Intranasal formulation of foralumab

Abstract

The present invention relates to therapeutic, diagnostic and / or prophylactic formulations and dosages and regimens of anti-CD3 antibodies, as well as methods of using such formulations and dosages and regimens.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 341,856, filed May 13, 2022, which is incorporated by reference in its entirety.

[0002] Incorporation by reference of sequence listing The Sequence Listing associated with this application has been provided electronically in XML format and is incorporated herein by reference. The XML file containing the Sequence Listing XML is named "TIZI-033_001WO_SeqList_ST26". The XML file is 10,714 bytes, was created on May 15, 2021, and has been submitted electronically via the USPTO Patent Center.

[0003] The present invention relates to intranasal formulations, dosages, and administration regimens of the anti-CD3 antibody foralamuab. [Background technology]

[0004] Antibodies against the CD3 epsilon signaling molecule of the T cell receptor complex have been demonstrated to be useful as immunosuppressants and in the treatment of autoimmune disorders, thereby providing improved methods for preparing anti-CD3 antibodies, methods for purifying anti-CD3 antibodies, and pharmaceutical formulations containing anti-CD3 antibodies. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure provides formulations, dosages, and dosing regimens for monoclonal antibodies specifically directed against CD3. The formulations of the present disclosure include the anti-CD3 antibody, foralumab. The anti-CD3 antibody formulations are intranasal formulations. [Means for solving the problem]

[0006] In some embodiments, the anti-CD3 antibody of the formulations of the disclosure comprises a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO:1), a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO:3), a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO:4), a light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO:5), a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence DASNRSAT (SEQ ID NO:6), and a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO:7).

[0007] In some embodiments, the anti-CD3 antibody of the formulations of the disclosure comprises a variable heavy chain (VH) amino acid sequence comprising the amino acid sequence of SEQ ID NO: 8, and a variable light chain (VL) amino acid sequence comprising the amino acid sequence of SEQ ID NO: 9. In other aspects, the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10, and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 11.

[0008] Provided by the disclosure is a formulation for nasal delivery comprising 0.25 mg / mL or 0.5 mg / mL foralaumab, 3.4 mg / mL sodium acetate, 0.20 mg / mL polysorbate 80, and 7.31 mg / mL sodium chloride.

[0009] In another aspect, the disclosure provides a unit dose device for intranasal administration of foralumab. In some embodiments, the unit dose device comprises a nasal spray device and contains 0.25 mg / mL or 0.5 mg / mL foralumab, 3.4 mg / mL sodium acetate, 0.20 mg / mL polysorbate 80, and 7.31 mg / mL sodium chloride.

[0010] In some embodiments, the unit dose device is capable of delivering 100 μL of the formulation. In some embodiments, the nasal spray device is an Aptar UniDose device. In some embodiments, the nasal administration device delivers droplet sizes of the formulation of about 10 μm to 100 μm.

[0011] In some embodiments, the nasal spray device is a Gerresheimer device. In some embodiments, the nasal administration device delivers a droplet size of the formulation of about 25 μm to 250 μm.

[0012] In some embodiments, the concentration of foralumab in the formulation is within 3%, within 5%, or within 10% of the starting concentration after storage at about 2° C. to about 8° C. for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0013] In some embodiments, the formulation contains less than 1%, less than 1.5%, less than 2%, or less than 2.5% aggregation after storage at about 2° C. to about 8° C. for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0014] In some embodiments, the pH of the formulation remains within 0.1, 0.2, or 0.3 of the starting value after storage at about 2° C. to about 8° C. for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0015] In some embodiments, the potency of foralumab in a nasal anti-CD3 antibody formulation remains within 10%, 15%, 20%, 25%, 30%, or 35% of the initial potency after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0016] The present invention further provides a method of treating or alleviating symptoms of an autoimmune disease, an inflammatory disorder, a neurodegenerative disease, or cancer by administering to a subject in need thereof a formulation according to the present invention. For example, the present disclosure provides a method of treating or alleviating symptoms of multiple sclerosis or Alzheimer's disease.

[0017] Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present disclosure provides intranasal formulations and dosing of monoclonal antibodies, such as fully human monoclonal antibodies that specifically bind to the CD3 epsilon chain (CD3ε). Specifically, the present disclosure provides intranasal formulations of anti-CD3ε antibodies that are useful for targeting tissue-specific immune regulation. Unlike systemic (e.g., intravenous) administration of anti-CD3 antibodies, the formulations of the present invention are believed to minimize off-target immune suppression. The formulations provided herein are useful for treating or alleviating symptoms of autoimmune diseases, inflammatory disorders, neurodegenerative disorders, and cancer.

[0019] CD3 antibody The present disclosure provides formulations for the nasal delivery of forlamab, an antibody that specifically binds to the CD3 epsilon chain (CD3ε).

[0020] Foralumab comprises heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO:1), heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO:3), heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO:4), light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO:5), light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence DASNRSAT (SEQ ID NO:6), and light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO:7).

[0021] Foralumab comprises a variable heavy chain amino acid sequence that comprises QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS (SEQ ID NO: 8), and a variable light chain amino acid sequence that comprises EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK (SEQ ID NO: 9).

[0022] Foralumab is QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPEEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ and a variable light chain amino acid sequence comprising: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

[0023] Foralumab is also referred to herein as NI-0401 or 28F11-AE. (See, e.g., Dean Y, Depis F, Kosco-Vilbois M. "Combination therapies in the context of anti-CD3 antibodies for the treatment of autoimmune diseases." Swiss Med Wkly. (2012), the contents of which are incorporated by reference in their entireties.)

[0024] In some embodiments, the anti-CD3 antibody is a fully human antibody or a humanized antibody. In some embodiments, the anti-CD3 antibody formulation comprises a full-length anti-CD3 antibody. In alternative embodiments, the anti-CD3 antibody formulation comprises an antibody fragment that specifically binds to CD3. In some embodiments, the anti-CD3 antibody formulation comprises a combination of a full-length anti-CD3 antibody and an antigen-binding fragment that specifically binds to CD3.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof that binds to CD3 is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab') 2 fragments, scFv, SCab, dAb, single domain heavy chain antibodies, or single domain light chain antibodies. In some embodiments, the antibodies or antigen-binding fragments thereof that bind CD3 are murine, other rodent, chimeric, humanized, or fully human monoclonal antibodies.

[0026] Optionally, the anti-CD3 antibody or antigen-binding fragment thereof used in the formulations of the present disclosure comprises at least one amino acid mutation. Typically, the mutation is in the constant region. In some embodiments, the mutation results in an antibody with altered effector function. The effector function of the antibody is altered by modifying, i.e., enhancing or decreasing, the affinity of the antibody for an effector molecule, such as an Fc receptor or a complement component. For example, the mutation may result in an antibody capable of reducing cytokine release from T cells. In some embodiments, the mutation is in amino acid residues 234, 235, 265, or 297, or a combination thereof (numbered according to the Kabat numbering scheme for immunoglobulins), in the heavy chain. Preferably, the mutation results in an alanine residue at position 234, 235, 265, and / or 297, or a glutamic acid residue at position 235, or a combination thereof. Examples of mutations that may be present in the anti-CD3 antibody used in the formulations described herein include L 234 L 235 →A 234 E 235 ,L 234 L235 →A 234 A 235 ,L 235 →E 235 ,N 297 →A 297 , and D 265 →A 265 Examples include:

[0027] Preferably, the anti-CD3 antibodies used in the formulations provided herein contain one or more mutations that prevent heavy chain constant region-mediated release of one or more cytokines in vivo.

[0028] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof used in the formulations of the present disclosure are fully human antibodies. Fully human anti-CD3 antibodies used in the formulations provided herein include, for example, L 234 L 235 →A 234 E 235 Mutations may be included in the Fc region that significantly reduce or eliminate cytokine release upon exposure to anti-CD3 antibodies. 234 L 235 →A 234 E 235 The mutations appear to reduce or eliminate cytokine release when anti-CD3 antibodies are exposed to human leukocytes, whereas other mutations (L 234 L 235 →A 234 A 235 ,L 235 →E 235 ,N 297 →A 297 , and D 265 →A 265 Antibodies containing the L-terminal end of the Fc region are believed to reduce or eliminate cytokine release when exposed to human leukocytes. 234 L 235 →A 234 E 235 The release of cytokines upon exposure to anti-CD3 antibodies carrying the mutations was compared with that of antibodies carrying no mutations or one or more other mutations (L 234L 235 →A 234 A 235 ,L 235 →E 235 ,N 297 →A 297 , and D 265 →A 265 The level of cytokine release upon exposure to another anti-CD3 antibody having the same or similar properties can be determined by comparing it to the level of cytokine release upon exposure to another anti-CD3 antibody having the same or similar properties.

[0029] The term "cytokine" refers to all human cytokines known in the art that bind to extracellular receptors expressed on the cell surface and thereby regulate cellular function, including, but not limited to, IL-2, IFN-γ, TNF-α, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13.

[0030] The anti-CD3 antibody used in the formulations described herein may be of any class of immunoglobulin, i.e., the anti-CD3 antibody may be an IgG, IgM, IgA, IgE, or IgD antibody. The anti-CD3 antibody may also be of any subclass of immunoglobulin, e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the anti-CD3 antibody is an IgG1 antibody.

[0031] formulation The anti-CD3 antibody formulations of the present disclosure may be liquid. For example, the liquid formulation may be aqueous.

[0032] The anti-CD3 antibody formulations of the present disclosure may include one or more salts (buffer salts), one or more polyols, and / or one or more excipients. The formulations of the present disclosure may also contain a buffering agent and / or a preservative. The anti-CD3 antibody formulation may be buffered in a solution at a pH in the range of about 4-8, about 4-7, about 4-6, about 5-6, or about 5.5-6.5. In a preferred embodiment, the antibody formulation is buffered in a solution at a pH of about 5.5.

[0033] Examples of salts that may be present in the formulations provided herein include those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, etc. Such salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts. Examples of buffering agents that may be present in the formulations of the present disclosure include phosphate salts, citrate salts, acetate salts, and 2-(N-morpholino)ethanesulfonic acid (MES).

[0034] The formulations of the present invention may include a buffer system. As used in this application, the term "buffering agent" or "buffer system" refers to a compound that, usually in combination with at least one other compound, is capable of neutralizing, within limits, either an acid or a base (alkali) with relatively little or no change in the original pH.

[0035] The buffering agent that can be present in the formulation of the present disclosure includes borate buffer, phosphate buffer, calcium buffer, and combinations and mixtures thereof.The borate buffering agent that can be present in the formulation of the present disclosure includes, for example, boric acid and its salt, for example, sodium borate or potassium borate.The borate buffering agent also includes compounds that generate boric acid or its salt in solution, such as potassium tetraborate or potassium metaborate.

[0036] Phosphate buffer systems include one or more monobasic phosphates, dibasic phosphates, and the like. Particularly useful phosphate buffers are those selected from alkali metal and / or alkaline earth metal phosphates. Examples of suitable phosphate buffers that may be present in the formulations of the present disclosure include one or more of dibasic sodium phosphate (Na2HPO4), monobasic sodium phosphate (NaH2PO4) and monobasic potassium phosphate (KH2PO4). Phosphate buffer components may be used in amounts of 0.01% to 0.5% (w / v), calculated as phosphate ions.

[0037] Other known buffer compounds, including, for example, citrate, sodium bicarbonate, TRIS, etc., can be optionally added to the formulations of the present disclosure. Other ingredients in the formulation have other functions, but can also affect buffer capacity. For example, EDTA, which is often used as a complexing agent, can have a significant effect on the buffer capacity of a solution.

[0038] Preferred salts that may be used in the formulations of the present disclosure include sodium chloride, sodium acetate, sodium acetate trihydrate, and sodium citrate.

[0039] In some embodiments, the concentration of the salt in a formulation according to the present disclosure is from about 10 mM to about 500 mM, from about 25 nM to 250 mM, or from about 25 nM to 150 mM.

[0040] In some embodiments, sodium acetate trihydrate is present in the formulations provided herein at a concentration ranging from about 10 mM to about 100 mM. For example, sodium acetate trihydrate can be present in the formulations provided herein at a concentration of about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 9 mM5, or about 100 mM. In some embodiments, sodium acetate trihydrate is present in the formulations provided herein at a concentration of 25 mM.

[0041] In some embodiments, sodium chloride is present in the formulations provided herein at a concentration ranging from about 50 mM to about 500 mM. In some embodiments, sodium chloride is present in the formulations provided herein at a concentration of about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, about 475 mM, or about 500 mM. In some embodiments, sodium chloride is present in the formulations provided herein at a concentration of about 125 mM.

[0042] In some embodiments, sodium citrate is present in the formulations provided herein at a concentration ranging from about 10 mM to about 100 mM. In some embodiments, sodium citrate is present in the formulations provided herein at a concentration of about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. In some embodiments, sodium citrate is present in the formulations provided herein at a concentration ranging from about 25 mM to about 50 mM.

[0043] In some embodiments, the formulations provided herein comprise sodium acetate trihydrate at a concentration ranging from about 25 mM to about 100 mM, and sodium chloride at a concentration ranging from about 150 mM to about 500 mM, in some embodiments, the formulation comprises about 25 mM sodium acetate trihydrate and about 150 mM sodium chloride.

[0044] In some embodiments, the formulation comprises one or more polyols as bulking agents and / or stabilizing excipients. Polyols include, for example, trehalose, mannitol, maltose, lactose, sucrose, sorbitol, or glycerol. The polyols are present in a concentration ranging from about 0.1% to about 50% or from about 5% to about 25%. For example, the polyols may be present in the formulations provided herein at a concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

[0045] In some embodiments, the formulation includes one or more excipients and / or surfactants to inhibit or otherwise reduce antibody aggregation. Suitable excipients for reducing antibody aggregation include, for example, surfactants such as polysorbate 20 or polysorbate 80. In some embodiments, polysorbate 20 or polysorbate 80 is present at a concentration ranging from about 0.01% to 1% or from about 0.01% to about 0.05%. In some embodiments, polysorbate 20 or polysorbate 80 is present in the formulations provided herein at a concentration of about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1.0%.

[0046] In some embodiments, the surfactant is polysorbate 80 and is present in the formulations described herein at a concentration ranging from about 0.01% to about 0.05%. In some embodiments, polysorbate 80 is present in the formulations provided herein at a concentration of about 0.02%.

[0047] In some embodiments, the formulations provided herein include one or more excipients to improve the stability of the antibody. In some embodiments, the excipient to improve stability is human serum albumin. In some embodiments, the human serum albumin is present in the formulations provided herein at a concentration ranging from about 1 mg to about 5 mg.

[0048] Suitable amino acids that may be present in the formulations described herein include, for example, leucine, arginine, histidine, or combinations thereof.

[0049] Examples of antimicrobial agents that may be present in the formulations described herein include, for example, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride, cresol, chlorobutanol, dehydroacetic acid, ethylparaben, methylparaben, phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate, potassium sorbate, propylparaben, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid, thimersol, thymosin, or mixtures thereof.

[0050] Examples of antioxidants that may be present in the formulations described herein include, for example, ascorbic acid, BHA, BHT, EDTA, or mixtures thereof.

[0051] In some embodiments, the anti-CD 3 antibody formulation described herein is a nasal formulation. In some embodiments, the nasal anti-CD 3 antibody formulation is an aerosol formulation. In some embodiments, the nasal anti-CD 3 antibody formulation is suitable for once-daily administration. In some embodiments, the nasal anti-CD 3 antibody formulation provides an aerosol of anti-CD 3 antibody at a dose ranging from about 10 μg to 100 μg per dose. In some embodiments, the nasal anti-CD 3 antibody formulation provides an aerosol delivery of anti-CD 3 antibody fragment at a dose ranging from about 25 μg to about 50 μg per dose. In some embodiments, the dose is administered to one nostril or split between both nostrils.

[0052] In some embodiments, a formulation for nasal delivery comprises 0.25 mg / mL foralumab, 3.4 mg / mL sodium acetate, 0.20 mg / mL polysorbate 80, and 7.31 mg / mL sodium chloride. In some embodiments, a formulation for nasal delivery consists essentially of 0.25 mg / mL foralumab, 3.4 mg / mL sodium acetate, 0.20 mg / mL polysorbate 80, and 7.31 mg / mL sodium chloride.

[0053] In some embodiments, a formulation for nasal delivery comprises 0.5 mg / mL foralaumab, 3.4 mg / mL sodium acetate, 0.20 mg / mL polysorbate 80, and 7.31 mg / mL sodium chloride. In some embodiments, a formulation for nasal delivery consists essentially of 0.5 mg / mL foralaumab, 3.4 mg / mL sodium acetate, 0.20 mg / mL polysorbate 80, and 7.31 mg / mL sodium chloride.

[0054] In some embodiments, the osmolality of the formulation is about 800-950 (eg, about 825-925) mOsm / kg.

[0055] In some embodiments, the average droplet size of the delivered formulation is between 10 μm and 250 μm. For example, the droplet size can be between 10 μm and 100 μm or between 25 μm and 250 μm.

[0056] In some embodiments, the nasal anti-CD3 antibody formulation is suitable for storage at about 2° C. to about 4° C. In some embodiments, the nasal anti-CD3 antibody formulation is suitable for storage at about 2° C. to about 8° C. In some embodiments, the nasal anti-CD3 antibody formulation is suitable for storage at about 5° C. In some embodiments, the nasal anti-CD3 antibody formulation is stored in a sealed vial or other suitable container. In some embodiments, the nasal anti-CD3 antibody formulation is stored at about 2° C. to about 4° C. in a sealed vial or other suitable container.

[0057] In some embodiments, the antibody concentration in a nasal anti-CD3 antibody formulation is within 3% of the starting concentration after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years. "Starting concentration or label concentration" in this context refers to the concentration after manufacture but before storage.

[0058] In some embodiments, the antibody concentration in the nasal anti-CD3 antibody formulation is within 5% of the starting concentration after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0059] In some embodiments, the antibody concentration in a nasal anti-CD3 antibody formulation is within 10% of the starting concentration after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0060] In some embodiments, the nasal anti-CD3 antibody formulation contains less than 1.5% aggregation after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0061] In some embodiments, the nasal anti-CD3 antibody formulation contains less than 2% aggregation after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0062] In some embodiments, the nasal anti-CD3 antibody formulation contains less than 2.5% aggregation after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0063] In some embodiments, the nasal anti-CD3 antibody formulation contains less than 3% aggregation after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0064] In some embodiments, the pH of the nasal anti-CD3 antibody formulation remains within 0.1 of the initial pH after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years. "Initial pH" in this context means the pH after manufacture but prior to storage.

[0065] In some embodiments, the pH of the nasal anti-CD3 antibody formulation remains within 0.2 of the starting pH after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0066] In some embodiments, the pH of the nasal anti-CD3 antibody formulation remains within 0.3 of the starting pH after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0067] In some embodiments, the pH of the nasal anti-CD3 antibody formulation remains within 0.4 of the starting pH after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0068] In some embodiments, the pH of the nasal anti-CD3 antibody formulation remains within 0.5 of the starting pH after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0069] In some embodiments, the appearance (including, e.g., haze and color) of the nasal anti-CD3 antibody formulation remains similar to its appearance immediately after manufacture after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0070] In some embodiments, impurities present in the nasal anti-CD3 antibody remain within 1% of the starting impurity level after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years, where "starting impurity level" refers to the concentration of the impurity measured after manufacture but prior to storage.

[0071] In some embodiments, the total aerobic microbial count in the nasal anti-CD3 antibody formulation remains below 100 cfu / g after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0072] In some embodiments, the total yeast and mold count in the nasal anti-CD3 antibody formulation remains below 10 cfu / g after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0073] In some embodiments, the total Staphylococcus aureus count in the nasal anti-CD3 antibody formulation remains less than 1 gram after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0074] In some embodiments, the total Pseudomonas aeruginosa count in the nasal anti-CD3 antibody formulation remains less than 1 gram after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0075] In some embodiments, the average pI value of the main cIEF peak of the nasal anti-CD3 antibody formulation remains within 0.05 of the starting pI value after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years, where "starting pI value" refers to the pI value of the main peak after manufacture but prior to storage.

[0076] In some embodiments, the average pI value of the major cIEF peak of the nasal anti-CD3 antibody formulation remains within 0.1 of the onset pI value after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0077] In some embodiments, the average pI value of the major cIEF peak of the nasal anti-CD3 antibody formulation remains within 0.2 of the onset pI value after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0078] In some embodiments, the potency of the antibody in the nasal anti-CD3 antibody formulation remains within 10% of the starting potency after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years. Here, "starting potency" refers to the antibody potency measured after manufacture but before storage. Antibody potency can be determined by lymphocyte proliferation assay or cell activation assay.

[0079] In some embodiments, the potency of the antibody in the nasal anti-CD3 antibody formulation remains within 15% of the initial potency after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0080] In some embodiments, the potency of the antibody in the nasal anti-CD3 antibody formulation remains within 20% of the starting potency after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years. In some embodiments, the potency of the antibody in the nasal anti-CD3 antibody formulation remains within 25% of the starting potency after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0081] In some embodiments, the potency of the antibody in the nasal anti-CD3 antibody formulation remains within 30% of the initial potency after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0082] In some embodiments, the potency of the antibody in the nasal anti-CD3 antibody formulation remains within 35% of the initial potency after storage at about 2° C. to about 8° C. (e.g., about 5° C.) for at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, or at least about 5 years.

[0083] A nasal formulation for nasal delivery having 0.25 mg / mL or 0.5 mg / mL foralaumab, 3.4 mg / mL sodium acetate, 0.20 mg / mL polysorbate 80, and 7.31 mg / mL sodium chloride, when stored at about 2° C. to about 8° C. for 3 months, 6 months, or 12 months, has the following characteristics: substantially free of microbial contamination; substantially free of protein aggregates; retains at least 65% lymphoproliferative activity compared to a control. Optionally, the formulation retains at least 70% of T cell activation activity compared to a reference; has a protein concentration that is at least 90% of the starting concentration; and has a pH of 5.5±0.2.

[0084] Free from microbial contamination means free from mycoplasma, endotoxins and microbial (eg, aerobic, anaerobic and fungal) contamination.

[0085] Substantially free of endotoxin means less endotoxin per dose than allowed by the FDA for biologics, which is 5 EU / kg body weight total endotoxin per day, or 350 EU per total dose for an average 70 kg human.

[0086] Substantially free of mycoplasma and microbial contamination means a negative reading for generally accepted tests known to those skilled in the art. For example, mycoplasma contamination is determined by subculturing a formulation sample in broth medium and distributing it on agar plates on days 1, 3, 7 and 14 at 37°C along with appropriate 30 positive and negative controls. The appearance of the formulation sample is compared microscopically to that of the positive and negative controls at 100x magnification. Additionally, an inoculum of indicator cell culture is incubated for 3 and 5 days and tested for the presence of mycoplasma by epifluorescence microscopy using a DNA-binding fluorescent dye at 600x magnification. If the agar and / or broth medium procedures and the indicator cell culture procedures show no evidence of mycoplasma contamination, the product is deemed satisfactory. Sterility testing to ensure that the product is free of microbial contamination is based on the United States Pharmacopeia.

[0087] Substantially free of protein aggregates means that there are less than 5%, 4%, 3%, 2%, 1% protein aggregates present, as measured by any method known in the art, such as UPLC.

[0088] Therapeutic Administration The therapeutic formulations provided herein, including the anti-CD3 antibody formulations disclosed herein, can be used to treat or alleviate symptoms associated with immune-related disorders, such as, for example, autoimmune diseases or inflammatory disorders. The anti-CD3 antibody formulations disclosed herein can also be used to treat or alleviate symptoms associated with neurodegenerative disorders or cancer.

[0089] Autoimmune diseases that may be treated by administering the formulations described herein include, for example, acquired immune deficiency syndrome (AIDS, a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune labyrinthitis (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue dermatitis (AIDS); chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, dermatomyositis-juvenile, discoid lupus erythematosus, essential mixed cryoglobulinemia, experimental autoimmune encephalomyelitis (EAE), fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, among others. Myocardial Infarction, ... These include globulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (also called progressive systemic sclerosis (PSS) or systemic sclerosis (SS)), Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0090] Inflammatory disorders that can be treated by administering the formulations described herein include, for example, chronic and acute inflammatory disorders.Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemia, inflammatory bowel disease (IBD), non-alcoholic fatty liver disease (NAFLD), osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy and ventilator-induced lung injury.

[0091] The anti-CD3 antibody formulations described herein may be administered to a subject suffering from an immune-related disorder, such as an autoimmune disease or an inflammatory disorder, a neurodegenerative disorder, or cancer. Subjects suffering from an autoimmune disease, an inflammatory disorder, a neurodegenerative disorder, or cancer may be identified by methods known in the art.

[0092] Administration of an anti-CD3 antibody formulation to a patient suffering from an immune-related disorder, such as an autoimmune disease, an inflammatory disorder, a neurodegenerative disorder, or cancer, can be considered successful if any of a variety of experimental or clinical results are achieved. For example, in some embodiments, administration of an anti-CD3 antibody formulation to a patient suffering from an immune-related disorder, such as an autoimmune disease or an inflammatory disorder, is considered successful if one or more of the symptoms associated with the disorder are alleviated, reduced, inhibited, or do not progress to a further or worse state. In some embodiments, administration of an anti-CD3 antibody formulation to a patient suffering from an immune-related disorder, such as an autoimmune disease or an inflammatory disorder, is considered successful if the disorder, e.g., an autoimmune disorder, goes into remission or does not progress to a further or worse state.

[0093] In another embodiment, the anti-CD3 antibody formulations provided herein are used in the treatment or diagnosis of multiple sclerosis (MS). MS is a chronic inflammatory autoimmune disease that affects the central nervous system (CNS). Symptoms of MS include, for example, sensory changes, vision problems, muscle weakness, depression, coordination and speech difficulties, and pain. The anti-CD3 antibody formulations provided herein can be administered to a subject suffering from, diagnosed with, suspected of having, or predisposed to MS. The anti-CD3 antibody formulations provided herein can be administered in a dosage sufficient to alleviate at least one symptom of MS, treat MS, and / or prevent MS from progressing to a further disease state in a subject. In some embodiments, administration of the anti-CD3 antibody formulations described herein reduces the incidence of relapse in relapsing-remitting MS. In some embodiments, administration of the anti-CD3 antibody formulations described herein delays the time to relapse in relapsing-remitting MS. In some embodiments, administration of an anti-CD3 antibody formulation described herein slows the progression of primary progressive MS.

[0094] In yet another embodiment, the anti-CD3 formulations provided herein are administered to a human individual to activate mucosal immunity and immunomodulation.

[0095] In some embodiments, the anti-CD3 antibody formulations provided herein are used to activate regulatory T cells (Tregs).

[0096] In another embodiment, the anti-CD3 antibody formulations provided herein are administered to a human subject to prevent, reduce or decrease immune cell recruitment to human tissue. The anti-CD3 antibodies used herein may be administered to a subject in need thereof to prevent and / or treat conditions associated with aberrant or unregulated immune cell recruitment to tissue sites of human disease.

[0097] In another embodiment, the anti-CD3 antibody formulations provided herein are administered to a human subject to prevent, reduce or decrease immune cell extravasation and leakage into human tissue. Thus, the anti-CD3 antibodies used herein can be administered to prevent and / or treat conditions associated with abnormal or unregulated immune cell infiltration into tissue sites of human disease.

[0098] In another embodiment, the anti-CD3 antibody formulations provided herein are administered to a human subject to prevent, reduce or decrease effects mediated by the release of cytokines in the human body. The term "cytokine" refers to all human cytokines known in the art that bind to extracellular receptors on the cell surface and thereby regulate cell function, including, but not limited to, IL-2, IFN-g, TNF-α, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13.

[0099] In another embodiment, the anti-CD3 antibody formulations provided herein are administered to a human subject to prevent, reduce or decrease the effects mediated by the release of cytokine receptors in the human body. The term "cytokine receptor" refers to any human cytokine receptor in the art that binds to one or more cytokines as defined herein, including but not limited to the receptors of the aforementioned cytokines. Thus, the anti-CD3 antibody used herein can be administered to treat and / or prevent conditions mediated by the abnormal activation, binding or ligation of one or more cytokine receptors in the human body. It is further envisioned that administration of the anti-CD3 antibody in vivo can deplete the intracellular signaling mediated by the cytokine receptor in such a human subject.

[0100] In one aspect, an anti-CD3 antibody formulation provided herein is administered to a subject upon a decline in pancreatic beta cell function. In one embodiment, an individual is tested for beta cell function, insulin secretion, or c-peptide levels using methods known in the art. Thereafter, upon determination of a decline in beta cell function, insulin secretion, or c-peptide levels, an anti-CD3 antibody formulation provided herein is administered to the subject in a dosage sufficient to prevent further progression of autoimmune destruction of beta cell function.

[0101] definition Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0102] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0103] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include polyclonal, monoclonal, chimeric, single chain, F ab , F ab ' and F (ab’)2 Fragments, and F ab "Specifically binds" or "immunoreacts with" refers to an antibody that reacts with one or more antigenic determinants of a desired antigen and does not react with (i.e., does not bind to) other polypeptides or has a much lower affinity (K d >10 -6 ) means to join.

[0104] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is primarily responsible for effector function. Human light chains can be classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, with the heavy chain also containing a "D" region of about 10 additional amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)). The variable regions of each light / heavy chain pair form the antibody binding site.

[0105] The term "monoclonal antibody" (mAb) or "monoclonal antibody composition" as used herein refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of an antigen characterized by a unique binding affinity for it.

[0106] Generally, antibody molecules obtained from humans are associated with one of the distinct classes IgG, IgM, IgA, IgE and IgD, depending on the nature of the heavy chains present in the molecule. 1 , IgG 2 etc. Furthermore, in humans, the light chain can be either a kappa chain or a lambda chain.

[0107] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An antibody is said to specifically bind an antigen if the dissociation constant is ≦1 μM; preferably ≦100 nM, most preferably ≦10 nM.

[0108] As used herein, the terms "immunological binding" and "immunological binding properties" and "specific binding" refer to non-covalent interactions of the type that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction is determined by the dissociation constant (K d ) and smaller K drepresents a greater affinity. The immunological binding properties of selected polypeptides are quantified using methods known in the art. One such method involves measuring the rates of formation and dissociation of antigen-binding site / antigen complexes, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on-rate constant" (K on ) and "off rate constant" (K off Both the K and the KD can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). off / K on The ratio of the dissociation constant K d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473.) The equilibrium binding constant (K d An antibody of the invention is said to specifically bind a CD3 epitope if the binding affinity of the antibody to the CD3 epitope is 1M≦μ, preferably ≦100 nM, more preferably ≦10 nM, and most preferably ≦100 pM to about 1 pM, as measured by an assay such as a radioligand binding assay or similar assay known to one of skill in the art.

[0109] Conservative amino acid substitution refers to the interchangeability of residues with similar side chains.For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine and isoleucine, the group of amino acids with aliphatic hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine and tryptophan, the group of amino acids with basic side chains is lysine, arginine and histidine, and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine.Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic-aspartic, and asparagine-glutamine.

[0110] As discussed herein, minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated to be encompassed by the present invention as long as the variations in the amino acid sequence are maintained at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally grouped into families: (1) acidic amino acids are aspartic acid, glutamic acid; (2) basic amino acids are lysine, arginine, histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include: (i) the aliphatic hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine.

[0111] The term "agent" is used herein to denote a chemical compound, a mixture of compounds, a biological macromolecule, or an extract made from biological material.

[0112] The term patient includes human and veterinary subjects. The terms "subject" and "patient" are used interchangeably herein.

[0113] The present disclosure also provides v , F ab, F ab and F (ab’)2 This includes anti-CD3 antibody fragments, single chain anti-CD3 antibodies, bispecific anti-CD3 antibodies, heteroconjugate anti-CD3 antibodies, trispecific antibodies, immunoconjugates and fragments thereof.

[0114] A bispecific antibody is an antibody that has binding specificities for at least two different antigens. In the present case, one of the binding specificities is for CD3. The second binding target is any other antigen, preferably a cell surface protein or a receptor or receptor subunit.

[0115] All publications and patent documents are incorporated herein by reference to the same extent as if each such publication or patent document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is relevant prior art, and does not constitute any admission as to its contents or date. Although the present disclosure has been described by way of written description, those skilled in the art will recognize that the present disclosure can be implemented in various embodiments, and that the foregoing description and the following examples are intended to be illustrative and not limiting of the scope of the following claims.

[0116] Other embodiments While the present disclosure has been described in conjunction with the detailed description, the foregoing description is intended to be illustrative, and not limiting, of the scope of the present disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. EXAMPLES

[0117] The examples described in this section are provided for illustrative purposes only and are not intended to limit the invention in any way.

[0118] Example 1: Evaluation of Metered Dose Delivery of Foralumab Using the Aptar Device Foralumab formulations were formulated to the intended dosing concentration by dilution with acetate buffered saline, pH 5.5.

[0119] The formulation is as follows: [Table 1]

[0120] The Aptar UniDose device comes pre-filled with the formulation. Intranasal administration is accomplished by manually pressing the bottom of the unit directly through one nostril. The process is repeated for the second nostril.

[0121] The objective of the study described in this document was to document the following attributes of a nasal product considered important for the nasal delivery of forlamab: 1) Delivered dose 2) Droplet size distribution of the delivered dose. 3) Spray pattern 4) Plume Geometry

[0122] Due to the limited number of samples available, only the drug product will be evaluated. The placebo will be evaluated at a separate time.

[0123] Dose content uniformity Dose content uniformity (shot weight) is used to determine the repeatability of individual sprays from each device. The unit is weighed. The unit is manually actuated for a single dose. The unit is reweighed to determine the amount of liquid dispensed in milligrams. This procedure is then repeated for a total of 10 separate devices. The results are shown in Table 2. [Table 2]

[0124] The results show that the delivered dose is uniform as shown by the %RSD. These results are within the USP requirements for dose content uniformity. The delivered dose of the drug product will be tested using a drug-specific assay if this device is selected for further testing.

[0125] Droplet Size Distribution Droplet size was measured using laser diffraction. A Malvern Spraytec unit was used for the analysis. Five separate devices were placed 1 inch under the laser beam and manually sprayed with Malvern and the results were averaged.

[0126] Malvern parameters are as follows: Measurement and setting equipment: Spraytec-Open Spray Lens = 300 mm Path length = 12.0 (mm) Particle refractive index = 1.33 + 0.000i Start of scattering = 9 Dispersant refractive index = 1.00 End of scattering = 36 Particle density = 1.00 (gm / cc) Scattering threshold = 1 Residual = 0.36 (%) Minimum size = 0.10 (μm) Extinction analysis = Off Maximum size = 900.00 (μm) Actuator distance = 0 Multiple Scattering = Off Actuator Angle = 0 Software = v3.30.016 Serial Number: Device=MAL1225354 Detector=08CX-CDL SOP Name = Nasal Spray[2].ssop

[0127] The results are shown in Tables 3 and 4. [Table 3] [Table 4]

[0128] The results shown above are highly consistent and reproducible.

[0129] Spray Pattern The spray pattern is a cross-sectional representation of the plume upon activation. The TLC plate was suspended 3 cm above the nosepiece and the device was activated. The maximum diameter, minimum diameter and shape of the pattern are recorded in centimeters. The results are shown in Tables 5 and 6. [Table 5] [Table 6]

[0130] Plume Geometry For plume geometry, the angle of the plume is measured. A picture of the plume was taken while the device was running. The edge of the plume was determined and a straight line was drawn until both sides of the plume intersected. The resulting angle is recorded in Tables 7 and 8 below. [Table 7] [Table 8]

[0131] Example 2: Evaluation of Metered Dose Delivery of Foralumab Using the Gerresheimer Device A unit dose device was developed. The device consists of two separate parts: ● 1 mL Type I, glass syringe with plunger, manufacturer Gerresheimer. This item is listed in the Gerresheimer DMF submitted to the FDA. Nasal spray device (NAD 300), manufacturer Teleflex. This item is listed in the Teleflex DMF submitted to the FDA.

[0132] The foralumab drug product is formulated to the intended dosing concentration by dilution with acetate buffered saline, pH 5.5, and pre-filled into Type I glass Gerresheimer syringes. The formulation is as follows: [Table 9]

[0133] Intranasal administration is accomplished by attaching a Teleflex Nasal Atomization Device to a loaded syringe (Gerresheimer Device). The dose is then administered directly through one nostril. The process is repeated for the second nostril.

[0134] The objective of the study described in this document was to document the following attributes of a nasal product considered important for the nasal delivery of forlamab: 1) Delivered dose 2) Droplet size distribution of the delivered dose. 3) Spray pattern 4) Plume Geometry

[0135] Dose content uniformity Dose content uniformity (shot weight) is used to determine the repeatability of each spray from each syringe. The unit is weighed. The unit is manually actuated for a single dose. The unit is reweighed to determine the amount of liquid dispensed in milligrams. This procedure is then repeated for a total of 10 separate syringes connected to 10 separate sprayers. The results are shown in Table 10. [Table 10]

[0136] The results show that the delivered dose is uniform as shown by the %RSD. These results are within the USP requirements for dose content uniformity. The delivered dose of the drug product will be tested using a drug-specific assay if this device is selected for further testing.

[0137] Droplet Size Distribution Droplet size was measured using laser diffraction. A Malvern Spraytec unit was used for the analysis. Ten separate syringes with ten separate nasal spray devices were placed one inch under the laser beam and manually sprayed with Malvern, and the results were averaged.

[0138] Malvern parameters are as follows: Measurements and Settings ●Equipment = Spraytec, Open Spray ● Lens = 300mm ●Path length = 12.0 (mm) ●Fine particle refractive index = 1.33 + 0.000i ●Start of scattering = 9 ●Dispersion refractive index = 1.00 ● End of scattering = 36 ●Particle density=1.00(gm / cc) Scattering threshold = 1 ●Residual = 0.36(%) ●Minimum size = 0.10 (μm) ● Quenching analysis = Off ●Maximum size = 900.00 (μm) ● Actuator distance = 0 Multiple scattering = off ● Actuator angle = 0 Software = v3.30.016 ●Serial number: I ●Equipment=MAL1225354 ●Detector=08CX-CDL ● SOP name = Nasal spray [2].ssop

[0139] The results are shown in Tables 11, 12 and 13 below. [Table 11] [Table 12] [Table 13]

[0140] There appears to be variability between devices, most likely due to device design and limitations.

[0141] Spray Pattern The spray pattern is a cross-sectional representation of the plume upon actuation. The TLC plate was suspended 3 cm above the nosepiece and the device was operated manually. The maximum diameter, minimum diameter and shape of the pattern are recorded in centimeters. The results are shown in Tables 14, 15 and 16. [Table 14] [Table 15] [Table 16]

[0142] There appears to be variability between devices, most likely due to device design and limitations.

[0143] Plume Geometry For plume geometry, the angle of the plume is measured. A picture of the plume was taken while the device was running. The edge of the plume was determined and a straight line was drawn until both sides of the plume intersected. The resulting angle is recorded in Tables 17, 18 and 19 below. [Table 17] [Table 18] [Table 19]

[0144] Example 3: Stability of nasal foralamuab formulations The stability of intranasal formulations containing 25 μg or 50 μg of foralumab, 3.4 mg / mL sodium acetate, 0.20 mg / mL polysorbate 80, and 7.31 mg / mL sodium chloride in a total volume of 100 μL was analyzed. Stability was determined after storage at approximately 5° C. (2° C. to 8° C.) for 1 month, 3 months, 6 months, or 12 months. Stability was assessed by the following readouts: appearance, protein concentration, impurities, pH, capillary isoelectric focusing (cIEF), bioburden, and potency. Appearance, visual; protein concentration, UV absorbance at 280 nm; assay and impurities by reversed-phase UPLC; cIEF, capillary isoelectric focusing; pH, potentiometric titration; bioburden, USP <6162> ; potency by either T cell proliferation assays or NAFT activation in pan T cells.

[0145] The results are shown in Tables 20, 21, 22 and 23.

[0146] Actuation data, samples are collected after spraying from the Aptar device to test for any effect of shear from the spray on important quality attributes. Non-actuation data, samples are collected directly from the Type 1 glass / Aptar plunger container closure system without spraying and therefore without pressure. [Table 20] 1. The sample's main peak pI value is within ±0.10 of the average pI value of the main peak of the reference standard. The sample's electrophoretic profile is comparable to that of the reference standard. [Table 21] 1. The sample's main peak pI value is within ±0.10 of the average pI value of the main peak of the reference standard. The sample's electrophoretic profile is comparable to that of the reference standard. [Table 22] 1. The sample's main peak pI value is within ±0.10 of the average pI value of the main peak of the reference standard. The sample's electrophoretic profile is comparable to that of the reference standard. [Table 23] 1. The sample's main peak pI value is within ±0.10 of the average pI value of the main peak of the reference standard. The sample's electrophoretic profile is equivalent to that of the reference standard.

Claims

1. A unit dose device for intranasal administration of a liquid formulation containing an anti-CD3 antibody, wherein 10 μg to 100 μg of the anti-CD3 antibody is delivered per dose in a volume of approximately 100 μL of the formulation, and the size of the delivered droplet is (a) 10 μm to 100 μm or (b) 25 μm to 250 μm. Here, the anti-CD3 antibody Heavy chain complementarity determination region (CDRH) 1, containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 3, CDRH3 containing the amino acid sequence of SEQ ID NO: 4, Light chain complementarity determining region (CDRL) 1, containing the amino acid sequence of SEQ ID NO: 5 CDRL2 containing the amino acid sequence of SEQ ID NO: 6, and CDRL3 containing the amino acid sequence of SEQ ID NO: 7, A unit dose device, including one.

2. A unit dose device according to claim 1, which delivers a drug dose unit with a relative standard deviation (RSD) of 10% or less.

3. The unit dose device according to claim 1, wherein the size of the delivered droplet is 10 μm to 100 μm.

4. The unit dose device according to claim 3, wherein the size of the delivered droplet is 25 μm to 250 μm.

5. The unit dose device according to claim 1, wherein the anti-CD3 antibody concentration in the liquid formulation is within 10% of the starting concentration after storage at approximately 2°C to approximately 8°C for at least approximately one month.

6. A unit dose device according to claim 1, comprising less than 2.5% aggregates after being stored at approximately 2°C to approximately 8°C for at least approximately one month.

7. The unit dose device according to claim 1, wherein the pH of the liquid formulation remains within 0.3 of the starting value after being stored at approximately 2°C to approximately 8°C for at least approximately one month.

8. The unit dose device according to claim 1, wherein the efficacy of the anti-CD3 antibody remains within 35% of the initial efficacy after being stored at approximately 2°C to approximately 8°C for at least approximately one month.

9. The unit dose device according to claim 1, wherein the unit dose device delivers 25 μg of anti-CD3 antibody per dose.

10. The unit dose device according to claim 1, wherein the unit dose device delivers 50 μg of anti-CD3 antibody per dose.

11. The unit dose device according to claim 1, wherein the pH of the liquid formulation is about 5 to 6.

12. The unit dose device according to claim 1, wherein the unit dose device is operated for the delivery of a single dose.

13. The unit dose device according to claim 1, wherein the anti-CD3 antibody comprises a variable heavy chain amino acid sequence including the sequence of SEQ ID NO: 8 and a variable light chain amino acid sequence including the sequence of SEQ ID NO:

9.

14. The unit dose device according to claim 1, wherein the anti-CD3 antibody is foralmab.

15. The unit dose device according to claim 1, wherein the liquid formulation is substantially free of protein aggregates.

16. A unit dose device for intranasal administration of a liquid formulation containing an anti-CD3 antibody to a target, wherein 10 μg to 100 μg of the anti-CD3 antibody is delivered per dose in a volume of approximately 100 μL of the formulation, and the size of the delivered droplet is (a) 10 μm to 100 μm or (b) 25 μm to 250 μm. Here, the anti-CD3 antibody Heavy chain complementarity determination region (CDRH) 1, containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 3, CDRH3 containing the amino acid sequence of SEQ ID NO: 4, Light chain complementarity determining region (CDRL) 1, containing the amino acid sequence of SEQ ID NO: 5 CDRL2 containing the amino acid sequence of SEQ ID NO: 6, and CDRL3 containing the amino acid sequence of SEQ ID NO: 7, A unit dose device, including one.

17. The unit dose device according to claim 16, wherein the subject is suffering from Alzheimer's disease.

18. The unit dose device according to claim 16, wherein the subject is suffering from multiple sclerosis (MS).

19. A nasal liquid formulation for treating a target neurodegenerative disease or disorder, or for alleviating the symptoms of a neurodegenerative disease, comprising an anti-CD3 antibody, about 10 mM to about 100 mM sodium acetate trihydrate, about 0.01% to about 1% polysorbate 80, and about 50 mM to about 500 mM sodium chloride, wherein 10 μg to 100 μg of the anti-CD3 antibody is delivered per dose.

20. The nasal liquid formulation according to claim 19, wherein the neurodegenerative disease or disorder is an inflammatory disease or disorder.

21. The nasal liquid formulation according to claim 19, wherein the disease or disorder is multiple sclerosis (MS).

22. The nasal liquid formulation according to claim 19, wherein the disease or disorder is Alzheimer's disease.

23. The nasal liquid formulation according to any one of claims 19 to 22, wherein 25 μg to 50 μg of the anti-CD3 antibody is administered.

24. The nasal liquid formulation according to any one of claims 19 to 23, which is delivered in droplet size of 10 μm to 100 μm.

25. The nasal liquid formulation according to any one of claims 19 to 24, which is delivered in droplet size of 25 μm to 250 μm.

26. The nasal liquid formulation according to any one of claims 19 to 25, wherein the concentration of anti-CD3 antibody in the nasal liquid formulation is within 10% of the starting concentration after being stored at approximately 2°C to approximately 8°C for at least approximately one month.

27. ​​The nasal liquid formulation according to any one of claims 19 to 26, wherein the nasal liquid formulation contains less than 2.5% aggregates after being stored at approximately 2°C to approximately 8°C for at least approximately one month.

28. The nasal liquid formulation according to any one of claims 19 to 27, wherein the pH of the nasal liquid formulation remains within 0.3 of the starting value after being stored at approximately 2°C to approximately 8°C for at least approximately one month.

29. The nasal liquid formulation according to any one of claims 19 to 28, wherein the efficacy of the anti-CD3 antibody remains within 35% of the initial efficacy after being stored at approximately 2°C to approximately 8°C for at least one month.

30. The nasal liquid formulation according to any one of claims 19 to 29, which delivers 25 μg of anti-CD3 antibody per dose.

31. The nasal liquid formulation according to any one of claims 19 to 30, which delivers 50 μg of anti-CD3 antibody per dose.

32. The nasal liquid preparation according to any one of claims 19 to 31, wherein the pH of the nasal liquid preparation is about 5 to 6.

33. The nasal liquid preparation according to any one of claims 19 to 32, wherein the nasal liquid preparation is administered using a unit dose device.

34. The nasal liquid preparation according to any one of claims 19 to 33, wherein the nasal liquid preparation is administered into one nostril.

35. The nasal liquid preparation according to any one of claims 19 to 33, wherein the nasal liquid preparation is administered into both nostrils.

36. The anti-CD3 antibody is Heavy chain complementarity determination region (CDRH) 1, containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 3, CDRH3 containing the amino acid sequence of SEQ ID NO: 4, Light chain complementarity determining region (CDRL) 1, containing the amino acid sequence of SEQ ID NO: 5 CDRL2 containing the amino acid sequence of SEQ ID NO: 6, and CDRL3 containing the amino acid sequence of SEQ ID NO: 7, A nasal liquid preparation according to any one of claims 19 to 35, including the above.

37. The nasal liquid formulation according to any one of claims 19 to 36, wherein the anti-CD3 antibody comprises a variable heavy chain amino acid sequence including the sequence of SEQ ID NO: 8 and a variable light chain amino acid sequence including the sequence of SEQ ID NO:

9.

38. The nasal liquid formulation according to any one of claims 19 to 37, wherein the anti-CD3 antibody is foralumab.

39. The nasal liquid formulation according to any one of claims 19 to 38, wherein the nasal liquid formulation comprises 0.25 mg / mL of anti-CD3 antibody, 3.4 mg / mL of sodium acetate, 0.20 mg / mL of polysorbate 80, and 7.31 mg / mL of sodium chloride.

40. The nasal liquid formulation according to any one of claims 19 to 39, wherein the nasal liquid formulation comprises 0.5 mg / mL of anti-CD3 antibody, 3.4 mg / mL of sodium acetate, 0.20 mg / mL of polysorbate 80, and 7.31 mg / mL of sodium chloride.

41. A nasal liquid formulation for treating a target neurodegenerative disease or disorder, or for alleviating the symptoms of a neurodegenerative disease, comprising an anti-CD3 antibody, about 10 mM to about 100 mM sodium acetate trihydrate, about 0.01% to about 1% polysorbate 80, and about 50 mM to about 500 mM sodium chloride, wherein 25 μg or 50 μg of the anti-CD3 antibody is delivered per dose, wherein the anti-CD3 antibody is, Heavy chain complementarity determination region (CDRH) 1, containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 3, CDRH3 containing the amino acid sequence of SEQ ID NO: 4, Light chain complementarity determining region (CDRL) 1, containing the amino acid sequence of SEQ ID NO: 5 CDRL2 containing the amino acid sequence of SEQ ID NO: 6, and CDRL3 containing the amino acid sequence of SEQ ID NO: 7, A nasal liquid formulation containing [this ingredient].