CDK inhibitors conjugated to EGFR targeting moieties

Abstract

The invention relates to cancer, and in particular, to novel conjugates, compositions, therapies and methods for treating, preventing or ameliorating cancer. The invention is especially concerned with conjugates comprising a targeting moiety and a CDK inhibitor, and compositions comprising such conjugates, and methods of making the compositions. The invention relates particularly, although not exclusively, to immunoconjugates, such as, antibody drug conjugates (ADC) comprising an antibody or an antigen-binding fragment thereof conjugated to a CDK inhibitor. The invention extends to the use of such conjugates in treating, preventing or ameliorating cancer.

Description

[0001] CDK INHIBITORS CONJUGATED TO EGFR TARGETING MOIETIES

[0002] The present invention relates to cancer, and in particular, to novel conjugates, compositions, therapies and methods for treating, preventing or ameliorating cancer. The invention is especially concerned with conjugates comprising a targeting moiety and a CDK inhibitor, and compositions comprising such conjugates, and methods of making the compositions. The invention relates particularly, although not exclusively, to immunoconjugates, such as, antibody drug conjugates (ADC) comprising an antibody or an antigen-binding fragment thereof conjugated to a CDK inhibitor. The invention extends to the use of such conjugates in treating, preventing or ameliorating cancer.

[0003] Triple-negative breast cancer (TNBC) comprises a heterogeneous disease group defined by lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression, and is often associated with increased genomic instability, high mitotic rates and poor prognosis [1, 2], Historically, treatment options were limited to surgery, adjuvant chemotherapy and radiotherapy. However, recent advancements have led to the approval of several targeted therapies, including the anti-programmed death-ligand 1 (PD-L1) antibody atezolizumab as immunotherapy in combination with nab-paclitaxel chemotherapy for advanced-stage TNBC [3], and olaparib for adjuvant treatment of patients with deleterious or suspected deleterious germline BRCA variants [4],

[0004] Epidermal growth factor receptor (EGFR)-targeting therapies, such as monoclonal antibodies and tyrosine kinase inhibitors, directed to its complex signalling network have been explored in clinical trials

[0013] . The anti-EGFR antibodies cetuximab and panitumumab have shown limited response rates in TNBCs and unselected patient populations [16, 17], The anti-TROP2 antibody drug conjugate (ADC) sacituzumab govitecan, with sacituzumab coupled to topoisomerase I inhibitor SN-38, was recently approved for the treatment of relapsed or metastatic TNBCs, highlighting the promise of ADC therapies [5]. However, challenges remain for patients with intrinsic or acquired resistance-driving mechanisms [6-8].

[0005] ADCs combine the specificity of an antibody with a potent cytotoxic warhead or payload molecule. These properties allow highly selective recognition and killing of malignant cells, while, in principle, sparing healthy cells, depending on the normal tissue distribution of the target, and avoiding systemic exposure to payloads

[0021] , Preclinical anti-tumour activities of EGFR-targeted ADCs bearing auristatin payloads have been evaluated in solid tumours [19, 20, 23]. However, these ADCs carry classical payloads targeting DNA or microtubules, which in principle could impact any proliferating cell and therefore normal organ function, limiting selective anti-tumour effects and therapeutic window.

[0006] The present invention arises from the inventors' work in attempting to overcome the problems associated with the prior art.

[0007] The inventors' work focused on a combined targeting approach against EGFR and Gl / S-phase cell cycle molecules by generating a novel EGFR targeting moiety (e.g. an anti-EGFR antibody) conjugated to a CDK inhibitor to directly attack EGFR-expressing cells and their micro-environment.

[0008] Hence, in a first aspect of the invention, there is provided a conjugate comprising an Epidermal Growth Factor Receptor (EGFR)-targeting moiety, and a cyclin-dependent kinase (CDK) inhibitor.

[0009] Epidermal growth factor receptor or "EGFR" is a member of the ErbB family of receptor tyrosine kinases found in both normal and tumour cells, and represents a known targetable cancer-associated marker that may serve to define patient subgroups potentially suitable for EGFR-directed therapy approaches. Surprisingly, the inventors have found that EGFR levels were significantly higher in triple-negative breast cancer (TNBC) than non-TNBC, and in basal-like PAM50 molecular subtype (Example 1).

[0010] As discussed in Example 5, the inventors have surprisingly shown improved antitumour effects (selectively killing EGFR-expressing cancer cells) for a conjugate comprising an EGFR-targeting moiety and a CDK inhibitor when compared to using the unconjugated targeting moiety with or without free CDK inhibitor (see, for example, Figure 5B). Furthermore, the inventors found that the cancer cells were in fact largely resistant to the use of the EGFR-targeting moiety alone, and when the unconjugated EGFR-targeting moiety was used in combination with free CDK inhibitor, the combination did not potentiate any anti-cancer effects above those of free CDK inhibitor alone. Therefore, the inventors have demonstrated that conjugating the CDK inhibitor to the EGFR-targeting moiety to form an immunoconjugate significantly improved the efficacy of the treatment, which was totally unexpected.

[0011] The EGFR-targeting moiety may comprise an antibody, an antibody fragment, a nucleic acid based molecule, a carbohydrate, a peptide, a modified peptide or a small molecule ligand. In some embodiments, the EGFR-targeting moiety comprises an anti-EGFR antibody, or an antigen-binding fragment thereof. Accordingly, the conjugate may be an Antibody-Drug Conjugate (ADC).

[0012] The skilled person will appreciate that an anti-EGFR antibody of the present invention can be any antibody that selectively binds to EGFR. Typically, the EGFR to which the targeting moiety binds is human EGFR having Database accession number P00533. Examples of EGFR-directed monoclonal antibodies include, for example, Mab A13, cetuximab, Depatuxizumab, Duligotuzumab, Futuximab, GC1118, Imgatuzumab, matuzumab, Necitumumab, Nimotuzumab, panitumumab, Zalutumumab, HumMRl and Tomuzotuximab. Details of these antibodies can be found in Cai et al. ("The Latest Battles Between EGFR Monoclonal Antibodies and Resistant Tumor Cells". Front Oncol. 2020 Jul 24; 10: 1249. doi : 10.3389 / fonc.2020.01249. PMID: 32793499; PMCID: PMC7393266).

[0013] In one embodiment, the antibody or antigen-binding fragment thereof binds to the extracellular region of EGFR. The extracellular region of EGFR comprises four subdomains, I to IV, also referred to as large EGF binding domain 1 (LI), cysteine-rich domain 1 (CR1), L2 and CR2.

[0014] In one embodiment, the antibody or antigen-binding fragment thereof binds to EGFR domain I, EGFR domain II, EGFR domain III and / or EGFR domain IV.

[0015] In one embodiment, the antibody or antigen-binding fragment thereof binds EGFR domain II and / or EGFR domain III.

[0016] In one embodiment, the antibody or antigen-binding fragment thereof binds to EGFR domain II. In such embodiments, the antibody is Depatuxizumab, or an antigenbinding fragment thereof.

[0017] In one embodiment, the antibody or antigen-binding fragment thereof binds to EGFR domain III. EGFR domain III is an extracellular domain. EGFR-extracellular domain III (ECDIII) harbours the binding site for currently approved therapeutic monoclonal antibodies, including cetuximab, panitumumab, nimotuzumab, and necitumumab. EGFR-ECDIII spans residues 310-480 and contains two disulfide bonds (C313 / C338, C446 / C475) that are important for maintaining its structure. Large amounts of EGFR- ECDIII in a soluble and active form could thus accelerate the search for EGFR-binding compounds with anti-cancerous properties. In some embodiments, therefore, the antibody is Cetuximab, Mab A13, Cetuximab, Futuximab, GC1118, Matuzumab, Necitumumab, Nimotuzumab and / or Panitumumab, or an antigen-binding fragment thereof.

[0018] Typically, the antibody is cetuximab or an antigen-binding fragment thereof. The skilled person would appreciate that Cetuximab is, for example, available under the trade name ERBITUX (Merck).

[0019] The invention extends to both whole antibodies, as well as to antigen-binding fragments or regions of the corresponding full-length antibody. The antibody or antigen-binding fragment thereof may be monovalent, divalent or polyvalent. Monovalent antibodies are dimers (HL) comprising a heavy (H) chain associated by a disulphide bridge with a light chain (L). Divalent antibodies are tetramer (H2L3) comprising two dimers associated by at least one disulphide bridge. Polyvalent antibodies may also be produced, for example by linking multiple dimers. The basic structure of an antibody molecule consists of two identical light chains and two identical heavy chains which associate non-covalently and can be linked by disulphide bonds. Each heavy and light chain contains an amino-terminal variable region of about 110 amino acids, and constant sequences in the remainder of the chain. The variable region includes several hypervariable regions, or Complementarity Determining Regions (CDRs), that form the antigen-binding site of the antibody molecule and determine its specificity for the antigen or variant or fragment thereof (e.g. an epitope). On either side of the CDRs of the heavy and light chains is a framework region, a relatively conserved sequence of amino acids that anchors and orients the CDRs. Antibody fragments may include a bi-specific antibody (BsAb) or a chimeric antigen receptor (CAR).

[0020] The constant region consists of one of five heavy chain sequences (p, y, , a, or E) and one of two light chain sequences (K or A). The heavy chain constant region sequences determine the isotype of the antibody and the effector functions of the molecule.

[0021] The antibody or antigen-binding fragment thereof may be isolated or purified.

[0022] In one embodiment, the antibody or antigen-binding fragment thereof comprises a polyclonal antibody, or an antigen-binding fragment thereof. The antibody or antigenbinding fragment thereof may be generated in a rabbit, mouse or rat. In another embodiment, the antibody or antigen-binding fragment thereof comprises a monoclonal antibody or an antigen-binding fragment thereof. Typically, the antibody is a human antibody. As used herein, the term "human antibody" can mean an antibody, such as a monoclonal antibody, which comprises substantially the same heavy and light chain CDR amino acid sequences as found in a particular human antibody exhibiting immunospecificity. An amino acid sequence, which is substantially the same as a heavy or light chain CDR, exhibits a considerable amount of sequence identity when compared to a reference sequence. Such identity is definitively known or recognizable as representing the amino acid sequence of the particular human antibody. Substantially the same heavy and light chain CDR amino acid sequence can have, for example, minor modifications or conservative substitutions of amino acids.

[0023] The term "human monoclonal antibody" can include a monoclonal antibody with substantially or entirely human CDR amino acid sequences produced, for example by recombinant methods such as production by a phage library, by lymphocytes or by hybridoma cells.

[0024] The term "humanised antibody" can mean an antibody from a non-human species (e.g. mouse or rabbit) whose protein sequences have been modified to increase their similarity to antibodies produced naturally in humans.

[0025] The antibody may be a recombinant antibody. The term "recombinant human antibody" can include a human antibody produced using recombinant DNA technology.

[0026] The term "antigen-binding region" can mean a region of the antibody having specific binding affinity for its target antigen or a variant or fragment thereof. Typically, the fragment is an epitope. The binding region may be a hypervariable CDR or a functional portion thereof. The term "functional portion" of a CDR can mean a sequence within the CDR which shows specific affinity for the target antigen. The functional portion of a CDR may comprise a ligand which specifically binds to the target antigen or a fragment thereof.

[0027] The term "CDR" can mean a hypervariable region in the heavy and light variable chains. There may be one, two, three or more CDRs in each of the heavy and light chains of the antibody. Normally, there are at least three CDRs on each chain which, when configured together, form the antigen-binding site, i.e. the three-dimensional combining site with which the antigen binds or specifically reacts. It has however been postulated that there may be four CDRs in the heavy chains of some antibodies. The definition of CDR also includes overlapping or subsets of amino acid residues when compared against each other. The exact residue numbers which encompass a particular CDR or a functional portion thereof will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0028] The term "functional fragment" of an antibody can mean a portion of the antibody which retains a functional activity. A functional activity can be, for example antigen binding activity or specificity. A functional activity can also be, for example, an effector function provided by an antibody constant region. The term "functional fragment" is also intended to include, for example, fragments produced by protease digestion or reduction of a human monoclonal antibody and by recombinant DNA methods known to those skilled in the art. Human monoclonal antibody functional fragments include, for example, individual heavy or light chains and fragments thereof, such as VL, VH and Fd; monovalent fragments, such as Fv, Fab, and Fab', single domain Ab, bivalent fragments such as F(ab')2, single chain Fv (scFv), bivalent or trivalent scFvs, scFv-Fc- scFv, an IgG-scFv, an IgG-dAb, KIH-IgG, kl-BODY, KIH-Fc Fab / scFv, tri / tetraspecific, KIH tri / tetra-specific, or a tandem scFv, bispecific antibody (BsAb), and Fc fragments. The Fc fragment of the antibody may be disabled by introducing amino acid substitutions into the Fc region, which silence or reduce the effector function of the antibody.

[0029] Thus, the antigen-binding domain may comprise a domain selected from the group consisting of: an antibody or antigen-binding fragment thereof, CDR, VL, VH and Fd; Fv, Fab, and Fab'; bivalent fragments such as F(ab')2; single chain Fv (scFv); bivalent or trivalent scFvs, scFv-Fc-scFv, an IgG-scFv, an IgG-dAb, KIH-IgG, kl-BODY, KIH-Fc Fab / scFv, tri / tetraspecific, KIH tri / tetra-specific, or a tandem scFv, bispecific antibody (BsAb), and Fc fragments.

[0030] The term "VL fragment" can mean a fragment of the light chain of a human monoclonal antibody which includes all or part of the light chain variable region, including the CDRs. A VL fragment can further include light chain constant region sequences. The term "VH fragment" can means a fragment of the heavy chain of a human monoclonal antibody which includes all or part of the heavy chain variable region, including the CDRs.

[0031] The term "Fd fragment" can mean the heavy chain variable region coupled to the first heavy chain constant region, i.e. VH and CH-1. The "Fd fragment" does not include the light chain, or the second and third constant regions of the heavy chain.

[0032] The term "Fv fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains. The variable regions of the heavy and light chains include, for example, the CDRs. For example, an Fv fragment includes all or part of the amino terminal variable region of about 110 amino acids of both the heavy and light chains.

[0033] The term "Fab fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than an Fv fragment. For example, a Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains. Thus, a Fab fragment additionally includes, for example, amino acid residues from about 110 to about 220 of the heavy and light chains.

[0034] The term "Fab1fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment can additionally include some or all of amino acid residues 220 to 330 of the heavy chain.

[0035] The term "F(ab')2 fragment" can mean a bivalent antigen-binding fragment of a human monoclonal antibody. An F(ab')2 fragment includes, for example, all or part of the variable regions of two heavy chains-and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.

[0036] The term "single chain Fv (scFv)" can mean a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide.

[0037] The term "bispecific antibody (BsAb)" can mean a bispecific antibody comprising two scFv linked to each other by a shorter linked peptide. One skilled in the art knows that the exact boundaries of a fragment of an antibody are not important, so long as the fragment maintains a functional activity. Using well- known recombinant methods, one skilled in the art can engineer a polynucleotide sequence to express a functional fragment with any endpoints desired for a particular application. A functional fragment of the antibody may comprise or consist of a fragment with substantially the same heavy and light chain variable regions as the human antibody.

[0038] The antigen-binding fragment thereof may comprise or consist of any one of the antigen binding region sequences of the VL, any one of the antigen binding region sequences of the VH, or a combination of VL and VH antigen binding regions of a human antibody. The appropriate number and combination of VH and VL antigen binding region sequences may be determined by those skilled in the art depending on the desired affinity and specificity and the intended use of the antigen-binding fragment. Functional fragments or antigen-binding fragments of antibodies may be readily produced and isolated using methods well known to those skilled in the art. Such methods include, for example, proteolytic methods, recombinant methods and chemical synthesis. Proteolytic methods for the isolation of functional fragments comprise using human antibodies as a starting material. Enzymes suitable for proteolysis of human immunoglobulins may include, for example, papain, and pepsin. The appropriate enzyme may be readily chosen by one skilled in the art, depending on, for example, whether monovalent or bivalent fragments are required. For example, papain cleavage results in two monovalent Fab' fragments that bind antigen and an Fc fragment. Pepsin cleavage, for example, results in a bivalent F (ab1) fragment. An F (ab')2 fragment of the invention may be further reduced using, for example, DTT or 2-mercaptoethanol to produce two monovalent Fab' fragments.

[0039] Functional or antigen-binding fragments of antibodies produced by proteolysis may be purified by affinity and column chromatographic procedures. For example, undigested antibodies and Fc fragments may be removed by binding to protein A. Additionally, functional fragments may be purified by virtue of their charge and size, using, for example, ion exchange and gel filtration chromatography. Such methods are well known to those skilled in the art.

[0040] The antibody or antigen-binding fragment thereof may be produced using techniques well known in the art. For example, by recombinant methodology (see US Pat. No. 4,816,567), hydridoma technology (Kohler et. al., Nature, 1975, 256, 495), phage display technologies (for example, see Clackson et. al., Nature, 1991, 352, 624 and Marks et. al., J. Mol. Biol., 1991, 222, 581), synthetic technologies or combinations of such technologies. Normally, one initially isolates a polynucleotide encoding desired regions of the antibody heavy and light chains. Such regions may include, for example, all or part of the variable region of the heavy and light chains. Generally, such regions can particularly include the antigen binding regions of the heavy and light chains, ideally the antigen binding sites, most typically the CDRs.

[0041] The polynucleotide encoding the antibody or antigen-binding fragment thereof according to the invention may be produced using methods known to those skilled in the art. The polynucleotide encoding the antibody or antigen-binding fragment thereof may be directly synthesized by methods of oligonucleotide synthesis known in the art. Alternatively, smaller fragments may be synthesized and joined to form a larger functional fragment using recombinant methods known in the art. Antibodies of use may be commercially obtained from a wide variety of known sources e.g. the American Type Culture Collection (ATCC, Manassas, Va.). A large number of antibodies against a wide variety of disease targets and tumour-associated antigens have been deposited at the ATCC and / or have published variable region sequences and are available for use in the claimed methods and compositions.

[0042] Cysteine-engineered antibodies have been designed as Fab antibody fragments (ThioFab) and expressed as full-length IgG monoclonal (thioMab) antibodies (US. Pat. 7,521,541). ThioFab and ThioMab antibodies have been conjugated through linkers at the newly introduced cysteine thiols to prepare site-specific antibody-drug conjugates (US. Pat. 7521541, US2008 / 0050310, W02008 / 052187).

[0043] Polytherics have described a method for bridging a pair of sulfhydryl groups contained in antibody proteins derived from reduction of a native disulfide hinge (Badescu et. al., Bioconjugate Chem., 2014, 25, 1124-1136) to synthesise homogenous drug-loaded ADCs. Similar methods have been described by Concords (US Patent 0105540, April 26 2015), Thiologics (Schumacher et. al., Org Biomol. Chem., 2014, 12, 7261-7269) and Igenica (Behrens et. al., Mol. Pharm., 2015, 12, 3986-3998). Related methods have been described in Frigerio et. al., Curr. Top. Med. Chem., 2018, 18, 1-32.

[0044] Other methods that have been used to target homogeneous drug-loaded ADCs include the incorporation of unnatural amino acids such as selenocysteine (Hofer, T. et. al., Biochem., 2009, 48, 12047-12057) or formyl glycine (Drake, P.M. et. al., Bioconj. Chem., 2014, 25, 1331-1341) groups into antibodies. Glycoengineering has been used to introduce sialic acid residues at specific sites (Zhou, Q. et. al., Bioconj. Chem., 2014, 25, 510-520) and transglutaminases used to enzymatically conjugate primary amine-containing linker / payloads to glutamine residues (Dorywalska, M. et. al., Bioconj. Chem., 2015, 26, 650-659). These, and other, methods are described in Sochaj, A.M. et. al., Biotech. Adv., 2015, 33, 775-784.

[0045] As used herein, the term "immunospecificity" can mean the binding region is capable of immunoreacting with the target antigen, or a variant or fragment thereof, by specifically binding therewith. The antibody or antigen-binding fragment thereof can selectively interact with an antigen with an affinity constant of approximately IO-5to 1013M1, typically IO-6to IO-9M1, even more typically, IO-10to 1012M1.

[0046] The term "immunoreact" can mean the binding region is capable of eliciting an immune response upon binding with the target antigen, or an epitope thereof.

[0047] The term "epitope" can mean any region of an antigen with the ability to elicit, and combine with, a binding region of the antibody or antigen-binding fragment thereof. The epitope may be linear. This can mean that the antibody interacts with a plurality of continuous amino acids of the antigen, and so the epitope can consist of these defined amino acids. Alternatively, the epitope may be conformational, i.e. non-linear or discontinuous. This can mean that the antibody interacts with multiple, distinct segments from the primary amino acid sequence of the antigen.

[0048] Thus, the antibody or antigen-binding fragment thereof may comprise a heavy chain. The heavy chain may be selected from the group consisting of IgA; IgD; IgE; IgG and IgM.

[0049] In one embodiment, the heavy chain is an IgG. In one embodiment, the heavy chain is IgGl, IgG2, IgG3 and / or IgG4. The heavy chain may be IgG4.

[0050] Typically, however, the heavy chain is IgGl.

[0051] In another embodiment, the heavy chain is an IgA.

[0052] In one embodiment, the targeting moiety may comprise a nucleic acid based molecule which targets EGFR. The nucleic acid based molecule may be an aptamer. Aptamers are nucleic acid or peptide molecules that assume a specific, sequence-dependent shape and bind to specific target ligands based on a lock-and-key fit between the aptamer and ligand. Typically, aptamers may comprise either single- or doublestranded DNA molecules (ssDNA or dsDNA) or single-stranded RNA molecules (ssRNA). Peptide aptamers consist of a short variable peptide domain, attached at both ends to a protein scaffold. Aptamers may be used to bind both nucleic acid and non-nucleic acid targets.

[0053] Suitable aptamers may be selected from random sequence pools, from which specific aptamers may be identified which bind to the selected antigen with high affinity. Methods for the production and selection of aptamers having desired specificity are well known to those skilled in the art, and include the SELEX (systematic evolution of ligands by exponential enrichment) process. Briefly, large libraries of oligonucleotides are produced, allowing the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction. Preferred methodologies for producing aptamers include those disclosed in WO 2004 / 042083.

[0054] In an alternative embodiment, the targeting moiety comprises a peptide or a modified peptide, which targets EGFR. In an alternative embodiment, the targeting moiety comprises a carbohydrate or a modified carbohydrate molecule, which targets EGFR. In another embodiment, the target moiety comprises a small molecule ligand, which targets EGFR.

[0055] Cyclin-dependent kinases (CDKs) are protein kinases that play important roles in the cell cycle and transcriptional regulation. In particular, CDK1, CDK2, CDK3, CDK4, CDK5 and CDK6 are directly related to the regulation of cell-cycle events. CDK7 is indirectly related to the regulation of cell-cycle events by phosphorylating CDK2. Targeting dysregulated cancer cell cycle-associated pathways offers advantages over the classical payloads (which target DNA or microtubules) with regards to: a) specific delivery of the inhibitor to tumour cells with a likely perturbed cell cycle transition axis, and b) systemic cytotoxicity reduction.

[0056] In an embodiment, the CDK inhibitor is an inhibitor of a CDK involved in the cell cycle and / or transcriptional regulation. In one embodiment, the CDK inhibitor is a selective inhibitor of CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, and / or CDK7. In one embodiment, the CDK inhibitor is a selective inhibitor of CDK2 and / or CDK7.

[0057] The CDK inhibitor may not be a selective inhibitor of CDK8. The CDK inhibitor may not be a selective inhibitor of CDK9.

[0058] In one embodiment, the CDK inhibitor is a selective inhibitor of CDK2.

[0059] The inventors have surprisingly found an association of EGFR with the early-phase cell cycle genes, and in particular CDK2 and its cyclin partners (cyclin A and cyclin E), upregulated in TNBCs (Examples 1 and 2).

[0060] The skilled person will appreciate that the structural homology between CDK proteins renders the identification of CDK2 inhibitors that do not possess some affinity for other kinases very challenging. It will be appreciated that 'selectivity' or 'potency' of a compound can be determined based on half maximal inhibitory concentration (IC50) values. Low IC50 value means that the drug is selective or potent at low concentrations. The skilled person would appreciate that IC50 values can be measured using well-known cell-based assays, for example, MTT assay.

[0061] Thus, the term "selective inhibitor" can mean that that the inhibitor is more selective and thus has lower IC50 values for one or more CDKs compared to other CDKs. A selective CDK inhibitor may be defined by having a higher selectivity ( / .e. lower IC50 values) for one or more CDKs when compared to one or more different CDKs.

[0062] Accordingly, in one embodiment, the selective CDK2 inhibitor has a higher selectivity for CDK2 over CDK1, CDK4 and / or CDK6.

[0063] In one embodiment, the CDK2 inhibitor is at least 2-fold, 4-fold, 6-fold, or 8-fold selective over CDK1, CDK4 and / or CDK6. In one embodiment, the CDK2 inhibitor is at least 10-fold or 15-fold selective over CDK1, CDK4 and / or CDK6. In one embodiment, the CDK2 inhibitor is at least 20-fold selective over CDK1, CDK4 and / or CDK6.

[0064] Examples of CDK2 inhibitors include, for example, NKT3447 (NIKang Therapeutics), INX-315 (Incyclix Bio), BLU-222 (Blueprint Medicines), PF-07104091 (Pfizer), BG- 6850 (Beigene), Fadraciclib (Cyclacel), ARTS-021 (Allorion Therapeutics), AZD-8421 (AstraZeneca), PF-06873600 (selleckchem), SNS-032 (BMS-387032; selleckchem) and Tagtociclib (PF-07104091; selleckchem). Thus, the CDK2 inhibitor is NKT3447, INX-315, BLU-222, PF-07104091, BG-6850, Fadraciclib, ARTS-021, AZD-8421, PF-06873600, SNS-032 and / or Tagtociclib.

[0065] Typically, the CDK2 inhibitor is SNS-032.

[0066] It will be appreciated that SNS-032 has the structure represented herein as Formula I, as follows:

[0067] SNS-032 is a selective inhibitor of CDK2 with IC50 of 48 nM in cell-free assays and is 10- and 20-fold selective over CDK1 and CDK4, with little effect on CDK6.

[0068] The CDK inhibitor may be conjugated to the targeting moiety (e.g. the antibody or antigen-binding fragment thereof) directly. Alternatively, the CDK inhibitor may be conjugated to the targeting moiety (e.g. the antibody or antigen-binding fragment thereof) via a linker.

[0069] Cetuximab IgGl was stochastically conjugated to the CDK inhibitor SNS-032 payload (Tocris Bioscience) via a MC-Val-Ala-PAB linker (Cambridge Bioscience) to produce a cetuximab-SNS-032 ADC. MC-Val-Ala-PAB is a cleavable ADC linker featuring a maleimide group, a Vai-Ala dipeptide, and a para-aminobenzyl (PAB) spacer.

[0070] Maleimide is a thiol-specific covalent linker which forms disulfide bonds with cysteine residues of proteins. The payload was labelled with the standard stochastic conjugation to cysteine residues that are reduced from the interchain disulfide bonds of the antibody. Val-Ala-PAB is a protease-cleavable linker which is designed for efficient payload release upon proteolysis by cathepsin.

[0071] Thus, the conjugate may comprise a linker by which the targeting moiety is conjugated to the CDK inhibitor.

[0072] The linker may be a cleavable linker. In one embodiment, the linker comprises a maleimide-based linker (see Figure 3C] .

[0073] The inventors have surprisingly shown that conjugation with CDK inhibitors using a maleimide-based linker does not affect an antibody's capacity for endocytosis and late lysosomal localization for payload release (Example 4). Thus, in an embodiment, the conjugate is capable of being endocytosed.

[0074] In one embodiment, the linker comprises a peptide, such as a Val-Ala dipeptide.

[0075] In an embodiment, the linker comprises a Val-Ala maleimide-based linker.

[0076] In some embodiments, the linker comprises a spacer, which may be a paraaminobenzyl (PAB) spacer.

[0077] Typically, therefore, the conjugate of the invention has the structure substantially as represented herein as Formula II, as follows:

[0078] The conjugate may comprise from 1 to 10, 2 to 7, or 3 to 6, or 4 to 5 CDK inhibitors per EGFR-targeting moiety. In some embodiments, therefore, the number of drug / linker moieties conjugated per antibody molecule ranges from 1 to 10. The drug antibody ratio (DAR) is typically from 1 to 10, and may be from 2 to 7, or 3 to 6, or 4 to 5. Typically, the DAR may be about 4.4.

[0079] In some embodiments, therefore, the conjugate comprises an antibody or antigenbiding fragment thereof, conjugated to a CDK2 inhibitor. Typically, the conjugate comprises an ADC comprising cetuximab, or an antigen-biding fragment thereof, conjugated to a CDK2 inhibitor, which is typically SNS-032. One embodiment of the conjugate of the first aspect is as illustrated in Figure 3C.

[0080] The inventors have shown in the Examples that the conjugates of the invention are useful for treating cancer, and in particular triple negative breast cancer.

[0081] Thus, in a second aspect, there is provided the conjugate according to the first aspect, for use as a medicament.

[0082] In addition, in a third aspect, there is provided the conjugate according to the first aspect, for use in treating, ameliorating or preventing cancer.

[0083] In a fourth aspect, there is provided a method of treating, preventing or ameliorating cancer in a subject, the method comprising administering to a subject in need of such treatment, a therapeutically effective amount of the conjugate according to the first aspect.

[0084] The cancer may be a solid tumour or solid cancer. The cancer may be bowel cancer, brain cancer, breast cancer, endometrial cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.

[0085] In one embodiment, the cancer is breast cancer. The breast cancer may be triple negative breast cancer (TNBC). The breast cancer may be basal-like TNBC.

[0086] It will be understood that breast cancer subtypes can be classified using Prediction Analysis of Microarray 50 (PAM50). PAM50 is a 50-gene signature that classifies breast cancer into five molecular intrinsic subtypes: Luminal A, Luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, Basal-like and Normal-like.

[0087] It will be appreciated that the conjugate described herein, may be used in a medicament which may be used in a monotherapy (i.e. use of the conjugate of the first aspect alone), for treating, ameliorating, or preventing cancer. Alternatively, the conjugate may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing cancer, such as breast cancer.

[0088] In one embodiment, the conjugate may be used in combination with a drug that damages DNA. Examples of DNA-damaging compounds used in the treatment of cancer include, for example, cisplatin, carboplatin, oxaliplatin, methotrexate, doxorubicin and daunorubicin. Thus, in one embodiment, the drug that damages DNA is cisplatin, carboplatin, oxaliplatin, methotrexate, doxorubicin and / or daunorubicin.

[0089] Known breast cancer drugs, which may be administered with the conjugate of the invention, include: Capecitabine (Xeloda), Carboplatin, Docetaxel (Taxotere), EC, EC- T, Eribulin (Halaven), Gemcitabine (Gemzar), Paclitaxel (Taxol) and Vinorelbine (Navelbine).

[0090] In one embodiment, the conjugate may be used in combination with drugs that block checkpoint proteins from binding with their partner proteins and thus allowing immune cells to target cancer cells. Accordingly, the conjugate may be used in combination a checkpoint inhibitor. The checkpoint inhibitor may be a programmed cell death protein 1 (PD-1) inhibitor, a programmed death-ligand 1 (PD-L1) inhibitor and / or a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitor. In one embodiment, the checkpoint inhibitor is a PD-1 inhibitor. Typically, the PD-1 inhibitor is Pembrolizumab.

[0091] In one embodiment, the checkpoint inhibitor is a PD-L1 inhibitor. Typically, the PD-L1 inhibitor is avelumab.

[0092] Alternatively, or additionally, the ADC comprising a CDK inhibitor may be used in combination with ionising radiation that damages DNA.

[0093] Medicaments comprising the conjugate described herein may be used in a number of ways. Compositions comprising the conjugate of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use.

[0094] For instance, creams or ointments may be applied to the skin.

[0095] The conjugate may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given. The conjugate according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with the conjugate used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).

[0096] The conjugate and compositions according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment, for example into a cancerous tumour (e.g. breast cancer) or into the blood stream adjacent thereto. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), intradermal (bolus or infusion) or intramuscular (bolus or infusion).

[0097] In an embodiment, the conjugate is administered orally. Accordingly, conjugate may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid.

[0098] It will be appreciated that the amount of the conjugate that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the inhibitor, and whether it is being used as a monotherapy, or in a combined therapy. The frequency of administration will also be influenced by the half-life of the inhibitor within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular conjugate in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the cancer. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

[0099] The conjugate may be administered before, during or after onset of the cancer to be treated. Daily doses may be given as a single administration. However, typically the conjugate is given two or more times during a day, and most preferably twice a day.

[0100] Generally, a daily dose of between O.Olpg / kg of body weight and 500mg / kg of body weight of the conjugate according to the invention may be used for treating, ameliorating, or preventing cancer. More preferably, the daily dose is between O.Olmg / kg of body weight and 400mg / kg of body weight, more preferably between O. lmg / kg and 200mg / kg body weight, and most preferably between approximately Img / kg and lOOmg / kg body weight.

[0101] A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, a slow release device may be used to provide optimal doses of the conjugate according to the invention to a patient without the need to administer repeated doses.

[0102] Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations comprising the conjugate according to the invention and precise therapeutic regimes (such as daily doses of the conjugate and the frequency of administration). The inventors believe that they are the first to describe a pharmaceutical composition for treating cancer, based on the use of the conjugate.

[0103] Hence, in a fifth aspect of the invention, there is provided a pharmaceutical composition comprising the conjugate according to the first aspect, and a pharmaceutically acceptable vehicle.

[0104] The pharmaceutical composition can be used in the therapeutic amelioration, prevention or treatment in a subject of cancer.

[0105] The invention also provides, in a sixth aspect, a process for making the pharmaceutical composition according to the fifth aspect, the process comprising contacting a therapeutically effective amount of the conjugate according to the first aspect, and a pharmaceutically acceptable vehicle.

[0106] A "subject" may be a vertebrate, mammal, or domestic animal. Hence, the conjugate, inhibitor, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications. Typically, however, the subject is a human being.

[0107] A "therapeutically effective amount" of the conjugate is any amount which, when administered to a subject, is the amount of drug that is needed to treat the cancer, such as breast cancer, e.g. TNBC. For example, the therapeutically effective amount of the conjugate used may be from about 0.01 mg to about 800 mg, and preferably from about 0.01 mg to about 500 mg. It is considered that the amount of the conjugate is an amount from about 0.1 mg to about 250 mg, or from about 0.1 mg to about 20 mg.

[0108] A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0109] In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tabletdisintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents (i.e. the conjugate) according to the invention. In tablets, the conjugate may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the conjugate. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

[0110] However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The conjugate according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0111] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The conjugate may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

[0112] The conjugate and compositions of the invention may be administered in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The conjugate used according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0113] All features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0114] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which :-

[0115] Figure 1 shows basal-like / TNBC is associated with upregulated EGFR and Gl / S-phase cell cycle genes. (A) Gene expression analysis of EGFR, cyclin A (CCNA1), cyclin E (CCNE1) and CDK2 were stratified according to their IHC-defined receptor status from five published databases of total 6173 primary tumour samples: Guy's cohort (TNBC vs HER2+ vs ER+, n = 131 vs 32 vs 14), SCAN-B cohort (n = 165 vs 420 vs 2425), METABRIC cohort (n = 101 vs 117 vs 347), TCGA cohort (n = 112 vs 158 vs 426), and ICGC cohort (n = 73 vs 4 vs 182). Cyclin A data in the ICGC breast cancer cohort was unavailable. The cohorts were analyzed by PAM50 classification (Basal-like (Basal), HER2, luminal A (LumA), luminal B (LumB) and normal-like (Normal) : Guy's cohort (n=95, 28, 11, 10, 7), SCAN-B cohort (n=339, 327, 1657, 729, 221), METABRIC cohort (n=237, 181, 483, 383, 93) and TCGA cohort (n=232, 153, 345, 263, 91). PAM50 information from ICGC cohort is unavailable. All P-values are compared against the Basal-like subtype. (B) Expression of the three Gl / S-phase cell cycle genes were compared between low and high EGFR-expressing breast cancer patients based on quartile ranges of gene expression values (EGFR-low, first quartile =Q1; EGFR-high, fourth quartile =Q4). Median-centered gene expression Iog2 values are shown. P- values were determined using a Mann-Whitney U test. Significant P-values are indicated with an asterisk whereas * P < 0.05; ** P < 0.005; *** P < 0.0005. (C) Gene expression was compared between matched pre-treatment and post- neoadjuvant chemotherapy (post-NAC)-resistant TNBC samples (King's College London cohort: (n = 8); Royal Marsden Hospital cohort (n=9); The Netherlands Cancer Institute cohort (n = 10)). (D) EGFR expression was measured by flow cytometry in human peripheral blood mononuclear cells (PBMC) (n = 3) following Fc-receptor blocking solution. Negligible EGFR expression was found on T cells, B cells, monocytes and NK cells.

[0116] Figure 2 shows single-cell RNA sequencing and spatial transcriptomic analyses reveal cellular and spatial co-expression of EGFR with CDK2 / cyclin E in TNBCs. (A) Dimensionality reduction t-SNE map of combined scRNA-seq transcriptomes of total 150290 cells from 27 untreated primary tumours (TNBC, n = 8 samples, 54819 cells; HER2+, n = 6 samples, 31917 cells; ER+, n = 13 samples, 63554 cells) colored by cell cluster

[0034] , EpCAM expression revealed two prominent EpCAM-i- tumour epithelial clusters in each cancer subtype (highlighted by dotted lines). t-SNE plots showing the expression level of EGFR, CCNE1 and CDK2 genes for the same clusters. Red : high expression, gray: not-detected. Overall expression of CCNA1 was too low and was excluded from the analysis. (B) Analyses of transcriptomic data above to evaluate expression of EGFR, CCNE1 and CDK2 genes per patient and for each patient cohort (TNBC, HER2+, ER+). Heatmap (top panel) with color boxes indicating normalized expression level of genes, each column represents a patient tumour. Quantitative analysis (bottom panel) was calculated by the number of cells expressing EGFR, CCNE1 or CDK2, in proportion to EpCAM-i- cells (left panels) or EpCAM + EGFR+ cells (right panels). Higher expression of EGFR and CCNE1 were observed in TNBC compared with HER2+ and ER+ tumours (left panels), while co-expression of CCNE1 and CDK2 with EpCAM + EGFR+ cells were also stronger in TNBC (right panels). P- values were determined by two-tailed unpaired t test with significant P-values indicated with an asterisk, * P < 0.05; ** P < 0.005; *** P < 0.0005. (C) Spatial transcriptomic analysis of EGFR, CCNE1 and CDK2 expression in EpCAM-i- cell clusters of 43 tumour sections from 22 TNBC patients

[0035] , where 26 sections were from 13 treatment-naive patients and 17 sections were from 9 post-NAC residual diseases. Tissue architecture integrity in these sections was confirmed by H&E staining (left). Color scale represents log -transformed normalized gene expression (red highest, blue lowest). Representative spatial-mapping revealed a consistent pattern of EGFR expression in chemo-naive and post-NAC-resistant TNBC patient tumours, and its spatial relationships with CCNE1 and CDK2 co-expression in tumour sections. Venn diagrams illustrate quantitative analyses for the relationships among EGFR, cyclin E and CDK2 co-expression (numbers in black represent the number of spatial clusters for all patients in the cohort, and % marked in red represent the proportion of these spatial clusters within all EpCAM-i- clusters).

[0117] Figure 3 shows stochastic conjugation of cetuximab to CDK inhibitor and ADC internalization in live breast cancer cells. (A) Surface EGFR was evaluated by flow cytometry using unconjugated-cetuximab in a panel of cell lines (TNBC model : MDA- MB-468, HCC1143, HCC1806, MDA-MB-231, HCC1937, SUM149 and CAL51; HER2+ model : SKBR3; ER+ model : MCF7, T47D; non-tumourigenic epithelial cell model: MCF10A; immune cell model : human B lymphocytes RPMI8866, RPMI8226 and monocytic cell line U937; human primary melanocyte model : melanocyte). (B) Gene expression analysis of EGFR mRNA expression from CCLE database showed positive correlation with surface EGFR measured by flow cytometry in Figure 3A (Spearman's rank coefficient, r =0.723). High level of correlation was found between EGFR and cyclin E (r =0.738), but not with cyclin A or CDK2. Non-significant P-values are marked as NS. (C) Top panel : Schematic diagram of stochastic ADC conjugation by antibody reduction with TCEP, then conjugation to SNS-032 payload via maleimide- dipeptide linker MC-Val-Ala-PAB. Middle panel : analytical data of hydrophobic interaction chromatography (HIC) analysis confirm an average DAR of 4.4. Bottom panel : size exclusion chromatography (SEC) trace indicates negligible aggregation of the ADC and minimal free linker-payload (less than 0.8%). (D) Surface Plasmon Resonance (SPR) analysis demonstrated similar binding affinity (KD) for cetuximab (0.73 nM) and ADC (1.28 nM). Isotype IgGl and isotype-ADC showed no measurable binding to human recombinant EGFR. (E) Monitoring internalization of Fabfluor-pH labeled cetuximab, ADC or isotype IgGl control (10 nM) in high and low EGFR- expressing cell models by Incucyte live-cell imaging. Phase and red fluorescence images were captured every hour for 24 hours. Images of internalized antibody display in cytosolic, low pH lysosomal vesicle-associated red fluorescence in cells. Time course data show rapid increase of internalization in EGFR-high cells treated with labeled cetuximab or ADC, but not with isotype control, while EGFR-low models display no / little intracellular fluorescence. Scale bar, 0.2 mm. (F) Cancer cells were seeded in Matrigel for five days allowing formation of spheroids. Fabfluor-pH labeled antibody or ADC (10 nM) were introduced in the Matrigel and time course data showed rapid increase of internalization in EGFR-high MDA-MB-468 and MDA-MB-231, while EGFR- low CAL51 displayed little red fluorescence signals. Low level of internalization was observed for isotype or isotype-ADC controls throughout the 24 hr time course. Scale bar, 0.5 mm. The data represent the mean ± SEM of three independent experiments. *** P < 0.0005 by two-tailed unpaired t test compared to isotype control.

[0118] Figure 4 shows spatiotemporal analysis of ADC internalization in lysosomal clusters and inhibition of cell cycle progression. (A) Images represent monitoring treatment of MDA-MB-468 cells with 10 nM Alexa-Fluor-647-labeled ADC (magenta) for 0, 3 and 24 hr and the colocalization with lysosomes. Following incubation, live MDA-MB-468 and CAL51 cells were stained with low pH lysosome dye (orange) followed by Hoechst 3342 (blue). Scale bar, 5 pm. Very few cell surface binding and uptake of ADC was shown by EGFR-low CAL51 cells at any time-point compared to high EGFR-expressing MDA-MB-468 model. Image J colocalization analysis demonstrated moderate level of colocalization with low pH lysosomes at 3 hr treatment time-point in MDA-MB-468 (cetuximab vs lysosome, Pearson's correlation r =0.342, n=45 cells; ADC vs lysosome, r =0.281, n = 34 cells), while the correlation was strong at 24 hr (cetuximab vs lysosome, r =0.630, n = 64 cells; ADC vs lysosome, r =0.600, n = 28 cells). Right panel white box: zoom-in images showing individual channel of a representative area of colocalization between ADC and lysosomal staining. (B) 3D reconstruction image of a live MDA-MB-468 cell. The Z-stack images showed spatial information of ADC colocalization within lysosome clusters at 24 hr. Top panel : merged Z-stack 3D image; bottom panels: 2D images of individual channel. Scale bar, 10 pm. (C) Confocal images showing high level of colocalization of internalized ADC in lysosome clusters within the endoplasmic reticulum (ER) in close proximity to nucleus. Scale bar, 5 pm. (D) Quantitative analyses on the distribution of cell cycle phases by flow cytometry in MDA-MB-468 and CAL51 cells after 72 hr of cetuximab-ADC treatment, in comparison to free SNS-032 payload, isotype-ADC and unconjugated-cetuximab controls.

[0119] Significant cell cycle inhibition (G1 arrest) was observed with SNS-032 (at 10, 100, 1000 nM) in both TNBC models, while only the high EGFR-expressing MDA-MB-468 demonstrated significant cell cycle inhibition by the ADC (at 10 nM), but not in EGFR- low CAL51 in three independent experiments. ** P < 0.005; *** P < 0.0005 by chi- squared test against untreated control.

[0120] Figure 5 shows anti-EGFR ADC reduces breast cancer cell activities and demonstrated bystander killing effects. (A) Viability of different cell types was determined by MTT assay following 96 hr incubation with SNS-032, cetuximab, isotype-ADC or ADC. Top panel : EGFR-high breast cancer models. Bottom panels: EGFR-low breast cancer models, and EGFR-low immune cell models. (B) Cancer cell viability assessment of MDA-MB-468 showed that addition of SNS-032 does not re-sensitize cells to EGFR inhibition by cetuximab alone, while reduced cancer cell viability was detected only when SNS-032 was conjugated to cetuximab as an ADC, suggesting that inhibition of cancer cell viability was induced by ADC internalization and subsequent drug release within cancer cells. The efficacy of SNS-032 also improve by conjugating the inhibitor as an ADC instead of treating as free drug. (C) Left and middle panel: EGFR expression was measured by flow cytometry and compared between parental and EGFR-knockdown cells using siRNA, and both MDA-MB-468 and MDA-MB-231 demonstrated reduction of surface EGFR levels. Right panel: cell viability was compared between parental and knockdown cells after 96 hr of ADC treatment (10 nM). (D) Confluence of cell growth was measured using live-cell time-lapse imaging with Incucyte microscopy. All five cell lines were treated with isotype control (lOnM), cetuximab (1 or 10 nM) and ADC (1 or lOnM). Representative phase images were shown with EGFR-high MDA-MB-468 model. Scale bar, 0.2 mm. (E) TNBC spheroids in Matrigel were treated with 10 nM cetuximab, ADC or isotype controls and confluence measured for 7 days using Incucyte live-cell microscopy. ADC treated MDA-MB-231 and MDA-MB-468 cells showed reduced spheroid growth, while the ADC had a less potent effect on the low EGFR-expressing CAL51. Scale bar, 0.5 mm. (F) MDA-MB-468 cancer cells were transduced with a lentiviral expression vector encoding a mCherry fluorescent protein tag (mChery-MDA-MB-468). Bystander killing effect of the ADC was accessed by co-cultures of high and low EGFR-expressing cells in a one-to-one ratio. High EGFR-expressing mCherry-MDA-MB-468 and either low EGFR-expressing MCF7 or CAL51 cells were plated alone as mono-culture or as co-culture, and treated with 1 nM ADC or unconjugated-isotype control antibody. Cell count was measured by Incucyte microscopy after washing at 120 hr. No effect of the ADC was seen with MCF7 or CAL51 when plated alone. However, bystander killing effect was measured in both co-cultures with mChery-MDA-MB-468, with significantly reduction of MCF7 and CAL51 cell counts. Scale bar, 0.5 mm. The data represent the mean ± SEM values of three independent experiments. * P < 0.05; ** P < 0.005; *** P < 0.0005 by two- tailed unpaired t test. Figure 6 shows ADC growth inhibition of orthotopic TNBC xenografts in vivo. (A) Staining for EGFR was confirmed using FFPE blocks of human breast cancer cell line xenografts of known EGFR expression pattern (see Figure 3A) (EGFR-high / positive: MDA-MB-468, HCC1143, MDA-MB-231 and SUM 149; EGFR-low: CAL51 and MCF7), in comparison to human normal breast glandular epithelium and normal tonsil. Scale bar, 100 pm. (B) Effects of ADC treatment on tumour growth in vivo, (i) Orthotopic tumour growth of MDA-MB-468 (n=4 mice per group) and CAL51 (n=4 mice per group) xenografts treated with vehicle, SNS-032 (5 mg / kg), cetuximab, isotype-ADC or ADC (7.5 mg / kg, weekly injection for a total of four weeks). One-way ANOVA reports P- value of * P < 0.05; ** P < 0.005. (II) Body weight measurement over the course of treatments. (C) Immunohistochemical (IHC) evaluation of EGFR expression in a TMA consisting of 38 PDX samples established from a total of 35 breast cancer patients (28 TNBC, five ER+ and two HER2+ tumours).. Representative images showing various staining intensities across PDX samples (Scale bar, 100 pm). A pathologist scored the TMA spots for EGFR positivity on a scale of 0 to 3, where: score 0 = no membrane staining; score 1 = weak incomplete membrane staining in >10% tumour cells; score 2 = moderate complete membrane staining in >10% tumour cells or strong complete membrane staining in < 10% tumour cells; score 3 = strong (intense and uniform) complete membrane staining in >10% tumour cells. (D) Pie chart showing out of 38 PDX samples in the TMA: 47.4% (n = 18, grey) of samples were negative for membrane EGFR expression; 39.5% (n = 15, yellow) were score 1; 10.5% (n=4, orange) were score 2; and 2.6% (n = l, red) is highly positive for membrane EGFR expression (score 3). (E) Microarray-based EGFR mRNA expression was compared to membrane EGFR staining positivity tested by IHC staining. * P < 0.05 by two-tailed unpaired t test. (F) Effects of ADC treatment on KCL004 PDX tumour growth in vivo. Left panels, representative tumour images after two doses of isotype-ADC or ADC treatment, where no visible residual tumour can be found in any mice treated with the cetuximab-ADC in the timeframe of the experiment, (i) Orthotopic tumour growth of KCL004 PDX (n = 5 mice per group) treated with isotype-ADC or ADC (10 mg / kg, total two doses). (II) Body weight measurements. One-way ANOVA reports P-value of *** P < 0.0005.

[0121] Examples

[0122] The inventors have investigated a cetuximab-based antibody drug conjugate (ADC) carrying a CDK inhibitor selected based on oncogene dysregulation, alongside patient subgroup stratification, to provide EGFR-targeted delivery. The inventors have surprisingly shown that the ADC inhibited cell cycle progression, induced cytotoxicity against high EGFR-expressing tumour cells and bystander killing of neighbouring EGFR-low tumour cells, but minimal effects on immune cells. Despite carrying a small fraction of the drug, the ADC restricted EGFR-expressing spheroid and cell line / patient-derived xenograft tumour growth.

[0123] Materials & Methods

[0124] Gene expression data of human breast cancers:

[0125] Detailed descriptions of bulk transcriptomic cohorts (Guy's hospital (n = 177), SCAN-B (n = 3273), METABRIC (n = 1380), TCGA (n = 1084), ICGC (n=259) [28-32]), matched baseline-residual TNBC cohort (n = 27)

[0033] , single-cell RNA sequencing (scRNA-seq) cohort (n=27)

[0034] and spatial transcriptomic cohort (n = 16)

[0035] have been published previously.

[0126] Cell culture conditions:

[0127] All breast cancer, immune and non-tumourigenic epithelial cell models were obtained from King's College London (KCL) Breast Cancer Now Unit or St. John's Institute of Dermatology. Cell lines were authenticated by short tandem repeat profiling, and used once tested negative for mycoplasma and used up to 30 passages.

[0128] Flow cytometric analysis:

[0129] Immunofluorescence staining was performed using cetuximab (Merck Serono), followed by FITC-labeled secondary antibody (2BScientific). Propidium Iodide Flow Cytometry Kit (Abeam) was used to monitor cell cycle and acquired using FACSCanto II (BD Biosciences).

[0130] ADC production and characterization:

[0131] Cetuximab was stochastically conjugated to SNS-032 via a maleimide linker (MC-Val- Ala-PAB) to produce cetuximab-SNS-032 ADC, characterized by hydrophobicity interaction chromatography (HIC) with an average 4.4 drug :antibody ratio (DAR).

[0132] The interchain disulfides of cetuximab were partially reduced with the reducing agent TCEP for 90-180 min. The reduced antibody was diluted with 2 mM EDTA-PBS to 2 mg / ml. Linker-payload was dissolved in 10 mM DMSO. Conjugation of the antibodies was achieved by addition of an excess of the linker-payload to a 1 : 1 mixture of the reduced antibody and propylene glycol, at a final protein concentration of 1 mg / ml. The antibody and the linker-payload were incubated at room temperature for 1 hr to form the ADC. Reaction was quenched with an excess of N-acetylmaleimide. The ADC was further diluted 1 : 1 with PBS 3% cyclodextrin, then bound to a Protein A resin. The resin-bound ADC was washed with PBS 3% cyclodextrin to remove excess smallmolecule impurities, then released from the resin. The ADC was formulated through G25 desalting into PBS 3% cyclodextrin and 0.2 pm filtered prior to aliquoting and - 80°C storage.

[0133] Size exclusion chromatography (SEC) was then used to detect aggregation. Surface plasmon resonance (SPR) binding experiments were performed using Biacore T200 (GE Healthcare).

[0134] In vitro cell viability and live-cell imaging:

[0135] Cell viability was detected by CellTiter-96-AQueous reagent (Promega). Internalization assays were carried out using Incucyte S3 Zoom system (Essen Bioscience). Confocal microscopy studies were imaged using Spinning Disk Super-Resolution by Optical Pixel Reassignment (SoRa) confocal microscopy and processed in NIS-Elements.

[0136] Patient-derived xenograft (PDX) tissue microarray (TMA) preparation and immunohistochemical (IHC) analyses of EGFR:

[0137] To generate PDX models, patients were consented (REC number: 13 / LO / 1248, IRAS ID 131133) for sample collection, whereas tumour fragments were orthotopically- implanted into mammary fat pads of NSG® mice (NOD SCID gamma NSG; (NOD.Cg- PrkdcSCIDII2rgtmlWjl / SzJ). Formalin-fixed paraffin-embedded (FFPE) TMA was constructed from 35 patients. IHC staining was performed using EGFR. 113, NCL-L- EGFR antibody (Leica Biosystems). Digital images were collected by NanoZoomer (Hamamatsu) and analyzed by trained histopathologists.

[0138] In vivo xenograft studies:

[0139] Female NSG® mice were orthotopically injected into the mammary fat pad with 2x 106 CAL51 or 1x 106 MDA-MB-468 cells in 50 pl Matrigel (day 1). Once tumour was palpable, mice received intravenous injection of vehicle, SNS-032 (5 mg / kg), cetuximab, isotype-ADC or ADC (7.5 mg / kg), weekly injections for four weeks. For PDX KCL004, mice were implanted with 2mm pieces of viably frozen tumour tissue (from a single mother tumour) into mammary fat pad via a trocar. Once tumours were established, mice were given intravenous injections of isotype-ADC / ADC (10 mg / kg) twice. Tumours were measured with calipers and volumes calculated (nxlengthxwidth2 / 6). Mice were terminated before tumours reached < 525 mm3.

[0140] Statistical analyses: GraphPad Prism and R Statistical Software were used for statistical analyses. Data were presented as mean ± standard error of the mean (SEM) values of three or more independent experiments. P-values < 0.05 were considered significant.

[0141] The inventors investigated whether EGFR may be a potential target for ADC therapy by interrogating gene expression in 6173 primary tumours from five breast cancer datasets. When specimens were stratified by IHC-defined status, in concordance with previous studies [36, 37], EGFR levels were significantly higher in TNBC than non- TNBC, and in basal-like PAM50 molecular subtype (Figure 1A).

[0142] In the same cohorts, the inventors also investigated the key Gl / S-phase cell cycle regulators, namely CDK4 / 6 / cyclin D (Gl-phase), CDK2 / cyclin E (Gl / S-phase transition) and CDK2 / cyclin A (S-phase), which are activated sequentially. Transcriptional regulators CDK7 / cyclin H and CDK9 / cyclin T control RNA polymerase II activity, while CDK7 also actively phosphorylates the CDK2 / cyclin E complex for Gl / S- phase progression

[0038] . Dysregulation of these axes can lead to cancer [26, 27], The inventors found significant upregulation of cyclin A, cyclin E and CDK2 in basal- like / TNBCs (Figure 1A). While CDK4 / 6 are upregulated, their binding partner cyclin D is downregulated. CDK7 / cyclin H and CDK9 / cyclin T are also downregulated or unchanged in basal-like / TNBCs (Data not shown).

[0143] Next, the inventors compared the expression of CDK2 and cyclin A / E between high and low EGFR-expressing specimens based on quartile ranges of expression values. In every cohort, the inventors found higher expression of both cyclins in EGFR-high compared with EGFR-low tumours, while CDK2 expression was significantly higher in the EGFR-high group in TCGA (Figure IB). Given the dysregulation of CDK2 / cyclin A / E axis, CDK2 inhibitors may offer a specific approach as ADC warheads. Moreover, compared with matched pre-treatment samples, EGFR, CCNA1, CCNE1 and CDK2 genes were retained in post-neoadjuvant chemotherapy (post-NAC)-resistant TNBCs (n = 27) (Figure 1C, although cyclin A expression was low across datasets), highlighting that these molecules may be relevant targets in primary as well as in residual disease settings.

[0144] A number of ADCs cause treatment-related hematotoxicity reactions, with doselimiting adverse events such as lymphopenia, neutropenia and thrombocytopenia, caused by apoptosis of megakaryocyte progenitors or disruption of microtubule function during bone marrow mitosis

[0039] . ADC binding to target antigen-expressing immune cells may increase the potential risk for toxicity. The inventors, therefore, investigated EGFR expression on human PBMCs by flow cytometric analysis and unexpectedly found negligible expression on the main immune cell types tested (Figure ID).

[0145] These findings confirm dysregulated expression of CDK2, cyclin A and cyclin E in basal-11 ke / TNBCs, and co-expression with EGFR-high tumours, including post-NAC- resistant TNBCs.

[0146] Example 2: scRNA-sea and spatial transcriptomic analyses reveal co-expression of EGFR with CDK2 / cvclin E in TNBCs

[0147] Next, the inventors explored transcriptional co-expression of these targets within TNBC tumour architecture using published datasets [34, 35]. The inventors applied dimensionality reduction (tSNE) to single-cell transcriptomes of 150290 cells in 27 treatment-naive tumours

[0034] , Cell clustering identified two malignant tumour cell populations defined by EpCAM-i- epithelial cells (Figure 2A). HER2 and ER expression were evaluated as internal controls for the TNBC subtype (Data not shown). The inventors identified EGFR, CDK2 and CCNE1 expression while overall CCNA1 expression was too low and therefore excluded from analysis.

[0148] Distinct cell clusters of EGFR-high and CCNEl-high cells were found predominantly in the EpCAM-i- clusters in TNBC (Figure 2B, left panels), where EGFR was expressed in 23.1± 1.6% of EpCAM-i- cells, compared to 4.4±0.7% in HER2+ and 3.5±0.5% in ER+ tumour cells. CCNE1 was measured in 10.3±0.8% of EpCAM-i- TNBC, compared to 2.0±0.4% in HER2+, and 2.2±0.2% in ER+ cells. When the inventors investigated CCNE1 and CDK2 expression in EpCAM + EGFR+ cells (Figure 2B, right panels), they found higher level of co-expression in TNBC (cyclin E: 15.6± 1.0% versus 3.2± 1.2% in HER2+ and 3.4±0.5% in ER+ cells; CDK2: 22.5± 1.7% versus 4.4±1.1% in HER2+ and 9.3± 1.2% in ER+ cells). Additionally, only CDK4, but not cyclin D or CDK6, showed correlation with EpCAM+EGFR+ cells (Data not shown).

[0149] Next, the inventors interrogated the transcriptional states of spatially-resolved intratumoural cellular populations for their expression of EGFR, CCNE1 and CDK2 by spatial transcriptomic analyses (Figure 2C). The inventors analyzed 43 tumour sections from 22 TNBC patients

[0035] , including treatment-naive and post-NAC residual tumours. Tissue architecture is maintained in these sections and provides context for high-dimension transcriptional measurements within EpCAM-i- clusters. Spatial- mapping revealed a consistent pattern of EGFR expression in chemotherapy-naive patients, and its spatial relationships with CCNE1 and CDK2 co-expression in the same cell clusters. These spatial associations were retained in post-NAC-resistant TNBC showing similar levels of co-expression of EGFR with CDK2 / CCNE1. Moreover, the inventors measured an increase in EGFR expression in post-NAC samples (76.7% of clusters) compared to pre-treatment samples (58.2% of clusters).

[0150] Advanced scRNA-seq and spatial molecular profiling in tumour specimens supported EGFR expression and higher expression of CDK2 / cyclin E in TNBC compared with other breast cancer types across primary and post-NAC-resistant TNBCs, and higher levels of co-expression and spatial colocalization in TNBCs. These data identified EGFR and CDK2 pathways as potential targets of combined therapy for aggressive TNBCs.

[0151] Example 3: Generation of cetuximab-SNS-032 ADC and internalization studies Despite significant EGFR expression in TNBCs, cetuximab does not engender significant direct cell signaling inhibition against TNBC cells. Based on combined high EGFR and CDK2 / cyclin E expression in TNBCs, the inventors aimed to develop an EGFR-targeted approach by developing a cetuximab-based ADC bearing a CDK inhibitor. To identify suitable cellular models, the inventors evaluated EGFR expression in cell lines by flow cytometry using cetuximab (Figure 3A), and confirmed correlation of cell surface protein with mRNA expression levels (r =0.723) (Figure 3B). The inventors also found a strong correlation between EGFR and CCNE1 mRNA levels in these cell line models (r =0.738) (Figure 3B) consistent with the correlation data in patient samples (Figure IB), but no correlation with CCNA1 or CDK2 expression.

[0152] The inventors then generated an ADC by conjugating SNS-032, known to have selective inhibition of CDK2 / 7 / 9 over CDK4 / 6

[0027] , to cetuximab, through a maleimide-based linker, using the antibody as a vehicle to specifically deliver the inhibitor to cancer cells. The payload was labelled with the standard stochastic conjugation to cysteine residues that are reduced from the interchain disulfide bonds of the antibody. HIC analysis confirmed average DAR of 4.4, and SEC analysis indicated negligible ADC aggregation and 0.8% free drug (Figure 3C). Furthermore, SPR studies showed comparable affinities (KD) of the ADC (1.28 nM) and cetuximab (0.78 nM) to EGFR, consistent with published literature

[0040] , while isotype IgGl and isotype-ADC showed no measurable binding (Figure 3D).

[0153] Cetuximab has been reported to internalize into EGFR-expressing cells

[0041] , The inventors aimed to test if the ADC maintained a similar internalization rate after conjugation with the hydrophobic inhibitors. The inventors detected internalization using a pH-sensitive Fabfluor-pH red fluorophore, which displays fluorescence only when sequestered into acidic environments. Incucyte microscopy was employed to enable real-time, kinetic evaluation of internalization. Cetuximab and ADC each displayed comparable and time-dependent increases in cytoplasmic fluorescence, demonstrating EGFR-dependent internalization, with the highest EGFR-expressing model MDA-MB-468 showing rapid internalization rate within the first hour (Figure 3E). Low levels of uptake were also seen in EGFR-low T47D and CAL51, while low levels of target-independent, non-specific endocytosis of the isotype control antibody were also observed in all cell lines in later time points. In concordance with data from the corresponding monolayer cultures, ADC internalization rates in MDA-MB-468 and MDA-MB-231 spheroids were comparable to cetuximab and higher than isotype controls (Figure 3F).

[0154] These data show that cetuximab and the corresponding cetuximab-ADC demonstrated similar cell binding properties and EGFR-dependent internalization into acidic compartments of tumour cells.

[0155] The primary purpose of the ADC is to increase the tumour selectivity and thus relative efficacy versus toxicity of cytotoxic treatments by releasing the payload within the acidic and protease-rich late lysosomal compartment inside tumour cells. Having shown cytoplasmic ADC uptake with low-resolution live-cell imaging, the inventors next monitored the intracellular uptake and fate of the ADC using super-resolution confocal microscopy. Alexa-Fluor-647-labelled ADC (magenta signal) was shown to associate with MDA-MB-468 cell membranes within 15 minutes of time-lapse imaging, then observed to bud inward into endosomes and transported into the cytoplasm. By 45 minutes, fluorescence signal accumulated intracellularly while the signal on the surface membrane weakened, suggesting ADC endocytosis.

[0156] The efficacy of this ADC relies on internalization and release of payloads within acidic intracellular compartments via linker cleaved by proteases in late lysosomes. After demonstrating internalization within an hour in live MDA-MB-468 cells, the inventors investigated if this ADC colocalizes with lysosomes at later stages. Cells were treated with Alexa-Fluor-647-labeled cetuximab / ADC for 3 and 24 hours. BioTracker Lysosome Dye (orange) was used to visualize lysosomes (pH 5.0). High-resolution imaging allowed visualization of individual organelles that contained the ADC (Figure 4A). Very low surface binding and ADC uptake were shown by EGFR-low CAL51 (Data not shown). In MDA-MB-468, correlation analysis showed weak colocalization of cetuximab with lysosomes at 3 hours (r =0.342) or ADC (r =0.281), indicating that the internalized antibodies were within early endosomes but not yet located in acidic compartments for payload release. After 24 hours, the inventors found a strong correlation of lysosome markers with both cetuximab (r =0.630) and ADC (r =0.600). Late lysosomes contain mature cysteine proteases, hence, conjugated cytotoxic payloads are expected to be released at this final stage of endocytosis. 3D reconstruction of Z-stack images confirmed spatial colocalization of ADC in individual vesicles within lysosome clusters (Figure 4B). The inventors further demonstrated that the ADC predominantly accumulated in late lysosomes located in the endoplasmic reticulum (ER) adjacent to the nucleus (Figure 4C).

[0157] These data demonstrate ADC internalization and colocalization within the endosomal and later within the low pH and protease-rich late lysosomal compartment, a critical attribute required for effective release of the payload. These findings suggest similar kinetic patterns for ADC and cetuximab and indicate that conjugation with CDK inhibitors did not affect the antibody's capacity for endocytosis and late lysosomal localization for payload release.

[0158] The ADC payload SNS-032 can be a potent selective inhibitor targeting the CDK2 / cyclin A / cyclin E complexes and inhibit Gl / S-phase progression

[0027] , Treatment with SNS-032 (10, 100 and 1000 nM) significantly induced Gl-phase arrest in MDA- MB-468 and CAL51 (Figure 4D), and proportionally decreased S-phase and G2 / M- phase compared to untreated controls. The ADC induced Gl-phase arrest in EGFR- high MDA-MB-468 at 10 nM, but not in EGFR-low CAL51, indicating specific effects of the ADC. Consistent with the reported resistance of these TNBC cells to anti-EGFR inhibition [18, 22], unconjugated-cetuximab (and isotype-ADC) showed no significant effects on cell cycle distribution at the dosages tested. These findings confirm that the ADC can inhibit cell cycle progression.

[0159] Example 5: ADC restricts cell viability and growth, and exhibits bystander killing effects

[0160] Next, the inventors evaluated whether the ADC could selectively kill EGFR-expressing cells. Despite comparable binding properties between cetuximab and ADC, all ten cell lines tested in viability assays were largely resistant to cetuximab (grey line, up to 500 nM), regardless of EGFR expression (Figure 5A). Isotype-ADC (blue line) exerted cytotoxicity after 96 hr, only, with IC50 much higher than cetuximab-ADC (red line) (MDA-MB-468 IC50 of ADC = 0.79 nM, isotype-ADC or cetuximab = >10000 nM; MDA-MB-231 IC50 of ADC = 6.72 nM, isotype-ADC = 3805 nM, cetuximab was unmeasurable). The IC50 of SNS-032 (black line) ranged from 94.6 - 370.8 nM, consistent with a previous report

[0027] , The inventors furthermore observed modest on-target ADC activity in some low EGFR-expressing (CAL51 and SKBR3) cells. Furthermore, treatment with cetuximab plus free SNS-032 at similar molar concentrations to those in the ADC did not potentiate any anti-cancer effects above those of free inhibitor alone, and further viability reduction was only detected when SNS-032 was conjugated in an ADC format (Figure 5B). The efficacy of SNS-032 was thus improved by conjugating the inhibitor to cetuximab as an ADC, rather than by additional treatment with free drug (IC50 in MDA-MB-468: ADC = 0.79 nM, SNS-032 = 309 nM, SNS-032+cetuximab = 299 nM). These data suggest that inhibition was most likely induced by ADC internalization and subsequent drug release, but not through any inhibitor-potentiated direct Fab-mediated cell signaling effect by cetuximab.

[0161] The inventors generated EGFR-knockdown cells showing >77% decrease in EGFR expression (mean fluorescence intensity). Compared with parental cells, EGFR- knockdown cells showed reduced sensitivity to ADC (Figure 5C), confirming that the ADC performed in an antigen-specific manner.

[0162] Next, the inventors measured cell growth by Incucyte time-lapse imaging for 120 hours (Figure 5D). The ADC impacted the growth of MDA-MB-468 (% confluency: 1 nM, 27±2%; 10 nM, 22±3%) and MDA-MB-231 (1 nM, 13±5%; 10 nM, 11±3%) compared to isotype control-treated cells. In contrast, at the same dose range unconjugated-cetuximab showed little effect (minimum 86% confluency). The ADC showed partial growth inhibition in EGFR-low models MCF7 (1 nM, 79±8%; 10 nM, 56±6%) and CAL51 (1 nM, 62± 13%; 10 nM, 51± 16%). Inhibition effects on non- malignant primary human melanocytes which do not express EGFR were minimal (1 nM, 108± l%; 10 nM, 92±9%) (Figure 3A, flow cytometry data). Monitoring spheroid growth in Matrigel for 7 days showed significant inhibition of MDA-MB-468 and MDA- MB-231 growth by the ADC, but not by isotype-ADC (Figure 5E). There was some on- target ADC uptake in EGFR-low CAL51, but growth inhibition effects of the ADC were not significantly higher than controls.

[0163] In addition to EGFR-expressing cancer cells being targeted, bystander killing effects may contribute to ADC efficacy through the release of the membrane permeable payloads from the internalizing cell following linker cleavage, or by payload release following cancer cell death. The payloads could then be taken up by neighboring cells which do not express sufficient EGFR levels, as a potential mechanism based on our observations of EGFR and CDK2 / cyclin E co-expression and spatial colocalization in TNBC. To interrogate the potential of bystander killing effects, the inventors developed co-cultures of mCherry-transfected MDA-MB-468 (red) with EGFR-low MCF7 / CAL51 (colorless) cells. In mono-cultures, ADC treatment (1 nM) reduced mCherry-MDA-MB- 468 cell count to <25% compared to control-treated wells after 120 hours, while inhibition effects were small in MCF7 and CAL51 (Figure 5F). However, the ADC exhibited significant bystander killing on MCF7 and CAL51 when each was co-cultured with mCherry-MDA-MB-468: MCF7 cell count reduced to 35.1± 13.3% (68.5±14.3% in mono-culture); CAL51 reduced to 20.2±8.1% (84.6±21.7% in mono-culture).

[0164] These data suggest that cetuximab-SNS-032 ADC can impair EGFR-expressing cell and spheroid growth and exert bystander cytotoxicity of neighboring EGFR-low cancer cells.

[0165] Example 6: ADC restricts orthotopic xenograft growth

[0166] Next, the inventors evaluated ADC effects in orthotopic xenografts grown in the mouse mammary fat pad to partly recapitulate the complexity of human disease

[0042] , EGFR expression is a pre-requisite for EGFR-specific ADC therapy. The inventors confirmed EGFR expression in paraffin-embedded xenografts (Figure 6A), in line with FACS evaluations (Figure 3A). In concordance with Human Protein Atlas dataset (proteinatlas.org, accessed on February 2024)

[0043] , the inventors found restricted EGFR expression in normal human breast glandular epithelium and specific staining in the stratified squamous compartment of human tonsil. The inventors measured significant tumour growth delay in ADC-treated mice (day 46: 58.8±8.7 mm3) compared to vehicle (308.1±34.12 mm3), SNS-032 (187.9±34.7 mm3), cetuximab (141.1± 11.2 mm3) or isotype-ADC (193.0±3.4 mm3) for the cetuximab-resistant MDA-MB-468 xenografts (Figure 6Bi). The ADC effect was notable considering that the conjugated SNS-032 dose measured only a molar fraction (1.65%) of that of the free uncoupled inhibitor treatment. None of the treatments induced weight loss (Figure 6Bii) or any signs of overt toxicity. Meanwhile, the ADC did not exert any significant tumour restriction on EGFR-low CAL51 xenografts compared with the same controls.

[0167] To select a patient-derived model for efficacy studies, EGFR expression was examined by IHC on a TMA consisting of 38 PDX samples established from 35 patients (28 TNBC, five ER+ and two HER2+ tumours). Surface EGFR staining was detected in 52.6% of the PDX (score 1 : 39.5% (n = 15); score 2: 10.5% (n=4); score 3: 2.6% (n = 1)) (Figure 6C-6D). PDX surface EGFR scores correlated with corresponding mRNA expression in the same samples (Figure 6E). KCL004 has a score 2 EGFR expression when grown as a PDX. Moreover, when previously-treated in vivo with olaparib, this xenograft subsequently showed resistance, giving rise to residual disease which retained EGFR expression levels (Figure 6C). Two ADC doses of 10 mg / kg given to animals with established parental KCL004 tumours greatly inhibited xenograft growth (Figure 6F). Xenografts in all five mice reduced in size after ADC treatment, and no palpable residual tumour was reformed in the timeframe of the experiment (day 50: isotype-ADC: 106.4±22.7 mm3; ADC: 0±0 mm3).

[0168] These findings demonstrate therapeutic potential of the ADC in xenograft models. Despite bearing only a fraction of the payloads compared to the free drug administered, ADC treatment was significantly more potent than inhibitor alone. These studies suggest that the ADC restricts TNBC tumour growth, and may benefit patients with treatment-resistant disease.

[0169] Discussion

[0170] By selecting a cell cycle-targeted inhibitor, and cetuximab to direct the payload to EGFR-expressing cancer cells, the inventors generated cetuximab-SNS-032 ADC, bearing a small fraction of the drug alone dose required to engender anti-tumour effects, and the inventors evaluated this as a treatment against TNBC. This ADC restricted EGFR-expressing cancer cell growth in vitro and in xenografts, while showing safe in vivo administration and low effects against EGFR-low breast cancer and immune cell models.

[0171] There are twelve ADCs approved for solid tumours and hematological malignancies, with over 100 ADCs at various stages of clinical testing, reflecting a fast-rising interest. However, approved ADCs are broadly based on two classes of payload, tubulin inhibitors (DM-1, DM-4, MMAE, MMAF) that disrupt microtubule formation in the cytosol, and DNA-interactive damaging agents (calicheamicin, DXd, SN-38 and PBD)

[0021] , Here, the inventors have demonstrated an ADC strategy, using a cell cycle inhibitor for targets identified based on the frequency of combination of a target surface antigen expression and druggable oncogenic pathway in selected patient cohorts.

[0172] Cetuximab binds to extracellular domain of EGFR with higher affinity than its ligands (EGF, TGFa, amphiregulin, epiregulin) and is designed to block the autophosphorylation of its tyrosine kinase-dependent signaling pathway

[0013] . Anti- EGFR antibodies have shown limited activity in TNBCs that are not exclusively dependent on EGFR signaling for survival [18, 22], An alternative ADC approach that does not depend on antibody-mediated inhibition of EGFR signaling could help redefine EGFR as a therapeutic target for patient populations who do not respond to cetuximab.

[0173] The inventors surprisingly found that EGFR levels alongside those of CDK2 and its cyclin partners were upregulated in primary basal-like / TNBCs. Furthermore, both CDK2 cyclin partners were expressed at higher levels in EGFR-high tumours. Additionally, the inventors demonstrated co-expression and colocalization of EGFR and the cell cycle genes in the tumour microenvironment by scRNA-seq and spatial transcriptomic analyses. These prompted the study of EGFR as a potential target using an ADC coupled with CDK inhibitors.

[0174] The structural homology between CDK proteins renders the identification of CDK2 inhibitors that do not possess some affinity for other kinases very challenging. In the absence of a highly-specific CDK2 inhibitor, SNS-032 is reported to be potent against CDK2 [45, 46], but also to inhibit RNA Polymerase II activity by targeting CDK7 / 9. However, alongside their binding partners, CDK7 / 9 are often downregulated in basal- like / TNBCs, hence TNBCs may not rely on CDK7 / 9 activity. Moreover, the molecular structure of SNS-032 contains a piperidine group, where its amine bridge (-NH) can react with the dipeptide linker for linker-payload synthesis, rendering this compound a suitable candidate for conjugation.

[0175] The inventors generated the ADC by conjugating cetuximab with SNS-032 via thiol- maleimide reaction, using the antibody as a vehicle to specifically deliver the payload to cancer cells. This linker is readily cleavable by cysteine cathepsins in lysosomes while remaining reasonably stable in plasma, of interest for the design of this ADC for which localization in lysosomal compartments was an important attribute. Moreover, overexpression of lysosomal proteases is frequently found in breast cancers

[0048] , and this offers another advantage for therapies using cathepsin-sensitive ADCs. Using super-resolution confocal microscopy, it was possible to visualize individual intracellular vesicles and to obtain spatiotemporal information of ADC-lysosome colocalization. Lysosomes are dynamic organelles with high mobility driven by motor proteins

[0049] that travel throughout the cell in response to nutrient levels and lipid distribution in membranes, while a relatively immobile perinuclear lysosome pool forms near the ER, where it controls directional transportation of lipid cholesterol, protein, and in this case, cleaved payloads from the ADCs. In this study it was necessary to stain only the acidic lysosomes as any other organelles (early / late endosomes) often contain pre-mature environments which are less acidic, with premature cathepsins, and are distant from ER / nucleus, and thus likely insufficient for payload release. Our live-cell spatiotemporal data showed high level of colocalization of the internalized ADC in lysosome clusters within ER and in close proximity to nucleus, an attribute crucial for drug release with the cleavable linkers.

[0176] The ADC, but not cetuximab alone, could induce cell cycle arrest and engender cytotoxic functions specifically against EGFR-high tumour cells, with subsequent release of free payloads to trigger bystander cytotoxicity against neighboring EGFR- low cells in the heterogenic tumour microenvironments.

[0177] Despite the in vivo-administered ADC carrying only a small molar fraction (1.65%) of the free SNS-032, the tumour-restricting effects of the ADC were superior to the much higher SNS-032 doses required to exert tumour growth restriction. The inventors observed no weight loss or signs of overt toxicity with ADC administration in vivo. Furthermore, in vitro assays showed no / low cytotoxic effects of the ADC on human cutaneous melanocytes or immune cells which, if targeted, could result in myelosuppression.

[0178] Accordingly, this study introduces an ADC strategy based on analyses of the frequency of combination of a target surface antigen expression and druggable oncogenic signaling activity in selected patient cohorts. Cetuximab-SNS-032 ADC inhibited cell cycle, restricted cellular growth and viability, exerted bystander killing effects on local EGFR-low cancer cells, and, despite carrying a small fraction of the inhibitor dose needed to exert anti-tumour effects, the ADC inhibited orthotopic TNBC xenograft growth in vivo.

[0179] Conclusions

[0180] Exploiting EGFR overexpression, and dysregulated cell cycle in aggressive and treatment-refractory tumours, a cetuximab-CDK inhibitor ADC can provide selective and efficacious delivery of cell cycle-targeted agents to basal-like / TNBCs, including chemotherapy-resistant residual disease.

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Claims

Claims1. A conjugate comprising an Epidermal Growth Factor Receptor (EGFR)- targeting moiety and a cyclin-dependent kinase (CDK) inhibitor.

2. The conjugate according to claim 1, wherein the EGFR-targeting moiety is an antibody or an antigen-binding fragment thereof.

3. The conjugate according to either claim 1 or claim 2, wherein the EGFR- targeting moiety binds to EGFR domain III.

4. The conjugate according to any preceding claim, wherein the EGFR- targeting moiety is Cetuximab, Mab A13, Futuximab, GC1118, Matuzumab, Necitumumab, Nimotuzumab and / or Panitumumab, or an antigen-binding fragment thereof.

5. The conjugate according to any preceding claim, wherein the EGFR- targeting moiety is Cetuximab, or an antigen-binding fragment thereof.

6. The conjugate according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises an IgGl, IgG2, IgG3 and / or IgG4 heavy chain, optionally wherein the heavy chain is IgGl.

7. The conjugate according to any preceding claim, wherein the CDK inhibitor is an inhibitor of a CDK involved in the cell cycle and / or transcriptional regulation.

8. The conjugate according to any preceding claim, wherein the CDK inhibitor is a selective inhibitor of CDK1, CDK2, CDK3, CDK4, CDK5, CDK6 and / or CDK7.

9. The conjugate according to any preceding claim, wherein the CDK inhibitor is a selective inhibitor of CDK2.

10. The conjugate according to any preceding claim, wherein the CDK inhibitor has a higher selectivity for CDK2 over CDK1, CDK4 and / or CDK6.

11. The conjugate according to claim 10, wherein the CDK2 inhibitor is at least 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 15-fold, or 20-fold selective over CDK1, CDK4 and / or CDK6.

12. The conjugate according to any preceding claim, wherein the CDK inhibitor is SNS-032, NKT3447, INX-315, BLU-222, PF-07104091, BG-6850, Fadraciclib, ARTS-021, AZD-8421, PF-06873600 and / or Tagtociclib.

13. The conjugate according to any preceding claim, wherein the CDK inhibitor is SNS-032.

14. The conjugate according to any preceding claim further comprising a linker, optionally wherein the linker is a cleavable linker.

15. The conjugate according to claim 14, wherein the linker comprises a maleimide-based linker.

16. The conjugate according to any one of claims 14 to 16, wherein the linker comprises a peptide, optionally a Val-Ala dipeptide.

17. The conjugate according to any preceding claim, wherein the conjugate comprises from 1 to 10, 2 to 7, or 3 to 6, or 4 to 5 CDK inhibitors per EGFR-targeting moiety.

18. The conjugate according to any preceding claim, wherein the conjugate comprises the structure substantially as represented as Formula II.

19. A conjugate according to any one of claims 1 to 18, for use as a medicament.

20. A conjugate according to any one of claims 1 to 18, for use in treating, ameliorating or preventing cancer.

21. The conjugate for use according to claim 20, wherein the cancer is a solid tumour or solid cancer.

22. The conjugate for use according to either claim 20 or claim 21, wherein the cancer is bowel cancer, brain cancer, breast cancer, endometrial cancer,gastric cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.

23. The conjugate for use according to claims 20-22, wherein the cancer is breast cancer.

24. The conjugate for use according to claim 23, wherein the breast cancer is triple negative breast cancer (TNBC), optionally basal-like TNBC.

25. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 18, and a pharmaceutically acceptable vehicle.

26. A process for making the pharmaceutical composition according to claim25, the process comprising contacting a therapeutically effective amount of the conjugate according to any one of claims 1 to 18, and a pharmaceutically acceptable vehicle.

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