8°C, each formulation showed robust stability with a main peak within 0.2–0.4% of the initial value. Freeze / thaw stability and shaking stress stability at 2–8°C were comparable for all test formulations. Incubation at 25°C for 12 weeks revealed that formulation 1 (10 mM histidine, 150 mM arginine, 150 mM glutamic acid, 0.01% PS20, pH 6.1) had slightly higher purity compared to the other formulations, but this was not the case after 3 weeks at 45°C, where a decrease of 1.3–1.6% in the main peak area was observed.

[0134] Example 5 Ion exchange chromatography separates molecules based on differences in accessible surface charge. Binding depends on the ionic attraction between molecules with opposite charges. The analysis applied here utilized a weak cation exchange (CEX-UPLC) column. Elution is achieved with an increasing pH gradient. pH gradient separation is performed using an ultrafast liquid chromatography system (Thermo Vanquish) with 280 nm UV detector monitoring (A280) and integrated software (Chromeleon ver. 7.2). In this specification, a Dionex ProPac WCX-10 column (4.0 x 250 mm) was used to separate and provide profiles of the measurable population of antibody-charged species during the stability testing described herein. CEX-UPLC was performed on anti-APRIL antibody stability samples. The sample was diluted to 1 mg / mL and 25 μg was injected into the column.

[0135] The tabular data obtained by CEX-UPLC is shown in Figure 7. After 12 weeks of incubation at 2–8°C and -70°C, CEX-UPLC showed a 0.1–0.3% change in the main peak, and no discernible differences or trends were observed among the four formulations. After incubation at 25°C and 45°C, a decrease in the main peak and basic peak levels, resulting in an increase in the acidic peak, was observed in all formulations during the stability test period. These differences were observed to be lower in the pH 6.1 formulation than in the pH 6.3 formulation. Formulation 11 (10 mM histidine, 75 mM arginine, 3% sorbitol, 0.01% PS20, pH 6.1) showed the best stability by CEX-UPLC. After 5 cycles of freeze / thaw or shaking stress at 2–8°C, no clear differences were observed among the four formulations.

[0136] After storage at -70°C and 2–8°C for 12 weeks, or after exposure to freeze / thaw and shaking stress conditions at 2–8°C, the obtained data were comparable to the T=0 result and within the assay variability range, so no significant changes were observed among any of the test formulations. After incubation at 25°C and 45°C, a decrease in the main peak and basic peak levels, resulting in an increase in the acidic peak, was observed in all formulations during the stability test period. These differences were observed to be lower at pH 6.1 than at pH 6.3 for the VH14_1G.VL15 formulation. Formulation 11 (10 mM histidine, 75 mM arginine, 3% sorbitol, 0.01% PS20, pH 6.1) showed the best stability by CEX-UPLC.

[0137] Example 6 A qualitative overview of the stability data shows that higher VH14_1G.VL15 stability was maintained in sample formulation 1 (10 mM histidine, 150 mM arginine, 150 mM glutamic acid, 0.01% PS20, pH 6.1) when measured by SEC sample purity, which was only observable under test conditions of 25°C and 45°C. The relationship between the reaction rate and its temperature is expressed by the Arrhenius equation. formula 1 ln kobs=(-Ea / R)T+ln A Here, k is the rate coefficient, and the pre-exponential coefficient A is a constant over a small temperature range, typically involving variables such as collision frequency and orientation. Ea is the activation energy, R is the universal gas constant, and T is the absolute temperature. This equation allows us to estimate reaction rates, such as decomposition at a specific temperature, assuming that decomposition is inherently thermal (as opposed to decomposition by light, for example) and that the decomposition mechanism does not change with temperature. If these criteria are met, the activation energy Ea and pre-exponential coefficient A should not change over a narrow temperature range, making extrapolation to other (often lower) temperatures easier.

[0138] Under these circumstances, experiments conducted under high-temperature (accelerated) conditions provide valuable information regarding the decomposition rate at low temperatures, while real-time degradation monitoring is extremely slow and costly. Accelerated testing provides quick insights into stability issues, but it is not a substitute for real-time stability testing.

[0139] To approach data analysis in a more rigorous manner, rate constants were determined for the loss of anti-APRIL(VH14_1G.VL15) antibody purity under each formulation and temperature condition, and the primary degradation rate was estimated. For test conditions of 2–8°C, 25°C, and 45°C, plots of ln purity against time were created using the percent antibody purity values ​​found by SE-UPLC analysis. The results are shown graphically in Figure 8. From the data, the slope is correlated with the degradation rate constant (-kobs). The temperature dependence results for vehicle and kobs are shown in Table 12 below. [Table 12]

[0140] Using the data in Table 12, Arrhenius plots (ln kobs vs. 1 / T) were created for each of the anti-APRIL antibody formulations at three stable temperatures (Figure 9). From this analysis, linear fitting parameters were obtained (Table 13). The good linear fit of ln kobs vs. 1 / T indicates similar degradation mechanisms operating within the tested temperature range. Furthermore, this means that the degradation follows Arrhenius behavior. [Table 13]

[0141] Using the Arrhenius relation in Equation 1, with slope = -Ea / R and intercept = lnA, the ln kobs for each vehicle at 5°C and 25°C were calculated using the data. Furthermore, from the kobs at 5°C, the linear form of the first-order kinetic velocity equation shown in Equation 2 is used. In the equation, k is the observed velocity constant (kobs), and Purity0 and Purity tThese values ​​represent the purity at time 0 and time t, respectively. t95% was calculated, corresponding to the number of incubation days until the purity of the VH14_1G.VL15 solution approached 95% of the overall sample composition (or 98.1% of the original purity). Solution stability was calculated to be 1.55–3.06 years at 5°C and 0.35–0.47 years at 25°C. The results are shown in Table 14. formula 2 ln(Purityt / Purity0)=kobst [Table 14]

[0142] Example 7 Following the same quantitative approach, the collected CEX-UPLC data can be used to estimate sample purity up to 95% of the original value. Using the CEX-UPLC data, ln purity was plotted against time, as shown in Figure 10. The lines generated using the data show a pseudo-first-order decomposition kinetics for at least the test samples at 45°C and 25°C, as shown in Table 15 below. From there, the slope correlates with the rate constant of decomposition (-kobs). Arrhenius plots were created for each of the VH14_1G.VL15 formulations at 45°C and 25°C (Figure 11). Data from 2–8°C were not included in the AR calculation because a non-pseudo-first-order change was observed. [Table 15]

[0143] Next, using the Arrhenius relation, ln kobs for each vehicle at 5°C and 25°C was calculated using the data in Table 15. Furthermore, from kobs, and the linear form of the velocity equation of first-order kinetics (where k is the observed velocity constant (kobs), Purity0 and Purity0) were derived. t Using (), the incubation period corresponds to the number of years until the purity of the VH14_1G.VL15 solution approaches 95% of the original sample purity. 95%The following was calculated (see Tables 16 and 17). The stability of the solution was calculated to be 2.83–5.95 years at 5°C and 0.12–0.19 years at 25°C. [Table 16] [Table 17]

[0144] Example 8 Dynamic light scattering (DLS) is used to determine the hydrodynamic radius of molecules. It measures the translational diffusion of molecules in solution by capturing fluctuations in scattered light intensity due to the Brownian motion of the molecules. Assuming the molecules are homogeneous spheres, the diffusion coefficient can be converted to the hydrodynamic radius of the molecule by the Stokes-Einstein relationship. DLS analysis was performed on VH14_1G.VL15 stable samples at each time point. Briefly, a buffer blank was first performed to measure scattering from each formulation. This value was subtracted from the test sample readings to minimize buffering effects. 10 μL of each undiluted and unfiltered test sample was added to a clean 1 μL NanoStar quartz cuvette and analyzed at 25°C with a measurement time of 50 seconds and acquisition time of 5 seconds. In this study, DLS intensity autocorrelation data for diffusion coefficient determination was analyzed using a regularization method. This provides estimates of the radius and relative abundance of all species present in the solution. As described in Wyatt Technology DYNAMIC 7.1.9, acceptable DLS data must have a smooth, continuous autocorrelation function that decays exponentially from a maximum intensity correlation value of 2 to a value of 1. All test samples showed acceptable decay.

[0145] Figure 12 summarizes the hydrodynamic radii of observable particle species. The data in the summary table are values ​​obtained from the initial measurement, which included 10 acquisitions. Detailed results for the main population of each measurement are shown in Tables 18-21 below. [Table 18] [Table 19] [Table 20] [Table 21]

[0146] At the start of the study, a bimodal distribution was observed in the T=0 sample, and the additional population was 10 2 The samples exhibited hydrodynamic radii in the range exceeding nm. The polydispersity, shown in Tables 18 to 21, ranged from 7.8% to 25.2% in the T=0 samples. At the start of the test, the main population represented the dominant species with % mass ≥ 99.3%. Overall, the particle size and relative abundance of the main population remained consistent throughout the test, regardless of storage conditions. DLS measurements revealed a hydrodynamic particle size range of 6.8–7.9 nm, depending on the formulation composition. A bimodal distribution was observed in the T=0 samples, with the additional population being 10 2 The hydrodynamic radius was observed in the range exceeding nm. At the start of the test, the main population represented the dominant species with % mass ≥ 99.3%. Overall, the particle size and relative abundance of the main population remained consistent throughout the test, regardless of storage conditions or formulation.

[0147] Example 9 Five anti-APRIL (VH14_1G.VL15) antibody preparations were prepared in histidine buffer, four at 20 mg / mL and one at 50 mg / mL, within a pH range of 6.0–6.5 (Table 22). 1.2 mL of test sample was aseptically placed into a 2 mL glass vial and then stoppered. At each required time point, the sample was removed from the conditions and examined for appearance in the original vial. A330 and pH were analyzed using unfiltered samples, while A280, SEC, and CEX were performed using 0.2 μm filtered samples from which particles that could interfere with the analysis had been removed. For HIAC analysis, a stable sample volume of 0.75 mL was diluted 1:1 with the corresponding buffer to obtain a sufficient volume for analysis. Osmolality and viscosity were performed only at time point T=0. All remaining sample volumes were kept at -70°C for backup testing. [Table 22]

[0148] The pH, concentration, viscosity, and gravimetric osmolality of the prepared test samples were recorded. The viscosity was 1.2 cP at 20 mg / mL for all four formulations. The 50 mg / mL formulation had a slightly higher viscosity of 1.5 cP. The gravimetric osmolality ranged from 293 to 377 mOsm. The results are provided in Table 23 below. [Table 23]

[0149] After stress incubation, the appearance, protein concentration, turbidity, and pH of the samples were recorded, as shown in Figure 13. No differences were observed in protein concentration and pH after different stress conditions. The appearance remained colorless and transparent, and no visible particles were present in any of the formulations after different stress conditions. Overall, the turbidity values ​​after incubation at 25°C and 45°C showed the greatest stability for the formulations containing arginine and sorbitol at pH 6.3. After shaking for 3 days at 5XF / T or 2–8°C, no discernible analytical changes were observed in any of the formulations (Table 24). [Table 24]

[0150] Example 10 This procedure applies to all liquid samples requiring particle sizing and counting tests performed in analytical and formulation development (AFD) laboratories using the HIAC9703 liquid particle counting system. The HIAC consists of a sampler, particle counter, and Royco sensor (HRLD400 sensor). The Royco sensor is capable of sizing and counting particles from 2 μm to 100 μm. This instrument can count particles at a rate of 10,000 counts / mL or less. This method is unvalidated and is for development purposes only.

[0151] In short, samples and controls are prepared in a biologically safe cabinet to prevent the addition of particles from the environment. A stable sample volume of 0.75 mL was diluted 1:1 with the corresponding buffer to obtain a sufficient volume for analysis. The control consists of purified water used as the system-compatible sample (used to ensure that no particles are introduced during preparation) and a placebo sample, which is essentially a buffer placed in the environment.

[0152] Samples and controls are degassed for 2 hours and analyzed using the HIAC system. The HIAC method consists of 6 consecutive runs with a volume of 0.2 mL from each sample. The first three runs are ignored as they are used to equilibrate the sensor, and the average particle count from the last three runs is averaged to obtain the particle count (number / mL).

[0153] The sizing and counting of particles measured by HIAC are shown in Figure 14. The obtained data were comparable to the results at t = 0 and were within the assay variability range, so no significant changes were observed among any of the test anti-APRIL (VH14_1G.VL15) antibody formulations. Therefore, they were all equally stable. Since the obtained data were comparable to the results at t = 0 and were within the assay variability range, no significant changes were observed among any of the test formulations when exposed to freeze / thaw and 2 - 8°C shaking stress conditions (Table 25). [Table 25]

[0154] Example 11 Size exclusion chromatography (also known as gel filtration chromatography) separates the molecular forms of proteins based on size. In this method, larger molecular species (e.g., aggregates, IgG dimers, and oligomers) elute earlier than the desired (monomeric) IgG species as a pre-peak. Smaller molecular species (e.g., degradation products and fragments) elute later as a post-peak. SE-UPLC was performed on anti-APRIL (VH14_1G.VL15) antibody stability samples according to the CMC13415 purity of item #5 - 0091 by the SE-UPLC method using a Waters Acquity UPLC BEH 200 SEC, 1.7 μm, 4.6 x 300 mm column and a mobile phase of 10 mM phosphate, 0.4 M NaCl, pH 7.0.

[0155] After storage at 45°C for 1, 2, and 4 weeks, the purity results showed that the formulation containing arginine / sorbitol at pH 6.0 had the lowest stability compared to the other three formulations with an equal concentration of 20 mg / mL (Figure 15). After incubation at 25°C and 45°C, when comparing two formulations containing arginine / sorbitol pH 6.5 with anti-APRIL antibodies at 20 mg / mL or 50 mg / mL, the high-concentration formulation at 50 mg / mL with low stability showed concentration-dependent stability. After storage at -70°C and 2 - 8°C for 12 weeks, no significant changes were observed among any of the test formulations.

[0156] Since the obtained data was comparable to the results at t = 0 and within the assay variability range, no significant changes were observed among any of the test formulations when subjected to freeze / thaw and shaking stress conditions at 2 - 8°C (Table 26).

Table 26

[0157] Example 12 Ion exchange chromatography separates molecules based on differences in accessible surface charges. Binding depends on the ionic attraction between molecules with opposite charges. In the analysis applied here, a weak cation exchange (CE-HPLC) column was utilized. Elution is achieved with a gradient of increasing pH. pH gradient separation was performed using a high-performance liquid chromatography system (Agilent 1260) equipped with a UV detector monitoring at 280 nm (A280) and integrated software (Chemstation ver. C.01.07 software package). In this specification, a Dionex ProPac WCX-10 column (4.0 x 250 mm) was used to separate and provide a profile of the measurable population of anti-APRIL antibody charge species during the stability tests described herein.

[0158] Following incubation at 25°C and 45°C, a decrease in main and basic peak levels was observed in all formulations during the stability test period, resulting in an increase in the acidic peak. These differences were observed in the formulations at lower pH 6.0 and pH 6.3 compared to higher pH 6.5. Concentration-dependent stability was observed after incubation at 25°C and 45°C, with higher stability observed in the 50 mg / mL formulation compared to the same formulation containing 20 mg / mL of anti-APRIL(VH14_1G.VL15) antibody. Interestingly, this differs from the results determined by SE-UPLC. After storage for 12 weeks at -70°C and 2–8°C, or after exposure to freeze / thaw and shaking stress conditions at 2–8°C, no significant changes were observed among any of the test formulations, as the obtained data were comparable to the t=0 results and within the assay variability range (Table 27). [Table 27]

[0159] Example 13 To approach data analysis in a more rigorous manner, rate constants were determined for the loss of anti-APRIL(VH14_1G.VL15) antibody purity for each formulation and temperature condition, and the primary degradation rate was estimated. For the 25°C and 45°C test conditions, the percentage values ​​of VH14_1G.VL15 purity detected by SEC-HPLC analysis were used to create a plot of ln purity versus time. The results are shown graphically in Figures 17 and 18. The straight lines generated using the data show a pseudo-primary degradation kinetics in which the trend correlates with the degradation rate constant (-kobs) (Table 28). [Table 28]

[0160] Using these data, Arrhenius plots (ln kobs vs 1 / T) were created for each of the anti-APRIL (VH14_1G.VL15) antibody preparations at three stable temperatures (Figure 19). Linear fitting parameters were obtained (Table 29). Note that while Arrhenius plots typically use at least three temperatures, this was not possible in this case, as no loss of purity was observed after 12 weeks at 2–8°C. The good linear fit of ln kobs vs 1 / T indicates a similar degradation mechanism operating within the tested temperature range. Furthermore, this means that the degradation follows Arrhenius behavior. [Table 29]

[0161] Using the Arrhenius relation in Equation 1, the ln kobs for each vehicle at 5°C were calculated using the data, and the number of incubation days corresponding to the time it takes for the purity of the anti-APRIL(VH14_1G.VL15) antibody to approach 95% of the overall sample composition was calculated. 95% The following calculations were performed (Table 30). The stability of the solution was calculated to be over 4600 days, or approximately over 12 years, for all formulations when stored at 5°C. [Table 30]

[0162] Example 14 Using the same approach, the purity of the samples can be estimated to 95% of the original value using the collected CE-HPLC data. For each test condition, a plot of ln purity against time was created using the anti-APRIL(VH14_1G.VL15) antibody purity percentage value found in the CECHPLC analysis. The straight lines generated using the data in Figures 20-22 show a pseudo-first-order degradation kinetics for test samples at least at 45°C and 25°C. The slope thereof correlates with the degradation rate constant (-kobs) (Table 31). [Table 31]

[0163] Using this data, for each formulation, Arrhenius plots (ln kobs vs 1 / T) were created at three stability temperatures (Figure 23). From this analysis, linear fit parameters were obtained, showing a good degree of correlation within the data, i.e., an R 2 value > 0.95. A good linear fit of ln kobs vs 1 / T indicates a similar degradation mechanism operating within the tested temperature range (Table 32). Furthermore, this means that the degradation follows Arrhenius behavior.

Table 32

[0164] From the Arrhenius relationship where slope = -Ea / R and intercept = lnA, using the data from Table 32, ln kobs for each vehicle at 5°C was calculated. Furthermore, from kobs at 5°C (where k is the observed rate constant (kobs), and Purity0 and Purity t are the purities at time 0 and time t respectively), using the linear form of the rate equation of first-order kinetics, t corresponding to the number of incubation days until the antibody purity approaches 95% of the original sample purity was calculated (Table 33). The stability of the antibody was calculated to be over 1200 days, or over about 3 years, for the formulation at pH 6.3, and even longer at pH 6.0. 95% was calculated (Table 33). The stability of the antibody was calculated to be over 1200 days, or over about 3 years, for the formulation at pH 6.3, and even longer at pH 6.0.

Table 33

[0165] Target concentrations of 20 mg / mL or 50 mg / mL were achieved with all test anti-APRIL(VH14_1G.VL15) antibody preparations, and colorless, clear, and particle-free preparations were obtained throughout the entire test period. No significant changes were observed in monomers or charged species among any of the test preparations, as the data obtained after 12 weeks of storage at -70°C and 2–8°C, freezing / thawing, or shaking stress conditions at 2–8°C were comparable to the t=0 results and within the assay variability range. Turbidity values ​​after incubation at 25°C and 45°C indicated maximum stability for preparations containing arginine and sorbitol at pH 6.3. When measured by SE-HPLC, higher stability of VH14_1G.VL15 was maintained in samples formulated in the pH 6.3–6.5 range compared to pH 6.0. This was clearly observed only under the 45°C test condition. Compared to the same formulation with 50 mg / mL of anti-APRIL(VH14_1G.VL15) antibody, higher stability was observed at a protein concentration of 20 mg / mL. After incubation at 25°C and 45°C, CE-HPLC evaluation of charged species revealed that formulations with pH 6.0 or pH 6.3 were preferable, as all charged species showed minimal changes. Based on degradation rates calculated using the Arrhenius relationship, the predicted monomer and charged species stability of the 10 mM histidine, 75 mM arginine, 3% sorbitol, 0.01% (w / v) PS20, pH 6.3 formulation is at least 3 years at 5°C.

[0166] Example 15 In one clinical study (see PCT / IB2020 / 000020), patients with multiple myeloma received the anti-APRIL antibody BION-1301 (also known herein as VH14_1G.VL15) intravenously at a maximum dose of 1350 mg once weekly, or at a maximum dose of 2700 mg every two weeks. BION-1301 inhibited serum APRIL levels in a dose-dependent manner at doses ranging from 50 to 2700 mg. At 450 mg, 95% target engagement (TE) was achieved near the peak exposure level, and at 1350 mg, 95% TE was maintained throughout the dosing interval. Exposure was nearly dose-linear across the evaluated dose range, and the incidence of anti-drug antibodies was low. Overall, the antibody was well-tolerated, no dose-limiting toxicities were reported, and a maximum tolerable dose has not been determined. One serious adverse reaction (SAR) (wheezing) was reported in a patient receiving a 50 mg dose, leading to discontinuation of treatment.

[0167] The following is a Phase 1 trial in volunteers administered BION-1301 intravenously to evaluate its safety, tolerability, PK, and PD, with single doses up to 1350 mg IV and multiple doses up to 450 mg IV. This trial will start in HV and will also be conducted in adults with IgAN. The trial design includes three parts, as shown in the trial scheme (Figure 24). BION-1301 is provided as a solution intended for intravenous (IV) administration. BION-1301 is diluted and administered by IV infusion over approximately 2 hours at the assigned dose level. BION-1301 is supplied in vials at 20 mg / mL in at least 5 mL of solution and diluted with 0.9% saline before infusion. For example, the number of vials required for a particular dose is determined as required volume = mg dose / 20 mg / mL, and this volume is divided by 5 mL / vial to determine the total number of vials required. When using a 250 mL 0.9% saline bag for dilution, remove a volume equal to the required amount of antibody solution from the saline bag before adding the antibody solution. Remove the required amount of antibody solution from each vial (single-use vial, use a new syringe for each vial) and add it to the saline bag. Part 3: After the initial dose, if the investigator assesses that there are no tolerable issues, the infusion time for subsequent doses can be reduced to 1 hour. The placebo is 0.9% saline for IV administration. The placebo is administered by IV infusion over approximately 2 hours (applicable to Parts 1 and 2 only). [Table 34] TIFF0007843248000035.tif235160TIFF0007843248000036.tif61160

[0168] Part 1 (SAD-HV) was a double-blind, randomized, placebo-controlled single-dose escalation (SAD) design in HV. Up to five dose cohorts could be evaluated. The dose levels were 10 mg, 50 mg, 150 mg, 450 mg, and 1350 mg. Within each cohort, HV was randomized in a 3:1 ratio and each received either BION-1301 or placebo. Sentinel dosing was employed within each cohort to check for any safety concerns that would justify discontinuation of treatment in the remaining HV within the cohort.

[0169] Part 2 (MAD-HV) was a double-blind, randomized, placebo-controlled, multi-stage dose-escalation (MAD) design conducted in HV patients. HV patients within each cohort were randomized in a 2:1 ratio to receive either BION-1301 or placebo. Each HV received BION-1301 or placebo every two weeks (Q2W) for a total of three doses (i.e., on days 1, 15, and 29) via IV infusion. Up to four dose cohorts could be evaluated. Expected levels per dose were 50 mg, 150 mg, 450 mg, and 1350 mg.

[0170] In Parts 1 and 2, dose escalation was discontinued if any of the following criteria were met: One or more subjects in the cohort developed a SAE that the principal investigator assessed as being related to BION-1301. Two or more subjects in the cohort have experienced a severe non-serious adverse event (grade 3 or higher according to the National Cancer Institute Common Terminology Criteria for Adverse Events [NCI-CTCAE] Version 5.0) that the principal investigator assessed as being related to BION-1301; Two or more subjects in the cohort receiving the investigational drug develop clinically significant abnormalities in the same organ class, such as abnormalities in clinical laboratory tests, ECG, or vital signs, and exhibit dose-limiting intolerance; Individual AUC (0-168h) and / or Cmax exceeding the mean AUC (24,000 μg*day / mL) or mean Cmax (5720 μg / mL) established as NOAEL in cynomolgus monkeys; or Two or more subjects in the same cohort exhibit signs of bacterial infection and have persistently low IgG levels of less than 1.5 g / L or less than 3.0 g / L.

[0171] Part 3 (MD-IgAN) is an ongoing, open-label, multi-dose design in adult subjects with IgAN. MD-IgAN will begin after the last MAD-HV cohort has been evaluated by the Safety Review Team (SRT). Part 3 will consist of at least two cohorts, with the option to add additional cohorts if alternative doses or dosing schedules are being explored. The total duration of dosing for cohorts 1, 2, and any subsequent cohorts (if applicable) will be up to two years. The sample size for cohort 1 is approximately 10 IgAN patients. For cohort 2 and any additional cohorts added, the sample size will be approximately 40 or fewer IgAN patients in total.

[0172] The first cohort will receive BION-1301 at a dose level of 450 mg via IV infusion every two weeks for up to one year. The second cohort will be enrolled after SRT recommendations and will demonstrate sufficient safety and tolerability based on available data from at least five subjects in MD-IgAN cohort 1. If SRT recommends initiating cohort 2, all patients will receive BION-1301 via SC injection (containing 10 mM histidine, 75 mM arginine, 3% sorbitol, and 0.01% (w / w) polysorbate 20, pH 6.3, formulated at 150 mg / mL) for up to one year. The selection of dose and dosing regimen for MD-IgAN cohort 2 will be based on all available safety, PK, and PD data from all subjects to date, including those in MD-IgAN cohort 1. If SRT recommends dose or schedule changes for future subjects, a new cohort will be formed. Beyond cohorts 1 and 2, one or more additional cohorts may be explored. The maximum dose of BION-1301 in Part 3 shall not exceed 1350 mg, and the maximum exposure to BION-1301 during the dosing interval shall not exceed the observed dose in the Phase 1 dose escalation study (ADU-CL-16; NCT03340883) conducted in subjects with multiple myeloma. For subjects receiving BION-1301 via SC injection, the dose shall not exceed 600 mg QW. All patients receiving BION-1301 via IV infusion must transition to the SC administration route after at least 24 weeks of IV administration. The dose or schedule for patients switching from IV to SC may be modified to account for differences in bioavailability between administration routes and shall be determined by the SRT in cooperation with the sponsor based on all available data.

[0173] In the case of Part 3 dose escalation, the SRT opinion is to stop dose escalation if two or more subjects in the cohort experience drug-related toxicity or TEAE that would prevent further administration of the subject, considering that this may be considered an acceptable risk for the study population.

[0174] Inclusion criteria: Health volunteers 1. Healthy male or female volunteers aged 18 to 55 at the time of screening. 2. Women are assumed to be unable to give birth (according to CTFG2014). 3. Men must agree to follow the contraception guidance specified in the protocol. 4. In screenings where the weight is at least 50 kg, the range is 18-35 kg / m². 2 They have a body mass index (BMI). 5. Non-smokers are defined as individuals who have never smoked before and / or have discontinued smoking, or who have discontinued the use of nicotine / nicotine-containing products (such as snuff, e-cigarettes, and similar products) at least three months prior to the screening visit, or whose medical history confirms this. 6. The patient's health status, as determined by medical history, physical examination, vital signs assessment, 12-lead electrocardiogram (ECG), and laboratory evaluation, is within the normal reference range (or outside the normal reference range as deemed clinically irrelevant by the principal investigator). 7. Total IgG > 10 g / L at screening 8. You can provide signed informed consent, including compliance with the requirements and limitations set out in the Informed Consent Form (ICF).

[0175] Exclusion criteria: Health volunteers Health volunteers who meet any of the following exclusion criteria are not eligible to participate. 1. You have a known or suspected allergy or hypersensitivity to any component of BION-1301, or a history of severe hypersensitivity reactions to any monoclonal antibody. 2. Regular alcohol consumption within the 6 months prior to screening (more than 7 drinks / week for women, more than 14 drinks / week for men, 1 drink = 5 ounces [150 mL] of wine or Use of 12 ounces [360 mL] of beer or 1.5 ounces [45 mL] of hard liquor, or use of soft drugs (such as marijuana) within 3 months prior to the screening visit, or use of hard drugs (such as cocaine and phencyclidine) within 1 year prior to the screening visit, and / or a positive result on a blood or urine test for abused drugs or alcohol at the time of the screening visit or hospitalization. 3. The patient donated blood three months prior to the first dose of the investigational drug, donated plasma seven days prior to the first dose of the investigational drug, or donated platelets six weeks prior to the first dose of the investigational drug. 4. You cannot refrain from strenuous exercise for 7 days prior to the first dose of the investigational drug until the final follow-up visit. 5. Within 28 days of the first dose of the investigational drug or within 5 half-lives (whichever is longer), use of any unprescribed systemic or topical medication, therapy or supplement, or any prescribed systemic or topical medication (except paracetamol up to 2 g / day, ibuprofen up to 800 mg / day, or hormone replacement therapy [HRT]). 6. When the principal investigator determined that there was insufficient venous access for the purpose of multiple blood draws and administration of the investigational drug, 7. Participating in another trial receiving a novel investigational drug or investigational device within 28 days of the first dose of the investigational drug in this study or within 5 half-lives (whichever is longer) 8. If you received an approved monoclonal antibody drug within 28 days prior to the first dose of the investigational drug in this study or within 5 half-lives (whichever is longer), 9. Any confirmed or suspected immunosuppressive or immunodeficiency condition, such as human immunodeficiency virus (HIV) infection or asplenia; -Within the six months prior to day 1, the patient had experienced a recurrent severe infection and chronic immunosuppressive therapy (28 days or more for inhaled / topical corticosteroids, or 1 week or more for systemic immunosuppression or corticosteroid therapy). 10. Serological tests for hepatitis A virus IgM antibody (anti-HAVIgM), hepatitis B surface antigen (HBsAg), and hepatitis C virus (HCV) antibody are positive at the time of screening. 11. The patient underwent major surgery or suffered a major trauma within 28 days prior to the first dose of the investigational drug. If the patient underwent major surgery more than 28 days prior to the first dose of the investigational drug, the patient must have fully recovered from any toxicity and / or complications from the intervention prior to the first dose of the investigational drug. 12. Having a history or evidence of a clinically significant disorder, condition, or disease that could pose a safety risk to the subject, interfere with the study, or disqualify the subject from participating, such as a respiratory disease, renal disease, hepatic disease, gastrointestinal disease, hematological disorder, lymphatic disorder, neurological disorder, cardiovascular disease, or psychiatric disorder. 13. At the time of screening, the QuantiFERON-TB Gold Plus test is positive. 14. You have received a live vaccine within 3 months prior to screening, or you are scheduled to receive a live vaccine within 3 months of your last dose of the investigational drug. 15. Women who are breastfeeding, or women who have a positive serum pregnancy test at the time of screening, or a positive urine pregnancy test on day -1, 16. Any other condition, in the opinion of the principal investigator or medical monitor, that is inappropriate for inclusion in the participant or that could impede the participant's ability to complete the study.

[0176] Selection Criteria for IgAN Patients An eligible individual must meet all of the following criteria: 1. Males or females aged 18 or older at the time of screening, 2. Women of childbearing potential (WOCBP; CTFG2014) must agree to follow the contraception guidance specified in the protocol throughout the entire study (from screening until approximately 5 half-lives (165 days) after the final dose of the investigational drug). 3. Men agree to follow the contraception guidance specified in the protocol throughout the entire study (from screening until approximately 5 half-lives (165 days) after the final dose of the investigational drug). 4. When screening for a weight of 50 kg or more, BMI is between 18 and 40 kg / m². 2 That is, 5. If, as determined by medical history, physical examination, vital signs assessment, 12-lead electrocardiogram, and clinical laboratory assessment, the patient is within the normal reference range or consistent with IgAN (or, if outside the normal reference range, is deemed clinically irrelevant by the principal investigator), 6. Diagnosed with IgAN, verified by a biopsy taken within the last 10 years. 7. Urine protein ≥ 0.5 g / 24 hours; or UPCR ≥ 0.5 g / g (or ≥ 50 mg / mmol) based on evaluation of 24-hour urine collected at the time of screening. 8. eGFR (according to the Chronic Kidney Disease Epidemiology Collaborative Study [CKD-EPI] formula) or measured GFR > 45 mL / min / 1.73 m² 2 This is the case; or, if there is no evidence of glomerular fibrosis in a renal biopsy performed within 2 years from day 1 (e.g., S1 according to the Oxford classification), then 30-45 mL / min / 1.73m 2 That is the case. 9. The patient is stable or intolerant to ACE / ARB at the optimal dose of angiotensin-converting enzyme (ACE) inhibitors and / or angiotensin receptor blockers (ARBs) for at least 3 months prior to screening. 10. Able to provide signed informed consent, including compliance with the requirements and limitations set forth in the ICF. 11. Blood pressure during the screening visit is <140 / 90 mmHg (systolic / diastolic),

[0177] Exclusion criteria for IgAN patients Individuals who meet any of the following exclusion criteria are not eligible to participate. 1. You have a known or suspected allergy or hypersensitivity to any component of BION-1301, or a history of severe hypersensitivity reactions to any monoclonal antibody. 2. The patient has donated blood during the three months prior to the first dose of the investigational drug; the patient has donated plasma during the seven days prior to the first dose of the investigational drug; or the patient has donated platelets during the six weeks prior to the first dose of the investigational drug. 3. When the principal investigator determined that there was insufficient venous access for the purpose of multiple blood draws and administration of the investigational drug, 4. Participants in other trials receiving a novel investigational drug or investigational device within 28 days of the first dose of the investigational drug in this study or within 5 half-lives (whichever is longer) 5. Any confirmed or suspected immunosuppressive or immunodeficient condition, such as HIV infection or asplenia; -Within the three months prior to day 1, the patient had experienced a recurrent severe infection and chronic immunosuppressive therapy (28 days or more for inhaled / topical corticosteroids, or 1 week or more for systemic immunosuppression or corticosteroid therapy). 6. Positive results on serological tests for anti-HAV IgM, HBsAg, HCV antibodies (if not receiving appropriate treatment for HCV), or antibodies against HIV-1 and / or HIV-2 at the time of screening. 7. The patient underwent major surgery or suffered a major trauma within 28 days prior to the first dose of the investigational drug. If the patient underwent major surgery more than 28 days prior to the first dose of the investigational drug, the patient must have fully recovered from the toxicity and / or complications of the intervention prior to the first dose of the investigational drug. 8. Having a history or evidence of a clinically significant impairment, condition, disease, or laboratory finding that could pose a safety risk to the subject, interfere with the study, or, in the judgment of the principal investigator, would disqualify the subject from participation. 9. Have you previously used any approved biological agents, such as monoclonal antibodies, within 28 days prior to the first dose of the investigational drug in this study, or within 5 half-lives (whichever is longer)? 10. Having a secondary form of IgAN as defined by the treating physician (e.g., IgA vasculitis with associated alcoholic cirrhosis), 11. Clinical suspicion of rapidly progressive glomerulonephritis (RPGN). 12. Having any renal condition that the principal investigator believes requires immediate treatment, such as requiring dialysis treatment or any planned kidney transplant during the trial. 13. Within three months prior to the first dose of the investigational drug, the patient received systemic corticosteroid therapy (prednisone or equivalent at a dose greater than 10 mg / day for at least 10 days) or any other form of immunosuppressive therapy. 14. You have type 1 or type 2 diabetes. 15. Uncontrolled cardiovascular disease as determined by the principal investigator, 16. Having a current malignant tumor or a history of malignant tumors within the past three years; except as follows: • Basal cell carcinoma that has been properly treated, • Squamous cell carcinoma of the skin, or cervical cancer in situ, • Low-risk prostate cancer (i.e., Gleason score < 7 and prostate-specific antigen < 10 ng / mL), 17. A patient has a life-threatening or clinically significant comorbidity (including an active infection) that the principal investigator assesses as potentially limiting compliance with the study requirements or posing an unacceptable risk to the patient's participation in the study. 18. At the time of screening, the QuantiFERON-TB Gold Plus test is positive. 19. Live vaccine was received within 3 months prior to screening and within 3 months after the last dose of the investigational drug. Non-replicating viral vector vaccines are acceptable. 20. Breastfeeding women, or women who have a positive serum pregnancy test at the time of screening, or a positive urine pregnancy test before the first day of administration.

[0178] The pharmacologically active dose (PAD) of BION-1301 is defined as a single dose predicted to cause a 95% reduction in free serum APRIL concentration over up to 24 hours and a minimal reduction in immunoglobulins (approximately 6% for IgG and approximately 15% for IgA). The estimated PAD is 50 mg, based on single and multiple-dose toxicity study data in cynomolgus monkeys using PK-PD modeling. This model accounts for the difference in APRIL concentrations between cynomolgus monkeys and humans, and allometric scaling was applied to convert between species rate constants (PK and PD) and volume (PK).

[0179] In the clinical trial (ADU-CL-16), a safety factor of 5 was applied to PAD to account for potential toxicity not yet observed in multiple myeloma patients and to explain the fact that the risk-benefit profiles differ between HVs and patients. In Part 1 of this trial, the initial cohort consisted of four HVs, starting at 10 mg. Three received BION-1301 and one received placebo (compared to the subsequent cohort, where six received BION-1301 and two received placebo). The reduction in the number of HVs in SAD-HV-1 is based on the assumption of expected minor pharmacological effects with respect to immunoglobulin concentration. Therefore, the usefulness of this data in characterizing the dose-exposure-response relationship of BION-1301 is limited.

[0180] In addition to the PAD-based considerations mentioned above, the starting dose of 10 mg is 600 to 2383 times less than the no-observed-adverse-effect level (NOAEL) of 100 mg / kg in cynomolgus monkeys. Therefore, in healthy adults, there is a sufficient safety factor to support the initial dose of BION-1301. [Table 35] a. A dose of 10 mg in humans corresponds to 0.17 mg / kg, assuming a person weighing 60 kg. b SFDose = Dosecyno / Dosehuman c SFAUC = AUCcyno / AUChuman.

[0181] On day 29 of the test, the sex-binding AUC0-168cyno at 100 mg / kg was 24,000 mg*day / L. The observed AUC0-inf in subjects with multiple myeloma administered 50 mg was 84 mg*day / L (582 nM / day). Assuming linear PK would result in an AUC0-inf of 16.8 mg*day / L at a dose of 10 mg. dSFCmax = (Cmax-cyno) / (Cmax-human), where C max-cyno is based on the observed mean Cmax-obs after 5 administrations on day 29. The sex-binding C max in cyno on day 29 is 5720 μg / mL. The observed C max in humans at 50 mgis 12 mg / L (85.0 nM); assuming linear PK would result in a C max of 2.4 mg / L at a dose of 10 mg.

[0182] Parts 1 and 2 were conducted, and the baseline demographics for Part 1 (SAD) and Part 2 (MAD) are shown in Table 36 below.

Table 36

[0183] BION-1301 was well tolerated in HV. No SAE, treatment discontinuation, or events meeting discontinuation criteria were reported. All patients received premedication prior to the first infusion, and in the MAD150 mg cohort, 1 infusion-related reaction was reported. The most common AEs occurring in more than 10% of subjects in the MAD cohort were headache, limb pain, elevated AST, and nasopharyngitis. The most common AE occurring in more than 10% of subjects in the SAD cohort was nasopharyngitis. This administration was associated with a low incidence of anti-drug antibodies and neutralizing antibodies.

[0184] Figure 25 shows the mean BION-1301 serum concentration (±SD) versus nominal time at the prescribed dose. While concentrations were similar within the cohort, individual variability was a result of fixed dose and variable body weight, likely influencing pharmacokinetics. Mean BION-1301 serum concentrations were generally dose-proportional at low doses but slightly greater than dose-proportional at high doses.

[0185] Figure 26 shows the mean free APRIL (fAPRIL) serum concentration + / -SD versus nominal time at the prescribed dose. As shown, BION-1301 exhibits a sustained dose-dependent increase in target occupancy, lasting for more than one month at high doses.

[0186] Figure 27 shows the mean percentage change (±SD) in serum immunoglobulin levels relative to baseline samples collected on day 1 prior to administration. Panels A–C show single-dose cohorts relative to time-course (day) baseline, and panels D–F show multiple-dose cohorts. BION-1301 dose-dependently and permanently reduces IgA and IgM, and reduces IgG to a low degree. This data is consistent with the possibility of monthly administration in patients. At single-dose levels of 1350 mg or multiple-dose levels of 450 mg, BION-1301 suppressed IgM levels to a low range of laboratory values, but there were no reports of treatment-related infections. The reduction of immunoglobulins mediated by BION-1301 may disrupt the stoichiometry of the IgA:IgG immune complex. As shown in Figures 28A and 28B, BION-1301 provides a pharmacodynamic window to utilize the reduction of IgA while mitigating its effect on IgG.

[0187] Figure 33 shows the reductions in serum IgA and Gd-IgA1 in single-dose escalation (SAD) and multiple-dose escalation (MAD) studies of BION-1301 administered by intravenous (IV) infusion in healthy human volunteers. As shown, BION-1301 resulted in dose-dependent proportional reductions in both serum IgA and Gd-IgA1 levels.

[0188] Preliminary data from Part 3 showed that five patients received 450 mg of BION-1301 via IV infusion for 12 weeks at Q2W. The treatment was well-tolerated, and there were no early terminations due to SAE adverse events. A rapid and sustained decrease in free april was observed, along with sustained decreases in Gd-IgA1, IgA, and IgM, and a smaller decrease in IgG. A clinically significant decrease in proteinuria (24-hour UPCR) was observed. The preliminary data provide early proof of concept regarding the potential for BION-1301 to deplete pathogenic Gd-IgA1 and reduce proteinuria in IgA nephropathy patients who remain at risk of progression with residual proteinuria despite optimized SOC treatment.

[0189] As a substitute for the presence of circulating pathogenic IgA-containing immune complexes, an ex vivo mesangial cell activation assay was used as a bioassay. IgA fractions were isolated by column chromatography from the plasma of the first two IgAN patients enrolled in this study at baseline, before BION-1301 administration and on days 29 and 85 of BION-1301 treatment. Primary human mesangial cells in culture were then stimulated with the IgA-containing fractions from these patients for 72 hours, and mesangial cell proliferation was measured three times by Brdu uptake. As shown in Figure 34, BION-1301 resulted in rapid APRIL neutralization in these IgAN patients, followed by decreased Gd-IgA1 depletion and mesangial cell activation, and subsequently decreased proteinuria.

[0190] Example 16 The following is a Phase 1 single-dose, parallel-group safety and bioavailability study of BION-1301 administered intravenously (IV) or subcutaneously (SC) to healthy adult volunteers. This study was conducted to define the bioavailability of BION-1301 when administered as SC injection, in order to help define the recommended Phase 2 dose (RP2D) that would enable chronic administration of SC for the proposed indication for the treatment of IgAN. [Table 37] [Table 38]

[0191] This study was a Phase I, open-label, randomized, single-dose, parallel-group safety and bioavailability study of BION-1301 administered intravenously (IV) and via seroconjunctival (SC) routes to healthy adult volunteers. The study enrolled approximately 34 subjects (17 per arm) in a two-arm, parallel-group, three-period design to achieve a total of 30 PK-evaluable subjects. Subjects were randomized in a 1:1 ratio to receive either BION-1301 via IV administration (Therapy Group A) or via SC administration (Therapy Group B). In both therapy arms, subjects received a single dose of 300 mg of BION-1301. The study was conducted over three defined study periods: a screening period, a treatment period, and a safety follow-up period. Written informed consent was obtained for participation in the study before any study-related procedures or evaluations were performed. Administration (SC or IV) was administered on day 1 in a clinic under the supervision of a qualified physician. Participants were closely monitored for the first 24 hours after administration on day 1 and throughout their hospital stay up to day 8. The study was completed when the last participant completed their final visit on day 57. The study period for any individual participant was a maximum of 14 weeks, including a 6-week screening period and treatment (1 day) and an 8-week safety follow-up period. Participants received a single dose of 300 mg of BION-1301 via one of two routes of administration: IV or SC. IV administration was prepared as described in Example 15, with a 20 mg / mL antibody formulation diluted in 0.9% saline, in this case using 3 x 5 mL vials to obtain 15 mL of antibody solution diluted in 235 mL of saline. SC administration was also provided in vials of the 150 mg / mL formulation, and the desired 2.0 mL volume was drawn with a syringe and injected directly into the abdomen.

[0192] Screening period: This period began when the Informed Consent Form (ICF) was signed. During this period, participants were evaluated to determine their eligibility to participate in the study. The screening period lasted a maximum of six weeks. Participants who met all eligibility criteria were enrolled in the study.

[0193] Hospitalization / Treatment Period: Treatment began with admission to a clinical facility on day 1 and ended with discharge from the clinical facility on day 8. Administration (SC or IV) was performed in the clinic under the supervision of a qualified person on day 1. Subjects were closely monitored for the first 24 hours after administration on day 1, and then carefully monitored until discharge on day 8. During the treatment period, subjects underwent safety monitoring and evaluation of PK and PD. After the completion of final PK and PD sample collection and safety evaluation on day 8, subjects entered the follow-up period.

[0194] Follow-up period: As described in the SOE, it began at discharge from the clinical facility and ended on day 57 with the End of Study (EOS) visit.

[0195] Participants who were eligible to take this exam had met all of the following criteria: Participants must be between 18 and 60 years of age at the time of signing the informed consent form. 1) - Screening for weight of 47 kg or more, 18-30 kg / m 2 They have a body mass index (BMI). 2) Non-smokers are defined as individuals who have never smoked before and / or have discontinued smoking, or who have discontinued the use of nicotine / nicotine-containing products (including, but not limited to, snuff, e-cigarettes, and similar products) at least three months prior to the screening visit, or who have been confirmed to have a medical history and negative cotinine levels. 3) The patient's health status, as determined by the principal investigator based on medical history, physical examination (PE), vital signs assessment, 12-lead ECG, and laboratory assessment, is within the normal reference range (or outside the normal reference range as the principal investigator considers clinically insignificant). 4) The principal investigator considers the subject to be likely to adhere to the protocol throughout their participation in the trial. 5) You are not currently taking any prescription or over-the-counter medications, including dietary supplements, unless deemed appropriate at the discretion of the Principal Investigator and / or Sponsor / Medical Monitor. 6) Participants may be male or female. Male participants were eligible to participate if they agreed to follow the contraception guidance provided during screening until 10 weeks after the final dose of the investigational drug. - It has been more than 3 months since the vasectomy, - Avoid sperm donation for 10 weeks after the final dose of the investigational drug and subsequent screening. Furthermore, one of the following: - Do not engage in heterosexual intercourse. or - You agree to use a male condom in conjunction with an additional, highly effective female barrier contraception during sexual intercourse. Female participants were eligible to participate if they agreed to follow the contraception guidance provided during screening until 10 weeks after the final dose of the investigational drug. - Not a woman of childbearing potential (WOCBP) or - The patient is a WOCBP (Wound, Ovarian, and Cushing's Box) and has used highly effective contraception from screening until 10 weeks after the last dose of the study treatment, and agrees not to donate eggs (eggs, oocytes) for reproductive purposes during this period. If the highly effective contraception is hormonal, the male partner must also use a barrier method (i.e., male condoms). The principal investigator will need to evaluate the effectiveness of the contraception in connection with the initiation of the study treatment. -WOCBP patients must have a negative result on a highly sensitive pregnancy test (serum as required by local regulations) within 24 hours prior to administration of the study treatment. 7) You are able to provide and understand signed informed consent and are willing to take the exam.

[0196] Participants were excluded from the study if they met any of the following criteria. 1) Regular alcohol consumption within the 6 months prior to screening (more than 7 drinks per week for women, more than 14 drinks per week for men, 1 drink = 5 ounces [150 mL] of wine or 12 ounces [360 mL] of beer or 1.5 ounces [45 mL] of hard liquor), or use of soft drugs (such as marijuana) within the 3 months prior to the screening visit, or use of hard drugs (such as cocaine and phencyclidine) within the 1 year prior to the screening visit and / or a positive result on a blood or urine test for abused drugs or alcohol at the time of the screening visit. 2) - The patient donated blood three months prior to a single dose of the investigational drug, donated plasma seven days prior to a single dose of the investigational drug, donated platelets six weeks prior to a single dose of the investigational drug, or donated blood or blood products within eight weeks prior to a single dose of the investigational drug. 3) - Within 28 days or 5 half-lives (whichever is longer) of the administration of the investigational drug, use of any prescription drug, unprescribed systemic or topical drug, treatment or supplement, or any prescribed systemic or topical drug (excluding paracetamol up to 2 g / day or ibuprofen up to 800 mg / day, hormone replacement therapy [HRT], or hormonal contraceptives), unless deemed acceptable by the principal investigator and medical monitor. 4) - When the principal investigator determined that there was insufficient venous access for the purpose of multiple blood draws and administration of the investigational drug, 5) Have you previously used an approved biological agent, such as a monoclonal antibody, within 28 days prior to a single dose of the investigational drug in this study, or within 5 half-lives (whichever is longer)? 6) - The patient has received a live vaccine within 3 months prior to screening, or is scheduled to receive a vaccine within 3 months after administration of the investigational drug. 7) Within the 8 weeks prior to day 1, any confirmed or suspected immunocompromised condition exists, or there is a recurrent, severe infection, or the patient is using a chronic systemic immunosuppressant. 8) A serological test is positive for hepatitis B (HBsAg) antibody, hepatitis C (HCV) antibody surface antigen (if not receiving appropriate treatment for HCV), or antibodies against HIV-1 and / or HIV-2 (human immunodeficiency virus). 9) A clinically significant impairment, condition, or disease that could pose a risk to the safety of the subject, interfere with the study, or disqualify the subject from participating, such as a history or evidence of clinically significant respiratory, renal, hepatic, gastrointestinal, hematological, lymphatic, neurological, cardiovascular, or psychiatric disorders. 10) - At the time of screening, the QuantiFERON-TB Gold Plus test is positive. 11) - Women who are breastfeeding, or who have a positive serum pregnancy test at the time of screening, or who have a positive serum pregnancy test on day 1, 12)-QTcF is defined as having an ECG duration greater than 450 msec in men, greater than 470 msec in women, or clinically significant ECG findings as determined by the principal investigator. 13) Measurement of vital signs including the following (one repeat measurement was possible): a. Systolic blood pressure (BP) > 140 mm / Hg or diastolic blood pressure > 90 mm / Hg. b. Systolic blood pressure < 90 mm / Hg or diastolic blood pressure < 50 mm / Hg. c. Pulse rate is over 100 bpm or less than 40 bpm. 14) If you participated in any other study in which you received the investigational drug or device within 28 days of administration of the investigational drug in this study or within 5 half-lives (whichever is longer), 15) A known or suspected allergy or hypersensitivity to any component of BION-1301, or a history of severe hypersensitivity reactions to any monoclonal antibody,

[0197] For the purpose of the analysis, the following analysis populations are defined in Table 37. [Table 39]

[0198] When the subject met all inclusion and exclusion criteria and was randomized to one of the treatment groups, the subject was considered enrolled.

[0199] An evaluable subject was defined as an enrolled subject who received at least one test treatment and provided a PK sample after baseline by day 15.

[0200] The Statistical Analysis Plan (SAP) was created and finalized before the database was locked, and the subject population included in the analysis and the procedures for explaining missing, unused, and spurious data were described in more detail.

[0201] All pharmacokinetic analyses were performed on the PK population using Phoenix® WinNonlin® (version 8.1). Analyses of all PK endpoints were provided separately for each treatment arm. PK parameters were estimated by non-compartmental analysis including but not limited to the following. -------------------------------------------------------------------------------- Parameter Definition -------------------------------------------------------------------------------- Serum concentration extrapolated to time zero C max Observed maximum serum concentration C min Observed minimum serum concentration t max C max Time corresponding to the occurrence of F Bioavailability AUC last Area under the serum concentration-time curve from time zero to the last observed time point AUC 0-t Area under the serum concentration-time curve from time zero to t AUC0-inf Area under the serum concentration-time curve from time 0 to infinity T max Time to reach maximum serum concentration t 1 / 2 Terminal phase disappearance half-life CL Clearance V ss Distribution in the steady state MRT average residence time

[0202] PK parameter measurements and estimates were aggregated and summarized for each arm using descriptive statistics (mean, standard deviation [SD], coefficient of variation (CV%), median, minimum, and maximum). PK summary statistics may include, where appropriate, reports of CV%, geometric mean, and geometric CV%. Graph displays include mean (±SD) serum concentration-time curves and, where possible, individual subject concentration-time curves over PK sampling time.

[0203] The specifications for the PK parameters for analysis; the statistical significance levels used, procedures for explaining missing and unused data, procedures for reporting deviations, and the selection of subjects to be included in the analysis population were presented to the SAP as needed. Comparisons of PK parameters within each treatment arm were performed.

[0204] This study was completed using AMRIGlasgow's BION-130 120 mg / mL solution lot number P197673-0004L001 / P02919 and 150 mg / mL solution lot number P217955-0001L001 / P04819. Samples of PK and PD were collected before administration, and at 0.25, 2, 4, and 8 hours after infusion / injection, and at 24 hours (day 2), 48 hours (day 3), 72 hours (day 4), 168 hours (day 8), 336 hours (day 15), 672 hours (day 29), 1008 hours (day 43), and 1344 hours (day 57) after administration. Of the 17 subjects who received a 300 mg intravenous dose via IV administration, 2 did not complete the study. Of the 17 subjects who received a 300 mg dose via SC administration, 3 did not complete the study and were considered lost to follow-up. Both routes of administration for a 300 mg dose were found to be safe and well-tolerated. Serum levels of BION-1301, free APRIL levels, and IgA, IgG, and IgM antibody levels were measured.

[0205] The mean (±SD) serum concentration levels of BION-1301 are shown in Figure 30. Time 0 is the administration time of BION-1301. Table 38 below provides the pharmacokinetic parameters of BION-1301 for IV versus SC dose administration. [Table 40]

[0206] The relative bioavailability between IV BION-1301 (reference) and SC BION-1301 (experimental) treatments was evaluated using ANOVA, and the results are shown in Table 39 below. The examination treatment was 300 mg of BION-1301 administered via SC. The reference treatment was 300 mg of BION-1301 administered intravenously. CI = confidence interval, CV = coefficient of variation, LSM = least squares mean, and N = number of observations. [Table 41]

[0207] Regarding the measurement of APRIL concentration, the mean (±SD) fAPRIL concentration after a single IV or SC dose is shown in Figure 31A, and the mean (±SD) percentage change of fAPRIL relative to baseline after a single IV or SC dose is shown in Figure 31B, where time 0 is the administration time of BION-1301. Table 40 below provides the pharmacodynamic parameters of fAPRIL after IV vs SC dose administration. Here, AUECBelowB 0-t R is the area under the response curve that falls below the baseline effect value from time 0 to time t, using the linear trapezoidal interpolation rule. min This is the minimum response observed after administration, and PBR min This is the minimum percentage change from the baseline response value after administration, and (R min -B) / Bx100 is calculated, and t min R min It is time for this. [Table 42]

[0208] Regarding the measurement of immunoglobulin levels, the mean percentage change in serum immunoglobulin levels relative to baseline over time for BION-1301 IV versus SC administration is shown in Figures 32A (IgA), 32B (IgG), and 32C (IgM).

[0209] Those skilled in the art will readily understand that the present invention is well adapted to perform its purpose and to obtain the purposes and benefits mentioned, as well as those unique to the invention. The examples provided herein are representative of preferred embodiments and are illustrative; they are not intended to limit the scope of the invention.

[0210] It should be understood that, in its application, the present invention is not limited to structural details and the arrangement of components described in the following description or shown in the drawings. The present invention is capable of embodiments beyond those described and can be practiced and implemented in a variety of ways. Furthermore, it should be understood that the language and terminology used herein, as well as the abstract, are for illustrative purposes only and should not be considered limiting.

[0211] Therefore, those skilled in the art will understand that the concepts underlying this disclosure can be readily used as the basis for designing other structures, methods, and systems to accomplish some of the objectives of the present invention. It is therefore important that the claims be deemed to include such equivalent configurations, as long as they do not depart from the spirit and scope of the present invention.

[0212] Although the present invention has been described and illustrated in sufficient detail for those skilled in the art to create and use it, various alternatives, modifications, and improvements will be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments and are illustrative, and are not intended to limit the scope of the invention. Modifications and other uses therein will arise for those skilled in the art. These modifications are incorporated into the spirit of the invention and are defined by the claims.

[0213] Those skilled in the art will readily see that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0214] All patent applications, patents, publications, and other references referenced herein represent the level of skill of those skilled in the art to whom the present invention relates and are incorporated herein by reference, respectively. None of the references cited herein are considered to be prior art of the claimed invention.

[0215] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention belongs. In case of any conflict, this specification, including its definitions, shall prevail.

[0216] In both this specification and the claims, the use of the articles “a,” “an,” and “the” shall be construed to encompass both singular and plural forms unless otherwise indicated herein or unless the context clearly contradicts this. The terms “comprising,” “having,” and “being of” shall be construed as open terms unless otherwise specified, such as “being of a chemical formula,” “including,” and “containing” (i.e., “including but not limited to”). Furthermore, whenever “comprising” or another open-ended term is used in an embodiment, it shall be understood that the same embodiment may be claimed more narrowly using the intermediate term “consisting essentially of” or the closed term “consisting of.”

[0217] The terms “approximately,” “about,” or “approximately,” when used in relation to a number, mean to include a set or range of values. For example, “approximately X” includes a range of values ​​of X that are ±20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1%, where X is a number. In one embodiment, the term “approximately” refers to a range of values ​​that are 10% more or less than a particular value. In another embodiment, the term “approximately” refers to a range of values ​​that are 5% more or less than a particular value. In yet another embodiment, the term “approximately” refers to a range of values ​​that are 1% more or less than a particular value.

[0218] The enumeration of value ranges is intended merely as a simple way to refer individually to the individual values ​​within the range unless otherwise indicated herein, and the individual values ​​are incorporated herein as if they were individually enumerated herein. As used herein, ranges include two limitations unless otherwise specified. For example, the terms “X to Y” and “range from X to Y” include X and Y and integers in between. On the other hand, where a set of individual values ​​is referred to in the disclosure, any range that includes either of the two individual values ​​as two endpoints is also assumed herein. For example, the expression “doses of about 100 mg, 200 mg, or 400 mg” could mean “doses in the range of 100 to 200 mg,” “doses in the range of 200 to 400 mg,” or “doses in the range of about 100 mg to 400 mg.”

[0219] The invention as described herein exemplary may be suitably implemented in the absence of any elements(s) or limitations(s) not specifically disclosed herein. Therefore, for example, in each example herein, any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with any of the other two terms. The terms and expressions used are for illustrative purposes only, not limitation, and the use of such terms and expressions is not intended to exclude any equivalent of the shown and described features or any part thereof, although it is recognized that various modifications are possible within the scope of the invention as described in the claims. Therefore, although the invention is specifically disclosed by preferred embodiments and optional features, it is understood that modifications and variations of the concepts disclosed herein may be utilized by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0220] Other embodiments are described in the following claims.

Claims

1. It is an antibody preparation, A humanized anti-APRIL antibody having a concentration of approximately 20 mg / mL to approximately 190 mg / mL, wherein the humanized anti-APRIL antibody includes a heavy chain variable region containing SEQ ID NO: 24 and a light chain variable region containing SEQ ID NO: 26; The aforementioned humanized anti-APRIL antibody is an IgG antibody, Approximately 10 mM L-histidine; Approximately 75 mM L-arginine; Approximately 3% by weight of sorbitol; Approximately 0.01% by weight of polysorbate 20; and It contains a pH of approximately 6.0 to 6.

6. The preparation is an antibody preparation having (i) a viscosity of about 16 cP or less, (ii) a weight osmolality of about 250 mOsm / kg to about 390 mOsm / kg, and (iii) an optical density (OD330) at 330 nm of less than about 1.

0.

2. The antibody preparation according to claim 1, wherein the preparation is suitable for intravenous or subcutaneous injection.

3. The antibody preparation according to claim 1 or 2, wherein, after being stored at 2 to 8°C for 9 months following the manufacture of the preparation, the preparation maintains the anti-APRIL antibody with a purity of at least 96%.

4. The antibody preparation according to any one of claims 1 to 3, wherein, after manufacturing the preparation, the preparation maintains the anti-APRIL antibody with a purity of at least 95% after being stored at 25°C for 6 months.

5. The antibody preparation according to any one of claims 1 to 4, wherein the anti-APRIL antibody in the preparation is concentrated at a concentration of approximately 150 mg / mL.

6. The antibody preparation according to claim 5, having a weight osmolality of approximately 290 mOsm / kg to approximately 390 mOsm / kg.

7. The antibody preparation according to claim 5 or 6, wherein the preparation has an OD330 of about 0.8 or less.

8. The antibody preparation according to any one of claims 1 to 7, wherein the preparation does not contain glutamic acid or its salts, glycine, carbonate, HEPES, phosphate, citrate, and acetate.

9. The antibody preparation according to any one of claims 1 to 9, wherein the preparation comprises about 10 mM L-histidine, about 75 mM L-arginine, about 3% by weight of sorbitol, and a pH of about 6.1 to about 6.

3.

10. The antibody preparation according to any one of claims 1 to 10, wherein the preparation comprises a humanized anti-APRIL antibody at a concentration of about 150 mg / mL, about 10 mM L-histidine, about 75 mM L-arginine, about 3% by weight sorbitol, about 0.01% by weight polysorbate 20, and a pH of about 6.1 or about 6.

3.

11. The antibody preparation according to any one of claims 1 to 10, wherein the humanized anti-APRIL antibody comprises the amino acid sequence of the heavy chain variable region of SEQ ID NO: 28 and the amino acid sequence of the light chain variable region of SEQ ID NO:

26.

12. A pharmaceutical formulation for the treatment of IgA nephropathy in an individual requiring administration of an anti-APRIL antibody, comprising the antibody formulation according to any one of claims 1 to 11.

13. The pharmaceutical preparation according to claim 12, which is administered to an individual by subcutaneous injection.

14. The pharmaceutical formulation according to claim 13, comprising repeating the administration on a schedule of at least weekly (QW), at least every two weeks (Q2W), at least every four weeks (Q4W), or monthly (QMT) for at least two administration cycles.

15. The pharmaceutical preparation according to any one of claims 12 to 14, wherein a total dose of approximately 10 mg to approximately 1350 mg of the anti-APRIL antibody is administered at each administration event.

16. The pharmaceutical formulation according to claim 15, wherein approximately 2 mL of the formulation having an anti-APRIL antibody concentration of approximately 150 mg / mL is delivered in a single administration, and each administration event comprises one or more of the said administrations, or approximately 4 mL of the formulation having an anti-APRIL antibody concentration of approximately 150 mg / mL is delivered in a single administration, and each administration event comprises one or more of the said administrations.

17. The pharmaceutical preparation according to any one of claims 12 to 16, wherein the preparation is administered subcutaneously to a site in the thigh, abdomen, or upper arm of the individual.

18. The pharmaceutical formulation according to any one of claims 12 to 17, wherein the individual requiring administration of an anti-APRIL antibody has a serum IgA level greater than 4 g / L.

19. A pharmaceutical formulation for the treatment of IgA nephropathy in an individual requiring administration of an anti-APRIL antibody, comprising the antibody formulation according to any one of claims 1 to 11, the pharmaceutical formulation comprising administering the anti-APRIL antibody by a loading / maintenance administration protocol.

20. The pharmaceutical formulation according to claim 19, wherein the loading / maintenance administration protocol includes a loading component administered at a higher concentration than the maintenance administration protocol.

21. The pharmaceutical formulation according to claim 19 or 20, wherein the loading / maintenance administration protocol includes a loading component administered at a higher frequency than the maintenance component.

22. A pharmaceutical preparation according to any one of claims 19 to 21, wherein the loading component of the loading / maintenance administration protocol is administered once or more times via a route different from the administration route of the anti-APRIL antibody in the maintenance component of the loading / maintenance administration protocol.

23. A pharmaceutical preparation according to any one of claims 19 to 22, wherein the loading component of the loading / maintenance administration protocol comprises the anti-APRIL antibody administered intravenously once or more times, and the maintenance component of the loading / maintenance administration protocol comprises the anti-APRIL antibody administered subcutaneously once or more times.

24. The pharmaceutical preparation according to any one of claims 19 to 23, wherein a total dose of approximately 10 mg to approximately 1350 mg of the anti-APRIL antibody is administered at each administration event.

Images