Methods and compositions for treating obesity, obesity-related disorders, fatty liver, and fatty liver-related disorders using anti-CD24 agents - Patents.com

Abstract

Methods and compositions are provided for treating obesity, obesity-related disorders, fatty liver, and fatty liver-related disorders. 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 for use in practicing embodiments of the methods are also provided.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 344,410, filed May 20, 2022, which is incorporated by reference in its entirety.

[0002] Inclusion by reference of sequence listing provided as a sequence listing Xml file The Sequence Listing is provided herewith as 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. [Background technology]

[0003] Introduction Obesity and obesity-related diseases are the main causes of morbidity and mortality worldwide. One of the main diseases associated with obesity is fatty liver disease. In addition to obesity, fatty liver disease can result from other causes. For example, it can be the result of excessive alcohol consumption, genetic predisposition, viral hepatitis, exposure to chemicals such as perfluorooctanoic acid (PFA), and metabolic syndrome. It is estimated that one billion people worldwide and about one-third of the US population, i.e., 100 million Americans, have 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.

[0004] In addition, 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 may lead to increased mortality and morbidity in liver transplant patients. Summary of the Invention

[0005] Methods and compositions are provided for treating obesity, obesity-related disorders, fatty liver, and fatty liver-related disorders. Aspects of the methods include administering to a subject in need thereof an effective amount of an anti-CD24 agent to treat the subject. Methods and compositions are provided for reducing steatosis (hepatic steatosis). Aspects of the methods include administering to a subject in need thereof an effective amount of an anti-CD24 agent to reduce steatosis (hepatic steatosis). Compositions for use in practicing embodiments of the methods are also provided.

[0006] The inventors have discovered that anti-CD24 agents can reduce steatosis (hepatic steatosis) and therefore can be used to treat many diseases / 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., G7 scFv), can be used to reduce steatosis. Thus, any binding agent that targets / binds CD24 will function to reduce steatosis. To this end, the inventors have demonstrated that known small molecules screened for the ability to bind to CD24 can indeed reduce steatosis and therefore can be used to treat diseases (e.g., fatty liver and obesity-related diseases) (see Examples below). The inventors have recognized that any anti-CD24 agent is useful for reducing steatosis / useful for treatment, i.e., the treatment is not limited to any particular anti-CD24 antibody or anti-CD24 antibody, instead, any anti-CD24 agent, including any CD24 binding agent, functions to reduce steatosis. For example, the inventors have recognized 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 oligos, LNA, etc.), CRISPR agents (e.g., CRISPR nuclease / cleavage activity, CRISPRi, etc.), are also active in reducing steatosis.

[0007] Methods of reducing steatosis in an individual are provided, the methods comprising administering to an individual having steatosis a therapeutically effective dose of an anti-CD24 agent 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).

[0008] In some embodiments, the anti-CD24 agent is an anti-CD24 antibody. In some cases, the anti-CD24 antibody comprises an Fc with one or more silencing 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 the three heavy chain CDRs and the three light chain CDRs of SWA11, ML5, SN3, or G7. In some cases, the anti-CD24 antibody is a humanized antibody. In some cases, 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 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 sequence identity to a humanized variable light chain sequence of any one of SEQ ID NOs: 19-31. In some cases, the anti-CD24 agent is a small molecule that binds to CD24.

[0009] In some embodiments, the course of treatment for an individual ranges from 1 to 8 weeks. In some cases, administration of the anti-CD24 agent is combined with another treatment modality, such as dietary modification and / or weight loss surgery. In some cases, the anti-CD24 agent is co-administered with another therapeutic agent.

[0010] In some embodiments, the individual is a liver transplant patient and the administration is to enhance liver graft function and / or prolong liver graft survival, hi some embodiments, the individual has fatty liver, a fatty liver-related disease, obesity, or an obesity-related disease. [Brief description of the drawings]

[0011] [Figure 1A] Figure 1A-1D: Relative lipid accumulation of Huh 7D cells when cultured on fat medium and treated with various anti-CD24 agents. Agents were added at 1 μg / mL unless otherwise specified in the Fab. MOSW1, SWA11, ML5, SN3: mouse anti-human CD24 antibody, MOSW2: SWA11 antibody with point mutations to silent complement activity, MOSW3: SWA11 antibody with point mutations to silent complement activity, antibody-dependent cellular phagocytosis (ADCC) and antibody-dependent cellular phagocytosis (ADCP), Fab: Fab of SWA11 antibody, G7: 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-lipidogenic activity of SWA-11, Fab, TW2, TW6 and G7S; Figure 1B: Anti-lipidogenic activity of V2, V3, V4, V6, V7, V8, V9, V10, V14, V15, V16; Figure 1C: Anti-lipidogenic activity of SWA11, Fab, ML5, SN3, MOSW1, MOSW2, MOSW3; Figure 1D: Anti-lipidogenic activity of three small molecules that bind to CD24. [Figure 1B] Please see the legend to FIG. 1A. [Figure 1C] Please see the legend to FIG. 1A. [Figure 1D] Please see the legend to FIG. 1A. [Diagram 2] Figures 2A-2B: H&E staining of livers from mice on HFD treated with IgG control (Figure 2A) versus anti-CD24 agent (Figure 2B). [Diagram 3] Liver ultrasound of mice on a high fat diet treated with IgG control (top) compared to anti-CD24 agent (bottom). [Figure 4] Figures 4A-4B: H&E staining of livers from ob / ob mice on HFD treated for 1 week with IgG control (Figure 4A) versus anti-CD24 agent (Figure 4B). [Diagram 5] Figures 5A-5B: H&E staining of livers from ob / ob mice returned to normal chow after a HFD and treated for 1 week with IgG control (Figure 5A) versus anti-CD24 agent (Figure 5B). [Figure 6] Serum liver chemistry in HFD-induced ob / ob mice treated with isotype IgG or anti-CD24 agents. [Figure 7] 7A-7B: Metabolism of clemizole in ob / ob mice treated with IgG control or anti-CD24 agents. [Figure 8] Figures 8A-8B: H&E staining of livers from HFD-induced ob / ob mice on a normal diet and treated for 1 week with IgG control (Figure 8A) versus ADCC-silent anti-CD24 antibody (Figure 8B). [Figure 9] Figures 9A-9C: Weekly serum human albumin in mice with humanized livers treated with MOSW3 IgG control for 3 weeks after a 6-week HFD (Figure 9A) and corresponding CT scans of livers after 3 weeks of IgG control treatment (Figure 9B) or MOSW3 treatment (Figure 9C). [Figure 10] 10A-10B: Body weights of mice treated with IgG control or anti-CD24 agents. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] definition The following description utilizes several terms that are conventionally used in the art. 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.

[0013] 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 a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0014] 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 a naturally occurring amino acid. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., 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 a naturally occurring amino acid. An "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.

[0015] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic 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.

[0016] The term "sample" with respect to a patient includes blood and other liquid samples of biological origin, solid tissue samples, such as biopsy specimens or tissue cultures, or cells and their progeny derived therefrom. This definition also includes specimens that have been manipulated in any way after procurement, such as by treatment with reagents, washing, or enrichment for certain cell populations. This definition also includes samples enriched for certain types of molecules, e.g., nucleic acids, polypeptides, etc.

[0017] The term "biological sample" encompasses clinical samples, including tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, aspirates, and the like. A "biological sample" includes a sample containing target cells and / or normal control cells, or suspected of containing such 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 containing infected cells, etc. from a patient, can also include non-infected cells.

[0018] The term "diagnosis" is used herein to refer to the identification of a molecular or pathological state, disease or condition.

[0019] The term "prognosis" is used herein to refer to a prediction of the likelihood of disease progression (eg, progression of an infection), including relapse, drug resistance, and the like.

[0020] 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 physician may predict the likelihood that a patient will survive.

[0021] As used herein, the term "specific binding member" refers to a member of a specific binding pair (i.e., two molecules, usually 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, via non-covalent means).

[0022] The term "specific binding" refers to a direct association between two molecules, for example due to covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bonding interactions (including interactions such as salt bridges and water bridges). A specific binding member describes a member of a pair of molecules that has binding specificity for one another. Members of a specific binding pair may be naturally occurring or may be wholly or partially synthetically produced. One member of the pair of molecules has an area, or cavity, on its surface that specifically binds to, and is therefore complementary to, a particular spatial and polar organization of the other member of the pair of molecules. Thus, the members of the pair have the property of specifically binding to one another. Examples of specific binding member pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. Specific binding members of a binding pair exhibit high affinity and binding specificity for binding to one another. Typically, the affinity between the specific binding members of a pair is greater than 10 -8 M or less, e.g., 10 -15 Including M and below, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 10 including M and below -7 M and under, etc. -6 K below M d (dissociation constant). "Affinity" refers to the strength of binding, with increased binding affinity being correlated with a lower KD.

[0023] In one embodiment, affinity is determined by surface plasmon resonance (SPR), for example, as used by the Biacore system. The affinity of one molecule to another molecule is determined by measuring the on-rate of the interaction, for example, the on-rate at 25°C. "Affinity" refers to the strength of binding, and an increase in binding affinity is correlated with a lower KD. In one embodiment, affinity is determined by surface plasmon resonance (SPR), for example, as used by the Biacore system. The affinity of one molecule to another molecule is determined by measuring the on-rate of the interaction, for example, the on-rate at 25°C.

[0024] The methods described herein may include multiple steps. Each step may be performed after a predetermined time between steps, as desired. Thus, the time between 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, including 5 hours or more. In certain embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting time after the completion of the previous step, for example, a waiting time of several minutes to overnight.

[0025] As used herein, the terms "evaluating," "determining," "measuring," and "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

[0026] The term "separating" as used herein refers to the physical separation of two elements (e.g., by size or affinity, etc.) as well as the disintegration of one element while leaving the other intact.

[0027] Detailed Description Methods and compositions are provided for treating obesity, obesity-related disorders, fatty liver, and fatty liver-related disorders. 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 for use in practicing embodiments of the methods are also provided.

[0028] Before the present invention is described in more detail, it is to be understood that the invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0029] Where a range of values ​​is provided, unless the context clearly indicates otherwise, it is understood that each intervening value is included, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value in that stated range. The upper and lower limits of these smaller ranges may individually be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0030] Certain ranges are presented herein with the term "about" preceding the numerical values. In this specification, the term "about" is used to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is close to or approximately a specifically recited number, the near or approximate unrecited number may be a number that provides the substantial equivalent of the specifically recited number in the context in which it is presented.

[0031] 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 this 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 exemplary methods and materials are now described.

[0032] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the date of the present application 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 publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0033] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely" and "only" in connection with the recitation of claim elements, or for the use of a "negative" limitation.

[0034] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0035] Although the apparatus and methods have been or will be described in functional descriptions for grammatical fluidity, it is expressly understood that the claims should not be construed as necessarily limited in any way by the syntax of "means" or "step" limitations unless expressly recited under 35 U.S.C. 112, but should be accorded the full scope of meaning and equivalents of the definitions provided by the claims under the doctrine of judicial equivalents, and that the claims, when expressly recited under 35 U.S.C. 112, should be accorded the full statutory equivalents under 35 U.S.C. 112.

[0036] Methods and compositions are provided for treating obesity, obesity-related disorders, fatty liver, and fatty liver-related disorders. 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 for use in practicing embodiments of the methods are also provided.

[0037] "Treatment" means that at least an improvement in one or more symptoms associated with the target condition afflicting the subject is achieved, and improvement is used in a broad sense to refer to at least a reduction in the parameters, e.g., the magnitude of symptoms associated with the target condition being treated. Thus, treatment also includes situations in which a pathological condition or at least the symptoms associated therewith are completely inhibited, e.g., prevented from occurring, or stopped, e.g., terminated, such that the adult mammal is no longer afflicted with the target condition or at least the symptoms that characterize the disorder. In some instances, "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents the disease or its symptoms, and / or therapeutic, in that it partially or completely cures the disease and / or the adverse effects caused by the disease. "Treatment" may be any treatment of a disease in a mammal, including (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, i.e., halting its development, or (c) relieving the disease, i.e., causing regression of the disease. Treatment may result in a variety of different physical symptoms, such as modulation of gene expression, rejuvenation of tissues or organs, etc. Treatment of ongoing disease (treatment stabilizes or reduces undesirable clinical symptoms in the patient) is performed in some embodiments. Such treatment may be performed prior to complete loss of function in the affected tissue. The subject therapies may be administered during, and in some cases after, the symptomatic stage of the disease.

[0038] A therapeutic treatment is one in which the subject is afflicted prior to administration, and a prophylactic treatment is one in which the subject is not afflicted prior to administration. In some embodiments, the subject has an increased likelihood of becoming afflicted or is suspected of being afflicted prior to treatment. In some embodiments, the subject is suspected of having an increased likelihood of becoming afflicted.

[0039] 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 with the methods include dogs and cats, horses, cows, sheep, etc., as well as primates, including humans. The subject methods, compositions, and reagents can also be applied to animal models, including small mammals, e.g., murines, lagomorphs, etc., for example, in experimental investigations. The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic 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. In some embodiments, the individual is not a human (e.g., a non-human mammal).

[0040] Anti-CD24 Agents. As summarized above, the method embodiments 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) for a target CD24, such as human CD24, sufficient to provide the desired treatment. As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents, and "affinity" can be expressed as a dissociation constant (Kd). The affinity may be at least 1-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 20-fold higher, at least 30-fold higher, at least 40-fold higher, at least 50-fold higher, at least 60-fold higher, at least 70-fold higher, at least 80-fold higher, at least 90-fold higher, at least 100-fold higher, or at least 1000-fold higher, or more, than the affinity of the antibody for an unrelated amino acid sequence. The affinity of a specific binding member for a target protein may 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 more. The term "binding" refers to a direct association between two molecules by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. In some embodiments, the anti-CD24 agent binds to human CD24 with nanomolar or picomolar affinity. In some embodiments, the anti-CD24 agent binds to human CD24 with a Kd of less than about 100 nM, less than 50 nM, less than 20 nM, less than 20 nM, or less than 1 nM. In some cases, the anti-CD24 agent is an antibody. In some embodiments, the antibody binds to human CD24 with nanomolar or picomolar affinity. In some embodiments, the antibody binds to human CD24 with a Kd of less than about 100 nM, less than 50 nM, less than 20 nM, less than 20 nM, or less than 1 nM.

[0041] Anti-CD24 agents include, but are not limited to, antibodies against CD24 (with or without Fc effector functions), fragment antigen binding regions (Fabs) against CD24, single chain variable fragments (scFvs) against CD24, peptides that bind to CD24, RNA interference agents (siRNA / shRNA agents) against CD24, oligonucleotide antisense agents against CD24 (e.g., locked nucleic acid (LNA) or any other oligonucleotide antisense targeting CD24), CRISPR agents targeting CD24 (e.g., CRISPR nuclease / cleavage activity to reduce CD24 expression at the DNA level via genome editing, CRISPRi agents to reduce CD24 expression, etc.), and small molecules that bind to CD24 (e.g., see compounds A, B, and C in Figure 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.

[0042] Examples of CD24-specific binding members include CD24 antibodies and binding fragments thereof. Non-limiting examples of such antibodies include antibodies directed against any epitope of CD24. Bispecific antibodies, i.e. antibodies in which each of the two binding domains recognizes a different binding epitope, are also included.

[0043] CD24 is a two-chain glycosylphosphatidylinositol (GPI)-anchored glycoprotein that is expressed at multiple stages of B cell development, beginning with the bone marrow pro-B cell compartment and continuing through mature surface Ig-positive B cells. Expression on plasma cells is very low or negative. It is also expressed in the majority of 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 Genbank NP_001278666, NP_001278667, NP_001278668, NP_037362, NP_001346013.

[0044] Antibodies known to bind human CD24 are known and commercially available, including, but not limited to, SWA11 (Creative Biolab), MA5-11833, 12-0247-42, anti-CD24 clone ML5 (Biolegend), SN3A5-2H10 (also called SN3), ALB9, EPR19925, EPR3006(N), SWA21, SWA22, OKB2, and the like. Anti-CD24 agents can include, for example, antibodies that bind human CD24, such as SN3 or ML5. G7 is a known scFv against human and mouse CD24. Anti-CD24 antibodies can, for example, bind to the LAP (leucine-alanine-proline) motif, as known in the art. Alternatively, antibodies specific for human CD24 can be generated.

[0045] Humanized anti-CD24 antibodies may be generated de novo, such as those disclosed herein, or those known in the art, for example, those described by Weber et al, Clinical Exp Immunol, 1993; Shapira et al. in: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016; 76(14 Suppl): Abstract nr 3805 and Sun et al. (2017) Oncotarget Vol. 8, (No. 31), pp: 51238-51252, each of which is specifically incorporated herein by reference. Humanized anti-CD24 antibodies include, for example, the humanized SWA11 antibody (described in Arber Patent US8,614,301B2; incorporated herein by reference for its teachings regarding humanized antibodies, e.g., the SWA11 antibody), and the humanized anti-CD24 antibody described in International Patent Publication No. WO 2019 / 222082 (CN103819561A).

[0046] In some cases, the anti-CD24 agent comprises an antigen-binding region (of an antibody). In some cases, the antigen-binding region comprises three heavy chain CDRs and three light chain CDRs (e.g., IMGT CDRs) of SWA11, ML5, SN3, or G7 (see, e.g., the IMGT CDRs of SWA11 in Table 1). In some cases, the antigen-binding region comprises three heavy chain CDRs and three light chain CDRs (e.g., IMGT CDRs) of SWA11. In some cases, the antigen-binding region comprises three heavy chain CDRs and three light chain CDRs (e.g., IMGT CDRs) of ML5. In some cases, the antigen-binding region comprises three heavy chain CDRs and three light chain CDRs (e.g., IMGT CDRs) of SN3. In some cases, the antigen-binding region comprises three heavy chain CDRs and three light chain CDRs (e.g., IMGT CDRs) of G7.

[0047] In some cases, the anti-CD24 agent comprises an antigen-binding region comprising an amino acid sequence having the three heavy chain IMGT CDRs of SWA11 and having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity with the SWA11 variable heavy chain sequence of SEQ ID NO: 1, and an amino acid sequence having the three light chain IMGT CDRs of SWA11 and having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity with 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 the three heavy chain IMGT CDRs of SWA11 and having 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity with the SWA11 variable heavy chain sequence of SEQ ID NO:1, and an amino acid sequence having the three light chain IMGT CDRs of SWA11 and having 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, 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 the three heavy chain IMGT CDRs of SWA11 and having 98% or more (e.g., 99% or more, or 100%) sequence identity with the SWA11 variable heavy chain sequence of SEQ ID NO: 1, and an amino acid sequence having the three light chain IMGT CDRs of SWA11 and having 98% or more (e.g., 99% or more, 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.

[0048] In some cases, the anti-CD24 agent comprises an antigen-binding region comprising an amino acid sequence having the three heavy chain IMGT CDRs of SWA11 and having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 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 more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 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 more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, 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 more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, 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 the three heavy chain IMGT CDRs of SWA11 and having 98% or more (e.g., 99% or more, 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 98% or more (e.g., 99% or more, 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 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.

[0049] The term "antibody" herein is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain only antibodies, triple chain antibodies, single chain Fvs, single domain antibodies, nanobodies, etc., including antibody fragments, with or without PEGylation, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. Antibodies, also referred to as immunoglobulins, traditionally comprise at least one heavy chain and one light chain, with the amino terminal domains of the heavy and light chains being variable in sequence and thus commonly referred to as variable region domains, or variable heavy (VH) domains or variable light (VL) domains. The two domains traditionally associate to form a specific binding region.

[0050] A "functional" or "biologically active" antibody or antigen-binding molecule is one that is capable of exerting one or more of its native activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule may have the ability to specifically bind to an antigen, which binding may then trigger or alter cellular or molecular events such as signal transduction or phagocytosis. A functional antibody may also block ligand activation of a receptor, and may act as an agonist or antagonist, or as an allosteric regulator.

[0051] The term antibody may refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptides, i.e., a polypeptide that contains an antigen binding site that immunospecifically binds to an antigen or portion thereof of a target of interest, including, but not limited to, infected cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may include any suitable Fc region, including, but not limited to, IgG, IgE, IgM, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, or subclasses of immunoglobulin molecules, including hybrid Ig, hybrid Fc, and engineered subclasses with modified Fc portions that reduce or enhance effector cell activity, from humans or other mammals, such as cynomolgus monkeys. The immunoglobulins may be derived from any species.

[0052] The term "variable" refers to the fact that certain portions of the variable domains vary widely in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments, called hypervariable regions, in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FRs that largely adopt a beta-sheet configuration, connected by three hypervariable regions that form loops that connect and, in some cases, form part of the beta-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). 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).

[0053] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are involved in antigen binding. A hypervariable region may comprise amino acid residues from the "complementarity determining regions" or "CDRs" and / or those residues from the "hypervariable loops". "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0054] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method.

[0055] Antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567, and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies that include variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0056] As used herein, an "intact antibody chain" is one that includes a full-length variable region and a full-length constant region. An intact "conventional" antibody includes an intact light chain and an intact heavy chain, as well as the light chain constant domain (CL) and the heavy chain constant domains, CH1, hinge, CH2, and CH3, for secreted IgG. Other isotypes, such as IgM or IgA, may have different CH and CL domains. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Intact antibodies may have one or more "effector functions," which refer to those biological activities attributable to the Fc constant region of an antibody (native sequence Fc region or amino acid sequence variant Fc region). 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 down-regulation of cell surface receptors. Constant region variants include those that alter the effector profile and binding to Fc receptors.

[0057] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact immunoglobulin antibodies, IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations 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; US2005 / 0048572; US2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0058] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary effector functions include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular phagocytosis (ADCC), antibody-dependent cellular phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require the Fc region to interact with a receptor, e.g., FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and the recycling receptor, FcRn, and may be assessed, for example, using the various assays disclosed in the definitions herein.

[0059] A "dead" or silenced Fc is one that has been mutated to retain activity, for example, in terms of extending serum half-life, but does not bind to or activate low and high affinity Fc receptors. As known to those skilled in the art, various mutations can be used to silence an Fc for a particular function, and any convenient mutation can be used as needed. Examples of silencing Fc mutations include, but are not necessarily limited to, the following: ●Human IgG1: L234A; L235A (LALA mutation) ● Human IgG1: L234A, L235A, P329G- ●Human IgG4: S228P ● Human IgG4: S228P, F234A, L235A Human IgG4 S228P, L235E Human IgG4 S228P, L235E, P329G Mouse IgG2a: K320A, K322A Mouse IgG2a: L234A, L235A, K320A, K322A, P329G

[0060] Antibody specific binding members that may be used include intact antibodies or immunoglobulins of any isotype, as well as fragments of antibodies that retain specific binding to the antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. Antibodies may be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, and the like. Antibodies may be further conjugated to other moieties, such as members of specific binding pairs, such as biotin (a member of the biotin-avidin specific binding pair). Also encompassed by the term are Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to the antigen, as well as monoclonal antibodies. Antibodies may be monovalent or bivalent.

[0061] "Antibody fragments" include intact antibodies, e.g., a portion of 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 (see Zapata et al., Protein Eng. 8(10):1057-1062 (1995)), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking to antigen.

[0062] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight non-covalent association. In this structure, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site.

[0063] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0064] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequences of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclassifications (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0065] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of 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 the desired structure for antigen binding. For a general review of sFvs, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0066] Thus, antibodies that may be used in connection with the present disclosure may include 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. Furthermore, the antibody molecule may be a fully human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody molecule is a monoclonal fully human antibody.

[0067] The antibodies that can be used in connection with the present disclosure can include any antibody variable region, mature or unprocessed, linked to any immunoglobulin constant region. When a light chain variable region is linked to a constant region, it can be a kappa chain constant region. When a heavy chain variable region is linked to a constant region, it can be a human gamma 1, gamma 2, gamma 3 or gamma 4 constant region, more preferably gamma 1, gamma 2 or gamma 4, even more preferably gamma 1 or gamma 4.

[0068] In some embodiments, fully human monoclonal antibodies against CD24 are generated using transgenic mice carrying parts of the human immune system rather than the mouse system.

[0069] Minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are encompassed by the present invention, provided that the variations in the amino acid sequence maintain at least 75%, e.g., at least 80%, 90%, 95%, or 99% of the sequence. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Fragments (or analogs) of an antibody or immunoglobulin molecule can be readily prepared by one of skill in the art. Preferred amino and carboxy termini of fragments or analogs occur near the boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods are known to identify protein sequences that fold into known three-dimensional structures. The sequence motifs and structural conformations can be used to define structural and functional domains in accordance with the present invention.

[0070] In yet another embodiment, the anti-CD24 agent is an agent that modulates, e.g., inhibits, CD24 activity by binding to CD24. 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 numerous chemical classes, e.g., organic molecules, e.g., small organic compounds with a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents contain functional groups for structural interaction with proteins, particularly hydrogen bonding, typically at least an amine, carbonyl, hydroxyl, or carboxyl group, 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 with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Such molecules may be identified by using, among others, the screening protocols described below.

[0071] Figure ID shows the anti-adipose efficacy of three small molecules (compounds A, B, and C) identified by screening for molecules that bind to CD24 (see Examples below). This finding (combined with other findings described herein, such as Fc region silenced antibodies and antibodies without Fc regions also function as anti-adipose agents) demonstrates 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) can be used in the methods described herein (e.g., as an anti-adipose agent). TIFF2025517381000002.tif71128TIFF2025517381000003.tif146128

[0072] In embodiments in which an active agent is administered to a subject, the active agent can be administered to the subject using any convenient administration protocol that can bring about the desired activity.Accordingly, the agent can be incorporated into various formulations, for example, pharma-ceutically acceptable vehicles, for therapeutic administration.More specifically, the agent of the present invention can be formulated into pharmaceutical compositions by combining with suitable pharma-ceutically acceptable carriers or diluents, and can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments (e.g., skin creams), solutions, suppositories, injections, inhalants and aerosols.Accordingly, administration of the agent can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, etc. administration.

[0073] In pharmaceutical dosage forms, the agents may be administered in the form of their pharma- ceutically acceptable salts, or they may be used alone or in suitable association and combination with other pharma- ceutically active compounds. The following methods and excipients are merely illustrative and in no way limiting.

[0074] For oral preparations, the agents may be used alone or in combination with suitable excipients to produce tablets, powders, granules or capsules, for example, with conventional excipients such as lactose, mannitol, corn starch or potato starch, with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatin, with disintegrating agents such as corn starch, potato starch or sodium carboxymethylcellulose, with lubricants such as talc or magnesium stearate, and, where appropriate, with diluents, buffers, wetting agents, preservatives and flavorings.

[0075] The agent can be formulated into a preparation for injection by dissolving, suspending, or emulsifying in an aqueous or non-aqueous solvent such as vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, and conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifying agents, stabilizers, and preservatives can be used as necessary.

[0076] The agents can be utilized in aerosol formulations to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0077] Furthermore, the agent can be made into a suppository by mixing with various bases such as emulsifying bases or water-soluble bases. The compound of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowax, and polyethylene glycol, which melt at body temperature but solidify at room temperature.

[0078] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, with each unit dosage form, e.g., teaspoon, tablespoon, tablet, or suppository, containing a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may contain the inhibitors in a composition as a solution in sterile water, saline, or another pharma- ceutically acceptable carrier.

[0079] The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined amount of a compound of the present invention calculated in association with a pharma- ceutically acceptable diluent, carrier, or vehicle, in an amount sufficient to produce the desired effect. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0080] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. Moreover, pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are readily available to the public.

[0081] If the agent is a polypeptide, polynucleotide, analog or mimic thereof, the agent may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described in Furth et al., Anal Biochem. (1992) 205: 365-368. DNA may be coated onto gold microparticles and delivered intradermally by particle bombardment devices, or by "gene guns" as described in the literature (see, for example, Tang et al., Nature (1992) 356: 152-154). Here, gold microprojectiles are coated with DNA and then shot into skin cells. For nucleic acid therapeutic agents, several different delivery vehicles are used, including viral and non-viral vector systems, as known in the art.

[0082] Those of skill in the art will readily appreciate that dosage levels can vary depending on the particular compound, the nature of the delivery vehicle, etc. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0083] An "effective amount" or "effective dose" refers to an amount that can ameliorate or slow the progression of a disease, degenerative, or damaging condition. An effective amount can be determined on an individual basis and is based, in part, on a consideration of the condition being treated and the results desired. An effective amount can be determined by one of ordinary skill in the art using such factors and no more than routine experimentation.

[0084] In some cases, an effective amount of an anti-CD24 agent reduces steatosis (hepatic steatosis). In some cases, the progression of steatosis is blocked, and in some cases steatosis is reduced (i.e., the amount of steatosis observed after administration of an anti-CD24 agent is less than the amount observed before administration). In some cases, steatosis will be reduced by 10% or more (e.g., 20% or more, 35% or more, 50% or more, 70% or more, 85% or more) (i.e., the amount observed will be 90% of the amount present before administration). 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.

[0085] A "therapeutically effective dose" or "therapeutic dose" or "therapeutically effective amount" or "therapeutic amount" is an amount sufficient to produce a desired clinical outcome (i.e., achieve 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, 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, or less than 0.01 mg / kg. The therapeutic dose may 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.

[0086] In some embodiments, the anti-CD24 agent is an antibody specific for human CD24, optionally a humanized or fully human monoclonal antibody, hi 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 may 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.

[0087] In some embodiments, the anti-CD24 agent is an antibody specific for human CD24 that is Fc silent (i.e., Fc dead). Silencing mutations are known to those skilled in the art, and any convenient silencing mutation can be used. Examples of silencing Fc mutations include, but are not 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).

[0088] In some embodiments, Fc-silenced anti-CD24 antibodies may be administered at higher doses than non-Fc-silenced anti-CD24 antibodies, such as at doses 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, 0.1-5 mg / kg, 0.25-5 mg / kg, 0.5-5 mg / kg, 0.75-5 mg / kg, 1-5 mg / kg, or 0.1-2.5 mg / kg, 0.25-2.5 mg / kg, 0.5-2.5 mg / kg, 0.7-2.5 mg / kg, 0.1-1 mg / kg, 0.25-1 mg / kg, 0.5-1 mg / kg, 0.75-1 mg / kg, etc. In some embodiments, the anti-CD24 agent is a small molecule (see, e.g., Compounds A, B, and C). An effective dose can be expected to be in the range (for humans) of about 0.001 to about 100 mg / kg body weight per day, in some cases about 0.1 to about 50 mg / kg body weight per day, in some cases about 1 to about 50 mg / kg body weight per day, in some cases about 5 to about 40 mg / kg body weight per day, in some cases about 2 to about 15 mg / kg body weight per day, and in some cases about 25 to about 40 mg / kg body weight per day.

[0089] Dosage and frequency may vary depending on the half-life of the anti-CD24 agent. One of skill in the art will appreciate that such guidelines are adjusted for the molecular weight of the active agent, e.g., the use of antibody fragments, the use of antibody conjugates, the use of anti-CD24 agents, etc. Dosage may also vary by local administration, e.g., intranasal, inhalation, etc., or systemic administration, e.g., im, ip, iv, sc, etc.

[0090] The treatment course may be less than about 12 weeks, less than about 8 weeks, less than about 4 weeks, e.g., 1-12 weeks, 2-12 weeks, 4-12 weeks, 4-8 weeks, etc. Administration may be once a week, twice a week, every other day, daily, twice a day, every two weeks, etc., and in some embodiments, once a week. In some embodiments, two or more treatment courses are administered. In some cases, the treatment course is in the range of 1-24 weeks (e.g., 1-8 weeks, 1-4 weeks, about 1 week, about 2 weeks, or about 3 weeks). In some cases, the treatment course is in the range of 1 day to 8 weeks (e.g., 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 (eg, 3 or more days, 4 or more days, 5 or more days, 6 or more days, or 7 or more days).

[0091] The anti-CD24 agent can be administered by any suitable means, including topical, oral, parenteral, intrapulmonary, and intranasal. Parenteral infusion includes intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal, or subcutaneous administration. The anti-CD24 agent can be administered in any medically acceptable manner. This may include injection by parenteral routes, such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumoral, intraperitoneal, intraventricular, intradural, or other, and oral, nasal, ocular, rectal, or topical. Sustained release administration is also specifically included in the present disclosure, by means such as depot injection or erodible implant. Local delivery is specifically contemplated, by means such as delivery via a catheter to one or more arteries, such as the renal artery or blood vessels supplying a local tumor.

[0092] The administration of anti-CD24 agents may be combined with other therapeutic methods to reduce fatty liver. Such modes may include, but are not limited to, alcohol intake control, dietary changes, exercise, metformin, vitamin E, and other agents for treating fatty liver. The terms "co-administration", "co-administration" and "in combination with" include administration of two or more therapeutic agents (e.g., an anti-CD24 agent and an additional agent, two different anti-CD24 agents, etc.) simultaneously, in parallel, or sequentially within an unspecified time limit. In one embodiment, the agents are present in the cell or in the subject's body 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 before), in parallel, or sequentially (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) administration of 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 optionally, but not necessarily, formulated with one or more agents that enhance activity or otherwise increase therapeutic effect. These are generally used in the same dosages and routes of administration as used herein, or at 1-99% of the dosages previously used. In some embodiments, treatment is accomplished by administering a combination (co-administration) of the subject anti-CD24 agent and / or another agent that opsonizes the target cells.

[0093] Treatment may also be combined with other active agents such as antibiotics, cytokines, etc. Antibiotic classes include penicillins, such as penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins in combination with β-lactamase inhibitors, cephalosporins, such as cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycin; polymyxins; sulfonamides; quinolones; chloramphenicol; metronidazole; spectinomycin; trimethoprim; vancomycin, etc. Cytokines may also be included, such as interferon gamma, tumor necrosis factor alpha, interleukin 12, etc.

[0094] Examples of diseases / conditions that can be treated with anti-CD24 agents include, but are not limited to, the following: (1) obesity; (2) obesity-related diseases such as hypertension, dyslipidemia, coronary heart disease, gallbladder disease, osteoarthritis, sleep apnea and respiratory disorders, psychiatric diseases 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), microvascular disease, eye disease, retinal disease, Diabetes-related complications including neuropathy, and renal lesions; cardiovascular disease (including heart failure, coronary artery insufficiency, and hypertension), arteriosclerosis, atherosclerosis, stroke, syndrome X, forms of cancer such as uterine, breast, colon, kidney, and gallbladder, and high cholesterol levels; (3) fatty liver (e.g., nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH)); and (4) fatty liver-related diseases such as obesity with high rates of abdominal fat, hypertension, diabetes mellitus (type II), high cholesterol, high triglyceride levels, and sleep apnea.

[0095] In some embodiments, the individual has undergone a liver transplant and the anti-CD24 agent is used to enhance liver graft function and / or prolong graft survival.

[0096] kit Also provided are kits for use in the subject methods. The kits of the present invention 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 context of a kit, the anti-CD24 agent can be provided in liquid or solid form in any convenient packaging (e.g., stick pack, dose pack, etc.). The agents of the kit may be in the same or separate containers. The agents may also be in the same container. In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, such as on a piece of paper on which the information is printed, in the kit packaging, in a package insert, etc. Yet another form of these instructions is a computer readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a flash drive, etc. Yet another form in which these instructions may be present is a website address.

[0097] Exemplary Non-Limiting Aspects of the Disclosure The above aspects, including the embodiments of the subject matter, may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the above description, certain non-limiting aspects of the present disclosure are provided below (see Set A and Set B). Each individually numbered aspect may be used or combined with any of the individually numbered aspects that precede or follow it, as will be apparent to a person skilled in the art upon reading this disclosure. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below. It will be apparent to a person skilled in the art that various changes and modifications may be made without departing from the spirit or scope of the present invention.

[0098] Set A 1. A method of treating obesity using an anti-CD24 agent. 2. A method of treating obesity-related diseases using an anti-CD24 agent. 3. A method for treating fatty liver and fatty liver-related diseases using an anti-CD24 agent. 4. The method of 2, wherein the obesity-related disease is nonalcoholic fatty liver disease (NAFLD). 5. The method according to 3, wherein the fatty liver and fatty liver-related diseases are nonalcoholic fatty liver disease (NAFLD). 6. A method of reducing steatosis in an individual, comprising contacting the individual with a therapeutically effective dose of an anti-CD24 agent for a period of time sufficient to reduce steatosis. 7. A method of administering anti-CD24 agents to patients undergoing liver transplantation to enhance liver graft function. 8. A method of administering anti-CD24 agents to patients undergoing liver transplantation to prolong graft survival. 9. Anti-CD24 agents are Antibodies against CD24, antibodies against CD24 engineered such that their Fc has silent ADCC and / or ADCP and / or complement binding activity, fragment antigen binding regions (Fab) against CD24 or PEGylated Fab fragments against CD24, single chain variable fragments (scFv) against CD24, peptides that bind to CD24, small molecules that bind to CD24 Any of methods 1 to 8 selected from the above. 10. Any of methods 1 to 9, wherein the treatment course is 1 to 24 weeks. 11. Any of methods 1 to 9, wherein the treatment course is 1 to 8 weeks. 12. Any of methods 1 to 9, wherein the treatment course is 1 to 4 weeks. 13. Any of methods 1 to 9, wherein the treatment course is one week. 14. Any of the methods 1-9, wherein the anti-CD24 agent can be used in combination with another treatment modality, including but not limited to dietary modification, weight loss surgery, or other therapies. 15. Any of the methods of 1-14, wherein the anti-CD24 agent is an anti-CD24 antibody that binds to the leucine-alanine-proline motif of CD24. 16. Any of the methods of 1-14, wherein the anti-CD24 agent is a humanized anti-CD24 antibody.

[0099] Set B 1. A method of reducing adiposity in an individual, comprising administering to an individual having adiposity a therapeutically effective dose of an anti-CD24 agent for a period of time sufficient to reduce adiposity. 2. The method of 1, wherein the subject has nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). 3. The method of 1 or 2, wherein the anti-CD24 agent is an anti-CD24 antibody. 4. The method of 3, wherein the anti-CD24 antibody comprises an Fc having one or more silencing mutations. 5. The method of 3 or 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 3 or 4, wherein the anti-CD24 antibody is an anti-CD24 single chain variable fragment (scFv). 7. Any one of the methods 3-6, wherein the anti-CD24 antibody comprises the three heavy chain CDRs and the three light chain CDRs of SWA11, ML5, SN3, or G7. 8. Any one of the methods 3 to 7, wherein the anti-CD24 antibody is a humanized antibody. 9. Any one of the methods 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 more sequence identity to any one of the humanized variable heavy chain sequences of SEQ ID NOs: 2 to 14, and an amino acid sequence having the three light chain IMGT CDRs of SWA11 and having 80% or more sequence identity to any one of the humanized variable light chain sequences of SEQ ID NOs: 19 to 31. 10. The method of 1 or 2, wherein the anti-CD24 agent is a small molecule that binds to CD24. 11. Any of methods 1 to 10, wherein the treatment course is 1 to 8 weeks. 12. Any of the methods 1 to 11, wherein the administration is in combination with dietary modification and / or weight loss surgery. 13. Any of the methods 1-12, wherein the anti-CD24 agent is co-administered with another therapeutic agent. 14. Any of methods 1 to 13, wherein the individual is a liver transplant patient and the administration is to enhance liver graft function and / or prolong liver graft survival. 15. Any of the methods 1 to 14, wherein the individual has fatty liver, a fatty liver-related disease, obesity, or an obesity-related disease.

[0100] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0101] The following examples are presented to provide those 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 numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0102] General methods in molecular and cellular biochemistry are described in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001), Short Protocols in Molecular Biology, 4th Ed. (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 & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press, 1997), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons Methods for the preparation of recombinant human cloning vectors and / or recombinant human cloning vectors can be found in standard textbooks such as Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and / or other sources, including ...

[0103] When incubated with fatty acids, cultured cells may have increased fatty infiltration, also known as steatosis. Here, we have shown that this cellular fatty infiltration can be reversed when treated with anti-CD24 agents. Anti-CD24 agents may include, but are not limited to, antibodies against CD24, fragment antigen binding regions (Fabs) against CD24, single chain variable fragments (scFvs) against CD24, peptides that bind to CD24, siRNA / shRNA against CD24, oligonucleotide antisense against CD24 such as locked nucleic acid (LNA) targeting CD24 or any other oligonucleotide antisense, CRISPR against CD24, and small molecules that bind to CD24. We then sought to determine the therapeutic efficacy of anti-CD24 agents in various in vivo models of fatty liver. Mice can develop fatty liver when placed on a high-fat diet. Mice with certain genetic mutations, such as those homozygous for the obese spontaneous mutation (ob), also known as ob / ob mice (Jackson Laboratory), develop steatosis on a normal diet. In addition, these ob / ob mice rapidly gain weight and exhibit features of metabolic syndrome such as hyperglycemia and glucose intolerance. When placed on a high-fat diet, hepatic steatosis can be further accelerated and made more severe in ob / ob mice. Using either in vivo model, we have shown that anti-CD24 agents have remarkable therapeutic efficacy in reducing hepatic steatosis. In addition, such treatment can result in a decrease in liver enzymes such as ALT, AST, alkaline phosphatase, and GGT, leading to improved liver function. To further demonstrate the therapeutic efficacy of anti-CD24 agents in an in vivo model that can more physiologically recapitulate human fatty liver disease, we utilized an in vivo model in which NSG mice can generate human hepatocytes. It was shown that treatment with anti-CD24 was also able to counter steatosis and improve graft function and graft survival when these mice were placed on a high-fat diet.

[0104] Administration of anti-CD24 agents inhibits the development of hepatic steatosis in cell cultures We identified the anti-steatosis efficacy of therapeutic agents that bind to CD24 in a cell culture system. Specifically, hepatoma cells, Huh7 cells, were cultured in 1% DMSO (Huh7D cells) and then supplemented with adipose medium to induce steatosis. Adipose medium was prepared by a 1:1 mixture of oleic acid (Sigma Aldrich) and palmitic acid (Sigma Aldrich) and 10% FFA-free bovine serum albumin (BSA) solution (Sigma Aldrich) in PBS according to previous literature (35). Briefly, 100 mM oleic acid and palmitic acid were prepared in 99% methanol. Palmitic acid was prepared by shaking at 500 rpm for 20 min at 50 °C. 10 mM oleic acid and palmitic acid were conjugated with 10% BSA by incubating at 50 °C for 30 min. 100 μM of the FFA mixture was dissolved in hepatocyte medium. Cells were incubated in adipose medium for 4 days to induce hepatic steatosis. Cells were then treated with anti-CD24 agents for 3 days in the presence of adipose medium. To determine the degree 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. Treatment was then performed with a wide variety of anti-CD24 agents, including antibodies against CD24, fragment antigen binding (Fab) against CD24, humanized antibodies against CD24, and small molecules that bind to CD24, all of which showed anti-steatosis efficacy (see Figure 1).

[0105] Table 1: Sequences of antibodies used in Figure 1 (IMGT CDRs are bold and underlined) TIFF2025517381000004.tif38164TIFF2025517381000005.tif216164TIFF2025517381000006.tif222164TIFF2025517381000007.tif48164

[0106] For small molecules (compounds A, B, and C in Figure 1D): Virtual screening of a diverse chemical library was performed against human CD24. Briefly, the sequence corresponding to the mature extracellular domain of human CD24 was folded using alphafold 2. Docking was performed using the Schrodinger software suite (Maestro, version 12.4, Schrodinger New York, NY, USA) following well-established approaches. The diverse compound library was extracted into SDF format, imported into Maestro, and prepared for docking using LigPrep. Ionizer was used to generate ionization states for all compounds at the target pH 7 ± 2. The folded human CD24 sequence was prepared using the Protein Preparation Wizard, where hydrogen and bond orders were assigned, heteroatom states were generated, and restrained minimization with the OPLS_2005 force field was performed. High-throughput docking was performed and ligand poses were scored using the GlideScore scoring function. The binding of the top candidate compounds discovered by virtual screening to human CD24 was then functionally assayed. Figure 1D shows the anti-adiposis efficacy of three small molecules thus identified (compounds A, B, and C). This finding demonstrates 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 an anti-adiposis agent). TIFF2025517381000008.tif162128TIFF2025517381000009.tif58128

[0107] We then sought to determine the therapeutic effect of targeting CD24 on fatty liver disease in vivo.

[0108] Administration of anti-CD24 antibody inhibits hepatic steatosis Six to eight week old Rag-deficient (T-cell and B-cell deficient) mice were subcutaneously administered 25 μg of rat IgG2b isotype control or rat anti-mouse CD24 antibody twice / week for 3 to 4 weeks. One day after the first administration, mice were fed a high fat diet (60% kcal from fat). Livers were collected after 4 weeks and liver histology was obtained in the form of hematoxylin and eosin (H&E) staining. All mice tolerated the antibody well. As shown in the representative liver histology slides in Figure 2, mice on a high fat diet developed significant hepatic steatosis (A), whereas mice on a high fat diet treated with anti-CD24 Ab showed essentially little or no hepatic steatosis.

[0109] In addition to histological improvement, improvement in hepatic steatosis can be monitored via ultrasound. As shown from the representative images in Figure 3, liver ultrasound of mice on a HFD treated with control IgG (top) showed increased echogenicity (aka hyperechoic, i.e. the liver appears whiter) compared to mice treated with anti-CD24 (bottom).

[0110] Administration of anti-CD24 antibody reverses hepatic steatosis within one week Eight-week-old ob / ob mice were fed a HFD (60% kcal from fat) for 1 week to accelerate the development of steatosis. They were then maintained on the HFD and administered 50 μg of IgG control or anti-CD24 Ab subcutaneously daily for 6 days. Interestingly, as shown in the representative histological images in Figure 4, mice on the high-fat diet developed severe hepatic steatosis (A), whereas mice on the high-fat diet treated with anti-CD24 Ab for only 1 week showed significantly more normal liver architecture and significantly less steatosis (B).

[0111] Anti-CD24 antibody treatment can more effectively reverse hepatic steatosis when combined with dietary modification Ob / ob mice on a HFD treated with anti-CD24 agents for 1 week showed significantly lower steatosis compared to control-treated mice, but still had significant steatosis. Alternatively, we hypothesize that combined treatment of anti-CD24 agents with dietary modification (i.e., returning ob / ob mice to a normal chow (4% kcal from fat) while treating them with anti-CD24 agents) could more effectively reverse hepatic steatosis. To test this hypothesis, after 1 week of HFD administration, ob / ob mice were returned to a normal chow and treated with either IgG control or anti-CD24 agents for 6 days. Indeed, as shown in the representative histological images in Figure 5, mice treated with IgG control still showed significant steatosis (A), whereas mice treated with anti-CD24 agents showed almost no steatosis.

[0112] In addition to histological improvement of steatosis, administration of anti-CD24 antibody resulted in improvement in serum liver chemistry as demonstrated by reductions in serum ALT, AST, alphos, GGT, and cholesterol (Figure 6) and was well tolerated.

[0113] Administration of anti-CD24 antibody improves liver function in mice with fatty liver. Having demonstrated that anti-CD24 agents can show improvement in liver histology and improve serum liver chemistry in mice that develop fatty liver disease, we sought to determine whether anti-CD24 agents could show improvement 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 severe fatty livers may have impaired drug metabolism 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 administered 25 μg / day of IgG control or anti-CD24 subcutaneously daily while maintained on the HFD. Eight days after the first dose of antibody, mice were orally administered 25 mg / kg of clemizole-HCl (vehicle: water). Thirty minutes after dosing, mice were euthanized and serum was collected and analyzed by quantitative mass spectrometry for clemizole and its major metabolite in mice (M14). 1 We determined serum concentrations of clemizole. Indeed, as shown in FIG. 7, ob / ob mice on a HFD treated with IgG exhibited impaired hepatic metabolism, as indicated by much higher levels of serum clemizole in IgG-treated mice compared to anti-CD24-treated mice (A). This is corroborated by a concomitant increase in serum concentrations of a major mouse metabolite (M14) in mice treated with anti-CD24 versus IgG control (B). Taken together, this demonstrates improved liver function with anti-CD24 treatment.

[0114] Administration of anti-CD24 antibodies that are Fc-silenced for ADCC activity still effectively inhibits steatosis Since some antibodies may exert undesirable toxicity due to antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), we sought to determine whether an anti-CD24 antibody whose Fc lacks ADCC and / or ADCP and thus has reduced toxicity could still retain anti-steatosis activity. To test this hypothesis, we utilized an anti-CD24 antibody whose Fc domain has been engineered to contain point mutations that abolish binding to Fc gamma receptors. These mutations result in silent ADCC activity (Absolute antibody). After placing ob / ob mice on a HFD for 1 week, followed by 1 week of IgG or ADCC silent anti-CD24 antibody (anti-CD24 [YBM5.10.4], mouse IgG2a, Fc silent kappa, product code: Ab00210-2.3 from Absolute antibody) on a normal diet, livers were harvested for histology. Indeed, as shown in the representative histology images in Figure 8, mice treated with the IgG control still exhibited significant steatosis (A), whereas mice treated with the ADCC-silent anti-CD24 antibody exhibited much less steatosis.

[0115] Administration of anti-CD24 improves human liver engraftment, maintains human albumin, and reduces steatosis in mice bearing humanized livers To determine the effect of anti-CD24 agents in a more physiological model of human fatty liver disease, we used a mouse model in which human livers can be engrafted. Briefly, NSG mice with humanized livers were prepared using the RAAPID-TKG method. These mice contain chimeric livers with both mouse and human hepatocytes, and the degree of human liver engraftment can be confirmed by measuring serum human albumin. Two mice with similar degrees of human liver engraftment, as measured by baseline serum human albumin, were placed on a HFD for 6 weeks and then treated with either an IgG control or an anti-CD24 antibody engineered such that its Fc contained point mutations that eliminated ADCC, ADCP, and complement binding activity, i.e., an Fc-silent antibody (termed MOSW3). The antibody was administered at 25 μg twice weekly. Interestingly, as shown in Figure 9A, treatment with MOSW3 showed longer graft survival as evidenced by the maintenance of serum human albumin, while mice treated with the IgG control showed progressive loss of the human liver graft as shown by a steady decrease in serum human albumin. This indicates that treatment with anti-CD24 improves the liver's synthetic function, i.e., albumin production. Three weeks after treatment, a CT scan was also performed, and in contrast to the ultrasound, fat appears as dark areas. IgG-treated mice showed higher levels of steatosis (Figure 9B) compared to MOSW3-treated mice (Figure 9C).

[0116] The results indicate that therapeutic administration of anti-CD24 agents can reduce steatosis and increase human liver graft survival. Anti-CD24 agents can be used to treat fatty liver before it is transplanted or after it is in the liver transplant recipient.

[0117] Administration of anti-CD24 antibodies can slow weight gain during a high-fat diet All mice treated with anti-CD24 agents at doses that show therapeutic benefit against fatty liver disease do not show weight loss or other signs of toxicity, but in some cases, therapeutic administration of anti-CD24 agents can slow the rate of weight gain in mice on HFD, which was observed in both Rag- / - and ob / ob- mice. This suggests that anti-CD24 agents may have therapeutic benefit in other obesity-related diseases. (See FIG. 10).

[0118] In at least some of the above embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment as long as the substitution is technically feasible. It will be understood 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 variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0119] Those skilled in the art will understand that the terms used herein in general, and in the appended claims in particular (e.g., the body 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.). It will be further understood by those skilled in the art that if a particular number of introduced claim recitations are intended, such intention will be expressly recited in the claim, and that in the absence of such recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, even if the same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an," the use of such phrases should not be interpreted as meaning that the introduction of a claim statement with the indefinite article "a" or "an" limits any particular claim that includes such an introduced claim statement to embodiments that include only one such statement (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"), and the same is true for the use of definite articles used to introduce claim statements. In addition, even if a particular number of introduced claim recitations are explicitly recited, one of ordinary skill in the art will recognize that such recitation should be interpreted as meaning at least the number recited (e.g., a bare recitation of "two recitations" without other modifiers means at least two recitations, or more than two recitations).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). Those skilled in the art will further appreciate that virtually any disjunctive word and / or disjunctive phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0120] In addition, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure therefore also is described in terms of any individual members of the Markush group or any subgroups of members.

[0121] As will be understood by those skilled in the art, for all purposes, e.g., in terms of providing a written description, all ranges disclosed herein also encompass any possible subranges and combinations of those subranges. Any listed range can be readily recognized as fully indicating and allowing the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, an upper third, etc. As will also be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," etc. refer to ranges that are inclusive of the numbers recited and that can subsequently be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, etc.

[0122] 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 readily apparent to those skilled in the art that, in light of the teachings of this invention, certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0123] Thus, the above merely illustrates the principles of the invention. It will be appreciated that those skilled in the art can devise various arrangements that embody the principles of the invention and are within its spirit and scope, although not expressly described or shown herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts contributed by the inventor to further advance the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein that describe principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be made available to the public, regardless of whether such disclosure is expressly recited in the claims.

[0124] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is 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 invoked for a limitation in a claim only when the precise phrase "means for" or the precise phrase "step for" is recited at the beginning of such limitation in the claim, and if such precise phrase is not used in the limitation in the claim, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

Claims

1. A pharmaceutical composition for reducing steatosis in an individual, comprising a therapeutically effective dose of an anti-CD24 agent.

2. The pharmaceutical composition according to claim 1, wherein the individual has non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

3. The pharmaceutical composition according to claim 1, wherein the anti-CD24 agent is an anti-CD24 antibody.

4. The pharmaceutical composition according to claim 3, wherein the anti-CD24 antibody comprises one or more Fc having silencing mutations.

5. The pharmaceutical composition according to claim 3, wherein the anti-CD24 antibody is an anti-CD24 fragment antigen-binding region (Fab) or an anti-CD24 PEG-modified Fab fragment.

6. The pharmaceutical composition according to claim 3, wherein the anti-CD24 antibody is an anti-CD24 single-strand variable fragment (scFv).

7. The pharmaceutical composition according to claim 3, wherein the anti-CD24 antibody comprises three heavy chain CDRs and three light chain CDRs, namely SWA11, ML5, SN3, or G7.

8. The pharmaceutical composition according to claim 3, wherein the anti-CD24 antibody is a humanized antibody.

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

10. The pharmaceutical composition according to claim 1, wherein the anti-CD24 agent is a small molecule that binds to CD24.

11. The pharmaceutical composition according to claim 1, wherein the treatment course is 1 to 8 weeks.

12. The pharmaceutical composition according to claim 1, administered to the individual in combination with dietary improvement and / or weight-loss surgery.

13. The pharmaceutical composition according to claim 1, wherein the anti-CD24 agent is co-administered to the individual with another therapeutic agent.

14. The pharmaceutical composition according to claim 1, wherein the individual is a liver transplant patient, and the pharmaceutical composition is administered to the individual to enhance liver graft function and / or extend liver graft survival time.

15. The pharmaceutical composition according to claim 1, wherein the individual has fatty liver, fatty liver-related disease, obesity, or obesity-related disease.

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