Methods of treating cancer and inflammation-related diseases with leronlimab

Abstract

A method of modulating CCR5 cellular pathway to treating or preventing a cancer or inflammation-related disease or facilitating transplant of HIV-negative donor cells in HIV-positive patents comprising administering an effective amount of leronlimab or a leronlimab variant to a patient in need thereof.

Description

[0001] METHODS OF TREATING CANCER AND INFLAMMATION-RELATED DISEASES WITH LERONLIMAB

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to a method of administering leronlimab to a patient to modulate CCR5 cellular pathways to treat or prevent a cancer or inflammation-related disease or to facilitate transplant of HIV-negative donor cells in HIV-positive patents. In particular embodiments, the CCR5 pathways are regulated in a cancer cell or immune system cell. In some embodiments, the cancer is colorectal cancer. In some embodiments, the inflammation-related disease is Alzeimer’s disease, metabolic dysfunction-associated steatohepatitis (MASH), or long -CO VID. In some embodiments, normal CCR5 function is dysregulated prior to leronlimab administration, while in other embodiments normal CCR5 function is intentionally modified by administration of leronlimab. In some embodiments leronlimab is provided as monotherapy, while in other embodiments leronlimab forms part of a combination therapy.

[0004] BACKGROUND

[0005] CCL5 (C-C chemokine ligand 5), an inflammatory chemokine also known as regulated upon activation and normal T cell expressed and secreted (RANTES), and its receptor, CCR5 play an important role in the development, control, and progression of a number of diseases. In particular, CCR5 serves as the primary viral receptor for human immunodeficiency virus (HIV). CCR5 also plays an important role in inflammation and in diseases resulting from dysregulation of inflammation.

[0006] CCL5 acts as a key regulator of T-cell migration to inflammatory sites, directing migration of T cells to damaged or infected sites. CCL5 also regulates T-cell differentiation.

[0007] The CCR5 receptor is a C-C chemokine G-coupled protein receptor expressed on lymphocytes, monocytes, macrophages, dendritic cells, a subset of T cells, etc. The CCR5 receptor spans the cellular plasma membrane seven times in a serpentine manner. The extracellular portions represent potential targets for HIV-inhibitory mAbs and comprise an amino-terminal domain (Nt) and three extracellular loops (ECL1, ECL2, and ECL3). The extracellular portions of CCR5 comprise just 90 amino acids distributed over four domains. The largest of these domains are at the Nt and ECL2 at approximately 30 amino acids each.

[0008] The formation of the CCL5 ligand and CCR5 receptor complex causes a conformational change in the receptor that activates the subunits of the G-protein, inducing signaling and leading to changed levels of cyclic AMP (cAMP), inositol triphosphate, intracellular calcium and tyrosine kinase activation. These signaling events cause cell polarization and translocation of the transcription factor NF-kB, which results in the increase of phagocytic ability, cell survival, and transcription of proinflammatory genes. Once G-protein dependent signaling occurs, the CCL5 / CCR5 receptor complex is internalized via endocytosis.

[0009] Evidence suggests that CCL5 / CCR5 axis signaling may be preferentially activated in certain types of cancers and that such signaling facilitates disease progression. Exploratory efforts using anti-CCR5 binding agents to alter CCL5 / CCR5 signaling in connection with some cancer types have been made.

[0010] Leronlimab (PROMO) is a monoclonal antibody that binds the CCR5 receptor, blocking CCL5 binding and HIV binding, but sometimes resulting in CCR5 activity that may differ from that induced by CCL5 binding.

[0011] DETAILED DESCRIPTION

[0012] The present disclosure relates to a method of administering leronlimab or a leronlimab variant to a patient to modulate CCR5 cellular pathways to treat or prevent a cancer or inflammation-related disease or to facilitate transplant of HIV-negative donor cells in HIV-positive patents. In particular embodiments, the CCR5 pathways are regulated in a cancer cell or immune system cell. In some embodiments, the cancer is colorectal cancer. In some embodiments, the inflammation-related disease is Alzeimer’s disease, metabolic dysfunction-associated steatohepatitis (MASH), or long -COVID. In some embodiments, normal CCR5 function is dysregulated prior to leronlimab or leronlimab variant administration, while in other embodiments normal CCR5 function is intentionally modified by administration of leronlimab or a leronlimab variant. In some embodiments leronlimab or a leronlimab variant is provided as monotherapy, while in other embodiments leronlimab or a leronlimab variant forms part of a combination therapy.

[0013] The present disclosure relates to a method of treating CCR5 dysregulation or modifying normal CCR5 function by administering leronlimab or a leronlimab variant to a patient in an amount effective to treat or prevent at least one symptom of the CCR5 dysregulation, or to modify normal CCR5 function. The symptom treated or prevented or ways in which normal CCR5 function is modified may vary depending on the disease to be treated or prevent.

[0014] Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. Additional definitions are set forth throughout this disclosure.

[0015] In the present description, the term “about” means + 20% of the indicated range, value, or structure, unless otherwise indicated. The term “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms “include” and “have” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting. The term “comprise” means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. Any ranges provided herein include all the values and narrower ranges in the ranges.

[0016] As used herein, “chemokine” refers to a low-molecular weight cytokine that can stimulate recruitment of leukocytes. Chemokines have cysteine residues in conserved locations that are key to forming their 3 -dimensional shape. Chemokines may be classified into four main subfamilies: Cys-Cys (C-C), Cys-X-Cys (CXC), CX3C, and XC depending on the spacing of their first two amino terminal cysteine residues. Chemokines may also be grouped according to their function, such as whether they are inflammatory or homeostatic. There are 47 known chemokines, including but not limited to CCL5 (also known as RANTES), MIP-la, MIP-ip, or SDF-1, or another chemokine which has similar activity.

[0017] As used herein, “C-C chemokine receptor 5,” also known as “CCR5” or “CD 195” refers to a G protein-coupled receptor expressed on lymphocytes (e.g., NK cells, B cells, T cells), monocytes, dendritic cells, eosinophils, and microglia, which functions as a chemokine receptor for the C-C chemokine group. CCR5’s 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 an amino acid sequence provided in NCBI Reference Sequence: NP_000570.1 (SEQ ID NO: 15).

[0018] As used herein, “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, z.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0019] As used herein, “mutation” refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).

[0020] As used herein, “protein” or “polypeptide” as used herein refers to a compound made up of amino acid residues that are covalently linked by peptide bonds. The term “protein” may be synonymous with the term “polypeptide” or may refer, in addition, to a complex of two or more polypeptides. A polypeptide may further contain other components (e.g., covalently bound), such as a tag, a label, a bioactive molecule, or any combination thereof. In certain embodiments, a polypeptide may be a fragment. As used herein, a “fragment” means a polypeptide that is lacking one or more amino acids that are found in a reference sequence. A fragment can comprise a binding domain, antigen, or epitope found in a reference sequence. A fragment 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 amino acids of the amino acid sequence of the reference sequence.

[0021] As described herein, a “variant” polypeptide species has 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 relative to a reference polypeptide as presented herein. In certain embodiments, “variant” means a polypeptide having a substantially similar activity (e.g., enzymatic function, immunogenicity) or structure relative to a reference polypeptide). A variant 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 for the reference polypeptide as determined by sequence alignment programs and parameters known in the art. The variant can result from, for example, a genetic polymorphism or human manipulation. Conservative substitutions of amino acids are well known and may occur naturally or may be introduced when a protein is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a 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). Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide- directed mutagenesis may be used to prepare polypeptide variants (see, e.g, Sambrook et al., supra).

[0022] A “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution 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 (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and He. Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0023] The terms “identical” or “percent identity,” in the context of two or more polypeptide or nucleic acid molecule sequences, means two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same over a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using methods known in the art, such as a sequence comparison algorithm, by manual alignment, or by visual inspection. The algorithm used herein for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, 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. Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. “Default values” mean any set of values or parameters which originally load with the software when first initialized.

[0024] The term “epitope” or “antigenic epitope” includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0025] As used herein, “specifically binds” or “specific for” may in some embodiments refer to an association or union of a binding protein (e.g., an anti-CCR5 antibody) or a binding domain (or fusion protein thereof) to a target molecule with an affinity or Ka(z.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M'1(which equals the ratio of the on-rate [kon] to the off-rate [koff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Binding domains (or fusion proteins thereof) may be classified as “high affinity” binding domains (or fusion proteins thereof) and “low affinity” binding domains (or fusion proteins thereof). “High affinity” binding domains refer to those binding domains with a Kaof at least 108M’1, at least 109M’1, at least IO10M’1, at least 1011M’1, at least 1012M’1, or at least 1013M’1, preferably at least 108M'1or at least 109M’1. “Low affinity” binding domains refer to those binding domains with a Kaof up to 108M’1, up to 107M’1, up to 106M’1, up to 105M’1. Alternatively, affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 10'5M to 10'13M), (which equals the ratio of the off-rate [koir] to the on-rate [kon] for this association reaction).

[0026] A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain or fusion protein affinities, such as Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 57:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).

[0027] Terms understood by those in the art of antibody technology are each given the meaning acquired in the art, unless expressly defined differently herein. The term “antibody” refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected 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 the antigen target molecule recognized by the intact antibody. Thus, the term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody). The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multi specific, e.g., bi specific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and sub-classes thereof (IgGl, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0028] An anti-CCR5 monoclonal antibody or antigen-binding portion thereof for use in the methods disclosed herein may be non-human (e.g., murine), chimeric, humanized, or human. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

[0029] The terms “VL” and “VH” refer to the variable binding region from an antibody light chain and an antibody heavy chain, respectively. The variable binding regions comprise discrete, well-defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs). The terms “complementarity determining region,” and “CDR,” are synonymous with “hypervariable region” or “HVR,” and refer to sequences of amino acids within antibody variable regions, which, in general, together confer the antigen specificity and / or binding affinity of the antibody, wherein consecutive CDRs (i.e., CDR1 and CDR2, CDR2 and CDR3) are separated from one another in primary amino acid sequence by a framework region. There are three CDRs in each variable region (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also referred to as CDRHs and CDRLs, respectively). In certain embodiments, an antibody VH comprises four FRs and three CDRs as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3- FR4; and an antibody VL comprises four FRs and three CDRs as follows: FR1-LCDR1- FR2-LCDR2-FR3-LCDR3-FR4. In general, the VH and the VL together form the antigen-binding site through their respective CDRs.

[0030] Numbering of CDR and framework regions may be determined according to any known method or scheme, such as the Kabat, Chothia, EU, IMGT, and AHo numbering schemes (see, e.g., Kabat et al., “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5thed.; Chothia and Lesk, J. Mol. Biol. 796:901-917 (1987)); Lefranc et al., Dev. Comp. Immunol. 27'.55, 2003; Honegger and Pltickthun, J. Mol. Bio. 309.657-67® (2001)). Equivalent residue positions can be annotated and for different molecules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300). Accordingly, identification of CDRs of an exemplary variable domain (VH or VL) sequence as provided herein according to one numbering scheme is not exclusive of an antibody comprising CDRs of the same variable domain as determined using a different numbering scheme.

[0031] A “leronlimab variant” is an antibody or antigen-binding fragment thereof that specifically binds to the same CCR5 epitope as leronlimab. In some embodiments, the leronlimab variant may have a binding KD for the CCR5 epitope that is at least 80%, at least 90%, at least 95%, or at least 99%, or in a range between 80%, 90%, 95%, or 99% and 99.99% of the KD of leronlimab. In some embodiments, the leronlimab variant comprises at least the CDRH3 of leronlimab. In some embodiments, the leronlimab variant comprises at least the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of leronlimab. In some further embodiments, the leronlimab variant may further comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of leronlimab with conservative substitutions. In some embodiments, the leronlimab variant comprises the VH of leronlimab, optionally with conservative substitutions. In some further embodiments, the leronlimab variant may comprise conservative substitutions only in the framework region of the VH. In some further embodiments, the leronlimab variant may have a VH comprising of consisting of the VH of leronlimab. In some embodiments, the leronlimab variant comprises the VL of leronlimab, optionally with conservative substitutions. In some further embodiments, the leronlimab variant may comprise conservative substitutions only in the framework region of the VL. In some further embodiments, the leronlimab variant may have a VL comprising of consisting of the VL of leronlimab. In some embodiments, the variant of leronlimab may have a VH and a VL both according to at least one of the VH or VL described above. In some embodiments, the leronlimab variant may further comprise the Fc of leronlimab, optionally with conservative substitutions or other substitutions known to modify biological half-life or Fc function.

[0032] As used herein, a “fusion protein” comprises a single chain polypeptide having at least two distinct domains, wherein the domains are not naturally found together in a protein. A nucleic acid molecule encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be made synthetically. A fusion protein may further contain other components (e.g., covalently bound), such as a tag, linker, transduction marker, or bioactive molecule.

[0033] A “nucleic acid molecule” or “polynucleotide” refers to a polymeric compound containing nucleotides that are covalently linked by 3’-5’ phosphodiester bonds. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes genomic DNA, mitochondrial DNA, cDNA, or vector DNA. A nucleic acid molecule may be double stranded or single stranded, and if single stranded, may be the coding strand or non-coding (anti-sense strand). A nucleic acid molecule may contain natural subunits or non-natural subunits. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.

[0034] Variants of the polynucleotides of this disclosure are also contemplated. Variant polynucleotides are at least 80%, 85%, 90%, 95%, 99%, or 99.9% identical to a reference polynucleotide as described herein, or that hybridizes to a reference polynucleotide of defined sequence under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65°-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. The polynucleotide variants retain the capacity to encode an immunoglobulin-like binding protein or antigen-binding fragment thereof having the functionality described herein.

[0035] The term “isolated” means that the material is removed from its original environment (e.g., 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, separated from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotide could be part of a vector and / or such polynucleotide or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.

[0036] As used herein, the term “engineered,” “recombinant,” or “non-natural” refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous or heterologous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (z.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding functional RNA, proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene, or operon.

[0037] As used herein, “heterologous” or “exogenous” nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from 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 is added (z.e., not endogenous or native) to a host cell or host genome by, for example, conjugation, transformation, transfection, electroporation, or the like, wherein the added molecule may integrate into the host genome or exist as extra-chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may be present in multiple copies. In addition, “heterologous” refers to a non-native enzyme, protein, or other activity encoded by an exogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity.

[0038] As used herein, the term “endogenous” or “native” refers to a gene, protein, or activity that is normally present in a host cell. Moreover, a gene, protein or activity that is mutated, overexpressed, shuffled, duplicated or otherwise altered as compared to a parent gene, protein or activity is still considered to be endogenous or native to that particular host cell. For example, an endogenous control sequence from a first gene e.g., promoter, translational attenuation sequences) may be used to alter or regulate expression of a second native gene or nucleic acid molecule, wherein the expression or regulation of the second native gene or nucleic acid molecule differs from normal expression or regulation in a parent cell.

[0039] As used herein, the term “expression”, refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, posttranslational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

[0040] As used herein, the term “operably linked” refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (z.e., the coding sequence is under the transcriptional control of the promoter). “Unlinked” means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.

[0041] As used herein, “expression vector” refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, “plasmid,” “expression plasmid,” “virus” and “vector” are often used interchangeably.

[0042] As used herein, the term “host” refers to a cell (e.g., T cell, Chinese Hamster Ovary (CHO) cell, HEK293 cell, B cell, or the like) or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., a CCR5 antibody of the present disclosure). In certain embodiments, a host cell may optionally already possess or be modified to include other genetic modifications that confer desired properties related or unrelated to, e.g., biosynthesis of the heterologous protein (e.g., inclusion of a detectable marker; deleted, altered or truncated endogenous BCR).

[0043] As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule (e.g., a heavy chain and a light chain of an antibody), as a single nucleic acid molecule encoding a protein (e.g, a heavy chain of an antibody), or any combination thereof. When two or more heterologous nucleic acid molecules are introduced 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 any combination thereof. The number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.

[0044] As used herein, the term “introduced” in the context of inserting a nucleic acid sequence into a cell, means “transfection”, or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0045] “Treat” or “treatment” or “ameliorate” refers to medical management of a disease, disorder, or condition of a patient (e.g, a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat). In general, an appropriate dose or treatment regimen comprising leronlimab is administered in an amount sufficient to elicit a therapeutic effect or therapeutic benefit. Therapeutic effect or therapeutic benefit includes improved clinical outcome; modulation of immune response to lessen, reduce, or dampen counterproductive inflammatory cytokine activity; modulation of immune response to normalize counterproductive inflammatory cytokine activity; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any combination thereof. Patients who have been treated may also be referred to as “dosed,” whereas patients who have not been treated may be referred to as “non-dosed.”

[0046] A prophylactic treatment meant to “prevent” a disease or condition (e.g., coronavirus induced respiratory illness in a patient or patient) is a treatment administered to a patient who does not exhibit signs of a disease or exhibits only early signs, for the purpose of decreasing the risk of developing pathology or further advancement of the early disease. For example, if an individual at risk of developing a coronavirus induced respiratory illness is treated with the methods of the present disclosure and does not later develop coronavirus induced respiratory illness, then the disease has been prevented, at least over a period of time, in that individual. A prophylactic treatment can mean preventing recurrence of a disease or condition in a patient that has previously been treated for the disease or condition, e.g., by preventing relapse or recurrence of coronavirus induced respiratory illness.

[0047] A “therapeutically effective amount” or “effective amount” of leronlimab refers to an amount of leronlimab sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; modulating immune response to lessen, reduce, or dampen counterproductive inflammatory cytokine activity; modulating immune response to normalize counterproductive inflammatory cytokine activity; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner. When referring to an individual active ingredient or a cell expressing a single active ingredient, administered alone, a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously.

[0048] As used herein, “relative reduction” or “relative risk reduction” refers to the percent reduction in a parameter (e.g. mortality, time to recovery) in the treated group (Y) compared to the control group (X). RR = 1- (Y / X) x 100%.

[0049] As used herein, “absolute reduction” or “absolute risk reduction” refers to the percent reduction between the control group (X) and the treatment group (Y). AR = X-Y.

[0050] The term “inhibit” or “inhibitor” refers to a diminishing, blunting, reduction, masking, interrupting, blocking, mitigation, or slowing directly or indirectly, in the expression, amount or activity of a target or signaling pathway relative to (1) a control, endogenous or reference target or pathway, or (2) the absence of a target or pathway, wherein the diminishing, blunting, reduction, masking, interrupting, blocking, mitigation, or slowing is statistically, biologically, or clinically significant. For example, leronlimab or a leronlimab variant may diminish, blunt, reduce, mask, interrupt, block, mitigate, or slow CCR5 signaling activity induced by CCL5 binding by about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more as compared to untreated CCR5. CCR5 activity induced by CCL5 binding may be measured by detecting, for example, a decrease in cAMP, cell migration, or both.

[0051] The term “pharmaceutically acceptable excipient or carrier” or “physiologically acceptable excipient or carrier” refer to biologically compatible vehicles, e.g., physiological saline, which are described in greater detail herein, that are suitable for administration to a human or other non-human mammalian patient and generally recognized as safe or not causing a serious adverse event.

[0052] Additional definitions are provided in the sections below.

[0053] Leronlimab

[0054] The present disclosure provides for use of leronlimab, or antigen binding fragment thereof, in treating or preventing a CCR5-mediated disease.

[0055] In some embodiments, the present disclosure provides use of the monoclonal antibody PA14, produced by the hybridoma cell line designated PA14 (ATCC Accession No. HB-12610), or an antigen binding fragment thereof, or an antibody that competes with monoclonal antibody PA- 14 in binding to CCR5.

[0056] In some embodiments, the present disclosure provides use of leronlimab (PROMO) antibody or antigen binding fragment thereof. Leronlimab (PROMO) is a humanized IgG4 monoclonal antibody that binds to CCR5 described in US Pat. Nos. 7,122,185 and 8,821,877, which are incorporated herein by reference, in their entirety. Leronlimab (PRO 140) is a humanized version of the murine monoclonal antibody, PAM, which was 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. VlROL., 73: 4145-4155. (1999). PRO 140 binds to CCR5 expressed on the surface of a cell, and potently inhibits HIV-1 entry and replication at concentrations that do not affect CCR5 chemokine receptor activity 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. VlROL., 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. VlROL., 75: 579-588 (2001).

[0057] Leronlimab does not downregulate CCR5 surface expression or deplete CCR5- expressing cells, but does prevent CCL5-induced calcium mobilization in CCR5+ cells with an IC50 of 45 pg / ml. In some embodiments, a CCR5 binding agent does not downregulate CCR5 surface expression, deplete CCR5-expressing cells, or both. In some embodiments, a CCR5 binding agent inhibits CCL5-induced calcium mobilization of CCR5+ cells with an IC50 of 45 pg / ml. In some embodiments, the CCR5 binding agent is leronlimab.

[0058] Leronlimab (PRO 140) binds to CCR5 and blocks viral entry by interfering with the final phase of viral binding to the cell surface prior to fusion of the viral and cell membranes. Leronlimab (PRO 140) has been administered intravenously or subcutaneously to more than 750 healthy and HIV-1 infected individuals in Phase I / II / III studies. The drug has been well tolerated following intravenous administration of single doses of 0.5 to 10 mg / kg or up to 700 mg weekly doses as subcutaneous (SC) injection. Overall, 324 patients have been exposed to leronlimab (PRO 140) 350 mg SC weekly dose with the longest duration of exposure lasting 4 years. Similarly, more than 250 and 150 patients have been exposed to leronlimab (PRO 140) 525 mg and 700 mg SC weekly dose, respectively.

[0059] In some embodiments, the present disclosure provides use of an anti-CCR5 antibody that binds to the same epitope as that to which leronlimab binds or competes with leronlimab in binding to CCR5. Leronlimab binds to a discontinuous epitope spanning multiple extracellular domains on CCR5, which include the N-terminus and second extracellular loop (ECL2) of CCR5 (Trkola et al. J. Virol. 75:579-588, incorporated by reference in its entirety). Leronlimab directly blocks binding of HIV Env to the CCR5 coreceptor via a competitive mechanism. Leronlimab binding at least requires amino acid residues D2 in the N-terminus and R168 and Y176 in the ECL2; mutation of amino acids D95 and C101 in the ECL1, and C178 in ECL2 also affect leronlimab binding, e.g., by conformational perturbation (Olson et al. J. Virol. 73:4145-4155, incorporated by reference in its entirety). Targeted loss-of-function mutagenesis and subsequent photo- cross-linking using genetically encoded unnatural amino acids method was also used to map antibody-GPCR complexes and identified residues 174 and 175 at the aminoterminal end of ECL2 as forming the strongest links with leronlimab (Ray-Saha et al., Biochem. 53:1302-13010).

[0060] CCR5 amino acid residues that are involved in CCL5 (RANTES) binding include KI, D2, DI 1, El 8, K26 in the N-terminus, D95 in the ECL1, and KI 71, KI 91, and R274 in the ECL2 (Navenot et ai. J. Mol. Biol. 313: 1181-1193, incorporated by reference in its entirety).

[0061] Nucleic acids encoding heavy and light chains of the humanized PA14 antibodies have been deposited with the ATCC. Specifically, the plasmids designated pVK- HuPRO140, pVg4-HuPRO140 (mut B+D+I) and pVg4-HuPRO140 HG2, respectively, were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty with the ATCC, Manassas, Va., U.S.A. 20108, on Feb. 22, 2002, under ATCC Accession Nos. PTA 4097, PTA 4099, and PTA 4098, respectively. The American Type Culture Collection (ATCC) is now located at 10801 University Boulevard, Manassas, Va. 20110- 2209. The plasmids designated pVK-HuPRO140 and pVg4-HuPRO140 HG2 encode the light chain and heavy chain, respectively, of leronlimab.

[0062] The HCDR1-3 and LCDR1-3 amino acid sequences of leronlimab are set forth in SEQ ID NOS: 12-14 and 9-11, respectively. The VH and VL sequences of leronlimab 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 heavy chain and light chain sequences of leronlimab are set forth in SEQ ID NOS:7 and 8, respectively.

[0063] In some embodiments, the leronlimab or leronlimab variant has 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 leronlimab or leronlimab variant has a light chain variable antibody region that is 70%- 100% identical to SEQ ID NO: 1, 75%-100% identical to SEQ ID NO: 1, 80%-100% identical to SEQ ID NO: 1, 85%-100% identical to SEQ ID NO: 1, 90%-100% identical to SEQ ID NO: lor 91%-100% identical to SEQ ID NO: 1.

[0064] In some embodiments, the leronlimab or leronlimab variant has 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 leronlimab or leronlimab variant has a light chain variable antibody region that is 70%-100% identical to amino acids 20-131 of SEQ ID NO: 1, 75%-100% identical to amino acids 20-131 of SEQ ID NO: 1, 80%-100% identical to amino acids 20-131 of SEQ ID NO: 1, 85%-100% identical to amino acids 20-131 of SEQ ID NO: 1, 90%-100% identical to amino acids 20-131 of SEQ ID NO: lor 91%-100% identical to amino acids 20-131 of SEQ ID NO: 1.

[0065] In some embodiments, the leronlimab or leronlimab variant has a heavy chain variable region (VH) that is at least 70% identical to SEQ ID NO:3, at least 75% identical to SEQ ID NO:3, at least 80% identical to SEQ ID NO:3, at least 85% identical to SEQ ID NO:3, or at least 90% identical to SEQ ID NO:3. In some embodiments, the leronlimab or leronlimab variant has a heavy chain antibody variable region that is 70%-100% identical to SEQ ID NO: 3, 75%-100% identical to SEQ ID NO: 3, 80%-100% identical to SEQ ID NO: 3, 85%-100% identical to SEQ ID NO: 3, 90%-100% identical to SEQ ID NO: 3, or 91%-100% identical to SEQ ID NO:3.

[0066] In some embodiments, t the leronlimab or leronlimab variant has a heavy chain variable region (VH) that is at least 70% identical to amino acids 20-141 of SEQ ID NO:3, at least 75% identical to amino acids 20-141 of SEQ ID NO:3, at least 80% identical to amino acids 20-141 of SEQ ID NO:3, at least 85% identical to amino acids 20-141 of SEQ ID NO:3, or at least 90% identical to amino acids 20-141 of SEQ ID NO:3. In some embodiments, the leronlimab or leronlimab variant has a heavy chain antibody variable region that is 70%-100% identical to amino acids 20-141 of SEQ ID NO: 3, 75%-100% identical to amino acids 20-141 of SEQ ID NO: 3, 80%-100% identical to amino acids 20-141 of SEQ ID NO: 3, 85%-100% identical to amino acids 20-141 of SEQ ID NO: 3, 90%-100% identical to amino acids 20-141 of SEQ ID NO: 3, or 91%-100% identical to amino acids 20-141 of SEQ ID NO:3.

[0067] In some embodiments, t the leronlimab or leronlimab variant has 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, t the leronlimab or leronlimab variant has a heavy chain variable antibody region that is 70%- 100% identical to SEQ ID NO: 5, 75%-100% identical to SEQ ID NO: 5, 80%-100% identical to SEQ ID NO: 5, 85%-100% identical to SEQ ID NO: 5, 90%-100% identical to SEQ ID NO: 5, or 91%-100% identical to SEQ ID NO: 5.

[0068] In some embodiments, the leronlimab or leronlimab variant has a heavy chain variable region (VH) that is at least 70% identical to amino acids 20-141 of SEQ ID NO:5, at least 75% identical to amino acids 20-141 of SEQ ID NO: 5, at least 80% identical to amino acids 20-141 of SEQ ID NO: 5, at least 85% identical to amino acids 20-141 of SEQ ID NO: 5, or at least 90% identical to amino acids 20-141 of SEQ ID NO: 5. In some embodiments, the leronlimab or leronlimab variant has a heavy chain variable antibody region that is 70%-100% identical to amino acids 20-141 of SEQ ID NO: 5, 75%-100% identical to amino acids 20-141 of SEQ ID NO: 5, 80%-100% identical to amino acids 20-141 of SEQ ID NO: 5, 85%-100% identical to amino acids 20-141 of SEQ ID NO: 5, 90%-100% identical to amino acids 20-141 of SEQ ID NO: 5, or 91%-100% identical to amino acids 20-141 of SEQ ID NO: 5.

[0069] In some embodiments, the leronlimab or leronlimab variant comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the 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 the VL comprises a light chain CDR1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NOV, 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, the VH comprises an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:3 or amino acids 20-141 of SEQ ID NO:3, and a VL comprises an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 1 or amino acids 20-131 of SEQ ID NO: 1, provided that the amino acid sequences of the VH-CDRs (SEQ ID NOS: 12-14) and VL-CDRs (SEQ ID NOS:9-11) are unchanged; or the VH comprises an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:5 or amino acids 20-141 of SEQ ID NO:5, and a VL comprises an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 1 or amino acids 20-131 of SEQ ID NO: 1, provided that the amino acid sequences of the VH-CDRs (SEQ ID NOS: 12-14) and VL-CDRs (SEQ ID NOS:9-11) are unchanged.

[0070] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant comprising: (a) a VH comprising an amino acid sequence of SEQ ID NO:3 or amino acids 20-141 of SEQ ID NO:3, and a VL comprising an amino acid sequence of SEQ ID NO: 1 or amino acids 20-131 of SEQ ID NO: 1; or (b) a VH comprising an amino acid sequence of SEQ ID NO:5 or amino acids 20-141 of SEQ ID NO:5, and a VL comprising an amino acid sequence of SEQ ID NO: 1 or amino acids 20- 131 of SEQ ID NO: 1.

[0071] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant 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 derives, a heavy chain constant region may comprise CHI, CH2, and CH3 domains (IgA, IgD, IgG), or CHI, CH2, CH3, and CH4 domains (IgE, IgM). In some embodiments, the heavy chain constant region comprises a human IgGl, 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, a CL comprises a C kappa (“CK”) constant region. In some embodiments, a CL comprises a C lambda (Ck) constant region. In some embodiments, an anti-CCR5 antibody of the present disclosure comprises two heavy chains and two light chains, held together covalently by disulfide bridges.

[0072] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant comprising a Fc region portion. As used herein, “Fc region portion” refers to the heavy chain constant region segment of the Fc fragment (the “fragment crystallizable” region or Fc region) from an antibody, which can include one or more constant domains, such as CH2, CH3, CH4 or any combination thereof. In some embodiments, an 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, a CH2CH3 or a CH3CH4 structure has sub-region domains from the same antibody isotype and are human, such as human IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, or IgM (e.g., CH2CH3 from human IgGl). By way of background, an Fc region is responsible for the effector functions of an antibody, such as ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent cytotoxicity) and complement fixation, binding to Fc receptors (e.g., CD16, CD32, FcRn), greater half-life in vivo relative to a polypeptide lacking an Fc region, protein A binding, and perhaps even placental transfer (see Capon et al. Nature 337: 525, 1989). In some embodiments, a Fc region portion in an antibody or antigen-binding fragment of the present disclosure is capable of mediating one or more of these effector functions. In some embodiments, a Fc region portion in an antibody or antigen-binding fragment of the present disclosure has normal effector function, meaning having less than 20%, 15%, 10%, 5%, 1% difference in effector function (e.g., ADCC, CDC, half-life or any combination thereof) as compared to a wild type IgGl antibody.

[0073] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant comprising a Fc region portion having an increase in one or more of these effector functions by way of, for example, one or more amino acid substitutions or deletions in the Fc region portion known in the art. An antibody or antigen-binding fragment having a mutated or variant Fc region portion having increased effector function means that the antibody exhibits an increase of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% 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 mutated or variant Fc region portion exhibits increased binding to FcRn, FcyRI (CD64), FcyRIIA (CD32), FcyRIIIA (CD 16a), FcyRIIIB (CD16b), or any combination thereof. In some embodiments, the Fc region portion in an antibody or antigen-binding fragment of the present disclosure is a variant Fc region portion having increased ADCC, CDC, half-life, or any combination thereof.

[0074] Amino acid modifications (e.g., substitutions) to modify (e.g., improve, reduce, or ablate) Fc functionalities include, for example, the 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 mutations are summarized and annotated in “Engineered Fc Regions”, published by InvivoGen (2011) and 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 are incorporated herein by reference.

[0075] In some embodiments, the present disclosure provides use leronlimab or a leronlimab variant comprising a Fc region portion having a reduction in one or more of these effector functions or lack one or more effector functions by way of, for example, one or more amino acid substitutions or deletions in the Fc region portion known in the art. An antibody or antigen-binding fragment having a mutated or variant Fc region portion having reduced effector function means that the antibody exhibits a decrease of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% 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 mutated or variant Fc region portion exhibits decreased binding to FcRn, FcyRI (CD64), FcyRIIA (CD32), FcyRIIIA (CD 16a), FcyRIIIB (CD 16b), or any combination thereof. In some embodiments, the Fc region portion in an antibody or antigen-binding fragment of the present disclosure is a variant Fc region portion having reduced ADCC, CDC, half-life, or any combination thereof. In some embodiments, the Fc region portion is a variant IgGl Fc region portion comprising a mutation corresponding to amino acid E233P, L234V, L234A, L235A, L235E, AG236, G237A, E318A, K320A, K322A, A327G, P329G, A330S, P331S, or any combination thereof, as numbered according to the EU set forth in Kabat. For example, amino acid substitutions L234A, L235E, G237A introduced into an IgGl Fc region portion reduces binding to FcyRI, FcyRIIa, and FcyRIII receptors, and A330S and P331S introduced into an IgGl Fc region portion reduces Clq-mediated complement fixation.

[0076] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant comprising a Fc region portion having an increase in one or more of these effector functions by way of, for example, one or more amino acid substitutions or deletions in the Fc region portion known in the art. An antibody or antigen-binding fragment having a mutated or variant Fc region portion having increased effector function means that the antibody exhibits an increase of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% in FcR binding, ADCC, CDC, or any combination thereof, as compared to an antibody having a wildtype Fc region portion. In some embodiments, the mutated or variant Fc region portion exhibits increased binding to FcRn, FcyRI (CD64), FcyRIIA (CD32), FcyRIIIA (CD 16a), FcyRIIIB (CD 16b), or any combination thereof. In some embodiments, the Fc region portion in an antibody or antigen-binding fragment of the present disclosure is a variant Fc region portion having increased ADCC, CDC, half-life, or any combination thereof.

[0077] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant that is glycosylated. 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 an antibody or antigen-binding fragment of the present disclosure comprises a N297 as numbered according to EU set forth in Kabat. In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant that comprises a mutation that alters glycosylation at N297 in the Fc region portion, optionally wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G. In some embodiments, an antibody or antigen-binding fragment thereof comprising a N297A, N297Q, or N297G mutation exhibits reduced Fc interaction with one or more low affinity FcyR(s), reduced CDC, reduced ADCC, or any combination thereof.

[0078] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant that comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 7, and the LC comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:8

[0079] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant that comprises a HC comprising an amino acid sequence that has the amino acid sequence of SEQ ID NO:7, and a LC comprising an amino acid sequence that has the amino acid sequence of SEQ ID NO: 8.

[0080] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant that comprises a Fc region or a fragment thereof, including a CH2 (or a fragment thereof), a CH3 (or a fragment thereof), or a CH2 and a CH3, wherein the CH2, the CH3, or both can be of any isotype and may contain amino acid substitutions or other modifications as compared to a corresponding wild-type CH2 or CH3, respectively. In certain embodiments, a Fc region of the present disclosure comprises two CH2-CH3 polypeptides that associate to form a dimer.

[0081] As used herein, unless otherwise provided, a position of an amino acid residue in the constant region of human IgGl heavy chain is numbered assuming that the variable region of human IgGl is composed of 128 amino acid residues according to the Kabat numbering convention. The numbered constant region of human IgGl heavy chain is then used as a reference for numbering amino acid residues in constant regions of other immunoglobulin heavy chains. A position of an amino acid residue of interest in a constant region of an immunoglobulin heavy chain other than human IgGl heavy chain is the position of the amino acid residue in human IgGl heavy chain with which the amino acid residue of interest aligns. Alignments between constant regions of human IgGl heavy chain and other immunoglobulin heavy chains may be performed using software programs 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, although human IgG2 CH2 region may have an amino acid deletion near its amino-terminus compared with other CH2 regions, the position of the “N” located at 296 in human IgG2 CH2 is still considered position 297 because this residue aligns with “N” at position 297 in human IgGl CH2.

[0082] In addition, the present disclosure provides use of leronlimab or a leronlimab variant that comprises a hinge sequence that is typically situated between the Fab and Fc region (but a lower section of the hinge may include an amino-terminal portion of the Fc region). By way of background, an immunoglobulin hinge acts as a flexible spacer to allow the Fab portion to move freely in space. In contrast to the constant regions, hinges are structurally diverse, varying in both sequence and length between immunoglobulin classes and even among subclasses. For example, a human IgGl hinge region is freely flexible, which allows the Fab fragments to rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. By comparison, a human IgG2 hinge is relatively short and contains a rigid poly-proline double helix stabilized by four inter-heavy chain disulfide bridges, which restricts the flexibility. A human IgG3 hinge differs from the other subclasses by its unique extended hinge region (about four times as long as the IgGl hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix and providing greater flexibility because the Fab fragments are relatively far away from the Fc fragment. A human IgG4 hinge is shorter than IgGl but has the same length as IgG2, and its flexibility is intermediate between that of IgGl and IgG2. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

[0083] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant that is chimeric, humanized, or human. Chimeric and humanized forms of non-human (e.g., murine) antibodies 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 antibodies), which can contain sequences derived from non-human immunoglobulin. In general, in the 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 corresponding amino acids derived from human immunoglobulin molecules. In one embodiment of the humanized forms of the antibodies, some, most, or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most, or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. A humanized antibody can also comprise at least a portion of a human immunoglobulin constant region (Fc). Suitable human immunoglobulin molecules for use in humanizing a non-human antibody would include IgGl, IgG2, IgG3, IgG4, IgA, and IgM molecules. A “humanized” antibody would retain a similar antigenic specificity as the original antibody, e.g., in the present disclosure, the ability to bind CCR5.

[0084] “Human antibodies” can include antibodies having, for example, the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that typically do not express endogenous immunoglobulins. Human antibodies can be produced using transgenic mice incapable of expressing functional endogenous immunoglobulins, but capable of expressing human immunoglobulin genes. Completely human antibodies that recognize a selected epitope can be generated using guided selection. In this approach, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.

[0085] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant that is a part of a multispecific antibody, e.g., a bispecific antibody or a dual variable domain antibody (DVD). Bispecific and DVD antibodies are monoclonal, often human or humanized, antibodies that have binding specificities for at least two different antigens, one of which is CCR5.

[0086] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant that are derivatized or otherwise modified. For example, derivatized antibodies can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or the like.

[0087] In any of the aforementioned embodiments, the leronlimab or leronlimab variant is conjugated to a small molecule drug to form an antibody drug conjugate.

[0088] In some embodiments, the present disclosure provides use of an anti-CCR5 antibody comprising: (i) two light chains, each light chain comprising the expression product of the plasmid designated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two heavy chains, each heavy chain comprising the expression product of either the plasmid designated pVg4:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (ATCC Deposit Designation PTA-4099).

[0089] In some embodiments, the present disclosure provides use of an anti-CCR5 antibody comprising: (i) two light chains, each light chain comprising the light chain variable (VL) and constant (CL) regions encoded by the plasmid designated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two heavy chains, each heavy chain comprising the heavy chain variable (VH) and constant (CH) regions encoded either by the plasmid designated pVg4:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (ATCC Deposit Designation PTA-4099).

[0090] Various CCR5 binding agents are known. In some embodiments, the present disclosure provides use of a CCR5 binding agent that is maraviroc. Maraviroc is a negative allosteric modulator of CCR5. Competitive binding studies involving the CCR5 receptor and various anti-CCR5 binding agents including its natural ligand CCL5, and PRO 140 and maraviroc demonstrate that each of these components has a different binding capacity, and each binds to one or more distinct portions of the CCR5 receptor. PRO 140 binds to extracellular portions of the CCR5 receptor and effectively diminishes the downstream immunomodulatory effects of CCL5 binding on the CCL5 receptor. Also, unlike maraviroc, PRO 140 is shown to have no CCL5 receptor agonist activity when bound to CCR5 with respect to cAMP levels or cell migration. Accordingly, PRO 140 is shown to provide an advantageous application and gives rise to new uses for this CCR5 receptor competitive inhibitor to inhibit, interrupt, block, mitigate, dampen, slow the progress of, and / or therapeutically treat conditions resulting, in whole or in part, from the downstream immunomodulatory effects induced by CCL5 ligand binding on the CCL5 receptor.

[0091] In some embodiments, the present disclosure provides use of a CCR5 binding agent that is vicriviroc, a non-competitive allosteric CCR5 antagonist. In some embodiments, the present disclosure provides use of a CCR5 binding agent that is cenicriviroc. In some embodiments, the present disclosure provides use of a CCR5 binding agent that is TAK-779. In some embodiments, the present disclosure provides use of a CCR5 binding agent that is Met-CCL5 (Met-RANTES). In some embodiments, the present disclosure provides use of a CCR5 binding agent that is anibamine. In some embodiments, the present disclosure provides use of a CCR5 binding agent that is GSK706769. In some embodiments, the present disclosure provides use of a CCR5 binding agent that is INCB009471. In some embodiments, the present disclosure provides use of a CCR5 binding agent that is DT-13. In some embodiments, the present disclosure provides use of a CCR5 binding agent that is aplaviroc.

[0092] In some embodiments, the present disclosure provides use of a CCR5 binding agent that is the CCR5mAb004 antibody or an antigen binding fragment thereof. CCR5mAb004 is a fully human monoclonal IgG4 antibody generated using the Abgenix XenoMouse ® technology. See Yadavalli et al., J Infect Dis. 2008;197:721-727; Roschke et al., Characterization of a Panel of Novel Human Monoclonal Antibodies That Specifically Antagonize CCR5 and Block HIV Entry, 44th Annual Interscience CONFERENCE ON ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Washington, D.C., Oct. 30-Nov. 2, 2004 (2004); HGS Press Release, Human Genome Sciences Characterizes Panel of Novel Human Monoclonal Antibodies That Specifically Antagonize the CCR5 Receptor and Block HIV-1 Entry, Nov. 2, 2004 (2004); HGS Press Release, Human Genome Sciences Begins Dosing of Patients in a Phase 1 Clinical Trial of CCR5 mAb in Patients Infected With HIV-1, Mar. 30, 2005 (2005).

[0093] In some embodiments, the present disclosure provides use of a CCR5 binding agent that is the RoAbl3 antibody or an antigen binding fragment thereof. RoAbl3 is a mouse, anti-human CCR5 monoclonal antibody that does not show agonist activity or cause internalization of CCR5 (Ji et al. Antiviral Res. 74: 125-37).

[0094] In some embodiments, the present disclosure provides use of a CCR5 binding agent that is a competitive inhibitor to CCR5. The term “competitive inhibitor” as used herein refers to a molecule that competes with a reference molecule for binding to a target, and thereby blunts, inhibits, dampens, reduces, or blocks the effects of the reference molecule on the target. Thus a competitive inhibitor to CCR5 would compete with CCL5 for binding to CCR5.

[0095] In some embodiments, a competitive inhibitor to CCR5 is leronlimab or a leronlimab variant that comprises:

[0096] (i) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the 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 the VL comprises a light chain CDR1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NOV, 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;

[0097] (ii) 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;

[0098] (iii) a VH comprises the amino acid sequence of SEQ ID NO:5 or amino acids 20- 141 of SEQ ID NO:5, and a VL comprises the amino acid sequence of SEQ ID NO: 1 or amino acids 20-131 of SEQ ID NO: 1; or

[0099] (iv) a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:8.

[0100] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant comprising: (a) a VH comprising an amino acid sequence of SEQ ID NO:3 or amino acids 20-141 of SEQ ID NO:3, and a VL comprising an amino acid sequence of SEQ ID NO: 1 or amino acids 20-131 of SEQ ID NO: 1; or (b) a VH comprising an amino acid sequence of SEQ ID NO:5 or amino acids 20-141 of SEQ ID NO:5, and a VL comprising an amino acid sequence of SEQ ID NO: 1 or amino acids 20- 131 of SEQ ID NO: 1.

[0101] In some embodiments, the present disclosure provides use of leronlimab or a leronlimab variant that is a competitive inhibitor to CCR5 and does not have CCL5 agonist activity upon binding to CCR5. CCL5 agonist activity may be detected by measuring a decrease in cAMP; induced cell migration; or both cAMP decrease and induced cell migration triggered in response to CCL5-CCR5 axis activity. Some CCR5 binding agents even while acting to inhibit, interrupt, block, mitigate, dampen, slow the progress of, or eliminate the triggering of the downstream effects of CCL5 on CCR5 receptor positive cells also give rise to independent and separate CCL5 agonistic downstream CCL5 / CCR5 axis signaling effects that may counteract or diminish the effectiveness of these CCR5 competitive inhibitors for the purposes of immunomodulatory regulation, alteration, or control for therapeutic purposes. In some embodiments, a competitive inhibitor to CCR5 that does not have CCL5 agonist activity is leronlimab or a leronlimab variant that comprises:

[0102] (i) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the 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 the VL comprises a light chain CDR1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NOV, 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;

[0103] (ii) 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;

[0104] (iii) a VH comprises the amino acid sequence of SEQ ID NO:5 or amino acids 20- 141 of SEQ ID NO:5, and a VL comprises the amino acid sequence of SEQ ID NO: 1 or amino acids 20-131 of SEQ ID NO: 1; or

[0105] (iv) a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:8.

[0106] Nucleic Acids, Vectors, and Host Cells

[0107] In another aspect, the present disclosure provides an isolated nucleic acid that encodes leronlimab or a leronlimab variant as described herein. In some embodiments, the isolated nucleic acid encodes the VH, the VL, or both the VH and VL of the antibody or antigen binding fragment thereof. In some embodiments, the isolated nucleic acid encodes the heavy chain, the light chain, or both the heavy and light chain of the antibody or antigen binding fragment thereof. In some embodiments, the nucleic acid encoding the leronlimab or a leronlimab variant is codon optimized to enhance or maximize expression in certain types of cells (e.g., Scholten et al., Clin. Immunol. 119'. 135-145, 2006). As used herein a “codon optimized” polynucleotide is a heterologous polypeptide having codons modified with silent mutations corresponding to the abundances of host cell tRNA levels.

[0108] In some embodiments, a nucleic acid molecule encoding leronlimab or a leronlimab variant comprises a nucleic acid sequence for a heavy chain or VH region and a light chain or VL, respectively, wherein the heavy chain or VH region is separated from the light chain or VL region by a 2A self-cleaving peptide. In some embodiments, the 2A self-cleaving peptide is a porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), foot-and-mouth disease virus (F2A), or any combination thereof (see, e.g., Kim et al., PLOS One 6:el8556, 2011, which 2A nucleic acid and amino acid sequences are incorporated herein by reference in their entirety).

[0109] In another aspect, an expression construct comprising a nucleic acid encoding leronlimab or a leronlimab variant as described herein is provided. In some embodiments, a nucleic acid may be operably linked to an expression control sequence (e.g., expression construct). As used herein, “expression construct” refers to a DNA construct containing a nucleic acid molecule that is operably-linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. An expression construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. The term “operably linked” refers to the association of two or more nucleic acids on a single polynucleotide fragment so that the function of one is affected by the other. For example, a promoter is operably-linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The term “expression control sequence” (also called a regulatory sequence) refers to nucleic acid sequences that effect the expression and processing of coding sequences to which they are operably linked. For example, expression control sequences may include transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion.

[0110] In some embodiments, a nucleic acid or an expression construct encoding leronlimab or a leronlimab variant is present in a vector. A “vector” is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. Exemplary vectors are those capable of autonomous replication (episomal vector) or expression of nucleic acids to which they are linked (expression vectors). Exemplary viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). In some embodiments, a vector is a plasmid. In some other embodiments, a vector is a viral vector. In some such embodiments, the viral vector is a lentiviral vector or a y-retroviral vector.

[0111] In a further aspect, the present disclosure also provides an isolated host cell comprising a nucleic acid, expression construct, or vector encoding leronlimab or a leronlimab variant. As used herein, the term “host” refers to a cell or microorganism targeted for genetic modification with a heterologous or exogenous nucleic acid molecule to produce leronlimab or a leronlimab variant. In certain embodiments, a host cell may optionally already possess or be modified to include other genetic modifications that confer desired properties related or unrelated to biosynthesis of the heterologous or exogenous protein (e.g., inclusion of a selectable marker). More than one heterologous or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof. When two or more exogenous nucleic acid molecules are introduced into a host cell, it is understood that the two more exogenous 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. The number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.

[0112] Examples of host cells include, but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli. In some embodiments, the cells are mammalian cells. In some embodiments, the host cell is a human embryonic kidney (HEK293) cell, YO cell, Sp2 / 0 cell, NSO murine myeloma cell, PER.C6® human cell, baby hamster kidney cell (BHK), COS cell, or Chinese hamster ovary (CHO) cell. In some embodiments, the host cell is a CH0-K1 cell. In some embodiments, the host cell is a CH0K1SV cell. Host cells are cultured using methods known in the art.

[0113] In yet another aspect, the present disclosure provides a process for making leronlimab or a leronlimab variant as described herein, comprising culturing a host cell of the present disclosure, under suitable conditions and for a sufficient time to express leronlimab or a leronlimab variant, and optionally isolating the a leronlimab or a leronlimab variant from the culture. Purification of soluble antibodies or antigen binding fragments thereof may be performed according to methods known in the art. Pharmaceutical Compositions

[0114] In another aspect, the present disclosure provides use of pharmaceutical compositions comprising leronlimab or a leronlimab variant for administration to a patient in need thereof. Pharmaceutical compositions can comprise the CCR5 binding agents described herein and one or more pharmaceutically acceptable carrier, diluent, or excipient, 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.), 18thEdition, 1990) and in 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, glidant, diluent, preservative, dye / colorant, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, emulsifier, or any combination thereof. For example, sterile saline and phosphate buffered saline at physiological pH can be suitable pharmaceutically acceptable carriers. Preservatives, stabilizers, dyes or the like may also be provided in the pharmaceutical composition. In addition, antioxidants and suspending agents may also be used. Pharmaceutical compositions 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 (e.g., glucose, sucrose, dextrins), chelating agents (e.g., EDTA), glutathione, and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary diluents.

[0115] Pharmaceutical compositions comprising leronlimab or a leronlimab variant can be manufactured, for example, by lyophilizing the leronlimab or a leronlimab variant, mixing, dissolving, emulsifying, encapsulating or entrapping the leronlimab or a leronlimab variant.

[0116] A 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 here are lyophilized or in powder form for re-constitution with a suitable vehicle, e.g., sterile water, before use. In some embodiments, the pharmaceutical compositions described herein is a suspension, solution, or emulsion. The pharmaceutical compositions and formulations can be sterilized. Sterilization can be accomplished by filtration through sterile filtration.

[0117] The pharmaceutical compositions described herein can be formulated for oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal administration. The term “parenteral”, as used herein, includes subcutaneous, intravenous, intramuscular, intrasternal, and intratumoral injection or infusion techniques. In some embodiments, the pharmaceutical compositions described herein are formulated for administration as an injection, e.g., an intravenous or subcutaneous injection. Nonlimiting examples of formulations for injection can include a sterile suspension, solution or emulsion in oily or aqueous vehicles. 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 can contain substances which increase the viscosity of the suspension. The suspension can also contain suitable stabilizers. Alternatively, the pharmaceutical compositions described herein can be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0118] The leronlimab or a leronlimab variant can be formulated for administration in a unit dosage form in association with a pharmaceutically acceptable vehicle. Such vehicles can be inherently nontoxic, and non-therapeutic. A vehicle can be water, 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 minor amounts of additives such as substances that enhance isotonicity and chemical stability (e.g., buffers and preservatives).

[0119] In some embodiments, an aqueous formulation of a leronlimab or a leronlimab variant, such as for subcutaneous administration, has a pH from 4-5.7. The aqueous formulation may comprise one or more excipients, such as, for example, one or more buffering agents, one or more lyoprotectants, and the like. In some embodiments, the pH of the formulation is from 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 comprises at least one buffer. In various embodiments, the buffer may be selected from histidine, citrate, aspartate, acetate, phosphate, lactate, tromethamine, gluconate, glutamate, tartrate, succinate, malic acid, fumarate, a-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-30 mM, 15 mM-25 mM, or 20 mM.

[0120] In some embodiments, the aqueous formulation comprises at least one lyoprotectant. In some such embodiments, the at least one lyoprotectant is selected from sucrose, arginine, glycine, sorbitol, glycerol, trehalose, dextrose, alpha-cyclodextrin, hydroxypropyl beta-cyclodextrin, hydroxypropyl gamma-cyclodextrin, proline, methionine, albumin, mannitol, maltose, dextran, and combinations thereof. In some embodiments, the lyoprotectant is sucrose. In some embodiments, the total concentration of lyoprotectant in the aqueous formulation is 3-12%, such as 5-12%, 6-10%, 5-9%, 7-9%, or 8%.

[0121] 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%. In some embodiments, pharmaceutical compositions of the present invention are formulated in a single dose unit or in a form comprising a plurality of dosage units. Methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).

[0122] In some embodiments, the concentration of the leronlimab or a leronlimab variant t in the aqueous formulation is 1 mg / mL-250 mg / mL, such as 10 mg / mL-220 mg / mL, 10 mg / mL-200 mg / mL 10 mg / mL-175 mg / mL, 10 mg / mL-150 mg / mL, 10 mg / mL-100 mg / mL, 20 mg / mL-200 mg / mL, 20 mg / mL-175 mg / mL, 20 mg / mL-150 mg / mL, 20 mg / mL- 125 mg / mL, 20 mg / mL-100 mg / mL, 30 mg / mL-200 mg / mL, 30 mg / mL-175 mg / mL, 30 mg / mL-150 mg / mL, 30 mg / mL-125 mg / mL, 30 mg / mL-100 mg / mL, 40 mg / mL-200 mg / mL 40 mg / mL-175 mg / mL, 40 mg / mL-150 mg / mL, 40 mg / mL-125 mg / mL, 40 mg / mL-100 mg / mL, 50 mg / mL-200 mg / mL 50 mg / mL-175 mg / mL, 50 mg / mL-150 mg / mL, 50 mg / mL-125 mg / mL, 50 mg / mL-100 mg / mL, 60 mg / mL-200 mg / mL, 60 mg / mL-175 mg / mL, 60 mg / mL-150 mg / mL, 60 mg / mL-125 mg / mL, 60 mg / mL-100 mg / mL, 70 mg / mL-200 mg / mL, 70 mg / mL-175 mg / mL, 70 mg / mL-150 mg / mL, 70 mg / mL-125 mg / mL, 80 mg / mL-200 mg / mL, 80 mg / mL-175 mg / mL, 80 mg / mL-150 mg / mL, 80 mg / mL-125 mg / mL, 100 mg / mL-200 mg / mL, 125 mg / mL-200 mg / mL, 150 mg / mL-200 mg / mL, or 160 mg / mL- 190 mg / mL, 170 mg / mL- 180 mg / mL, or 175 mg / mL. In some embodiments, the concentration of the leronlimab or a leronlimab variant in the aqueous formulation is 100 mg / mL-200 mg / mL. In some embodiments, the concentration of the leronlimab or a leronlimab variant in the aqueous formulation is 175 mg / mL.

[0123] In some embodiments, the leronlimab or a leronlimab variant is formulated in a high protein concentration. High protein concentration formulations containing leronlimab or a leronlimab variant are described in US Patent 9,956,165 (incorporated by reference in its entirety).

[0124] In some embodiments, the leronlimab or a leronlimab variant is in a formulation comprising concentrated leronlimab or a leronlimab variant in an amount greater than about 100 mg / mL and less than about 200 mg / mL; a tonicifier consisting essentially of a sodium salt and a histidine and glycine buffer present in a combined amount of from about 110 mM to about 120 mM and wherein the buffer is present in an amount of about 10 mM to about 25 mM; and a surfactant, wherein the formulation is hypotonic and has a total salt concentration of less than 100 mM.

[0125] In some embodiments, the leronlimab or a leronlimab variant is in a formulation comprising: concentrated leronlimab or a leronlimab variant 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 stabilizing agent or non-salt tonicifier in an amount of about 0.05% w / v to about 1.8% w / v; wherein the formulation has an osmolality of about 250 to about 280 mOsm and has a total salt concentration of less than 100 mM.

[0126] In some embodiments, the leronlimab or a leronlimab variant is formulated in a low viscosity, hypotonic formulation, comprising: (a) concentrated anti-CCR5 antibody or antigen binding fragment in an amount greater than about 100 mg / mL and less than about 200 mg / mL; (b) a sodium salt in an amount selected from about 90 mM or about 95 mM; (c) a histidine and glycine buffer in an amount of about 20 mM; (d) a surfactant in an amount of 0.005% to 0.2% w / v; and optionally (e) a stabilizing agent or non-salt tonicifier in an amount sufficient to provide an osmolality of the formulation of about 260-280 mOs / kg; wherein the formulation has a total salt concentration of less than 100 mM.

[0127] In some embodiments, the leronlimab or a leronlimab variant is in a low viscosity hypotonic formulation, comprising: (a) concentrated anti-CCR5 antibody or antigen binding fragment in an amount 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) a histidine and glycine buffer in an amount of about 20 mM; (d) a surfactant in an amount of about 0.005% to about 0.2% w / v, wherein the surfactant is a polysorbate, a poloxamer, or a pluronic; and (e) a stabilizing agent or non-salt tonicifier present in an amount sufficient to provide an osmolality of the formulation of about 230 mOs / kg to about 280 mOs / kg, wherein the stabilizing agent or non-salt tonicifier is selected from a sugar alcohol, a monosaccharide, a disaccharide, or a combination thereof; wherein the formulation has a total salt concentration of less than 100 mM.

[0128] In some embodiments, the leronlimab or a leronlimab variant is formulated in a composition comprising anti-CCR5 antibody or antigen binding fragment in an amount greater than about 100 mg / mL and less than about 200 mg / mL, a tonicifier comprising a sodium salt present in a concentration of greater than about 90 mM and a histidine and glycine buffer present in a combined amount of from 110 mM to 120 mM and a surfactant present in an amount of from about 0.001% to about 0.2% w / v, wherein the composition has an osmolality of about 230 to about 290 mOs / kg and a total salt concentration of less than 100 mM.

[0129] In some embodiments, the leronlimab or a leronlimab variant is provided as an article of manufacture comprising a container and a formulation comprising anti-CCR5 antibody or antigen binding fragment in a concentration of greater than 100 mg / mL and less than 200 mg / mL, a tonicifier of a sodium salt present in a concentration of greater than about 90 mM and a histidine and glycine buffer present in a combined amount of from 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 from about 0.005% to about 0.2%, and instructions for use.

[0130] In some embodiments, the leronlimab or a leronlimab variant is administered in a dose of 700 mg of leronlimab or a leronlimab variant, such as 175 mg / mL delivered as two injections of 2 mL each and administered subcutaneously on opposite sides of the abdomen. Each vial of the leronlimab or a leronlimab variant product may contain ~1.4 mL antibody at a concentration of 175mg / mL. In other embodiments, the leronlimab or a leronlimab variant may be administered in a dose of 1000 mg, 650 mg, 600 mg, 550 mg, 500 mg, 450 mg, 400 mg, 350 mg, or 300 mg. Injection volumes and concentrations may be adjusted to facilitate the dose to be administered.

[0131] In some embodiments, the leronlimab or leronlimab variant may be formulated with Resmetirom.

[0132] Methods of Use

[0133] The CCR5 receptor is a C-C chemokine G-coupled protein receptor expressed on lymphocytes (e.g., NK cells, B cells), monocytes, monocytes, dendritic cells, a subset of T cells, etc. The extracellular portions represent potential targets for antibodies targeting CCR5, and comprise an amino-terminal domain (Nt) and three extracellular loops (ECL1, ECL2, and ECL3). The extracellular portions of CCR5 comprise just 90 amino acids distributed over four domains. The largest of these domains are at the Nt and ECL2 at approximately 30 amino acids each (Olson et al., Curr. Opin. HIV AIDS, March, 4(2): 104-111 (2009)).

[0134] 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 immunologic mechanisms such as controlling cell recruitment and activation in basal and inflammatory circumstances. CCL5 acts as a key regulator of CCR5+ cell (e.g., monocyte and T cell) migration to inflammatory sites, directing migration of monocytes and T cells to damaged or infected sites. CCR5 also plays a crucial role in differentiation and activation of CD8+ T cells. Many biologic effects of chemokines are mediated by their interaction with chemokine receptors on cell surfaces. The most relevant known receptor for CCL5 is the CCR5 receptor; however, CCR1 and CCR3 are also known 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).

[0135] The formation of the CCL5 ligand and CCR5 receptor complex causes a conformational change in the receptor that activates the subunits of the G-protein, inducing signaling and leading to changed levels of cyclic AMP (cAMP), inositol triphosphate, intracellular calcium and tyrosine kinase activation. These signaling events cause cell polarization and translocation of the transcription factor NF-kB, which results in the increase of phagocytic ability, cell survival, and transcription of proinflammatory genes.

[0136] Inflammatory conditions resulting from CCR5 dysregulation include long-COVID. Symptoms of long-COVID include cough, stuffy / runny nose, shortness of breath, tightness of chest, feeling of fast heartbeat, fatigue, muscle aches / cramps, muscle weaknessjoint pain / swelling, chills / shivering, feeling hot or feverish, difficulty in concentration, sleep disturbance / insomnia, headache, dizziness, tingling / numbness, decreased sense of taste, decreased sense of smell, sore throat, exertional malaise, anxiety, nausea, vomiting, diarrhea, confusion, heart palpitations, and any combinations thereof.

[0137] Patients may be administered leronlimab or a leronlimab variant at the time of diagnosis with SARS-CoV-2 infection, upon exhibiting acute or long-COVID symptoms, or at any point thereafter. The clinical endpoint data from a CD 15 long-COVID trial was recently published in the Journal of Infection. That study enrolled 56 patients with long- COVID treated for 8 weeks with either leronlimab or placebo. The study results showed clinical improvement in 19 of the 24 endpoints that were evaluated.

[0138] CCR5 additionally plays a response in tumor growth by preventing Tregs from entering the tumor microenvironment where they suppress the immune response against cancer cells. In addition, blocking CCR5 can prevent the conversion of macrophages into tumor-associated macrophages, which promote angiogenesis and tumor growth. Thus, dysregulation of CCR5 can lead to an environment in which cancer cells can proliferate and metastasize and reversing this dysregulation can hamper cancer growth or metastasis. This may in particular allow other therapeutics, which may be administered concurrently with the leronlimab or leronlimab variant to kill the cancer cells. Cancer immunotherapies may, in particular, show improvements in efficacy when administered concurrently with the leronlimab or leronlimab variant, as dysregulation of CCR5 may cause immune effects that impede the abilities of immunotherapies to induce an immune response to cancer cells.

[0139] In some embodiments, leronlimab or a leronlimab variant may be administered to patients with relapsed colorectal cancer. Colorectal cancer is among the most common and deadly forms of cancer and, unfortunately, appears to be increasing in incidence, especially among younger people. Leronlimab has been demonstrated to inhibit metastasis in a humanized mouse model of colon cancer. Further, the clinical observation that 4 of 6 patients with colon cancer in a basket trial showed the patients were either stable or partially responsive disease up to 11 months after starting leronlimab. A clinical study will evaluate leronlimab in patients with colorectal cancer who have received at least 1 but no more than 2 previous lines of treatment. Leronlimab will be paired with an established salvage regimen and administered at both 350 and 700 mg dose levels.

[0140] CCR5 and CCR2 dysregulation may play a further role in the growth and development of other cancers in which CCR5 has been identified as influential, such as astrocytomas, gliomas, and glioblastomas see e.g., Lah Tumsek T, Jiao X, Novak M, Jammula S, Cicero G, Ashton AW, Joyce D, Pestell RG. An Update on Glioblastoma Biology, Genetics, and Current Therapies: Novel Inhibitors of the G Protein-Coupled Receptor CCR5. International Journal of Molecular Sciences. 2021; 22(9):4464. https: / / doi.org / 10.3390 / ijms22094464; Novak M, Koprivnikar Krajnc M, Hrastar B, et al. CCR5-Mediated Signaling Is Involved in Invasion of Glioblastoma Cells in Its Microenvironment. Int J Mol Sci. 2020;21(12):4199. Published 2020 Jun 12. doi: 10.3390 / ijms21124199; and Felsenstein M, Blank A, Bungert AD, et al. CCR2 of Tumor Microenvironmental Cells Is a Relevant Modulator of Glioma Biology. Cancers (Basel). 2020;12(7): 1882. Published 2020 Jul 13. doi: 10.3390 / cancersl2071882; (Guo, X., Pan, Y., Xiong, M. et al. Midkine activation of CD8+ T cells establishes a neuron- immune-cancer axis responsible for low-grade glioma growth. Nat Commun 11, 2177 (2020). https: / / doi.org / 10.1038 / s41467-020-15770-3; Kranjc MK, Novak M, Pestell RG, Lah TT. Cytokine CCL5 and receptor CCR5 axis in glioblastoma multiforme. Radiol Oncol. 2019 Nov 20;53(4):397-406. doi: 10.2478 / raon-2019-0057. PMID: 31747383; PMCID: PMC6884928; and Tumsek et al., Int. J. Mol. Sci. 2021, 22(9), 4464;

[0141] (doi.org / 10.3390 / ijms22094464).

[0142] Alzheimer’s disease is increasingly being understood as an inflammatory-related disease. In Alzheimer’s, CCR5 appears to be essential for the transendothelial migration of T cells across the blood- brain barrier and thus may be essential for the infiltration of non-phagocytic peripheral immune cells to the brain of Alzheimer’s patents. A clinical study will enroll 20 patients with Alzheimer’s Disease who will treated with leronlimab at either 350 or 700 mg doses weekly and followed for 12 weeks with a primary neuroradiology endpoint. *

[0143] CCR5 has also been strongly implicated in the development of metabolic dysfunction-associated steatohepatitis (MASH) (previously known as nonalcoholic steatohepatitis (NASH)) and related fibrosis, which is also an inflammatory-related condition. (See e.g. 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.) Accordingly CCR5 dysregulation may play a significant role in the development of this condition, such that treatment with leronlimab or a leronlimab variant may be beneficial.

[0144] A prior MASH study demonstrated a statistically significant benefit of leronlimab at a 350 mg dose, but not at the 700 mg dose level. In order to clarify the optimal dosing regimen for MASH and efficiently evaluate the potential for combination therapy, a preclinical study will evaluate both 350 and 700 mg dose levels alone and in combination with Resmetirom, a drug recently approved by the FDA for the treatment of MASH. The study will evaluate leronliamb alone and in combination with Resmetriom, in both preventing and reversing fibrosis in a mouse model of MASH.

[0145] Leronlimab or a leronlimab variant may also be used generally to treat inflammation and other inflammatory conditions, particularly by affecting C-Reactive Protein (CRP) levels. Clinical studies will be performed to clarify the specific effect of blocking CCR5 with leronlimab on downstream signaling and biomarkers of inflammation. Clinical studies will also determine what the optimal dose of leronlimab is, when used in the non-HIV and non-antiviral setting, such as in treating or preventing inflammation and inflammation-related diseases.

[0146] Data from prior studies, including the prior MASH study, where the effect on markers of inflammation in the subgroup of patients that entered that study with elevated levels of CRP at baseline was determined, has been reexamined. CRP is widely understood to be a key marker of inflammation. The analysis indicated an important dose dependent reduction in CRP when leronlimab was dosed at both 350 and 700 mg compared to placebo.

[0147] The main goal of the inflammation clinical study will be to statistically confirm this effect of leronlimab on this key measure of inflammation, determine optimal dose, and clarify the underlying mechanism of action. The study will enroll 90 HIV+ subjects who have chronic inflammation as demonstrated by an elevated high sensitivity CRP at a prescreening visit confirmed at a screening visit at least 2 weeks apart. These study participants will be treated for 6 months with weekly SQ leronlimab at either 350 mg, 700 mg or placebo. This study will evaluate CRP as the primary endpoint with a host of other inflammatory biomarkers as secondary endpoints.

[0148] CCR5 also plays a role in a number of other inflammatory conditions, which may also be prevented or treated using CCR5 binding agents. (See e.g., Ruytinx P, Proost P, Van Damme J, Struyf S. Chemokine-Induced Macrophage Polarization in Inflammatory Conditions. Front Immunol. 2018;9: 1930, doi:10.3389 / fimmu.2018.01930; and Rautenbach A, Williams AA. Metabolomics as an Approach to Characterise the Contrasting Roles of CCR5 in the Presence and Absence of Disease. Int J Mol Sci. 2020;21(4): 1472. Published 2020 Feb 21. doi: 10.3390 / ijms21041472.)

[0149] Finally, leronlimab or a leronlimab variant may be used in conjunction with bone marrow transplant for treatment of HIV. A LATCH (Leronlimab in Allogenic Stem Cell Transplant to Cure HIV) study will evaluate the use of leronlimab to facilitate an HIV cure in HIV+ subjects undergoing stem cell transplantation. Previous reports of HIV+ patients achieving a cure after stem cell transplantation have occurred when rare homozygous, CCR5 double negative individuals have been identified to provide donor stem cells for the transplantation. This study will evaluate the possibility that leronlimab could extend the list of potential donors to include the much larger pool of CCR5-positive individuals. Leronlimab will be administered following transplantation for 6 months during the engraftment period to protect the HIV-negative donor Cells from becoming HIV-infected. The donor cells, themselves protected from HIV infection by leronliamb, are expected to eliminate HIV from the reservoir of the transplant recipient.

[0150] Patients or patients that can be treated by leronlimab or a leronlimab variant include, but are not limited to, a mammal, such as human or non-human primates (e.g., monkeys and apes). In embodiments, the patient is human. The patient can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric. In embodiments, the patient is > 65 years. In some embodiments, the patient is < 65 years. In embodiments, the patient is > 18 years and < 65 years.

[0151] Patients may also be administered leronlimab or a leronlimab variant upon initial detection of the condition or the propensity to develop such as condition in order to treat or prevent the development to at least one symptom of the condition.

[0152] Administration may be once or may continue for a set period of time, 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, until the propensity to develop the condition has resolved, or until at least one symptom of the condition has resolved.

[0153] An appropriate dose, suitable duration, and frequency of administration of the leronlimab or leronlimab variant will be determined by such factors as the condition of the patient, size, weight, body surface area, age, sex, type and severity of the disease, particular therapy to be administered, particular form of the active ingredient, time and the method of administration, and other drugs being administered concurrently, which can readily be determined by a person skilled in the art.

[0154] Dosages can range from 0.1 to 100,000 pg / kg. Based upon the composition, the dose can be delivered continuously, such as by continuous pump, or at periodic intervals, e.g., on one or more separate occasions. Desired time intervals of multiple doses of a particular composition can be determined without undue experimentation by one skilled in the art.

[0155] In some embodiments, leronlimab or a leronlimab variant is administered to the patient a plurality of times and each administration delivers from 0.01 mg per kg body weight to 50 mg per kg body weight of the antibody or binding fragment thereof to the patient. In another embodiment, each administration delivers from 0.05 mg per kg body weight to 25 mg per kg body weight of leronlimab or a leronlimab variant to the patient. In a further embodiment, each administration delivers from 0.1 mg per kg body weight to 10 mg per kg body weight of leronlimab or a leronlimab variant to the patient. In a still further embodiment, each administration delivers from 0.5 mg per kg body weight to 5 mg per kg body weight of leronlimab or a leronlimab variant to the patient. In another embodiment, each administration delivers from 1 mg per kg body weight to 3 mg per kg body weight of t leronlimab or a leronlimab variant to the patient. In another embodiment, each administration delivers about 2 mg per kg body weight of leronlimab or a leronlimab variant to the patient, leronlimab or a leronlimab variant may be administered once, twice, or a plurality of times, with a first administration is separated from the subsequent administration by an interval of less than one week. In another embodiment, the first administration is separated from the subsequent administration by an interval of at least one week. In a further embodiment, the first administration is separated from the subsequent administration by an interval of one week. In another embodiment, the first administration is separated from the subsequent administration by an interval of two to four weeks. In another embodiment, the first administration is separated from the subsequent administration by an interval of two weeks. In a further embodiment, the first administration is separated from the subsequent administration by an interval of four weeks. In yet another embodiment, leronlimab or a leronlimab variant is administered a plurality of times, and a first administration is separated from the subsequent administration by an interval of at least one month. In yet another embodiment, leronlimab or a leronlimab variant is administered once a week for two weeks. In yet another embodiment, t leronlimab or a leronlimab variant is administered once a week for four weeks. In yet another embodiment, leronlimab or a leronlimab variant is administered once per week as long as needed.

[0156] In a further embodiment, leronlimab or a leronlimab variant is administered to the patient via intravenous infusion. In another embodiment, leronlimab or a leronlimab variant is administered to the patient via subcutaneous injection. In another embodiment, leronlimab or a leronlimab variant is administered to the patient via intramuscular injection.

[0157] In some embodiments, particularly involving dysregulations in the central nervous system, leronlimab or a leronlimab variant may be administered by intracranial or intrathecal injection to allow access to the central nervous system.

[0158] In some embodiments, leronlimab or a leronlimab variant is administered at a once weekly dose of 350mg to 1400 mg, or about 525 mg or about 700 mg or about 1050 mg. In some embodiments, leronlimab or a leronlimab variant is administered at a twice weekly dose of 350mg to 1400 mg, or about 525 mg or about 700 mg or about 1050 mg. In some embodiments, leronlimab or a leronlimab variant is administered at a dose of about 700 mg or 350 mg, once weekly.

[0159] In some embodiments, leronlimab or a leronlimab variant may be administered in a manner and dose similar to that used to treat HIV infection.

[0160] The safety profile of leronlimab (PRO 140) has been extensively evaluated in clinical trials. Leronlimab (PRO 140) has been administered intravenously or subcutaneously to more than 750 healthy and HIV-1 infected individuals in Phase I / II / III studies. The drug has been well tolerated following intravenous administration of single doses of 0.5 to 10 mg / kg or up to 700 mg weekly doses as subcutaneous (SC) injection. Overall, 324 patients have been exposed to leronlimab (PRO 140) 350 mg SC weekly dose with the longest duration of exposure lasting 4 years. Similarly, more than 250 and 150 patients have been exposed to leronlimab (PRO 140) 525 mg and 700 mg SC weekly dose, respectively. In some embodiments, leronlimab or a leronlimab variant is administered in a dose of 700 mg (175 mg / mL) delivered as two subcutaneous injections of 2 mL each on opposite sides of the abdomen.

[0161] The disclosure provides the following numbered embodiments, which may be combined with one another and with other disclosure of the present specification:

[0162] 1. A method of modulating a CCR5 cellular pathway to treating or preventing a cancer or inflammation-related disease or facilitating transplant of HIV-negative donor cells in HIV-positive patents comprising administering an effective amount of leronlimab or a leronlimab variant to a patient in need thereof.

[0163] 2. The method of embodiment 1, wherein the effective amount of leronlimab or a leronlimab variant is 700 mg.

[0164] 3. The method of embodiment 1, wherein the effective amount of leronlimab or a leronlimab variant is 525 mg.

[0165] 4. The method of embodiment 1, wherein the effective amount of leronlimab or a leronlimab variant is 350 mg.

[0166] 5. The method of embodiment 1, wherein the effective amount of leronlimab or a leronlimab variant is between 350 mg and 700 mg.

[0167] 6. The method of any one of embodiments 1-5, wherein the leronlimab or a leronlimab variant is administered subcutaneously.

[0168] 7. The method of any one of embodiments 1-6, wherein the leronlimab or a leronlimab variant is administered once weekly.

[0169] 8. The method of any one of embodiments 1-7, wherein the method is a method of modulating a CCR5 cellular pathway.

[0170] 9. The method of any one of embodiments 1-8, wherein the method is a method or treating or preventing cancer.

[0171] 10. The method of any one of embodiments 1-8, wherein the method is a method of treating or preventing an inflammation-related disease.

[0172] 11. The method of any one of embodiments 1-8, wherein the method is a method of facilitating transplant of HIV-negative donor cells in HIV-positive patents.

[0173] 12. The method of embodiment 11, wherein the leronlimab or a leronlimab variant is administered prior to transplant of the HIV-negative donor cells.

[0174] 13. The method of embodiment 12, wherein the leronlimab or leronlimab variant is administered at least one week prior to transplant of the HIV-negative donor cells.

[0175] 14. The method of any one of embodiments 11-13, wherein the leronlimab or leronlimab variant is administered contemporaneously with or within 24 hours before or after transplant of the HIV-negative donor cells.

[0176] 15. The method of any one of embodiments 11-14, wherein the leronlimab or leronlimab variant is first administered prior to, contemporaneously with or within 24 hours before or after transplant of the HIV-negative donor cells.

[0177] 16. The method of any one of embodiments 11-15, wherein leronlimab is administered repeatedly for at least one month after transplant of the HIV-negative donor cells.

[0178] 17. The method of any one of embodiments 11-16, wherein leronlimab is administered repeatedly for at least three months after transplant of the HIV-negative donor cells

[0179] 18. The method of any one of embodiments 11-17, wherein leronlimab is administered repeatedly for at least six months after transplant of the HIV-negative donor cells.

[0180] 19. The method of any one of embodiments 11-18, wherein leronlimab is administered repeatedly for at least one year after transplant of the HIV-negative donor cells.

[0181] 20. The method of any one of embodiments 11-19, wherein leronlimab administration ceases when donor cells are determined to be successfully accepted by the patient according to medically accepted transplant guidelines and the presence of HIV using a medically accepted assay has not been detected in the patient for at least six months.

[0182] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and / or listed in the Application Data Sheet, including U.S. Provisional Patent Application No. 63 / 653,149, filed May 29, 2025, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

[0183] SEQUENCE LISTING

[0184] >SEQ ID NO:1 VL protein sequence; signal peptide at amino acids 1-19; CDRs underlined

[0185] MKLPVRLLVLMFWIPASSSDIVMTQSPLSLPVTPGEPASISCRSSQRLLSSYGHTYL HWYLQKPGOSPOLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCS QSTHVPLTFGOGTKVEIK

[0186] >SEQ ID NO:3 PRO#2 VH protein sequence; signal peptide at amino acids 1-19; CDRs underlined

[0187] MEWSGVFIFLLSVTAGVHSEVQLVESGGGLVKPGGSLRLSCAASGYTFSNYWIG WVROAPGKGLEWIGDIYPGGNYIRNNEKFKDKTTLSADTSKNTAYLOMNSLKTE DTAVYYCGSSFGSNYVFAWFTYWGOGTLVTVS S

[0188] >SEQ ID NO:5 PRO#1 VH protein sequence; signal peptide at amino acids 1-19; CDRs underlined

[0189] MEWSGVFIFLLSVTAGVHSQVQLVQSGPDVKKPGTSMKMSCKTSGYTFSNYWIG WVROAPGOGLEWIGDIYPGGNYIRNNEKFKDKTTLTADTSTSTAYMQLGSLRSED TAVYYCGSSFGSNYVFAWFTYWGOGTLVTVSS

[0190] >SEQ ID NO:7 heavy chain protein sequence

[0191] EVQLVESGGG LVKPGGSLRL SCAASGYTFS NYWIGWVRQA PGKGLEWIGD IYPGGNYIRNNEKFKDKTTL SADTSKNTAY LQMNSLKTED TAVYYCGSSF GSNYVFAWFT YWGQGTLVTVSSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQSSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPS CPAPEFLGGPSVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEMTKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQEGNVFSCSVM HEALHNHYTQ KSLSLSLGK >SEQ ID NO:8 light chain protein sequence

[0192] DIVMTQSPLS LPVTPGEPAS ISCRSSQRLL SSYGHTYLHW YLQKPGQSPQ LLIYEVSNRFSGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCSQSTHVP LTFGQGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSLSSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

[0193] >SEQ ID NO:9 LCDR1 amino acid sequence - RSSQRLLSSYGHTYLH

[0194] >SEQ ID NO: 10 LCDR2 amino acid sequence - EVSNRFS

[0195] >SEQ ID NO: 11 LCDR3 amino acid sequence - SQSTHVPLT

[0196] >SEQ ID NO: 12 HCDR1 amino acid sequence - NYWIG

[0197] >SEQ ID NO: 13 HCDR2 amino acid sequence - DIYPGGNYIRNNEKFKD

[0198] >SEQ ID NO: 14 HCDR3 amino acid sequence - SFGSNYVFAWFTY

[0199] >SEQ ID NO: 15 Homo sapiens CCR5 , NCBI Reference Sequence: NP 000570.1

[0200] MDYQVSSPIYDINYYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNMLVI LILINCKRLKSMTDIYLLNLAISDLFFLLTVPFWAHYAAAQWDFGNTMCQLLTGL YFIGFFSGIFFIILLTIDRYLAVVHAVFALKARTVTFGVVTSVITWVVAVFASLPGIIF TRSQKEGLHYTCSSHFPYSQYQFWKNFQTLKIVILGLVLPLLVMVICYSGILKTLL RCRNEKKRHRAVRLIFTIMIVYFLFWAPYNIVLLLNTFQEFFGLNNCSSSNRLDQA MQVTETLGMTHCCINPIIYAFVGEKFRNYLLVFFQKHIAKRFCKCCSIFQQEAPER ASSVYTRSTGEQEISVGL

Claims

CLAIMS1. A method of modulating a CCR5 cellular pathway to treating or preventing a cancer or inflammation-related disease or facilitating transplant of HIV-negative donor cells in HIV-positive patents comprising administering an effective amount of leronlimab or a leronlimab variant to a patient in need thereof.

2. The method of claim 1, wherein the effective amount of leronlimab or a leronlimab variant is 700 mg.

3. The method of claim 1, wherein the effective amount of leronlimab or a leronlimab variant is 525 mg.

4. The method of claim 1, wherein the effective amount of leronlimab or a leronlimab variant is 350 mg.

5. The method of claim 1, wherein the effective amount of leronlimab or a leronlimab variant is between 350 mg and 700 mg.

6. The method of any one of claims 1-5, wherein the leronlimab or a leronlimab variant is administered subcutaneously.

7. The method of any one of claims 1-6, wherein the leronlimab or a leronlimab variant is administered once weekly.

8. The method of any one of claims 1-7, wherein the method is a method of modulating a CCR5 cellular pathway.

9. The method of any one of claims 1-8, wherein the method is a method or treating or preventing cancer.

10. The method of any one of claims 1-8, wherein the method is a method of treating or preventing an inflammation-related disease.

11. The method of any one of claims 1-8, wherein the method is a method offacilitating transplant of HIV-negative donor cells in HIV-positive patents.

12. The method of claim 11, wherein the leronlimab or a leronlimab variant is administered prior to transplant of the HIV-negative donor cells.

13. The method of claim 12, wherein the leronlimab or leronlimab variant is administered at least one week prior to transplant of the HIV-negative donor cells.

14. The method of any one of claims 11-13, wherein the leronlimab or leronlimab variant is administered contemporaneously with or within 24 hours before or after transplant of the HIV-negative donor cells.

15. The method of any one of claims 11-14, wherein the leronlimab or leronlimab variant is first administered prior to, contemporaneously with or within 24 hours before or after transplant of the HIV-negative donor cells.

16. The method of any one of claims 11-15, wherein leronlimab is administered repeatedly for at least one month after transplant of the HIV-negative donor cells.

17. The method of any one of claims 11-16, wherein leronlimab is administered repeatedly for at least three months after transplant of the HIV-negative donor cells18. The method of any one of claims 11-17, wherein leronlimab is administered repeatedly for at least six months after transplant of the HIV-negative donor cells.

19. The method of any one of claims 11-18, wherein leronlimab is administered repeatedly for at least one year after transplant of the HIV-negative donor cells.

20. The method of any one of claims 11-19, wherein leronlimab administration ceases when donor cells are determined to be successfully accepted by the patient according to medically accepted transplant guidelines and the presence of HIV using a medically accepted assay has not been detected in the patient for at least six months.