WO2022003156A1 - Ccr8 non-blocking binders - Google Patents

Abstract

The present invention relates CCR8 binders having cytotoxic activity, wherein the CCR8 binder is a non-blocking binder of CCR8. Such binders are particularly useful for the depletion of intra-tumoural regulatory T-cells and immunotherapy in general tumour.

Description

CCR8 NON-BLOCKING BINDERS

FIELD OF THE INVENTION

The present invention relates CCR8 binders having cytotoxic activity, wherein the CCR8 binder is a non-blocking binder of CCR8. Such binders are particularly useful for the depletion of intra- tumoural regulatory T-cells and immunotherapy in general.

BACKGROUND OF THE INVENTION

Regulatory T (Treg) cells are one of the integral components of the adaptive immune system whereby they contribute to maintaining tolerance to self-antigens and preventing auto-immune diseases. However, Treg cells are also found to be highly enriched in the tumour microenvironment of many different cancers (Colombo and Piconese, 2007; Nishikawa and Sakaguchi, 2014; Roychoudhuri et al., 2015). In the tumour microenvironment, Treg cells contribute to immune escape by reducing tumour-associated antigen (TAA)-specific T-cell immunity, thereby preventing effective anti-tumour activity. High tumour infiltration by Tregs is hence often associated with an invasive phenotype and poor prognosis in cancer patients (Shang et al., 2015; Piltas et al., 2016).

Acknowledging the significance of tumour-infiltrating Treg cells and their potential role in inhibiting anti-tumour immunity, multiple strategies have been proposed to modulate Treg cells in the tumour microenvironment. Several studies have demonstrated that modulating Tregs has the potential to offer significant therapeutic benefit (Elpek et al, 2007).

However, one major challenge associated with Treg modulation is that systematic removal or inhibition of Treg cells may elicit autoimmunity. It is therefore critical to specifically deplete tumour-infiltrating Treg cells while preserving tumour-reactive effector T cells and peripheral Treg cells (e.g. circulating blood Treg cells) in order to prevent autoimmunity.

The G protein-coupled CC chemokine receptor protein CCR8 (CKRL1/CMKBR8/CMKBRL2) and its natural ligand CCL1 have been known to be implicated in cancer and specifically in T- cell modulation in the tumour environment. Eruslanov et al. (Clin Cancer Res 2013, 17:1670- 80) showed upregulation of CCR8 expression in human cancer tissues and demonstrated that primary human tumours produce substantial amounts of the natural CCR8 ligand CCL1 . This indicates that CCL1/CCR8 axis contributes to immune evasion and suggest that blockade of CCR8 signals is an attractive strategy for cancer treatment. Hoelzinger et al. (J Immunol 2010, 184:8633-42) similarly show that blockade of CCL1 inhibits Treg suppressive function and enhances tumour immunity without affecting Treg responses. Wang et al. (PloSONE 2012, e30793) reported increased expression of CCR8 on tumour-infiltrating FoxP3+ T-cells and suggested that blocking CCR8 may lead to the inhibition of migration of T regs into the tumours. Due to the high and relatively specific expression of CCR8 on tumour-infiltrating Tregs, neutralizing monoclonal antibodies against CCR8 have been used for the modulation and depletion of this Treg population in the treatment of cancer (EP3431105 A1 and WO2019/157098 A1). WO2018/181425 suggests that, in mice, a neutralizing anti-CCR8 mAb is able to deplete T reg cells in tumour tissues by antibody-dependent cell-mediated cytotoxicity (ADCC), and thereby enhance tumour immunity. Through their neutralizing activity, these antibodies inhibit Treg migration into the tumour, reverse the suppressive function of Tregs and deplete intratumoural Tregs (WO2019/157098 A1 ). Recently, Wang et al. (Cancer Immunol Immonother 2020, https://doi.org/10.1007/s00262-020-02583-y) showed that CCR8 blockade could destabilize intratumoural Tregs into a fragile phenotype accompanied with reactivation of the antitumour immunity and augment anti-PD-1 therapeutic benefits.

However, alternative strategies for intratumoural Treg modulation are still required, especially strategies that reduce the risks of side effects associated with existing therapies.

SUMMARY OF THE INVENTION

The inventors have now surprisingly found that a non-blocking binder of CCR8 having cytotoxic activity as detailed in the claims fulfils the above-mentioned need. In particular, the inventors have surprisingly found that a non-blocking binder of CCR8 having cytotoxic activity allows for the efficient depletion of tumour-infiltrating regulatory T-cells (Tregs). Surprisingly, the absence of functional CCR8 blockade, suggested in the prior art to reduce Treg infiltration into the tumour and to inhibit or revert the immunosuppressive function of intra-tumoural Tregs, does not reduce therapeutic efficacy. The non-blocking CCR8 binders of the invention therefore provide an efficacious tumour therapy, while displaying an improved safety profile.

It is thus an object of the invention to provide non-blocking CCR8 binders having cytotoxic activity. Therefore in a first embodiment, the present invention provides a CCR8 binder having cytotoxic activity, wherein said CCR8 binder is a non-blocking binder of CCR8. Preferably, the cytotoxic activity is caused by the presence of a cytotoxic moiety that induces antibody-dependent cellular cytotoxicity (ADCC), induces complement-dependent cytotoxicity (CDC), induces antibody-dependent cellular phagocytosis (ADCP), binds to and activates T- cells, or comprises a cytotoxic payload.

In a further embodiment, the cytotoxic moiety comprises a fragment crystallisable (Fc) region moiety. Preferably, the Fc region can be engineered to increase ADCC, CDC, and/or ADCP activity, for example through afucosylation or by comprising an ADCC, CDC, and/or ADCP- increasing mutation or mutations.

In still another embodiment, the CCR8 binder comprises at least one single domain antibody moiety that binds to CCR8. Preferably, the CCR8 binder comprises at least one Fc region moiety and at least two single domain antibody moieties that bind to CCR8.

In still another embodiment, the CCR8 binder inhibits signalling of CCR8 by less than 90%, preferably less than 80%, more preferably less than 70%, still more preferably less than 60%, most preferably less than 50%.

Another object of the present invention is to provide nucleic acids encoding the CCR8 binder.

Yet another object of the present invention is to provide non-blocking CCR8 binders having cytotoxic activity for use as a medicine.

A further object of the present invention is to provide non-blocking CCR8 binders having cytotoxic activity for use in the treatment of a tumour. Preferably, the tumour is selected from the group consisting of breast cancer, uterine corpus cancer, lung cancer, stomach cancer, head and neck cancer, squamous cell carcinoma, skin cancer, colorectal cancer, and kidney cancer.

In a particular embodiment of the invention, the CCR8 binder for use comprises (a) an Fc region moiety that has ADCC, CDC and/or ADCP activity, and (b) at least one single domain antibody moiety that bind to CCR8.

Preferably, the administration of the CCR8 binder leads to the depletion of tumour-infiltrating regulatory T-cells (Tregs).

In yet a further embodiment, the treatment further comprises administration of a checkpoint inhibitor. A checkpoint inhibitor is a compound that blocks checkpoint proteins from binding to their partner proteins thereby activating the immune system function. Preferably the checkpoint inhibitor blocks proteins selected from the group consisting of PD-1 , PD-L1 , CTLA-4, B7-1 and B7-2. More preferably the checkpoint inhibitor blocks PD-1 or PD-L1 .

BRIEF DESCRIPTION OF FIGURES

Figure 1 illustrates the evaluation by flow cytometry of two VHHs (VHH-01 and VHH-06) derived from llama immunization with mouse CCR8 for their binding to full-length mouse CCR8 versus N-terminal deletion mouse CCR8 overexpressed in Hek293 cells.

Figure 2 presents a schematic representation of the VHH-Fc fusions VHH-Fc-14, VFIFI-Fc-25, VHH-Fc-41 and VHH-Fc-43.

Figure 3 illustrates the evaluation of VFIFI-Fc-14 and VFIFI-Fc-25 for their potential to functionally inhibit the protective activity of ligand CCL1 against dexamethasone-induced apoptosis in BW5147 cells.

Figure 4 shows the effects on intratumoural Treg depletion by VFIFI-Fc-43, which is a CCR8 blocking Fc fusion with ADCC activity, and VFIFI-Fc-41 , which lacks ADCC activity, as well as isotype control.

Figure 5 shows the effects on circulating Tregs by VFIFI-Fc-43 and VFIFI-Fc-41 and isotype control.

Figure 6 illustrates the effects on intestinal Treg levels by VFIFI-Fc-43 and VFIFI-Fc-41 and isotype control.

Figure 7 shows the in vivo effects of VFIFI-Fc-25 on tumour growth in comparison to isotvoe and VFIFI-Fc-14 in LLC-OVA tumors.

Figure 8 shows the in vivo effects of VFIFI-Fc-25 on tumour growth in comparison to isotvoe and VFIFI-Fc-14 in MC38 tumors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described in the following with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art.

As described herein before, the present invention provides a CCR8 binder having cytotoxic activity, wherein said CCR8 binder is a non-blocking binder of CCR8. Such compounds are particularly useful due to their ability to bind to CCR8 expressed on a cell, such as a regulatory T-cell, particularly an intra-tumoural regulatory T-cell, and to deplete such cells through their cytotoxic activity. CCR8 is a member of the beta-chemokine receptor family which is predicted to be a seven transmembrane protein similar to G-coupled receptors. Identified ligands of CCR8 include its natural cognate ligand CCL1 (I-309).

Binders

As described herein, the term “binder” of a specific antigen denotes a molecule capable of specific binding to said antigen. Specifically, a CCR8 binder as used herein refers to a molecule capable of specifically binding to CCR8. Such a binder is also referred to herein as a ”CCR8 binder”.

“Specific binding”, “bind specifically”, and “specifically bind” is particularly understood to mean that the binder has a dissociation constant (K_d) for the antigen of interest of less than about 10^-6 M, 10^-7 M, 10^-8 M, 10^-9 M, 10-¹° M, 10-¹¹ M, 10 ¹² M or 10^~13 M. In a preferred embodiment, the dissociation constant is less than 10^_8 M, for instance in the range of 10^_9 M, 10^_1° M, 10^-11 M, 10^-12 M or 10^-13 M. Binder affinities towards membrane targets may be determined by a surface plasmon resonance based assay (such as the BIAcore assay as described in PCT Application Publication No. W02005/012359) using viral like particles; cellular enzyme- linked immunoabsorbent assay (ELISA); and fluorescent activated cell sorting (FACS) read outs for example. A preferred method for determining apparent Kd or EC50 values is by using FACS at 21 °C with cells overexpressing huCCR8.

As will be understood by the skilled person, in principle any type of binder that binds to CCR8 can be used in the present invention and different types of binders are readily available to the skilled person or can be generated using the typical knowledge in the art. In a particular embodiment, the binding moiety of the CCR8 binder is proteinaceous, more particularly a CCR8 binding polypeptide. In a further embodiment, the binding moiety of the CCR8 binder is antibody based or non-antibody based, preferably antibody based. Non-antibody based binders include, but are not limited to, affibodies, Kunitz domain peptides, monobodies (adnectins), anticalins, designed ankyrin repeat domains (DARPins), centyrins, fynomers, avimers; affilins; affitins, peptides and the like.

As described herein, the terms “antibody”, “antibody fragment” and “active antibody fragment” refer to a protein comprising an immunoglobulin (Ig) domain or an antigen-binding domain capable of specifically binding the antigen, in this case the CCR8 protein. “Antibodies” can further be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies may be multimers, such as tetramers, of immunoglobulin molecules. In a preferred embodiment, the binder comprises a CCR8 binding moiety that is an antibody or active antibody fragment. In a further aspect of the invention, the binder is an antibody. In a further aspect of the invention the antibody is monoclonal. The antibody may additionally or alternatively be humanised or human. In a further aspect, the antibody is human, or in any case an antibody that has a format and features allowing its use and administration in human subjects. Antibodies may be derived from any species, including but not limited to mouse, rat, chicken, rabbit, goat, bovine, non human primate, human, dromedary, camel, llama, alpaca, and shark.

The term “antigen-binding fragment” is intended to refer to an antigen-binding portion of said intact polyclonal or monoclonal antibodies that retains the ability to specifically bind to a target antigen or a single chain thereof, fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. The antigen-binding fragment comprises, but not limited to Fab; Fab'; F(ab')2; a Fc fragment; a single domain antibody (sdAb or dAb) fragment. These fragments are derived from intact antibodies by using conventional methods in the art, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). As used herein, antigen-binding fragment also refers to fusion proteins comprising heavy and/or light chain variable regions, such as single-chain variable fragments (scFv).

As used herein, the term “monoclonal antibody” refers to an antibody composition having a homogeneous antibody population. It is understood that monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional antibody (polyclonal) preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The binders of the invention preferably comprise a monoclonal antibody moiety that binds to CCR8.

As used herein, the term “humanized antibody” refers to an antibody produced by molecular modeling techniques to identify an optimal combination of human and non-human (such as mouse or rabbits) antibody sequences, that is, a combination in which the human content of the antibody is maximized while causing little or no loss of the binding affinity attributable to the variable region of the non-human antibody. For example, a humanized antibody, also known as a chimeric antibody comprises the amino acid sequence of a human framework region and of a constant region from a human antibody to "humanize" or render non- immunogenic the complementarity determining regions (CDRs) from a non-human antibody.

As used herein, the term “human antibody” means an antibody having an amino acid sequence corresponding to that of an antibody that can be produced by a human and/or which has been made using any of the techniques for making human antibodies known to a skilled person in the art or disclosed herein. It is also understood that the term “human antibody” encompasses antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides.

In one aspect of the invention, the binder comprises an active antibody fragment. The term “active antibody fragment” refers to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more antigen-binding sites, e.g. complementary-determining-regions (CDRs), accounting for such specificity. Non-limiting examples include immunoglobulin domains, Fab, F(ab)’2, scFv, heavy- light chain dimers, immunoglobulin single variable domains, single domain antibodies (sdAb or dAb), Nanobodies^®, and single chain structures, such as complete light chain or complete heavy chain, as well as antibody constant domains that have been engineered to bind to an antigen. An additional requirement for the “activity” of said fragments in the light of the present invention is that said fragments are capable of binding CCR8. The term “immunoglobulin (Ig) domain” or more specifically “immunoglobulin variable domain” (abbreviated as “IVD”) means an immunoglobulin domain essentially consisting of framework regions interrupted by complementary determining regions. Typically, immunoglobulin domains consist essentially of four “framework regions” which are referred in the art and below as “framework region 1” or “FR1 as “framework region 2” or “FR2”; as “framework region 3” or “FR3”; and as “framework region 4” or “FR4”, respectively; which framework regions are interrupted by three “complementarity determining regions” or “CDRs”, which are referred in the art and herein below as “complementarity determining region 1 ” or “CDR1 as “complementarity determining region 2” or “CDR2”; and as “complementarity determining region 3” or “CDR3”, respectively. Thus the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs) that confer specificity to an antibody for the antigen by carrying the antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VFI) and a light chain variable domain (VL) interact to form an antigen binding site. In this case the complementary determining regions (CDRs) of both VFI and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab’)2 fragment, an Fv fragment such as a disulphide linked Fv or scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, with binding to the respective epitope of an antigen by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VFI-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen. A single domain antibody (sdAb) as used herein, refers to a protein with an amino acid sequence comprising 4 framework regions (FR) and 3 complementarity determining regions (CDRs) according to the format FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. Single domain antibodies of this invention are equivalent to “immunoglobulin single variable domains” (abbreviated as “ISVD”) and refers to molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets single domain antibodies apart from “conventional” antibodies or their fragments, wherein two immunoglobulin domains, in particular two variable domains interact to form an antigen binding site. The binding site of a single domain antibody is formed by a single V H/ V H H or VL domain. Flence, the antigen binding site of a single domain antibody is formed by no more than 3 CDRs. As such a single domain may be a light chain variable domain sequence (e.g. a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a VFI-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of a single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).

Thus, in one embodiment, the CCR8 binder having cytotoxic activity as detailed above, comprises at least one single domain antibody moiety. Preferably, the CCR8 binder having cytotoxic activity comprises at least two single domain antibody moieties. In a further embodiment of the present invention, the CCR8 binder, as detailed above comprises at least one Fc region moiety and at least two single domain antibody moieties that bind to CCR8. Preferably, the CCR8 binder is a genetically engineered polypeptide that comprises at least one Fc region moiety and at least two single domain antibody moieties that bind to CCR8, joined together by a peptide linker. The amino acid sequence of the Fc region moiety and/or the single domain antibody moiety region(s) may be humanized to reduce immunogenicity for humans.

In particular, the single domain antibody may be a Nanobody^® (as defined herein) or a suitable fragment thereof (Note: Nanobody^®, Nanobodies^® and Nanoclone^® are registered trademarks of Ablynx N.V., a Sanofi Company). For general description of Nanobodies^® reference is made to the further description below, and described in the prior art such as e.g. W02008/020079. “VHH domains”, also known as VHHs, VHH antibody fragments and VHH antibodies, have originally been described as the antigen binding immunoglobulin (Ig) (variable) domain of “ heavy chain antibodies” (i.e. of “antibodies devoid of light chains”; see e.g. Hamers-Casterman et al., Nature 363:446-8 (1993)). The term “VHH domain” has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4- chain antibodies (which are referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VL domains”). For a further description of VHHs and Nanobodies^®, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001 ), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591 , WO 99/37681 , WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301 , EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1433793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551 , WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. As described in these references, Nanobody^® (in particular VHH sequences and partially humanized Nanobody^®) can in particular be characterized by the presence of one or more “Hallmark residues” in one or more of the framework sequences. A further description of the Nanobody^®, including humanization and/or camelization of Nanobody, as well as other modifications, parts or fragments, derivatives or “Nanobody fusions”, multivalent or multispecific constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of the Nanobody^® and their preparations can be found e.g. in WO 08/101985 and WO 08/142164. VHHs and Nanobodies^® are among the smallest antigen binding fragment that completely retains the binding affinity and specificity of a full-length antibody (see e.g. Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh- Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)).

Furthermore, as for full-size antibodies, single variable domains such as VHHs and Nanobodies^® can be subjected to humanization, i.e. increase the degree of sequence identity with the closest human germline sequence. In particular, humanized immunoglobulin single variable domains, such as VHHs and Nanobodies^® may be single domain antibodies in which at least one single amino acid residue is present (and in particular, at least one framework residue) that is and/or that corresponds to a humanizing substitution (as defined further herein). Potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence and the resulting humanized VHH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person.

Humanized single domain antibodies, in particular VHHs and Nanobodies^®, may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. By humanized is meant mutated so that immunogenicity upon administration in human patients is minor or non-existent. The humanizing substitutions should be chosen such that the resulting humanized amino acid sequence and/or VHH still retains the favourable properties of the VHH, such as the antigen-binding capacity. Based on the description provided herein, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring VHH domains on the other hand. Such methods are known by the skilled addressee. A human consensus sequence can be used as target sequence for humanization, but also other means are known in the art. One alternative includes a method wherein the skilled person aligns a number of human germline alleles, such as for instance but not limited to the alignment of IGHV3 alleles, to use said alignment for identification of residues suitable for humanization in the target sequence. Also a subset of human germline alleles most homologous to the target sequence may be aligned as starting point to identify suitable humanisation residues. Alternatively, the VHH is analyzed to identify its closest homologue in the human alleles, and used for humanisation construct design. A humanisation technique applied to Camelidae VHHs may also be performed by a method comprising the replacement of specific amino acids, either alone or in combination. Said replacements may be selected based on what is known from literature, are from known humanization efforts, as well as from human consensus sequences compared to the natural VHH sequences, or the human alleles most similar to the VHH sequence of interest. As can be seen from the data on the VHH entropy and VHH variability given in Tables A-5-A-8 of WO 08/020079, some amino acid residues in the framework regions are more conserved between human and Camelidae than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions. For instance, a human-like class of Camelidae single domain antibodies contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by other substitutions at position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation. Indeed, some Camelidae VHH sequences display a high sequence homology to human VH framework regions and therefore said VHH might be administered to patients directly without expectation of an immune response therefrom, and without the additional burden of humanization.

Suitable mutations, in particular substitutions, can be introduced during humanization to generate a polypeptide with reduced binding to pre-existing antibodies (reference is made for example to WO 2012/175741 and WO2015/173325), for example at at least one of the positions: 11 , 13, 14, 15, 40, 41 , 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, or 108. The amino acid sequences and/or VHH of the invention may be suitably humanized at any framework residue(s), such as at one or more Hallmark residues (as defined below) or at one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof. Depending on the host organism used to express the amino acid sequence, VHH or polypeptide of the invention, such deletions and/or substitutions may also be designed in such a way that one or more sites for posttranslational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation.

In some cases, at least one of the typical Camelidae hallmark residues with hydrophilic characteristics at position 37, 44, 45 and/or 47 is replaced (see W02008/020079 Table A-03). Another example of humanization includes substitution of residues in FR 1 , such as position 1 , 5, 11 , 14, 16, and/or 28; in FR3, such as positions 73, 74, 75, 76, 78, 79, 82b, 83, 84, 93 and/or 94; and in FR4, such as position 10 103, 104, 108 and/or 111 (see W02008/020079 Tables A-05 -A08; all numbering according to the Kabat).

The binders of the present invention may be monospecific, bispecific, or multispecific. “Multispecific binders” may be specific for different epitopes of one target antigen or polypeptide, or may contain antigen-binding domains specific for more than one target antigen or polypeptide (Kufer et al. Trends Biotechnol 22:238-44 (2004)).

In one aspect of the invention, the binder is a monospecific binder. As discussed further below, in an alternative aspect the binder is a bispecific binder.

As used herein, “bispecific binder” refers to a binder having the capacity to bind two distinct epitopes either on a single antigen or polypeptide, or on two different antigens or polypeptides.

Bispecific binders of the present invention as discussed herein can be produced via biological methods, such as somatic hybridization; or genetic methods, such as the expression of a non native DNA sequence encoding the desired binder structure in a cell line or in an organism; chemical methods (e.g. by chemical coupling, genetic fusion, noncovalent associated or otherwise to one or more molecular entities, such as another binder of fragment thereof); or combination thereof.

The technologies and products that allow producing monospecific or bispecific binders are known in the art, as extensively reviewed in the literature, also with respect to alternative formats, binder-drug conjugates, binder design methods, in vitro screening methods, constant regions, post-translational and chemical modifications, improved feature for triggering cancer cell death such as Fc domain engineering (Tiller K and Tessier P, Annu Rev Biomed Eng. 17:191-216 (2015); Speiss C et al., Molecular Immunology 67:95-106 (2015); Weiner G, Nat Rev Cancer, 15:361 -370 (2015); Fan G et al., J Hematol Oncol 8:130 (2015)). As used herein, “epitope” or “antigenic determinant” refers to a site on an antigen to which a binder, such as an antibody, binds. As is well known in the art, epitopes can be formed both from contiguous amino acids (linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of a protein (conformational epitopes). Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes are well known in the art and include, for example, x-ray crystallography and 2-D nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

Non-blocking binders

As discussed above, neutralizing CCR8 binders having cytotoxic activity and leading to the depletion of tumour-infiltrating regulatory T-cells (Tregs) have already been described in the prior art. However, it was further demonstrated that using such binders as therapeutics could lead to systemic side-effects and autoimmunity. Benefits of the binders of the invention may include reduced side effects on the intestinal and/or skin T reg populations. In addition, the non- blocking binders of the invention may include the absence of or a lowered inhibition of dendritic cell migration towards lymph nodes. It has furthermore been observed that Treg depletion using blocking CCR8 binders, especially in combination with checkpoint inhibition such as PD- 1/PD-L1 inhibitors, increases neutrophils in the tumour microenvironment. The non-blocking binders of the invention may have a lesser effect on neutrophil increase, thereby providing a greater anti-tumour efficacy.

The inventors have surprisingly found that CCR8 binders having cytotoxic activity characterized in that the CCR8 binder is a non-blocking binder of CCR8 can nonetheless specifically deplete tumour-infiltrating regulatory T-cells (Tregs), obtaining the same and higher efficacies, while reducing unwanted systemic side effects, as evidenced by the examples below.

A “non-blocking” binder of CCR8 means that it does not block or substantially block the binding of a CCR8 ligand to CCR8, in particular, the binder does not block the binding of at least one ligand selected from CCL1 , CCL8, CCL16, andCCL18 to CCR8, in particular it does not block binding of CCL1 or CCL18 to CCR8, preferably it does not block the binding of CCL1 to CCR8. Blockade of ligand binding to CCR8 may be determined by methods known in the art. Examples thereof include, but are not limited to, the measurement of the binding of a ligand such as CCL1 to CCR8, the migration of CCR8-expressing cells towards a ligand such as CCL1 , increase in intracellular Ca²⁺ levels by a CCR8 ligand such as CCL1 , rescue from dexamethasone-induced apoptosis by a ligand such as CCL1 , and variation in the expression of a gene sensitive to CCR8 ligand stimulation, such as CCL1 stimulation. References to “non- blocking”, “non-ligand blocking”, “does not block” or “without blocking” and the like (with respect to the non-blocking of CCR8 ligand binding to CCR8 in the presence of the CCR8 binder) include embodiments wherein the CCR8 binder of the invention does not block or does not substantially block the signalling of CCR8 ligand via CCR8, in particular the signalling of CCL1 via CCR8. That is, the CCR8 binder inhibits less than 50% of ligand signalling compared to ligand signalling in the absence of the binders. In particular embodiments of the invention as described herein, the CCR8 binder inhibits less than 40%, 35%, 30%, preferably less than about 25% of ligand signalling compared to ligand signalling in the absence of the binders. In a particular embodiment, the percentage of ligand signalling is measured at a CCR8 binder molar concentration that is at least 10, in particular at least 50, more in particular at least 100 times the binding EC50 of the CCR8 binder to CCR8. In another embodiment, the percentage of ligand signalling is measured at a CCR8 binder molar concentration that is at least 10, in particular at least 50, more in particular at least 100 times the molar concentration of the ligand. Non-blocking CCR8 binders allow binding of CCR8 without interfering with the binding of at least one ligand to CCR8, or without substantially interfering with the binding of at least one ligand to CCR8. Ligand signalling, such as CCL1 signalling, via CCR8 may be measured by methods as discussed in the Examples and as known in the art. Comparison of ligand signalling in the presence and absence of the CCR8 binder can occur under the same or substantially the same conditions.

In some embodiments, CCR8 signalling can be determined by measuring the cAMP release. Specifically, CHO-K1 cells stably expressing recombinant (human) CCR8 receptor (such as FAST-065C available from EuroscreenFAST) are suspended in an assay buffer of KRH: 5 mM KCI, 1.25 mM MgS04, 124 mM NaCI, 25 mM HEPES, 13.3 mM Glucose, 1.25 mM KH2P04, 1.45 mM CaCI2, 0.5 g/l BSA, supplemented with 1 mM IBMX. The CCR8 binder is added at a concentration of 100nM and incubated for 30 minutes at 21 °C. A mixture of 5mM forskolin and (human) CCL1 in assay buffer is added to reach a final assay concentration of 5 nM CCL1 . The assay mixture is then incubated for 30 minutes at 21 °C. After addition of a lysis buffer and 1 hour incubation, the concentration of cAMP is measured. cAMP can be measured by e.g. determining fluorescence levels, such as with the HTRF kit from Cisbio using manufacturer assay conditions (catalogue #62AM9PE). A non-blocking binder leads to a change of less than 50% of the amount of cAMP compared to a control that lacks the binder. In particular less than 40%, more in particular less than 30%, such as less than 20%. Preferably, a non-blocking binder leads to a change of less than 10%, more preferably less than 5% of cAMP compared to control.

Techniques for generating non-blocking CCR8 binders are available to the person skilled in the art. As non-limiting example, antibodies can be generated through immunization using CCR8 antigens comprising full length CCR8 or CCR8 fragments and generated antibodies can be screened for the absence of CCR8 blocking activity. In a particular embodiment, antibodies are generated through immunization using CCR8 fragments that are not involved in ligand binding, especially CCL1 binding. Non-blocking antibodies may be obtained through immunization with CCR8 fragments derived from the N-terminal region, in particular the N- terminal extracellular region which is not located between transmembrane domains. Therefore, in a particular embodiment, the binder of the invention binds CCR8 at said N-terminal region of CCR8. In one particular embodiment, the binder binds to the N-terminal region of CCR8 and one or more extracellular loops located between the transmembrane domains of CCR8. In another embodiment, the binder binds to the N-terminal region of CCR8 and doesn’t bind to extracellular loops located between the transmembrane domains of CCR8. In yet another particular embodiment, the binder binds to one or more extracellular loops located between the transmembrane domains of CCR8. In another particular embodiments, the epitope(s) of the binder are located in said N-terminal region. In yet another embodiment, the epitope(s) of the binder are not located in the extracellular loops between the transmembrane domains.

Cytotoxicity

Further according to the invention, the CCR8 binder has cytotoxic activity. “Cytotoxicity” or “cytotoxic activity” as used herein refers to the ability of a binder to be toxic to a cell that it is bound to. As is clear to the skilled person from the description of the invention, any type of cytotoxicity can be used in the context of the invention. Of importance is the ability of the binder of the invention to bind CCR8 in a non-blocking manner and to cause toxicity to the cell that it is bound to. Cytotoxicity can be direct cytotoxicity, wherein the binder itself directly damages the cell (e.g. because it comprises a chemotherapeutic payload) or it can be indirect, wherein the binder induces extracellular mechanisms that cause damage to the cell (e.g. an antibody that induces antibody-dependent cellular activity). More in particular, the binder of the invention can signal the immune system to destroy or eliminate the cell it is bound to or the binder can carry a cytotoxic payload to destroy the cell it is bound to. In particular, the cytotoxic activity is caused by the presence of cytotoxic moiety. Examples of such cytotoxic moieties includes moieties which induce antibody-dependent cellular activity (ADCC), induce complement- dependent cytotoxicity (CDC), induce antibody-dependent cellular phagocytosis (ADCP), bind to and activate T-cells, or comprise a cytotoxic payload. Most preferably, said cytotoxic moiety induces antibody-dependent cellular activity (ADCC).

Antibody-dependent cellular cytotoxicity (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells that express Fc receptors recognize binders on a target cell and subsequently cause lysis of the target cell. Examples of non-specific cytotoxic cells that express Fc receptors include natural killer cells, neutrophils and macrophages.

Complement-dependent cytotoxicity (CDC) refers to the lysis of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a binder complexed with a cognate antigen.

Antibody-dependent cellular phagocytosis (ADCP) refers to a cell-mediated reaction in which phagocytes (such as macrophages) that express Fc receptors recognize binders on a target cell and thereby lead to phagocytosis of the target cell.

CDC, ADCC and ADCP can be measured using assays that are known in the art (Vafa et al. Methods 2014 Jan 1 ;65(1 ):114-26 (2013)).

Binding to and activation of T-cells refers to the binding of a T-cell marker that is distinct from CCR8 and the resulting activation of said T-cell. Activation of the T-cell induces the cytotoxic activity of the T-cell against the cell on which the binder of the invention is bound. Therefore, in a particular embodiment, the binder of the invention binds to CCR8 and binds to and activates T-cells. For example, the cytotoxic moiety may bind to CD3. In a further embodiment, the cytotoxic moiety comprises an antibody or antigen-binding fragment thereof that binds to CD3. Thus, the binder of the invention may bind to CCR8 and CD3. Such a binder binds to intratumoural Tregs and directs the cytotoxic activity of T-cells to these Tregs, thereby depleting them from the tumour environment. In a particular embodiment, the binder of the invention comprises a moiety that binds to CCR8 and a moiety that binds to CD3, wherein at least one moiety is antibody based, particularly wherein both moieties are antibody based. Therefore, in a particular embodiment, the present invention provides a bispecific construct comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 and an antibody or antigen-binding fragment thereof that specifically binds to CD3. A cytotoxic payload refers to any molecular entity that causes a direct damaging effect on the cell that is contacted with the cytotoxic payload. Cytotoxic payloads are known to the persons skilled in the art. In a particular embodiment, the cytotoxic payload is a chemical entity. Particular examples of such cytotoxic payloads include toxins, chemotherapeutic agents and radioisotopes or radionuclides. In a further embodiment, the cytotoxic payload comprises an agent selected from the group consisting of alkylating agents, anthracyclines, cytoskeletal disruptors, epothilones, histone deacetylase inhibitors, inhibitors of topoisomerase I, inhibitors of topoisomerase II, kinase inhibitors, nucleotide analogues and precursor analogues, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids and derivatives, peptide or small molecule toxins, and radioisotopes. Chemical entities can be coupled to proteinaceous inhibitors, e.g. antibodies or antigen-binding fragments, using techniques known in the art. Such coupling can be covalent or non-covalent and the coupling can be labile or reversible.

As is well known in the field, the Fc region of IgG antibodies interacts with several cellular Fey receptors (FcyR) to stimulate and regulate downstream effector mechanisms. There are five activating receptors, namely FcyRI (CD64), FcyRIla (CD32a), FcyRIIc (CD32c), FcyRIIIa (CD16a) and FcyRIIIb (CD16b), and one inhibitory receptor FcyRIIb (CD32b). The communication of IgG antibodies with the immune system is controlled and mediated by FcyRs, which relay the information sensed and gathered by antibodies to the immune system, providing a link between the innate and adaptive immune systems, and particularly in the context of biotherapeutics (Flayes J et al., 2016. J Inflamm Res 9: 209-219).

IgG subclasses vary in their ability to bind to FcyR and this differential binding determines their ability to elicit a range of functional responses. For example, in humans, FcyRIIIa is the major receptor involved in the activation of antibody-dependent cell-mediated cytotoxicity (ADCC) and lgG3 followed closely by lgG1 display the highest affinities for this receptor, reflecting their ability to potently induce ADCC. Whilst lgG2 have been shown to have weaker binding for this receptor binders having the human lgG2 isotype have also been found to efficiently deplete Tregs.

In a preferred embodiment of the invention, the binder of the invention induces antibody effector function, in particular antibody effector function in human. In a particular embodiment, the binder of the invention binds FcyR with high affinity, preferably an activating receptor with high affinity. Preferably the binder binds FcyRI and/or FcyRIla and/or FcyRIIIa with high affinity. Particularly preferably, the binder binds to FcyRIIIa. In a particular embodiment, the binder binds to at least one activating Fey receptor with a dissociation constant of less than about 10^-6 M, 10^-7 M, 10^-8 M, 10^-9 M, 10-¹° M, 1Q-¹¹ M, 1 Q-¹² M or 1 Q-¹³ M. FcyR binding can be obtained through several means. For example, the cytotoxic moiety may comprise a fragment crystallisable (Fc) region moiety or it may comprise a binding part, such as an antibody or antigen-binding part thereof that specifically binds to an FcyR.

Therefore, in one embodiment, the cytotoxic moiety comprises a fragment crystallisable (Fc) region moiety. Within the context of the present invention the term “fragment crystallisable (Fc) region moiety” refers to the crystallisable fragment of an immunoglobulin molecule composed of the constant regions of the heavy chains and responsible for the binding to antibody Fc receptors and some other proteins of the complement system, thereby inducing ADCC, CDC, and/or ADCP activity.

In one embodiment, the Fc region moiety has been engineered to increase ADCC, CDC and/or ADCP activity.

ADCC may be increased by methods that reduce or eliminate the fucose moiety from the Fc moiety glycan and/or through introduction of specific mutations on the Fc region of an immunoglobulin, such as lgG1 (e.g. S298A/E333/K334A, S239D/I332E/A330L or G236A/S239D/A330L/I332E) (Lazar et al. Proc Natl Acad Sci USA 103:2005-2010 (2006); Smith et al. Proc Natl Acad Sci USA 209:6181-6 (2012)). ADCP may also be increased by the introduction of specific mutations on the Fc portion of human IgG (Richards et al. Mol Cancer Ther 7:2517-27 (2008)). Methods for engineering binders for increased ADCC, CDC and ADCP activity have been described in Saunders (Frontiers in Immunology 2019, 1296) and Wang et al. (Protein Cell 2019, 9:63-73).

In a particular embodiment of the present invention, the binder comprising an Fc region moiety is optimized to elicit an ADCC response, that is to say the ADCC response is enhanced, increased or improved relative to other CCR8 binders comprising an Fc region moiety, including those that do not inhibit the binding of CCL1 to CCR8 and, for example, unmodified anti-CCR8 monoclonal antibodies. In a preferred embodiment, the CCR8 binder has been engineered to elicit an enhanced ADCC response.

In a preferred embodiment of the present invention, the binder comprising an Fc region moiety is optimized to elicit an ADCP response, that is to say the ADCP response is enhanced, increased or improved relative to other CCR8 binders comprising an Fc region moiety, including those that do not inhibit the binding of CCL1 to CCR8 and, for example, unmodified anti-CCR8 monoclonal antibodies. In another embodiment, the cytotoxic moiety comprises a moiety that binds to an Fc gamma receptor. More in particular binds to and activates an FcyR, in particular an activating receptor, such as FcyRI and/or FcyRIla and/or FcyRIIIa, especially FcyRIIIa. The moiety that binds to an FcyR may be antibody based or non-antibody based as described herein before. If antibody based, the moiety may bind the FcyR through its variable region.

In a further embodiment, the present invention provides nucleic acid molecules encoding CCR8 binders as defined herein. In some embodiments, such provided nucleic acid molecules may contain codon-optimized nucleic acid sequences. In another embodiment, the nucleic acid is included in an expression cassette within appropriate nucleic acid vectors for the expression in a host cell such as, for example, bacterial, yeast, insect, piscine, murine, simian, or human cells. In some embodiments, the present invention provides host cells comprising heterologous nucleic acid molecules (e.g. DNA vectors) that express the desired binder.

In a particular embodiment, the binder of the invention is administered as a therapeutic nucleic acid. The term “therapeutic nucleic acid” used herein refers to any nucleic acid molecule that have a therapeutic effect when introduced into a eukaryotic organism (e.g., a mammal such as human) and includes DNA and RNA molecules encoding the binder of the invention. As is known to the skilled person, the nucleic acid may comprise elements that induce transcription and/or translation of the nucleic acid or that increases ex and/or in vivo stability of the nucleic acid.

In some embodiments, the present invention provides methods of preparing an isolated CCR8 binder as defined above. In some embodiments, such methods may comprise culturing a host cell that comprises nucleic acids (e.g. heterologous nucleic acids that may comprise and/or be delivered to the host cell via vectors). Preferably, the host cell (and/or the heterologous nucleic acid sequences) is/are arranged and constructed so that the binder is secreted from the host cell and isolated from cell culture supernatants.

Treatment

A CCR8 binder presenting the features as described herein represents a further object of the invention. The CCR8 binder can be used as a medicine. In a further embodiment the invention provides a method for treating a disease in a subject comprising administering a CCR8 binder having cytotoxic activity that does not inhibit the binding of CCL1 to CCR8 or signalling of CCL1 via CCR8. Preferably the disease is a cancer, in particular the treatment of solid tumours. In a preferred embodiment of the present invention, the subject of the aspects of the invention as described herein, is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, hamster, mouse, rat, rabbit, or guinea pig, but most preferably the subject is a human. Thus in all aspects of the invention as described herein the subject is preferably a human.

As used herein, the terms “cancer”, “’’cancerous”, or “malignant” refer to or describe the physiological condition on mammals that is typically characterized by unregulated cell growth.

As used herein, the term “tumour” as it applies to a subject diagnosed with, or suspected of having, a cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size, and includes primary tumours and secondary neoplasms. The terms “cancer”, “malignancy”, “neoplasm”, “tumour” and “carcinoma” can also be used interchangeably herein to refer to tumours and tumour cells that exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In general, cells of interest for treatment include precancerous (e.g. benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. The teachings of the present disclosure may be relevant to any and all tumours.

Examples of tumours include but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More particular examples of such cancers include squamous cell carcinoma, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (HCC), hodgkin's lymphoma, non-hodgkin's lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer.

In one aspect, the tumour involves a solid tumour. Examples of solid tumours are sarcomas (including cancers arising from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, vascular, hematopoietic, or fibrous connective tissues), carcinomas (including tumours arising from epithelial cells), mesothelioma, neuroblastoma, retinoblastoma, etc. Tumours involving solid tumours include, without limitations, brain cancer, lung cancer, stomach cancer, duodenal cancer, esophagus cancer, breast cancer, colon and rectal cancer, renal cancer, bladder cancer, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, mouth cancer, sarcoma, eye cancer, thyroid cancer, urethral cancer, vaginal cancer, neck cancer, lymphoma, and the like. In another particular embodiment, the tumour is selected from the group consisting of breast invasive carcinoma, colon adenocarcinoma, head and neck squamous carcinoma, stomach adenocarcinoma, lung adenocarcinoma (NSCLC), lung squamous cell carcinoma (NSCLC), kidney renal clear cell carcinoma, skin cutaneous melanoma, esophageal cancer, cervical cancer, hepatocellular carcinoma, merkel cell carcinoma, small Cell Lung Cancer (SCLC), classical Hodgkin Lymphoma (cHL), urothelial Carcinoma, Microsatellite Instability-High (MSI- H) Cancer and mismatch repair deficient (dMMR) cancer.

In a further embodiment, the tumour is selected from the group consisting of a breast cancer, uterine corpus cancer, lung cancer, stomach cancer, head and neck squamous cell carcinoma, skin cancer, colorectal cancer, and kidney cancer. In an even further embodiment, the tumour is selected from the group consisting of breast invasive carcinoma, colon adenocarcinoma, head and neck squamous carcinoma, stomach adenocarcinoma, lung adenocarcinoma (NSCLC), lung squamous cell carcinoma (NSCLC), kidney renal clear cell carcinoma, and skin cutaneous melanoma. In one aspect, the cancers involve CCR8 expressing tumours, including but not limited to breast cancer, uterine corpus cancer, lung cancer, stomach cancer, head and neck squamous cell carcinoma, skin cancer, colorectal cancer, and kidney cancer. In one particular embodiment, the tumour is selected from the group consisting of breast cancer, colon adenocarcinoma, and lung carcinoma.

As used herein, the term “administration” refers to the act of giving a drug, prodrug, antibody, or other agent, or therapeutic treatment to a physiological system (e.g. a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplary routes of administration to the human body can be through the mouth (oral), skin (transdermal), oral mucosa (buccal), ear, by injection (e.g. intravenously, subcutaneously, intratumourally, intraperitoneally, etc.) and the like. The term administration of the binder of the invention includes direct administration of the binder as well as indirect administration by administering a nucleic acid encoding the binder such that the binder is produced from the nucleic acid in the subject. Administration of the binder thus includes DNA and RNA therapy methods that result in in vivo production of the binder.

Reference to “treat” or “treating” a tumour as used herein defines the achievement of at least one therapeutic effect, such as for example, reduced number of tumour cells, reduced tumour size, reduced rate to cancer cell infiltration into peripheral organs, or reduced rate of tumour metastasis or tumour growth. As used herein, the term “modulate” refers to the activity of a compound to affect (e.g. to promote or treated) an aspect of the cellular function including, but not limited to, cell growth, proliferation, invasion, angiogenesis, apoptosis, and the like. Positive therapeutic effects in cancer can be measured in a number of ways (e.g. Weber (2009) J Nucl Med 50, 1S-10S). By way of example, with respect to tumour growth inhibition, according to National Cancer Institute (NCI) standards, a T/C<42% is the minimum level of anti-tumour activity. A T/C<10% is considered a high anti-tumour activity level, with T/C (%) = Median tumour volume of the treated/Median tumour volume of the controlxlOO. In some embodiments, the treatment achieved by a therapeutically effective amount is any of progression free survival (PFS), disease free survival (DFS) or overall survival (OS). PFS, also referred to as “Time to Tumour Progression” indicates the length of time during and after treatment that the cancer does not grow, and includes the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease. DFS refers to the length of time during and after treatment that the patient remains free of disease. OS refers to a prolongation in life expectancy as compared to naive or untreated individuals or patients.

Reference to “prevention” (or prophylaxis) as used herein refers to delaying or preventing the onset of the symptoms of the cancer. Prevention may be absolute (such that no disease occurs) or may be effective only in some individuals or for a limited amount of time.

In a preferred aspect of the invention the subject has an established tumour, that is the subject already has a tumour, e.g. that is classified as a solid tumour. As such, the invention as described herein can be used when the subject already has a tumour, such as a solid tumour. As such, the invention provides a therapeutic option that can be used to treat an existing tumour. In one aspect of the invention the subject has an existing solid tumour. The invention may be used as a prevention, or preferably as a treatment in subjects who already have a solid tumour. In one aspect the invention is not used as a preventative or prophylaxis.

In one aspect, tumour regression may be enhanced, tumour growth may be impaired or reduced, and/or survival time may be enhanced using the invention as described herein, for example compared with other cancer treatments (for example standard-of care treatments for the a given cancer).

In one aspect of the invention the method of treatment or prevention of a tumour as described herein further comprises the step of identifying a subject who has tumour, preferably identifying a subject who has a a solid tumour.

The dosage regimen of a therapy described herein that is effective to treat a patient having a tumour may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the therapy to elicit an anti-cancer response in the subject. Selection of an appropriate dosage will be within the capability of one skilled in the art. For example 0.01 , 0.1 , 0.3, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 mg/kg. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).

The binder according to any aspect of the invention or the nucleic acid encoding it as described herein may be in the form of a pharmaceutical composition which additionally comprises a pharmaceutically acceptable carrier, diluent or excipient. As used herein, the term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity. Pharmaceutically acceptable carriers enhance or stabilize the composition or can be used to facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, as is known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329; Remington: The Science and Practice of Pharmacy, 21st Ed. Pharmaceutical Press 2011 ; and subsequent versions thereof). Non limiting examples of said pharmaceutically acceptable carrier comprise any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents.

These compositions include, for example, liquid, semi-solid and solid dosage formulations, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, or liposomes. In some embodiments, a preferred form may depend on the intended mode of administration and/or therapeutic application. Pharmaceutical compositions containing the binder or the nucleic acid of the invention can be administered by any appropriate method known in the art, including, without limitation, oral, mucosal, by-inhalation, topical, buccal, nasal, rectal, or parenteral (e.g. intravenous, infusion, intratumoural, intranodal, subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal, or other kinds of administration involving physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue). Such a formulation may, for example, be in a form of an injectable or infusible solution that is suitable for intradermal, intratumoural or subcutaneous administration, or for intravenous infusion. In a particular embodiment, the binder or nucleic acid is administered intravenously. The administration may involve intermittent dosing. Alternatively, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time, simultaneously or between the administration of other compounds.

Formulations of the invention generally comprise therapeutically effective amounts of a binder of the invention. “Therapeutic levels”, “therapeutically effective amount” or “therapeutic amount” means an amount or a concentration of an active agent that has been administered that is appropriate to safely treat the condition to reduce or prevent a symptom of the condition.

In some embodiments, the binder can be prepared with carriers that protect it against rapid release and/or degradation, such as a controlled release formulation, such as implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used.

Those skilled in the art will appreciate, for example, that route of delivery (e.g., oral vs intravenous vs subcutaneous vs intratumoural, etc) may impact dose amount and/or required dose amount may impact route of delivery. For example, where particularly high concentrations of an agent within a particular site or location (e.g., within a tumour) are of interest, focused delivery (e.g., in this example, intratumoural delivery) may be desired and/or useful. Other factors to be considered when optimizing routes and/or dosing schedule for a given therapeutic regimen may include, for example, the particular cancer being treated (e.g., type, stage, location, etc.), the clinical condition of a subject (e.g., age, overall health, etc.), the presence or absence of combination therapy, and other factors known to medical practitioners.

The pharmaceutical compositions typically should be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the binder in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations as discussed herein. Sterile injectable formulations may be prepared using a non-toxic parenterally acceptable diluent or solvent. Each pharmaceutical composition for use in accordance with the present invention may include pharmaceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers are non-toxic to the subjects at the dosages and concentrations employed. Preferably, such a composition can further comprise a pharmaceutically acceptable carrier or excipient for use in the treatment of cancer that that is compatible with a given method and/or site of administration, for instance for parenteral (e.g. sub-cutaneous, intradermal, or intravenous injection), intratumoural, or peritumoural administration.

While an embodiment of the treatment method or compositions for use according to the present invention may not be effective in achieving a positive therapeutic effect in every subject, it should do so in a using pharmaceutical compositions and dosing regimens that are consistently with good medical practice and statistically significant number of subjects as determined by any statistical test known in the art such as the Student's t-test, the X²-test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), Jonckheere-Terpstra test and the Wilcoxon-test.

Where hereinbefore and subsequently a tumour, a tumour disease, a carcinoma or a cancer is mentioned, also metastasis in the original organ or tissue and/or in any other location are implied alternatively or in addition, whatever the location of the tumour and/or metastasis is.

As discussed herein, the present invention relates to depleting regulatory T cells (T regs). Thus, in one aspect of the invention, treatment with the non-blocking CCR8 binder having cytotoxic activity depletes or reduces regulatory T cells, especially tumour-infiltrating regulatory T cells. In one aspect, the depletion is via ADCC. In another aspect, the depletion is via CDC. In a further aspect, the depletion is via ADCP.

As such, the invention provides a method for depleting regulatory T cells in a tumour in a subject, comprising administering to said subject a non-blocking CCR8 binder having cytotoxic activity. In a preferred embodiment Tregs are depleted in a solid tumour. By “depleted” it is meant that the number, ratio or percentage of Tregs is decreased relative to when the non- blocking CCR8 binder having cytotoxic activity, is not administered. In particular embodiments of the invention as described herein, over about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% of the tumour-infiltrating regulatory T cells are depleted.

As used herein, “regulatory T cells” (“Treg”, “Treg cells”, or “Tregs”) refer to a lineage of CD4+ T lymphocytes specialized in controlling autoimmunity, allergy and infection. Typically, they regulate the activities of T cell populations, but they can also influence certain innate immune system cell types. Tregs are usually identified by the expression of the biomarkers Cd3, CD4, CD25, and CD127 or Foxp3. Naturally occurring T reg cells normally constitute about 5-10% of the peripheral CD4+ T lymphocytes. However, within a tumour microenvironment (i.e. tumour- infiltrating Treg cells), they can make up as much as 20-30% of the total CD4+ T lymphocyte population. Activated human Treg cells may directly kill target cells such as effector T cells and APCs through perforin- or granzyme B-dependent pathways; cytotoxic T-lymphocyte-associated antigen 4 (CTLA4+) Treg cells induce indoleamine 2,3-dioxygenase (IDO) expression by APCs, and these in turn suppress T-cell activation by reducing tryptophan; Treg cells, may release interleukin-10 (IL-10) and transforming growth factor (TQRb) in vivo, and thus directly inhibit T-cell activation and suppress APC function by inhibiting expression of MHC molecules, CD80, CD86 and IL-12. Treg cells can also suppress immunity by expressing high levels of CTLA4 which can bind to CD80 and CD86 on antigen presenting cells and prevent proper activation of effector T cells.

In a preferred embodiment of the present invention the ratio of effector T cells to regulatory T cells in a solid tumour is increased after administration of the binder of the invention. In some embodiments, the ratio of effector T cells to regulatory T cells in a solid tumour is increased to over 5, 10, 15, 20, 40 or 80.

An immune effector cell refers to an immune cell which is involved in the effector phase of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils.

Immune effector cells involved in the effector phase of an immune response express specific Fc receptors and carry out specific immune functions. An effector cell can induce antibody- dependent cell-mediated cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes which express FcaR are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. An effector cell can also phagocytose a target antigen, target cell, or microorganism. As discussed herein, antibodies according to the present invention may be optimised for ability to induce ADCC.

In preferred embodiments, the methods and compositions for depleting Tregs are specific for Tregs with limited to no impact on other T cells. In further embodiments, the methods and compositions of the present invention deplete tumour-infiltrating Tregs to a greater extent than other Tregs. In a further embodiment, the methods and compositions of the present invention deplete tumour-infiltrating Tregs to a greater extent than circulating Tregs. In yet another embodiment, the methods and compositions of the present invention deplete tumour-infiltrating Tregs to a greater extent than normal tissue-infiltrating Tregs, such as intestinal Tregs. Comparing the extent of depletion of cell populations is preferably performed by comparing the percentage decrease of the cell population without and with treatment, such as shown in the examples.

In a further particular embodiment, the methods and compositions of the invention decrease the ratio of Tregs over T-cells, in particular the ratio of Tregs over T-cells in a tumour. In a further embodiment, the methods and compositions of the invention decrease the ratio of T regs over T-cells in the tumour to a greater extent than the ratio of Tregs over T-cells outside of the tumour. In another embodiment, the methods and compositions of the invention decrease the ratio of Tregs over T-cells in the tumour to a greater extent than the ratio of Tregs over T-cells in normal tissue, in particular in intestinal tissue.

In some embodiments, a different agent against cancer may be administered in combination with the binder of the invention via the same or different routes of delivery and/or according to different schedules. Alternatively or additionally, in some embodiments, one or more doses of a first active agent is administered substantially simultaneously with, and in some embodiments via a common route and/or as part of a single composition with, one or more other active agents. Those skilled in the art will further appreciate that some embodiments of combination therapies provided in accordance with the present invention achieve synergistic effects; in some such embodiments, dose of one or more agents utilized in the combination may be materially different (e.g., lower) and/or may be delivered by an alternative route, than is standard, preferred, or necessary when that agent is utilized in a different therapeutic regimen (e.g., as monotherapy and/or as part of a different combination therapy).

In some embodiments, where two or more active agents are utilized in accordance with the present invention, such agents can be administered simultaneously or sequentially. In some embodiments, administration of one agent is specifically timed relative to administration of another agent. For example, in some embodiments, a first agent is administered so that a particular effect is observed (or expected to be observed, for example based on population studies showing a correlation between a given dosing regimen and the particular effect of interest). In some embodiments, desired relative dosing regimens for agents administered in combination may be assessed or determined empirically, for example using ex vivo, in vivo and/or in vitro models; in some embodiments, such assessment or empirical determination is made in vivo, in a patient population (e.g., so that a correlation is established), or alternatively in a particular patient of interest.

In another aspect of the invention, a non-blocking CCR8 binder having cytotoxic activity has improved therapeutic effects when combined with an immune checkpoint inhibitor. A combination therapy with a non-blocking CCR8 binder having cytotoxic activity and an immune checkpoint inhibitor can have synergistic effects in the treatment of established tumours. As such, the interaction between the PD-1 receptor and the PD-L1 ligand may be blocked, resulting in “PD-1 blockade”. In one aspect, the combination may lead to enhanced tumour regression, enhanced impairment or reduction of tumour growth, and/or survival time may be enhanced using the invention as described herein, for example compared with administration of the checkpoint inhibitor alone. Therefore, in a particular aspect of the invention, the present invention provides a CCR8 binder of the invention for use in the treatment of a tumour, wherein the treatment further comprises administration of an immune checkpoint inhibitor.

As used herein, “immune checkpoint” or “immune checkpoint protein” refer to proteins belonging to inhibitory pathways in the immune system, in particular for the modulation of T- cell responses. Under normal physiological conditions, immune checkpoints are crucial to preventing autoimmunity, especially during a response to a pathogen. Cancer cells can alter the regulation of the expression of immune checkpoint proteins in order to avoid immune surveillance.

Examples of immune checkpoint proteins include but are not limited to PD-1 , CTLA-4, BTLA, KIR, CD155, B7H4, VISTA and TIM3, and also 0X40, GITR, 4-1 BB and HVEM. Immune checkpoint proteins may also refer to proteins which bind to other immune checkpoint proteins. Such proteins include PD-L1 , PD-L2, CD80, CD86, HVEM, LLT1 , and GAL9.

“Immune checkpoint protein inhibitor”, “immune checkpoint inhibitor”, or “checkpoint inhibitor” refers to any molecule that can interfere with the signalling and/or protein-protein interactions mediated by an immune checkpoint protein. In one aspect of the invention the immune checkpoint protein is PD-1 or PD-L1 . In a preferred aspect of the invention as described herein the immune checkpoint inhibitor interferes with PD-1/PD-L1 interactions via anti-PD-1 or anti PD-L1 antibodies.

In another particular embodiment, the immune checkpoint is CTLA-4 (also known as CTLA4, cytotoxic T-lymphocyte-associated protein 4 or CD152) and the immune checkpoint inhibitor is an inhibitor of CTLA-4. In a particular embodiment, the binder of the invention is used in the treatment of a tumour, wherein the treatment further comprises administration of a CTLA-4 inhibitor, in particular an anti-CTLA-4 antibody, particularly a blocking anti-CTLA-4 antibody. Anti-CTLA-4 antibodies of the instant invention can bind to an epitope on human CTLA-4 so as to inhibit CTLA-4 from interacting with a human B7 counter-receptor. Because interaction of human CTLA-4 with human B7 transduces a signal leading to inactivation of T-cells bearing the human CTLA-4 receptor, antagonism of the interaction effectively induces, augments or prolongs the activation of T cells bearing the human CTLA-4 receptor, thereby prolonging or augmenting an immune response. Anti-CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811 ,097; 5,855,887; 6,051 ,227; in PCT Application Publication Nos. WO 01/14424 and WO 00/37504; and in U.S. Patent Publication No. 2002/0039581. Each of these references is specifically incorporated herein by reference for purposes of description of anti-CTLA-4 antibodies. An exemplary clinical anti-CTLA-4 antibody is human monoclonal antibody 10D1 as disclosed in WO 01/14424 and U.S. patent application Ser. No. 09/644,668. Antibody 10D1 has been administered in single and multiple doses, alone or in combination with a vaccine, chemotherapy, or interleukin-2 to more than 500 patients diagnosed with metastatic melanoma, prostate cancer, lymphoma, renal cell cancer, breast cancer, ovarian cancer, and HIV. Other anti-CTLA-4 antibodies encompassed by the methods of the present invention include, for example, those disclosed in: WO 98/42752; WO 00/37504; U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95(17):10067-10071 ; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. 58:5301-5304. In certain embodiments, the methods of the instant invention comprise use of an anti-CTLA-4 antibody that is a human sequence antibody, preferably a monoclonal antibody and in another embodiment is monoclonal antibody 10D1. In another particular embodiment, the CTLA-4 inhibitor is ipilimumab or tremelimumab.

PD-1 (Programmed cell Death protein 1), also known as CD279, is a cell surface receptor expressed on activated T cells and B cells. Interaction with its ligands has been shown to attenuate T-cell responses both in vitro and in vivo. PD-1 binds two ligands, PD-L1 and PD- L2. PD-1 belongs to the immunoglobulin superfamily. PD-1 signaling requires binding to a PD- 1 ligand in close proximity to a peptide antigen presented by major histocompatibility complex (MHC) (Freeman, Proc Natl Acad Sci USA 105, 10275-6 (2008)). Therefore, proteins, antibodies or small molecules that prevent co-ligation of PD-1 and TCR on the T cell membrane are useful PD-1 antagonists.

In one embodiment, the PD-1 receptor antagonist is an anti-PD-1 antibody, or an antigen binding fragment thereof, which specifically binds to PD-1 and blocks the binding of PD-L1 to PD-1 . The anti-PD-1 antibody may be a monoclonal antibody. The anti-PD-1 antibody may be a human or humanised antibody. An anti-PD-1 antibody is an antibody capable of specific binding to the PD-1 receptor. Anti-PD-1 antibodies known in the art and suitable for the invention include nivolumab, pembrolizumab, pidilizumab, BMS-936559, and toripalimab.

PD-1 antagonists of the present invention also include compounds or agents that either bind to and/or block a ligand of PD-1 to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or bind directly to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor. In particular PD-1 antagonists include small molecules inhibitors of the PD-1/PD-L1 signaling pathway. Alternatively, the PD-1 receptor antagonist can bind directly to the PD-1 receptor without triggering inhibitory signal transduction and also binds to a ligand of the PD-1 receptor to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and/or amount of ligands that bind to PD- 1 receptor and trigger the transduction of an inhibitory signal, fewer cells are attenuated by the negative signal delivered by PD-1 signal transduction and a more robust immune response can be achieved.

In one embodiment, the PD-1 receptor antagonist is an anti-PD-L1 antibody, or an antigen binding fragment thereof, which specifically binds to PD-L1 and blocks the binding of PD-L1 to PD-1. The anti-PD-L1 antibody may be a monoclonal antibody. The anti-PD-L1 antibody may be a human or humanized antibody, such as atezolizumab (MPDL3280A) or avelumab.

Any aspect of the invention as described herein may be performed in combination with additional therapeutic agents, in particular additional cancer therapies. In particular, the CCR8 binder and, optionally, the immune checkpoint inhibitor according to the present invention may be administered in combination with co-stimulatory antibodies, chemotherapy and/or radiotherapy (by applying irradiation externally to the body or by administering radio- conjugated compounds), cytokine-based therapy, targeted therapy, monoclonal antibody therapy, or any combination thereof.

A chemotherapeutic entity for combination therapy as used herein refers to an entity which is destructive to a cell, that is the entity reduces the viability of the cell. The chemotherapeutic entity may be a cytotoxic drug. A chemotherapeutic agent contemplated includes, without limitation, alkylating agents, anthracyclines, epothilones, nitrosoureas, ethylenimines/methylmelamine, alkyl sulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophylotoxins, enzymes such as L-asparaginase; biological response modifiers such aslFN-g, IL-2, IL-12, and G-CSF; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin, anthracenediones, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,r'-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide.

The additional cancer therapy may be other antibodies or small molecule reagents that reduce immune regulation in the periphery and within the tumour microenvironment, for example molecules that target TGFbeta pathways, IDO (indoleamine deoxigenase), Arginase, and/or CSF1 R.

‘In combination’ or treatments comprising administration of a further therapeutic may refer to administration of the additional therapy before, at the same time as or after administration of any aspect according to the present invention. Combination treatments can thus be administered simultaneous, separate or sequential.

In another embodiment, the invention provides a kit comprising any of the binders as described above. In some embodiments, the kit further contains a pharmaceutically acceptable carrier or excipient of it. In other related embodiments, any of the components of the above combinations in the kit are present in a unit dose, in particular the dosages as described herein. In a yet further embodiment, the kit includes instructions for use in administering any of the components or the above combinations to a subject. In one particular embodiment, the kit comprises a CCR8 binder as described herein and an immune checkpoint inhibitor, such as a PD-1 or PD-L1 inhibitor. The CCR8 binder and the immune checkpoint inhibitor can be present in the same or in a different composition.

In one particular embodiment, the present invention provides a package comprising a binder as described herein, wherein the package further comprises a leaflet with instructions to administer the binder to a tumour patient that also receives treatment with an immune checkpoint inhibitor.

The invention will now be further described by way of the following Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention, with reference to the drawings. EXAMPLES

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not construed as limiting the scope thereof.

Example 1. Generation of CCR8-targeting single domain antibody moieties

CCR8 DNA Immunization

Immunization of llamas and alpacas with CCR8 DNA was performed essentially as disclosed in Pardon E., et al. (A general protocol for the generation of Nanobodies for structural biology, Nature Protocols, 2014, 9(3), 674-693) and Henry K.A. and MacKenzie C.R. eds. (Single- Domain Antibodies: Biology, Engineering and Emerging Applications. Lausanne: Frontiers Media). Briefly, animals were immunized four times at two week intervals with 2 mg of DNA encoding mouse CCR8 inserted into the expression vector pVAX1 (ThermoFisher Scientific Inc., V26020), after which blood samples were taken. Three months later, all animals received a single administration of 2 mg the same DNA, after which blood samples were taken.

Phage display library preparation

Phage display libraries derived from peripheral blood mononuclear cells (PBMCs) were prepared and used as described in Pardon E., et al. (A general protocol for the generation of Nanobodies for structural biology, Nature Protocols, 2014, 9(3), 674-693) and Henry K.A. and MacKenzie C.R. eds. (Single-Domain Antibodies: Biology, Engineering and Emerging Applications. Lausanne: Frontiers Media). The VHH fragments were inserted into a M13 phagemid vector containing MYC and His6 tags. The libraries were rescued by infecting exponentially-growing Escherichia coli TG1 [(F’ traD36 proAB laclqZ DM15) supE thi-1 A(lac- proAB) A(mcrB-hsdSM)5(rK- mK-)] cells followed by surinfection with VCSM13 helper phage.

Phage display libraries were subjected to two consecutive selection rounds on HEK293T cells transiently transfected with mouse CCR8 inserted into pVAX1 followed by CHO-K1 cells transiently transfected with mouse CCR8 inserted into pVAX1 . Polyclonal phagemid DNA was prepared from E. coli TG1 cells infected with the eluted phages from the second selection rounds. The VHH fragments were amplified by means of PCR from these samples and subcloned into an E. co// expression vector, in frame with N-terminal PelB signal peptide and C-terminal FLAG3 and His6 tags. Electrocompetent E. coli TG1 cells were transformed with the resulting VHH-expression plasmid ligation mixture and individual colonies were grown in 96-deep-well plates. Monoclonal VHHs were expressed essentially as described in Pardon E., et al. (A general protocol for the generation of Nanobodies for structural biology, Nature Protocols, 2014, 9(3), 674-693). The crude periplasmic extracts containing the VHHs were prepared by freezing the bacterial pellets overnight followed by resuspension in PBS and centrifugation to remove cellular debris.

Example 2. Screening for CCR8 selection outputs

Recombinant cells expressing CCR8 were recovered using cell dissociated non-enzymatic solution (Sigma Aldrich, C5914-100mL) and resuspended to a final concentration of 1.0 x 10⁶ cells/ml in FACS buffer. Dilutions (1 :5 in FACS buffer) of crude periplasmic extracts containing VHHs were incubated with mouse anti-FLAG biotinylated antibody (Sigma Aldrich, F9291- 1 MG) at 5 pg/ml in FACS buffer for 30 min with shaking at room temperature. Cell suspensions were distributed into 96-well v-bottom plates and incubated with the VFIFI/antibody mixture with one hour with shaking on ice. Binding of VHHs to cells was detected with streptavidin R-PE (Invitrogen, SA10044) at 1 :400 dilution (0.18 pg/ml) in FACS buffer, incubated for 30 minutes in the dark with shaking on ice. Surface expression of mCCR8 on transiently transfected cell lines was confirmed by means of PE anti-mouse CCR8 (Biolegend, 150311 ) antibody at 2 pg/ml.

VHH clones resulting from the mouse CCR8 immunization and selection campaign were screened by means of flow cytometry for binding to HEK293 cells previously transfected with mCCR8 or with N-terminal deletion mouse CCR8 (delta16-3XHA) plasmid DNA, in comparison to mock-transfected control cells. Comparison of the binding (median fluorescent intensity) signal of a given VHH clone across the three cell lines enabled classification of said clone as an N-terminal mouse CCR8 binder (i.e. binding on mCCR8 cells, but not on mouse CCR8 (delta16-3XHA) or control cells) or as an extracellular loop mCCR8 binder (i.e. binding on mCCR8 cells and on mouse CCR8 (delta16-3XHA), but not on control cells).

Example 3. Purification and evaluation of monovalent VHHs

Synthetic DNA fragments encoding CCR8-binding VHHs were subcloned into an E. coli expression vector under control of an IPTG-inducible lac promoter, infra me with N-terminal PelB signal peptide for periplasmic compartment-targeting and C-terminal FLAG3 and His6 tags. Electrocompetent E. coli TG1 cells were transformed and the resulting clones were sequenced. VHH proteins were purified from these clones by IMAC chromatography followed by desalting, essentially as described in Pardon E., etal. (A general protocol for the generation of Nanobodies for structural biology, Nature Protocols, 2014, 9(3), 674-693).

Two purified VHHs (VHH-01 and VHH-06, herein after) obtained from the mouse CCR8 immunization campaign were selected and evaluated by flow cytometry for their binding to mCCR8 as compared with N-terminal deletion mCCR8. The results of this assessment are summarized in Figure 1 . VHH-01 binds to both full-length and N-terminal deletion mouse CCR8 whereas VHH-06 only binds to full-length mouse CCR8.

Example 4. Binding and functional characterization for monovalent VHHs cAMP Homogenous Time Resolved Fluorescence (HTRF) assay

The two selected monovalent VHHs (VHH-01 and VHH-06) were evaluated for their potential to functionally inhibit mouse CCL1 signalling on CHO-K1 cells displaying mouse CCR8 in cAMP accumulation experiments.

CHO-K1 cells stably expressing recombinant mouse CCR8 were grown prior to the test in media without antibiotic and detached by flushing with PBS-EDTA (5 mM EDTA), recovered by centrifugation and resuspended in KHR buffer (5 mM KCI, 1.25 mM MgSC>4, 124 mM NaCI, 25 mM HEPES, 13.3 mM Gluclose, 1.25 mM KH₂P0₄, 1.45 mM CaCI₂, 0.5 g/l BSA, supplemented with 1 mM IBMX). Twelve microliters of cells were mixed with six microliters of VHH (final concentration: 1 mM) in triplicate and incubated for 30 minutes. Thereafter, six microliters of a mixture of forskolin and mouse CCL1 (R&D Systems, 845-TC) was added at a final concentration corresponding to its EC80 value. The plates were then incubated for 30 min at room temperature. After addition of the lysis buffer and 1 hour incubation, fluorescence ratios were measured with the HTRF kit (Cisbio, 62AM9PE) according to the manufacturer’s specification.

At 1 mM, VHH-01 inhibited CCL1 action on cAMP levels, whereas VHH-06 did not alter cAMP levels over the control (PBS). These data indicate that VHH-01 is a blocking binder of CCR8, while VHH-06 is a non-blocking binder.

Ca²⁺ release assay

The potential of VHH-01 to functionally inhibit mouse CCL1 signalling on CHO-K1 cells displaying mCCR8 was further evaluated in Ca²⁺ release experiments.

Recombinant cells (CHO-K1 mt-aequorin stably expressing mouse CCR8) were grown 18 hours in media without antibiotics and detached gently by flushing with PBSEDTA (5 mM EDTA), recovered by centrifugation and resuspended in assay buffer (DMEM/HAM’s F12 with HEPES + 0.1% BSA protease free). Cells were then incubated at room temperature for at least 4 hours with Coelenterazine h (Molecular Probes). Thirty minutes after the first injection of 100 mI of a mixture e of cells and VHHs (final concentration: 1 mM), 100 mI of mouse CCL1 (R&D Systems, 845-TC) was added at a final concentration corresponding to its EC80 value and injected into the mixture. The resulting spectral emission was recorded using a Functional Drug Screening System 6000 (FDSS 6000, Hamamatsu).

VHH-01 indeed led to a strong inhibition of Ca²⁺ release by 94%, confirming that VHH-01 is a blocking binder of CCR8.

Example 5. Synthesis and purification of blocking and non-blocking VHH-Fc fusions

In order to compare the effects of a non-blocking CCR8 binder with a blocking CCR8 binder, two VHH-Fc constructs (VHH-Fc-14 and VHH-Fc-25) were generated by combining anti-CCR8 VHHs to the mouse lgG2a Fc domain, separated by flexible GlySer linkers (10GS). Construct VHH-Fc-25 contains two VHH-06 binders, whereas VHH-Fc-14 contains two VHH-01 binders in addition to two VHH-06 binders. A schematic representation of the VHH-Fc-14 and VHH- Fc-25 constructs is provided in Figure 2. Thus, VHH-Fc-25 is a non-blocking CCR8 binder with cytotoxic activity (ADCC) derived from the Fc domain. VHH-Fc-14 is identical to VHH-Fc-25, except for the additional blocking CCR8 domains.

The constructs were cloned in a pcDNA3.4 mammalian expression vector, in frame with the mouse Ig heavy chain V region 102 signal peptide to direct the expressed recombinant proteins to the extracellular environment. DNA synthesis and cloning, cell transfection, protein production in Expi293F cells and protein A purification were done by Genscript (GenScript Biotech B.V., Leiden, Netherlands).

Example 6. Confirmation of CCR8 binding by VHH-Fc fusions

The multivalent VHH-Fc fusions VHH-Fc-14 and VHH-Fc-25 were evaluated for their ability to bind to mouse CCR8 endogenously expressed on BW5147 cells by means of flow cytometry experiments. Cells were incubated with different concentrations of the multivalent VHH-Fc fusions for 30 minutes at 4°C, followed by two washes with FACS buffer, followed by 30 minutes incubation at 4°C with AF488 goat anti-mouse IgG (Life Technologies, A11029) or AF488 donkey anti-rat IgG (Life Technologies, A21208), followed by two washing steps. Dead cells were stained using TOPR03 (Thermo Fisher Scientific, T3605).

The binding of VHH-Fc-14 and VHH-Fc-25 to mouse CCR8 are highly comparable, with pEC50 values of respectively 9.14 ± 0.39 M (n=6) and9.49 ± 0.17 M (n=3) (mean ± standard deviation).

Example 7. Functional inhibition by blocking and non-blocking VHH-Fc fusions

Apoptosis assay

VHH-Fc-14 and VHH-Fc-25 fusions were compared in an apoptosis assay for their ability to functionally inhibit the action of the agonistic ligand CCL1 .

Dexamethasone induces cell death in mouse lymphoma BW5147 cells that endogenously express CCR8. The dexamethasone-induced cell death can be reversed by addition of the antagonist ligand CCL1 (Van Snick et al., 1996, Journal of immunology, 157, 2570-2576; Louahed et al., 2003, European Journal of Immunology, 33, 494-501 ; Spinetti et al., 2003, Journal of Leukocyte Biology, 73, 201-207; Denis et al., 2012, PLOS One, 7, e34199). 50 mI of cells (seeded at 2.75 x 10⁴ cells/ml in Iscove-Dulbecco’s medium + 10% FBS, 50 mM 2-ME, 1 .25 mM l-glutamine) were incubated with 30 mI of serial dilutions of the VHH-Fc fusions and incubated for 30 minutes at 37°C. Next, a 20 mI mixture of dexamethasone (Sigma-Aldrich, D4902) and human CCL1 (Biolegend, 582706) was added to a final concentration of 10 nM each. After 48 hours incubation at 37°C, cell viability was quantified using the ATPlite 1 -step lit according to the manufacturer’s instructions (Perkin Elmer, 6016736). These results of this assessment are depicted in Figure 3.

The VHH-Fc fusion VHH-Fc-14 that carries both building blocks VHH-01 (blocking) and VHH- 06 (non-blocking) provides strong functional inhibition in the assay with a plC50 value of 9.29 ± 0.22 M (n=9) (mean ± standard deviation). By contrast, the VHH-Fc fusion VHH-Fc-25, carrying two copies of building block VHH-06, does not impart functional inhibition. These data confirm that VHH-Fc-25 is a non-blocking CCR8 binder, while the addition of blocking VHH-01 domains in VHH-Fc-14 introduces blocking activity. cAMP assay

VHH-Fc-14 was tested in the cAMP assay as described in example 4. VHH-Fc-14 provides for a 100% inhibition of the cAMP signal at a concentration of 50 nM and higher, with a plC50 value of 8.54 M, again confirming that it is a blocking CCR8 binder. Example 8: Blocking VHH-Fc fusions affect intestinal Treg levels

In order to study the effects of cytotoxic blocking CCR8 binders on intratumoural and other Treg levels, VHH-Fc-14 was modified to obtain VHH-Fc fusions with increased and abolished ADCC activity. Increased ADCC activity was obtained through a-fucosylation of VHH-Fc-14 (VHH-Fc-43). Alternatively, ADCC activity was abolished in VHH-Fc-14 through insertion of the LALAPG Fc mutations (VHH-Fc-41) (Lo et al., 2017, Journal of Biological Chemistry, 292, 3900-3908). Constructs were cloned in mammalian expression vector pQMCF vector in frame with a secretory signal peptide and transfected to CHOEBNALT85 1 E9 cells, followed by expression, protein A and gel filtration chromatography (lcosagen Cell Factory, Tartu, Estonia). Versions with a-fucosylated N-glycans in the CH2 domain of the Fc moiety were obtained from expressions in a CHOEBNALT85 cell line that carries GlymaxX technology (ProBioGen AG, Berlin, Germany) (lcosagen Cell Factory, Tartu, Estonia). Proteins were 0.22 mm sterile filtrated. Protein concentration was determined by measurement of absorbance at 280 nm and purity was determined by SDS-PAGE and size exclusion chromatography. Endotoxin levels were assessed by LAL test (Charles-River Endochrome). The control, mlgG2a isotype, was purchased from BioXCell. VHH-Fc-41 (pEC50 value of 9.33 M (n=1)) and VHH-Fc-43 (pEC50 value of 9.23 ± 0.17 M (n=2)) bind comparably to CCR8 on BW5147 cells. In addition, both VHH-Fc-41 (plC50 value of 9.51 ± 0.02 M (n=2)) and VHH-Fc-43 (plC50 value of 9.39 ± 0.11 M (n=4)) (mean ± standard deviation) potently inhibit the action of CCL1 in the BW147 apoptosis assay. All values are show as mean ± standard deviation.

To test the effects of these blocking CCR8 VHH-Fc fusions with and without ADCC activity, 3 x 10⁶ cells LLC-OVA cells (200mI) were subcutaneously injected in female C57BL/6 mice (6- 12 weeks). At day 4, mice were treated with 200pg of anti-CCR8 VHH-Fc (VHH-Fc-41 or VHH- Fc-43) or mouse lgG2a (control) once weekly (i.e. day 4, 11) (n_mice /grou_P=5).

At day16 mice were sacrificed and tumour, blood and intestines were harvested from each mouse.

Tumour single cell suspensions were obtained by cutting the tissues in small pieces, followed by treatment with 10 U ml-1 collagenase I, 400 U ml-1 collagenase IV and 30 U ml-1 DNasel (Worthington) for 25 minutes at 37°C. The tissues were subsequently squashed and filtered (70pm). The obtained cell suspensions were removed of red blood cells using erythrocyte lysis buffer (155mM NH4CI, 10mM KHC03, 500mM EDTA), followed by neutralization with RPMI. Blood was depleted of red blood cells through repeated rounds of incubation for 5 minutes in erythrocyte lysis buffer until only leukocytes remained. Intestinal single cell suspensions were prepared as previously described (C. C. Bain, A. Mcl. Mowat, CD200 receptor and macrophage function in the intestine, Immunobiology 217, 643-651 (2012) ). After erythrocyte lysis, the obtained single cell suspensions were resuspended in FACS buffer (PBS enriched with 2% FCS and 2mM EDTA) and counted. All single cell suspensions were pre-incubated with rat anti-mouse CD16/CD32 (2.4G2; BD Biosciences) or anti-human Fc block reagent (Miltenyi) for 15 minutes prior to staining. After washing, the samples were stained with fixable viability dye eFluor506 (eBioscience) (1 :200) for 30 minutes at 4°C and in the dark. Subsequently, the samples were washed and stained for 30 minutes at 4°C and in the dark. The intracellular staining of cytokines/chemokines and transcription factors was done according to the manufacturers protocol (Cat N° 554715; BD Biosciences) and (Cat N° 00- 5523; Invitrogen), respectively. FACS data were acquired using the BD FACSCantoll (BD Biosciences) and analyzed using FlowJo (TreeStar, Inc.).

As is shown in Fig. 4, Tregs are depleted in the tumour by VHH-Fc-43, which is a CCR8 blocking Fc fusion with ADCC activity, while no intratumoural Treg depletion is observed for VHH-Fc-41 , which lacks ADCC activity. No depletion of circulating Tregs was observed for either construct (Fig. 5). Reduced Treg levels, however, were observed in the intestines with both VHH-Fc molecules (with ADCC and without ADCC- functionality), showing that this observed reduction in Treg levels in the intestines is due to functionally blocking CCR8 rather than cytotoxic effects of the CCR8 binder (Fig. 6). This indicates that a non-blocking CCR8 binder with cytotoxic activity is preferred and avoids side effects on Treg populations outside of the tumour environment.

Example 9: Effects of cytotoxic non-blocking CCR8 binders on tumour growth in syngeneic LLC-OVA mouse model

To confirm the efficacy of cytotoxic non-blocking CCR8 binders for tumour treatment, the syngeneic mouse LLC-OVA model was used.

3 x 10⁶ cells LLC-OVA cells (200mI) were subcutaneously injected in female C57BL/6 mice (6- 12 weeks). At day 4, mice were treated with 200pg of anti-CCR8 VHH-Fc (VHH-Fc-14 or VHH- Fc-25) or mouse lgG2a (control) once weekly (i.e. day 4, 11 ) (n_mice /_grou_P=5). Tumours were calipered in two dimensions to monitor growth.

Tumour size, in mm³, was calculated using the following formula:

Tumor Volume = n(w²x l)/6 where w= width and Mength, in mm, of the tumour.

The median tumour size (in mm³) for all the different cohorts is described in Fig. 7.

The cohorts treated with a VFIFI-Fc-14 and VFIFI-Fc-25 showed from day 11 a lower tumour size in comparison with the isotype control. The non-blocking CCR8 binder VFIFI-Fc-25 shows the same efficacy in comparison to blocking CCR8 binder VFIFI-Fc-14. These data show that cytotoxic non-blocking CCR8 binders are efficacious for tumour treatment, while having a safer profile than blocking CCR8 Treg depleters.

Example 10: Effects of cytotoxic non-blocking CCR8 binders on tumour growth in MC38 syngeneic mouse model

To confirm the efficacy of cytotoxic non-blocking CCR8 binders for tumour treatment, the mouse MC38 model was used.

5x10⁵ MC38 cells (1 OOmI) were subcutaneously injected in female C57BL/6J mice (7-9 weeks). At day 7 (tumours average size =118 mm³) mice were sorted into different groups. The different cohorts consist of 10 mice for each condition, and each group of mice was intraperitoneal injected with 200pg of mouse lgG2a (control), VHH-Fc-14 or VHH-Fc-25 biweekly for 3 weeks. Bodyweight and tumour size were measured biweekly.

Tumours were calipered in two dimensions to monitor growth. Tumour size, in mm³, was calculated using the following formula:

Tumor Volume = (w²x l)x 0.52 where w= width and Mength, in mm, of the tumour.

The median tumour size (in mm³) for all the different cohorts is described in Fig. 8. The cohorts treated with a VFIFI-Fc-14 and VFIFI-Fc-25 showed from day 18 a significantly lower tumour size in comparison with the isotype control, leading to tumour stasis or regression in a part of the mice treated with the CCR8 binders with ADCC activity.

Surprisingly, despite the indications in the prior art that CCR8 blockade is important for tumour treatment, the non-blocking CCR8 binder VFIFI-Fc-25 shows the same and even slightly higher efficacy in comparison to blocking CCR8 binder VFIFI-Fc-14. These data show that cytotoxic non-blocking CCR8 binders are efficacious for tumour treatment, while having a safer profile than blocking CCR8 Treg depleters.

Claims

1. A CCR8 binder having cytotoxic activity, wherein said CCR8 binder is a non-blocking binder of CCR8.

2. The CCR8 binder according to claim 1 , wherein the cytotoxic activity is caused by the presence of a cytotoxic moiety that

- induces antibody-dependent cellular cytotoxicity (ADCC),

- induces complement-dependent cytotoxicity (CDC),

- induces antibody-dependent cellular phagocytosis (ADCP),

- binds to and activates T-cells, or

- comprises a cytotoxic payload.

3. The CCR8 binder according to claim 1 or 2, wherein the cytotoxic moiety comprises a fragment crystallisable (Fc) region moiety.

4. The CCR8 binder according to claim 3, wherein the Fc region moiety has been engineered to increase ADCC, CDC, and/or ADCP activity, such as through afucosylation or by comprising an ADCC, CDC and/or ADCP-increasing mutation.

5. The CCR8 binder according to any one of the preceding claims, wherein the CCR8 binder comprises at least one single domain antibody moiety that binds to CCR8.

6. The CCR8 binder according to any one of the preceding claims, wherein the CCR8 binder comprises at least one Fc region moiety and at least two single domain antibody moieties that bind to CCR8.

7. The CCR8 binder according to any one of the preceding claims, wherein the CCR8 binder inhibits signalling of CCR8 by less than 50%.

8. A nucleic acid encoding the CCR8 binder according to any one of the previous claims.

9. The CCR8 binder according to any one of claims 1 to 7, or the nucleic acid according to claim 8, for use as a medicine.

10. The CCR8 binder according to any one of claims 1 to 7, or the nucleic acid according to claim 8, for use in the treatment of a tumour.

11 . The CCR8 binder or the nucleic acid for use according to claim 10, wherein the CCR8 binder comprises (a) an Fc region moiety that has ADCC, CDC and/or ADCP activity, and (b) at least one single domain antibody moiety that binds to CCR8; wherein the CCR8 binder is a non-blocking binder of CCR8.

12. The CCR8 binder or the nucleic acid for use according to claim 10 or 11 , wherein the tumour is selected from the group consisting of a breast cancer, uterine corpus cancer, lung cancer, stomach cancer, head and neck squamous cell carcinoma, skin cancer, colorectal cancer, and kidney cancer.

13. The CCR8 binder or the nucleic acid for use according to any one of claims 9 to 12, wherein administration of the CCR8 binder leads to the depletion of tumour-infiltrating regulatory T-cells (Tregs).

14. The CCR8 binder or the nucleic acid for use according to any one of claims 9 to 13, wherein the treatment further comprises administration of a checkpoint inhibitor.

15. The CCR8 binder or the nucleic acid for use according to claim 14, wherein the checkpoint inhibitor blocks PD-1 or PD-L1 .