Methods for treating NAFLD and NASH

Abstract

The present disclosure provides CCR5 binders that are useful for preventing or treating dysregulation of CCR5 and CCR2 and the resulting conditions and symptoms, such as NASH and NAFLD.

Description

Technical Field

[0001] The present disclosure relates to a method of treating or preventing non-alcoholic fatty liver disease (NAFLD), particularly non-alcoholic steatohepatitis (NASH), by administering an anti-CCR5 antibody or an antigen-binding fragment, such as leronlimab or an antigen-binding fragment thereof.

Background Art

[0002] Background The role of C-C motif chemokine ligand 5 (CCL5) (also known as regulated on activation, normal T cell expressed and secreted (RANTES)) in binding to CCR5, and the role of C-C motif chemokine ligand 2 in binding to CCR2 and subsequent cell signaling have been well characterized. CCR5 / RANTES binding related to signaling supports chemotaxis of pro-inflammatory cells, while CCR2 / CCL2 signaling via IL-10 supports chemotaxis of immunosuppressive cells, such as M2 monocytes, bone marrow-derived suppressors, and dendritic cells, that regulate / prevent the transition from innate immune response (Th0) to adaptive immune response (Th1 / Th2). CCR2 signaling via CCL2 is very well characterized and is associated with the formation of a tumorigenic environment.

Disclosure of the Invention

[0003] Summary The present disclosure provides a method of treating or preventing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in a patient by administering to the patient an effective amount of leronlimab to treat or prevent NAFLD or NASH. This method further provides the following: wherein leronlimab is administered by injection; wherein leronlimab is administered weekly; Here, Relonumab is administered in an amount effective to treat or prevent NASH-related liver fibrosis; Here, Relonumab is administered at a dose of 350 mg; Here, Relonumab is administered at a dose of 525 mg; Here, Relonumab is administered at a dose of 700 mg;

[0004] Here, the patient is evaluated for CCR5 haplotype, and if the patient has a CCR5 haplotype not associated with increased CCR5 cell surface expression, the patient is administered 350 mg of Relonumab weekly; Here, the patient is evaluated for CCR5 haplotype, and if the patient has a CCR5 haplotype not associated with increased CCR5 cell surface expression, the patient is administered 525 mg of Relonumab weekly; Here, the CCR5 haplotype not associated with increased CCR5 cell surface expression does not include HHE or HHG;

[0005] Here, the patient is evaluated for CCR5 haplotype, and if the patient has a CCR5 haplotype associated with increased CCR5 cell surface expression, the patient is administered 525 mg of Relonumab weekly; Here, the patient is evaluated for CCR5 haplotype, and if the patient has a CCR5 haplotype associated with increased CCR5 cell surface expression, the patient is administered 700 mg of Relonumab weekly; Here, the CCR5 haplotype associated with increased CCR5 cell surface expression includes HHE or HHG;

[0006] Here, after administration of at least one dose of Relonumab, the level of a biomarker indicating liver function or inflammation is measured, and if the level does not change by a set amount compared to the baseline level or a previous level, or if the level does not exceed or fall below a set value, the dose or dosing frequency of Relonumab is adjusted; Here, the biomarker is one or more of RANTES, CCL2, CCL3, CCL11, CCL18, VCAM, and EN RAGE; Here, the biomarker is RANTES, and if its serum level is not low enough such that it has no clinical inflammatory effect on NASH, the dose or dosing frequency of relonremab is increased;

[0007] Here, the dose is increased from 350 mg per week to 700 mg per week; Here, after administration of at least one dose of relonremab, the level of the MRI index of NASH is measured, and if the level does not change by a set amount compared to the baseline level or a previous level, or if the level does not exceed or fall below a set value, the dose or dosing frequency of relonremab is adjusted; Here, the MRI index of NASH is PDFF or cT1; Here, if PDFF or cT1 does not fall below a set level or has not decreased compared to a previous measurement for the patient, the dose of relonremab is increased from 350 mg per week to 700 mg per week; And any compatible combination thereof.

Brief Description of the Drawings

[0008] The patent file or application file includes at least one color drawing. A copy of this patent or the published patent application with color drawings is provided by the Patent Office upon request and payment of the necessary fees. This disclosure can be better understood by referring to the following figures.

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[0012] Detailed Description The present disclosure relates to a method of treating NAFLD, particularly NASH, and further disorders resulting as related symptoms and consequences, by i) treating abnormal regulation of CCR5 and CCR2, ii) administering an anti-CCR5 antibody or antigen-binding fragment, such as leronlimab or an antigen-binding fragment, or iii) both. Before describing the present disclosure in more detail, it may be useful to provide definitions of certain terms used herein. Unless defined otherwise, 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. Additional definitions are set forth throughout the present disclosure.

[0013] As used herein, the term “about” means ±20% of the indicated range, value, or composition, unless otherwise indicated. The term “consisting essentially of” limits the scope of a claim to the specified substance or process or those that do not materially affect the basic and novel characteristics of the invention recited in the claim. The terms “a” and “an” as used herein should be understood to refer to “one or more” of the recited components. The use of an alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof. As used herein, the terms “include” and “have” are used synonymously, and these terms and their variations are intended to be construed non-limitingly. The term “comprise” means the presence of the features, integers, steps, or components recited in the claim, but does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. Any range provided herein includes all values within the range and narrower ranges.

[0014] As used herein, "chemokine" refers to a low molecular weight cytokine that can stimulate the recruitment of leukocytes. Chemokines have cysteine residues at conserved positions that form the key to their three-dimensional shape. Chemokines can be classified into four major subfamilies: Cys-Cys (C-C), Cys-X-Cys (CXC), CX3C, and XC, depending on the spacing between the first two cysteine residues at the amino terminus. Chemokines can also be classified by their function, such as whether they are inflammatory or constitutive. Forty-seven chemokines are known, including, but not limited to, CCL5 (also known as RANTES), MIP-1α, MIP-1β, or SDF-1, or another chemokine with similar activity.

[0015] As used herein, "C-C chemokine receptor 5" is also known as "CCR5" or "CD195" and refers to a G protein-coupled receptor that is expressed on lymphocytes (e.g., NK cells, B cells, T cells), monocytes, dendritic cells, eosinophils, and microglia that function as receptors for chemokines of the C-C chemokine group. CCR5 cognate ligands include CCL3, CCL4, CCL3L1, and CCL5. In some embodiments, CCR5 refers to human CCR5. In some embodiments, CCR5 refers to a protein having the amino acid sequence provided in NCBI reference sequence: NP_000570.1 (SEQ ID NO: 15).

[0016] As used herein, "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, examples of which include hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, examples of which are homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have an R group (e.g., norleucine) or a modified peptide backbone but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0017] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or a polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. Mutations can cause several different types of sequence changes, including substitution, insertion, or deletion of nucleotide(s) or amino acid(s).

[0018] As used herein, "protein" or "polypeptide" refers to a compound composed of amino acid residues covalently linked by peptide bonds. The term "protein" may be synonymous with the term "polypeptide" or, additionally, may refer to a complex of two or more polypeptides. A polypeptide may further contain other components (e.g., covalently attached), such as tags, labels, bioactive molecules, or any combination thereof. In certain embodiments, the polypeptide may be a fragment. As used herein, "fragment" means a polypeptide that lacks one or more amino acids found in a reference sequence. A fragment can include a binding domain, antigen, or epitope found in the reference sequence. Fragments of a reference polypeptide can have at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the amino acids of the amino acid sequence of the reference sequence.

[0019] As described herein, "variant" polypeptide species have one or more non-natural amino acids, one or more amino acid substitutions, one or more amino acid insertions, one or more amino acid deletions, or any combination thereof, at one or more sites, compared to the reference polypeptides presented herein. In certain embodiments, a "variant" means a polypeptide having substantially similar activity (e.g., enzymatic function, immunogenicity) or structure compared to a reference polypeptide. Variants of a reference polypeptide can have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence of the reference polypeptide as determined by sequence alignment programs and parameters known in the art. Such variants can arise, for example, from genetic polymorphisms or from artificial manipulations. Conservative substitutions of amino acids are well known and can occur naturally or can be introduced when the protein is recombinantly produced. Amino acid substitutions, deletions, and additions can be introduced into the protein using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY, 2001). Modified polynucleotides can be provided in which a particular codon has been altered according to a desired substitution, deletion, or insertion, using oligonucleotide-directed site-specific (or segment-specific) mutagenesis procedures. Alternatively, polypeptide variants can also be prepared using random or saturation mutagenesis techniques such as alanine scanning mutagenesis, mutagenic polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis (see, e.g., Sambrook et al., supra).

[0020] "Conservative substitution" refers to an amino acid substitution that does not significantly affect or alter the binding properties of a particular protein. Generally, a conservative substitution is a substitution in which the substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include substitutions found in one of the following groups: Group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and Group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally, or alternatively, amino acids can be classified into conservative substitution groups by similar function, chemical structure, or composition (by way of example, acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, the aliphatic group can include Gly, Ala, Val, Leu, and Ile for the purposes of substitution. Other conservative substituents include the following: sulfur-containing: Met and cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0021] The terms "identical" or "percent identity" in the context of the sequences of two or more polypeptides or nucleic acid molecules refer to sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) over a comparison window or specified region, as measured by methods known in the art such as sequence comparison algorithms, manual alignment, or visual inspection, when aligned and compared to maximize correspondence over the comparison window or specified region. The algorithms used herein to determine percent sequence identity and sequence similarity are the BLAST 2.0 algorithms, as described in Altschul et al. “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs“, Nucleic Acids Res. 2007, 25, 3389-3402. In the context of the present disclosure, when sequence analysis software is used for analysis, it is understood that the analysis results are based on the "default values" of the program being referenced. "Default values" refer to the set of values or parameters that are initially loaded when the software is first initialized.

[0022] As used herein, "fusion protein" includes a single-chain polypeptide having at least two different domains, where the domains are not naturally found together in a protein. Nucleic acid molecules encoding fusion proteins can be constructed using, for example, PCR, recombinant manipulation, etc., or such fusion proteins can be made synthetically. Fusion proteins may further contain other components (e.g., covalent linkages), such as tags, linkers, transduction markers, or bioactive molecules.

[0023] "Nucleic acid molecule" or "polynucleotide" refers to a macromolecular compound containing nucleotides covalently linked by 3'-5' phosphodiester bonds. Nucleic acid molecules include polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), which include genomic DNA, mitochondrial DNA, cDNA, or vector DNA. Nucleic acid molecules may be double-stranded or single-stranded, and in the case of single-stranded molecules, they may be coding strands or non-coding strands (antisense strands). Nucleic acid molecules may contain natural or non-natural subunits. Nucleic acid molecules encoding an amino acid sequence include all nucleotide sequences encoding the same amino acid sequence. Some versions of the nucleotide sequence may also include introns (singular or plural) to the extent that the introns are removed through co-transcriptional or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as a result of the redundancy or degeneracy of the genetic code or through splicing.

[0024] Variants of the polynucleotides of the present disclosure are also contemplated. Variant polynucleotides are at least 80%, 85%, 90%, 95%, 99%, or 99.9% identical to the reference polynucleotide described herein, or hybridize to the reference polynucleotide of a sequence defined under stringent hybridization conditions of about 65°C to 68°C in 0.015 M sodium chloride, 0.0015 M sodium citrate, or about 42°C in 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide. Polynucleotide variants retain the ability to encode an immunoglobulin-like binding protein or an antigen-binding fragment thereof having the functionality described herein.

[0025] The term "isolated" means that a substance has been removed from its original environment (for example, the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide is isolated when it is separated from some or all of the substances that coexist in the natural system. Such a polynucleotide may be part of a vector and / or such a polynucleotide or polypeptide may be part of a composition (for example, a cell lysate), and such a vector or composition is still isolated in that it is not part of the natural environment of the nucleic acid or polypeptide.

[0026] As used herein, the terms "engineered," "recombinant," or "non-natural" refer to an organism, microorganism, cell, nucleic acid molecule, or vector that contains at least one genetic modification or has been modified by the introduction of an exogenous or heterologous nucleic acid molecule, where such modification or modification is introduced by genetic engineering (i.e., human intervention). Genetic modifications include, for example, modifications that introduce an expressible nucleic acid molecule encoding a functional RNA, protein, fusion protein, or enzyme, the addition, deletion, substitution, or other functional disruption of the genetic material of a cell of another nucleic acid molecule. Additional modifications include, for example, non-coding regulatory regions where the modification alters the expression of a polynucleotide, gene, or operon.

[0027] As used herein, the terms "heterologous" or "exogenous" nucleic acid molecule, construct or sequence refer to a nucleic acid molecule or a portion of a nucleic acid molecule that is not native to the host cell, but may be homologous to a nucleic acid molecule or a portion of a nucleic acid molecule of the host cell. The source of the heterologous or exogenous nucleic acid molecule, construct or sequence may be from a different genus or species. In certain embodiments, a heterologous or exogenous nucleic acid molecule (i.e., not endogenous or native) is added to the host cell or host genome, for example, by conjugation, transformation, transfection, electroporation, etc., where the added molecule can be integrated into the host genome or exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector) and may be present in multiple copies. Additionally, "heterologous" refers to a non-native enzyme, protein, or other activity encoded by an exogenous nucleic acid molecule introduced into a host cell, even if the host cell encodes a homologous protein or activity.

[0028] As used herein, the terms "endogenous" or "native" refer to a gene, protein, or activity that is normally present in the host cell. Moreover, a gene, protein, or activity that has been altered by mutation, overexpression, shuffling, duplication, or other means, as compared to the parental gene, protein, or activity, is considered to be endogenous or native to that particular host cell. For example, an endogenous regulatory sequence (e.g., a promoter, translational attenuation sequence) from a first gene can be used to alter or regulate the expression of a second native gene or nucleic acid molecule, where the expression or regulation of the second native gene or nucleic acid molecule is different from the normal expression or regulation in the parental cell.

[0029] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule such as a gene. This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. The nucleic acid molecule to be expressed is typically operably linked to an expression control sequence (e.g., a promoter).

[0030] As used herein, the term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it can affect the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Not linked" means that the related genetic elements are not closely related to each other and the function of one does not affect the other.

[0031] As used herein, an "expression vector" refers to a DNA construct containing a nucleic acid molecule operably linked to suitable control sequences that can effect the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter that effects transcription, any operator sequence that controls such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that controls the termination of transcription and translation. The vector may be a plasmid, phage particle, virus, or simply a potential genomic insert. When transformed into a suitable host, the vector can replicate and function independently of the host genome or, in some cases, can be integrated into the genome itself. In this specification, the terms "plasmid", "expression plasmid", "virus" and "vector" are often used interchangeably.

[0032] As used herein, the term "host" refers to a cell that is the target of genetic modification by a heterologous nucleic acid molecule and produces a polypeptide of interest (e.g., a CCR5 antibody of the present disclosure) (e.g., T cells, Chinese hamster ovary (CHO) cells, HEK293 cells, B cells, etc.). In certain embodiments, the host cell optionally already has or may be modified to include other genetic modifications that confer desired properties (e.g., inclusion of a detectable marker, deletion, modification or cleavage of an endogenous BCR), whether or not related to the biosynthesis of a heterologous protein.

[0033] As described herein, two or more heterologous nucleic acid molecules can be introduced into a host cell as separate nucleic acid molecules, as multiple individually controlled genes, as a polycistronic nucleic acid molecule (e.g., the heavy and light chains of an antibody), as a single nucleic acid molecule encoding a protein (e.g., the heavy chain of an antibody), or as any combination thereof. When introducing two or more heterologous nucleic acid molecules into a host cell, it is understood that the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or in any combination thereof. The number of heterologous nucleic acid molecules or protein activities referred to refers to the number of encoding nucleic acid molecules or protein activities, and not to the number of separate nucleic acid molecules introduced into the host cell.

[0034] As used herein, the term "introduced" means "transfection", or "transformation" or "transduction" in the context of inserting a nucleic acid sequence into a cell, and includes reference to incorporating a nucleic acid sequence into a eukaryotic or prokaryotic cell, where the nucleic acid molecule may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0035] "To treat", "treatment", or "to improve" refers to the medical management of a disease, disorder, or condition in a patient (e.g., a human or non-human mammal such as a primate, horse, cat, dog, goat, mouse, or rat). Generally, an appropriate dosage or treatment regimen, including Relonlimab, is administered in an amount sufficient to induce a therapeutic effect or therapeutic benefit. Therapeutic effects or therapeutic benefits include improvement in clinical outcome, reduction, decrease, or suppression of inflammatory cytokine activity of adverse effects by modulation of the immune response, normalization of inflammatory cytokine activity of adverse effects by modulation of the immune response, reduction or alleviation of symptoms associated with the disease, decrease in the occurrence of symptoms, improvement in quality of life, prolongation of disease-free state, reduction in the extent of the disease, stabilization of the disease state, delay in disease progression, remission, survival, prolongation of survival period, or any combination thereof. A treated patient is also referred to as "administered", and an untreated patient is also referred to as "non-administered".

[0036] Preventive treatment aimed at "preventing" a disease or condition (e.g., a disease or condition in a patient with coronavirus-induced respiratory disease) is treatment administered to a patient who shows no signs of the disease or only initial signs, for the purpose of reducing the risk of onset of the disease state or further progression of the initial disease. For example, if an individual at risk of developing coronavirus-induced respiratory disease is treated by the methods of the present disclosure and later does not develop coronavirus-induced respiratory disease, the disease has been prevented in that individual for at least a certain period of time. Preventive treatment may mean preventing recurrence or recurrence of a disease or condition in a patient who has previously received treatment for the disease or condition, for example, by preventing recurrence of coronavirus-induced respiratory disease.

[0037] The "therapeutically effective amount" or "effective amount" of Relonlimab means an amount of Relonlimab sufficient to produce a therapeutic effect, including improvement of clinical outcome, reduction or alleviation of symptoms associated with a disease, reduction, diminution, or suppression of inflammatory cytokine activity of an adverse effect by modulation of the immune response, normalization of inflammatory cytokine activity of an adverse effect by modulation of the immune response, decrease in the occurrence of symptoms, improvement in quality of life, prolongation of disease-free state, reduction in the scope of a disease, stabilization of a disease state, delay in disease progression, remission, survival, or prolongation of survival in a statistically significant manner. When administering an individual active ingredient or cells expressing a single active ingredient alone, the therapeutically effective amount refers to the effect of that ingredient or the cells expressing only that ingredient. When referring to a combination, the therapeutically effective amount refers to the total amount of the active ingredients, or the total amount of the accessory active ingredients in combination with the cells expressing the active ingredients that produce a therapeutic effect, regardless of whether administered continuously or simultaneously.

[0038] As used herein, "relative reduction" or "relative risk reduction" refers to the percentage reduction of a parameter (e.g., mortality, time to recovery) in a treatment group (Y) compared to a control group (X). RR = 1 - (Y / X) x 100%. As used herein, "absolute reduction" or "absolute risk reduction" refers to the percentage reduction between a control group (X) and a treatment group (Y). AR = X - Y.

[0039] The term "pharmaceutically acceptable excipient or carrier" or "physiologically acceptable excipient or carrier" refers to a biologically compatible vehicle, such as physiological saline, which is described in more detail herein and is suitable for administration to human or other non-human mammalian patients and is generally recognized as safe or not causing serious adverse events. Additional definitions are provided in the following section.

[0040] Relonlimab The present disclosure provides the use of Relonlimab or an antigen-binding fragment thereof in treating or preventing NAFLD or NASH. Terms understood by those skilled in the art of antibody technology are given their respective meanings obtained in the art, unless explicitly defined otherwise herein. The term "antibody" refers to an intact antibody that includes at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody, such as an scFv, Fab, or Fab'2 fragment, that has or retains the ability to bind to an antigen target molecule recognized by the intact antibody. Thus, the term "antibody" as used herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, which include fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments (including single-chain variable fragments (scFv)), and single-domain antibodies (e.g., sdAb, sdFv, nanobodies), as well as intact antibodies and their functional (antigen-binding) antibody fragments. This term includes genetically engineered and / or otherwise modified forms of immunoglobulins, such as, for example, intracellular antibodies (intrabodies), peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, tetrabodies, tandem diabodies, and tandem trispecific antibodies. Unless stated otherwise, the term "antibody" should be understood to include its functional antibody fragments. This term also includes intact or full-length antibodies, including any class or subclass of antibodies, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0041] The terms "VL" and "VH" refer to the variable binding regions from an antibody light chain and an antibody heavy chain, respectively. The variable binding regions are composed of discrete and well-defined sub-regions known as "complementary determining regions" (CDRs) and "framework regions" (FRs). The terms "complementary determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and refer to the amino acid sequences within the antibody variable regions that generally confer together the antigen specificity and / or binding affinity of the antibody, where the contiguous CDRs (i.e., CDR1 and CDR2, CDR2 and CDR3) are separated from each other within the primary amino acid sequence by the framework regions. Each variable region has three CDRs (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also referred to as CDRH and CDRL, respectively). In one embodiment, the antibody VH contains four FRs and three CDRs as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and the antibody VL contains four FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. Generally, VH and VL together form an antigen binding site via their respective CDRs.

[0042] The numbering of CDRs and framework regions can be determined according to any known method or scheme, such as, for example, the Kabat, Chothia, EU, IMGT, and AHo numbering schemes (see, for example, Kabat et al., “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; Honegger and Plueckthun, J. Mol. Bio. 309:657-670 (2001)). By using the Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300), equivalent residue positions can be annotated and different molecules can be compared. Thus, the identification of CDRs of exemplary variable domain (VH or VL) sequences provided herein according to one numbering scheme does not exclude antibodies containing CDRs of the same variable domain determined using different numbering schemes.

[0043] In some embodiments, the present disclosure provides the use of an anti-CCR5 antibody or an antigen-binding fragment thereof having a light chain variable region (VL) that is at least 70% identical to SEQ ID NO:1, at least 75% identical to SEQ ID NO:1, at least 80% identical to SEQ ID NO:1, at least 85% identical to SEQ ID NO:1, or at least 90% identical to SEQ ID NO:1. In some embodiments, the present disclosure provides the use of an anti-CCR5 antibody or an antigen-binding fragment thereof having a light chain variable antibody region that is 70% to 100% identical to SEQ ID NO:1, 75% to 100% identical to SEQ ID NO:1, 80% to 100% identical to SEQ ID NO:1, 85% to 100% identical to SEQ ID NO:1, 90% to 100% identical to SEQ ID NO:1, or 91% to 100% identical to SEQ ID NO:1.

[0044] In some embodiments, the present disclosure provides the use of an anti-CCR5 antibody or an antigen-binding fragment thereof having a light chain variable region (VL) that is at least 70% identical to amino acids 20-131 of SEQ ID NO:1, at least 75% identical to amino acids 20-131 of SEQ ID NO:1, at least 80% identical to amino acids 20-131 of SEQ ID NO:1, at least 85% identical to amino acids 20-131 of SEQ ID NO:1, or at least 90% identical to amino acids 20-131 of SEQ ID NO:1. In some embodiments, the present disclosure provides the use of an anti-CCR5 antibody or an antigen-binding fragment thereof having a light chain variable antibody region that is 70% to 100% identical to amino acids 20-131 of SEQ ID NO:1, 75% to 100% identical to amino acids 20-131 of SEQ ID NO:1, 80% to 100% identical to amino acids 20-131 of SEQ ID NO:1, 85% to 100% identical to amino acids 20-131 of SEQ ID NO:1, 90% to 100% identical to amino acids 20-131 of SEQ ID NO:1, or 91% to 100% identical to amino acids 20-131 of SEQ ID NO:1.

[0045] In some embodiments, the present disclosure provides the use of an anti-CCR5 antibody or an antigen-binding fragment thereof having a heavy chain variable region (VH) that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, or at least 90% identical to SEQ ID NO: 3. In some embodiments, the present disclosure provides the use of an anti-CCR5 antibody or an antigen-binding fragment thereof having a heavy chain antibody variable region that is 70% to 100% identical, 75% to 100% identical, 80% to 100% identical, 85% to 100% identical, 90% to 100% identical, or 91% to 100% identical to SEQ ID NO: 3.

[0046] In some embodiments, the present disclosure provides the use of an anti-CCR5 antibody or an antigen-binding fragment thereof having a heavy chain variable region (VH) that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, or at least 90% identical to amino acids 20 to 141 of SEQ ID NO: 3. In some embodiments, the present disclosure provides the use of an anti-CCR5 antibody or an antigen-binding fragment thereof having a heavy chain antibody variable region that is 70% to 100% identical, 75% to 100% identical, 80% to 100% identical, 85% to 100% identical, 90% to 100% identical, or 91% to 100% identical to amino acids 20 to 141 of SEQ ID NO: 3.

[0047] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody having a heavy chain variable region (VH) that is at least 70% identical to SEQ ID NO:5, at least 75% identical to SEQ ID NO:5, at least 80% identical to SEQ ID NO:5, at least 85% identical to SEQ ID NO:5, or at least 90% identical to SEQ ID NO:5. In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody having a heavy chain variable antibody region that is 70% to 100% identical to SEQ ID NO:5, 75% to 100% identical to SEQ ID NO:5, 80% to 100% identical to SEQ ID NO:5, 85% to 100% identical to SEQ ID NO:5, 90% to 100% identical to SEQ ID NO:5, or 91% to 100% identical to SEQ ID NO:5.

[0048] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody having a heavy chain variable region (VH) that is at least 70% identical to amino acids 20 to 141 of SEQ ID NO:5, at least 75% identical to amino acids 20 to 141 of SEQ ID NO:5, at least 80% identical to amino acids 20 to 141 of SEQ ID NO:5, at least 85% identical to amino acids 20 to 141 of SEQ ID NO:5, or at least 90% identical to amino acids 20 to 141 of SEQ ID NO:5. In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody having a heavy chain variable antibody region that is 70% to 100% identical to amino acids 20 to 141 of SEQ ID NO:5, 75% to 100% identical to amino acids 20 to 141 of SEQ ID NO:5, 80% to 100% identical to amino acids 20 to 141 of SEQ ID NO:5, 85% to 100% identical to amino acids 20 to 141 of SEQ ID NO:5, 90% to 100% identical to amino acids 20 to 141 of SEQ ID NO:5, or 91% to 100% identical to amino acids 20 to 141 of SEQ ID NO:5.

[0049] In some embodiments, the present disclosure provides the use of an anti-CCR5 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises a heavy chain CDR1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 (VH-CDR2) comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 (VH-CDR3) comprising the amino acid sequence of SEQ ID NO: 14; and VL comprises a light chain CDR1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 (VL-CDR2) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain CDR3 (VL-CDR3) comprising the amino acid sequence of SEQ ID NO: 11. In some such embodiments, VH comprises the amino acid sequence of SEQ ID NO: 3 or amino acids 20-141 of SEQ ID NO: 3 and an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and VL comprises the amino acid sequence of SEQ ID NO: 1 or amino acids 20-131 of SEQ ID NO: 1 and an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, provided that the amino acid sequences of VH-CDR (SEQ ID NOs: 12-14) and VL-CDR (SEQ ID NOs: 9-11) are not altered; or VH comprises the amino acid sequence of SEQ ID NO: 5 or amino acids 20-141 of SEQ ID NO: 5 and an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and VL comprises the amino acid sequence of SEQ ID NO: 1 or amino acids 20-131 of SEQ ID NO: 1 and an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, provided that the amino acid sequences of VH-CDR (SEQ ID NOs: 12-14) and VL-CDR (SEQ ID NOs: 9-11) are not altered.

[0050] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody or an antigen-binding fragment thereof, comprising: (a) a VH comprising the amino acid sequence of SEQ ID NO: 3 or amino acids 20-141 of SEQ ID NO: 3, and a VL comprising the amino acid sequence of SEQ ID NO: 1 or amino acids 20-131 of SEQ ID NO: 1; or (b) a VH comprising the amino acid sequence of SEQ ID NO: 5 or amino acids 20-141 of SEQ ID NO: 5, and a VL comprising the amino acid sequence of SEQ ID NO: 1 or amino acids 20-131 of SEQ ID NO: 1.

[0051] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody comprising a heavy chain (HC) and a light chain (LC). The heavy chain typically comprises a VH and a heavy chain constant region (CH). Depending on the antibody isotype from which it is derived, the heavy chain constant region may comprise CH1, CH2, and CH3 domains (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4 domains (IgE, IgM). In some embodiments, the heavy chain constant region comprises a human IgG1, IgG2, IgG3, or IgG4 constant region. In some embodiments, the constant region of the anti-CCR5 antibody is an IgG4 constant region. The light chain typically comprises a VL and a light chain constant region (CL). In some embodiments, the CL comprises a C kappa ("CK") constant region. In some embodiments, the CL comprises a C lambda (Cλ) constant region. In some embodiments, the anti-CCR5 antibody of the present disclosure comprises two heavy chains and two light chains covalently held together by disulfide bridges.

[0052] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody that includes an Fc region portion. As used herein, "Fc region portion" refers to a heavy chain constant region segment of an Fc fragment (the "crystallizable fragment" region or Fc region) from an antibody and may include one or more constant domains such as CH2, CH3, CH4, or any combination thereof. In some embodiments, the Fc region portion includes the CH2 and CH3 domains of an IgG, IgA, or IgD antibody, or any combination thereof, or the CH3 and CH4 domains of an IgM or IgE antibody, and any combination thereof. In some embodiments, the CH2CH3 or CH3CH4 structure has sub-region domains from the same antibody isotype and is of human origin such as human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM (e.g., CH2CH3 from human IgG1). By way of background, the Fc region is involved in effector functions of antibodies such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and complement fixation, binding to Fc receptors (e.g., CD16, CD32, FcRn), having a longer in vivo half-life relative to an Fc region-deficient polypeptide, protein A binding, and possibly also placental transfer (see Capon et al. Nature 337: 525, 1989). In some embodiments, the Fc region portion in the antibody or antigen-binding fragment of the present disclosure can mediate one or more of these effector functions. In some embodiments, the Fc region portion in the antibody or antigen-binding fragment of the present disclosure has normal effector functions, which means having a difference of less than 20%, 15%, 10%, 5%, 1% in effector function (e.g., ADCC, CDC, half-life, or any combination thereof) compared to a wild-type IgG1 antibody.

[0053] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody comprising an Fc region portion in which one or more effector functions are increased, for example, by substitution or deletion of one or more amino acids in the Fc region portion known in the art. An antibody or antigen-binding fragment having a mutated or variant Fc region portion with increased effector function means that the antibody exhibits at least a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% increase in FcR binding, ADCC, CDC, or any combination thereof compared to an antibody having a wild-type Fc region portion. In some embodiments, the mutated or variant Fc region portion exhibits increased binding to FcRn, FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), or any combination thereof. In some embodiments, the Fc region portion in the antibody or antigen-binding fragment of the present disclosure is a variant Fc region portion in which ADCC, CDC, half-life, or any combination thereof is increased.

[0054] Amino acid modifications (such as substitutions) for modifying (e.g., improving, reducing, or removing) Fc functionality include, for example, T250Q / M428L, M252Y / S254T / T256E, H433K / N434F, M428L / N434S, E233P / L234V / L235A / G236+A327G / A330S / P331S, E333A, S239D / A330L / I332E, P257I / Q311, K326W / E333S, S239D / I332E / G236A, N297Q, K322A, S228P, L235E+E318A / K320A / K322A, L234A / L235A, and L234A / L235A / P329G mutations, which are summarized and annotated in "Engineered Fc Regions" (2011) published by InvivoGen, which is available online at www.invivogen.com / PDF / review / review-Engineered-Fc-Regions-invivogen.pdf?utm_source=review&utm_medium=pdf&utm_campaign=review&utm_content=Engineered-Fc-Regions and is incorporated herein by reference.

[0055] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody comprising an Fc region portion in which one or more effector functions are reduced or absent, for example, by substitution or deletion of one or more amino acids in the Fc region portion known in the art. An antibody or antigen-binding fragment having a mutant or variant Fc region portion with reduced effector function means that the antibody exhibits at least a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% decrease in FcR binding, ADCC, CDC, or any combination thereof compared to an antibody having a wild-type Fc region portion. In some embodiments, the mutant or variant Fc region portion exhibits a decrease in binding to FcRn, FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), or any combination thereof. In some embodiments, the Fc region portion in the antibody or antigen-binding fragment of the present disclosure is a variant Fc region portion in which ADCC, CDC, half-life, or any combination thereof is reduced. In some embodiments, the Fc region portion is a variant IgG1 Fc region portion comprising a mutation corresponding to amino acid E233P, L234V, L234A, L235A, L235E, ΔG236, G237A, E318A, K320A, K322A, A327G, P329G, A330S, P331S, or any combination thereof, numbered according to EU as described in Kabat. For example, the amino acid substitutions L234A, L235E, G237A introduced into the IgG1 Fc region portion reduce binding to the FcγRI, FcγRIIa, and FcγRIII receptors, and A330S and P331S introduced into the IgG1 Fc region portion reduce the complement binding reaction mediated by C1q.

[0056] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody comprising an Fc region portion in which one or more effector functions are increased, for example, by substitution or deletion of one or more amino acids in the Fc region portion known in the art. An antibody or antigen-binding fragment having a mutant or variant Fc region portion with increased effector function means that the antibody exhibits at least a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% increase in FcR binding, ADCC, CDC, or any combination thereof as compared to an antibody having a wild-type Fc region portion. In some embodiments, the mutant or variant Fc region portion exhibits increased binding to FcRn, FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), or any combination thereof. In some embodiments, the Fc region portion in the antibody or antigen-binding fragment of the present disclosure is a variant Fc region portion in which ADCC, CDC, half-life, or any combination thereof is increased.

[0057] In some embodiments, the present disclosure provides for the use of a glycosylated anti-CCR5 antibody. IgG subtype antibodies contain a conserved glycosylation site at amino acid N297 in the CH2 domain of the Fc region portion. In some such embodiments, the Fc region portion in the antibody or antigen-binding fragment of the present disclosure includes N297 numbered according to EU as described in Kabat. In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody comprising a mutation that alters glycosylation at N297 of the Fc region portion, optionally, the mutation that alters glycosylation includes N297A, N297Q, or N297G. In some embodiments, an antibody or antigen-binding fragment thereof comprising the N297A, N297Q, or N297G mutation exhibits a reduction in Fc interaction with one or more low-affinity FcγRs, a reduction in CDC, a reduction in ADCC, or any combination thereof.

[0058] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 7; and the LC comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 8.

[0059] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody comprising an HC comprising an amino acid sequence having the amino acid sequence of SEQ ID NO: 7, and an LC comprising an amino acid sequence having the amino acid sequence of SEQ ID NO: 8. In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody comprising an Fc region or a fragment thereof, including CH2 (or a fragment thereof), CH3 (or a fragment thereof), or CH2 and CH3, wherein CH2, CH3, or both may be of any isotype and may each contain an amino acid substitution or other modification as compared to the corresponding wild-type CH2 or CH3. In one embodiment, the Fc region of the present disclosure comprises two CH2-CH3 polypeptides that associate to form a dimer.

[0060] As used herein, unless otherwise specified, the positions of amino acid residues in the constant region of the human IgG1 heavy chain are numbered assuming that the variable region of human IgG1 consists of 128 amino acid residues according to the Kabat numbering rule. The numbered constant region of the human IgG1 heavy chain is then used as a reference for the numbering of amino acid residues in the constant regions of other immunoglobulin heavy chains. The position of a target amino acid residue in the constant region of an immunoglobulin heavy chain other than the human IgG1 heavy chain is the position of the amino acid residue in the human IgG1 heavy chain to which the target amino acid residue aligns. The alignment between the constant region of the human IgG1 heavy chain and the constant regions of other immunoglobulin heavy chains can be performed using a software program known in the art, such as the Megalign program (DNASTAR Inc.) using the Clustal W method with default parameters. According to the numbering system described herein, for example, the human IgG2 C H2 region may have an amino acid deletion near its amino terminus compared to other C H2 regions, but the position of "N" located at 296 of human IgG2 C H2 is still considered to be at position 297 because this residue aligns with "N" at position 297 of human IgG1 C H2 .

[0061] In addition, the present disclosure provides the use of an anti-CCR5 antibody that typically includes a hinge sequence located between the Fab region and the Fc region (although the lower part of the hinge may include the amino-terminal portion of the Fc region). As background, the hinge of an immunoglobulin acts as a flexible spacer that allows the Fab portions to move freely in space. In contrast to the constant regions, the hinge is structurally diverse, with both sequence and length varying between immunoglobulin classes and even between subclasses. For example, the human IgG1 hinge region is sufficiently flexible to allow the Fab fragment to rotate around its axis of symmetry and move within the sphere centered on the first of two inter-heavy chain disulfide bridges. In comparison, the human IgG2 hinge is relatively short and contains a rigid poly-proline double helix stabilized by four inter-heavy chain disulfide bridges that limit its flexibility. The human IgG3 hinge is distinct from other subclasses by its unique extended hinge region (about four times the length of the IgG1 hinge), contains 62 amino acids (including 21 prolines and 11 cysteines), forms a non-flexible poly-proline double helix, and provides greater flexibility due to the Fab fragment being relatively far from the Fc fragment. The human IgG4 hinge is shorter than IgG1 but has the same length as IgG2, and its flexibility is intermediate between that of IgG1 and IgG2. The structure and function of immunoglobulins are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

[0062] In some embodiments, the disclosure provides for the use of an anti-CCR5 antibody or antigen-binding fragment thereof that is chimeric, humanized, or human. Chimeric and humanized forms of non-human (e.g., mouse) antibodies can contain sequences derived from non-human immunoglobulins and can be intact (full-length) chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof (such as Fv, Fab, Fab’, F(ab’)2, or other target-binding subdomains of an antibody). Generally, in a humanized antibody or antigen-binding fragment thereof, most or all of the amino acids outside the CDR regions (e.g., the framework (FR) regions) are replaced with the corresponding amino acids from a human immunoglobulin molecule. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR regions are replaced with amino acids from a human immunoglobulin molecule, while some, most, or all of the amino acids within one or more CDR regions are not altered. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are tolerated so long as they do not impair the ability of the antibody to bind to a given antigen. A humanized antibody can also include at least a portion of a human immunoglobulin constant region (Fc). Suitable human immunoglobulin molecules for use in humanizing non-human antibodies would include IgG1, IgG2, IgG3, IgG4, IgA, and IgM molecules. A “humanized” antibody will retain the same antigen specificity as the original antibody, e.g., the ability to bind to CCR5 in the context of the present disclosure.

[0063] "Human antibody" can include, for example, an antibody having the amino acid sequence of human immunoglobulin, and an antibody isolated from a human immunoglobulin library, or an antibody isolated from an animal in which one or more human immunoglobulins are transgenic and typically do not express endogenous immunoglobulin. Human antibodies can be produced using transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. A fully human antibody that recognizes a selected epitope can be generated using guide selection. In this approach, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope.

[0064] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody or an antigen-binding fragment thereof that is part of a multispecific antibody, such as a bispecific antibody or a dual variable domain (DVD) antibody. Bispecific antibodies and DVD antibodies are monoclonal antibodies that have binding specificity for at least two different antigens, one of which is CCR5, and are often human antibodies or humanized antibodies. In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody or an antigen-binding fragment thereof that has been derivatized or otherwise modified. For example, a derivatized antibody can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and the like.

[0065] In any of the foregoing embodiments, the anti-CCR5 antibody or an antigen-binding fragment thereof is conjugated to a small molecule drug to form an antibody-drug conjugate. In some embodiments, the present disclosure provides for the use of monoclonal antibody PA14, an antigen-binding fragment thereof, or an antibody that competes with monoclonal antibody PA-14 in binding to the CCR5 receptor, produced by the hybridoma cell line designated as PA14 (ATCC accession number HB-12610).

[0066] In some embodiments, the present disclosure provides for the use of the PRO140 antibody or an antigen-binding fragment thereof. PRO140 is a humanized IgG4 monoclonal antibody that binds to CCR5, as described in U.S. Pat. Nos. 7,122,185 and 8,821,877, which are hereby incorporated by reference in their entirety. PRO140 is a humanized version of the murine monoclonal antibody PA14 generated against CD4 + CCR5 + cells. Olson et al., Differential Inhibition of Human Immunodeficiency Virus Type 1 Fusion, gp 120 Binding and CC-Chemokine Activity of Monoclonal Antibodies to CCR5, J. VIROL., 73: 4145-4155. (1999). PRO140 binds to CCR5 expressed on the cell surface and potently inhibits HIV-1 entry and replication at concentrations that do not affect CCR5 chemokine receptor activity, both in vitro and in the hu-PBL-SCID mouse model of HIV-1 infection. Olson et al., Differential Inhibition of Human Immunodeficiency Virus Type 1 Fusion, gp 120 Binding and CC-Chemokine Activity of Monoclonal Antibodies to CCR5, J. VIROL., 73: 4145-4155. (1999); Trkola et al., Potent, Broad-Spectrum Inhibition of Human Immunodeficiency Virus Type 1 by the CCR5 Monoclonal Antibody PRO 140, J. VIROL., 75: 579-588 (2001).

[0067] LERONLIMAB does not downregulate CCR5 surface expression, does not deplete CCR5-expressing cells, but prevents CCL5-induced calcium mobilization in CCR5+ cells at an IC of 45 μg / ml. 50 In some embodiments, the CCR5 binder does not downregulate CCR5 surface expression, does not deplete CCR5-expressing cells, or does neither. In some embodiments, the CCR5 binder inhibits CCL5-induced calcium mobilization in CCR5+ cells at an IC of 45 μg / ml. 50 In some embodiments, the CCR5 binder is LERONLIMAB.

[0068] LERONLIMAB (PRO 140) binds to CCR5 and blocks virus entry by interfering with the final step of virus binding to the cell surface prior to virus-cell membrane fusion. LERONLIMAB (PRO 140) has been administered intravenously or subcutaneously to over 750 healthy HIV-1-infected individuals in Phase I / II / III trials. The drug has shown good tolerability following single intravenous doses of 0.5–10 mg / kg or subcutaneous (SC) injections up to 700 mg weekly. Overall, 324 patients received a weekly dose of 350 mg SC of LERONLIMAB (PRO 140) for up to 4 years. Similarly, weekly SC doses of 525 mg and 700 mg of LERONLIMAB (PRO 140) were administered to over 250 and over 150 patients, respectively.

[0069] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody that binds to the same epitope to which leronlimab binds or that competes with leronlimab in binding to CCR5. Leronlimab binds to a discontinuous epitope spanning multiple extracellular domains on CCR5, including the N-terminus and the second extracellular loop (ECL2) of CCR5 (see Trkola et al. J. Virol. 75:579-588, which is incorporated by reference in its entirety). Leronlimab directly blocks the binding of HIV Env to the CCR5 co-receptor via a competitive mechanism. Binding of leronlimab requires at least the N-terminal amino acid residue D2, and R168 and Y176 of ECL2; mutations of amino acids D95 and C101 of ECL1 and C178 of ECL2 also affect the binding of leronlimab, for example, by conformational perturbation (see Olson et al. J. Virol. 73:4145-4155, which is incorporated by reference in its entirety). Targeted loss-of-function mutagenesis followed by photocrosslinking using genetically engineered unnatural amino acids has also been used to map the antibody-GPCR complex, and it has been identified that residues 174 and 175 at the amino terminus of ECL2 form the strongest binding to leronlimab (Ray-Saha et al., Biochem. 53:1302-13010).

[0070] CCR5 amino acid residues involved in CCL5 (RANTES) binding include K1, D2, D11, E18, K26 at the N-terminus, D95 of ECL1, K171, K191, and R274 of ECL2 (see Navenot et al. J. Mol. Biol. 313:1181-1193, which is incorporated by reference in its entirety).

[0071] The nucleic acids encoding the heavy and light chains of the humanized PA140 antibody have been deposited with the ATCC. Specifically, the plasmids designated as pVK-HuPRO140, pVg4-HuPRO140(mut B+D+I), and pVg4-HuPRO140 HG2, respectively, were deposited with the ATCC (Manassas, Va., U.S.A. 20108) on February 22, 2002, as ATCC accession numbers PTA 4097, PTA 4099, and PTA 4098, respectively, in accordance with and to meet the requirements of the Budapest Treaty. The American Type Culture Collection (ATCC) is currently located at 10801 University Boulevard, Manassas, Va. 20110-2209. The plasmids designated as pVK-HuPRO140 and pVg4-HuPRO140 HG2 encode the light and heavy chains of remlimumab, respectively.

[0072] The amino acid sequences of the HCDR1-3 and LCDR1-3 of remlimumab are set forth in SEQ ID NOs: 12-14 and 9-11, respectively. The sequences of the VH and VL of remlimumab are set forth in amino acids 20-141 of SEQ ID NO: 3 and amino acids 20-131 of SEQ ID NO: 1, respectively. The sequences of the heavy and light chains of remlimumab are set forth in SEQ ID NOs: 7 and 8, respectively.

[0073] In some embodiments, the present disclosure provides for the use of an anti-CCR5 antibody comprising: (i) two light chains, wherein each light chain comprises the expression product of a plasmid designated as pVK: HuPRO140-VK (ATCC deposit designation PTA-4097), and (ii) two heavy chains, wherein each heavy chain comprises the expression product of either a plasmid designated as pVg4: HuPRO140 HG2-VH (ATCC deposit designation PTA-4098) or a plasmid designated as pVg4: HuPRO140(mut B+D+I)-VH (ATCC deposit designation PTA-4099).

[0074] In some aspects, the present disclosure provides for the use of an anti-CCR5 antibody comprising: (i) two light chains, where each light chain comprises a variable region (V L ) and a constant region (C L ) encoded by a plasmid designated as pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two heavy chains, where each heavy chain comprises a variable region (V H ) and a constant region (C H ) encoded by either a plasmid designated as pVg4: HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or a plasmid designated as pVg4: HuPRO140(mut B+D+I)-VH (ATCC Deposit Designation PTA-4099).

[0075] Pharmaceutical Composition In another aspect, the present disclosure provides for the use of a pharmaceutical composition comprising leronlimab or a fragment thereof, and another anti-CCR5 antibody or a fragment thereof described herein, for administration to a patient in need thereof. The pharmaceutical composition can comprise an antibody or antigen-binding fragment described herein and one or more pharmaceutically acceptable carriers, diluents, or excipients suitable for administration by a selected route. Pharmaceutically acceptable carriers for diagnostic and therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington’s Pharmaceutical Sciences, Mack Publishing Co. A.R.Gennaro (Ed.), 18 thIt is described in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, ed., 18th ed., 1990) and CRC Handbook of Food, Drug, and Cosmetic Excipients, CRC Press LLC (S. C. Smolinski, ed., 1992). Exemplary pharmaceutically acceptable carriers include any adjuvant, carrier, excipient, lubricant, diluent, preservative, dye / colorant, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, emulsifying agent, or any combination thereof. For example, sterile physiological saline at physiological pH and phosphate buffered saline can be suitable pharmaceutically acceptable carriers. Preservatives, stabilizers, dyes, etc. may also be provided in the pharmaceutical composition. In addition, antioxidants and suspending agents may also be used. The pharmaceutical composition may also contain diluents such as water, buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates (by way of example, glucose, sucrose, dextrin), chelating agents (by way of example, EDTA), glutathione, and other stabilizers and excipients. Neutral buffered saline, or saline mixed with non-specific serum albumin, are exemplary diluents.

[0076] A pharmaceutical composition comprising an antibody or antigen-binding fragment can be manufactured, for example, by lyophilizing the antibody or antigen-binding fragment and then mixing, dissolving, emulsifying, encapsulating or enclosing the antibody or antigen-binding fragment. The pharmaceutical composition can also comprise the antibodies or antigen-binding fragments described herein in free base form or in a pharmaceutically acceptable salt form.

[0077] The pharmaceutical composition may be formulated in the form of a solid, semi - solid or liquid composition. Solid compositions may include powders and tablets. In some embodiments, the pharmaceutical compositions described herein are lyophilized or in powder form for reconstitution with a suitable vehicle, such as sterile water, before use. In some embodiments, the pharmaceutical compositions described herein are suspensions, solutions, or emulsions. The pharmaceutical compositions and formulations can be sterilized. Sterilization can be achieved by filtration by sterile filtration.

[0078] The pharmaceutical compositions described herein can be formulated for oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal administration. As used herein, the term "parenteral" includes subcutaneous injection, intravenous injection, intramuscular injection, intracardiac injection, and intratumoral injection, or infusion techniques. In some embodiments, the pharmaceutical compositions described herein are formulated for administration as an injection solution, such as by intravenous or subcutaneous injection. Non - limiting examples of injectable formulations include sterile suspensions, solutions, or emulsions in an oily or aqueous vehicle. Suitable oily vehicles can include, but are not limited to, lipophilic solvents or vehicles such as fatty oils or synthetic fatty acid esters, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension. The suspension can also contain suitable stabilizers. Alternatively, the pharmaceutical compositions described herein are lyophilized or in powder form for reconstitution with a suitable vehicle, such as sterile pyrogen - free water, before use.

[0079] An anti-CCR5 antibody or an antigen-binding fragment thereof can be formulated for administration in a unit dosage form in combination with a pharmaceutically acceptable vehicle. Such a vehicle is essentially non-toxic and can be non-therapeutic. The vehicle can be water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate can also be used. The vehicle can contain small amounts of additives such as substances that enhance isotonicity and chemical stability (such as buffers and preservatives).

[0080] In some embodiments, for example, an aqueous formulation of the anti-CCR antibody or antigen-binding fragment provided herein for subcutaneous administration has a pH of 4 to 5.7. The aqueous formulation may include one or more excipients such as, for example, one or more buffers, one or more lyoprotectants, etc. In some embodiments, the pH of the formulation is 4.0 - 6.0, 4.1 - 5.1, 4.2 - 5.1, 4.3 - 5.1, 4.4 - 5.1, 4.5 - 5.1, 4 - 5, 4.1 - 5, 4.2 - 5, 4.3 - 5, 4.4 - 5, 4.5 - 5, or about 4.5 - 5.5, about 5.3, about 5.4, about 5.5, about 5.6, or about 5.7. In some embodiments, the formulation includes at least one buffer. In various embodiments, the buffer can be selected from histidine, citrate, aspartate, acetate, phosphate, lactate, tromethamine, gluconate, glutamate, tartrate, succinate, malate, fumarate, α-ketoglutarate, and combinations thereof. In some embodiments, the buffer is at least one buffer selected from histidine, citrate, aspartate, acetate, and combinations thereof. In some embodiments, the buffer is a combination of histidine and aspartate. In some embodiments, the total concentration of the buffer in the aqueous formulation is 10 mM to 40 mM, such as 15 mM to 30 mM, 15 mM to 25 mM, or 20 mM, etc.

[0081] In some embodiments, the aqueous formulation comprises at least one cryoprotectant. In some such embodiments, the at least one cryoprotectant is selected from sucrose, arginine, glycine, sorbitol, glycerol, trehalose, dextrose, α-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, proline, methionine, albumin, mannitol, maltose, dextran, and combinations thereof. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the total concentration of the cryoprotectant in the aqueous formulation is 3-12%, such as 5-12%, 6-10%, 5-9%, 7-9%, or 8%.

[0082] In some embodiments, the aqueous formulation comprises at least one surfactant. Exemplary surfactants include polysorbate 80, polysorbate 20, poloxamer 88, and combinations thereof. In some embodiments, the aqueous formulation comprises polysorbate 80. In some embodiments, the total concentration of the at least one surfactant is 0.01%-0.1%, such as 0.01%-0.05%, 0.01%-0.08%, or 0.01%-0.06%, 0.01%-0.04%, 0.01%-0.03%, or 0.02%.

[0083] In some embodiments, the pharmaceutical composition of the present invention is formulated in single-dose units or in forms comprising multiple dosage units. Methods for preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).

[0084] In some embodiments, the concentration of the anti-CCR5 antibody or antigen-binding fragment in the aqueous formulation is from 1 mg / mL to 250 mg / mL, such as from 10 mg / mL to 220 mg / mL, from 10 mg / mL to 200 mg / mL, from 10 mg / mL to 175 mg / mL, from 10 mg / mL to 150 mg / mL, from 10 mg / mL to 100 mg / mL, from 20 mg / mL to 200 mg / mL, from 20 mg / mL to 175 mg / mL, from 20 mg / mL to 150 mg / mL, from 20 mg / mL to 125 mg / mL, from 20 mg / mL to 100 mg / mL, from 30 mg / mL to 200 mg / mL, from 30 mg / mL to 175 mg / mL, from 30 mg / mL to 150 mg / mL, from 30 mg / mL to 125 mg / mL, from 30 mg / mL to 100 mg / mL, from 40 mg / mL to 200 mg / mL, from 40 mg / mL to 175 mg / mL, from 40 mg / mL to 150 mg / mL, from 40 mg / mL to 125 mg / mL, from 40 mg / mL to 100 mg / mL, from 50 mg / mL to 200 mg / mL, from 50 mg / mL to 175 mg / mL, from 50 mg / mL to 150 mg / mL, from 50 mg / mL to 125 mg / mL, from 50 mg / mL to 100 mg / mL, from 60 mg / mL to 200 mg / mL, from 60 mg / mL to 175 mg / mL, from 60 mg / mL to 150 mg / mL, from 60 mg / mL to 125 mg / mL, from 60 mg / mL to 100 mg / mL, from 70 mg / mL to 200 mg / mL, from 70 mg / mL to 175 mg / mL, from 70 mg / mL to 150 mg / mL, from 70 mg / mL to 125 mg / mL, from 80 mg / mL to 200 mg / mL, from 80 mg / mL to 175 mg / mL, from 80 mg / mL to 150 mg / mL, from 80 mg / mL to 125 mg / mL, from 100 mg / mL to 200 mg / mL, from 125 mg / mL to 200 mg / mL, from 150 mg / mL to 200 mg / mL, or from 160 mg / mL to 190 mg / mL, from 170 mg / mL to 180 mg / mL, or 175 mg / mL. In some embodiments, the concentration of the CCR5 binder in the aqueous formulation is from 100 mg / mL to 200 mg / mL. In some embodiments, the concentration of the CCR5 binder in the aqueous formulation is 175 mg / mL.

[0085] In some embodiments, the anti-CCR5 antibody or antigen-binding fragment thereof is formulated at a high protein concentration. Exemplary high protein concentration formulations containing anti-CCR5 antibodies are described in U.S. Patent No. 9,956,165, which is incorporated herein by reference in its entirety.

[0086] In some embodiments, the anti-CCR5 antibody or antigen-binding fragment is a formulation comprising: a concentrated anti-CCR5 antibody or antigen-binding fragment thereof in an amount greater than about 100 mg / mL and less than about 200 mg / mL, about 110 mM ~ An isotonic agent consisting essentially of sodium salts, histidine, and glycine buffer present in a total amount of about 120 mM (where the buffer is present in an amount of about 10 mM ~ About 25 mM), and a surfactant; wherein the formulation is hypotonic and has a total salt concentration of less than 100 mM.

[0087] In some embodiments, the anti-CCR5 antibody or antigen-binding fragment is a formulation comprising: a concentrated anti-CCR5 antibody or antigen-binding fragment thereof in an amount greater than about 100 mg / mL and less than about 200 mg / mL, a sodium salt in an amount greater than about 90 mM and less than 100 mM, a histidine and glycine buffer in an amount greater than about 5 mM and less than about 25 mM, a surfactant in an amount greater than about 0.001% w / v and less than about 0.2% w / v, and optionally, a stabilizer or non-salt isotonic agent in an amount of about 0.05% w / v to about 1.8% w / v; wherein the formulation has an osmotic pressure of about 250 to about 280 mOsm and a total salt concentration of less than 100 mM.

[0088] In some embodiments, the anti-CCR5 antibody or antigen-binding fragment is formulated in a low-viscosity, hypotonic formulation comprising: (a) an amount of concentrated anti-CCR5 antibody or antigen-binding fragment greater than about 100 mg / mL and less than about 200 mg / mL, (b) an amount of sodium salt selected from about 90 mM or about 95 mM, (c) an amount of histidine and glycine buffer of about 20 mM, (d) a surfactant in an amount of 0.005% - 0.2% w / v, and optionally, (e) an amount of stabilizer or non-salt isotonicity agent sufficient to provide a formulation osmotic pressure of about 260 - 280 mOs / kg; wherein the formulation has a total salt concentration of less than 100 mM.

[0089] In some embodiments, the anti-CCR5 antibody or antigen-binding fragment is a low-viscosity, hypotonic formulation and comprises: (a) an amount of concentrated anti-CCR5 antibody or antigen-binding fragment greater than about 100 mg / mL and less than about 200 mg / mL, (b) a salt in an amount selected from about 90 mM or about 95 mM, wherein the salt is selected from sodium chloride, sodium gluconate, or sodium lactate, (c) an amount of histidine and glycine buffer of about 20 mM, (d) a surfactant in an amount of about 0.005% - about 0.2% w / v, wherein the surfactant is polysorbate, poloxamer, or pluronic acid, (e) a stabilizer or non-salt isotonicity agent present in an amount sufficient to provide a formulation osmotic pressure of about 230 mOs / kg - about 280 mOs / kg, wherein the stabilizer or non-salt isotonicity agent is selected from sugar alcohols, monosaccharides, disaccharides, or combinations thereof; wherein the formulation has a total salt concentration of less than 100 mM.

[0090] In some embodiments, the anti-CCR5 antibody or antigen-binding fragment is formulated in a composition comprising: an anti-CCR5 antibody or antigen-binding fragment in an amount greater than about 100 mg / mL and less than about 200 mg / mL, a sodium salt present at a concentration greater than about 90 mM, a histidine and glycine buffer present in a total amount of 110 mM to 120 mM, and a surfactant present in an amount of about 0.001% to about 0.2% w / v; wherein the composition has an osmotic pressure of about 230 to about 290 mOs / kg and a total salt concentration of less than 100 mM.

[0091] In some embodiments, the anti-CCR5 antibody or antigen-binding fragment is provided as a manufactured product comprising a container and the formulation, the formulation comprising: an anti-CCR5 antibody or antigen-binding fragment at a concentration greater than 100 mg / mL and less than 200 mg / mL, an isotonic agent of a sodium salt present at a concentration greater than about 90 mM and a histidine and glycine buffer present in a total amount of about 110 mM to about 120 mM; and the formulation has a total salt concentration of less than 100 mM, a surfactant in an amount of about 0.005 to about 0.2%, and instructions for use.

[0092] In some embodiments, the anti-CCR5 antibody or antigen-binding fragment is administered at a dose of 700 mg of the anti-CCR5 antibody or antigen-binding fragment (175 mg / mL), delivered as two injections of 2 mL each, and administered subcutaneously on opposite sides of the abdomen. Each vial of the anti-CCR5 antibody or antigen-binding fragment product may contain about 1.4 mL of the antibody at a concentration of 175 mg / mL. In any of the aforementioned pharmaceutical compositions, the anti-CCR5 antibody may be leronlimab.

[0093] Method of Use The CCR5 receptor is a C-C chemokine G-protein coupled receptor that is expressed in lymphocytes (such as NK cells, B cells), monocytes, macrophages, dendritic cells, subsets of T cells, etc. The extracellular portion presents potential targets for antibodies targeting CCR5 and contains an amino-terminal domain (Nt) and three extracellular loops (ECL1, ECL2, and ECL3). The extracellular portion of CCR5 contains only 90 amino acids distributed over four domains. The largest of these domains are Nt and ECL2, each approximately 30 amino acids (Olson et al., Curr. Opin. HIV AIDS, March, 4(2): 104-111 (2009)).

[0094] The CCR5 receptor binds to a chemokine known as CCL5 (C-C chemokine ligand 5), which is an inflammatory chemokine that plays an important role in immune mechanisms such as the control of cell recruitment and activation in basal and inflammatory states. CCL5 acts as a major regulator of the migration of CCR5+ cells (such as monocytes and T cells) to the site of inflammation and dictates the migration of monocytes and T cells to the site of injury or infection. CCR5 also plays an important role in the differentiation and activation of CD8+ T cells. Many of the biological actions of chemokines are mediated by their interaction with chemokine receptors on the cell surface. The best-known receptor associated with CCL5 is the CCR5 receptor, although CCR1 and CCR3 are also known as CCL5 receptors, and CCR4 and CD44 are auxiliary receptors. Tamamis et al., Elucidating a Key Anti-HIV-1 and Cancer-Associated Axis: The Structure of CCL5 (Rantes) in Complex with CCR5, SCIENTIFIC REPORTS, 4: 5447 (2014).

[0095] The formation of the complex of CCL5 ligand and CCR5 receptor causes a structural change in the receptor that activates the subunits of the G protein, induces signal transduction, and results in changes in the levels of cyclic AMP (cAMP), inositol trisphosphate, intracellular calcium, and tyrosine kinase activation. These signal transduction events cause cell polarization and translocation of the transcription factor NF-kB, resulting in increased phagocytic ability, cell survival, and transcription of inflammatory genes.

[0096] Treatment with a CCR5 binder, such as leronlimab, can affect major anti-inflammatory mediators or other biomarkers in patients with NASH and NAFLD, as disclosed herein. In particular, the production of CCR5L (RANTES) can increase as a direct result of CCR5 stabilization on the surface of immune cells by a CCR5 binder such as leronlimab. The binding and signal transduction of CCR5 / RANTES are important mediators in the activation of CD8+ T cells.

[0097] In several reports, CCR5 and CCR2 have been shown to have the ability to form heterodimers, and heterodimerization has been shown to preferentially alter signal transduction outcomes from chemotaxis to cell adhesion. Treatment with a CCR5 binder such as leronlimab significantly improves the surface stability of CCR5 and increases the likelihood of heterodimerization with CCR2 on cells that express both receptors. Heterodimerization of CCR5 / CCR2 does not prevent intracellular migration, but a CCR5 binder such as leronlimab-bound CCR5 prevents intracellular migration, which is thought to be an important mechanism by which CCR5 binders such as leronlimab restore vascular integrity and immune function.

[0098] Non-alcoholic fatty liver disease (NAFLD) is a spectrum of conditions in which fat deposits form in the liver. NAFLD encompasses non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NAFL is characterized by the accumulation of fat without substantial inflammation or liver injury. NAFL may cause pain in patients but rarely causes clinically relevant liver injury. NASH is characterized by the presence of both fat deposits and inflammation in the liver. This inflammation can lead to liver fibrosis and other complications such as cirrhosis or liver cancer. CCR5 and CCR2 are deeply involved in the development of NASH and associated fibrosis. (See, for example, Lefere S, Devisscher L, Tacke F. Targeting CCR2 / 5 in the treatment of nonalcoholic steatohepatitis (NASH) and fibrosis: opportunities and challenges. Expert Opin Investig Drugs. 2020 Feb;29(2):89-92. doi: 10.1080 / 13543784.2020.1718106. Epub 2020 Jan 20. PMID: 31952447). Thus, dysregulation of CCR5 and CCR2 may play an important role in the development of these conditions, and treatment with CCR5 binders may be beneficial. Details of methods and uses for treating NASH and associated symptoms or further disorders such as fibrosis and other scarring with the CCR5 binder leronlimab are provided in the Examples. Details of these methods and uses, including dosage and administration regimens and target patient populations, can be combined with any of the present disclosure regarding the modulation of CCR5 and CCR2 and the administration of CCR5 binders. In particular, CCR5 binders such as leronlimab can reduce liver inflammation. Leronlimab may be administered at any dose and on any schedule, but in a specific embodiment, it may be administered by intravenous injection once a week at a dose of 350 mg or 700 mg. Liver inflammation can be measured by circulating cytokine levels in the patient's blood or by biopsy.Reduction of inflammation, fat deposits, fibrosis, scarring, or cirrhosis can also be measured by MRI or other medical imaging methods, liver function tests, or any method clinically used to evaluate these conditions. MRI-based evaluations can include (Proton Density Fat Fraction) PDFF and iron-corrected T1 mapping (cT1) evaluations. Treatment efficacy can be measured by the decrease in hepatic fat fraction, as evaluated by MRI-PDFF, from the pre-treatment baseline. This change can be evaluated 6 weeks, 12 weeks, 14 weeks, 20 weeks, or 24 weeks after the first administration of the CCR5 binder. Treatment efficacy can also be measured by the decrease in fibroinflammatory activity in the liver, as evaluated by cT1, from the pre-treatment baseline. This change may also be evaluated 6 weeks, 12 weeks, 14 weeks, 20 weeks, or 24 weeks after the first administration of the CCR5 binder.

[0099] The present disclosure further provides a method of treating NASH, NAFLD, and related symptoms and disorders by administering reloramab to a patient with NASH or NAFLD, or a patient at high risk of developing NASH or NAFLD. Patients with NAFLD may be at increased risk of developing NASH. Patients with NAFLD with elevated levels of biomarkers related to inflammation may be at increased risk of developing NASH. Patients with abnormal results of PDFF and / or CT1 MRI may be at increased risk of developing NASH.

[0100] CCR5 haplotype pairs (genotypes) based on the -2459 position CCR5 promoter polymorphism are known. The single nucleotide polymorphism (SNP) at -2135 is 100% linked to the SNP at -2459 position, with -2459G always linked to -2135T and -2459A always linked to -2135C. CCR5 haplotypes not associated with increased CCR5 cell surface expression may include the type A / A of SNP2459. The HHE and HHG haplotypes are often associated with increased CCR5 cell surface expression. The effect of CCR5 haplotypes in HIV is described in Catano G, Chykarenko ZA, Mangano A, et al. Concordance of CCR5 genotypes that influence cell-mediated immunity and HIV-1 disease progression rates. J Infect Dis. 2011;203(2):263-272. doi:10.1093 / infdis / jiq023, which is incorporated herein by reference.

[0101] Patients having CCR5 haplotypes associated with overexpression of CCR5 and high surface levels of CCR5, particularly the HHE and HHG haplotypes, may be at high risk of developing NASH. Patients may include mammals such as humans or non-human primates. In one aspect, the patient is human. The patient can be male or female and can be of any suitable age including infants, juveniles, young adults, adults, and the elderly, although the likelihood of NASH is typically higher in older patients.

[0102] In patients, reloramab may be administered once, or for a certain period such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 6 months, 9 months, 1 year, 2 years, etc., until the tendency to develop NASH is eliminated, until at least one symptom of NASH is eliminated, or until at least one MRI image index or biomarker of NASH returns to a normal level or a level not associated with the presence of NASH. In some embodiments, reloramab may be administered until the serum RANTES level is low enough not to have a clinical inflammatory effect on NASH. Administration may be recommended when the MRI or biomarker levels progress in a direction indicating recurrence or exacerbation of NASH, or when the RANTES level is no longer low enough not to have a clinical inflammatory effect on NASH.

[0103] In the case of NASH patients, the optimized RANTES level reflects the intermediate CCR5 binding by reloramab. This RANTES level is very different from the desired level in HIV patients; in the case of HIV patients, it is desirable for as much CCR5 binding by reloramab as possible to prevent HIV binding and cell infection. In NASH, if reloramab is administered in excess, CCR5 binds excessively and the immune response increases. At a low dose of reloramab, intermediate CCR5 binding occurs, causing a change in immune function but not an increase in the immune response.

[0104] The appropriate dosage, preferred period, and frequency of administration of reloramab are determined by factors such as the patient's condition, size, weight, body surface area, age, gender, type and severity of the disease, the specific treatment being administered, the specific form of the active ingredient, the time and method of administration, and other drugs administered simultaneously, which can be easily determined by those skilled in the art. The dosage can be in the range of 0.1 to 100,000 μg / kg. Based on the composition, the dosage may be delivered continuously, such as by a continuous pump, or at regular intervals, for example, on separate occasions one or more times. The desired time intervals for multiple dosages of a particular composition can be determined by those skilled in the art without undue experimentation.

[0105] In some embodiments, Relonumab is administered to a patient multiple times, and each administration delivers to the patient an antibody or a binding fragment thereof in an amount of 0.01 mg / kg body weight to 50 mg / kg body weight. In another embodiment, each administration delivers to the patient Relonumab in an amount of 0.05 mg / kg body weight to 25 mg / kg body weight. In a further embodiment, each administration delivers to the patient Relonumab in an amount of 0.1 mg / kg body weight to 10 mg / kg body weight. In yet a further embodiment, each administration delivers to the patient Relonumab in an amount of 0.5 mg / kg body weight to 5 mg / kg body weight. In another embodiment, each administration delivers to the patient Relonumab in an amount of 1 mg / kg body weight to 3 mg / kg body weight. In another embodiment, each administration delivers to the patient Relonumab in an amount of about 2 mg / kg body weight.

[0106] Relonumab may be administered once, twice, or multiple times. In one aspect, lenolutamide is administered multiple times, and the first administration is separated from subsequent administrations by an interval of less than one week. In another aspect, the first administration is separated from subsequent administrations by an interval of at least one week. In a further aspect, the first administration is separated from subsequent administrations by an interval of one week. In another aspect, the first administration is separated from subsequent administrations by an interval of 2 to 4 weeks. In another aspect, the first administration is separated from subsequent administrations by an interval of 2 weeks. In a further aspect, the first administration is separated from subsequent administrations by an interval of 4 weeks. In yet another aspect, lenolutamide is administered multiple times, and the first administration is separated from subsequent administrations by an interval of at least one month. In yet another aspect, lenolutamide is administered once a week for 2 weeks. In yet another aspect, lenolutamide is administered once a week for 4 weeks. In yet another aspect, lenolutamide is administered once a week as needed.

[0107] In a further aspect, lenolutamide is administered to a patient via intravenous infusion. In another aspect, lenolutamide is administered to a patient via subcutaneous injection. In another aspect, lenolutamide is administered to a patient via intramuscular injection.

[0108] In some aspects, lenolutamide is administered as a once-weekly dose of 350 mg to 1400 mg, or about 525 mg or about 700 mg or about 1050 mg. In some aspects, lenolutamide is administered as a twice-weekly dose of 350 mg to 1400 mg, or about 525 mg or about 700 mg or about 1050 mg. In some aspects, lenolutamide is administered once a week at a dose of about 700 mg. In some aspects, lenolutamide is administered in a manner and at a dose similar to that used to treat HIV infection.

[0109] Relonlimab (PRO 140) is currently approved or in development for the indications of HIV, graft-versus-host disease (GVHD), metastatic triple-negative breast cancer (mTNBC), metastatic colorectal cancer (mCRC), and acute or long-term COVID. The safety profile of Relonlimab (PRO 140) has been widely evaluated in clinical trials. Relonlimab (PRO 140) has been administered intravenously or subcutaneously to more than 750 healthy HIV-1 infected individuals in Phase I / II / III trials. This drug has shown good tolerability after single intravenous doses of 0.5 - 10 mg / kg or up to 700 mg per week by subcutaneous (SC) injection. Overall, 324 patients received a SC weekly dose of 350 mg of Relonlimab (PRO 140) with a maximum treatment duration of 4 years. Similarly, more than 250 and more than 150 patients received SC weekly doses of 525 mg and 700 mg of Relonlimab (PRO 140), respectively.

[0110] In some embodiments, Relonlimab can be administered at a dose of 700 mg (175 mg / mL) by two 2 mL subcutaneous injections, administered on opposite sides of the abdomen. In some embodiments, Relonlimab can be administered at a dose of 525 mg or 350 mg by one or two 2 mL subcutaneous injections, and if two injections are used, they can be administered on opposite sides. In some embodiments, the dose of Relonlimab is determined by the patient's CCR5 haplotype, and patients with CCR5 haplotypes associated with increased cell surface expression of CCR5, such as the HHE or HHG haplotypes, receive a higher dose of Relonlimab or more frequent dosing compared to patients without such haplotypes. For example, patients with CCR5 haplotypes associated with high cell surface levels of CCR5 can receive a weekly dose of 525 mg or 700 mg of Relonlimab, or a dose of 350 mg or 535 mg twice a week.

[0111] In some embodiments, determining an appropriate Relonabuvir dosage is done by measuring the baseline level of an MRI metric or biomarker, administering a low dosage of Relonabuvir, such as 350 mg per week, for a short period, such as one week, two weeks, three weeks, one month, or two months, then measuring the MRI metric or biomarker to determine any changes. If the changes do not exceed a pre-set level, or if the overall level of the MRI metric or biomarker does not meet a set parameter (e.g., serum RANTES level is about zero, which could be less than about 0.01 ng / mL), the dosage of Relonabuvir may be increased to 525 mg or 700 mg per week, or the frequency of administration may be increased, for example, to twice a week for a short period, and then the MRI metric or biomarker may be measured again and compared to the pre-set change or overall level. If the set parameter is not met, the dosage or frequency may be further increased, for example, to a dosage of 700 mg once a week. Once an effective dosing regimen is determined, the patient may continue that dosing regimen or periodically test a lower dosing regimen and measure the effect on the MRI metric or biomarker to determine if the lower dosing regimen is effective at that time. Some patients may be able to maintain clinical benefit at a lower Relonabuvir dosage or may even be able to completely discontinue Relonabuvir as inflammation decreases.

[0112] Specific biomarkers for NASH include (i) chemokines, (ii) CCL, particularly CCL2, 3, 5, 11, and 18, (iii) interleukins, (iv) adhesion markers such as VCAM, (v) apoptosis and necrosis markers, particularly mitochondrial CK18 m30 and 65 apoptosis and necrosis. Biomarker testing may be performed on one or more members of one of these groups, or on one or more members of more than one, two or more, three or more, four or more, or all of these groups.

Example

[0113] Example 1: Preclinical Study of Elonremab for the Treatment of NASH The potential of elonremab in the treatment of NASH was demonstrated in a preclinical model of fatty liver disease. Immunodeficient NOD scid IL-2 receptor gamma knockout mice (NSG) were fed a high-fat NASH-inducing diet, transplanted with human stem cells to regenerate the defective immune system, and treated with elonremab or an Ig control. Sixteen male mice were first humanized by intravenous inoculation of normal human umbilical cord blood cells. After 5 weeks of feeding with normal mouse chow, the mice were successfully humanized, with >25% human CD45 cells observed in the peripheral blood. The mice were switched to a high-fat (52%), high-cholesterol (1.25%) diet. (FPC diet: fructose, palmitate, cholesterol, trans fat; Envigo-Teklad TD.160785). Elonremab and a control antibody (normal human IgG, Sigma) were delivered by intraperitoneal (i.p.) administration at a dose of 2 mg i.p. twice a week to n = 8 mice / group. The mice were euthanized 16 weeks after the start of the high-fat, high-cholesterol diet.

[0114] Liver sections were analyzed for markers of NAFLD. Representative 20x microscopic images of elonremab- and generic IgG-treated mice are shown in Figure 1A. Mice treated with elonremab showed a marked reduction in the presence of liver fat, as shown by Oil Red O staining. Regions of interest were identified in the micrographs, digitized using an Aperio AT2 slide scanner (Leica Biosystems), and analyzed using QuPath v0.2.01 imaging software. The results obtained from all mice used in this study are shown in Figure 1B. This graph shows the mean and standard error (SE) of Oil Red O-positive pixels for the entire region of interest (ROI) calculated for both treatment groups. The positive rate for the IgG treatment group was 9.751 ± 1.789. The positive rate for the elonremab treatment group was 3.207 ± 1.515. Student's t-test p = 0.014. Steatosis was numerically scored according to semi-quantitative pathological criteria. The results indicate that elafibranor inhibited the development of fatty liver, a key feature of early-stage NASH.

[0115] Example 2: Clinical study of elafibranor for the treatment of NASH Study objective A clinical study was conducted to evaluate the potential of elafibranor in the treatment of NASH. This trial, called CDI-NASH-01, demonstrated that elafibranor can be used to treat NASH and the symptoms and further disorders resulting from NASH, as shown below.

[0116] Study design CDI-NASH-01 was designed as a multi-center, concurrent Phase 2a trial and was subsequently changed to an exploratory study to evaluate the dose, efficacy, and safety of elafibranor at doses of 700 mg and 350 mg for the treatment of NASH. Biomarkers were also measured to assist in the design of future trials and the understanding of the potential mechanism of action of elafibranor. The primary objective of Part 1 of the study was to evaluate the efficacy of 700 mg of elafibranor (n = 22) compared to placebo (n = 28) in improving NASH tests in adult patients diagnosed with NASH / NAFLD. Subsequently, Part 2 was added to evaluate the efficacy of 350 mg of elafibranor in improving NASH / NAFLD tests in adult patients diagnosed with NASH (n = 22). The secondary objective of this study was to evaluate the safety and tolerability of elafibranor compared to placebo in adult patients diagnosed with NASH. The summary and related timeline of this study are as follows:

[0117] Part 1: - Week - 4 to Week 0 - Screening Treatment assignment (elafibranor vs. placebo) was performed at the beginning of Week 0 Week 0 to Week 1 - Double-blind, placebo-controlled trial (randomized 1:1) Week 1 to Week 14 - Treatment is administered weekly (±1 day from the final dose) Week 13 to Week 14 - End of treatment visit Week 14 to Week 18 - Follow-up

[0118] Part 2 - Week 4 to Week 0 - Screening Treatment assignment (relonremab) was done at the beginning of Week 0 Week 0 to Week 14 - Single-group, open-label treatment is administered weekly (±1 day from the final dose) Week 13 to Week 14 - End of treatment visit Week 14 to Week 18 - Follow-up In both Part 1 and Part 2, relonremab was administered subcutaneously. In both Part 1 and Part 2, follow-up was 28 (±) 3 days after the end of treatment (EOT) or early termination (ET) visit.

[0119] Eligible patients included adults aged 18 to 75 years with a proven phenotype of non-alcoholic steatohepatitis (NASH). Patients had a body mass index (BMI) of 28 kg / m 2 or more, and at screening, it was necessary to demonstrate the presence of liver fat content defined as MRI - (proton density fat fraction) PDFF of 8% or more and iron-corrected T1 mapping (cT1) of 800 milliseconds (msec) or more. A stable weight (±5%) was required for 6 months prior to screening. Exclusion criteria included, but were not limited to, HIV, autoimmune hepatitis, excessive alcohol consumption, viral hepatitis, and past or planned liver transplantation. There were no patients on combination treatment with semaglutide in the 350 mg trial, but 18% of the patients in the placebo group were on combination treatment with semaglutide. Patient demographics are summarized in Figure 2. Analysis was performed in the full analysis set (n = 72), of which 44% were Hispanic or Latino ethnicity and 58% had moderate to severe fibroinflammation (cT1 ≥ 875 ms) at baseline.

[0120] Study treatment Subjects who met all eligibility criteria according to the data collected during the screening period were eligible for registration. All subjects who did not meet the eligibility criteria were considered screening failures and the study was terminated without further evaluation. This study consisted of the following two parts: In Part 1 of the study, eligible subjects were randomly assigned 1:1 to one of two study groups and received either 700 mg of relonremab (Group A) or placebo (Group B) once a week (±1 day) for up to 13 weeks during the treatment period at the study site (maximum of 60 participants).

[0121] In Part 2 of the study, eligible subjects were enrolled and received 350 mg of relonremab in an open-label manner once a week (±1 day) for up to 13 weeks during the treatment period at the study site (maximum of 28 participants). There were 21 patients for cT1 analysis and 22 patients for PDFF analysis. One patient had PDFF obtained at the end of treatment, but despite repeated attempts, the MRI for cT1 could not be evaluated for quality (due to three incisions), so cT1 results were not obtained for that patient. Since this patient had severe cT1 at baseline, the total number of patients who received 350 mg of relonremab, the number of patients with moderate NASH who received 350 mL of relonremab, and the number of patients with severe NASH who received 350 mL of relonremab were all reduced from the numbers seen with PDFF.

[0122] The primary efficacy objective of this study was the change from baseline in liver fat content, which was evaluated at week 14 by magnetic resonance imaging proton density fat fraction (MRI-PDFF). The secondary efficacy goal was the change from baseline in hepatic fibroinflammatory activity, evaluated at week 14 by cT1 (corrected T1). cT1 was obtained by multiparametric magnetic resonance imaging of the liver and is a quantitative measure for assessing the composite of hepatic inflammation and fibrosis, expressed in milliseconds (msec). Additional goals were the changes from baseline to week 14 in LFT, chemokine, and cytokine levels, and in major biomarkers of inflammation. In particular, the changes in serum cK18 and K18 by M30-M65 ELISA were evaluated at week 14.

[0123] Over-transcription of CCR5 by the HHG allele is associated with an increase in surface CCR5 on T cells but not on monocytes, and is associated with reduced T cell immunity, reduced T cell proliferation, and reduced T cell differentiation. Patients with over-transcription of CCR5 may have delayed hypersensitivity responses and Th1 impairment. A pathway that may be associated with these effects is shown in FIG. 12. The effect of CCR5 haplotypes in HIV is described in Catano G, Chykarenko ZA, Mangano A, et al. Concordance of CCR5 genotypes that influence cell-mediated immunity and HIV-1 disease progression rates. J Infect Dis. 2011;203(2):263-272. doi:10.1093 / infdis / jiq023, which is incorporated herein by reference.

[0124] CCR5 haplotypes were investigated in five patients. Haplotypes with very high transcription, and thus high cell surface expression of CCR5, include HHE / HHE and HHG1 / HHG1. When the high-transcription allele is combined with the null allele delta 32 or HHG2, it defaults to a heterozygous allele by default, and HHE / HHG2 and HHG1 / HHG2 are also high transcribers of CCR5. Other combinations including HHE / HHG1 were also included in the patient population showing high transcription.

[0125] Results Treatment with leronlimab was generally well tolerated in both Part 1 and Part 2 of the study (see Figures 3 and 4). There were no drug-related treatment-emergent adverse events of grade 3 or higher. Injection site reactions and mild diarrhea occurred more frequently with leronlimab than with placebo but were not associated with discontinuation. Part 1: Leronlimab 700 mg did not reduce the mean changes in proton density fat fraction (PDFF) and cT1 from baseline to week 14 compared to placebo. Part 2: Leronlimab 350 mg significantly reduced the mean changes in PDFF and cT1 from baseline to 14 weeks compared to placebo.

[0126] Pooled Part 1 and 2: In the pooled 350 mg and 700 mg groups, PDRR and cT1 were also significantly reduced compared to placebo. CCR5 haplotype analysis in a small sample size (n = 5) suggests that certain haplotypes may be more suitable for the 700 mg dose of leronlimab. In the analysis, specific patients were grouped by other differentiating factors, such as i) the presence of moderate NASH defined by baseline cT1 ≥ 875 ms, or the presence of severe NASH defined by baseline cT1 ≥ 950 ms, and ii) CCR5 haplotype.

[0127] MRI Analysis : The results of MRI PDFF and cT1 are shown in Figure 6A. Representative MRI images are shown in Figure 6B. The mean rate of change from baseline PDFF was significantly reduced in the 350 mg group compared to placebo (-5.94% vs +9.85%, p = 0.008), but not in the 700 mg group (+3.75% vs +9.85%, p = 0.135). (See Tables 1 and 2). The mean change in cT1 was significantly reduced in the 350 mg group compared to placebo (-24.38 ms vs +27.64 ms, p = 0.021), but not in the 700 mg group (-2.73 ms vs +27.64 ms, p = 0.059). In the 350 mg subgroup with baseline cT1 ≥ 875 ms, significant reductions were seen in both PDFF and cT1 compared to placebo (-4.37% vs +9.85%, p = 0.020 and -42.00 ms vs +27.64 ms, p = 0.011, respectively). In subjects with baseline cT1 ≥ 950 ms, PDFF and cT1 were significantly reduced with 350 mg compared to placebo (-9.39% vs +9.85%, p = 0.027 and -68.85 ms vs +27.64, p = 0.009, respectively). (See Table 1). In post hoc analysis, the mean PDFF rate and mean cT1 were significantly reduced in the pooled 350 + 700 mg group compared to placebo (-1.09% vs +9.85%, p = 0.014 and -13.30 ms vs +27.64 ms, p = 0.013), and in the 700 mg group with a genetic haplotype known to overproduce CCR5, significantly reduced compared to placebo (-27.9% vs +9.85%, p = 0.006 and -45.4 ms vs +27.64 ms, p = 0.013). Overall, when fibroinflammation was more prominent, patients showed a more prominent reduction in cT1, with up to a -69 ms reduction in patients with severe NASH.

[0128] Serum analysis: Figure 5 shows a heatmap of changes in blood chemistry, including cytokines and other markers, in the patients of the study. Additional data analyzing the patients individually based on cT1 levels are shown in Table 1. The average change in M65 ELISA (cK18 and K18) from baseline to week 14 decreased in the 350 mg group (340.55 - 332.4 U / L, -8.18), while it increased in the placebo group (301.96 - 411.64 U / L, +109.78).

[0129] Reductions in the general liver function biomarkers ALT, AST, and alkaline phosphatase were observed in the 350 mg group and the cT1 lower group compared to the placebo. Specifically, in patients with high ALT at baseline (n = 8), when administered 350 mg of leronlimab, ALT decreased compared to the placebo (mean 83.7 - 54.3; -29.3 U / L vs. 87.4 - 83.2; -4.2 U / L), and accordingly, PDFF (-18% vs. 6%) and cT1 (-69 ms vs. 5 ms) also decreased.

[0130] Reductions in the chemotactic proteins CCL2, CCL3, CCL11, and CCL18 were also observed in patients administered 350 mg of leronlimab, while in patients administered 700 mg of leronlimab, the balance of these biomarker levels or an increase was observed (however, different results may be expected in patients with a haplotype corresponding to an increase in surface CCR5 expression). VCAM and EN RAGE levels were also reduced by the administration of 350 mg of leronlimab. The potential mechanisms of action suggested by these reductions are shown in Figure 8.

[0131] Levels of IL-1 beta, IL-1 IRA, IL-6, IL-8, and TNF receptor 2 were also reduced in patients administered 350 mg of leronlimab. The potential mechanisms of action suggested by these reductions are shown in Figure 9. Cardiovascular biomarkers were also positively affected by relugolix. In patients administered 350 mg of relugolix, favorable increases in apolipoprotein A1S and HDL levels, a reduction in the neutrophil-to-lymphocyte ratio, a reduction in TIMP-1 (which may correlate with reduced activation of the fibrogenic pathway), and reductions in EN-RAGE and VCAM (which are highly likely to have systemic effects such as endothelial inflammation) were observed. The potential mechanisms of action of the cardiovascular effects of relugolix suggested by these biomarkers are shown in Figure 11.

[0132]

Table 1

[0133] Additional data, including individual measurements of patients administered placebo in combination with semaglutide, are shown in Table 2. Favorable increases in apolipoprotein A1 and HDL were observed in patients administered 350 mg of relugolix, but not in patients administered semaglutide + placebo or in the placebo group. Reductions in the inflammatory biomarkers IL-1RA, IL-6, and sTNFR-2 were observed in the group administered 350 mg of relugolix, but not in the group administered semaglutide + placebo or in the placebo group. Levels of the biomarkers CCL5, CCL11, CCL18, VCAM, CCL2, CCL3, and VEGF were also reduced in patients administered 350 mg of relugolix compared to the placebo group. These biomarkers did not increase significantly in the group administered semaglutide + placebo compared to the placebo group.

[0134]

Table 2

[0135] The CCR5 promoter region has been shown to play an important role in the transcriptional control of CCR5 in the progression of diseases such as HIV, HBV, Chagas disease, heart disease, and cancer. The transcriptional activity of CCR5 and disease progression can be delayed or accelerated. Surface CCR5 expression levels are also involved in the progression of NASH disease, and specific CCR5 haplotypes were correlated with dose and treatment outcomes. Data on patients with CCR5 haplotypes with increased surface expression when administered 700 mg of elafibranor are included in Figure 5 as "700 mg HM" and in Figure 6A as "700 mg haplotype".

[0136] Specifically, certain haplotypes with high cell surface levels of CCR5 on T cells but insufficient T cell regulation were shown to exhibit different responses to 350 mg and 700 mg of elafibranor compared to patients with low cell surface levels of CCR5 but more normal T cell regulation. Patients with haplotypes correlated with increased CCR5 expression required a high dose of elafibranor to benefit from treatment. Specifically, patients with haplotypes correlated with low to normal surface expression of CCR5 showed a negative effect when administered 700 mg of elafibranor but benefited at a dose of 350 mg, while patients with haplotypes correlated with high surface expression of CCR5 showed a positive effect when administered 700 mg or 350 mg of elafibranor, with a greater positive effect at a dose of 700 mg. All patients appeared to benefit from a dose that reduced RANTES (CCL5) levels to nearly zero.

[0137] Patients with haplotypes associated with increased CCR5 expression showed a significant increase in HDL and a reduction in CK18 when administered 700 mg of elafibranor, indicating that CCR5 binding is optimized for surface CCR5 mitochondrial changes that include a reduction in apoptosis and necrosis. In these patients, improvement in apolipoprotein A1 and steatosis was also observed with 700 mg of elafibranor. PDFF decreased by 28%.

[0138] Figure 10 shows the possible mechanisms of action in patients with increased CCR5 cell surface expression. Helper T cells use glycolysis and the glutaminolysis system for energy, while regulatory T cells (Tregs) use fatty acids. Tregs have a longer lifespan than helper T cells. Therefore, the new cells produced after administration of leronlimab maintain a rebalance of Treg and helper T cell levels because the new cells use less glucose and glutamine for energy production. Since leronlimab also reduces IL-6 and IL-1 beta levels, the new CD4 cells use more efficient energy sources such as fatty acids.

[0139] The conversion of the TH response from TH17 / TH1 is caused by the reduction of TGF-beta and IL-6, which changes the FasL-induced mitochondrial permeability. This externally changes the activation of caspases via T lymphocytes (FasL), reducing apoptosis and necrosis. Consistent with this, patients who received 700 mg of leronlimab and showed an increase in CCR5 cell surface levels showed a statistically significant reduction in CK18, M30, and M65. This mechanism provides further evidence of the effect of CCR5 on steatosis and metabolism when optimized with haplotype-matched dosing. Insulin resistance may also be improved by a more dominant TH2 / TH22 response and Treg balance, in contrast to the high insulin resistance associated with a dominant TH1 / TH17 and Tc (cytotoxic) T cell response.

[0140] Conclusion These results demonstrate that leronlimab significantly reduced the mean change from baseline to week 14 of PDFF, cT1, CCL3, CCL18, VCAM, and other inflammatory markers in the 350 mg group. The reduction was also observed in the moderate to severe fibroinflammatory subgroup in which the relevant biomarker tests were performed. The mechanism of action of leronlimab appears to be multifactorial, including competitive binding of CCR5, which affects metabolic and fibroinflammatory parameters leading to a systemic reduction in vascular permeability, atherosclerosis, and oxidative stress, as well as VCAM, CCL2, CCL3, CCL11, and CCL18.

[0141] In particular, CCR5 is thought to dimerize with itself and other chemokine receptors, and these heterodimers may be able to signal through the native ligand of either receptor. Leronlimab stabilizes the cell surface level of CCR5. The data in this example suggest that CCR5 / CCR2 heterodimerization causes a reduction in CCL2 and CCL18 in serum, while the reduction in serum CCL3 and CCL11 is likely due to the competitive antagonism of leronlimab-induced CCR5. This pathway and effect are shown in Figure 7.

[0142] The reduction in serum VCAM levels observed in patients administered 350 mg of leronlimab is thought to be specific to leronlimab and differentiates the effect of the antibody from other CCR2 / CCR5 agents. The reduction in CCL2 observed is also thought to be specific to leronlimab and differentiates leronlimab from other CCR5 antagonists. This data also suggests that when administering GLP-1 agents for the treatment of NASH, in addition to metabolic control, control of inflammation may be required.

[0143] This data also suggests that CCR5 haplotypes may be considered when determining the appropriate dose of leronlimab for patients with NASH or other inflammatory diseases, and higher doses may be prescribed to patients with haplotypes with more CCR5 transcription and cell surface expression. This data also suggests that when the patient's haplotype has not been measured, the dose of leronlimab can be adjusted so that the RANTES level is nearly zero or to the level of another biomarker similarly associated with a reduction in inflammation.

[0144] The various aspects described above can be combined to provide further aspects. U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the application data sheet, including U.S. Provisional Application No. 63 / 354,664, filed on June 22, 2022, are hereby incorporated by reference in their entirety. Aspects of the embodiments can be modified, if desired, to employ concepts from various patents, applications, and publications to provide yet further additional embodiments.

Claims

1. A method for treating or preventing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in a patient by administering to the patient an effective amount of relonatilumab for treating or preventing NAFLD or NASH.

2. The method according to claim 1, wherein relonatilumab is administered by injection.

3. The method according to claim 1 or claim 2, wherein relonatilumab is administered weekly.

4. The method according to any one of the preceding claims, wherein relonatilumab is administered in an amount effective for treating or preventing NASH-related liver fibrosis.

5. The method according to any one of the preceding claims, wherein relonatilumab is administered at a dose of 350 mg.

6. The method according to any one of the preceding claims, wherein relonatilumab is administered at a dose of 525 mg.

7. The method according to any one of the preceding claims, wherein relonatilumab is administered at a dose of 700 mg.

8. The method according to any one of the preceding claims, wherein the patient is evaluated for CCR5 haplotype, and if the patient has a CCR5 haplotype not associated with increased CCR5 cell surface expression, 350 mg of relonatilumab is administered to the patient weekly.

9. The method according to any one of claims 1 to 7, wherein the patient is evaluated for CCR5 haplotype, and if the patient has a CCR5 haplotype not associated with increased CCR5 cell surface expression, 525 mg of relonatilumab is administered to the patient weekly.

10. The method according to claim 8 or claim 9, wherein the CCR5 haplotype not associated with increased CCR5 cell surface expression does not include HHE or HHG.

11. The method according to any one of claims 1 to 7, wherein the patient is evaluated for CCR5 haplotype, and if the patient has a CCR5 haplotype associated with increased CCR5 cell surface expression, 525 mg of relonatilumab is administered to the patient weekly.

12. The method according to any one of claims 1 to 7, wherein the patient is evaluated for CCR5 haplotype, and if the patient has a CCR5 haplotype associated with increased CCR5 cell surface expression, 700 mg of relonatilumab is administered to the patient weekly.

13. The method according to any one of claims 11 to 12, wherein the CCR5 haplotype associated with increased CCR5 cell surface expression includes HHE or HHG.

14. The method according to any one of claims 1 to 7, wherein after administration of at least one dose of elonremab, the level of a biomarker indicating liver function or inflammation is measured, and when the level has not changed by a set amount compared to the baseline level or a previous level, or when the level is not above or below a set value, the dose or dosing frequency of elonremab is adjusted.

15. The method according to claim 14, wherein the biomarker is one or more of RANTES, CCL2, CCL3, CCL11, CCL18, VCAM, and EN RAGE.

16. The method according to claim 15, wherein the biomarker is RANTES and when its serum level is not low enough to have no clinical inflammatory effect on NASH, the dose or dosing frequency of elonremab is increased.

17. The method according to claim 16, wherein the dose is increased from 350 mg per week to 700 mg per week.

18. The method according to any one of claims 1 to 7, wherein after at least one administration of elonremab, the level of an MRI index of NASH is measured, and when the level has not changed by a set amount compared to the baseline level or a previous level, or when the level is not above or below a set value, the dose or dosing frequency of elonremab is adjusted.

19. The method according to claim 18, wherein the MRI index of NASH is PDFF or cT1.

20. The method according to claim 19, wherein when PDFF or cT1 is not below a set level or has not decreased compared to a previous measurement for the patient, the dose of elonremab is increased from 350 mg per week to 700 mg per week.

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