Formulations for anti-insulin receptor antibodies and their uses

Abstract

The present disclosure relates generally to formulations comprising the anti-insulin receptor antibody RZ 358 and their use to treat hyperinsulinemic disorders.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 344,286, filed May 20, 2022, which is incorporated herein by reference in its entirety.

[0002] Field of Disclosure The present disclosure relates generally to the formulation of antibodies specific for the insulin receptor in the treatment and prevention of hypoglycemia and conditions associated with hyperinsulinemia, such as congenital hyperinsulinemia.

[0003] INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY The sequence listing, which is part of this disclosure, is submitted as a text file at the same time as the specification. The name of the text file containing the sequence listing is "57847_Seqlisting.html", which was created on May 16, 2023, and is 9,676 bytes in size. The subject matter of the sequence listing is incorporated herein by reference. [Background technology]

[0004] background Insulin is the main hormone for lowering blood glucose levels. The first step of insulin action is the binding of the hormone to the insulin receptor (INSR), an integral membrane glycoprotein also called CD220 or HHF5. When insulin binds to INSR, the receptor is activated by tyrosine autophosphorylation, and the INSR tyrosine kinase phosphorylates various effector molecules, including insulin receptor substrate-1 (IRS-1), resulting in the hormone's action (Ullrich et al, Nature 313:756-761, 1985 (Non-Patent Document 1); Goldfine et al, Endocrine Reviews 8:235-255, 1987 (Non-Patent Document 2); White and Kahn, Journal Biol. Chem. 269:1-4, 1994 (Non-Patent Document 3)). The binding and phosphorylation of IRS-1 ultimately leads to an increase in high-affinity glucose transporter (Glut4) molecules on the outer membrane of insulin-responsive tissues, including muscle cells and adipose tissue, and an increase in glucose uptake from the blood into these tissues. Glut4 mediates the transport of glucose into cells and the reduction of blood glucose levels.

[0005] Abnormally increased insulin secretion can result in hypoglycemia, or low blood sugar, which can lead to serious medical conditions including epilepsy and brain damage. Drug-induced hypoglycemia can result from the administration of sulfonylureas or from taking too much insulin. Several rare medical conditions are characterized by non-drug-induced endogenous hyperinsulinemic hypoglycemia, i.e., hypoglycemia caused by the body's excessive production of insulin. These conditions include congenital hyperinsulinism, insulinoma, and hyperinsulinemic hypoglycemia after gastric bypass surgery.

[0006] RZ358 is a monoclonal antibody specific for the insulin receptor (INSR), which regulates insulin binding to the insulin receptor and binds allosterically to the INSR without blocking insulin binding to the insulin receptor. In a phase 1 study, RZ358 was shown to reduce blood glucose levels in healthy volunteers (Johnson et al., J Clin Endocrinol Metab. 2017 102(8):3021-3028 (Non-Patent Document 4)), and later studies showed a reduction in daily periods of hypoglycemia and correction of nocturnal hypoglycemia in patients with post-gastric bypass hypoglycemia (PGBH) (Hu et al., Journal of the Endocrine Society, Volume 5, Issue Supplement 1, April-May 2021, Pages A328-A329 (Non-Patent Document 5)). A recent phase 2b trial demonstrated improvement in hypoglycemia in patients with congenital hyperinsulinism (CHI) receiving antibody therapy (Thornton et al., RZ358 in Congenital Hyperinsulinism: Results from a Multi-Center, Global, Phase 2b Study (RIZE), Pediatric Endocrine Society Annual Meeting 2022 (Non-Patent Document 6)). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Ullrich et al,Nature 313:756-761,1985 [Non-Patent Document 2] Goldfine et al, Endocrine Reviews 8:235-255,1987 [Non-Patent Document 3] White and Kahn,Journal Biol.Chem.269:1-4,1994 [Non-Patent Document 4] Johnson et al.,J Clin Endocrinol Metab.2017 102(8):3021-3028 [Non-Patent Document 5] Hu et al.,Journal of the Endocrine Society,Volume 5,Issue Supplement 1,April-May 2021,Pages A328-A329 [Non-Patent Document 6] Thornton et al.,RZ358 in Congenital Hyperinsulinism:Results from a Multi-Center,Global,Phase 2b Study(RIZE),Pediatric Endocrine Society Annual Meeting 2022 Summary of the Invention

[0008] overview Contemplated herein are formulations for anti-INSR antibodies, such as RZ 358, that provide stability of the antibody over time under different storage conditions.

[0009] The present disclosure provides a composition comprising an antibody that specifically binds to the insulin receptor (INSR), at least one amino acid or a salt thereof, a surfactant, and a sugar alcohol, wherein the anti-INSR antibody comprises: (A) a light chain variable domain comprising (i) a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6, (ii) a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7, and (iii) a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8; and (B) a heavy chain variable domain comprising (i) a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3, (ii) a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5.

[0010] In various embodiments, the anti-INSR antibody comprises (A) a light chain variable domain comprising a sequence of amino acids at least 80% identical to SEQ ID NO:2, or (B) a heavy chain variable domain comprising a sequence of amino acids that is at least 80% identical to SEQ ID NO:1, or (C) the light chain variable domain of (A) and the heavy chain variable domain of (B). In various embodiments, the heavy chain variable region amino acid sequence is at least 85%, 90%, or 95% or more identical to SEQ ID NO:1. In various embodiments, the light chain variable region amino acid sequence is at least 85%, 90%, or 95% or more identical to SEQ ID NO:2.

[0011] In various embodiments, the anti-INSR antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0012] In various embodiments, the anti-INSR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:10.

[0013] In various embodiments, the anti-INSR antibody is an IgG2 antibody.

[0014] In various embodiments, the anti-INSR antibody is RZ 358. In various embodiments, RZ358 comprises (A) a light chain variable domain comprising (i) a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6, (ii) a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7, and (iii) a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8, and (B) a heavy chain variable domain comprising (i) a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3, (ii) a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5, or a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2, or a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:10.

[0015] In various embodiments, the amino acid or salt thereof is selected from the group consisting of histidine, histidine HCL, methionine, and arginine. In various embodiments, the amino acid is histidine. In various embodiments, the histidine is at a concentration of about 4 mM to about 25 mM, or about 10 mM to about 20 mM. In various embodiments, the histidine is at a concentration of about 4±1 mM.

[0016] In various embodiments, the amino acid is histidine HCL. In various embodiments, the histidine HCL is at a concentration of about 4 mM to about 25 mM, or about 10 mM to about 20 mM. In various embodiments, the histidine HCL is at a concentration of about 6±1 mM.

[0017] In various embodiments, the amino acid is methionine. In various embodiments, the methionine is at a concentration of about 4 mM to about 25 mM, or about 10 mM to about 20 mM. In various embodiments, the methionine is at a concentration of about 10±2 mM.

[0018] In various embodiments, the surfactant is a polysorbate. In various embodiments, the polysorbate is polysorbate 20 or polysorbate 80, or a mixture thereof. In various embodiments, the surfactant is at a concentration of about 0.002% (w / v) to about 0.02% (w / v). In various embodiments, the composition comprises about 0.005% (w / v), 0.010% (w / v), 0.015% (w / v), or 0.02% (w / v) of the surfactant. In various embodiments, the composition comprises about 0.01% (w / v) ± 0.0025% (w / v) of the surfactant, optionally, the surfactant is polysorbate 20 or polysorbate 80, or a mixture thereof.

[0019] In various embodiments, the sugar alcohol is selected from the group consisting of sucrose, sorbitol, and mannitol.

[0020] In various embodiments, the sugar alcohol is sorbitol. In various embodiments, the sorbitol is at a concentration of about 100 mM to about 350 mM, or about 200 mM to about 300 mM. In various embodiments, the sorbitol is at a concentration of about 270±30 mM.

[0021] In various embodiments, the sugar alcohol is mannitol. In various embodiments, the mannitol is at a concentration of about 100 mM to about 350 mM, or about 200 mM to about 300 mM. In various embodiments, the mannitol is at a concentration of about 270±30 mM.

[0022] In various embodiments, the anti-INSR antibody is present in the composition at a concentration of about 20 mg / ml to about 200 mg / mL. In various embodiments, the anti-INSR antibody is present in the composition at a concentration of about 50 mg / ml to about 150 mg / mL. In various embodiments, the anti-INSR antibody is present in the composition at a concentration of about 80 mg / mL to about 120 mg / mL.

[0023] In various embodiments, the composition is a liquid. In various embodiments, the composition is lyophilized. In various embodiments, the composition is a liquid reconstituted from a lyophilized form.

[0024] In various embodiments, the pH is less than about 6.5. In various embodiments, the pH is from about 5.0 to about 6.5. In various embodiments, the pH is from about 5.5 to about 5.9. In various embodiments, the pH is about 5.8.

[0025] In various embodiments, the composition is characterized by a viscosity of about 2 cP to about 10 cP at 25° C. and the concentration of anti-INSR antibody is about 100 mg / ml or less.

[0026] In various embodiments, the composition is isotonic or has an osmolality ranging from about 200 mOsm / kg to about 500 mOsm / kg, or from about 225 mOsm / kg to about 400 mOsm / kg, or from about 250 mOsm / kg to about 400 mOsm / kg.

[0027] In various embodiments, the compositions contain less than about 10% impurities as determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis after about 24 months to about 36 months of storage at 2° C. to 8° C. The impurities include degradation products, high molecular weight species, low molecular weight species, oxidized species, or aggregates.

[0028] In various embodiments, less than 10% of the antibody is degraded as determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis after about 24 to about 36 months of storage at 2° C. to 8° C. In various embodiments, the composition contains less than about 5% degradation products as determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis after about 24 to about 36 months of storage at 2° C. to 8° C.

[0029] In various embodiments, less than 15% of the antibodies are oxidized or aggregated after about 3 months of storage at 40° C. as determined by hydrophobic interaction chromatography (HIC) or size exclusion chromatography (SEC).

[0030] In various embodiments, less than 30% of the antibody is detected in the acidic peak after about 24 to about 36 months of storage at 2° C. to 8° C. as determined by cIEX-UHPLC analysis. In various embodiments, approximately 8-20% of the antibody is detected in the acidic peak after about 24 to about 36 months of storage at 2° C. to 8° C. as determined by cIEX-UHPLC analysis. In various embodiments, less than 22% of the antibody is detected in the basic peak after about 24 to about 36 months of storage at 2° C. to 8° C. as determined by cIEX-UHPLC analysis. In various embodiments, approximately 5-20% of the antibody is detected in the basic peak after about 24 to about 36 months of storage at 2° C. to 8° C. as determined by cIEX-UHPLC analysis.

[0031] In various embodiments, the composition contains less than 5% high molecular weight species as determined by SE-UHPLC after about 24 months to about 36 months of storage at 2° C. to 8° C. In various embodiments, the composition contains less than 5% low molecular weight species as determined by SE-UHPLC after about 24 months to about 36 months of storage at 2° C. to 8° C.

[0032] In various embodiments, the composition contains less than 18% of the antibody in an oxidized form after about 24 months to about 36 months of storage at 2° C. to 8° C. as determined by HIC-HPLC analysis.

[0033] In various embodiments, the potency of the antibody composition, as determined by a bioassay, is at least about 75% to about 120% after about 24 months to about 36 months of storage at 2° C. to 8° C. In various embodiments, the bioassay is a phosphorylated AKT (pAKT) assay.

[0034] Provided herein is a composition comprising about 20-100 mg / mL of an anti-INSR antibody, 0.01% (w / v) polysorbate 20, about 250 mM to about 300 mM sorbitol, about 5 mM to about 15 mM methionine, and about 5 mM to about 15 mM histidine, the composition having a pH of about 5.8. In various embodiments, the anti-INSR antibody is RZ358.

[0035] Further contemplated are articles of manufacture comprising the compositions described herein, optionally comprising from about 0.5 mL to about 5 mL, or from about 1 mL to about 3 mL of the composition.

[0036] Also contemplated is a method for treating a condition associated with hyperinsulinemia or excessive insulin signaling in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-INSR antibody as described herein. In various embodiments, the condition is selected from the group consisting of hypoglycemia, insulin sensitivity, cancer, insulinoma, Kaposi's sarcoma, insulin overdose, islet cell hyperplasia (KATP-Hl diffuse disease, KATP-Hl focal disease, or "PHHI"), GDH-Hl (hyperinsulinemia / hyperammonemia syndrome (HI / HA), leucine-sensitive hypoglycemia, diazoxide-sensitive hypoglycemia, islet cell dysregulation syndrome, idiopathic hypoglycemia of infants, persistent hyperinsulinemic hypoglycemia of infants (PHHI), congenital hyperinsulinism, acute hypoglycemia due to renal failure, chronic hypoglycemia due to renal failure, and hypoglycemia due to chronic kidney disease. In various embodiments, the disease is congenital hyperinsulinism.

[0037] In various embodiments, the composition is administered daily, every 2 days, every 3 days, weekly, every 2 weeks, every 3 weeks, twice a month, monthly, every 2 months, every 3 months, or every 6 months. In various embodiments, the composition is administered for at least 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, a year, or more.

[0038] In various embodiments, the composition is administered intravenously, hi various embodiments, the composition is administered subcutaneously.

[0039] The present disclosure also provides a composition comprising an anti-INSR antibody as described herein for treating hyperinsulinemia or a condition associated with excessive insulin signaling in a subject in need thereof.

[0040] It is understood that each feature or embodiment or combination described herein is a non-limiting, illustrative example of any of the aspects of the invention and is therefore intended to be combinable with any other feature or embodiment or combination described herein. For example, when a feature is described with words such as "one embodiment," "some embodiments," "certain embodiments," "further embodiments," "particular exemplary embodiments," and / or "another embodiment," each of these types of embodiments is a non-limiting example of the feature that is intended to be combined with any other feature or combination of features described herein, without the need to recite all possible combinations. Such features or combinations of features apply to any of the aspects of the invention. When examples of values ​​falling within a range are disclosed, any of these examples are contemplated as possible endpoints of the range, and any and all numerical values ​​between such endpoints are contemplated, with any and all combinations of upper and lower endpoints being envisioned.

[0041] The headings herein are for the reader's convenience and are not intended to be limiting. Additional aspects, embodiments, and variations of the invention will become apparent from the detailed description and / or drawings and / or claims. [Brief description of the drawings]

[0042] [Figure 1] 1 shows DSC thermograms of RZ 358 at different pH in citrate buffered saline formulations. [Diagram 2] SEC chromatograms (UV 214 nm) of freeze-thaw stressed samples in citrate buffered saline formulations at pH 4.0 (top panel) and 7.0 (bottom panel) are shown. [Diagram 3] SEC (top panel) and HIC (bottom panel) chromatograms (UV 280 nm) for 3 month -40°C stability samples formulated at different pHs from pH 5.0 to pH 7.0 in citrate buffered saline are shown. [Figure 4]1 shows WCX chromatograms for RZ 358 stability samples at different pH formulations, pH 5.0 to pH 7.0, stored at 40° C. for 1 month (top panel) and at 30° C. for 3 months (bottom panel). [Diagram 5] 1 shows the change in total acidic species (top graph) and total basic species (bottom graph) at 30° C. for RZ 358 stability samples formulated at different pH values ​​ranging from pH 5.0 to pH 7.0. [Figure 6] SEC (top panel) and HIC-HPLC (bottom panel) chromatograms for RZ 358 stability samples at different pH formulations, pH 5.0 to pH 7.0, stored at 40° C. for 3 months. [Figure 7] 1 provides the results of an agitation study showing SEC-HPLC chromatograms of RZ 358 samples formulated in 0.002% (top panel) and 0.01% (bottom panel) PS20. (Samples were shaken at 1,000 RPM / 25° C. for up to 8 days.) [Figure 8] Particle concentrations (counts per mL) for 10 μm (top panel) and 25 μm (bottom panel) of samples with different polysorbate 20 concentrations stressed by up to 5 cycles of freeze-thaw (−70° C. / RT) are shown. [Figure 9] 4 shows DSC chromatograms of XMET D samples formulated in different formulations at pH 6 (Cit / NaCl=10 mM NaCit. 150 mM NaCl; H / R=10 mM L-histidine, 150 mM L-arginine; Mannitol=10 mM L-histidine, 10 mM L-methionine, 270 mM mannitol; Sorbitol=10 mM L-histidine, 10 mM L-methionine, 270 mM sorbitol). [Figure 10] Figure 1 shows the formation of a "pre-main HIC peak" (oxidation) at 40°C for RZ 358 stability samples formulated in three different formulations (Arg. = 10 mM L-histidine, 150 mM L-arginine; Man. = 10 mM L-histidine, 10 mM L-methionine, 270 mM mannitol; Sorb. = 10 mM L-histidine, 10 mM L-methionine, 270 mM sorbitol). [Figure 11] Chromatograms of stability samples stored at 40° C. for 3 months are shown (top panel=SEC, bottom panel=HIC; (1) blue=10 mM His, 150 mM L-arg.; (2) red=10 mM His, 10 mM Met, 270 mM mannitol; (3) green=10 mM His, 10 mM Met, 270 mM sorbitol). [Figure 12] Chromatograms of stability samples stored for 2 months at 40 °C in different formulations with various concentrations of sorbitol, from 90 to 270 mM, are shown (upper panel = SEC, lower panel = HIC). [Figure 13] Chromatograms are shown for stability samples stored for 3 months at 40° C. in formulations with different concentrations of methionine, 0-10 mM (upper panel=HIC, lower panel=SEC). [Figure 14A] 14A-14B show the long-term stability of RZ358 at 80 mg / mL in 10 mM L-histidine, 10 mM L-methionine, 270 mM sorbitol, 0.01% (w / w) polysorbate 20 at pH 5.4 to pH 6.3 at 2° C. to 8° C. for up to 60 months (FIG. 14A) and the effect on protein concentration at pH 5.8 (FIG. 14B). [Figure 14B] See legend to Figure 14A. [Figure 15A] 15A-15B show total impurities over time for 80 mg / mL RZ358 in 10 mL histidine, 10 mM L-methionine, 270 mM sorbitol, 0.01% (w / w) polysorbate 20 at pH 5.8 under reducing (FIG. 15A) and non-reducing (FIG. 15B) conditions. [Figure 15B] See legend to Figure 15A. [Figure 16A] FIG. 16A shows reduced CE-SDS analysis of the main peak fraction. [Figure 16B] 16B-16C show cation exchange chromatography analysis of the antibodies detected in the main peaks M1 and M2, and the ratio of M1 / M2 is shown in FIG. 16D. [Figure 16C]See legend to Figure 16B. [Figure 16D] See legend to Figure 16B. [Figure 17A] FIG. 17A shows the CEX-UHPLC acidic peak fraction of RZ358 at 80 mg / mL in 10 mM L-histidine, 10 mM L-methionine, 270 mM sorbitol, 0.01% (w / w) polysorbate 20. [Figure 17B] FIG. 17B shows the CEX-UHPLC basic peak fractions. [Figure 18A] Figures 18A-18C show the degradation of antibodies during 60 months of storage at 2-8°C as shown by SE-UHPLC analysis of monomeric species (Figure 18A), % high molecular weight species (Figure 18B), or % low molecular weight species (Figure 18C). [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 18D] FIG. 18D shows the % oxidized species under the same storage conditions. [Figure 19] 1 shows the potency of antibody formulations after 60 months of storage at 2-8°C as assessed by cell bioassay. [Figure 20] 1 shows the number of particles per container ≧10 μm for RZ358 DP at 2° C.-8° C. formed over a 60 month period. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Detailed Description The present disclosure provides formulations of a monoclonal antibody specific for the insulin receptor, RZ358, that are stable over extended storage periods, with minimal impurities formed and the relative potency of the antibody maintained during storage. Such stable antibody formulations are useful for treating disorders associated with insulin resistance and conditions associated with hyperinsulinemia.

[0044] definition The foregoing description has been given for clarity of understanding only, and no unnecessary limitations should be understood therefrom, since modifications within the scope of the invention may be apparent to those skilled in the art.

[0045] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise", and variations such as "comprises" and "comprising", will be understood to mean the inclusion of a specified integer or step, or group of integers or steps, but not to the exclusion of any other integers or steps or group of integers or steps.

[0046] Throughout this specification, when a composition is described as comprising a component or material, it is contemplated that the composition also consists essentially of, or may consist of, any combination of the recited components or materials, unless otherwise stated. Similarly, when a method is described as comprising particular steps, it is contemplated that the method also consists essentially of, or may consist of, any combination of the recited steps, unless otherwise stated. The inventions illustratively disclosed herein may be practiced in the absence of any element or step not specifically disclosed herein.

[0047] The implementation of the methods disclosed herein, and their individual steps, may be performed manually and / or with the aid of electronic equipment or with automation provided by electronic equipment. Although the process is described with reference to specific embodiments, one of ordinary skill in the art will readily appreciate that other ways of performing the operations associated with the methods may be used. For example, unless otherwise noted, the order of various steps may be changed without departing from the scope or spirit of the methods. Furthermore, some of the individual steps may be combined, omitted, or further subdivided into additional steps.

[0048] The compositions and methods are contemplated to include embodiments that include any combination of one or more of the additional optional elements, features, and steps further described below (including those shown in the figures), unless otherwise stated.

[0049] In jurisdictions that prohibit the patenting of methods performed on the human body, the meaning of "administering" a composition to a human subject shall be limited to formulating a controlled substance that the human subject self-administers by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation consistent with the statute or regulation defining patentable subject matter is intended. In jurisdictions that do not prohibit the patenting of methods performed on the human body, "administering" a composition includes both the method performed on the human body and the aforementioned activities.

[0050] Every maximum numerical limitation given throughout this specification should be understood to include as alternative embodiment ranges formed with every corresponding lower numerical limitation, as if such ranges were expressly written. Every minimum numerical limitation given throughout this specification will include as alternative embodiment ranges formed with every higher numerical limitation, as if such ranges were expressly written. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Dimensions and values ​​disclosed herein should be understood to include disclosure of both the recited value and the corresponding exact numerical value, for example, a value described as "about 10 mM" should be understood to include "10 mM" as an alternative disclosure.

[0051] All patents, publications, and references cited herein are hereby incorporated by reference in their entirety. In the event of a conflict between this disclosure and the incorporated patents, publications, and references, this disclosure shall control.

[0052] Unless otherwise stated, the following terms used in this Application, including the specification and claims, have the definitions given below.

[0053] As used in this specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural and singular referents unless the context clearly indicates otherwise.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this disclosure, including, but not limited to: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d Ed. 1994), THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker Ed., 1988), THE GLOSSARY OF GENETICS, 5th Ed., R. Rieger et al. (Eds.), Springer Verlag (1991), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).

[0055] The term "about" or "approximately" refers to a margin of error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term "about" or "approximately" appears before a first number in a series of two or more numbers, it is understood that the term "about" or "approximately" applies to every single one of the numbers in the series.

[0056] The term "antibody" is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments capable of binding antigen (e.g., Fab', F'(ab)2, Fv, single chain antibodies, diabodies), and recombinant peptides containing the above, so long as they exhibit the desired biological activity. An "immunoglobulin" or "tetrameric antibody" is a tetrameric glycoprotein consisting of two heavy chains and two light chains, each containing a variable region and a constant region. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antibody fragments or antigen-binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), complementarity determining region (CDR) fragments, CDR-grafted antibodies, single chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, linear antibodies, chelating recombinant antibodies, tribodies or bibodies, intrabodies, nanobodies, small modular immunopreparations (SMIPs), antigen-binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies, or variants or derivatives thereof, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide, such as one, two, three, four, five, or six CDR sequences, so long as the antibody retains the desired biological activity.

[0057] A "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations.

[0058] As used herein, "antibody variant" refers to an antibody polypeptide sequence that contains at least one amino acid substitution, deletion, or insertion in the variable region of a native antibody variable region domain. The variant may be substantially homologous or substantially identical to the unmodified antibody.

[0059] "Chimeric antibody," as used herein, refers to an antibody that contains sequences derived from two different antibodies, typically originating from different species (see, e.g., U.S. Patent No. 4,816,567). Most typically, chimeric antibodies contain human and rodent antibody fragments, generally comprising a human constant region and a murine variable region.

[0060] A "neutralizing antibody" is an antibody molecule that can eliminate or significantly reduce a biological function of the antigen to which it binds. Thus, a "neutralizing" antibody can eliminate or significantly reduce a biological function such as enzymatic activity, ligand binding, or intracellular signaling.

[0061] As used herein, "heavy chain variable region" refers to the region of an antibody molecule that contains at least one complementarity determining region (CDR) of the antibody heavy chain variable domain. A heavy chain variable region may contain one, two, or three CDRs of the antibody heavy chain.

[0062] As used herein, "light chain variable region" refers to the region of an antibody molecule that contains at least one complementarity determining region (CDR) of the antibody light chain variable domain. The light chain variable region may contain one, two, or three CDRs of the antibody light chain, which may be either a kappa or lambda light chain, depending on the antibody.

[0063] As used herein, an antibody that "specifically binds" refers to an antibody or polypeptide binding agent as used herein that is "antigen-specific," "specific" for an antigen target, or "immunoreactive" with an antigen and binds to the antigen with higher affinity than other antigens of similar sequence. In one aspect, an antibody, or fragment, variant, or derivative thereof, binds to a human antigen with higher affinity compared to its binding affinity to the analogous antigen of other, i.e., non-human, species, although polypeptide binding agents that recognize and bind to orthologues of the target are within the scope of the present methods.

[0064] For example, a polypeptide binding agent that is an antibody or fragment thereof "specific" for its cognate antigen will show that the variable region of the antibody recognizes and binds the desired antigen with detectable preference (e.g., if the desired antigen is a polypeptide, the variable region of the antibody can distinguish the antigen polypeptide from other known polypeptides of the same family by measurable differences in binding affinity, despite the possibility of localized sequence identity, homology, or similarity between family members). It will be understood that certain antibodies may also interact with other proteins (e.g., S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibody, particularly in the constant region of the molecule. Screening assays to determine the binding specificity of polypeptide binding agents, e.g., antibodies, for use in the methods herein are well known and routinely performed in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6. Antibodies for use in the present methods may be produced using any method known in the art.

[0065] The term "epitope" refers to a portion of any molecule capable of being recognized and bound by a selective binding agent at one or more of the antigen-binding regions. Epitopes usually consist of chemically active surface groupings of molecules, such as amino acids or carbohydrate side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. As used herein, epitopes can be continuous or discontinuous.

[0066] The term "sample" or "biological sample" refers to a specimen obtained from a subject for use in the methods, and includes urine, whole blood, plasma, serum, saliva, sputum, tissue biopsy, and cerebrospinal fluid.

[0067] The term "therapeutically effective amount" is used herein to refer to an amount of a target-specific composition effective to ameliorate or alleviate a symptom or sign of a disease associated with aberrant (e.g., abnormally high or abnormally low) signaling of a signaling complex.

[0068] The terms "treat", "treating" and "treatment" refer to eliminating, reducing, inhibiting or ameliorating, either temporarily or permanently, either partially or completely, the clinical symptoms, signs or progression of an event, disease or condition associated with an inflammatory disorder as described herein. As recognized in the relevant art, a drug used as a therapeutic agent may reduce the severity of a given disease state, but need not eliminate all symptoms of the disease to be considered a useful therapeutic agent. Similarly, a treatment administered prophylactically need not be completely effective in preventing the onset of a condition to constitute a viable prophylactic agent. It is sufficient to merely reduce the impact of the disease (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or to reduce the likelihood of the disease occurring or worsening in a subject. One embodiment of the present disclosure is directed to a method for determining the effectiveness of a treatment, comprising administering a therapeutic agent to a patient in an amount and for a time sufficient to induce a sustained improvement over baseline of an index reflecting the severity of a particular disorder.

[0069] Anti-INSR antibody Provided herein is a composition comprising an anti-INSR antibody, e.g., RZ358, in a stable pharmaceutical formulation. Anti-INSR antibodies that negatively regulate the activity of insulin on the insulin receptor are disclosed in U.S. Patent Nos. 9,944,698, 10,253,101, and 11,261,247. These negative regulator antibodies allosterically bind to the insulin receptor at a site that does not prevent insulin binding, but weakens insulin binding to the receptor and reduces signaling through the receptor. This mechanism increases and normalizes blood glucose levels in hyperinsulinemic patients.

[0070] Monoclonal antibodies can be modified for use as therapeutic or diagnostic agents. One embodiment is a "chimeric" antibody in which a portion of the heavy (H) and / or light (L) chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass. Also included are fragments of such antibodies, so long as they exhibit the desired biological activity. See U.S. Patent No. 4,816,567; Morrison et al., 1985, Proc. Natl. Acad. Sci. 81:6851-55.

[0071] In another embodiment, the monoclonal antibody is a "humanized" antibody. Methods for humanizing non-human antibodies are well known in the art. See U.S. Patent Nos. 5,585,089 and 5,693,762. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Humanization can be performed, for example, by replacing at least a portion of a rodent complementarity determining region with the corresponding region of a human antibody using methods described in the art (Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1998, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-36).

[0072] Chimeric, CDR-grafted, and humanized antibodies and / or antibody variants are typically produced by recombinant methods. Nucleic acid encoding the antibody is introduced into a host cell and expressed using the materials and procedures described herein. In a preferred embodiment, the antibody is produced in a mammalian host cell, such as a CHO cell. Monoclonal (e.g., human) antibodies can be produced by expression of recombinant DNA in a host cell or by expression in a hybridoma cell as described herein.

[0073] In various embodiments, the disclosure provides an antibody or fragment thereof comprising three heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 3 to 5, and three light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 6 to 8. In various embodiments, the antibody or fragment thereof binds to (i) the insulin receptor, or (ii) a complex comprising insulin and the insulin receptor, or both (i) and (ii).

[0074] In various embodiments, the antibody comprises a polypeptide having an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain variable region set forth in SEQ ID NO:1, and an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain variable region set forth in SEQ ID NO:2, and the antibody further comprises at least one, two, three, four, five, or all of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 set forth in SEQ ID NOs:3-8.

[0075] In various embodiments, the anti-INSR antibody or fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO:1 and the light chain variable region amino acid sequence set forth in SEQ ID NO:2.

[0076] In an illustrative example, an anti-INSR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:10.

[0077] Also provided is an antibody or antibody variant that binds to INSR, comprising: (A) a heavy chain variable domain selected from the group consisting of (i) a sequence of amino acids at least 80% (e.g., about 85%, about 90%, about 95%, more than 95%) identical to SEQ ID NO:1; (ii) a sequence of amino acids encoded by a polynucleotide sequence encoding a polypeptide that is at least 80% (e.g., about 85%, about 90%, about 95%, more than 95%) identical to SEQ ID NO:1; (iii) a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complementary strand of a polynucleotide encoding a polypeptide consisting of SEQ ID NO:1; and (B) (i) an amino acid sequence at least 80% (e.g., about 85%, about 90%, about 95%, more than 95%) identical to SEQ ID NO:2. or (C) a light chain variable domain of (A) and a heavy chain variable domain of (A), wherein the antibody or antibody variant specifically binds to (i) an insulin receptor, or (ii) a complex comprising insulin and the insulin receptor, or both (i) and (ii).

[0078] In various embodiments, the antibody or antibody fragment is a Fab fragment.

[0079] In various embodiments, the antibody or antibody variant thereof is bivalent and is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody. In various embodiments, the anti-INSR antibody is an IgG2 antibody.

[0080] An exemplary sequence of a human IgG2 constant region is available from the Uniprot database as Uniprot number P01859, and is incorporated herein by reference. Information including sequence information of heavy and light chain constant regions of other antibodies is also publicly available via the Uniprot database as well as other databases well known in the art of antibody engineering and production.

[0081] In various embodiments, the antibody, antibody variant, or fragment thereof binds at least 10 to (i) the insulin receptor, or (ii) a complex comprising insulin and the insulin receptor, or both (i) and (ii). -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 M, 10 -13 M, 10 -14 M or 10 -15 M or less, which can attenuate the binding affinity between insulin and the insulin receptor by at least about 1.5-fold, and optionally up to 1000-fold. In certain embodiments, the antibody can attenuate the binding affinity between said insulin and the insulin receptor by about 2-fold to 500-fold. In various embodiments, the antibody can optionally attenuate the EC50 of insulin signaling activity in a pAKT assay. 50 Increases by approximately 2 to 1000 times.

[0082] formulation The present disclosure provides formulations of the anti-INSR antibody RZ358 that are stable over extended periods of storage and under stress conditions, which are useful in the methods described herein.

[0083] It is contemplated that the antibody formulation is in liquid or lyophilized form. The formulation may also be a liquid reconstituted from a lyophilized form.

[0084] In various embodiments, the composition of the present disclosure is liquid. In certain aspects, the composition has a pH of less than about 6.5. In some aspects, the pH is about 5.0 to about 6.0, about 5.1 to about 5.8, about 5.5 to about 5.9, such as about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0.

[0085] In various embodiments, the formulation comprises one or more of a surfactant, a stabilizer, an amino acid, an antioxidant, and / or a buffering agent to minimize aggregation, oxidation, and other proteolytic degradation during storage. Exemplary stabilizers include surfactants and sugar alcohols. Exemplary antioxidants include methionine, sugar alcohols, and histidine. Exemplary buffering agents include histidine HCl.

[0086] In various embodiments, the composition includes histidine. In various embodiments, the histidine is at a concentration of about 4 mM to about 25 mM, for example, about 5 mM to about 20 mM, about 10 mM to about 20 mM, or about 8 mM to 15 mM, or about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM. In various embodiments, the histidine is at a concentration of about 4±1 mM.

[0087] In certain embodiments, the composition comprises histidine HCl. In various embodiments, the histidine HCl is at a concentration of about 4 mM to about 25 mM, for example, about 5 mM to 20 mM, about 10 mM to about 20 mM, or about 8 mM to 15 mM, or about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM. In various embodiments, the histidine HCl is at a concentration of about 6±1 mM.

[0088] In various embodiments, the composition includes methionine. In various embodiments, the methionine is at a concentration of about 4 to about 25 mM, for example, about 5 mM to 20 mM, about 10 mM to about 20 mM, or about 8 mM to 15 mM, or about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM. In various embodiments, the methionine is at a concentration of about 10±2 mM.

[0089] The compositions herein may further comprise a sugar alcohol. Exemplary sugar alcohols include sorbitol, mannitol, trehalose, and sucrose. In various embodiments, the formulation comprises a sugar alcohol that is sorbitol. In various embodiments, the sorbitol is at a concentration of about 100 mM to about 350 mM, about 150 mM to 350 mM, about 100 mM to 300 mM, about 100 mM to 200 mM, or about 200 mM to about 300 mM. The sorbitol may be at a concentration of about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, or about 350 mM. In various embodiments, the sorbitol is at a concentration of about 270±30 mM.

[0090] In various embodiments, the formulation comprises a sugar alcohol that is mannitol. In various embodiments, the mannitol is at a concentration of about 100 mM to about 350 mM, about 150 mM to about 350 mM, about 100 mM to about 300 mM, about 100 to about 200 mM, or about 200 mM to about 300 mM. The mannitol can be at a concentration of about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, or about 350 mM. In various embodiments, the mannitol is at a concentration of about 270±30 mM.

[0091] In some embodiments, the compositions of the present disclosure include a surfactant. A surfactant is a surface active agent that is amphiphilic (having a polar head and a hydrophobic tail). Surfactants preferentially accumulate at interfaces, resulting in a reduction in interfacial tension. The use of surfactants can also help mitigate the formation of large proteinaceous particles. In some aspects, the surfactant present in the compositions of the present disclosure is an amphiphilic and / or non-ionic surfactant. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 80), alkylaryl polyethers such as oxyethylated alkylphenols (e.g., Triton™ X-100), and poloxamers (e.g., Pluronics®, e.g., Pluronic® F68), as well as combinations of any of the foregoing, either within or between surfactant classes. Polysorbate 20 and polysorbate 80 (and optionally mixtures thereof) are contemplated herein. In illustrative examples, the surfactant is present in the composition at a concentration of about 0.002% (w / v) to about 0.02% (w / v) or less. For example, the formulation may contain about 0.005% (w / v) to about 0.015% (w / v) surfactant, e.g., about 0.005% (w / v), about 0.006% (w / v), about 0.007% (w / v), about 0.008% (w / v), about 0.009% (w / v), about 0.010% (w / v), about 0.011% (w / v), about 0.012% (w / v), about 0.013% (w / v), about 0.014% (w / v), about 0.015% (w / v), about 0.016% (w / v), about 0.017% (w / v), about 0.018% (w / v), about 0.019% (w / v), or about 0.020% (w / v). In exemplary embodiments, the formulation comprises about 0.010% (w / v), 0.015% (w / v), or 0.02% (w / v) of a surfactant.

[0092] It is contemplated that the formulations herein contain an anti-INSR antibody at a concentration of about 20 mg / ml to about 200 mg / ml, about 50 mg / ml to about 150 mg / ml, or about 80 mg / ml to about 120 mg / ml. The formulations may contain an anti-INSR antibody (e.g., RZ358) at a concentration of about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 110 mg / ml, about 120 mg / ml, about 130 mg / ml, about 140 mg / ml, about 150 mg / ml, about 160 mg / ml, about 170 mg / ml, about 180 mg / ml, about 190 mg / ml, or about 200 mg / ml.

[0093] In various embodiments, the composition comprises about 20-100 mg / mL of an anti-INSR antibody, 0.01% (w / w) polysorbate 20, about 250 to about 300 mM sorbitol, about 5 mM to about 15 mM methionine, and about 5 mM to about 15 mM histidine, and the composition has a pH of about 5.8. In various embodiments, the composition comprises about 20-100 mg / mL of an anti-INSR antibody, 0.01% (w / w) polysorbate 20, about 270 mM sorbitol, about 10 mM methionine, and about 10 mM histidine, and the composition has a pH of about 5.8. In various embodiments, the antibody is RZ358.

[0094] When the formulation is in a form intended for parenteral administration, it may be isotonic with blood (osmolality of about 300 mOsm / kg). The osmolality of the composition is contemplated in some aspects to be in the range of about 200 mOsm / kg to about 500 mOsm / kg, or about 225 mOsm / kg to about 400 mOsm / kg, or about 250 mOsm / kg to about 400 mOsm / kg, about 250 mOsm / kg to about 350 mOsm / kg, or about 275 to about 375 mOsm / kg. In various embodiments, the formulation has an osmolality of about 275 to about 375 mOsm / kg.

[0095] In various embodiments, the formulation has a low viscosity, hi some embodiments, the formulation is characterized by a viscosity of about 2 cP to about 10 cP at 25° C. and the concentration of anti-INSR antibody is about 100 mg / ml or less.

[0096] In various aspects, the compositions of the present disclosure are stable and can withstand long-term storage at refrigerated temperatures. It is contemplated that less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%) of the antibodies are degraded after about 1 month to about 3 months of storage at about 2°C to about 8°C (e.g., about 2°C, about 4°C, about 6°C, about 8°C). In various embodiments, less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%) of the antibodies are degraded after 36 months, 48 ​​months, or 60 months of storage at about 2°C to about 8°C as determined by CE-SDS. In various embodiments, greater than about 90%, or about 95% of the antibodies are intact after 24 months of storage at about 2°C to about 8°C as determined by CE-SDS.

[0097] In various embodiments, the compositions of the present disclosure are stable and can withstand short-term storage under stressed storage conditions. Optionally, less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%) of the antibody is degraded after about 1 month to about 3 months of storage at about 38°C to about 42°C (e.g., about 38°C, about 39°C, about 40°C, about 41°C, about 42°C).

[0098] In various embodiments, after extended storage at about 2° C. to about 8° C., at least 90%, or at least about 95% of the antibody is in monomeric form, optionally as detected by SE-HPLC. In various embodiments, less than about 3%, 2.5%, 2%, 1.5%, or 1% of the impurities are high molecular weight species, optionally as detected by SE-HPLC. In various embodiments, after extended storage, less than about 15% of the antibody are oxidized species, optionally as detected by HIC-HPLC.

[0099] In various embodiments, after extended storage at about 2° C. to about 8° C., the antibody maintains 80-120% relative potency compared to the reference antibody, optionally as measured by a pAKT assay. In various embodiments, the reference antibody has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 2. In various embodiments, the reference antibody has a heavy chain amino acid sequence set forth in SEQ ID NO: 9 and a light chain amino acid sequence set forth in SEQ ID NO: 10.

[0100] In various embodiments, the compositions are provided for storage or use in, for example, disposable vials, disposable syringes, or glass, glass-lined, or glass-coated primary containers. In various embodiments, the compositions are contained in glass vials or syringes for storage, for example, long-term storage at about 2° C. to about 8° C., or storage at higher temperatures (e.g., about 25° C., about 30° C., about 40° C.).

[0101] In various embodiments, the composition is administered subcutaneously to a subject and is isotonic to the intended site of administration.

[0102] Method of preparation Methods for making the compositions of the present disclosure are further provided herein.Using transgenic animals (e.g., mice) that can produce a repertoire of human antibodies in the absence of endogenous immunoglobulin production, such antibodies are produced by immunization with polypeptide antigens (i.e., having at least six consecutive amino acids), optionally conjugated to a carrier.See, for example, Jakobovits et al., 1993, Proc.Natl.Acad.Sci.90:2551-55; Jakobovits et al., 1993, Nature 362:255-58; Bruggermann et al., 1993, Year in Immuno.7:33.See also PCT Application Nos. PCT / US96 / 05928 and PCT / US93 / 06926. Additional methods are described in U.S. Patent No. 5,545,807, PCT Application Nos. PCT / US91 / 245 and PCT / GB89 / 01207, and European Patent Nos. 546073B1 and 546073A1. Human antibodies may be produced by expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.

[0103] Chimeric, CDR-grafted, and humanized antibodies and / or antibody variants are typically produced by recombinant methods. Nucleic acid encoding the antibody is introduced into a host cell and expressed using the materials and procedures described herein. In a preferred embodiment, the antibody is produced in a mammalian host cell, such as a CHO cell. Monoclonal (e.g., human) antibodies can be produced by expression of recombinant DNA in a host cell or by expression in a hybridoma cell.

[0104] For recombinant production of an antibody or antibody fragment, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can bind specifically to genes encoding the antibody heavy and light chains). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selectable marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0105] Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotic, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example, Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, and the like. Examples of suitable E. coli cloning hosts include Enterobacteriaceae, such as Bacillus subtilis and B. licheniformis (e.g., B. licheniformis 41P, disclosed in DD 266,710 published April 12, 1989), Pseudomonas, such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.

[0106] Eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, Schizosaccharomyces pombe has been shown to be effective against Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus, as well as Yarrowia (EP 402,226), Pichia pastors (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa, Schwanniomyces occidentalis (EP 244,234), Several other genera, species, and strains, such as Schwanniomyces, e.g., A. occidentalis, and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. niger, are commonly available and useful herein.

[0107] Suitable host cells for the expression of glycosylated antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori, as well as corresponding permissive insect host cells, have been identified. Various viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as viruses herein in accordance with the present disclosure, particularly for transfection of Spodoptera frugiperda cells.

[0108] Examples of useful mammalian host cell lines include CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al., J. Gen Virol. 36:59, 1977), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli cells (TM4, Mather, (Biol. Reprod. 23:243-251, 1980), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (HepG2, HB 8065), mouse mammary carcinoma (MMT 060562, ATCC CCL 51), TRI cells (Mather et al., Annals N.Y.Acad.Sci.383:44-68(1982)), MRC5 cells, FS4 cells, and a human hepatoma line (HepG2).

[0109] Host cells are transformed or transfected with expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as necessary for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In addition, novel vectors and transfected cell lines having multiple copies of the transcription unit separated by a selectable marker are particularly useful and preferred for expressing antibodies that bind to a desired antigen.

[0110] How to use Provided herein is the use of anti-INSR antibody formulations, including, for example, RZ358, in the treatment of hyperinsulinism / hyperinsulinemic disorders resulting from abnormal insulin / INSR signaling and low blood glucose levels.

[0111] Exemplary hyperinsulinemic disorders include hypoglycemia, insulin sensitivity, cancer, insulinoma, Kaposi's sarcoma, insulin overdose, islet cell hyperplasia (KATP-Hl diffuse disease, KATP-Hl focal disease, or "PHHI"), GDH-Hl (hyperinsulinemia / hyperammonemia syndrome (HI / HA), leucine-sensitive hypoglycemia, diazoxide-sensitive hypoglycemia, islet cell dysregulation syndrome, idiopathic hypoglycemia of infants, persistent hyperinsulinemic hypoglycemia of infants (PHHI), congenital hyperinsulinism, acute hypoglycemia due to renal failure, chronic hypoglycemia due to renal failure, and hypoglycemia due to chronic kidney disease.

[0112] Congenital hyperinsulinism (CHI) comprises a group of genetic disorders characterized by recurrent episodes of hyperinsulinemic hypoglycemia due to uncontrolled secretion of insulin by pancreatic β cells (Arnoux J., et al. Orphanet Journal of Rare Diseases 6:63(2011); Yorifuji T., Ann Pediatr Endocrinol Metab 19:57-68(2014). CHI is the most common cause of hyperinsulinemic hypoglycemia in neonates, infants, and children and is usually diagnosed within the first 2 years of life. Histopathologically, CHI can be demonstrated in either a diffuse or focal form. In the diffuse form, all pancreatic β cells are affected, whereas in the focal form, abnormal β cell pathology is (usually) limited to a small area of ​​the pancreas. The most common known cause of CHI is loss-of-function mutations in the genes encoding SUR1 and Kir6.2, subunits of the ATP-sensitive potassium channel (KATP channel) involved in the secretion of insulin in pancreatic β cells.

[0113] Currently, there are only a few therapeutic approaches for persistent congenital hyperinsulinemia (CHI) [Arnoux J., et al. Orphanet Journal of Rare Diseases 6:63 (2011), Yorifuji T., Ann Pediatr Endocrinol Metab. 19:57-68 (2014)]. Diazoxide, a KATP channel activator, inhibits insulin secretion in pancreatic β cells. The most frequent adverse effect is hypertrichosis (hirsutism). Other side effects include sodium and fluid retention, which can lead to congestive heart failure. Diazoxide is generally ineffective in those patients with CHI due to KATP channel mutations, one of the most common causes of CHI. Octreotide is a somatostatin analogue that inhibits insulin release. Although not approved for CHI, octreotide is utilized for diazoxide-nonresponsive CHI. It is administered subcutaneously (SC) or intravenously (IV) via multiple daily injections or continuously via pump due to its short half-life (1-2 hours). Common adverse events include gastrointestinal symptoms and gallbladder complications. In cases of localized disease, partial pancreatectomy is an option and may be curative in the majority of cases. However, lesions are not always visible or palpable at the site indicated by preoperative imaging. Patients with diffuse morphology are primarily treated via continuous glucose supply or off-label drug therapy. Near total pancreatectomy has been considered as a treatment, but this is characterized by a high risk of diabetes.

[0114] Iatrogenic hypoglycemia describes the state and effects of hypoglycemia caused by the administration of either excess insulin or its analogs, or drugs that stimulate endogenous insulin secretion. Iatrogenic hypoglycemia is primarily, but not exclusively, the result of treatment with insulin secretagogues or insulin, and is the major limiting factor in the glycemic control of diabetes. Iatrogenic hypoglycemia causes recurrent morbidity and is sometimes fatal in most people with T1DM and many with advanced T2DM. Recurrent episodes of hypoglycemia impair the body's defense against subsequent declines in plasma glucose concentration, thus creating a vicious cycle of recurrent hypoglycemia.

[0115] Hypoglycemia can result in a variety of symptoms including loss of muscle coordination, confusion, loss of consciousness, seizures, and even death.

[0116] Most episodes of mild hypoglycemia are effectively self-treated by ingestion of glucose tablets or other carbohydrates, including drinks or snacks. More severe symptomatic hypoglycemia may also be treated with oral carbohydrate ingestion. However, parenteral therapy is necessary when a hypoglycemic patient is unable to take oral glucose supplements because of confusion, unconsciousness, or other reasons. As a non-hospital rescue procedure, injections of the hyperglycemic hormone, glucagon, are sometimes used either subcutaneously or intramuscularly, either by the patient themselves or by a relative of the patient who is trained to recognize and treat severe hypoglycemia. In the medical setting, intravenous glucose is the standard parenteral therapy.

[0117] Administration and Dosing Also contemplated by the present disclosure are methods of administering the antibody compositions described herein to treat the hyperinsulinemic disorders described herein.

[0118] The disclosed methods are carried out using any medically acceptable means for directly or indirectly introducing a therapeutic agent into a mammalian subject, including, but not limited to, injection, infusion, oral ingestion, intranasal, topical, transdermal, parenteral, inhalation spray, vaginal, or rectal administration. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, and intracisternal injections, as well as catheter or infusion techniques. Administration by intradermal, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection, and / or surgical implantation at a specific site is also contemplated. Suitable delivery devices may include those developed for the delivery of insulin (see, e.g., Owens et al Diabetic Med. 20(11):886-898,2003, US2014 / 0128803, and Peyser et al;., Annals NY Acad Sci,1311:102-123,2014).

[0119] The antibody compositions described herein may be administered daily, every 2 days, every 3 days, weekly, every 2 weeks, every 3 weeks, twice a month, monthly, every 2 months, every 3 months, or every 6 months. In various embodiments, the compositions are administered for a period of at least 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, a year, or more.

[0120] In one embodiment, administration is performed at the site of the affected tissue requiring treatment, by direct injection at the site or via a sustained delivery or sustained release mechanism capable of delivering the formulation internally. For example, biodegradable microspheres or capsules or other biodegradable polymeric constructs capable of sustained delivery of the composition (e.g., soluble polypeptides, antibodies, or small molecules) may be included in the formulations useful for the present disclosure that are implanted at the site.

[0121] The therapeutic composition can also be delivered to the patient at multiple sites. Multiple doses can be given simultaneously or over a period of time. In certain cases, it is beneficial to provide a continuous flow of the therapeutic composition. Additional therapy can be administered on a periodic basis, for example, hourly, daily, weekly, biweekly, triweekly, or monthly.

[0122] The amount of antibody composition in a given dosage will vary depending on the size of the individual being treated, as well as the characteristics of the disorder being treated. Exemplary treatments may require administration of about 0.1 to about 25 mg / kg, or about 0.05 mg / kg to about 10 mg / kg, about 0.3 mg / kg to about 6 mg / kg, or about 0.1 mg / kg to about 3 mg / kg per dose or day. Exemplary doses include 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 0.75 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, or 25 mg / kg. Other dosage amounts include 1 mg / day, 2.5 mg / day, 5 mg / day, 10 mg / day, 20 mg / day, 25 mg / day, 50 mg / day, 75 mg / day, 100 mg / day, 150 mg / day, 200 mg / day, 250 mg / day, 500 mg / day, or 1000 mg / day. These concentrations may be administered as a single dosage form or as multiple doses, or continuously.

[0123] The method also contemplates administration of multiple agents, such as an antibody composition described herein in conjunction with a second agent described herein. A composition comprising an antibody described herein can be administered to a human or mammal suffering from or predisposed to suffering from a treated condition or disorder associated with the target polypeptide.

[0124] Concurrent administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, so long as there is an overlap in the period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

[0125] The second agent may be another therapeutic agent, such as an anti-diabetic agent, a cytokine, a growth factor, another anti-inflammatory agent, an anticoagulant, an agent that lowers or reduces blood pressure, an agent that reduces cholesterol, triglycerides, LDL, VLDL, or lipoprotein(a) or increases HDL, an agent that increases or decreases the level of a cholesterol-regulating protein, an anti-tumor agent or molecule.

[0126] Exemplary agents include, but are not limited to, insulin, glucagon, acarbose, octreotide, verapamil, diazoxide, and other agents useful for treating hypoglycemia or side effects associated with hypoglycemia. In various embodiments, the antibodies described herein are optionally administered with insulin and / or glucagon in a delivery device, such as a smart delivery device (see, e.g., US2014 / 0128803) or a dual sensor / pump (e.g., the Bionic Pancreas System).

[0127] Any of the foregoing antibodies or fragments thereof described herein may be administered as adjunctive therapy, simultaneously with one or more second agents that are anti-diabetic agents known in the art or described herein.

[0128] Sulfonylureas (e.g., glimepiride, glizentide, sulfonylurea, AY31637), biguanides (e.g., metformin), alpha-glucosidase inhibitors (e.g., acarbose, miglitol), thiazol-idinediones (e.g., troglitazone, pioglitazone, rosiglitazone, glipizide, balaglitazone, rivoglitazone, netoglitazone, troglitazone, englitazone, AD5075, T 174, YM 268, R 102380, NC 2100, NIP 223, NIP 221, MK 0767, ciglitazone, adaglitazone, CLX 0921, darglitazone, CP 92768, BM Many antidiabetic agents are known in the art, including, but not limited to, glycemic control (GDC) and GLP analogs or agonists of the GLP-1 receptor (e.g., exendins) or stabilizers thereof (e.g., DPP4 inhibitors such as sitagliptin), insulin or analogs or mimetics thereof (e.g., lispro, aspart, glulisine, detemir insulin, egludec insulin, insulin glargine, LANTUS®), and sodium-glucose cotransporter-2 (SGLT2) inhibitors (e.g., canagliflozin, dapagliflozin, and empagliflozin).

[0129] It is contemplated that the antibody and second agent may be given simultaneously in the same formulation. It is further contemplated that the agents may be administered in separate formulations and administered simultaneously, which refers to agents administered within 30 minutes of each other.

[0130] In another embodiment, the second agent is administered prior to administration of the antibody composition. Prior administration refers to administration of the second agent within a week prior to treatment with the antibody and up to 30 minutes prior to administration of the antibody. It is further contemplated that the second agent is administered after administration of the antibody composition. Subsequent administration is intended to describe administration from 30 minutes after antibody treatment to up to 1 week after antibody administration.

[0131] It is further contemplated that other supportive therapies may be administered as needed. For example, the patient may also be administered a diet or food plan designed for hypoglycemic patients, surgical therapy, or radiation therapy as needed.

[0132] It will also be apparent that dosing may be modified when conventional therapeutic agents are administered in combination with the antibody formulations described herein.

[0133] kit The present disclosure also provides kits that include the compositions described herein, together with a package insert, package label, instructions, or other labeling that indicates or discloses any of the methods or embodiments disclosed herein. In certain embodiments, the present disclosure provides kits for making single-dose administration units. In certain embodiments of the present disclosure, kits that contain single and multi-chamber pre-filled syringes (e.g., liquid syringes) are included.

[0134] Additional aspects and details of the present disclosure will become apparent from the following examples, which are intended as illustrative rather than limiting. EXAMPLES

[0135] Example 1 - Formulation of RZ358 Antibody RZ358 is a fully human IgG2 monoclonal antibody (mAb) that binds with high affinity to the insulin receptor (INSR). The RZ 358 antibody has a molecular weight of approximately 149 kD and a pI at pH of 7.6. Described herein is the development of a formulation of RZ 358 for in vivo administration and stability studies, as well as the selection of a unique combination of parenterally approved GRAS excipients aimed at stabilizing RZ 358 during liquid storage at 2°C-8°C for up to 2 years. Initially, a pH was selected to minimize aggregation and optimize conformational stability.

[0136] method Buffer exchange: Buffer exchange was performed using a VIVASPIN® ultrafiltration device (PES membrane, 30k MWCO, Sartorius Stedim). 2 The extinction coefficient in mg / g was determined by UV absorption measurement at 280 nm. The adjusted concentration solutions were filtered with a syringe-driven filter unit (Millex-MP, PES, 0.22 μm, Millipore). The filtered solutions were filled into appropriate containers and placed under different stress conditions for stability testing.

[0137] Mechanical stress: RZ 358 solution was filled into 2 cc glass vials at half capacity (1.5 ml per vial) and sealed with rubber stoppers. The vials were placed horizontally in a THERMOMIXER®R and shaken at 1,000 RPM / 25° C. for up to 1 week or until visual changes were observed. Freeze-thaw: The filled samples were placed in a −70° C. freezer for at least 2 hours for freezing, then placed on the lab bench until completely thawed. The cycle was repeated 5 times.

[0138] Heat Stress: RZ 358 specimens were placed at 5°C, 30°C, and 40°C and pulled at specific time points for analysis (usually 2 weeks, 1 month, 2 months, 3 months, and 6 months).

[0139] Differential Scanning Calorimetry (DSC): DSC analysis was performed on a TA Instrument NanoDSC. The capillary cell was thoroughly washed with water and sample buffer. Samples / buffers were degassed in a MicroCal ThermoVac Sample Degassing and Thermostat unit at 18°C ​​for at least 5 minutes before loading into the capillary cell. Samples / buffers were scanned from 15°C to 95°C at a scan rate of 1°C / min.

[0140] Analysis of subvisible particulate matter: Analysis was performed using a Z2 Coulter Particle Count and Size Analyzer. The 100μ aperture lobe was calibrated with a "CC L10 size standard" and counting accuracy was checked with the "concentration reference" provided by the counter. The sample solution was gently swirled and shaken to ensure the solution was homogenous, after which 0.8 mL of sample solution was transferred to a clean "3cc" glass vial (volume approximately 4.1 mL) containing 3.2 mL of 0.2 M NaCl solution. Each sample was measured at least twice. The lower and upper thresholds were set at 10μ and 25μ, respectively.

[0141] Dynamic Light Scattering (DLS): DLS analysis was performed using a DYNAPRO® Plate Reader (Wyatt Technologies, Santa Barbara, CA). Samples (30 μL each) were loaded into 384-well non-treated assay plates (Corning, Cat. No. 3540, polystyrene, black with clear flat bottom). The instrument temperature was set to 25° C.

[0142] All HPLC analyses were performed using an Agilent 1100 or 1200 system. Data acquisition and analysis were performed using ChemStation software (Rev. B.03.02). Detection was by UV at wavelengths of 280 nm and 214 nm.

[0143] WCX-HPLC: Weak cation exchange HPLC was performed using a Dionex PROPAC™ WCX10 column (4×250 mm). The injected sample was eluted using a pH gradient. Buffer A was 15 mM NaH2PO4 and Buffer B was 15 mM Na 2 HPO 4The percentage of buffer B was increased from 25 at the start to 95 at 30 min. The column was washed with 95% B for 5 min, then reduced to 25% B and equilibrated for 15 min before a new injection. The flow rate was maintained at 1 ml / min. The eluted peak was monitored at UV wavelengths of 280 nm and 214 nm. The 280 nm signal was used for integration and quantitative calculations.

[0144] SEC-HPLC: SEC-HPLC was performed on a Tosoh TSK gel SWxl G3000 7.8x300mm column. The injected sample was eluted isocratically with the following elution buffer: 20 mM sodium phosphate, 0.2 M ammonium sulfate, pH 6.8, at a flow rate of 0.7 ml / min. The run time was 25 min. Peaks were detected at 280 nm.

[0145] HIC-HPLC: Hydrophobic interaction chromatography is performed on a Dionex PROPAC™ HIC-10 (5 μm, 4.6×100 mm) column. Buffer A is 0.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7, and buffer B is 10% (v / v) acetonitrile in 50 mM sodium phosphate, pH 7. The flow rate is 1 mL / min and the run time is 30 (45) min with the following gradient: TIFF2025517443000001.tif9128

[0146] To understand the effect of formulation pH on aggregate formation, RZ 358 antibody was formulated at 1 mg / mL in 10 mM sodium citrate, 150 mM sodium chloride at pH 4.0, 5.0, 5.5, 6.0, 6.3, and 7.0.

[0147] Conformational stability was assessed by DSC. At acidic pH 4, RZ 358 in the excipients showed a lower thermal transition at 50.7°C due to unfolding of the more flexible CH2 domain, and a major thermal transition at 64.8°C for the Fab (Figure 1). As formulation pH increased, the CH2 transition temperature increased rapidly and merged with the main Fab transition peak at pH 5.5 and above. The unfolding temperature of the Fab reached a maximum value when the formulation pH was above 6. The DSC data indicate that RZ 358 is conformationally more stable when the solution pH was near or above pH 6.

[0148] RZ 358 was subjected to five cycles of freeze-thaw and storage conditions at 40°C. Upon freeze-thaw, RZ 358 formed soluble dimers at acidic pH conditions (pH 4.0 and 5.0) (Figure 2, top panel) and high molecular weight (HMW) species at neutral pH conditions (Figure 2, bottom panel). More optimal stability upon freeze-thaw was exhibited in the pH range of 5.5-6.3.

[0149] RZ 358 was also incubated at 40 °C for up to 3 months. At pH 4.0, RZ 358 formed soluble aggregates, as indicated by the loss of signal by SEC-HPLC at 2 weeks and the appearance of a gel-like precipitate after 2 weeks of storage. As the pH of the solution increased to 5.0 or higher, the formation of fragmented degradants became predominant (Figure 3, top panel). The aggregation levels of the stressed 40 °C stability samples (pH 5 to pH 7) at 3 months showed no significant differences.

[0150] As detected by SEC-HPLC, RZ 358 fragmentation occurred as the pH deviated from pH 5.5 to both lower (pH 5) and higher pH (7.0). Oxidation of RZ 358 as measured by degradation patterns from HIC-HPLC (Figure 3, bottom panel) showed a similar trend, with the pH 5.5 formulation showing the least amount of the pre-main peak.

[0151] pH studies showed that the optimum pH for controlling aggregation / fragmentation / oxidation of RZ 358 was approximately pH 5.5.

[0152] Further pH studies were performed based on charge variant distribution. 5 mg / mL RZ 358 was formulated in 10 mM L-histidine / HCl, 10 mM methionine, 270 mM sorbitol, containing 10 mM L-histidine (free base), 10 mM methionine, 270 mM sorbitol at pH 5.0, 5.5, 5.8, 6.4, and 7.0. Formulation buffer exchange was achieved using a VivaSpin ultracentrifugation device. Samples were incubated at 40° C. for 1 month and 30° C. for 3 months and analyzed via WCX-HPLC for charge variant distribution. Higher pH samples showed faster growth of acidic species. Basic species increased under slightly acidic conditions (FIG. 4).

[0153] A slightly acidic pH was good for controlling acidic species, and the optimal pH for controlling the formation of basic species was around pH 5.8 (Figure 5). The pH 5.8 formulation was chosen to minimize fragmentation (Figure 6, top panel), oxidation (Figure 6, bottom panel), and aggregation (Figure 6, top panel). It is noteworthy that the lower pH formulation produced more species, shown as a back shoulder of the main HIC peak.

[0154] Example 2 - Analysis of Buffer Solutions for RZ358 Formulations Surfactants may enhance physical stability during freeze-thaw and agitation. Experiments were performed to determine which surfactants provide sufficient stability for RZ358 during freeze-thaw. RZ358 was formulated at 2.2 mg / mL in 10 mM L-histidine, 270 mM sorbitol, pH 5.9 with 0, 0.002%, 0.005%, 0.01%, and 0.03% polysorbate 20. Each formulated solution of RZ358 was filled at 1.5 mL into 2 cc Schott glass vials and sealed with West 4432 / 50 rubber stoppers. The 1.5 mL fill volume was half the maximum volume of a 2 cc glass vial and was chosen to maximize the air / liquid surface.

[0155] RZ 358 filled vials were exposed to agitation stress by shaking at 1000 RPM for up to 8 days at room temperature. The solution became cloudy after less than 3 days of shaking in the absence of PS20, but remained clear for 8 days in the presence of 0.002% or more PS20. SE-HPLC detected less than 2% RZ 358 after 3 days of shaking in the absence of PS20. Soluble aggregates increased with shaking time when PS20 concentrations were 0.002% or 0.005% (Figure 7). Soluble aggregates showed minimal growth at 0.01% PS20 or above (Figure 7).

[0156] In the absence of surfactant, freeze-thaw produced numerous visible particles less than 25 μm in size, with very few particles greater than 25 μm in diameter. The number of subvisible particles less than 10 μm was significantly reduced at PS20 concentrations of 0.002% and above. No significant effect of PS20 was observed for particles less than 25 μm (FIG. 8). The minimum effective PS20 concentration for prevention of freeze-thaw induced particle formation appeared to be 0.002%. To mitigate agitation and freeze-thaw induced aggregation, a PS20 concentration of 0.01% was selected for the formulation.

[0157] Oxidation of amino acid side chains during purification and storage of protein drug products can lead to instability and destruction of therapeutic drugs over time. RZ 358 oxidation control employed the use of a scavenger (sugar alcohol) and a free radical sink (methionine) in the formulation. Mannitol and sorbitol were tested in the presence of methionine for their effectiveness in minimizing oxidation of RZ 358 when tested by HIC.

[0158] RZ 358 showed better conformational stability in the sugar alcohol / methionine formulation as indicated by the higher thermal denaturation temperature in the sugar alcohol / methionine formulation than in the arginine formulation (Figure 9). No significant difference was found between the mannitol and sorbitol formulations in terms of thermal denaturation. The oxidation rate was significantly reduced in the sugar alcohol / methionine formulation compared to the arginine formulation (Figures 10 and 11, bottom panels). The two sugar alcohols showed roughly equal effectiveness in controlling oxidation. Consistent with the better conformational stability indicated by the DSC data, the two sugar alcohol formulations showed less aggregation and less fragmentation in the stressed temperature storage stability samples as indicated by the SEC-HPLC data (Figure 11, top panel). Sorbitol was selected as the excipient for oxidation reduction because freeze-thaw cycles resulted in mannitol precipitation.

[0159] The effect of different ratios of sorbitol to arginine concentration on the stability of RZ358 was evaluated in 10 mM L-histidine and 10 mM L-methionine at pH 6. RZ358 was formulated at 5 mg / mL in four different sorbitol / arginine formulations in 10 mM histidine / methionine at pH 6: [1] 150 mM arginine, [2] 90 mM sorbitol / 100 mM arginine, [3] 180 mM sorbitol / 50 mM arginine, and [4] 270 mM sorbitol. Stability data from samples incubated at 40° C. for 2 months showed that higher sorbitol concentrations were more effective in controlling oxidation, aggregation, and fragmentation ( FIG. 12 ).

[0160] The effect of different methionine concentrations on the stability of RZ 358 was also studied at a concentration of 5 mg / mL RZ358 in 10 mM L-histidine, 270 mM sorbitol at pH 6 with 0, 2.5 mM, 5 mM, 7.5 mM, and 10 mM L-methionine. Formulated samples were stored at 40° C. for up to 3 months. Accelerated stability data showed that higher methionine concentrations resulted in less oxidation ( FIG. 13 , top panel) and aggregation ( FIG. 13 , bottom panel) of RZ 358.

[0161] Example 3 - Formulation of RZ358 at higher antibody concentrations Higher concentration RZ358 formulations were tested to evaluate long-term stability and efficacy. Long-term stability of 80 mg / mL RZ358 in 10 mM L-histidine, 10 mM L-methionine, 270 mM sorbitol, 0.01% (w / w) polysorbate 20, pH 5.8 at 2°C to 8°C was measured. Analysis showed that the two experimental lots were stable between pH 5.4 and pH 6.3 for up to 60 months (Figure 14A). Protein concentration was also stable at the same storage conditions at 70-90 mg / kg (Figure 14B). Osmolality measurements of the same lots over the same time frame showed that osmolality was stable at approximately 320-340 mOsm / kg and potentially stable at 286-366 mOsm / kg.

[0162] Evaluation of % light chain and % heavy chain of antibody lots after 60 months storage at 2° C.-8° C. was assessed by CE-SDS (reduced). The amount of light chain was in the range of 27%-36%, while the amount of heavy chain was within the acceptance criteria of 62%-71%.

[0163] The antibody formulations were measured for impurities and degradation products after 60 months of storage at 2°C-8°C. Total impurities assessed by reduced CE-SDS showed less than 3% impurities in either of the two lots detected over the course of the storage period (Figure 15A). Analysis of impurities by non-reduced CE-SDS showed less than 9% impurities over the storage period (Figure 15B). Reduced CE-SDS identified the majority of the antibody detectable in the main peak, within the acceptance criteria of 90-100% (Figure 16A).

[0164] Cation exchange chromatography analysis showed antibodies detected in the main peaks (approximately 40-50% M1 and approximately 30% M2) (Figures 16B, 16C), with a subset of proteins detectable in the acidic peak fractions (less than 20%) (Figure 17A) and basic peak fractions (less than 22%) (Figure 17B).

[0165] The monomeric, high and low molecular weight, and oxidized species of the 80 mg / ml RZ358 formulation were also determined. SE-HPLC detection of monomeric species indicates that the antibody was greater than 95% in monomeric form, within the 90% acceptance criteria (Figure 18A). In addition, less than 1.5% appeared to be high molecular weight species (Figure 18B), while little to no low molecular weight species were detected (Figure 18C). Detection of oxidized species by HIC-HPLC indicates that less than 15% of the species were oxidized during storage (Figure 18D).

[0166] The potency of the stored materials was also determined using a cell-based potency assay. Briefly, the bioassay measures the dose-dependent inhibition of phosphorylated AKT by RZ 358 by utilizing CHO-K1 hulNS cells engineered to express the human insulin receptor. First, CHO-K1 hulNS cells are insulin starved overnight. After starvation, the cells are exposed to dose-dependent levels of RZ 358 and then stimulated with insulin. The cells are lysed, and then phosphorylated Akt and total Akt are quantified by a sandwich immunoassay utilizing MSD plates pre-coated with capture antibodies against phospho-AKT (Ser473) and total Akt. The plates are read and the data for the reference standard, test samples, and controls are fitted to a four-parameter curve-fitting model. The final results are reported as % relative potency to the reference standard. FIG. 19 shows that stored RZ 358 maintained good potency over time, with approximately 80-120% relative potency measured over 60 months of storage.

[0167] The number of particles formed over time was also measured across storage conditions, with little to no particles detected in either the 25 μM RZ 358 or 10 μM R358 batches (FIG. 20).

[0168] Evaluation of surfactant level on formulation stability: RZ 358 was formulated at 100 mg / mL in 10 mM L-histidine, 10 mM methionine, 270 mM sorbitol, pH 5.8 with 0%, 0.002%, 0.004%, 0.006%, and 0.01% polysorbate 20 (formulations F1-F5, respectively). Each formulated solution of RZ 358 was sterile filtered and filled at 1.3 mL into 2 cc Schott glass vials and sealed with West 4110 / 40 rubber stoppers.

[0169] RZ 358 filled vials were exposed to agitation stress by shaking at 300 RPM for up to 48 hours at room temperature followed by shaking at 1000 RPM for 1 hour. In a separate arm of the study, samples were exposed to 5 freeze-thaw cycles from -20°C to room temperature. Solutions were analyzed for the presence of subvisible particles by appearance and by HIAC. The study found that the presence of PS20 in RZ358 formulations at concentrations of 0.004% or higher mitigated turbidity and visible particle formation induced by agitation and freeze-thaw stress, while the presence of PS20 levels of 0.002% or higher controlled visible particulation in non-stressed RZ358 samples at T=0 (Table 1). Under the current experimental conditions, all RZ358 formulations tested passed the USP threshold for subvisible particles regardless of the presence of stress, but a significant reduction in particle counts was seen in both T=0 and stressed samples in the presence of any level of polysorbate (Table 1).

[0170] Table 1. RZ358 appearance and subvisible particle counts as a function of agitation and freeze-thaw stress. TIFF2025517443000002.tif101147

[0171] Preferred embodiments of the present disclosure are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors intend for the present disclosure to be carried out in ways other than as specifically described herein. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A composition comprising an antibody that specifically binds to the insulin receptor (INSR), at least one amino acid or a salt thereof, a surfactant, and a sugar alcohol, wherein the anti-INSR antibody is (A) A light chain variable domain comprising (i) a light chain CDR1 sequence containing the amino acid sequence described in SEQ ID NO: 6, (ii) a light chain CDR2 sequence containing the amino acid sequence described in SEQ ID NO: 7, and (iii) a light chain CDR3 sequence containing the amino acid sequence described in SEQ ID NO: 8, (B) A heavy chain variable domain comprising (i) a heavy chain CDR1 sequence containing the amino acid sequence described in SEQ ID NO: 3, (ii) a heavy chain CDR2 sequence containing the amino acid sequence described in SEQ ID NO: 4, and (iii) a heavy chain CDR3 sequence containing the amino acid sequence described in SEQ ID NO:

5. The composition comprising the above.

2. The composition according to claim 1, wherein the at least one amino acid or salt thereof is selected from the group consisting of histidine, histidine HCl, methionine, and arginine.

3. (i) When the amino acid is histidine, the histidine is at a concentration of about 4 mM to about 25 mM, or about 10 mM to about 20 mM, or about 4 ± 1 mM. (ii) When the amino acid is histidine HCl, the concentration of the histidine HCl is about 4 mM to about 25 mM, or about 10 mM to about 20 mM, or about 6 ± 1 mM, (iii) When the amino acid is methionine, the concentration of the methionine is approximately 4 mM to approximately 25 mM, or approximately 10 mM to approximately 20 mM, or approximately 10 ± 2 mM. The composition according to claim 2.

4. The composition according to claim 1, wherein the surfactant is a polysorbate.

5. The composition according to claim 4, wherein the polysorbate is polysorbate 20, polysorbate 80, or a mixture thereof.

6. The composition according to claim 1, comprising the surfactant in a concentration of approximately 0.002% (w / v) to approximately 0.02% (w / v), or approximately 0.005% (w / v), 0.010% (w / v), 0.015% (w / v), or 0.02% (w / v), or approximately 0.01% (w / v) ± 0.0025% (w / v).

7. The composition according to claim 1, wherein the sugar alcohol is selected from the group consisting of sucrose, sorbitol, and mannitol.

8. (i) When the sugar alcohol is sorbitol, the sorbitol is concentrated at a concentration of about 100 mM to about 350 mM, or about 200 mM to about 300 mM, or about 270 ± 30 mM, or (ii) When the sugar alcohol is mannitol, the concentration of the mannitol is approximately 100 mM to approximately 350 mM, or approximately 200 mM to approximately 300 mM, or approximately 270 ± 30 mM. The composition according to claim 7.

9. The aforementioned anti-INSR antibody (A) A light chain variable domain containing an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, or (B) A heavy chain variable domain containing an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, or (C) Light chain variable domain of (A) and heavy chain variable domain of (B) A composition according to any one of claims 1 to 8, comprising:

10. The composition according to any one of claims 1 to 8, wherein the anti-INSR antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO:

10.

11. The composition according to any one of claims 1 to 8, wherein the anti-INSR antibody is present in the composition at a concentration of about 20 mg / ml to about 200 mg / mL, about 50 mg / ml to about 150 mg / mL, or about 80 mg / mL to about 120 mg / mL.

12. A composition according to any one of claims 1 to 8, which is a liquid.

13. The composition according to claim 12, wherein the pH is less than approximately 6.5, or approximately 5.0 to approximately 6.0, or approximately 5.5 to approximately 5.9, or approximately 5.

8.

14. The composition according to claim 12, characterized by a viscosity of about 2 cP to about 10 cP at 25°C, and having a concentration of the anti-INSR antibody of about 100 mg / ml or less.

15. The composition according to claim 12, which is isotonic or has a weight osmolality in the range of about 200 mOsm / kg to about 500 mOsm / kg, or about 225 mOsm / kg to about 400 mOsm / kg, or about 250 mOsm / kg to about 400 mOsm / kg.

16. (i) As determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis, after storage at 2°C to 8°C for about 24 months to about 36 months, the composition contains less than 10% impurities. (ii) When determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis, less than 10% of the antibody has degraded after storage at 2°C to 8°C for approximately 24 to 36 months. (iii) When determined by hydrophobic interaction chromatography (HIC) or size exclusion chromatography (SEC), less than 15% of the antibody is oxidized or aggregated after storage at 40°C for approximately 3 months. (iv) When determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis, after storage at 2°C to 8°C for about 24 months to about 36 months, the composition contains less than about 10% degradation products. (v) When determined by IEX-UHPLC analysis, less than 30% of the antibodies are detected at an acidic peak after storage at 2°C to 8°C for approximately 24 to 36 months. (vi) When determined by cIEX-UHPLC analysis, approximately 8-20% of the antibodies are detected as acidic peaks after storage at 2°C to 8°C for approximately 24 to 36 months. (vii) When determined by IEX-UHPLC analysis, less than 22% of the antibody is detected with a basic peak after storage at 2°C to 8°C for approximately 24 to 36 months. (viiii) When determined by IEX-UHPLC analysis, approximately 5-20% of the antibody is detected with a basic peak after storage at 2°C to 8°C for approximately 24 to 36 months. (ix) As determined by SE-UHPLC, after storage at 2°C to 8°C for about 24 to 36 months, the composition contains less than 5% high molecular weight species. (x) When determined by SE-UHPLC, after storage at 2°C to 8°C for about 24 to 36 months, the composition contains less than 5% low molecular weight species. (xi) When determined by HIC-HPLC analysis, after storage at 2°C to 8°C for about 24 to 36 months, the composition contains the antibody in an oxidized form of less than 18%, and / or (xi) When the potency of the antibody composition is determined by a bioassay, it is at least about 75% to about 120% after storage at 2°C to 8°C for about 24 months to about 36 months. The composition according to claim 12.

17. A composition comprising approximately 20 to 100 mg / mL of anti-INSR antibody, 0.01% (w / v) of polysorbate 20, approximately 250 mM to approximately 300 mM of sorbitol, approximately 5 mM to approximately 15 mM of methionine, and approximately 5 mM to approximately 15 mM of histidine, wherein the composition has a pH of approximately 5.

8.

18. A pharmaceutical composition comprising the composition according to any one of claims 1 to 8 for treating a condition related to hyperinsulinemia or excessive insulin signaling in a subject.

19. The pharmaceutical composition according to claim 18, wherein the aforementioned condition is selected from the group consisting of hypoglycemia, insulin sensitivity, cancer, insulinoma, Kaposi's sarcoma, insulin overdose, islet cell proliferation (KATP-HL diffuse disease, KATP-HL focal disease, or "PHHI"), GDH-HL (hyperinsulinemia / hyperammonemia syndrome (HI / HA), leucine-sensitive hypoglycemia, diazoxide-sensitive hypoglycemia, islet cell dysregulation syndrome, idiopathic hypoglycemia in infants, persistent hyperinsulinic hypoglycemia in infants (PHHI), congenital hyperinsulinemia, acute hypoglycemia due to renal failure, chronic hypoglycemia due to renal failure, and hypoglycemia due to chronic kidney disease.

20. The pharmaceutical composition according to claim 18, wherein the disease is congenital hyperinsulinism.

21. The pharmaceutical composition according to claim 18, which is administered intravenously or subcutaneously.

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