CCL5 variants for modulating GPR75

Abstract

The disclosure provides methods and uses of CCL5 variants for modulating GPR75 signaling in a cell or subject, treating a GPR75-related disease or disorder in a subject, treating obesity, hypertension, metabolic syndrome, metabolic dysfunction-associated steatohepatitis, and / or diabetes in a subject, and inducing weight loss and / or preventing weight gain in a subject, wherein the CCL5 variant comprises a variant N-terminal portion and a C-terminal portion with at least 70% identity to the corresponding portion of wild type CCL5.

Description

CCL5 VARIANTS FOR MODULATING GPR75

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority from US Provisional Application 63 / 556,613 filed February 22, 2024, and US Provisional Application 63 / 672,021 filed July 16, 2024, both of which are incorporated herein by reference in their entirety.

[0003] FIELD

[0004] The present invention relates to CCL5 variants for modulating GPR75 signaling and for therapeutic use in the treatment of GPR75-related disorders.

[0005] BACKGROUND

[0006] G protein-coupled receptors are found on numerous cell types. Binding between a G protein-coupled receptor and an agonist ligand induces signaling via activation of guanine nucleotide binding proteins (G proteins). G protein-coupled receptor 75 (GPR75) is a G protein- coupled receptor for which the ligand was long unknown. It was recently discovered that 20- Hydroxy eicosatetraenoic acid (20-HETE) binds to GPR75 and activates distinct signaling cascades in endothelial and vascular smooth muscle cells, leading to the activation of vascular angiotensin-converting enzyme expression, endothelial dysfunction, contractility, remodeling, and hypertension (Garcia et al., 2017, Circulation Research, 120(11): 1776-1788). Modulation of GPR75 signaling has been proposed for use in the treatment of obesity and for stimulating the proliferation of beta islet cells in the treatment of diabetes (US 2022 / 0042101, US 2023 / 0364192).

[0007] CCL5 has been implicated as a ligand and modulator of GPR75 in several previous studies ((i) Ignatov et al., 2006, Br. J. Pharmacol., 149(5):490-497; (ii) Liu et al., 2013, Diabetologia, 56(11):2467-2476 (iii) Dedoni et al., 2018, Journal of Neurochemistry, 146(5):526-539) but no consensus has been reached about the nature of its modulatory effects and indeed its identification as a direct modulator of GPR75 has been challenged (Garcia et al., 2017, Circulation Research, 120(11): 1776-1788). It has been reported that the chemokine ligand CCL5, which is an agonist of the chemokine receptor CCR5, also binds to GPR75. Although CCL5 is an agonist of CCR5, it was unexpectedly reported that CCL5 is a low affinity antagonist of GPR75. Binding of CCL5 was reported to prevent activation of GPR75 by the high affinity agonist 20-HETE. Blocking of 20-HETE agonism was reported despite evidence that CCL5 binds to a different region of the receptor than 20-HETE (Pascale et al., 2021, Br. J. Pharmacol., 178(18):3813-3282). Nevertheless, the full nature of the CCL5-GPR75 interaction is not fullyunderstood. CCL5 interacts very differently with GPR75 than with CCR5, and past studies of CCL5-CCR5 interactions are not predictive of how CCL5 interacts with other receptors such as GPR75.

[0008] SUMMARY

[0009] The present invention relates to CCL5 variants for modulating GPR75 signaling and for therapeutic use in the treatment of GPR75-related disorders.

[0010] In an embodiment, there is provided a method for modulating GPR75 signaling in a cell, comprising contacting the cell with a CCL5 variant.

[0011] In an embodiment, there is provided a use of a CCL5 variant for modulating GPR75 signaling in a cell.

[0012] In an embodiment, there is provided a CCL5 variant for use in modulating GPR75 signaling in a cell.

[0013] In an embodiment, there is provided a method for modulating GPR75 signaling in a subject, comprising administering a CCL5 variant to the subject.

[0014] In an embodiment, there is provided a use of a CCL5 variant for modulating GPR75 signaling in a subject.

[0015] In an embodiment, there is provided a CCL5 variant for use in modulating GPR75 signaling in a subject.

[0016] In an embodiment, there is provided a method for treating a GPR75-related disease or disorder in a subject, comprising administering to the subject a CCL5 variant.

[0017] In an embodiment, there is provided a use of a CCL5 variant for treating a GPR75 -related disease or disorder in a subject.

[0018] In an embodiment, there is provided a CCL5 variant for use in treating a GPR75- related disease or disorder in a subject.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Embodiments will be described, by way of example only, with reference to the accompanying figures.

[0021] Figure 1. Intracellular calcium flux in response to 20-HETE or solvent control in GPR75 -expressing EA.hy926 cells or in GPR75 -knockdown (KD) EA.hy926 cells. Results are presented as the integrated area of the calcium flux response (AU) measured every second for 2 minutes (mean ± s.e.m. for 8 technical replicates).

[0022] Figure 2. Intracellular calcium flux in response to 20-HETE in the presence or absence of OB-002 or CCL5. Results are presented as the integrated area of the calcium flux response (AU) measured every second for 2 minutes (mean ± s.e.m. for 8 technical replicates).

[0023] Figure 3. Weight gain over time (Delta body weight (BW) measured in grams (g)) over time in mice given a high fat diet (HFD) and administered saline control or OB-002 at a low dose (LD; 10 mg / kg) or at a high dose (HD; 100 mg / kg).

[0024] Figure 4. Intake of food from a high fat diet (HFD) over time (measured in grams (g)) in mice administered saline control or OB-002 at a low dose (LD; 10 mg / kg) or at a high dose (HD; 100 mg / kg).

[0025] DETAILED DESCRIPTION

[0026] N-terminal variations in the sequence of CCL5 results in changes in chemokine receptor interaction and subsequent biological effects. Although CCL5 variants have been investigated for their interactions with CCR5, the receptor site and mechanism by which CCL5 interacts with GPR75 is not known and may be very different from that established for CCR5. Thus, CCL5 variants may interact differently with GPR75 than their interactions on CCR5 would indicate. Some CCL5 variants such as 5P12-RANTES (also known as OB-002, QGPPLMATQS, SEQ ID NO: 19) are not agonists of CCR5, and thus the use of non-agonist CCL5 variants to modulate GPR75 signaling in vivo would advantageously not induce off-target CCR5 signaling.

[0027] As demonstrated herein, it has been discovered that CCL5-variant CCR5- antagonists act as antagonists of GPR75 and are capable of limiting weight gain while preserving lean mass and without suppressing appetite in a mouse model of diet-induced obesity (DIO).

[0028] CCL5 variants

[0029] The present invention comprises methods and uses of CCL5 variants. As used herein, the term “ligand” carries the ordinary meaning in the art of a molecule that specifically binds to one or more receptors. As used herein, the term “CCL5 variant” refers to a modified CCL5 polypeptide containing one or more structural differences compared to the wild typeCCL5 polypeptide. As used herein, the term “wild type CCL5” refers to the naturally occurring structure of CCL5 as found in an organism. As used herein, a “wild type CCL5” is a CCL5 polypeptide that is isolated from an organism or a CCL5 polypeptide that is produced synthetically and has the structure and / or function of CCL5 as found in an organism. The terms “naturally occurring CCL5”, “native CCL5”, and “wild type CCL5” may be used interchangeably.

[0030] The CCL5 variants contain mutations, substitutions, additions, deletions, or other structural variations compared to wild type CCL5. In some embodiments, the CCL5 variants contain mutations or other structural variations to the receptor-binding region of wild type CCL5.

[0031] In some embodiments, the CCL5 variant is an antagonist of GPR75. In some embodiments, the CCL5 variant exhibits at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 15-fold, or 20-fold higher antagonism of GPR75 compared to the wild-type CCL5.

[0032] As used herein, an “antagonist” is a ligand that inhibits one or more biological activities induced by the agonism, activation, or signaling of a receptor. As used herein, “antagonism” refers to the activity of antagonist. In some embodiments, the CCL5 variant is an antagonist that inhibits one or more biological activities of GPR75 as described herein. The activity of an antagonist may be measured in the terms of IC50 values obtained from an inhibitory potency assay. The IC50 is typically defined as the concentration of the agonist at which 50 % of a signal elicited via the receptor by a reference agonist is inhibited. IC50 may sometimes be reported as pICso, which is the negative log of the IC50 value in moles per liter (molar or M).

[0033] As used herein, an “agonist” is a ligand that activates a receptor to induce one or more biological activities of the receptor. As used herein, “agonism” refers to the activity of an agonist. Engagement of G protein-coupled receptors with an agonist leads to activation of a repertoire of intracellular signaling pathways, broadly defined as G protein-dependent and G protein-independent signaling pathways. One manifestation of G protein-dependent signaling is the induction of intracellular calcium flux. One manifestation of G protein-independent signaling is the induction of receptor internalization by arrestin recruitment, leading to intracellular receptor sequestration. In some embodiments, the CCL5 variant is an agonist that induces one or more biological activities of GPR75 as described herein. The activity of an agonist is typically measured by its EC50 value. The EC50 can be measured for a given agonist by determining the concentration of agonist needed to elicit half of the maximum biological response of the receptorwhen agonized by a reference agonist. Smaller EC50 values, as measured in concentration of agonist, indicate increased agonism because a lower concentration of the agonist is required to elicit the maximum biological response. Alternatively, agonism may be measured by signaling efficacy. Signaling efficacy is usually measured by determining the maximum biological activity elicited by the ligand (Emax).

[0034] In some embodiments, the CCL5 variant may bind with higher affinity to GPR75 compared to wild-type CCL5. In some embodiments, the CCL5 variant may bind to GPR75 with at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold higher affinity compared to the wild-type CCL5. In some embodiments, the CCL5 variant may bind with higher affinity to GPR75 compared to wild-type CCL5 without exhibiting increased GPR75 agonism compared to wild-type CCL5. In some embodiments, the CCL5 variant may bind with higher affinity to GPR75 compared to wild-type CCL5 while exhibiting increased GPR75 antagonism compared to wild-type CCL5. Binding affinity may be measured by techniques known in that art such as, for example, surface plasmon resonance. Binding affinity between a ligand and a receptor is usually measured by dissociation constant (Ka) in pM, nM, pM, or mM units. Alternatively, binding affinity may be measured by binding capacity. Binding capacity is usually measured by determining the maximum biological activity of the receptor at receptor saturation with the ligand (Bmax)

[0035] Receptors may exhibit more than one biological activity when activated by an agonist. The magnitude of the agonism or antagonism of a ligand may be measured with respect to one or more of the biological activities exhibited by the activated receptor. Agonism may be measured by one or more of increased G protein signaling through the receptor when bound by the ligand; increased arrestin recruitment to the receptor when bound by the ligand; increased induction of intracellular calcium flux when the receptor is bound by the ligand; and / or increased receptor internalization when the receptor is bound by the ligand. In some embodiments, the CCL5 variant may provide at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 15-fold, or 20-fold higher antagonism as measured by the biological activities described above as compared to wild-type CCL5. The measurements of agonism and antagonism as described herein may be determined using routine methods known in the art.

[0036] In some embodiments, the CCL5 variant is as disclosed in Gaertner et al., 2008, PNAS, 105(46): 17706-17711 or as disclosed in WO 2008 / 012689.

[0037] Methionine residues can be conservatively substituted with non-oxidizable amino acid analogs or amino acid derivatives to reduce complications from methionine oxidationduring synthesis. In some embodiments, one or more methionine residues in the CCL5 variants of the present invention are conservatively substituted with an amino acid analog or an amino acid derivative such as, but not limited to, norleucine (Nle). It is known in the art the N-terminal glutamine or glutamic acids can spontaneously convert to pyroglutamate in various in vitro and in vivo conditions (Cao et al., 2022, J. Pharm. Sci., I l l :335-344). In some embodiments, the N- terminal residue designated herein as glutamine (Q) is present as a pyroglutamate.

[0038] In some embodiments, the CCL5 variant comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to CCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS (SEQ ID NO: 81), or to CCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEXS (SEQ ID NO: 82) wherein the ‘X’ at position 58 in SEQ ID NO: 82 denotes a norleucine.

[0039] In some embodiments, the CCL5 variant comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 81 or 82, wherein the variant sequence is: QGP[P or L][L or G or S or M][M or D or S or Q or G]; QGP[P or L][L or G][M or D or S]; QGP[P or L][L or G or S or M][M or D or S or Q or G]XX[Q or G or L or A or T or S]X, wherein X denotes any natural or modified amino acid; QGP[P or L][L or G][M or D or S]XX[Q or L]X, wherein X denotes any natural or modified amino acid; QGP[P or L]LM (SEQ ID NO: 1); QGPPG[D or S] (SEQ ID NO: 2); QGPPLM (SEQ ID NO: 3); or QGPPGD (SEQ ID NO: 4).

[0040] In some embodiments, the CCL5 variant comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 81 or 82, wherein the variant sequence is: QGP[P or L][L or M][M or Q][A or W or G or Q or N]X[Q or G or L][S or V or T or G], wherein X denotes any natural or modified amino acid; QGP[P or L][L or M][M or Q][A or W or G or Q or N][L or T or M or S or G or Q or R or Y][Q or G or L][S or V or T or G]; QGP[P or L]LM[A or W][L or T or M][Q or G][S or V or T or G] (SEQ ID NO: 5); or QGPPLM[A or W][L or T or M][Q or G][S or V or T or G] (SEQ ID NO: 6).

[0041] In some embodiments, the CCL5 variant comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 81 or 82, wherein the variant sequence is: QGP[P or L][L or G or S][D or S or G or Q]XX[L or A or T or Q][W or A or V], wherein X denotes any natural or modified amino acid; QGP[P or L][L or G or S][D or S or G or Q][T or I or S or W or Q][V or L or A or S or G][L or A or T or Q][W or A or V]; QGPPG[D or S][T or I]VL[W or A] (SEQ ID NO: 7); or QGPPGD[T or I]VL[W or A] (SEQ ID NO: 8).

[0042] In some embodiments, the CCL5 variant comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 81 or 82, wherein the variant sequence is: QGPP[G or L][M or Q]XX[Q or S][S or V] (SEQ ID NO: 9), wherein X denotes any natural or modified amino acid; QGPP[G or L][M or Q][S or G or W or A or T][L or F or T or S or G or Y][Q or S][S or V] (SEQ ID NO: 10); or QGPPLM[S or G][L or F or T]Q[S or V] (SEQ ID NO: 11).

[0043] In some embodiments, the CCL5 variant comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 81 or 82, wherein the variant sequence is selected from the group QGPPLMALQS (SEQ ID NO: 12), QGPPLMWMQV (SEQ ID NO: 13), QGPPLMWLQV (SEQ ID NO: 14), QGPPLMWTQS (SEQ ID NO: 15), QGPPLMWLQT (SEQ ID NO: 16), QGPPLMWTQV (SEQ ID NO: 17), QGPPLMWMQS (SEQ ID NO: 18), QGPPLMATQS (SEQ ID NO: 19), QGPPLMWLQS (SEQ ID NO: 20), QGPPLMALQV (SEQ ID NO: 21), QGPPLMWLGG (SEQ ID NO: 22), QGPPLMWRGS (SEQ ID NO: 23), QGPLLMWLQV (SEQ ID NO: 24), QGPPLMQTTP (SEQ ID NO: 25), QGPPLSWLQV (SEQ ID NO: 26), QGPPLSWLQS (SEQ ID NO: 27), QGPPGQWSQV (SEQ ID NO: 28), QGPPMMAGLS (SEQ ID NO: 29), QGPPLSWQQS (SEQ ID NO: 30), QGPPGMWSQS (SEQ ID NO: 31), QGPPLQWRQS (SEQ ID NO: 32), QGPPLMGTQS (SEQ ID NO: 33), QGPPLMQLQV (SEQ ID NO: 34), QGPPLSWSQV SEQ ID NO: 35), QGPPMSWSQS (SEQ ID NO: 36), QGPPLMNLQV (SEQ ID NO: 37), QGPPMSAYQV (SEQ ID NO: 38), QGPPMQGGLS (SEQ ID NO: 39), QGPPGDTVLW (SEQ ID NO: 40), QGPPGDIVLA (SEQ ID NO: 41), QGPPGSYDYS (SEQ ID NO: 42),QGPPGDGGSV (SEQ ID NO: 43), QGPLSGQSTP (SEQ ID NO: 44), QGPPGDWLQV (SEQ ID NO: 45), QGPPLMSLAV (SEQ ID NO: 46), QGPPLMSLTV (SEQ ID NO: 47), QGPLSGWAQV (SEQ ID NO: 48), QGPLSQSSQV (SEQ ID NO: 49), QGPLSSQSQV (SEQ ID NO: 50), QGPLGQQGQV (SEQ ID NO: 51), QGPPLMSFQS (SEQ ID NO: 52), QGPPLMSTQS (SEQ ID NO: 53), QGPPLMSLQV (SEQ ID NO: 54), QGPPLMGLQV (SEQ ID NO: 55), QGPLSGWLQV (SEQ ID NO: 56), QGPPLQWFQV (SEQ ID NO: 57), QGPPLQWTQV (SEQ ID NO: 58), QGPPLMALSV (SEQ ID NO: 59), QGPPLMWSQV (SEQ ID NO: 60), QGPPGQWGQV (SEQ ID NO: 61), QGPPGSWSQV (SEQ ID NO: 62), QGPPLMSSQS (SEQ ID NO: 63), QGPPLMGLSV (SEQ ID NO: 64), QGPPLMTLQV (SEQ ID NO: 65), QGPPGQWYQS (SEQ ID NO: 66), QGPPLMSVLA (SEQ ID NO: 67), QGPPGSWSSV (SEQ ID NO: 68), QGPPLGSMGP (SEQ ID NO: 69), QGPPLQWMQA (SEQ ID NO: 70), QGPPLQWMQV (SEQ ID NO: 71), QGPPLMSTQV (SEQ ID NO: 72), QGPPLMSLSV (SEQ ID NO: 73), QGPPLMSLQS (SEQ ID NO: 74), QGPPLMSLQA (SEQ ID NO: 75), QGPPLMSVQS (SEQ ID NO: 76), QGPPLMSAQS (SEQ ID NO: 77), QGPPLMSGQS (SEQ ID NO: 78) and QGPPLMSGQV (SEQ ID NO: 79).

[0044] In some embodiments, the CCL5 variant comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises the amino acid QGPPLMATQS (SEQ ID NO: 19) and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 81 or 82. In some embodiments, the CCL5 variant comprises an N- terminal portion and a C-terminal portion, wherein the N-terminal portion comprises the amino acid QGPPLMATQS (SEQ ID NO: 19) and the C-terminal portion comprises the amino acid sequence of SEQ ID NO: 81 or 82.

[0045] In some embodiments, the CCL5 variant comprises an N-terminal portion comprising the sequence PSC-SSDTTP (SEQ ID NO: 80), wherein PSC is N(alpha)(n- nonanoyl)-des-Ser(l)-[ L-thioprolyl(2), L-cyclohexylglycyl(3)], and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 81 or 82.

[0046] In some embodiments, the CCL5 variant is as disclosed in Hartley et al., 2004, PNAS, 101(47): 16460-16465.

[0047] In some embodiments, the N-terminal portion of the CCL5 variant comprises a moiety as shown in Table 1, and the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to any one of SEQ ID NOs: 81-90.Table 1

[0048] In some embodiments, the N-terminal portion of the CCL5 variant comprises the NNY-RANTES moiety as shown in Table 1, the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 83 or 84, and the C-terminal portion comprises (i) a substitution of the amino acid at position 2 of SEQ ID NO: 83 or 84 with a substituent as shown in Table la; or (ii) a substitution of the amino acid at position 3 of SEQ ID NO: 83 or 84 with a substituent as shown in Table lb; or (iii) a substitution of the amino acids at positions 2 and 3 with a substituent as shown in Table 1c.Table laTable lbTable 1c

[0049] In some embodiments, the N-terminal portion of the CCL5 variants described herein may consist of no more than 15, 14, 13, 12, 11, or 10 amino acids. In some embodiments, said N-terminal portion consists of 10 amino acids. In some embodiments, the N-terminus of the C-terminal portion may adjoin directly to the C-terminus of the N-terminal portion, meaning that the N-terminal portion and the C-terminal portion are directly adjoined. The C-terminal portion of CCL5 variants described in herein may have more than 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9% or 100% sequence identity to SEQ ID NO: 81 or 82. The variant sequence of the CCL5 variants described herein may be located near the N-terminus of the polypeptide. For example, the variant sequence may be located such that the beginning of the variant sequence lies within 15, 12, 10, 8, 6, 5, 4, 3, 2, or 1 residue of the N-terminus of the polypeptide. Herein, the expression “the beginning of the signature sequence” refers to the N-terminus of the signature sequence. The signature sequence may also be located at the extreme N-terminus of the polypeptide, i.e., the N-termini of the polypeptide as a whole and the signature sequence may coincide.

[0050] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two nucleic acid (polynucleotide) or two amino acid (polypeptide) sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions / total number of positions multiplied by 100%). The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin, S. and Altschul, S., 1990, PNAS, 87(6):2264-2268, modified in Karlin, S. and Altschul, S., 1993, PNAS, 90(12):5873-5877. Such an algorithm is incorporated into the BLAST programs. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a given nucleic acid molecule. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score=50, wordlength=3 to obtain amino acid sequences homologous to a given polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res., 25(17):3389-3402. Alternatively, PSLBLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSLBlast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers, E. and Miller, W ., 1988, Bioinformatics, 4(1): 11-17. Such an algorithm is incorporated in the ALIGN program which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0051] A sequence is also said to bear similarity to or to be a homologue of a reference sequence if it contains one or more conservative substitutions with respect to the reference sequence. Conservative substitutions are substitutions in the sequence of a peptide or polypeptide that do not lead to a significant loss of function or which lead only to a small loss of function.Such a loss of function due to one or more conservative substitutions may be considered not to be significant if said loss amounts to less than 20 %, less than 15 %, less than 10 %, less than 6 %, or less than 4 % with respect to the function of the polypeptide having the unsubstituted sequence. Conservative substitutions are often substitutions wherein an amino acid side chain is replaced by an amino acid side chain that is related, or similar in physicochemical properties, to the replaced residue. Such conservative substitutions may be made, for example, using one of the 20 natural amino acids according to Table 2 wherein amino acids in the same block in the middle column and preferably in the same line in the right-hand column may be substituted for each other. Conservative substitutions may also be made using amino acid analogs or amino acid derivatives such as, for example, a substitution of methionine with norleucine. fable 2

[0052] As used herein, the term “amino acid” is used to describe any amino acid, natural or otherwise, that can be incorporated into a polypeptide. Amino acids are small molecules comprising an amine (-NH2) group, a carboxyl (-COOH), and a variable side chain (R-group) specific to each amino acid. Amino acids are covalently linked by peptide bonds between the amine group of one amino acid to the carboxyl group of another amino acid to form polypeptides. Amino acids within a polypeptide are often referred to in the art as “residues”.

[0053] Polypeptides, including CCL5 variants of the present invention, may comprise modifications such as, for example, phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, acylation, prenylation, alkylation, oxidation, or other modifications known in the art. In some embodiments, the CCL5 variants described herein comprise one or more modifications selected from phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, acylation, prenylation, alkylation, oxidation, or other modifications known in the art.

[0054] Polypeptides may comprise amino acid analogs. As used herein, the term “amino acid analogs” describes artificial, synthetic, or unnatural amino acids known in the art beyond the 20 genetically-encoded amino acids. Examples of amino acid analogs that can be incorporated into polypeptides, peptides, and conjugates of the present invention include, but are not limited to, norleucine, P-amino acids, homo-amino acids, synthetic proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, N-methyl amino acids, and amino acids with synthetic R-groups. Polypeptides and peptides may also comprise amino acid derivatives. As used herein, the term “amino acid derivatives” describes amino acids that have been derived from the modification of one of the 20 genetically-encoded amino acids. Amino acid derivatives can be synthetic, such as by chemical reaction, or they can be naturally occurring in organisms, such as in vivo metabolites. An example of an amino acid derivative is pyroglutamate / pyroglutamic acid, a cyclized derivative of glutamine in which the free amino group of glutamic acid cyclizes to form a lactam.

[0055] Nucleic Acid Molecules and Vectors

[0056] In some embodiments, the CCL5 variants may be provided in the form of nucleic acid molecules that encode the CCL5 variants. For example, a nucleic acid molecule encoding a CCL5 variant may be provided to a cell in vitro or in vivo to induce expression of the CCL5 variant polypeptide. In some embodiments, the nucleic acid molecules encoding CCL5 variants for use in the present invention are RNA or DNA. The skilled person is able to design or identify nucleic acid molecules encoding the CCL5 variants of the present invention using methods known in the art. In some embodiments, the nucleic acid molecules encoding CCL5 variants of the present invention are incorporated into a vector, such as a plasmid, episome, artificial chromosome, virus, or a viral vector.

[0057] Pharmaceutical Compositions

[0058] The CCL5 variants described herein may be incorporated into pharmaceutical compositions for administration to a subject in need thereof. Said pharmaceutical compositions may comprise a pharmaceutically acceptable carrier, excipient, and / or stabilizer. The pharmaceutical compositions may be provided for use as a medicament. The preparation of pharmaceutical compositions is well known to the person skilled in the art.

[0059] A pharmaceutical composition according to the present invention may be administered to a subject in a therapeutically effective amount. As used herein, a "therapeuticallyeffective amount" means an amount of the pharmaceutical composition or CCL5 variant therein effective to provide a therapeutic, prophylactic or diagnostic benefit to a subject. In some embodiments, a therapeutically effective amount of the pharmaceutical composition is an amount capable of inducing a clinical response in a subject in the treatment of a particular disease or disorder. Determination of a therapeutically effective amount of the pharmaceutical composition is well within the capability of those skilled in the art, especially in light of the disclosure provided herein. The therapeutically effective amount may vary according to a variety of factors such as the subject’s condition, weight, sex and age.

[0060] Pharmaceutical compositions provided herein may be prepared in various pharmaceutical dosage forms, such as an instant release, controlled release, sustained release, or target drug-delivery system. Commonly used dosage forms include, for example, solutions and suspensions, (micro-) emulsions, ointments, gels, creams, pastes, foams, suppositories, ovules, implants, patches, liposomes, tablets, dragees, lozenges, soft or hard shell capsules, amorphous or crystalline powders, effervescent powders or tablets, aerosols, and lyophilized formulations. Depending on the route of administration used, special devices may be required for application or administration of a dosage form, such as syringes and needles, inhalers, pumps, injection pens, applicators, special flasks, or other devices for administration, which may also be implanted within a body. Pharmaceutical dosage forms provided herein may be manufactured by any of the methods well-known in the art, such as, for example, by conventional mixing, sieving, dissolving, melting, granulating, dragee-making, tabletting, suspending, extruding, spray-drying, levigating, emulsifying, (nano / micro-) encapsulating, entrapping, or lyophilization processes.

[0061] Pharmaceutical compositions provided herein may further comprise a pharmaceutically acceptable carrier, excipient, and / or stabilizer as known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations, and may comprise, for example but not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such asEDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (such as Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0062] Methods and Uses

[0063] In some embodiments, the CCL5 variant is used for modulating GPR75 signaling in a cell. Modulating GPR75 signaling comprises antagonism of GPR75 as described herein. In some embodiments, the cell is, but not limited to, an endothelial cell, a smooth muscle cell, or a beta islet cell. In some embodiments, the cell is a primary cell or transformed cell that natively expresses GPR75 or is genetically modified to express GPR75. In some embodiments, the cell is contacted with a CCL5 variant according to the present invention in vitro. In some embodiments, the cell is contacted with a CCL5 variant as described herein in vivo or ex vivo.

[0064] In some embodiments, the CCL5 variant is used for modulating GPR75 signaling in a subject. Modulating GPR75 signaling comprises antagonism of GPR75 as described herein. In such embodiments, the subject is administered a CCL5 variant as described herein or a pharmaceutical composition as described herein comprising the CCL5 variant. In vivo binding of the CCL5 variant to GPR75 -expressing cells in the subject will modulate the GPR75 signaling in the subject. GPR75 -expressing cells in the subject may include, but are not limited to, endothelial cells, smooth muscle cells, cancer cells, and / or beta islet cells. In some embodiments, the subject is human.

[0065] In some embodiments, the CCL5 variant is used for treating a GPR75-related disease or disorder in a subject. As used herein, a “GPR75 -related disease or disorder” refers to a disease or disorder for which modulation of GPR75 signaling (e.g. antagonism) in the subject leads to complete or partial treatment or prevention of the disease or disorder. In some embodiments, the GPR75-realted disease or disorder is a metabolic disease. In some embodiments, the GPR75-related disease or disorder is obesity, hypertension, metabolic syndrome, metabolic dysfunction-associated steatohepatitis (MASH), GPR75-mediated cancer, GPR75 -expressing cancer, or diabetes (e.g. Type I or Type II). In some embodiments, the subject is human.

[0066] In some embodiments, the CCL5 variant is used for treating a metabolic disease in a subject. In some embodiments, the CCL5 variant is used for treating obesity, hypertension, metabolic syndrome, metabolic dysfunction-associated steatohepatitis (MASH), or diabetes (e.g. Type I or Type II) in a subject. In some embodiments, the subject is human.

[0067] In some embodiments, the CCL5 variant is used for inducing weight loss and / or preventing weight gain in a subject. In some embodiments, the weight loss is a loss of non-lean body mass and / or the weight gain is a gain of non-lean body mass. In some embodiments, the CCL5 variant is used for inducing loss of non-lean body mass and / or preventing gain of non-lean body mass in a subject, while preserving (z.e. maintaining or not significantly decreasing) and / or not preventing gain (z.e. allowing for an increase) of lean body mass in the subject. In some embodiments, the lean body mass is maintained or is not significantly decreased compared to lean body mass at the start of treatment of the subject with the CCL5 variant. In some embodiments, the lean body mass is maintained or is increased in a subject compared to lean body mass of the subject at the start of treatment of the subject with the CCL5 variant.

[0068] As described herein, it was surprisingly discovered that CCL5 variants were capable of inhibiting weight gain without suppressing appetite. In some embodiments the CCL5 variant is used for inducing loss of body mass (e.g. non-lean body mass) and / or preventing gain of body mass (e.g. non-lean body mass) in a subject, while preserving (z.e. maintaining or not significantly decreasing) appetite in the subject. In some embodiments, appetite is maintained or is not significantly decreased in a subject compared to appetite of the subject at the start of treatment of the subject with the CCL5 variant. Appetite may be measured by methodologies known in the art. In some embodiments, appetite is measured by food intake of the subject over a defined period of time (e.g. at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 6 months, or at least 1 year). In some embodiments, food intake of the subject as measured by the mass of consumed food in a defined a period of time, or as measured by calories of consumed food in a defined period of time, is maintained or is not significantly decreased compared to food intake of the subject at the start of treatment of the subject with the CCL5 variant. In some embodiments, food intake of the subject as measured by the mass of consumed food in a defined a period of time is maintained or is decreased by less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 10%, less than 15%, or less than 20% compared to food intake of the subject at the start of treatment of the subject with the CCL5 variant.

[0069] “Treating” or “treatment”, or “preventing” or “prevention”, as used herein, refers to an approach for obtaining beneficial or desired results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, dimini shment of extent of disease, stabilisation of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression, suppression of disease, delay or slowing of disease onset, and amelioration or palliation of thedisease state. “Treating” or “preventing” can also mean prolonging survival of a patient beyond that expected in the absence of treatment and can also mean inhibiting the progression of disease temporarily or preventing the occurrence of disease. “Treating” may be distinguished from “preventing” in that “treating” typically occurs in a subject who already has a disease or disorder, whereas “preventing” typically occurs in a subject who does not have a disease or disorder. As will be appreciated, there may be overlap in treatment and prevention. For example, it is possible to be “treating” a disease in a subject, while at same time “preventing” symptoms or progression of the disease.

[0070] Sequences referred to herein by SEQ ID NO are set forth in Table 3 :Table 3

[0071] In some embodiments, the present invention relates to:1. A method for modulating GPR75 signaling in a cell, comprising contacting the cell with a CCL5 variant.2. Use of a CCL5 variant for modulating GPR75 signaling in a cell.3. A CCL5 variant for use in modulating GPR75 signaling in a cell.4. The method, use, or CCL5 variant for use of any one of embodiments 1-3, wherein the modulation of GPR75 signaling is antagonism.5. The method, use, or CCL5 variant for use of embodiment 4, wherein the antagonism is characterized by one or more of:- decreased G protein signaling through GPR75;- decreased arrestin recruitment to GPR75; and- decreased calcium flux.6. The method, use, or CCL5 variant for use of any one of embodiments 1-5, wherein the cell is an endothelial cell, a smooth muscle cell, a cancer cell, or a beta islet cell.7. The method, use, or CCL5 variant for use of any one of embodiments 1-6, wherein the cell is in vitro or ex vivo.8. The method, use, or CCL5 variant for use of any one of embodiments 1-6, wherein the cell is in vivo.9. A method for modulating GPR75 signaling in a subject, comprising administering a CCL5 variant to the subject.10. Use of a CCL5 variant for modulating GPR75 signaling in a subject.11. A CCL5 variant for use in modulating GPR75 signaling in a subject.12. The method, use, or CCL5 variant for use of any one of embodiments 9-11, wherein the modulation of GPR75 signaling is antagonism.13. The method, use, or CCL5 variant for use of embodiment 12, wherein the antagonism is characterized by one or more of:- decreased G protein signaling through GPR75;- decreased arrestin recruitment to GPR75; and- decreased calcium flux.14. A method for treating a GPR75-related disease or disorder in a subject, comprising administering to the subject a CCL5 variant.15. Use of a CCL5 variant for treating a GPR75-related disease or disorder in a subject.16. A CCL5 variant for use in treating a GPR75-related disease or disorder in a subject.17. The method, use, or CCL5 variant for use of any one of embodiments 14-16, wherein the GPR75-related disease or disorder is selected from obesity, hypertension, metabolic syndrome, metabolic dysfunction-associated steatohepatitis, GPR75-mediated cancer, GPR75 -expressing cancer, and diabetes.18. A method for treating obesity, hypertension, metabolic syndrome, and / or diabetes in a subject, comprising administering to the subject a CCL5 variant.19. Use of a CCL5 variant for treating obesity, hypertension, metabolic syndrome, metabolic dysfunction-associated steatohepatitis, and / or diabetes in a subject.20. A CCL5 variant for use in treating obesity, hypertension, metabolic syndrome, metabolic dysfunction-associated steatohepatitis, and / or diabetes in a subject.21. A method for inducing weight loss and / or preventing weight gain in a subject, comprising administering to the subject a CCL5 variant.22. Use of a CCL5 variant for inducing weight loss and / or preventing weight gain in a subject.23. A CCL5 variant for use in inducing weight loss and / or preventing weight gain in a subject.24. The method, use, or CCL5 variant for use of any one of embodiments 21-23, wherein the weight loss is a loss of non-lean body mass and / or the weight gain is a gain of non-lean body mass.25. The method, use, or CCL5 variant for use of any one of embodiments 21-24, wherein appetite is maintained or is not significantly decreased.26. The method, use, or CCL5 variant for use of any one of embodiments 14-25, wherein the subject is human.27. The method, use, or CCL5 variant for use of any one of embodiments 1-26, wherein the CCL5 variant comprises an N-terminal portion and a C-terminal portion.28. The method, use, or CCL5 variant for use of embodiment 27, wherein the N-terminal portion comprises the sequence QGP[P or L], and wherein the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 81 or 82.29. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S or M][M or D or S or Q or G],30. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G][M or D or S],31. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S or M][M or D or S or Q or G]XX[Q or G or L or A or T or S]X, wherein X denotes any natural or modified amino acid.32. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G][M or D or S]XX[Q or G or L]X, wherein X denotes any natural or modified amino acid.33. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGP[P or L]LM (SEQ ID NO: 1) or QGPPGfD or S] (SEQ ID NO: 2).34. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGPPLM (SEQ ID NO: 3) or QGPPGD (SEQ ID NO: 4).35. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGP[P or L][L or M][M or Q][A or W or G or Q or N]X[Q or G or L][S or V or T or G],36. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGP[P or L][L or M][M or Q][A or W or G or Q or N][L or T or M or S or G or Q or R or Y][Q or G or L][S or V or T or G],37. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGP[P or L]LM[A or W][L or T or M][Q or G][S or V or T or G] (SEQ ID NO: 5).38. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGPPLMfA or W][L or T or M][Q or G][S or V or T or G] (SEQ ID NO: 6).39. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S][D or S or G or Q]XX[L or A or T or Q][W or A or V],40. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S][D or S or G or Q][T or I or S or W or Q][V or L or A or S or G][L or A or T or Q][W or A or V],41. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGPPGfD or S][T or I]VL[W or A] (SEQ ID NO: 7).42. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGPPGDfT or I]VL[W or A] (SEQ ID NO: 8).43. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGPP[G or L][M or Q]XX[Q or S][S or V] (SEQ ID NO: 9).44. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGPP[G or L][M or Q][S or G or W or A or T][L or F or T or S or G or Y][Q or S][S or V] (SEQ ID NO: 10).45. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGPPLMfS or G][L or F or T]Q[S or V] (SEQ ID NO: 11).46. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMALQS (SEQ ID NO: 12), QGPPLMWMQV (SEQ ID NO: 13), QGPPLMWLQV (SEQ ID NO: 14),QGPPLMWTQS (SEQ ID NO: 15), QGPPLMWLQT (SEQ ID NO: 16), QGPPLMWTQV (SEQ ID NO: 17), QGPPLMWMQS (SEQ ID NO: 18), QGPPLMATQS (SEQ ID NO: 19), QGPPLMWLQS (SEQ ID NO: 20), QGPPLMALQV (SEQ ID NO: 21), QGPPLMWLGG (SEQ ID NO: 22), QGPPLMWRGS (SEQ ID NO: 23), QGPLLMWLQV (SEQ ID NO: 24), QGPPLMQTTP (SEQ ID NO: 25), QGPPLSWLQV (SEQ ID NO: 26), QGPPLSWLQS (SEQ ID NO: 27), QGPPGQWSQV (SEQ ID NO: 28), QGPPMMAGLS (SEQ ID NO: 29), QGPPLSWQQS (SEQ ID NO: 30), QGPPGMWSQS (SEQ ID NO: 31), QGPPLQWRQS (SEQ ID NO: 32), QGPPLMGTQS (SEQ ID NO: 33), QGPPLMQLQV (SEQ ID NO: 34), QGPPLSWSQV SEQ ID NO: 35), QGPPMSWSQS (SEQ ID NO: 36), QGPPLMNLQV (SEQ ID NO: 37), QGPPMSAYQV (SEQ ID NO: 38), QGPPMQGGLS (SEQ ID NO: 39), QGPPGDTVLW (SEQ ID NO: 40), QGPPGDIVLA (SEQ ID NO: 41), QGPPGSYDYS (SEQ ID NO: 42), QGPPGDGGSV (SEQ ID NO: 43), QGPLSGQSTP (SEQ ID NO: 44), QGPPGDWLQV (SEQ ID NO: 45), QGPPLMSLAV (SEQ ID NO: 46), QGPPLMSLTV (SEQ ID NO: 47), QGPLSGWAQV (SEQ ID NO: 48), QGPLSQSSQV (SEQ ID NO: 49), QGPLSSQSQV (SEQ ID NO: 50), QGPLGQQGQV (SEQ ID NO: 51), QGPPLMSFQS (SEQ ID NO: 52), QGPPLMSTQS (SEQ ID NO: 53), QGPPLMSLQV (SEQ ID NO: 54), QGPPLMGLQV (SEQ ID NO: 55), QGPLSGWLQV (SEQ ID NO: 56), QGPPLQWFQV (SEQ ID NO: 57), QGPPLQWTQV (SEQ ID NO: 58), QGPPLMALSV (SEQ ID NO: 59), QGPPLMWSQV (SEQ ID NO: 60), QGPPGQWGQV (SEQ ID NO: 61), QGPPGSWSQV (SEQ ID NO: 62), QGPPLMSSQS (SEQ ID NO: 63), QGPPLMGLSV (SEQ ID NO: 64), QGPPLMTLQV (SEQ ID NO: 65), QGPPGQWYQS (SEQ ID NO: 66), QGPPLMSVLA (SEQ ID NO: 67), QGPPGSWSSV (SEQ ID NO: 68), QGPPLGSMGP (SEQ ID NO: 69), QGPPLQWMQA (SEQ ID NO: 70), QGPPLQWMQV (SEQ ID NO: 71), QGPPLMSTQV (SEQ ID NO: 72), QGPPLMSLSV (SEQ ID NO: 73), QGPPLMSLQS (SEQ ID NO: 74), QGPPLMSLQA (SEQ ID NO: 75), QGPPLMSVQS (SEQ ID NO: 76), QGPPLMSAQS (SEQ ID NO: 77), QGPPLMSGQS (SEQ ID NO: 78) and QGPPLMSGQV (SEQ ID NO: 79).47. The method, use, or CCL5 variant for use of embodiment 28, wherein the N-terminal portion comprises the sequence QGPPLMATQS (SEQ ID NO: 19).48. The method, use, or CCL5 variant for use of any one of embodiments 27-47, wherein the N-terminal portion consists of no more than 15 amino acids.49. The method, use, or CCL5 variant for use of any one of embodiments 27-47, wherein the N-terminal portion consists of 10 amino acids.50. The method, use, or CCL5 variant for use of embodiment 27, wherein the N-terminal portion comprises the sequence PSC-SSDTTP (SEQ ID NO: 80), wherein PSC is N(alpha)(n-nonanoyl)-des-Ser(l)-[ L-thioprolyl(2), L-cyclohexylglycyl(3)], and wherein the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 81 or 82.51. The method, use, or CCL5 variant for use of embodiment 27, wherein the N-terminal portion comprises a moiety as shown in Table 1, and the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to any one of SEQ ID NOs: 81-90.52. The method, use, or CCL5 variant for use of embodiment 27, wherein the N-terminal portion comprises the NNY-RANTES moiety as shown in Table 1, the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 83 or 84, and wherein the C-terminal portion comprises:(i) a substitution of the amino acid at position 2 of SEQ ID NO: 83 or 84 with a substituent as shown in Table la; or(ii) a substitution of the amino acid at position 3 of SEQ ID NO: 83 or 84 with a substituent as shown in Table lb; or(iii) a substitution of the amino acids at positions 2 and 3 with a substituent as shown in Table 1c.53. The method, use, or CCL5 variant for use of any one of embodiments 27-51, wherein the amino acid sequence of the C-terminal portion comprises the amino acid sequence of SEQ ID NO: 81 or 82.54. The method, use, or CCL5 variant for use of any one of embodiments 27-53, wherein the N-terminal portion is located at the extreme N terminus.

[0072] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0073] As used herein, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. In embodiments comprising an “additional” or “second” component, the second component as used herein is different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0074] All publications and patents cited herein are incorporated by reference in their entirety as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In the case of any conflict between a definition of a term in the present disclosure and a definition in a cited publication or patent, the definition provided in the present disclosure is to be used in describing the present invention.

[0075] The present invention will now be described by way of non-limiting examples having regard to the appended drawings.

[0076] Example 1

[0077] Experiments were performed that confirm 20-HETE as an agonist of GPR75 and reveal that a CCL5 variant that is a CCR5 agonist, exemplified by OB-002 (QGPPLMATQS, SEQ ID NO: 19), is a potent antagonist of GPR75.

[0078] Methods and assays for the analysis of GPR75 agonism and antagonism were carried out essentially as described in Pascale et al., 2021, Br. J. Pharmacol., 178(18):3813- 3282.

[0079] Agonism of GPR75 in response to 20-HETE was measured by intracellular calcium flux. Antagonism of GPR75 was measured by the reduction of calcium flux in response to 20-HETE in the presence of either wild-type human CCL5 (synthetic or recombinant origin) or the exemplary CCL5 variant OB-002. Experiments were carried out with a GPR75 -expressing human endothelial cell line (EA.hy926), using either the parental cell line (EA.hy926) or a variant in which the gene for GPR75 has been knocked down (GPR75KD EA.hy926).

[0080] Calcium signaling measurements were conducted using the FLIPR Calcium 6 Assay Kit (R8190, Molecular Devices, San Jose, CA) or an equivalent kit according to the manufacturer's protocol.

[0081] Cells were plated onto clear bottom 96-well plates and grown to near confluency, then washed and starved in incomplete medium prior to the addition of the FLIPR Calcium 6 dyeor equivalent reconstituted in physiological buffer. Cells were then incubated at 37°C, 5% CO2 for a period of 10 min to 3 h.

[0082] Measurements were made with an appropriate microplate reader to determine the calcium assay signal. Fluorescence readings (excitation wavelength 484 nm, emission wavelength 525 nm when performed with FLIPR Calcium 6 Assay Kit or equivalent) were acquired before and during treatment with the agonist GPR75 ligand 20-HETE (or the solvent used for 20-HETE as a solvent control), or CCL5 or the exemplary CCL5 variant OB-002 (or the solvent used for CCL5 and variants thereof as a solvent control). Results were presented as the integrated area of the calcium flux response (AU) measured every second for 2 minutes (mean ± s.e.m. for 8 technical replicates).

[0083] The experiments determined the baseline responses of the EA.hy926 cell line to 20-HETE and the extent to which 20-HETE-induced responses are inhibited by the presence of CCL5 or the exemplary CCL5 variant OB-002. The inclusion of the GPR75 knockdown (KD) variant of EA.hy926 provided experimental demonstration that the observed 20-HETE signaling and inhibition by CCL5 or the exemplary CCL5 variant OB-002 were acting via GPR75.

[0084] As shown in Figure 1, 20-HETE induced significant calcium flux in EA.hy926 cells. In GPR75 knockdown (KD) EA.hy926 cells, the level of calcium flux in response to 20- HETE was reduced to the level of the solvent control, confirming that 20-HETE signals through GPR75.

[0085] As shown in Figure 2, the calcium flux induced in response to 20-HETE was reduced by the presence of wild type CCL5, thereby blocking GPR75 activation. The calcium flux induced in response to 20-HETE was even further reduced by the presence of the exemplary CCL5 variant OB-002, revealing this molecule as a potent GPR75 antagonist.

[0086] Example 2

[0087] Experiments were performed that reveal OB-002 as a GPR75 antagonist that inhibits weight gain in mice without suppressing appetite.

[0088] OB-002 was tested in a diet-induced obesity (DIO) model in diet-induced obeseC57BL / 6J mice. The mice were given a high fat diet (HFD) and either saline control or OB-002 at a low dose (LD; lOmg / kg) or at a high dose (HD; 100 mg / kg) starting at Day 0. Mice were administered saline control or OB-002 daily by intraperitoneal injection. Data points represent mean ± s.e.m. for 5 mice.

[0089] As shown in Figure 3, mice treated with saline control gradually gained weight when provided the high fat diet (HFD). By contrast, mice treated with OB-002 gained less weight (low dose) or essentially maintained their initial weight (high dose) during the course of the high fat diet (HFD).

[0090] As shown in Figure 4, mice treated with OB-002 had similar (week 1) or equivalent (week 2) food intakes (measured in grams of the high fat diet cumulatively consumed by one cage of mice per week) compared to mice treated with the saline control.

[0091] These data show that OB-002 is able to inhibit weight gain and preserve lean mass while allowing consistent food intake.

[0092] The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching. The embodiments described were chosen and described in order to best explain the principles of the invention and its practical application, and to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions or substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but is intended to cover the application or implementation without departing from the spirit or scope of the claims of the present invention.

Claims

CLAIMS:

1. A CCL5 variant for use in modulating GPR75 signaling in a cell.

2. The CCL5 variant for use of claim 1, wherein the cell is an endothelial cell, a smooth muscle cell, a cancer cell, or a beta islet cell.

3. A CCL5 variant for use in modulating GPR75 signaling in a subject.

4. The CCL5 variant for use of any one of claims 1-3, wherein the modulation of GPR75 signaling is antagonism.

5. The CCL5 variant for use of claim 4, wherein the antagonism is characterized by one or more of- decreased G protein signaling through GPR75;- decreased arrestin recruitment to GPR75; and- decreased calcium flux.

6. A CCL5 variant for use in treating a GPR75-related disease or disorder in a subject.

7. The CCL5 variant for use of claim 6, wherein the GPR75-related disease or disorder is selected from obesity, hypertension, metabolic syndrome, metabolic dysfunction-associated steatohepatitis, GPR75-mediated cancer, GPR75 -expressing cancer, and diabetes.

8. A CCL5 variant for use in treating obesity, hypertension, metabolic syndrome, metabolic dysfunction-associated steatohepatitis, and / or diabetes in a subject.

9. A CCL5 variant for use in inducing weight loss and / or preventing weight gain in a subject.

10. The CCL5 variant for use of claim 9, wherein the weight loss is a loss of non-lean body mass and / or the weight gain is a gain of non-lean body mass.

11. The CCL5 variant for use of claim 9 or 10, wherein appetite is maintained or is not significantly decreased.

12. The CCL5 variant for use of any one of claims 3-11, wherein the subject is human.

13. The CCL5 variant for use of any one of claims 1-12, wherein the CCL5 variant comprises an N-terminal portion and a C-terminal portion.

14. The CCL5 variant for use of claim 13, wherein the N-terminal portion comprises the sequence QGP[P or L], and wherein the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 81 or 82.

15. The CCL5 variant for use of claim 14, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMALQS (SEQ ID NO: 12), QGPPLMWMQV (SEQ ID NO: 13), QGPPLMWLQV (SEQ ID NO: 14), QGPPLMWTQS (SEQ ID NO: 15), QGPPLMWLQT (SEQ ID NO: 16), QGPPLMWTQV (SEQ ID NO: 17),QGPPLMWMQS (SEQ ID NO: 18), QGPPLMATQS (SEQ ID NO: 19), QGPPLMWLQS (SEQ ID NO: 20), QGPPLMALQV (SEQ ID NO: 21), QGPPLMWLGG (SEQ ID NO: 22), QGPPLMWRGS (SEQ ID NO: 23), QGPLLMWLQV (SEQ ID NO: 24), QGPPLMQTTP (SEQ ID NO: 25), QGPPLSWLQV (SEQ ID NO: 26), QGPPLSWLQS (SEQ ID NO: 27), QGPPGQWSQV (SEQ ID NO: 28), QGPPMMAGLS (SEQ ID NO: 29), QGPPLSWQQS (SEQ ID NO: 30), QGPPGMWSQS (SEQ ID NO: 31), QGPPLQWRQS (SEQ ID NO: 32), QGPPLMGTQS (SEQ ID NO: 33), QGPPLMQLQV (SEQ ID NO: 34), QGPPLSWSQV SEQ ID NO: 35), QGPPMSWSQS (SEQ ID NO: 36), QGPPLMNLQV (SEQ ID NO: 37), QGPPMSAYQV (SEQ ID NO: 38), QGPPMQGGLS (SEQ ID NO: 39), QGPPGDTVLW (SEQ ID NO: 40), QGPPGDIVLA (SEQ ID NO: 41), QGPPGSYDYS (SEQ ID NO: 42), QGPPGDGGSV (SEQ ID NO: 43), QGPLSGQSTP (SEQ ID NO: 44), QGPPGDWLQV (SEQ ID NO: 45), QGPPLMSLAV (SEQ ID NO: 46), QGPPLMSLTV (SEQ ID NO: 47), QGPLSGWAQV (SEQ ID NO: 48), QGPLSQSSQV (SEQ ID NO: 49), QGPLSSQSQV (SEQ ID NO: 50), QGPLGQQGQV (SEQ ID NO: 51), QGPPLMSFQS (SEQ ID NO: 52), QGPPLMSTQS (SEQ ID NO: 53), QGPPLMSLQV (SEQ ID NO: 54), QGPPLMGLQV (SEQ ID NO: 55), QGPLSGWLQV (SEQ ID NO: 56), QGPPLQWFQV (SEQ ID NO: 57), QGPPLQWTQV (SEQ ID NO: 58), QGPPLMALSV (SEQ ID NO: 59), QGPPLMWSQV (SEQ ID NO: 60), QGPPGQWGQV (SEQ ID NO: 61), QGPPGSWSQV (SEQ ID NO: 62), QGPPLMSSQS (SEQ ID NO: 63), QGPPLMGLSV (SEQ ID NO: 64), QGPPLMTLQV (SEQ ID NO: 65), QGPPGQWYQS (SEQ ID NO: 66), QGPPLMSVLA (SEQ ID NO: 67), QGPPGSWSSV (SEQ ID NO: 68), QGPPLGSMGP (SEQ ID NO: 69), QGPPLQWMQA (SEQ ID NO: 70), QGPPLQWMQV (SEQ ID NO: 71), QGPPLMSTQV (SEQ ID NO: 72), QGPPLMSLSV (SEQ ID NO: 73), QGPPLMSLQS (SEQ ID NO: 74), QGPPLMSLQA (SEQ ID NO: 75), QGPPLMSVQS (SEQ ID NO: 76), QGPPLMSAQS (SEQ ID NO: 77), QGPPLMSGQS (SEQ ID NO: 78) and QGPPLMSGQV (SEQ ID NO: 79).

16. The CCL5 variant for use of claim 14, wherein the N-terminal portion comprises the sequence QGPPLMATQS (SEQ ID NO: 19).

17. The CCL5 variant for use of claim 13, wherein the N-terminal portion comprises the sequence PSC-SSDTTP (SEQ ID NO: 80), wherein PSC is N(alpha)(n-nonanoyl)-des-Ser(l)- [ L-thioprolyl(2), L-cyclohexylglycyl(3)], and wherein the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 81 or 82.

18. The CCL5 variant for use of claim 13, wherein the N-terminal portion comprises a moiety as shown in Table 1, and the C-terminal portion is at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to any one of SEQ ID NOs: 81-90.

19. The CCL5 variant for use of claim 13, wherein the N-terminal portion comprises the NNY-RANTES moiety as shown in Table 1, the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 83 or 84, and wherein the C-terminal portion comprises:(i) a substitution of the amino acid at position 2 of SEQ ID NO: 83 or 84 with a substituent as shown in Table la; or(ii) a substitution of the amino acid at position 3 of SEQ ID NO: 83 or 84 with a substituent as shown in Table lb; or(iii) a substitution of the amino acids at positions 2 and 3 with a substituent as shown in Table 1c.

20. The CCL5 variant for use of any one of claims 13-18, wherein the amino acid sequence of the C-terminal portion comprises the amino acid sequence of SEQ ID NO: 81 or 82.

Images