Methods and compositions for treating obesity, obesity-related diseases, fatty liver, and fatty liver-related diseases with anti-CD24 agents

Abstract

Methods and compositions for treating obesity, obesity-related diseases, fatty liver, and fatty liver-related diseases are provided. Aspects of the methods include administering to a subject in need thereof an effective amount of an anti-CD24 agent to treat the subject. Compositions useful for implementing embodiments of the methods are also provided.

Description

[0001] Cross-references

[0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 344,410, filed on May 20, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporating by reference the sequence listing provided as a sequence listing xml file

[0004] The Sequence Listing is therefore provided as a Sequence Listing XML "STAN-1959WO_SEQ_LIST.xml" created on May 15, 2023 and having a size of 35,582 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. Technical Field Background Art

[0005] Obesity and obesity-related diseases are the leading causes of morbidity and mortality worldwide. One of the main obesity-related diseases is fatty liver disease. Fatty liver disease can also be caused by other reasons besides obesity. For example, it can be the result of excessive alcohol consumption, genetic predisposition, viral hepatitis, exposure to chemicals such as perfluorooctanoic acid (PFAS), and metabolic syndrome. It is estimated that 1 billion people worldwide and about one-third of the U.S. population or 100 million Americans suffer from non-alcoholic fatty liver disease (NAFLD). In many patients, NAFLD can progress to non-alcoholic steatohepatitis (NASH), which can eventually lead to liver failure and / or liver cancer.

[0006] Furthermore, many patients with end-stage liver disease (ESLD) require liver transplantation as a life-saving therapy. The presence of significant steatosis in the donor liver increases the risk of graft failure and leads to increased mortality and morbidity in liver transplant patients. Summary of the invention

[0007] Methods and compositions for treating obesity, obesity-related diseases, fatty liver, and fatty liver-related diseases are provided. Aspects of the method include administering an effective amount of an anti-CD24 agent to a subject in need thereof to treat the subject. Methods and compositions for reducing steatosis (liver steatosis) are provided. Aspects of the method include administering an effective amount of an anti-CD24 agent to a subject in need thereof to reduce steatosis (liver steatosis). Compositions of embodiments for implementing the method are also provided.

[0008] The inventors have found that anti-CD24 agents reduce steatosis (liver fatty degeneration) and are therefore useful for treating a variety of ailments / diseases (e.g., fatty liver and obesity-related diseases). The inventors have demonstrated that many different anti-CD24 antibodies, including those with fully functional Fc regions, "dead" Fc regions, or no Fc regions at all (e.g., G7s scFv), can be used to reduce steatosis. Therefore, any binding agent that targets / binds to CD24 should act to reduce steatosis. To this end, the inventors have demonstrated (see the working examples below) that small molecules known to be screened for their ability to bind to CD24 do in fact reduce steatosis and are therefore useful for treating diseases (e.g., fatty liver and obesity-related diseases). The inventors recognize that any anti-CD24 agent is useful / useful for treatment to reduce steatosis, i.e., treatment is not limited to certain anti-CD24 antibodies, or is not limited to anti-CD24 antibodies at all, on the contrary, any anti-CD24 agent, including any CD24 binding agent, will act to reduce steatosis. For example, the inventors recognize that agents that reduce CD24 expression (also referred to herein as anti-CD24 agents) such as RNAi agents (shRNA / siRNA), antisense agents (e.g., antisense oligonucleotides, LNA, etc.), CRISPR agents (e.g., CRISPR nuclease / cleavage activity, CRISPRi, etc.) will also be active in reducing steatosis.

[0009] A method of reducing steatosis in an individual is provided, wherein the method comprises administering a therapeutically effective dose of an anti-CD24 agent to an individual with steatosis for a period of time sufficient to reduce steatosis. In some cases, the subject has non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0010] In some embodiments, the anti-CD24 agent is an anti-CD24 antibody. In some cases, the anti-CD24 antibody includes an Fc with one or more silent mutations. In some cases, the anti-CD24 antibody is an anti-CD24 fragment antigen binding region (Fab) or an anti-CD24 PEGylated Fab fragment. In some cases, the anti-CD24 antibody is an anti-CD24 single-chain variable fragment (scFv). In some cases, the anti-CD24 antibody comprises 3 heavy chains and 3 light chain CDRs of SWA11, ML5, SN3 or G7s. In some cases, the anti-CD24 antibody is a humanized antibody. In some cases, the anti-CD24 antibody comprises an amino acid sequence having 3 heavy chain IMGT CDRs of SWA11 and having 80% or greater sequence identity with the humanized variable heavy chain sequence of SEQ ID NO: 2-14; and an amino acid sequence having 3 light chain IMGT CDRs of SWA11 and having 80% or greater sequence identity with the humanized variable light chain sequence of SEQ ID NO: 19-31. In some instances, the anti-CD24 agent is a small molecule that binds to CD24.

[0011] In some embodiments, the course of treatment for an individual is in the range of 1 to 8 weeks. In some cases, administration of an anti-CD24 agent is combined with another form of treatment such as dietary modification and / or bariatric surgery. In some cases, an anti-CD24 agent is co-administered with another therapeutic agent.

[0012] In some embodiments, the individual is a liver transplant patient, and the administration is for enhancing liver graft function and / or prolonging liver graft survival. In some embodiments, the individual suffers from fatty liver, fatty liver-related diseases, obesity, or obesity-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figures 1A to 1D : Relative lipid accumulation of Huh 7D cells when cultured on adipogenic medium and treated with various anti-CD24 agents. Agents were added at 1 μg / mL unless otherwise specified in Fab. MOSW1, SWA11, ML5, SN3: mouse anti-human CD24 antibodies; MOSW2: SWA11 antibody with point mutations that silence complement activity; MOSW3: SWA11 antibody with point mutations that silence complement activity, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP); Fab: Fab of SWA11 antibody; G7s: scFv against human and mouse CD24; TW2, TW6, V2, V3, V4, V6, V7, V8, V9, V10, V14, V15, V16: different humanized anti-CD24 antibodies; hIgG: human IgG isotype control. Figure 1A: Anti-steatosis activity of SWA-11, Fab, TW2, TW6, and G7S; Figure 1B : Anti-steatosis activity of V2, V3, V4, V6, V7, V8, V9, V10, V14, V15, and V16; Figure 1C : Anti-fat degeneration of SWA11, Fab, ML5, SN3, MOSW1, MOSW2, and MOSW3; FIG.1D: Anti-fat degeneration of three small molecules that bind to CD24.

[0014] FIG. 2A to FIG. 2B : HFD, IgG control ( Figure 2A ) compared to anti-CD24 agents ( Figure 2B ) H&E staining of the livers of mice treated with .

[0015] Figure 3 : Ultrasound of the liver of mice fed a high-fat diet treated with IgG control (top) compared with anti-CD24 treatment (bottom).

[0016] FIG. 4A to FIG. 4B : IgG control ( Figure 4A ) and anti-CD24 agents ( Figure 4B ) H&E staining of the liver of ob / ob mice treated with HFD for 1 week.

[0017] FIG. 5A to FIG. 5B : IgG control ( Figure 5A ) and anti-CD24 agents ( Figure 5B ) H&E staining of livers from ob / ob mice treated with HFD for 1 week and then returned to regular diet.

[0018] Figure 6 : Serum liver chemistries of HFD-induced ob / ob mice treated with isotype IgG or anti-CD24 agents.

[0019] FIG. 7A to FIG. 7B : Metabolism of clemizole in ob / ob mice treated with IgG control or anti-CD24 agents.

[0020] FIG. 8A to FIG. 8B : IgG control ( Fig. 8A ) and ADCC silent anti-CD24 antibody ( Figure 8B ) H&E staining of livers of HFD-induced ob / ob mice treated for 1 week while on a regular diet.

[0021] 9A to 9C : HFD for 6 weeks, followed by 3 weeks of treatment with MOSW3 IgG control ( Fig. 9A ) of mice with humanized livers, and in IgG control-treated ( Fig. 9B ) or MOSW3 treatment ( Fig. 9C ) Corresponding CT scan of the liver 3 weeks after operation.

[0022] FIG. 10A to FIG. 10B : Body weight of mice treated with IgG control or anti-CD24 agents. DETAILED DESCRIPTION

[0023] definition

[0024] In the following description, a number of terms commonly used in the art are used. In order to provide a clear and consistent understanding of the specification and claims, and the scope to be given to such terms, the following definitions are provided.

[0025] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0026] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.

[0027] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is a human.

[0028] The term "sample" in relation to a patient encompasses liquid samples of blood and other biological origin, solid tissue samples such as biopsy specimens or tissue cultures, or cells derived therefrom and their progeny. The definition also includes samples that have been manipulated in any way after being obtained, such as treated with an agent; washed; or enriched for certain cell populations. The definition also includes samples that have been enriched for a particular type of molecule (e.g., nucleic acids, polypeptides, etc.).

[0029] The term "biological sample" encompasses clinical samples, and also includes tissues obtained by surgical resection, tissues obtained by biopsy, cultured cells, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, etc. "Biological sample" includes samples containing target cells and / or normal control cells, or samples suspected of containing these cells. The definition includes biological fluids derived therefrom (e.g., infected cells, etc.), such as samples containing polynucleotides and / or polypeptides obtained from such cells (e.g., cell lysates or other cell extracts containing polynucleotides and / or polypeptides). Biological samples from patients containing infected cells, etc. may also include uninfected cells.

[0030] The term "diagnosis" is used herein to refer to the identification of a molecular or pathological state, disease or condition.

[0031] The term "prognosis" is used herein to refer to the prediction of the likelihood of disease progression (eg, progression of infection, etc.) (including relapse, drug resistance, etc.).

[0032] The term "prediction" is used herein to refer to the act of predicting or estimating, based on observation, experience, or scientific reasoning. In one example, a doctor might predict the likelihood that a patient will survive.

[0033] As used herein, the term "specific binding member" refers to a member of a specific binding pair (i.e., two molecules, typically two different molecules, in which one of the molecules (e.g., a first specific binding member) specifically binds to the other molecule (e.g., a second specific binding member) by non-covalent means).

[0034] The term "specific binding" refers to the direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges). Specific binding members describe members of a pair of molecules that have binding specificity to each other. The members of a specific binding pair can be naturally derived or produced in whole or in part synthetically. One member of the pair of molecules has an area or cavity on its surface that specifically binds to and is therefore complementary to the specific spatial and polar organization of the other member of the pair of molecules. Therefore, the members of the pair have the property of specifically binding to each other. Examples of paired specific binding members are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The specific binding members of the binding pair exhibit high affinity and binding specificity for binding to each other. Typically, the affinity between a pair of specific binding members is characterized by 10 -6 M or smaller, such as 10 -7 M or smaller, including 10 -8 M or smaller, e.g. 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 10 -13 M or smaller, 10 -14 M or smaller, including 10 -15 M or smaller K d (Dissociation constant). "Affinity" refers to the strength of binding, with increased binding affinity correlating to lower KD.

[0035] In one embodiment, affinity is determined by surface plasmon resonance (SPR), e.g., as used by a Biacore system. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g., at 25°C. "Affinity" refers to the strength of binding, with increased binding affinity being associated with a lower KD. In one embodiment, affinity is determined by surface plasmon resonance (SPR), e.g., as used by a Biacore system. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g., at 25°C.

[0036] Method described herein may include multiple steps. As required, each step may be performed after a predetermined amount of time has passed between each step. Therefore, the time for performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, and include 5 hours or more. In certain embodiments, each subsequent step may be performed immediately after the previous step is completed. In other embodiments, a step may be performed after the incubation or waiting time (e.g., a few minutes to overnight waiting time) after the previous step is completed.

[0037] As used herein, the terms "assess," "determine," "measure," "assess," and "measure" are used interchangeably and include both quantitative and qualitative determinations.

[0038] As used herein, the term "separated" refers to the physical separation of two elements (eg, by size or affinity, etc.) as well as the degradation of one element, leaving the other element intact.

[0039] Specific description

[0040] Methods and compositions for treating obesity, obesity-related diseases, fatty liver and fatty liver-related diseases are provided. Aspects of the method include administering an effective amount of an anti-CD24 agent to a subject in need thereof to treat the subject. Compositions of embodiments for implementing the method are also provided.

[0041] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific embodiments described, as such can certainly vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

[0042] Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range (based on one-tenth of the unit of the lower limit unless the context clearly dictates otherwise) and any other stated or intervening value in the stated range are included within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any explicitly excluded limits in the stated range. Where a stated range includes one or both limits, ranges excluding one or both of these included limits are also included within the invention.

[0043] Certain ranges appearing herein are presented by preceding the numerical value with the term "about". The term "about" is used herein to provide literal support for the exact number preceding it as well as a number that is close to or approximately the number preceding the term. In determining whether a number is close to or approximately a specifically cited number, the close or approximate unquoted number may be a number that provides a substantial equivalent of the specifically cited number in the context in which it appears.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0045] All publications and patents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and discloses and describes the methods and / or materials related thereto. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0046] It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology, such as "only," "only," and the like, in connection with the recitation of claim elements or as a "negative" limitation.

[0047] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0048] Although the apparatus and method have been or will be described for the sake of grammatical fluency and functional interpretation, it should be clearly understood that unless expressly stated in accordance with 35 U.S.C. §112, the claims should not be interpreted as necessarily being limited in any way to the "means" or "step" limitations, but should be consistent with the full scope of the meaning and equivalents provided by the claims in accordance with the judicial doctrine of equivalents, and in the event that the claims are expressly stated in accordance with 35 U.S.C. §112, they will be given full legal equivalents in accordance with 35 U.S.C. §112.

[0049] Methods and compositions for treating obesity, obesity-related diseases, fatty liver and fatty liver-related diseases are provided. Aspects of the method include administering an effective amount of an anti-CD24 agent to a subject in need thereof to treat the subject. Compositions of embodiments for implementing the method are also provided.

[0050] "Treatment" means at least achieving an improvement in one or more symptoms associated with a target condition afflicting a subject, wherein improvement refers broadly to a reduction in at least the magnitude of a parameter (e.g., a symptom associated with the target condition being treated). Thus, treatment also includes situations where a pathological condition or at least a symptom associated therewith is completely suppressed (e.g., prevented from occurring) or stopped (e.g., terminated), such that the adult mammal no longer suffers from the target condition, or at least no longer suffers from symptoms characterizing the injury. In some cases, "treatment", "treating", etc. refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or adverse effects attributable to a disease. "Treatment" may be any treatment of a disease in a mammal, and includes: (a) preventing a disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; or (c) alleviating the disease, i.e., causing regression of the disease. Treatment may result in a variety of different physical manifestations, such as modulation of gene expression, rejuvenation of tissues or organs, etc. In some embodiments, treatment of an ongoing disease occurs, wherein the treatment stabilizes or reduces the patient's undesirable clinical symptoms. Such treatment can be performed before the function of the affected tissue is completely lost. The subject therapy can be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0051] Therapeutic treatment is treatment in which the subject is ill prior to administration, whereas prophylactic treatment is treatment in which the subject is not ill prior to administration. In some embodiments, the subject is ill or suspected of having an increased likelihood of having an illness prior to treatment. In some embodiments, the subject is suspected of having an increased likelihood of having an illness.

[0052] The methods described herein can be used to treat any type of subject in need of treatment. Subjects include mammalian species. Mammalian species that can be treated by the methods of the present invention include canines and felines; horses; bovines; sheep, etc. and primates, including humans. The subject methods, compositions and reagents can also be applied to animal models, such as small mammals, such as rodents, lagomorphs, etc., in experimental studies. The terms "recipient", "individual", "subject", "host" and "patient" are used interchangeably herein and refer to any mammalian subject in need of diagnosis, treatment or therapy, especially humans. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock and farm animals, and zoos, sports or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, etc. In some embodiments, the mammal is a human. In some embodiments, the individual is not a human (e.g., a non-human mammal).

[0053] Anti-CD24 agents. As summarized above, aspects of the method include administering an anti-CD24 agent to a subject. Anti-CD24 agents of interest include specific binding members for CD24. The specific binding members exhibit an affinity (Kd) sufficient to provide the desired treatment for target CD24 (such as human CD24). As used herein, the term "affinity" refers to the equilibrium constant for reversible binding of two agents; "affinity" can be expressed as a dissociation constant (Kd). Affinity can be at least 1 times greater than the affinity of the antibody for an unrelated amino acid sequence, at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, at least 100 times greater, or at least 1000 times greater or greater than the affinity of the antibody for an unrelated amino acid sequence. The affinity of the specific binding member to the target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or greater. The term "binding" refers to the direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic and ionic and / or hydrogen bond interactions (including interactions such as salt bridges and water bridges). In some embodiments, the anti-CD24 agent binds to human CD24 with nanomolar affinity or picomolar affinity. In some embodiments, the anti-CD24 agent binds to human CD24 with a Kd of less than about 100 nM, 50 nM, 20 nM, 20 nM or 1 nM. In some cases, the anti-CD24 agent is an antibody. In some embodiments, the antibody binds to human CD24 with nanomolar affinity or picomolar affinity. In some embodiments, the antibody binds human CD24 with a Kd of less than about 100 nM, 50 nM, 20 nM, 20 nM, or 1 nM.

[0054] Anti-CD24 agents include, but are not limited to, anti-CD24 antibodies (with or without Fc effector functions), anti-CD24 fragment antigen binding regions (Fab), anti-CD24 single-chain variable fragments (scFv), peptides that bind to CD24, anti-CD24 RNA interference agents (siRNA / shRNA agents), anti-CD24 oligonucleotide antisense agents (e.g., locked nucleic acids (LNA) targeting CD24 or any other oligonucleotide antisense agents), CRISPR agents targeting CD24 (e.g., CRISPR nuclease / cleavage activity that reduces CD24 expression at the DNA level via genome editing, CRISPRi agents that reduce CD24 expression, etc.), and small molecules that bind to CD24 (see, e.g., compounds A, B, and C of FIG. 1 ). In some cases, the anti-CD24 agent is an antibody. In some cases, it is a humanized antibody. Small molecules, peptides, DNA or RNA aptamers, etc. that target CD24 are also considered anti-CD24 agents.

[0055] Examples of CD24-specific binding members include CD24 antibodies and binding fragments thereof. Non-limiting examples of such antibodies include antibodies to any epitope of CD24. Bispecific antibodies, i.e. antibodies in which each of the two binding domains recognizes a different binding epitope, are also contemplated.

[0056] CD24 is a double-chain glycosylphosphatidylinositol (GPI) anchored glycoprotein expressed at multiple stages of B cell development, starting from the pre-B cell compartment of the bone marrow and continuing to mature surface Ig-positive B cells. Plasma cells express very low or negative. It is also expressed on most B-lineage acute lymphoblastic leukemias, B-cell CCLs, and B-cell non-Hodgkin's lymphomas. CD24 may play a role in regulating B cell proliferation and maturation. Protein reference sequences include gene bank NP_001278666; NP_001278667; NP_001278668; NP_037362; NP_001346013.

[0057] Antibodies known to bind to human CD24 are known and commercially available, including but not limited to SWA11 (Creative Biolabs); MA5-11833; 12-0247-42; anti-CD24 clone ML5 (Bio-Legend), SN3 A5-2H10 (also known as SN3); ALB9, EPR19925; EPR3006 (N); SWA21; SWA22; OKB2, etc. Anti-CD24 agents may include, for example, antibodies that bind to human CD24, such as SN3 or ML5. G7s is a known scFv against human and mouse CD24. As is known in the art, anti-CD24 antibodies may bind, for example, to a LAP (Leucine-Alanine-Proline) motif. Alternatively, antibodies specific for human CD24 may be produced.

[0058] Humanized anti-CD24 antibodies can be generated de novo, such as those disclosed herein, or those known in the art, including, for example, those described in Weber et al., Clinical Exp Immunol, 1993; Shapira et al., In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; April 2016, 16-20; Philadelphia, New Orleans, Louisiana (PA): AACR; Cancer Research 2016; 76(14 Suppl): Abstract No. 3805 and Sun et al. (2017) Oncotarget, Vol. 8(No. 31), pp: 51238-51252, each of which is expressly incorporated herein by reference. Humanized anti-CD24 antibodies include, for example, humanized SWA11 antibodies (as described in Arber patent US8,614,301B2; its teachings on humanized antibodies, such as SWA11 antibodies, are incorporated herein by reference), and humanized anti-CD24 antibodies as described in International Patent Publication WO2019222082 (CN103819561A).

[0059] In some cases, the anti-CD24 agent includes an antigen binding region (of an antibody). In some cases, the antigen binding region includes 3 heavy chains and 3 light chain CDRs (e.g., IMGT CDRs) of SWA11, ML5, SN3, or G7s (see, e.g., IMGT CDRs of SWA11 in Table 1). In some cases, the antigen binding region includes 3 heavy chains and 3 light chain CDRs (e.g., IMGTCDRs) of SWA11. In some cases, the antigen binding region includes 3 heavy chains and 3 light chain CDRs (e.g., IMGT CDRs) of ML5. In some cases, the antigen binding region includes 3 heavy chains and 3 light chain CDRs (e.g., IMGT CDRs) of SN3. In some cases, the antigen binding region includes 3 heavy chains and 3 light chain CDRs (e.g., IMGT CDRs) of G7s.

[0060] In some instances, an anti-CD24 agent includes an antigen binding region comprising an amino acid sequence having three heavy chain IMGT CDRs of SWA11 and having 80% or greater (e.g., 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) sequence identity to the SWA11 variable heavy chain sequence of SEQ ID NO:1; and an amino acid sequence having three light chain IMGT CDRs of SWA11 and having 80% or greater (e.g., 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) sequence identity to the SWA11 variable light chain sequence of SEQ ID NO:18 (see, e.g., Table 1). In some cases, the antigen binding region comprises an amino acid sequence having 3 heavy chain IMGT CDRs of SWA11 and having 90% or greater (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) sequence identity with the SWA11 variable heavy chain sequence of SEQ ID NO:1; and an amino acid sequence having 3 light chain IMGT CDRs of SWA11 and having 90% or greater (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) sequence identity with the SWA11 variable light chain sequence of SEQ ID NO:18. In some cases, the antigen binding region comprises an amino acid sequence having 3 heavy chain IMGTCDRs of SWA11 and having 98% or greater (e.g., 99% or greater or 100%) sequence identity with the SWA11 variable heavy chain sequence of SEQ ID NO: 1; and an amino acid sequence having 3 light chain IMGT CDRs of SWA11 and having 98% or greater (e.g., 99% or greater or 100%) sequence identity with the SWA11 variable light chain sequence of SEQ ID NO: 18. In some cases, the antigen binding region comprises the SWA11 variable heavy chain sequence of SEQ ID NO: 1; and the SWA11 variable light chain sequence of SEQ ID NO: 18.

[0061] In some instances, an anti-CD24 agent includes an antigen binding region comprising an amino acid sequence having the three heavy chain IMGT CDRs of SWA11 and having 80% or greater (e.g., 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) sequence identity to a humanized variable heavy chain sequence of any one of SEQ ID NOs: 2-14; and an amino acid sequence having the three light chain IMGT CDRs of SWA11 and having 80% or greater (e.g., 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) sequence identity to a humanized variable light chain sequence of any one of SEQ ID NOs: 19-31 (see, e.g., Table 1). In some cases, the antigen binding region comprises an amino acid sequence having the three heavy chain IMGT CDRs of SWA11 and having 90% or greater (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) sequence identity to a humanized variable heavy chain sequence of any one of SEQ ID NOs: 2-14; and an amino acid sequence having the three light chain IMGT CDRs of SWA11 and having 90% or greater (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) sequence identity to a humanized variable light chain sequence of any one of SEQ ID NOs: 19-31. In some cases, the antigen binding region comprises an amino acid sequence having 3 heavy chain IMGT CDRs of SWA11 and having 98% or greater (e.g., 99% or greater or 100%) sequence identity with a humanized variable heavy chain sequence of any one of SEQ ID NOs: 2-14; and an amino acid sequence having 3 light chain IMGT CDRs of SWA11 and having 98% or greater (e.g., 99% or greater or 100%) sequence identity with a humanized variable light chain sequence of any one of SEQ ID NOs: 19-31. In some cases, the antigen binding region comprises a humanized variable heavy chain sequence of any one of SEQ ID NOs: 2-14; and a humanized variable light chain sequence of any one of SEQ ID NOs: 18-31.

[0062] The term "antibody" is used in the broadest sense herein, and specifically encompasses monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain antibodies only, triabodies, single-chain Fvs, single domain antibodies, nanobodies, etc., and also includes antibody fragments with or without pegylation, as long as they exhibit the desired biological activity (Miller et al. (2003) Journal of Immunology, 170: 4854-4861). Antibodies can be murine, human, humanized, chimeric or from other species. Antibodies, also known as immunoglobulins, typically comprise at least one heavy chain and one light chain, wherein the amino terminal domains of the heavy and light chains are variable in sequence, and are therefore typically referred to as variable region domains, or variable heavy (VH) or variable light (VL) domains. The two domains typically associate to form a specific binding region.

[0063] A "functional" or "biologically active" antibody or antigen binding molecule is one that is able to exert one or more of its natural activities in a structural, regulatory, biochemical or biophysical event. For example, a functional antibody or other binding molecule may have the ability to specifically bind to an antigen, and the binding may in turn initiate or alter a cellular or molecular event, such as signal transduction or phagocytosis. A functional antibody may also block ligand activation of a receptor, either as an agonist or antagonist, or as an allosteric modulator.

[0064] The term antibody can refer to a full-length heavy chain, a full-length light chain, a complete immunoglobulin molecule; or an immunologically active portion of any of these polypeptides, i.e., a polypeptide comprising an antigen binding site that immunospecifically binds to an antigen or a portion thereof of a target of interest, such targets including but not limited to infected cells or cells producing autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may comprise any suitable Fc region, including but not limited to humans or other mammals, such as Cynomorium, IgG, IgE, IgM, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 or subclasses of immunoglobulin molecules, including hybrid Igs, hybrid Fcs and engineered subclasses with altered Fc portions that provide reduced or enhanced effector cell activity. Immunoglobulins may be derived from any species.

[0065] The term "variable" refers to that some parts of the variable domain are very different in sequence between antibodies, and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed in the variable domain of the antibody. It is concentrated in the three segments called hypervariable regions in both the light chain and the heavy chain variable domain. The more highly conserved part of the variable domain is called the framework region (FR). The variable domains of the natural heavy chain and light chain each contain four FRs, mainly adopting a β-folded configuration, connected by three hypervariable regions, forming a loop connecting the β-folded structure, and forming a part of the β-folded structure in some cases. The hypervariable region in each chain is closely held together by FR, and together with the hypervariable region from another chain, it helps to form the antigen binding site of the antibody (see Kabat et al. (1991) "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service Center, National Institutes of Health, Bethesda, Maryland). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0066] The term "hypervariable region" refers herein to the amino acid residues of an antibody that are responsible for antigen binding. A hypervariable region may comprise amino acid residues from a "complementarity determining region" or "CDR", and / or those residues from a "hypervariable loop". "Framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0067] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible naturally occurring mutations that may be present in small amounts, the individual antibodies constituting the population are identical. Monoclonal antibodies have a high degree of specificity for a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to its specificity, the advantage of monoclonal antibodies is that they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" represents the characteristics of an antibody such as obtained from a substantially uniform antibody population, and should not be construed as requiring antibodies to be produced by any particular method.

[0068] The monoclonal antibodies herein specifically include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity. (U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sic. USA, 81: 6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies, which comprise variable domain antigen binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0069] As used herein, a "complete antibody chain" is an antibody chain comprising a full-length variable region and a full-length constant region. A complete "conventional" antibody comprises a complete light chain and a complete heavy chain, as well as a light chain constant domain (CL) and a heavy chain constant domain, CH1, hinge, CH2, and CH3 of a secretory IgG. Other isotypes, such as IgM or IgA, may have different CH and CL domains. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. A complete antibody may have one or more "effector functions," which refer to those biological activities attributed to the Fc constant region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP) phagocytosis; and downregulation of cell surface receptors. Constant region variants include those that change effector distribution, binding to Fc receptors, and the like.

[0070] Depending on the amino acid sequence of the constant domain of its heavy chain, intact antibodies can be assigned to different "classes". There are five major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ and μ, respectively. The subunit structure and three-dimensional configuration of the different classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms (Roux et al. (1998) J. Immunol 161:4083-4090; Lund et al. (2000) Eur. J. Biochem. 267:7246-7256; US 2005 / 0048572; US 2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains.

[0071] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary effector functions include C1q binding; complement dependent cytotoxicity (CDC); Fc-receptor binding; antibody dependent cellular cytotoxicity (ADCC); antibody dependent cellular phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require the Fc region to interact with receptors, such as FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors and recycling receptor FcRn; and can be assessed using various assays disclosed, for example, in the definitions herein.

[0072] A "dead" or silent Fc is an Fc that has been mutagenized to retain activity with respect to, for example, extending serum half-life, but does not bind to or activate low and high affinity Fc receptors. As will be known to those of ordinary skill in the art, a variety of mutations can be used to silence an Fc for a particular function, and any convenient mutation can be used when desired. Examples of silent Fc mutations include, but are not necessarily limited to:

[0073] Human IgG1: L234A; L235A (LALA mutation)

[0074] Human IgG1: L234A; L235A; P329G

[0075] Human IgG4: S228P

[0076] Human IgG4: S228P, F234A, L235A

[0077] Human IgG4 S228P, L235E

[0078] Human IgG4 S228P, L235E, P329G

[0079] Mouse IgG2a: K320A, K322A

[0080] Mouse IgG2a: L234A, L235A, K320A, K322A, P329G

[0081] The antibody specific binding members that can be used include whole antibodies or immunoglobulins of any isotype, and antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies and fusion proteins comprising the antigen-binding portion of antibodies and non-antibody proteins. Antibodies can be detectably labeled, such as with radioisotopes, enzymes that produce detectable products, fluorescent proteins and the like. Antibodies can be further conjugated with other moieties, such as members of specific binding pairs, such as biotin (members of biotin-avidin specific binding pairs), etc. The term also encompasses Fab', Fv, F(ab')2 and / or other antibody fragments that retain specific binding to antigens, and monoclonal antibodies. Antibodies can be monovalent or divalent.

[0082] "Antibody fragments" comprise a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name that reflects the ability to crystallize readily. Pepsin treatment produces F(ab')2 fragments, which have two antigen-binding sites and are still able to cross-link antigen.

[0083] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain that are tightly non-covalently associated. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. In general, these six CDRs give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three CDRs specific to an antigen) has the ability to recognize and bind to an antigen, although the affinity is lower than the entire binding site.

[0084] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment in that a few residues are added at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab' herein in which the cysteine ​​residues of the constant domains carry free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0085] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Immunoglobulins can be assigned to different classes, depending on the amino acid sequences of the constant domains of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0086] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form a desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer, New York, pp. 269-315 (1994).

[0087] Therefore, the antibodies that can be used in conjunction with the present disclosure can encompass monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments (e.g., VH or VL), single-chain Fv antibody fragments, and dsFv antibody fragments. In addition, the antibody molecule can be a fully human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody molecule is a monoclonal fully human antibody.

[0088] Antibodies that can be used in conjunction with the present disclosure can include any mature or unprocessed antibody variable region connected to any immunoglobulin constant region. If the light chain variable region is connected to the constant region, it can be a kappa chain constant region. If the heavy chain variable region is connected to the constant region, it can be a human γ1, γ2, γ3 or γ4 constant region, more preferably γ1, γ2 or γ4, and even more preferably γ1 or γ4.

[0089] In some embodiments, fully human monoclonal antibodies to CD24 are generated using transgenic mice that carry parts of the human immune system rather than the mouse system.

[0090] The present invention encompasses minor changes in the amino acid sequence of an antibody or immunoglobulin molecule, provided that the change in the amino acid sequence maintains at least 75%, such as at least 80%, 90%, 95% or 99% of the sequence. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those occurring within a family of related amino acids in the side chain. By determining the specific activity of a polypeptide derivative, it is easy to determine whether an amino acid change produces a functional peptide. A person of ordinary skill in the art can easily prepare fragments (or analogs) of an antibody or immunoglobulin molecule. The preferred amino-terminal and carboxyl-terminal ends of a fragment or analog appear near the boundaries of a functional domain. By comparing nucleotide and / or amino acid sequence data with a public or proprietary sequence database, structural and functional domains can be identified. Preferably, a computerized comparison method is used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. According to the present invention, sequence motifs and structural conformations can be used to define structural and functional domains.

[0091] In yet other embodiments, the anti-CD24 agent is an agent that is regulated by binding to CD24, such as an agent that inhibits CD24 activity. For example, small molecules that bind to CD24 and inhibit its activity are of interest. Naturally occurring or synthetic small molecule compounds of interest include many chemical categories, such as organic molecules, such as small organic compounds with a molecular weight greater than 50 and less than about 2,500 Daltons. Candidate agents include functional groups for interacting with protein structures, particularly hydrogen bonds, and typically include at least one amine, carbonyl, hydroxyl or carboxyl, preferably at least two of the functional chemical groups. Candidate agents may include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted by one or more of the above-mentioned functional groups. Candidate agents have also been found in biomolecules, including peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. In addition to other methods, these molecules can be identified by using the following screening schemes.

[0092] Figure 1D The anti-fat degeneration efficacy of three small molecules (compounds A, B and C) identified by screening for molecules that bind to CD24 is shown (see the working examples below). This finding (combined with other findings described herein, for example, antibodies with silent Fc regions and antibodies without Fc regions can also be used as anti-fat degeneration agents) indicates that any agent that binds to CD24 or reduces CD24 expression (e.g., anti-CD24 CRISPR agents, such as CRISPR nucleases or CRISPRi, anti-CD24 RNAi agents, such as shRNA or siRNA, anti-CD24 oligonucleotide agents, such as LNA, etc.) can be used in the methods described herein (e.g., as anti-fat degeneration agents).

[0093] Compound A

[0094]

[0095] Compound B

[0096]

[0097] Compound C

[0098]

[0099] In those embodiments where an active agent is administered to a subject, any convenient administration regimen that can produce a desired activity can be used to administer the active agent to the subject. Therefore, the medicament can be incorporated into various formulations, such as pharmaceutically acceptable vehicles for therapeutic administration. More specifically, the medicament of the present invention can be formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into a preparation in the form of a solid, semisolid, liquid or gas, such as a tablet, capsule, powder, granule, ointment (e.g., skin cream), solution, suppository, injection, inhalant and aerosol. Therefore, the administration of the medicament can be achieved in various ways, including oral, oral, rectal, parenteral, intraperitoneal, intradermal, percutaneous, intrathecal, etc. administration.

[0100] In pharmaceutical dosage forms, the agents may be administered in the form of their pharmaceutically acceptable salts, or they may be used alone or in appropriate association, and in combination with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and non-limiting.

[0101] For oral formulations, the medicament can be used alone or in combination with suitable additives to make tablets, powders, granules or capsules, for example, with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch or gelatin; with disintegrants such as corn starch, potato starch or sodium carboxymethyl cellulose; with lubricants such as talc or magnesium stearate; and, if necessary, with diluents, buffers, wetting agents, preservatives and flavoring agents.

[0102] The agent can be formulated into an injection preparation by dissolving, suspending or emulsifying it in an aqueous or non-aqueous solvent (such as vegetable oil or other similar oils, synthetic fatty acid glycerides, higher fatty acid esters or propylene glycol); and if necessary, adding conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0103] The medicament can be used in an aerosol formulation for administration by inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

[0104] In addition, the medicament can be made into a suppository by mixing with a variety of bases (such as an emulsifying base or a water-soluble base). The compounds of the present invention can be administered rectally via a suppository. The suppository may include a vehicle (such as cocoa butter, carbowax, and polyethylene glycol) that melts at body temperature but solidifies at room temperature.

[0105] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, wherein each dosage unit (e.g., teaspoon, tablespoon, tablet, or suppository) contains a predetermined amount of a composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may contain the inhibitor in a composition as a solution in sterile water, physiological saline, or another pharmaceutically acceptable carrier.

[0106] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit doses for human and animal subjects, each unit containing a predetermined amount of the compound of the present invention, a calculated amount sufficient to produce the desired effect in combination with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications of the novel unit dosage form of the present invention depend on the specific compound employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0107] Pharmaceutically acceptable excipients (such as vehicles, adjuvants, carriers or diluents) are readily available. In addition, pharmaceutically acceptable auxiliary substances (such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, etc.) are readily available.

[0108] When the agent is a polypeptide, polynucleotide, its analog or mimetic, it can be introduced into tissues or host cells by a variety of routes, including viral infection, microinjection or vesicle fusion. Jet injection can also be used for intramuscular administration, such as Furth et al. in "Analytical Biochemistry (Anal Biochem.)", (1992) 205: 365-368. DNA can be coated on gold particles and delivered intradermally by a particle bombardment device or a "gene gun" described in the following literature (see, for example, Tang et al., "Nature (Nature)" (1992) 356: 152-154), wherein the gold particles are coated with DNA and then bombarded into skin cells. For nucleic acid therapeutics, many different uses of delivery vehicles have been found, including viral and non-viral vector systems known in the art.

[0109] Those skilled in the art will readily appreciate that dosage levels may vary depending on the function of the particular compound, the nature of the delivery vehicle, etc. The preferred dosage for a given compound is readily determined by those skilled in the art in a variety of ways.

[0110] An "effective amount" or "effective dose" refers to an amount that can improve or slow the progression of a diseased, degenerative or impaired condition. An effective amount can be determined on an individual basis and will be based in part on considerations of the symptoms to be treated and the results sought. An effective amount can be determined by one of ordinary skill in the art using such factors and only routine experimentation.

[0111] In some cases, an effective amount of an anti-CD24 agent will reduce steatosis (liver steatosis). In some cases, the progression of steatosis will be blocked, and in some cases steatosis will be reduced (i.e., the amount of steatosis observed after administration of the anti-CD24 agent will be less than that observed before administration). In some cases, steatosis will be reduced by 10% (i.e., the amount observed will be 90% of the amount present before administration) or more (e.g., 20% or more, 35% or more, 50% or more, 70% or more, 85% or more). In some cases, steatosis will be reduced by 30% (i.e., the amount observed will be 70% of the amount present before administration) or more (e.g., 50% or more, 70% or more, 85% or more). In some cases, steatosis will be reduced by 50% or more.

[0112] A "therapeutically effective dose" or "therapeutic dose" or "therapeutically effective amount" or "therapeutic amount" is an amount sufficient to achieve a desired clinical outcome (i.e., to achieve a therapeutic efficacy). A therapeutically effective dose can be administered in one or more administrations. In some embodiments, the anti-CD24 agent is an antibody specific for human CD24, which is optionally a chimeric or humanized monoclonal antibody. In some embodiments, the anti-CD24 antibody is administered at a dose of less than 8 mg / kg body weight, less than 2.5 mg / kg, less than 1 mg / kg, less than 0.75 mg / kg, less than 0.5 mg / kg, less than 0.25 mg / kg, less than 0.1 mg / kg, less than 0.05 mg / kg, less than 0.01 mg / kg. The therapeutic dose can be, for example, 0.1 to 5 mg / kg, 0.25 to 5 mg / kg, 0.5 to 5 mg / kg, 0.75 to 5 mg / kg, 1 to 5 mg / kg; or 0.1 to 2.5 mg / kg, 0.25 to 2.5 mg / kg, 0.5 to 2.5 mg / kg, 0.75 to 2.5 mg / kg; 0.1 to 1 mg / kg, 0.25 to 1 mg / kg, 0.5 to 1 mg / kg, 0.75 to 1 mg / kg, etc.

[0113] In some embodiments, the anti-CD24 agent is an antibody specific for human CD24, which is optionally a humanized or fully human monoclonal antibody. In some embodiments, the anti-CD24 antibody is administered at a dose of less than 10 mg / kg body weight, less than 8 mg / kg, less than 2.5 mg / kg, less than 1 mg / kg, less than 0.75 mg / kg, less than 0.5 mg / kg, less than 0.25 mg / kg, less than 0.1 mg / kg, less than 0.5 mg / kg, less than 0.1 mg / kg. The therapeutic dose can be, for example, 0.1 to 5 mg / kg, 0.25 to 5 mg / kg, 0.5 to 5 mg / kg, 0.75 to 5 mg / kg, 1 to 5 mg / kg; or 0.1 to 2.5 mg / kg, 0.25 to 2.5 mg / kg, 0.5 to 2.5 mg / kg, 0.7 to 2.5 mg / kg; 0.1 to 1 mg / kg, 0.25 to 1 mg / kg, 0.5 to 1 mg / kg, 0.75 to 1 mg / kg, etc.

[0114] In some embodiments, the anti-CD24 agent is an antibody specific for human CD24, whose Fc has been silenced (i.e., its Fc has been dead). Silent mutations are known to those of ordinary skill in the art, and any convenient silent mutation can be used. Examples of silent Fc mutations include, but are not necessarily limited to: L234A / L235A (LALA mutation) (human IgG1), L234A / L235A / P329G (human IgG1), S228P (human IgG4), S228P / F234A / L235A (human IgG4), S228P / L235E (human IgG4), S228P / L235E / P329G (human IgG4), K320A / K322A (mouse IgG2a), and L234A / L235A / K320A / K322A / P329G (mouse IgG2a).

[0115] In some embodiments, an Fc-silent anti-CD24 antibody can be administered at a higher dose than a non-Fc-silent anti-CD24 antibody, such that it can be administered at a dose of less than 100 mg / kg body weight, less than 90 mg / kg body weight, less than 80 mg / kg body weight, less than 70 mg / kg body weight, less than 60 mg / kg body weight, less than 50 mg / kg, less than 40 mg / kg body weight, less than 30 mg / kg body weight, less than 20 mg / kg body weight, less than 10 mg / kg body weight, less than 8 mg / kg, less than 2.5 mg / kg, less than 1 mg / kg, less than 0.75 mg / kg, less than 0.5 mg / kg, less than 0.25 mg / kg, less than 0.1 mg / kg, less than 0.5 mg / kg, less than 0.1 mg / kg. The therapeutic dose can be, for example, 0.1 to 5 mg / kg, 0.25 to 5 mg / kg, 0.5 to 5 mg / kg, 0.75 to 5 mg / kg, 1 to 5 mg / kg; or 0.1 to 2.5 mg / kg, 0.25 to 2.5 mg / kg, 0.5 to 2.5 mg / kg, 0.7 to 2.5 mg / kg; 0.1 to 1 mg / kg, 0.25 to 1 mg / kg, 0.5 to 1 mg / kg, 0.75 to 1 mg / kg, etc. In some embodiments, the anti-CD24 agent is a small molecule (see, e.g., Compounds A, B, and C). An effective dosage can be expected to be in the range of about 0.001 to about 100 mg / kg body weight / day (for humans), in some cases about 0.1 to about 50 mg / kg body weight / day, in some cases about 1 to about 50 mg / kg body weight / day, in some cases about 5 to about 40 mg / kg body weight / day, in some cases about 2 to about 15 mg / kg body weight / day, and in some cases about 25 to about 40 mg / kg body weight / day.

[0116] Dosage and frequency can vary depending on the half-life of the anti-CD24 agent. Those skilled in the art will appreciate that these guidelines will be adjusted for the molecular weight of the active agent, for example, in the use of antibody fragments, the use of antibody conjugates, the use of anti-CD24 agents, etc. Dosages can also vary for local administration, such as intranasal, inhalation, etc., or for systemic administration, such as intramuscular, intraperitoneal, intravenous, subcutaneous, etc.

[0117] The course of treatment can be less than about 12 weeks, less than about 8 weeks, less than about 4 weeks, and can be, for example, 1 to 12 weeks, 2 to 12 weeks, 4 to 12 weeks, 4 to 8 weeks, etc. Administration can be once a week, twice a week, every other day, every day, twice a day, once every two weeks, etc., and in some embodiments, once a week. In some embodiments, more than one course of treatment is administered. In some cases, the course of treatment is in the range of 1 to 24 weeks (for example, 1 to 8 weeks, 1 to 4 weeks, about 1 week, about 2 weeks or about 3 weeks). In some cases, the course of treatment is in the range of 1 day to 8 weeks (for example, 1 day to 6 weeks, 1 day to 4 weeks, 1 day to 2 weeks, 1 day to 7 days, 3 days to 8 weeks, 3 days to 6 weeks, 3 days to 4 weeks, 3 days to 2 weeks or 3 days to 7 days). In some cases, the compound is administered for 2 or more days (for example, 3 or more days, 4 or more days, 5 or more days, 6 or more days, or 7 or more days).

[0118] Anti-CD24 agents can be administered by any suitable means, including topical, oral, parenteral, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal, or subcutaneous administration. Anti-CD24 agents can be administered in any medically acceptable manner. This can include injection by a parenteral route, such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumoral, intraperitoneal, intraventricular, intradural, or other routes, as well as oral, intranasal, intraocular, rectal, or topical. Sustained release administration is also specifically included in the present disclosure, by means such as long-acting injections or erodible implants. Local delivery is particularly contemplated, by means such as delivery to one or more arteries (such as the renal artery or a blood vessel supplying a localized tumor) via a catheter.

[0119] The administration of anti-CD24 agents can be combined with other therapeutic approaches to reduce fatty liver. This approach may include, but is not limited to, regulating alcohol intake, dietary changes, exercise, other agents for the treatment of fatty liver, such as metformin, vitamin E, etc. The terms "co-administered", "co-administered" and "combined with..." include the simultaneous, parallel or sequential administration of two or more therapeutic agents (e.g., anti-CD24 agents and additional agents, two different anti-CD24 agents, etc.) without specific time limits. In one embodiment, the agents are present in cells or subjects at the same time, or exert their biological or therapeutic effects at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, the first agent may be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the second therapeutic agent. Administration of the anti-CD24 agent may be combined with co-administration of any number of additional agents. The anti-CD24 agent is not required, but is optionally formulated with one or more agents that enhance activity or otherwise increase the therapeutic effect. These are generally used in the same dosages and administration routes as used herein, or 1% to 99% of the dosages heretofore used.In some embodiments, treatment is accomplished by administering to the subject a combination (co-administration) of an anti-CD24 agent and / or another agent that modulates target cells.

[0120] Treatment can also be combined with other active agents, such as antibiotics, cytokines, etc. Classes of antibiotics include penicillins, such as penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins combined with β-lactamase inhibitors, cephalosporins (e.g., cefaclor, cefazolin, cefuroxime, ramoxef, etc.); carbapenems; monoamide rings; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; chloramphenicol; metronidazole; spectinomycin; trimethoprim; vancomycin, etc. Cytokines, such as interferon gamma, tumor necrosis factor alpha, interleukin 12, etc., can also be included.

[0121] Examples of diseases / disorders that can be treated with anti-CD24 agents include, but are not limited to: (1) obesity; (2) obesity-related diseases such as hypertension, dyslipidemia, coronary heart disease, gallbladder disease, osteoarthritis, sleep apnea and breathing problems, psychiatric disorders such as clinical depression, anxiety and other psychiatric disorders, ischemic cardiovascular disorders, hyperlipidemia, hyperuricemia, insulin resistance, non-insulin-dependent diabetes mellitus (NIDDM or type II diabetes), insulin-dependent diabetes mellitus (IDDM or type I diabetes), diabetes-related complications (including microangiopathy, ocular lesions, retinopathy, neuropathy and nephropathy), cardiovascular disease (including heart failure, coronary artery failure, and hypertension), atherosclerosis, atherosclerotic disease, stroke, syndrome X, forms of cancer (such as uterine cancer, breast cancer, colorectal cancer, kidney cancer, and gallbladder cancer), and high cholesterol levels, (3) fatty liver (e.g., non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)); and (4) fatty liver-related diseases such as obesity with high abdominal fat content, hypertension, diabetes (type 2), high cholesterol, high triglyceride levels, and sleep apnea.

[0122] In some embodiments, the subject is a subject undergoing a liver transplant, and the anti-CD24 agent is used to enhance liver graft function and / or prolong graft survival.

[0123] Reagent test kit

[0124] A kit for the method is also provided. The subject kit may include an anti-CD24 agent. In some embodiments, the anti-CD24 agent is provided in a dosage form (e.g., a therapeutically effective dosage form). In the case of a kit, the anti-CD24 agent may be provided in any convenient packaging (e.g., strip packaging, dosage packaging, etc.) in liquid or solid form. The agents of the kit may be present in the same or separate containers. The agents may also be present in the same container. In addition to the above components, the subject kit may further include (in certain embodiments) instructions for practicing the subject method. These instructions may be present in the subject kit in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may exist is as printed information on a suitable medium or substrate, such as one or more sheets of paper with information printed thereon, in the packaging of the kit, in a package insert, and the like. Another form of these instructions is a computer-readable medium with information recorded thereon, such as a disk, a compact disk (CD), a flash drive, etc. Another form of these instructions that may exist is a website address.

[0125] EXEMPLARY NON-LIMITING ASPECTS OF THE DISCLOSURE

[0126] Aspects (including embodiments) of the above-mentioned subject matter of the present invention may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the present disclosure are provided below (see setting A and setting B). As will be understood by those of ordinary skill in the art after reading this disclosure, each of the separately numbered aspects can be used or combined with any preceding or following separately numbered aspects. This is intended to provide support for the combination of all such aspects, and is not limited to the combination of aspects explicitly provided below. It is obvious to those of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the present invention.

[0127] Setting A

[0128] 1. A method for treating obesity using an anti-CD24 agent.

[0129] 2. A method for treating obesity-related diseases using an anti-CD24 agent.

[0130] 3. A method for treating fatty liver and fatty liver related diseases using an anti-CD24 agent.

[0131] 4. The method according to 2, wherein the obesity-related disease is non-alcoholic fatty liver disease (NAFLD)

[0132] 5. The method according to 3, wherein fatty liver and fatty liver-related diseases are non-alcoholic fatty liver disease (NAFLD)

[0133] 6. A method of reducing steatosis in an individual, the method comprising: contacting the individual with a therapeutically effective dose of an anti-CD24 agent for a period of time sufficient to reduce steatosis.

[0134] 7. A method for enhancing liver graft function by administering an anti-CD24 agent to a patient receiving a liver transplant

[0135] 8. A method of administering an anti-CD24 agent to a patient receiving a liver transplant to prolong graft survival.

[0136] 9. The method according to any one of 1 to 8, wherein the anti-CD24 agent is selected from: an anti-CD24 antibody, an anti-CD24 antibody whose Fc is engineered to have silent ADCC and / or ADCP and / or complement binding activity, an anti-CD24 fragment antigen binding region (Fab) or an anti-CD24 pegylated Fab fragment, an anti-CD24 single-chain variable fragment (scFv), a peptide that binds to CD24, and a small molecule that binds to CD24.

[0137] 10. The method according to any one of 1 to 9, wherein the course of treatment is 1 to 24 weeks.

[0138] 11. The method according to any one of 1 to 9, wherein the course of treatment is 1 to 8 weeks.

[0139] 12. The method according to any one of 1 to 9, wherein the course of treatment is 1 to 4 weeks.

[0140] 13. The method according to any one of 1 to 9, wherein the course of treatment is 1 week.

[0141] 14. The method according to any one of 1 to 9, wherein the anti-CD24 agent can be combined with another treatment modality including but not limited to: dietary modification, bariatric surgery or other therapy.

[0142] 15. The method according to any one of 1 to 14, wherein the anti-CD24 agent is an anti-CD24 antibody that binds to the Leucine-Alanine-Proline motif of CD24.

[0143] 16. The method according to any one of 1 to 14, wherein the anti-CD24 agent is a humanized anti-CD24 antibody.

[0144] Setting B

[0145] 1. A method of reducing steatosis in an individual, the method comprising: administering to an individual with steatosis a therapeutically effective dose of an anti-CD24 agent for a period of time sufficient to reduce the steatosis.

[0146] 2. The method according to 1, wherein the subject suffers from non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0147] 3. The method according to 1 or 2, wherein the anti-CD24 agent is an anti-CD24 antibody.

[0148] 4. The method of claim 3, wherein the anti-CD24 antibody comprises an Fc having one or more silent mutations.

[0149] 5. The method according to 3 or 4, wherein the anti-CD24 antibody is an anti-CD24 fragment antigen binding region (Fab) or an anti-CD24 PEGylated Fab fragment.

[0150] 6. The method according to 3 or 4, wherein the anti-CD24 antibody is an anti-CD24 single-chain variable fragment (scFv).

[0151] 7. The method of any one of 3 to 6, wherein the anti-CD24 antibody comprises 3 heavy chain and 3 light chain CDRs of SWA11, ML5, SN3 or G7s.

[0152] 8. The method according to any one of 3 to 7, wherein the anti-CD24 antibody is a humanized antibody.

[0153] 9. The method according to any one of 3 to 6, wherein the anti-CD24 antibody comprises an amino acid sequence having 3 heavy chain IMGT CDRs of SWA11 and having 80% or greater sequence identity with the humanized variable heavy chain sequence of SEQ ID NOs: 2-14; and an amino acid sequence having 3 light chain IMGT CDRs of SWA11 and having 80% or greater sequence identity with the humanized variable light chain sequence of SEQ ID NOs: 19-31.

[0154] 10. The method according to 1 or 2, wherein the anti-CD24 agent is a small molecule that binds to CD24.

[0155] 11. The method according to any one of 1 to 10, wherein the course of treatment is 1 to 8 weeks.

[0156] 12. The method according to any one of 1 to 11, wherein the administration is combined with dietary modification and / or bariatric surgery.

[0157] 13. The method of any one of 1 to 12, wherein the anti-CD24 agent is co-administered with another therapeutic agent.

[0158] 14. The method according to any one of 1 to 13, wherein the individual is a liver transplant patient, and the administration is for enhancing liver graft function and / or prolonging liver graft survival.

[0159] 15. The method according to any one of 1 to 14, wherein the individual suffers from fatty liver, a fatty liver-related disease, obesity, or an obesity-related disease.

[0160] The following examples are offered by way of illustration and not limitation.

[0161] Examples

[0162] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure.

[0163] General methods of molecular and cellular biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd edition, (Sambrook et al., Cold Spring Harbor Laboratory Press, 2001); Short Protocols in Molecular Biology, 4th edition, (Ausubel et al., eds., John Wiley & Sons, 1999); Protein Methods (Bollag et al., John Wiley & Sons, 1996); Nonviral Vectors for Gene Therapy (Wagner et al., eds., Academic Press, 1999); Viral Vectors (Kaplift and Loewy, Academic Press, 1995); Immunology Methods Manual (I. Lefkovits, ed., Academic Press, 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology. Culture: Laboratory Procedures in Biotechnology (Doyle and Griffiths, John Wiley & Sons, 1998), the disclosure of which is incorporated herein by reference. The agents, cloning vectors, cells and kits used in the methods mentioned or involved in the present disclosure are available from commercial suppliers such as Bio-Rad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Biotech, etc., and repositories such as, for example, Adyne, American Type Culture Collection (ATCC), etc.

[0164] When incubated with fatty acids, the cultured cells can have increased fat infiltration, also known as steatosis. Here, we show that this cell fat infiltration can be reversed when treated with anti-CD24 agents. Anti-CD24 agents may include, but are not limited to, anti-CD24 antibodies, anti-CD24 fragment antigen binding regions (Fab), anti-CD24 single-chain variable fragments (scFv), peptides bound to CD24, anti-CD24 siRNA / shRNA, anti-CD24 oligonucleotide antisense, such as locked nucleic acids (LNA) targeting CD24 or any other oligonucleotide antisense, anti-CD24 CRISPR and small molecules bound to CD24. Then, we tried to determine the therapeutic efficacy of anti-CD24 agents in various in vivo models of fatty liver. When placed on a high-fat diet, mice can be made to suffer from fatty liver. Mice with certain genetic mutations, such as mice homozygous for the obesity spontaneous mutation (ob), also known as ob / ob mice (Jackson Laboratory), develop steatosis under a regular diet. In addition, these ob / ob mice gain weight rapidly and show features of metabolic syndrome, such as hyperglycemia and glucose intolerance. In ob / ob mice, when they are placed on a high-fat diet, liver steatosis is further accelerated and becomes more severe. Using either in vivo model, we show that anti-CD24 agents have significant therapeutic efficacy in alleviating liver steatosis. In addition, this treatment can lead to a decrease in liver enzymes such as ALT, AST, alkaline phosphatase and GGT, and improve liver function. In order to further demonstrate the therapeutic efficacy of anti-CD24 agents in an in vivo model that can more physiologically reproduce human fatty liver disease, we utilized an in vivo model in which NSG mice can be transplanted with human hepatocytes. When these mice were placed on a high-fat diet, we found that treatment with anti-CD24 was also able to counteract steatosis and improve graft function and graft survival.

[0165] Administration of anti-CD24 agents inhibits the development of hepatic steatosis in cell culture

[0166] We identified the anti-steatosis efficacy of therapeutic agents bound to CD24 in a cell culture system. Specifically, Huh7 cells (which are liver hepatoma cells) were cultured in 1% DMSO (Huh7D cells) and then supplemented with adipose medium to induce steatosis. According to previous literature (35), adipose medium was prepared by a 1:1 mixture of oleic acid (Sigma Aldrich) and palmitic acid (Sigma Aldrich) with 10% FFA-free bovine serum albumin (BSA) solution (Sigma Aldrich) in PBS. In brief, 100mM oleic acid and palmitic acid were prepared in 99% methanol. Palmitic acid was prepared by shaking at 500rpm for 20 minutes at 50°C. 10mM oleic acid and palmitic acid were conjugated with 10% BSA by incubation at 50°C for 30 minutes. 100μM FFA mixture was dissolved in hepatocyte culture medium. Cells were incubated in adipose medium for 4 days to induce fatty liver. The cells were then treated with anti-CD24 agents for 3 days in the presence of adipose medium. To determine the extent of steatosis, cells were stained with 1 mg / ml Nile Red solution (Sigma-Aldrich) in cell culture medium at 37°C for 15 minutes and the fluorescent signal was measured. The cells were then treated with a variety of anti-CD24 agents, including anti-CD24 antibodies, anti-CD24 fragment antigen binding (Fab), anti-CD24 humanized antibodies, and small molecules that bind to CD24, all of which showed anti-steatosis efficacy (see Figure 1).

[0167] Table 1: Antibody sequences used in Figure 1 (IMGT CDRs are bold and underlined)

[0168]

[0169]

[0170]

[0171] About small molecules ( Figure 1DCompounds A, B and C): Virtual screening of different chemical libraries was performed for human CD24. In short, alphafold2 was used to fold the sequence corresponding to the mature extracellular domain of human CD24. According to the established method, Schrödinger software suite (Maestro, version 12.4, Schrödinger, New York, New York, USA) was used for docking. Different compound libraries were extracted into SDF format and imported into Maestro, and docking was prepared using LigPrep. The ionization state of all compounds was generated at the target pH 7±2 using an ionizer. The folded human CD24 sequence was prepared with a protein preparation wizard, in which the order of hydrogen and bonds was specified, heteroatom states were generated, and constrained minimization was performed with the OPLS_2005 force field. High-throughput docking was performed, and the ligand posture was scored using the GlideScore scoring function. Subsequently, the binding of the top candidate compounds found by virtual screening for human CD24 was functionally determined. Figure 1D The anti-steatosis efficacy of three small molecules identified in this manner (compounds A, B, and C) was demonstrated. This finding suggests that any agent that binds to CD24 or reduces CD24 expression (e.g., an anti-CD24 CRISPR agent, such as a CRISPR nuclease or CRISPRi, an anti-CD24 RNAi agent, such as shRNA or siRNA, an anti-CD24 oligonucleotide agent, such as LNA, etc.) can be used in the methods described herein (e.g., as an anti-steatosis agent).

[0172] Compound A

[0173]

[0174] Compound B

[0175]

[0176] Compound C

[0177]

[0178] We then sought to determine the therapeutic effect of targeting CD24 on fatty liver disease in vivo.

[0179] Administration of anti-CD24 antibody inhibits hepatic steatosis

[0180] 6-8 week old Rag-deficient (T-cell and B-cell deficient) mice were administered 25 μg of rat IgG2b isotype control or rat anti-mouse CD24 antibody 2x / week subcutaneously for 3-4 weeks. One day after the first dose, mice were placed on a high-fat diet (60% kcal from fat). Liver was collected 4 weeks later and liver histology obtained in the form of hematoxylin and eosin (H&E) staining. All mice tolerated the antibody well. As shown in representative liver histology sections in Figure 2, mice on a high-fat diet developed significant hepatic steatosis (A), while mice on a high-fat diet treated with anti-CD24 antibody developed essentially little or no hepatic steatosis.

[0181] In addition to histological improvements, improvements in hepatic steatosis can be monitored by ultrasound. Figure 3 As shown in representative images in , liver ultrasound of mice treated with control IgG on a HFD (top) showed increased echogenicity (aka hyperechoic or the liver appears whiter) compared to mice treated with an anti-CD24 agent (bottom).

[0182] Administration of anti-CD24 antibody reversed hepatic steatosis within 1 week

[0183] 8-week-old ob / ob mice were placed on a HFD (60% kcal from fat) for 1 week to accelerate the development of steatosis. They were then maintained on a HFD and 50 μg of IgG control or anti-CD24 antibody was administered subcutaneously daily for 6 days. Excitingly, as shown in the representative histological images in Figure 4, mice on a high-fat diet developed severe hepatic steatosis (A), while mice on a high-fat diet treated with anti-CD24 antibody for only 1 week showed significantly more normal liver architecture and significantly less steatosis (B).

[0184] Administration of anti-CD24 antibodies may be more effective in reversing hepatic steatosis when combined with dietary modification.

[0185] Although ob / ob mice treated with anti-CD24 for 1 week on HFD showed significantly less steatosis compared to control-treated mice, significant steatosis was still present. Alternatively, we hypothesized that a combination treatment of anti-CD24 and dietary changes (i.e., returning ob / ob mice to a normal diet (4% kcal from fat) while treating them with anti-CD24) could more effectively reverse hepatic steatosis. To test this hypothesis, after 1 week on HFD, ob / ob mice were returned to regular chow and treated with either IgG control or anti-CD24 for 6 days. Indeed, as shown in the representative histological images in Figure 5, mice treated with IgG control still showed significant steatosis (A), while mice treated with anti-CD24 showed little steatosis.

[0186] In addition to the histological improvement of steatosis, administration of anti-CD24 antibody also resulted in improvement in serum liver chemistries as evidenced by decreases in serum ALT, AST, Alphos, GGT, cholesterol ( Figure 6 ) and was well tolerated.

[0187] Administration of anti-CD24 antibody improves liver function in mice with fatty liver

[0188] Having demonstrated that anti-CD24 agents can show improvements in liver histology in mice that develop fatty liver disease, and improve serum liver chemistry, we wanted to determine if anti-CD24 agents could show improvements in liver function. One such function of the liver is drug metabolism. One known drug that is extensively metabolized by the liver is clemizole. We hypothesized that drug metabolism in severely steatotic livers might be impaired compared to normal livers, and that anti-CD24 agents could restore liver function in addition to improving liver histology and serum chemistry. To test this hypothesis, ob / ob mice were placed on a HFD (60% kcal from fat) for 1 week. One week after fat induction, mice were subcutaneously administered 25 μg / day of IgG control or anti-CD24 daily while still on a HFD. Eight days after the first dose of antibody, mice were PO administered 25 mg / kg of clemizole-HCl (vehicle: water). Thirty minutes after administration, mice were euthanized, serum was collected, and clemizole and the major mouse metabolite (M14) were determined via quantitative mass spectrometry. 1 Indeed, as shown in Figure 7, ob / ob mice treated with IgG on a HFD showed impairment of liver metabolism, as shown by the much higher serum clemizole levels in IgG-treated mice compared to anti-CD24-treated mice (A). This was confirmed by the simultaneous increase in serum concentrations of the major mouse metabolite (M14) in mice treated with anti-CD24 compared to IgG controls (B). Taken together, this suggests that anti-CD24 treatment improves liver function.

[0189] Administration of anti-CD24 antibody whose Fc was silenced for ADCC activity still effectively inhibited steatosis

[0190] Since some antibodies may produce unwanted toxicity due to antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), we sought to determine whether anti-CD24 antibodies whose Fc lacks ADCC and / or ADCP and therefore has reduced toxicity can still maintain anti-steatosis activity. To test this hypothesis, we utilized anti-CD24 antibodies whose Fc domains have been engineered to contain point mutations that abolish binding to Fcγ receptors. These mutations resulted in silent ADCC activity (Absolute Antibodies). Ob / ob mice were placed on a HFD for 1 week and then administered IgG or ADCC-silent anti-CD24 antibodies (anti-CD24 [YBM 5.10.4], mouse IgG2a, Fc-silent κ; product code: Ab00210-2.3, from Absolute Antibodies) while on a regular diet for 1 week, after which the livers were removed for histological examination. Indeed, as shown in representative histological images in FIG8 , mice treated with IgG control still showed significant steatosis (A), whereas mice treated with ADCC-silencing anti-CD24 antibody showed much less steatosis.

[0191] In mice with humanized livers, administration of anti-CD24 agents improved human liver engraftment, maintained human albumin, and Reduce fatty degeneration

[0192] To determine the effect of anti-CD24 agents in a more physiological model of human fatty liver disease, we used a mouse model that can transplant human livers. Briefly, NSG mice with humanized livers were prepared using the RAAPID-TKG method. These mice contain chimeric livers containing both mouse and human hepatocytes, and the extent of human liver engraftment can be determined by measuring serum human albumin. Two mice with similar degrees of human liver engraftment (measured by baseline serum human albumin) were placed on a HFD for 6 weeks and then treated with an IgG control or an anti-CD24 antibody (which we call MOSW3) whose Fc has been engineered to contain point mutations that eliminate ADCC, ADCP, and complement binding activity, i.e., an Fc-silent antibody. The antibody was administered twice a week at 25 μg. Excitingly, as Fig. 9A As shown, treatment with MOSW3 showed longer graft survival, as evidenced by maintenance of serum human albumin, while mice treated with IgG control showed progressive loss of human liver graft, as evidenced by a steady decline in serum human albumin. This suggests that treatment with anti-CD24 agents improves the synthetic function of the liver, i.e., albumin production. CT scans were also performed 3 weeks after treatment, in which, in contrast to ultrasound, fat appears as dark areas. Compared to mice treated with MOSW3 ( Fig. 9C ) mice treated with IgG showed higher levels of steatosis ( Fig. 9B ).

[0193] The results suggest that therapeutic administration of anti-CD24 agents can reduce steatosis and increase survival of human liver transplants. Anti-CD24 agents may be used to treat fatty liver prior to transplantation or after transplantation in liver transplant recipients.

[0194] Administration of anti-CD24 antibodies can slow the rate of weight gain during a high-fat diet.

[0195] Although all mice treated with anti-CD24 agents at doses showing therapeutic benefit for fatty liver disease showed no signs of weight loss or other toxicity, we observed that in some cases, therapeutic administration of anti-CD24 agents could slow the rate of weight gain in mice on a HFD, and this was observed in both Rag- / - and ob / ob- mice. This suggests that anti-CD24 agents have therapeutic benefits for other obesity-related diseases. (See Figure 10).

[0196] In at least some of the previously described embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions, and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter as defined in the appended claims.

[0197] Those skilled in the art will understand that, in general, the terms used herein, especially in the appended claims (e.g., the bodies of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "includes" should be interpreted as "including but not limited to", etc.). Those skilled in the art will further understand that if a specific number of claim recitations is intended to be introduced, such intent will be explicitly stated in the claim, and in the absence of such a statement, such intent is absent. For example, to aid understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be interpreted as implying that the introduction of a claim recitation by the indefinite article "a" and / or "an" will limit any particular claim that includes such introduced claim recitation to multiple embodiments that include only one such recitation, even if the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article such as "a" and / or "an" (e.g., "a" and / or "an" should generally be interpreted as meaning "at least one" or "one or more"); the same applies to the use of definite articles used to introduce claim recitations. Furthermore, even if a specific number of an introduced claim reference is explicitly referenced, one skilled in the art will recognize that such reference should be interpreted as indicating at least the number of references (e.g., a simple reference of "two references" without other modifiers means at least two references, or two or more references). Furthermore, where a convention similar to “at least one of A, B, and C, etc.” is used, generally, those skilled in the art will use such construction in the sense of understanding the convention (e.g., “a system having at least one of A, B, and C” will include, but is not limited to, A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Where a convention similar to “at least one of A, B, or C, etc.” is used, generally, those skilled in the art will use such construction in the sense of understanding the convention (e.g., “a system having at least one of A, B, or C” will include, but is not limited to, A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further understand that, whether in the specification, claims, or drawings, virtually any disjunctive word and / or phrase indicating two or more alternative terms should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase “A or B” will be understood to include the possibility of “A” or “B” or “A and B”.

[0198] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0199] As will be understood by those skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily identified as fully describing and enabling the same range to be decomposed into at least equal halves, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled in the art, all languages ​​such as "up to", "at least", "greater than", "less than", etc. include the cited numbers and refer to the ranges that can be subsequently decomposed into sub-ranges as described above. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group with 1 to 3 articles refers to a group with 1, 2 or 3 articles. Similarly, a group with 1 to 5 articles refers to a group with 1, 2, 3, 4 or 5 articles, etc.

[0200] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0201] Therefore, the above only illustrates the principle of the present invention.It should be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown in this article, embody the principle of the present invention and are included in its spirit and scope.In addition, all examples and conditional language described in this article are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventor to promote this field, and should be interpreted as not being limited to such specific examples and conditions.In addition, all statements describing the principles, aspects and embodiments of the present invention and their specific examples are intended to cover both their structural equivalents and functional equivalents.In addition, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, that is, any element that performs the same function developed, regardless of the structure.In addition, regardless of whether such disclosure is explicitly described in the claims, any content disclosed herein is not intended to be dedicated to the public.

[0202] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being cited for limitation in the claims only when the exact phrase "means for..." or the exact phrase "step for..." is cited at the beginning of such limitation in the claims; if such exact phrase is not used in the limitation in the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is not cited.

Claims

1. A method of reducing steatosis in an individual, the method comprising: A therapeutically effective dose of an anti-CD24 agent is administered to an individual having steatosis for a period of time sufficient to reduce the steatosis.

2. The method of claim 1, wherein the subject suffers from non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

3. The method of claim 1 or claim 2, wherein the anti-CD24 agent is an anti-CD24 antibody.

4. The method of claim 3, wherein the anti-CD24 antibody comprises an Fc having one or more silent mutations.

5. The method of claim 3 or claim 4, wherein the anti-CD24 antibody is an anti-CD24 fragment antigen binding region (Fab) or an anti-CD24 PEGylated Fab fragment.

6. The method of claim 3 or claim 4, wherein the anti-CD24 antibody is an anti-CD24 single chain variable fragment (scFv).

7. The method of any one of claims 3 to 6, wherein the anti-CD24 antibody comprises three heavy chain and three light chain CDRs of SWA11, ML5, SN3 or G7s.

8. The method according to any one of claims 3 to 7, wherein the anti-CD24 antibody is a humanized antibody.

9. The method according to any one of claims 3 to 6, wherein the anti-CD24 antibody comprises an amino acid sequence having the three heavy chain IMGT CDRs of SWA11 and having 80% or greater sequence identity with the humanized variable heavy chain sequence of SEQ ID NOs: 2-14; and an amino acid sequence having the three light chain IMGT CDRs of SWA11 and having 80% or greater sequence identity with the humanized variable light chain sequence of SEQ ID NOs: 19-31.

10. The method of claim 1 or claim 2, wherein the anti-CD24 agent is a small molecule that binds to CD24.

11. The method according to any one of claims 1 to 10, wherein the course of treatment is 1 to 8 weeks.

12. The method according to any one of claims 1 to 11, wherein the administration is combined with dietary modification and / or bariatric surgery.

13. The method of any one of claims 1 to 12, wherein the anti-CD24 agent is co-administered with another therapeutic agent.

14. The method according to any one of claims 1 to 13, wherein the individual is a liver transplant patient, and the administration is for enhancing liver graft function and / or prolonging liver graft survival.

15. The method of any one of claims 1 to 14, wherein the subject suffers from fatty liver, a fatty liver-related disease, obesity, or an obesity-related disease.

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