Formulations for anti-insulin receptor antibodies and uses thereof

Abstract

The present disclosure relates generally to formulations comprising an anti-insulin receptor antibody RZ 358 and their use for the treatment of hyperinsulinemia disorders.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 344,286, filed on May 20, 2022, which is incorporated herein in its entirety. Technical Field

[0003] The present disclosure generally relates to formulations of antibodies specific for the insulin receptor for use in the treatment and prevention of hypoglycemia and conditions associated with hyperinsulinemia, such as congenital hyperinsulinemia.

[0004] Incorporation by Reference of Electronically Submitted Materials

[0005] The sequence listing, which is part of the present disclosure, is filed concurrently with the specification as a text file. The name of the text file containing the sequence listing is "57847_Seqlisting.hml", the file 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 in its entirety. Background Art

[0006] Insulin is the main hormone used to lower blood sugar levels. The first step in insulin action is the binding of the hormone to the insulin receptor (INSR), which is an integral membrane protein also known as CD220 or HHF5. When insulin binds to the INSR, the receptor is activated by tyrosine autophosphorylation, and the INSR tyrosine kinase phosphorylates various effector molecules, including insulin receptor substrate-1 (IRS-1), thereby causing the hormone to act (Ullrich et al., Nature 313: 756-761, 1985; Goldfine et al., Endocrine Reviews 8: 235-255, 1987; White and Kahn, Journal Biol. Chem 269: 1-4, 1994). IRS-1 binding and phosphorylation ultimately lead 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 lead to an increase in glucose uptake from the blood by these tissues. Glut4 mediates glucose transport into cells and lowers blood glucose levels.

[0007] Abnormal increase in insulin secretion can lead to hypoglycemia or low blood sugar, a state that may cause significant morbidity including epilepsy and brain damage. Drug-induced hypoglycemia can be caused by the administration of sulfonylurea drugs or excessive insulin. Many rare medical conditions are characterized by non-drug-induced endogenous hyperinsulinemic hypoglycemia, i.e., hypoglycemia caused by excessive production of insulin by the body. These conditions include congenital hyperinsulinemia, insulinoma, and hyperinsulinemic hypoglycemia after gastric bypass surgery.

[0008] RZ358 is a monoclonal antibody specific for the insulin receptor (INSR), which regulates the binding of insulin to the insulin receptor and binds to the INSR allosterically without blocking the binding of insulin to the insulin receptor. In a Phase 1 trial, RZ358 was shown to reduce glucose levels in healthy volunteers (Johnson et al., "Journal of Clinical Endocrinol Metabolism (J Clin Endocrinol Metab.)" 2017 102 (8): 3021-3028), and in subsequent studies, it was shown to reduce the time period of daily hypoglycemia in patients with post-gastric bypass hypoglycemia (PGBH) and correct nocturnal hypoglycemia (Hu et al., "Journal of the Endocrine Society (Journal of the Endocrine Society)", Vol. 5, Supplement 1, April-May 2021, Pages A328-A329). A recent Phase 2b trial demonstrated improved hypoglycemia in patients with congenital hyperinsulinemia (CHI) who received 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). Summary of the invention

[0009] Contemplated herein are formulations for anti-INSR antibodies (eg, RZ 358) that provide stability of the antibody over time under different storage conditions.

[0010] The present disclosure provides a composition comprising an antibody that specifically binds to an 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 shown in SEQ ID NO:6; (ii) a light chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO:7; and (iii) a light chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO:8; and (B) a heavy chain variable domain comprising: (i) a heavy chain CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO:3; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO:4; and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO:5.

[0011] In various embodiments, the anti-INSR antibody comprises: (A) a light chain variable domain comprising an amino acid sequence at least 80% identical to SEQ ID NO: 2; or (B) a heavy chain variable domain comprising an amino acid sequence 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.

[0012] 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.

[0013] 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.

[0014] In various embodiments, the anti-INSR antibody is an IgG2 antibody.

[0015] In various embodiments, the anti-INSR antibody is RZ358. In various embodiments, RZ358 comprises (A) a light chain variable domain comprising: (i) a light chain CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO:6; (ii) a light chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO:7; and (iii) a light chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO:8; and (B) a heavy chain variable domain comprising: (i) a heavy chain CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO:3; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO:4; and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO:5; or 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; or 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.

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

[0017] In various embodiments, the amino acid is histidine HCL. In various embodiments, the concentration of the histidine HCL is about 4 mM to about 25 mM or about 10 mM to about 20 mM. In various embodiments, the concentration of the histidine HCL is about 6±1 mM.

[0018] In various embodiments, the amino acid is methionine. In various embodiments, the concentration of methionine is about 4 mM to about 25 mM or about 10 mM to about 20 mM. In various embodiments, the concentration of methionine is about 10 ± 2 mM.

[0019] In various embodiments, the surfactant is a polysorbate. In various embodiments, the polysorbate is polysorbate 20, polysorbate 80, or a mixture thereof. In various embodiments, the concentration of the surfactant is 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) surfactant. In various embodiments, the composition comprises about 0.01% (w / v) ± 0.0025% (w / v) surfactant, optionally wherein the surfactant is polysorbate 20 or polysorbate 80, or a mixture thereof.

[0020] In various embodiments, the sugar alcohol is selected from the group consisting of sucrose, sorbitol, and mannitol.

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

[0022] In various embodiments, the sugar alcohol is mannitol. In various embodiments, the concentration of the mannitol is about 100mM to about 350mM or about 200mM to about 300mM. In various embodiments, the concentration of the mannitol is about 270±30mM.

[0023] 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.

[0024] 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.

[0025] In various embodiments, the pH is less than about 6.5. In various embodiments, the pH is about 5.0 to about 6.5. In various embodiments, the pH is about 5.5 to about 5.9. In various embodiments, the pH is about 5.8.

[0026] In various embodiments, the composition is characterized by a viscosity of about 2 cP to about 10 cP at 25°C, wherein the concentration of the anti-INSR antibody is about 100 mg / ml or less.

[0027] In various embodiments, the composition is isotonic or has an osmotic pressure within a 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.

[0028] In various embodiments, the composition comprises less than about 10% impurities after storage for about 24 months to about 36 months at 2° C. to 8° C. as determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis. Impurities include degradation products, high molecular weight species, low molecular weight species, oxidized species, or aggregates.

[0029] In various embodiments, after storage for about 24 months to about 36 months at 2°C to 8°C, less than 10% of the antibody is degraded as determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis. In various embodiments, after storage for about 24 months to about 36 months at 2°C to 8°C, the composition comprises less than about 5% degradation products as determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis.

[0030] In various embodiments, after storage at 40°C for about 3 months, less than 15% of the antibody is oxidized or aggregated as determined by hydrophobic interaction chromatography (HIC) or size exclusion chromatography (SEC).

[0031] In various embodiments, less than 30% of the antibody is detected in the acidic peak after storage at 2°C to 8°C for about 24 months to about 36 months, as determined by cIEX-UHPLC analysis. In various embodiments, approximately 8%-20% of the antibody is detected in the acidic peak after storage at 2°C to 8°C for about 24 months to about 36 months, as determined by cIEX-UHPLC analysis. In various embodiments, less than 22% of the antibody is detected in the basic peak after storage at 2°C to 8°C for about 24 months to about 36 months, as determined by cIEX-UHPLC analysis. In various embodiments, approximately 5%-20% of the antibody is detected in the basic peak after storage at 2°C to 8°C for about 24 months to about 36 months, as determined by cIEX-UHPLC analysis.

[0032] In various embodiments, the composition comprises less than 5% high molecular weight species as determined by SE-UHPLC after storage for about 24 months to about 36 months at 2°C to 8°C. In various embodiments, the composition comprises less than 5% low molecular weight species as determined by SE-UHPLC after storage for about 24 months to about 36 months at 2°C to 8°C.

[0033] In various embodiments, the composition comprises less than 18% of the antibody in oxidized form after storage at 2°C to 8°C for about 24 months to about 36 months as determined by HIC-HPLC.

[0034] In various embodiments, the potency of the antibody composition is at least about 75% to about 120% after storage at 2°C to 8°C for about 24 months to about 36 months as determined by a bioassay. In various embodiments, the bioassay is a phosphorylated AKT (pAKT) assay.

[0035] 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, wherein the pH of the composition is about 5.8. In various embodiments, the anti-INSR antibody is RZ358.

[0036] Further contemplated is an article comprising a composition as 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.

[0037] Also contemplated is a method for treating a condition associated with hyperinsulinemia or excess insulin signaling in a subject in need thereof, the method 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 excess, islet cell proliferation disorders (KATP-HI diffuse disease, KATP-HI focal disease or "PHHI"), GDH-HI (hyperinsulinemia / hyperammonemia syndrome (HI / HA), leucine-sensitive hypoglycemia, diazoxide-sensitive hypoglycemia, islet cell dysregulation syndrome, idiopathic hypoglycemia of infancy, persistent hyperinsulinemic hypoglycemia of infancy (PHHI), congenital hyperinsulinemia, acute hypoglycemia caused by renal failure, chronic hypoglycemia caused by renal failure, and hypoglycemia caused by chronic kidney disease. In various embodiments, the disease is congenital hyperinsulinemia.

[0038] In various embodiments, the composition is administered every day, every 2 days, every 3 days, every week, every 2 weeks, every 3 weeks, twice a month, every month, every 2 months, every 3 months, or every 6 months. In various embodiments, the composition is administered for a period of at least 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 1 year, or longer.

[0039] In various embodiments, the composition is administered intravenously. In various embodiments, the composition is administered subcutaneously.

[0040] The present disclosure also provides a composition comprising an anti-INSR antibody as described herein for use in treating a condition associated with hyperinsulinemia or excess insulin signaling in a subject in need thereof.

[0041] It should be understood that each feature or embodiment or combination described herein is a non-limiting illustrative example of any aspect of the present invention and is therefore intended to be combined with any other feature or embodiment or combination described herein. For example, in the case of describing features with language such as "one embodiment", "some embodiments", "certain embodiments", "other embodiments", "specific exemplary embodiments" and / or "another embodiment", each of these types of embodiments is a non-limiting example of features intended to be combined with any other feature or feature combination described herein, without necessarily listing every possible combination. Such features or combinations of features are applicable to any aspect of the present invention. In the case of disclosing examples of values ​​within a range, it is envisioned that any of these examples are possible endpoints of the range, any and all numerical values ​​between such endpoints are envisioned, and any and all combinations of upper and lower endpoints are envisioned.

[0042] The headings herein are for the convenience of the reader 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

[0043] Figure 1 Depicted are DSC thermograms of RZ 358 in citrate buffered saline formulation at different pH.

[0044] Figure 2 Shown are SEC chromatograms (UV 214 nm) of samples subjected to freeze-thaw stress in pH 4.0 (upper panel) and 7.0 (lower panel) citrate buffered saline formulations.

[0045] Figure 3Shown are SEC (upper panel) and HIC (lower panel) chromatograms (UV 280 nm) of three month 40°C stability samples formulated at different pHs from pH 5.0 to pH 7.0 in citrate buffered saline.

[0046] Figure 4 Shown are WCX chromatograms of RZ 358 stability samples in different pH formulations (pH 5.0 to pH 7.0) stored at 40°C for 1 month (upper panel) and at 30°C for 3 months (lower panel).

[0047] Figure 5 Shown are the changes in total acidic species (upper graph) and total alkaline species (lower graph) at 30°C for RZ 358 stability samples formulated at different pH values ​​(pH 5.0 to pH 7.0).

[0048] Figure 6 Shown are SEC (upper panel) and HIC-HPLC (lower panel) chromatograms of RZ 358 stability samples in different pH formulations (pH 5.0 to pH 7.0) stored at 40°C for 3 months.

[0049] Figure 7 Results of agitation studies are provided showing SEC-HPLC chromatograms of RZ 358 samples formulated in 0.002% (upper panel) and 0.01% (lower panel) PS20 (samples were shaken at 1,000 RPM / 25°C for up to 8 days).

[0050] Figure 8 Shown are the particle concentrations (counts per mL) of 10 μm (upper graph) and 25 μm (lower graph) for samples with different polysorbate 20 concentrations subjected to up to 5 freeze-thaw cycles (-70°C / room temperature) stress.

[0051] Fig. 9 Depicted are DSC thermograms 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).

[0052] Fig.10Shown is the formation of the "main pre-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).

[0053] Fig.11 Chromatograms of stability samples stored at 40°C for 3 months are shown (upper panel = SEC; lower 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).

[0054] Fig.12 Shown are chromatograms of stability samples stored for 2 months at 40°C in different formulations with different concentrations of sorbitol (90-270 mM) (upper panel = SEC; lower panel = HIC).

[0055] Fig.13 Shown are chromatograms of stability samples stored for 3 months at 40°C in formulations with different concentrations (0-10 mM) of methionine (upper panel = HIC; lower panel = SEC).

[0056] Figure 14A-14B 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 (between pH 5.4 and pH 6.3) at 2°C to 8°C for up to 60 months is shown ( Fig.14A ) and the effect on protein concentration at pH 5.8 ( Fig. 14B ).

[0057] Figure 15A-15B It is shown that in the restoration ( Fig.15A ) and non-reduced ( Fig. 15B Total impurities of 80 mg / mL RZ358 in 10 mM L-histidine, 10 mM L-methionine, 270 mM sorbitol, 0.01% (w / w) polysorbate 20 over time at pH 5.8.

[0058] Fig.16A Reduced CE-SDS analysis of the main peak fractions is shown. Figures 16B-16CCation exchange chromatography analysis of the antibodies detected in the main peaks M1 and M2 is shown, and the M1 / M2 ratio is shown in Fig.16D middle.

[0059] Fig.17A The CEX-UHPLC acidic peak fraction of 80 mg / mL RZ358 in 10 mM L-histidine, 10 mM L-methionine, 270 mM sorbitol, 0.01% (w / w) polysorbate 20 is shown, and Fig. 17B CEX-UHPLC basic peak fractions are shown.

[0060] Figures 18A-18C The degradation of the antibody during storage at 2-8°C for 60 months is shown, as shown by the monomeric species ( Fig.18A ), high molecular weight species%( Fig.18B ) or low molecular weight species% ( Fig. 18C ) as shown by SE-UHPLC analysis. Fig.18D Shown are the % of oxidized species under the same storage conditions.

[0061] Fig.19 Shown is the potency of the antibody formulations after storage for 60 months at 2-8°C, as assessed by cellular bioassay.

[0062] Fig. 20 Shown are the number of particles (≥10 μm) formed per container for RZ358 DP at 2°C to 8°C over 60 months. DETAILED DESCRIPTION

[0063] The present disclosure provides a preparation of a monoclonal antibody specific for the insulin receptor RZ358, which is stable in long-term storage, forms minimal impurities and maintains the relative potency of the antibody during storage. Such stable antibody preparations can be used to treat disorders associated with insulin resistance and conditions associated with hyperinsulinemia.

[0064] definition

[0065] The foregoing description has been given only for clearness of understanding, and no unnecessary limitations should be understood therefrom, since modifications within the scope of the invention may be obvious to one skilled in the art.

[0066] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or groups of integers or steps but not the exclusion of any other integer or step or groups of integers or steps.

[0067] Throughout the specification, unless otherwise described, when a composition is described as comprising a component or material, it is contemplated that the composition may also consist essentially of or consist of any combination of said components or materials. Similarly, unless otherwise described, when a method is described as comprising specific steps, it is contemplated that the method may also consist essentially of or consist of any combination of said steps. The invention illustratively disclosed herein may be suitably practiced in the absence of any element or step not specifically disclosed herein.

[0068] The practice of the method disclosed herein and its individual steps can be performed manually and / or by means of an electronic device or an automation provided by an electronic device. Although each process has been described with reference to a specific embodiment, it will be readily understood by those skilled in the art that other ways of performing the behavior associated with the method can be used. For example, unless otherwise described, the order of the various steps can be changed without departing from the scope or spirit of the method. In addition, some of the individual steps can be combined, omitted or further subdivided into other steps.

[0069] Unless otherwise stated, it is contemplated that the compositions and methods include embodiments comprising any combination of one or more of the additional optional elements, features, and steps further described below (including those shown in the Figures).

[0070] In jurisdictions that prohibit patenting of methods practiced on human subjects, the meaning of "administering" a composition to a human subject shall be limited to providing a controlled substance that a human subject may self-administer by any technique (e.g., oral, 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 patenting of methods practiced on human subjects, "administering" a composition includes both methods practiced on human subjects as well as the aforementioned activities.

[0071] It should be understood that each maximum numerical limit given throughout this specification includes the range formed by each corresponding lower numerical limit as an alternative aspect, as if these ranges are clearly written. Each minimum numerical limit given throughout this specification will include the range formed by each higher numerical limit as an alternative aspect, as if these ranges are clearly written. Each numerical range given throughout this specification will include each narrower numerical range falling within such a wider numerical range, as if such a narrower numerical range is clearly written in this article. The sizes and values ​​disclosed herein should be understood to include disclosures of both the values ​​and the corresponding exact numerical values, for example, a value described as "about 10 mM" should be understood to include "10 mM" disclosed as an alternative.

[0072] All patents, publications, and references cited herein are hereby incorporated by reference in their entirety. In the event of a conflict between the present disclosure and the incorporated patents, publications, and references, the present disclosure shall control.

[0073] Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below.

[0074] As used in this specification and the appended claims, the indefinite articles "a", "an" and "the" include plural as well as singular referents, unless the context clearly dictates otherwise.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. The following references provide general definitions of many terms used in the present disclosure for those skilled in the art, including but not limited to: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2nd edition 1994); Cambridge Dictionary of Science and Technology (THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY) (Walker edited, 1988); Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale and Marham, Dictionary of Harper Collins Biology (THEHARPER COLLINS DICTIONARY OF BIOLOGY) (1991).

[0076] The term "about" or "approximately" means the acceptable error of a particular value as determined by one of ordinary skill in the art, which will depend in part on how to measure or determine the value. 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" is before the first numerical value in a series of two or more numerical values, it is understood that the term "about" or "approximately" is applicable to each numerical value in the numerical value in that series.

[0077] 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 that can bind to antigens (e.g., Fab', F'(ab)2, Fv, single-chain antibodies, bifunctional antibodies), and recombinant peptides comprising the foregoing, as long as they exhibit the desired biological activity. An "immunoglobulin" or "tetrameric antibody" is a tetrameric glycoprotein composed of two heavy chains and two light chains, each comprising a variable region and a constant region. The antigen binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antibody fragments or antigen-binding portions include, among others, Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), complementarity determining region (CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv), single-chain antibody fragments, chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, mini antibodies, linear antibodies; chelated recombinant antibodies, tribodies or bibodies, intracellular antibodies, nanobodies, small modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies or their variants or derivatives, and polypeptides, which 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, as long as the antibody retains the desired biological activity.

[0078] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0079] As used herein, "antibody variant" refers to an antibody polypeptide sequence containing 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.

[0080] As used herein, "chimeric antibodies" refer to antibodies containing sequences derived from two different antibodies (see, e.g., U.S. Patent No. 4,816,567), which are generally derived from different species. Most typically, chimeric antibodies comprise human and rodent antibody fragments, generally human constant regions and mouse variable regions.

[0081] "Neutralizing antibodies" are antibody molecules that can eliminate or significantly reduce the biological function of the antigen to which the antibody binds. Thus, "neutralizing" antibodies can eliminate or significantly reduce biological functions, such as enzyme activity, ligand binding, or intracellular signaling.

[0082] As used herein, "heavy chain variable region" refers to a region of an antibody molecule that contains at least one complementarity determining region (CDR) of the antibody heavy chain variable domain. The heavy chain variable region may contain one, two or three CDRs of the antibody heavy chain.

[0083] As used herein, "light chain variable region" refers to the region of an antibody molecule comprising at least one complementarity determining region (CDR) of the antibody light chain variable domain. A light chain variable region can contain one, two or three CDRs of the antibody light chain, which may be a kappa or lambda light chain depending on the antibody.

[0084] As used herein, an antibody that "specifically binds to" is "antigen-specific," "specific for" an antigenic target, or "immunoreactive" to an antigen refers to an antibody or polypeptide binding agent, as used herein, that binds to an antigen with greater affinity than other antigens of similar sequence. In one aspect, an antibody or fragment, variant, or derivative thereof binds to a human antigen with greater affinity than to a similar antigen from another species (i.e., a non-human species), but polypeptide binding agents that recognize and bind to orthologs of the target are also within the scope of the present method.

[0085] For example, a polypeptide binding agent that is "specific" for its cognate antigen as an antibody or fragment thereof indicates that the variable region of the antibody recognizes and binds to the desired antigen with a detectable preference (e.g., where the desired antigen is a polypeptide, the variable region of the antibody is able to distinguish the antigen polypeptide from other known polypeptides of the same family by means of measurable differences in binding affinity, regardless of the local sequence identity, homology or similarity that may exist between family members). It will be understood that a particular antibody may also interact with other proteins (e.g., Staphylococcus aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibody and particularly within the constant region of the molecule. Screening assays for determining the binding specificity of polypeptide binding agents (e.g., antibodies) used in the methods herein are well known and routinely practiced 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 these methods can be produced using any method known in the art.

[0086] The term "epitope" refers to a portion of any molecule that can be recognized and bound by a selective binding agent at one or more locations of an antigen binding region. An epitope is usually composed of chemically active surface groups of a molecule, such as amino acids or carbohydrate side chains, and has specific three-dimensional structural characteristics as well as specific charge characteristics. As used herein, an epitope can be continuous or non-continuous.

[0087] The term "sample" or "biological sample" refers to a specimen obtained from a subject for use in the methods of the present invention, and includes urine, whole blood, plasma, serum, saliva, sputum, tissue biopsy, and cerebrospinal fluid.

[0088] The term "therapeutically effective amount" is used herein to indicate an amount of a target-specific composition that is effective in ameliorating or alleviating symptoms or signs of a disease associated with abnormal (eg, abnormally high or abnormally low) signaling of a signaling complex.

[0089] The terms "treat," "treating," and "treatment" refer to temporarily or permanently, partially or completely eliminating, reducing, inhibiting, or ameliorating clinical symptoms, manifestations, or progression of events, diseases, or conditions associated with the inflammatory conditions described herein. As recognized in the relevant art, drugs used as therapeutic agents can reduce the severity of a given disease state, but do not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, treatments administered prophylactically do not need to be completely effective in preventing the onset of the disease in order to constitute a viable prophylactic. It is sufficient to simply 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 reduce the likelihood of the disease occurring or worsening in a subject. One embodiment of the present disclosure relates to a method for determining the efficacy of a treatment, the method comprising administering an amount of a therapeutic agent to a patient and continuing for a period of time sufficient to induce a sustained improvement in an indicator reflecting the severity of a particular condition relative to a baseline.

[0090] Anti-INSR Antibodies

[0091] Provided herein are compositions comprising anti-INSR antibodies, such as 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. Patents 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 interfere with insulin binding but weakens the binding of insulin to the receptor and reduces signaling through the receptor. This mechanism increases and normalizes blood glucose levels in hyperinsulinemic patients.

[0092] 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 chain (H) and / or light chain (L) is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. Fragments of such antibodies are also included as 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.

[0093] 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. Typically, a humanized antibody has one or more amino acid residues introduced thereto from a non-human source. For example, 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) can be used to replace the corresponding region of a human antibody with at least a portion of a rodent complementary determining region.

[0094] Chimeric antibodies, antibodies transplanted with CDRs and humanized antibodies and / or antibody variants are usually produced by recombinant methods. Nucleic acids encoding antibodies are introduced into host cells and expressed using materials and procedures described herein. In a preferred embodiment, the antibody is produced in mammalian host cells such as CHO cells. As described herein, monoclonal (e.g., human) antibodies can be produced by expressing recombinant DNA in host cells or by expressing in hybridoma cells.

[0095] In various embodiments, the present disclosure provides antibodies or fragments thereof, comprising three heavy chain CDRs having the amino acid sequences shown in SEQ ID NOs: 3-5 and three light chain CDRs having the amino acid sequences shown in SEQ ID NOs: 6-8. In various embodiments, the antibodies or fragments thereof bind to (i) an insulin receptor or (ii) a complex comprising insulin and an insulin receptor, or both (i) and (ii).

[0096] 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 shown 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 shown 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 shown in SEQ ID NO:3-8.

[0097] In various embodiments, the anti-INSR antibody or fragment thereof comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:1 and the light chain variable region amino acid sequence shown in SEQ ID NO:2.

[0098] In an exemplary case, 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.

[0099] Also contemplated are antibodies or antibody variants that bind to INSR, the antibodies or antibody variants 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%, greater than 95%) identical to SEQ ID NO: 1; (ii) a sequence of amino acids encoded by a polynucleotide sequence that encodes a polypeptide at least 80% (e.g., about 85%, about 90%, about 95%, greater 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 the complement of a polynucleotide encoding a polypeptide consisting of SEQ ID NO: 1; and (B) a light 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%, greater than 95%) identical to SEQ ID NO: 2; (ii) a sequence of amino acids encoded by a polynucleotide sequence that encodes a polypeptide at least 80% (e.g., about 85%, about 90%, about 95%, greater than 95%) identical to SEQ ID NO: 1. NO:2 is at least 80% (e.g., about 85%, about 90%, about 95%, greater than 95%) identical; (iii) a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions with the complement of a polynucleotide encoding a polypeptide consisting of SEQ ID NO:2; or (C) the light chain variable domain of (A) and the heavy chain variable domain of (A), wherein the antibody or antibody variant specifically binds to (i) the insulin receptor or (ii) a complex comprising insulin and the insulin receptor, or both (i) and (ii).

[0100] In various embodiments, the antibody or fragment thereof is a Fab fragment.

[0101] 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 bifunctional antibody, a trifunctional antibody, a tetrafunctional antibody, 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.

[0102] For example, an exemplary sequence of a human IgG2 constant region can be obtained from the Uniprot database at Uniprot Accession No. P01859, which is incorporated herein by reference. Information including sequence information of other antibody heavy and light chain constant regions can also be publicly available through the Uniprot database and other databases well known to those skilled in the art of antibody engineering and production.

[0103] In various embodiments, the antibody, antibody variant or fragment thereof is present in an amount of at least 10-5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M or 10 -15 M or less equilibrium dissociation constant Kd binds to (i) insulin receptor or (ii) a complex comprising insulin and insulin receptor, wherein the Kd is capable of reducing the binding affinity between insulin and insulin receptor by at least about 1.5 times, optionally up to 1000 times. In certain embodiments, the antibody is capable of reducing the binding affinity between insulin and insulin receptor by about 2 times to 500 times. In various embodiments, the antibody reduces the EC of insulin signaling activity by about 1.5 times. 50 An increase of about 2-fold to 1000-fold, optionally in a pAKT assay.

[0104] preparation

[0105] The present disclosure provides formulations of the anti-INSR antibody RZ358 that are stable under conditions of long-term storage and stress that can be used in the methods described herein.

[0106] It is contemplated that the antibody formulations are in liquid or lyophilized form. The formulation may also be a liquid reconstituted from a lyophilized form.

[0107] In various embodiments, the compositions of the present disclosure are liquids. In some aspects, the pH of the composition is less than about 6.5. In some aspects, the pH is from about 5.0 to about 6.0 or from about 5.1 to about 5.8, or from 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.

[0108] In various embodiments, the formulation comprises one or more of a surfactant, a stabilizer, an amino acid, an antioxidant and / or a buffer to minimize aggregation, oxidation and other protein degradation during storage. Exemplary stabilizers include surfactants and sugar alcohols. Exemplary antioxidants include methionine, sugar alcohols and histidine. Exemplary buffers include histidine HCl.

[0109] In various embodiments, the composition comprises histidine. In various embodiments, the concentration of histidine is about 4mM to about 25mM, such as about 5mM to 20mM, about 10mM to about 20mM, or about 8mM to 15mM, or about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM or about 20mM. In various embodiments, the concentration of the histidine is about 4±1mM.

[0110] In certain embodiments, the composition comprises histidine HCl. In various embodiments, the concentration of histidine HCl is about 4mM to about 25mM, such as about 5mM to 20mM, about 10mM to about 20mM, or about 8mM to 15mM, or about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM or about 20mM. In various embodiments, the concentration of the histidine HCL is about 6±1mM.

[0111] In various embodiments, the composition comprises methionine. In various embodiments, the concentration of methionine is about 4mM to about 25mM, such as about 5mM to 20mM, about 10mM to about 20mM, or about 8mM to 15mM, or about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM or about 20mM. In various embodiments, the concentration of methionine is about 10±2mM.

[0112] The compositions herein may further include sugar alcohols. Exemplary sugar alcohols include sorbitol, mannitol, trehalose and sucrose. In various embodiments, the preparation includes sugar alcohols, and the sugar alcohol is sorbitol. In various embodiments, the concentration of sorbitol is about 100mM to about 350mM, about 150mM to 350mM, about 100mM to 300mM, about 100mM to 200mM or about 200mM to about 300mM. The concentration of sorbitol can be about 100mM, about 125mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM, about 300mM, about 325mM or about 350mM. In various embodiments, the concentration of sorbitol is about 270 ± 30mM.

[0113] In various embodiments, preparation comprises sugar alcohol, and described sugar alcohol is mannitol.In various embodiments, the concentration of mannitol is about 100mM to about 350mM, about 150mM to about 350mM, about 100mM to about 300mM, about 100mM to about 200mM or about 200mM to about 300mM.The concentration of mannitol can be about 100mM, about 125mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM, about 300mM, about 325mM or about 350mM.In various embodiments, the concentration of described mannitol is about 270 ± 30mM.

[0114] In some embodiments, the compositions of the present disclosure include surfactants. The surfactant is an amphiphilic surfactant (having a polar head and a hydrophobic tail). The surfactant preferentially aggregates at the interface, causing the interfacial tension to decrease. The use of surfactants also helps to mitigate the formation of large protein particles. In some aspects, the surfactant present in the compositions of the present disclosure is an amphiphilic and / or nonionic surfactant. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 80), alkyl aryl polyethers (e.g., oxyethylated alkylphenols (e.g., Triton TM X-100) and poloxamers (e.g., For example F68), and combinations of any of the foregoing, within a class of surfactants or between multiple classes of surfactants. Polysorbate 20 and polysorbate 80 (and optional mixtures thereof) are contemplated herein. In exemplary cases, the surfactant is present in the composition at a concentration of less than or about 0.002% (w / v) to about 0.02% (w / v). For example, the formulation can contain about 0.005% (w / v) to about 0.015% (w / v) surfactant, such as 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 aspects, the formulation comprises about 0.010% (w / v), 0.015% (w / v), or 0.02% (w / v) surfactant.

[0115] It is contemplated that the formulations herein comprise 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 formulation may comprise 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.

[0116] In various embodiments, the composition comprises about 20-100 mg / mL of an anti-INSR antibody, 0.01% (w / w) 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, wherein the pH of the composition is 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, wherein the pH of the composition is about 5.8. In various embodiments, the antibody is RZ358.

[0117] If the formulation is in a form intended for parenteral administration, it can be isotonic with blood (about 300mOsm / kg osmotic pressure). In some aspects, it is envisioned that the osmotic pressure of the composition is in the range of about 200mOsm / kg to about 500mOsm / kg, or about 225mOsm / kg to about 400mOsm / kg, or about 250mOsm / kg to about 400mOsm / kg, about 250mOsm / kg to about 350mOsm / kg, or about 275mOsm / kg to about 375mOsm / kg. In various embodiments, the osmotic pressure of the formulation is about 275mOsm / kg to about 375mOsm / kg.

[0118] In various embodiments, the formulation has a low viscosity. In some embodiments, the formulation is characterized by a viscosity of about 2 cP to about 10 cP at 25°C, wherein the concentration of the anti-INSR antibody is about 100 mg / ml or less.

[0119] In various aspects, the compositions of the present disclosure are stable and can be stored for a long time at refrigerated temperatures. It is envisioned that 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), 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. In various embodiments, after 36 months, 48 ​​months or 60 months of storage at about 2 ° C to about 8 ° C, 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 antibody is degraded, as determined by CE-SDS. In various embodiments, after 24 months of storage at about 2 ° C to about 8 ° C, more than about 90% or about 95% of the antibody is intact, as determined by CE-SDS.

[0120] In various embodiments, the compositions of the present disclosure are stable and can be stored for a short period of time under storage conditions subject to stress. 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 storage for about 1 month to about 3 months 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).

[0121] In various embodiments, at least 90% of the antibody or at least about 95% of the antibody is in monomeric form after long-term storage at about 2°C to about 8°C, 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, optimally as detected by SE-HPLC. In various embodiments, less than about 15% of the antibody is oxidized species after long-term storage, optionally as detected by HIC-HPLC.

[0122] In various embodiments, the antibody maintains 80%-120% relative potency compared to a reference antibody after long-term storage at about 2°C to about 8°C, optionally as measured by a pAKT assay. In various embodiments, the reference antibody has 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. In various embodiments, the reference antibody has the heavy chain amino acid sequence set forth in SEQ ID NO: 9 and the light chain amino acid sequence set forth in SEQ ID NO: 10.

[0123] In various embodiments, compositions are provided for storage or use, for example, in 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, at about 2°C to about 8°C for long-term storage or at higher temperatures (e.g., about 25°C, about 30°C, about 40°C).

[0124] In various embodiments, the composition is administered to the subject subcutaneously and is isotonic with the intended site of administration.

[0125] Preparation method

[0126] Further provided herein is a method for preparing the compositions of the present disclosure. Using transgenic animals (e.g., mice) capable of producing a range of human antibodies in the absence of endogenous immunoglobulin production, such antibodies are produced by immunizing with a polypeptide antigen (i.e., having at least 6 consecutive amino acids) optionally conjugated to a carrier. See, for example, Jakobovits et al., 1993, Proceedings of the National Academy of Sciences of the United States of America 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. Pat. No. 5,545,807, PCT Application Nos. PCT / US91 / 245 and PCT / GB89 / 01207, and European Patent Nos. 546073B1 and 546073A1. As described herein, human antibodies can be produced by expressing recombinant DNA in host cells or by expression in hybridoma cells.

[0127] Chimeric antibodies, antibodies transplanted with CDRs and humanized antibodies and / or antibody variants are usually produced by recombinant methods. Nucleic acids encoding antibodies are introduced into host cells and expressed using materials and procedures described herein. In a preferred embodiment, the antibody is produced in mammalian host cells such as CHO cells. Monoclonal (e.g., human) antibodies can be produced by expressing recombinant DNA in host cells or by expressing in hybridoma cells.

[0128] In order to recombinantly produce an antibody or antibody fragment, the nucleic acid encoding the antibody or antibody fragment is separated and inserted into a reproducible vector for further cloning (amplification of DNA) or for expression. The DNA encoding the monoclonal antibody can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can be specifically bound to the heavy chain and light chain of the encoding antibody). 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 selective marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0129] Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast cells, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, and Bacilli, such as B. subtilis and B. licheniformis (e.g., DD 12, April 1989). 266,710 disclosed in Bacillus licheniformis 41P), Pseudomonas (Pseudomonas), such as Pseudomonas aeruginosa (P.aeruginosa) and Streptomyces (Streptomyces). A preferred E. coli cloning host is E. coli 294 (ATCC 31,446), but 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 and non-restrictive.

[0130] Eukaryotic microorganisms 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 of the lower eukaryotic host microorganisms. However, many other genera, species and strains are commonly used and suitable for use herein, such as Schizosaccharomyces pombe; 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; Yarrowia (EP 402,226); Pichia pastors (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts (such as A. nidulans and A. niger).

[0131] Suitable host cells for expressing glycosylated antibodies are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. A variety of baculovirus strains and variants and corresponding permissive insect host cells have been identified, and the permissive insect host cells are derived from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori. A variety of viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain variant of Bombyx mori NPV, and such viruses can be used as viruses herein according to the present disclosure, particularly for transfecting Spodoptera frugiperda cells.

[0132] Examples of useful mammalian host cell lines are Chinese hamster ovary cells, including 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 CRL 1651); 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 (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0133] Host cells are transformed with expression or cloning vectors for antibody production and cultured in conventional nutrient media modified to be suitable for inducing promoters, selecting transformants or amplifying genes encoding desired sequences. In addition, novel vectors with multiple copies of transcription units separated by selective markers and transfected cell lines are particularly useful and preferred for expressing antibodies that bind to desired antigens.

[0134] How to use

[0135] Provided herein are uses of anti-INSR antibody formulations, e.g., comprising RZ358, for treating hyperinsulinism / hyperinsulinemia caused by aberrant insulin / INSR signaling and low blood glucose levels.

[0136] Exemplary hyperinsulinemia disorders include hypoglycemia, insulin sensitivity, cancer, insulinoma, Kaposi's sarcoma, insulin excess, islet cell proliferation disorders (KATP-HI diffuse disease, KATP-HI focal disease or "PHHI"), GDH-HI (hyperinsulinemia / hyperammonemia syndrome (HI / HA), leucine-sensitive hypoglycemia, diazoxide-sensitive hypoglycemia, islet cell dysregulation syndrome, idiopathic hypoglycemia of infancy, persistent hyperinsulinemic hypoglycemia of infancy (PHHI), congenital hyperinsulinemia, acute hypoglycemia due to renal failure, chronic hypoglycemia due to renal failure, and hypoglycemia due to chronic kidney disease.

[0137] Congenital hyperinsulinemia (CHI) comprises a group of genetic disorders characterized by recurrent episodes of hyperinsulinemic hypoglycemia due to unregulated insulin secretion from pancreatic beta cells (Arnoux et al., Orphanet Journal of Rare Diseases 6:63 (2011); Yorifuji T., Ann Pediatr Endocrinol Metabolism 6:64 (2011); 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 two years of life. Histopathologically, CHI can present in diffuse or focal forms. In the diffuse form, all pancreatic beta cells are affected, while in the focal form, lesions of abnormal beta cells are (usually) confined to small areas of the pancreas. The most common known causes of CHI are loss-of-function mutations in genes encoding SUR1 and Kir6.2, which are subunits of the ATP-sensitive potassium channel (KATP channel) and are involved in insulin secretion in pancreatic beta cells.

[0138] There are currently only a few treatments for persistent congenital hyperinsulinemia (CHI) [Arnoux J. et al., Journal of Orphan Drugs for Rare Diseases 6:63 (2011); Yorifuji T., Annals of Pediatric Endocrinology and Metabolism, 19:57-68 (2014)]. The KATP channel activator diazoxide inhibits insulin secretion in pancreatic β cells. The most common adverse effect is excessive hair growth (hirsutism). Other side effects include sodium and fluid retention, which may induce congestive heart failure. Diazoxide is generally ineffective for those patients with CHI caused by KATP channel mutations, one of the most common causes of CHI. Octreotide is a somatostatin analog that inhibits insulin release. Although not approved for CHI, octreotide is used for CHI that is unresponsive to diazoxide. Due to its short half-life (1 to 2 hours), octreotide is administered subcutaneously (SC) or intravenously (IV) as multiple daily injections or continuously with a pump. Common adverse events include gastrointestinal symptoms and gallbladder complications. Partial pancreatectomy is the option for focal lesions and is curative in most cases. However, lesions are not always visible or palpable at the site specified by preoperative imaging. Patients with diffuse forms are primarily treated with continuous glucose feeds or off-label medications. Near-total pancreatectomy has been considered as a treatment but is characterized by a high risk of diabetes.

[0139] Iatrogenic hypoglycemia describes the symptoms and effects of hypoglycemia caused by the administration of excessive insulin or its analogs or drugs that stimulate endogenous insulin secretion. Iatrogenic hypoglycemia, which is essentially but not exclusively a result of treatment with insulin secretagogues or insulin, is a major limiting factor in the glycemic management of diabetes. Iatrogenic hypoglycemia causes recurrent episodes in most people with T1DM and many people with advanced T2DM, and is sometimes fatal. Repeated episodes of hypoglycemia impair the body's defenses against subsequent declines in plasma glucose concentrations, and thus lead to a vicious cycle of recurrent hypoglycemia.

[0140] Hypoglycemia causes a variety of symptoms, including: lack of coordination, confusion, loss of consciousness, seizures, and even death.

[0141] Most of the mild hypoglycemia episodes can be effectively self-treated by taking glucose lozenges or other beverages or snacks containing carbohydrates. More serious symptomatic hypoglycemia can also be treated with oral intake of carbohydrates. However, when hypoglycemic patients cannot take oral glucose supplements due to confusion, unconsciousness or other reasons, parenteral therapy is needed. As a non-hospital rescue procedure, injection of high blood sugar hormone, glucagon, is sometimes adopted, and the injection is carried out subcutaneously or intramuscularly by the patient himself or the companion of the patient who has been trained to identify and treat severe hypoglycemia. In the medical environment, intravenous glucose is a standard parenteral therapy.

[0142] Administration and dosing

[0143] The present disclosure also contemplates methods of administering the antibody compositions as described herein to treat the hyperinsulinemia disorders described herein.

[0144] The method of the present disclosure is performed using any medically acceptable way of directly or indirectly introducing therapeutic agents into mammalian subjects, including but not limited to injection, infusion, oral ingestion, intranasal, topical, percutaneous, parenteral, inhalation spray, vaginal or rectal administration. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular and intracisternal injections, as well as catheter or infusion techniques. It is also envisioned that it is administered by intradermal, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and or in a specific site surgical implantation. Suitable delivery devices can include those developed for insulin delivery (see, for example, Owens et al. Diabetic Med. 20 (11): 886-898, 2003; US20140128803; and Peyser et al., Annals NY Acad Sci, 1311: 102-123, 2014).

[0145] Antibody compositions as described herein can be administered every day, every 2 days, every 3 days, every week, every 2 weeks, every 3 weeks, twice a month, every month, every 2 months, every 3 months or every 6 months. In various embodiments, the composition is administered for a period of at least 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 1 year or more.

[0146] In one embodiment, administration is performed at a site in need of treatment by direct injection into the affected tissue location or by a sustained delivery or sustained release mechanism that can deliver the formulation internally. For example, biodegradable microspheres or capsules or other biodegradable polymer configurations capable of sustained delivery of a composition (e.g., a soluble polypeptide, antibody, or small molecule) may be included in the formulations useful for the present disclosure implanted at the site.

[0147] Therapeutic composition can also be delivered to the patient at multiple positions. Multiple administrations can be performed simultaneously or can be administered over a period of time. In some cases, it is useful to provide a continuous flow of therapeutic composition. Other therapies may be administered on a time period basis, for example, hourly, daily, weekly, every 2 weeks, every 3 weeks or monthly.

[0148] The amount of the antibody composition in a given dose will vary depending on the size of the individual to whom the therapy is to be administered and the nature of the condition being treated. In exemplary treatments, it may be necessary to administer from about 0.1 mg / kg to about 25 mg / kg, or from about 0.05 mg / kg to about 10 mg / kg, from about 0.3 mg / kg to about 6 mg / kg, or from about 0.1 mg / kg to about 3 mg / kg per dose or per day. Exemplary dosages 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 dosages 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.

[0149] The administration of multiple agents is also contemplated in the methods of the invention, such as the combination of an antibody composition described herein and a second agent described herein. Compositions comprising antibodies described herein can be administered to a human or mammal suffering from or susceptible to a condition or disorder to be treated associated with a target polypeptide.

[0150] Concurrent administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is overlap during the time period during which the agents exert their therapeutic effects. Concurrent or sequential administration, such as administration on different days or weeks, is contemplated.

[0151] The second agent can be other therapeutic agents, such as antidiabetic agents, cytokines, growth factors, other anti-inflammatory agents, anticoagulants, agents that lower / reduce blood pressure, agents that lower cholesterol, triglycerides, LDL, VLDL or lipoprotein (a) or increase HDL, agents that increase or decrease the level of cholesterol regulating proteins, anti-tumor drugs or molecules.

[0152] Exemplary agents include, but are not limited to, insulin, glucagon, acarbose, octreotide, verapamil, diazoxide, and other agents that can be used to treat hypoglycemia or the side effects associated with hypoglycemia. In various embodiments, the antibodies described herein are optionally administered in a delivery device, such as an intelligent delivery device (see, e.g., US20140128803) or dual sensors / pumps (e.g., bionic pancreatic systems) together with insulin and / or glucagon.

[0153] Any of the foregoing antibodies or fragments thereof described herein may be administered concurrently with one or more second agents as an adjunct therapy, wherein the second agent is an anti-diabetic agent known in the art or described herein.

[0154] Many antidiabetic agents are known in the art, including, but not limited to, sulfonylureas (e.g., glimepiride, glisentide, sulfonylureas, AY31637); biguanides (e.g., metformin); α-glucosidase inhibitors (e.g., acarbose, miglitol); thiazolidinediones (e.g., troglitazone, pioglitazone, rosiglitazone, glipizide, balaglitazone, rivoglitazone, netoglitazone, troglitazone, englitazone, AD 5075, T 174, YM268, R 102380, NC 2100, NIP 223, NIP 221, MK 0767, ciglitazone, adaglitazone, CLX 0921, darglitazone, CP 92768, BM 152054); glucagon-like peptide (GLP) and GLP-1 receptor GLP analogs or agonists (e.g., exendin) or stabilizers thereof (e.g., DPP4 inhibitors such as sitagliptin); insulin or its analogs or mimetics (e.g., lispro, aspart, glulisine, detemir, degludec, glargine, ); and sodium-glucose co-transporter-2 (SGLT2) inhibitors (e.g., canagliflozin, dapagliflozin, and empagliflozin).

[0155] It is contemplated that the antibody and the second agent can be administered simultaneously in the same formulation. It is further contemplated that the agents are administered in separate formulations and administered simultaneously, wherein simultaneously means that the agents are administered within 30 minutes of each other.

[0156] On the other hand, the second agent is administered before the antibody composition is administered. Pre-administration refers to the administration of the second agent within a week before the antibody treatment, at most 30 minutes before the administration of the antibody. It is further envisioned that the second agent is administered after the antibody composition is administered. Subsequent administration is intended to describe administration from 30 minutes after the antibody treatment to at most one week after the administration of the antibody.

[0157] It is further contemplated that other adjunctive therapies may be administered where appropriate. For example, a diet or food regimen designed for hypoglycemic patients, surgical therapy, or radiotherapy may also be administered to the patient where appropriate.

[0158] It will also be apparent that dosages may be altered if conventional therapy is administered in conjunction with the antibody formulations described herein.

[0159] Reagent test kit

[0160] The disclosure also provides a kit, which includes compositions described herein and package inserts, package labels, instructions or other labels that instruct or disclose any method or embodiment disclosed herein. In certain embodiments, the disclosure provides a kit for producing a single-dose administration unit. In certain embodiments of the disclosure, a kit containing a single-chamber and multi-chamber pre-filled syringe (e.g., a liquid syringe) is included.

[0161] Further aspects and details of the present disclosure will become apparent from the following examples, which are intended to be illustrative rather than limiting.

[0162] Examples

[0163] Example 1 - Formulation of RZ358 Antibody

[0164] RZ358 is a fully human IgG2 monoclonal antibody (mAb) that binds to the insulin receptor (INSR) with high affinity. The molecular weight of the RZ 358 antibody is approximately 149 kD and has a pI at pH 7.6. The development of the formulation of RZ358 for in vivo administration and stability studies and the selection of a unique combination of GRAS excipients approved for parenteral administration are described herein, with the aim of stabilizing RZ 358 in liquid storage at 2-8°C for up to 2 years. Initially, a pH that minimizes aggregation and optimizes conformational stability was selected.

[0165] method

[0166] Buffer exchange: use Buffer exchange was performed using an ultrafiltration device (PES membrane, 30k MWCO, Sartorius Stedim). Concentrations were determined by UV absorption measurements at 280 nm with an extinction coefficient of 1.47 cm 2 / mg. The concentration-adjusted solution was filtered using a syringe-driven filter unit (Millex-MP, PES, 0.22 μm, Millipore). The filtered solution was filled into suitable containers and placed under different stress conditions for stability studies.

[0167] Mechanical stress: Fill 2cc glass vials with RZ 358 solution to half capacity (1.5 ml per vial) and seal with a rubber stopper. Place the vial horizontally on R and shake at 1,000 RPM / 25°C for up to 1 week or until visual changes are observed. Freeze-thaw: Place the filled sample into a -70°C freezer for at least 2 hours and then place on the lab bench until completely thawed. Repeat this cycle 5 times.

[0168] Thermal stress: RZ 358 samples were placed at 5°C, 30°C, and 40°C and pulled at specific time points (usually 2 weeks, 1 month, 2 months, 3 months, and 6 months) for analysis.

[0169] Differential Scanning Calorimetry (DSC): DSC analysis was performed with a TA Instruments NanoDSC. The capillary cell was rinsed thoroughly with water and sample buffer. The sample / buffer was degassed with a MicroCal ThermoVac sample degassing and thermostat unit at 18°C ​​for at least 5 minutes before loading into the capillary cell. The sample / buffer was scanned from 15°C to 95°C at a scan rate of 1°C / min.

[0170] Subvisible particulate matter analysis: The analysis was performed using a Z2 Coulter particle counter and particle size analyzer. The 100μ aperture probe was calibrated with the "CC L10 size standard", and the counting accuracy was checked with the "concentration control" provided by Coulter. Before transferring 0.8 ml of the sample solution into a clean "3cc" glass vial (capacity of approximately 4.1 mL) containing 3.2 mL of 0.2M NaCl solution, the sample solution was gently swirled and shaken to ensure that the solution was uniform. Each sample was measured at least 2 times. The lower and upper thresholds were set to 10μ and 25μ, respectively.

[0171] Dynamic light scattering (DLS): DLS analysis was performed using a plate reader (Wyatt Technologies, Santa Barbara, CA). Samples (30 μL each) were loaded into 384-well untreated assay plates (Corning, catalog number 3540, polystyrene, black, clear flat bottom). The instrument temperature was set to 25°C.

[0172] All HPLC analyses were performed using an Agilent 1100 or 1200 system. Data acquisition and analysis were performed using ChemStation software (version B.03.02). UV detection was performed using wavelengths of 280 nm and 214 nm.

[0173] WCX-HPLC: Using Dionex PROPAC TM WCX10 post (4x 250mm) carries out weak cation exchange HPLC.Use pH gradient elution through the sample of injection.Buffer A is 15mM NaH2PO4, and buffer B is 15mM Na2HPO4.The percentage of buffer B increases from 25% at the beginning to 95% at 30 minutes.Post is washed 5 minutes under 95%B, and then is reduced to 25%B and balances 15 minutes before carrying out new injection.Flow velocity remains on 1 ml / min.Under the UV wavelength of 280nm and 214nm, monitor the elution peak.280nm signal is used for integration and quantitative calculation.

[0174] SEC-HPLC: SEC-HPLC was performed using a Tosoh TSK gel SWxl G3000 7.8 x 300 mm column. The injected sample was eluted isocratically with 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 minutes. The peak was detected at 280 nm.

[0175] HIC-HPLC: The Dionex PROPAC TM Hydrophobic interaction chromatography was performed on a HIC-10 (5 μm, 4.6 x 100 mm) column. Buffer A was 0.5 M ammonium sulfate, 50 mM sodium phosphate at pH 7, and buffer B was 50 mM sodium phosphate containing 10% (v / v) acetonitrile at pH 7. The flow rate was 1 ml / min and the run time was 30 (45) min with the following gradient:

[0176] Time, minutes 0 2 25 33 33.1 45 B％ 0 0 100 100 0 0

[0177] To understand the effect of formulation pH on aggregate formation, RZ358 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.

[0178] Conformational stability was assessed by DSC. At acidic pH 4, RZ 358 showed a lower temperature transition at 50.7 °C in the above excipients due to the unfolding of the more flexible CH2 domain and the main thermal transition of the Fab at 64.8 °C ( Figure 1 ). As the pH of the formulation increases, the CH2 transition temperature increases rapidly and merges into the main Fab transition peak at pH 5.5 and above. Once the formulation pH is above 6, the Fab unfolding temperature reaches a maximum. The DSC data indicate that RZ 358 is more stable in conformation when the solution pH is about pH 6 or above.

[0179] RZ 358 was subjected to five freeze-thaw cycles and stored at 40°C. After freeze-thaw, RZ 358 formed soluble dimers ( Figure 2 , upper figure), and at neutral pH conditions form high molecular weight (HMW) species ( Figure 2 , bottom). After freeze-thaw, it showed better stability in the pH range of 5.5 to 6.3.

[0180] RZ 358 was also incubated at 40°C for up to 3 months. At pH 4.0, RZ 358 formed soluble aggregates, as evidenced by the loss of signal detected by SEC-HPLC at 2 weeks and the appearance of a gel-like precipitate after 2 weeks of storage. As the solution pH increased to 5.0 and above, the formation of fragmented degradation products became dominant ( Figure 3 , upper panel). At 3 months, the aggregation levels of the 40°C stability samples subjected to stress (pH 5 to pH 7) showed no significant differences.

[0181] As detected by SEC-HPLC, RZ 358 fragmentation occurs when the pH shifts from pH 5.5 to both lower (pH 5) and higher pH (7.0). As measured by the degradation pattern from HIC-HPLC, RZ 358 oxidation ( Figure 3 , lower panel) showed a similar trend, with the pH 5.5 formulation showing the least amount of major pre-peak.

[0182] pH studies indicated that the optimal pH for controlling RZ 358 aggregation / fragmentation / oxidation is approximately pH 5.5.

[0183] Additional 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 and 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. The samples were incubated at 40 ° C for one month and at 30 ° C for 3 months, and the distribution of charge variants was analyzed by WCX-HPLC. The higher the pH of the sample, the faster the acidic species grew. Under slightly acidic conditions, alkaline species increased ( Figure 4 ).

[0184] A slightly acidic pH is more suitable for controlling acidic species, and the optimal pH for controlling the formation of basic species is about pH 5.8 ( Figure 5 The pH 5.8 formulation was selected to minimize fragmentation ( Figure 6 , above), oxidation ( Figure 6, below) and clustering ( Figure 6 , top). Notably, the lower pH formulation produced more species, as indicated by the back shoulder of the main HIC peak.

[0185] Example 2 - Buffer Analysis of RZ358 Formulations

[0186] Surfactant can enhance the physical stability during freeze-thaw and stirring. Experiments were conducted to determine the surfactant providing sufficient stability for RZ358 during freeze-thaw. RZ 358 was deployed to 10mM L-histidine, 270mM sorbitol containing 0, 0.002%, 0.005%, 0.01% and 0.03% polysorbate 20 at pH 5.9 with 2.2mg / mL. Every kind of deployed solution of RZ358 was filled into 2cc Schott (Schott) glass vials with 1.5mL and sealed with West 4432 / 50 rubber stopper. The filling volume of 1.5mL is half of the maximum capacity of 2cc glass vials and is selected for maximizing air / liquid surface.

[0187] The vials filled with RZ 358 were exposed to agitation stress by shaking at 1000 RPM for up to 8 days at room temperature. In the absence of PS20, the solution became turbid within less than 3 days of shaking, while in the presence of 0.002% or more PS20, the solution remained clear for 8 days. After shaking for 3 days in the absence of PS20, SE-HPLC detected less than 2% RZ358. If the PS20 concentration was 0.002% or 0.005%, the soluble aggregates did increase with shaking time ( Figure 7 At 0.01% PS20 or higher, soluble aggregates showed minimal growth ( Figure 7 ).

[0188] In the absence of surfactant, freeze-thaw produced a large number of visible particles with a size less than 25 μm and very few visible particles with a diameter greater than 25 μm. When the PS20 concentration was 0.002% or higher, the sub-visible particle counts of ≤10 μm were significantly reduced. No significant effect of PS20 on particles ≤25 μm was observed ( Figure 8 ). The minimum effective PS20 concentration to prevent freeze-thaw induced particle formation appears to be 0.002%. In order to mitigate agitation and freeze-thaw induced aggregation, a PS20 concentration of 0.01% was selected for the formulation.

[0189] During purification and storage of protein drug products, oxidation of amino acid side chains may lead to instability and decomposition of the therapeutic over time. RZ 358 oxidation control uses scavengers (sugar alcohols) and free radical sinks (methionine) in the formulation. Mannitol and sorbitol were tested for their effectiveness in minimizing RZ 358 oxidation in the presence of methionine, as tested by HIC.

[0190] RZ 358 showed better conformational stability in the sugar alcohol / methionine formulation, as indicated by a higher thermal unfolding temperature in the sugar alcohol / methionine formulation than in the arginine formulation ( Fig. 9 ). There appeared to be no significant difference between the mannitol and sorbitol formulations in terms of thermal unfolding. The oxidation rate was significantly reduced in the sugar alcohol / methionine formulation compared to the arginine formulation ( Fig.10 and Fig.11 , lower figure). Both sugar alcohols showed almost the same effectiveness in controlling oxidation. Consistent with the better conformational stability as indicated by the DSC data, both sugar alcohol formulations showed lower aggregation and lower fragmentation in the stressed temperature storage stability samples, as demonstrated by the SEC-HPLC data ( Fig.11 , top). Sorbitol was chosen as the redox excipient because freeze-thaw cycles lead to precipitation of mannitol.

[0191] The effect of different ratios of sorbitol relative to arginine concentration on the stability of RZ 358 was evaluated in 10 mM L-histidine and 10 mM L-methionine at pH 6. RZ 358 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. Based on stability data from samples incubated at 40°C for 2 months, higher sorbitol concentrations were more effective in controlling oxidation, aggregation, and fragmentation ( Fig.12 ).

[0192] The effect of different methionine concentrations on the stability of RZ 358 was also studied at a concentration of 5 mg / mL RZ 358 in 10 mM L-histidine, 270 mM sorbitol containing 0, 2.5 mM, 5 mM, 7.5 mM, and 10 mM L-methionine at pH 6. The formulated samples were stored at 40°C for up to 3 months. The accelerated stability data indicated that higher methionine concentrations resulted in lower RZ 358 oxidation ( Fig.13 , above) and clustering ( Fig.13 , figure below).

[0193] Example 3 - Formulation of RZ358 with higher antibody concentration

[0194] Formulations of RZ358 at higher concentrations were tested and long-term stability and efficacy were evaluated. The 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. The analysis showed that 2 experimental batches remained stable between pH 5.4 and pH 6.3 for up to 60 months ( Fig.14A ). Under the same storage conditions, the protein concentration is also stable between 70-90 mg / kg ( Fig. 14B ). Osmotic pressure measurements of the same batch over the same time frame showed that the osmotic pressure stabilized at approximately 320-340 mOsm / kg and could stabilize between 286 mOsm / kg and 366 mOsm / kg.

[0195] The light chain % and heavy chain % of the antibody batches after 60 months storage at 2°C-8°C were assessed by CE-SDS (reduced). The amount of light chain ranged from 27% to 36%, while the amount of heavy chain was within the accepted criteria of 62% to 71%.

[0196] Impurities and degradation products of the antibody formulations were measured 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 batches tested during the storage time ( Fig.15A Impurity analysis by non-reducing CE-SDS showed less than 9% impurities during storage ( Fig. 15B Reducing CE-SDS identified the majority of detectable antibodies in the main peak, within the 90%-100% acceptance criteria ( Fig.16A ).

[0197] Cation exchange chromatography analysis showed that antibodies were detected in the main peak (about 40%-50% M1 and about 30% M2) ( Fig. 16B , 16C ), and the acidic peak fraction (less than 20%) ( Fig.17A ) and basic peak fraction (less than 22%) ( Fig. 17B ) is a subset of proteins detectable in .

[0198] The monomeric species, high molecular weight species, low molecular weight species, and oxidized species of the 80 mg / ml RZ358 formulation were also determined. SE-HPLC detection of monomeric species showed that more than 95% of the antibody was in monomeric form ( Fig.18A ), within the 90% acceptance criteria. Additionally, less than 1.5% appeared to be high molecular weight species ( Fig.18B), while little or no low molecular weight species were detected ( Fig. 18C Detection of oxidized species by HIC-HPLC showed that less than 15% of the species were oxidized during storage ( Fig.18D ).

[0199] The efficacy of the stored material is also determined using a cell-based efficacy assay. In short, the bioassay measures the dose-dependent inhibition of phosphorylated AKT by RZ 358 using CHO-K1 hulNS cells engineered to express human insulin receptor. First, CHO-K1 hulNS cells are insulin starved overnight. After starvation, cells are exposed to dose-dependent levels of RZ 358 and then stimulated with insulin. Cells are lysed and then phosphorylated AKT and total Akt are quantified by sandwich immunoassays, which utilize MSD plates pre-coated with capture antibodies of phospho-AKT (Ser 473) and total Akt. Plates are read and the data of reference standards, test samples and controls are fitted to a 4-parameter curve fitting model. The final results are reported as relative efficacy % relative to the reference standard. Fig.19 Stored RZ 358 was shown to maintain good potency over time, with relative potencies of approximately 80%-120% measured over a 60 month storage period.

[0200] The number of particles formed over time was also measured across storage conditions. Almost no particles were detected in the 25 μM RZ 358 batch or the 10 μM R358 batch ( Fig. 20 ).

[0201] Evaluation of surfactant levels relative to formulation stability: RZ 358 was formulated at 100 mg / mL into 10 mM L-histidine, 10 mM methionine, 270 mM sorbitol, pH 5.8, containing 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 into 2 cc Schott glass vials at 1.3 mL and sealed with West 4110 / 40 rubber stoppers.

[0202] By shaking at room temperature at 300RPM for up to 48 hours, then shaking at 1000RPM for one hour, the vial filled with RZ358 is exposed to stirring stress. In the independent group of this study, the sample is exposed to 5 freeze-thaw cycles from -20°C to room temperature. The solution is analyzed by appearance and the presence of sub-visible particles is analyzed by HIAC. The study found that the presence of PS20 in the RZ358 preparation at a concentration of 0.004% and above alleviates the turbidity and visible particle formation induced by stirring and freeze-thaw stress, while at T = 0, the presence of PS20 at a level of 0.002% and above in the RZ358 sample that has not been subjected to stress controls the formation of visible particles (Table 1). Under current experimental conditions, all RZ358 preparations examined, whether or not there is stress, have passed the sub-visible particle USP threshold, however, in both T = 0 and the samples subjected to stress, in the presence of any level of polysorbate, the particle count is significantly reduced (Table 1).

[0203] Table 1. Changes in RZ358 appearance and subvisible particle counts with stirring and freeze-thaw stress

[0204]

[0205]

[0206] Preferred embodiments of the present disclosure are described herein. After reading the foregoing description, the variations of those preferred embodiments may become apparent to those of ordinary skill in the art. The inventors anticipate that the skilled person may adopt such variations when appropriate, and the inventors intend that the present disclosure be practiced in other ways different from the ways specifically described herein. Therefore, where permitted by applicable law, the present disclosure includes all modifications and equivalents of the subject matter recited in the appended claims. In addition, unless otherwise indicated herein or otherwise clearly contradictory to the context, the present disclosure encompasses any combination of the above-mentioned elements in all possible variations thereof.

Claims

1. A composition comprising an antibody that specifically binds to an 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 shown in SEQ ID NO: 6; (ii) a light chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO: 7; and (iii) a light chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO: 8; and (B) a heavy chain variable domain, comprising: (i) a heavy chain CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO:3; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO:4; and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO:

5.

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

3. The composition of claim 1 or 2, wherein the amino acid is histidine.

4. The composition of claim 2 or 3, wherein the concentration of histidine is about 4 mM to about 25 mM or about 10 mM to about 20 mM.

5. The composition according to any one of claims 2 to 4, wherein the concentration of histidine is about 4±1 mM.

6. The composition according to claim 1 or 2, wherein the amino acid is histidine HCL.

7. The composition of claim 2 or 6, wherein the concentration of the histidine HCL is about 4 mM to about 25 mM or about 10 mM to about 20 mM.

8. The composition of claim 2, 6 or 7, wherein the concentration of histidine HCL is about 6±1 mM.

9. The composition of claim 1 or 2, wherein the amino acid is methionine.

10. The composition of claim 2 or 9, wherein the concentration of methionine is about 4 mM to about 25 mM or about 10 mM to about 20 mM.

11. The composition of claim 2, 9 or 10, wherein the concentration of methionine is about 10±2 mM.

12. The composition according to any one of claims 1 to 11, wherein the surfactant is a polysorbate.

13. The composition of claim 12, wherein the polysorbate is polysorbate 20, polysorbate 80, or a mixture thereof.

14. The composition of any one of claims 1 to 13, comprising the surfactant at a concentration of about 0.002% (w / v) to about 0.02% (w / v).

15. The composition of claim 14, comprising about 0.005% (w / v), 0.010% (w / v), 0.015% (w / v) or 0.02% (w / v) surfactant.

16. The composition according to any one of claims 1 to 15, comprising about 0.01% (w / v) ± 0.0025% (w / v) surfactant, optionally wherein the surfactant is polysorbate 20 or polysorbate 80 or a mixture thereof.

17. The composition according to any one of claims 1 to 16, wherein the sugar alcohol is selected from the group consisting of sucrose, sorbitol and mannitol.

18. The composition according to any one of claims 1 to 17, wherein the sugar alcohol is sorbitol.

19. The composition of claim 18, wherein the concentration of sorbitol is from about 100 mM to about 350 mM or from about 200 mM to about 300 mM.

20. The composition of claim 18 or 19, wherein the concentration of sorbitol is about 270±30 mM.

21. The composition of any one of claims 1 to 17, wherein the sugar alcohol is mannitol.

22. The composition of claim 21, wherein the concentration of mannitol is about 100 mM to about 350 mM or about 200 mM to about 300 mM.

23. The composition of claim 21 or 22, wherein the concentration of mannitol is about 270±30 mM.

24. The composition of any one of claims 1 to 23, wherein the anti-INSR antibody comprises: (A) a light chain variable domain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2; or (B) a heavy chain variable domain comprising an amino acid sequence 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).

25. The composition of any one of claims 1 to 24, wherein 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.

26. The composition of any one of claims 1 to 25, wherein the anti-INSR antibody is an IgG2 antibody.

27. The composition of any one of claims 1 to 26, wherein the anti-INSR antibody is present in the composition at a concentration of about 20 mg / ml to about 200 mg / mL.

28. The composition of claim 27, wherein the anti-INSR antibody is present in the composition at a concentration of about 50 mg / ml to about 150 mg / mL.

29. The composition of claim 28, wherein the anti-INSR antibody is present in the composition at a concentration of about 80 mg / mL to about 120 mg / mL.

30. The composition of any one of claims 1 to 29, wherein the composition is a liquid.

31. The composition of any one of claims 1 to 30, wherein the pH is less than about 6.

5.

32. The composition of claim 31, wherein the pH is from about 5.0 to about 6.

33. The composition of claim 32, wherein the pH is from about 5.5 to about 5.

9.

34. The composition of claim 33, wherein the pH is about 5.

8.

35. A composition according to any one of claims 30 to 34, characterised in that The viscosity at 25°C is about 2 cP to about 10 cP, wherein the concentration of the anti-INSR antibody is about 100 mg / ml or less.

36. The composition of any one of claims 30 to 35, wherein the composition is isotonic or has an osmotic pressure within 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.

37. The method of any of the preceding claims, wherein the composition comprises less than about 10% impurities after storage at 2°C to 8°C for about 24 months to about 36 months as determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis.

38. The composition of any of the preceding claims, wherein less than 10% of the antibody is degraded after storage at 2°C to 8°C for about 24 months to about 36 months as determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis.

39. The composition of any of the preceding claims, wherein after storage at 40°C for about 3 months, less than 15% of the antibody is oxidized or aggregated as determined by hydrophobic interaction chromatography (HIC) or size exclusion chromatography (SEC).

40. The composition of any of the preceding claims, wherein the composition comprises less than about 10% degradation products after storage at 2°C to 8°C for about 24 months to about 36 months as determined by reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis.

41. The composition of any of the preceding claims, wherein less than 30% of the antibody is detected in the acidic peak after storage at 2°C to 8°C for about 24 months to about 36 months as determined by cIEX-UHPLC analysis.

42. The composition of claim 41, wherein approximately 8%-20% of the antibody is detected in the acidic peak after storage at 2°C to 8°C for about 24 months to about 36 months as determined by cIEX-UHPLC analysis.

43. The composition of any of the preceding claims, wherein less than 22% of the antibody is detected in the basic peak after storage at 2°C to 8°C for about 24 months to about 36 months as determined by cIEX-UHPLC analysis.

44. The composition of claim 43, wherein about 5%-20% of the antibody is detected in the basic peak after storage at 2°C to 8°C for about 24 months to about 36 months as determined by cIEX-UHPLC analysis.

45. The composition of any of the preceding claims, wherein the composition comprises less than 5% high molecular weight species as determined by SE-UHPLC after storage at 2°C to 8°C for about 24 months to about 36 months.

46. ​​The composition of any of the preceding claims, wherein the composition comprises less than 5% low molecular weight species as determined by SE-UHPLC after storage at 2°C to 8°C for about 24 months to about 36 months.

47. The composition of any of the preceding claims, wherein after storage at 2°C to 8°C for about 24 months to about 36 months, the composition comprises less than 18% of the antibody in oxidized form as determined by HIC-HPLC.

48. The composition of any of the preceding claims, wherein the potency of the antibody composition is at least about 75% to about 120% after storage at 2°C to 8°C for about 24 months to about 36 months as determined by a bioassay.

49. 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, wherein the pH of the composition is about 5.

8.

50. An article comprising the composition of any preceding claim, optionally comprising from about 0.5 mL to about 5 mL or from about 1 mL to about 3 mL of the composition.

51. A method for treating a condition associated with hyperinsulinemia or excess insulin signaling in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 1 to 49.

52. The method of claim 51, wherein the condition is selected from the group consisting of hypoglycemia, insulin sensitivity, cancer, insulinoma, Kaposi's sarcoma, insulin excess, islet cell proliferation disorders (KATP-HI diffuse disease, KATP-HI focal disease or "PHHI"), GDH-HI (hyperinsulinemia / hyperammonemia syndrome (HI / HA), leucine-sensitive hypoglycemia, diazoxide-sensitive hypoglycemia, islet cell dysregulation syndrome, idiopathic hypoglycemia of infancy, persistent hyperinsulinemic hypoglycemia of infancy (PHHI), congenital hyperinsulinemia, acute hypoglycemia due to renal failure, chronic hypoglycemia due to renal failure, and hypoglycemia due to chronic kidney disease.

53. The method of claim 51 or 52, wherein the disease is congenital hyperinsulinemia.

54. The method of any one of claims 51 to 53, wherein 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.

55. The method of any one of claims 51 to 54, wherein the composition is administered intravenously.

56. The method of any one of claims 51 to 54, wherein the composition is administered subcutaneously.

57. A composition according to any one of claims 1 to 49 for use in treating a condition associated with hyperinsulinemia or excess insulin signalling in a subject in need thereof.

58. The composition of claim 57, wherein the condition is selected from the group consisting of hypoglycemia, insulin sensitivity, cancer, insulinoma, Kaposi's sarcoma, insulin excess, islet cell proliferation disorders (KATP-HI diffuse disease, KATP-HI focal disease or "PHHI"), GDH-HI (hyperinsulinemia / hyperammonemia syndrome (HI / HA), leucine-sensitive hypoglycemia, diazoxide-sensitive hypoglycemia, islet cell dysregulation syndrome, idiopathic hypoglycemia of infancy, persistent hyperinsulinemic hypoglycemia of infancy (PHHI), congenital hyperinsulinemia, acute hypoglycemia due to renal failure, chronic hypoglycemia due to renal failure, and hypoglycemia due to chronic kidney disease.

59. The composition of claim 57 or 58, wherein the disease is congenital hyperinsulinemia.

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