Intranasal p40 monomer and Anti-p40 homodimer mab for type 1 diabetes

Abstract

Methods of treating type 1 diabetes mellitus are provided including administering a recombinant p40 monomer and / or a p402 mAb intranasally to a subject in need.

Description

[0001] Intranasal p40 monomer and anti-p40 homodimer mAh for type 1 diabetes

[0002]

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0003]

[0002] This International Patent Application claims the benefit of priority to U.S. Provisional Application No. 63 / 657,619, filed June 7, 2024, the entire contents of which are incorporated herein by reference.

[0004]

[0003] INCORPORATION BY REFERENCE SEQUENCE LISTING

[0005]

[0004] A Sequence Listing compliant with the WIPO Standard ST.26 standards is submitted and incorporated herein by reference in its entirety. The Sequence Listing is identified as RUM01-00172.xml, containing 3,000 bytes and generated on June 7, 2025.

[0006]

[0005] BACKGROUND

[0007]

[0006] Type 1 diabetes mellitus (T1DM) is an autoimmune disorder associated with T-cell mediated destruction of pancreatic [3-cells. Currently, the only effective drug treatment available for subjects constitutes daily insulin injections. Insulin injections combined with a low carbohydrate diet is the standard therapy for T1DM subjects. However, there are no effective therapeutic options that can stop autoimmune progression of the disease as well as protect pancreatic [3-cells.

[0008]

[0007] SUMMARY

[0009]

[0008] The disclosure herein describes the use of a recombinant p40 monomer cytokine and monoclonal antibodies (mAb) for p40 homodimer (p402) cytokine. It was discovered that T1DM subjects have lower levels of the p40 monomer cytokine and higher levels of the p40 homodimer cytokine. The disclosure includes methods of treating T1DM in a subject comprising administering a recombinant p40 monomer and / or an antibody against p40 homodimer (p402 mAb). Also described are methods of intranasally administering a composition comprising a recombinant p40 monomer to inhibit the progression of diabetes. Additionally disclosed herein are methods of intranasally administering a composition comprising an anti-p40 homodimer mAb a3-ld to inhibit the progression of diabetes.

[0010]

[0009] Further, disclosed herein are methods of producing the p40 monomer and the p402 mAb. In some embodiments, the recombinant p40 monomer comprises or consists essentially of an amino acid sequence of SEQ ID NO: 1. This disclosure contemplates both the nucleic acid sequence and the amino acid sequence of the disclosed SEQ ID NO: 1. The disclosed p4Ch mAb is available to the public and capable of being reproduced from a hybridoma deposited with the American Type Culture Collection (ATCC).

[0011]

[0010] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] [Oi l] FIGURE 1 : Level of p40 monomer (p40) in serum of type 1 diabetes (T1DM) subjects (n=8) and age-matched healthy controls (n=8). Serum samples obtained from Discovery Life Sciences (Los Osos, CA) were analyzed for IL-12 by sandwich ELISA. **p< 0.01, ***p< 0.001.

[0013]

[0012] FIGURE 2: Level of p40 monomer (p40) in serum of T1DM subjects (n=8) and age- matched healthy controls (n=8). Serum samples were analyzed for IL-23 sandwich ELISA. **p< 0.01, ***p< 0.001.

[0014]

[0013] FIGURE 3 : Level of p40 monomer (p40) in serum of T1DM subjects (n=8) and age- matched healthy controls (n=8). Serum samples were analyzed for p402 by sandwich ELISA. **p< 0.01, ***p< 0.001.

[0015]

[0014] FIGURE 4: Level of p40 monomer (p40) in serum of T1DM subjects (n=8) and age- matched healthy controls (n=8). Serum samples were analyzed for p40 by sandwich ELISA. **p< 0.01, ***p< 0.001.

[0016]

[0015] FIGURE 5: Intranasal p40 monomer and mAb (a3- 1 d) against p40 homodimer enriches Tregs in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks. Separate groups of mice also received 25 ng (nanogram)Zmouse p40 homodimer mAb a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A).

[0017]

[0016] FIGURE 6: Intranasal p40 monomer and mAb (a3- 1 d) against p40 homodimer enriches Tregs in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks. Separate groups of mice also received 25 ng (nanogram)Zmouse p40 homodimer mAb a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A) and dead cells excluded with Zombie Aqua Fixable Viability Kit.

[0018]

[0017] FIGURE 7: Intranasal p40 monomer and mAb (a3- 1 d) against p40 homodimer enriches Tregs in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks.

[0019] Separate groups of mice also received 25 ng (nanogram) / mouse p40 homodimer mAb a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A), dead cells excluded with Zombie Aqua Fixable Viability Kit and singlets were selected from the FSC-H versus FSC-A dot plot.

[0020]

[0018] FIGURE 8: Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer enriches Tregs in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks.

[0021] Separate groups of mice also received 25 ng (nanogram)Zmouse p40 homodimer mAb a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A), dead cells excluded with Zombie Aqua Fixable Viability Kit and singlets were selected from the FSC-H versus FSC-A dot plot, then divided into T cells on the basis of surface expression of CD3.

[0022]

[0019] FIGURE 9: Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer enriches Tregs in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks.

[0023] Separate groups of mice also received 25 ng (nanogram)Zmouse p40 homodimer mAb a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A), dead cells excluded with Zombie Aqua Fixable Viability Kit and singlets were selected from the FSC-H versus FSC-A dot plot, then divided into T cells on the basis of surface expression of CD3. The dot plot on the lower right and upper left show a clear demarcation of CD4 and CD8 cell populations.

[0024]

[0020] FIGURE 10: Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer enriches Tregs in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks.

[0025] Separate groups of mice also received 25 ng (nanogram)Zmouse p40 homodimer mAb a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A), dead cells excluded with Zombie Aqua Fixable Viability Kit and singlets were selected from the FSC-H versus FSC-A dot plot, then divided into T cells on the basis of surface expression of CD3. Foxp3+CD4+population is shown. Age-matched female C57 / BL6 mice were used as controls for FIGURES 5 - 10.

[0021] FIGURE 1 1 : Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer suppresses Thl7 response in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks. Separate groups of mice also received 25 ng / mouse p40 homodimer mAh a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A).

[0026]

[0022] FIGURE 12: Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer suppresses Thl7 response in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks. Separate groups of mice also received 25 ng / mouse p40 homodimer mAb a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A) and dead cells excluded with Zombie Aqua Fixable Viability Kit.

[0027]

[0023] FIGURE 13: Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer suppresses Thl7 response in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks. Separate groups of mice also received 25 ng / mouse p40 homodimer mAb a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A), dead cells excluded with Zombie Aqua Fixable Viability Kit, and singlets were selected from the FSC-H versus FSC-A dot plot.

[0028]

[0024] FIGURE 14: Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer suppresses Th 17 response in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks. Separate groups of mice also received 25 ng / mouse p40 homodimer mAb a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A), dead cells excluded with Zombie Aqua Fixable Viability Kit, and singlets were selected from the FSC-H versus FSC-A dot plot, then divided into T cells on the basis of surface expression of CD3.

[0029]

[0025] FIGURE 15: Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer suppresses Thl7 response in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks. Separate groups of mice also received 25 ng / mouse p40 homodimer mAb a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A), dead cells excluded with Zombie Aqua Fixable Viability Kit, and singlets were selected from the FSC-H versus FSC-A dot plot, then divided into T cells on the basis of surface expression of CD3. The dot plot on the lower right and upper left show a clear demarcation of CD4 and CD8 cell populations.

[0030]

[0026] FIGURE 16: Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer suppresses Thl7 response in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week for 14 weeks. Separate groups of mice also received 25 ng / mouse p40 homodimer mAb a3-ld intranasally twice a week for 14 weeks. Then splenocytes were gated based on side scatter (SSC-A) versus forward scatter (FSC-A), dead cells excluded with Zombie Aqua Fixable Viability Kit, and singlets were selected from the FSC-H versus FSC-A dot plot, then divided into T cells on the basis of surface expression of CD3. The dot plot on the lower right and upper left show a clear demarcation of CD4 and CD8 cell populations. IL-17+CD4+population is shown. Age-matched female C57 / BL6 mice were used as controls for FIGURES 11 - 16.

[0031]

[0027] FIGURE 17: Intranasal p40 monomer treatment reduces inflammatory infdtration into the islets of Langerhans of NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week. Briefly, 50 ng recombinant human p40 was dissolved in 2 pl (microliter) normal saline, mice were held in supine position and 1 pl volume was delivered into each nostril using a pipetman. One group of NOD mice also received the same amount of saline intranasally. After 14 weeks of treatment, islets underwent H&E (hematoxylin and eosin) staining. Age-matched female C57 / BL6 mice were used as controls.

[0032]

[0028] FIGURE 18: Intranasal p40 homodimer mAb a3-ld treatment reduces inflammatory infdtration into the islets of Langerhans of NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 25 ng / mouse p40 homodimer mAb a3-ld intranasally twice a week. Briefly, 25 ng p402 mAb was dissolved in 2 pl normal saline, mice were held in supine position and 1 pl volume was delivered into each nostril using a pipetman. One group of NOD mice also received the same amount of control IgG (immunoglobin G) intranasally. After 14 weeks of treatment, islets underwent H&E staining. Age-matched female C57 / BL6 mice were used as controls.

[0033]

[0029] FIGURE 19: Intranasal p40 monomer and mAb (a3- 1 d) against p40 homodimer reduce the infiltration of CD4 T cells into the islets of Langerhans of NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week. Separate groups of mice also received 25 ng / mouse p40 homodimer mAb a3-ld intranasally twice a week. After 14 weeks of treatment, pancreatic sections were double-labeled for insulin & CD4 (p40 monomer). Age-matched female C57 / BL6 mice were used as controls.

[0034]

[0030] FIGURE 20: Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer reduces the infiltration of CD4 T cells into the islets of Langerhans of NOD mice. Twelve- weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week. Separate groups of mice also received 25 ng / mouse p40 homodimer mAb a3-ld intranasally twice a week. After 14 weeks of treatment, pancreatic sections were double-labeled for insulin & CD4 (p40 homodimer mAb a3-ld). Age-matched female C57 / BL6 mice were used as controls.

[0035]

[0031] FIGURE 21 : Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer inhibits the level of IL-ip in pancreas of NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week. Separate groups of mice also received 25 ng / mouse p40 homodimer mAb a3-ld intranasally twice a week. After 14 weeks of treatment, the level of IL-ip protein was monitored in pancreas by Western blot (p40 monomer). Age-matched female C57 / BL6 mice were used as controls.

[0036]

[0032] FIGURE 22: Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer inhibits the level of IL-ip in pancreas of NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week. Separate groups of mice also received 25 ng / mouse p40 homodimer mAb a3-ld intranasally twice a week. After 14 weeks of treatment, the level of IL-1 P protein was monitored in pancreas by Western blot (p40 homodimer mAb a3-ld). Age-matched female C57 / BL6 mice were used as controls.

[0033] FIGURE 23: Intranasal p40 monomer and mAb (a3-ld) against p40 homodimer reduces blood glucose level in NOD mice. Twelve-weeks-old female NOD / ShiLtJ mice (n=3) received 50 ng / mouse recombinant human p40 monomer intranasally twice a week. Separate groups of mice also received 25 ng / mouse p40 homodimer mAb a3-ld intranasally twice a week. After 14 weeks of treatment, blood glucose level was measured in mg / dl (milligram per deciliter) using a One Touch Ultra Glucometer. Age-matched female C57 / BL6 mice were used as controls.

[0037]

[0034] DETAILED DESCRIPTION

[0038]

[0035] Type 1 diabetes mellitus (T1DM) is an autoimmune condition associated with the T-cell- mediated destruction of pancreatic P-cells. Among young subjects, it is the most common autoimmune disease that is caused by the damage of pancreatic endocrine P-cells capable of producing insulin in specific areas of the pancreas, known as islets of Langerhans. Despite intense investigations, there is no effective drug or medication to cure type 1 diabetes mellitus or to inhibit or reduce the impact of the autoimmune response by the body on the pancreatic cells. Insulin injections and low carbohydrate diets are the current standard of care for T1DM. Therefore, alternative therapeutic options for stopping the autoimmune progression of T1DM and protecting pancreatic beta cells is an important area of research. Disclosed herein are two new therapeutic options for T1DM. In some embodiments, a method of treating T1DM comprises administering a recombinant p40 monomer to a subject in need thereof. In some embodiments, a method of treating T1DM comprises administering an anti-p40 homodimer mAb (monoclonal antibody). The methods disclosed herein include administering a recombinant p40 monomer to a subject in need thereof in addition to administering insulin and / or a low carbohydrate diet. The methods disclosed herein may comprise administering an anti-p40 homodimer (p4Ch) mAb to a subject in need thereof in addition to administering insulin and / or a low carbohydrate diet. In some embodiments, the methods for treating T1DM in a subject comprise administering a recombinant p40 monomer and / or an anti-p402 mAb. In some embodiments, the method of treating T1DM in a subject comprises administering a recombinant p40 monomer and an anti-p402 mAb.

[0039]

[0036] As demonstrated herein, the recombinant p40 monomer and / or anti-p40 homodimer mAb can be an effective treatment for type 1 diabetes mellitus, alone or in combination with insulin, low or controlled carbohydrate diet, and / or exercise. Without being limited by theory, the recombinant p40 monomer and anti-p40 homodimer mAh may protect insulinproducing cells to stabilize blood glucose in type 1 diabetes mellitus.

[0040]

[0037] An “antibody” refers to a binding agent that is a polypeptide comprising a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, such as those recognized by an immune cell. The term “antibody” includes antigen binding fragments thereof. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies) and antigen binding fragments thereof. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997. The antibody or immunologically active fragment thereof may be a monoclonal antibody (mAb) or an immunologically active fragment of a monoclonal antibody. In other embodiments, the antibody or immunologically active fragment thereof is a polyclonal, monoclonal, human, humanized, and chimeric antibody; a single chain antibody or an epitope-binding antibody fragment of such an antibody. In another embodiment, the antibody or immunologically active fragment thereof is a humanized antibody or an immunologically active fragment thereof.

[0041]

[0038] As used herein, the term “amount” refers to “an amount effective” or “therapeutically effective amount” of a composition, e.g., antibody or peptide, to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results. A “therapeutically effective amount” of a composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or peptide to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects.

[0042]

[0039] The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a subject). When a therapeutic amount is indicated, the precise amount of the compositions of the present disclosure to be administered may be determined by a physician with consideration of individual differences in age, weight, extent of the disease’s progression, levels of biomarkers in the subject’s body fluids, and the overall condition of the subject.

[0043]

[0040] As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0044]

[0041] Recombinant p40 monomer

[0045]

[0042] In some embodiments, the method comprises administering to a subject diagnosed with or suspected of having T1DM a therapeutically effective amount of a first composition comprising a recombinant p40 monomer. In some embodiments, the method does not include administering a composition comprising a p4Ch mAb.

[0046]

[0043] The recombinant p40 monomer may be produced using an expression vector. An expression vector may comprise a nucleic acid sequence encoding the recombinant p40 monomer (i.e., the recombinant protein). Additionally, the vector may include regulatory elements such as promoters, enhancers, and terminators, which are configured to facilitate the transcription and translation processes within a host cell. The introduction of the expression vector into the host cell may be achieved through various methodologies, including transformation, transfection, or electroporation. Post-introduction, the expression vector may either integrate into the host genome or remain as an episomal element, contingent upon the vector's design and the type of host cell employed.

[0047]

[0044] The host cell may be selected from a variety of cell types, including bacterial, yeast, insect, or mammalian cells. Each cell type may offer distinct advantages concerning protein expression levels, post-translational modifications, and scalability. The selection of the host cell may be influenced by the specific requirements of the recombinant protein to be expressed. Within the host cell, the expression vector's regulatory elements may initiate the transcription of the nucleic acid sequence into messenger RNA (mRNA), which is subsequently translated into the recombinant protein. The recombinant protein may undergo folding and post-translational modifications to achieve its functional conformation.

[0045] The recombinant protein expressed may be isolated from the host cell culture using various purification techniques, such as affinity chromatography, ion exchange chromatography, or size exclusion chromatography, to yield a purified protein product suitable for subsequent applications.

[0048]

[0046] An amino acid sequence of the recombinant p40 monomer is provided herein as SEQ ID NO: 1.

[0049]

[0047] SEQ ID NO: 1 is a recombinant human amino acid sequence:

[0050]

[0048] MHPQQLVVSW F SLVLLASPI VAIWELEKNV YVVELDWYPD APGETVVLTC DTPEEDGITW TSDQSSEVLG SGKTLTIQVK EFGDAGQYTC HKGGEALSRS LLLLHKKEDG IWSTDILKDQ KEPKAKSFLK CEAKDYSGHF TCWWLTAIST DLKFSVKSSR GSSDPRGVTC EAASLSAEKV SVDHREYNKY TVECQEGSTC PAAEESLLIE VVVEAVHKLK YENYTSSFFI RDIIKPDPPK NLQLKPLKNS RQVEVSWEYP DTWSTPHSYF SLTFCVQVQG KNKREKKLFM DQTSAKVTCH KDANVRVQAR DRYYSSFWSE WASVSCS

[0051]

[0049] In some embodiments, the p40 monomer is purified from a cell culture using an expression vector containing a nucleic acid sequence that would result in a recombinant protein comprising an amino acid sequence of SEQ ID NO: 1.

[0052]

[0050] p402 Monoclonal Antibody

[0053]

[0051] In some embodiments, the method comprises administering to a subject diagnosed with or suspected of having T1DM a therapeutically effective amount of a second composition comprising a p402 mAb. In some embodiments, the method does not include administering a composition comprising a recombinant p40 monomer.

[0054]

[0052] The p402 mAb may be produced any number of ways including using a hybridoma cell culture. Methods of generating a hybridoma cell culture to produce monoclonal antibodies is well known in the art. For example, a person of ordinary skill in the art starts by immunizing a host animal, such as a mouse, with an antigen of interest. The immunization may be performed using multiple injections over a period of time to elicit a robust immune response. The antigen may be administered in conjunction with an adjuvant to enhance the immune response. The antigen used in this method is purified p40 homodimer protein. Following immunization, the host animal is sacrificed, and the spleen is harvested. Spleen cells, which include B lymphocytes, are isolated from the spleen tissue. The isolation process may involve mechanical disruption of the spleen followed by filtration to obtain a single-cell suspension. Next, the isolated spleen cells may be fused with myeloma cells to form hybridoma cells. Myeloma cells are selected for their ability to proliferate indefinitely in culture. The fusion process may be facilitated by the use of a fusogenic agent, such as polyethylene glycol (PEG), which promotes the merging of cell membranes. Then, the resulting cell mixture is subjected to a selection process to isolate hybridoma cells. This may involve culturing the cells in a selective medium, such as hypoxanthine-aminopterin- thymidine (HAT) medium, which allows only the hybridoma cells to survive and proliferate. Non-fused spleen and myeloma cells are unable to survive in this medium. Finally, the hybridoma cells are screened for the production of antibodies specific to the antigen of interest. Screening may be conducted using techniques such as enzyme-linked immunosorbent assay (ELISA) or flow cytometry to identify hybridomas secreting the desired monoclonal antibodies. Positive hybridoma cells are cloned to ensure monoclonality. Cloning may be achieved through limiting dilution or other cloning techniques to isolate single hybridoma cells, which are then expanded to produce a homogeneous population. The cloned hybridoma cells are expanded in culture to produce large quantities of monoclonal antibodies. The antibodies may be harvested from the culture supernatant and purified using standard purification techniques, such as protein A / G affinity chromatography. Of course, the process may be adapted or modified to accommodate different antigens or host animals, as required.

[0055]

[0053] In some embodiments, the p402 mAb comprises, consists essentially of, or is the monoclonal antibody produced from the hybridoma deposited with the ATCC and assigned deposit number PTA-126900.

[0056]

[0054] Under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure, Applicant has deposited biological material comprising twenty -five (25) vials of hybridoma with the designation of antibody (mAb)a3-ld with the International Depositary Authority, American Type Culture Collection (ATCC) of 1080 University Blvd., Manassas, Va. 20110-2209, on Dec. 3, 2020 and assigned deposit number PTA-126900. The deposited hybridoma is capable of producing a monoclonal antibody against the p402 homodimer which is identified as mAb-p402 a3-ld.

[0055] In some embodiments, a method of treating T1DM is provided comprising administering to a subject in need thereof a first composition comprising a recombinant p40 monomer and / or a second composition comprising a monoclonal antibody against p402. In some embodiments, the first composition comprises SEQ ID NO: 1. In some embodiments, the second composition comprises antibodies produced from a hybridoma cell line identified as mAb-p402 a3-ld and stored with the ATCC.

[0057]

[0056] Formulation and Mode of Administration

[0058]

[0057] Formulations of therapeutic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman et al., (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

[0059]

[0058] Selecting an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain embodiments, an administration regimen maximizes the amount of therapeutic delivered to the subject consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert et al, (2003) New Engl. J. Med. 348:601-608; Milgrom et al, (1999) New Engl. J. Med. 341 : 1966-1973; Slamon et al, (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al, (2000) New Engl. J. Med. 342:613-619; Ghosh et al, (2003) New Engl. J. Med. 348:24-32; Lipsky et al, (2000) New Engl. J. Med. 343: 1594-1602).

[0060]

[0059] Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.

[0061]

[0060] Actual dosage levels of the active ingredients in the compositions as used herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level may depend upon a variety of pharmacokinetic factors including for example the activity of the particular biologic employed, the route of administration, the time of administration, the rate of excretion of the particular biologic being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular biologies employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors known in the medical arts. Compositions comprising binding agents such as antibodies or fragments thereof can be provided by continuous infusion, or by doses at intervals of, e g., one day, one week, or 1-7 times per week. Doses may be provided intravenously, subcutaneously, orally, nasally, intramuscular, or by inhalation. A specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. A total weekly dose may be at least 0.05 pg / kg body weight, at least 0.2 pg / kg, at least 0.5 pg / kg, at least 1 pg / kg, at least 10 pg / kg, at least 100 pg / kg, at least 0.2 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 10 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 40 mg / kg or at least 50 mg / kg (see, e.g., Yang et al, (2003) New Engl. J. Med. 349:427-434; Herold et al, (2002) New Engl. J. Med. 346: 1692- 1698; Portielji et al, (2003) Cancer Immunol. Immunother. 52: 133-144). The desired dose of antibodies, fragments thereof, or monomers may be based on a moles / kg body weight. The desired plasma concentration of the p402 mAb, fragments thereof, or recombinant p40 monomer may be based on a moles / kg body weight. The dose may be at least about 15 pg at least 20 ig, at least 25 pig, at least 30 pig, at least 35 pig, at least 40 pig, at least 45 pig, at least 50 pig, at least 55 pig, at least 60 pig, at least 65 pig, at least 70 pig, at least 75 pig, at least 80 pig, at least 85 pig, at least 90 pig, at least 95 pig, or at least 100 pig. The doses administered to a subject may number at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or more per day, per week, or per month. For antibodies, fragments thereof, or monomers as used herein, the dosage administered to a subject may be about 0.0001 mg / kg to 100 mg / kg of the subject's body weight. The dosage may be between about 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.10 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.25 mg / kg or 0.01 to 0.10 mg / kg of the subject's body weight.

[0062]

[0061] The dosage of the antibodies, fragments thereof, or monomers as used herein may be calculated using the subject's weight in kilograms (kg) multiplied by the dose to be administered in mg / kg. The dosage of the antibodies, fragments thereof, or monomers of the invention may be about 150 pg / kg or less, 125 pg / kg or less, 100 pg / kg or less, 95 pg / kg or less, 90 pg / kg or less, 85 pg / kg or less, 80 pg / kg or less, 75 pg / kg or less, 70 pg / kg or less, 65 pg / kg or less, 60 pg / kg or less, 55 pg / kg or less, 50 pg / kg or less, 45 pg / kg or less, 40 pg / kg or less, 35 pg / kg or less, 30 pg / kg or less, 25 pg / kg or less, 20 pg / kg or less, 15 pg / kg or less, 10 pg / kg or less, 5 pg / kg or less, 2.5 pg / kg or less, 2 pg / kg or less, 1.5 pg / kg or less, 1 pg / kg or less, 0.5 pg / kg or less, or 0.5 pg / kg or less of a subject's body weight.

[0063]

[0062] A unit dose of the antibodies, fragments thereof, or monomer as used herein may be about 0. 1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 60 mg, 0.25 mg to 40 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0064]

[0063] The dosage of the antibodies, fragments thereof, or monomer as used herein may achieve a serum titer of at least about 0.1 pg / ml, at least 0.5 pg / ml, at least 1 pg / ml, at least 2 pg / ml, at least 5 pg / ml, at least 6 pg / ml, at least 10 pg / ml, at least 15 pg / ml, at least 20 pg / ml, at least 25 pg / ml, at least 50 pg / ml, at least 100 pg / ml, at least 125 pg / ml, at least 150 pg / ml, at least 175 pg / ml, at least 200 pg / ml, at least 225 pg / ml, at least 250 pg / ml, at least 275 pg / ml, at least 300 pg / ml, at least 325 pg / ml, at least 350 pg / ml, at least 375 pg / ml, or at least 400 pg / ml in a subject. Alternatively, the dosage of the antibodies, fragments thereof, or monomer as used herein may achieve a serum titer of at least 0.1 pg / ml, at least 0.5 pg / ml, at least 1 pg / ml, at least, 2 pg / ml, at least 5 pg / ml, at least 6 pg / ml, at least 10 pg / ml, at least 15 pg / ml, at least 20 pg / ml, at least 25 pg / ml, at least 50 pg / ml, at least 100 pg / ml, at least 125 pg / ml, at least 150 pg / ml, at least 175 pg / ml, at least 200 pg / ml, at least 225 pg / ml, at least 250 pg / ml, at least 275 pg / ml, at least 300 pg / ml, at least 325 pg / ml, at least 350 pg / ml, at least 375 pg / ml, or at least 400 pg / ml in the subject.

[0065]

[0064] The route of administration may include pulmonary administration, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference their entirety.

[0066]

[0065] In some embodiments, the first and / or second compositions are formulated to be delivered to a subject intranasally for example via inhalation. In this manner, the first and / or second compositions may be nebulized or formulated into a spray or gas that can be inhaled by the subject. In some embodiments, the antibodies, fragments thereof, or monomer are formulated in a liquid form that may be sprayed into a subject’s nose and absorbed into the body via the nasal or buccal tissues.

[0067]

[0066] While it may not be easy to predict whether a therapeutic will be effective when administered intranasally, a person of skill in the art understands the general methods for formulating a therapeutic for intranasal delivery. For example, a method for formulating a drug for inhalation may comprise the steps of selecting an active pharmaceutical ingredient (API) (e.g., a p40 monomer and / or a p402 mAb), reducing the size of the clusters or particles comprising the API, optionally combining with excipients, including the API in an inhalation device, performing quality control and testing, and scaling up for commercial distribution.

[0068]

[0067] Specifically, first an active pharmaceutical ingredient is selected based on its therapeutic efficacy for inhalation delivery. The API may be in the form of a fine powder or a liquid solution, depending on the desired delivery mechanism and the physicochemical properties of the compound. Next, the API is subjected to a particle size reduction process to achieve a particle size distribution suitable for inhalation. Techniques such as micronization or nano- milling may be employed to produce particles with a mean aerodynamic diameter typically ranging from 1 to 5 micrometers, which is optimal for deep lung deposition. Then, the API is combined with pharmaceutically acceptable excipients to enhance stability, dispersibility, and delivery efficiency. Excipients may include carriers such as lactose or mannitol, which aid in the uniform distribution of the API within the formulation. The choice of excipients may be influenced by factors such as compatibility with the API and the intended inhalation device. Next, the API and excipients are blended to achieve a homogeneous mixture. The blending process may involve the use of high-shear mixers or tumbling blenders to ensure uniform distribution of the API throughout the formulation. The blending parameters, such as time and speed, may be optimized to prevent agglomeration and ensure consistency.

[0069]

[0068] Following blending, the formulated drug is filled into an appropriate inhalation device, such as a dry powder inhaler (DPI) or a metered-dose inhaler (NIDI). The filling process may involve precise dosing mechanisms to ensure accurate delivery of the therapeutic dose with each actuation. Then the final formulation is subjected to quality control testing to ensure compliance with regulatory standards. Tests may include particle size analysis, content uniformity, and aerodynamic performance assessments using cascade impaction or laser diffraction techniques. Stability testing may also be conducted to evaluate the shelf-life of the formulation under various storage conditions. Finally, the formulation process may be optimized and scaled up for commercial production. This may involve adjustments to the formulation components, processing parameters, or device design to enhance manufacturability and subject compliance. Of course, the process may be adapted or modified to accommodate different APIs or inhalation devices, as required.

[0070]

[0069] In some embodiments, the method of treatment comprises administering a recombinant p40 monomer and / or p402 mAb to a subject having or suspected of having T1DM, wherein the administration is delivered intranasally. For example, the subject may receive the treatment in a liquid formulation capable of being absorbed into the buccal or nasal passage tissues.

[0071]

[0070] The compositions may be administered to a subject at the same time or separately. For example, the first composition may be administered before the second composition. Alternatively, the second composition may be delivered before the first composition. In some embodiments, the first or second composition may be administered at least 30 minutes before the other composition (i.e., the first or second composition) is administered. In some embodiments a patient having T1DM is administered a composition comprising a recombinant p40 monomer or a p40 mAb in a therapeutic effective amount, but not both.

[0072]

[0071] In some embodiments, the subject is a human. In some embodiments, the subject is identified in an age category of infant, toddler, child, pre-teen, teenager, young adult, adult, older adult, or senior. In some embodiments, the subject is bom with T1DM. In some embodiments, the subject develops T1DM after birth. In some embodiments, the subject is suspected of having T1DM.

[0073]

[0072] EXAMPLES

[0074]

[0073] Actives p40 monomer and p402 mAb.

[0075]

[0074] Materials and Methods for producing the p40 monomer antibodies e.g., a3-3a and a3-7g, as well as characterization is described in non-patent literature to Dasgupta, S. et al., “Generation of Functional Blocking Monoclonal Antibodies Against Mouse Interleukin- 12 p40 Homodimer and Monomer,” Hybridoma. 2008 27(3) pp. 141-151, the entire contents of which are incorporated herein by reference.

[0076]

[0075] Under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purpose of Patent Procedure, Applicant has deposited biological material comprising twenty -five (25) vials of hybridoma with the designation of antibody (mAb) a3-3a and twenty-five (25) vials of hybridoma with the designation of antibody (mAb) a3-7g with the International Depository Authority, American Type Culture Collection (ATCC) of 1080 University Blvd., Manassas, Va. 20110-2209. The mAb a3-3a was deposited on Aug. 18, 2021 and assigned deposit number PTA-127109. The mAb a3-7g was deposited on Sep. 15, 2021 and assigned deposit number PT A- 127131. The deposited hybridomas comprise monoclonal antibodies against the p40 monomer which comprises mAb a3-3a or mAb a3-7g. The p402 mAb deposited with the ATCC identified as a3-ld was used in the following studies. Methods of producing and characterizing a3-ld are also described in Dasgupta, S. et al.

[0077]

[0076] The p40 family of cytokines has four members comprising of IL-12( p35-p40), p40 monomer, p40 homodimer (p402), and the IL-23 (pl9-p40), only heterodimers (IL- 12 and IL- 23) were thought to have biological effects. Since all four members contain p40 in different forms, it is important to use specific monoclonal antibodies (mAb) to characterize these molecules.

[0078]

[0077] Therefore, functional blocking mAbs against p40 monomer (i.e., a3-3a and a3-7g) and p402 (a3-ld) were generated. By using such mAb, the selective loss of p40 monomer, but not IL-12, IL-23 and p402, in serum of T1DM subjects as compared to healthy controls (FIG. 1 - 4) was delineated. Since the level of p40 monomer was less in T1DM subjects as compared to healthy controls, the effect of p40 monomer supplementation on the disease process of T1DM in the NOD mouse model was tested. On the other hand, since the level of p40 homodimer was elevated in T1DM subjects compared to healthy controls, the effect of p40 homodimer neutralization on the disease process of T1DM in NOD (non-obese diabetic) mice was examined. As a result, described herein are two new therapeutic options for T1DM: one involving the recombinant p40 monomer and the other with anti-p40 homodimer mAb.

[0079]

[0078] Intranasal administration of recombinant human p40 monomer inhibits the progression of prediabetic NOD mice to diabetic stage. Here, 12-weeks-old female NOD mice were treated with recombinant human p40 monomer intranasally twice a week at a dose of 50 ng / mouse. Briefly, 50 ng (nanogram) recombinant human p40 monomer was dissolved in 2 pl (microliter) normal saline, mice were held in a supine position and 1 pl volume was delivered into each nostril using a pipetman. The NOD mice displayed loss of Foxp3 -positive regulatory T cells (Tregs) (FIG. 5 - 10) and upregulation of IL- 17-positive Thl7 cells (FIG.

[0080] 11 - 16) in the spleen as compared to control mice. After 14 weeks of treatment, p40 monomer protected Tregs (FIG. 5 - 10) and reduced Thl7 cells (FIG. 11 - 16) in the spleen of NOD mice.

[0081]

[0079] Accordingly, remarkable infiltration of mononuclear cells (FIG. 17) and CD4 T cells (FIG. 19 - 20) was observed into the islets of Langerhans of NOD mice as compared to control mice. However, intranasal p40 monomer treatment reduced the infiltration of mononuclear cells (FIG. 17) as well as CD4 T cells (FIG. 19) into the islets of Langerhans of NOD mice. Inflammatory infiltration also caused inflammation in the pancreas as evident from the upregulation of IL-ip in the pancreas of NOD mice in comparison to control mice (FIG. 21). Finally, NOD mice exhibited an elevation of blood glucose as compared to control mice (FIG. 23). Consistent to the upregulation of Tregs, suppression of Thl7 cells, decrease in inflammatory infiltration, and inhibition of insulitis, p40 monomer treatment normalized blood glucose level in NOD mice (FIG. 23).

[0082]

[0080] Intranasal administration of anti-p40 homodimer mAb a3-ld hinders the progression of prediabetic NOD mice to diabetic stage. Here as well, 12-weeks-old female NOD mice were treated with anti-p40 homodimer mAb a3-ld intranasally twice a week at a dose of 25 ng / mouse. Briefly, 25 ng p402 mAb a3-ld was dissolved in 2 pl normal saline, mice were held in a supine position and 1 pl volume was delivered into each nostril using a pipetman. After 14 weeks of treatment, mAb a3-ld restored / upregulated Tregs (FIG. 5 - 10) and decreased Thl7 cells (FIG. 11 - 16) in the spleen of NOD mice. The mAb a3-ld treatment also inhibited the infiltration of mononuclear cells (FIG. 18) and CD4 T cells (FIG. 20) into islets, suppressed the islet expression of proinflammatory cytokine IL-ip (FIG. 22), and stabilized blood glucose level in NOD mice (FIG. 23).

Claims

We claim:

1. A method of treating type 1 diabetes mellitus (T1DM) in a subject, comprising administering a therapeutically effective amount of a first composition comprising a recombinant p40 monomer and / or a second composition comprising an anti-p40 homodimer monoclonal antibody (p402 mAb) to the subject.

2. The method of claim 1, wherein the recombinant p40 monomer is produced using an expression vector comprising a nucleic acid sequence encoding the recombinant p40 monomer.

3. The method of claim 2, wherein the recombinant p40 monomer comprises an amino acid sequence of SEQ ID NO: 1.

4. The method of claim 1, wherein the p402 mAb is produced using a hybridoma cell culture.

5. The method of claim 4, wherein the p4C>2 mAb comprises the antibody a3-ld produced from a hybridoma cell culture identified as (mAb)a3-ld deposited with the American Type Culture Collection (ATCC) and assigned deposit number PTA-126900.

6. The method of claim 1, wherein the first composition and second composition are administered together.

7. The method of claim 1, wherein the p40 monomer is administered intranasally.

8. The method of claim 7, wherein the p40 monomer is administered via inhalation.

9. The method of claim 1, wherein the p4C>2 mAb is administered intranasally.

10. The method of claim 1, wherein the p402 mAb is administered via inhalation.

11. The method of claim 1, wherein the subject is a human.

12. The method of claim 11, wherein the human is selected from the group consisting of infant, toddler, child, pre-teen, teenager, young adult, adult, older adult, and senior.

13. The method of claim 1, wherein the subject is diagnosed with or suspected of having T1DM.

14. The method of claim 1, wherein the first composition and the second composition are administered separately.

15. A method of treating type 1 diabetes mellitus in a subject comprising administering a therapeutically effective amount of a recombinant p40 monomer intranasally and administering a therapeutically effect amount of a p402 mAb intranasally.

16. The method of claim 15, wherein the recombinant p40 monomer is administered before the p4C>2 mAb.

17. The method of claim 15, wherein the p402 mAb is administered before the recombinant p40 monomer.

18. The method of claim 15, wherein the recombinant p40 monomer comprises an amino acid sequence of SEQ ID NO: 1.

19. The method of claim 15, wherein the p4C>2 mAb is the a3-dl antibody produced from a hybridoma cell culture identified as (mAb)a3-ld deposited with the American Type Culture Collection (ATCC) and assigned deposit number PTA-126900.

20. The method of claim 15, wherein the recombinant p40 monomer comprises an amino acid sequence of SEQ ID NO: 1 and the p402 mAb is the a3-dl antibody produced from a hybridoma cell culture identified as (mAb)a3-ld deposited with the American Type Culture Collection (ATCC) and assigned deposit number PTA-126900.

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