Novel selective ACKR3 modulators and their uses

Abstract

The present application discloses a selective ACKR3 modulating peptide comprising the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is F or W; X2 is K, V or F; and wherein the peptide has a length of up to 15 amino acids; fusion proteins comprising the peptides as taught herein; nucleic acids encoding the peptides as taught herein; nucleic acid expression cassettes and vectors comprising the nucleic acids as taught herein; and pharmaceutical compositions comprising the peptides as taught herein or the nucleic acids as taught herein. Further provided are peptides as taught herein for use as pharmaceuticals; methods for in vitro or ex vivo diagnosis, prediction, prognosis and / or monitoring of diseases or conditions characterized by abnormal levels of ACKR3 polypeptides using the peptides as taught herein; and in vitro methods for identifying agents for use as therapeutic agents using the peptides as taught herein.

Description

Technical Field

[0001] The present invention relates broadly to the field of medicine and provides novel atypical chemokine receptor 3 (ACKR3) modulatory molecules, both per se and as fusion proteins with other agents, that can be used in various fields including diagnosis and therapy, and further provides methods and uses of the ACKR3 modulatory molecules. Background Art

[0002] Opioid receptors are G protein-coupled receptors (GPCRs) expressed by the central nervous system and immune cells that play a central role in regulating analgesia, reward processing, and stress, anxiety, or depression. The opioid receptor family consists of three classical receptors: μ (μ or MOR), δ (δ or DOR), and κ (κ or KOR); and the non-classical nociceptive peptide receptor (NOP, or orphan peptide FQ receptor).

[0003] All endogenous opioid peptides are derived from protease cleavage of large protein precursors and are produced primarily in the central nervous system (CNS), but also in the adrenal and pituitary glands and by several types of immune cells. With some exceptions, these ligands trigger downstream signaling reactions via G proteins, followed by recruitment of beta-arrestin, leading to receptor desensitization and internalization. Opioid receptors can also be modulated by non-peptide opioids such as morphine, fentanyl or naloxone. Opioid receptors are attractive targets for drugs, and opioid receptor modulators remain the most widely used analgesics in clinical practice. However, the use of these drugs is often associated with tolerance, dependence and various adverse effects (e.g., respiratory depression) or misuse.

[0004] The expression, signal transduction and desensitization of opioid receptors are also affected by their interactions with other GPCRs, particularly chemokine receptors. Chemokine receptors bind to chemokines, which are small (8-14 kDa) secreted chemoattractant cytokines, chemokines that regulate cellular processes such as migration, adhesion and growth, thereby playing a key role in inflammatory and developmental processes. To date, nearly 50 chemokines and 20 classical receptors have been identified in humans. Similar to the opioid receptor-ligand network, many chemokine receptors recognize multiple chemokines, and vice versa, many chemokines activate more than one receptor. Recently, a new family called atypical chemokine receptors (ACKRs) has emerged as a small subgroup of chemokine receptors. ACKRs bind to chemokines without triggering G protein signaling, but instead participate in chemotactic events by transporting or capturing chemokines or internalizing and degrading ligands to resolve inflammatory processes or form appropriate chemokine gradients.

[0005] ACKR3, formerly known as CXCR7, is expressed in various cell types, including B and T lymphocytes, neurons, and endothelial cells, and plays a key role in numerous processes, including cardiovascular and neuronal development, as well as the migration and homing of hematopoietic stem / progenitor cells. A growing body of research implicates ACKR3 in cardiovascular disease and numerous cancers. ACKR3 is expressed in various cancer cell types and in tumor-associated vasculature, with increasing evidence suggesting its involvement in metastatic development. ACKR3 has also been shown to be upregulated following infection with several oncogenic viruses, including HHV-8, EBV, and HTLV-1, and to play a crucial role in cell transformation and proliferation. Due to its unusual biology, it has recently been classified as an atypical chemokine receptor. Indeed, ACKR3 binds two endogenous chemokines, CXC-motif chemokine 12 (CXCL12) and CXC-motif chemokine 11 (CXCL11), which are also recognized by CXC-motif chemokine receptor 4 (CXCR4) and CXC-motif chemokine receptor 3 (CXCR3), respectively. However, unlike traditional chemokine receptors, ACKR3 does not activate the canonical G protein pathway and is proposed to trigger β-arrestin-dependent signaling. Furthermore, through its continuous cycling between the plasma membrane and endosomal compartments and its ability to efficiently internalize and degrade chemokines, ACKR3 functions as a scavenger receptor, regulating the availability of CXCL12 and CXCL11 to CXCR4 and CXCR3. Furthermore, ACKR3 is proposed to regulate CXCR4 activity by forming heterodimers or competing with intracellular effector proteins involved in signal transduction.

[0006] In view of the above, there is an urgent need to explore new approaches to modulate diseases involving ACKR3. Summary of the Invention

[0007] The present inventors have found that the atypical chemokine receptor ACKR3 binds to a large number of endogenous opioid peptides found in the central nervous system (CNS) and immune cells, including those from the enkephalin, dynorphin and nociceptin families. This broad-spectrum selectivity is atypical and unique among opioid receptors. In addition, the present inventors have found that, contrary to known opioid receptors and Ikeda et al. (Ikeda et al., 2013, Modulation of circadian glucocorticoid oscillation through ad renal opioid-CXCR7 signaling alters emotional behavior, Cell. 155 (6): 1323-1336), ACKR3 is unable to activate downstream signaling pathways, such as through G proteins or β-arrestins, in response to endogenous opioid peptides, which are present in different cellular compartments and act as scavengers, regulating their local and / or systemic concentrations, thereby regulating the availability of classic opioid receptors.

[0008] The present inventors have discovered that ACKR3 can be modulated to alter the levels of endogenous opioid peptides with a potentially improved safety profile in the treatment of diseases associated with dysregulation of endogenous opioid peptides, such as distress dysfunctional diseases or conditions, such as depression or chronic pain.

[0009] To this end, the present inventors have developed selective ACKR3 modulators. More specifically, these selective ACKR3 modulators are peptides having the consensus sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is F or W, and X2 is K, V or F, preferably having a length of up to 15 amino acids, which have high affinity and selectivity for ACKR3 and no regulatory (e.g., agonistic or antagonistic) activity for any other type of tested receptor. In addition, novel peptides with high affinity and selectivity for ACKR3 can act as ACKR3 agonists and can induce β-arrestin-1 and / or β-arrestin-2 to ACKR3. Furthermore, when the peptides as taught herein have a length of up to 15 amino acids, these peptides take advantage of low production costs, which is very important for maintaining competitiveness and allowing for use by the majority of patients.

[0010] In view of the above, a first aspect provides a selective ACKR3 modulating peptide comprising the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is F or W; X2 is K, V or F; and the peptide has a length of up to 15 amino acids.

[0011] In certain embodiments, X1 is F.

[0012] In a specific embodiment, X2 is K.

[0013] In a specific embodiment, the peptide comprises the amino acid sequence FGGX1MRRX2X3 (SEQ ID NO: 2), wherein X3 can be any amino acid, preferably wherein X3 is R or A.

[0014] In a specific embodiment, the peptide comprises an amino acid sequence selected from the group consisting of FGGFMRRK (SEQ ID NO:3), FGGFMRRKR (SEQID NO:4), FGGFMRRVR (SEQ ID NO:5) and FGGMRRK (SEQ ID NO:6), preferably wherein the peptide comprises the amino acid sequence FGGFMRRK (SEQ ID NO:3), preferably wherein the C-terminus of the peptide is substituted by NH2.

[0015] Another aspect provides a fusion protein comprising a peptide as taught herein.

[0016] Another aspect provides a nucleic acid encoding a peptide as taught herein or a fusion protein as taught herein.

[0017] Another aspect provides a nucleic acid expression cassette comprising a nucleic acid as taught herein operably linked to a promoter and / or transcriptional and translational regulatory signals.

[0018] Another aspect provides a vector, such as a viral vector, comprising a nucleic acid as taught herein or a nucleic acid expression cassette as taught herein.

[0019] Another aspect provides a pharmaceutical composition comprising a peptide as taught herein, a fusion peptide as taught herein, a nucleic acid as taught herein, a nucleic acid expression cassette as taught herein or a vector as taught herein, and optionally a pharmaceutically acceptable carrier.

[0020] On the other hand, a peptide as taught herein, a fusion protein as taught herein, a nucleic acid as taught herein, a nucleic acid expression cassette as taught herein, a vector as taught herein or a pharmaceutical composition as taught herein is provided for use as a medicament, preferably for treating a disease or condition in a subject, the disease or condition being selected from the group consisting of: adverse stress dysfunction diseases or conditions, cancer, atherosclerotic vascular disease, cardiovascular disease, fibrosis (e.g. cardiac fibrosis), inflammatory or autoimmune diseases and conditions, conditions of excessive or abnormal vascularization (e.g. wound healing and HIV infectivity), stem cell differentiation and mobilization disorders, brain and neuronal dysfunction (e.g. Alzheimer's disease, multiple sclerosis and demyelinating diseases), renal dysfunction, renal insufficiency, pre-eclampsia and obesity.

[0021] Another aspect provides a method for in vitro or ex vivo diagnosis, prediction, prognosis and / or monitoring of a disease or condition characterized by abnormal levels of an ACKR3 polypeptide, comprising the steps of:

[0022] - obtaining a biological sample from a subject,

[0023] - contacting said biological sample with a peptide as taught herein, wherein said peptide is fused to a detectable label,

[0024] - determining the level of ACKR3 polypeptide in said biological sample by detecting said peptide, and

[0025] - Diagnosing, predicting, prognosing and / or monitoring a disease or condition based on the levels of ACKR3 polypeptide.

[0026] Another aspect provides a therapeutic or prophylactic agent for treating an adverse stress dysfunction disease or condition in a subject, wherein the therapeutic or prophylactic agent is capable of modulating the recruitment of β-arrestin-1 and / or β-arrestin-2 to an atypical chemokine receptor 3 (ACKR3) polypeptide and not to any other receptor polypeptide.

[0027] Another aspect provides an in vitro method for identifying an agent useful as a therapeutic agent, the method comprising determining whether the test agent is capable of inducing recruitment of β-arrestin-1 and / or β-arrestin-2 to an ACKR3 polypeptide and is unable to induce recruitment of β-arrestin-1 and / or β-arrestin-2 to any other receptor polypeptide.

[0028] Another aspect provides an in vitro method for identifying an agent useful as a therapeutic agent, the method comprising determining whether the test agent is capable of inhibiting the recruitment of β-arrestin-1 and / or β-arrestin-2 to an ACKR3 polypeptide by a selective ACKR3 modulating peptide as taught herein.

[0029] Another aspect provides a kit for diagnosing, predicting, prognosing, and / or monitoring a disease or condition characterized by abnormal levels of an ACKR3 polypeptide in a subject, the kit comprising:

[0030] (a) a peptide as taught herein; and

[0031] (b) a reference value for the level of ACKR3 polypeptide, wherein the reference value represents a known diagnosis, prediction and / or prognosis of a disease or condition characterized by abnormal levels of ACKR3 polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Opioid peptide library screening, hit confirmation, and comparison with classic opioid receptors for ACKR3. (A) The ability of 58 compounds (including natural opioid peptides from four opioid families, their variants, and small molecule opioid receptor modulators) to induce β-arrestin-2 recruitment to ACKR3, CXCR4, and CXCR3 in U87 cells at a concentration of 5 μM. A positive control chemokine was used at a concentration of 300 nM. Results are expressed as the fold change for vehicle-treated cells and are expressed as the mean ± SD of two replicates. (BF) Comparison of the potency and efficacy of ACKR3 activating peptides in inducing β-arrestin-1 recruitment to opioid receptors MOR (B), DOR (C), KOR (D), NOP (E), and ACKR3 (F) in U87 cells. Results are expressed as the percentage of the indicated agonist response. (G) Competition of ACKR3-activating peptides with Alexa Fluor 647-labeled CXCL12 (5 nM) on U87-ACKR3 cells, as determined by flow cytometry. (H) Sequences of opioid peptides representing four families, along with their potency and efficacy in inducing β-arrestin-1 recruitment to ACKR3 or competing with Alexa Fluor 647-labeled CXCL12. Results for BH are presented as mean ± SEM (n ≥ 3).

[0033] Figure 2 Specific activation of ACKR3 by opioid peptides. The agonist activity of opioid peptides (3 μM) representing four opioid families against 21 classical and four atypical chemokine receptors was assessed in a β-arrestin-1 recruitment assay in U87 cells. For each receptor, 100 nM of a known agonist chemokine listed in the IUPHAR library of chemokine receptor ligands was added as a positive control. Results are expressed as fold change relative to vehicle and are presented as means (n ​​≥ 3).

[0034] Figure 3Structure-activity relationship analysis of adrenorphin variants at ACKR3 and classical opioid receptors. (A) Comparison of the effects of substitution, truncation, extension, D-amino acid replacement, or dimerization on the agonist activity of adrenorphin at ACKR3, MOR, DOR, KOR, and NOP. Agonist activity or each variant was assessed in a β-arrestin-1 recruitment assay in U87 cells and expressed as fold change relative to wild-type adrenorphin activity. (B, E) Comparison of the potency and efficacy of adrenorphin (B) and its variants bearing mutations Y1F (C), M5L (D), and R6A (E) in inducing β-arrestin-1 recruitment to ACKR3 and the opioid receptors KOR, MOR, DOR, and NOP in U87 cells. "~" indicates similar effects of the modification on the peptide's potency at ACKR3 and the indicated opioid receptors. Results represent mean ± SEM (n≥3) (F) Comparison of the potency of peptide variants combining mutant Y1F with other modifications (mutations and / or extensions) in inducing β-arrestin-1 recruitment to ACKR3. Modified amino acids relative to the parent peptide (Y1F adrenorphin FGGFMRRV) are underlined and in bold.

[0035] Figure 4Activity of Classical Opioid Modulators at ACKR3 and Development of LIH383 as a Subnanomolar ACKR3 Selective Agonist. (A) Agonist and antagonist activity of opioid modulators (10 μM, 1 μM, and 100 nM) commonly used for research purposes or clinically at ACKR3, and comparison with the opioid receptors MOR, DOR, KOR, and NOP monitored in a β-arrestin-1 recruitment assay. Antagonist activity was measured for MOR, DOR, KOR, NOP, and ACKR3 following the addition of BAM22 (50 nM), methionine enkephalin (70 nM), dynorphin A (50 nM), nociceptin (70 nM), and CXCL12 (4 nM). (B and E) Agonist activity of LIH383 against human (B) and mouse (E) ACKR3, as well as comparisons with other endogenous ACKR3 chemokine ligands and a control peptide (MRRKFGGF) consisting of eight amino acids arranged in different arrangements to construct LIH383. (C and D) LIH383 selectivity was assessed by comparing β-arrestin-1 recruitment to ACKR3 and opioid receptors MOR, DOR, KOR, and NOP (C) or all other known chemokine receptors (3 μM) (D). (F) Selective binding of fluorescently labeled LIH383 (LIH383-Cy5) to ACKR3-expressing cells. All assays were performed in U87 cells. Results represent mean ± SEM (n ≥ 3). (G) Competition of increasing concentrations of the ACKR3-activating chemokine, adrenorphin, or LIH383 with Alexa Fluor 647-labeled CXCL12 (5 nM) binding on U87-ACKR3 cells, as measured by flow cytometry. (H) Activation of the serum response element (SRE) associated with the ERK1 / 2 signaling cascade in U87 cells (-) or U87 expressing ACKR3, CXCR4, or the classical opioid receptors (MOR, DOR, KOR, or NOP) in response to LIH383 or a positive control (positive control, 30 nM PMA, 10% FBS). Bars for untreated cells (medium) serve as a baseline reference. (I) Dose-dependent antagonist activity of LIH383 (3000 nM–12.3 nM) against the classical opioid receptors DOR, MOR, KOR, and NOP, as monitored by β-arrestin-1 recruitment. For the DOR, MOR, and KOR, naloxone (1000 nM–4.1 nM) or buprenorphine (27000 nM–111 nM) was used as a positive control antagonist for NOP. Antagonist activity was measured for MOR, DOR, KOR, and NOP after the addition of BAM22 (50 nM), methionine enkephalin (70 nM), dynorphin A (50 nM), and nociceptin (70 nM). Results are expressed as mean ± SEM (n ≥ 3).

[0036] Figure 5 Absence of ACKR3 signaling in response to opioid and chemokine ligands. (A–D) DMR profiles of U87 cells or U87 cells expressing ACKR3, CXCR4, or classical opioid receptors (KOR and NOP) stimulated with the chemokine CXCL12 (200 nM) (A) or the opioid peptides dynorphin A (B), adrenorphin (C), and nociceptin 1-13 (500 nM). Left panel: Representative DMR profiles determined over 3000 seconds. Right panel: Area under the curve (AUC) ± SEM from at least three independent experiments. (E) Comparison of small Gi recruitment to ACKR3, CXCR4, and classical opioid receptors (MOR, DOR, KOR, or NOP) monitored in U87 cells in response to CXCL12 and opioid peptides. (F) Kinetic analysis of ERK1 / 2 phosphorylation in U87 cells stably expressing ACKR3 or CXCR4 stimulated by CXCL12 and opioid peptides. EGF was used as a positive control. (G) Expression of SRE (ERK1 / 2) and NFAT-RE (Ca2+) in U87 cells (-) or expressing ACKR3, CXCR4, or classical opioid receptors (MOR, DOR, KOR, or NOP) in response to chemokines CXCL12 and CXCL11 (200 nM) or opioid peptides (500 nM) or positive controls (30 nM PMA, 10% FBS for SRE and 30 nM PMA, 1 μM ionomycin, 10% FBS for NFAT-RE). 2+ ) Comparison of activation of signaling cascades. Results represent mean ± SEM (n≥3).

[0037] Figure 6ACKR3 exhibits efficient uptake of various opioid peptides and atypical localization and trafficking properties compared to classical opioid receptors. (A) Uptake of fluorescently labeled dynorphin A (1-13) by cells expressing ACKR3 was visualized by imaging flow cytometry. U87, U87-ACKR3, or U87-ACKR3 cells pretreated with LIH383 (3 μM) were stimulated with 250 nM (FAM)-labeled dynorphin A (1-13) for 40 minutes at 37°C. Dead cells were excluded using a viability dye. For each condition, the percentage of cells with a given number of distinguishable vesicle-like structures (puncta) was determined, as was the geometric mean fluorescence intensity (MFI) of the green channel (FAM labeling). Data shown are representative of three independent experiments. (B) Uptake of fluorescently labeled opioid peptides (dynorphin A(1-13)-FAM (250 nM), big dynorphin-Cy5 (400 nM), BAM22-Cy5 (400 nM), or nociceptin-FAM (1 μM)) representing different families (dynorphin, enkephalin, or nociceptin) by U87 cells (NT) or U87 cells transfected with ACKR3 or the corresponding preferred classical opioid receptors (KOR, MOR, or NOP) analyzed by imaging flow cytometry as described in (A). (C) ACKR3-mediated depletion of extracellular dynorphin A was monitored by its ability to activate KOR. U87 or U87-ACKR3 cells pretreated for 15 minutes with LIH383 (400 nM) or a control peptide (LIH383 control) (left panel) and CXCL12 (200 nM) or the negative control chemokine CXCL10 (right panel) were incubated with dynorphin A for 25 minutes. The cell supernatant was then added to U87 cells expressing SmBiT-tagged KOR and LgBiT-β-arrestin-1 (left panel) or LgBiT-miniGi (right panel). (Inset): EC expressed as a bar graph 50(D) Opioid peptide-induced delivery of receptor inhibitory proteins to endosomes was monitored by β-galactosidase complementation assay in U2OS cells stably expressing ACKR3. (E-G) Kinetics of opioid peptide-induced internalization of ACKR3 and classical opioid receptors monitored by HiBiT technology (E and G) and flow cytometry (F). (E) U87 cells expressing N-terminally HiBiT-tagged receptors were stimulated with opioid peptides including dynorphin A, dynorphin B, big dynorphin, dynorphin A1-13, adrenorphin, BAM22, methionine enkephalin, endomorphin 1, endomorphin 2, and β-endorphin (1 μM) or CXCL12 (300 nM) for the indicated times, and the remaining membrane receptors were quantified using soluble LgBiT protein. (F) ACKR3 and KOR internalization and recycling profiles in U87 cells following ligand stimulation (1 μM for opioid peptides including dynorphin A, dynorphin B, big dynorphin, dynorphin A1-13, adrenorphin, BAM22, methionine enkephalin, nociceptin, nociceptin 1-13, and FG nociceptin 1-13, and 300 nM for CXCL12) monitored by flow cytometry followed by acid wash. (G) Effects of bafilomycin A1 (1.5 μM) on endosomal trafficking / recycling of ACKR3 and KOR after 180 minutes of stimulation with various opioid peptides (1 μM), as monitored by HiBiT technology. Results are expressed as mean (A) or mean ± SEM (B, G) (n ≥ 3). *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA with Bonferroni correction (B), by two-way ANOVA: interaction between cell line and ligand treatment with Tukey post hoc test (C), and by two-tailed unpaired t-test (G).

[0038] Figure 7ACKR3-mediated regulation of opioid peptide availability by classical receptors in neural progenitor cells and brain opioid centers. (AC) Relative gene expression of ACKR3 and classical opioid receptors in smNPCs and different brain regions corresponding to centers important for opioid peptide activity. (A) RNA-Seq RPKM (reads per kilobase per million) values ​​from an open-source database (brainspan.org), containing data from 16–22 donors. (B and C) mRNA expression determined by qPCR in five adult brains (B) or two smNPC samples (C), normalized to the arithmetic mean of PPIA and GAPDH, a stable housekeeping gene. (D) Extracellular and intracellular expression of ACKR3 monitored by flow cytometry using an ACKR3-specific mAb (11G8) or a matched isotype control (MG1-45) and a PE-conjugated secondary antibody compared to unstained cells. (E) Uptake of fluorescently labeled dynorphin A (1-13) by smNPCs assessed by imaging flow cytometry. smNPCs pretreated with LIH383 or LIH383 control (3 μM) for 15 minutes were incubated with 250 nM (FAM)-labeled dynorphin A (1-13) for 40 minutes at 37°C and analyzed by imaging flow cytometry. Results represent mean ± SEM (n ≥ 3). (F) Activation of the SRE (ERK1 / 2) signaling cascade in smNPCs in response to various opioid peptides (500 nM) or 10% FBS as a positive control. Results represent mean ± SEM (n ≥ 3). (G) ACKR3-mediated depletion of extracellular dynorphin A monitored by its ability to activate KOR. smNPCs pretreated for 15 minutes with LIH383, LIH control (1.5 μM), CXCL12, or CXCL10 (300 nM) were incubated with dynorphin A (3 μM) for 4 hours. Residual dynorphin A activity in cell supernatants was probed in U87 cells expressing SmBiT-labeled KOR and LgBiT-labeled small Gi. Representative 30x supernatant dilutions are shown. Results are expressed as mean ± SEM (n ≥ 3). (H and I) Inhibition of isolated rat locus coeruleus neuronal depolarization induced by LIH383 alone, dynorphin A in the presence or absence of naloxone (H), or increasing concentrations of dynorphin A in the presence or absence of LIH383 (I). Data and EC 50 Values ​​(inset) are expressed as mean ± SEM and are based on six independent depolarization experiments for each condition. *p < 0.05, **p < 0.01 by one-way ANOVA with Bonferroni correction (E and G) and Kruskal-Wallis by Dunn's test (I).

[0039] illustrate

[0040] As used herein, the singular forms "a," "an," and "the" include singular and plural referents unless the context clearly dictates otherwise.

[0041] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," "contains," and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps. These terms also include "consisting of" and "essentially consisting of," which have recognized meanings in patent terminology.

[0042] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the corresponding ranges, as well as the recited endpoints.

[0043] As used herein, the terms "about" or "approximately" when referring to a measurable value such as a parameter, an amount, a duration of time, etc., are intended to encompass variations from the specified value and variations therefrom, for example, variations of + / - 10% or less, preferably + / - 5% or less, more preferably + / - 1% or less, more preferably + / - 0.1% or less, to the extent such variations are suitable for performing the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically and preferably disclosed.

[0044] Although the term "one or more" or "at least one," e.g., one or more members or at least one member of a group of members, is self-explanatory, by way of further illustration, the term specifically includes reference to any one of the members, or to any two or more of the members, e.g., any ≥3, ≥4, ≥5, ≥6, or ≥7 of the members, etc., and up to all of the members. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7, or more.

[0045] This discussion of the background to the invention is included to explain the context of the invention. This should not be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in any country as at the priority date of any claim.

[0046] In this disclosure, various publications, patents, and published patent specifications are cited by identifying citations. All documents cited in this specification are incorporated herein by reference in their entirety. In particular, the teachings or sections of such documents specifically mentioned herein are incorporated by reference.

[0047] Unless otherwise defined, all terms, including technical and scientific terms, used to disclose the present invention have the meanings commonly understood by those of ordinary skill in the art to which the invention belongs. By way of further guidance, term definitions are included to better understand the teachings of the present invention. When a particular term is defined in connection with a particular aspect of the invention or a specific embodiment of the invention, unless otherwise defined, such meaning is intended to apply throughout the specification, i.e., also in the context of other aspects or embodiments of the invention.

[0048] In the following paragraphs, different aspects or embodiments of the present invention are defined in more detail. Unless explicitly stated to the contrary, each aspect or embodiment so defined may be combined with any other aspect or embodiment. In particular, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous.

[0049] References in this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner, as will be apparent to those skilled in the art from this disclosure. Furthermore, although some embodiments described herein include some features that are not included in other features in other embodiments, the combination of features from different embodiments is intended to be within the scope of the present invention and to form different embodiments, as will be understood by those skilled in the art. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0050] The present inventors identified the atypical chemokine receptor ACKR3 (also known as CXCR7) as a novel key regulator of the opioid system.

[0051] More specifically, the present inventors discovered that ACKR3 is abundantly expressed in the brain along with classical opioid receptors, and demonstrated that, in addition to BAM22 (Ikeda et al., 2013, Modulation of circadian glucocorticoid oscillation through adrenal opioid-CXCR7 signaling alters emotional behavior, Cell. 155(6):1323-1336), ACKR3 binds to a large number of other endogenous opioid peptides found in the central nervous system (CNS) and immune cells, including those from the enkephalin, dynorphin, and nociceptin families. This broad spectrum of selectivity is atypical and unique among opioid receptors. Furthermore, the present inventors found that, contrary to known opioid receptors and those proposed by Ikeda et al. (Ikeda et al., 2013, Modulation of circadian glucocorticoid oscillation through adrenal opioid-CXCR7 signaling alters emotional behavior, Cell. 155(6): 1323-1336), ACKR3 is unable to activate downstream signaling pathways such as through G proteins or β-arrestins in response to endogenous opioid peptides, is present in different cellular compartments and acts as a scavenger, regulating their local and / or systemic concentrations, thereby regulating the availability of classical opioid receptors.

[0052] The present inventors discovered that ACKR3 acts as a scavenger for this family of neuromodulators, regulating their availability to signaling opioid receptors. ACKR3 thus serves as a new broad-spectrum receptor for opioid peptides. More specifically, the present inventors demonstrated in an in vitro rat model that blocking ACKR3 increased the availability and signaling induced by opioid peptides through classical receptors. The present inventors also demonstrated that inhibition of neuronal firing could not be achieved through specific activation of ACKR3, but only through activation of classical opioid receptors, and that neutralization of the scavenging capacity of ACKR3 clearly demonstrated the enhanced efficacy of dynorphin A at its classical receptors. Opioid peptide scavenging was further confirmed in neural precursor cells (smNPCs) and U87 cells.

[0053] Therefore, in the treatment of diseases associated with endogenous opioid peptide dysregulation (such as adverse stress dysfunction diseases or conditions), such as depression or chronic pain, ACKR3 can be used to regulate and / or restore normal levels of endogenous opioid peptides with potentially improved safety. To this end, the present inventors developed a novel antagonist with the consensus sequence FGGX1MRRX2 (SEQ ID NO:1), wherein X1 is F or W, and X2 is K, V or F, preferably having a length of up to 15 amino acids, which has high affinity and selectivity for ACKR3, and for μ (μ) type opioid receptor (MOR), δ (δ) type opioid receptor (DOR), κ (κ) type opioid receptor (KOR) and non-classical nociceptive peptide receptor (NOP), or CC chemokine receptor type 1 (CCR1), CC chemokine receptor type 2A (CCR2A), CC chemokine receptor type 2B (CCR2B), CC chemokine receptor type 3 (CCR3), CC chemokine receptor type 4 (CCR4), CC chemokine receptor type 5 (CCR5), CC chemokine receptor type 6 (CCR6), CC chemokine receptor type 7 (CCR7), CC chemokine receptor type 8 (CCR8), CC chemokine receptor type 9 (CCR9), CXC motif chemokine receptor 1 (CXCR1), atypical chemokine receptor 2 (ACKR2), atypical chemokine receptor 4 (ACKR4), CXC motif chemokine receptor 5 (CXCR5), CXC motif chemokine receptor 6 (CXCR6), CXC motif chemokine receptor 8 (CXCR8)), XC motif chemokine receptor 1 (XCR1), C-X3-C motif chemokine receptor 1 (CX3CR1), atypical chemokine receptor 1 (ACKR1), atypical chemokine receptor 2 (ACKR2), and atypical chemokine receptor 4 (ACKR4). Furthermore, novel peptides with high affinity and selectivity for ACKR3 can act as ACKR3 agonists and can induce the recruitment of β-arrestin-1 and / or β-arrestin-2 to ACKR3.

[0054] Furthermore, when the peptides as taught herein have a length of up to 15 amino acids, these peptides take advantage of low production costs, which is very important to remain competitive and allow for use by the majority of patients.

[0055] Thus, a first aspect provides a peptide comprising, consisting essentially of or consisting of the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is F or W, X2 is K, V or F, and optionally, wherein the peptide has a length of up to 15 amino acids.

[0056] The term "peptide" as used throughout this specification preferably refers to a polypeptide as used herein that essentially consists of 50 amino acids or less, such as 45 amino acids or less, preferably 40 amino acids or less, such as 35 amino acids or less, more preferably 30 amino acids or less, such as 25 or less, 20 or less, 15 or less, 10 or less or 5 or less amino acids.

[0057] In particular embodiments, the peptide is non-naturally occurring, eg, not found in nature or isolated from nature (eg, naturally or endogenously produced or expressed by and optionally isolated from a cell or tissue).

[0058] In certain embodiments, the peptide is recombinant, i.e., produced by recombinant DNA technology, and / or may be partially or completely chemically or biochemically synthesized. Without limitation, the peptide may be recombinantly produced by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or recombinantly produced by cell-free translation or cell-free transcription and translation, or abiotic peptide synthesis.

[0059] In a specific embodiment, the peptide consists of 20 amino acids or less, 15 amino acids or less, such as 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, preferably 10 amino acids or less, such as 9 amino acids or 8 amino acids. In a specific embodiment, the peptide consists of 8 to 20 amino acids, 8 to 15 amino acids, 8 to 14 amino acids, 8 to 13 amino acids, 8 to 12 amino acids, 8 to 11 amino acids, 8 to 10 amino acids, or 8 or 9 amino acids, preferably 8 to 15 amino acids, more preferably 8 to 10 amino acids.

[0060] In a specific embodiment, the peptide comprises, consists essentially of, or consists of the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is F or W, preferably F, and X2 is K or V, preferably K.

[0061] In a preferred embodiment, when X2 is located at the C-terminus of the peptide, X2 of SEQ ID NO: 1 is NH2 substituted.

[0062] In a specific embodiment, the peptide comprises, consists essentially of, or consists of the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is W and X2 is K or V, preferably K.

[0063] In a specific embodiment, the peptide comprises, consists essentially of, or consists of the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is F and X2 is K or V, preferably K.

[0064] In a specific embodiment, if X2 is V, the peptide has a length of at most 15 amino acids.

[0065] In a specific embodiment, a peptide as taught herein is not a homoserine O-acetyltransferase from Chromohalobacter salexigens as annotated under NCBI Genbank Accession No. WP_110061560.1.

[0066] In a specific embodiment, the peptide as taught herein is not BAM-22, wherein the N-terminal tyrosine (Y) is replaced by phenylalanine (F), as described by Iked et al. (Ikeda et al., 2013, Modulation of circadianglucocorticoid oscillation through adrenal opioid-CXCR7 signaling altersemotional behavior, Cell. 155(6): 1323-1336).

[0067] In a specific embodiment, the peptide comprises, consists essentially of, or consists of the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is F.

[0068] In a specific embodiment, the peptide comprises, consists essentially of, or consists of the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is W.

[0069] In a specific embodiment, the peptide comprises, consists essentially of, or consists of the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X2 is K.

[0070] The present inventors have found that in particular a peptide comprising, consisting essentially of or consisting of, preferably consisting of, the amino acid sequence FGGFMRRK (SEQ ID NO: 3; NH2-substituted at the C-terminus) (also referred to herein as "LIH383") is a highly potent (i.e. EC in the subnanomolar range) inhibitor of ACKR3. 50) and selective peptide modulators, even 20 times more potent than known ACKR3 agonists, such as compound 18 described in Menhaji-Klotz et al., Discovery of a novel small-molecule modulator of C-XC chemokine receptor type 7 as a treatment of cardiac fibrosis, J. Med. Chem, 2018, 61(8): 3685-3696, which has an EC of 11 nM 50 .

[0071] Thus, in a preferred embodiment, the peptide comprises, consists essentially of or consists of the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is F and X2 is K. In other words, in a preferred embodiment, the peptide comprises, consists essentially of or consists of the amino acid sequence FGGFMRRK (SEQ ID NO: 3), and in an even more preferred embodiment, the peptide comprises, consists essentially of or consists of the amino acid sequence FGGFMRRK (SEQ ID NO: 3; NH2-substituted at the C-terminus).

[0072] In a specific embodiment, the peptide comprises at least one (e.g., one, two, three or four, preferably one or two, more preferably one) amino acid at the C-terminus of the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is F or W and X2 is K, V or F. In other words, in a specific embodiment, the peptide comprises, consists essentially of or consists of, preferably consists of the following amino acid sequence: FGGX1MRRX2X3 (SEQ ID NO: 2) or FGGX1MRRX2X3X4 (SEQ ID NO: 7), FGGX1MRRX2X3X4X5 (SEQ ID NO: 8), FGGX1MRRX2X3X4X5X6 (SEQ ID NO: 9), wherein X1 is F or W, X2 is K, V or F and X3, X4, X5 and X6 can be any amino acid. In a preferred embodiment, when X3, X4, X5 or X6 is located at the C-terminus of the peptide, respectively, X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 7, X5 of SEQ ID NO: 8 or X6 of SEQ ID NO: 9 is substituted with NH2. In a specific embodiment, the peptide comprises, consists essentially of or consists of the following amino acid sequence: FGGX1MRRX2X3 (SEQ ID NO: 2), wherein X1 is F or W, X2 is K, V or F, and X3 is R or A.

[0073] In a specific embodiment, at the C-terminus of the amino acid sequence FGGX1MRRX2 (SEQ ID NO: 1), the peptide comprises the amino acid sequence G, GR, GRP, GRPE (SEQ ID NO: 73), GRPEW (SEQ ID NO: 74), GRPEWW (SEQ ID NO: 75) or GRPEWWM (SEQ ID NO: 76), wherein X1 is F or W, and X2 is K, V or F.

[0074] In a specific embodiment, the peptide comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of FGGFMRRK (SEQ ID NO:3), FGGFMRRKR (SEQ ID NO:4), FGGFMRRVR (SEQ ID NO:5), FGGWMRRK (SEQ ID NO:6), FGGWMRRVR (SEQ ID NO:10), FGGWMRRKR (SEQ ID NO:11), FGGWMRRV (SEQ ID NO:68), FGGFMRRF (SEQ ID NO:69), FGGFMRRFR (SEQ ID NO:70), FGGWMRRFR (SEQ ID NO:71), or FGGWMRRF (SEQ ID NO:72), preferably selected from the group consisting of FGGFMRRK (SEQ ID NO:3), FGGFMRRKR (SEQ ID NO:4), FGGFMRRVR (SEQ ID NO:5), FGGWMRRK (SEQ ID NO:6), FGGWMRRVR (SEQ ID NO:10), FGGWMRRKR (SEQ ID NO:11), FGGWMRRV (SEQ ID NO:68), FGGFMRRF (SEQ ID NO:69), FGGFMRRFR (SEQ ID NO:70), FGGWMRRFR (SEQ ID NO:71), or FGGWMRRF (SEQ ID NO:72). NO:10) or FGGWMRRKR (SEQ ID NO:11), more preferably FGGFMRRK (SEQ ID NO:3), FGGFMRRKR (SEQ ID NO:4), FGGFMRRVR (SEQ ID NO:5), FGGWMRRK (SEQ ID NO:6), even more preferably FGGFMRRK (SEQ ID NO:3).

[0075] In a preferred embodiment, the C-terminus of a peptide as taught herein is NH2-substituted. If an NH2-substituted form of the C-terminus of a peptide as taught herein is contemplated, the peptide will be represented by its SEQ ID NO followed by the phrase "NH2-substituted at the C-terminus." For example, the sequence of LIH383 would be represented as "(SEQ ID NO: 3; NH2-substituted at the C-terminus)."

[0076] Thus, in a specific embodiment, the peptide comprises, consists essentially of, or consists of, preferably consists of, an amino acid sequence selected from the group consisting of: FGGFMRRK (SEQ ID NO: 3; NH2-substituted at the C-terminus) wherein the C-terminal K is NH2-substituted, FGGFMRRKR (SEQ ID NO: 4; NH2-substituted at the C-terminus) wherein the C-terminal R is NH2-substituted, FGGFMRRVR (SEQ ID NO: 5; NH2-substituted at the C-terminus) wherein the C-terminal R is NH2-substituted, FGGWMRRK (SEQ ID NO: 6; NH2-substituted at the C-terminus) wherein the C-terminal K is NH2-substituted, FGGWMRRVR (SEQ ID NO: 10; NH2-substituted at the C-terminus) wherein the C-terminal R is NH2-substituted, FGGWMRRKR (SEQ ID NO: 7; NH2-substituted at the C-terminus) wherein the C-terminal K is NH2-substituted, NO: 11; NH2-substituted at the C-terminus) wherein the C-terminal R is NH2-substituted, FGGWMRRV (SEQ ID NO: 68; NH2-substituted at the C-terminus) wherein the C-terminal V is NH2-substituted, FGGFMRRF (SEQ ID NO: 69; NH2-substituted at the C-terminus) wherein the C-terminal F is NH2-substituted, FGGFMRRFR (SEQ ID NO: 70; NH2-substituted at the C-terminus) wherein the C-terminal R is NH2-substituted, FGGWMRRFR (SEQ ID NO: 71; NH2-substituted at the C-terminus) wherein the C-terminal R is NH2-substituted or FGGWMRRF (SEQ ID NO: 72; NH2-substituted at the C-terminus) wherein the C-terminal F is NH2-substituted, preferably selected from the group consisting of: FGGFMRRK (SEQ ID NO:3; NH2-substituted at the C-terminus) wherein the C-terminal K is NH2-substituted, FGGFMRKR (SEQ ID NO:4; NH2-substituted at the C-terminus) wherein the C-terminal R is NH2-substituted, FGGFMRRVR (SEQ ID NO:5; NH2-substituted at the C-terminus) wherein the C-terminal R is NH2-substituted, FGGWMRRK (SEQ ID NO:6; NH2-substituted at the C-terminus) wherein the C-terminal K is NH2-substituted, FGGWMRRVR (SEQ ID NO:10; NH2-substituted at the C-terminus) wherein the C-terminal R is NH2-substituted or FGGWMRRKR (SEQ ID NO:11; NH2-substituted at the C-terminus) wherein the C-terminal R is NH2-substituted, more preferably FGGFMRRK (SEQ ID NO:3;NH2-substituted at the C-terminus) wherein the C-terminal K is NH2-substituted, FGGFMRRKR (SEQ ID NO: 4; NH2-substituted at the C-terminus) wherein the C-terminal K is NH2-substituted, FGGFMRRVR (SEQ ID NO: 5; NH2-substituted at the C-terminus) wherein the C-terminal R is NH2-substituted, FGGWMRRK (SEQ ID NO: 6; NH2-substituted at the C-terminus) wherein the C-terminal K is NH2-substituted, and even more preferably FGGFMRRK (SEQ ID NO: 3; NH2-substituted at the C-terminus) wherein the C-terminal K is NH2-substituted. In certain embodiments, the peptide is capable of specifically binding to ACKR3.

[0077] As used throughout this specification, the terms "bind," "interact," "specifically bind," or "specifically interact" refer to an agent that binds to or affects one or more desired molecules or analytes to the substantial exclusion of random or unrelated other molecules, and optionally to the substantial exclusion of structurally related other molecules. These terms do not necessarily require that an agent binds exclusively to its intended target. For example, an agent may be considered to be a target if its affinity for such intended target under binding conditions is at least about 2-fold greater, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, still more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, even more preferably at least about 100-fold greater or more, e.g., at least about 1000-fold greater or more, at least about 1 x 10 4 times or more, or at least about 1x10 5 fold or more, then the agent can be said to specifically bind to the target.

[0078] The binding or interaction between the agent and its intended target can be covalent (i.e., mediated by one or more chemical bonds involving the sharing of electron pairs between atoms) or, more generally, non-covalent (i.e., mediated by non-covalent forces, such as hydrogen bridges, dipole interactions, van der Waals interactions, etc.). Preferably, the agent can bind to the target in such a manner that the binding K A ≥1×10 6 M -1 , more preferably K A ≥1×10 7 M -1 , and more preferably K A ≥1×10 8 M -1 , even more preferably K A ≥1×10 9 M -1 , and more preferably K A ≥1×10 10 M -1 or K A ≥1×1011 M -1 The affinity constant (KA) of a protein binds or interacts with its intended target, where K A =[A_T] / [A][T], A represents the drug and T represents the expected target. K A The determination of can be performed by methods known in the art, for example, using equilibrium dialysis and Scatchard plot analysis.

[0079] A peptide is said to "specifically bind" to a particular target when it has affinity for, specificity for, and / or is specific for a target (ie, for at least a portion or fragment thereof).

[0080] The "specificity" of a peptide as taught herein can be determined based on affinity. The "affinity" of a polypeptide is represented by the equilibrium constant for dissociation between the peptide and ACKR3, preferably human ACKR3 (e.g., annotated under NCBI Genbank Accession No. NP_064707.1). The lower the KD value, the stronger the binding strength between the peptide and ACKR3. Alternatively, affinity can also be represented by an affinity constant (KA), which corresponds to 1 / KD. KD values ​​greater than about 1 millimolar are generally considered to indicate non-binding or non-specific binding.

[0081] The binding of an agent (e.g., a peptide as described herein) to a target and the affinity and specificity of the binding can be determined by any method known in the art. Non-limiting examples include binding competition assays using fluorescently labeled or radioactively labeled ligands (e.g., fluorescently labeled or radioactively labeled chemokines, such as CXCL12), co-immunoprecipitation, bimolecular fluorescence complementation, affinity electrophoresis, label transfer, phage display, proximity ligation assays (PLA), tandem affinity purification (TAP), in silico docking and calculation of the predicted Gibbs binding energy, and competitive binding assays.

[0082] The present inventors have discovered that peptides as taught herein have the ability to recruit beta-arrestin-1 and beta-arrestin-2 to the ACKR3 receptor when used at nanomolar or even sub-nanomolar concentrations.

[0083] In a specific embodiment, the peptides as taught herein have potency against ACKR3, characterized by EC 50An EC of 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.95 nM or less, 0.90 nM or less, 0.85 nM or less, 0.80 nM or less, 0.75 nM or less, 0.70 nM or less or 0.65 nM or less, preferably 5 nM or less 50 , more preferably an EC of 1 nM or less 50 For example, the peptides taught herein have an EC of 0.61 nM for ACKR3. 50 EC in the context of the present invention 50 Is determined based on a β-arrestin recruitment assay. β-arrestin recruitment can be determined by any method known in the art, such as by a nanoluciferase complementation assay (e.g., NanoBiT, Promega), for example using ACKR3 fused to the C-terminus with SmBiT and β-arrestin fused to the N-terminus with LgBiT.

[0084] In certain embodiments, the peptides as taught herein are capable of binding to the binding pocket of ACKR3.

[0085] In certain embodiments, the peptides as taught herein inhibit, reduce and / or prevent the interaction between ACKR3 and an endogenous or exogenous ligand for ACKR3, such as an endogenous opioid peptide (e.g., BAM-22), an endogenous chemokine (e.g., CXCL12 or CXCL11), or an exogenous opioid peptide.

[0086] In certain embodiments, the peptides as taught herein inhibit, reduce and / or prevent the interaction between ACKR3 and endogenous opioid peptides, such as those derived from proenkephalin, prodynorphin, proopiomelanocortin or prepronociceptin.

[0087] Preferably, the endogenous opioid peptide is selected from the group consisting of BAM-22, BAM-18, peptide E, adrenorphin, dynorphin A or fragments thereof (eg dynorphin 1-13, dynorphin 2-17), dynorphin B, big dynorphin or fragments thereof, nociceptin or fragments thereof.

[0088] In specific embodiments, the peptides as taught herein inhibit, reduce and / or prevent the interaction between ACKR3 and an endogenous chemokine selected from the group consisting of CXCL12 (eg having Uniprot Accession No. P48061) and CXCL11 (eg having Uniprot Accession No. 014625).

[0089] The inhibition, reduction and / or prevention of the interaction between ACKR3 and endogenous ACKR3 ligands by the peptides as taught herein can be determined by any means known in the art, such as competitive binding assays or displacement assays. In a specific embodiment, the peptide is capable of displacing labeled CXCL12 at a concentration of less than 1 μM, more particularly less than 100 nM or less than 10 nM. As previously mentioned, the present inventors have found that, contrary to known opioid receptors and those proposed by Ikeda et al. (Ikeda et al., 2013, Modulation of circadian glucocorticoid oscillation through adrenal opioid-CXCR7 signaling alters emotional behavior, Cell. 155(6): 1323-1336), ACKR3 is unable to activate downstream signaling pathways, such as through G proteins or β-arrestins, in response to endogenous opioid peptides, but instead acts as a scavenger, regulating their local and / or systemic concentrations, thereby regulating the availability of classical opioid receptors. The absence or presence of downstream signaling pathway activation can be determined using methods known in the art, such as whole-cell biosensing methods based on dynamic mass redistribution, determining the recruitment of small G proteins to receptors, determining the phosphorylation level of ERK1 / 2, and determining the phosphorylation of SRE (ERK1 / 2) and NFAT-RE (Ca 2+ )Activation of signaling cascades.

[0090] Thus, consistent with the inventors' findings that ACKR3 acts as a scavenger in response to endogenous opioid peptides, in certain embodiments, the peptides as taught herein do not induce G protein-mediated signaling mediated by ACKR3. The presence or absence of G protein-mediated signaling can be determined using methods known in the art, such as determining the recruitment of small G proteins to receptors, determining the phosphorylation level of ERK1 / 2, whole-cell biosensing methods based on dynamic mass redistribution, and determining the expression of SRE (ERK1 / 2) and NFAT-RE (Ca 2+ )Activation of signaling cascades.

[0091] In more specific embodiments, the peptides taught do not induce small G proteins (mG) (e.g., G αs , G αi / o , G αq / 11 and / or G α12 / 13) to ACKR3. The recruitment of mG to ACKR3 (or its absence) can be determined by any established analytical technique for determining protein-protein binding, such as co-immunoprecipitation, bimolecular fluorescence complementation, label transfer, tandem affinity purification, chemical cross-linking, fluorescence resonance energy transfer, and nanoluciferase complementation assays (e.g., NanoBiT, Promega), for example, using ACKR3 fused to SmBiT at the C-terminus and mG fused to LgBiT at the N-terminus. Protein binding assays can be performed in cell-free systems or cell lysates or in isolated or cultured cells or in isolated or cultured tissues.

[0092] In specific embodiments, the peptides as taught herein do not activate any signaling pathways (e.g., cAMP signaling and / or MAPK / ERK signaling pathways) as a result of recruiting β-arrestin- and / or β-arrestin-2 to the ACKR3 receptor.

[0093] In a specific embodiment, the peptides as taught herein cannot interact with and / or activate μ (μ)-type opioid receptors (MOR), δ (δ)-type opioid receptors (DOR), κ (κ)-type opioid receptors (KOR) and non-classical nociceptive peptide receptors (NOP). In a more specific embodiment, the peptides as taught herein do not reduce the recruitment of beta-arrestin 1 and beta-arrestin 2 to MOR, DOR, KOR and / or NOP receptors induced by known ligands of MOR, DOR, KOR and / or NOP receptors, respectively. In a more specific embodiment, the peptides as taught herein do not induce G protein-mediated signal transduction by MOR, DOR, KOR and / or NOP receptors. In a more specific embodiment, the peptides as taught herein cannot induce the recruitment of beta-arrestin 1 and beta-arrestin 2 to MOR, DOR, KOR and / or NOP receptors. The absence of G protein-mediated signaling can be determined by any method known in the art, such as determining the level of phosphorylation of ERK1 / 2 upon contacting the agent with ACKR3, wherein the absence of phosphorylated ERK1 / 2 indicates the absence of G-protein-mediated signaling.

[0094] In a specific embodiment, the peptides as disclosed herein cannot induce the recruitment of β-arrestin-1 and β-arrestin-2 to MOR, DOR, KOR or NOP receptors. In a more specific embodiment, the peptides as disclosed herein do not enhance or even reduce the recruitment of β-arrestin-1 or β-arrestin-2 to MOR, DOR, KOR or NOP receptors compared to the baseline β-arrestin-1 or β-arrestin-2 recruitment or background β-arrestin-1 or β-arrestin-2 recruitment induced by a neutral substance or a negative control. As described elsewhere herein, the recruitment of β-arrestin-1 and β-arrestin-2 to MOR, DOR, KOR or NOP receptors can be measured by nano-luciferase complementation assays.

[0095] Any existing, available or conventional separation, detection and quantification methods can be used herein to measure the presence or absence (e.g., readout of presence vs. absence; or detectable vs. undetectable amount) and / or quantity (e.g., readout of absolute or relative amount, such as absolute or relative concentration) of a peptide, polypeptide, protein in a sample. For example, such methods can include biochemical assays, immunoassays, mass spectrometry, or chromatography, or a combination thereof.

[0096] In a particular embodiment, the peptides as taught herein are not able to induce the recruitment of β-arrestin-1 and β-arrestin-2 to any other chemokine receptors than ACKR3, more particularly a chemokine receptor selected from the group consisting of: CC chemokine receptor type 1 (CCR1) (e.g. UniProt accession number P32246), CC chemokine receptor type 2 (CCR2) (e.g. UniProt accession number P41597) such as CCR2A type (CCR2A) or CCR2B type (CCR2B), CC chemokine receptor type 3 (CCR3) (e.g. UniProt accession number P516 77), CC chemokine receptor type 4 (CCR4) (e.g. UniProt accession number P51679), CC chemokine receptor type 5 (CCR5) (e.g. UniProt accession number P51681), CC chemokine receptor type 6 (CCR6) (e.g. UniProt accession number P51684), CC chemokine receptor type 7 (CCR7) (e.g. UniProt accession number P32248), CC chemokine receptor type 8 (CCR8) (e.g. UniProt accession number P51685), CC chemokine receptor type 9 (CCR9) (e.g. UniProt accession number P51 686), CC chemokine receptor type 10 (CCR10) (e.g. UniProt accession number P46092), CXC motif chemokine receptor 1 (CXCR1) (e.g. UniProt accession number P25024), CXC motif chemokine receptor 2 (CXCR2) e.g. having UniProt accession number P25025), CXC motif chemokine receptor 3 (CXCR3) (e.g. having UniProt accession number P49682) e.g. CXCR3A type (CXCR3A) and CXCR3B type (CXCR3B), CXC motif chemokine receptor 4 (CXCR4) (e.g. having UniProt accession number P49683) e.g. such as UniProt accession number P61073), CXC motif chemokine receptor 5 (CXCR5) (such as UniProt accession number P32302), CXC motif chemokine receptor 6 (CXCR6) (such as UniProt accession number O00574), CXC motif chemokine receptor 8 (CXCR8) (such as UniProt accession number Q9HC97), XC motif chemokine receptor 1 (XCR1) (such as UniProt accession number P46094), C-X3-C motif chemokine receptor 1 (CX3CR1) (such as UniProt accession number P49238),Atypical chemokine receptor 1 (ACKR1) (e.g. UniProt accession number Q16570), atypical chemokine receptor 2 (ACKR2) (e.g. UniProt accession number O00590) and atypical chemokine receptor 4 (ACKR4) (e.g. UniProt accession number Q9NPB9).

[0097] In specific embodiments, the peptides as disclosed herein are unable to induce recruitment of beta-arrestin-1 and beta-arrestin-2 to the CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, or ACKR4 receptors. In an even more specific embodiment, the peptides as disclosed herein do not enhance or reduce the recruitment of β-arrestin-1 or β-arrestin-2 to the CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2 or ACKR4 receptors compared to baseline β-arrestin-1 or β-arrestin-2 recruitment or background β-arrestin-1 or β-arrestin-2 recruitment induced by a neutral substance or a negative control. As described elsewhere herein, recruitment of β-arrestin-1 and β-arrestin-2 to CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, or ACKR4 receptors can be measured by nanoluciferase complementation assays.

[0098] By way of additional guidance, atypical chemokine receptor 3 (ACKR3) is also referred to in the art as chemokine receptor 7 (CXCR7). As an example, human ACKR3 mRNA is annotated under NCBI Genbank accession number NM_020311.2. Human ACKR3 polypeptide is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number NP_064707.1 and Uniprot accession number P25106.

[0099] By way of additional guidance, the μ(μ)-type opioid receptor (MOR) is also known in the art as OPRM, LMOR, or MOP. As an example, the human MOR protein is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number AY521028.1 and Uniprot accession number P35372.

[0100] By additional guidance, delta (δ)-type opioid receptor (DOR) is also referred to in the art as OPRD or DOP. For example, the human DOR protein is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number NM_000911.4 and Uniprot accession number P41143.

[0101] By way of additional guidance, the kappa (κ)-type opioid receptor (KOR) is also referred to in the art as OPRK or KOP. For example, the human KOR protein is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number AF498922.1 and Uniprot accession number P41145.

[0102] By way of additional guidance, the nonclassical nociceptin receptor (NOP) is also known in the art as orphanin FQ receptor, OPRL, and opioid-related nociceptin receptor 1. As an example, the human NOP protein is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number AY268428.1 and Uniprot accession number P41146.

[0103] The skilled artisan will appreciate that any sequence represented in a sequence database or in this specification may be a precursor of the corresponding peptide, polypeptide, protein or nucleic acid and may include portions that are processed away from the mature molecule.

[0104] Reference to any peptide, polypeptide, protein or nucleic acid refers to the respective peptide, polypeptide, protein or nucleic acid as commonly known in the art under the respective name. More specifically, reference to "ACKR3", "MOR", "DOR", "KOR", "NOP", "CCR1", "CCR2A", "CCR2B", "CCR3", "CCR4", "CCR5", "CCR6", "CCR7", "CCR8", "CCR9", "CCR10", "CXCR1", "CXCR2", "CXCR3A", "CXCR3B", "CXCR4", "CXCR5", "CXCR6", "CXCR8", "XCR1", "CX3CR1", "ACKR1", "ACKR2" or "ACKR4" refers to the respective peptide, polypeptide, protein or nucleic acid as commonly known in the art under the respective name, as is apparent from the context.

[0105] The term includes a peptide, polypeptide, protein, or nucleic acid when it forms part of an organism, organ, tissue, or cell, when it forms part of a biological sample, and when it is at least partially isolated from these sources. The term also includes a peptide, polypeptide, protein, or nucleic acid when it is produced by recombinant or synthetic means.

[0106] Reference to any peptide, polypeptide, protein or nucleic acid includes any organism in which such peptide, polypeptide, protein or nucleic acid is found, particularly animals, preferably warm-blooded animals, more preferably vertebrates, still more preferably mammals, including humans and non-human mammals, more preferably humans.

[0107] Thus, in certain embodiments, one or more, and preferably all, of ACKR3, MOR, DOR, KOR, NOP, CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, and ACKR4 as used herein are of animal origin, preferably of warm-blooded animal origin, more preferably of vertebrate origin, still more preferably of mammalian origin, including human origin and non-human mammalian origin, more preferably of human origin.

[0108] For biological peptides, polypeptides, proteins or nucleic acids, due to the genetic divergence between these species, the native sequences between different species may be different. In addition, due to the normal genetic diversity (variation) in a given species, the native sequences between different individuals of the same species or inside may be different. In addition, due to somatic mutation, post-transcriptional or post-translational modification, the native sequences between different individuals of the same species or even inside may be different. Any such variant or isoform of a peptide, polypeptide, protein or nucleic acid is expected in this article. Therefore, the sequences of all peptides, polypeptides, proteins or nucleic acids found in nature or derived from nature are considered to be "natural".

[0109] Unless the context indicates otherwise, reference herein to any peptide, polypeptide, protein or nucleic acid also includes modified forms of the peptide, polypeptide, protein or nucleic acid, such as forms with post-expression modifications including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteineylation, sulfonation, glutathionylation, acetylation, oxidation of methionine to methionine sulfoxide or methionine sulfone, and the like.

[0110] As used throughout this specification, the term "protein" generally includes macromolecules comprising one or more polypeptide chains (i.e., polymeric chains of amino acid residues linked by peptide bonds). The term can include proteins produced naturally, recombinantly, semisynthetically, or synthetically. The term also includes proteins that carry one or more co-expression or post-expression modifications of the polypeptide chains, such as, but not limited to, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of a zymogen or prohormone to an active form, and the like. The term also includes protein variants or mutants that carry amino acid sequence variations relative to the corresponding native protein, such as, for example, amino acid deletions, additions, and / or substitutions. The term encompasses full-length proteins and protein portions or fragments, such as naturally occurring portions of proteins produced by processing of such full-length proteins.

[0111] As used throughout this specification, the term "polypeptide" generally includes a polymeric chain of amino acid residues linked by peptide bonds. Thus, particularly when a protein consists of only a single polypeptide chain, the terms "protein" and "polypeptide" are used interchangeably herein to refer to such a protein. The term is not limited to any minimum length of the polypeptide chain. The term can include naturally occurring, recombinant, semisynthetic, or synthetically produced polypeptides. The term also includes polypeptides that carry one or more co-expression or post-expression modifications of the polypeptide chain, such as, but not limited to, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of a zymogen or prohormone to an active form, and the like. The term also includes polypeptide variants or mutants that carry amino acid sequence variations relative to the corresponding native polypeptide, such as amino acid deletions, additions, and / or substitutions. The term encompasses full-length polypeptides and polypeptide portions or fragments, such as naturally occurring polypeptide portions produced by processing of such full-length polypeptides.

[0112] The polypeptide or protein can be naturally occurring, e.g., present in nature or isolated from nature, e.g., naturally or endogenously produced or expressed by a cell or tissue and optionally isolated therefrom. The polypeptide or protein can be recombinant, i.e., produced by recombinant DNA technology, and / or can be partially or completely chemically or biochemically synthesized. Without limitation, the polypeptide or protein can be recombinantly produced by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or recombinantly produced by cell-free translation or cell-free transcription and translation, or abiotic polypeptide or protein synthesis.

[0113] In certain embodiments, a peptide as taught herein may be fused to a pharmaceutical agent.

[0114] In the context of the present invention, the term "coupled" as used herein is synonymous with "connected", "bound", "fused", "joined" and refers to a physical connection between at least two elements or components.

[0115] As used herein, the term "agent" refers broadly to any chemical (e.g., inorganic or organic), biochemical or biological substance, molecule or macromolecule (e.g., biomacromolecule), combination or mixture thereof, sample of undefined composition, or extract made from biological material such as bacteria, fungi, plant or animal cells or tissues. Preferred but non-limiting "agents" include nucleic acids, oligonucleotides, ribozymes, peptides, polypeptides, proteins, peptidomimetics, antibodies, antibody fragments, antibody-like protein scaffolds, aptamers, photoaptamers, spiegelmers, chemical substances, preferably organic molecules, more preferably small organic molecules, lipids, carbohydrates, polysaccharides, etc., and any combination thereof.

[0116] In certain embodiments, a peptide as taught herein may be coupled to an agent selected from the group consisting of a chemical, an antibody, an antibody fragment, an antibody-like protein scaffold, a protein or polypeptide and peptide, a peptide mimetic, an aptamer, a photoaptamer, a spiegelmer, and a nucleic acid.

[0117] As used herein, the term "chemical substance" is used in its broadest sense and generally refers to any substantially pure substance with a constant chemical composition and characteristic properties. The chemical substance can be an organic molecule, preferably an organic small molecule. The term "small molecule" refers to a compound, preferably an organic compound, whose size is comparable to those organic molecules commonly used in medicine. The term does not include biological macromolecules (e.g., proteins, peptides, nucleic acids, etc.). The size range of preferred organic small molecules is up to about 5000Da, for example up to about 4000, preferably up to 3000Da, more preferably up to 2000Da, even more preferably up to about 1000Da, for example up to about 900, 800, 700, 600 or up to about 500Da.

[0118] The term "antibody" is used in its broadest sense herein and generally refers to any immunobinder, such as a complete antibody, including but not limited to chimeric antibodies, humanized antibodies, human antibodies, recombinant antibodies, transgenic antibodies, transplanted antibodies and single-chain antibodies, or any fusion proteins, conjugates, fragments or derivatives comprising one or more domains that selectively bind to a target antigen. Therefore, the term antibody includes complete immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies or any immunologically effective fragments thereof. Therefore, the term specifically includes complete monoclonal antibodies, polyclonal antibodies, multivalent (e.g., 2-, 3- or more valences) and / or multispecific antibodies (e.g., bi- or more-specific antibodies) formed by at least two complete antibodies, and antibody fragments as long as they exhibit desired biological activity (particularly, the ability to specifically bind to a target antigen), and the multivalent and / or multispecific complexes of these fragments. The term "antibody" includes not only antibodies produced by methods including immunity, but also any polypeptide, such as recombinantly expressed polypeptides, which are made to include at least one complementary determining region (CDR) that can specifically bind to an epitope on a target antigen. Thus, the term applies to such molecules regardless of whether they are produced in vitro, in cell culture, or in vivo.

[0119] The term "antibody fragment" or "antigen binding portion" comprises a portion or region of a full-length antibody, generally the antigen binding domain or its variable domain. Examples of antibody fragments include Fab, Fab', F(ab)2, Fv, scFv fragments, single domain (sd) Fv, such as V H domain, V Ldomain and V HH domains, diabodies, linear antibodies, single-chain antibody molecules, in particular heavy chain antibodies; and multivalent and / or multispecific antibodies formed from antibody fragments, such as diabodies, triabodies, and multibodies. The above-mentioned designations Fab, Fab', F(ab')2, Fv, scFv, etc. are intended to have their established meanings in the art.

[0120] In a more specific embodiment, the peptides as taught herein can be coupled to an agent selected from the group consisting of a detectable label (e.g., a fluorescent protein or an enzyme), an immunoglobulin Fc region (e.g., IgG2a Fc), a protoxin, a toxin, or a drug, preferably the group consisting of a fluorescent protein, an enzyme, an immunoglobulin Fc region, a protoxin, or a toxin.

[0121] In certain embodiments, the peptides as taught herein may be fused to a detectable label.

[0122] The term "label" refers to any atom, molecule, moiety or biomolecule that can be used to provide a detectable and preferably quantifiable readout or property and that can be attached to or become part of a target entity, such as a peptide as taught herein. Labels can be suitably detected, for example, by mass spectrometric, spectroscopic, optical, colorimetric, magnetic, photochemical, biochemical, immunochemical or chemical methods. Labels include, but are not limited to, dyes; such as 32 P. 33 P. 35 S. 125 I. 13 1I radiolabel; electron-dense reagents; enzymes (e.g., horseradish peroxidase or alkaline phosphatase commonly used in immunoassays); binding moieties, such as biotin-streptavidin; haptens, such as digoxigenin; luminescent, phosphorescent, or fluorescent moieties; mass tags; and fluorescent dyes (e.g., fluorophores, such as fluorescein, carboxyfluorescein (FAM), tetrachlorofluorescein, TAMRA, ROX, Cy3, Cy3.5, Cy5, Cy5.5, Texas Red, etc.), alone or in combination with moieties that may inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET).

[0123] In some embodiments, peptides as taught herein may be equipped with a tag that allows detection with another agent (e.g., with a probe binding partner). Such tags may be, for example, biotin, streptavidin, a his-tag, a myc tag, maltose, maltose binding protein, or any other type of tag with a binding partner known in the art. Examples of associations that can be used in probe:binding partner arrangements may be any and include, for example, biotin:streptavidin, his-tag:metal ion (e.g., Ni 2+), maltose: maltose binding protein, etc.

[0124] In certain embodiments, the tag may be Large BiT (LgBiT) or Small BiT (SmBiT) or HiBiT of NanoBiT Technology (NanoBiT).

[0125] The peptides as taught herein can be associated or attached to a detection agent to facilitate detection. Examples of detection agents include, but are not limited to, luminescent labels; colorimetric labels, such as dyes; fluorescent labels (e.g., green fluorescent protein (GFP)); or chemical labels, such as electroactive agents (e.g., ferrocyanide); enzymes; radioactive labels; or radiofrequency labels. The detection agent can be a particle. Examples of such particles include, but are not limited to, colloidal gold particles; colloidal sulfur particles; colloidal selenium particles; colloidal barium sulfate particles; colloidal ferric sulfate particles; metal iodate particles; silver halide particles; silica particles; colloidal metal (hydrated) oxide particles; colloidal metal sulfide particles; colloidal lead selenide particles; colloidal cadmium selenide particles; colloidal metal phosphate particles; colloidal metal ferrite particles; any of the above colloidal particles coated with an organic or inorganic layer; protein or peptide molecules; liposomes; or organic polymer latex particles, such as polystyrene latex beads.

[0126] In certain embodiments, a peptide as taught herein may be coupled to a pharmaceutical agent via one or more linkers.

[0127] As used herein, the term "linker" refers to a connecting element used to connect other elements. The linker can be a rigid linker or a flexible linker. In a specific embodiment, the linker is a covalent linker, which realizes a covalent bond. The term "covalent" or "covalent bond" refers to a chemical bond involving the sharing of one or more electron pairs between two atoms. For many molecules, the sharing of electrons allows each atom to obtain the equivalent of a complete outer electron shell, corresponding to a stable electronic configuration. Covalent bonds include different types of interactions, including σ-bonds, π-bonds, metal-metal bonds, agostic interactions, bent bonds, and three-center two-electron bonds.

[0128] In a particular embodiment, the linker is a (poly)peptide linker or a non-peptide linker, such as a non-peptide polymer, such as a non-biological polymer. Preferably, the bond between the peptide as taught herein and the second peptide, protein or polypeptide can be a hydrolytically stable bond, i.e., substantially stable in water at useful pH values, including in particular under physiological conditions, for an extended period of time, such as several days. In a particular embodiment, the linker is a peptide linker of one or more amino acids.

[0129] The term "amino acid" includes naturally occurring amino acids, naturally encoded amino acids, non-naturally encoded amino acids, non-naturally occurring amino acids, amino acid analogs, and amino acid mimetics that function in a manner similar to naturally occurring amino acids, all in their D and L stereoisomers, provided that their structure allows such stereoisomeric forms. Amino acids are referred to herein by their names, their commonly known three-letter symbols, or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The term "naturally occurring" generally refers to materials found in nature and not manipulated by humans. The terms "non-naturally occurring," "non-natural," etc. generally refer to materials not found in nature or that have been structurally modified, semi-synthesized, or artificially synthesized by humans. The term includes, but is not limited to, amino acids that appear through modification (e.g., post-translational modification) of naturally encoded amino acids, but which themselves are not naturally incorporated into a growing polypeptide chain by the translation complex. Amino acid analogs are also included in which one or more individual atoms are replaced by different atoms, isotopes of the same atom, or different functional groups. Also included are the non-natural amino acids and amino acid analogs described in Ellman et al. Methods Enzymol. 1991, Vol. 202, 301-36. It may be advantageous to incorporate non-natural amino acids into proteins or polypeptides in a variety of different ways. For example, polypeptides containing D-amino acids exhibit increased stability in vitro or in vivo compared to their L-amino acid counterparts. More specifically, polypeptides containing D-amino acids may be more resistant to endogenous peptidases and proteases, thereby providing improved bioavailability of pharmaceutical agents and extended in vivo service life.

[0130] More specifically, the peptide linker can be 1 to 50 amino acids long or 2 to 50 amino acids long or 1 to 45 amino acids long or 2 to 45 amino acids long, preferably 1 to 40 amino acids long or 2 to 40 amino acids long or 1 to 35 amino acids long or 2 to 35 amino acids long, more preferably 1 to 30 amino acids long or 2 to 30 amino acids long. More preferably, the linker can be 5 to 25 amino acids long or 5 to 20 amino acids long. Particularly preferably, the linker can be 5 to 15 amino acids long or 7 to 15 amino acids long. Therefore, in certain embodiments, the linker can be 1, 2, 3 or 4 amino acids long. In other embodiments, the linker can be 5, 6, 7, 8 or 9 amino acids long. In further embodiments, the linker can be 10, 11, 12, 13 or 14 amino acids long. In other embodiments, the linker can be 15, 16, 17, 18 or 19 amino acids long. In further embodiments, the linker can be 20, 21, 22, 23, 24 or 25 amino acids long. In certain embodiments, the linker is 4-10 or 5-9 or 6-8 or 7 amino acids long. In other embodiments, the linker is 12-18 or 13-17 or 14-16 or 15 amino acids long.

[0131] The nature of the amino acids constituting the linker is of no particular relevance, provided that the biological activity of the polypeptide fragments thus linked is not substantially impaired and that the linker provides the desired spatial separation of the peptide and the second peptide, protein or polypeptide as taught herein. Preferred linkers are substantially non-immunogenic and / or not susceptible to protease cleavage.

[0132] In some preferred embodiments, the peptide linker can comprise amino acid or be substantially composed of amino acid or be composed of amino acid, and the group that described amino acid is selected from by glycine, serine, alanine, threonine and combination thereof is formed.In even more preferred embodiments, the linker can comprise amino acid or be substantially composed of amino acid or be composed of amino acid, and the group that described amino acid is selected from by glycine, serine and combination thereof is formed.This linker provides especially good flexibility.In some embodiments, the linker can only be composed of glycine residues.In some embodiments, the linker can only be composed of serine residues.

[0133] In a specific embodiment, the linker is a non-peptide linker. In a preferred embodiment, the non-peptide linker may comprise a non-peptide polymer, be essentially composed of a non-peptide polymer, or be composed of a non-peptide polymer. As used herein, the term "non-peptide polymer" refers to a biocompatible polymer comprising two or more repeating units, which are linked to each other by covalent bonds that do not include peptide bonds. For example, the non-peptide polymer may be 2 to 200 units long or 2 to 100 units long or 2 to 50 units long or 2 to 45 units long or 2 to 40 units long or 2 to 35 units long or 2 to 30 units long or 5 to 25 units long or 5 to 20 units long or 5 to 15 units long. The non-peptide polymer can be selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyethylene ethyl ether, biodegradable polymers such as PLA (polylactic acid) and PLGA (polylactic-co-glycolic acid), lipid polymers, chitin, hyaluronic acid and combinations thereof. Particularly preferred is poly (ethylene glycol) (PEG). The molecular weight of the non-peptide polymer can preferably be between 1 and 100 kDa, preferably between 1 and 20 kDa. The non-peptide polymer can be a combination of a polymer or different types of polymers. The non-peptide polymer has a reactive group capable of combining a peptide and a second peptide, protein or polypeptide as taught herein, which will form a conjugate. Preferably, the non-peptide polymer has a reactive group at each end. Preferably, the reactive group is selected from the group consisting of reactive aldehyde, propionaldehyde, butyraldehyde, maleimide and succinimide derivatives. The succinimide derivative can be succinimidyl propionate, hydroxy succinimidyl ester, succinimidyl carboxymethyl or succinimidyl carbonate. The reactive groups at both ends of the non-peptide polymer can be the same or different. In certain embodiments, the non-peptide polymer has reactive aldehyde groups at both ends. For example, the non-peptide polymer can have a maleimide group at one end and an aldehyde group, a propionaldehyde group or a butyraldehyde group at the other end. When polyethylene glycol (PEG) having reactive hydroxyl groups at both ends is used as the non-peptide polymer, the hydroxyl groups can be activated into various reactive groups by known chemical reactions, or PEG having commercially available modified reactive groups can be used to prepare protein conjugates.

[0134] In a particular embodiment, the agent is fused to the C-terminus of a peptide as taught herein.A related aspect provides a fusion protein comprising a peptide as taught herein.

[0135] The terms "fusion protein" or "fusion polypeptide" and "protein conjugate" or "polypeptide conjugate" refer to hybrid or chimeric molecules comprising at least two peptides, proteins or polypeptides linked, connected or joined together in a manner not normally found in nature. The molecule may suitably be represented as an amino acid-based compound, i.e., as a substance or molecule comprising primarily, but not necessarily exclusively, amino acid residues. Any recombinant, semisynthetic or synthetically produced fusion or conjugate is included. If desired, the fusion or conjugate may be modified by glycosylation, phosphorylation, sulfonation, methylation, acetylation, lipidation, pegylation, and the like.

[0136] More specifically, the term "fusion protein" or "fusion polypeptide" refers to a genetic fusion, whereby two or more peptides, proteins, polypeptides, or variants or fragments thereof are linked by colinearity, covalent bonds through their respective polypeptide backbones, by the genetic expression of a single contiguous polynucleotide molecule encoding a fusion product. Typically, to produce a contiguous polynucleotide molecule encoding a fusion product, two or more open reading frames (ORFs), each encoding a given polypeptide segment, are linked to form a continuous, longer ORF in a manner that maintains the correct reading frame of each original ORF. In the resulting recombinant fusion polypeptide, the two or more polypeptide fragments encoded by the original ORFs are linked in the same polypeptide molecule, whereas they are not typically so linked in nature. Although the reading frame is thus continuous throughout the fused genetic fragments, the polypeptide fragments so fused can be physically or spatially separated by, for example, in-frame polypeptides or peptide linkers.

[0137] In certain embodiments, the fusion protein further comprises an agent as described elsewhere herein, wherein the agent is a peptide, protein or polypeptide, preferably a detectable label or tag; or an immunoglobulin Fc region.

[0138] In certain embodiments, the fusion protein further comprises one or more linkers as described elsewhere herein, wherein the linker is positioned between the peptide as taught herein and the agent as described elsewhere herein.

[0139] Another aspect provides a nucleic acid encoding a peptide as taught herein or a fusion protein as taught herein.

[0140] As used herein, the term "nucleic acid" generally refers to an oligomer or polymer (preferably a linear polymer) of any length that is essentially composed of nucleotides. Nucleotide units generally include a heterocyclic base, a sugar group and at least one, such as one, two or three phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases can especially include purine and pyrimidine bases, such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) that are widely present in naturally occurring nucleic acids, other naturally occurring bases (such as xanthine, inosine, hypoxanthine) and chemically or biochemically modified (such as methylated), non-natural or derived bases. Sugar groups can especially include pentose (pentofuranosyl) groups, such as ribose and / or 2-deoxyribose commonly found in naturally occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, and modified or substituted sugar groups. Nucleic acids as referred to herein can include naturally occurring nucleotides, modified nucleotides or their mixtures. The modified nucleotides may include modified heterocyclic bases, modified sugar moieties, modified phosphate groups, or combinations thereof. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful properties. The term "nucleic acid" further preferably includes DNA, RNA, and DNA / RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA, or DNA / RNA hybrids. Nucleic acid may be naturally occurring, such as present in nature or separated from nature; or it may be non-naturally occurring, such as recombinant, i.e., produced by recombinant DNA technology, and / or partially or entirely chemically or biochemically synthesized. "Nucleic acid" may be double-stranded, partially double-stranded, or single-stranded. In the case of a single strand, the nucleic acid may be a sense strand or an antisense strand. In addition, the nucleic acid may be circular or linear.

[0141] "Encoding" refers to a nucleic acid sequence or portion thereof that corresponds to a specific amino acid sequence according to the genetic code of the organism in question, such as the amino acid sequence of one or more desired proteins or polypeptides, or another nucleic acid sequence in a template-transcription product (e.g., RNA or RNA analog) relationship.

[0142] To allow expression of a nucleic acid encoding a peptide or fusion protein as taught herein, the nucleic acid can be inserted into a nucleic acid expression cassette and / or vector, as is well known in the art.

[0143] Another aspect provides a nucleic acid expression cassette comprising a nucleic acid as taught herein operably linked to a promoter and / or transcriptional and translational regulatory signals.

[0144] As used herein, the term "nucleic acid expression cassette" refers to a nucleic acid molecule, typically DNA, into which a nucleic acid segment can be inserted for expression, wherein the nucleic acid molecule comprises one or more nucleic acid sequences that control the expression of the nucleic acid segment. Non-limiting examples of such further nucleic acid sequences that control the expression of the nucleic acid segment include a promoter sequence, an open reading frame, and a transcription terminator.

[0145] Preferably, the nucleic acid expression cassette may comprise one or more open reading frames (ORFs) encoding the one or more proteins, polypeptides or peptides. An "open reading frame" or "ORF" refers to a series of coding nucleotide triplets (codons) that begin with a translation initiation codon and end with a translation termination codon known per se, and does not comprise any internal in-frame translation termination codons, and is potentially capable of encoding a protein, polypeptide or peptide. Therefore, the term can be synonymous with a "coding sequence" as used in the art.

[0146] "Operably linked" is a connection in which a regulatory sequence and a sequence for which expression is sought are linked in a manner that permits such expression. For example, sequences (e.g., a promoter and an ORF) are said to be operably linked if the nature of the connection between the sequences does not: (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter to direct transcription of the ORF, or (3) interfere with the ability of the ORF to be transcribed from the promoter sequence. Thus, "operably linked" can refer to incorporation into a genetic construct such that an expression control sequence, such as a promoter, effectively controls the transcription / expression of a sequence of interest.

[0147] The precise nature of the transcriptional and translational regulatory sequences or elements required for expression may vary between expression environments, but generally include a transcription terminator and, optionally, an enhancer.

[0148] Reference to "promoter" is made in its broadest context, including the transcriptional regulatory sequences required for precise transcription initiation, and, where applicable, precise spatial and / or temporal control of gene expression or thereof to, for example, internal or external (e.g., exogenous) stimulation. More specifically, a "promoter" can describe a region on a nucleic acid molecule, preferably a DNA molecule, to which RNA polymerase binds and initiates transcription. A promoter is preferably, but not necessarily, located upstream, i.e., 5', of the sequence that controls transcription. Typically, in prokaryotes, a promoter region may include the promoter itself and a sequence (e.g., Shine-Dalgarno sequence) that signals the start of protein synthesis when transcribed into RNA. A promoter sequence may also include an "enhancer region," which is one or more DNA regions that can bind to proteins (i.e., trans-acting factors) to enhance the transcriptional level of genes in a gene cluster. Although an enhancer is typically located at the 5' end of the coding region, it may also be separated from the promoter sequence, e.g., it may be located within an intron region of a gene or 3' of a gene coding region.

[0149] In an embodiment, the promoter considered herein can be constitutive or inducible. A constitutive promoter is understood to be a promoter whose expression is constant under standard culture conditions. An inducible promoter is a promoter that responds to one or more induction cues. For example, an inducible promoter can be chemically regulated (for example, a promoter whose transcriptional activity is regulated by the presence or absence of a chemical inducer such as alcohol, tetracycline, steroid, metal or other small molecules) or physically regulated (for example, a promoter whose transcriptional activity is regulated by the presence or absence of a physical inducer such as light or high temperature or low temperature). Inducible promoters can also be indirectly regulated by one or more transcription factors, which themselves are directly regulated by chemical or physical cues. Non-limiting examples of promoters include T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter and EF1α promoter.

[0150] The term "terminator" or "transcription terminator" generally refers to a sequence element at the end of a transcription unit that signals the termination of transcription. For example, a terminator is typically located downstream, i.e., 3', of the ORF encoding the polypeptide of interest. For example, when a recombinant nucleic acid contains two or more ORFs, e.g., sequenced sequentially and together forming a multicistronic transcription unit, the transcription terminator may advantageously be located 3' of the most downstream ORF.

[0151] In particular embodiments, the nucleic acid expression cassette comprises a nucleic acid encoding a peptide or fusion protein as disclosed herein, operably linked to one or more promoters, enhancers, ORFs, and / or transcription terminators.

[0152] Another aspect provides a vector, such as a viral vector, comprising a nucleic acid as taught herein or a nucleic acid expression cassette as taught herein.

[0153] The term "vector" or "expression vector" used in this application refers to a nucleic acid molecule, such as double-stranded DNA, in which another nucleic acid molecule (insertion nucleic acid molecule) such as, but not limited to, a cDNA molecule may have been inserted. The vector is used to transport the insertion nucleic acid molecule into a suitable host cell. The vector may include the necessary elements that allow transcription of the insertion nucleic acid molecule and optionally translation of the transcript into a peptide, protein, or polypeptide. The insertion nucleic acid molecule may be derived from a host cell, or may be derived from different cells or organisms. Once entering the host cell, the vector may be independent of the host chromosomal DNA or replicate simultaneously with the host chromosomal DNA, and may produce multiple copies of the vector and the nucleic acid molecule inserted therein. The vector may be an additional vector (i.e., a vector that is not integrated into the host cell genome), or may be a vector that is integrated into the host cell genome. Therefore, the term "vector" may also be defined as a gene delivery vehicle that promotes gene transfer into a target cell. This definition includes non-viral and viral vectors. Non-viral vectors include, but are not limited to, cationic lipids, liposomes, nanoparticles, PEG, PEI, plasmid vectors (e.g., pUC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof without bacterial sequences (minicircles)), transposon-based vectors (e.g., PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), and the like. Viral vectors are derived from viruses and include, but are not limited to, retroviral vectors, lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, herpes virus vectors, hepatitis virus vectors, or the like. Typically, but not necessarily, viral vectors are replication-defective because they have lost the ability to propagate in a given cell because the viral genes necessary for replication have been eliminated from the viral vector. However, some viral vectors may also be suitable for specific replication in a given cell (e.g., cancer cell) and are commonly used to trigger (cancer) cell-specific (tumor) lysis. Virions are non-limiting examples of vectors comprising viral and non-viral elements, particularly they combine liposomes with inactivated HIV or influenza viruses. Another example includes viral vectors mixed with cationic lipids.

[0154] A peptide or fusion protein as taught herein may suitably be obtained by expression in a host cell or host organism transformed with an expression construct encoding and configured for expression of the peptide or fusion protein in said host cell or host organism, followed by purification of the peptide or fusion protein.

[0155] Thus, in another aspect there is provided a host cell comprising a nucleic acid, nucleic acid expression cassette or vector as taught herein.

[0156] In certain embodiments, the host cell can be a bacterial cell, a yeast cell, an animal cell, or a mammalian cell.

[0157] The terms "host cell" and "host organism" may suitably refer to cells or organisms including prokaryotes (e.g., bacteria) and eukaryotes (e.g., yeasts, fungi, protozoa, plants, and animals). In particular, unicellular organisms are contemplated as host cells, such as bacteria (e.g., E. coli, Salmonella tymphimurium, Serratia marcescens, or Bacillus subtilis), yeasts (e.g., Saccharomyces cerevisiae, or Pichia pastoris), (cultured) plant cells (e.g., from Arabidopsis thaliana or Nicotiana tobaccum), and (cultured) animal cells (e.g., vertebrate cells, mammalian cells, primate cells, human cells, or insect cells). Considered as host organisms are in particular multicellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrates, still more preferably mammals, yet more preferably primates; non-human such animals and animal classes are particularly considered.

[0158] Such proteins, polypeptides or peptides can be suitably isolated and / or purified.Purified nucleic acids, proteins, polypeptides or peptides can be obtained by known methods, including, for example, laboratory or recombinant synthesis, chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification, etc.

[0159] In certain embodiments, the vector comprising a nucleic acid as described herein is a viral vector, preferably a viral vector specific for the central and / or peripheral nervous system (eg, a brain-specific viral vector).

[0160] In a specific embodiment, the nucleic acid encoding the agent as taught herein may be contained in a vector that provides a signal peptide. Depending on the host cell used to produce the agent as taught herein, the signal peptide may be a homologous or heterologous signal peptide. In addition, for prokaryotic expression of the agent as taught herein, a protease cleavage site motif may be present at the C-terminus of the signal peptide and the N-terminus of the agent as taught herein.

[0161] Another aspect provides a pharmaceutical composition comprising a peptide as taught herein, a fusion peptide as taught herein, a nucleic acid as taught herein, a nucleic acid expression cassette as taught herein or a vector as taught herein, and optionally a pharmaceutically acceptable carrier.

[0162] As used herein, the term "pharmaceutically acceptable" is consistent with the art and means compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.

[0163] As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (e.g., neutral buffered saline or phosphate buffered saline), solubilizers, colloids, dispersion media, excipients, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavorings, fragrances, thickeners, agents for achieving storage effects, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical actives is well known in the art. Unless any conventional media or agents are incompatible with the active substance, their use in therapeutic compositions may be considered.

[0164] Exemplary, non-limiting carriers for formulating pharmaceutical compositions include, for example, oil-in-water or water-in-oil emulsions, aqueous compositions with or without organic co-solvents suitable for intravenous (IV) use, liposomes or vesicles containing a surfactant, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to those of ordinary skill in the art.

[0165] The pharmaceutical compositions referred to herein can be formulated for essentially any route of administration, such as, but not limited to, oral administration (e.g., oral ingestion or inhalation), intranasal administration (e.g., intranasal inhalation or intranasal mucosal application), parenteral administration (e.g., subcutaneous, intravenous (IV), intramuscular, intraperitoneal, or intrasternal injection or infusion), transdermal or transmucosal (e.g., oral, sublingual, intranasal) administration, topical administration, rectal, vaginal, or intratracheal instillation, etc. In this manner, the therapeutic effect obtainable by the methods and compositions can be, for example, systemic, local, tissue-specific, etc., depending on the specific needs of a given application.

[0166] In a preferred embodiment, the peptide, fusion protein, nucleic acid encoding the peptide or fusion protein, nucleic acid expression cassette comprising the nucleic acid, vector comprising the nucleic acid or nucleic acid expression cassette, host cell or pharmaceutical composition as taught herein is administered parenterally. More preferably, the peptide, fusion protein, nucleic acid encoding the peptide or fusion protein, nucleic acid expression cassette comprising the nucleic acid, vector comprising the nucleic acid or nucleic acid expression cassette, host cell or pharmaceutical composition as taught herein is administered intravenously, for example by infusion.

[0167] Optionally in combination with one or more other active compounds to be administered, the dosage or amount of the medicament taught herein depends on individual circumstances, and as a rule, adapts to individual circumstances to achieve optimal results. Therefore, the unit dose and regimen depend on the nature and severity of the disease to be treated, and also depend on factors such as the species, sex, age, body weight, overall health, diet, mode and time of administration of the human or animal to be treated, immune status and individual reactivity, efficacy, metabolic stability and duration of action of the compound used, depending on whether the treatment is acute, chronic or preventive, or on whether other active compounds are administered in addition to the medicament of the present invention. In order to optimize therapeutic efficacy, the peptides, fusion proteins, nucleic acids encoding peptides or fusion proteins, nucleic acid expression cassettes comprising nucleic acids, vectors, host cells or pharmaceutical compositions as taught herein can first be administered with different dosing regimens. Typically, suitable screening tests can be used as part of a clinical testing program to monitor the level of the medicament in the tissue, for example, to determine the efficacy of a given treatment regimen. The frequency of administration is within the skill and clinical judgment of the physician (e.g., doctor, veterinarian or nurse). Typically, a dosing regimen is established through clinical trials, and clinical trials can establish optimal administration parameters. However, the practitioner may vary such administration regimens depending on one or more of the above factors, such as the subject's age, health, weight, sex, and medical condition. The frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic.

[0168] The toxicity and therapeutic efficacy of a medicament as described herein or a pharmaceutical composition comprising the medicament can be determined by known pharmaceutical procedures in, for example, cell cultures or experimental animals. These procedures can be used, for example, to determine LD50 (a dose lethal to 50% of a population) and ED50 (a dose effective for the treatment of 50% of a population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as a ratio LD50 / ED50. Pharmaceutical compositions showing a high therapeutic index are preferred. Although pharmaceutical compositions showing toxic and side effects can be used, it should be noted that the delivery system designed to target such compounds to the affected tissue site is to minimize potential damage to normal cells (e.g., non-target cells) and, thereby, to reduce side effects.

[0169] The data obtained from cell culture experiments and animal studies can be used to formulate a dosage range for appropriate subjects. The dosage of such pharmaceutical compositions is generally within a circulating concentration range including the ED50, with little or no toxicity. Depending on the dosage form employed and the route of administration employed, the dosage can vary within this range. For the pharmaceutical compositions used as described herein, a therapeutically effective dose can be initially estimated from cell culture experiments. Dosages can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the pharmaceutical composition that achieves half-maximal inhibition of symptoms) determined in cell culture. Such information can be used to more accurately determine a useful dose in the human body. For example, the level in plasma can be measured by high performance liquid chromatography.

[0170] In certain embodiments, a peptide or fusion protein as taught herein is the major or sole active ingredient of a pharmaceutical composition.

[0171] The selectivity and high affinity of the peptides as taught herein for ACKR3 provide for many valuable in vitro, ex vivo and in vivo applications of the peptides as taught herein.

[0172] Another aspect provides the use of a peptide as described herein for stabilizing an ACKR3 polypeptide, for example, during nuclear magnetic resonance (NMR) analysis. The conformational flexibility of the receptor can be an obstacle to protein production and crystallographic studies. Because the peptides taught herein specifically recognize ACKR3 polypeptides, the peptides can be used to specifically target agents, such as detectable labels, drugs, or toxins, to ACKR3 polypeptides.

[0173] Thus, another aspect provides the use of a peptide or fusion protein as described herein for targeted delivery of an agent, such as a drug or toxin, to an ACKR3 polypeptide.

[0174] For example, ACKR3 is expressed in various cell types such as B and T lymphocytes, neurons, and endothelial cells, and plays a role in multiple types of cancer, cardiovascular and neuronal development, cardiac and immune pathophysiology, and the migration and homing of hematopoietic stem / progenitor cells. ACKR3 is expressed in various cancer cell types (e.g., colorectal cancer, breast cancer, prostate cancer, lung cancer, liver cancer, lymphoma, leukemia, glioblastoma, and head and neck cancer) and in tumor-associated vasculature, and is involved in metastatic development. ACKR3 is also upregulated following infection with several oncogenic viruses, including HHV-8, EBV, and HTLV-1, and plays an important role in cell transformation and proliferation.

[0175] Thus, in certain embodiments, peptides fused to toxins as taught herein are used in the treatment of cancer.

[0176] A related aspect provides the use of the peptides described herein as peptide tracers, e.g., for in vivo, ex vivo, or in vitro imaging. For example, the peptides taught herein, when fused to a detectable label, can be used to visualize cells, tissues, and / or organs (e.g., certain types of cancer cells) that express ACKR3. Thus, the peptides as taught herein, when fused to a detectable label, can also be used to visualize diseases or conditions associated with ACKR3, such as cancer, diseases or conditions involving excessive or abnormal angiogenesis, and inflammatory or autoimmune diseases and conditions (e.g., arthritis). More specifically, the peptides as taught herein, when fused to a detectable label, can be used to visualize cancer, atherosclerotic vascular disease, cardiac fibrosis, or brain and neuronal dysfunction (e.g., Alzheimer's disease, multiple sclerosis, and demyelinating diseases) in vivo, ex vivo, or in vitro. Non-limiting examples of cancers that can be visualized using the peptides taught herein include carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, prostate cancer, Burkitt lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, bladder cancer, penile cancer, urethral cancer, testicular cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin lymphoma, and non-Hodgkin lymphoma.

[0177] Another aspect provides a method for detecting and / or determining the level of an ACKR3 polypeptide in a biological sample in vitro or ex vivo, comprising the following steps:

[0178] - obtaining a biological sample from a subject,

[0179] - contacting said biological sample with a peptide as taught herein, wherein said peptide is fused to a detectable label,

[0180] - Detecting and / or determining the level of ACKR3 polypeptide in said biological sample by detecting a peptide as taught herein.

[0181] The terms "level," "quantity," and "amount" are synonymous and generally well understood in the art. As used herein, the terms may refer, inter alia, to the absolute quantification of a molecule or analyte in a sample, or the relative quantification of a molecule or analyte in a sample, i.e., relative to another value, such as a reference value as taught herein, or relative to a range of values ​​indicating baseline expression of the molecule or analyte. These values ​​or ranges may be obtained from a single patient or a group of patients.

[0182] A related aspect provides a peptide as taught herein for use in methods of diagnosis, prediction, prognosis and / or monitoring of a disease or condition characterized by abnormal levels of an ACKR3 polypeptide in a subject, and corresponding methods of use, wherein the peptide is fused to a detectable label.

[0183] Unless otherwise indicated, the terms "subject" or "patient" are used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and particularly include human patients and non-human mammals and primates. Preferred subjects are human subjects. The terms "subject" or "patient" include subjects in need of treatment, more specifically subjects who will benefit from treatment for a given condition. Such subjects may include, but are not limited to, those who have been diagnosed with the condition, those who are susceptible to developing the condition, and / or those in whom the condition is to be prevented.

[0184] The absolute amount of a molecule or analyte in a sample may advantageously be expressed as a weight or molar amount, or more generally as a concentration, such as weight / volume or moles / volume.

[0185] In sample, the relative amount of molecule or analyte can be advantageously expressed as an increase or decrease or increase multiple or decrease multiple relative to the other value, such as relative to a reference value as taught herein. Performing relative comparison between the first and second parameters (such as, the first and second amounts) can but need not first determine the absolute values ​​of the first and second parameters. For example, measuring method can produce quantifiable readings (such as, such as signal intensity) for the first and second parameters, wherein the reading is a function of the parameter value, and wherein the reading can be directly compared to produce the relative value of the first parameter to the second parameter, without first converting the reading into the absolute value of the corresponding parameter.

[0186] The terms "predicting" or "prediction", "diagnosing" or "diagnosis" and "prognosticating" or "prognosis" are commonplace and well understood in medicine and clinical practice. It will be understood that the phrase "a method for diagnosing, predicting and / or prognosing a given disease or condition" may also be interchanged with phrases such as "a method for diagnosing, predicting and / or prognosing said disease or condition" or "a method for making (or determining or establishing) a diagnosis, prediction and / or prognosis of said disease or condition", etc.

[0187] By further explanation and without limitation, "prediction" or "prediction" generally refers to a pre-statement, indication or prophecy of a disease or condition of a subject who has not yet (yet) suffered from the disease or condition. For example, the prediction of a disease or condition of a subject can indicate the probability, chance or risk that the subject will develop the disease or condition, for example, within a certain time period or to a certain age. The probability, chance or risk can especially be expressed as an absolute value, range or statistical data, or can be relative to a suitable control subject or subject population (for example, relative to a general, normal or healthy subject or subject population). Therefore, relative to a suitable control subject or subject population, the probability, chance or risk that a subject will develop a disease or condition can advantageously be indicated as an increase or decrease, or an increase multiple or a decrease multiple. As used herein, "prediction" (term) of a condition or disease as taught herein in a subject can also particularly mean that the subject has a "positive" prediction of this type, i.e., the subject has a risk of suffering from such a condition or disease (for example, compared to a control subject or subject population, the risk is significantly increased). As described herein, "prediction of absence" (the term) of a disease or condition as taught herein in a subject can specifically mean that the subject has a "negative" prognosis for such, i.e., the subject does not have a significantly increased risk of having such disease or condition relative to a control subject or population of subjects.

[0188] The term "diagnosing" or "diagnosis" generally refers to the process or behavior of identifying, determining, or summarizing a disease or condition in a subject based on symptoms and signs and / or results from various diagnostic procedures (e.g., by understanding the presence, absence, and / or quantity of one or more biomarkers characteristic of the diagnosed disease or condition). As used herein, "diagnosis" of a disease or condition as taught herein in a subject may particularly mean that the subject has such a disease or condition, and is therefore diagnosed as having such a disease or condition. "Diagnosis-free" of a disease or condition as taught herein in a subject may particularly mean that the subject does not have such a disease or condition, and is therefore diagnosed as not having such a disease or condition. Despite exhibiting one or more conventional symptoms or signs reminiscent of such a disease or condition, a subject may be diagnosed as not having such a disease or condition.

[0189] The term "prediction" or "prognosis" generally refers to an expectation of the prospects (e.g., probability, duration, and / or degree) for the progression and recovery of a disease or condition. A good prognosis for a disease or condition as taught herein may generally include an expectation of satisfactory partial or complete recovery from the disease or condition, preferably within an acceptable time period. A good prognosis for such a disease or condition may more generally include an expectation that such a disease or condition will not further deteriorate or worsen, preferably within a given time period. A poor prognosis for a disease or condition as taught herein may generally include substandard recovery and / or unsatisfactory slow recovery, or substantially no expectation of recovery or even further deterioration of such a disease or condition.

[0190] Thus, prediction or prognosis of a disease or condition may allow, inter alia, the prediction or prognosis of the occurrence of a disease or condition, or the prediction or prognosis of the progression, worsening, remission or recurrence of a disease or condition, or the response to treatment or other external or internal factors, circumstances or stressors.

[0191] Furthermore, monitoring a disease or condition can, inter alia, allow for the prediction of the onset of the disease or condition, or for monitoring the progression, worsening, remission, or recurrence of the disease or condition, or its response to treatment or other external or internal factors, circumstances, or stressors. Advantageously, monitoring can be applied during the course of a subject's medical treatment, preferably a medical treatment intended to alleviate the monitored disease or condition. Such monitoring can be included, for example, in deciding whether a patient can be discharged from the hospital, requires a change in treatment, or requires further hospitalization. As used herein, reference to monitoring a disease or condition also specifically includes monitoring the probability, risk, or chance of a subject developing the disease or condition, i.e., monitoring changes in said probability, risk, or chance over time.

[0192] A related aspect provides a method for in vitro or ex vivo diagnosis, prediction, prognosis and / or monitoring of a disease or condition characterized by abnormal levels of an ACKR3 polypeptide, comprising the steps of

[0193] - obtaining a biological sample from a subject,

[0194] - contacting said biological sample with a peptide as taught herein, wherein said peptide is fused to a detectable label,

[0195] - determining the level of ACKR3 polypeptide in said biological sample by detecting a peptide as taught herein, and

[0196] - Diagnosing, predicting, prognosing and / or monitoring a disease or condition based on the level of ACKR3 protein.

[0197] The term "in vitro" generally means outside or external to the body (e.g., an animal or human body). The term also includes "ex vivo." An example of "in vitro" is tissue cell culture.

[0198] As used herein, the term "sample" or "biological sample" includes any biological sample obtained and separated from a subject. Samples may include, but are not limited to, organ tissue (i.e., tumor tissue, more specifically breast tumor tissue), whole blood, plasma, serum, whole blood cells, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), saliva, urine, feces (i.e., feces), tears, sweat, sebum, nipple aspirates, ductal lavage, tumor exudate, synovial fluid, cerebrospinal fluid, lymph fluid, fine needle aspirates, amniotic fluid, any other body fluid, cell lysate, cell secretion products, inflammatory fluid, semen, and vaginal secretions. Preferably, the sample can be easily obtained by minimally invasive methods such as blood collection or tissue biopsy, thereby allowing the sample to be removed / separated / provided from the subject. As used herein, the term "tissue" encompasses all types of human cells, including organ cells, but also includes blood and the other body fluids mentioned above.

[0199] As used herein, the term "contact" or "contacting" refers to bringing one or more first components (e.g., one or more molecules, biological entities, cells, or materials) and one or more second components (e.g., one or more molecules, biological entities, cells, or materials) together in such a manner that the first component can (if it is capable of) bind to or modulate the second component, or the second component can (if it is capable of) bind to or modulate the first component. Such modulation can occur directly, i.e., through direct interaction between the first and second components; or indirectly, for example, when the first component interacts with or modulates one or more other components, one or more of which in turn interacts with or modulates the second component, or vice versa. Depending on the context, the term "contacting" can be synonymous with "exposing," "incubating," "mixing," "reacting," "treating," and the like.

[0200] In certain embodiments, the peptides or methods used as taught herein may include the steps of comparing the level of an ACKR3 polypeptide in a biological sample from a subject to a given reference value; finding a deviation or lack of deviation between the level of the ACKR3 polypeptide in the biological sample from the subject and the reference value; and attributing the finding of deviation or lack of deviation to a particular diagnostic, predictive, or prognostic step for a disease or condition characterized by abnormal levels of the ACKR3 polypeptide.

[0201] Such comparisons may generally include any means for determining the presence or absence of at least one difference and, optionally, the presence or absence of the magnitude of such a difference between the values ​​or profiles being compared. Comparisons may include visual inspection, arithmetic or statistical comparisons of measurements. Such statistical comparisons include, but are not limited to, application of algorithms.

[0202] Reference values ​​for ACKR3 polypeptide levels can be established using known procedures previously used for other biomarkers. For example, a reference value for the amount of ACKR3 polypeptide for a specific diagnosis, prediction, prognosis, and / or monitoring of a proliferative disease as taught herein can be established by determining the amount or expression level of the ACKR3 polypeptide in a sample from an individual or a population of individuals characterized for the specific diagnosis, prediction, prognosis, and / or monitoring of the disease or condition. Such a population can include, but is not limited to, ≥2, ≥10, ≥100, or even hundreds of individuals or more.

[0203] The skilled artisan will appreciate that the reference value depends on whether the diagnosis, prediction, prognosis, and / or monitoring of a disease or condition characterized by abnormal levels of an ACKR3 polypeptide is envisioned. For example, different reference values ​​may represent the diagnosis of a disease or condition characterized by abnormal levels of an ACKR3 polypeptide versus the absence of a disease or condition characterized by abnormal levels of an ACKR3 polypeptide (e.g., healthy or recovered from a disease or condition characterized by abnormal levels of an ACKR3 polypeptide).

[0204] A "deviation" of a first value from a second value may generally include any direction (e.g., increase: first value > second value; or decrease: first value < second value) and any degree of change. Preferably, a deviation may refer to a statistically significant observed change. For example, the deviation may include, but is not limited to, an increase of at least about 10% (about 1.1 times or more), or at least about 20% (about 1.2 times or more), or at least about 30% (about 1.3 times or more), or at least about 40% (about 1.4 times or more), or at least about 50% (about 1.5 times or more), or at least about 60% (about 1.6 times or more), or at least about 70% (about 1.7 times or more), or at least about 80% (about 1.8 times or more), or at least about 90% (about 1.9 times or more), or at least about 100% (about 2 times or more), or at least about 150% (about 2.5 times or more), or at least about 200% (about 3 times or more), or at least about 500% (about 6 times or more), or at least about 700% (about 8 times or more), or the like, in a first value relative to a second value being compared.

[0205] In a further embodiment, a deviation can be inferred if the observed change exceeds a given threshold or cutoff value. Such a threshold or cutoff value can be selected as is known in the art to increase the sensitivity and / or specificity of the selected prediction method.

[0206] In the methods provided herein, observation of a deviation between the level of an ACKR3 polypeptide in a biological sample from a subject and a reference value can lead to a conclusion that the diagnosis, prediction, and / or prognosis of the proliferative disease in the subject is different from that represented by the reference value. Similarly, when no deviation is observed between the amount or expression level of an ACKR3 polypeptide in a biological sample from a subject and a reference value, the absence of such a deviation can lead to a conclusion that the diagnosis, prediction, and / or prognosis of the proliferative disease in the subject is substantially the same as that represented by the reference value.

[0207] ACKR3 polypeptides are preferentially expressed in cancer cells compared to normal (non-cancerous) cells.

[0208] Thus, in certain embodiments, the disease characterized by abnormal levels of an ACKR3 polypeptide is a proliferative disease, preferably cancer, more preferably a cancer selected from the group consisting of carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, prostate cancer, Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, bladder cancer, penile cancer, urethral cancer, testicular cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid tumor, bone cancer, skin cancer, retinoblastoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. In further embodiments, the disease characterized by abnormal levels of an ACKR3 polypeptide is fibrosis. In a further embodiment, the disease characterized by abnormal levels of ACKR3 polypeptide is atherosclerosis or atherosclerotic plaque formation.

[0209] Another aspect of the present invention relates to a kit for diagnosing, predicting, prognosing and / or monitoring a disease or condition characterized by abnormal levels of an ACKR3 polypeptide in a subject, the kit comprising:

[0210] (a) a peptide as taught herein, preferably wherein the peptide is fused to a detectable label; and

[0211] (b) a reference value for the level of an ACKR3 polypeptide, wherein the reference value represents a known diagnosis, prediction, and / or prognosis of a disease or condition characterized by abnormal levels of an ACKR3 polypeptide, e.g., wherein the reference value corresponds to the level of an ACKR3 polypeptide in a tissue not affected by the disease or condition characterized by abnormal levels of an ACKR3 polypeptide, e.g., in healthy tissue, or wherein the reference value corresponds to the level of an ACKR3 polypeptide in a tissue affected by the disease or condition characterized by abnormal levels of an ACKR3 polypeptide.

[0212] The kit for diagnosing, predicting, prognosing and / or monitoring a disease or condition characterized by abnormal levels of ACKR3 polypeptide in a subject may also include a ready-to-use substrate solution, a wash solution, a dilution buffer and instructions. The diagnostic kit may also include positive and / or negative control samples.

[0213] Preferably, the instructions included in the diagnostic kit are clear, concise and understandable to a person skilled in the art. The instructions typically provide information about the contents of the kit, how to collect tissue samples, methods, experimental readouts and their interpretation, as well as precautions and warnings.

[0214] In certain embodiments, the kit further comprises means for detecting said peptide as taught herein.

[0215] The means for measuring the level of ACKR3 polypeptide in a tissue sample from a subject can include binding agents discussed elsewhere herein and / or supports that allow visualization and / or qualitative readout of the measurement, for example, by spectrophotometry. Alternatively, these supports allow for cascade testing. Non-limiting examples of supports are translucent microtiter plates, translucent strip wells, or translucent tubes.

[0216] Another aspect provides an in vitro method for identifying an agent useful as a therapeutic agent, the method comprising determining whether the test agent is capable of modulating (ie, inducing or inhibiting) recruitment of β-arrestin-1 and / or β-arrestin-2 to an ACKR3 polypeptide.

[0217] Another aspect provides an in vitro method for identifying an agent useful as a therapeutic agent, the method comprising determining whether the test agent is capable of inducing recruitment of β-arrestin-1 and / or β-arrestin-2 to an ACKR3 polypeptide and is not capable of inducing recruitment of β-arrestin-1 and / or β-arrestin-2 to any other receptor polypeptide, e.g., to any opioid receptor polypeptide selected from the group consisting of MOR, DOR, KOR, and NOP receptors, or to any chemokine receptor polypeptide selected from the group consisting of CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR1, CXCR8, ACKR1, ACKR2, or ACKR4.

[0218] As used herein, the term "test agent" refers to any chemical (e.g., inorganic or organic), biochemical or biological substance, molecule or macromolecule (e.g., biomacromolecule), combination or mixture thereof, sample of undefined composition, or extract made from biological material (e.g., bacterial, fungal, plant or animal cells or tissues) for which it is desired to determine whether it specifically binds to and activates an ACKR3 polypeptide.

[0219] Determination of induction (or absence thereof) of beta-arrestin-1 and / or beta-arrestin-2 recruitment to an ACKR3, MOR, DOR, KOR, NOP, CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, or ACKR4 polypeptide can be determined as described elsewhere herein.

[0220] When reference is made herein to the recruitment of β-arrestin-1 and / or β-arrestin-2 to a receptor, this refers to the recruitment of β-arrestin-1 and / or β-arrestin-2 to the receptor induced or mediated by the binding of the agent to the same receptor. Thus, it does not include any indirect effects on other receptors caused by the binding of the agent to the receptor, for example, as a result of the scavenger function of the receptor. For example, the recruitment of β-arrestin-1 and / or β-arrestin-2 to the MOR polypeptide does not include the recruitment of β-arrestin-1 and / or β-arrestin-2 to the MOR polypeptide caused by the binding of a peptide as taught herein to the ACKR3 receptor, resulting in increased availability of endogenous opioid peptides to the MOR polypeptide.

[0221] In certain embodiments, an in vitro method for identifying an agent disclosed herein for use as a therapeutic agent comprises contacting the test agent with a cell capable of recruiting β-arrestin-1 and β-arrestin-2 to an ACKR3 polypeptide, and selectively measuring the recruitment of β-arrestin-1 and / or 1-arrestin-2 to an ACKR3 polypeptide, and contacting the test agent with a cell capable of recruiting β-arrestin-1 and / or 1-arrestin-2 to a MOR, DOR, KOR, NOP, CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CX ... R4, CXCR5, CXCR6, CXCR8, XCR1, CX3RC1, ACKR1, ACKR2 and / or ACKR4 polypeptides, and selectively measures the recruitment of said beta-arrestin-1 and / or beta-arrestin-2 to MOR, DOR, KOR, NOP, CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2 and / or ACKR4 polypeptides.

[0222] Selective measurement of β-arrestin-1 and / or β-arrestin-2 recruitment to a receptor can be performed as described elsewhere herein. For example, ACKR3 C-terminally fused to SmBi and β-arrestin N-terminally fused to LgBiT can be used to selectively measure β-arrestin-1 and / or β-arrestin-2 recruitment to an ACKR3 polypeptide. Similarly, selective measurement of β-arrestin-1 and / or β-arrestin-2 recruitment to a MOR, DOR, KOR, NOP, CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, or ACKR4 polypeptide can be performed using The results were performed by C-terminally fused to a MOR, DOR, KOR, NOP, CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, or ACKR4 polypeptide of SmBi and N-terminally fused to a β-arrestin of LgBiT. Alternatively, an ACKR3, MOR, DOR, KOR, NOP, CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CRCX5, CRCX6, CRCX8, XCR1, CX3CR1, ACKR1, ACKR2, or ACKR4 polypeptide can be C-terminally fused to LgBiT and β-arrestin can be C-terminally or N-terminally fused to SmBiT. In these examples, the interaction between a SmBiT-tagged receptor polypeptide and LgBiT-tagged β-arrestin, or the interaction between a SmBiT-tagged β-arrestin and a LgBiT-tagged receptor polypeptide was determined.

[0223] Cells capable of recruiting β-arrestin-1 and β-arrestin-2 to the ACKR3 receptor are typically cells that contain β-arrestin-1 and β-arrestin-2 in their cytosol and express ACKR3 polypeptides on their plasma membrane. Cells capable of recruiting β-arrestin-1 and β-arrestin-2 to the MOR, DOR, KOR and / or NOP receptors are typically cells that contain β-arrestin-1 and β-arrestin-2 in their cytosol and express MOR, DOR, KOR and / or NOP polypeptides on their plasma membrane. Cells that are able to recruit beta-arrestin-1 and beta-arrestin-2 to the CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CRCX4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, and / or ACKR4 receptors are typically The gel contains cells that contain β-arrestin-1 and β-arrestin-2 and express CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2 and / or ACKR4 polypeptides on their plasma membrane.

[0224] In a specific embodiment, when the recruitment of β-arrestin-1 and / or β-arrestin-2 to a receptor is selectively measured by a nanoluciferase complementation assay (e.g., NanoBiT, Promega), the cells capable of recruiting β-arrestin-1 and β-arrestin-2 to the ACKR3 receptor are typically cells that express LgBiT-tagged or SmBiT-tagged β-arrestin-1 and / or LgBiT-tagged or SmBiT-tagged β-arrestin-2 in their cytosol and SmBiT-tagged or LgBiT-tagged ACKR3 polypeptide on their plasma membrane. Cells capable of recruiting β-arrestin-1 and β-arrestin-2 to MOR, DOR, KOR and / or NOP receptors are typically cells that contain LgBiT-tagged or SmBiT-tagged β-arrestin-1 and / or LgBiT-tagged or SmBiT-tagged β-arrestin-2 in their cytosol and express SmBiT-tagged or LgBiT-tagged MOR, DOR, KOR and / or NOP polypeptides on their plasma membrane. Cells capable of recruiting β-arrestin-1 and β-arrestin-2 to CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CRCX4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, and / or ACKR4 receptors typically contain LgBiT-tagged or SmBiT-tagged β-arrestin in the cytosol. In some embodiments, the present invention relates to cells that express a β-arrestin-1 and / or β-arrestin-2 tagged with LgBiT or SmBiT and that express a SmBiT-tagged or LgBiT-tagged CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, and / or ACKR4 polypeptide on their plasma membrane. The skilled artisan will understand that in this embodiment, if β-arrestin-1 and / or β-arrestin-2 are tagged with LgBiT, the receptor polypeptide must be tagged with SmBiT, and vice versa. As described elsewhere herein, the absence or non-comprising or non-containing of a receptor polypeptide in this context does not in itself mean the complete absence of the receptor polypeptide on the cell membrane, but rather means that the amount of the receptor polypeptide cannot be detected by a protein assay known to those skilled in the art or is below the sensitivity range of a protein assay known to those skilled in the art.

[0225] Primary screening can be performed by screening the test agent for its ability to bind to the ACKR3 polypeptide.

[0226] In certain embodiments, in vitro methods for identifying agents useful as therapeutic agents as disclosed herein comprise determining whether the test agent is capable of binding to an ACKR3 polypeptide, and optionally determining whether the test agent is capable of binding to any other receptor polypeptide, such as a MOR, DOR, KOR, NOP, CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CRCX5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2 and / or ACKR4 polypeptide.

[0227] Binding assays may involve contacting an ACKR3 polypeptide, a MOR polypeptide, a DOR polypeptide, a KOR polypeptide, a NOP polypeptide, a CCR1 polypeptide, a CCR2A polypeptide, a CCR2B polypeptide, a CCR3 polypeptide, a CCR4 polypeptide, a CCR5 polypeptide, a CCR6 polypeptide, a CCR7 polypeptide, a CCR8 polypeptide, a CCR9 polypeptide, a CCR10 polypeptide, a CXCR1 polypeptide, a CXCR2 polypeptide, a CXCR3A polypeptide, a CXCR3B polypeptide, a CXCR4 polypeptide, a CXCR5 polypeptide, a CXCR6 polypeptide, a CXCR8 polypeptide, an XCR1 polypeptide, a CX3CR1 polypeptide, an ACKR1 polypeptide, an ACKR2 polypeptide, or an ACKR4 polypeptide with a test agent and allowing sufficient time for the test agent to react. Sufficient time is allowed for the test agent and the ACKR3 polypeptide, MOR polypeptide, DOR polypeptide, KOR polypeptide, NOP polypeptide, CCR1 polypeptide, CCR2A polypeptide, CCR2B polypeptide, CCR3 polypeptide, CCR4 polypeptide, CCR5 polypeptide, CCR6 polypeptide, CCR7 polypeptide, CCR8 polypeptide, CCR9 polypeptide, CCR10 polypeptide, CXCR1 polypeptide, CXCR2 polypeptide, CXCR3A polypeptide, CXCR3B polypeptide, CXCR4 polypeptide, CXCR5 polypeptide, CXCR6 polypeptide, CXCR8 polypeptide, XCR1 polypeptide, CX3CR1 polypeptide, ACKR1 polypeptide, ACKR2 polypeptide, or ACKR4 polypeptide to form a binding complex. Formation of the binding complex can be detected using any established analytical technique for determining protein-protein binding, such as binding competition assays using fluorescently or radioactively labeled ligands (e.g., fluorescently or radioactively labeled chemokines, such as CXCL12), co-immunoprecipitation, bimolecular fluorescence complementation, label transfer, tandem affinity purification, chemical cross-linking, and fluorescence resonance energy transfer. Protein binding assays can be performed in cell-free systems or cell lysates or in isolated or cultured cells or in isolated or cultured tissues.

[0228] In a binding assay, one or more of the agent, ACKR3 polypeptide, MOR polypeptide, DOR polypeptide, KOR polypeptide, NOP polypeptide, CCR1 polypeptide, CCR2A polypeptide, CCR2B polypeptide, CCR3 polypeptide, CCR4 polypeptide, CCR5 polypeptide, CCR6 polypeptide, CCR7 polypeptide, CCR8 polypeptide, CCR9 polypeptide, CCR10 polypeptide, CXCR1 polypeptide, CXCR2 polypeptide, CXCR3A polypeptide, CXCR3B polypeptide, CXCR4 polypeptide, CXCR5 polypeptide, CXCR6 polypeptide, CXCR8 polypeptide, XCR1 polypeptide, CX3CR1 polypeptide, ACKR1 polypeptide, ACKR2 polypeptide, and / or ACKR4 polypeptide can be conjugated to a label that can directly or indirectly provide a detectable signal. Non-limiting examples of such labels or signals include radioisotopes, fluorescent labels or signals, chemiluminescent labels or signals, enzymes, specific binding molecules, or particles (e.g., magnetic particles).

[0229] In certain embodiments, the in vitro methods for identifying agents useful as therapeutic agents as disclosed herein may further comprise the use of one or more reagents that improve assay efficiency, promote optimal protein-protein binding, and / or reduce nonspecific or background interactions. Non-limiting examples of such reagents are salts, neutral proteins (e.g., albumin), detergents, protease inhibitors, nuclease inhibitors, and antimicrobial agents.

[0230] In specific embodiments, the level of recruitment of β-arrestin-1 and / or 1-arrestin-2 to the ACKR3 polypeptide induced by the test agent can be compared to the level of recruitment of β-arrestin-1 and / or 1-arrestin-2 to the ACKR3 polypeptide induced by a peptide as taught herein.

[0231] In certain embodiments, the binding affinity of a test agent to an ACKR3 polypeptide can be compared to the binding affinity of a peptide as taught herein to an ACKR3 polypeptide.

[0232] In certain embodiments, a test agent can be identified as an agent for use as a therapeutic agent disclosed herein if the agent induces at least 1.5-fold greater, at least 2-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 4.5-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, or at least 30-fold greater recruitment of β-arrestin-1 and / or β-arrestin-2 to the ACKR3 polypeptide as compared to baseline β-arrestin-1 and / or β-arrestin-2 recruitment or background β-arrestin-1 and / or β-arrestin-2 recruitment to the ACKR3 polypeptide induced by a neutral substance or a negative control, e.g., as measured in an assay described elsewhere herein.

[0233] In certain embodiments, a test agent can be identified as an agent useful as a therapeutic agent as disclosed herein if:

[0234] if the agent induces recruitment of beta-arrestin-1 and / or beta-arrestin-2 to the ACKR3 polypeptide that is equal to or greater than recruitment of beta-arrestin-1 and / or beta-arrestin-2 to the ACKR3 polypeptide induced by a peptide taught herein, e.g., at least 1.1-fold greater, at least 1.2-fold greater, at least 1.3-fold greater, at least 1.4-fold greater, at least 1.5-fold greater, at least 2-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 4.5-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, or at least 30-fold greater, e.g., as measured in an assay described elsewhere herein; and / or

[0235] - If the agent binds to the ACKR3 polypeptide with an affinity that is equal to or greater than the binding affinity of a peptide as taught herein to the ACKR3 polypeptide, e.g., at least 1.1-fold greater, at least 1.2-fold greater, at least 1.3-fold greater, at least 1.4-fold greater, at least 1.5-fold greater, at least 2.-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 4.5-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, or at least 30-fold greater.

[0236] Selective ACKR3 modulating peptides as taught herein can be used in competitive binding studies to identify ACKR3 antagonists or positive / negative allosteric modulators. For example, ACKR3 can be pre-treated with a test agent prior to exposing it to a selective ACKR3 modulating peptide as taught herein. If the recruitment of β-arrestin-1 and / or β-arrestin-2 to the ACKR3 polypeptide is inhibited following exposure to a selective ACKR3 modulating peptide as taught herein, the test agent can be identified as an antagonist or positive / negative allosteric modulator of the ACKR3 polypeptide.

[0237] Thus, another aspect provides an in vitro method for identifying an agent useful as a therapeutic agent, the method comprising determining whether the test agent is capable of modulating, preferably inhibiting, the recruitment of β-arrestin-1 and / or β-arrestin-2 to an ACKR3 polypeptide by a selective ACKR3 modulating peptide taught herein.

[0238] In certain embodiments, in vitro methods for identifying agents useful as therapeutic agents as disclosed herein comprise determining whether a test agent is capable of specifically binding to an ACKR3 polypeptide.

[0239] The present inventors have discovered that ACKR3 acts as a scavenger of endogenous opioid peptides, regulating local and / or systemic concentrations, thereby modulating the availability of classical opioid receptors. Therefore, all agents that specifically bind to ACKR3 polypeptides and / or specifically induce the recruitment of β-arrestin-1 and / or β-arrestin-2 to ACKR3 polypeptides can be used to modulate the availability of endogenous opioid peptides to other opioid receptors, including MOR, KOR, DOR, and NOP polypeptides.

[0240] Thus, another aspect provides a therapeutic or prophylactic agent for treating an adverse stress dysfunction disease or condition in a subject, wherein the therapeutic or prophylactic agent is capable of modulating (e.g., inducing or antagonizing), preferably capable of inducing, the recruitment of beta-arrestin-1 and / or beta-arrestin-2 to an ACKR3 polypeptide, and is incapable of inducing the recruitment of beta-arrestin-1 and / or beta-arrestin-2 to any other receptor polypeptide, including any opioid receptor polypeptide selected from the group consisting of MOR, DOR, KOR, and NOP receptors, and any chemokine receptor polypeptide selected from the group consisting of CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, and ACKR4.

[0241] The term "treat" or "treatment" includes therapeutic treatment of an already developed disease or condition, as well as prophylactic or preventative measures, wherein the purpose is to prevent or reduce the chance of an undesirable ailment occurring. Beneficial or desired clinical results may include, but are not limited to, relief of one or more symptoms or one or more biomarkers, reduction in the extent of the disease, a stable (i.e., non-worsening) state of the disease, a delay or slowing of disease progression, improvement or alleviation of the disease state, and the like. "Treatment" may also mean prolonging survival compared to the expected survival if not receiving treatment.

[0242] In a specific embodiment, the adverse stress dysfunction disease or condition is selected from the group consisting of anxiety, depression, anger, insomnia, mood disorders, substance and behavioral addictions (e.g., opiate, cocaine or alcohol abuse and / or dependence) and eating disorders (e.g., anorexia). In a preferred embodiment, the adverse stress dysfunction disease or condition is selected from the group consisting of anxiety and depression.

[0243] In a specific embodiment, the therapeutic or prophylactic agent is selected from the group consisting of a chemical, an antibody, an antibody fragment, an antibody-like protein scaffold, a protein or polypeptide, a peptide, a peptide mimetic, an aptamer, a photoaptamer, a spiegelmer and a nucleic acid, preferably wherein the agent is a protein or polypeptide or peptide.

[0244] Methods for determining whether the agent is capable of inducing recruitment of β-arrestin-1 and / or β-arrestin-2 to the ACKR3 polypeptide, and is unable to induce recruitment of β-arrestin-1 and / or β-arrestin-2 to any other receptor polypeptide are known to those of skill in the art, as described elsewhere herein, wherein the any other receptor polypeptide includes any opioid receptor polypeptide selected from the group consisting of MOR, DOR, KOR, and NOP receptors, and any chemokine receptor polypeptide selected from the group consisting of CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, and ACKR4.

[0245] A related aspect provides a method for treating an adverse stress dysfunction disease or condition in a subject, comprising administering to the subject a therapeutically and / or prophylactically effective amount of a therapeutic or prophylactic agent, wherein the therapeutic or prophylactic agent is capable of inducing recruitment of β-arrestin-1 and / or β-arrestin-2 to an ACKR3 polypeptide and is incapable of inducing recruitment of β-arrestin-1 and / or β-arrestin-2 to any other receptor polypeptide, wherein the any other receptor polypeptide includes any opioid receptor polypeptide selected from the group consisting of MOR, DOR, KOR, and NOP receptors, and any chemokine receptor polypeptide selected from the group consisting of CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, and ACKR4.

[0246] As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic agent that will elicit the biological or medical response in a subject that is being sought by a surgeon, researcher, veterinarian, medical doctor, or other clinician, which may include, among other things, alleviation of the symptoms of the disease or condition being treated. The term "prophylactically effective amount" refers to the amount of a prophylactic agent that will inhibit or delay the onset of a condition in a subject that is being sought by a researcher, veterinarian, medical doctor, or other clinician. Methods for determining a therapeutically and / or prophylactically effective amount of the therapeutic or prophylactic agents described herein are known in the art.

[0247] The peptides as taught herein can be used to treat diseases or disorders that are at least in part dependent on ACKR3 activity.

[0248] ACKR3 acts as a scavenger of chemokines, regulating local and / or systemic concentrations, thereby regulating the availability of other chemokine receptors. Thus, the peptides taught herein can be used to regulate the availability of endogenous (e.g., CXCL11 or CXCL12) or exogenous (e.g., vCCL2 (vMIP-II)) chemokines to other chemokine receptors, wherein the other chemokine receptors include CCR1, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR8, XCR1, CX3CR1, ACKR1, ACKR2, and ACKR4 polypeptides. Thus, a peptide as taught herein, a fusion protein as taught herein, a nucleic acid encoding an agent or fusion protein as taught herein, a nucleic acid expression cassette as taught herein, a vector as taught herein, or a pharmaceutical composition as taught herein can be used to treat diseases or conditions in which these endogenous or exogenous chemokines play a role.

[0249] Thus, another aspect provides a peptide as taught herein, a fusion protein as taught herein, a nucleic acid encoding an agent or fusion protein as taught herein, a nucleic acid expression cassette as taught herein, a vector as taught herein or a pharmaceutical composition as taught herein for use as a medicament.

[0250] ACKR3 plays a key role in controlling angiogenesis, such as in cancer. Thus, the present invention encompasses reducing angiogenesis in any subject in need thereof (e.g., a subject suffering from a disease or disorder involving excessive or abnormal angiogenesis) by administering a peptide as taught herein.

[0251] On the other hand, a peptide as taught herein, a fusion protein as taught herein, a nucleic acid encoding an agent or fusion protein as taught herein, a nucleic acid expression cassette as taught herein, a vector as taught herein, or a pharmaceutical composition as taught herein for use in treating a disease or condition in a subject, wherein the disease or condition is selected from the group consisting of adverse stress dysfunction diseases or conditions, cancer, atherosclerotic vascular disease (or atherosclerosis), cardiovascular disease, fibrosis (e.g., cardiac fibrosis), inflammatory or autoimmune diseases and conditions, conditions of excessive or abnormal vascularization (e.g., wound healing and HIV infection), stem cell differentiation and mobilization disorders, brain and neuronal dysfunction (e.g., Alzheimer's disease, multiple sclerosis, and demyelinating diseases), renal dysfunction, renal insufficiency, pre-eclampsia, and obesity, preferably a disease or condition selected from the group consisting of adverse stress dysfunction diseases or conditions, cancer, atherosclerotic vascular disease, cardiovascular disease, and fibrosis.

[0252] Non-limiting examples of inflammatory or autoimmune diseases and disorders include inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, polyarthritis, nephritic disease, multiple sclerosis, colitis, allergic diseases, psoriasis, atopic dermatitis, and asthma. A related aspect provides a method of treating a disease or condition in a subject comprising administering a therapeutically and / or prophylactically effective amount of a peptide as taught herein, a fusion protein as taught herein, a nucleic acid encoding an agent or fusion protein as taught herein, a nucleic acid expression cassette as taught herein, a vector as taught herein, or a pharmaceutical composition as taught herein, wherein the disease or condition is selected from the group consisting of adverse stress dysfunction diseases or conditions, cancer, atherosclerotic vascular disease (or atherosclerosis), cardiovascular disease, fibrosis (e.g., cardiac fibrosis), inflammatory or autoimmune diseases and conditions, conditions of excessive or abnormal vascularization (e.g., wound healing and HIV infection), stem cell differentiation and mobilization disorders, brain and neuronal dysfunction (e.g., Alzheimer's disease, multiple sclerosis, and demyelinating diseases), renal dysfunction, renal insufficiency, pre-eclampsia, and obesity, preferably selected from the group consisting of adverse stress dysfunction diseases or conditions, cancer, atherosclerotic vascular disease, cardiovascular disease, and fibrosis.

[0253] Another aspect provides the use of a peptide as taught herein, a fusion protein as taught herein, a nucleic acid encoding an agent or fusion protein as taught herein, a nucleic acid expression cassette as taught herein, a vector as taught herein, or a pharmaceutical composition as taught herein for reducing tumor cell proliferation, tumor formation, tumor vascularization, and metastasis.

[0254] Another aspect provides the use of a peptide as taught herein, a fusion protein as taught herein, a nucleic acid encoding an agent or fusion protein as taught herein, a nucleic acid expression cassette as taught herein, a vector as taught herein or a pharmaceutical composition as taught herein for increasing T cell recruitment in a subject.

[0255] Another aspect provides the use of a peptide as taught herein, a fusion protein as taught herein, a nucleic acid encoding an agent or fusion protein as taught herein, a nucleic acid expression cassette as taught herein, a vector as taught herein or a pharmaceutical composition as taught herein for reducing viral multiplication in a subject.

[0256] In specific embodiments, peptides as taught herein, fusion proteins as taught herein, nucleic acids encoding agents or fusion proteins as taught herein, nucleic acid expression cassettes as taught herein, vectors as taught herein, or pharmaceutical compositions as taught herein are used in combination with agents known to treat the aforementioned diseases or conditions. While the present invention has been described in conjunction with specific embodiments thereof, it is apparent that many alternatives, modifications, and variations will be apparent to those skilled in the art based on the foregoing description. Therefore, it is intended that all such alternatives, modifications, and variations as follows be encompassed within the spirit and broad scope of the appended claims.

[0257] The following non-limiting examples further support the aspects and embodiments of the invention disclosed herein. Example

[0258] Example 1. Materials and methods used in Examples 2 to 7.

[0259] 1.1. Peptides and chemokines

[0260] Unlabeled chemokines CXCL12, CXCL11, and vCCL2 were purchased from PeproTech. Alexa Fluor 647-labeled CXCL12 (CXCL12-AF647) was purchased from Almac. The opioid peptide library and all opioid peptides, as well as FAM-labeled dynorphin A (1-13) and FAM-labeled nociceptin, were from Phoenix Pharmaceuticals. BAM22 and large dynorphin labeled with Cy5 were generated using the Amersham QuickStain Cy5 kit according to the manufacturer's protocol. Adrenorphin-derived peptides were synthesized by JPT. These peptides contain a free amine at the N-terminus and an amide group at the C-terminus to avoid additional negative charges. All non-peptide opioids were obtained from Tocris, except for levallorphan, which was purchased from Sigma.

[0261] Cell culture

[0262] U87 cells, derived from human glioblastoma, were obtained through the NIH AIDS Reagent Program of Dr. Deng and Dr. Littman. U87 cells stably expressing ACKR3 and CXCR4 were generated as previously described by Szpakowska, M. et al. The different contributions of chemokine N-terminal features demonstrate distinct ligand binding modes and a preference for ACKR3 / CXCR7 activation compared to CXCR4 and CXCR3. Br J Pharmacol 175, 1419-1438 (2018). U87 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum and penicillin / streptomycin (100 units / ml and 100 μg / ml). U87-ACKR3 and U87-CXCR4 were maintained under puromycin (1 μg / ml) selection pressure. smNPCs (small-molecule neural precursor cells) from a healthy donor (C1-1) who obtained informed consent were grown on Geltrex™-coated surfaces in N2B27 medium supplemented with 0.5 μM 2,6,9-trisubstituted purine (Purmorphamine), 3 μM CHIR99021, and 150 μM ascorbic acid. N2B27 medium consists of a 50:50 ratio of DMEM / F12 and NeuroBasal medium, containing 0.5% N2 supplement, 1% B27 supplement lacking vitamin A, 1% GlutaMAX, and 1% penicillin / streptomycin. Medium was refreshed every other day.

[0263] 1.3. Binding competition assay

[0264] U87-ACKR3 cells were distributed into 96-well plates (1.5 × 10 cells per well). 5 Cells) were incubated on ice with a mixture of 5 nM CXCL12-AF647 and the indicated concentrations of unlabeled chemokines or opioid peptides for 90 minutes, then washed twice with FACS buffer (PBS, 1% BSA, 0.1% NaN3) at 4°C. Dead cells were excluded using Zombie Green viability dye (BioLegend). ACKR3-negative U87 cells were used to assess nonspecific binding of CXCL12-AF647. 0% receptor binding of CXCL12-AF647 was defined as the signal obtained after adding 1 μM unlabeled CXCL12. The signal obtained for CXCL12-AF647 in the absence of unlabeled chemokines was used to define 100% binding. Ligand binding was quantified by mean fluorescence intensity on a BD FACS Fortessa cytometer (BD Biosciences).

[0265] Nanoluciferase complementation assay

[0266] As previously described by Szpakowska, M. et al., ligand-induced recruitment of β-arrestin to chemokine and opioid receptors was monitored by the NanoLuc complementation assay (NanoBiT, Promega). Mutational analysis of the extracellular disulfide bonds of the atypical chemokine receptor ACKR3 / CXCR7 revealed its diverse binding and activation modes for chemokines and endogenous non-chemokine agonists. Biochem Pharmacol 153, 299-309 (2018). Briefly, 1.2×10 6 U87 cells were seeded in 10 cm culture dishes and co-transfected 48 hours later with pNBe vectors encoding a GPCR tagged C-terminally to SmBiT and N-terminally to LgBiT fused to β-arrestin-1 or -2 or a small G protein (mG, an engineered GTPase domain of the Gα subunit). Cells were harvested 48 hours after transfection, incubated with 200-fold diluted Nano-Glo Live Cell substrate at 37°C for 25 minutes, and then distributed into white 96-well plates (5 × 10 cells per well). 4 cells). Ligand-induced recruitment of β-arrestin and mG to GPCRs was assessed for 20 minutes using a Mithras LB940 luminometer (Berthold Technologies). For concentration-response curves, the signal recorded at the saturating concentration of the full agonist for each receptor was set to 100%. To assess the antagonist properties of the ligands, full agonists for each receptor (50 nM BAM22 for MOR, 50 nM dynorphin A for KOR, 70 nM methionine enkephalin for DOR, 70 nM nociceptin for NOP, and 4 nM CXCL12 for ACKR3) were added after 20 minutes of incubation with the ligands. The signal from the wells treated with the full agonist only was defined as 0% inhibition, while the signal from the wells not treated with the agonist was used to set 100% inhibition. For the dynorphin A clearance experiment using U87 cells, 1.5 × 10 5 U87 or U87.ACKR3 cells were incubated with 400 nM LIH383 or LIH383 control peptide (200 nM CXCL12 or CXCL10) for 15 min at 37°C, and then dynorphin A was added at concentrations ranging from 0.15 nM to 3 μM and incubated at 37°C for 25 min. 4U87 cells, co-transfected with SmBiT-labeled KOR and LgBiT-labeled β-arrestin-1 or small Gi 48 hours before the experiment and pre-incubated with Nano-Glo Live substrate for 25 minutes, were added to each well and the signal was measured for 20 minutes. 6 smNPCs were pretreated with 1.5 μM LIH383 or LIH383 control peptide (300 nM CXCL12 or CXCL10) for 15 minutes and then incubated for 4 hours with 3 μM dynorphin A. The cells were centrifuged and the activity of the dynorphin A remaining in serially diluted supernatants was determined on U87 cells expressing SmBiT-tagged KOR and LgBiT-tagged small Gi proteins.

[0267] 1.5. Label-free dynamic mass redistribution (DMR) assay

[0268] Dynamic mass redistribution (DMR) experiments were performed using the Corning Epic (Corning) biosensor system as previously described in Deconvolution of complex Gprotein-coupled receptorsignaling in live cells using dynamic mass redistribution measurements. NatBiotechnol 28, 943-949 (2010) and et al. Applying label-free dynamic mass redistribution technology to frame sigaling of G protein-coupled receptors noninvasively in living cells. Nat Protoc. 6(11): 1748-1760(2011). In short, 6×10 5 U87 cells were seeded in 6 cm culture dishes. 24 hours later, cells were transfected with pcDNA3.1-based expression plasmids encoding the corresponding chemokine (ACKR3, CXCR4) or opioid (KOR, NOP) receptors using polyethyleneimine (PEI) reagent (Polysciences). 24 hours after transfection, 1 × 10 4The cells were transferred to a 384-well Epic biosensor plate and incubated overnight in (37°C, 5% CO2). The cells were then washed twice with Hanks balanced salt solution (HBSS) (Life Technologies) containing 20mM HEPES (Life Technologies) and subsequently incubated for 1.5 hours in a DMR reader to reach temperature equilibrium (37°C). After the baseline DMR trace was balanced for five minutes, the compound was added to the biosensor plate. The changes in ligand-induced DMR were monitored for at least 3000 seconds. GraphPad Prism 7.05 (GraphPad Inc) was used to process and analyze the raw data. For quantification, the negative and positive areas of the area under the curve (AUC) between 0 and 3000 seconds were used.

[0269] 1.6. Extracellular signal-regulated kinase 1 and 2 (ERK1 / 2) phosphorylation assay based on homogeneous time-resolved fluorescence (HTRF)

[0270] Homogeneous time-resolved fluorescence (HTRF)-based phosphorylated ERK1 / 2 and total ERK1 / 2 assays were performed using the Phospho-ERK1 / 2 (Thr202 / Tyr204) and Total ERK1 / 2 Cell Kits (Cisbio International). Briefly, to quantify total and phosphorylated ERK1 / 2 protein, U87 cells stably expressing (or not expressing) ACKR3 or CXCR4 were plated at 3.5 × 10 cells per well. 4 Cells were seeded at a density of 100 cells per well into 96-well poly-D-lysine (PDL)-coated microtiter plates (Sigma-Aldrich). After overnight incubation, cells were starved for 4 hours at 37°C in serum-free medium. Cells were then stimulated with chemokines or opioid ligands for the indicated time intervals, terminated by replacing the supernatant with lysis buffer and incubating for 1.5 hours on an orbital shaker. Lysates were transferred to white 384-well plates, and D2-labeled anti-phospho-ERK1 / 2 and Eu were added to each well. 3+ - Cryptate-labeled anti-phospho-ERK1 / 2 antibody was used for phosphorylated ERK1 / 2 assay. D2-labeled anti-total ERK1 / 2 and Eu 3+ -Cryptate-labeled anti-total ERK1 / 2 antibody was used to quantify total ERK1 / 2 abundance. After incubation for at least 2 hours (phospho-ERK1 / 2) or 24 hours (total ERK1 / 2), time-resolved FRET signals were measured using a Mithras LB 940 multimode reader (Berthold Technologies) equipped with a 320 nm excitation filter and 620 nm (donor) and 665 nm (acceptor) emission filters.

[0271] 1.7. Transcriptional Nanoluciferase Reporter Gene Assay

[0272] The serum response element (SRE) nanoluciferase reporter gene assay was used to assess the activation of the MAPK / ERK signaling pathway. The nuclear factor of activated T cell response element (NFAT-RE) nanoluciferase reporter gene assay was used to assess the activation of the calcium-dependent signaling pathway. For both assays, 1.2 × 10 6 U87 cells were seeded in 10-cm culture dishes and 48 h later co-transfected with pNanoLuc / SRE or pNanoLuc / NFAT-RE vectors (Promega), which contained the Nanoluciferase gene downstream of SRE or NFAT-RE and pcDNA3.1 encoding the respective chemokine or opioid receptor. 24 h later, 2.5 × 10 4 cells / well (for smNPC, 2.5×10 5 ) were seeded in white 96-well plates. After 24 hours, the culture medium was replaced with serum-free and phenol red-free DMEM (serum-free DMEM / F12 for smNPC) and incubated for a further two hours. Opioid peptides (500nM) and chemokines (200nM) were then added to the cells and incubated for six hours. 30nM porphyrinol 12-myristate 13-acetate (PMA), 10% FBS or 30nM PMA, 1uM ionomycin, 10% FBS were used as positive controls for SRE and NFAT-RE determinations, respectively. Nano-Glo Live Cell substrate (Promega) was then added and luminescence was read on a MithrasLB940 microplate reader (Berthold Technologies) for 20 minutes.

[0273] 1.8. Visualization of Fluorescently Labeled Opioid Peptide Uptake by Imagestream

[0274] Cells were distributed into 96-well plates (2 × 10 for U87 and U87-ACKR3 in Opti-MEM). 5 cells / well, and for smNPCs, 3 × 10 5 After incubation with LIH383 (3 μM) or Opti-MEM alone at 37°C for 15 minutes, FAM-labeled dynorphin A (1-13) (250 nM), BAM22-Cy5 (400 nM), big dynorphin-Cy5 (400 nM), or nociceptin-FAM (1 μM) were added, incubated at 37°C for 40 minutes, and washed twice with FACS buffer. To compare the uptake of labeled opioid peptides by ACKR3 or classical opioid receptors, 1.2 × 106 U87 cells were seeded in 10-cm dishes and transfected 48 hours later with 4 μg of pcDNA3.1 plasmid encoding ACKR3, KOR, MOR, or NOP. 48 hours after transfection, cells were harvested and treated as described above. Dead cells were excluded using Zombie NIR or Zombie Green viability dye (BioLegend) for FAM-labeled peptides and Cy5-labeled peptides, respectively. 1 × 10 cells were acquired using an ImageStream MKII imaging flow cytometer (Amnis) at 40× magnification (60× magnification for smNPCs). 4 In-focus live single-cell images. Samples were analyzed using Ideas 6.2 software. The number of dots per cell was determined using a mask-based software wizard.

[0275] 1.9. Depolarization of isolated rat neurons

[0276] 1.9.1. Animals

[0277] Adult male Wistar rats (6 to 8 weeks old) were housed in groups of three or four at room temperature on a 12:12 h light-dark cycle. All animals had ad libitum access to food and water. All procedures were performed in accordance with the guidelines of the European Communities Council Directive of 24 November 1986 (86 / 609 / EE) and were approved by the Ethical Committee on the Use of Animals of the University of Liège (protocol 2061). All efforts were made to minimize animal suffering.

[0278] 1.9.2. Brain Explant Preparation and Recording Procedure

[0279] Rats were anesthetized with chloral hydrate (400 mg / kg, ip) and placed under a cap in oxygenated air (95% O2, 5% CO2) two minutes before decapitation. Following decapitation, the brain was rapidly removed and placed in ice-cold (~2°C) oxygenated artificial cerebrospinal fluid (aCSF) with the following composition: NaCl 130 mM, KCl 3.5 mM, NaH2PO4 1.25 mM, NaHCO3 24 mM, glucose 10 mM, CaCl2 2 mM, MgSO4 1.25 mM. The tissue block containing the pons was placed in a vibrating blade microtome (Vibratome 1000 Plus, Slicing Systems), and a coronal section (400 μm thick) was cut containing the locus coeruleus (LC) adjacent to the fourth ventricle and seventh nerve, which served as an anatomical landmark. The slice was placed on a nylon mesh in a recording chamber (volume: 0.5 ml), where oxygenated aCSF (34.0 ± 0.5°C) was perfused at a rate of 2 to 3 ml / min. During transillumination, the LC was identified as a translucent area located lateral to the fourth ventricle. All experiments were performed in oxygenated aCSF with synaptic blockers consisting of 10 μM CNQX, 10 μM SR95531, 1 μM MK801, and 1 μM CGP55845, which block AMPA / Kainate, GABAA, NMDA, and GABAB receptors, respectively. This ensured that the spontaneous firing of neurons was solely due to their endogenous pacemaking.

[0280] Extracellular single-cell recordings of LC neurons were performed using glass microelectrodes filled with aCSF (resistance 10-20 MΩ). Signals were amplified 1000-fold using a homemade amplifier via an impedance adapter. They were displayed on a Fluke Combiscope oscilloscope and fed into an analog-to-digital interface (CED1401, Cambridge Electronic Design, Cambridge, UK) connected to a computer. Data were collected and analyzed using Spike2 software (Cambridge Electronic Design). The firing rate of all recorded neurons was 0.5 to 3 Hz with good regularity (coefficient of variation of the interspike interval was 0.13 ± 0.01, N = 18), and firing ceased during application of the α2-adrenergic receptor agonist clonidine (10-20 nM). The duration of the extracellularly recorded action potential was 2-3 milliseconds. Drugs and peptides were applied for at least 10 minutes.

[0281] The average firing rate over 1 minute was calculated for each condition. Next, the inhibition of firing by the peptides and drugs used (LIH383 and dynorphin) was quantified as a percentage of the total inhibition. To this end, the inventors considered the average firing rate during the last minute of each condition (control, LIH383 alone, LIH383 plus a given concentration of dynorphin). The Hill equation (E / E) in GraphPad Prism (version 6) was used. max =[dynorphin] / EC 50 (dynorphin) + [dynorphin] to obtain the EC of dynorphin 50 EC values ​​under different conditions were compared using the Kruskal-Wallis test. 50 One outlier (559 nM in the LIH383 3 μM group, which differed by >2 SD from the mean of the group) was omitted.

[0282] RNA extraction and quantitative PCR on human brain samples and smNPCs

[0283] As reported in Cao-Lei, L. et al. Glucocorticoid receptor gene expression and promoter CpG modifications throughout the human brain. J Psychiatr Res 47, 1597-1607 (2013), postmortem samples from six brain regions of 5 patients with fatal non-head trauma were collected within 2-10 hours after death. Total RNA was extracted from biopsies using the AllPrep DNA / RNA micro kit (Qiagen), or from smNPCs using the RNeasy micro kit (Quiagen) and stored at -80 ° C until cDNA synthesis. The first chain synthesis was carried out in two steps. Initially, the sample was incubated with RNaseOUT (Invitrogen) at 65 ° C for 5 minutes. Subsequently, reverse transcription reaction was performed at 55 ° C for 60 minutes using Superscript III RT (Invitrogen) and 2 μM dT20 primers (Eurogentec). Quantitative PCR was performed on a CFX96 thermal cycler (Bio-Rad). Thermal cycling was performed as follows: denaturation at 95°C for 15 minutes, 40 cycles of 15 seconds at 95°C, annealing for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 10 minutes. For each primer, amplification specificity was verified by melting curve analysis and visualization of PCR products on agarose gels using SYBR Safe (Invitrogen). Relative PCR quantification was performed using the comparative threshold cycle method using the arithmetic mean of PPIA and GAPDH as a stable housekeeping gene. Samples with Ct values ​​greater than three standard deviations from the mean were excluded from further analysis.

[0284] 1.11. Brainbank database analysis of gene expression

[0285] CNS gene expression data were extracted from the Allen Institute, BrainSpan: A developing human brain atlas (http: / / www.brainspan.org / static / download.html, file: RNA-SeqGencodev10summarizedtogenes). This dataset contains RNA-Seq RPKM (reads per kilobase per million) values ​​averaged to genes. For detailed instructions on sample preparation, tissue selection criteria, and data normalization, please refer to the technical white paper Developmental Transcriptome (http: / / help.brain-map.org / display / devhumanbrain / Documentation). Gene expression data were extracted from 16-22 donors aged from 4 months to 40 years old, depending on brain region. Please note that prenatal samples from the database were excluded (pcw8-37). Brain samples from male and female donors were equally representative.

[0286] 1.12. Detection and localization of receptors by flow cytometry

[0287] Intracellular and surface ACKR3 levels were analyzed by flow cytometry using ACKR3-specific mAb (12.5 μg / ml, clone 11G8 (R&D Systems) or a matched isotype control (12.5 μl / ml, clone MG1-45, BioLegend) and phycoerythrin-conjugated F(ab')2 fragment anti-mouse IgG (Jackson Immuno Research). Dead cells were excluded using Zombie NIR fixable viability dye (BioLegend). For intracellular staining, cells were treated with the BD Cytofix / Cytoperm fixation / permeabilization solution kit (BD Biosciences) according to the manufacturer's recommendations. Fluorescence intensity was quantified on a Novocyte Quanteon flow cytometer (ACEA Biosciences).

[0288] 1.13 Opioid peptide-induced arrestin-dependent ACKR3 delivery to endosomes

[0289] The PathHunter eXpress ACKR3-activated GPCR internalization assay (DiscoverX) was used to monitor opioid peptide-induced delivery of the receptor-arrestin complex to endosomes via β-galactosidase complementation. Briefly, U2OS cells stably expressing ACKR3, β-arrestin-2 fused to the enzyme receptor for β-galactosidase, and an endosomal marker fused to the β-galactosidase ProLink donor peptide were incubated at 1 × 10 4 Cells were seeded at a density of 10 cells / well in a 96-well plate. Opioid peptides (3 μM and 300 nM) were then added. After incubation at 37°C for 4 hours, a luminescent signal was generated by adding 55 μl of β-galactosidase substrate (PathHunter detection reagent). After incubation at room temperature for 1 hour, the chemiluminescent signal was measured on a Mithras LB940 microplate reader (Berthold Technologies).

[0290] 1.14 Monitoring opioid peptide-induced changes in receptor cell surface levels

[0291] Determination of receptor surface expression levels for NanoLuc complementation assays was performed using the Nano-Glo HiBiT Extracellular Detection System (Promega) according to the manufacturer's protocol. 6 U87 cells were seeded on 10 cm culture dishes and 48 h later were transfected with 100 ng of plasmid encoding ACKR3 or the corresponding opioid receptor N-terminally tagged with HiBiT. A small amount of nanoluciferase has a high affinity for LgBiT. 4 Cells / well were seeded in 96-well plates and stimulated with CXCL12 (300 nM) or opioid peptides (1 μM) at 37°C for the indicated times. The cells were then incubated with HiBiT extracellular reagent, which consists of nanoluciferase extracellular substrate and LgBiT protein in HiBiT buffer. Light emission from LgBiT protein complementation of HiBiT with the remaining surface receptor fusion was measured on a Mithras LB940 microplate reader (Berthold Technologies). The signal was normalized to the measurement recorded at t = 1 minute. Of note, due to significant LgBiT protein cross-complementation with nociceptin, the effects of nociceptin and its derivatives could not be determined in this assay. Where indicated, cells were treated with bafilomycin A1 (1.5 μM in 0.15% DMSO) (Santa Cruz Biotechnology) or 0.15% DMSO for 45 minutes before and during ligand stimulation (180 minutes).

[0292] In order to determine the receptor surface expression level by flow cytometry, U87-ACKR3 or U87-KOR cells were stimulated with opioid peptides (1 μM) or CXCL12 (300 nM) at 37 ° C for 60 minutes. The remaining surface-bound ligands were then removed by briefly washing with 150 mM NaCl, 50 mM glycine, pH 3 and FACS buffer twice. Where indicated, the cells were incubated for another 120 minutes to allow surface receptors to recover. ACKR3 and or KOR cell surface levels were then measured by flow cytometry using receptor-specific mAbs (clone 11G8 for ACKR3 and 387301 for KOR, R&D Systems) and secondary phycoerythrin-conjugated F (ab') 2 fragment anti-mouse IgG (Jackson Immuno Research). Dead cells were excluded using Zombie NIR fixable viability dyes (BioLegend). Mean fluorescence intensity was quantified on a Novocyte Quanteon flow cytometer (ACEA Biosciences).

[0293] Data Analysis

[0294] Concentration-response curves were fitted to a four-parameter Hill equation using iterative least squares (GraphPad Prism version 8.0.1). All curves were fitted to data points generated from the average of at least three independent experiments.

[0295] Example 2. Activation of ACKR3 by a wide range of opioid peptides from different families

[0296] In a recent study, the present inventors proposed that the proenkephalin-derived peptide BAM22 shares structural and functional features that are important / involved in the N-terminal binding and activation of ACKR3 by chemokine ligands. Given that all endogenous opioid peptides display significant sequence homology, including the F / YGGFL / M (SEQ ID NO: 12) motif at their N-termini, as well as several positively charged residues throughout the sequence, the present inventors wondered whether BAM22 and related peptides are the only opioid peptides capable of activating ACKR3. Figure 1H; Table 1a). Therefore, the inventors screened a library of 58 opioid peptides for their ability to induce recruitment of β-arrestin-2 to ACKR3 and additionally to CXCR4 and CXCR3 (two classical chemokine receptors that share CXCL12 and CXCL11, respectively, as ligands for ACKR3, as negative controls). In addition to BAM22, BAM18, and peptide E, which were previously reported as ACKR3 ligands, the inventors' screening revealed that many other opioid peptides were able to induce recruitment of β-arrestin-2 to ACKR3. These included adrenorphin, another proenkephalin-derived peptide, but also peptides from the nociceptin and dynorphin families ( Figure 1 A). However, endorphins and endomorphins did not activate ACKR3. None of these peptides acted as ACKR3 antagonists (data not shown) or induced β-arrestin-2 recruitment to CXCR4 or CXCR3 ( Figure 1 A).

[0297] The inventors next analyzed and compared the potency and efficacy of different hits targeting ACKR3 and classical opioid receptors in β-arrestin-1 and β-arrestin-2 recruitment ( Figure 1 BF and H, Table 1b, data for β-arrestin-2 not shown). ACKR3 was activated by several endogenous opioid peptides such as dynorphin A, dynorphin A1-13, big dynorphin, BAM22, or adrenorphin at low concentrations, comparable to their activity at classical opioid receptors. Higher concentrations of dynorphin B, nociceptin, or nociceptin 1-13 amide were required for ACKR3 activation. Surprisingly, ACKR3 was also activated by the NOP antagonist Phe1ψ(CH2-NH)-Gly2-nociceptin-1-13 amide (FG nociceptin 1-13) (SEQ ID NO: 13; NH2-substituted at the C-terminus) as well as by the endogenous truncated dynorphin variants, dynorphin 2-13 (SEQ ID NO: 20; NH2-substituted at the C-terminus) and dynorphin 2-17 (SEQ ID NO: 19) ( Figure 1 H, Table 1b), which do not activate classical opioid receptors but have been shown to have physiological effects. ACKR3 appears to show a degree of selectivity, as several peptides such as endorphins, short endomorphins, and Leu-enkephalin or Met-enkephalin do not trigger β-arrestin recruitment. These data were further confirmed in HEK293T and CHO-K1 cell backgrounds (data not shown) and in binding competition studies, showing that all identified ligands were able to compete with and displace AlexaFluor 647-labeled CXCL12 from ACKR3. Figure 1G) Taken together, these data indicate that ACKR3, unlike CXCR3 or CXCR4, is selectively activated by various endogenous opioid peptides from different families at concentration ranges similar to those observed for activation and signaling through opioid receptors.

[0298] ACKR3 is the only chemokine receptor activated by opioid peptides

[0299] Like the classic opioid receptors, many chemokine receptors have multiple ligands, which they often share with other receptors. Therefore, the inventors wondered whether ACKR3 was the only member of the chemokine receptor family that was activated by opioid peptides. To this end, the inventors tested all chemokine receptors for arrestin recruitment in response to different ACKR3-bound opioid peptide ligands at saturating concentrations using the same nanoluciferase complementation assay. None of the peptides induced similar recruitment of β-arrestin-1 or β-arrestin-2 to any of the other 24 chemokine receptors ( Figure 2 (Data for β-arrestin-2 not shown). For several receptors treated with big dynorphin, such as CX3CR1, CXCR3, and CCR3, a weak induction of β-arrestin recruitment could be detected. However, in contrast to ACKR3 or opioid receptors, their responses to big dynorphin were severely reduced compared to those achieved with their cognate chemokines (data not shown). These data provide strong support for the view that the ability to recruit arrestins in response to endogenous opioid peptides is unique and distinct for ACKR3 among all chemokine receptor family members.

[0300] Similarities and differences between the binding pockets of ACKR3 and classical opioid receptors: a structure-activity relationship study of adrenorphin

[0301] To further understand the binding and activation mode of ACKR3 compared to classical opioid receptors, the present inventors conducted structure-activity relationship studies based on the octapeptide adrenorphin (YGGFMRRV (SEQ ID NO: 32; NH2-substituted at the C-terminus), formerly methyl octapeptide amide). Adrenorphin triggers arrestin recruitment to ACKR3, MOR, DOR, and KOR with approximately the same potency. Figure 3 B), providing a suitable basis for studying the activation mode of the four receptors. The inventors performed alanine scanning on adrenorphin and introduced substitutions with closely related amino acids or other modifications (such as N- and C-terminal extensions, D-amino acid substitutions or dimerization). The inventors evaluated the ability of these modified peptides to activate ACKR3 and opioid receptors in a β-arrestin-1 recruitment assay ( Figure 3A). Interestingly, the "message" and "address" sequences of ACKR3 differ somewhat from those of the classical opioid receptors, despite similar trends in potency. ACKR3 appears to be more tolerant to modifications of the N-terminal tyrosine residue, which is essential for activation of the classical opioid receptors. Indeed, variants displaying either leucine or phenylalanine retained parental activity, and a Y1F mutation, mimicking the nociceptin N-terminus, resulted in a tenfold increase in potency ( Figure 3 A and C). However, similar to the classical receptors, the phenylalanine at position 4 of the YGGF-L / M (SEQ ID NO: 15) core was found to be critical for ACKR3 binding, as any mutation except F4W was deleterious to receptor activation. Substitution of methionine to leucine at position 5, which mimics peptides of the dynorphin family, improved binding to KOR but significantly reduced binding to MOR and ACKR3 ( Figure 3 D), whereas mutation of position 6 in the twin-arginine motif abolished the activity towards KOR and ACKR3 but greatly enhanced the activity towards MOR ( Figure 3 E).

[0302] Based on the SAR analysis described above, the present inventors concluded that the interaction pattern of ACKR3 with opioid peptides differs in some respects from that of the classical opioid receptors, and that ACKR3 shares important interaction determinants with all four other receptors. This feature may explain its ability to bind and respond to peptides from different families.

[0303] ACKR3 is unresponsive to alkaloid opioids and synthetic opioid drugs

[0304] Based on its ability to respond to different families of endogenous opioid peptides and its similarity in binding mode to classical opioid receptors, the inventors wondered whether ACKR3 could also respond to non-endogenous opioid ligands that are commonly used to activate or inhibit classical opioid receptors. In addition to prototypical opioid tool compounds such as D-Ala 2 , D-Leu 5 -enkephalin (DADLE) (SEQ ID NO: 16) or D-Ala 2 、N-MePhe 4 , Gly-ol]-enkephalin (DAMGO) (SEQ ID NO: 17), the inventors tested approved analgesics such as morphine, fentanyl or buprenorphine in a β-arrestin recruitment assay. All molecules showed their expected agonist or antagonist activity at their respective opioid receptors ( Figure 4A). For morphine, consistent with previous reports, only weak recruitment of β-arrestin to the MOR was observed. At high concentrations, many of these molecules, although designed to specifically target one receptor, showed some activity at other opioid receptors, similar to that observed with endogenous ligands. However, ACKR3 did not respond to any of the molecules tested, even at high concentrations. Weak activation of ACKR3 was observed using the KOR agonist U50488 and the antagonist nor-binaltrophimine, but the potency was much weaker than that observed for KOR.

[0305] These results suggest that, although ACKR3 shares several endogenous opioid peptides with classical opioid receptors, it is not modulated by opiate analgesics or synthetic opioid drugs that target classical opioid receptors.

[0306] Example 3 Development of LIH383, an adrenorphin-derived octapeptide with high specificity and subnanomolar agonist activity for ACKR3

[0307] To develop highly potent and selective ACKR3 modulators, the present inventors utilized adrenorphin SAR data to design a second generation of peptides. The present inventors used the adrenorphin Y1F variant (SEQ ID NO: 35; NH2 substitution at the C-terminus) as a scaffold because it exhibited a 10-fold increase in potency against ACKR3 and a greater than 100-fold decrease in potency against the classical opioid receptors MOR, DOR, and KOR compared to WT adrenorphin. Figure 3 A and C). Additional mutations that increase potency against ACKR3 were further combined, including F4W, V8F, V8K, or 9R, and the resulting peptides were tested in the β-arrestin recruitment assay ( Figure 3 F). Among all the combinations tested, the octapeptide FGGFMRRK (SEQ ID NO: 3; NH2 substitution at the C-terminus) (designated LIH383) was the most potent ACKR3 agonist, EC 50 Notably, LIH383 was more potent than the full-length chemokine ligands CXCL12 or CXCL11 (EC 50 1.2nM and 2.2nM, respectively) are more effective ( Figure 4 B). Importantly, no activation or inhibition of any other opioid receptors or any other chemokine receptors could be detected after LIH383 treatment, even at concentrations as high as 3 μM ( Figure 4 C, D, H and I). LIH383 also has the same activity against human and mouse ACKR3 (mACKR3) ( Figure 4 B and E). LIH383 directly competes with CXCL12-AF647 for ACKR3 binding at low nanomolar concentrations ( Figure 4 G). In addition, Cy-5-labeled LIH383 bound to U87 cells expressing ACKR3, but not to native or CXCR4-expressing U87 cells ( Figure 4 F), making this peptide a potentially valuable and versatile tool for specific ACKR3 modulation or detection in ACKR3-expressing cells.

[0308] Example 4. ACKR3 does not signal in response to endogenous opioid peptides

[0309] To decipher the function and impact of ACKR3 on the opioid system, the inventors tested the ability of opioid peptides to trigger downstream signaling through ACKR3 in U87 cells. The inventors first applied a whole-cell optical biosensing method based on dynamic mass redistribution (DMR), which is capable of detecting multiple downstream signaling events including all four major G protein pathways. Consistent with other studies, the inventors did not detect any ACKR3-dependent signaling upon chemokine stimulation ( Figure 5 A). Similarly, no difference in DMR signal was observed between ACKR3-transfected and untransfected cells in response to opioid peptides ( Figure 5 In contrast, U87 cells expressing G protein signaling-competent CXCR4, KOR, or NOP did show robust DMRs in response to CXCL12, dynorphin A / adrenorphin, or nociceptin 1-13, respectively, consistent with robust activation of downstream signaling pathways through these receptors ( Figure 5 Consistent with these observations, in contrast to CXCR4 or classical opioid receptors, which both efficiently recruit mGi, the present inventors did not detect any interaction of ACKR3 with small G (mG) proteins (mGi, mGs, mGq, or mG12 / 13) following chemokine or opioid peptide treatment ( Figure 5 E, data for mG, mGq, and mG12 / 13 not shown). Furthermore, ERK phosphorylation levels monitored by homogeneous time-resolved fluorescence (HTRF) remained unchanged after ligand stimulation of cells stably expressing ACKR3, whereas a robust increase in ERK phosphorylation was observed between 2 and 120 min after CXCL12 stimulation of cells stably expressing CXCR4 ( Figure 5 F). In contrast to the robust signal increase in CXCR4 or classical opioid receptor expressing cells after stimulation with the corresponding ligands, in ACKR3 positive U87 cells ( Figure 5These results were further corroborated by the absence of MAPK / ERK-dependent serum response element (SRE) activation following opioid peptide or chemokine stimulation in HEK293T and CHO-K1 cells (G, left panel) or in HEK293T and CHO-K1 cells (data not shown). A similar absence of signaling through ACKR3 in response to opioid or chemokine ligands was shown for the calcium-dependent nuclear factor of activated T cell response element (NFAT-RE) ( Figure 5 G, right).

[0310] These data demonstrate that opioid peptides induce β-arrestin recruitment to ACKR3 but are inactive in a canonical G protein-driven readout, suggesting that ACKR3 may act as a scavenger of opioid peptides.

[0311] Example 5. ACKR3 mediates endogenous opioid peptide uptake, which can be modulated or blocked by the peptides of the present invention

[0312] To investigate the ability of ACKR3 to clear opioid peptides, the present inventors used imaging flow cytometry to measure the uptake of fluorescently labeled opioid peptides of different families by cells expressing ACKR3 or the corresponding classical opioid receptors. For dynorphin-A (1-13), mono-5 (6)-carboxyfluorescein (FAM)-labeled dynorphin A (1-13), hereinafter referred to as dynorphin A-FAM, was used, and its uptake by U87-ACKR3 cells was measured using imaging flow cytometry. The present inventors observed significant intracellular accumulation of the fluorescently labeled peptide after 40 minutes of stimulation, with a significantly higher number of distinguishable vesicle-like structures and mean fluorescence intensity ( mFI) compared to U87 cells or U87-ACKR3 cells pre-incubated with saturating concentrations of LIH383. Figure 6 A), demonstrating that ACKR3 is capable of mediating the uptake of opioid peptides. Furthermore, ACKR3 is more effective for the uptake of dynorphin A(1-13) than KOR, the major classical opioid receptor for this peptide, although dynorphin A(1-13) is less potent for ACKR3 ( Figure 6 B). Similar observations were made with labeled big dynorphin A (the precursor of dynorphins A and B) and BAM22 (a peptide from the enkephalin family). Indeed, despite similar potency at both receptors, ACKR3-positive cells internalized significantly more BAM22 than MOR-positive cells. The low-affinity ligand nociceptin was also internalized by ACKR3 to an extent comparable to that of the corresponding classical opioid receptor, NOP ( Figure 6B). Importantly, this ACKR3-driven intracellular accumulation of opioid peptides is also associated with a reduced availability of them in the extracellular space. For example, the present inventors found that the apparent efficacy of dynorphin A in activating KOR was impaired in the presence of cells expressing ACKR3. This effect was reversed when ACKR3-expressing cells were pretreated with saturating concentrations of LIH383 or CXCL12, but not with an irrelevant control peptide (LIH383ctrl) or the irrelevant chemokine CXCL10, suggesting a plausible clearance function for ACKR3. Figure 6 C).

[0313] Example 6. ACKR3 exhibits atypical localization and recycling patterns comparable to classical opioid receptors

[0314] Consistent with this clearance function, ACKR3 exhibits atypical cellular localization, internalization, and trafficking patterns compared to classical opioid receptors. Consistent with previous reports, the present inventors observed a higher proportion of ACKR3 present within the cell compared to the cell surface. In contrast, classical opioid receptors MOR, DOR, KOR, and NOP are primarily localized to the plasma membrane (data not shown). Furthermore, despite the efficient uptake and delivery of various opioid peptides to early endosomes ( Figure 6 D), but the overall reduction in cell surface ACKR3 was much less pronounced than that seen for classical opioid receptors, likely reflecting the rapid cycling of ACKR3 between the plasma membrane and intracellular compartments ( Figure 6 E). Similar to what has been reported for chemokines, we found that removal of the agonist after stimulation resulted in a gradual increase in ACKR3 at the plasma membrane, whereas no such recovery was observed for classical receptors like KOR ( Figure 6 F). This was further confirmed by the results obtained with bafilomycin A1, a vacuolar H+-ATPase inhibitor. Previous studies have shown that low endosomal pH is required for the dissociation of chemokines from ACKR3 and for efficient receptor recycling and resensitization. The present inventors observed that treatment with bafilomycin A1, a vacuolar H+-ATPase inhibitor, resulted in a reduction in receptor recovery at the plasma membrane after stimulation with various opioid peptides, while it had no effect on the surface levels of KOR ( Figure 6 G). Taken together, these results suggest that ACKR3 can support the rapid and efficient uptake of different families of opioid peptides through continuous receptor recycling between intracellular compartments and the plasma membrane, leading to the gradual depletion of extracellular opioid peptides, thereby limiting their availability to classical receptors.

[0315] Example 7. Modulation of the availability of endogenous opioid peptides in the CNS opioid center using the peptides of the invention

[0316] To establish the physiological relevance of the observed opioid peptide clearance capacity of ACKR3, the present inventors then analyzed the gene expression profiles of classical opioid receptors in different brain regions (corresponding to important centers for opioid signaling / activity) using the Brainspan database (www.brainspan.org). Interestingly, ACKR3 is not only expressed in many regions such as the amygdala, hippocampus, or medial prefrontal cortex, but its expression is often higher (up to 100-fold) than that of MOR (OPRM1), KOR (OPRK1), DOR (OPRD1), and NOP (OPRL1) in the same regions ( Figure 7 A). These data were further confirmed by qPCR on human brain samples, where additional opioid centers such as the dentate gyrus or locus coeruleus showed similarly high ACKR3 expression ( Figure 7 B).

[0317] Given that ACKR3 is expressed in the same CNS regions as classical opioid receptors and its ability to efficiently internalize opioid peptides without inducing downstream signaling, particularly G protein-mediated signaling, the inventors wondered whether ACKR3 might influence classical opioid receptor signaling by modulating the availability of its ligands. To test this hypothesis and the inability of ACKR3 to trigger signaling in a more physiological context, the inventors used small-molecule neural precursor cells (smNPCs), which endogenously express ACKR3 but not classical opioid receptors ( Figure 7 C). The present inventors demonstrated that, like U87-ACKR3 cells, smNPCs expressed a higher proportion of ACKR3 intracellularly compared to the cell surface ( Figure 7 D), and they are able to do so without activating the ERK signaling pathway ( Figure 7 F) Accumulation of labeled dynorphin A(1-13) ( Figure 7 E). This uptake was significantly reduced when smNPCs were pretreated with LIH383, but not with the LIHctrl peptide ( Figure 7 E). Consistent with the U87 cell results, this uptake was also associated with a reduction in extracellular dynorphin A concentrations and, therefore, the ability to signal through its corresponding classical opioid receptor ( Figure 7 G).

[0318] To finally confirm the opioid peptide clearance function of ACKR3, the present inventors monitored the inhibition of spontaneous neuronal depolarization (i.e., neuronal firing) mediated by dynorphin A in vitro in rat locus coeruleus (one of the brain regions where ACKR3 is found together with KOR and MOR). Figure 7B). Treatment with dynorphin A resulted in a dose-dependent inhibition of neuronal depolarization (i.e., neuronal firing), with 1 μM achieving total inhibition ( Figure 7 H). However, when pretreated with naloxone, the same concentration of dynorphin A did not result in changes in neuronal firing rate, suggesting that the inhibition of dynorphin A-induced firing can be attributed to classical opioid receptors. However, treatment with LIH383 at concentrations up to 3 μM did not result in significant inhibition of neuronal firing, further confirming that ACKR3 is unable to trigger classical G protein signaling in this region of the CNS ( Figure 7 H).

[0319] Interestingly, pretreatment of locus coeruleus neurons with LIH383 (1 or 3 μM) to selectively block the clearance capacity of ACKR3 resulted in a significant increase in depolarizing inhibition and a markedly enhanced potency of dynorphin A at its classical receptor ( Figure 7 I). This observation is consistent with our in vitro data and suggests that also in vivo, under a more physiological environment and endogenous receptor abundance, ACKR3 can exert its scavenging function to form an opioid gradient, thereby fine-tuning the signaling of opioid peptides through their classical opioid receptors.

Claims

1. A selective ACKR3 regulatory peptide having an amino acid sequence of FGGX1MRRX2 (SEQ ID NO: 1), wherein X1 is F or W; and X2 is K, V or F. The peptide according to claim 1 , wherein X 1 is F.

3. The peptide according to claim 1 or 2, wherein X2 is K.

4. A selective ACKR3 regulatory peptide having an amino acid sequence of: FGGX1MRRX2X3 (SEQ ID NO: 2), wherein X1 is F or W; X2 is K, V or F; and X3 is R. 5 . The peptide according to claim 1 , wherein the amino acid sequence of the peptide is selected from the group consisting of FGGFMRRK (SEQ ID NO: 3) and FGGWMRRK (SEQ ID NO: 6). The peptide according to claim 1 , wherein the amino acid sequence of the peptide is FGGFMRRK (SEQ ID NO: 3).

7. The peptide according to claim 4, wherein the amino acid sequence of the peptide is FGGFMRRKR (SEQ ID NO: 4) or FGGFMRRVR (SEQ ID NO: 5).

8. The peptide according to any one of claims 5 to 7, wherein the C-terminus of the peptide is substituted with NH2.

9. A fusion protein consisting of the peptide according to any one of claims 1 to 8 and a tag, a detectable marker or an immunoglobulin Fc region.

10. The fusion protein according to claim 9, wherein the peptide according to any one of claims 1 to 8 is fused to a detectable marker or tag.

11. A nucleic acid encoding the peptide according to any one of claims 1 to 8 or the fusion protein according to claim 9 or 10.

12. A nucleic acid expression cassette comprising the nucleic acid according to claim 11 operably linked to a promoter and / or transcriptional and translational regulatory signals.

13. A vector comprising the nucleic acid according to claim 11 or the nucleic acid expression cassette according to claim 12. The vector according to claim 13 , wherein the vector is a viral vector. 15 . A pharmaceutical composition comprising the peptide according to claim 1 , the fusion protein according to claim 9 or 10 , the nucleic acid according to claim 11 , the nucleic acid expression cassette according to claim 12 , or the vector according to claim 13 or 14 . The pharmaceutical composition according to claim 15 , further comprising a pharmaceutically acceptable carrier.

17. Use of the peptide according to any one of claims 1 to 8, the fusion protein according to claim 9 or 10, the nucleic acid according to claim 11, the nucleic acid expression cassette according to claim 12, the vector according to claim 13, or the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating a disease or condition in a subject selected from the group consisting of adverse stress dysfunction diseases or conditions, chronic pain, cardiovascular disease, fibrosis, and inflammatory diseases and conditions.

18. Use according to claim 17, wherein the cardiovascular disease is atherosclerotic vascular disease.

19. Use of a peptide according to any one of claims 1 to 8, wherein the peptide is fused to a detectable label, for the preparation of a diagnostic reagent for in vitro or ex vivo diagnosis, prediction, prognosis and / or monitoring of a disease or condition characterized by abnormal levels of an ACKR3 polypeptide.

20. A kit for diagnosing, predicting, prognosing and / or monitoring a disease or condition characterized by abnormal levels of an ACKR3 polypeptide in a subject, the kit comprising: (a) the peptide according to any one of claims 1 to 8; and (b) a reference value for the level of ACKR3 polypeptide, wherein the reference value represents a known diagnosis, prediction and / or prognosis of a disease or condition characterized by abnormal levels of ACKR3 polypeptide.

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