Methods and compositions

Abstract

The present application relates to chemokine (C-C motif) receptor 8 (CCR8) binding polypeptides and their uses in chimeric antigen receptor (CAR) proteins, in the form of bi- and multi-specific fusion proteins, and / or as ligand-drug conjugates. The application also relates to methods, uses and kits for such CCR8 binding polypeptides.

Description

[0001] METHODS AND COMPOSITIONS

[0002] Field

[0003] The present invention is in the field of immunotherapy.

[0004] Background

[0005] Regulatory T cells (Treg cells) are specialised immunoregulatory cells which accumulate to high frequencies in tumours and powerfully suppress immune responses to cancer7-16. Treg cells are also required for immune homeostasis1-3. There is significant medical interest in selectively targeting the immunosuppressive function of Treg cells within tumours without disrupting their systemic anti-inflammatory functions. It has been shown that high levels of expression of the chemokine receptor CCR8 discriminate Treg cells within tumours from those within systemic lymphoid tissues (Figure la)4-6. It has been shown that CCR8 expression marks highly suppressive Treg cells within tumours but is not required for Treg migration or function within tumours, suggesting that depletion of CCR8+cells rather than blockade of CCR8 function is required for cancer therapy4. It is now also known that PD-1+Treg cells undergo proliferative expansion and activation upon anti-PD-1 checkpoint inhibitor therapy, contributing to immunotherapy resistance and disease 'hyper-progression' after treatment17-19. Treg cells are therefore important therapeutic targets in cancer. However, efforts to deplete Treg cells using antibody approaches have been clinically ineffective in part due to poor antibody penetration and impaired antibody-dependent cellular cytotoxicity in human tumours, necessitating alternate approaches20-26.

[0006] Aside from an impaired ability to deplete Treg cells in tumours, there is a concern that systemic depletion of Treg cells would result in unwanted autoimmune side-effects. Moreover, a majority of molecules expressed on Treg cells are also expressed on highly activated conventional T cells limiting the potential efficacy of depleting therapies targeting such molecules. This limits the number of potential molecules that can be targeted with efficacy in Treg depleting approaches in cancer.

[0007] Brief Summary of the Invention

[0008] Against this background, the present inventors have developed a novel and advantageous approach for targeting CCR8 expressing cells that overcomes many of the problems and difficulties associated with prior art approaches (e.g. the issues facing the monoclonal depleting antibody approach to selectively deplete CCR8+Treg cells). As discussed in detail herein, the present inventors have found that alternative formats of CCR8 binding domains can be leveraged, such as the natural CCR8 ligand,chemokine (C-C motif) ligand 1 (CCL1), as novel targeting moieties for cell therapy-based or biologic-based approaches (Figure Ib-d). As shown herein, engineered chimeric antigen receptor (CAR) T cells using CCL1 as the extracellular, CCR8 engaging domain, can successfully direct cytotoxic activity of CD8+T cells against CCR8+cells. These data show that alternative CCR8 binding domains, such as targeting CCR8 with a CCL1 ligand, lead to increased cytotoxic activity, compared with that of a traditional anti-CCR8 CAR, demonstrating that alternative CCR8 binding domains, such as CCL1, can be used to redirect the cytotoxic function of T cells to kill CCR8-expressing cells, either using CAR T cell therapy, or using biologic approaches such as bispecific T-cell engagers (BiTEs). The invention is also suitable for use in the context of ligand-drug conjugates (LDCs).

[0009] While chimeric antigen receptors employing endogenous protein ligands as binders are known, the present invention is specifically directed to CCR8 to deplete intratumoural FOXP3+Tregs and thereby relieve tumour-microenvironment immunosuppression. In contrast to CARs targeting CCR3 or other receptors, the disclosed CCL1-CCR8 CARs exhibit: (i) significant depletion of CCR8+Tregs isolated from tumours, and (ii) no depletion of CCR8-splenic Tregs, under matched conditions, thereby sparing systemic Treg homeostasis. The invention further demonstrates enhanced cytotoxicity versus an anti-CCR8 scFv CAR, indicating a surprising technical effect of the ligand-based CCR8 binder in this setting. Indeed, the embodiments of the invention are unified by a common technical problem of selectively modulating CCR8+Tregs in the tumour microenvironment.

[0010] The invention provides a CCR8 binding polypeptide comprising or consisting of at least:

[0011] a) a first domain that is a CCR8 binding domain that is a CCL1 protein or derivative thereof; and

[0012] b) a second domain that:

[0013] i) together with the first domain forms a CCR8-binding CAR protein; ii) comprises a transmembrane domain, a hinge domain, and / or at least one intracellular signalling domain;

[0014] iii) is a T-cell binding domain; and / or

[0015] iv) is a drug or toxic agent.

[0016] Preferences for the CCR8 binding domain; features of the CAR protein such as transmembrane domain, hinge domain and intracellular signalling domain; T-cell binding domain; and the drug or toxic agent are as set out elsewhere herein.In a first aspect, the invention provides a CAR protein comprising a CCR8 binding domain that is a CCL1 protein or derivative thereof.

[0017] In a further aspect, the invention provides a CCR8 binding polypeptide comprising a CCR8 binding domain as an extracellular domain of a CAR protein. Such embodiments may be referred to as "a CCR8 binding polypeptide in the form of a CAR protein comprising a CCR8 binding domain as an extracellular domain" or a " CAR protein comprising a CCR8 binding domain as an extracellular domain". In some embodiments, the CAR protein further comprises a transmembrane domain, a hinge domain, and / or at least one intracellular signalling domain. In some embodiments, the CCR8 binding polypeptide comprises or consists of a CCR8 binding domain and at least one intracellular signalling domain where the CCR8 binding domain is not an antibody-based domain. In some embodiments, the CCR8 binding polypeptide comprises or consists of a CCR8 binding domain and at least one intracellular signalling domain, where the CCR8 binding domain is not an antibody-based domain, and also comprises a transmembrane domain and a hinge domain.

[0018] In all embodiments and aspects of the invention described herein, the extracellular domain is not an antibody-based domain with binding specificity for CCR8. By "antibody-based domain", we include the meaning of any domains derived from antibodies that are used in conventional CAR proteins, for example a single chain variable fragment (scFv). Thus, in some embodiments, the extracellular domain is not an scFv with binding specificity for CCR8. Preferences for the CCR8 binding domain, such as preferably being a CCL1 protein, are as set out elsewhere herein. Accordingly, the CCR8 binding domain in all embodiments and aspects of the invention may be referred to as a "non-antibody-based CCR8 binding domain".

[0019] In some embodiments, the CCR8 binding polypeptide can be considered to be a CAR (chimeric antigen receptor) with specificity for CCR8.

[0020] In a further aspect, the invention provides a CCR8 binding polypeptide comprising or consisting of a CCR8 binding domain conjugated to at least one T-cell binding domain. The CCR8 binding polypeptide conjugated to at least one T-cell binding domain may be a fusion protein comprising or consisting of a first protein domain that is a CCR8 binding domain and a second domain that is a T-cell binding domain. In this instance, the two domains can be expressed as a single transcript and translated into a single protein. In other instances, the CCR8 binding domain and the at least one T-cell binding domain are not a fusion protein, and are separate entities that are conjugatedusing various means. For example, in some instances, the CCR.8 binding domain may be a protein such as the CCL1 ligand, and the T-cell binding domain may be a nucleic acid aptamer.

[0021] Preferences for the CCR.8 binding domain, such as preferably being a CCL1 protein, are as set out elsewhere herein.

[0022] In a further aspect, the invention provides a CCR8 binding polypeptide comprising or consisting of a CCR8 binding domain conjugated to a drug or toxic agent. As used herein, this aspect may be referred to as a ligand-drug conjugate (LDC). As above, where the CCR8 binding domain and the drug or toxic agent are both proteinaceous components, they may be expressed as a single protein entity. In other instances, where the drug or toxic agent and / or the CCR8 binding domain are not proteinaceous, the two components may be separate components that are conjugated by various means.

[0023] Preferences for the CCR8 binding domain, such as preferably being a CCL1 protein, are as set out elsewhere herein.

[0024] By " CCR8 binding domain", we include the meaning of a domain that provides the means to specifically bind to CCR8, i.e. has preferential binding to CCR8 over that of non-targets (also referred to as "off-targets"). In some embodiments, the means to specifically bind to CCR8 excludes antibody-based domains (e.g. scFv). In preferred embodiments of any aspect of the invention, the CCR8 binding domain is not an antibody-based domain, for example is not an scFv domain.

[0025] The terms "polypeptide" and "protein" are considered to be interchangeable herein unless otherwise specified, such that reference to a CCR8 binding polypeptide may also be in reference to a CCR8 binding protein. In some embodiments, the CCR8 binding polypeptide or CCR8 binding protein does not comprise an antibody-based domain with binding specificity for CCR8.

[0026] In some preferred embodiments, the CCR8 binding domain is a C-C motif chemokine ligand 1 (CCL1) protein or derivate thereof. CCL1 is a glycoprotein that interacts with CCR8. By "derivative", we include the meaning of a "mutant" or "variant" of CCL1, which may comprise one or more differences in the sequence or structure (e.g. of the amino acid sequence and / or nucleic acid sequence), as compared with a native or wildtype CCL1 structure. For example, when deployed in a therapeutic context in a firstorganism, the CCL1 protein may be from a second organism. In this instance, the CCL1 protein is a "wild-type" protein but is not native to the first organism into which the therapeutic protein is to be administered. Such situations are contemplated by the present invention. Differences in the structure may be substitutions and / or deletions and / or insertions. Preferably, the derivative maintains a similar property as the original sequence from which it derives. In the present case, the derivative must retain the ability to bind to CCR8. By "similar", we include the derivative maintains at least 50% of the property or activity (e.g. cell killing, levels of activation markers, or otherwise described herein and shown in the Examples) in comparison with the corresponding wildtype sequence, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. A comparison can readily be made with a variant and its wildtype sequence to determine whether a particular property of a variant is within X% of that observed with the wildtype sequence.

[0027] The required sequence identity is, in some contexts, situation specific. For example, where the CCR.8 binding domain is for use in a human subject and so must have binding affinity to human CCR.8, then the CCR8 binding domain may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the relevant human CCR8 binding domain, such as human CCL1. In instances where the agents of the invention are for use in a mouse, then the CCR8 binding domain may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the relevant mouse CCR8 binding domain, such as murine CCL1.

[0028] Accordingly, in some instances, the CCR8 binding domain has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a CCR8 binding domain known to bind to the corresponding CCR8 protein.

[0029] The skilled person will appreciate that some sequence divergence is tolerable, provided that the CCR8 binding domain of the protein of the invention is able to bind to the corresponding or required CCR8, for example to bind to the human CCR8.

[0030] For the purpose of this invention, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in a firstsequence for optimal alignment with a second sequence). The nucleotide or amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the nucleotides or amino acids are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence x 100).

[0031] Typically, the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given ("test") sequence is 95% identical to SEQ ID NO: 6, SEQ ID NO: 6 would be the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 6 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 6, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 6. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 6. If the sequence is shorter than SEQ ID NO: 6, the gaps or missing positions should be considered to be nonidentical positions.

[0032] The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For example, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0033] A variant of human CCL1 is described in Denis et al., 2012 (represented as SEQ ID NO: 15), which lacks three of the C-terminal amino acids, yet retains binding to CCR8. Accordingly, a derivative thereof as described herein maintains sufficient binding to CCR8. By "sufficient binding", we include the meaning that the derivative maintains binding specificity for CCR8. In some embodiments, the derivative has higher binding affinity (for example, at least 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 115%, 120%, 125% or 130% binding affinity) in comparison with native CCL1. In some embodiments, the derivative has identical binding affinity incomparison with native CCL1. In some embodiments, the derivative has lower binding affinity (for example, at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75% or 70% binding affinity) in comparison with native CCL1.

[0034] Murine CCL1 is 69 amino acids in length (represented as SEQ ID NO: 6), while human CCL1 is 73 amino acids in length (represented as SEQ ID NO: 8). Accordingly, in some embodiments, the derivative thereof comprises at least 48 of the corresponding amino acids of mCCLl, for example at least 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68 of the corresponding amino acids. Alternatively, or additionally, in some embodiments, the derivative comprises at least 51 of the corresponding amino acids of hCCLl, for example at least 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 of the corresponding amino acids. In some embodiments, the corresponding amino acids are contiguous with the reference sequence (i.e. native mCCLl or native hCCLl), starting from the N-terminus. Alternatively, the derivative comprises at least 70% sequence homology with a native CCL1 sequence (e.g. native mCCLl or native hCCLl), for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology. In some embodiments, the derivative thereof is hCCLl variant 1-70 (SEQ ID NO: 15).

[0035] Other derivatives may include versions in which minimal amino acids are mutated compared with the wild-type sequences. Thus, any of the sequences described herein may have, for example, a CCL1 protein having one or more amino acid substitutions, insertions, and / or deletions relative to the wild-type sequence, provided that CCR8-binding activity is retained. For example, the CCL1 protein may include 1-5 (e.g. 1-2) conservative substitutions (e.g. Lys-»Arg, Ser- Thr) or non-conservative substitutions that enhance stability, reduce proteolysis, modulate receptor affinity, and / or reduce receptor activation (i.e. G protein signalling) whilst maintaining internalisation (which may avoid potential activation of a Treg cell). In some embodiments, the variant comprises up to 1, 2, 3, 4 or 5 amino acid changes, preferably within non-critical regions outside the CCR8-binding interface. In other embodiments, the variant includes engineered cysteine residues to enable site-specific conjugation for ligand-drug conjugates or PEGylation. Preferably, the variant maintains at least 70%, 80%, 90%, or 95% CCR8-binding activity compared to wild-type CCL1, as determined by the assays described herein, e.g. flow cytometry or ligand-binding assays. Such variants may also be assessed for functional activity in CAR T cell killing assays or BiTE-mediated cytotoxicity as described herein.In some embodiments, the derivative thereof has a similar binding affinity as a native CCL1. For example, murine CCL1 has a nanomolar affinity of Kd = 1.2 nM for murine CCR8. In some embodiments, the CCR.8 binding domain and / or the derivative thereof has a nanomolar affinity for CCR.8, for example <10 nM, <9 nM, <8 nM, <7 nM, <6 nM, <5 nM, <4 nM, <3 nM, <2 nM, <1.9 nM, <1.8 nM, <1.7 nM, <1.6 nM, <1.5 nM, <1.4 nM, <1.3 nM, or <1.2 nM.

[0036] For a CAR protein as described herein, a derivative may be assessed with respect to a function of a CAR cell. For example, a co-culture and killing assay with CCR8-expressing cells (as described in the Examples and shown in Figure 3) may be performed to assess whether a derivative of the CCR8 binding domain, for instance a derivative of CCL1, of a CAR protein maintains killing capacity. Therefore, in some embodiments, a CAR cell expressing a CAR protein of the invention comprising a derivative of the CCR8 binding domain, for instance comprising a derivative of CCL1, has at least 70% of the killing capacity compared with a CAR cell expressing a CAR protein comprising a native or wild-type CCL1 as the binding domain, for example at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% killing capacity. Alternatively, or additionally, killing capacity may be compared with a conventional anti-CCR8 scFv-based CAR cell, and preferably maintains the improved activity observed herein. For example, the killing capacity may be at least 10% higher than a conventional CAR cell, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, or 600% higher than a conventional CAR cell.

[0037] Alternatively, or additionally, a derivative of the CCR8 binding domain may be assessed with respect to T cell activation markers, for example CD69 (as described in the Examples and shown in Figure 2). Methods to determine activation markers are known in the art, for example by flow cytometry, immunohistochemistry, confocal microscopy, etc. In some embodiments, a cell (e.g. a T cell) expressing a CAR protein comprising a CCR8 binding domain that is a derivative of a wild-type or native CCR8 binding domain, such as a native or wild-type CCL1 protein, has higher activation levels of CD69 compared with cells (e.g. T cells) that do not express the CAR.

[0038] The above aspects are combinable, such that the invention provides embodiments in which a cell may comprise: (i) a CCR8 binding polypeptide comprising a CCR8 binding domain as an extracellular domain of a CAR protein; (ii) a CCR8 binding polypeptide comprising a CCR8 binding domain conjugated to at least one T-cell binding domain;and / or (iii) a CCR8 binding polypeptide comprising a CCR8 binding domain conjugated to a drug or toxic agent.

[0039] As set out above, the CCR8 binding polypeptide comprising or consisting of a CCR8 binding domain conjugated to at least one T-cell binding domain may be a fusion protein of a CCR8 binding domain, such as CCL1, and a T-cell binding domain.

[0040] The CCR8 binding domain may be any suitable CCR8 binding domain. However, as set out elsewhere herein, there are advantages with using binding domains as described herein rather than antibody-based CCR8 binding domains. Accordingly, in some embodiments, the CCR8 binding domain is a protein-based binding domain, or is a nucleic acid based binding domain, for example is an aptamer. Where the CCR8 binding domain is a protein-based binding domain, it may be expressed as a fusion protein with the rest of the CCR8 binding protein or polypeptide of the invention, or may be expressed separately from the remainder of the CCR8 protein or polypeptide of the invention and conjugated to the remainder of the CCR8 protein or polypeptide of the invention.

[0041] The protein based CCR8 binding domain may be (or is derived from) a mammalian CCR8 binding domain. For example, the CCR8 binding domain may be selected from a human, a non-human primate, a murine, or a canine CCR8 binding domain. The CCR8 binding domain may depend on the intended recipient. For example, in the case of a human subject, it may be preferable to use a human CCR8 binding domain. However, it may also be possible to use a CCR8 binding domain derived from a different species to the intended recipient (such as using a murine CCR8 binding domain in a human subject). In some embodiments, the CCR8 binding domain corresponds to the natural (also referred to as wildtype) sequence for a particular CCR8 binding domain. Human, non-human primate, murine and canine sequences for CCR8 binding domain are known in the art, and so can be readily prepared based on those known sequences. The same applies the other components of CCR8 binding polypeptides (e.g. other domains within a CAR protein) as described herein.

[0042] As set out elsewhere herein, preferably, in all embodiments, the CCR8 binding domain is the CCL1 protein or derivative or variant thereof. The sequence of the human CCL1 protein is set out in SEQ ID NO: 7 (which includes a signal sequence); the sequence of human CCL1 excluding the signal sequence, which may be removed for use in the present invention, is set out in SEQ ID NO: 8; the sequence of a variant of the human CCL1 protein is set out in SEQ ID NO: 15; and the sequence of the murine CCL1 is setout in SEQ ID NO: 5; and the sequence of the murine CCL1 excluding the signal peptide is set out in SEQ ID NO: 6.

[0043] The CCR.8 binding domain may have a sequence that has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to any of SEQ ID NO: 7, 8, 15, 5, or 6.

[0044] Any of these sequences and variants or derivatives thereof are considered to be useful in the present invention as an appropriate CCR.8 binding domain. The skilled person will appreciate that in some instances, for example where the CCL1 protein is being used in the context of a CAR, the signal peptide present in the wild-type protein is preferably deleted.

[0045] In some embodiments, the CCR8 binding domain comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 15, and 6. Preferably, the sequence selected matches the intended recipient. I.e. for a human subject, it is preferable to select SEQ ID NOs: 8 and / or 15.

[0046] In some embodiments, the CCR8 binding domain is selected from the group consisting of CCL1, CCL16, CCL18, derivates thereof (i.e. CCL1 derivatives, CCL16 derivatives, and / or CCL18 derivatives), and combinations thereof. In preferable embodiments, the CCR8 binding domain is CCL1.

[0047] In some embodiments, the CCR8 binding domain is a mammalian chemokine ligand (e.g. CCL1, CCL16 and / or CCL18), for example a human, non-human primate, murine, or canine chemokine ligand. Preferably, the chemokine ligand is selected to match the intended recipient (i.e. a subject-matched CCL). For example, in human therapy, it is preferable to use a human CCL.

[0048] In other embodiments, the protein based CCR8 binding domain is a CCR8-binding domain obtained from a virus, for example is a MC148 protein, vMIP-II protein, or a vMIP-I protein (e.g. SEQ ID NOs: 9, 16 and 18, with a signal sequence respectively; or SEQ ID NOs: 10, 17 and 19, without a signal sequence respectively).

[0049] The CCR8 binding domain may have a sequence that has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to any of SEQ ID NO: 10, 17 or 19.In a further aspect, the invention provides a CAR protein comprising a CCR8 binding domain as an extracellular domain. In some embodiments, the extracellular domain binds to a human CCR8. In some embodiments, the extracellular domain binds to a non-human primate CCR8. In some embodiments, the extracellular domain binds to a murine CCR8. In some embodiments, the extracellular domain binds to a canine CCR8.

[0050] In some embodiments, a CAR protein or a cell comprising a CAR protein of the invention further comprises one or more further extracellular domains (for example, as a further CAR protein co-expressed on the cell surface, such as a dual CAR expressing two CAR proteins; or as a tandem CAR whereby a single CAR molecule has multiple binding domains, such as multiple binding domains capable of binding to multiple targets). Alternatively, or additionally, a cell of the invention may be used in combination with one or more further cells that comprise one or more distinct further extracellular domains. The further extracellular domain may have specificity to an antigen, optionally a tumour antigen. In some embodiments, a tandem CAR comprises a CCR8-binding domain (e.g. CCL1) and a second binding domain specific for a tumour antigen (e.g. GPC3), yielding cytotoxic activity against either CCR8+cells or tumour antigen* cells in a ratio-dependent manner. It will be understood that the tumour antigen is adaptable based on the cancer in question.

[0051] By "extracellular domain", we include the meaning of an "extracellular ligand binding domain" and "targeting domain". In some embodiments comprising multiple extracellular domains, the further extracellular domain(s) is / are in the form of an antibody or antigen-binding fragment thereof (which may also be referred to herein as an antibody domain or antigen-binding domain thereof). The antigen-binding fragment may comprise an immunoglobulin domain, a single chain variable fragment (scFv), a variable domain derived from an antibody (e.g. heavy chain variable domain (VH) or a nanobody (VHH)), a ligand or receptor (also referred to as a natural ligand or natural receptor), a DARPin, D-Domain, or a Bcl2 domain. ScFvs comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) of an immunoglobulin, connected by a short linker peptide. In some embodiments, the further extracellular domain is a ligand that binds to a cell surface marker on a target cell (e.g. a tumour cell).

[0052] By "specificity", we include "binding specificity", which indicates the propensity for the extracellular domain to preferentially attach to its target over that of non-intendedtargets. The CCR8 extracellular binding domain of the proteins or polypeptide of the invention, or one or more further extracellular domains is preferably able to bind to the target antigen (for example, CCR.8) in vivo, i.e. under the physiological conditions in which target antigen exists within body of a subject (e.g. a human subject). The extracellular domain, in some cases, does not bind to any other protein in vivo. Alternatively, or additionally, specificity may mean that the extracellular domain is capable of binding to the target antigen ex vivo or in vitro. Such binding specificity may be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blots and flow cytometry, and using cells expressing the target antigen (such as cells transduced to express the target antigen). The extracellular domain may be capable of binding selectively to the target protein, i.e. it binds at least 10-fold more strongly to the target protein than to any other proteins. In some embodiments, the extracellular domain is cross-reactive to human, murine, canine and / or cynomolgus monkey target antigen.

[0053] By "tumour antigen", we include "tumour-associated antigen" (TAA). A tumour antigen or TAA may be selected from, for example, CD19, IL13Ro2, CD20, CD22, BCMA, GPRC5D, CD38, CD138, CD30, CD34, CD40, GD2, B7-H3, NKGD2, Mesothelin, Mucl, SLAMF7 (CS1), CD5, CD7, FLT3, CD45, CD70, CD123, ErbB2 (HER2 / neu), epithelial cell adhesion molecule (EpCAM), Epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), disialoganglioside GD2, disialoganglioside GD3, RORI, CD33 / IL3Ra, C-Met, PSMA, Glycolipid, F77, NY-ESO-1 TOR, melanoma-associated antigen (MAGE), A3 TOR, melanoma-associated antigen (MAGE) Al TOR, alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial tumour antigen (ETA), tyrosinase, CA15-3, CA27-29, CA19-9, calcitonin, calretinin CD99MIC2, CD7, chromogranin, cytokeratin, desmin, CD31, FL1, glial fibrillary acidic protein, gross cystic disease fluid protein, HMB-45, human chorionic gonadotropin inhibin, MART-1, Myo DI, neuron-specific enolase, placental alkaline phosphatase, prostate specific antigens, PSCA, PTPRC, S100 protein, synaptophysin, thyroglobulin, thyroid transcription factor 1, tumour M2-PK, vimentin, human telomerase reverse transcriptase (hTERT), surviving, mouse double minute 2 homolog (MDM2), kappa-light chain, LeY, LI cell adhesion molecule, oncofoetal antigen (h5T4), TAG-72, VEGF-R2, GPC3, CLDN6, CLDN18.2, and combinations thereof.

[0054] In some embodiments, the CAR protein further comprises a cytoplasmic domain. The cytoplasmic domain may comprise at least one (preferably, at least two) intracellular signalling domain, such as a signalling domain of CD3 (zeta), Fc receptor gamma, Fc receptor beta, CD3γ (gamma), CD3δ (delta), CD3e (epsilon), CD5, CD22, CD28,CD66d, CD79a, CD79b, and / or 41BB (CD137). In some embodiments, the intracellular signalling domain comprises immunoreceptor tyrosine-based activation motifs domains (ITAM domains), which are considered to be stimulatory. In other embodiments, the intracellular signalling domain comprises immunoreceptor tyrosine-based inhibitory motif domains (ITIM domains) which are considered to be inhibitory.

[0055] In some embodiments, the CAR protein further comprises one or more costimulatory domain. The costimulatory domain may comprise one or more functional signalling domain (also known as an activation domain) obtained from a protein selected from the group consisting of CD28, 41BB (CD137), 0X40 (CD134), ICOS, CD27, LIGHT (TNFSF14), HVEM, DAP10, DAP12, CD3, and CD3e. In some embodiments, the cytoplasmic domain comprises at least a CD28 domain and a CD3 domain. In some embodiments, the cytoplasmic domain comprises at least a 41BB domain and a CD3 domain.

[0056] Different generations of CARs are known in the art. For example, the CAR may be a first generation CAR comprising a CD3 functional domain; a second generation CAR comprising a CD3 functional domain and a CD28 functional domain (also referred to as a CD28z domain), or a 41BB functional domain and a CD3 functional domain (also referred to as a 41BBz domain); and a third generation CAR comprises a CD3 functional domain, a CD28 functional domain, and a 41BB functional domain. A key feature of the present invention is the inclusion of the CCR8 binding domains as set out herein. It is considered that these CCR8 binding domains, such as CCL1, are appropriate for use with any generation of CAR, and any intracellular signalling domain or combinations thereof.

[0057] More information on the various domains of CAR proteins that may be compatible with the CCR8 binding domains of the invention can be seen in Jayaraman et al., 2020.

[0058] The intracellular signalling domain of a CAR typically activates at least one of effector function or response in the cell, which may lead to cell activation and cytokine secretion.

[0059] In some embodiments, the CAR protein further comprises one or more hinge domain. The hinge domain may refer to an extracellular portion of the CAR spanning the extracellular domain and the transmembrane domain. The hinge may be derived from a protein selected from the group consisting of CD28, CD8o, CD4, CD3, and / or Ig.CAR proteins further comprise a transmembrane domain (TMD). A TMD may be selected from the group consisting of CD28, CD8o, CD4, the alpha, beta or zeta chain of the T-cell receptor, CD3e, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIR2DS2, and combinations thereof.

[0060] In a further aspect, the invention provides a use of a CCR8 binding polypeptide of the invention to produce a CAR protein, wherein the CCR8 binding polypeptide is as defined herein. Other components of a CAR protein are known in the art, as described herein. Therefore, the use may be to combine a CCR8 binding polypeptide of the invention with the other necessary components to create a CAR protein. This means that known CAR proteins can be adapted to contain the beneficial properties described herein (e.g. with respect to enhanced killing) by swapping the original extracellular / binding domain for one of the CCR8 binding polypeptides of the invention.

[0061] In a further aspect, the invention provides a fusion protein comprising at least two binding domains, wherein the first binding domain is a CCR8 binding domain, such as CCL1, and the second binding domain is a T-cell binding domain. In some embodiments, the T-cell binding domain is selected from the group consisting of CD3, CD28, 41BB, and combinations thereof.

[0062] Conventional Bispecific T-cell Engagers (BiTEs) consist of two scFvs directed to distinct targets. The fusion protein may therefore be in the form of a BiTE. Although conventional BiTEs consisting of two scFvs, the term " BiTE" is used herein to include embodiments in which the CCR8 binding domain is not an scFv. For example, the first binding domain (i.e. CCR8 binding domain) may be a CCL1 or derivate thereof, an aptamer, or a CCR8-binding viral domain.

[0063] Preferably, the CCR8 binding domain is not an antibody based domain such as an scFv. Accordingly, in some embodiments, the fusion protein of the invention comprises a CCR8 binding domain that is not an antibody based domain or an scFv. Preferably the CCR8 binding domain of the fusion protein is a CCL1 protein.

[0064] In some embodiments, the fusion protein is tri-specific, comprising three binding domains: a first binding domain that is a CCR8 binding domain, and two further binding domains. The further binding domains in this embodiment may be additional CCR8 binding domains and / or T-cell binding domains as described herein. For example, the tri-specific fusion protein may comprise a CCR8 binding domain, a CD3 binding domain, and a CD28 binding domain. In cases where the fusion protein includes multiplebinding domains, it will be understood that the domains can be referred to as a first domain, second domain, third domain, etc, in any order.

[0065] In a further aspect, the invention provides a ligand-drug conjugate (LDC), wherein the ligand is a CCR.8 binding domain, for example CCL1. In some embodiments, the drug in the conjugate is selected from the group consisting of maytansinoids, auristatins, calicheamicins, duocarmycins, pyrrolobenzodiazepines (PBDs), amatoxins, taxanes, and combinations thereof. By "drug", we include the meaning of a "toxic agent" or "cytotoxic payload" or "immunomodulatory agent". In some embodiments, the drug is capable of inducing death of a target cell, for example by inducing apoptosis or necrosis of a Treg (such as a CCR8+Treg). In some embodiments, the drug disturbs the function of Tregs, for example by using a p60 peptide to inhibit FOXP3, which can result in the conversion of Tregs into anti-tumour effector cells. By "disturbs", we include the meaning that the function of the Treg (e.g. its immunosuppressive function) is reduced compared with a Treg that has not been exposed to the drug, or that the function ceases entirely. The drug may utilise streptavidin-biotin interactions to combine with the CCR.8 binding domain. For example, streptavidin-saporin may be used to form an LDC, wherein the CCR8 binding domain comprises biotin.

[0066] Preferences for the CCR8 binding domain are as set out elsewhere herein, and preferably does not comprise or consist of an antibody-based CCR8 binding domain such as an scFv, and preferably is CCL1 or a variant or derivative thereof.

[0067] In some embodiments, the LDC further comprises a linker. For example, the linker may be between the CCR8 binding domain (ligand) and the drug. The linker provides the means to connect the ligand and the drug to each other, for example via a covalent bond. The linker may be a functionalised amino acid that is part of the ligand and / or the drug. The linker may be a string of one or more amino acids that facilitates a connection between the ligand and the drug. The linker may be a flexible linker as is known to the skilled person.

[0068] In some embodiments, the linker is cleavable (i.e. a cleavable linker), which liberates the drug from the ligand under certain conditions (e.g. pH-dependency and / or exposure to an enzyme). Linkers may provide stability in the blood of a subject, and allow release of the drug after cellular internalisation and trafficking to endosomes and / or lysosomes. In some embodiments, the drug is a peptide- or protein-based drug, which advantageously allows for the LDC to be produced by a T cell (e.g. a CAR T cell) in vivo.In some embodiments, the drug is not a peptide- or protein-based drug (i.e. the drug may be a small molecule or radioisotope). An advantage of an LDC that is based on native CCL1 in combination with a non-protein-based drug is that the LDC has no foreign protein sequences, which may otherwise be susceptible to neutralising antibodies generated within a subject following treatment.

[0069] In some embodiments, the drug is a peptide or protein-based drug.

[0070] In some embodiments, the CCR.8 binding domain is modified, for example by PEGylation to enhance pharmacokinetic and pharmacodynamic properties. PEGylation may involve covalent attachment of polyethylene glycol (PEG) chains of varying molecular weights (e.g. 2kDa to 40kDa) to the ligand and / or linker region. The PEG moiety may be linear or branched and may be introduced via N-terminal amine, lysine side chains, or engineered cysteine residues using maleimide, NHS ester, or click chemistry approaches. In some embodiments, PEGylation is employed to: (i) reduce immunogenicity; (ii) increase solubility and stability; (iii) prolong serum half-life; and / or (iv) modulate biodistribution to favour tumour penetration. Cleavable PEG linkers (e.g. pH-sensitive or enzyme-sensitive) may also be used to allow controlled release of the active agent at the tumour site. PEGylation may be combined with other modifications such as glycosylation or Fc-fusion for additional stability and therapeutic benefit.

[0071] In some embodiments, the CCR.8 binding domain is engineered to include an Fc region with mutations that modulate effector functions. For example, an Fc domain comprising L234A and L235A substitutions (LALA mutant) can be employed to substantially reduce or abolish Fey receptor binding and complement activation, thereby minimising antibody-dependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC). This modification is advantageous where selective depletion of CCR8+cells is desired without triggering systemic immune activation. Additional Fc variants such as LS (M428L / N434S) for extended half-life, YTE (M252Y / S254T / T256E) for enhanced neonatal Fc receptor (FcRn) binding, or effector-silenced variants (e.g. aglycosylated Fc) may also be used to tailor pharmacokinetics and safety profiles. Fc mutants may be combined with PEGylation or glycoengineering strategies to further optimise therapeutic performance.

[0072] In some embodiments, the CAR T cells of the invention are further engineered to secrete CCR8-targeted biologies, such as ligand-drug conjugates (LDCs) or bispecificT-cell engagers (BiTEs) described herein, into the tumour microenvironment. This may be achieved by incorporating an additional expression cassette within the CAR T cell vector encoding a secreted form of the CCR8 binding domain (e.g. CCL1 or a derivative thereof) operably linked to: (i) a cytotoxic payload and / or an immunomodulatory agent for LDC embodiments; or (ii) a CD3-binding domain for BiTE embodiments. The secretion signal may comprise a leader peptide (e.g. IgK signal sequence) to ensure efficient extracellular release. In some embodiments, secretion is constitutive; in other embodiments, it is inducible, for example under N FAT-responsive promoters activated upon CAR engagement. This dual-function design enables CART cells to mediate direct cytotoxicity and / or immunogenicity via CAR engagement while simultaneously delivering soluble agents that recruit bystander immune cells or exert additional cytotoxic and / or immunogenic effects on CCR8+targets, thereby amplifying therapeutic potency within the tumour microenvironment.

[0073] In a further aspect, the invention provides a nucleic acid sequence encoding a CCR8 binding polypeptide of the invention or a polynucleotide with a nucleic acid sequence encoding a CCR8 binding polypeptide of the invention, a CAR protein of the invention, a fusion protein of the invention, and / or the ligand in an LDC of the invention. The polynucleotide may be an RNA, such as an mRNA, or a DNA. References to a nucleic acid sequence of the invention are intended to refer to a polynucleotide that has the said nucleic acid sequence.

[0074] Preferences for this aspect of this invention are as set out elsewhere herein, for example preferably the CCR8 binding domain is not an antibody based domain, and is preferably CCL1 or a variant or derivative thereof.

[0075] In a further aspect, the invention provides a vector comprising one or more polynucleotides of the invention or the nucleic acid sequence of the invention. In some embodiments, the vector further comprises one or more polynucleotides for encoding one or more further CAR protein domain. In some embodiment, the vector further comprises one or more polynucleotides for encoding a T-cell binding domain. For example, the T-cell binding domain may be selected from the group consisting of CD3, CD28, 41BB, and combinations thereof. In some embodiments, the vector further comprises a nucleic acid (such as a polynucleotide) encoding a linker sequence between the CCR8 binding domain and the T-cell binding domain. The vector may be a DNA vector or an RNA vector. The vector may be a viral vector, such as a gamma-retroviral, lentiviral, adenoviral, an adeno-associated virus (AAV) and a herpes simplex virus (HSV) vector. The vector may be non-viral, for example a transposon based system(such as the PiggyBac or Sleeping Beauty systems). The polynucleotide may also be introduced into the host cell genome through viral or non-viral CRISPR technologies. The vector preferably carries sufficient genetic information to allow expression of a CAR protein, fusion protein, and / or LDC of the invention. Alternatively, the vector may comprise a CCR8 binding domain of the invention, which may be combined with other vectors to prepare a CAR protein, fusion protein, and / or LDC comprising the CCR8 binding domain of the invention.

[0076] Preferences for this aspect of this invention are as set out elsewhere herein, for example preferably the CCR8 binding domain is not an antibody based domain, and is preferably CCL1 or a variant or derivative thereof.

[0077] In a further aspect, the invention provides a cell comprising at least one CCR8 binding polypeptide of the invention, at least one CAR protein of the invention, at least one fusion protein of the invention, and / or at least one LDC of the invention. Alternatively, or additionally, the cell may comprise at least one polynucleotide and / or at least one vector that encodes the CCR8 polypeptide, CAR protein, fusion protein and / or LDC (or ligand part of the LDC, configured to be attachable to a drug or toxic agent).

[0078] Preferences for this aspect of this invention are as set out elsewhere herein, for example preferably the CCR8 binding domain is not an antibody based domain, and is preferably CCL1 or a variant or derivative thereof.

[0079] In some embodiments, the cell is an effector cell (also referred to herein as an "immune effector cell"). In some embodiments, the cell is a CAR cell. By " CAR cell", we include the meaning that the cell expresses or is capable of expressing one or more CAR proteins on its cell surface. In some embodiments, the cell is selected from the group consisting of a T cell, an NK cell, and a macrophage. In some embodiments, the cell is a T cell selected from the group consisting of a αβ T cell, a γδT cell, and a NK-T cell. The cell may be derived from an induced pluripotent stem cell (iPSC). For example, iPSCs may be cultured into a type of T cell, in which case the cell may be referred to as an iPSC-derived T cell. It will be understood that these terms may be combinable, such that a CAR protein being expressed in a T cell may be referred to as a " CAR T cell" or " CAR-T cell".

[0080] In some embodiments, where the cell is a T cell, the T cell is a CD8+T cell, which may be referred to as a cytotoxic T lymphocyte (CLT). In some embodiments, the T cell is a CD4+T cell, such as a T helper 1 (TH1) cell, TH2 cell, TH17 cell, TH9 cell, T follicularhelper (TFH) cell, or a regulatory T cell (Treg). The T cell may be a naive, effector, memory, effector memory, central memory, or memory stem T cell.

[0081] In some embodiments, where the cell is an NK cell, the NK cell may be derived from an NK cell line (e.g. the NK-92® cell line, marketed as CRL-2407™, which is an IL2 dependent NK cell that is commercially available).

[0082] In some embodiments, where the cell is a macrophage, the macrophage may be characterised as an Ml macrophage (or having an Ml phenotype), which are a pro-inflammatory phenotype that fights infections. Alternatively, the macrophage may be characterised as an M2 macrophages (or having an M2 phenotype), which are an anti-inflammatory / regenerative phenotype that promotes tissue repair.

[0083] The cell may be modified in ways that enhance relevant functions for the intended use. For example, the cell may be genetically modified in a way that enhances T cell function, which may contribute towards anti-cancer activity. The cell may be engineered to not express CCR.8 (e.g. CCR.8 KO, or siRNA). Since some T cell may express CCR8 at very low levels, reducing or removing that expression may avoid selfreactivity.

[0084] The cell may be isolated from a sample selected from the group consisting of peripheral blood, peripheral blood mononuclear cells (PBMCs), cord blood, spleen tissue, bone marrow, lymph nodes tissue, thymus tissue, tissue from the site of infection, ascites, pleural effusion, and / or tumours. For example, a leukapheresis product (where white cells are separated from red cells) may be collected from a large volume of blood where the pool of collected white cells (mostly PBMCs) contain T cells that may be used to produce CAR-T cells, for example. In some embodiments, the sample is taken from the subject to which the cells will be administered. For example, cells (e.g. T cells) may be obtained from a subject's spleen or portion thereof. In some embodiments, the cell is an autologous cell. In some embodiments, the cell is an allogeneic cell. By "autologous", we mean that the cell derives from the same subject to which the cell is intended to be administered. We also include the meaning of "syngeneic" in the case of the cell deriving from an identical twin, for example. By "allogeneic", we mean that the cell derives from a donor that differs from the subject to which the cell is intended to be administered. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In the case of allogeneic cells, it is preferable that the cell is HLA-matched with respect to the donor and recipient.However, it is possible to use a cell that is partially HLA-mismatched with respect to the donor and recipient.

[0085] The terms "subject" and "patient" can be used interchangeably herein.

[0086] In some embodiments, the cell is a mammalian cell, optionally a human cell, nonhuman primate cell, murine cell or canine cell. Preferably, the cell is selected to match the intended recipient, such that a human cell is used for treating a human subject.

[0087] The cell may express more than one CAR protein (e.g. a dual CAR), wherein at least one of the CAR proteins is a CAR protein of the invention. For example, the cell may express 2, 3, 4, 5 or more CAR proteins, where one or more correspond to a CAR protein of the invention. By "express", we include the meaning that the cell is engineered to express the CAR protein(s). One or more of the further CAR proteins may target a tumour antigen. In some embodiments, the cell does not comprise an anti-mesothelin (MSLN) CAR protein (i.e. a CAR protein with binding specificity to MSLN). In some embodiments, the cell only expresses (i.e. the cell is engineered only to express) one or more CAR proteins of the invention, and no other types of CAR protein.

[0088] In a further aspect, the invention provides a method of producing a cell of the invention. In some embodiments, the method includes the step of inducing a cell to express at least one CCR8 binding polypeptide of the invention, at least one CAR protein of the invention, at least one fusion protein of the invention, at least one LDC of the invention, at least one polynucleotide of the invention, and / or at least one vector of the invention. The method may further comprise the step of obtaining a sample of cells from a subject and / or a donor. By "obtaining", we include the meaning of "providing" a sample. The obtention or provision of a sample may or may not include a step of surgery on a subject or donor. For example, the method may provide a sample that has already been obtained previously from a subject or donor, such that the method starts with the sample, and excludes a surgical step. Alternatively, or additionally, the method may obtain a sample directly from a subject and / or donor.

[0089] Preferences for this aspect of this invention are as set out elsewhere herein, for example preferably the CCR8 binding domain is not an antibody based domain, and is preferably CCL1 or a variant or derivative thereof.In a further aspect, the invention provides a method of producing a cell. In some embodiments, the method comprises introducing a CAR protein or a polynucleotide encoding a CAR protein as described herein into a cell; and / or the method comprises introducing a vector as described herein into a cell. By "introducing", we include the meaning that the CAR protein is directly or indirectly (e.g. via a polynucleotide and / or vector) expressed at the cell surface of the cell. In the case of a CAR protein being introduced, it may be introduced via a connectable linker to the surface of the cell, or it may be loaded into the cell in such a way that the cell expresses it at the surface. In the case of a polynucleotide and / or vector, the cell may be transfected with the polynucleotide and / or vector, which translates the CAR protein within the cell before it is trafficked to the cell surface for expression. Methods for introducing foreign proteins or nucleic acids and / or vectors to a cell are known in the art. The invention therefore also provides a cell comprising or capable of encoding a CAR protein comprising a CCR8 binding domain. Accordingly, the cell may be referred to as a transformed cell, a genetically engineered cell, an engineered cell, or a modified cell. Transformation may be transfection or transduction. A cell may also be prepared by genetically engineering it to express a CAR protein, for example by using CRISPR technologies using either viral or non-viral transfer.

[0090] Preferences for this aspect of this invention are as set out elsewhere herein, for example preferably the CCR8 binding domain is not an antibody based domain, and is preferably CCL1 or a variant or derivative thereof.

[0091] Reference to "a cell" is interchangeable herein with reference to "cells" or "a population of cells".

[0092] By "transfection", we include the meaning of non-virus-mediated nucleic acid transfer. The cells may be transfected using any method known in the art. Transfection may be in vitro, ex vivo, and / or in vivo (e.g. using mRNA loaded in a lipid nanoparticle (LNP) that is administered to a subject). Any vector capable of transfecting cells may be used, such as conventional plasmid DNA or RNA transfection, including mRNA transfection. A human artificial chromosome and / or naked RNA may be used to transfect the cell with the polynucleotide or nucleic acid construct. Human artificial chromosomes are described in e.g. Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014). Alternative non-viral delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Methods of non-viral delivery of nucleic acids include lipofection, microinjection, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, artificial virions, and agent-enhanced uptake of DNA. Other techniques, such as electroporation and nanoparticle delivery systems, may also be used.

[0093] By "transduction", we include the meaning of virus mediated nucleic acid transfer. A viral vector may be used to transduce the cell with the one or more constructs. Conventional viral based expression systems could include gamma-retroviral, lentiviral, adenoviral, adeno-associated virus (AAV) and herpes simplex virus (HSV) for gene transfer. Non-viral transduction vectors include transposon-based systems including PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are known in the art. The vector is preferably a vector described herein. Cells may be transduced using any method known in the art. Transduction may be in vitro, ex vivo, and / or in vivo, (e.g. using mRNA loaded in a lipid nanoparticle (LNP) that is administered to a subject).

[0094] Reference to a cell, fusion protein, or LDC may be to a "dosage unit", meaning that it is at a suitable concentration to be administered to a subject in need thereof. A physician can readily determine a suitable dose for an intended recipient. Clinical efficacy for CAR therapy, for example, has been observed at doses between 50-100 million cells for anti-CD19 CAR-T cells, and >100 million cells for anti-BCMA CAR-T cells (see Rotte et al., 2022). Accordingly, in some embodiments, the population of cells or dosage unit comprises at least 1 million cells, 10 million cells, such as 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, 100 million, 110 million, 120 million, 130 million, 140 million, 150 million, 160 million, 170 million, 180 million, 190 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, or 5 billion cells. The dose may vary depending on the age and size of a subject, as well as on the disease, conditions and route of administration. The dosage may be reflected based on kg of a patient.

[0095] It will be understood that multiple doses may be administered in order to reach clinical efficacy. In some embodiments, a subject in need thereof may be administered with at least one dosage unit, for example, 2 dosage unit, 3 dosage unit, 4 dosage unit, 5 dosage unit, 6 dosage unit, 7 dosage unit, 8 dosage unit, 9 dosage unit, 10 or more dosage unit. Thus, the therapy may be administered as a single dose, or in a multiple dose regimen. For example, the initial dose may be followed by administration of a second or plurality of subsequent doses. The second and subsequent doses may be separated by an appropriate time. For example, the doses may be administered onceabout every week, once about every 2 weeks, once about every 3 weeks, once about every four weeks, or once about every month.

[0096] Prior to administration, the subject may undergo lymphodepletion. Lymphodepletion may be achieved via administration to the subject with fludarabine, cyclophosphamide and / or bendamustine. Lymphodepletion may be carried out for at least about one day, such as about 2 days, about 3 days, about 4 days, or about 5 days. Lymphodepletion is typically given 5, 4 and 3 days before CAR-T cell infusion to reduce immunosuppression and provide space for the CAR-T cells to expand in vivo. By giving lymphodepletion a few days before CAR-T cell infusion, the lymphodepleting drugs can be largely cleared out before the CAR-T cells are administered not to negatively affect the infused CAR-T cells. In some embodiments, the subject does not undergo lymphodepletion.

[0097] In a further aspect, the invention provides a pharmaceutical composition comprising at least one cell, at least one fusion protein, and / or at least one LDC of the invention. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The term "pharmaceutical composition", "composition", and "therapeutic composition" may be used interchangeably herein. The cell or population of cells may be at least 1% of the total cells in the composition, such as at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.9%, or 100% of the total cells in the composition.

[0098] Preferences for this aspect of this invention are as set out elsewhere herein, for example preferably the CCR8 binding domain is not an antibody based domain, and is preferably CCL1 or a variant or derivative thereof.

[0099] Suitable pharmaceutically acceptable carriers comprise aqueous carriers, diluents or excipients. Examples of suitable carriers include all aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers and solutes, which render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorption agents and the like. It will be understood that compositions of the invention may also include other supplementary physiologically active agents. In veterinary uses, such carriers may be referred to as "veterinarially acceptable carriers".By "acceptable", we include the meaning that the pharmaceutically acceptable carrier is compatible with the other ingredients in the composition and not harmful to the subject (i.e. does not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate). Compositions include those suitable for parenteral administration, including subcutaneous, intramuscular, intravenous, intradermal, intratumoural, intraventricular, intranasal, and retro-orbital administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods include preparing the carrier for association with, for example, isolated T cells. In general, the compositions are prepared by uniformly and intimately bringing into association any active ingredients with liquid carriers. The composition described herein may be prepared in a manner known in the art and are those suitable for parenteral administration to mammals, particularly humans, comprising a therapeutically effective amount of the composition with one or more pharmaceutically acceptable carriers or diluents.

[0100] The invention also provides associated therapeutic uses and methods of treatment that use the various CCR8 binding proteins, polypeptides, fusion proteins, cells, nucleic acids, polynucleotides and vectors as set out herein.

[0101] In a further aspect, the invention provides a CCR.8 binding polypeptide of the invention, a CAR of the invention, a cell of the invention, a fusion protein of the invention, an LDC of the invention, and / or a pharmaceutical composition of the invention, for use in medicine.

[0102] In a further aspect, the invention provides a CCR8 binding polypeptide of the invention, a CAR of the invention, a cell of the invention, a fusion protein of the invention, an LDC of the invention, and / or a pharmaceutical composition of the invention, for use in prophylaxis or treatment of cancer in a subject in need thereof.

[0103] In a further aspect, the invention provides a CCR8 binding polypeptide of the invention, a CAR of the invention, a cell of the invention, a fusion protein of the invention, an LDC of the invention, and / or a pharmaceutical composition of the invention, for use in the treatment of a disease characterised by the presence of unwanted cells that express CCR8 on the cell surface.In a further aspect, the invention provides use of a CCR8 binding polypeptide of the invention, a CAR of the invention, a cell of the invention, a fusion protein of the invention, an LDC of the invention, and / or a pharmaceutical composition of the invention, for the manufacture of a medicament for prophylaxis or treatment of cancer.

[0104] In a further aspect, the invention provides a method of prophylaxis or treatment of cancer in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a CCR8 binding polypeptide of the invention, a CAR of the invention, a cell of the invention, a fusion protein of the invention, an LDC of the invention, and / or a pharmaceutical composition of the invention.

[0105] By "a subject in need thereof", we include the meaning of a subject who has been diagnosed with cancer and / or as having symptoms thereof. Preferably, the cancer is a solid cancer or a solid tumour. In some embodiments, the subject or patient has been determined to have a solid tumour that comprises CCR8+cells (e.g. CCR8+Tregs), or comprises a significant number of CCR8+cells (e.g. CCR8+Tregs).

[0106] For veterinary use, a compound of the invention is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

[0107] By "treatment" or "treating", we include both therapeutic and prophylactic or preventive treatment of the subject. The terms "preventive" or "prophylactic" are used to encompass the use of the CCR8 binding polypeptide to either prevent or reduce the likelihood of a disease, disorder or condition as described herein. The term "prophylactic" also encompasses preventing recurrence of the disease, disorder or condition in a patient who has previously been treated for any of the diseases, disorders or conditions described herein. Accordingly, as used herein, reference to a method of treatment also includes a method of prevention and / or a method of prophylaxis.

[0108] The use or method may be to alleviate a disease, disorder or condition as described herein (or symptoms associated therewith). By "alleviate" or "alleviation", we mean, without being limited by it, decreased symptoms or processes associated with a disease disorder, or condition, i.e. resulting in a milder disease, disorder or condition.In some embodiments, the use or method is a means for counteracting immunosuppression, for example Treg-mediated immunosuppression in cancer.

[0109] In some embodiments, the use or method comprises the following steps:

[0110] (a) obtaining a cell or population of cells (for example, T cells, NK cells and / or macrophages); and

[0111] (b) introducing a CAR protein or a polynucleotide with a nucleic acid sequence encoding a CAR protein to the cell or population of cells, configured to express the CAR on the cell surface.

[0112] In some embodiments, the use or method further comprises the following steps:

[0113] (c) administering the cell or population of cells to a subject in need thereof.

[0114] In embodiments where autologous cells are used, then step (a) may include the obtention of cells from the intended recipient. It will be understood that the method may or may not include any surgical step on the human or animal body, but merely uses cells that have formerly been obtained from the subject. Thus, "obtaining" or "obtained", as used herein, does not necessarily imply the direct obtention from a subject for the method. By "obtaining", we include the meaning of "providing", in that the cells are provided by an alternative source, and thus act as a starting material for the method.

[0115] Alternatively, the use or method may include a step of obtaining the cell or population of cells from a subject. For example, a cell (e.g. T cell) or population of cells (e.g. T cells) may be obtained from a subject's spleen.

[0116] In some embodiments, the cancer is a solid cancer (also referred to herein as a solid tumour). In some embodiments, the solid tumour has a microenvironment (also referred to as a tumour microenvironment) comprising CCR8+Tregs. The solid tumour may be selected from the group consisting of breast cancer, gastric cancer, lung cancer, prostate cancer, colorectal cancer, pancreatic cancer, kidney cancer, bladder cancer, head and neck cancer, osteosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, glioblastoma, astrocytoma, medulloblastoma, ependymoma, testicular cancer, ovarian cancer, and melanoma.

[0117] In some embodiments, the cancer is a haematological cancer (also referred to herein as a haematological tumour). In some embodiments, the haematological cancer is a T cell lymphoma or a B cell lymphoma. For example, the haematological cancer maybe selected from the group consisting of leukaemia (e.g. Acute Lymphocytic Leukaemia (ALL); B-cell Acute Lymphoblastic Leukaemia (B-ALL); Acute Myeloid Leukaemia (AML); Chronic Lymphocytic Leukaemia (CLL); Chronic Myeloid Leukaemia (CML)); Lymphoma (e.g. Hodgkin Lymphoma; Non-Hodgkin Lymphoma (NHL); Diffuse Large B-Cell Lymphoma (DLBCL); Follicular Lymphoma; Mantle Cell Lymphoma (MCL); Burkitt Lymphoma; Mantel Cell Lymphoma; Marginal Zone Lymphoma); Multiple Myeloma; Myelodysplastic Syndromes (MDS); Myeloproliferative Neoplasms (MPNs); Polycythemia Vera (PV); Essential Thrombocythemia (ET); and Primary Myelofibrosis (PMF). In some embodiments, the cancer is a T cell lymphoma associated with CCR.8 expression. By "associated with CCR.8 expression", we include the meaning that a cell obtained from the leukaemia or lymphoma expresses CCR8 on its cell surface. Such a CCR8+cancerous cell may therefore be targeted by the therapies described herein.

[0118] By "cancer", we include targeting secondary cancer (i.e. a secondary malignancy or secondary tumour).

[0119] In some embodiments, the use or method at least partially depletes CCR8+Tregs in the subject, preferably wherein the depletion occurs in the tumour microenvironment (also referred to as intratumoural CCR8+Tregs). In some embodiments, the use or method depletes CCR8+Tregs in the subject to a non-detectable level, for example in a serum sample and / or tumour biopsy obtained from the subject. By depleting the CCR8+Tregs in the tumour microenvironment, immunosuppression is alleviated, which may therefore increase anti-cancer activity (i.e. of the native immune system and / or any other anti-cancer agents used in combination). In some embodiments, the use of method selectively depletes CCR8+regulatory T cells in tumours, while substantially sparing CCR8“ regulatory T cells derived from systemic lymphoid tissue, thereby reducing tumour-associated immunosuppression without materially perturbing systemic immune homeostasis.

[0120] In some embodiments, the use or method achieves a reduction of intratumoural CCR8+Tregs by at least about 30%, such as > 40%, > 50%, > 60%, or > 70%, and in some embodiments up to about 80%. In some embodiments, the use or method has limited depletion of CCR8-splenic Tregs to < 10%, such as < 8%, < 5%, or < 2%. The level of Tregs may be assessed by flow cytometry or immunohistochemistry. In some embodiments, CAR T cells of the invention exhibit at least a 2-fold increase in targetcell lysis compared with an anti-CCR8 scFv CAR, and in some embodiments > 3-fold, > 4-fold, or > 5-fold, under matched effector-to-target (E: T) ratios ranging from about 1:8 to about 10:1, including intermediate ratios such as 1:4, 1:2, and 2:1.For ligand-drug conjugates (LDCs), some embodiments may achieve > 30% reduction in viability of human CCR8+target cells, such as > 40%, > 50%, or > 60%, and in some embodiments > 70%, together with at least a 2-fold decrease in live-cell counts relative to human-CCR8-negative controls (murine CCR8+) at equimolar doses; in some embodiments, the reduction in live-cell counts may be > 3-fold or > 4-fold.

[0121] In embodiments where the drug of the LDC is an immunomodulatory agent (rather than a cytotoxic payload), therapeutic benefit may be achieved without depleting CCR8+cells. In such cases, CCR8+target cells can remain present and viable (and may continue to express CCR8), but exhibit a modified immunological phenotype (e.g. a phenotype indicative of reduced suppressive function and / or altered effector behaviour). Non-limiting readouts include one or more of: (i) reduced Treg suppressive activity in standard Treg: responder co-culture assays (e.g. >20-80% restoration of responder T-cell proliferation versus baseline); (ii) down-modulation of suppressive markers and cytokines (e.g. decreased FOXP3 mean fluorescence intensity, Helios, CTLA-4, CD39 / CD73, TGF-p, and / or IL-10), such as > 20%, > 30%, > 50% reductions from baseline; (iii) phenotypic skewing toward a less suppressive or effector-like state (e.g. increased T-bet or IFNy in formerly suppressive cells, increased CD80 / CD86 on myeloid targets, or reduced arginase-l / IDO where applicable); (iv) checkpoint rebalancing (e.g. decreased PD-l / PD-Ll / Tim-3 / Lag-3 expression or function); and / or (v) altered chemokine / chemokine-receptor signalling consistent with reduced intratumoural retention or suppressive niche formation. Accordingly, for immunomodulatory LDC embodiments, preferred performance criteria may comprise maintenance of > 70-100% viability of CCR8+targets with significant decreases in suppressive phenotype and / or functional suppression as defined above, optionally in the absence of a > 2-fold drop in live-cell counts. These endpoints may be used alone or in combination with the viability-based criteria set out for cytotoxic LDCs.

[0122] In some embodiments, the use or method further comprises a step of assessing the presence of CCR8+cells (e.g. CCR8+Tregs). For example, an assessment may be made of a sample (e.g. serum and / or tumour biopsy) obtained / provided from a subject, which may indicate that the subject is particularly suitable for the therapy. In one embodiment, the assessment includes the determination of at least one further marker of Tregs, for example CD25 and / or Foxp3. In some embodiments, the assessment is based on mRNA and / or protein expression of the markers. In some embodiments, the assessment is carried out by flow cytometry (FACS), masscytometry (CyTOF), immunohistochemistry (IHC), immunofluorescence (IF), and / or qPCR. Preferably, the assessment is performed on FACS sorted cells.

[0123] The present disclosure also contemplates the combination of one or more cells of the invention, fusion proteins of the invention, LDCs of the invention, and / or pharmaceutical compositions of the invention with other active agents and / or in addition to other treatment regimens or modalities (for example, radiation therapy and / or surgery), i.e. the use or method may comprise administration of at least one further agent. When used in combination with known active agents, the combination may be administered either in sequence (either continuously or broken up by periods of no treatment), concurrently, or as an admixture. I.e. administration may be simultaneously or sequential. The other agent may be selected based on the intended use.

[0124] The further agent may be, for example, a CAR protein or CAR cell directed to a distinct target as the therapy of the invention. I.e. the further CAR protein may be expressed in the same cell as a CAR protein of the invention. In the case of treating cancer, the other agent may be a further anti-cancer agent (including embodiments of the invention). For example, the use or method may include administration in combination with an alkylating agent (such as mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide cisplatin, or platinum-containing alkylating agents such as cisplatin, carboplatin and oxaliplatin), an anti-metabolite (such as a purine or pyrimidine analogue or an anti-folate agent, such as azathioprine and mercaptopurine), an anthracycline (such as daunorubicin, doxorubicin, epirubicin and idarubicin, valrubicin, mitoxantrone or anthracycline analog), a plant alkaloid (such as a vinca alkaloid or a taxane, such as vincristine, vinblastine, vinorelbine, vindesine, paclitaxel or docetaxel), a topoisomerase inhibitor (such as a type I or type II topoisomerase inhibitor), a podophyllotoxin (such as etoposide or teniposide), a tyrosine kinase inhibitor (such as imatinib mesylate, nilotinib or dasatinib), an adenosine receptor inhibitor (such as A2aR inhibitors, SCH58261, CPI-444, SYN115, ZM241385, FSPTP or A2BR inhibitors such as PSB-1115), adenosine receptor agonists (such as CCPA, IB-MECA and CI-IB-MECA), a checkpoint inhibitor, including those of the PDL-1: PD-1 axis, nivolumab, pembrolizumab, atezolizumab, BMS-936559, MEDI4736, MPDL33280A or MSB0010718C), an inhibitor of the CTLA-4 pathway (such as ipilimumab and tremelimumab), an inhibitor of the TIM-3 pathway or an agonist monoclonal antibody that is known to promote T cell function (including anti-OX40, such as MEDI6469; and anti-41BB, such as PF-05082566).The therapeutic methods and uses described herein may comprise inhibiting the disease state (e.g. the cancer), for example by arresting its development and / or causing regression of the disease state until a desired end point is reached. The therapeutic methods and uses of the invention may comprise achieving a partial response or a full response of the cancer. The therapeutic methods and uses of the invention may achieve remission of the cancer. The therapeutic methods and uses described herein may delay the growth of the cancer, arrest the growth of the cancer and / or reverse the growth of the cancer. The therapeutic methods and uses of the invention may reduce the size of the cancer by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by 100%.

[0125] Uses or methods of the invention that use a cell may be referred to as a cell therapy. By "cell therapy" or "cell therapies", we include the meaning of "adoptive cell therapy" (ACT). ACT is a form of immunotherapy that involves modifying and using a patient's own immune cells (autologous uses), or a donor's immune cells (allogeneic uses) to fight, for example, cancer. The key components include immune cells (typically T cells) that may be collected from a patient or donor; modification and / or expansion of the cells (including genetic modification, for example to enhance their ability to recognise and attack cancer cells, and / or to reduce cytokine output); and reinfusion or infusion of the modified cells (also referred to herein as "engineered cells") to the patient to destroy the target cells (e.g. target cancer cells). Accordingly, in some embodiments, the cell or pharmaceutical composition of the invention is for use in adoptive cell therapy.

[0126] In a further aspect, the invention provides a kit (e.g. a kit for preparing a cell, such as a CAR cell, of the invention; and / or for performing a method or use (i.e. therapeutic method or therapeutic use) as described herein). The kit may comprise a CCR8 binding domain, wherein the CCR8 binding domain is in the form of, or configurable for preparing, a CAR protein, a cell comprising a CAR protein, a fusion protein, and / or an LDC of the invention. By "configurable for preparing", we include the meaning that the CCR8 binding domain may be combined with the other components / domains necessary (as described herein) to form the CAR protein, the cell comprising a CAR protein, the fusion protein, and / or the LDC. For example, in the case of a CAR protein, the CCR8 binding domain may be in the form of a polynucleotide having a nucleic acid sequence in a vector or expression cassette, along with encoding sequences for the other domains required of a CAR protein (e.g. TMDs, cytoplasmic domains, costimulatorydomains and / or hinge domains), such that a CAR protein comprising all required domains may be expressed within a cell.

[0127] In some embodiments, the kit comprises at least one CCR8 binding polypeptide of the invention, at least one CAR protein of the invention, at least one cell (e.g. CAR cell) of the invention, at least one fusion protein of the invention, at least one LDC of the invention (or ligand configurable for an LDC of the invention, e.g. provided in combination with the drug or toxic agent), at least one polynucleotide of the invention, and / or at least one vector of the invention.

[0128] In some embodiments, the kit comprises a CCR8 binding domain as described herein, optionally in the form of a series of amino acid sequences to other components of a CAR, and / or as a polynucleotide (such as a vector) that encodes a CCR8 binding polypeptide as described herein. In some embodiments, the kit comprises a cell or pharmaceutical composition of the invention, i.e. a cell that can express a CAR protein comprising a CCR8 binding domain. For example, the kit may comprise an aliquot of cells as a dosage unit for administration to a subject in need thereof, optionally wherein the kit further comprises media and other suitable agents for reconstituting the cells.

[0129] The kit may further comprise: a container holding aspects of the invention; and a label or package insert with instructions for use. The kit may further comprise reagents for reconstituting an amino acid sequence or a polypeptide such as a protein or peptide, or polynucleotide or nucleic acid sequence (e.g. if lyophilised), a buffer, and / or other components of a CAR protein, fusion protein, and / or LDC of the invention. The buffer may be in a container (e.g. a separate container to the protein, peptide, nucleic acid, composition or cell). The buffer may be a pharmaceutically acceptable buffer, such as bacteriostatic water for injection, phosphate-buffered saline, Ringer's solution and dextrose solution. The kit may further comprise other materials desirable from a commercial and user standpoint, which would be known to persons skilled in the art, suitable examples of which include other buffers, diluents, filters, needles, and syringes.

[0130] The invention also provides a kit comprising the means to determine if a sample obtained from a tumour from a subject comprises CCR8+cells (e.g. CCR8+Tregs) or comprises a significant number of CCR8+cells (e.g. CCR8+Tregs), and further comprises any one or more of the cells of the invention, fusion proteins of the invention, LDCs of the invention, and / or pharmaceutical compositions of the invention.In another aspect, the invention provides a method, a use, or a kit, substantially as described herein with reference to the accompanying claims, description, examples and / or figures.

[0131] It is to be understood that different applications of the disclosed CARs, fusion proteins, LDCs, cells, and / or pharmaceutical compositions of the invention may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0132] In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "a CCR8 binding polypeptide" includes two or more " CCR8 binding polypeptides".

[0133] Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention.

[0134] The invention also provides the following numbered embodiment paragraphs:

[0135] 1. A Chimeric Antigen Receptor (CAR) protein comprising a chemokine (C-C motif) receptor 8 (CCR8) binding domain that is a chemokine (C-C motif) ligand 1 (CCL1) protein or derivative thereof.

[0136] 2. A CCR8 binding polypeptide comprising a CCR8 binding domain as an extracellular domain of a CAR protein; wherein the CAR protein further comprises a transmembrane domain, a hinge domain, and / or at least one intracellular signalling domain.

[0137] 3. A CCR8 binding polypeptide comprising a CCR8 binding domain conjugated to at least one T-cell binding domain.

[0138] 4. A CCR8 binding polypeptide comprising a CCR8 binding domain conjugated to a drug or toxic agent.

[0139] 5. The CCR8 binding polypeptide according to any preceding embodiment, wherein the CCR8 binding domain is selected from the group consisting of a proteinbinding domain, an aptamer, a CCR8-binding viral domain, and combinations thereof.

[0140] The CCR8 binding polypeptide according to any preceding embodiment, wherein the CCR8 binding polypeptide:

[0141] a. comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 15 and 6;

[0142] b. is selected from the group consisting of chemokine (C-C motif) ligand 1 (CCL1), CCL16, CCL18, and combinations thereof; or

[0143] c. is a mammalian chemokine ligand (e.g. CCL1, CCL16 and / or CCL18), for example a human, non-human primate, murine, or canine chemokine ligand.

[0144] The CCR8 binding polypeptide according to any preceding embodiment, wherein the CCR8 binding polypeptide is not an antibody-based domain.

[0145] A CAR protein comprising a CCR8 binding domain, wherein the CAR protein further comprises:

[0146] a. a transmembrane domain (TMD), optionally wherein the TMD is selected from the group consisting of CD28; CD8o; CD4; the alpha, beta or zeta chain of the T-cell receptor; CD3 epsilon; CD5; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; and KIR2DS2;

[0147] b. a cytoplasmic domain, optionally comprising at least one intracellular signalling domain, such as a signalling domain of CD3 zeta, Fc receptor gamma, Fc receptor beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD28, CD66d, CD79a, CD79b, and / or 41BB (CD137);

[0148] c. one or more costimulatory domains, optionally comprising one or more functional signalling domains obtained from a protein selected from the group consisting of CD28, 41BB (CD137), 0X40 (CD134), ICOS, CD27, LIGHT (TNFSF14), HVEM, DAP10, DAP12, CD3 (zeta), CD3E (epsilon), and CD27; and / or

[0149] d. one or more hinge domains, optionally a hinge derived from a protein selected from the group consisting of CD28, CD8o, CD4, CD3 zeta, and / or Ig.

[0150] The CAR protein of embodiment 8, wherein the CCR8 binding domain:a. comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 15 and 6;

[0151] b. is selected from the group consisting of chemokine (C-C motif) ligand 1 (CCL1), CCL16, CCL18, and combinations thereof; or

[0152] c. is a mammalian chemokine ligand (e.g. CCL1, CCL16 and / or CCL18), for example a human, non-human primate, murine, or canine chemokine ligand.

[0153] A fusion protein comprising at least two binding domains, wherein a first binding domain is a CCR8 binding domain, and a second binding domain is a T-cell binding domain, optionally wherein the T-cell binding domain is selected from the group consisting of CD3, CD28, 41BB, and combinations thereof.

[0154] A ligand-drug conjugate (LDC), wherein the ligand is a CCR8 binding polypeptide, optionally wherein the LDC further comprises a linker.

[0155] A polynucleotide that has a nucleic acid sequence encoding the CCR8 binding polypeptide according to any of embodiments 1-7, the CAR protein according to embodiment 8 or 9, the fusion protein according to embodiment 10, and / or the ligand in the LDC according to embodiment 11.

[0156] A vector comprising the polynucleotide of embodiment 12; optionally further comprising:

[0157] a. one or more polynucleotides encoding one or more further CAR protein domain; and / or

[0158] b. one or more polynucleotides for encoding a T-cell binding domain, optionally wherein the T-cell binding domain is selected from the group consisting of CD3, CD28, 41BB, and combinations thereof; optionally further comprising polynucleotide encoding a linker sequence between the CCR8 binding and the T-cell binding domain.

[0159] A cell comprising at least one CCR8 binding polypeptide according to any of embodiments 1-7, at least one CAR protein according to embodiment 8 or 9, at least one fusion protein according to embodiment 10, and / or at least one ligand in the LDC according to embodiment 11; optionally wherein the cell is a T cell, NK cell or macrophage.15. A cell comprising at least one polynucleotide according to embodiment 12, and / or at least one vector according to embodiment 13.

[0160] 16. A method of producing a cell, wherein the method includes the step of inducing a cell to express at least one CCR8 binding polypeptide according to any of embodiments 1-7, at least one CAR protein according to embodiment 8 or 9, at least one fusion protein according to embodiment 10, at least one ligand in the LDC according to embodiment 11, at least one polynucleotide according to embodiment 12, and / or at least one vector according to embodiment 13.

[0161] 17. A cell comprising a CAR protein according to embodiment 8 or 9, a fusion protein according to embodiment 10, and / or an LDC according to embodiment 11, for use in medicine.

[0162] 18. A cell comprising a CAR protein according to embodiment 8 or 9, a fusion protein according to embodiment 10, and / or an LDC according to embodiment 11, for use in prophylaxis or treatment of cancer in a subject in need thereof.

[0163] 19. A method of prophylaxis or treatment of cancer in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a cell comprising a CAR protein according to embodiment 8 or 9, a fusion protein according to embodiment 10, and / or an LDC according to embodiment 11.

[0164] 20. A cell, fusion protein and / or LDC for use according to embodiment 18, or the method of treating cancer of embodiment 19, wherein the cancer is:

[0165] a. a solid tumour; optionally wherein the solid tumour has a microenvironment comprising CCR8+Treg cells; or

[0166] b. a haematological tumour associated with CCR8 expression.

[0167] 21. A cell, fusion protein and / or LDC for use according to embodiment 18 or 20, or the method of treating cancer according to embodiment 19 or 20, wherein the use / method at least partially depletes CCR8+Tregs in the subject; preferably wherein the depletion occurs in the tumour microenvironment.

[0168] 22. A cell, fusion protein and / or LDC for use according to any of embodiment 18, 20 and 21, or the method of treating cancer according to any of embodiment 19-21, wherein the solid tumour has been assessed for the presence of CCR8+Tregs; optionally wherein the assessment is made based on a biopsy of the solid tumour, and / or wherein the assessment includes at least one further marker of Tregs (e.g. CD25 and / or Foxp3).

[0169] 23. A cell, fusion protein and / or LDC for use according to any of embodiment 18 and 20-22, or the method of treating cancer according to any of embodiment 19-22, wherein the use / method comprises administration of at least one further agent.

[0170] 24. A kit comprising:

[0171] a. a CCR8 binding domain, wherein the CCR8 binding domain is in the form of, or configurable for preparing, a CAR protein or a cell comprising a CAR protein according to embodiment 8 or 9, a fusion protein according to embodiment 10, and / or an LDC according to embodiment 11; and / or b. at least one CCR8 binding polypeptide according to any of embodiments 1-7, at least one CAR protein according to embodiment 8 or 9, at least one fusion protein according to embodiment 10, at least one ligand in the LDC according to embodiment 11, at least one polynucleotide according to embodiment 12, and / or at least one vector according to embodiment 13.FIGURES

[0172] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying figures, in which:

[0173] Figure 1: a, Schematic showing the selective expression of CCR8 on Tregs within tumours, b, Schematic showing how a CCL1 ligand-BiTE approach could be used to selectively deplete CCR8+cells, c, Schematic showing how a CCL1 ligand CAR approach could be used to selectively deplete CCR8+cells, d, Schematic showing how a CCL1 ligand-drug conjugate could be used to selectively deplete CCR8+cells.

[0174] Figure 2: a, Representative flow cytometry (left) and replicate measurements (right) of CCR8 antibody staining on Treg within MC38 tumours of WT and Ccr8- / - animals at day 21 following tumour implantation, b, CCR8 marks highly suppressive FOXP3+Treg cells within MC38 colorectal adenocarcinoma tumours. Representative flow cytometry (left) of CTV-labelled naive CD4+Tconv cells incubated with no Treg cells, or at a 4:1 ratio with CCR8-Treg cells or CCR8+Treg cells from Foxp3 EFGP-DTR mice after 4 days incubation, and replicate measurements of Tconv cell division (right), c, Flow cytometry measurements of CCR8 expression on indicated CD4+and CD8+T cell subsets within tumours and spleens of MC38 tumour bearing animals at day 21 following tumour implantation, d, Systemic loss of CCR8 does not affect growth of subcutaneously implanted MC38 or B16-F10 tumours in contrast to total Treg ablation. Volume of heterotopic MC38 colorectal adenocarcinoma tumours at indicated time points following implantation into FoxP3 EFGP-DTR animals which were administered with PBS or DTx on days 7, 9, 11 and 14 (left). Volume of heterotopic MC38 colorectal adenocarcinoma tumours at indicated timepoints following implantation into animals of the indicated genotypes (right).

[0175] Figure 3: CD8+CAR T cells were transduced with anti-CCR8 scFv-based CAR, murine CCL1 ligand-CAR or No CAR (empty vector) control and co-cultured with indicated ratios of murine CCR8-expressing target cells. CD8+T cells transduced with mCCL1 ligand CAR, or mCCR8 CAR show dose dependent activation, as indicated by CD69 expression (top graph). Murine CCL1 ligand CAR exhibits enhanced cytotoxic activity when cultured with CCR8-expressing target cells (bottom graph).

[0176] Figure 4: Sequence alignment of various CCR8 binding polypeptides.Figure 5: Sequence alignment of canine, human and murine CCL1. 18.1% sequence identity across all three sequences; 37.2% sequence identity, and 56.4% sequence similarity between human and murine; 43.4% sequence identity, and 61.8% sequence similarity between human and canine; and 32.9% sequence identity, and 59.2% sequence similarity between murine and canine.

[0177] Figure 6: CD8+CAR T cells were transduced with human CCL1 ligand-CAR, or an empty vector control (i.e. having no CAR) and co-cultured with indicated ratios of human CCR8-expressing target cells (a control for no cells is also included). Human CCL1 ligand CAR exhibits enhanced cytotoxic activity when cultured with CCR8-expressing target cells.

[0178] Figure 7: CCR8-positive and CCR8-negative Treg cells were isolated by fluorescence-activated cell sorting (FACS) from the tumours and spleens of MC38 tumour-bearing mice using the FOXP3-GFP reporter mouse strain. FACS sorted Treg populations were then co-cultured for 48 hours with CAR T cells transduced with either a non-targeting control CAR, an anti-CCR8 scFv-based CAR, or the CCL1 ligand CAR. Following coculture, cells were stained and analysed by flow cytometry. A, CCL1 ligand CART cells significantly depleted CCR8 positive Treg cells isolated from tumours, reducing both total Treg counts and CCR8+Treg counts compared to both the anti CCR8 scFv and the non-targeting CAR control (p<0.01). B, CCR8-negative Treg cells from the spleens of tumour-bearing mice were used as targets. The CCL1 ligand CAR T cells showed no depletion of these Tregs, demonstrating the selectivity of this approach for CCR8-expressing cells.

[0179] Figure 8: A, The tandem CAR construct was designed by inserting CCL1 at the N terminus of the anti-GPC3 scFv, connected via a flexible linker. B-D, Murine CCR8+target cells (300-19) were co-cultured with CAR T cells at various effector-to-target ratios (1:2, 2:1, and 10:1) for 48 hours. Transduced CAR T cells were cultured in triplicates, and after the incubation period, cells were stained for key markers and analysed by flow cytometry. E-F, GPC3+target cells were co-cultured with CAR T cells under identical conditions. Cells were stained for key markers and analysed by flow cytometry.

[0180] Figure 9: Human CCR8-expressing 300-19 cells were treated with human CCL1 alone, toxin alone (streptavidin-saporin conjugate, ATS IT-27), or the complete hCCL1-toxin conjugate (hCCL1-biotin conjugated to streptavidin-saporin). Murine CCR8 expressing300-19 cells were used as a negative control. Cells were co-cultured for 48 hours at 37°C in a 5% CO2 incubator, before analysis by flow cytometry. A-B, hCCR8 and mCCR8 target cell viability after 48 hour co-culture. C, hCCR8+live target cell counts, assessed by flow cytometry.

[0181] Figure 10: Exemplary BiTE configuration.EXAMPLES

[0182] Example 1: CCR8 as a therapeutic target for selective depletion of Tregs cells in cancer

[0183] It has been shown that high levels of expression of the chemokine receptor CCR8 discriminate Treg cells within tumours from those within systemic lymphoid tissues (Figure 2a-b)4-6. CCR8 expression marks highly suppressive Treg cells within tumours (Figure 2c) but is not required for Treg-mediated immunosuppression, since tumours grow with similar kinetics in wildtype (WT) and CCR8-deficient animals (Figure 2d).

[0184] These findings suggest that depletion of CCR8 expressing Treg cells, rather than functional inhibition of CCR8, is the relevant approach to targeting Treg cells in solid cancer.

[0185] Antibodies targeting CCR8 using ADCC / ADP-dependent approaches have mediated robust depletion of tumour-associated Treg cells, and tumour rejection in mouse subcutaneous heterotopic tumour models27-28, models featuring robust ADCC / ADCP29-30. However, heterogeneous and typically poor infiltration of human tumours with NK cells and macrophages, and suppression of ADCC and ADP within human tumours has contributed to clinical failure of prior Treg depletion attempts clinically2-3'29'30, necessitating alternative approaches.

[0186] Therefore, new and improved therapies are needed.

[0187] 1.1 Use of CCL1 as a natural targeting moiety for depletion of CCR8-expressinq Treg cells in tumours

[0188] CCL1 is one of four known ligands of CCR8 which binds with low nanomolar affinity to CCR8 (Kd = 1.2nM)31. A chimeric antigen receptor (CAR) utilising murine CCL1 has been designed and tested as the binding moiety for murine CCR8. This novel strategy allows for therapeutic targeting of CCR8-expressing Treg cells using its natural ligand. This brings benefits including the potential of preferential binding dynamics that could lead to increased efficacy of cellular and antibody therapies.

[0189] Furthermore, a common limitation of current antibody-based therapeutics is the generation of neutralising antibody responses to foreign protein epitopes. By using native human CCL1 as the binder, this novel approach significantly reduces the risk of such immune responses, potentially improving the long-term efficacy and safety of the therapy. Additionally, the smaller size of CCL1 is considered to help to improve cellular expression as well as facilitating the migration of antibodies to target tumour sites.1.2 CD8+T cells expressing a CCL1 ligand CAR show enhanced cytotoxic activity against CCR8-expressing target cells

[0190] CD8+T cells have been successfully engineered to express a chimeric antigen receptor (CAR protein) where CCL1 is fused via a linker to the intracellular signalling domains of CD3 and CD28. T cells were isolated, transduced with a murine CCL1 ligand CAR, a traditional anti-CCR8 scFv-based CAR, or empty vector (denoted as "no CAR"). After 48 hours, a co-culture was set up at 4:1, 1:1 and 1:4 ratios with murine CCR8-expressing target cells. CCL1 ligand CAR T cells efficiently lysed CCR8+cells in vitro.

[0191] Activation of CD8+T cells was observed expressing both the ligand CAR and scFv-based CAR in a manner dependent upon the dose of cocultured CCR8-expressing cells, as indicated by expression of the activation marker CD69 (Figure 3, top). Enhanced ability of CCL1 ligand-CAR to kill CCR8-expressing target cells was observed over that of traditional anti-CCR8 scFv-based CAR transduced T cells or T cells expressing no CAR (Figure 3, bottom).

[0192] Heightened activation and enhanced levels of killing exhibited by the ligand CAR compared with the scFv-based CAR suggests that targeting using the CCL1 moiety enhances therapeutic efficacy over conventional scFv-based targeting approaches.

[0193] Killing efficacy was also confirmed for a human CCL1 ligand CAR in a co-culture setup at 2: 1, 1:2 and 1:8 ratios with human CCR8-expressing target cells (Figure 6). These data demonstrate that the approach of using a natural CCL1 ligand works for multiple species, and therefore can be applied to (for example) a human setting.

[0194] Example 2: Use of CCL1 as a binding moiety for biological therapeutics aimed at depleting CCR8 positive Treg cells

[0195] These data also support broader use of CCL1 ligand as a binding moiety to redirect the cytotoxic function of CD8+T cells to CCR8-expressing cells. For instance, CCL1 ligandbased approach could be used in biological therapeutics.

[0196] 2.1 CCL1 ligand-based bispecific T cell engagers

[0197] Bispecific T cell engagers (BiTEs) are artificial molecules designed to direct T cells against other cells by simultaneously binding CD3 on T cells and a target cell-expressed antigen. The CD19: CD3 BiTE blinatumomab has shown impressive efficacy in CD19+B cell malignancies, inducing complete remissions in relapsed / refractory B-cell acute lymphoblastic leukaemia in a high freguency of individuals32.Ligand BiTEs offer an alternative to antibody-based BiTEs, exploiting the natural receptor binding activity of cognate ligands to lessen the induction of neutralising antibodies directed against the BiTE, which can limit the duration of effective treatment33. In addition, the ligand CCL1 is smaller than an scFv, potentially improving tumour penetration.

[0198] 2.2 CCL1 ligand-drug conjugates (LDCs)

[0199] An additional approach is a ligand-drug conjugate (LDC), involving a fusion of CCL1 to a cytotoxic payload capable of killing Treg cells. The advantage of this approach is its small size, and the option for a complete absence of foreign protein sequences to act as substrate for a neutralising antibody response. Another potential advantage is the ligand driven internalisation of the receptor-LDC complex.

[0200] Conclusion

[0201] In conclusion, the novel CCLl-based targeting approach presents a useful strategy for selectively depleting CCR8+tumour-infiltrating regulatory T cells. These findings demonstrate that CCL1 ligand chimeric antigen receptor (CAR) T-cells exhibit enhanced cytotoxicity and activation compared to traditional anti-CCR8 CARs, indicating a more efficient and selective targeting mechanism. The use of a natural ligand such as CCL1 offers advantages including reduced immune rejection and improved therapeutic efficacy.

[0202] Example 3: Sequences, materials and methods

[0203] 3.1 Comparator anti-hCCR8

[0204] Anti-hCCR8 (SEQ ID NO: 1):

[0205] DIVMTQTPLSLSVTPGQPASISCRSSQSLFHSSGNTYLHWYLQKPGQPPQLLIYKVSNRFSGVPDR FSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPFTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLV QSGAEVKKPGASVKVSCKASGYTFTDSEMHWVRQATGQGLEWMGAIQPETGGTAYNQKFKARVTMT RDTSISTAYMELSSLRSEDTAVYYCARRRRNFDYWGQGTLVTVSS

[0206] 3.2 Comparator anti-mCCR8

[0207] VL (SEQ ID NO: 2):

[0208] QSVLTQPPSASGTPGQRVTISCSGSSFNIGSHFVYWYQQLPGTAPKLLIYKNNQRPSGVPDRFSGS KSGTSASLAISGLRSEDEADYYCAAWDDSLNGPVFGGGTKLTVL

[0209] Linker (SEQ ID NO: 3):

[0210] GGGGSGGGGSGGGGS VH (SEQ ID NO: 4):EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGVHWVRQAPGKGLEWVSGVSWNGSRTHYADSVKG RFT I S RDN S KNT L YLQMN S LRAEDTAVYYCVT RGAWGQGT LVT VS S

[0211] 3.3 mCCLl

[0212] Taken from the UniProt references (P10146). Signal peptide removed in the CAR construct.

[0213] mCCLl (SEQ ID NO: 5):

[0214] MKFTAMAFLCFFFAAWLQWLSKSMLTVSNSCCLNTLKKELPLKFIQCYRKMGSSCPDPPAWFR LNKGRESCASTNKTWVQNHLKKVNPC

[0215] mCCLl (excluding signal sequence) (SEQ ID NO: 6):

[0216] KSMLTVSNSCCLNTLKKELPLKFIQCYRKMGSSCPDPPAWFRLNKGRESCASTNKTWVQNHLKKV NPC

[0217] 3.4 hCCLl

[0218] Sequence obtained from human CCL1 gene (CCDS11282.1). Signal sequence removed in the CAR construct.

[0219] hCCLl full sequence (SEQ ID NO: 7):

[0220] MQJJTTAFVCFFFAG REWnSKSMQVPFSRCCFSFAEQEIPLRAILCYRNTSSICSNEGLIFKL KRGKEACALDTVGWVQRHRKMLRHCPSKRK

[0221] hCCLl (excluding signal sequence) (SEQ ID NO: 8):

[0222] KSMQVPFSRCCFSFAEQEIPLRAILCYRNTSSICSNEGLIFKLKRGKEACALDTVGWVQRHRKMLR HCPSKRK

[0223] 3.5 mCD8a signal peptide (SEO ID NO: 11)

[0224] MASPLTRFLSLNLLLLGESIILGSGEA

[0225] 3.6 Hinge and Transmembrane domain (mCD8) (SEO ID NO: 12)

[0226] TTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLI CYHSVL

[0227] 3.74-1BB (SEO ID NO: 13)

[0228] KWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQEEEGGGGGYEL

[0229] 3.8 CD3z (SEO ID NO: 14)

[0230] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKD KMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR3.9 CAR architecture

[0231] A similar CAR architecture as that of the APRIL CAR (Camviel et al.) was followed. Architecture consisted of:

[0232] mCD8a signal peptide

[0233] - CCR8 Binding Moiety (m / hCCLl, MC148)

[0234] 1 x G4S linker

[0235] CD8 Hinge and TM domain

[0236] 4-1BB, CD3z signalling domain

[0237] The scFv CAR sequences consisted of a replacement of the CCR8 binding moiety with the anti-CCR8 scFv. The G4S linker was removed when using an scFv, and a (G4S)s linker placed between the VH and VL domains.

[0238] 3.10 CCL1 Variants

[0239] An exemplary CCL1 variant (referred to as the hCCLl variant 1-70) is described in Denis eta / ., 2012 (PLOS One, C-Terminal Clipping of Chemokine CCL1 / I-309 Enhances CCR8-Mediated Intracellular Calcium Release and Anti-Apoptotic Activity).

[0240] hCCLl variant 1-70 (excluding signal sequence) (SEQ ID NO: 15):

[0241] KSMQVPFSRCCFSFAEQEIPLRAILCYRNTSSICSNEGLIFKLKRGKEACALDTVGWVQRHRKMLR HOPS

[0242] The above variant has been shown to augment intracellular calcium release mediated by CCR8. It is hypothesised, therefore, that the CCL1 protein can tolerate a level of variation while maintaining (or even improving) certain properties.

[0243] 3.11 Viral chemokines

[0244] Viral chemokines that may be workable as CCR8 binding domains are described in, for example:

[0245] 1. HHV8-encoded vMIP-I Selectively Engages Chemokine Receptor CCR8: AGONIST AND ANTAGONIST PROFILES OF VIRAL CHEMOKINES, Daniel J. Dairaghi, Rong A. Fan, Brian E. McMaster, Michael R. Hanley, Thomas J. Schall.

[0246] 2. Crystal Structure of Viral Macrophage Inflammatory Protein I Encoded by Kaposi's Sarcoma-associated Herpesvirus at 1.7 A, John G. Luz, Minmin Yu, Ying Su, Zining Wu, Zhou Zhou, Ren Sun, Ian A. Wilson.

[0247] 3. Luttichau HR, Stine J, Boesen TP, Johnsen AH, Chantry D, Gerstoft J, Schwartz TW. A highly selective CC chemokine receptor (CCR)8 antagonist encoded bythe poxvirus molluscum contagiosum. J Exp Med. 2000 Jan 3;191(1): 171-80. doi: 10.1084 / jem.191.1.171. PMID: 10620615; PMCID: PMC2195798.

[0248] 4. Luttichau HR, Gerstoft J, Schwartz TW. MC148 encoded by human molluscum contagiosum poxvirus is an antagonist for human but not murine CCR8. J Leukoc Biol. 2001 Aug;70(2):277-82. PMID: 11493620.

[0249] MC148:

[0250] Sequence obtained from MC148 (UniProt: Q98314). Signal peptide removed (peptide identified through H. R. Luttichau et al, 2000, JEM) for addition into the CAR construct. MC148 is a viral peptide that is a highly selective, high affinity antagonist for human (but not murine) CCR8.

[0251] MC148 full sequence (SEQ ID NO: 9):

[0252] MRGGWFASWFMFFFAFFRFGVSLARRKCCLNPTNRPIPNPLLQDLSRVDYQAIGHDCGREAFRV TLQDGRQGCVSVGNKSLLDWLRGHKDLCPQIWSGCESL

[0253] MC148 excluding signal sequence (SEQ ID NO: 10):

[0254] LARRKCCLNPTNRPIPNPLLQDLSRVDYQAIGHDCGREAFRVTLQDGRQGCVSVGNKSLLDWLRGH KDLCPQIWSGCESL

[0255] vMIP-I:

[0256] CCR8 agonist.

[0257] Highly selective for CCL1.

[0258] Also known as 'K6' protein, which is encoded by the K6 gene in human herpesvirus 8, as represented by UniProt: F5HET8.

[0259] vMIP-I full sequence (SEQ ID NO: 18):

[0260] MAFVWFCCVSVFFATFYILAPTESAGSLVSYTPNSCCYGFQQHPPPVQILKEWYPTSPACPKPGVI LLTKRGRQICADPSKNWVRQLMQRLPAIA

[0261] vMIP-I excluding signal sequence (SEQ ID NO: 19):

[0262] AGSLVSYTPNSCCYGFQQHPPPVQILKEWYPTSPACPKPGVILLTKRGRQICADPSKNWVRQLMQR LPAIA

[0263] vMIP-II:

[0264] CCR8 antagonist

[0265] Promiscuous binding

[0266] 40% identical to VMIP-1vMIP-II full sequence (SEQ ID NO: 16):

[0267] MDTKGLLVAVLTALLCLQSGDTLGASWHRPDKCCLGYQKRPLPQVLLSSWYPTSQLCSKPGVIFL TKRGRQVCADKSKDWVKKLMQQLPVTAR

[0268] vMIP-II excluding signal sequence (SEQ ID NO: 17):

[0269] GDTLGASWHRPDKCCLGYQKRPLPQVLLSSWYPTSQLCSKPGVIFLTKRGRQVCADKSKDWVKKLM QQLPVTAR

[0270] 3.14 Methods

[0271] 3.14.1 Vector Synthesis

[0272] Self-inactivating murine leukaemia virus reverse transcriptase (SIN-MMLV) retrovirus vectors were used as backbone for generating mCCR.8 CAR and mCCLl-ligand CAR. Subsequently, a constitutively active short EFla promoter and the open reading frame of the surface reporter Thyl.l were inserted downstream of the CAR region to mark successfully transduced cells.

[0273] 3.14.2 Splenocyte isolation and OT-I CD8 T cell expansion

[0274] Spleens were mashed through a 40 pm nylon cell strainer and the resulting splenocyte suspension was washed in phosphate-buffered saline (PBS). Erythrocytes were lysed by resuspending the splenocytes in ammonium-chloride-potassium (ACK) lysing buffer (Gibco) for 1 minute at room temperature (RT). The cells were washed in PBS and resuspended in RPMI complete medium (CM) (RPMI 1640 medium (Gibco), 10% v / v heat-inactivated foetal bovine serum (FBS) (Sigma), 1% v / v GlutaMAX (Gibco), 1% v / v penicillin-streptomycin (Gibco), 1% v / v minimum essential medium non-essential amino acids solution (Gibco), 0.1% v / v 2-mercaptoethanol (Gibco), 0.1% v / v amphotericin B (Gibco), 0.1% v / v gentamycin (Gibco)). For expanding OT-I+CD8 T cells, isolated splenocytes from OT-I mice were activated and expanded in RPMI CM in the presence of 100 lU / ml IL-2 and 10 pg / ml of soluble anti-CD3 (145-2C11, InVivoMab) and anti-CD28 (37.51, InVivoMab) at 37°C in a 5% CO2 incubator.

[0275] 3.14.3 Retroviral transduction

[0276] Retroviral transductions were performed as described in Huang et al34. In brief, Platinum-E retroviral packaging cells (Cell Biolabs) grown in DMEM CM were plated in tissue culture-treated T175 flasks (Thermo Scientific). At 60-80% confluency, cells were co-transfected with plasmid DNA of interest and pCL-Eco retroviral packaging plasmid (Addgene cat. 12371) in 60 pl / well OptiMEM medium (Invitrogen) and 1.8 pl / well TransIT-293 transfection reagent (Mirus). 24 and 48 hours after transfection,viral supernatant (VSN) was collected, centrifuged to remove cell debris and stored at -80°C, and fresh DMEM CM was added to the cells. OT-I+CD8+ T cells previously isolated and expanded for 24 hours with 10 mg / ml anti-CD3 and anti-CD28 were resuspended in VSN at IxlO6cells / ml and supplemented with 5 ng / ml rhIL-2 and 8 pg / ml polybrene. The cells were plated in a 24-well plate, centrifuged at 2,000 x g, for 2 hours at 37°C and resuspended in RPMI CM supplemented with 5 ng / ml IL-2. The medium was replaced every 1-2 days, and the cells were kept at 1-2 x 106cells / ml.

[0277] 3.14.4 Co- Culture

[0278] 300-19 CCR8+cells were cultured in RPMI CM and plated at 50,000 cells per well. Retrovirally transduced OT-1 CD8+T cells were culture in RPMI CM and plated at 200,000 per well, 50,000 per well or 12,500 per well for the respective conditions. The target cell and effector cell only conditions consisted of 50,000 cells per well. Cells were incubated at 37°C in a 5% CO2 incubator for 24 hours before proceeding to flow cytometry staining.

[0279] 3.14.5 Flow cytometry

[0280] Cells were washed in PBS and stained in PBS with surface antibodies and live / dead dye for 30 minutes at 4°C and protected from light. Cells were washed in fluorescence-activated cell sorting (FACS) buffer (PBS, 2% v / v heat-inactivated FBS (Sigma), 2 mM EDTA (Sigma), 0.6 g / l sodium azide (Sigma)). Cells were washed and resuspended in FACS buffer before analysis. Acquisition of data was performed using a Cytek Aurora. Data was analysed in FlowJo v.10.9.0 and statistical analyses were performed using GraphPad Prism 9.

[0281] Example 4: Selective Depletion of CCR8+Regulatory T Cells Using CCLl-Based CAR Constructs and Ligand-Drug Conjugates

[0282] 4.1 MC38 Tumour Model and Treg Isolation

[0283] MC38 colorectal adenocarcinoma cells were cultured in DMEM complete medium and implanted subcutaneously into the flanks of FOXP3-GFP CD45.1+ reporter mice on the C57BL / 6 background. Tumours were allowed to establish for 14-21 days until reaching approximately 150-200 mm3. Tumours were excised, mechanically dissociated, and enzymatically digested using a collagenase / DNase cocktail for 30 minutes at 37°C with agitation. Single-cell suspensions were filtered through 40 pm nylon cell strainers and washed in PBS. Lymphocytes were isolated by density gradient centrifugation using a lymphocyte separation medium. Spleens were harvested from the same tumourbearing mice and processed as described in section 3.14.2. Treg cells were identified as GFP+(FOXP3+) CD4+cells. CCR8 expression was determined by staining with ananti-CCR8 antibody conjugated to BV421. CCR8+and CCR8-Treg populations were isolated by fluorescence-activated cell sorting (FACS) using a BD FACSAria III or equivalent cell sorter.

[0284] 4.2 Ex Vivo Treg Depletion Assay

[0285] FACS-sorted CCR8+or CCR8-Treg cells were plated in 96-well round-bottom plates in RPMI complete medium (RPMI CM) supplemented with 100 lU / ml IL-2. CAR T cells transduced with non-targeting control CAR, anti-CCR8 scFv CAR, or CCL1 ligand CAR were added at defined effector-to-target ratios. Co-cultures were incubated for 48 hours at 37°C in a 5% CO2 incubator. Cells were harvested and stained for analysis by flow cytometry, as described in 3.14.5.

[0286] 4.3 Tandem CAR Construction

[0287] The CCL1-GPC3 tandem CAR construct was designed with the following architecture: mCD8o signal peptide - murine CCL1 (excluding signal sequence) - flexible linker (G4S)S - anti-human GPC3 scFv - CD8 hinge and transmembrane domain - 4-1BB costimulatory domain - CD3 signalling domain. Thyl.l was inserted upstream of a P2A self-cleaving peptide sequence as a transduction marker. The complete construct was synthesised and cloned into a self-inactivating murine leukaemia virus (SIN-MMLV) retroviral vector backbone. Control constructs included: CCL1 ligand CAR (as described previously), anti-GPC3 CAR (lacking the CCL1 moiety), both in the same backbone.

[0288] 4.4 Tandem CAR Cytotoxicity Assay

[0289] Murine CCR8-expressing target cells (300-19) or GPC3-expressing target cells were plated at 10,000 cells per well in 96-well plates. Retrovirally transduced CAR T cells (CCL1 ligand CAR, CCL1-GPC3 tandem CAR, anti-GPC3 CAR, or untransduced controls) were added at effector-to-target ratios of 1:2, 2:1, and 10:1, in triplicates. Co-cultures were incubated for 48 hours at 37°C in a 5% CO2 incubator. Following incubation, cells were harvested, stained for viability and relevant surface markers, and analysed by flow cytometry.

[0290] 4.5 Ligand-Drug Conjugate - Cytotoxicity Assay

[0291] Biotinylated hCCLl was conjugated to streptavidin-saporin (Advanced Targeting Systems IT-27) by incubation at a 1:1 molar ratio for 30 minutes at room temperature. The resulting hCCLl-toxin conjugate was used without further purification. Control conditions included: Biotinylated human CCL1 alone, and streptavidin-saporin toxin alone (not conjugated to CCL1). 300-19 cells stably expressing human CCR8 (hCCR8+) or murine CCR8 (mCCR8+, serving as a species-specificity control) were plated at 2,000cells per well in 96-well plates. Cells were treated with hCCLl alone, streptavidin-saporin toxin alone, or the hCCLl-toxin conjugate at equivalent molar concentrations. Cells were incubated for 48 hours at 37°C in a 5% CO2 incubator. Cells were stained and analysed by flow cytometry (as described in 3.14.5).

[0292] REFERENCES

[0293] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. All references listed below and throughout this application are incorporated by reference.

[0294] 1 Roychoudhuri, R., Eil, R. L. & Restifo, N. P. The interplay of effector and regulatory T cells in cancer. Curr Opin Immunol 33, 101-111 (2015). https: / / doi. Org / 10.1016 / j.coi.2015.02.003

[0295] 2 Stockis, J., Roychoudhuri, R. & Halim, T. Y. F. Regulation of regulatory T cells in cancer. Immunology 157, 219-231 (2019). https: / / doi.org / 10.llll / imm.13064 3 Gallimore, A., Quezada, S. A. & Roychoudhuri, R. Regulatory T cells in cancer: where are we now? Immunology 157, 187-189 (2019). https: / / doi.org / 10.llll / imm.13088

[0296] 4 Whiteside, S. K. et al. CCR8 marks highly suppressive Treg cells within tumours but is dispensable for their accumulation and suppressive function. Immunology 163, 512-520 (2021). https: / / doi.org / 10.llll / imm.13337

[0297] 5 Plitas, G. et al. Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer. Immunity 45, 1122-1134 (2016). https: / / doi. Org / 10.1016 / j.immuni.2016.10.032

[0298] 6 De Simone, M. et al. Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells. Immunity 45, 1135-1147 (2016). https: / / doi. Org / 10.1016 / j.immuni.2016.10.021

[0299] 7 Quezada, S. A., Peggs, K. S., Simpson, T. R. & Allison, J. P. Shifting the equilibrium in cancer immunoediting: from tumor tolerance to eradication. Immunol Rev 241, 104-118 (2011). https: / / doi. Org / 10.llll / j.1600-065X.2011.01007.x 8 Plitas, G. & Rudensky, A. Y. Regulatory T Cells in Cancer. Annual Review of Cancer Biology 4, 459-477 (2020). https: / / doi.org / 10.1146 / annurev-cancerbio-030419-033428

[0300] 9 DePeaux, K. & Delgoffe, G. M. Metabolic barriers to cancer immunotherapy. Nature reviews. Immunology (2021). https: / / doi.org / 10.1038 / s41577-021-00541-y10 Sakaguchi, S. et al. Regulatory T Cells and Human Disease. Annu Rev Immunol 38, 541-566 (2020). https: / / doi.org / 10.1146 / annurev-immunol-042718-041717 11 Sato, E. et al. Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+ / regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci U S A 102, 18538-18543 (2005). https: / / doi.org / 10.1073 / pnas.0509182102

[0301] 12 Curiel, T. J. et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10, 942-949 (2004). https: / / doi.org / 10.1038 / nml093

[0302] 13 Petersen, R. P. et al. Tumor infiltrating Foxp3+ regulatory T-cells are associated with recurrence in pathologic stage I NSCLC patients. Cancer 107, 2866-2872 (2006). https: / / doi.org / 10.1002 / cncr.22282

[0303] 14 Hiraoka, N., Onozato, K., Kosuge, T. & Hirohashi, S. Prevalence of FOXP3+ regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions. Clin Cancer Res 12, 5423-5434 (2006). https: / / doi.org / 10.1158 / 1078-0432. CCR-06-0369

[0304] 15 Sinicrope, F. A. etal. Intraepithelial effector (CD3+) / regulatory (FoxP3+) T-cell ratio predicts a clinical outcome of human colon carcinoma. Gastroenterology 137, 1270-1279 (2009). https: / / doi. Org / 10.1053 / j.gastro.2009.06.053

[0305] 16 Jordanova, E. S. et al. Human leukocyte antigen class I, MHC class I chain-related molecule A, and CD8+ / regulatory T-cell ratio: which variable determines survival of cervical cancer patients? Clin Cancer Res 14, 2028-2035 (2008). https: / / doi.org / 10.1158 / 1078-0432. CCR-07-4554

[0306] 17 Kamada, T. et al. PD-1(+) regulatory T cells amplified by PD-1 blockade promote hyperprogression of cancer. Proc Natl Acad Sci U S A 116, 9999-10008 (2019). https: / / doi.org / 10.1073 / pnas.1822001116

[0307] 18 Kumagai, S. etal. The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies. Nat Immunol 21, 1346-1358 (2020). https: / / doi.org / 10.1038 / s41590-020-0769-3

[0308] 19 Tan, C. L. et al. PD-1 restraint of regulatory T cell suppressive activity is critical for immune tolerance. J Exp Med 218 (2021). https: / / doi.org / 10.1084 / jem.20182232 20 Liu, C., Workman, C. J. & Vignali, D. A. Targeting regulatory T cells in tumors. FEBS J 283, 2731-2748 (2016). https: / / doi.org / 10.llll / febs.13656

[0309] 21 Jacobs, J. F. et al. Dendritic cell vaccination in combination with anti-CD25 monoclonal antibody treatment: a phase I / II study in metastatic melanoma patients. Clin Cancer Res 16, 5067-5078 (2010). https: / / doi.org / 10.1158 / 1078-0432. CCR-10-175722 Sampson, J. H. et al. A pilot study of IL-2Ralpha blockade during lymphopenia depletes regulatory T-cells and correlates with enhanced immunity in patients with glioblastoma. PLoS One 7, e31046 (2012). https: / / doi.org / 10.1371 / journal.pone.0031046

[0310] 23 Rech, A. J. et al. CD25 blockade depletes and selectively reprograms regulatory T cells in concert with immunotherapy in cancer patients. Sci Transl Med 4, 134ral62 (2012). https: / / doi.org / 10.1126 / scitranslmed.3003330

[0311] 24 Attia, P., Maker, A. V., Haworth, L. R., Rogers-Freezer, L. & Rosenberg, S. A. Inability of a fusion protein of IL-2 and diphtheria toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory T lymphocytes in patients with melanoma. J Immunother 28, 582-592 (2005). https: / / doi.org / 10.1097 / 01.cji.0000175468.19742.10

[0312] 25 Zamarin, D. et al. Mogamulizumab in Combination with Durvalumab or Tremelimumab in Patients with Advanced Solid Tumors: A Phase I Study. Clin Cancer Res 26, 4531-4541 (2020). https: / / doi.org / 10.1158 / 1078-0432. CCR-20-0328 26 Kurose, K. et al. Phase la Study of FoxP3+ CD4 Treg Depletion by Infusion of a Humanized Anti-CCR4 Antibody, KW-0761, in Cancer Patients. Clin Cancer Res 21, 4327-4336 (2015). https: / / doi.org / 10.1158 / 1078-0432. CCR-15-0357

[0313] 27 Kidani, Y. et al. CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory. Proc Natl Acad Sci U S A 119 (2022). https: / / doi.org / 10.1073 / pnas.2114282119

[0314] 28 Van Damme, H. et al. Therapeutic depletion of CCR8(+) tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy. J Immunother Cancer 9 (2021). https: / / doi.org / 10.1136 / jitc-2020-001749

[0315] 29 Gogesch, P., Dudek, S., van Zandbergen, G., Waibler, Z. & Anzaghe, M. The Role of Fc Receptors on the Effectiveness of Therapeutic Monoclonal Antibodies. Int J Mol Sci 22 (2021). https: / / doi.org / 10.3390 / ijms22168947

[0316] 30 Graziano, R. F. & Engelhardt, J. J. Role of FcgammaRs in Antibody-Based Cancer Therapy. Curr Top Microbiol Immunol 423, 13-34 (2019). https: / / doi.org / 10.1007 / 82_2019_150

[0317] 31 Roos, R. S. et al. Identification of CCR8, the receptor for the human CC chemokine 1-309. J Biol Chem 272, 17251-17254 (1997). https: / / doi.org / 10.1074 / jbc.272.28.17251

[0318] 32 Kantarjian, H. et al. Blinatumomab versus Chemotherapy for Advanced Acute Lymphoblastic Leukemia. N Engl J Med 376, 836-847 (2017). https: / / doi.org / 10.1056 / NEJMoal60978333 Godbersen, C. et al. NKG2D Ligand-Targeted Bispecific T-Cell Engagers Lead to Robust Antitumor Activity against Diverse Human Tumors. Mol Cancer Ther 16, 1335-1346 (2017). https: / / doi.org / 10.1158 / 1535-7163. MCT-16-0846

[0319] 34 Huang, B., Johansen, K. H. & Schwartzberg, P. L. Efficient CRISPR / Cas9-Mediated Mutagenesis in Primary Murine T Lymphocytes. Current Protocols in Immunology 124 (2019). https: / / doi.org / 10.1002 / cpim.62

[0320] 35 Luz JG, Yu M, Su Y, Wu Z, Zhou Z, Sun R, Wilson IA. Crystal structure of viral macrophage inflammatory protein I encoded by Kaposi's sarcoma-associated herpesvirus at 1.7A. J Mol Biol. 2005 Oct 7;352(5): 1019-28. doi: 10.1016 / j.jmb.2005.08.011.

Claims

CLAIMS1. A CCR8 binding polypeptide comprising at least:a) a first domain that is a CCR8 binding domain that is a CCL1 protein or derivative thereof; andb) at least a second domain that:i) together with the first domain forms a CCR8-binding Chimeric Antigen Receptor (CAR) protein;ii) comprises a transmembrane domain, a hinge domain, and / or at least one intracellular signalling domain;iii) is a T-cell binding domain; and / oriv) is a drug or toxic agent.

2. The CCR8 binding polypeptide of claim 1 where the second domain is a domain that together with the first domain forms a CCR8-binding Chimeric Antigen Receptor (CAR) protein.

3. The CCR8 binding polypeptide of claim 1 where the second domain comprises a transmembrane domain, a hinge domain, and / or at least one intracellular signalling domain.

4. The CCR8 binding polypeptide of claim 1 where the second domain is a T-cell binding domain.

5. The CCR8 binding polypeptide of claim 1 where the second domain is a drug or toxic agent.

6. The CCR8 binding polypeptide of any of claims 1-5 wherein the first and second domain and protein domains.

7. The CCR8 binding polypeptide of claim 6 where the first and second domains are expressed as a single transcript.

8. The CCR8 binding polypeptide of any of claims 1-6 wherein the first and second domains are conjugated to one another.

9. The CCR8 binding polypeptide according to any preceding claims, wherein the CCR8 binding domain is selected from the group consisting of a protein binding domain, an aptamer, a CCR8-binding viral domain, and combinations thereof.

10. The CCR8 binding polypeptide according to any preceding claims, wherein the CCR8 binding polypeptide:a. comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 15 and 6 or a sequence with at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to any of SEQ ID NO: 8, 15, or 6;b. is selected from the group consisting of chemokine (C-C motif) ligand 1 (CCL1), CCL16, CCL18, and combinations thereof; orc. is a mammalian chemokine ligand (e.g. CCL1, CCL16 and / or CCL18), for example a human, non-human primate, murine, or canine chemokine ligand.

11. The CCR8 binding polypeptide according to any preceding claim, wherein the CCR8 binding polypeptide is not an antibody-based domain.

12. A CAR protein comprising a CCR8 binding domain, wherein the CAR protein further comprises:a. a transmembrane domain (TMD), optionally wherein the TMD is selected from the group consisting of CD28; CD8o; CD4; the alpha, beta or zeta chain of the T-cell receptor; CD3 epsilon; CD5; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; and KIR2DS2;b. a cytoplasmic domain, optionally comprising at least one intracellular signalling domain, such as a signalling domain of CD3 zeta, Fc receptor gamma, Fc receptor beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD28, CD66d, CD79a, CD79b, and / or 41BB (CD137);c. one or more costimulatory domains, optionally comprising one or more functional signalling domains obtained from a protein selected from the group consisting of CD28, 41BB (CD137), 0X40 (CD134), ICOS, CD27,LIGHT (TNFSF14), HVEM, DAP10, DAP12, CD3 (zeta), CD3e (epsilon), and CD27; and / ord. one or more hinge domains, optionally a hinge derived from a protein selected from the group consisting of CD28, CD8o, CD4, CD3 zeta, and / or Ig.

13. The CAR protein of claim 12, wherein the CCR8 binding domain:a. comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 15 and 6 or a sequence with at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to any of SEQ ID NO: 8, 15, or 6;b. is selected from the group consisting of chemokine (C-C motif) ligand 1 (CCL1), CCL16, CCL18, and combinations thereof; orc. is a mammalian chemokine ligand (e.g. CCL1, CCL16 and / or CCL18), for example a human, non-human primate, murine, or canine chemokine ligand.

14. A fusion protein comprising at least two binding domains, wherein a first binding domain is a CCR8 binding domain, and a second binding domain is a T-cell binding domain, optionally wherein the T-cell binding domain is selected from the group consisting of CD3, CD28, 41BB, and combinations thereof.

15. A ligand-drug conjugate (LDC), wherein the ligand is a CCR8 binding polypeptide, optionally wherein the LDC further comprises a linker.

16. A polynucleotide with a nucleic acid sequence encoding the CCR8 binding polypeptide according to any of claims 1-11, the CAR protein according to claim 12 or 13, the fusion protein according to claim 14, and / or the ligand in the LDC according to claim 15.

17. A vector comprising the polynucleotide of claim 16; optionally further comprising:a. one or more nucleic acid sequence for encoding one or more further CAR protein domain; and / orb. one or more nucleic acid sequences for encoding a T-cell binding domain, optionally wherein the T-cell binding domain is selected from the group consisting of CD3, CD28, 41BB, and combinations thereof; optionallyfurther comprising a nucleic acid encoding a linker sequence between the CCR8 binding and the T-cell binding domain.

18. A cell comprising at least one CCR8 binding polypeptide according to any of claims 1-11, at least one CAR protein according to claim 12 or 13, at least one fusion protein according to claim 14, and / or at least one ligand in the LDC according to claim 15; optionally wherein the cell is a T cell, NK cell or macrophage.

19. A cell comprising at least one polynucleotide according to claim 16, and / or at least one vector according to claim 17.

20. A method of producing a cell, wherein the method includes the step of inducing a cell to express at least one CCR8 binding polypeptide according to any of claims 1-11, at least one CAR protein according to claim 12 or 13, at least one fusion protein according to claim 14, at least one ligand in the LDC according to claim 15, at least one polynucleotide according to claim 16, and / or at least one vector according to claim 17.

21. A cell comprising a CAR protein according to claim 12 or 13, a fusion protein according to claim 14, and / or an LDC according to claim 15, for use in medicine.

22. A cell comprising a CAR protein according to claim 12 or 13, a fusion protein according to claim 14, and / or an LDC according to claim 15, for use in prophylaxis or treatment of cancer in a subject in need thereof.

23. A method of prophylaxis or treatment of cancer in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a cell comprising a CAR protein according to claim 12 or 13, a fusion protein according to claim 14, and / or an LDC according to claim 15.

24. A cell, fusion protein and / or LDC for use according to claim 22, or the method of treating cancer of claim 23, wherein the cancer is:a. a solid tumour; optionally wherein the solid tumour has a microenvironment comprising CCR8+Treg cells; orb. a haematological tumour associated with CCR8 expression.

25. A cell, fusion protein and / or LDC for use according to claim 22 or 24, or the method of treating cancer according to claim 23 or 24, wherein the use / method atleast partially depletes CCR8+Tregs in the subject; preferably wherein the depletion occurs in the tumour microenvironment.

26. A cell, fusion protein and / or LDC for use according to any of claims 22, 24 or 25 or the method of treating cancer according to any of claims 23-25, wherein the solid tumour has been assessed for the presence of CCR8+Tregs; optionally wherein the assessment is made based on a biopsy of the solid tumour, and / or wherein the assessment includes at least one further marker of Tregs (e.g. CD25 and / or Foxp3).

27. A cell, fusion protein and / or LDC for use according to any of claim 22 and 24-26, or the method of treating cancer according to any of claim 23-26, wherein the use / method comprises administration of at least one further agent.

28. A kit comprising:a. a CCR8 binding domain, wherein the CCR8 binding domain is in the form of, or configurable for preparing, a CAR protein or a cell comprising a CAR protein according to claim 12 or 13, a fusion protein according to claim 14, and / or an LDC according to claim 15; and / orb. at least one CCR8 binding polypeptide according to any of claims 1-11, at least one CAR protein according to claim 12 or 13, at least one fusion protein according to claim 14, at least one ligand in the LDC according to claim 15, at least one polynucleotide according to claim 16, and / or at least one vector according to claim 17.

Images