Variant ligand conjugates for payload delivery

Abstract

The present invention relates to conjugates comprising a payload conjugated to a variant ligand, wherein the variant ligand is capable of binding to a receptor, wherein the variant ligand binds with higher affinity to the receptor compared to a naturally occurring ligand of the receptor and / or wherein the variant ligand provides increased agonism of the receptor compared to a naturally occurring ligand of the receptor.

Description

VARIANT LIGAND CONJUGATES FOR PAYLOAD DELIVERYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application Nos. 63 / 437,548, filed January 6, 2023, and 63 / 437,878 filed January 9, 2023.FIELD

[0002] The present invention relates to conjugates comprising a variant ligand conjugated to a payload.BACKGROUND

[0003] The targeted delivery of drugs to specific organs, tissues, or cells provides many advantages over systemic delivery. For example, targeted delivery of drugs that are toxic or have side-effects can reduce adverse reactions. For some therapies, antibody-drug conjugates have been developed, wherein a drug is conjugated to an antibody that specifically binds to a molecule on target cells (Dumontet et al. 2023). However, antibodies are relatively large molecules formed by multiple polypeptides and may cause unwanted antibody-related effects such as modulation of immune cells by binding to Fc receptors. Therefore, researchers continue to search for alternative strategies to target “payloads” such as drugs to specific cell types.SUMMARY

[0004] The present invention relates to conjugates that provide effective delivery of a payload to a target. The conjugates of the present invention comprise a variant ligand conjugated to a pay load, wherein the variant ligand binds to a receptor allowing for the delivery of the payload to target cells expressing the receptor.

[0005] In an embodiment, there is provided a conjugate comprising a payload conjugated to a variant ligand, wherein the variant ligand is capable of binding to a receptor, and wherein the variant ligand binds with higher affinity to the receptor compared to a naturally occurring ligand of the receptor.

[0006] In an embodiment, there is provided a conjugate comprising a payload conjugated to a variant ligand, wherein the variant ligand is capable of binding to a receptor, and wherein thevariant ligand provides increased agonism of the receptor compared to a naturally occurring ligand of the receptor.

[0007] In an embodiment, there is provided a nucleic acid molecule encoding the conjugate as described herein.

[0008] In an embodiment, there is provided a vector comprising the nucleic acid molecule as described herein.

[0009] In an embodiment, there is provided a host cell comprising the nucleic acid molecule or the vector as described herein.

[0010] In an embodiment, there is provided a pharmaceutical composition comprising the conjugate, the nucleic acid molecule, or the vector as described herein, and a pharmaceutically acceptable carrier, excipient, and / or stabilizer.

[0011] In an embodiment, there is provided a conjugate or pharmaceutical composition as described herein for use in delivering a payload to a cell.

[0012] In an embodiment, there is provided a conjugate or pharmaceutical composition as described herein for use in killing or modifying a target cell.

[0013] In an embodiment, there is provided a method for delivering a payload to a cell, comprising the step of contacting the cell with the conjugate or the pharmaceutical composition as described herein.

[0014] In an embodiment, there is provided a method for delivering a payload to a cell in a subject, comprising the step of administering the conjugate or the pharmaceutical composition as described herein to the subject.

[0015] In an embodiment, there is provided a method for treating cancer in a subject, comprising the step of administering the conjugate or the pharmaceutical composition as described herein to the subject.

[0016] In an embodiment, there is provided a method for killing or modifying a target cell, comprising the step of contacting the cell with the conjugate or the pharmaceutical composition as described herein.

[0017] In an embodiment, there is provided a method for killing or modifying a target cell in a subject, comprising the step of administering the conjugate or the pharmaceutical composition as described herein to the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Embodiments will be described, by way of example only, with reference to the accompanying figures.

[0019] FIG. 1. Analytical hplc chromatogram obtained for purified folded azido-CCL5. Observed mass = 7982 Da, expected mass = 7983 Da.

[0020] FIG. 2. Analytical hplc chromatogram obtained for purified folded azido-6P4- CCL5. Observed mass = 7978 Da, expected mass = 7978 Da.

[0021] FIG. 3. Synthesis step 1.

[0022] FIG. 4. Synthesis step 2.

[0023] FIG. 5. Synthesis step 3.

[0024] FIG. 6. Analytic hplc chromatogram obtained for the DBCO-PEG4-VA-PAB-MMAE linker. Observed mass: [M+2H]+2786.9 Da; [M+H+Na]+2797.8 Da; [M+H+K]+2805.8 Da; [M+H+2Na]+2808.9 Da; expected mass: [M+H]+1571.90 Da; [M+2H]+2786.45 Da;[M+H+Na]+2797.45 Da; [M+H+K]+2805.45 Da; [M+H+2Na]+2808.95 Da.

[0025] FIG. 7. Analytic hplc chromatogram obtained for the CCL5-MMAE conjugate. Observed mass = 9554 Da, expected mass = 9554 Da.

[0026] FIG. 8. Analytic hplc chromatogram obtained for the 6P4-CCL5-MMAE conjugate. Observed mass = 9551 Da, expected mass = 9550 Da.

[0027] FIG. 9. Cytotoxicity assay performed on CCR5-postive (closed circle, HEK- CCR5) and CCR5-negative cells (open circle, Parental HEK) using MMAE alone (9 A), MMAE conjugated to native CCL5 (9B) and MMAE conjugated to 6P4-CCL5 (9C).DETAILED DESCRIPTION

[0028] In the search for alternative strategies to target payloads to specific cell types, the present inventors have investigated the use of ligands attached to a payload for the purpose of targeting the payload to cells expressing a receptor that binds to the ligand. This has been tested in the present disclosure using the exemplary cytotoxic drug Monomethyl auristatin E (MMAE), the ligand CCL5, and the receptor CCR5. MMAE inhibits cell division by blocking polymerisation of tubulin. MMAE is cell-permeable and, as shown in FIG. 9A, it is able to kill cells in vitro. CCL5 is a polypeptide that is not cell-permeable. The conjugation of MMAE to CCL5 should therefore inhibit MMAE from diffusing into cells through the cell membrane. It would have been expected that the CCL5-MMAE conjugate would bind to CCR5 on CCR5- expressing cells, thereby specifically targeting the drug to CCR5 -expressing cells. Surprisingly, as shown in FIG.9B, it was found that CCL5-MMAE did not enhance the killing of CCR5- expressing cells and killed CCR5-negative similarly to CCR5 -expressing cells. This was unexpected because, like most ligand-receptor pairs, CCL5 and CCR5 have evolved to bind to each other to effect biological functions. However, it was discovered that a synthetic variant of CCL5 (6P4-CCL5) provided effective targeted delivery of MMAE to CCR5 -expressing cells. As shown in FIG.9C, 6P4-CCL5-MMAE killed 50% of HEK-CCR5 cells at a lower concentration compared to CCL5-MMAE or MMAE alone, thus demonstrating increased potency. As also shown in FIG.9C, 6P4-CCL5-MMAE was selective for HEK-CCR5 cells, and did not cause measurable killing of parental HEK cells (that did not express CCR5) at the concentrations that significantly killed HEK-CCR5 cells. 6P4-CCL5 (also known as 6P4-RANTES) is a synthetic variant of CCL5 that is known to be a super agonist that binds to the receptor CCR5 with high affinity (Gaertner et al. 2008). Further variant ligands with improved binding and / or agonism of their receptors are also known (Gaertner et al. 2008) and are disclosed herein. The present inventors have thus discovered that variant ligands with increased receptor agonism and / or higher binding affinity to the receptor provide enhanced targeting of payloads to target cells. Such variant ligands can thus be ligated to payloads to provide effective targeting conjugates.

[0029] Sequences, compositions, and methods for carrying out the invention are presented in terms of examples and embodiments in the present disclosure. However, the invention is not limited to the described examples and embodiments, and a person skilled in the art will appreciate that many other embodiments of the invention are possible without deviating from the basic concept of the invention, and that any such work around will also fall under scopeof this invention. It is envisioned that other styles and configurations of the present invention can be easily incorporated into the teachings of the present invention, and the configurations shall be shown and described for purposes of clarity and disclosure and not by way of limitation of scope.

[0030] Conjugates

[0031] Conjugates of the present invention comprise a payload conjugated to a variant ligand.

[0032] In some embodiments, the variant ligand and / or the payload comprise a polypeptide. Polypeptides may be produced by methods known in the art such as transgenic expression in a host cell followed by isolation of the expressed polypeptide. Alternatively, polypeptides may be produced by artificial synthesis. Polypeptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge 1995; Merrifield 1997; Ollivier 2010; Raibaut 2015). Solid-phase peptide synthesis can employ either Foe or Bmoc chemistries as known in the art (Jaradat 2018). Furthermore, automated synthesis may be achieved using, for example but not limited to, the ABI 433 Peptide Synthesizer (Applied Biosystems), the Prelude® synthesizer (Protein Technologies Inc.), or the MultiPep RSi 384-well peptide synthesizer (Intavis) in accordance with the instructions provided by the manufacturer. Polypeptides for use in the invention can be synthesized in parallel by other methods known in the art, including laser-based techniques (Loeffler 2016) or flow-based techniques (Mijalis 2017). Methods for the synthesis of polypeptides as known in the art are disclosed in WO / 2020 / 070587.

[0033] Conjugation of the variant ligand to the payload can be performed by ligation methods known in the art. Ligation can be performed by a number of techniques known in the art including imine capture, pseudoproline ligation, Staudinger ligation, thioester capture ligation, and hydrazine formation ligation (Tam 2001). Various methods of click chemistry ligation are known in the art (Hein 2008; Zhang 2021). Ligation of polypeptides can be performed by native chemical ligation as known in the art (Dawson 1994; Raibaut 2015; Engelhard 2016). Native chemical ligation allows the covalent assembly of two or more unprotected peptide segments to produce a larger polypeptide. Native chemical ligation reactions can occur as soluble ligations, in which the polypeptides to be conjugated are in solution, or as solid-phase ligations, in which the N-terminal polypeptide fragment is covalently attached to a solid-phase resin via a detachablelinker (Canne US 7,094,871; Low W02004105685). Ligation of polypeptides can be performed by SEA native peptide ligation as known in the art (Ollivier 2010). In this method, ligation occurs between the C-terminal / ? / .s(2-sulfanylethyl)amido (SEA) group of one peptide and the N- terminal cysteine of another peptide. Similarly to native chemical ligation, SEA native peptide ligation can occur as a soluble or as a solid-phase ligation reaction. Methods for the ligation of polypeptides as known in the art are disclosed in WO / 2020 / 070587.

[0034] In some embodiments, the variant ligand may be conjugated to the payload via a linker. In some embodiments, the structure of the linker will depend on the type of ligation chemistry employed in the ligation step. Linkers known in the art for the conjugation of polypeptides to payloads may be used in the present invention for linking the variant ligand to the payload (Sheyi 2022). In some embodiments, the linker comprises a peptide linker, a hydrazone linker, or a disulfide linker. In some embodiments, the linker comprises VA-PAB.

[0035] In some embodiments, the linker is a cleavable linker, such as a linker that is cleaved in the intracellular or endosomal environment when the conjugate is internalized by a cell. Cleavable linkers are known in the art and may be used in the present invention (Bargh 2019).

[0036] Payload

[0037] Conjugates of the present invention comprise a payload. As used herein, the term “payload” carries the ordinary meaning in the art of an agent such as a drug or a detectable marker that is targeted to a specific tissue or cell type by the conjugate to achieve a therapeutic and / or diagnostic effect.

[0038] In some embodiments, the payload is a toxin such as a toxin that enables the killing of a receptor-expressing cell or the killing of a pathogen inside a receptor-expressing cell. In some embodiments, the toxin is a cytotoxic agent, an anti-tumor agent, a chemotherapeutic agent, an antiviral agent, or an antibacterial agent. In some embodiments, the toxin is pseudomonas exotoxin A, diptheria toxin, a ribosome inactivating protein, or saporin.

[0039] In some embodiments, the payload is a carrier protein such as albumin. In some embodiments, the payload is a polymer such as polyethylene glycol. In some embodiments, the payload is a lipid.

[0040] In some embodiments, the payload is a small molecule drug. The term “small molecule drug” refers an organic or inorganic compound that may be used to treat, cure, prevent or diagnose a disease, disorder or condition. A small molecule drug typically has a molecular weight of less than 2000 Daltons (Da), such as less than 1500 Da, less than 1000 Da, less than 900 Da, less than 800 Da, less than 700 Da, less than 600 Da or less than 500 Da.

[0041] In some embodiments, the small molecule drug is a substance or compound that regulates a biological process in the body of a subject, and more particularly within a cell. The small molecule drug may exert its activity in the form in which it is administered, or the small molecule drug may be a prodrug. In this regard, the term “small molecule drug”, as used herein, encompasses both the active form and the prodrug. The term “prodrug” refers to a compound or substance that, under physiological conditions, is converted into the therapeutically active agent. In an embodiment, a prodrug is a compound or substance that, after administration, is metabolized in the body of a subject or within a cell into the pharmaceutically active form (e.g. by enzymatic activity). A common method for making a prodrug is to include selected moieties that are hydrolyzed under physiological conditions to reveal the pharmaceutically active form.

[0042] In some embodiments, the small molecule drug is a cytotoxic agent, an anticancer agent, an anti-tumor agent, a chemotherapeutic agent, an anti-neoplastic agent, an antiviral agent, an antibacterial agent, an anti-inflammatory agent, an immunomodulatory agent (e.g. an immune enhancer or suppressor), an immune response checkpoint agent, a biological response modifier, a prodrug, a vitamin, or a steroid.

[0043] The small molecule drug may be any of those described herein, or may be a pharmaceutically acceptable salt thereof. As used herein, the term “pharmaceutically acceptable salt(s)” refers to any salt form of small molecule drug described herein that are safe and effective for administration to a subject of interest, and that possess the desired biological, pharmaceutical and / or therapeutic activity. Pharmaceutically acceptable salts include salts of acidic or basic groups. Pharmaceutically acceptable acid addition salts may include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate(i.e., l,l'-methylene-bis-(2 -hydroxy-3 -naphthoate)) salts. Suitable base salts may include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.

[0044] In some embodiments, the small molecule is a cytotoxic drug. In an embodiment, the cytotoxic drug is a glucocorticoid, a cytostatic (e.g. alkylating agents, antimetabolites), a drug acting on immunophilins, an opioid, or a TNF binding protein. Cytotoxic drugs include, without limitation, nitrogen mustards (e.g. cyclophosphamide), nitrosoureas, platinum compounds, folic acid analogs (e.g. methotrexate), purine analogs (e.g. azathioprine and mercaptopurine), pyrimidine analogs (e.g. fluorouracil), protein synthesis inhibitors, cytotoxic antibiotics (e.g. dactinomycin, anthracyclines, mitomycin C, bleomycin and mithramycin), cyclosporine, tacrolimus, sirolimus / rapamycin, everolimus, prednisone, dexamethasone, hydrocortisone, mechlorethamine, clorambucil, mycopholic acid, fmgolimod, myriocin, and etanercept.

[0045] In some embodiments, the small molecule drug is cyclophosphamide, ifosfamide, afosfamide, melphalan, bendamustine, uramustine, palifosfamide, chlorambucil, busulfan, 4-hydroxycyclophosphamide, bis-chloroethylnitrosourea (BCNU), mitomycin C, yondelis, procarbazine, dacarbazine, temozolomide, cisplatin, carboplatin, oxaliplatin, acyclovir, gemcitabine, 5 -fluorouracil, cytosine arabinoside, ganciclovir, camptothecin, topotecan, irinotecan, doxorubicin, daunorubicin, epirubicin, idarubicin, etoposide, teniposide, mitoxantrone or pixantrone, or a pharmaceutically acceptable salt of any one thereof In some embodiments, the small molecule drug is Monomethyl auristatin E (MMAE).

[0046] In some embodiments, the payload is a detectable marker such as a radiopharmaceutical, a radioisotope, a radioisotope chelator, a dye, or a fluorescent marker (e.g. green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or an AlexaFluor™). Conjugates of the present invention may target detectable markers to target cells, tissues, or organs for diagnostic purposes.

[0047] In some embodiments, the payload is a radiotherapeutic, such as a radiopharmaceutical, a radioisotope, or a radioisotope chelator. Conjugates of the present invention may target radiotherapeutics to target cells, tissues, or organs for therapeutic purposes.

[0048] In some embodiments, the payload is a polynucleotide. In some embodiments, the polynucleotide comprises a transgene, an mRNA, an interfering RNA (e.g. miRNA, siRNA, shRNA), or an RNA for association with a CRISPR-associated protein (e.g. gRNA, sgRNA, crRNA / tracrRNA).

[0049] In some embodiments, the payload is an enzyme. In some embodiments, the enzyme is a DNA-editing enzyme such as, for example, a Zinc-finger nuclease, a transcription activator-like effector nuclease (TALEN), or a CRISPR-associated protein (e.g. Cas9, Casl2a, Cascade).

[0050] Variant Ligand

[0051] Conjugates of the present invention comprise a variant ligand. As used herein, the term “ligand” carries the ordinary meaning in the art of a molecule that specifically binds to one or more receptors. As used herein, the term “variant ligand” refers to a ligand derived from a naturally occurring ligand and contains one or more structural differences compared to the naturally occurring ligand. As used herein, the term “naturally occurring ligand” refers to the wild-type structure of the ligand as found in an organism. As used herein, a “naturally occurring ligand” is a ligand that is isolated from an organism or a ligand that is produced synthetically and has the structure and / or function of the ligand as found in an organism. The terms “naturally occurring ligand”, “native ligand”, and “wild-type ligand” may be used interchangeably.

[0052] The variant ligand contains mutations, substitutions, additions, deletions, or other structural variations compared to the naturally occurring version of the ligand from which the variant ligand is derived. In some embodiments, the variant ligand contains mutations or other structural variations to the receptor-binding region of the ligand.

[0053] In some embodiments, the variant ligand binds with higher affinity to one or more of the receptors that bind the ligand compared to the naturally occurring ligand. In some embodiments, the variant ligand binds to one or more of the receptors with at least 2-fold, 3 -fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold higher affinity compared to the naturally occurring ligand. In some embodiments, the variant ligand binds with higher affinity to one or more of the receptors compared to the naturally occurring ligand, without exhibiting higher receptor agonism compared to the naturally occurring ligand. In someembodiments, the variant ligand binds with higher affinity to one or more of the receptors compared to the naturally occurring ligand, and is an antagonist of the one or more receptors. Binding affinity may be measured by techniques known in that art. Binding affinity between a ligand and a receptor is usually measured by dissociation constant (Ka) in pM, nM, pM, or mM units. Alternatively, binding affinity may be measured by binding capacity. Binding capacity is usually measured by determining the maximum biological activity of the receptor at receptor saturation with the ligand (Bmax).

[0054] In some embodiments, the variant ligand provides increased agonism of one or more receptors that bind the ligand compared to the naturally occurring ligand. As used herein, the term “agonism” carries the ordinary meaning in the art of activating a receptor to induce a biological function of the receptor. A ligand that activates a receptor is an agonist. An antagonist is a ligand that inhibits one or more biological activities induced by the agonism, activation, or signaling of a receptor. The amount of agonism provided by a ligand is typically measured by its EC50 value. The EC50 can be measured for a given agonist ligand by determining the concentration of agonist ligand needed to elicit half of the maximum biological response of the receptor when agonized by a reference ligand. Smaller EC50 values, as measured in concentration of agonist ligand, indicate increased agonism because a lower concentration of the agonist ligand is required to elicit the maximum biological response. By contrast, the antagonism of an antagonist ligand may be measured in the terms of IC50 values obtained from an inhibitory potency assay. The IC50 is typically defined as the concentration of the agent at which 50 % of a signal elicited via the receptor by a natural agonist is inhibited by the agent. IC50 may sometimes be reported as pICso, which is the negative log of the IC50 value in moles per liter (molar or M). Alternatively, agonism may be measured by signaling efficacy. Signaling efficacy is usually measured by determining the maximum biological activity elicited by the ligand (Emax).

[0055] Receptors may exhibit more than one biological response when activated by an agonist ligand. The magnitude of the agonism or antagonism of a ligand may be measured with respect to one or more of the biological responses exhibited by the activated receptor. In some embodiments, agonism is measured by one or more of increased phosphorylation of the receptor when bound by the ligand; increased G protein signaling through the receptor when bound by the ligand; increased arrestin recruitment to the receptor when bound by the ligand; increasedinduction of intracellular calcium flux when the receptor is bound by the ligand; and / or increased receptor internalization when the receptor is bound by the ligand. In some embodiments, the variant ligand provides at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 15-fold, or 20-fold higher agonism as measured by the biological responses described above as compared to the naturally occurring ligand. The measurements of agonism as described herein may be determined using routine methods known in the art.

[0056] In some embodiments, the variant ligand is a ligand of a receptor tyrosine kinase.

[0057] In some embodiments, the variant ligand is a ligand of a G protein-coupled receptor.

[0058] In some embodiments, the variant ligand is a variant of a chemokine and is a ligand of a chemokine receptor. Chemokines are a specific class of cytokines that recruit cells to specific locations by inducing chemotaxis by signaling through chemokine receptors.Chemokines are classified into four groups (C chemokines, CC chemokines, CXC chemokines, and CXXXC chemokines). In some embodiments, the variant ligand is a variant of a C chemokine, CC chemokine, CXC chemokine, or a CXXXC chemokine. In some embodiments, the variant ligand is a variant of XCL1, CX3CL1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, or CXCL17. Variant chemokines may be produced using methods known in the art (Paolini-Bertrand 2018).

[0059] As disclosed herein, the exemplary CCL5 variant 6P4-CCL5 provides effective delivery and targeted killing of CCR5 -expressing cells in a 6P4-CCL5-MMAE conjugate, especially compared to the natural ligand CCL5 which was surprisingly ineffective at targeted delivery (FIG. 9). 6P4-CCL5 (also known as 6P4-RANTES) was previously discovered in the study of Gaertner et al. 2008 as a super agonist of CCR5 with potent efficacy for inhibiting HIV infection. In this study, 6P4-CCL5 exhibited similar receptor agonism and binding strength compared to another known super agonist PSC-RANTES (Table 1 of Gaertner et al. 2008). This study also identified a number of related CCL5 variants sharing the synthetic N-terminal QGPmotif that also exhibited strong receptor agonism (as measured by induced calcium flux or receptor internalization) and / or strong binding of the CCR5 receptor (as evidenced by a low IC50 in a CCR5-tropic cell fusion assay). For example, this study discovered the 5P12-CCL5 variant (sharing the N-terminal QGP motif) that, although being an inhibitor of CCR5 that did not induce strong signaling, nevertheless bound strongly to CCR5 and exhibited an IC50 in a cell fusion assay equivalent to the super agonist PSC-RANTES. Another study solved the crystal structure of the 5P7-CCL5 variant (also sharing the N-terminal QGP motif) when bound to CCR5, showing that its increased anti -HIV potency was due to stronger binding to the receptor resulting from near-complete occupancy of the binding pocket and a dense network of intermolecular hydrogen bonds (Zheng et al. 2017). In addition to CCL5 variants, Example 2 of the present disclosure provides variants of the ligand CCL2 that also exhibit increased receptor binding as evidenced by low IC50 values in a calcium flux assay. There are thus numerous ligand variants exhibiting strong receptor agonism and / or strong receptor binding that, in view of the surprising discovery of the present disclosure, can be used as the variant ligand in conjugates of the present invention.

[0060] In some embodiments, the variant ligand is a variant of CCL5. In some embodiments, the variant ligand is a CCL5 variant as disclosed in Gaertner et al. 2008 or as disclosed in WO 2008 / 012689. CCL5, also known as RANTES (regulated on activation, normal T cell expressed and secreted), has been shown to interact with CCR1, CCR3, CCR4, and CCR5. CCR5 exists in at least two conformations at the cell surface, and among the possible explanations for their existence is one in which one conformation consists of G protein-coupled CCR5 and the other G protein-uncoupled CCR5. Engagement of endogenous CCR5 agonists leads to activation of a repertoire of intracellular signaling pathways, broadly defined as G protein-dependent and G protein-independent signaling pathways. One manifestation of G protein-dependent signaling is the induction of intracellular calcium flux. One manifestation of G protein-independent signaling is the induction of receptor internalization by arrestin recruitment, leading to intracellular receptor sequestration. CCR5 is expressed by certain cancer cells, where its activation is suggested to provide survival and metastatic signals. In addition, CCR5 is expressed on immunomodulatory leukocytes (M2 macrophages and regulatory T cells) that infiltrate the tumor and suppress anti-tumor immune responses.

[0061] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is: QGP[P or L][L or G or S or M][M or D or S or Q or G]; QGP[P or L][L or G][M or D or S]; QGP[P or L][L or G or S or M][M or D or S or Q or G]XX[Q or G or L or A or T or S]X, wherein X denotes any natural or modified amino acid; QGP[P or L][L or G][M or D or S]XX[Q or L]X, wherein X denotes any natural or modified ammo acid; QGP[P or L]LM (SEQ ID NO: 150); QGPPG[D or S] (SEQ ID NO: 151); QGPPLM (SEQ ID NO: 152); or QGPPGD (SEQ ID NO: 153). As described herein, methionine residues can be conservatively substituted with non-oxidizable amino acid analogs or amino acid derivatives to reduce complications from methionine oxidation during synthesis. In some embodiments, one or more methionine residues in the polypeptides, peptides, and conjugates of the present invention are conservatively substituted with an amino acid analog or an amino acid derivative such as, but not limited to, norleucine (Nle). It is known in the art the N-terminal glutamine or glutamic acids can spontaneously convert to pyroglutamate in various in vitro and in vivo conditions (Cao et al. 2022). In some embodiments, the N-terminal residue designated herein as glutamine (Q) is present as a pyroglutamate.

[0062] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is: QGP[P or L][L or M][M or Q][A or W or G or Q or N]X[Q or G or L][S or V or T or G], wherein X denotes any natural or modified amino acid; QGP[P or L] [L or M] [M or Q] [A or W or G or Q or N] [L or T or M or S or G or Q or R or Y] [Q or G or L][S or V or T or G]; QGP[P or L]LM[A or W][L or T or M][Q or G][S or V or T or G] (SEQ ID NO: 154); or QGPPLM[A or W][L or T or M][Q or G][S or V or T or G] (SEQ ID NO: 155).

[0063] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is: QGP[P or L][L or G or S][D or S or G or Q]XX[L or A or T or Q][W or A or V], wherein X denotes any natural or modified amino acid; QGP[P or L][L or G or S][D or S or G or Q][T or I or S or W or Q][V or L or A or S or G][L or A or T or Q][W or A or V]; QGPPG[D or S][T or I]VL[W or A] (SEQ ID NO: 156); or QGPPGD[T or I]VL[W or A] (SEQ ID NO: 157).

[0064] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is: QGPP[G or L][M or Q]XX[Q or S][S or V] (SEQ ID NO: 158), wherein X denotes any natural or modified amino acid; QGPP[G or L][M or Q][S or G or W or A or T][L or F or T or S or G or Y][Q or S][S or V] (SEQ ID NO: 159); or QGPPLM[S or G][L or F or T]Q[S or V] (SEQ ID NO: 160).

[0065] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is selected from the group QGPPLMALQS (SEQ ID NO: 161), QGPPLMWMQV (SEQ ID NO: 162), QGPPLMWLQV (SEQ ID NO: 163), QGPPLMWTQS (SEQ ID NO: 164), QGPPLMWLQT (SEQ ID NO: 165), QGPPLMWTQV (SEQ ID NO: 166), QGPPLMWMQS (SEQ ID NO: 167), QGPPLMATQS (SEQ ID NO: 168), QGPPLMWLQS (SEQ ID NO: 169), QGPPLMALQV (SEQ ID NO: 170), QGPPLMWLGG (SEQ ID NO: 171), QGPPLMWRGS (SEQ ID NO: 172), QGPLLMWLQV (SEQ ID NO: 173), QGPPLMQTTP (SEQ ID NO: 174), QGPPLSWLQV (SEQ ID NO: 175), QGPPLSWLQS (SEQ ID NO: 176), QGPPGQWSQV (SEQ ID NO: 177), QGPPMMAGLS (SEQ ID NO: 178), QGPPLSWQQS (SEQ ID NO: 179), QGPPGMWSQS (SEQ ID NO: 180), QGPPLQWRQS (SEQ ID NO: 181), QGPPLMGTQS (SEQ ID NO: 182), QGPPLMQLQV (SEQ ID NO: 183), QGPPLSWSQV SEQ ID NO: 184), QGPPMSWSQS (SEQ ID NO: 185), QGPPLMNLQV(SEQ ID NO: 186), QGPPMSAYQV (SEQ ID NO: 187) and QGPPMQGGLS (SEQ ID NO: 188).

[0066] In some embodiments, the variant ligand comprises an N-terminal portion and a C -terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is selected from the group QGPPLMALQS (SEQ ID NO: 161), QGPPLMWMQV (SEQ ID NO: 162), QGPPLMWLQV (SEQ ID NO: 163), QGPPLMWTQS (SEQ ID NO: 164), QGPPLMWLQT (SEQ ID NO: 165), QGPPLMWTQV (SEQ ID NO: 166), QGPPLMWMQS (SEQ ID NO: 167), QGPPLMATQS (SEQ ID NO: 168), QGPPLMWLQS (SEQ ID NO: 169), QGPPLMALQV (SEQ ID NO: 170), QGPPLMWLGG (SEQ ID NO: 171), QGPPLMWRGS (SEQ ID NO: 172), QGPLLMWLQV (SEQ ID NO: 173), and QGPPLMQTTP (SEQ ID NO: 174).

[0067] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is selected from the group QGPPGDTVLW (SEQ ID NO: 189), QGPPGDIVLA (SEQ ID NO: 190), QGPPGSYDYS (SEQ ID NO: 191), QGPPGDGGSV (SEQ ID NO: 192), QGPLSGQSTP (SEQ ID NO: 193), QGPPGDWLQV (SEQ ID NO: 194), QGPPLMSLAV (SEQ ID NO: 195), QGPPLMSLTV (SEQ ID NO: 196), QGPLSGWAQV (SEQ ID NO: 197), QGPLSQSSQV (SEQ ID NO: 198), QGPLSSQSQV (SEQ ID NO: 199) and QGPLGQQGQV (SEQ ID NO: 200).

[0068] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is selected from the group QGPPGDTVLW (SEQ ID NO: 189), QGPPGDIVLA (SEQ ID NO: 190), QGPPGSYDYS (SEQ ID NO: 191), QGPPGDGGSV(SEQ ID NO: 192), QGPLSGQSTP (SEQ ID NO: 193), and QGPPGDWLQV (SEQ ID NO: 194).

[0069] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is selected from the group QGPPLMSFQS (SEQ ID NO: 201), QGPPLMSTQS (SEQ ID NO: 202), QGPPLMSLQV (SEQ ID NO: 203), QGPPLMGLQV (SEQ ID NO: 204), QGPLSGWLQV (SEQ ID NO: 205), QGPPLQWFQV (SEQ ID NO: 206), QGPPLQWTQV (SEQ ID NO: 207), QGPPLMALSV (SEQ ID NO: 208), QGPPLMWSQV (SEQ ID NO: 209), QGPPGQWGQV (SEQ ID NO: 210), QGPPGSWSQV (SEQ ID NO: 211), QGPPLMSSQS (SEQ ID NO: 212), QGPPLMGLSV (SEQ ID NO: 213), QGPPLMTLQV (SEQ ID NO: 214) and QGPPGQWYQS (SEQ ID NO: 215).

[0070] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is selected from the group QGPPLMSFQS (SEQ ID NO: 201), QGPPLMSTQS (SEQ ID NO: 202), QGPPLMSLQV (SEQ ID NO: 203), QGPPLMGLQV (SEQ ID NO: 204), and QGPLSGWLQV (SEQ ID NO: 205).

[0071] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is selected from the group QGPPLMSVLA (SEQ ID NO: 216), QGPPGSWSSV (SEQ ID NO: 217), QGPPLGSMGP (SEQ ID NO: 218), QGPPLQWMQA (SEQ ID NO: 219), QGPPLQWMQV (SEQ ID NO: 220), QGPPLMSTQV (SEQ ID NO: 221), QGPPLMSLSV (SEQ ID NO: 222), QGPPLMSLQS (SEQ ID NO: 223), QGPPLMSLQA (SEQ ID NO: 224), QGPPLMSVQS (SEQ ID NO: 225), QGPPLMSAQS(SEQ ID NO: 226), QGPPLMSGQS (SEQ ID NO: 227) and QGPPLMSGQV (SEQ ID NO: 228).

[0072] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is selected from the group QGPPLMSVLA (SEQ ID NO: 216), QGPPGSWSSV (SEQ ID NO: 217), and QGPPLGSMGP (SEQ ID NO: 218).

[0073] In some embodiments, the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises a variant sequence and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235, wherein the variant sequence is QGPPGDIVLA (SEQ ID NO: 190).

[0074] In some embodiments, the variant ligand comprises the amino acid sequence of any one of SEQ ID NO: 229-232.

[0075] In some embodiments, the variant ligand comprises an N-terminal portion comprising the sequence PSC-SSDTTP (SEQ ID NO: 236), wherein PSC is N(alpha)(n- nonanoyl)-des-Ser(l)-[ L-thioprolyl(2), L-cyclohexylglycyl(3)], and the amino acid sequence of the C-terminal portion is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 234 or 235.

[0076] In some embodiments, the N-terminal portion of the CCL5 variant ligands described herein may consist of no more than 15, 14, 13, 12, 11, or 10 amino acids. In some embodiments, said N-terminal portion consists of 10 amino acids. In some embodiments, the N- terminus of the C-terminal portion may adjoin directly to the C-terminus of the N-terminal portion, i.e. the N-terminal portion and the C-terminal portion are directly adjoined. The C- terminal portion of CCL5 variant ligands described in herein may have more than 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9% or 100% sequence identity to SEQ ID NO: 234 or 235. The variant sequence of the CCL5 variant ligands described herein may be located near the N- terminus of the polypeptide. For example, the variant sequence may be located such that thebeginning of the variant sequence lies within 15, 12, 10, 8, 6, 5, 4, 3, 2, or 1 residue of the N- terminus of the polypeptide. Herein, the expression “the beginning of the signature sequence” refers to the N-terminus of the signature sequence. The signature sequence may also be located at the extreme N-terminus of the polypeptide, i.e., the N-termini of the polypeptide as a whole and the signature sequence may coincide.

[0077] In some embodiments, the variant ligand exhibits an an IC50 of less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 90 pM, less than 80 pM, less than 70 pM, less than 60 pM, less than 50 pM, less than 40 pM, or less than 30 pM in a CCR5-tropic cell fusion assay. As used herein, a CCR5-tropic cell fusion assay measures the inhibition of fusion between two cell types mediated by binding of CCR5 on the cell membrane, wherein a lower IC50 value indicates a higher potency in inhibiting cell fusion. CCR5 -tropic cell fusion assays are known in the art, and may be performed as disclosed in WO 2008 / 012689, as disclosed in Gaertner et al. 2008, or as disclosed in Sabbe et al. 2001. In an exemplary CCR5 -tropic cell fusion assay, the cell lines HeLa-P5L and HeLa-Env-ADA are used. HeLa-P5L cells are seeded in 96-well plates (104cells per well in 100 pl). Twenty- four hours later, medium is removed and medium containing 104HeLa-Env-ADA cells per well plus variant ligands are added to a final volume of 200 pl. After a further 24 h, cells are washed once in phosphate-buffered saline and lysed in 50 pl of phosphate-buffered saline-0.5% NP-40 for 15 min at room temperature. Lysates are then assayed for P-galactosidase activity by the addition of 50 pl 2 CPRG (chlorophenol red-P-d-galactopyranoside) substrate (16 mM CPRG 120 mM Na2HPO4, 80 mM NalLPCh, 20 mM KC1, 20 mM MgSCL, 10 mM P-mercaptoethanol) followed by incubation for 1 to 2 h in the dark at room temperature. The 75 is then read on a Labsystems microplate reader. The reaction is stopped when the 575 for the positive control wells (no chemokine) reaches 0.5 to 1, and results are expressed as 100 x (mean absorbance [treated] - mean absorbance [no envelope cells]) / (mean absorbance [no chemokine] - mean absorbance [no envelope cells]).

[0078] In some embodiments, the variant ligand induces at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the calcium flux signaling through CCR5 induced by PSC-RANTES. As used herein, a calcium flux assay measures the calcium flux in a cell induced by agonism of the CCR5 receptors on thecell when bound by a ligand, such as the native ligand CCL5 or the synthetic super agonist PSC- RANTES. Calcium flux assays using CCR5 are known in the art, and may be performed as disclosed in WO 2008 / 012689 or as disclosed in Gaertner et al. 2008. In an exemplary calcium flux assay, THP-1 cells are seeded 20000 cells / well) to wells of black- walled clear-bottom 384- well plates. Test samples of variant ligands are diluted in PBS supplemented with 1% BSA and 25 mM HEPES to generate dilution series for dose-response experiments: 12-point doseresponse starting at 688 nM with a 2.5-fold dilution interval for each treatment). THP-1 cells are loaded with a calcium-sensitive fluorescent dye (Screen Quest™ Fluo-8 No Wash Calcium Assay Kit, AAT Bioquest) according to the manufacturer’s instructions, then a first addition of either test sample dilutions or vehicle alone were added. 5 minutes later, cells are stimulated with 100 nM test or reference samples and fluorescence signals (ex. 490 nm, em. 525 nm) are recorded.

[0079] In some embodiments, the variant ligand induces sequestration of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of CCR5 receptors in a CCR5 sequestration assay. As used herein, a sequestration assay measures the internalization and / or down-regulation of a surface receptor on a cell in response to exposure of the cell to a ligand of the receptor. Sequestration assays using CCR5 are known in the art, and may be performed as disclosed in WO 2008 / 012689 or as disclosed in Gaertner et al. 2008. In an exemplary CCR5 sequestration assay, CHO-CCR5 cells are seeded at a density of 80,000 cells / well. After overnight incubation, medium is removed and replaced with medium containing variant ligands, and cells are incubated for 1 h at 37°C. Medium is then removed and cells are fixed with 4% paraformaldehyde and washed twice with PBS. Cells are then labeled with either phycoerythrin-conjugated anti-CCR5 antibody (clone 3A9, PharMingen) or phycoerythrin- conjugated anti-CCRl antibody (clone 53504, R&D Systems; negative control) in PBS supplemented with 1% BSA (PBS-1% BSA) on ice for 1 h. Plates are washed three times with PBS-1% BSA and fluorescence values for each well are determined using a FLEXstation fluorimeter (Molecular Devices). Results are expressed as percentage control level of surface CCR5: 100 (mean fluorescence [chemokine added, anti-CCR5] - mean negative control fluorescence [anti-CCRl ]) / (mean positive control fluorescence [no chemokine added, anti- CCR5] - mean fluorescence [anti-CCRl]).

[0080] In some embodiments, the variant ligand is a variant of CCL2. The chemokine CCL2 (monocyte chemoattractant protein 1, MCP-1) binds preferentially to the receptor CCR2 and mediates cellular behaviors including monocyte chemotaxis. Studies have implicated CCL2- mediated monocyte infiltration in pain, cancer, and a range of inflammatory diseases. The variant of CCL2 is derived from CCL2, for example human CCL2 (SEQ ID NO: 1) or mouse CCL2 (SEQ ID NO: 73), in which the N-terminal portion comprises a variant sequence and the C- terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 2 or 3. In some embodiments, the C-terminal portion comprises an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 71, 72, 74, or 75. In some embodiments, the N-terminal portion comprises or consists of an amino acid sequence with 0, 1, 2, 3, or 4 amino acid substitutions relative to any one of SEQ ID NO: 9-70 and 76-145. In some embodiments, the N-terminal portion comprises or consists of the amino acid sequence of any one of SEQ ID NO: 146-149. As described herein, methionine residues can be conservatively substituted with non-oxidizable amino acid analogs or amino acid derivatives to reduce complications from methionine oxidation during synthesis. In some embodiments, one or more methionine residues in the polypeptides, peptides, and conjugates of the present invention are conservatively substituted with an amino acid analog or an amino acid derivative such as, but not limited to, norleucine (Nle). In some embodiments, the C-terminal portion of the CCL2 variant comprises a norleucine (Nle) residue at position 54 relative to SEQ ID NO: 74 (position 64 relative to SEQ ID NO: 1). In some embodiments, the C-terminal portion of the CCL2 variant comprises a norleucine (Nle) residue at one or more of positions 9, 12, and 62 relative to SEQ ID NO: 72. It is known in the art the N- terminal glutamine or glutamic acids can spontaneously convert to pyroglutamate in various in vitro and in vivo conditions (Cao et al. 2022). In some embodiments, the N-terminal residue designated herein as glutamine (Q) is present as a pyroglutamate.

[0081] In some embodiments, the variant ligand comprises an N-terminal portion and a C terminal portion, wherein said N-terminal portion comprises an amino acid sequence with 0, 1, 2, 3, or 4 amino acid substitutions relative to any one of SEQ ID NO: 9-70 and 76-145 or comprises the amino acid sequence of any one of SEQ ID NO: 146-149, and the C-terminal portion comprises an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NO: 71, 72, 74, or75. In some embodiments, the N-terminal portion comprises the amino acid sequence FTNPTWXXXX (SEQ ID NO: 146), wherein X is any natural or modified amino acid, and the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 71, 72, 74, or 75. In some embodiments, the N-terminal portion comprises the amino acid sequence FTNPTW[A or D or R or S or K or Q] [P or A or T or G or S or Q or R or H or E] [V or F or Q or G or S or H or L or Y][T or V or Q or S or A] (SEQ ID NO: 147) and the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 71, 72, 74, or 75. In some embodiments, the N-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 40 and 76-99 and the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 71, 72, 74, or 75. In some embodiments, the N-terminal portion comprises the amino acid sequence FPX1DGWX2X3X4X5 (SEQ ID NO: 148), wherein Xi is Methionine or Norleucine (Nle), and X2-X5 is any natural or modified amino acid, and the C- terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 71, 72,74, or 75. In some embodiments, the N-terminal portion comprises the amino acid sequence FPXiDGW[A or R or G or H or V or Q] [P or S or G or E] [V or R or L or E or T or G or Q] [T or V or Q] (SEQ ID NO: 149), wherein Xi is Methionine or Norleucine (Nle), and the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 71, 72, 74, or75. In some embodiments, the N-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 41 and 100-145 and the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 71, 72, 74, or 75.

[0082] In some embodiments, the N-terminal portion of the CCL2 variant ligands described herein consists of no more than 12 amino acids, such as no more than 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids. In some embodiments, the N-terminal portion consists of 11 amino acids, consists of 10 amino acids, or consists of 8 amino acids. In an embodiment, the N- terminus of the C-terminal portion adjoins directly to the C-terminus of the N-terminal portion, i.e. the N-terminal portion and the C terminal portion are directly adjoined. In some embodiments, the N-terminal portion is adjoined to the C-terminal portion via a peptide linker. In some embodiments, the N-terminal portion is located at the extreme N terminus of the polypeptide.

[0083] In some embodiments, the conjugate inhibits CCR2 with an IC50 of less than 300 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 30 nM, less than 10 nM, or less than 3 nM. Inhibition of CCR2 can be measured by IC50 using a calcium flux assay as disclosed herein.

[0084] The term “sequence identity,” as used herein, has the standard meaning in the art. As is known in the art, a number of different programs can be used to identify whether a polynucleotide or polypeptide has sequence identity or similarity to a known sequence.Sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith 1981, by the sequence identity alignment algorithm of Needleman 1970, by the search for similarity method of Pearson 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux 1984, preferably using the default settings, or by inspection. A sequence is also said to bear similarity to or to be a homologue of a reference sequence if it contains one or more conservative substitutions with respect to the reference sequence. Conservative substitutions are substitutions in the sequence of a peptide or polypeptide that do not lead to a significant loss of function or which lead only to a small loss of function. Such a loss of function due to one or more conservative substitutions may be considered not to be significant if said loss amounts to less than 20 %, less than 15 %, less than 10 %, less than 6 %, or less than 4 % with respect to the function of the polypeptide having the unsubstituted sequence. Conservative substitutions are often substitutions wherein an amino acid side chain is replaced by an amino acid side chain that is related, or similar in physicochemical properties, to the replaced residue. Such conservative substitutions may be made, for example, using one of the 20 natural amino acids according to Table 2 wherein amino acids in the same block in the middle column and preferably in the same line in the right-hand column may be substituted for each other. Conservative substitutions may also be made using amino acid analogs or amino acid derivatives such as, for example, asubstitution of methionine with norleucine.Fable 2

[0085] In some embodiments, the variant ligands are polypeptides. Polypeptides and peptides are polymers that comprise amino acids linked by peptide bonds. As used herein, the term “amino acid” is used to describe any amino acid, natural or otherwise, that can be incorporated into a polypeptide or a peptide. Amino acids are small molecules comprising an amine (-NH2) group, a carboxyl (-COOH), and a variable side chain (R-group) specific to each amino acid. Amino acids are covalently linked by peptide bonds between the amine group of one amino acid to the carboxyl group of another amino acid to form polypeptides. Amino acids within a polypeptide are often referred to in the art as “residues”.

[0086] Polypeptides and peptides may comprise post-translational modifications such as, for example, phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, acylation, prenylation, alkylation, oxidation, or other modifications known in the art.

[0087] Polypeptides and peptides may comprise amino acid analogs. As used herein, the term “amino acid analogs” describes artificial, synthetic, or unnatural amino acids beyond the 20 genetically-encoded amino acids, such as for example the amino acid analogs described in Zou 2018. Examples of amino acid analogs that can be incorporated into polypeptides, peptides, and conjugates of the present invention include, but are not limited to, norleucine, P-amino acids, homo-amino acids, synthetic proline and pyruvic acid derivatives, 3 -substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, N-methyl amino acids, and amino acids with synthetic R-groups. Polypeptidesand peptides may also comprise amino acid derivatives. As used herein, the term “amino acid derivatives” describes amino acids that have been derived from the modification of one of the 20 genetically-encoded amino acids. Amino acid derivatives can be synthetic, e.g. made in vitro by chemical reaction, or they can be naturally occurring in organisms, e.g. in vivo metabolites. An example of an amino acid derivative is pyroglutamate / pyroglutamic acid, a cyclized derivative of glutamine in which the free amino group of glutamic acid cyclizes to form a lactam.

[0088] Nucleic Acid Molecules. Vectors, and Host Cells

[0089] The present invention provides nucleic acid molecules encoding the conjugates as described herein. In some embodiments, the nucleic acid molecules encoding conjugates of the present invention are RNA or DNA. The skilled person is able to design or identify nucleic acid molecules encoding the conjugates of the present invention using methods known in the art. In some embodiments, the nucleic acid molecules encoding conjugates of the present invention are incorporated into a vector, such as a plasmid, episome, artificial chromosome, virus, or a viral vector. In some embodiments, the nucleic acid molecules encoding polypeptides, peptides, and / or conjugates of the present invention or the vector comprising said nucleic acid molecule are comprised within a host cell to enable expression of the conjugates of the present invention. In some embodiments, the host cell is a bacterial cell (e.g. E.coli), a yeast cell (e.g.Saccharomyces cerevisiae), or a mammalian cell (e.g. a human cell, a mouse cell, a CHO cell, a HEK cell, a HeLa cell).

[0090] Pharmaceutical Compositions

[0091] The present invention provides pharmaceutical compositions comprising conjugates as described herein and a pharmaceutically acceptable carrier, excipient, and / or stabilizer. The pharmaceutical compositions of the present invention may be provided for use as a medicament. The preparation of pharmaceutical compositions is well known to the person skilled in the art.

[0092] A pharmaceutical composition of the present invention may be administered to a subject in a therapeutically effective amount. As used herein, a "therapeutically effective amount" means an amount of the composition or therapeutic agent effective to provide a therapeutic, prophylactic or diagnostic benefit to a subject. In some embodiments, atherapeutically effective amount of the composition is an amount capable of inducing a clinical response in a subject in the treatment of a particular disease or disorder. Determination of a therapeutically effective amount of the composition is well within the capability of those skilled in the art, especially in light of the disclosure provided herein. The therapeutically effective amount may vary according to a variety of factors such as the subject’s condition, weight, sex and age.

[0093] Pharmaceutical compositions provided herein may be prepared in various pharmaceutical dosage forms, such as an instant release, controlled release, sustained release, or target drug-delivery system. Commonly used dosage forms include, for example, solutions and suspensions, (micro-) emulsions, ointments, gels, creams, pastes, foams, suppositories, ovules, implants, patches, liposomes, tablets, dragees, lozenges, soft or hard shell capsules, amorphous or crystalline powders, effervescent powders or tablets, aerosols, and lyophilized formulations. Depending on the route of administration used, special devices may be required for application or administration of a dosage form, such as syringes and needles, inhalers, pumps, injection pens, applicators, special flasks, or other devices for administration, which may also be implanted within a body. Pharmaceutical dosage forms provided herein may be manufactured by any of the methods well-known in the art, such as, for example, by conventional mixing, sieving, dissolving, melting, granulating, dragee-making, tabletting, suspending, extruding, spray-drying, levigating, emulsifying, (nano / micro-) encapsulating, entrapping, or lyophilization processes.

[0094] Pharmaceutical compositions provided herein may further comprise a pharmaceutically acceptable carrier, excipient, and / or stabilizer (Remington 2000). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations, and may comprise, for example but not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides,and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0095] Methods and Uses

[0096] Conjugates and pharmaceutical compositions of the present invention may be used for the delivery of the payload to a target cell that expresses a receptor that binds the variant ligand in the conjugate. In some embodiments, the cell is a dendritic cell, a monocyte, a plasma cell, a macrophage, a Kupffer cell, a Langerhans cell, a T cell, a B cell, an erythroid cell, a hepatic stellate cell, a cholangiocyte, a type 2 alveolar cell, a gastric mucus-secreting cell, an NK cell, a hepatocyte, a Hofbauer cell, a spermatid, a fibroblast, a myeloid-derived suppressor cell, a neutrophil, an osteoclast, a stem cell, a basal keratinocyte, a cardiomyocyte, an endothelial cell, a breast glandular cell, a breast myoepithelial cell, a glandular cell, a luminal cell, a theca cell, a spermatogonium, a cytotrophoblast, a smooth muscle cell, an adipocyte, an immortalized cell, or a cancer cell. In some embodiments, the cell is a receptor-expressing cancer cell from a cancer such as leukemia such as acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chromic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia; myelodysplastic syndrome (MDS); a lymphoma such as Hodgkin's disease, a malignant lymphoma, non- Hodgkin’s lymphoma, Burkitt's lymphoma; multiple myeloma; Kaposi's sarcoma; colorectal cancer such as colorectal carcinoma; pancreatic cancer such as pancreatic carcinoma; renal cell carcinoma; breast cancer; prostate cancer; cervical cancer; ovarian cancer; liver cancer; kidney cancer; stomach cancer; bladder cancer; ling cancer; oesophageal cancer; nasopharyngeal carcinoma; malignant histiocytosis; paraneoplastic syndrome / hypercalcemia of malignancy; solid tumors; adenocarcinoma such as lung adenocarninoma; squamous cell carcinomas such as squamous epithelial cell and basal squamous epithelial cell; sarcoma such as osteosarcoma; malignant melanoma; melanoma; thyroid carcinoma; salivary adenoid cystic carcinoma, glioma, or haemangioma. In some embodiments, the cancer cell is a cancerous dendritic cell, monocyte, plasma cell, macrophage (e.g. a Kupffer cell or a Langerhans cell), T cell, B cell, NK cell, myeloid-derived suppressor cell, or neutrophil. By “cancerous” cell it is meant that the cell isderived from a normal cell lineage and contains one or more genetic abnormalities leading to transformation into a cancer cell

[0097] Conjugates and pharmaceutical compositions of the present invention may be used therapeutically in the diagnosis, prevention, and / or treatment of diseases and disorders such as, for example, cancer. In an embodiment, the cancer is leukemia such as acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chromic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia; myelodysplastic syndrome (MDS); a lymphoma such as Hodgkin's disease, a malignant lymphoma, non- Hodgkin’s lymphoma, Burkitt's lymphoma; multiple myeloma; Kaposi's sarcoma; colorectal cancer such as colorectal carcinoma; pancreatic cancer such as pancreatic carcinoma; renal cell carcinoma; breast cancer; prostate cancer; cervical cancer; ovarian cancer; liver cancer; kidney cancer; stomach cancer; bladder cancer; ling cancer; oesophageal cancer; nasopharyngeal carcinoma; malignant histiocytosis; paraneoplastic syndrome / hypercalcemia of malignancy; solid tumors; adenocarcinoma such as lung adenocarninoma; squamous cell carcinomas such as squamous epithelial cell and basal squamous epithelial cell; sarcoma such as osteosarcoma; malignant melanoma; melanoma; thyroid carcinoma; salivary adenoid cystic carcinoma, glioma, or haemangioma.

[0098] One or more embodiments of conjugates of the present invention may be administered to a subject. When more than one is administered, they may be administered together (as an admixture or separately though substantially simultaneously) or sequentially. They may be administered in combination with one or more other pharmaceutical or therapeutic agent that are not comprised the conjugates of the present invention. The conjugates of the present invention may then also be administered together (as an admixture or separately though substantially simultaneously) with said one or more other pharmaceutical or therapeutic agent, or sequentially.

[0099] “Treating” or “treatment of’, or “preventing” or “prevention of’, as used herein, refers to an approach for obtaining beneficial or desired results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilisation of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression,suppression of disease, delay or slowing of disease onset, conferring protective immunity against a disease-causing agent and amelioration or palliation of the disease state. “Treating” or “preventing” can also mean prolonging survival of a patient beyond that expected in the absence of treatment and can also mean inhibiting the progression of disease temporarily or preventing the occurrence of disease, such as by preventing infection in a subject.

[0100] “Treating” may be distinguished from “preventing” in that “treating” typically occurs in a subject who already has a disease or disorder, whereas “preventing” typically occurs in a subject who does not have a disease or disorder. As will be appreciated, there may be overlap in treatment and prevention. For example, it is possible to be “treating” a disease in a subject, while at same time “preventing” symptoms or progression of the disease.

[0101] Conjugates and pharmaceutical compositions of the present invention may be used to kill or modify target cells that express a receptor that binds the variant ligand in the conjugate. For example, the payload is a cytotoxic drug, and the conjugate may be used to deliver the cytotoxic drug to specific receptor-expressing cells, thereby killing the target cells. In another example, the payload is a transgene, mRNA, or an interfering RNA, and the conjugate may be used to deliver the polynucleotide payload to specific receptor-expressing cells, thereby modifying gene expression and / or polypeptide translation in the target cells. In another example, the pay load is a gene-editing enzyme, and the conjugate may be used to deliver the gene-editing enzyme to specific receptor-expressing cells, thereby modifying the genome of the target cells.

[0102] Particular embodiments of the disclosure include, without limitation, the following:1. A conjugate comprising a payload conjugated to a variant ligand, wherein the variant ligand is capable of binding to a receptor, and wherein the variant ligand binds with higher affinity to the receptor compared to a naturally occurring ligand of the receptor.2. A conjugate comprising a payload conjugated to a variant ligand,wherein the variant ligand is capable of binding to a receptor, and wherein the variant ligand provides increased agonism of the receptor compared to a naturally occurring ligand of the receptor.3. The conjugate of embodiment 2, wherein the increased agonism comprises increased receptor phosphorylation.4. The conjugate of embodiment 2 or 3, wherein the increased agonism comprises increased G protein signaling through the receptor.5. The conjugate of any one of embodiments 2-4, wherein the increased agonism comprises increased arrestin recruitment to the receptor.6. The conjugate of any one of embodiments 2-5, wherein the increased agonism comprises increased induction of intracellular calcium flux.7. The conjugate of any one of embodiments 2-6, wherein the increased agonism comprises increased receptor internalization.8. The conjugate of any one of embodiments 1-7, wherein the receptor comprises a polypeptide.9. The conjugate of any one of embodiments 1-8, wherein the receptor is a G protein-coupled receptor.10. The conjugate of any one of embodiments 1-9, wherein the receptor is a chemokine receptor.11. The conjugate of any one of embodiments 1-10, wherein the naturally occurring ligand is a naturally occurring chemokine, and wherein the variant ligand is a variant chemokine.12. The conjugate of any one of embodiments 1-11, wherein the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises the sequence QGP[P or L], and wherein the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 234 or 235.13. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S or M][M or D or S or Q or G],14. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G][M or D or S],15. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S or M][M or D or S or Q or G]XX[Q or G or L or A or T or S]X, wherein X denotes any natural or modified amino acid.16. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G][M or D or S]XX[Q or G or L]X, wherein X denotes any natural or modified amino acid.17. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGP[P or L]LM (SEQ ID NO: 150) or QGPPG[D or S] (SEQ ID NO: 151).18. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGPPLM (SEQ ID NO: 152) or QGPPGD (SEQ ID NO: 153).19. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or M][M or Q][A or W or G or Q or N]X[Q or G or L][S or V or T or G],20. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or M][M or Q][A or W or G or Q or N][L or T or M or S or G or Q or R or Y][Q or G or L][S or V or T or G],21. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGP[P or L]LM[A or W][L or T or M][Q or G][S or V or T or G] (SEQ ID NO: 154).22. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGPPLM[A or W][L or T or M][Q or G][S or V or T or G] (SEQ ID NO: 155).23. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S][D or S or G or Q]XX[L or A or T or Q][W or A or V],24. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S][D or S or G or Q][T or I or S or W or Q][V or L or A or S or G][L or A or T or Q][W or A or V],25. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGPPG[D or S][T or I]VL[W or A] (SEQ ID NO: 156).26. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGPPGD[T or I]VL[W or A] (SEQ ID NO: 157).27. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGPP[G or L][M or Q]XX[Q or S] [S or V] (SEQ ID NO: 158).28. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGPP[G or L][M or Q] [S or G or W or A or T][L or F or T or S or G or Y][Q or S] [S or V] (SEQ ID NO: 159).29. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGPPLM[S or G][L or F or T]Q[S or V] (SEQ ID NO: 160).30. The conjugate of embodiment 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMALQS (SEQ ID NO: 161), QGPPLMWMQV (SEQ ID NO: 162), QGPPLMWLQV (SEQ ID NO: 163), QGPPLMWTQS (SEQ ID NO: 164), QGPPLMWLQT (SEQ ID NO: 165), QGPPLMWTQV (SEQ ID NO: 166), QGPPLMWMQS (SEQ ID NO: 167), QGPPLMATQS (SEQ ID NO: 168), QGPPLMWLQS (SEQ ID NO: 169), QGPPLMALQV (SEQ ID NO: 170), QGPPLMWLGG (SEQ ID NO: 171), QGPPLMWRGS (SEQ ID NO: 172), QGPLLMWLQV (SEQ ID NO: 173), QGPPLMQTTP (SEQ ID NO: 174), QGPPLSWLQV (SEQ ID NO: 175), QGPPLSWLQS (SEQ ID NO: 176), QGPPGQWSQV (SEQ ID NO: 177), QGPPMMAGLS (SEQ ID NO: 178), QGPPLSWQQS (SEQ ID NO: 179), QGPPGMWSQS (SEQ ID NO: 180), QGPPLQWRQS (SEQ ID NO: 181), QGPPLMGTQS (SEQ ID NO: 182), QGPPLMQLQV (SEQ ID NO: 183), QGPPLSWSQV SEQID NO: 184), QGPPMSWSQS (SEQ ID NO: 185), QGPPLMNLQV (SEQ ID NO: 186), QGPPMSAYQV (SEQ ID NO: 187) and QGPPMSAYQV (SEQ ID NO: 187).31. The conjugate of embodiment 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMALQS (SEQ ID NO: 161), QGPPLMWMQV (SEQ ID NO: 162), QGPPLMWLQV (SEQ ID NO: 163), QGPPLMWTQS (SEQ ID NO: 164), QGPPLMWLQT (SEQ ID NO: 165), QGPPLMWTQV (SEQ ID NO: 166), QGPPLMWMQS (SEQ ID NO: 167), QGPPLMATQS (SEQ ID NO: 168), QGPPLMWLQS (SEQ ID NO: 169), QGPPLMALQV (SEQ ID NO: 170), QGPPLMWLGG (SEQ ID NO: 171), QGPPLMWRGS (SEQ ID NO: 172), QGPLLMWLQV (SEQ ID NO: 173) and QGPPLMQTTP (SEQ ID NO: 174).32. The conjugate of embodiment 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMALQS (SEQ ID NO: 161), QGPPLMWMQV (SEQ ID NO: 162), QGPPLMWLQV (SEQ ID NO: 163), QGPPLMWTQS (SEQ ID NO: 164), QGPPLMWLQT (SEQ ID NO: 165), QGPPLMWTQV (SEQ ID NO: 166), QGPPLMWMQS (SEQ ID NO: 167), QGPPLMATQS (SEQ ID NO: 168), QGPPLMWLQS (SEQ ID NO: 169), QGPPLMALQV (SEQ ID NO: 170), QGPPLMWLGG (SEQ ID NO: 171), QGPPLMWRGS (SEQ ID NO: 172), QGPLLMWLQV (SEQ ID NO: 173) and QGPPLMQTTP (SEQ ID NO: 174).33. The conjugate of embodiment 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPGDTVLW (SEQ ID NO: 189), QGPPGDIVLA (SEQ ID NO: 190), QGPPGSYDYS (SEQ ID NO: 191), QGPPGDGGSV (SEQ ID NO: 192), QGPLSGQSTP (SEQ ID NO: 193), QGPPGDWLQV (SEQ ID NO: 194), QGPPLMSLAV (SEQ ID NO: 195), QGPPLMSLTV (SEQ ID NO: 196), QGPLSGWAQV (SEQ ID NO: 197), QGPLSQSSQV (SEQ ID NO: 198), QGPLSSQSQV (SEQ ID NO: 199) and QGPLGQQGQV (SEQ ID NO: 200).34. The conjugate of embodiment 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPGDTVLW (SEQ ID NO: 189), QGPPGDIVLA (SEQ ID NO: 190), QGPPGSYDYS (SEQ ID NO: 191), QGPPGDGGSV (SEQ ID NO: 192), QGPLSGQSTP (SEQ ID NO: 193) and QGPPGDWLQV (SEQ ID NO: 194).35. The conjugate of embodiment 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMSFQS (SEQ ID NO: 201), QGPPLMSTQS (SEQ ID NO: 202), QGPPLMSLQV (SEQ ID NO: 203), QGPPLMGLQV (SEQ ID NO: 204), QGPLSGWLQV (SEQ ID NO: 205), QGPPLQWFQV (SEQ ID NO: 206), QGPPLQWTQV (SEQ ID NO: 207), QGPPLMALSV (SEQ ID NO: 208), QGPPLMWSQV (SEQ ID NO: 209), QGPPGQWGQV (SEQ ID NO: 210), QGPPGSWSQV (SEQ ID NO: 211), QGPPLMSSQS (SEQ ID NO: 212), QGPPLMGLSV (SEQ ID NO: 213), QGPPLMTLQV (SEQ ID NO: 214) and QGPPGQWYQS (SEQ ID NO: 215).36. The conjugate of embodiment 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMSFQS (SEQ ID NO: 201), QGPPLMSTQS (SEQ ID NO: 202), QGPPLMSLQV (SEQ ID NO: 203), QGPPLMGLQV (SEQ ID NO: 204), and QGPLSGWLQV (SEQ ID NO: 205).37. The conjugate of embodiment 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMSVLA (SEQ ID NO: 216), QGPPGSWSSV (SEQ ID NO: 217), QGPPLGSMGP (SEQ ID NO: 218), QGPPLQWMQA (SEQ ID NO: 219), QGPPLQWMQV (SEQ ID NO: 220), QGPPLMSTQV (SEQ ID NO: 221), QGPPLMSLSV (SEQ ID NO: 222), QGPPLMSLQS (SEQ ID NO: 223), QGPPLMSLQA (SEQ ID NO: 224), QGPPLMSVQS (SEQ ID NO: 225), QGPPLMSAQS (SEQ ID NO: 226), QGPPLMSGQS (SEQ ID NO: 227) and QGPPLMSGQV (SEQ ID NO: 228).38. The conjugate of embodiment 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMSVLA (SEQ ID NO: 216), QGPPGSWSSV (SEQ ID NO: 217) and QGPPLGSMGP (SEQ ID NO: 218).39. The conjugate of embodiment 12, wherein the N-terminal portion comprises the sequence QGPPGDIVLA (SEQ ID NO: 190).40. The conjugate of embodiment 11 or 12, wherein the variant ligand comprises the sequence of SEQ ID NO: 229.41. The conjugate of any one of embodiments 12-40, wherein the N-terminal portion consists of no more than 15 amino acids.42. The conjugate of any one of embodiments 12-41, wherein the N-terminal portion consists of 10 amino acids.43. The conjugate of any one of embodiments 1-11, wherein the N-terminal portion comprises the sequence PSC-SSDTTP (SEQ ID NO: 236), wherein PSC is N(alpha)(n- nonanoyl)-des-Ser(l)-[ L-thioprolyl(2), L-cyclohexylglycyl(3)].44. The conjugate of any one of embodiments 12-43, wherein the amino acid sequence of the C-terminal portion comprises the amino acid sequence of SEQ ID NO: 234 or 235.45. The conjugate of any one of embodiments 12-44, wherein the N-terminal portion is located at the extreme N terminus.46. The conjugate of any one of embodiments 12-45, wherein the naturally occurring ligand is CCL5.47. The conjugate of any one of embodiments 12-46, wherein the naturally occurring ligand comprises the amino acid sequence of SEQ ID NO: 230 or 233.48. The conjugate of any one of embodiments 12-47, wherein the receptor is CCR1, CCR3, CCR4, and / or CCR5.49. The conjugate of any one of embodiments 12-48, wherein the variant ligand exhibits an an IC50 of less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 90 pM, less than 80 pM, less than 70 pM, less than 60 pM, less than 50 pM, less than 40 pM, or less than 30 pM for inhibition of cell fusion in a CCR5 -tropic cell fusion assay.50. The conjugate of any one of embodiments 12-49, wherein the variant ligand induces at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the calcium flux signaling through CCR5 induced by PSC- RANTES.51. The conjugate of any one of embodiments 12-50, wherein the variant ligand induces sequestration of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of CCR5 receptors in a CCR5 sequestration assay.52. The conjugate of any one of embodiments 1-11, wherein the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises an amino acid sequence with 0, 1, 2, 3, or 4 amino acid substitutions relative to any one of SEQ ID NO: 40-70 and 76-145, and wherein the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 71, 72, 74 or 75.53. The conjugate of embodiment 52, wherein the amino acid substitution is a conservative substitution.54. The conjugate of embodiment 52, wherein the N-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 40-70 and 76-145.55. The conjugate of embodiment 52, wherein the N-terminal portion comprises the amino acid sequence FTNPTWXXXX (SEQ ID NO: 146), wherein X is any natural or modified amino acid.56. The conjugate of embodiment 55, wherein the N-terminal portion comprises the amino acid sequence FTNPTW[A or D or R or S or K or Q] [P or A or T or G or S or Q or R or H or E][V or F or Q or G or S or H or L or Y][T or V or Q or S or A] (SEQ ID NO: 147).57. The conjugate of embodiment 55, wherein the N-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 40 and 76-99.58. The conjugate of embodiment 52, wherein the N-terminal portion comprises the amino acid sequence FPX1DGWX2X3X4X5 (SEQ ID NO: 148), wherein Xi is Methionine or Norleucine, and X2-X5 is any natural or modified amino acid.59. The conjugate of embodiment 58, wherein the N-terminal portion comprises the amino acid sequence FPXiDGW[A or R or G or H or V or Q] [P or S or G or E] [V or R or L or E or T or G or Q][T or V or Q] (SEQ ID NO: 149), wherein Xi is Methionine or Norleucine.60. The conjugate of embodiment 58, wherein the N-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 41 and 100-145.61. The conjugate of any one of embodiments 52-60, wherein the N-terminal portion consists of 8-11 amino acids.62. The conjugate of any one of embodiments 52-61, wherein the C-terminal portion comprises the amino acid sequence of SEQ ID NO: 71, 72, 74, or 75.63. The conjugate of any one of embodiments 52-62, wherein the conjugate inhibits CCR2 with an IC50 of less than 300 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 30 nM, less than 10 nM, or less than 3 nM.64. The conjugate of any one of embodiments 52-63, wherein the naturally occurring ligand is CCL2.65. The conjugate of any one of embodiments 52-64, wherein the naturally occurring ligand comprises the amino acid sequence of SEQ ID NO: 1 or 73.66. The conjugate of any one of embodiments 52-65, wherein the receptor is CCR2 and / or CCR4.67. The conjugate of any one of embodiments 1-66, wherein the payload comprises a polynucleotide, an enzyme, a toxin, a drug, a carrier protein, a polymer, an antibody, and / or a detectable marker.68. The conjugate of embodiment 67, wherein the drug is a small molecule drug.69. The conjugate of embodiment 68, wherein the small molecule drug is Monomethyl auristatin E (MMAE).70. The conjugate of any one of embodiments 1-69, wherein the variant ligand is conjugated to the payload via a linker.71. The conjugate of embodiment 70, wherein the linker is a cleavable linker.72. The conjugate of embodiment 70 or 71, wherein the linker comprises a peptide linker, a hydrazone linker, or a disulfide linker.73. The conjugate of embodiment 70 or 71, wherein the linker comprises VA-PAB.74. A nucleic acid molecule encoding the conjugate of any one of embodiments 1-73.75. A vector comprising the nucleic acid molecule of embodiment 74.76. A host cell comprising the nucleic acid molecule of embodiment 74 or the vector of embodiment 75.77. A pharmaceutical composition comprising the conjugate of any one of embodiments 1-73, the nucleic acid molecule of embodiment 74, or the vector of embodiment 75, and a pharmaceutically acceptable carrier, excipient, and / or stabilizer.78. A conjugate as defined in any one of embodiments 1-73 or the pharmaceutical composition as defined in embodiment 77 for use in delivering a payload to a cell.79. The conjugate or the pharmaceutical composition for use of embodiment 78, wherein the use is for the treatment of cancer in a subject.80. A conjugate as defined in any one of embodiments 1-73 or the pharmaceutical composition as defined in embodiment 77 for use in killing or modifying a target cell.81. A method for delivering a payload to a cell, comprising the step of contacting the cell with the conjugate of any one of embodiments 1-73 or the pharmaceutical composition of embodiment 77.82. A method for delivering a pay load to a cell in a subject, comprising the step of administering the conjugate of any one of embodiments 1-73 or the pharmaceutical composition of embodiment 77 to the subject.83. A method for treating cancer in a subject, comprising the step of administering the conjugate of any one of embodiments 1-73 or the pharmaceutical composition of embodiment 77 to the subject. 184. A method for killing or modifying a target cell, comprising the step of contacting the cell with the conjugate of any one of embodiments 1-73 or the pharmaceutical composition of embodiment 77.85. A method for killing or modifying a target cell in a subject, comprising the step of administering the conjugate of any one of embodiments 1-73 or the pharmaceutical composition of embodiment 77 to the subject

[0103] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0104] All publications and patents cited herein are incorporated by reference in their entirety as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In the case of any conflict between a definition of a term in the present disclosure and a definition in a cited publication or patent, the definition provided in the present disclosure is to be used in describing the present invention.

[0105] The present invention will now be described by way of non-limiting examples having regard to the appended drawings.

[0106] Example 1

[0107] Methods - Synthesis of native ligand and variant ligand with azido functionality

[0108] Fmoc Solid phase peptide synthesis (Prelude synthesizer, Fmoc-Lys(N3)-2CT resin, 16 pmol at 0.157 mmol / g) was used to generate linear peptides corresponding to Azido- CCL5:SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVR EYINSLE-Nle-S-Lys(N3) (SEQ ID NO: 232); or Azido-6P4-CCL5:ZGPPGDIVLACCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWV REYINSLE-Nle-S-Lys(N3) (SEQ ID NO: 231); where Z is pyroglutamate (which forms spontaneously through the conversion of a glutamine residue located at the N-terminus of a polypeptide chain), Nle is norleucine (replacing methionine) and Lys(N3) is L-azidolysine (included as part of the Fmoc Solid phase peptide synthesis after cleavage from the resin). Cleaved peptides were purified by reverse-phase hplc and folded (48 h, 0.2 mg / mL) in 2 M guanidine, 0.1 M Tris, reduced / oxidized glutathione (0.5 mM / 0.3 mM), methioinine 10 mM, pH8.0. Folded peptides were purified by reverse-phase hplc with final products analyzed for purity and integrity by reverse-phase hplc and mass spectrometry (FIGs 1 and 2).

[0109] Methods - Synthesis of payload-linker moiety

[0110] Step 1 (FIG. 3). Fmoc-VC-PAB-PNP (35 mg, 51.5 pmol, 1 equiv.), monomethyl auristatin E (MMAE, 48 mg, 67 pmol, 1.3 equiv.), HOBt (1.4 mg, 10.3 pmol, 0.2 equiv.) and pyridine (124.4 pL, 30 eq) were dissolved in 8.0 mL of dimethylformamide and the mixture was stirred at ambient temperature for 24 h.

[0111] Step 2 (FIG. 4). 20% piperidine in DMF (1.6 mL) was added to the reaction mixture and stirred for 20 min at ambient temperature. The product (NH2-VA-PAB-MMAE) was then purified by reverse-phase hplc and lyophilized to obtain a white powder (14.3 mg).

[0112] Step 3 (FIG. 5). NH2-VA-PAB-MMAE (12.43 pmol, 1 eq) was dissolved in dimethylformamide (1 mL) with DBCO-PEG4-NHS solution (177.6 pL, 50 mg / mL stock; 13.67 pmol, 1.1 eq) and A, Y-Di isopropyl ethylamine (10.83 pL, 62.15 pmol, 5 eq) and stirred overnight at ambient temperature. The product (DBCO-PEG4-VA-PAB-MMAE linker) was purified by reverse-phase hplc and lyophilized to obtain pure product (6.8 mg) (FIG. 6).

[0113] Methods - Conjugation of azido-functionalized ligands with payload-linker moiety

[0114] 1 mg of azido-CCL5 or azido-6P4-CCL5 (0.125 pmol, 1 eq) dissolved in 125 pL6 M guamdine-HCl (pH 4.3) (1 mM) was added to 18.8 pL of 10 mM DBCO-PEG4-VA-PAB- MMAE linker (DMSO, 1.5 eq) and stirred at ambient temperature for 24 h. Ligand-conjugates were purified by size exclusion using AMICON IkD 0.5 mL kits according to the manufacturer’s protocol. Eluates were then desalted using solid-phase extraction resin (Cl 8 Chromabond resin; Macherey-Nagel, 130 mg / well) according to the manufacturer’s protocol, and eluates were lyophilized and solubilized in pure water (200 pL). Purity and integrity were assessed by reverse-phase hplc and mass spectrometry (FIG.s 7 and 8). Concentrations, determined using calculated extinction coefficients at 280 nm and purity estimated by reversephase hplc, were adjusted to make stock solutions of conjugate at 137 pM (CCL5-MMAE conjugate) and 200 pM (6P4-CCL5-MMAE conjugate).

[0115] Results - Cytotoxicity Assay

[0116] HEK-CCR5, HEK-WT (2000 cells, 50 uL / well) were seeded overnight into black, transparent, flat-bottom 96-well plates. Cells were then treated with serial dilutions (1 uM to 0.5 pM; 8-fold dilution steps; 8 data points) of either MMAE alone, CCL5-MMAE conjugate or 6P4-CCL5-MMAE conjugate diluted in added in 50 uL of DMEM supplemented with FCS and antibiotics. Cells were incubated at 37°C, 5% CO2 for 3 hours, then medium was removed and replaced with fresh, drug-free medium. 72 h later, cell viability was determined using a Cell TiterGlo™ kit according to manufacturer instructions. The resulting bioluminescence signal was acquired with a 96-well luminometer (POLARstar Omega ). Results were expressed as % normalized viability: 100 x (Xn-Xzero viabiiity) / (XfuiiViabiiity-Xzero viability); where Xn= luminescence value obtained for a given treatment condition, Xzero viability = lumiescence value obtained for Cells treated with 10% DMSO, Xfuii viability = non-treated cells. The results are shown in FIG. 9.

[0117] As expected MMAE alone shows an equally high level of toxicity against CCR5- positive (target cells) and CCR5-negative (bystander cells).

[0118] Unexpectedly, conjugation of MMAE to the native CCR5 ligand CCL5 did not lead to increased discrimination between CCR5 -positive and CCR5 -negative cells, but instead lowered the potency of the MMAE-conjugate compared to MMAE alone.

[0119] Unexpectedly, conjugation of MMAE to the variant ligand 6P4-CCL5 led to (i) a very high level of discrimination between CCR5 -positive and CCR5 -negative cells and (ii) a clear increase in the potency of the MMAE conjugate on CCR5-positive cells compared to MMAE used alone.

[0120] Example 2

[0121] CCL2 variant ligands with strong binding to CCR2 were produced.

[0122] Methods - Materials and Protocols

[0123] CCR2 inhibitory potency assay was determined using the calcium flux method. Ca2+flux measurements were performed using an FDSS micro-cell device (HAMAMATSU). On the day of the experiment, THP-1 cells were seeded 20000 cells / well) to wells of black-walled clear-bottom 384-well plates. Synthesized test samples (100 mM) were diluted in PBS supplemented with 1% BSA and 25 mM HEPES to generate dilution series for dose-response experiments: 12-point dose-response starting at 688 nM with a 2.5-fold dilution interval for each treatment). THP-1 cells were loaded with a calcium-sensitive fluorescent dye (Screen Quest™ Fluo-8 No Wash Calcium Assay Kit, AAT Bioquest) according to the manufacturer’s instructions, then a first addition of either test sample dilutions or vehicle alone were added. 5 minutes later, cells were stimulated with 100 nM CCL2. Fluorescence signals (ex. 490 nm, em. 525 nm) were recorded during the full course of the experiment.

[0124] Methods - Synthesis of Fmoc-Thr(tBu)-2-chlorotrityl resin

[0125] Synthesis of variant CCL2 ligands, such as the exemplary variant 1P2-CCL2 (Met64Nle OB-004), involved synthesis of Fmoc-Thr(tBu)-2-chloro trityl resin.

[0126] The synthesis of 1P2-CCL2 (Met64Nle OB-004) is detailed as an example of synthesis of a CCL2 variant ligand. 3 g of 2-chlorotrityl resin (2CT resin loading: 1.08 mmol / g) was weighed in a 50 mL peptide synthesis fritted syringe and swelled with 20 mL of DMF for 30 min at room temperature (RT). Fmoc-Thr(tBu)-OH (0.15 mmol, 178.9 mg) and DIPEA (392 pL, 0.45 mmol, 5 eq to amino acid) were dissolved in 12 mL of DMF and added to the pre-swelled resin, and the reaction mix was rotated at 6 rpm for 1.5 h at RT. The loading of the resin was determined by quantifying the Fmoc-Thr(tBu)-OH in the flow through using UV absorbance(UV 301 nm; s 7800 M-l cm-1), and was quantified at 0.149 mmol / g. After that, 1 mL of DIPEA (2 eq to the resin) and 1.5 mL MeOH were added to the reaction mixture sequentially and the reaction was rotated at 6 rpm for 10 min at RT to quench the unreacted trityl chloridegroups of the resin. Then the resin was washed three times with 12 mL of DMF and two times with 12 mL of MeOH and lyophilized.

[0127] Methods - Synthesis and purification of lP2-CCL2(Met64Nle OB-004) core fragment (11-76)

[0128] A C-terminal fragment of human CCL2 [11 -76 human CCL2], in which the methionine residue at position 64 relative to SEQ ID NO: 1) was replaced by norleucine (Nle), was synthesized on an automated peptide synthesizer (Prelude®, Protein Technologies, Inc.) at a 100 pmol scale on Lmoc-Thr(tBu)-2CT-resin (0.667 g; loading 0.15 mmol / g) using Fmoc- chemistry. Coupling was carried out twice for each amino acid [4 equivalents (equiv)] using HCTU (4 equiv) and DIPEA (10 equiv) in 4.5 mL of N,N - dimethylformamide (DMF) with nitrogen purging for 45 min. The resin was washed once with 4 mL of DMF, and unreacted amino groups were capped by incubation with 5% acetic anhydride and 0.5 M DIPEA in 8 mL of DMF with nitrogen purging for 5 min. The resin was washed nine times with 4 mL of DMF.Fmoc groups were removed by incubation twice with 4 mL of 20% (v / v) piperidine in DMF with nitrogen purging for 10 min. The resin was washed nine times with 4 mL of DMF. In all washing steps, the resin was purged with nitrogen for 30 seconds. At the end of the synthesis, the resin was washed three times with 8 mL of methanol manually and air dried for 1 h before peptide cleavage.

[0129] Peptides were cleaved from the resin, and the protecting groups were removed under reducing conditions by incubation in 30 mL of cleavage solution [86% trifluoroacetic acid (TFA), 5% H2O, 5% phenol, and 4% triisopropylsilane (TIS)] with shaking for 4 hours at room temperature (RT). The resin was removed by filtration and filtrate was distributed into 6 falcons (5 mL each). Peptide was precipitated with cold diethyl ether (45 mL), incubated for overnight at -20°C, and pelleted by centrifugation at 4000 g for 20 min.

[0130] The core fragment peptide was purified with a reverse phase high-performance liquid chromatography (HPLC) system (Prep 1525 HPLC, Waters) using a preparative C8reversed-phase column (10-15 pm, 250x22 mm; Vydac 208TP, GRACE), applying a flow rate of 15 mL / min and a linear gradient of 25 to 40% (v / v) solvent B for 30 min [solvent A: H2O with 0.1% (v / v) TFA; solvent B: 90% (v / v) acetonitrile (ACN) / H2O with 0.1% (v / v) TFA], Fractions containing the desired peptide were lyophilized.

[0131] Methods - Synthesis of !P2-CCL2(Met64Nle QB-004) N-terminal fragment (1-10)

[0132] The N-terminal fragment of 1P2-CCL2 (Met64Nle OB-004) was synthesized on an Intavis MultiPep RSi parallel peptide synthesizer by standard Fmoc chemistry on Fmoc- Cys(Trt)-SEA-2CP resin [loading 0.136 mmol / g; 14.7 mg / well; scale 2 pmol; SEA = bis(2- SulfanylEthyl) Amino], Coupling was carried out twice for each amino acid using HCTU and DIPEA (10 equiv) in 59 pL of N,N - dimethylformamide (DMF) without shaking for 30 min. The unreacted amino groups were capped by incubation with 5% acetic anhydride and 6% 2,6- lutidine in 50 pL of DMF for 10 min. The resin was washed six times with 150 pL of DMF, and Fmoc groups were removed by incubation twice with 50 pL of 20% (v / v) piperidine in DMF for 10 min. The resin was washed ten times with 150 pL of DMF. At the end of the synthesis, the resin was washed ten times with 150 pL of MeOH.

[0133] The peptide was cleaved from the resin, and the protecting groups were removed under reducing conditions by incubation in 0.4 mL of cleavage solution (86% TFA, 5% H2O, 5% phenol, and 4% TIS) with shaking at 400 rpm for 3 hours at RT. The peptide was then precipitated with cold diethyl ether (1 mL), incubated for overnight at -20°C, and pelleted by centrifugation at 1700 g for 30 min. This process was repeated three times to remove cleavage impurities, then the pellet was air-dried for 1 hour at RT. The pellet was then dissolved in 1 mL of 70% ACN / H2O (0.1% TFA) solution with the resin removed by filtration. The collected peptide solution was lyophilized, and the mass of the product was confirmed by MALDI- TOF / TOF using DHB (2,5 -Dihydroxybenzoic acid) matrix with linear positive ionization method.

[0134] Methods - Fragment assembly and purification of final product

[0135] 4.0 mg of C-terminal core fragment [0.47 pmol] was added to 18 equiv (8 pmol) of N-terminal SEA-peptide fragment in 470 pL ligation buffer (Final concentration of the core fragment = ~1 mM) and incubated the reaction at 37 °C with stirring for overnight [Ligationbuffer: 0.2 M sodium phosphate buffer which contains 6 M guanidine hydrochloride (Gn.HCl), 0.1 M TCEP.HC1, 0.1 M MPAA and 50 mM methionine, pH 7.5], Completion of the reaction was monitored by RP-HPLC. After completion of the reaction, the reaction mixture treated with 240 pL of 0.28 M TCEP.HC1 solution (6 M Gn.HCl, pH 5.5) for 30 min at RT and then crude product was purified on preparative HPLC using C8 column [Gradient method: 10-50% solvent B in 80 min & Flow rate: 15 mL / min], Pure fractions were collected and lyophilized. Ligation yield was 1.4 mg.

[0136] For folding, purified linear peptide was dissolved in folding buffer at a concentration of 0.2 mg / mL and left the solution at RT for 2-3 days with stirring (Folding buffer: 2 M Gn.HCl, 0.1 M Tris, 10 mM methionine, 0.5 mM GSH, 0.3 mM oxidized GSH, pH 8.0). The progress of the reaction monitored by RP-HPLC. After completion of the reaction, the reaction mixture acidified with 1 mL of 33% acetic acid solution and then crude product was purified on HPLC using semipreparative C8 column [Gradient method: 10-50% solvent B in 40 min & Flow rate: 4 mL / min], Pure fractions were collected and lyophilized.

[0137] Following final authentication by analytical RP-HPLC (FIG. 1) and mass spectrometry (FIG. 2 wherein MALDI-TOF / TOF m / z calc. avg. mass [M+H]+ 8772.15 and obs. avg. mass [M+H]+ 8771.22), lyophilized 1P2-CCL2 (Met64Nle OB-004) was dissolved in H20 to give a final concentration of 100 pM.

[0138] Results - CCR2 inhibitory activity

[0139] A number of variant CCL2 ligands, including 1P2-CCL2, were synthesized and identified as providing CCR2-inhibitory activity, by the methods described above. The peptides identified are detailed in Table 2 below, indicating the measured IC50 of CCR2 inhibition for each peptide.

[0140] Table 2

[0141] Further to the variant CCL2 ligands described in Table 2, further variants were based on the N-terminal portion 1P2 (SEQ ID NO: 40) or 2P6 (SEQ ID NO: 41) but with substitutions at positions 7-10, and were conjugated to the C-terminal fragment of human CCL2 [11-76 human CCL2], in which the methionine residue at position 64 relative to SEQ ID NO: 1) was replaced by norleucine (Nle). The peptides identified are detailed in Table 3 below, indicating the measured IC50 of CCR2 inhibition for each peptide using the methods described above.

[0142] Table 3

[0143] The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention and method of use to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching. The embodiments described were chosen and described in order to best explain the principles of the invention and its practical application, and to thereby enable others skilled in the art to best utilize the inventionand various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions or substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but is intended to cover the application or implementation without departing from the spirit or scope of the claims of the present invention.

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Claims

CLAIMS:

1. A conjugate comprising a payload conjugated to a variant ligand, wherein the variant ligand is capable of binding to a receptor, and wherein the variant ligand binds with higher affinity to the receptor compared to a naturally occurring ligand of the receptor.

2. A conjugate comprising a payload conjugated to a variant ligand, wherein the variant ligand is capable of binding to a receptor, and wherein the variant ligand provides increased agonism of the receptor compared to a naturally occurring ligand of the receptor.

3. The conjugate of claim 2, wherein the increased agonism comprises increased receptor phosphorylation.

4. The conjugate of claim 2 or 3, wherein the increased agonism comprises increased G protein signaling through the receptor.

5. The conjugate of any one of claims 2-4, wherein the increased agonism comprises increased arrestin recruitment to the receptor.

6. The conjugate of any one of claims 2-5, wherein the increased agonism comprises increased induction of intracellular calcium flux.

7. The conjugate of any one of claims 2-6, wherein the increased agonism comprises increased receptor internalization.

8. The conjugate of any one of claims 1-7, wherein the receptor comprises a polypeptide.

9. The conjugate of any one of claims 1-8, wherein the receptor is a G protein- coupled receptor.

10. The conjugate of any one of claims 1-9, wherein the receptor is a chemokine receptor.

11. The conjugate of any one of claims 1-10, wherein the naturally occurring ligand is a naturally occurring chemokine, and wherein the variant ligand is a variant chemokine.

12. The conjugate of any one of claims 1-11, wherein the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises the sequence QGP[P or L], and wherein the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 234 or 235.

13. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S or M][M or D or S or Q or G],14. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G][M or D or S],15. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S or M][M or D or S or Q or G]XX[Q or G or L or A or T or S]X, wherein X denotes any natural or modified amino acid.

16. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G][M or D or S]XX[Q or G or L]X, wherein X denotes any natural or modified amino acid.

17. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGP[P or L]LM (SEQ ID NO: 150) or QGPPG[D or S] (SEQ ID NO: 151).

18. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGPPLM (SEQ ID NO: 152) or QGPPGD (SEQ ID NO: 153).

19. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or M][M or Q][A or W or G or Q or N]X[Q or G or L][S or V or T or G],20. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or M][M or Q][A or W or G or Q or N][L or T or M or S or G or Q or R or Y] [Q or G or L] [S or V or T or G],21. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGP[P or L]LM[A or W][L or T or M][Q or G][S or V or T or G] (SEQ ID NO: 154).

22. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGPPLM[A or W][L or T or M][Q or G][S or V or T or G] (SEQ ID NO: 155).

23. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S][D or S or G or Q]XX[L or A or T or Q][W or A or V],24. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGP[P or L][L or G or S][D or S or G or Q][T or I or S or W or Q][V or L or A or S or G][L or A or T or Q][W or A or V],25. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGPPG[D or S][T or I]VL[W or A] (SEQ ID NO: 156).

26. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGPPGD[T or I]VL[W or A] (SEQ ID NO: 157).

27. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGPP[G or L][M or Q]XX[Q or S] [S or V] (SEQ ID NO: 158).

28. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGPP[G or L][M or Q] [S or G or W or A or T][L or F or T or S or G or Y][Q or S] [S or V] (SEQ ID NO: 159).

29. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGPPLM[S or G][L or F or T]Q[S or V] (SEQ ID NO: 160).

30. The conjugate of claim 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMALQS (SEQ ID NO: 161), QGPPLMWMQV(SEQ ID NO: 162), QGPPLMWLQV (SEQ ID NO: 163), QGPPLMWTQS (SEQ ID NO: 164), QGPPLMWLQT (SEQ ID NO: 165), QGPPLMWTQV (SEQ ID NO: 166), QGPPLMWMQS (SEQ ID NO: 167), QGPPLMATQS (SEQ ID NO: 168), QGPPLMWLQS (SEQ ID NO: 169), QGPPLMALQV (SEQ ID NO: 170), QGPPLMWLGG (SEQ ID NO: 171), QGPPLMWRGS (SEQ ID NO: 172), QGPLLMWLQV (SEQ ID NO: 173), QGPPLMQTTP (SEQ ID NO: 174), QGPPLSWLQV (SEQ ID NO: 175), QGPPLSWLQS (SEQ ID NO: 176), QGPPGQWSQV (SEQ ID NO: 177), QGPPMMAGLS (SEQ ID NO: 178), QGPPLSWQQS (SEQ ID NO: 179), QGPPGMWSQS (SEQ ID NO: 180), QGPPLQWRQS (SEQ ID NO: 181), QGPPLMGTQS (SEQ ID NO: 182), QGPPLMQLQV (SEQ ID NO: 183), QGPPLSWSQV SEQ ID NO: 184), QGPPMSWSQS (SEQ ID NO: 185), QGPPLMNLQV (SEQ ID NO: 186), QGPPMSAYQV (SEQ ID NO: 187) and QGPPMSAYQV (SEQ ID NO: 187).

31. The conjugate of claim 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMALQS (SEQ ID NO: 161), QGPPLMWMQV (SEQ ID NO: 162), QGPPLMWLQV (SEQ ID NO: 163), QGPPLMWTQS (SEQ ID NO: 164), QGPPLMWLQT (SEQ ID NO: 165), QGPPLMWTQV (SEQ ID NO: 166), QGPPLMWMQS (SEQ ID NO: 167), QGPPLMATQS (SEQ ID NO: 168), QGPPLMWLQS (SEQ ID NO: 169), QGPPLMALQV (SEQ ID NO: 170), QGPPLMWLGG (SEQ ID NO: 171), QGPPLMWRGS (SEQ ID NO: 172), QGPLLMWLQV (SEQ ID NO: 173) and QGPPLMQTTP (SEQ ID NO: 174).

32. The conjugate of claim 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMALQS (SEQ ID NO: 161), QGPPLMWMQV (SEQ ID NO: 162), QGPPLMWLQV (SEQ ID NO: 163), QGPPLMWTQS (SEQ ID NO: 164), QGPPLMWLQT (SEQ ID NO: 165), QGPPLMWTQV (SEQ ID NO: 166), QGPPLMWMQS (SEQ ID NO: 167), QGPPLMATQS (SEQ ID NO: 168), QGPPLMWLQS (SEQ ID NO: 169), QGPPLMALQV (SEQ ID NO: 170), QGPPLMWLGG (SEQ ID NO: 171), QGPPLMWRGS (SEQ ID NO: 172), QGPLLMWLQV (SEQ ID NO: 173) and QGPPLMQTTP (SEQ ID NO: 174).

33. The conjugate of claim 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPGDTVLW (SEQ ID NO: 189), QGPPGDIVLA(SEQ ID NO: 190), QGPPGSYDYS (SEQ ID NO: 191), QGPPGDGGSV (SEQ ID NO: 192), QGPLSGQSTP (SEQ ID NO: 193), QGPPGDWLQV (SEQ ID NO: 194), QGPPLMSLAV (SEQ ID NO: 195), QGPPLMSLTV (SEQ ID NO: 196), QGPLSGWAQV (SEQ ID NO: 197), QGPLSQSSQV (SEQ ID NO: 198), QGPLSSQSQV (SEQ ID NO: 199) and QGPLGQQGQV (SEQ ID NO: 200).

34. The conjugate of claim 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPGDTVLW (SEQ ID NO: 189), QGPPGDIVLA (SEQ ID NO: 190), QGPPGSYDYS (SEQ ID NO: 191), QGPPGDGGSV (SEQ ID NO: 192), QGPLSGQSTP (SEQ ID NO: 193) and QGPPGDWLQV (SEQ ID NO: 194).

35. The conjugate of claim 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMSFQS (SEQ ID NO: 201), QGPPLMSTQS (SEQ ID NO: 202), QGPPLMSLQV (SEQ ID NO: 203), QGPPLMGLQV (SEQ ID NO: 204), QGPLSGWLQV (SEQ ID NO: 205), QGPPLQWFQV (SEQ ID NO: 206), QGPPLQWTQV (SEQ ID NO: 207), QGPPLMALSV (SEQ ID NO: 208), QGPPLMWSQV (SEQ ID NO: 209), QGPPGQWGQV (SEQ ID NO: 210), QGPPGSWSQV (SEQ ID NO: 211), QGPPLMSSQS (SEQ ID NO: 212), QGPPLMGLSV (SEQ ID NO: 213), QGPPLMTLQV (SEQ ID NO: 214) and QGPPGQWYQS (SEQ ID NO: 215).

36. The conjugate of claim 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMSFQS (SEQ ID NO: 201), QGPPLMSTQS (SEQ ID NO: 202), QGPPLMSLQV (SEQ ID NO: 203), QGPPLMGLQV (SEQ ID NO: 204), and QGPLSGWLQV (SEQ ID NO: 205).

37. The conjugate of claim 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMSVLA (SEQ ID NO: 216), QGPPGSWSSV (SEQ ID NO: 217), QGPPLGSMGP (SEQ ID NO: 218), QGPPLQWMQA (SEQ ID NO: 219), QGPPLQWMQV (SEQ ID NO: 220), QGPPLMSTQV (SEQ ID NO: 221), QGPPLMSLSV (SEQ ID NO: 222), QGPPLMSLQS (SEQ ID NO: 223), QGPPLMSLQA (SEQ ID NO: 224), QGPPLMSVQS (SEQ ID NO: 225), QGPPLMSAQS (SEQ ID NO: 226), QGPPLMSGQS (SEQ ID NO: 227) and QGPPLMSGQV (SEQ ID NO: 228).

38. The conjugate of claim 12, wherein the N-terminal portion comprises a sequence selected from the group consisting of QGPPLMSVLA (SEQ ID NO: 216), QGPPGSWSSV (SEQ ID NO: 217) and QGPPLGSMGP (SEQ ID NO: 218).

39. The conjugate of claim 12, wherein the N-terminal portion comprises the sequence QGPPGDIVLA (SEQ ID NO: 190).

40. The conjugate of claim 11 or 12, wherein the variant ligand comprises the sequence of SEQ ID NO: 229.

41. The conjugate of any one of claims 12-40, wherein the N-terminal portion consists of no more than 15 amino acids.

42. The conjugate of any one of claims 12-41 , wherein the N-terminal portion consists of 10 amino acids.

43. The conjugate of any one of claims 1-11, wherein the N-terminal portion comprises the sequence PSC-SSDTTP (SEQ ID NO: 236), wherein PSC is N(alpha)(n- nonanoyl)-des-Ser(l)-[ L-thioprolyl(2), L-cyclohexylglycyl(3)].

44. The conjugate of any one of claims 12-43, wherein the amino acid sequence of the C-terminal portion comprises the amino acid sequence of SEQ ID NO: 234 or 235.

45. The conjugate of any one of claims 12-44, wherein the N-terminal portion is located at the extreme N terminus.

46. The conjugate of any one of claims 12-45, wherein the naturally occurring ligand is CCL5.

47. The conjugate of any one of claims 12-46, wherein the naturally occurring ligand comprises the amino acid sequence of SEQ ID NO: 230 or 233.

48. The conjugate of any one of claims 12-47, wherein the receptor is CCR1, CCR3, CCR4, and / or CCR5.

49. The conjugate of any one of claims 12-48, wherein the variant ligand exhibits an an ICso of less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 90 pM, less than 80 pM, less than 70 pM, less than 60 pM, less than 50 pM, less than 40 pM, or less than 30 pM for inhibition of cell fusion in a CCR5 -tropic cell fusion assay.

50. The conjugate of any one of claims 12-49, wherein the variant ligand induces at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the calcium flux signaling through CCR5 induced by PSC- RANTES.

51. The conjugate of any one of claims 12-50, wherein the variant ligand induces sequestration of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of CCR5 receptors in a CCR5 sequestration assay.

52. The conjugate of any one of claims 1-11, wherein the variant ligand comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises an amino acid sequence with 0, 1, 2, 3, or 4 amino acid substitutions relative to any one of SEQ ID NO: 40-70 and 76-145, and wherein the C-terminal portion comprises an amino acid sequence at least 70% identical to SEQ ID NO: 71, 72, 74 or 75.

53. The conjugate of claim 52, wherein the amino acid substitution is a conservative substitution.

54. The conjugate of claim 52, wherein the N-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 40-70 and 76-145.

55. The conjugate of claim 52, wherein the N-terminal portion comprises the amino acid sequence FTNPTWXXXX (SEQ ID NO: 146), wherein X is any natural or modified amino acid.

56. The conjugate of claim 55, wherein the N-terminal portion comprises the amino acid sequence FTNPTW[A or D or R or S or K or Q] [P or A or T or G or S or Q or R or H or E][V or F or Q or G or S or H or L or Y][T or V or Q or S or A] (SEQ ID NO: 147).

57. The conjugate of claim 55, wherein the N-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 40 and 76-99.

58. The conjugate of claim 52, wherein the N-terminal portion comprises the amino acid sequence FPX1DGWX2X3X4X5 (SEQ ID NO: 148), wherein Xi is Methionine or Norleucine, and X2-X5 is any natural or modified amino acid.

59. The conjugate of claim 58, wherein the N-terminal portion comprises the amino acid sequence FPXiDGW[A or R or G or H or V or Q] [P or S or G or E] [V or R or L or E or T or G or Q][T or V or Q] (SEQ ID NO: 149), wherein Xi is Methionine or Norleucine.

60. The conjugate of claim 58, wherein the N-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 41 and 100-145.

61. The conjugate of any one of claims 52-60, wherein the N-terminal portion consists of 8-11 amino acids.

62. The conjugate of any one of claims 52-61 , wherein the C-terminal portion comprises the amino acid sequence of SEQ ID NO: 71, 72, 74, or 75.

63. The conjugate of any one of claims 52-62, wherein the conjugate inhibits CCR2 with an IC50 of less than 300 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 30 nM, less than 10 nM, or less than 3 nM.

64. The conjugate of any one of claims 52-63, wherein the naturally occurring ligand is CCL2.

65. The conjugate of any one of claims 52-64, wherein the naturally occurring ligand comprises the amino acid sequence of SEQ ID NO: 1 or 73.

66. The conjugate of any one of claims 52-65, wherein the receptor is CCR2 and / orCCR4.

67. The conjugate of any one of claims 1-66, wherein the pay load comprises a polynucleotide, an enzyme, a toxin, a drug, a carrier protein, a polymer, an antibody, and / or a detectable marker.

68. The conjugate of claim 67, wherein the drug is a small molecule drug.

69. The conjugate of claim 68, wherein the small molecule drug is Monomethyl auristatin E (MMAE).

70. The conjugate of any one of claims 1-69, wherein the variant ligand is conjugated to the payload via a linker.

71. The conjugate of claim 70, wherein the linker is a cleavable linker.

72. The conjugate of claim 70 or 71, wherein the linker comprises a peptide linker, a hydrazone linker, or a disulfide linker.

73. The conjugate of claim 70 or 71, wherein the linker comprises VA-PAB.

74. A nucleic acid molecule encoding the conjugate of any one of claims 1-73.

75. A vector comprising the nucleic acid molecule of claim 74.

76. A host cell comprising the nucleic acid molecule of claim 74 or the vector of claim 75.

77. A pharmaceutical composition comprising the conjugate of any one of claims 1 - 73, the nucleic acid molecule of claim 74, or the vector of claim 75, and a pharmaceutically acceptable carrier, excipient, and / or stabilizer.

78. A conjugate as defined in any one of claims 1-73 or the pharmaceutical composition as defined in claim 77 for use in delivering a payload to a cell.

79. The conjugate or the pharmaceutical composition for use of claim 78, wherein the use is for the treatment of cancer in a subject.

80. A conjugate as defined in any one of claims 1 -73 or the pharmaceutical composition as defined in claim 77 for use in killing or modifying a target cell.

81. A method for delivering a payload to a cell, comprising the step of contacting the cell with the conjugate of any one of claims 1-73 or the pharmaceutical composition of claim 77.

82. A method for delivering a pay load to a cell in a subject, comprising the step of administering the conjugate of any one of claims 1-73 or the pharmaceutical composition of claim 77 to the subject.

83. A method for treating cancer in a subject, comprising the step of administering the conjugate of any one of claims 1 -73 or the pharmaceutical composition of claim 77 to the subject.

84. A method for killing or modifying a target cell, comprising the step of contacting the cell with the conjugate of any one of claims 1-73 or the pharmaceutical composition of claim 77.

85. A method for killing or modifying a target cell in a subject, comprising the step of administering the conjugate of any one of claims 1-73 or the pharmaceutical composition of claim 77 to the subject.

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