Novel selective ackr2 modulators

Abstract

The application discloses selective chimeric chemokines and their use for selectively targeting atypical chemokine receptor 2 (ACKR2) in the treatment of an autoimmune, inflammatory, neurological, cardiovascular or proliferative disease or disorder in a subject and / or for use in improving the response of a subject to anticancer immunotherapy. The application further discloses pharmaceutical compositions comprising such selective chimeric chemokines and further provides methods of production and uses of said chimeric cytokines.

Description

[0001] NOVEL SELECTIVE ACKR2 MODULATORS

[0002] FIELD

[0003] The invention is broadly in the medical field, and provides chimeric chemokines and their use for selectively targeting atypical chemokine receptor 2 (ACKR2) in detection, diagnostics, modulation and therapy, both as such and in combination with other agents, and further provides methods of production and uses of said chimeric cytokines.

[0004] BACKGROUND

[0005] The interactions between chemokines and their receptors orchestrate leukocyte mobilization, both in homeostatic and inflammatory conditions. Chemokine receptors represent one of the largest family of class A of the G protein-coupled receptor (GPCR) family, presenting a conserved seven transmembrane structure and binding small (8-14 kDa) soluble cytokines, termed chemokines.

[0006] Based on conserved cysteine motifs, chemokines are divided into four subfamilies: CC, CXC, XC and CX3C and the receptors are named according to the subfamily of chemokines they bind (CCR, CXCR, XCR and CX3CR). In addition, other receptors referred to as Atypical Chemokine Receptors (ACKR1- 4) can recognize chemokines and act as transporters, scavengers or signal through alternative pathways, further contributing to the complexity of the chemokine network

[0007] ACKR2 (formerly termed D6 or CCBP2) is one of the most promiscuous chemokine receptors, interacting with most of the CC chemokines (CCL2-8, CCL11-14, CCL17 and CCL22) but also the CXC chemokine CXCL10. This ability to act on a large spectrum of scavenged inflammatory chemokines makes ACKR2 one of the key players in the resolution phase of inflammation, preventing exacerbated immune responses. This is further corroborated by ACKR2 expression in placental trophoblasts, innate-like B cells, alveolar macrophages, myeloid progenitor-derived cells, blood endothelial cells and lymphatic endothelial cells.

[0008] In physiological conditions, ACKR2 plays an active role in pregnancy, as evidenced by the foetal loss of ACKR2 knockout mice exposed to LPS; in mammary gland development, shown by the premature development of these glands in ACKR2 -deficient mice; and in lymphatic regulation, where ACKR2 competes with CCR2, leading to efficient antigen presentation by DCs and regulated macrophage proximity to lymphatic vessels, respectively.

[0009] Still, most research on ACKR2 highlights its impact in different pathological conditions, such as autoimmune, cardiovascular and neurological diseases, and cancer. Nevertheless, the data currently available is dependent on the use of ACKR2 knockout mice or cell lines, which do not reflect the true impact of receptor modulation. The major challenge for chemokine receptor modulation has been the scarcity of antibodies and modulators able to selectively target membrane expression of these endogenous receptors. Specifically for ACKR2, there have been no reported attempts to create such a molecules due to its high promiscuity, especially towards CC chemokines. Moreover, the recent inclusion of CXC chemokines in the repertoire of chemokines bound by this receptor, further increased the challenge. Selective modulation of ACKR2 is believed to be promising for treatment of autoimmune, inflammatory, neurological, cardiovascular and proliferative diseases, and cancer. Therefore there is a great need for the development of small, selective binders and modulators for ACKR2.

[0010] SUMMARY

[0011] Present inventors have found chimeric chemokines which activate ACKR2 with high selectivity and with high affinity. Since ACKR2 is highly promiscuous, in that it binds a large array of chemokines, which themselves typically bind several other receptors, identifying specific binders has been considered challenging. The solution has been found by combining the N-terminal portion of CCL2, CCL7 or CCL8 with the core portion of CCL22. The chimeric chemokine obtained binds ACKR2 with high affinity and selectivity. This allows the use of the chimeric chemokine in investigations into ACKR2 function and as a diagnostic marker and also allows to modify ACKR2’s function. The chimeric chemokine of the invention in fact has a strong affinity to ACKR2. As a result, while ACKR2 acts as a scavenger for conventional chemokines in a number of diseases states, the provision of the chimeric chemokine will ensure that ACKR2 preferentially scavenges the chimeric chemokine, allowing the free conventional chemokines to bind to their cognate classical chemokine receptors.

[0012] Accordingly, a first aspect of the invention provides for a chimeric chemokine characterized in that it comprises a CCL2, CCL7 or CCL8 chemokine polypeptide fused to a CCL22 chemokine polypeptide. The chimeric chemokines of the invention are further characterized in that said chimeric chemokine selectively binds to ACKR2 and not to CCR2, CCR4 and / or ACKR4 or any other known chemokine receptor.

[0013] In particular embodiments, said chimeric chemokine comprises the N-terminal end of CCL2, CCL7 or CCL8, or comprises an amino acid sequence represented by any of SEQ ID NOs 34, 35, 36 or 37, and comprises the three-stranded-antiparallel beta sheet and C-terminal alpha helix of CCL22.

[0014] In particular embodiments, the N-terminal end of CCL2, CCL7 or CCL8 and the three -stranded- antiparallel beta sheet and C-terminal alpha helix of CCL22 originate from a different species, preferably selected from human and mouse.

[0015] More particularly, said chimeric chemokine comprises the N-terminal residues up to the Cys-Cys motif from the N-terminal end of the human CCL2, CCL7 or CCL8 sequence, or comprises an amino acid sequence represented by any of SEQ ID NOs 34, 35, 36 or 37, or a corresponding fragment of an orthologue thereof, fused to the C-terminal residues starting from the Cys-Cys motif to the end of the human CCL22 sequence or a corresponding sequence of an orthologue thereof.

[0016] More particularly, said chimeric chemokine comprises the N-terminal residues up to the Cys-Cys motif from the N-terminal end of the human CCL2 sequence represented by SEQ ID NO 1, the human CCL7 sequence represented by SEQ ID NO: 11, the human CCL8 sequence represented by SEQ ID NO: 17, or a corresponding fragment of an orthologue thereof and the C-terminal residues starting from the Cys- Cys motif to the end of the human CCL22 sequence represented by SEQ ID NO: 5 or a corresponding sequence of an orthologue thereof.

[0017] In particular embodiments, said chimeric chemokine comprises a polypeptide corresponding to the N terminal residues up to the Cys-Cys motif from the N-terminal end of the mouse CCL2 represented by SEQ ID NO 3, the N-terminal end of the mouse CCL7 represented by SEQ ID NO 14, the N-terminal end of the mouse CCL8 represented by SEQ ID NO 20, or a corresponding fragment of an orthologue thereof and the C-terminal residues starting from the Cys-Cys motif to the end of the mouse CCL22 polypeptide represented by SEQ ID NO 7 or a corresponding fragment of an orthologue thereof.

[0018] In particular embodiments, said chimeric chemokine comprises the N-terminal end of the human CCL2 sequence is represented by SEQ ID NO: 2, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of human CCL2; the N-terminal end of the human CCL7 sequence is represented by SEQ ID NO: 12, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of human CCL7; the N-terminal end of the human CCL8 sequence is represented by SEQ ID NO: 18, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of human CCL8, and / or the C-terminal end of the human CCL22 sequence is represented by SEQ ID NO 6 or a corresponding fragment starting from the Cys-Cys motif to the end of an orthologue of human CCL22.

[0019] In particular embodiments, said chimeric chemokine comprises the N-terminal end the mouse CCL2 is represented by SEQ ID NO 4, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of mouse CCL2, the N-terminal end of the mouse CCL7 sequence represented by SEQ ID NO: 15, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of mouse CCL7, the N-terminal end of the mouse CCL8 sequence represented by SEQ ID NO: 21, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of mouse CCL8, and / or the C-terminal end of the mouse CCL22 polypeptide represented by SEQ ID NO: 8.

[0020] In particular embodiments, the N terminal glutamine at the N terminal end of the chimeric chemokine of the invention comprises a pyruvate modification forming a pyroglutamic acid.

[0021] In particular embodiments, said chimeric chemokine further comprises a label or a functional moiety. In some embodiments the label is detectable label such as a fluorescent protein or enzyme. In some embodiments, the functional moiety is an immunoglobulin Fc region, a paratope -containing antibody fragment, a protoxin, a toxin, a nucleotide sequence or a pharmaceutical.

[0022] In a further aspect, the invention provides for a multimer comprising at least two, at least three, at least for or at least more chimeric chemokines, preferably wherein the chimeric chemokines are coupled through their C-terminus to a linker.

[0023] In particular embodiments, the multimer comprises two or three chimeric chemokines.

[0024] In particular embodiments, the multimer comprises two chimeric chemokines wherein the linker is a dimerization scaffold, such as the dimerization scaffold of the C4bp alpha or C4bp beta chain of the C4b binding protein (C4bp), the dimerization scaffold of the HNF-1 alpha or HNF-1 beta chain of the hepatocyte nuclear factor (HNF)l, an azido-PEG-azide, such as an azido-PEG5 -azide, azido-PEG5- azide, azido-PEG7-azide, azido-PEG8-azide, azido-PEGlO-azide, azido-PEG 11 -azide, azido-PEG12- azide, azido-PEG 19-azide, azido-PEG20-azide or azido-PEG23 -azide, preferably an azido-PEG5 -azide or azido-PEG 10-azide.

[0025] In particular embodiments, the multimer comprises three chimeric chemokines wherein the linker is a trimerization scaffold, such as the collagen-like region of ficolin-2, a T4 foldon, such as the T4 fibritin trimerization domain, the GCN4 trimerization motif or a 3 -arm -PEG- Azide.

[0026] A related aspect of the invention provides nucleic acids encoding the chimeric chemokines as provided herein.

[0027] A further related aspect of the invention provides the chimeric chemokines as envisaged herein for use as a medicament or diagnostic, i. e. for use in methods of treatment, prevention of disease and in methods of diagnosis. In particular embodiments, the invention provides a chimeric chemokine as described herein for use in the diagnosis of a disorder characterized by aberrant chemokine levels, more particularly aberrant levels of one or more chemokines selected from CCL2-8, CCL11-14, CCL17, CCL22, CCL26 and CXCL10. In particular embodiments the invention provides the chimeric chemokines as described herein for use in the treatment of an autoimmune, inflammatory, neurological, cardiovascular or proliferative disease or disorder in a subject.

[0028] In particular embodiments, said treatment comprises the administration of the selective chimeric chemokine as described herein in combination with anticancer immunotherapy, preferably in combination with one or more immune checkpoint inhibitors.

[0029] In particular embodiments, said autoimmune, inflammatory, neurological and cardiovascular disease or disorder is a disease or disorder selected from the group consisting of psoriasis, fibrosis, obesity, transplantation and Graft-Versus-Host-Disease, asthma, Alzheimer’s Disease, multiple sclerosis, myocardial infarction; and wherein said proliferative disease or disorder is a proliferative disease or disorder selected from the group consisting of skin cancer such as melanoma, colon cancer, rectal cancer, colorectal cancer, bladder cancer, neuroblastoma, squamous cell cancer, lung cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer such as gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatoma, breast cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, head cancer and neck cancer, preferably skin cancer or colorectal cancer.

[0030] In particular embodiments, said subject is diagnosed with or assumed to have a proliferative disease or disorder which is non-responsive to anticancer immunotherapy, preferably non-responsive to immune checkpoint blockade therapy.

[0031] A further related aspect of the invention provides pharmaceutical compositions comprising the chimeric chemokine selectively binding to ACKR2 described herein and a pharmaceutical excipient.

[0032] In particular embodiments, said pharmaceutical compositions comprise one or more immune checkpoint modulators, preferably one or more immune checkpoint inhibitors, and optionally a pharmaceutically acceptable carrier.

[0033] In particular embodiments, said one or more immune checkpoint modulators, preferably the one or more immune checkpoint inhibitors, are selected from the group consisting of a Programmed Death-ligand 1 (PDL-1) inhibitor, a Programmed Death 1 (PD-1) inhibitor, a Cytotoxic T -Lymphocyte- Associated protein 4 (CTLA-4) inhibitor, a cluster of differentiation 3 (CD3) inhibitor, a NKG2A inhibitor, a immunoglobulin-like receptors (KIR) inhibitor, a cluster of differentiation 47 (CD47) inhibitor, a CD24 inhibitor, a CD73 inhibitor, a tumor necrosis factor (TNF) receptor 2 (TNFR2) inhibitor, and a signal- regulatory protein alpha (SIRPa) inhibitor, preferably a PD-1 inhibitor. In particular embodiments, the use of the chimeric chemokine is envisaged as a targeting agent for extracellular membrane and cellular proteins.

[0034] Yet a further related aspect of the invention provides an in vitro method for determining whether a test agent is an ACKR2 specific ligand, said method comprising determining whether said test agent interferes with the selective binding of the chimeric chemokine to ACKR2. Such ACKR2 specific ligands can be useful for identifying an agent useful as a therapeutic, such as useful for the treatment of a proliferative disease or disorder in a subject.

[0035] In particular embodiments, said in vitro method comprises a step of determining whether said test agent interferes with the biological activity of ACKR2, such as but not limited to P-arrestin recruitment by ACKR2.

[0036] The invention further provides the chimeric chemokine of the invention fused or linked to a dye, or displayed on scaffold proteins or another functional moiety, such as an Fc part of an antibody. Further aspects relate to the use of the chimeric chemokines of the invention, more particularly the labelled chimeric chemokine in methods of detection of AKCR2 expressed at the cell surface and the in vitro methods described above and the use of the chimeric chemokines of the invention as targeting agents.

[0037] BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1: Activity of native chemokines towards ACKR2 and development of LIH222 as high- affinity ACKR2- selective modulator.

[0039] (A) P-arrestin- 1 recruitment to ACKR2 in response to all known human chemokines (100 nM) monitored by NanoBiT-based assay.

[0040] (B) Induction of P-arrestin- 1 recruitment to ACKR2 by CCL2, CCL5, CCL7, CCL8, CCL17, CCL22, CCL26, CXCL2 and CXCL10 monitored by NanoBiT, showing the concentration-response relationship. CXCL1 was used as negative control and 300 nM of CCL5 was considered as 100% of agonist response.

[0041] (C) Sequences of different chimeric chemokines developed based on the combination of N and C terminal regions of different native chemokines.

[0042] (D) Comparison of the potency and efficacy of chimeric chemokines developed. Highly potent chimeric chemokines are represented in black, medium-potency are represented in dark grey and low-potency or inactive are represented in light grey.

[0043] Data points represent mean ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001 by one-way ANOVA with Bonferroni post hoc tests. Figure 2: Assessment of the selectivity of all chimeric chemokines against the relevant CC chemokine receptors (A-E) (Activity of different chimeric chemokines against (A) CCR1, (B) CCR2,

[0044] (C) CCR4, (D) CCR5, (E) ACKR4, monitored by NanoBiT showing the concentration-response curves. 300nM of CCL5 (CCR1, CCR5), CCL2 (CCR2), CCL22 (CCR4), CCL19 (ACKR4) were considered as 100% of agonist response. All NanoBiT assays monitor P-arrestin recruitment to the receptor and were performed in HEK293T cells.

[0045] Figure 3: Specific activation of ACKR2 by LIH222.

[0046] (A) p-arrestin- 1 recruitment to ACKR2, CCR1, CCR2, CCR4, CCR5 and ACKR4 induced by LIH222 (left panel), and the native chemokines that compose the chimera, CCL2 (middle panel) and CCL22 (right panel) monitored by NanoBiT showing the concentration-response curves. 300nM of CCL5 (CCR1, CCR5), CCL2 (CCR2), CCL22 (CCR4), CCL19 (ACKR4) were considered as 100% of agonist response.

[0047] (B) P-arrestin- 1 recruitment induced by LIH222 on all known human chemokine receptors and the most relevant murine receptors, CCR2, CCR3 and CCR4.

[0048] (C) Comparison of the induction of P-arrestin- 1 recruitment to ACKR2 by unlabelled and AZ647- labelled LIH222. Labelling the chimeric chemokine does not affect its ability to modulate ACKR2.

[0049] (D) Selective binding of fluorescently labelled LIH222 (LIH222 AZ64?) to ACKR2 -expressing cells; no binding was observed for the main receptors of the native chemokines (CCR2 and CCR4) nor for the most promiscuous atypical chemokine receptors (ACKR1 and GPR182). Mean fluorescence intensity of 300 nM of LIH222 binding to ACKR2 was considered as 100%.

[0050] (E) LIH222 containing a pyruvate modification (pyroLIH222) that protects from dipeptidyl-peptidase IV (CD26) cleavage, is able to induce P-arrestin- 1 recruitment to ACKR2 to the same extent as the original chimeric chemokine.

[0051] (F) PyroLIH222 but not LIH222 is protected from CD26 cleavage. LIH222 shows a loss of potency consistent with the partial cleavage of the first two amino acids of the N terminal region of the chimera. 2 ALIH222 is LIH222 lacking the first two amino acids, mimicking the full cleavage by CD26, revealing the inability to bind and induce P-arrestin-1 recruitment to ACKR2. All assays were performed in HEK293T cells. Data points represent mean ± SEM of at least three independent experiments.

[0052] Figure 4: Modifications of the N terminal region of LIH222 may further improve its potency.

[0053] (A) Sequences of new chimeric chemokines developed by altering the N-terminal region of LIH222, and sequences of new chimeric chemokine modulators based on the chimerization of murine and human orthologs of CCL2 and CCL22. (B, C) Induction of P-arrestin-1 recruitment to ACKR2 by the (B) N-terminally altered chimeric chemokines and (C) the chimeric chemokines between human and mouse orthologs monitored by NanoBiT, showing the concentration-response relationship. 300 nM of CCL5 was considered as 100% of agonist response.

[0054] (D, E) Binding competition of unlabelled CCL22, CCL2, LIH222, LIHM222, LIH2M22 and LIHM2M22 against Alexa Fluor 647-labelled CCL5 (5 nM) on (D) human ACKR2 -expressing HEK cells or (E) mouse ACKR2 -expressing cells determined by flow cytometry. CXCL1 was used as a negative control. Data points represent mean ± SEM of at least three independent experiments.

[0055] Figure 5: Assessment of the selectivity N terminal-modified chimeric chemokines against the relevant CC chemokine receptors and ACKRs.

[0056] (A-E) Induction of P-arrestin-1 recruitment to (A) CCR1, (B) CCR2, (C) CCR4, (D) CCR5, as well as the selectivity against (E) the atypical chemokine receptor ACKR4, monitored by NanoBiT showing the concentration-response curves. 300 nM of CCL5 (CCR1, CCR5), CCL2 (CCR2), CCL22 (CCR4), CCL19 (ACKR4) were considered as 100% of agonist response. Data points represent mean ± SEM of at least three independent experiments.

[0057] Figure 6: LIH222 coupled to the Fc fragment of an immunoglobulin (LIH222-Fc) is active and able to selectively interact with ACKR2.

[0058] (A) Induction of P-arrestin-1 recruitment to ACKR2 by LIH222-Fc compared to LIH222. The response obtained with 300 nM of CCL5 was considered as 100% (B) Selective binding of LIH222-Fc to cells transiently transfected with vectors encoding the different relevant receptors or cells stably overexpressing ACKR2. No binding was observed for any of the receptors tested, except ACKR2.

[0059] Figure 7: Uptake competition of fluorescently labelled LIH222 by ACKR2-expressing cells visualized by imaging flow cytometry.

[0060] (A) HEK-ACKR2 were incubated for 1 h at 37°C solely with 5 nM LIH222AZ647 or with 5 nM LIH222AZ647 and either 100 nM CCL22, CCL2, CCL5, CXCL10, CXCL1 or CXCL11. ** p < 0.01, * * * * p < 0.0001 by one-way ANOVA with Dunnet post hoc tests. (B) Four representative cells for each condition are shown. BF means Brightlight: D / L means Dead or Live cell (in case of no staining, cells are alive); CK means chemokine. Scale bar: 7 pm.

[0061] Figure 8: Assessment of activity and selectivity of LIH222 in vivo.

[0062] A) Percentage of LIH222-positive blood and lymphatic endothelial cells in wildtype or ACKR2- deficient mice. B) Colocalization of LIH222 and ACKR2 in either endothelial cells of Ackr2L-TdT / flox CDH5-Cre-ERT2 mice (Het) or Ackr2L-TdT / L-TdT CDH5-Cre-ERT2 mice (KO). Data points represent mean ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001 by unpaired t-test.

[0063] Figure 9: LIH222 can be multimerized to increase its efficacy and binding potency towards ACKR2.

[0064] (A) P-arrestin-1 recruitment induced by monomeric, dimeric and trimeric LIH222. The response obtained with 300 nM of CCL5 was considered as 100 % (B) Binding competition of monomers, dimers and trimers of LIH222 against monomeric AZ594-labelled LIH222.

[0065] DESCRIPTION

[0066] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0067] The terms “comprising”, “comprises” and “comprised of’ as used herein 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. The terms also encompass “consisting of’ and “consisting essentially of’, which enjoy well-established meanings in patent terminology.

[0068] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0069] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / - 1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed.

[0070] Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0071] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to 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 of the priority date of any of the claims. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0072] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.

[0073] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0074] Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0075] The term “polypeptide” as used throughout this specification generally encompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms “protein” and “polypeptide” may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced polypeptides. The term also encompasses polypeptides that carry one or more co- or post-expressiontype modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes polypeptide variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions and / or substitutions. The term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g., naturally- occurring polypeptide parts that ensue from processing of such full-length polypeptides.

[0076] A polypeptide or protein can be naturally occurring, e.g., present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom. A polypeptide or protein can be recombinant, i.e., produced by recombinant DNA technology, and / or can be, partly or entirely, chemically or biochemically synthesised. Without limitation, a polypeptide or protein can be produced recombinantly 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 produced recombinantly by cell-free translation or cell-free transcription and translation, or non-biological peptide, polypeptide or protein synthesis.

[0077] The term “nucleic acid” as used herein typically refers to an oligomer or polymer (preferably a linear polymer) of any length composed essentially of nucleotides. A nucleotide unit commonly includes a heterocyclic base, a sugar group, and at least one, e.g. one, two, or three, phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Sugar groups may include inter alia pentose (pento furanose) groups such as preferably ribose and / or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2- deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides or mixtures thereof. A modified nucleotide may include a modified heterocyclic base, a modified sugar moiety, a modified phosphate group or a combination thereof. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term “nucleic acid” further preferably encompasses DNA, RNA and DNA / RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesised) DNA, RNA or DNA / RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature; or can be non-naturally occurring, e.g., recombinant, i.e., produced by recombinant DNA technology, and / or partly or entirely, chemically or biochemically synthesised. A “nucleic acid” can be double -stranded, partly double stranded, or single-stranded. Where single -stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

[0078] The reference to any polypeptides, proteins or nucleic acids encompass such polypeptides, proteins or nucleic acids of any organism where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and non-human mammals, still more preferably of humans.

[0079] Chemokines or chemotactic cytokines, are a family of small cytokines or signalling proteins secreted by cells that induce directional movement of leukocytes, as well as other cell types, including endothelial and epithelial cells. In addition to playing a major role in the activation of host immune responses, chemokines are important for biological processes, including morphogenesis and wound healing, as well as in the pathogenesis of diseases like cancers.

[0080] To date 47 chemokines and 19 classical receptors, forming a highly intricate and precisely regulated network, have been identified in humans. Based on conserved cysteine motifs, chemokines are divided into four subfamilies: CC, CXC, XC and CX3C and the receptors are named according to the subfamily of chemokines they bind (CCR, CXCR, XCR and CX3CR). In addition, other receptors referred to as Atypical Chemokine Receptors (ACKRs) can recognize chemokines and act as transporters, scavengers or signal through alternative pathways, further contributing to the complexity of the chemokine network.

[0081] ACKRs are a small family of membrane proteins comprising four members (ACKR1, 2, 3 and 4). In contrast to classical chemokine receptors, ACKRs are unable to initiate classical signaling pathways after chemokine binding but instead modulate chemokine bioavailability by transporting them through endosomal pathway to lysosomal compartment following the recruitment of -arrestin. ACKRs are therefore considered as scavenging receptors for chemokines and as major receptors orchestrating chemokine-driven immune and inflammatory responses. ACKR2 is a member of the ACKR family. Indeed, following chemokines binding, ACKR2 rapidly traffics from the cell surface to endosomes and chemokines can be dislodged from the receptor by the low pH in endosomes before being degraded in lysosomes. Chemokine-free ACKR2 traffics back to the cell surface to re-acquire chemokines. The repeated internalization and recycling leads to progressive depletion of extracellular chemokines. The ACKR2 internalizes through clathrin-coated pits, in a mechanism dependent on classical endosome signaling pathway involving Rab5, Vpsl5, Vps34, UVRAG and Beclinl proteins. ACKR2 is able to bind inflammatory chemokines exclusively belonging to the CC subfamily. Human ACKR2 is able to bind CCL2, CCL3, CCL3L1, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL17, CCL22 and CCL26, but also the CXC chemokine CXCL10. The atypical chemokine receptor 2 (ACKR2) is also known in the art as Chemokine -binding protein D6, chemokine receptor D6, chemokine-binding protein 2, chemokine receptor CCR-9, CMKBR9 or CCBP2. By means of an example, human ACKR2 gene is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) Gene ID 1238. Human ACKR2 mRNA is annotated under NCBI Genbank accession number NM_001296.5. Nucleotides 207 (start codon) to 1361 (stop codon) of NM_001296.5 constitute the ACKR2 coding sequence. Human ACKR2 protein sequence is annotated under NCBI Genbank accession number NP_001287.2, and Uniprot (www.uniprot.org) accession number 000590.

[0082] Although historically (i.e. before the year 2000), ACKR2 may have been given the name CCR10 at some point after its identification as a chemokine receptor (number 10 being the first available in the CCR subfamily), ACKR2 was excluded from the CCR nomenclature of classical chemokine receptors (among others because of its inability to trigger canonical G protein-dependent signalling) and the name CCR10 has been since reattributed to another chemokine receptor that binds to chemokines CCL27 and CCL28 and signals via G proteins. Accordingly, the person skilled in the art will understand that ACKR2 (e.g. with UniProt accession number 000590) having ligands CCL2, CCL3, CCL3L1, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL17, CCL22, CCL26 and CXCL10 is different from the classical chemokine receptor CCR10 (e.g. with UniProt accession number P46092) having ligands CCL27 and CCL28.

[0083] A skilled person can appreciate that any sequences represented in sequence databases or in the present specification may be of precursors of the respective peptides, polypeptides, proteins or nucleic acids and may include parts which are processed away from mature molecules.

[0084] After extensive experimentation, the present inventors found for the first time (a) selective and high- affinity modulator(s) of ACKR2, a clinically relevant receptor, for which no synthetic or ligand-based selective modulator has been described so far. More particularly, this selective ACKR2 modulator is a chimeric chemokine.

[0085] The term “chimeric chemokine” as used throughout this specification generally refers to a chimeric polypeptide comprising an N-terminal part of a chemokine fused to a C-terminal part of a second chemokine. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced proteins.

[0086] Given the huge number of chemokines binding to ACKR2, it was surprisingly found that the three chimera between CCL2, CCL7 and CCL8 on the one hand, and CCL22 on the other hand, all endogenous chemokine ligands, which chimera are also referred to as CCL2 / CCL22, CCL7 / CCL22, CCL8 / CCL22 provided the best results overreaching the properties of the original ligand molecules. The similarity of function between the 3 chimeras is likely due to their high similarity in the N-terminal region, in a 10 aa N-terminus, they share 5 aa, which is not the case for any other chemokine. The 3 chemokines CCL2, CCL7 and CCL8 share similarities, hence why they are also referred to as MCP-1 (CCL2), MCP-2 (CCL8) and MCP-3 (CCL7). One of these similarities is the pyroglutamate modification on the first position which protects them from CD26 cleavage. The resulting chimeric chemokines have a strong selectivity for ACKR2 compared to the parental chemokines CCL2 (which binds to CCR2 and ACKR2), CCL7 (which binds to CCR1, CCR2, CCR3 and CCR5), CCL8 (which binds to CCR1, CCR3 and CCR5) and CCL22 (which binds to CCR4, ACKR2 and ACKR4) while maintaining strong potency and agonist activity (as exemplified herein with specific embodiments, such as EC5o=1.8nM for human LIH222, 2.6nM for LIH722 and 1.9nM for LIH822).

[0087] Therefore, the invention relates to a chimeric chemokine, comprising a CCL22 polypeptide fused to a peptide having the motif QPXXXXXXXT, preferably corresponding to the motif QPDXXXXPXT (SEQ ID NO: 34) and / or motif QPXXINXXXT (SEQ ID NO: 35) and / or motif XPDXXXAPXX (SEQ ID NO: 36) and XPDXAPXX (SEQ ID NO: 37).

[0088] More particularly, the invention relates to chimeric chemokines selectively binding to ACKR2 comprising a CCL2, CCL7 or CCL8 chemokine polypeptide fused to a CCL22 chemokine polypeptide. More particularly, said chimeric chemokine selectively binds to ACKR2 and not to other known chemokine receptors. Indeed, while CCL2 binds to CCR2, and CCL22 binds CCR4 and ACKR4, this is not the case for the CCL2 / CCL22 (LIH222), as this binds only to ACKR2. Similarly, while CCL7 naturally binds to CCR1, CCR2, CCR3 and / or CCR5, this is not the case for CCL7 / CCL22 (LIH722), which binds only to ACKR2. Finally, while CCL8 binds to CCR1, CCR3 and / or CCR5, this is not the case for CCL8 / CCL22 (LIH822), which also binds selectively to ACKR2.

[0089] The term “chimeric chemokine” refers to a polypeptide which is composed of parts of chemokines thereby reconstituting a new chemokine having one or more of the original and / or new functions when compared to the functions of the chemokines from which it is made.

[0090] The term “CCL2 chemokine polypeptide”, “CCL7 chemokine polypeptide”, “CCL8 chemokine polypeptide” and “CCL22 chemokine polypeptide” as used herein refer to parts of the natural chemokine, i.e. polypeptides which represent a sequence of consecutive amino acids occurring within but shorter than the natural chemokine. The chemokines CCL2, CCL7, CCL8 and CCL22 have similar structural elements. Preferably, the chimeric chemokine of the invention combines different structural elements from these two chemokines.

[0091] In particular embodiments, the chimeric chemokine is made by switching corresponding parts of the two chemokine molecules at a conserved position in these molecules. For instance, “CC”chemokines have a conserved Cys-Cys (or CC) motif in the N-terminal end of their polypeptide sequence. The sequence before the Cys-Cys motif can be combined with the C-terminal end after the Cys-Cys motif of another chemokine, resulting in a chimera. It is clear that the Cys-Cys motif is present in both original chemokines. It will further be understood that the N- and C-terminal ends from the different chemokine s retain their original position with respect to the Cys-Cys motif in the chimera.

[0092] In particular embodiments, the chimeric chemokines described herein comprise a peptide corresponding to the motif QPXXXXXXXT, preferably corresponding to the motif QPDXXXXPXT (SEQ ID NO: 34) and / or motif QPXXINXXXT (SEQ ID NO: 35) and / or motif XPDXXXAPXX (SEQ ID NO: 36) and XPDXAPXX (SEQ ID NO: 37) linked through the Cys-Cys motif to the C-terminal part (after the Cys-Cys motif) of CCL22. In particular embodiments, the chimeric chemokines described herein comprises the N-terminal end before the Cys-Cys motif of CCL2, CCL7 or CCL8 and comprise the C- terminal part of CCL22 after the Cys-Cys motif, linked by the Cys-Cys motif. In particular embodiments, the chimeric cytokineq comprise the N-terminal end of CCL2, CCL7 or CCL8 and comprise the three-stranded-antiparallel beta sheet and C-terminal alpha helix of CCL22, linked by the Cys-Cys motif. More specific, the chimeric chemokine comprises the first amino acids of a CCL2, CCL7 or CCL8 polypeptide up to the Cys-Cys motif from the N-terminal end of the human CCL2, CCL7 or CCL8 sequence represented by SEQ ID NO 1, SEQ ID NO 11, SEQ ID NO 17, respectively, or a corresponding sequence of an orthologue thereof and the amino acids starting from the Cys-Cys motif to the end of the human CCL22 sequence represented by SEQ ID NO 4 or the corresponding sequence of an orthologue thereof.

[0093] An embodiment of a human CCL2 sequence is illustrated below, wherein the Cys-Cys motif is in bold underlined:

[0094] QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDS MDHLDKQTQTPKT (SEQ ID NO: 1)

[0095] An embodiment of a human CCL2 polypeptide sequence up to the Cys-Cys motif is:

[0096] QPDAINAPVT (SEQ ID NO: 2)

[0097] An embodiment of a human CCL22 is illustrated below, wherein the Cys-Cys motif is in bold underlined:

[0098] GPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWV KMILNKLSQ (SEQ ID NO: 3).

[0099] An embodiment of the C-terminal end of a human CCL22 polypeptide including the Cys-Cys motif is:

[0100] CCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVKMILNKLSQ (SEQ ID NO: 4). An embodiment of a human CCL7 sequence is illustrated below, wherein the Cys-Cys motif is in bold underlined:

[0101] QPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDF MKHLDKKTQTPKL (SEQ ID NO: 11).

[0102] An embodiment of a human CCL7 polypeptide sequence up to the Cys-Cys motif is:

[0103] QPVGINTSTT (SEQ ID NO: 12).

[0104] An embodiment of a human CCL8 sequence is illustrated below, wherein the Cys-Cys motif is in bold underlined:

[0105] QPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMK HLDQIFQNLKP (SEQ ID NO: 17).

[0106] An embodiment of a human CCL8 polypeptide sequence up to the Cys-Cys motif is:

[0107] QPDSVSIPIT (SEQ ID NO: 18).

[0108] The invention envisages that, in light of the exchangeability of CCL2, CCL7 and CCL8, in particular embodiments, CCL2, CCL7, CCL8 and CCL22 chemokine polypeptides can be used, which may differ slightly from the human CCL2, CCL7, CCL8 and CCL22 chemokine polypeptides disclosed herein. Indeed, polypeptides of a different origin may be used. In particular embodiments, the chimeric chemokines comprise CCL2, CCL7 CCL8, and CCL22 chemokine polypeptides originating from animals such as mice or other rodents. In further embodiments, the chimeric chemokine is composed of chemokine polypeptides originating from other animals such as non-human primates, donkey, pig, dog, cat, rabbit, rat or horse or from cattle, poultry, fish (e.g. zebra fish) or camelids (e.g. llama). In particular embodiments, the chimeric chemokines according to the invention comprise CCL2, CCL7, CCL8 and CCL22 chemokine polypeptides from mouse.

[0109] Accordingly, in particular embodiments, the chimeric chemokine described herein comprises a polypeptide corresponding to the N-terminal end of the mouse CCL2, CCL7, or CCL8 up to the Cys- Cys motif on the one hand, and the C-terminal end of the mouse CCL22 as from the Cys-Cys motif on the other hand, linked by the Cys-Cys motif. In particular embodiments, the N-terminal end of the mouse CCL2 is the N-terminal end of the sequence represented by SEQ ID NO: 3, for instance the N- terminal residues up to the Cys-Cys motif of the polypeptide represented by SEQ ID NO: 3, or the corresponding fragment of an orthologue thereof. In particular embodiments, the N-terminal end of the mouse CCL7 is the N-terminal end of the sequence represented by SEQ ID NO: 14, for instance the N- terminal residues up to the Cys-Cys motif of the polypeptide represented by SEQ ID NO: 14, or the corresponding fragment of an orthologue thereof. In particular embodiments, the N-terminal end of the mouse CCL8 is the N-terminal end of the sequence represented by SEQ ID NO: 20, for instance the N- terminal residues up to the Cys-Cys motif of the polypeptide represented by SEQ ID NO: 20, or the corresponding fragment of an orthologue thereof. In particular embodiments the C-terminal end of the mouse CCL22 as from the Cys-Cys motif corresponds to the C-terminal end of the mouse CCL22 polypeptide represented by SEQ ID NO: 7 as from the Cys-Cys motif, for instance starting from about position 11 to the end of the mouse CCL22 polypeptide represented by SEQ ID NO: 7, or a corresponding fragment of an orthologue thereof.

[0110] An embodiment of a mouse CCL2 sequence is provided below, wherein the Cys-Cys motif is in bold underlined:

[0111] QPDAVNAPLTCCYSFTSKMIPMSRLESYKRITSSRCPKEAVVFVTKLKREVCADPKKEWVQT YIKNLDRNQMRSEPTTLFKTASALRSSAPLNVKLTRKSEANASTTFSTTTSSTSVGVTSVTVN (SEQ ID NO: 5)

[0112] An embodiment of a mouse CCL2 polypeptide up to the Cys-Cys motif is:

[0113] QPDAVNAPLT (SEQ ID NO: 6)

[0114] An embodiment of a mouse CCL7 sequence is provided below, wherein the Cys-Cys motif is in bold underlined:

[0115] QPDGPNASTCCYVKKQKIPKRNLKSYRRITSSRCPWEAVIFKTKKGMEVCAEAHQKWVEEAI AYLDMKTPTPKP (SEQ ID NO: 14)

[0116] An embodiment of a mouse CCL7 polypeptide up to the Cys-Cys motif is:

[0117] QPDGPNAST (SEQ ID NO: 15)

[0118] An embodiment of a mouse CCL8 sequence is provided below, wherein the Cys-Cys motif is in bold underlined:

[0119] GPDKAPVTCCFHVLKLKIPLRVLKSYERINNIQCPMEAVVFQTKQGMSLCVDPTQKWVSEY MEILDQKSQILQP (SEQ ID NO: 20)

[0120] An embodiment of a mouse CCL8 polypeptide up to the Cys-Cys motif is:

[0121] GPDKAPVT (SEQ ID NO: 21)

[0122] An embodiment of a mouse CCL22 sequence is provided below, wherein the Cys-Cys motif is in bold underlined:

[0123] GPYGANVEDSICCQDYIRHPLPSRLVKEFFWTSKSCRKPGVVLITVKNRDICADPRQVWVKK

[0124] LLHKLS (SEQ ID NO: 7) An embodiment of a mouse CCL22 polypeptide corresponding to the C-terminal end including the Cys- Cys motif is:

[0125] CCQDYIRHPLPSRLVKEFFWTSKSCRKPGVVLITVKNRDICADPRQVWVKKLLHKLS (SEQ ID NO: 8).

[0126] In particular embodiments, the chimeric CCL2 / CCL22 chemokine comprises the N terminus of the human CCL2 chemokine (e.g. as annotated under UniProt accession number Pl 3500) and the C terminus of the human CCL22 chemokine polypeptide (e.g. as annotated under UniProt accession number 000626).

[0127] In other particular embodiments, the chimeric CCL2 / CCL22 chemokine comprises the N terminus of the mouse CCL2 chemokine (e.g. as annotated under UniProt accession number P10148) and the C terminus of the mouse CCL22 chemokine polypeptide (e.g. as annotated under UniProt accession number 088430).

[0128] An embodiment of a human chimeric chemokine is given in SEQ ID NO: 9 (LIH222) wherein the Cys- Cys motif is underlined:

[0129] QPDAINAPVTCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVKMI LNKLSQ (SEQ ID NO: 9)

[0130] An embodiment of a mouse chimeric chemokine is given in SEQ ID NO: 10 (LIHM2M22) wherein the Cys-Cys motif is underlined:

[0131] QPDAVNAPLTCCQDYIRHPLPSRLVKEFFWTSKSCRKPGVVLITVKNRDICADPRQVWVKKL LHKLS (SEQ ID NO: 10).

[0132] In particular embodiments, the chimeric CCL7 / CCL22 chemokine comprises the N terminus of the human CCL7 chemokine (e.g. as annotated under UniProt accession number P80098) and the C terminus of the human CCL22 chemokine polypeptide (e.g. as annotated under UniProt accession number 000626).

[0133] In other particular embodiments, the chimeric CCL7 / CCL22 chemokine comprises the N terminus of the mouse CCL7 chemokine (e.g. as annotated under UniProt accession number Q03366) and the C terminus of the mouse CCL22 chemokine polypeptide (e.g. as annotated under UniProt accession number 088430).

[0134] An embodiment of a human chimeric chemokine is given in SEQ ID NO: 13 (LIH722) wherein the Cys-Cys motif is underlined:

[0135] QPVGINTSTTCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVKMI

[0136] LNKLSQ (SEQ ID NO: 13). An embodiment of a mouse chimeric chemokine is given in SEQ ID NO: 16 (LIHM7M22) wherein the Cys-Cys motif is underlined:

[0137] QPDGPNASTCCYVKKQKIPKRNLKSYRRITSSRCPWEAVIFKTKKGMEVCAEAHQKWVEEAI AYLDMKTPTPKP (SEQ ID NO: 16) In particular embodiments, the chimeric CCL8 / CCL22 chemokine comprises the N terminus of the human CCL8 chemokine (e.g. as annotated under UniProt accession number P80075) and the C terminus of the human CCL22 chemokine polypeptide (e.g. as annotated under UniProt accession number 000626).

[0138] In other particular embodiments, the chimeric CCL8 / CCL22 chemokine comprises the N terminus of the mouse CCL8 chemokine (e.g. as annotated under UniProt accession number Q9Z121) and the C terminus of the mouse CCL22 chemokine polypeptide (e.g. as annotated under UniProt accession number 088430).

[0139] An embodiment of a human chimeric CCL8 / CCL22 chemokine is given in SEQ ID NO: 19 (LIH822) wherein the Cys-Cys motif is underlined:

[0140] QPDSVSIPITCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVKMIL NKLSQ (SEQ ID NO: 19).

[0141] An embodiment of a mouse chimeric chemokine CCL8 / CCL22 is given in SEQ ID NO: 22 (LIHM8M22) wherein the Cys-Cys motif is underlined:

[0142] GPDKAPVTCCQDYIRHPLPSRLVKEFFWTSKSCRKPGVVLITVKNRDICADPRQVWVKKLLH KLS (SEQ ID NO: 22)

[0143] The inventors have further found that quite strong binging affinity to ACKR2 can be obtained by the use of chimeric chemokines wherein the N-terminal end is from a different species as the C-terminal end. Accordingly, in still other particular embodiments, the chimeric chemokines comprise a CCL2, CCL7 or CCL8 chemokine polypeptide of one species combined with a CCL22 chemokine polypeptide of another species. More particularly, the chimeric chemokine comprises the N-terminal end of CCL2, CCL7 or CCL8 of one species and the C-terminal end of CCL22 of another species, more particularly the N-terminal end of CCL2, CCL7 or CCL8 of one species and the three stranded -antiparallel beta sheet and C-terminal alpha helix of CCL22 of another species. For instance, in particular embodiments, the chimeric chemokine comprises the N terminus of the human CCL2, CCL7 or CCL8 chemokine and the C terminus of the mouse CCL22 chemokine.

[0144] An embodiment of a human / mouse chimeric CCL2 / CCL22 chemokine comprising the N terminus of the human CCL2 and the C terminus of mouse CCL22 is given in SEQ ID NO: 23 wherein the Cys- Cys motif is underlined: QPDAINAPVTCCQDYIRHPLPSRLVKEFFWTSKSCRKPGVVLITVKNRDICADPRQVWVKKL LHKLS (SEQ ID NO: 23).

[0145] In still other particular embodiments, the chimeric chemokine comprises the N terminus of the mouse CCL2, CCL7 or CCL8 chemokine and the C terminus of the human CCL22 chemokine. An embodiment of a mouse / human chimeric CCL2 / CCL22 chemokine comprising the N terminus of the mouse CCL2 and the C terminus of human CCL22 is given in SEQ ID NO: 24 wherein the Cys- Cys motif is underlined:

[0146] QPDAVNAPLTCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVKM ILNKLSQ (SEQ ID NO: 24). Particular embodiments of chimeric chemokines of the invention have been made or can be envisaged based on the sequences above. These are referred to herein with the following references:

[0147] The consensus motifs of the N-terminal part of CCL2, CCL7 and CCL8 are thus obtained as follows wherein X is an undefined amino acid:

[0148] The invention thus provides a number of embodiments of chimeric chemokines according to the present invention. It will be clear to the skilled person that further embodiments according to the invention can be obtained by minor modifications of the sequences described herein, such as to obtain sequences having at least 70%, preferably at least 75%, more preferably at least 80%, 85% or up to 90%, most preferably having at least 95% sequence identity with one or more of the chimeric chemokines described herein, preferably with the sequences represented by SEQ ID NOs 9, 10, 13, 16, 19, 22, 23 or 24. Alternatively the skilled person will consider introducing one or more, such as two, three, four, five, six, seven, eight, nine or ten or more modifications, preferably conservative amino acid substitutions, in the sequences set out herein.

[0149] An important constraint in chemokine activity is CD26 cleavage, leading to inactivation of the chemokine. Previous studies have shown the sensitivity of CCL22 and CXCL10 to CD26. Specifically for CCL22, this chemokine has been shown to be completely unable to bind ACKR2 upon CD26 inactivation.

[0150] Therefore, in particular embodiments, in the chimeric chemokines herein described, the amino terminal or N-terminal glutamine comprises a pyruvate modification, forming pyroglutamate.

[0151] The thus resulting chimeric chemokines described herein are shown to be insensitive to proteolytic inhibition of CD26 cleavage. Similarly, the application envisages chimeras that carry one or more co- or post-expression-type modifications of the polypeptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native polypeptides, such as, e.g., amino acid deletions, additions and / or substitutions.

[0152] The term “selectively binding” as used herein when referring to a chemokine selectively binding to ACKR2 means that the chemokine binds exclusively to ACKR2.

[0153] The chimeric chemokines as described herein selectively bind to ACKR2, i.e. exclusively binds to ACKR2. Most particularly, the chimeric chemokines are not capable of binding to any other chemokine receptor than ACKR2. More particularly the chimeric chemokines are not capable of binding to a chemokine receptor selected from the group consisting of C-C chemokine receptor type 1 (CCR1) (e.g. with UniProt accession number P32246), C-C chemokine receptor type 2 (CCR2) (e.g. with UniProt accession number P41597) such as CCRtype 2A (CCR2A) or CCRtype 2B (CCR2B), C-C chemokine receptor type 3 (CCR3) (e.g. with UniProt accession number P51677), C-C chemokine receptor type 4 (CCR4) (e.g. with UniProt accession number P51679), C-C chemokine receptor type 5 (CCR5) (e.g. with UniProt accession number P51681), C-C chemokine receptor type 6 (CCR6) (e.g. with UniProt accession number P51684), C-C chemokine receptor type 7 (CCR7) (e.g. with UniProt accession number P32248), C-C chemokine receptor type 8 (CCR8) (e.g. with UniProt accession number P51685), C-C chemokine receptor type 9 (CCR9) (e.g. with UniProt accession number P51686), C-C chemokine receptor type 10 (CCR10) (e.g. with UniProt accession number P46092), C-X-C motif chemokine receptor 1 (CXCR1) (e.g. with UniProt accession number P25024), C-X-C motif chemokine receptor 2 (CXCR2) (e.g. with UniProt accession number P25025), C-X-C motif chemokine receptor 3 (CXCR3) (e.g. with UniProt accession number P49682) such as CXCRtype 3A (CXCR3A) and CXCR type 3B (CXCR3B), C-X-C motif chemokine receptor 4 (CXCR4) (e.g. with UniProt accession number P61073), C-X-C motif chemokine receptor 5 (CXCR5) (e.g. with UniProt accession number P32302), C-X-C motif chemokine receptor 6 (CXCR6) (e.g. with UniProt accession number 000574), C-X-C motif chemokine receptor 8 (CXCR8) (e.g. with UniProt accession number Q9HC97), X-C motif chemokine receptor 1 (XCR1) (e.g. with UniProt accession number P46094), C-X3-C motif chemokine receptor 1 (CX3CR1) (e.g. with UniProt accession number P49238), atypical chemokine receptor 1 (ACKR1) (e.g. with UniProt accession number QI 6570), atypical chemokine receptor 3 (ACKR3) (e.g. with UniProt accession number P25106), atypical chemokine receptor 4 (ACKR4) (e.g. with UniProt accession number Q9NPB9) and G protein-coupled receptor 182 (GPR182) (e.g. with UniProt accession number 015218). In particular embodiments, the selective chimeric chemokines as described herein are not capable of inducing the recruitment of P-arrestin-1 and P-arrestin-2 through another chemokine receptor than ACKR2

[0154] The term “modulate” broadly denotes a qualitative and / or quantitative alteration, change or variation in that which is being modulated. Where modulation can be assessed quantitatively - for example, where modulation comprises or consists of a change in a quantifiable variable such as a quantifiable property or where a quantifiable variable provides a suitable surrogate for the modulation - modulation specifically encompasses both increase (e.g., activation) or decrease (e.g., inhibition) in the measured variable. The term encompasses any extent of such modulation, e.g., any extent of such increase or decrease, and may more particularly refer to statistically significant increase or decrease in the measured variable. By means of example, modulation may encompass an increase in the value of the measured variable by at least about 10%, e.g., by at least about 20%, preferably by at least about 30%, e.g., by at least about 40%, more preferably by at least about 50%, e.g., by at least about 75%, even more preferably by at least about 100%, e.g., by at least about 150%, 200%, 250%, 300%, 400% or by at least about 500%, compared to a reference situation without said modulation; or modulation may encompass a decrease or reduction in the value of the measured variable by at least about 10%, e.g., by at least about 20%, by at least about 30%, e.g., by at least about 40%, by at least about 50%, e.g., by at least about 60%, by at least about 70%, e.g., by at least about 80%, by at least about 90%, e.g., by at least about 95%, such as by at least about 96%, 97%, 98%, 99% or even by 100%, compared to a reference situation without said modulation. Preferably, modulation may be specific or selective, hence, that which is being modulated may be changed or altered without modulated without substantially altering other (unintended, undesired, unrelated) targets, functions, properties or processes. The term "modulator" as used herein, refers to an agent that interacts with a target either directly or indirectly. The interactions include, but are not limited to, the interactions of an agonist, partial agonist, an inverse agonist, antagonist, degrader, or combinations thereof.

[0155] The chimeric chemokines of the invention are said to “specifically bind to” ACKR2 when these chimeric chemokines have affinity for, specificity for, and / or are specifically directed against ACKR2 (i.e., against at least one part or fragment thereof).

[0156] The “specificity” of the chimeric chemokines as described herein can be determined based on affinity. The “affinity” of a chimeric chemokine is represented by the equilibrium constant for the dissociation of the chimeric chemokine and ACKR2, preferably human ACKR2 (e.g. as annotated under NCBI Genbank accession number NP_001287.2). The lower the KD value, the stronger the binding strength between the chimeric chemokine and ACKR2. Alternatively, the affinity can also be expressed in terms of the affinity constant (KA), which corresponds to 1 / KD. A KD value greater than about 1 millimolar is generally considered to indicate non-binding or non-specific binding.

[0157] The binding of the chimeric chemokines, as described herein to ACKR2 and the affinity and specificity of said binding may be determined by any methods known in the art. Non-limiting examples thereof include binding competition assays using fluorescently labelled or radiolabelled ligands, coimmunoprecipitation, bimolecular fluorescence complementation, affinity electrophoresis, label transfer, phage display, proximity ligation assay (PLA), Tandem affinity purification (TAP), in-silico docking and calculation of the predicted Gibbs binding energy and competition binding assays.

[0158] In particular embodiments, the specific chimeric chemokines as described herein have a potency for ACKR2 that is characterized by an EC50 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 an EC50 of 5 nM or less, more preferably an EC50 of 1 nM or less. The EC50 in the context of the present invention can be determined based on P-arrestin recruitment assay. -arrestin recruitment can be determined by any methods known in the art such as by nanoluciferase complementation assays (e.g. NanoBiT, Promega or NanoBRET, Promega), for instance using ACKR2 C-terminally fused to SmBiT and the P-arrestin N-terminally fused to LgBiT.

[0159] The term “orthologue” when referring to a protein from a species, e.g. different from mus musculus. is a protein that shows strong similarity, i.e. sequence identity, to the specified protein of mus musculus and exhibits essentially the same function as the protein of mus musculus because both proteins are encoded by genes originating from a common ancestor. In particular the orthologue (or the sequence encoding said orthologue) will have at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a given sequence the respective orthologue refers to, e.g. over the whole sequence length.

[0160] A further related aspect of the invention provides for multimers of at least two, at least three, at least four or more chimeric chemokines. Chimeric chemokines are preferably coupled to each other through their C-terminus, preferably using a specific linker allowing the coupling of for instance two or three of the chimeric cytokines of the invention. Such multimers of chimeric cytokines may provide for an enhanced effect, such as a stronger affinity towards ACKR2.

[0161] In particular embodiments of the above aspects relating to multimers, the linkers used are linkers capable of coupling the chimeric cytokine through their C-terminus. The coupling may be either through a recombinant or a chemical coupling. The nature of the linker is not critical and several multimerization scaffolds are available in nature. Dimers can be formed using a recombinant construct, comprising a dimerization scaffold, such as the dimerization scaffold of the C4bp alpha or C4bp beta chain of the C4b binding protein (C4bp), the dimerization scaffold of the HNF-1 alpha or HNF-1 beta chain of the hepatocyte nuclear factor (HNF). Trimers can be formed using a trimerization scaffold, such as the collagen-like region of ficolin-2, a T4 foldon, such as the T4 fibritin trimerization domain, the GCN4 trimerization motif.

[0162] Linkers allowing dimerization or trimerization through chemical coupling are commercially available, for instance with Broadpharm. Chemical dimerization of chimeric cytokines can be performed using for instance an azido-PEG-azide, such as an azido-PEG5 -azide, azido-PEG5 -azide, azido-PEG7 -azide, azido-PEG8-azide, azido-PEGlO-azide, azido-PEG 11 -azide, azido-PEG12-azide, azido-PEG19-azide, azido-PEG20-azide or azido-PEG23 -azide, preferably an azido-PEG5 -azide or azido-PEGlO-azide.

[0163] Further related aspects of the invention, provide for envisaged uses of the chimeric chemokines or multimers of the invention. In particular embodiments the chimeric chemokines or multimers thereof described herein can be used as a medicament or diagnostic, they can be used for research or clinical purposes, can be used as soluble protein(s), fused to dyes, toxins or displayed on scaffold proteins to detect, target or modulate the function of ACKR2 expressed at the cell surface. In view of the broad involvement of ACKR2 in both physiological processes and disease conditions, analyzing the expression of ACKR2 can be useful both in research and as a biomarker for disease in body fluids and tissues.

[0164] In particular embodiments the invention relates to methods for detecting the presence of ACKR2, such as on the surface of a cell or cell-line using the chimeric chemokine or multimer of the invention. In particular embodiments, the method is in vitro or ex vivo, such as the detection in cells or tissue isolated from a living organism. In particular embodiments the absence or presence of ACKR2 on a cell surface can be indicative for a disease state, such as when compared with its expression on said cell surface in a healthy control cell or tissue. Accordingly, the invention also encompasses the use of the chimeric chemokine or multimer of the invention as a diagnostic, more particularly in methods of diagnosis which involve contacting a cell or tissue with the chimeric chemokines of the invention and determining the expression of ACKR2 by said cell or tissue based on the binding of the chimeric chemokines of the invention.

[0165] In this context, the chimeric chemokines or multimers of the invention may further comprise a detectable label (such as a fluorescent protein or enzyme) or another functional moiety such as an immunoglobulin Fc region, a protoxin, a toxin or a pharmaceutical. The term “functional moiety” as used herein refers to a chemical structure, such as but not limited to an amino acid sequence which ensures a specific function which can be but is not limited to functions which affect the chimeric chemokine, such as allowing the detection of the chimeric chemokine (in case of a detectable label), ensuring stabilization of the chimeric chemokine (such as is the case for an Fc region). Examples of functional moieties which ensuring a separate function independent of the chemokine are protoxins or toxins, binding agents, nucleotide sequence etc. Preferably, the chimeric chemokine is linked to a fluorescent protein, an enzyme, an immunoglobulin Fc region, a protoxin or a toxin. The inventors have surprisingly found that the chimeric chemokines of the invention, remain functional when linked to a functional moiety, more particularly when linked c-terminally to the chimeric chemokine, does not impact the binding to ACKR2, making it suitable for use as a fusion protein.

[0166] The term “detectable label” refers to any atom, molecule, moiety or biomolecule that may be used to provide a detectable and preferably quantifiable read-out or property, and that may be attached to or made part of an entity of interest, such as the peptide as taught herein. Labels may be suitably detectable by for example mass spectrometric, spectroscopic, optical, colourimetric, magnetic, photochemical, biochemical, immunochemical or chemical means. Labels include without limitation dyes; radiolabels such as32P,33P,35S,1251,131I; electron-dense reagents; enzymes (e.g., horse-radish peroxidase or alkaline phosphatase as commonly used in immunoassays); binding moieties such as biotin-streptavidin; haptens such as digoxigenin; luminogenic, phosphorescent or fluorogenic moieties; mass tags; and fluorescent dyes (e.g., fluorophores such as fluorescein, carboxyfluorescein (FAM), tetrachloro-fluorescein, TAMRA, ROX, Cy3, Cy3.5, Cy5, Cy5.5, Texas Red, etc.) alone or in combination with moieties that may suppress or shift emission spectra by fluorescence resonance energy transfer (FRET).

[0167] In some embodiments, the chimeric chemokines or multimers as taught herein may be provided with a tag that permits detection with another agent (e.g., with a probe binding partner). Such tags may be, for example, biotin, streptavidin, his-tag, myc tag, maltose, maltose binding protein or any other kind of tag known in the art that has a binding partner. Example of associations which may be utilised in the probe:binding partner arrangement may be any, and includes, for example biotin: streptavidin, his- tag:metal ion (e.g., Ni2+), maltose maltose binding protein, etc.

[0168] In particular embodiments, the label may be Large BiT (LgBiT) or Small BiT (SmBiT) or HiBiT of NanoLuc® Binary Technology (NanoBiT).

[0169] The chimeric chemokines or multimers as taught herein may be associated with 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 or a nucleotide sequence. The detection agent may be a particle. Examples of such particles include, but are not limited to, colloidal gold particles; colloidal sulphur particles; colloidal selenium particles; colloidal barium sulphate particles; colloidal iron sulphate particles; metal iodate particles; silver halide particles; silica particles; colloidal metal (hydrous) oxide 1 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- mentioned colloidal particles coated with organic or inorganic layers; protein or peptide molecules; liposomes; or organic polymer latex particles, such as polystyrene latex beads.

[0170] In particular embodiments, the chimeric chemokines as taught herein may be coupled to the agent by one or more linkers.

[0171] As used herein, the term “linker” refers to a connecting element that serves to link other elements. The linker may be a rigid linker or a flexible linker. In particular embodiments, the linker is a covalent linker, achieving a covalent bond. The terms “covalent” or “covalent bond” refer to a chemical bond that involves the sharing of one or more electron pairs between two atoms. For many molecules, the sharing of electrons allows each atom to attain the equivalent of a full outer electron shell, corresponding to a stable electronic configuration. Covalent bonds include different types of interactions, including o- bonds, n-bonds, metal-to-metal bonds, agostic interactions, bent bonds and three-center two-electron bonds.

[0172] In particular embodiments, the linker is a (poly) peptide linker or a non-peptide linker, such as a nonpeptide polymer, such as a non-biological polymer. Preferably, the linkage(s) between the peptide as taught herein and the second peptide, protein or polypeptide may be hydrolytically stable linkage(s), i.e., substantially stable in water at useful pH values, including in particular under physiological conditions, for an extended period of time, e.g., for days. In particular embodiments, the linker is a peptide linker of one or more amino acids.

[0173] It will be understood that where a multimer comprises a scaffold, the detectable label (such as a fluorescent protein or enzyme) or another functional moiety such as an immunoglobulin Fc region, a protoxin, a toxin or a pharmaceutical may also be linked to the scaffold.

[0174] The binding of the chimeric chemokines of the invention to ACKR2 can also be detected by other means such as labelled antibodies or other binding agents or by methods such as isothermal calorimetry (ITC), surface plasmon resonance (SPR), biologically modified field effect transistors (BioFET), fluorescence resonance energy transfer (FRET), differential optical scattering, electrophoretic mobility shift assays (EMSA) or electronic spectroscopy.

[0175] In particular embodiments, detection of ACKR2 may be in vivo. Accordingly, the invention also provides for the chimeric chemokines and multimers of the invention for use as a diagnostic.

[0176] In particular embodiments, the diagnostic is used in the detection of aberrant ACKR2 expression or functioning in an organism (or isolated cells or tissue thereof). Aberrant ACKR2 expression can affect chemokine functioning. For instance, over-expression of ACKR2 can lead to chemokine dysregulation as a result of excessive scavenging activity. Patients suffering from ACKR2 overexpression could be susceptible to treatment with ACKR2 modulators such as the chimeric chemokines of the present invention as will be detailed herein, or with ACKR2 inhibitors such as antisense RNA or gene editing. Accordingly, in particular embodiments, the chimeric chemokines of the invention are of interest for use as a diagnostic to determine the susceptibility of a patient to treatment by an ACKR2 modulator or inhibitor.

[0177] A further related aspect of the invention is the use of the chimeric chemokines of the invention (optionally in the form of a multimer) in therapy.

[0178] In particular embodiments the invention provides the use of the chimeric chemokines as described herein as a targeting agent. More particularly, the chimeric chemokines of the invention can be bound to a functional component (such as a toxin) or can be fused to a paratope-comprising molecule, to target cell surface proteins. For instance it has been shown that bispecific antibodies which combine a first paratope targeting a chemokine and a second paratope targeting a cell-surface protein can make use of the cytokine-mediated internalization of cognate receptors to ensure broad and efficient lysosomal delivery of both cell-surface and extracellular proteins. The chimeric chemokines of the present invention can be used in a similar way, by coupling them to one arm of a bispecific antibody and combining this with a second arm engineered to bind a target protein. Such methods are useful in the targeting of therapeutically relevant surface proteins, such as HER2 which is frequently upregulated in cancer or epidermal growth factor receptor, which has also been demonstrated to be a relevant target in the treatment of different types of cancer. Antibodies to cancer targets are known in the art, such as Herceptin (anti-HER2 antibody) or cetuximab (anti-EGFR).

[0179] Accordingly, another aspect of the invention relates to fusion proteins of the chimeric chemokines of the present invention to paratope-containing antibody fragments, more particularly bispecific antibody chimeric chemokine fusions. In particular embodiments, the fusion is a knob-in-hole antibody-chimeric chemokine fusion, whereby the chimeric chemokine is fused N-terminally to the knob Fc domain and a paratope binding a target protein, such as an extracellular or membrane protein.

[0180] The chimeric chemokines of the present invention (and nucleic acids encoding) them are also suitable for therapeutic use as such, more particularly as modulators of chemokine levels. A further related aspect thus provides a pharmaceutical composition comprising at least one of the selective chimeric chemokines as described herein, the nucleic acid encoding the selective chimeric chemokine, the nucleic acid expression cassette as described herein below or the vector as described herein below, and optionally a pharmaceutically acceptable carrier.

[0181] Generally, ACKR2 modulation is envisaged to be of interest where ACKR2 scavenging is detrimental, such as, but not limited to where there is aberrant ACKR2 expression, more particularly, ACKR2 overexpression, or where chemokine activity is critical, such as where an immune response is required. The use of the chimeric chemokines of the invention is envisaged to compete with the natural ligands of ACKR2, thereby affecting its function as a chemokine scavenger. This is of interest in diseases where the immune reaction is suppressed by (over) expression of ACKR2, such as in the tumor microenvironment, where CCL2 is necessary to attract NK cells. Accordingly, the chimeric chemokines of the present invention are particularly envisaged for use in cancer therapy, more particularly in combination with other medicaments that suppress cancer growth. In particular embodiments the pharmaceutical compositions of the invention further comprise one or more immune checkpoint modulators, preferably one or more immune checkpoint inhibitors, and optionally a pharmaceutically acceptable carrier.

[0182] The term “pharmaceutically acceptable” as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

[0183] As used herein, “carrier” or “excipient” includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fdlers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active substance, its use in the therapeutic compositions may be contemplated.

[0184] Illustrative, non-limiting carriers for use in formulating the pharmaceutical compositions include, for example, oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents suitable for intravenous (IV) use, liposomes or surfactant-containing vesicles, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to one of ordinary skill in the art.

[0185] Pharmaceutical compositions as intended herein may be formulated for essentially any route of administration, such as without limitation, oral administration (such as, e.g., oral ingestion or inhalation), intranasal administration (such as, e.g., intranasal inhalation or intranasal mucosal application), parenteral administration (such as, e.g., subcutaneous, intravenous (I.V.), intramuscular, intraperitoneal or intrastemal injection or infusion), transdermal or transmucosal (such as, e.g., oral, sublingual, intranasal) administration, topical administration, rectal, vaginal or intra-tracheal instillation, and the like. In this way, the therapeutic effects attainable by the methods and compositions can be, for example, systemic, local, tissue-specific, etc., depending of the specific needs of a given application. In particular embodiments, the compositions of the present invention are designed for tissue-specific delivery. Indeed, it can be of interest to limit the delivery of the chimeric chemokine to the environment and / or the vicinity of the tissues affected by the tumor or disease, in order to optimize the effect of the chimeric chemokine. Tissue-specificity can be achieved at different levels. To some extent, tissuespecificity can be achieved by local delivery of the compositions of the invention. Tissue-specific vectors and promoters are known in the art. Alternatively since the chimeric chemokine is a protein, targeting can be achieved through binding with a targeting agent such as an antibody.

[0186] The dosage or amount of the selective chimeric chemokines as taught herein, optionally in combination with one or more other active compounds to be administered, depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect. Thus, the unit dose and regimen depend on the nature and the severity of the disorder to be treated, and also on factors such as the species of the subject, the sex, age, body weight, general health, diet, mode and time of administration, immune status, and individual responsiveness of the human or animal to be treated, efficacy, metabolic stability and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the agent of the invention. In order to optimize therapeutic efficacy, the selective chimeric chemokine, the nucleic acid encoding the selective chimeric chemokine, the nucleic acid expression cassette, the vector, the host cell or the pharmaceutical composition as described or taught herein can be first administered at different dosing regimens. Typically, levels of the chimeric chemokine in a tissue can be monitored using appropriate screening assays as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. The frequency of dosing is within the skills and clinical judgement of medical practitioners (e.g., doctors, veterinarians or nurses). Typically, the administration regime is established by clinical trials which may establish optimal administration parameters. However, the practitioner may vary such administration regimes according to the one or more of the aforementioned factors, e.g., subject’s age, health, weight, sex and medical status. The frequency of dosing can be varied depending on whether the treatment is prophylactic or therapeutic.

[0187] Therapeutic efficacy of the chimeric chemokines as described herein or pharmaceutical compositions comprising the same can be determined by known pharmaceutical procedures in, for example, cell cultures or experimental animals. These procedures can be used, e.g., for determining the ED50 (the dose therapeutically effective in 50% of the population). Pharmaceutical compositions that exhibit high therapeutic indices are preferred.

[0188] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in appropriate subjects. The dosage of such pharmaceutical compositions lies generally within a range of circulating concentrations that include the ED50. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a pharmaceutical composition used as described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose 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 which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0189] In particular embodiments, the selective chimeric chemokines described herein having a high affinity and selectivity for ACKR2 can be used in the prevention or treatment of an autoimmune, inflammatory, neurological or cardiovascular disease or disorder in a subject. More specific a disease or disorder selected from the group consisting of psoriasis, fibrosis, obesity, transplantation and Graft-Versus-Host- Disease, asthma, Alzheimer’s Disease, multiple sclerosis and myocardial infarction.

[0190] A similar aspect provides a method of treating an autoimmune, inflammatory, neurological or cardiovascular disease or disorder in a subject comprising administering a therapeutically or prophylactically effective amount of a selective chemokine to said subject.

[0191] Indeed, as chemokines play an important role in diseases such as autoimmune, inflammatory, neurological or cardiovascular disease or disorder, the ability to modulate scavenging of chemokines is relevant in the treatment of these diseases. Indeed, the chimeric chemokines of the present invention bind with high affinity and selectivity to ACKR2, such they can be used to interfere with the scavenging function of ACKR2 in disease conditions.

[0192] In particular embodiments, the chimeric chemokines of the present invention are used to treat patients which are suffering from autoimmune, inflammatory, neurological or cardiovascular disease or disorder and are characterized by decreased chemokine and / or increased ACKR2 levels.

[0193] In particular embodiments, the selective chimeric chemokines described herein can be used in the treatment of a proliferative disease or disorder in a subject. More specific a disease or disorder selected from the group consisting of skin cancer such as melanoma, colon cancer, rectal cancer, colorectal cancer, bladder cancer, neuroblastoma, squamous cell cancer, lung cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer such as gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatoma, breast cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, head cancer and neck cancer, preferably skin cancer or colorectal cancer. In particular antibodies, targeting ACKR2 may be beneficial in the context of melanoma (skin cancer) (Noman et al. 2024, DOI: https: / / doi.Org / 10. l 101 / 2024,04.02.587761) and its dual use has been demonstrated in the context of primary and metastatic cancers by Massara et al., (2018)),

[0194] In particular embodiments, the chimeric chemokines of the invention are used to treat solid tumors.

[0195] Indeed, the present inventors have found that exclusively targeting ACKR2 with the chimeric chemokines of the invention decreases the volume and weight of tumors in a subject. The invention thus also provides a method of treating a proliferative disease or disorder in a subject comprising administering a therapeutically or prophylactically effective amount of a selective chemokine to said subject.

[0196] The terms “treat” or “treatment” encompass both the therapeutic treatment of an already developed disease or condition, as well as prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0197] In particular embodiments the chimeric chemokines of the invention, can be used in the prevention or treatment of proliferative diseases or disorders and to improve the response of a subject to anticancer immunotherapy, such as to one or more immune checkpoint modulators, such as to one or more immune checkpoint inhibitors.

[0198] In particular embodiments the chimeric chemokine is used in a subject who is diagnosed with or assumed to have a proliferative disease or disorder which is non-responsive to anticancer immunotherapy, preferably non-responsive to immune checkpoint blockade therapy.

[0199] In particular embodiments, the treatment comprises the administration of said selective chemokine in combination with anticancer immunotherapy, preferably with one or more checkpoint inhibitors.

[0200] In particular embodiments, the invention relates to the combined treatment of a patient with one of the selective chimeric chemokines of the invention and anticancer immunotherapy, such as a checkpoint modulator. Thus, in particular embodiments, the pharmaceutical composition as taught herein further comprises anticancer immunotherapy, preferably one or more immune checkpoint modulators, more preferably one or more immune checkpoint inhibitors. Immune checkpoint modulators and immune checkpoint inhibitors are known in the art.

[0201] In particular embodiments, the pharmaceutical composition as taught herein further comprises one or more (such as one, two, three or four) immune checkpoint inhibitors, preferably one or more selected from the group consisting of a Programmed Death-ligand 1 (PDL-1) inhibitor, a Programmed Death 1 (PD-1) inhibitor, a Cytotoxic T -Lymphocyte- Associated protein 4 (CTLA-4) inhibitor, a cluster of differentiation 3 (CD3) inhibitor, aNKG2A inhibitor, a immunoglobulin-like receptors (KIR) inhibitor, a cluster of differentiation 47 (CD47) inhibitor, a CD24 inhibitor, a CD73 inhibitor, a tumor necrosis factor (TNF) receptor 2 (TNFR2) inhibitor, and a signal-regulatory protein alpha (SIRPa) inhibitor, preferably a PD-1 inhibitor.

[0202] In particular embodiments, the pharmaceutical composition as taught herein further comprises one or more (such as one, two, three or four) immune checkpoint inhibitors of the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, tislelizumab, sintilimab, MEDI0680, and any combination thereof, preferably pembrolizumab.

[0203] In particular embodiment, the chimeric chemokine and optionally the one or more immune checkpoint inhibitors are the main or only active ingredients of the pharmaceutical composition.

[0204] The term “proliferative disease or disorder” as used herein generally refers to any disease or disorder characterized neoplastic cell growth and proliferation, whether benign (not invading surrounding normal tissues, not forming metastases), pre-malignant (pre-cancerous), or malignant (invading adjacent tissues and capable of producing metastases). The term proliferative disease generally includes all transformed cells and tissues and all cancerous cells and tissues. Proliferative diseases or disorders include, but are not limited to abnormal cell growth, benign tumors, premalignant or precancerous lesions, malignant tumors, and cancer. Examples of proliferative diseases and / or disorders are benign, pre-malignant, and malignant neoplasms located in any tissue or organ, such as in the prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, or urogenital tract. The term “proliferative disease or disorder” as used herein may be used as a synonym of a neoplastic disease or disorder.

[0205] The proliferative disease or disorder (i.e. neoplastic disease or disorder) may be a tumor or may be characterized by the presence of a tumor. As used herein, the terms “tumor” or “tumor tissue” refer to an abnormal mass of tissue that results from excessive cell division. A tumor or tumor tissue comprises “tumor cells” which are neoplastic cells with abnormal growth properties and no useful bodily function. Tumors, tumor tissue and tumor cells may be benign, pre-malignant or malignant, or may represent a lesion without any cancerous potential. A tumor or tumor tissue may also comprise “tumor-associated non-tumor cells”, e.g., vascular cells which form blood vessels to supply the tumor or tumor tissue. Non-tumor cells may be induced to replicate and develop by tumor cells, for example, the induction of angiogenesis in a tumor or tumor tissue. The proliferative disease or disorder (i.e. neoplastic disease or disorder) may be malignancy. As used herein, the term “malignancy” refers to a non-benign tumor or a cancer.

[0206] As used herein, the term “cancer” refers to a malignant neoplasm characterized by deregulated or unregulated cell growth. In certain embodiments, the proliferative disease or disorder may be selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, and lymphoid malignancies.

[0207] In certain further embodiments, the proliferative disease or disorder (i.e. neoplastic disease or disorder) may be selected from the group consisting of skin cancer such as melanoma, colon cancer, rectal cancer, colorectal cancer, bladder cancer, neuroblastoma, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung and large cell carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatoma, breast cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, head cancer and neck cancer, preferably skin cancer or colorectal cancer, more preferably melanoma or colorectal cancer, even more preferably melanoma (e.g. subcutaneous melanoma).

[0208] The term “cancer” includes primary malignant cells ortumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells ortumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).

[0209] The proliferative disease or disorder (i.e. neoplastic disease or disorder) may also be a premalignant condition. Premalignant conditions are known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell 1976 (Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79).

[0210] In particular embodiments, the proliferative disease or disorder (i.e. neoplastic disease or disorder) is a primary tumor.

[0211] The term “primary tumor” or “original tumor” or “primary lesion” as used herein refers to a tumor at the original site where the tumor first arose.

[0212] In particular embodiments, the proliferative disease or disorder (i.e. neoplastic disease or disorder) is not a metastasis (i.e. secondary tumor). In particular embodiments, the therapy using the specific chimeric chemokine is a local therapy, for example specifically directed to the tumor or tumor cells. This may be achieved by local administration of the specific ACKR2 modulators, local expression and / or local release of the specific ACKR2 modulator.

[0213] In particular embodiments, the method of treating a proliferative disease or disorder in a subject comprising administering a therapeutically or prophylactically effective amount of a specific chimeric chemokine described herein to said subject is combined with anticancer immunotherapy. Non-limiting examples of anticancer immunotherapy include immune checkpoint modulation therapy (e.g. immune checkpoint blockade therapy, such as a CD3-targeted bispecific antibody, anti-NKG2A-based immunotherapy, anti-KIR based immunotherapy, anti-CD47-based immunotherapy, anti-CD24-based immunotherapy, anti-CD73 -based immunotherapy, anti-TNFR2 -based immunotherapy, or anti-SIRPa- based immunotherapy), monoclonal antibodies (e.g. alemtuzumab or trastuzumab), chimeric antigen receptor (CAR)-T cell therapy, oncolytic viruses, vaccination with a cancer vaccine, adoptive cell transfer, and stimulator of interferon genes (STING)-based immunotherapy, such as treatment with a STING agonist.

[0214] In particular embodiments, the method of treating a proliferative disease or disorder in a subject comprises administering a therapeutically or prophylactically effective amount of a specific chimeric chemokine described herein to said subject in combination with a therapeutically or prophylactically effective amount of one or more anticancer immunotherapies selected from the group consisting of an immune checkpoint modulator (such as a CD3 -targeted bispecific antibody, anti-NKG2A antibody, anti-KIR antibody, anti-CD47 antibody, anti-CD24 antibody, anti-CD73 antibody, anti-TNFR2 antibody, or anti-SIRPa antibody), monoclonal antibodies (e.g. alemtuzumab or trastuzumab), chimeric antigen receptor T cells, an oncolytic virus, a cancer vaccine, a STING agonist and autologous or heterologous tumor-infiltrating lymphocytes to said subject.

[0215] In particular embodiments, the method of treating a proliferative disease or disorder in a subject comprises administering a therapeutically or prophylactically effective amount of a specific chimeric chemokine described herein to said subject in combination with a therapeutically or prophylactically effective amount of one or more of the group consisting of an immune checkpoint inhibitor (such as a CD3 -targeted bispecific antibody, anti-NKG2A antibody, anti-KIR antibody, anti-CD47 antibody, anti- CD24 antibody, anti-CD73 antibody, anti-TNFR2 antibody, or anti-SIRPa antibody), monoclonal antibodies (e.g. alemtuzumab or trastuzumab), chimeric antigen receptor T cells, an oncolytic virus, a cancer vaccine, a STING agonist and autologous or heterologous tumor-infiltrating lymphocytes to said subject. In particular embodiments, the method of treating a proliferative disease or disorder in a subject comprises administering a therapeutically or prophylactically effective amount of a specific chimeric chemokine described herein to said subject in combination with a therapeutically or prophylactically effective amount of one or more immune checkpoint modulators, preferably one or more immune checkpoint inhibitors.

[0216] The term “therapeutically effective amount” as used herein, refers to an amount of therapeutic agent that elicits the biological or medicinal response in a subject that is being sought by a surgeon, researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. The term “prophylactically effective amount” refers to an amount of the prophylactic agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician. Methods are known in the art for determining therapeutically and / or prophylactically effective amounts of the therapeutic or prophylactic agent as described herein.

[0217] A further aspect provides a specific chimeric chemokine for decreasing tumor growth, for decreasing tumor cell proliferation, for shrinking or decreasing the volume of a tumor (e.g. by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, preferably at least 50%) and / or for decreasing the weight of a tumor (e.g. by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, preferably at least 50%) in a subject.

[0218] Furthermore, cancer growth and progression are associated with immune suppression. Cancer cells have the ability to activate different immune checkpoint pathways that harbor immunosuppressive functions. Inhibiting the cancer cells’ ability to suppress the immune system is of interest in the treatment of cancer. Many of the immune checkpoints are initiated by ligand-receptor interactions, and therefore can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors, also known as immune checkpoint inhibitors.

[0219] Accordingly, also disclosed herein is a specific chimeric chemokine for use in improving the response of a subject to anticancer immunotherapy, preferably to an immune checkpoint modulator, more preferably to an immune checkpoint inhibitor.

[0220] The improvement of the response of a subject to anticancer immunotherapy, such as to an immune checkpoint modulator, such as an immune checkpoint inhibitor, may be determined by methods known in the art, such as by the conventional WHO criteria, for example as described in Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting results of cancer treatment.Cancer.l981;47:207-214, Response Evaluation Criteria in Solid Tumors (RECIST), for example as described in Therasse P, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst.2000;92:205-216 or Eisenhauer EA, et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1) Eur J Cancer.2009;45 :228- 247, by determining the level of biomarkers of immunotherapy response such as the expression level of PD-L1, or by determining the number of tumor-infdtrating lymphocytes, such as CD8+ T cells. More particularly, the improvement of the response of a subject to an immune checkpoint modulator, such as an immune checkpoint inhibitor, may be determined by determining the ability of the immune checkpoint modulator, such as an immune checkpoint inhibitor, to enhance the proliferation, migration, persistence and / or cytoxic activity of CD8+ T cells.

[0221] In particular embodiments, the specific chimeric chemokine improves the response of a subject to anticancer immunotherapy, such as to an immune checkpoint modulator, preferably to an immune checkpoint inhibitor, by at least 1%, at least 5%, at least 10%, at least 25% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or greater than 100%, compared to the response of the subj ect to the anticancer immunotherapy, such as to the immune checkpoint modulator, preferably to the immune checkpoint inhibitor, in absence of the specific chimeric chemokine.

[0222] Anticancer immunotherapy is known in the art and includes immune checkpoint modulation therapy (e.g. immune checkpoint blockade therapy, such as a CD3 -targeted bispecific antibody, anti-NKG2A- based immunotherapy, anti -KIR based immunotherapy, anti-CD47-based immunotherapy, anti-CD24 antibody, anti-CD73 antibody, tumor necrosis factor (TNF) receptor 2 (TNFR2) inhibitors (e.g. an anti- TNFR2 antibody), or anti-SIRPa-based immunotherapy), monoclonal antibodies (e.g. alemtuzumab or trastuzumab), chimeric antigen receptor (CAR)-T cell therapy, oncolytic viruses, vaccination with a cancer vaccine, adoptive cell transfer, and stimulator of interferon genes (STING)-based immunotherapy, such as treatment with a STING agonist.

[0223] The term "immune checkpoint modulator" as used herein generally refers to any compound inhibiting (e.g. reducing or fully blocking) or stimulating (e.g. increasing) the function of an immune inhibitory checkpoint protein. Non-limiting examples of stimulatory checkpoint modulators include agonists of CD27, OX-40, CD40, ICOS, 4-1BB and GITR.

[0224] The term "subject" or "patient" as used herein typically and preferably denotes humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, such as, e.g., non-human primates, rodents, canines, felines, equines, ovines, porcines, and the like. Particularly intended are subjects known or suspected to have an autoimmune, inflammatory, neurological, cardiovascular or a proliferative disease or disorder. Suitable subjects may include ones presenting to a physician for a screening for an autoimmune, inflammatory, neurological, cardiovascular or a proliferative disease or disorder and / or with symptoms and signs indicative of an autoimmune, inflammatory, neurological, cardiovascular or a proliferative disease or disorder.

[0225] In particular embodiments, the subject is diagnosed with or assumed to have a proliferative disease or disorder, preferably cancer.

[0226] In particular embodiments, the subject is diagnosed with or assumed to have a proliferative disease or disorder characterized by a low or non-existing tumor infiltration of NK cells, CD8+ T cells, CD4+ cells and / or CD4 effector cells, preferably characterized by a low or non-existing tumor infiltration of NK cells and / or CD8+ T cells.

[0227] In particular embodiments, the subject is diagnosed with or assumed to have melanoma characterized by a ratio of the number of CD8+ cells over the number of T regulator cells of less than 0.2, preferably less than 0.1.

[0228] In particular embodiments, the subject is diagnosed with or assumed to have a proliferative disease or disorder not responding to anticancer immunotherapy, preferably not responding to immune checkpoint modulation therapy, more preferably not responding to immune checkpoint blockade therapy, such as not responding to an immune checkpoint inhibitor.

[0229] As used herein the term "non-responder" in the context of the present disclosure refers to a subject that will not achieve a response, i.e. a subject where the proliferative disease or disorder is not eradicated, reduced or improved upon administration of the anticancer immunotherapy, such as immune checkpoint modulation therapy, such as immune checkpoint blockade therapy. The term "non-responder" may also include patients having a stabilized proliferative disease or disorder.

[0230] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims.

[0231] The selective chimeric chemokines, envisaged for use as disclosed herein may be an expressible molecule such as a protein or polypeptide. It shall be understood that the selective chimeric chemokines may be introduced to a subject by means of a recombinant nucleic acid comprising a sequence encoding the chimeric chemokine operably linked to one or more regulatory sequences allowing for expression of said sequence encoding the chimeric chemokine (e.g. for use in gene therapy or cell therapy).

[0232] Hence, the selective chimeric chemokines may be encoded by a recombinant nucleic acid comprising a sequence encoding one or more desired proteins, polypeptides, peptides operably linked to one or more regulatory sequences allowing for expression of said sequence or sequences encoding the proteins, polypeptides, or peptides, e.g., in vitro, in a host cell, host organ and / or host organism (expression constructs). Such recombinant nucleic acid may be comprised in a suitable vector.

[0233] Accordingly, a related aspect provides a nucleic acid encoding the selective chimeric chemokines of the invention.

[0234] By “encoding” is meant that a nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides.

[0235] A related aspect provides a nucleic acid expression cassette comprising the nucleic acid encoding the selective chimeric chemokines as described herein, operably linked to a promoter and / or transcriptional and translational regulatory signals.

[0236] The term “nucleic acid expression cassettes” as used herein refers to nucleic acid molecules, typically DNA, to which nucleic acid fragments may be inserted to be expressed, wherein said nucleic acid molecules comprise one or more nucleic acid sequences controlling the expression of the nucleic acid fragments. Non-limiting examples of such more nucleic acid sequences controlling the expression of the nucleic acid fragments include promoter sequences, open reading frames and transcription terminators.

[0237] Preferably, the nucleic acid expression cassette may comprise one or more open reading frames (ORF) encoding said one or more proteins, polypeptides or peptides. An “open reading frame” or “ORF” refers to a succession of coding nucleotide triplets (codons) starting with a translation initiation codon and closing with a translation termination codon known per se, and not containing any internal in-frame translation termination codon, and potentially capable of encoding a protein, polypeptide or peptide. Hence, the term may be synonymous with “coding sequence” as used in the art.

[0238] An “operable linkage” is a linkage in which regulatory sequences and sequences sought to be expressed are connected in such a way as to permit said expression. For example, sequences, such as, e.g., a promoter and an ORF, may be said to be operably linked if the nature of the linkage between said sequences does not: (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter to direct the transcription of the ORF, (3) interfere with the ability of the ORF to be transcribed from the promoter sequence. Hence, “operably linked” may mean incorporated into a genetic construct so that expression control sequences, such as a promoter, effectively control transcription / expression of a sequence of interest.

[0239] The precise nature of transcriptional and translational regulatory sequences or elements required for expression may vary between expression environments, but typically include a transcription terminator, and optionally an enhancer. Reference to a “promoter” is to be taken in its broadest context and includes transcriptional regulatory sequences required for accurate transcription initiation and where applicable accurate spatial and / or temporal control of gene expression or its response to, e.g. , internal or external (e.g. , exogenous) stimuli. More particularly, “promoter” may depict a region on a nucleic acid molecule, preferably DNA molecule, to which an RNA polymerase binds and initiates transcription. A promoter is preferably, but not necessarily, positioned upstream, z.e., 5’, of the sequence the transcription of which it controls. Typically, in prokaryotes a promoter region may contain both the promoter per se and sequences which, when transcribed into RNA, will signal the initiation of protein synthesis (e.g., Shine-Dalgamo sequence). A promoter sequence can also include “enhancer regions”, which are one or more regions of DNA that can be bound with proteins (namely the trans-acting factors) to enhance transcription levels of genes in a gene-cluster. The enhancer, while typically at the 5 ’ end of a coding region, can also be separate from a promoter sequence, e.g., can be within an intronic region of a gene or 3’ to the coding region of the gene.

[0240] In embodiments, promoters contemplated herein may be constitutive or inducible. A constitutive promoter is understood to be a promoter whose expression is constant under the standard culturing conditions. Inducible promoters are promoters that are responsive to one or more induction cues. For example, an inducible promoter can be chemically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a chemical inducing agent such as an alcohol, tetracycline, a steroid, a metal, or other small molecule) or physically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a physical inducer such as light or high or low temperatures). An inducible promoter can also be indirectly regulated by one or more transcription factors that are themselves directly regulated by chemical or physical cues. Non-limiting examples of promoters include T7, U6, Hl, retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the - actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.

[0241] In particular embodiments, the promoter is a cancer- or tumor- specific promoter. Non-limiting examples of cancer-specific promoters include ran, brmsl and mcm5 promoters. Non-limiting examples of tumor-specific promoters include E2F-1 promoter, HE4 promoter, LP promoter, and COX-2 promoter. In particular embodiments, the promoter is a melanoma-specific promoter, such as the tyrosinase promoter as described in Lillehammer T. et al., Melanoma-specific expression in first-generation adenoviral vectors in vitro and in vivo — use of the human tyrosinase promoter with human enhancers, Cancer Gene Ther. 2005, 12(1 l):864-72.

[0242] The terms “terminator” or “transcription terminator” refer generally to a sequence element at the end of a transcriptional unit which signals termination of transcription. For example, a terminator is usually positioned downstream of, z.e., 3’ of ORF(s) encoding a polypeptide of interest. For instance, where a recombinant nucleic acid contains two or more ORFs, e.g., successively ordered and forming together a multi-cistronic transcription unit, a transcription terminator may be advantageously positioned 3 ’ to the most downstream ORF.

[0243] In particular embodiments, the nucleic acid expression cassette comprises the nucleic acid encoding one of the selective chimeric chemokines as disclosed herein, operably linked to one or more promoters, enhancers, ORFs and / or transcription terminators.

[0244] A related aspect provides a vector comprising the nucleic acid encoding one of the selective chimeric chemokines or the nucleic acid expression cassette as described herein, such as a viral vector.

[0245] The term “vector” or “expression vector” as used in the application refers to nucleic acid molecules, e.g. double-stranded DNA, which may have inserted into it another nucleic acid molecule (the insert nucleic acid molecule) such as, but not limited to, a cDNA molecule. The vector is used to transport the insert nucleic acid molecule into a suitable host cell. A vector may contain the necessary elements that permit transcribing the insert nucleic acid molecule, and, optionally, translating the transcript into a peptide, protein or polypeptide. The insert nucleic acid molecule may be derived from the host cell, or may be derived from a different cell or organism. Once in the host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA, and several copies of the vector and its inserted nucleic acid molecule may be generated. The vectors can be episomal vectors (i.e., that do not integrate into the genome of a host cell), or can be vectors that integrate into the host cell genome. The term “vector” may thus also be defined as a gene delivery vehicle that facilitates gene transfer into a target cell. This definition includes both 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 that are devoid of bacterial sequences (minicircles)) transposons-based vectors (e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno- associated viral, adenoviral, herpes viral, hepatitis viral vectors or the like. Typically, but not necessarily, viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis. Virosomes are a non-limiting example of a vector that comprises both viral and non-viral elements, in particular they combine liposomes with an inactivated HIV or influenza virus (Yamada et al., 2003). Another example encompasses viral vectors mixed with cationic lipids.

[0246] The selective chimeric chemokines as described herein may be suitably obtained through expression by host cells or host organisms, transformed with an expression construct encoding and configured for expression of said selective chimeric chemokine in said host cells or host organisms, followed by purification of the selective chimeric chemokine.

[0247] Hence, a further related aspect provides a host cell comprising the nucleic acid, nucleic acid expression cassette or vector as described herein.

[0248] In certain embodiments, the host cell may be a bacterial cell, a yeast cell, an animal cell, or a mammalian cell.

[0249] The terms “host cell” and “host organism” may suitably refer to cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals. Contemplated as host cells are inter alia unicellular organisms, such as bacteria (e.g. , E. coli. Salmonella iymphimiiriiim. Serratia marcescens. or Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae or Pichia pasioris)' . (cultured) plant cells (e.g., from Arabidopsis thaliana or Nicotiana tobaccum) and (cultured) animal cells (e.g., vertebrate animal cells, mammalian cells, primate cells, human cells or insect cells). Contemplated as host organisms are inter alia multi-cellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrate animals, still more preferably mammals, yet more preferably primates; particularly contemplated are such animals and animal categories which are non-human.

[0250] The selective chimeric chemokines as described herein may be suitably isolated and / or purified. Purified proteins, polypeptides or peptides may be obtained by known methods including, for example, laboratory or recombinant synthesis, chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification, etc.

[0251] In particular embodiments, the vector comprising the nucleic acid as described herein is a viral vector, preferably a viral vector specifically directed towards cancer, preferably specifically directed towards melanoma or colorectal cancer. Melanoma-specific targeting of a viral vector may be achieved, for example, by fusion of a single chain antibody recognizing the high molecular-weight melanoma- associated antigen (HMWMAA), followed by a blocking peptide and a matrix metalloprotease cleavage site, to the amino terminus of the murine leukemia virus amphotropic strain envelope as described by Martin et al., Envelope-Targeted Retrovirus Vectors Transduce Melanoma Xenografts but Not Spleen or Liver, Molecular Therapy. 2002, 5(3): 269-274.

[0252] In particular embodiments, the nucleic acid encoding one of the selective chimeric chemokines as taught herein may be comprised within a vector providing for a signal peptide. The signal peptide may be a homologous or heterologous signal peptide, depending on the host cell used for production of the selective chimeric chemokine as described herein. Furthermore, for prokaryotic expression of one of the selective chimeric chemokines as described herein, a protease cleavage site motif may be present C-terminally of said signal peptide and N-terminally of the selective chimeric chemokine as described herein.

[0253] The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples.

[0254] EXAMPLES

[0255] Example 1. Materials and methods for Examples 2 to 8

[0256] Native and custom-made chemokines

[0257] All native chemokines were purchased from Peprotech or Protein Foundry, LLC. Custom-made chimeric chemokines, including the dimeric and trimeric chemokines were produced by Protein Foundry, LLC. Dimeric and trimeric chemokines were multimerized through their C-terminus using the commercially available (BroadPharm) linkers Azido-PEG 10- Azide (BP-23827) and 3-Arm PEG5- Azide (BP-29766), respectively.

[0258] Fluorescently labelled chemokines

[0259] CCL2, CCL5, CCL22, CXCL10, LIH222 and pyroLIH222 coupled to Alexa Fluor 647 were purchased from Protein Foundry, LLC.

[0260] Chemokine processing by dipeptidyl peptidase 4 (CD26)

[0261] CCL2, CCL22, LIH222 and pyroLIH222 (3 pM) were incubated with recombinant dipeptidyl peptidase 4 (200 U, Merck) in 60 pL Tris / HCl 50 mM pH 7.5 + 1 mM EDTA for 90 minutes at 37°C.

[0262] Cells and reagents

[0263] HEK293T cells (Abeam) were grown in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma) and penicillin / streptomycin (100 units / mL and 100 pg / mL, respectively, Gibco). HEK-ACKR2 cell lines stably expressing human or mouse ACKR2 (hACKR2 and mACKR2, respectively) were established using pIRES-hygromycin vector (Takara Bio). Cells were selected and grown in complete DMEM medium supplemented with 200 pg / mL hygromycin. P-arrestin recruitment assays

[0264] Ligand-induced P-arrestin recruitment to receptors was monitored by a split nanoluciferase complementation-based assay (NanoBiT, Promega). Briefly, 6 x 106HEK293T cells were plated in 10- cm dishes and cultured for 24 hours before co-transfection with pNBe vectors encoding chemokine receptors C-terminally tagged with SmBiT and P-arrestin- 1 N-terminally tagged with LgBiT. 24 hours after transfection, cells were harvested and incubated for 20 minutes at 37°C with coelenterazine H (Regis Technologies) in Opti-MEM™ I Reduced Serum Medium (Gibco). Cells were then distributed into white 96-well plates (1 x 105cells / well) and chemokine ligands were added at the indicated concentrations. Luminescence generated upon nanoluciferase complementation was measured with a Mithras LB940 luminometer (Berthold Technologies). Results shown correspond to average values acquired for 25 min, represented as percentage of maximum full agonist response.

[0265] Chemokine binding and competition

[0266] HEK293T cells transiently expressing ACKR2, CCR2B and CCR4 were incubated at 4°C on ice for 1 hour in FACS buffer - phosphate buffered saline (PBS, Gibco), 1% bovine serum albumin (BSA, Merck) and 0.1% NaNs (Merck) - containing the indicated concentrations of LIH222AZ647. Cells were washed once in PBS and dead cells were excluded using an incubation for 30 min at 4°C with Zombie Green viability dye (BioLegend). Untransfected HEK293T cells were used to evaluate non-specific binding of LIH222AZ647.

[0267] Competition binding assays were performed with HEK293T cells stably expressing human or mouse ACKR2. Cells were incubated at 4°C on ice for 1 hour in FACS buffer containing 5 nM of CCL5AF647 and concentrations of unlabelled chemokines ranging from 300 nM to 0.01 nM. The same protocol was followed to assess competition between LIH222AZ647 and native chemokines in cells transiently expressing human ACKR2. Untransfected HEK293T cells were used to evaluate non-specific binding of both labelled chemokines. The signal obtained for CCL5AF647 (5 nM) or LIH222AZ647 (5 nM) in the absence of unlabelled chemokines was used to define 100% binding. Ligand binding was quantified by mean fluorescence intensity on a BD LSRFortessa™ cytometer and analyzed with FlowJo software vlO.6.2 (both from BD Biosciences).

[0268] Chemokine uptake

[0269] Chemokine uptake using either labelled pyroLIH222Az647 (5 nM), CCL5AF647 (5 nM), CCL2AF647 (10 nM) or CXCL10AF647 (10 nM) was visualized by imaging flow cytometry. Briefly, HEK293T or HEK- hACKR2 cells were resuspended at a concentration of 3.75 x 106cells / mL in Opti-MEM + 0,5% BSA, and incubated for 1 hour at 37°C with labelled chemokines at the concentrations indicated, in the presence or absence of unlabelled competitors (100 nM). Cells were then washed with PBS and dead cells were excluded using Zombie Green viability dye. Images of 1 x 104in-focus living single cells were acquired with an Image Stream MKII imaging flow cytometer (Luminex) using 60 x magnification and extended depth field (EDF). Samples were analyzed using Ideas 6.2 software. The number of spots per cell was determined using a mask-based software wizard.

[0270] Data and statistical analysis

[0271] Concentration-response curves were fitted to the three-parameter Hill equation using an iterative, leastsquares method (GraphPad Prism version 9.3.1) to provide EC50, % maximum values and standard errors of the mean. All curves were fitted to the data points generated from the mean of at least three independent experiments. All statistical tests i.e. ordinary or repeated measures one way- and two-way ANOVA, unpaired t-tests, Kruskal-Wallis and Mann-Whitney tests were performed with GraphPad Prism 9.3.1.

[0272] Expression, purification and quantification of LIH222-Fc

[0273] HEK293T cells were transfected in 10-cm culture dishes and cultured for 48h in DMEM medium supplemented with 100 units / mL of penicillin and 100 pg / mL of streptomycin, 10% FBS, 1% L- glutamine (Gibco) and 5 pg / mL puromycin (SAS InvivoGen). The medium was replaced daily for 5-7 days in order to achieve proper selection. Cells were then expanded and seeded into 8-layer Celldisc TC Standard (Greiner Bio-one) and allowed to proliferate for 24 h. Then, DMEM medium was replaced with FreeStyle™ 293 Expression Medium (Gibco) and five days later, the supernatant was collected, centrifuged at 4000 xg at 4°C and stored at 4°C. DMEM medium was again added and 24 h later, it was replaced with Free Style™ medium and the process continued until 1 L of supernatant was collected. Subsequently, the supernatant was filtered using a 0.22 pM Bottle-Top Vacuum Filtration System (VWR) and purification initiated. Briefly, 500 pL of Protein G Sepharose 4 Fast Flow beads (Merck) were added to the supernatant and mixed in a rolling agitator at 4°C overnight. The following day, 1 mb polypropylene columns were washed with 15 mb of 25% EtOH, followed by another wash with 15 mb of PBS. Beads were collected from the supernatant by centrifugations at 4000 rpm at4°C and loaded into the columns. After beads were captured, columns were washed with 15 mb of PBS, and the protein was eluted using a phosphate citrate buffer into 2 mb fractions containing a neutralizing bicarbonate buffer. Fractions were then merged into an Amicon (Merck) and protein fractions containing LIH222- Fc were concentrated. Protein content was quantified using the Micro BCA™ Protein Assay Kit (ThermoFisher Scientific).

[0274] Mice

[0275] In vivo experiments were performed using C57B1 / 6J mice from Charles River Laboratories. UBC-Cre- ERT2 mice were purchased from The Jackson Laboratory and CDH5-Cre-ERT2 mice were purchased from Taconic. To induce Cre recombination, mice were fed for two weeks with Tamoxifen diet TAM400 / CreER (ENVIGO) followed by one washout week before experimental procedures. Experiments were performed using sex- and age-matched mice. Mice genotyping was performed by polymerase chain reaction (PCR). All mice were housed in specific pathogen-free conditions, in individually ventilated cages and food and water were provided ad libitum. All animal experiments were performed at Humanitas Clinical and Research Center (Rozzano, Milan, Italy) under the supervision of Professor Raffaella Bonecchi. Procedures involving animal handling and care were conformed to protocols approved by the Humanitas Clinical and Research Center in compliance with national and international law and policies. The study was approved by the Italian Ministry of Health. All efforts have been made to minimize the number of animals used and their suffering.

[0276] In vivo chemokine uptake

[0277] For flow cytometry analysis, 5 pg of fluorescently labelled pyroLIH222 (LIH222Az64?) were injected into either heterozygous or ACKR2 -deficient CDH5-Cre-ERT2 mice. Two hours later, mice were sacrificed, the lungs perfused with 15 mb of PBS and collected. Subsequently, the lungs were minced, digested for 1 hour in 1 mg / mL collagenase IV (Sigma) in PBS, and filtered using a 70-pm strainer. Red blood cells were lysed and the single cell suspension was stained with the antibodies listed in Table 1. To exclude dead cells from analysis, cells were stained with Zombie Aqua Fixable Viability kit (BioLegend). All antibodies were used according to the manufacturer’s instructions. Data was acquired using a FACS Symphony A5 analyzer (BD Biosciences) using FACSDiva software and representative images were generated using FlowJo Software vlO.8.2 (BD Biosciences).

[0278] Table 1 - Antibody list for lung staining Example 2. Activity of native chemokines towards ACKR2 and development of LIH222 as high- affinity ACKR2-selective modulator

[0279] The ability of all human chemokines to activate ACKR2 was assessed in HEK293T cells using the ultra-sensitive Nanoluciferase complementation-based assay (NanoBiT) based on -arrestin recruitment. All chemokines were tested at a lOOnM concentration. Present inventors’ screening revealed several CC chemokines as highly promising, as well as at least one CXC chemokine, CXCL10 (Figure 1 A). To further characterize the interactions of these chemokines with ACKR2 and to pinpoint the better candidates the inventors screened the ligands in a concentration-response curve (Figure 1 B). Considering the potencies, efficacies, protection from CD26 and the intrinsic selectivity of each native chemokine, CCL2, CCL22, CCL5 and CXCL10 were selected as potential scaffolds for the chimeric chemokines. Several chimeric chemokines were prepared (Figure 1 C) and tested.

[0280] The chimeric chemokines were named and constructed as follows:

[0281] SEQ ID NO 9: Chimeric LIH222: N-terminus human CCL2 and C-terminus human CCL22

[0282] QPDAINAPVTCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVKMI LNKLSQ

[0283] SEQ ID NO 25: Chimeric LIH052: N-terminus human CCL5 and C-terminus human CCL2

[0284] SPYSSDTTPCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMD HLDKQTQTPKT

[0285] SEQ ID NO 26: Chimeric LIH210: N-terminus human CCL2 and C-terminus human CXCL10

[0286] QPDAINAPVTCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNL LKAVSKERSKRSP

[0287] SEQ ID NO 27: Chimeric LIH510: N-terminus human CCL5 and C-terminus human CXCL10

[0288] SPYSSDTTPCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLL KAVSKERSKRSP

[0289] SEQ ID NO 28: Chimeric LIH522: N-terminus human CCL5 and C-terminus human CCL22

[0290] SPYSSDTTPCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVKMIL NKLSQ

[0291] SEQ ID NO 29: Chimeric LIH2222: N-terminus human CCL2 missing first two AA and C-terminus human CCL22

[0292] DAINAPVTCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVKMILN

[0293] KLSQ SEQ ID NO 30: Chimeric LIH2822: N-terminus human CCL2 missing first eight AA and C-terminus human CCL22

[0294] VTCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVKMILNKLSQ

[0295] SEQ ID NO 31 : Chimeric LIH222P2G: N-terminus human CCL2 with a mutation in the second amino acid to protect from CD26 and C-terminus human CCL22

[0296] QGDAINAPVTCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVKM ILNKLSQ

[0297] SEQ ID NO 32: Chimeric LIH2V1122: N-terminus human CCL2 with an extra V in position 11 to mimic length of human CCL22 and C-terminus human CCL22

[0298] QPDAINAPVTVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVK MILNKLSQ

[0299] SEQ ID NO 33: Chimeric LIHV222: N-terminus viral CCL2 and C-terminus human CCL22

[0300] LGASWHRPDKCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVK MILNKLSQ

[0301] LIH222 was identified as a highly potent (EC50= 1.78 nM) ACKR2 modulator, while the remaining chemokines were completely inactive or acted as partial agonists (Figure 1 D). To assess the selectivity of these chimeric chemokines, the inventors screened them against the most relevant chemokine receptors, in a concentration-response curve. Strikingly, LIH222 was completely unable to activate the receptors, confirming its selectivity towards ACKR2 (Figure 2A-E).

[0302] Example 3: Specific activation of ACKR2 by LIH222

[0303] The present inventors compared the affinity and selectivity of LIH222 and the native chemokines CCL2 and CCL22 against the relevant receptors CCR1, CCR2, CCR4, CCR5 and ACKR4. LIH222 was clearly highlighted by an increased potency against ACKR2 from the native chemokine CCL2, and presented selectivity against ACKR2 when compared to both native chemokines (Figure 3 A). Furthermore, the inventors screened LIH222 against all known human chemokine receptors and against murine CCR2, CCR3 and CCR4, where the high selectivity of LIH222 was confirmed (Figure 3 B). Fluorescently labelled LIH222 was able to induce arrestin recruitment to the same extent as the unlabelled version (Figure 3 C) and strongly bound to HEK293T cells expressing ACKR2, but not other receptors (Figure 3 D). A relevant fact for application in vivo is the protection against CD26 inactivation, as cleavage by this enzyme is detrimental towards the activity of several chemokines. Thus, the present inventors created LIH222 with a pyroglutamate modification, named pyroLIH222. This modulator retained the same characteristics as LIH222, was shown to activate ACKR2 to the same extent, with the additional benefit of being protected against CD26 post-translational modification (Figures 3 E-F).

[0304] Example 4: Modifications of the N terminal region of LIH222 may further improve its potency

[0305] N terminal modifications and differences between murine and human forms of the chemokines can have great impacts on modulator interaction. Thus, the present inventors made several modifications to LIH222 in an attempt to increase its affinity to ACKR2. Chimeric chemokines containing the N termini of CCL7 and CCL8 were created, under the name LIH722 and LIH822, as well as human and murine chimeric chemokine (Figure 4 A), s. LIH722 and LIH822 were also revealed as strong candidates, however their potency was slightly higher than that of LIH222 (Figure 4 B). Strikingly, the chimeric chemokine between mouse CCL2 and mouse CCL22, termed LIHM2M22, revealed the strongest potency of all the tested candidates (Figure 4 C). Furthermore, LIH222, LIHM222, LIH2M22 and LIHM2M22 were able to displace a high concentration of AF647-labelled CCL5, one of the strongest ACKR2 ligands, both in cells expressing the human and the mouse version of the receptor (Figure 4 D-E).

[0306] The present inventors then assessed the selectivity of these new ACKR2 modulators and reported that they do not activate any of the relevant receptors (Figure 5 A-E).

[0307] Example 5: LIH222 coupled to the Fc fragment of an immunoglobulin (LIH222fc) is active and able to selectively recognize ACKR2.

[0308] As further proof of concept, present inventors fused an Fc fragment of murine IgG2A C-terminally to LIH222 and assessed the ability of this novel fusion molecule to selectively bind ACKR2. This protein was expressed in HEK293T cells and after protein G-based purification, it was tested for its ability to induce ACKR2 activation monitored in a -arrestin recruitment assay. LIH222-Fc was able to bind ACKR2 (Figure 6 A). Subsequently, the inventors assessed its selectivity by evaluating its ability to recognize and stain in flow cytometry the cells expressing ACKR2 or the most relevant chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, ACKR1, ACKR4 and GPR182. The selectivity of LIH222-Fc was attested by the fact that it only bound to ACKR2, even at the high concentrations of 300 nM (Figure 6 B).

[0309] Example 6: Uptake competition.

[0310] To verify that LIH222 was able to outcompete native ligands in regards to ACKR2 -driven uptake, the inventors performed imaging flow cytometry assays. Briefly, ACKR2 -expressing cells were incubated with either solely 5 nM of labelled LIH222 or in combination with saturating concentrations (100 nM) of relevant ligands like CCL2, CCL5, CCL22 and CXCL10. CXCL1 and CXCL11 were used as negative controls. Consistently with binding results (Figure 4D), most relevant competitors were able to partially or fully compete for uptake with LIH222 (Figure 7A and 7B).

[0311] Example 7: pyroLIH222 is active in vivo and remains a selective modulator for ACKR2

[0312] Since ACKR2 is widely expressed in endothelial cells and lungs are enriched in these cell populations, the present inventors injected fluorescently labelled pyroLIH222 (LIH222AZ64?) in wildtype or ACKR2- deficient mice and assessed its binding in endothelial cells isolated from lung tissue. Interestingly, pyroLIH222 fluorescence was detected solely in the blood and lymphatic endothelial cells of wildtype mice, but not their knockout counterparts (Figure 8 A). To further confirm the selectivity towards ACKR2, LIH222AZ647 was injected into reporter mice where either one or both alleles of ACKR2 were replaced with TdTomato. Strikingly, pyroLIH222 fluorescence was detected in the endothelial cells of the heterozygous mice, where it colocalized with the TdTomato signal, indicating interaction with ACKR2. On the other hand, the same was not observed for the ACKR2 -deficient mice (Figure 8 B). Experiments were perfomed at Humanitas Clinical and Research Center (Rozzano, Milan, Italy) under the supervision of Professor Raffaella Bonecchi.

[0313] Example 8: Dimeric and trimeric LIH222 display stronger affinity and act as ACKR2 superagonists compared to monomeric LIH222.

[0314] The inventors generated dimeric (18.2 kDa) and trimeric (27.3 kDa) forms of LIH222 by fusing LIH222 monomers (8.8 kDa) through their C-terminal extremities using an azido-PEGlO-azide linker and a 3- Arm PEG5-azide linker, respectively. These multimeric modulators displayed a superagonist effect compared to monomeric LIH222 in -arrestin recruitment assay, with the dimeric form revealing a higher potency when compared to the monomeric form (Figure 9 A). Furthermore, the multimers were shown to be able to displace LIH222 with high affinity by competing for binding to ACKR2, highlighting the potential use of these forms (Figure 9 B).

Claims

CLAIMS1. A chimeric chemokine selectively binding to ACKR2 comprising the N-terminal end up to the Cys- Cys motif of a CCL2, CCL7 or CCL8 chemokine polypeptide, or comprising an amino acid sequence represented by any of SEQ ID NOs 34, 35, 36 or 37, fused to the C-terminal end starting from the Cys- Cys motif to the end of a CCL22 chemokine polypeptidecomprising the three-stranded-antiparallel beta sheet and C-terminal alpha helix and wherein said chimeric chemokine selectively binds to ACKR2 and not to any other chemokine receptor.

2. The chimeric chemokine according to claim 1 wherein the N-terminal end of CCL2, CCL7 or CCL8 and the three-stranded-antiparallel beta sheet and C-terminal alpha helix of CCL22 originate from a different species, preferably selected from human and mouse.

3. The chimeric chemokine according to claim 1 or 2, wherein the N-terminal end of the human CCL2 sequence is represented by SEQ ID NO: 2, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of human CCL2; the N-terminal end of the human CCL7 sequence is represented by SEQ ID NO: 12, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of human CCL7; the N-terminal end of the human CCL8 sequence is represented by SEQ ID NO: 18, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of human CCL8, and / or wherein the C-terminal end of the human CCL22 sequence is represented by SEQ ID NO 6 or a corresponding fragment starting from the Cys-Cys motif to the end of an orthologue of human CCL22.

4. The chimeric chemokine according to any one of claims 1 or 2 wherein the N-terminal end the mouse CCL2 is represented by SEQ ID NO 4, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of mouse CCL2, the N-terminal end of the mouse CCL7 sequence represented by SEQ ID NO: 15, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of mouse CCL7, the N-terminal end of the mouse CCL8 sequence represented by SEQ ID NO: 21, or a corresponding fragment comprising the N-terminal residues up to the Cyc-Cys motif of an orthologue of mouse CCL8, and / or wherein the C-terminal end of the mouse CCL22 polypeptide represented by SEQ ID NO: 8.

5. The chimeric chemokine according to any one of claims 1 to 4 wherein the N terminal glutamine of said chimeric chemokine comprises a pyruvate modification.

6. The chimeric chemokine according to any of claims 1 to 5 further comprising a detectable label or a functional moiety.

7. A multimer comprising at least two or at least three chimeric chemokines according to any of claims 1 to 6 wherein each of the chimeric chemokines is coupled through its C-terminus to a linker.

8. A nucleic acid encoding the chimeric chemokine according to any of claims 1 to 6.

9. The chimeric chemokine according to any one of claims 1 to 6, the multimer of claim 7 or the nucleic acid according to claim 8 for use as a medicament or diagnostic.

10. The chimeric chemokine, the multimer or the nucleic acid according to claim 9 for use in the treatment of an autoimmune, inflammatory, neurological, cardiovascular or proliferative disease or disorder in a subject.

11. Pharmaceutical composition comprising a chimeric chemokine selectively binding to ACKR2 according to any one of claims 1 to 6, the multimer of claim 7 or the nucleic acid according to claim 8 and optionally a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11 further comprising one or more immune checkpoint modulators, preferably one or more immune checkpoint inhibitors, and optionally a pharmaceutically acceptable carrier.

13. An in vitro method for determining whether a test agent is an ACKR2-specific ligand, said method comprising determining whether said test agent interferes with the selective binding of the chimeric chemokine of any one of claims 1 to 6 or the multimer of claim 7 to ACKR2.

14. The in vitro method according to claim 13, wherein the chimeric chemokine or multimer is fused or linked to a dye, or displayed on scaffold proteins, or fused to another moiety such as an Fc part of an antibody.

15. The in vitro method according to claim 13 or 14, wherein said method comprises a step of determining whether a test agent decreases or eliminates the biological activity of ACKR2.

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