Co-administration of antibody-drug conjugate

Abstract

The present invention relates to antibody-drug conjugates (ADCs) that target the CanAg antigen and co-administration of the ADC with unconjugated antibody. In particular, the present invention relates to compositions (e.g. pharmaceutical compositions) comprising the ADCs and unconjugated antibody and methods of using the same, including for the treatment of cancer.

Description

[0001] CO-ADMINISTRATION OF ANTIBODY-DRUG CONJUGATE

[0002] Field of the Invention

[0003] The present invention relates to antibody-drug conjugates (ADCs) and coadministration of the ADC with unconjugated antibody. In particular, the present invention relates to ADCs that target the CanAg antigen. Compositions comprising the ADCs and unconjugated antibody and methods of using the same, including for the treatment of cancer, are also provided.

[0004] Background of the Invention

[0005] Antibody-drug conjugate (ADC) technology is a target-oriented technology, which exploits the ability of an antibody to sensitively discriminate between healthy and diseased tissue to selectively deliver a cytotoxic payload. Three key elements define an ADC: the antibody, the cytotoxic drug (also called payload) and the linker connecting the drug to the antibody. ADCs are known for use as anticancer agents and function by using the antibody to target a specific antigen associated with cancerous cells and then releasing the drug payload under specific conditions to induce cell death. This enables the targeted delivery of a highly potent drug directly into the tumour, thereby reducing systemic exposure and toxicity to normal tissues. Accordingly, ADCs have significant potential to improve the treatment and survival of patients suffering from diseases such as cancer.

[0006] Despite the potential to use toxic payloads that are normally not tolerated by patients, a low therapeutic index (a ratio that compares toxic dose to efficacious dose) continues to be a problem and accounts for the discontinuance of many ADCs in clinical development. The selection of an appropriate target, antibody, cytotoxic payload, and the manner in which the antibody is linked to the payload have all been identified as key determinants of the safety and efficacy of ADCs. As such, the development of each ADC represents a unique challenge. In particular, it is known that the anti-cancer activity of an ADC is dependent on multiple factors such as the potency of the payload, tumour sensitivity to the cytotoxic drug, antigen expression levels in tumour tissues and their accessibility to the ADC, antigen internalisation and pharmacokinetics of the conjugate in plasma. Thus, delivering an optimal dose of payload in a manner that reaches and kills as many cancer cells as possible, while avoiding concentrations that are toxic in healthy tissues, remains a significant challenge.

[0007] The CanAg antigen has been suggested as one suitable target for selective antibody-based anticancer therapies based on its favourable expression pattern. CanAg is highly expressed in most pancreatic, biliary and colorectal cancers as well as in a significant proportion of gastric, uterine, non-small cell lung cancer, and bladder cancers. In contrast, only minimal expression of CanAg in normal tissue has been reported. Despite this, there are currently no anti-CanAg ADCs approved for use in the treatment of cancer. Cantuzumab mertansine and Cantuzumab ravtansine are two known ADCs that target CanAg, but neither compound has progressed further than Phase 2 clinical trials, possibly due to the limited efficacy observed against colorectal and pancreatic cancers.

[0008] Accordingly, there continues to be a need to identify and develop effective ADC combinations and therapies, in particular, having an acceptable safety profile, minimal off-target activity, and sufficient efficacy.

[0009] Summary of Invention

[0010] In a first aspect, there is provided a composition comprising: an anti-CanAg antibody-drug conjugate; and an unconjugated anti-CanAg antibody; wherein the anti-CanAg antibody-drug conjugate is represented by Formula I or a pharmaceutically acceptable salt or solvate thereof:

[0011] Formula I: Ab-(L-D)n wherein:

[0012] Ab is the anti-CanAg antibody or an antigen-binding fragment thereof;

[0013] L is a linker connecting Ab to D; n is an integer from 1 to 20; and D is a pyrrolobenzodiazepine dimer prodrug represented by Formula (II):

[0014] Formula (II)

[0015] The present inventors have identified specific combinations of antibody, linkers and drug, which together provide ADCs having improved safety and efficacy compared to existing ADCs that target the CanAg antigen. Advantageously, it has also been found that co-administration of the ADC with unconjugated antibody further improves anti-cancer activity. Without wishing to be bound by theory, the improvement in observed activity in vivo may result from improved conjugate distribution in tumour tissue, resulting in greater tumoral exposure.

[0016] ADCs utilised in the present invention comprise a pyrrolobenzodiazepine (PBD) dimer cytotoxic payload in the form of a prodrug according to Formula (II). PBDs are a known class of highly cytotoxic DNA cross-linking agents that exploit a different cellular target to the auristatin and maytansinoid tubulin inhibitor classes and a different mode of DNA damage to other DNA interacting payloads, such as calicheamicin. The prodrug form of the PBD dimer according to Formula (II) is more stable and exhibits lower cytotoxicity compared to conventional PBD drugs, which may suffer from poor stability in blood after administration. The prodrug is converted to an active form through cleavage of the glucuronic acid moieties by a - glucuronidase enzyme, which is known to be upregulated in cancer cells relative to surrounding normal tissues (Fishman, W.H., J. Biol. Chem., 1947, 169(2) p:449). This may result in higher tumour selectivity of the active form of the drug and reduce the occurrence of side effects caused by premature decomposition of the linker by normal cells.

[0017] Suitably, n of the antibody-drug conjugate may be 1 to 8. For example, n may be 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 2 to 4. In one aspect, n = 2.

[0018] Any antibody or an antigen-binding fragment thereof that can target CanAg may be used as a component of the ADC and / or as the unconjugated antibody in accordance with the invention. Suitably, the antibody is a humanised C242 antibody or an antigen binding-fragment thereof. The humanized C242 (HuC242 or Cantuzumab) binds to the CA242 epitope on the extracellular domain of the CanAg antigen. Examples of humanised C242 for use in the present invention may comprise one or more amino acid sequences from SEQ ID NO: 8-13 or 16-21. Suitably, the unconjugated antibody is a humanised antibody or an antigen bindingfragment thereof having an amino sequence which can be the same or different to the amino acid sequence of the antibody of the ADC. In one aspect, the unconjugated antibody has the same amino acid sequence as the antibody used in the ADC. References to "antibody", particularly in terms of defining optional components of the antibody, are used herein to refer to "the unconjugated antibody and / or the antibody in the ADC" unless specified otherwise.

[0019] The ratio of the anti-CanAg antibody-drug conjugate to unconjugated antibody may be in a range from 1:1 to 1 :50, optionally in a range from 1:5 to 1 :50 or 1 :10 to 1:20. For example, the ratio of the anti-CanAg antibody-drug conjugate to unconjugated antibody may be 1:1 , 1:5, 1:10, 1:20 or 1:50. The ratio of the anti-CanAg antibodydrug conjugate to unconjugated antibody can be tailored to optimise efficacy depending on the glycan epitope expression levels on the target tumour cells.

[0020] The Linker (L) (sometimes referred to as “linker” herein) is a bifunctional compound which can be used to link the drug and the antibody. Various examples have been described in the art for linking the antibody moiety to the payload. These linker systems can generally be categorised as either cleavable or non-cleavable. For cleavable ADC linker systems, the release mechanism is typically enzymatically driven, although chemically labile cleavable systems are also known. In non- cleavable ADC linker systems, drug release is effected by degradation within the cell after internalisation of the ADC.

[0021] Optionally, the linker may comprise a central portion represented by Formula III, IV, V, VI or VII:

[0022] Formula VI Formula VII wherein:

[0023] L1 comprises a first connecting portion connecting the central portion to Ab; and

[0024] L2 comprises a second connecting portion connecting the central portion to D.

[0025] The central portion of the linker connecting the antibody and the drug may comprise an O-substituted oxime according to Formula III. When the carbon atom of the oxime is substituted with a first connecting group that covalently links the oxime to the antibody, the oxygen atom of the oxime is substituted with a second connecting group that covalently links the oxime to the drug (D). Alternatively, when the carbon atom of the oxime is substituted with a connecting group that covalently links the oxime to the drug, the oxygen atom of the oxime is substituted with a connecting group that covalently links the oxime to the antibody.

[0026] In other embodiments, the central portion of the linker may comprise a substituted triazole according to formula IV, V, VI or VII, instead of an oxime. Advantageously, triazoles can be formed by click chemistry reactions carried out under mild conditions, which can be performed in the presence of an antibody without denaturing occurring. Further, an azide-alkyne click chemistry reaction, for example, may produce a triazole in a high yield and with high reaction specificity. Therefore, even though antibodies have various functional groups (for example, amines, carboxyls, carboxamides, and guanidiniums), a click chemistry reaction may be performed, for example, without affecting the amino acid side chains of the antibody. As would be appreciated by those skilled in the art, formulae VI and VII are regioisomers of formulae IV and V, respectively.

[0027] The first connecting portion L1 may comprise a reactive site e.g., an electrophilic group, that is reactive to a nucleophilic group present on the antibody. Useful nucleophilic groups on an antibody include but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group on the connecting group and forms a covalent bond thereto. Useful electrophilic groups include, but are not limited to, vinylpyridine, maleimide and haloacetamide groups. Alternatively, connecting group L1 may comprise a reactive site which has a nucleophilic group that is reactive to an electrophilic group present on the antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a linker unit can react with an electrophilic group on an antibody and form a covalent bond to the antibody. Useful nucleophilic groups on a linker unit include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. Alternatively, the antibody may be modified to include an azide group (-N3) and the connecting group may incorporate an azide reactive moiety, such as a cyclooctyne group, for production of the antibody-drug conjugate according to the present invention. In this way, the linker can be bound to the antibody through a clickreaction between the azide group in the antibody and the cyclooctyne group in the linker. Alternatively, the first connecting portion L1 may connect the central portion to D and the second connecting portion L2 may connect the central portion to Ab.

[0028] The first connecting portion may include at least one isoprenyl unit represented by Formula VIII:

[0029] Formula VIII

[0030] When the first connecting portion includes at least one isoprenyl unit represented by Formula VIII, the carbon atom (a) of the isoprenyl unit forms a thioether bond with a sulfur atom, preferably of a cysteine, of the antibody, thereby covalently linking the isoprenyl group and the antibody. The carbon atom (b) of the isoprenyl group covalently links the isoprenyl group to the central portion of the linker.

[0031] Advantageously, antibody prenylation of a C-terminal amino acid sequence to install a modified isoprenyl unit has been described that allows for attachment of a drug or other active agent to the antibody in a mild and site-specific manner. This allows for the preparation of homogeneous ADCs having a defined number of drugs, which is known to improve pharmacokinetics and efficacy and is more desirable from a regulatory perspective.

[0032] The antibody may comprise an amino acid motif, preferably at a C-terminus of the antibody that is recognized by an isoprenoid transferase. The amino acid motif may be a sequence selected from CXX, CXC, XCXC, XXCC, and CYYX, wherein C represents cysteine; Y, independently for each occurrence, represents an aliphatic amino acid; and X, independently for each occurrence, represents glutamine, glutamate, serine, cysteine, methionine, alanine, or leucine. Suitably, the thioether bond may comprise a sulfur atom of a cysteine of the amino acid motif. Optionally, the amino acid motif may be a sequence CYYX, and Y, independently for each occurrence, represents alanine, isoleucine, leucine, methionine, or valine. For example, the amino acid motif may be CVIM (SEQ ID NO: 1) or CVLL (SEQ ID NO: 2). At least one of the seven amino acids preceding the amino acid motif may be glycine. For example, at least three of the seven amino acids preceding the amino acid motif are each independently selected from glycine and proline. Suitably, each of the one, two, three, four, five, six, seven, eight, nine, or ten amino acids preceding the amino acid motif is glycine, preferably seven. Optionally, the antibody comprises the amino acid sequence GGGGGGGCVIM (SEQ ID NO: 3), preferably at a C- terminus.

[0033] Alternatively, the first connecting portion is represented by Formula IX:

[0034] Formula IX

[0035] Wherein: a denotes a point of attachment to Ab; b denotes a point of attachment to the central portion; and m is an integer from 1 to 10.

[0036] Advantageously, vinylpyridine-based linkers in accordance with Formula IX have been shown to react selectively and irreversibly with thiol groups on an antibody to form highly stable thioether bonds. As noted above, linker stability is critical to the efficacy and toxicity of ADCs.

[0037] Suitably, the second connecting portion may comprise at least one polyethylene glycol unit represented by Formula X:

[0038] Formula X -(CH2CH2O)O- wherein o is an integer from 1 to 10.

[0039] Alternatively, the second connecting portion may be represented by Formula XI:

[0040] Formula XI -(CH2)p-NHC(O)-(CH2CH2)O)q(CH2)r -C(O)-NH-(CH2)S-C(O)- wherein: p is an integer from 1 to 10; q is an integer from 1 to 20; r is an integer from 1 to 10; and s is an integer from 1 to 10.

[0041] Suitably, the antibody-drug conjugate may comprise a structure selected from: wherein, m is an integer from 0 to 20.

[0042] Additionally or alternatively, the antibody-drug conjugate may comprise a structure selected from:

[0043] wherein: m is an integer from 0 to 20; and o is an integer from 0 to 10.

[0044] In some aspects, the present invention excludes embodiments wherein the antibody drug-conjugate comprises vinylpyridine-based linkers, for example, in accordance with Formula IX. In one aspect, the present invention excludes embodiments wherein the antibody drug-conjugate is ADC-1 according to Example 3 of the present disclosure.

[0045] In a second aspect, there is provided a pharmaceutical composition comprising a composition according to the first aspect; and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0046] In a third aspect, there is provided a kit comprising:

[0047] (a) a first pharmaceutical composition comprising an anti-CanAg antibodydrug conjugate and one or more pharmaceutically acceptable excipient, diluents, or carriers; and io (b) a second pharmaceutical composition comprising an unconjugated anti- CanAg antibody and one or more pharmaceutically acceptable excipient, diluents, or carriers.

[0048] Suitably, the first pharmaceutical composition of the kit according to the third aspect comprises the anti-CanAg antibody-drug conjugate as defined in any of claims 1 to 10.

[0049] The pharmaceutical composition according to the second aspect, or the kit according to third aspect may be for use in the treatment of cancer.

[0050] Optionally, the cancer may be selected from the group consisting of lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, pancreatic cancer, biliary cancer, cervical cancer and uterine cancer. For example, the cancer may be pancreatic cancer.

[0051] In a fourth aspect there is provided a pharmaceutical combination for use in the treatment of cancer comprising:

[0052] (a) a first pharmaceutical composition comprising an anti-CanAg antibodydrug conjugate and one or more pharmaceutically acceptable excipient, diluents, or carriers; and

[0053] (b) a second pharmaceutical composition comprising an unconjugated anti- CanAg antibody and one or more pharmaceutically acceptable excipient, diluents, or carriers; wherein the compositions (a) and (b) are provided in separate dosage forms, which are administered simultaneously, sequentially or in the form of a mixture of compositions (a) and (b).

[0054] Optionally, the cancer may be selected from the group consisting of lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, pancreatic cancer, biliary cancer, cervical cancer and uterine cancer.

[0055] Suitably, the first and second pharmaceutical compositions may be separately packaged and available for sale independently of one another, but are co-marketed or co-promoted for simultaneous and / or subsequent administration, and / or administration as a mixture.

[0056] Suitably, the first pharmaceutical composition of the pharmaceutical combination comprises the anti-CanAg antibody-drug conjugate as defined in any of claims 1 to 10.

[0057] In a fifth aspect, there is provided a method of treating cancer in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to the second aspect or the pharmaceutical combination for use according to the fourth aspect.

[0058] Optionally, the cancer is selected from the group consisting of lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, pancreatic cancer, biliary cancer, cervical cancer, and uterine cancer.

[0059] Suitably, when the method comprises the step of administering, to the subject, a therapeutically effective amount of the pharmaceutical combination according to the fourth aspect, the first pharmaceutical composition and all or part of the second pharmaceutical composition may be administered simultaneously or separately.

[0060] Suitably, when the method comprises the step of administering, to the subject, a therapeutically effective amount of the pharmaceutical combination according to the fourth aspect, the first pharmaceutical composition and all or part of the second pharmaceutical composition may be premixed and administered as a mixture.

[0061] Optionally, the ratio of the anti-CanAg antibody-drug conjugate to unconjugated antibody administered to the subject may be in a range from 1 :1 to 1:50. Optionally, the range may be from 1:5 to 1:50 or 1:10 to 1:20. For example, the ratio of the anti- CanAg antibody-drug conjugate to unconjugated antibody may be 1:1, 1:5, 1:10, 1:20 or 1:50.

[0062] Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures. Brief Description of Figures

[0063] The accompanying drawings illustrate presently exemplary embodiments of the disclosure, and together with the general description given above and the detailed description of the embodiments given below, serve to explain, by way of example, the principles of the disclosure.

[0064] Figure 1 shows binding of humanised antibodies to CanAg positive Colo205 cells;

[0065] Figure 2 shows a PLRP chromatogram (A214 nm) of an ADC-1 with a DAR of 2.3. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain;

[0066] Figure 3 shows a SEC chromatogram (A214 nm) of an ADC-1 with a DAR of 2.3;

[0067] Figure 4 shows a HIC chromatogram (A214 nm) of ADC-2 with a DAR of 2 after conjugation of compound 2 to the prenylated HC1+LC1 intermediate and purification by semi-preparative HIC;

[0068] Figure 5 shows a SEC chromatogram (A214 nm) of an ADC-2 with a DAR of 2 after purification;

[0069] Figure 6 shows a HIC chromatogram (A214 nm) of an ADC-3 with a DAR of 2 after conjugation of compound 5 to the prenylated HC1 LC1 intermediate and purification by semi-preparative HIC;

[0070] Figure 7 shows a SEC chromatogram (A214 nm) of an ADC-3 with a DAR of 2 after purification;

[0071] Figure 8 shows SEC analysis of Cantuzumab ravtansine at 280 nm;

[0072] Figure 9 shows CanAg expression level on SNll-16, Colo-205, HT29, BxPC3 and NCI-N87 cells;

[0073] Figure 10 shows p-glucuronidase activity in Colo-205, HT29, BxPC3 and NCI-N87 cells;

[0074] Figure 11 shows the in vitro activity of ADC-2 and ADC non-binding control on Colo- 205 cells;

[0075] Figure 12 shows the in vitro activity of ADC-2 and ADC non-binding control on BxPC-3 cells;

[0076] Figure 13 shows the in vitro activity of ADC-2 and ADC non-binding control on HT- 29 cells;

[0077] Figure 14 shows the in vitro activity of ADC-2 and ADC non-binding control on N87 cells;

[0078] Figure 15 shows the in vitro activity of ADC-1 , ADC-2 and ADC-3 on Colo-205 cells; Figure 16 shows the in vitro activity of ADC-3, Cantuzumab ravtansine and ADC non-binding control on Colo-205 cells;

[0079] Figure 17 shows the in vivo activity of ADC-2 and ADC-3 in Colo-205 xenograft (A), with body weight changes shown in (B);

[0080] Figure 18 shows the in vivo activity of ADC-1, ADC-2 and ADC-3 in Colo-205 xenograft (A), with body weight changes shown in (B);

[0081] Figure 19 shows the in vivo activity of Cantuzumab ravtansine in Colo-205 xenograft (A), with body weight changes shown in (B);

[0082] Figure 20 shows the in vivo activity of ADC-2, ADC-3 and Cantuzumab ravtansine in BxPC-3 xenograft (A), with body weight changes shown in (B);

[0083] Figure 21 shows the in vivo activity of Cantuzumab ravtansine in BxPC-3 xenograft (A), with body weight changes shown in (B);

[0084] Figure 22 shows the in vivo activity of ADC-3 and non-binding ADC control in NCI- N87 xenograft (A), with body weight changes shown in (B);

[0085] Figure 23 shows the in vivo activity of Cantuzumab ravtansine (A) and Enhertu (B) ADCs in NCI-N87 xenograft, with body weight changes shown in (C) and (D), respectively;

[0086] Figure 24 shows a SEC chromatogram of anti-CanAg antibody;

[0087] Figure 25 shows ADC-3 in vitro activity with or without an excess of unconjugated anti-CanAg antibody on SNll-16 (A) and HT-29 (B) cell lines;

[0088] Figure 26 shows the in vivo activity of ADC-3 in the presence of unconjugated anti- CanAg antibody in a SNll-16 xenograft (A), with body weight changes shown in (B);

[0089] Figure 27 shows the in vivo activity of ADC-3 in the presence of unconjugated anti- CanAg antibody in a HT-29 xenograft (A), with body weight changes shown in (B); and

[0090] Figure 28 shows the alignment of the heavy chain (HC) and light chain (LC) sequences of humanised antibodies. CDR regions are shown underlined. Amino acids belonging to the signal peptide are shown in bold font.

[0091] Detailed Description

[0092] The present invention relates to antibody-drug conjugates (ADCs) and coadministration of the ADC with unconjugated antibody. In particular, the present invention relates to ADCs that target the CanAg antigen. Compositions comprising the ADCs and unconjugated antibody and methods of using the same, including for the treatment of cancer, are also provided.

[0093] The term "antibody" means an immunoglobulin molecule that recognises and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. References to antibodies include immunoglobulins whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein comprising an antigen binding domain. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. lgG1, lgG2, lgG3, lgG-4, lgA1 and lgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three- dimensional configurations.

[0094] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, nanobodies, single chain antibodies, bispecific and multispecific antibodies formed from antibody fragments.

[0095] “Prodrug” refers to a compound that is metabolised, for example hydrolysed, in the host after administration to form a biologically active molecule. Typical examples of prodrugs include compounds that have biologically labile or cleavable protecting groups on a functional moiety of the active compound. The "drug-antibody ratio" (DAR) in an antibody-drug conjugate of the invention is defined as the molar ratio between the drug moieties in the conjugate and the antibodies in the conjugate. Where an antibody has more than one site of attachment, more than one drug moiety may be linked to each antibody. In some instances, a mixture is obtained comprising more than one antibody-drug conjugate (ADC) molecules. The drug-antibody ratios of the antibody-drug conjugates can be measured by analytical methods know in the art, for example, as described below. In some embodiments, the antibody-drug conjugates have an average DAR of about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1.5 to about 3.5, or about 2 to about 4. In compositions comprising a mixture of antibody-drug conjugate and unconjugated antibody, the DAR of the composition as a whole will be diluted according to the ratio of antibody-drug conjugate to unconjugated antibody.

[0096] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterised by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, fallopian tube cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancers.

[0097] “Tumour" refers to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including precancerous lesions.

[0098] The term "subject" refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject. The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation can be sterile.

[0099] An "effective amount" as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.

[0100] The term "therapeutically effective amount" refers to an amount of an ADC or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumour size; inhibit (i.e., slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumour metastasis; inhibit, to some extent, tumour growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. See the definition of "treating" below. To the extent the drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0101] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In certain embodiments, a subject is successfully "treated" for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumour size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumour metastasis; inhibition or an absence of tumour growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumourigenicity, tumourigenic frequency, or tumourigenic capacity, of a tumour; reduction in the number or frequency of cancer stem cells in a tumour; differentiation of tumourigenic cells to a non-tumourigenic state; or some combination of effects.

[0102] The term "co-administration" refers to the administration of two or more therapeutic agents to treat a disease as described herein. Such administration includes coadministration of the therapeutic agents in a substantially simultaneous manner, for example, as a mixture with fixed ratios of the active ingredients. Alternatively, such administration includes co-administration for each active ingredient in multiple or in separate containers (eg tablets, capsules, powders and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dosage before administration. Furthermore, such administration also includes using each type of therapeutic agent in a sequential manner at about the same time. In either case, the treatment regimen will provide for the beneficial effect of the drug combination in treating the disorders or conditions described herein.

[0103] The disclosure provides antibody-drug conjugates of antibodies that bind to the CanAg antigen. CanAg is strongly expressed in most pancreatic, biliary, and colorectal cancers. It is also expressed in a substantial proportion of gastric cancers, uterine cancers, non-small cell lung cancers, and bladder cancers. In contrast, only minimal expression of CanAg in normal tissue has been reported. As such, CanAg appears to be a suitable candidate for mAb-based anticancer therapies. However, there are currently no marketed ADCs that target CanAg. Cantuzumab mertansine and cantuzumab ravtansine are two known ADCs that target CanAg, but neither compound progressed further than Phase 2 clinical trials. The present invention has surprisingly found that particular ADCs as claimed provide improved ADCs which target CanAg.

[0104] Any humanized antibody or an antigen-binding fragment thereof that can target CanAg may be used in accordance with the invention. In order to maintain binding affinity, the humanisation process may comprise identifying CDR regions and residues interacting with CDRs or in VH-VL interfaces and preserving these regions in the humanised antibody. Suitably, the antibody in accordance with the invention is a humanised antibody in which the CDRs underlined in Figure 28 are preserved, or an antigen binding-fragment including these regions. Examples of humanised C242 for use in the present invention may comprise one or more amino acid sequences from SEQ ID NO: 8-13 or 16-21 .

[0105] The compositions and combinations according to the present invention may be useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as the treatment of cancer. In certain embodiments, the agents are useful for inhibiting tumour growth, inducing differentiation, reducing tumour volume, and / or reducing the tumourigenicity of a tumour. The methods of use may be in vitro, ex vivo, or in vivo methods. In certain embodiments, the disease treated with the compositions or combinations is a cancer. In certain embodiments, the cancer is characterised by tumours expressing CanAg.

[0106] The present invention provides for methods of treating cancer comprising administering a therapeutically effective amount of the compositions or combinations to a subject (e.g., a subject in need of treatment). In certain embodiments, the cancer is a cancer selected from the group consisting of lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, pancreatic cancer, biliary cancer, cervical cancer and uterine cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the subject is a human.

[0107] The pharmaceutical compositions and combinations of the present invention can be administered in any number of ways for either local or systemic treatment. Administration can be pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal); oral; or parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration.

[0108] For the treatment of the disease, the appropriate dosage of an antibody or agent of the present invention depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the composition is administered for therapeutic or preventative purposes, previous therapy, patient's clinical history, and so on all at the discretion of the treating physician. The compositions can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g. reduction in tumour size). Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual antibody or agent. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates.

[0109] Experimental Data and Discussion

[0110] The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations to the scope of this invention as defined by the appended claims.

[0111] Example 1. Development of humanised anti-CanAg antibodies

[0112] The humanisation design of the parental antibody was performed using in silico analysis. A 3D structure of the parental antibody using homology modeling was generated. Acceptor frameworks were identified based on the overall sequence identity across the framework, matching interface position, and similarly classed CDR canonical positions. Three heavy chain (HC) frameworks and three light chain (LC) frameworks were selected for the humanisation design.

[0113] Humanised antibodies were designed by creating multiple hybrid sequences that fuse select parts of the parental antibody sequence with the human framework sequences. Using the 3D structure, these humanized sequences were methodically analysed by eye and computer modelling to isolate the sequences that would most likely retain antigen binding (focusing on key residues supporting CDR loop and VH- VL interface). The goal was to maximise the amount of human sequence in the final humanised antibodies while retaining the original antibody specificity. Three humanised VH and three humanised VL sequences were designed: “HV1-18 BM (HC1)," “HV1-46 BM (HC2),” and “HV1-8 BM (HC3)”; “KV2-40 BM (LC1) and “KV2-28 BM (LC2)," and “KV2D-29 BM (LC3)". The humanness score (T20 score) for the humanised antibodies was caiculated by analysing the primary sequences of the variable regions using the method described in Gao et al (Monoclonal antibody humanness score and its applications; BMC Biotechnology, 13:55, 2013).

[0114] The humanised heavy and light chains were then combined to create variant fully humanised antibodies. Nine (9) combinations of humanised heavy and light chains were tested for their expression level and antigen-binding affinity to identify antibodies that perform similarly to the chimeric parental antibody. The antibodies tested are shown in Table 1 below. A 0.01 L transient production (TunaCHO™ 7- day) for the nine humanised variants and chimeric parental antibody was performed. All clones were purified by Protein A (see Table 1 for production yield).

[0115] Table 1. Production yield of the antibodies in mg / L.

[0116] The affinity of the nine humanised antibody combinations for CanAg positive Colo- 205 cells was evaluated by flow cytometry as follows. 20x105Colo205 cells were aliquoted per well and washed once with FACS buffer (2% FBS in PBS). Humanised antibodies and isotype control antibodies (Anti-HEL-Human-lgG1(N297A), catalog: B109801, brand: BIOINTRON) were diluted with FACS buffer diluted (8 concentrations starting at 100 pg / ml with 2-fold dilutions), then 100 uL was added to each well with cells. Cells were incubated at 4 °C for 60 minutes followed by washing with FACS buffer twice. Secondary antibody AF488 Goat anti-human lgG(H+L) (catalog: A11013, brand: Invitrogen) was diluted 1:1000 with FACS buffer, then 100 uL added to each well and incubated at 4 °C for 30 minutes. After the incubation, cells were re-suspended in 200 uL FACS buffer for flow cytometry analysis. MFI (mean fluorescence intensity) was used to calculate EC50. The EC50 values for binding of humanised antibodies to Colo205 cells determined by FACS are shown in Table 2.

[0117] Table 2. EC50 values for binding of humanised antibodies to Colo205 cells determined by FACS.

[0118] As can be seen from Table 2, all humanised antibodies tested (HC1+LC1, HC1+LC2, HC1+LC3, HC2+LC1 , HC2+LC2, HC2+LC3, HC3+LC1, HC3+LC2 and HC3+LC3) exhibited comparable affinity and expression yield in TunaCHO™ cell transient expression system.

[0119] Example 2 - Antibody generation

[0120] Stable transfected CHOMGV cells with heavy and light chains (HC1+LC1) of anti- CanAg antibody were cultured in the bioreactor for 14 days and then harvested via depth filtration using the Millipore POD system. The clarified supernatant was then purified using Protein A chromatography (Toyopearl AF rProA HC 650F) followed by virus inactivation (via low pH hold step). The eluate was depth filtered and the filtrate was further purified using anion exchange chromatography (Poros 50 HQ) followed by cation exchange chromatography (Toyopearl Gigacap-S-650M). Virus reduction was achieved using Viresolve® Pro Modus 1.3. The concentration of the eluate and buffer exchange into the final formulation buffer was achieved using Pell icon 3 Ultrafiltration cassettes.

[0121] Determination of monomeric content of anti-CanAg antibody

[0122] The percentage monomer and relative amounts of high molecular weight (HMW) and low molecular weight (LMW) species of the purified antibody were determined using Size Exclusion Chromatography (SEC). Briefly, samples were diluted and loaded onto an Acquity UPLC Protein BEH, 200 column. The mobile phase was 200 mM Potassium Phosphate, 250 mM Potassium Chloride, pH 6.0.

[0123] A representative example is shown in Figure 24, which shows the percentage monomer and percentage of HMW and LMW species obtained for an HC1+LC1 antibody comprising a CAAX tag.

[0124] Example 3. ADC-1

[0125] ADC-1 was produced by vinylpyridine-mediated cysteine modification using compound 1 and compound 2 below.

[0126] Synthesis of compound 1

[0127] Lithium 3-(6-methyl-4-vinylpyridin-2-yl)propanoate (17.5 g, 87.6 mmol) and 2-(2-(2- azidoethoxy)ethoxy)ethan-1-amine (23.0 g, 87.6 mmol, commercially available from BroadPharm, catalogue number: BP-21615) were dissolved in dimethylformamide (525.0 mL). / V-(3-Dimethylaminopropyl)- / V'-ethylcarbodiimide hydrochloride (33.6 g, 175 mmol) was added portion-wise at 0°C. Diisopropylethylamine (61.0 mL, 350.0 mmol) was added dropwise and the mixture stirred for 12 hours. The reaction was quenched by addition of aqueous LiCI (1 .0 L, 5.0 % wt / vol) and washed with ethyl acetate (3 x 500mL). The organic extracts were combined, dried over MgSC and evaporated to dryness to afford crude A / -(2-(2-(2-azidoethoxy)ethoxy)ethyl)-3-(6- methyl-4-vinylpyridin-2-yl)propenamide (Compound 1). The crude product was purified by column chromatography (Si2O, dichloromethane:methanol gradient 98:2 - 96:4) to afford Compound 1 as an orange oil (21.5 g, 56 % yield).1H NMR (CDCL, 400 MHz) 5 ppm: 6.97 (2H, d) 6.53 (1 H, t), 5.90 (1H, d), 5.42 (1 H, d), 3.63 (14H, m), 3.44 (2H, d), 3,40 (4H, m), 3.04 (2H, t), 2.62 (2H, t), 2.45 (3H, s). LCMS (ESI+): Compound 1 (C21H33N5O5) Theoretical: 436.25 [M+1]1+. Found: 436.28 [M+1]1+.

[0128] Compound 2, shown below, was synthesised by the method described in WO2018182341, which is hereby incorporated by reference in its entirety.

[0129] Compound 2

[0130] ADC-1 generation

[0131] HC1+LC1 antibody was partially reduced with 2.8 molar equivalents of TCEP and conjugated to compound 1 in a 4-fold molar excess and in the presence of 1.2% (v / v) dimethylacetamide (DMA), at pH 7.4 for >18 hours at 25 °C. After isolation by desalting, the resulting intermediate was conjugated to compound 2 in a 2.5-fold molar excess and in the presence of 0.7% (v / v) dimethylacetamide (DMA), at pH 7.4 for >4 hours at 25 °C. The conjugate was purified by desalting column to remove excess free drug and solvent and re-buffered to phosphate-buffered saline (PBS), pH 7.4.

[0132] Determination of Drug Antibody Ratio (PAR) for ADC-1

[0133] Determination of average drug-load and drug-load distribution is crucial for ADC generation, as these factors effect the potency and pharmacokinetics of the ADC. DAR determination of ADC-1 was accomplished by Polymer-Linked Reverse-Phase (PLRP) chromatography with an Agilent PLRP-S (1000 A, 2.1 x 50 mm, 5 pm) column. Separation of dithiothreitol (DTT) reduced conjugate via a PLRP column afforded well resolved peaks corresponding to unconjugated or drug conjugated antibody light and heavy chains, as shown in Figure 2. The DAR value was determined to be 2.3 for ADC-1. Peak separation was performed using the following procedure; Buffer A (H2O + 0.1% TFA) and Buffer B (MeCN + 0.1% TFA); Gradient; 0-3 min 25% buffer B; 3-28 min = 25-50% buffer B; 28-31 min = 95% buffer B; 31-40 min = 25% buffer B.

[0134] Determination of monomeric content of ADC-1

[0135] Size-exclusion chromatography (SEC) was employed to determine the degree to which the conjugate had aggregated during conjugation using a MAbPac™ SEC-1 column (5 pM, 300 A, 7.8 x 300 mm). Elution was performed in 20 mM MES sodium salt, 150 mM NaCI, 5% MeCN, pH 6.0. ADC-1 shows more than 96% monomeric content, as shown in Figure 3.

[0136] Example 4. ADC-2

[0137] ADC-2 was prepared using protein prenylation of a C-terminal amino acid sequence to install a modified isoprenoid unit that allows for attachment of the drug to the antibody in a mild and site-specific manner. Such prenylation of an antibody is described, for example, in U.S. Patent Publication No. 2012 / 0308584, U.S. Patent No. 9,919,057, PCT Publication No. WO 2017 / 089890 and PCT Publication No. WO 2017 / 089895, the contents of which are fully incorporated by reference herein. In this example, the antibody was modified with an isoprenoid derivative functionalised with an azide group, shown below as Compound 3, for coupling with Compound 2 via click chemistry using the procedure described below. Compound 3 was prepared by the method described in LIS2012 / 0308584.

[0138] Compound 5

[0139] ADC-2 generation HC1 LC1 antibody with CAAX tag was prenylated with 8.3 molar equivalents of compound 3 in the presence of 0.2 pM FTase, 100 pM DTT, 50 mM Tris-HCI, 0.01 mM ZnCl2 and 5 mM MgCl2 at pH 7.4 for 4 hours at 30 °C. The resulting intermediate was desalted into PBS, pH 7.4. Prenylated intermediate was conjugated with 2.5 molar equivalents of compound 2 in the presence 0.45% DMA for 2 hours at 30 °C. ADC-2 was purified by semi-preparative HIC using a Phenyl phase HIC column (Tosoh Bioscience, L x I.D 7.5 cm x 7.5 mm) using the following procedure; Buffer A (50 mM potassium phosphate + 0.5 M ammonium sulphate, pH 7.0) and Buffer B (50 mM potassium phosphate + 30% (v / v) MeCN, pH 7.0); Gradient - 0-30 mins = 0- 100% buffer B; 30-32 min = 100% buffer B; 32-32.1 min = 100-0% buffer B; 32.1-49 min = 0% buffer B; Flow rate - 0.8 mL / min. Sample was desalted to phosphate buffered saline (PBS), pH 7.4.

[0140] Determination of Drug Antibody Ratio (PAR) for ADC-2

[0141] DAR determination for the ADC-2 was accomplished by Hydrophobic Interaction chromatography (HIC) with a Tosoh Biosciences HIC column (phase Butyl, L x I.D 3.5 cm 4 4.6 mm, 2.5 pM particle size). Separation of conjugate sample via a HIC column afforded one peak corresponding to the antibody conjugated to two drugs, as shown in Figure 4. Elution was performed using the following procedure: Buffer A (25 mM sodium phosphate, 1.5 M ammonium sulphate, pH 7.0) and buffer B (25 mM sodium phosphate, 25% isopropanol pH 7.0); Gradient - 0-30 min = 0-100% buffer B; 30-35 min = 0 % buffer B.

[0142] Determination of monomeric content of ADC-2

[0143] Size-exclusion chromatography (SEC) was employed to determine the degree to which the conjugate had aggregated during conjugation using a MAbPac™ SEC-1 column (5 pM, 300 A, 7.8 x 300 mm). Elution was performed in 20 mM MES sodium salt, 150 mM NaCI, 5% MeCN, pH 6.0. ADC-2 shows more than 99% monomeric content, as shown in Figure 5.

[0144] Example 5. ADC-3

[0145] In this example, the antibody was modified with an isoprenoid derivative functionalised with a ketone group, shown above as Compound 4, for coupling with Compound 5 via oxime-forming chemistry using the procedure described below. Compound 4 was prepared by the method described in US2012 / 0308584. Compound 5 was synthesised by the method described in WO2018182341.

[0146] ADC-3 generation

[0147] HC1LC1 antibody with CAAX tag was prenylated with 6.25 molar eguivalents of compound 4 in the presence of 0.2 pM FTase, 250 pM DTT, 50 mM Tris-HCI, 0.01 mM ZnCh and 5 mM MgCl2 at pH 7 for >18 hours at 30 °C. The resulting intermediate was desalted into PBS, pH 7.4. Prenylated intermediate was conjugated with 10 molar equivalents of compound 5 in the presence 10% DMSO for 6 hours at 30 °C. ADC-3 was purified by semi-preparative HIC using a Phenyl phase HIC column (Tosoh Bioscience, L x I.D 7.5 cm x 7.5 mm) using the following procedure; Buffer A (50 mM potassium phosphate + 0.5 M ammonium sulphate, pH 7.0) and Buffer B (50 mM potassium phosphate + 30% (v / v) MeCN, pH 7.0); Gradient - 0-30 mins = 0-100% buffer B; 30-32 min = 100% buffer B; 32-32.1 min = 100-0% buffer B; 32.1-49 min = 0% buffer B; Flow rate - 0.8 mL / min. Sample was desalted to phosphate buffered saline (PBS), pH 7.4.

[0148] Determination of Drug Antibody Ratio (PAR) for ADC-3

[0149] DAR determination for the ADC-3 was accomplished by Hydrophobic Interaction chromatography (HIC) with a Tosoh Biosciences HIC column (phase Butyl, L x I.D 3.5 cm 4 4.6 mm, 2.5 pM particle size). Separation of the conjugate sample via a HIC column afforded one peak corresponding to the antibody conjugated to two drugs, as shown in Figure 6. Elution was performed using the following procedure: Buffer A (25 mM sodium phosphate, 1.5 M ammonium sulphate, pH 7.0) and buffer B (25 mM sodium phosphate, 25% isopropanol pH 7.0); Gradient - 0-30 min = 0- 100% buffer B; 30-35 min = 0 % buffer B. Sample was desalted to phosphate buffered saline (PBS), pH 7.4.

[0150] Determination of monomeric content of ADC-3

[0151] Size-exclusion chromatography (SEC) was employed to determine the degree to which the conjugate had aggregated during conjugation using a MAbPac™ SEC-1 column (5 pM, 300 A, 7.8 x 300 mm). Isocratic elution was performed in 20 mM MES sodium salt, 150 mM NaCI, 5% MeCN, pH 6. 0. ADC-3 shows more than 99% monomeric content, as shown in Figure 7.

[0152] Example 6. Cantuzumab ravtansine comparator

[0153] As described above, cantuzumab ravtansine is a humanised antibody-drug conjugate targeting CanAg. Cantuzumab ravtansine comprises cantuzumab conjugated to the cytotoxic maytansinoid drug, ravtansine, shown as Compound 6 below.

[0154] Preparation of Cantuzumab ravtansine

[0155] HuC242 (Cantuzumab antibody) at 5.35mg / mL in PBS buffer was preconditioned for conjugation by the addition of 5% v / v 0.5 M borate 25 mM EDTA to achieve pH 8.2.

[0156] 6 equivalents of compound 6 over antibody were added as a 50 mM stock in DMA, with additional DMA pre-dosed to achieve final 5% v / v of DMA. The reaction was incubated for 3h at 20 °C before it was quenched with the addition of 6 equivalents of glycine over antibody added from a 50 mM stock solution in water. The conjugate at DAR 4.0 was purified by G25 desalting followed by 6 volumes of buffer exchange using a 30 KDa ViVa membrane concentrator into 25 mM Histidine / CI, pH 6.0, 200 mM sucrose. 2% w / v polysorbate 20 (PS20) was added to achieve final of 0.02% w / v of PS20 in 25 mM His / CI, pH 6.0, 200 mM sucrose.

[0157] Determination of Drug Antibody Ratio (DAR) for Cantuzumab ravtansine by UV-VIS

[0158] The DAR value for Cantuzumab ravtansine was determined using UV-VIS analysis at 252 nm, 280 nm and 320 nm. Dilutions were performed in formulation buffer using a 1 cm path length quartz cuvette with blank correction with formulation buffer alone . The molar concentration of DM4 in the ADC sample was calculated following equation 1 with [DM4] = DM4 molar concentration, A280 = absorbance at 280 nm - absorbance at 320 nm x dilution, A252 = absorbance at 252nm - absorbance at 320 nm x dilution, £DM4252= 26159 M’V, £DM428o = 5180 M’1C’1.

[0159] Equation 1: Calculation for determining concentration (mol / L) of DM4

[0160] The molar concentration of protein in the ADC sample was calculated following equation 2 with [Ab] = molar protein concentration, A280 = absorbance at 280 nm - absorbance at 320 nm x dilution, £DM42so = 5180 M’1C’1, £Ab2so = 223400 M’1C’1, [DM4] = molar concentration of DM4 in ADC sample, from equation 1.

[0161] Equation 2: Calculation for determining protein concentration (mol / L) for the ADC, subtracting DM4 contribution

[0162] To calculate the DAR by UV analysis, the molar concentration of DM4 in the ADC sample is divided by the molar protein concentration of the ADC, as in equation 3. [DM4] = molar concentration of DM4 in ADC sample, from equation 1 and [Ab] = molar protein concentration from equation 2. A DAR of 4.04 was calculated using equations 1 to 3 and the results shown in Table 3. Equation 3: DAR calculation by UV analysis, combining results from equations 1 and 2

[0163] Table 3 shows triplicate UV-vis readings of Cantuzumab ravtansine and summary of DAR value calculated based on equations 1, 2 and 3.

[0164] Determination of monomeric content of Cantuzumab ravtansine comparator

[0165] ADC was assessed for monomeric content and the presence of high molecular weight (HMW) aggregates, dimers, and fragments (LMW) using size exclusion chromatography (TOSOH TSKgel G3000SWXL 7.8 mm x 30 cm, 5 pmcolumn). Running conditions: Flow at 0.5 mL / min in 10% IPA, 0.2 M Potassium phosphate, 0.25 M Potassium chloride, pH 6.95. Analysis of Cantuzumab ravtansine showed 98.6 % monomeric ADC, as shown in Figure 8.

[0166] Example 7. Determination of CanAg expression levels on SNU-16, Colo205, BxPC3, HT29 and N87 cell lines using Flow Cytometry

[0167] The expression of CanAg in five different cell lines was evaluated by comparison of a Phycoerythrin (PE)-conjugated HC1LC1 antibody to a Phycoerythrin -conjugated Isotype-matched control antibody (Biolegend, No:403504), using a flow cytometrybased binding assay. Phycoerythrin-conjugated HC1 LC1 antibody was prepared using the PE I R-Phycoerythrin Conjugation Kit - Lightning-Link® kit (Abeam, ab102918) following manufacture instructions. The five cell lines tested were SNU- 16 (gastric cancer cell line), Colo-205 (human colorectal cancer cell line), BxPC-3 (human pancreatic epithelial adenocarcinoma cells), N87 (human gastric carcinoma) and HT29 (human colorectal adenocarcinoma cells). Cells were harvested and resuspended with FACS buffer and counted. 2 x 105cells were aliquoted and washed once with 3 ml of FACS buffer. Cells were then resuspended with 100 pL FACS buffer containing 2 pL (20 pg / ml) of HC1LC1 antibody-PE or the Isotype control-PE. Cells were incubated with PE-conjugates at 4 °C for 30 minutes followed by washing with 3 mL of FACS buffer twice. Cells were re-suspended in 500 pL FACS buffer for flow cytometry analysis. BD Quantibrite™ PE beads (BD Biosciences, 340495) were reconstituted with 0.5 mL 0.5% BSA and analysed following the manufacturer’s instructions.

[0168] The PE geometric means of the different HC1LC1 antibody-PE concentrations were exported and the Log10 values were calculated. Log10 values were also calculated for the number of PE molecules per bead, based on lot-specific values, provided by the manufacturer. A linear regression of Log10 values for PE geometric means against the number of PE molecules per bead was generated. To determine PE molecules per cell, Log10 PE geometric means were substituted into the equation and the anti-Log determined. As shown in Figure 9 and Table 4, SNU-16 and Colo- 205 cells were found to be the highest expressors of CanAg with an average of 11.5 million and 2.6 million PE molecules per cell. HT-29 showed moderate expression of CanAg with an average of 626,000 PE molecules per cell. BxPC-3 showed low expression of CanAg with an average of 75,000 PE molecules per cell. N87 cells showed very low expression of CanAg with an average of 2,500 PE molecules per cell.

[0169]

[0170] Table 4 shows calculations of PE molecules on SNU-16Colo205, BxPC3 and HT29 cell lines.

[0171] Example 8. Determination of p-glucuronidase activity in Colo205, BxPC3, HT29 and N87 cell lines using fluorometric assay

[0172] Activity of p-glucuronidase enzyme was determined in cell lines using p- glucuronidase Activity Assay Kit (Abeam, Ab234625). Cells were counted and 1 x 107cells were collected. Cells were washed once with 1 mL DPBS and centrifuged at 400 x g for 5 minutes. Supernatant was discarded and cells were lysed with 500 l assay buffer (Colo205, HT-29 and BxPC3 cells) or 300 pl assay buffer (N87 cells) and homogenized by ultrasonic cell disruptor. Lysate was centrifuged at 10,000 x g for 5 minutes at 4 °C and supernatant was collected. 50 pL of supernatant was added to wells of a black 96-well plate. The volume was adjusted to 90 pL with p- Glucuronidase Assay Buffer. 5 pL of the reconstituted Positive control was mixed with p-Glucuronidase Assay Buffer to have 90 pL solution. 200 pM solution of 4- Methylumbelliferone (4-Mll) standard was prepared in p-Glucuronidase Assay Buffer and 0, 0.5, 1 , 2, 4, 6, 8, 10 pL of 200 pM 4-Mll standard was added into a series of wells and the volume of each reaction was adjusted to 100 pL with p-Glucuronidase Assay Buffer to generate 0, 0.1 , 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0 nmol of 4-Mll per well respectively. Substrate solution was 10-fold diluted in p-Glucuronidase Assay Buffer. 10 pL of the Substrate was added the Positive control and test samples. Fluorescence (Ex / Em = 330 / 450 nm) was measured immediately after addition of substrate for 60 minutes at 37 °C recording every 2 min. The results are shown in Figure 10 with the p-glucuronidase level on 4 cell lines normalized to volume. HT29 cells were found to have the highest activity of - glucuronidase enzyme. Colo-205 and BxPC3 cells were found to have similar activity of p-glucuronidase, and N87 cells showed the lowest activity of the enzyme.

[0173] Example 9. In vitro activity assays

[0174] In vitro activity was assessed using the luminescence-based Cell Titre-Gio (CTG) assay (Promega, No: G7572), which quantitates the amount of ATP present as a measure of viable cells. Specificity of cell killing was shown by incubating cells with a non-binding ADC control composed of the same payload.

[0175] Anti-CanAg ADC in vitro activity was evaluated in the colorectal cancer cell lines, Colo-205 (ATCC: CCL-222) and HT-29 (ATCC: HTB-38), the pancreatic adenocarcinoma cell line, BxPC-3 (ATCC: CRL-1687), and N87 gastric carcinoma (ATCC: CRL-5822). The ADC non-binding control was an anti-CD19 ADC (DAR 2.0) composed of the same linker-drug combination as ADC-3.

[0176] Cells were trypsinised and seeded to 96 well microplates in appropriate complete medium (Colo-205, BxPC-3 and N87 - RPMI-1640 with 20% heat inactivated fetal bovine serum (FBS); HT-29 - McCoy’s-5A medium with 20% heat inactivated FBS) for 24 h at 37 °C, 5% CO2. Cells were seeded at density of 4000 (Colo-205) or 5000 (BxPC-3, HT-29, N87) cells per well, in a volume of 100 pL. After incubation, media was removed and replaced with 100 pL of fresh appropriate growth media. ADC-2 was 3-fold serially or 5- fold serially diluted in appropriate growth media to have a range of the following concentrations of ADC in 100 pL: BxPC-3 and HT-29 - 1000 nM, 333.3 nM, 111.1 nM, 37 nM, 12.34 nM, 4.1 nM, 1.37 nM, 0.45 nM, 0.15 nM, 0.051 nM; Colo-205 - 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, 0.0013 nM, 0.00026 nM, 0.000051 nM; N87 - 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.06 nM, 0.013 nM, 0.0026 nM, 0.0013 nM.

[0177] For the comparison of ADC-1 , ADC-2 and ADC-3, cells were trypsinised and seeded to 96 well microplates in appropriate complete medium (RPMI-1640 with 10% FBS) for 24 h at 37 °C, 5% CO2. Cells were seeded at a density of 4000 (Colo-205) cells per well, in a volume of 100 pL. After incubation, the media was removed and replaced with 100 pL of fresh appropriate growth media. ADCs were 5-fold serially diluted in appropriate growth media to have a range of the following concentrations of ADC in 100 pL: 50 nM, 10 nM, 2 nM, 0.40 nM, 0.08 nM, 0.016 nM, 0.0032 nM, 0.00064 nM 0.000128 nM, 0.0000256 nM, 0.0000051 nM, 0.000001 nM.

[0178] For the comparison of ADC-3 with Cantuzumab ravtansine and a non-binding ADC control cells were trypsinised and seeded to 96 well microplates in appropriate complete medium (RPMI-1640 with 10% FBS) for 24 h at 37 °C, 5% CO2. Cells were seeded at a density of 4000 (Colo-205) cells per well, in a volume of 100 pL. After incubation, the media was removed and replaced with 100 pL of fresh appropriate growth media. All ADCs were 3-fold serially diluted in appropriate growth media to have a range of the following concentrations of ADC in 100 pL: 50 nM, 16.67 nM, 5.56 nM, 1.85 nM, 0.62 nM, 0.206 nM, 0.069 nM, 0.023 nM, 0.0076 nM, 0.0025 nM, 0.00085 nM, 0.00028 nM. Cells were incubated with ADCs for 3 days (72 h). After ADC treatment, CTG reagent and cell plates were kept at RT for 30 minutes before CTG reagent addition. 100 pL of CTG reagent was added to each well and plates were shaken for 30s. Then the plate was incubated for 20 minutes at RT followed by luminescence measurements.

[0179] The results are shown in Figures 11 to 16. IC50 of ADC-2 correlates with CanAg expression level on Colo205, BxPC3 and N87 cells (see Figures 11, 12 and 14). Colo205 and BxPC3 cell lines have the same activity of p-glucuronidase (see Figure 10). N87 cells showed the lowest amount of p-glucuronidase activity and CanAg expression level (see Figure 10 and 9), and ADC-2 showed very limited activity on N87 cells (see Figure 14). The activity of ADC-2 on HT-29 was similar to the activity on Colo205 cells in spite of the lower expression level of CanAg on HT-29 (see Figure 13) but the p-glucuronidase activity was the highest on HT-29 (see Figure 10). This demonstrates that p-glucuronidase activity is also a factor contributing to the observed ADC activity. ADC-2 induced 100% cell killing on Colo-205, HT-29 and BcPC3. The incubation of cells with a non-binding ADC confirmed the specific activity of ADC-2 (see Figures 11 to 14).

[0180] As shown in Figure 15, ADC1 , ADC-2 and ADC-3 all showed similar potent activity on Colo 205 cells, indicating that ADC in vitro activity is independent of the conjugation chemistry used in this cell line. Significantly, Cantazumab ravtansine is less efficient in killing Colo205 cancer cells as only 75% of cells were killed (see Figure 16).

[0181] Example 10. In vivo efficacy studies in Colo-205 xenograft model

[0182] ADC1 , ADC-2, ADC-3 and Cantuzumab ravtansine were evaluated in female CB17 SCID mice bearing Colo-205 xenograft. Mice were subcutaneously inoculated into the right flank with 5 x 106Colo-205 cells in 0.2 mL of DPBS mixed 1 :1 with BD Matrigel. Tumour-bearing mice were randomized into groups of 5 animals each and treated with a single intravenous dose of ADC or alternatively with a vehicle solution (30 mM histidine, 200 mM sorbitol, 0.02% PS20 (w / v)) when the average tumour volume reached approximately 170 mm3(Cantuzumab ravtansine) or 190 mm3(ADC-1 , ADC-2 and ADC-3). Conjugate doses of 1 mg / kg, for ADC-1 , ADC-2 and ADC-3 (13 nmol drug / kg), and 2 mg / kg (53 nmol drug / kg) for Cantuzumab ravtansine were used for the Colo-205 xenograft study. Tumour size was measured thrice weekly in two dimensions using a calliper, and the volume was expressed in mm3using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively (see Figures 17 A, 18 A and 19 A). The tumour size was then used for calculations of TGI (%) values (see Table 5). TGI, representing antitumor effectiveness, were calculated using the formula TGI (%)=[1 -(Vtreat-t-Vtreat- i) / (Vcontroi-t-VControi-i)]x100, where Vtreat-i and Vcontroi-1 are the mean volumes of the treated and control groups on grouping day; Vtreat-t and VCOntroi-t are the mean volumes of the treated and control groups on a given day. Animals were euthanized when tumour volumes reached 2000 mm3. Body weight was also measured thrice weekly as a measure of compound toxicity (see Figures 17 B, 18 B and 19 B).

[0183] The in vivo effect of ADCs on Colo205 tumour xenograft is shown in Figure 17, 18 and 19. ADC-1, ADC-2 and ADC-3 induced substantial tumour growth inhibition (79%, 103% and 102% TGI, respectively) at 1 mg / kg (13 nmol of payload / kg (ADC-2 and ADC-3) or 15 nmol of payload / kg (ADC-1) with no observable toxicity, for example, see Figures 17 and 18 and Table 5. Cantuzumab ravtansine showed 40% tumour growth inhibition at 2 mg / kg (53 nmol of payload / kg) equivalent of 4 mg / kg of ADC-2 and ADC-3 as per drug load (see Figure 19 A and Table 5).

[0184] Table 5 shows Tumour Growth Inhibition (TGI) of ADCs tested in Colo-205 xenograft model on day 15 of the study. * TGI calculation for Cantuzumab ravtansine was based on day 16.

[0185] Example 11. In vivo efficacy studies in BxPC3 xenograft model

[0186] ADC-2, ADC-3 and Cantuzumab ravtansine were evaluated in female CB17 SCID mice bearing BxPC3 xenograft. Mice were subcutaneously inoculated into the right flank with 5 x 106BxPC3 cells in 0.2 mL of DPBS containing 50% BD Matrigel. Tumour-bearing mice were randomized into groups of 5 animals each and treated with a single intravenous dose of ADC or alternatively with a vehicle solution comprising PBS pH 7.4 (see Figure 20) or 30 mM histidine, 200 mM sorbitol, 0.02% PS20 (w / v) (see Figure 21) when the average tumour volume reached approximately 150-180 mm3. Conjugate doses of 0.4 mg / kg (5.3 nmol of conjugated drug / kg), 1 mg / kg (13 nmol of conjugated drug / kg) for ADC-2 and ADC-3, and 0.2 mg / kg (5.3 nmol of conjugated drug / kg) and 0.5 mg / kg (13 nmol of conjugated drug / kg) for Cantuzumab ravtansine were used. Tumour size was measured thrice weekly in two dimensions using a calliper, and the volume was expressed in mm3using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively (see Figures 20 A and 21 A). The tumour size was then used for calculations of TGI (%) values (see Table 6). TGI, representing antitumor effectiveness, were calculated using the formula TGI (%)=[1 -(Vtreat-t-Vtreat-i) / (Vcontroi-t- VControi-i)]x100, where Vtreat-i and VCOntroi-i are the mean volumes of the treated and control groups on grouping day; Vtreat-t and Vcontroi-t are the mean volumes of the treated and control groups on a given day. Animals were euthanized when tumour volumes reached 2000 mm3. Body weight was also measured thrice weekly as a measure of compound toxicity (see Figures 20 B and 21 B). The in vivo effect of ADCs on BxPC3 tumour xenograft is shown in Figures 20 to 21. ADC-2 induced tumour growth inhibition (66.7 and 107% TGI) at doses such as 0.4 mg / kg (5.3 nmol of conjugated drug / kg) and 1 mg / kg (13 nmol of conjugated drug / kg) (see Figure 20 A and Table 6). Treatment of BxPC3 xenograft with ADC-3 showed lower activity (57% and 101 % TGI) over ADC-2 at 0.4 mg / kg (5.3 nmol of conjugated drug / kg) and 1 mg / kg (13 nmol of conjugated drug / kg) doses. The Cantuzumab ravtansine comparator, 0.2 mg / kg (5.3 nmol of conjugated drug / kg) and 0.5 mg / kg (13 nmol of conjugated drug / kg) showed a weak anti-tumour response in comparison to the ADC-2 and ADC-3 treatment at the same doses (see Figure 20)

[0187] For the Cantuzumab ravtansine comparator, a dose of 2 mg / kg (53 nmol drug / kg) induced complete tumour inhibition but the anti-cancer activity was not maintained as tumour started to regrowth after 32 days (see Figure 21 A).

[0188] Table 6 shows Tumour Growth Inhibition (TGI) of ADCs tested in BxPC3 xenograft model on day 29 or 32 of the study.

[0189] Example 12. In vivo efficacy studies in NCI-N87 xenograft model

[0190] ADC-3, a ADC non-binding control, Cantuzumab ravtansine and Enhertu were evaluated in female CB17 SCID mice bearing NCI-N87 gastric cancer xenograft.

[0191] Enhertu is the brand name for Trastuzumab deruxtecan, which is an ADC consisting of the humanised anti-Her2 antibody trastuzumab (Herceptin) covalently linked to the topoisomerase I inhibitor deruxtecan (DAR 8.0). Enhertu has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of breast cancer or gastric or gastroesophageal adenocarcinoma

[0192] The ADC non-binding control was an anti-CD19 ADC (DAR 2.0) composed of the same linker-drug combination as ADC-3. Mice were subcutaneously inoculated into the right flank with 5 x 106NCI-N87 cells in 0.1 mL of DPBS containing 50% BD Matrigel. Tumour-bearing mice were randomized into groups of five animals each and treated with a single intravenous dose of ADC or alternatively with a vehicle solution (30 mM histidine, 200 mM sorbitol, 0.02% PS20 (w / v)) when the average tumour volume reached approximately 170-175 mm3. Conjugate doses of 0.2 mg / kg (2.6 nmol of conjugated drug / kg), 0.5 mg / kg (6.7 nmol of conjugated drug / kg) and 1 mg / kg (13 nmol of conjugated drug / kg) for ADC-3; 0.5 mg / kg (6.7 nmol of conjugated drug / kg) and 1 mg / kg (13 nmol of conjugated drug / kg) for ADC non-binding control; 0.2 mg / kg (10 nmol of conjugated drug / kg), 0.5 mg / kg (26 nmol of conjugated drug / kg) and 1 mg / kg (53 nmol of conjugated drug / kg) for Enhertu; 2 mg / kg (53 nmol of conjugated drug / kg) for Cantuzumab ravtansine were used. Tumour size was measured thrice weekly in two dimensions using a calliper, and the volume was expressed in mm3using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively (see Figures 22 A, 23 A and B). The tumour size was then used for calculations of TGI (%) values (see Table 7). TGI, representing antitumor effectiveness, were calculated using the formula TGI (%)=[1 - (Vtreat-t"Vtreat-i) / (Vcontroi-t"Vcontroi-i)]x100, where Vtreat-i and Vcontroi-1 are the mean volumes of the treated and control groups on grouping day; Vtreat-t and Vcontroi-t are the mean volumes of the treated and control groups on a given day. Animals were euthanized when tumour volumes reached 2000 mm3Body weight was also measured thrice weekly as a measure of compound toxicity (see Figures 22 B, 23 C and D).

[0193] The in vivo effect of ADCs on NCI-N87 tumour xenograft is shown in Figures 22 and 23. Interestingly, the in vitro analysis of ADC-2 on NCI-N87 cells described above showed no specific activity due to lack of CanAg expression on in vitro cell culture (see Figures 14 and 9). However, in this in vivo xenograft, ADC-3 showed targetspecific activity in NCI-N87 as compared to the response of ADC non-binding control, which indicates the differences in CanAg expression on N87 cells cultured in vitro and in vivo xenograft. ADC-3 induced 53% of specific tumour growth inhibition at a dose as low as 0.2 mg / kg (2.6 nmol of conjugated drug / kg) (see Figure 22 A and Table 7). In contrast, Cantuzumab ravtansine and Enhertu required higher doses (i.e 2 mg / kg (53 nmol of drug / kg) for Cantuzumab ravtansine; and 0.5 mg / kg (26 nmol drug / kg) for Enhertu) to achieve 43% and 46% tumour growth inhibition, respectively (see Figures 23 A-B, Table 7). Further, although 1 mg / kg dose of Enhertu induced 99% TGI, tumour regrowth was observed after 28 days. On the other hand, ADC-3 dose of 0.5 mg / kg (6.7 nmol of conjugated drug / kg) and ADC-3 dose of 1 mg / kg (13 nmol drug / kg) induced 100% TGI by day 40 with no subsequent tumour recurrence for the 1 mg / kg dose (see Figure 22 A and Table 7).

[0194] Table 7 shows Tumour Growth Inhibition (TGI) of ADCs tested in NCI-N87 xenograft model on day 33 of the study.

[0195] Accordingly, in all cell lines tested, ADCs according to the present invention demonstrated superior TGI compared to the comparator anti-CanAg ADC, Cantuzumab ravtansine. Additionally, ADC-3 demonstrated comparable TGI in a gastric cancer xenograft at a near 8-fold lower conjugated drug concentration compared to Enhertu, which is an FDA approved ADC for use in the treatment of gastric cancer. Moreover, while Enhertu induced 99% TGI at higher conjugated drug concentrations (i.e. 1 mg / kg, 53 nmol / kg), tumour re-growth was observed after 28 days following administration, whereas no observable tumour re-growth was detected at day 55 following administration of ADC-3 at a dose of 1mg / kg (13 nmol drug / kg). This result is even more surprising in view of the relative concentrations of conjugated drug used, i.e. 13 nmol / kg conjugated drug for ADC-3 versus 53 nmol / kg conjugated for Enhertu.

[0196] Example 13 - Co-administration with unconjugated antibody: in vitro activity assay

[0197] In vitro activity was assessed using the luminescence-based Cell Titre-Gio (CTG) assay (Promega, No: G7572), which quantitates the amount of ATP present as a measure of viable cells. The specificity of cell killing was shown by pre-incubating cells with unconjugated CanAg antibody.

[0198] ADC-3 in vitro activity was evaluated in the colorectal cancer cell line, HT-29 (ATCC: HTB-38) and the gastric carcinoma cell line, SNll-16 (ATCC: CRL-5974).

[0199] Cells were trypsinised and seeded to 96 well microplates in appropriate complete medium (SNll-16 - RPMI-1640 with 10% fetal bovine serum (FBS); HT-29 - McCoy’s-5A medium with 10% FBS) for 24 h at 37 °C, 5% CO2. Cells were seeded at a density of 3000 (cells per well, in a volume of 100 pL. ADC-3 was either 3-fold (SNll-16) or 5-fold (HT-29) serially diluted in appropriate growth media to have a range of the following concentrations of ADC in 100 pL: SNll-16 - 1 nM, 0.33 nM, 0.11 nM, 0.037 nM, 0.012 nM, 0.0041 nM, 0.0014 nM, 0.00046 nM and 0.00015 nM; HT-29 - 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, 0.0013 nM and 0.00026 nM.

[0200] Cells were either treated directly with ADC-3 or were pre-incubated with 1 pM unconjugated anti-CanAg antibody for 30 min at 37 °C, 5% CO2. As shown in Figure 25, the IC50 potency of ADC-3 on SNll-16 cells is 0.026 nM. In the presence of 1 pM anti-CanAg antibody, the potency of ADC-3 is inhibited 15-fold to an IC50 of 0.39 nM. The IC50 potency of ADC-3 on HT-29 cells is 0.7 nM. In the presence of 1 pM anti-CanAg antibody, an accurate IC50 could not be determined, but the potency of ADC-3 was inhibited >140 fold to >100 nM. Example 14 - Co-administration with unconjugated antibody: in vivo efficacy studies in Snu-16 xenograft model

[0201] ADC-3 in the presence of various doses of unconjugated anti-CanAg antibody was evaluated in female CB17 SCID mice bearing SNU-16 xenograft. Mice were subcutaneously inoculated into the right flank with 5 x 106SNU-16 cells in 0.2 mL of DPBS mixed 1 :1 with BD Matrigel. Tumour-bearing mice were randomized into groups of 5 animals and treated with a single intravenous dose of ADC-3, unconjugated anti-CanAg antibody or alternatively with a vehicle solution (50 mM histidine pH 6.5, 150 mM NaCI, 0.01% (w / w) polysorbate 80) when the average tumour volume reached approximately 180 mm3. ADC-3 dose of 0.4 mg / kg, ADC-3 of 0.4 mg / kg in the presence of 2, 4, 8 and 20 mg / kg of unconjugated anti-CanAg antibody and unconjugated anti-CanAg antibody at 20 mg / kg were used for the SNU- 16 xenograft study. ADC-3 was premixed with an unconjugated antibody before dosing. Additional group composed of 3 tumour-bearing mice was selected for the collection of tumour tissues for preparation of formalin-fixed, paraffin embedded (FFPE) blocks when tumour volumes reached approximately 180 mm3. These not dosed mice were euthanized, and tumour tissues were excised and placed in 10% neutral buffered formalin for 18-24 hours, followed by transfer into 70% ethanol. Formalin-fixed tissues were embedded in paraffin to create a FFPE blocks.

[0202] Tumour size was measured thrice weekly in two dimensions using a calliper, and the volume was expressed in mm3using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively (see Figure 26 A). The tumour size was then used for calculations of TGI (%) values (see Table 8). TGI, representing antitumor effectiveness, was calculated using the formula TGI (%)=[1 - (Vtreat-t"Vtreat-i) / (Vcontroi-t"Vcontroi-i)]x100, where Vtreat-i and Vcontroi-1 are the mean volumes of the treated and control groups on grouping day; Vtreat-t and Vcontroi-t are the mean volumes of the treated and control groups on a given day. Animals were euthanized when tumour volumes reached 2000 mm3Body weight was also measured thrice weekly as a measure of compound toxicity (see Figure 26 B).

[0203] ADC-3 in combination with an unconjugated anti-CanAg antibody induced substantial improvements in SN-16 tumour growth inhibition in comparison to ADC-3 alone. Co-administration of 2 mg / kg of unconjugated antibody with 0.4 mg / kg ADC-3 induced 67% tumour growth inhibition (TGI) in comparison to the ADC dosed without antibody (30% TGI). Further increasing the amount of co-administered antibody to 20 mg / kg resulted in 87% TGI by ADC-3 with no observable toxicity (see Figures 26 A-B). The unconjugated anti-CanAg antibody at 20 mg / kg had very limited efficacy in the tumour model by itself (19.37% TGI). Thus, it can be seen that the combination of ADC-3 and unconjugated antibody had a synergistic effect since the observed activity was greater than the combined efficacy of ADC-3 alone and unconjugated antibody alone.

[0204] Table 8 shows Tumour Growth Inhibition (TGI) of ADC-3 with and without unconjugated antibody tested in SNll-16 xenograft model on day 24 of the study.

[0205] Example 15 - Co-administration with unconjugated antibody: In vivo efficacy studies in HT-29 xenograft model

[0206] ADC-3 in the presence of various doses of unconjugated anti-CanAg antibody was evaluated in female CB17 SCID mice bearing HT-29 xenograft. Mice were subcutaneously inoculated into the right flank with 5 x 106Ht-29 cells in 0.2 mL of DPBS mixed 1:1 with BD Matrigel. Tumour-bearing mice were randomized into groups of 5 animals each and treated with a single intravenous dose of ADC-3, unconjugated anti-CanAg antibody or alternatively with a vehicle solution (50 mM histidine pH 6.5, 150 mM NaCI, 0.01 % (w / w) polysorbate 80) when the average tumour volume reached approximately 176 mm3. ADC-3 dose of 0.4 mg / kg, ADC-3 of 0.4 mg / kg in the presence of 2, 4, 8 and 20 mg / kg of unconjugated anti-CanAg antibody and unconjugated anti-CanAg antibody at 20 mg / kg were used for the HT- 29 xenograft study. ADC-3 was premixed with an unconjugated antibody before dosing. Additional group composed of 3 tumour-bearing mice was selected for the collection of tumour tissues for preparation of formalin-fixed, paraffin-embedded (FFPE) blocks when tumour volumes reached approximately 176 mm3. These not dosed mice were euthanized, and tumour tissues were excised and placed in 10% neutral buffered formalin for 18-24 hours, followed by transfer into 70% ethanol. Formalin-fixed tissues were embedded in paraffin to create a FFPE blocks.

[0207] Tumour size was measured thrice weekly in two dimensions using a calliper, and the volume was expressed in mm3using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively (see Figure 27 A). The tumour size was then used for calculations of TGI (%) values (see Table 9). TGI, representing antitumor effectiveness, was calculated using the formula TGI (%)=[1 - (Vtreat-t"Vtreat-i) / (Vcontroi-t"Vcontroi-i)]x100, where Vtreat-i and Vcontroi-1 are the mean volumes of the treated and control groups on grouping day; Vtreat-t and Vcontroi-t are the mean volumes of the treated and control groups on a given day. Animals were euthanized when tumour volumes reached 2000 mm3Body weight was also measured thrice weekly as a measure of compound toxicity (see Figure 27 B).

[0208] ADC-3 in combination with unconjugated anti-CanAg antibody induced improvements in HT-29 tumour growth inhibition in comparison to only ADC-3. Coadministration of 4 and 8 mg / kg of unconjugated antibody with fixed dose of ADC-3 induced -90% tumour growth inhibition (TGI) in comparison to the ADC dosed without antibody (61 % TGI). Further increasing amount of co-administered antibody to 20 mg / kg resulted in lower TGI (84%) by ADC-3 (see Figure 27 A). The unconjugated anti-CanAg antibody at 20 mg / kg had very limited efficacy in the HT29 tumour model (18.39% TGI). Thus, it can be seen that the combination of ADC-3 and unconjugated antibody had a synergistic effect since the observed activity was greater than the combined efficacy of ADC-3 alone and unconjugated antibody alone.

[0209] Table 9 shows Tumour Growth Inhibition (TGI) of ADC-3 with and without unconjugated anti-CanAg antibody tested in HT-29 xenograft model on day 20 of the study.

[0210] The examples described above demonstrate that the presence of unconjugated antibody improves anti-cancer activity of the already very potent ADC-3 conjugate. The unconjugated anti-CanAg antibody had very limited efficacy in the tumour models by itself (up to around 20% TGI at the highest tested dose). The efficacy of ADC-3, co-administered with unconjugated anti-CanAg antibody, was improved across cancer models with high (SNll-16) and moderate (HT-29) CanAg expression levels (see Tables 8-9 and Figure 9).

[0211] These results are surprising since it is considered in the art that the presence of unconjugated antibody will compete with the corresponding ADC for antigen binding sites, leading to the decreased anti-tumour activity of the conjugate. For example, this can be seen in the in vitro activity assay results reported in Figure 25, where preincubation with unconjugated antibody resulted in a substantial drop in ADC activity. Without wishing to be bound by theory, the improvement observed in in vivo activity when ADC-3 was co-administered with unconjugated antibody may be due to improved conjugate distribution in tumour tissue, resulting in greater overall tumoral exposure to ADC-3. In particular, the unconjugated antibody may compete with the ADC for CanAg receptors, thereby allowing more cells to receive the cytotoxic payload. Thus, a number of factors must be controlled to ensure that enough ADC is delivered into cancer cells to induce cell death in the presence of competing antibody. These include payload potency, target expression levels, ADC internalisation rates, and / or ratio of unconjugated antibody to ADC.

[0212] It will be appreciated by persons skilled in the art that the above embodiment has been described by way of example only and not in any limitative sense, and that various alterations and modifications are possible without departing from the scope of the invention as defined by the appended claims.

[0213] Sequence Listings

[0214] In the following sequence listings, amino acids belonging to the signal peptide are shown in bold.

[0215] 1. Murine parental sequence used for humanisation

[0216] SEQ ID NO: 4 - Murine c242 HC (CDR regions are shown underlined)

[0217] MDWLRNLLFLMAAAQSIQAQVQLVQSGPELKKPGETVKISCKASDYTFTYYGMN WVKQAPGKGLKWMGWIDTTTGEPTYAEDFKGRIAFSLETSASTAYLQIKNLKNEDT ATYFCARRGPYNWYFDVWGAGTTVTVSSAKTTPPSVYP

[0218] SEQ ID NO: 5 - Murine c242 LC (CDR regions are shown underlined)

[0219] MRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTY

[0220] LYWFLQRPGQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC

[0221] LQHLEYPFTFGPGTKLELKRADAAPTVT

[0222] 2. Murine c242 sequence in hlqG1 (G1m17) backbone

[0223] SEQ ID NO: 6 - pLEV123-Parental murine HC-hlgG1- murine Ab (Mu)

[0224] MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGPELKKPGETVKISCKASDYTFTYY

[0225] GMNWVKQAPGKGLKWMGWIDTTTGEPTYAEDFKGRIAFSLETSASTAYLQIKNLK

[0226] NEDTATYFCARRGPYNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA

[0227] ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQ

[0228] TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

[0229] SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

[0230] LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS

[0231] LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

[0232] GNVFSCSVMHEALHNHYTQKSLSLSPG*

[0233] SEQ ID NO: 7 - pLEV123-Parental murine LC-hKappa METDTLLLWVLLLWVPGSTGDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNT

[0234] YLYWFLQRPGQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYY

[0235] CLQHLEYPFTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

[0236] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

[0237] GLSSPVTKSFNRGEC*

[0238] 3. Humanised Antibodies for CanAq target

[0239] SEQ ID NO: 8 - pLEV123-HC1-hlgG1(G1m17)

[0240] MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASDYTFTY

[0241] YGINWVRQAPGQGLEWMGWIDTTTGEPNYAQKLQGRVTFTLDTSASTAYMELRSL

[0242] RSDDTAVYYCARRGPYNWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG

[0243] TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

[0244] TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT

[0245] LMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV

[0246] LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN

[0247] QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

[0248] QQGNVFSCSVMHEALHNHYTQKSLSLSPG*

[0249] SEQ ID NO: 9 - pLEV123-HC2-hlgG1(G1m17)

[0250] MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASDYTFTY

[0251] YGMNWVRQAPGQGLEWMGWIDTTTGEPSYAQKFQGRVTFTLDTSASTVYMELSS

[0252] LRSEDTAVYYCARRGPYNWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0253] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0254] GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

[0255] TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

[0256] VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK

[0257] NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

[0258] WQQGN VFSCSVM H EALH N H YTQKSLSLSPG*

[0259] SEQ ID NO: 10 - pLEV123-HC3-hlgG1(G1m17)

[0260] MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASDYTFTY

[0261] YGINWVRQATGQGLEWMGWIDTTTGEPTYAQKFQGRVTFTLETSISTAYMELSSL

[0262] RSEDTAVYYCARRGPYNWYFDVWGAGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT

[0263] QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

[0264] MISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[0265] TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ

[0266] VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ

[0267] QGNVFSCSVMHEALHNHYTQKSLSLSPG*

[0268] SEQ ID NO: 11 - pLEV123-LC1-hKappa

[0269] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGNT

[0270] YLYWYLQKPGQSPQLLIYRMSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVY

[0271] YCLQHLEYPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE

[0272] AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC*

[0273] SEQ ID NO: 12 - pLEV123-LC2-hKappa

[0274] METDTLLLWVLLLWVPGSTGDIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNT

[0275] YLYWYLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY

[0276] CLQHLEYPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

[0277] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC*

[0278] SEQ ID NO: 13 - pLEV123-LC3-hKappa

[0279] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSKSLLHSNGNT

[0280] YLYWYLQKPGQSPQLLIYRMSNLFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY

[0281] CLQHLEYPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

[0282] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

[0283] GLSSPVTKSFNRGEC* 4. HuC242 (Cantuzumab) antibody

[0284] SEQ ID NO: 14 - H-GAMMA-1

[0285] QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWIDTT

[0286] TGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFDVWG

[0287] QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

[0288] SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0289] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN

[0290] WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA

[0291] PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP

[0292] ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS

[0293] LSPGK

[0294] SEQ ID NO: 15 - L-KAPPA

[0295] DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRM

[0296] SNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTKLELK

[0297] RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQES

[0298] VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0299] 5. Humanised antibodies with CAAX tag (GGGGGGGCVIM - SEQ ID NO: 3) at LC

[0300] C-terminus

[0301] SEQ ID NO: 16 - pLEV123-HC1-hlgG1(G1m17)

[0302] MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASDYTFTY

[0303] YGINWVRQAPGQGLEWMGWIDTTTGEPNYAQKLQGRVTFTLDTSASTAYMELRSL

[0304] RSDDTAVYYCARRGPYNWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG

[0305] TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

[0306] TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT

[0307] LMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV

[0308] LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN

[0309] QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

[0310] QQGNVFSCSVMHEALHNHYTQKSLSLSPG* SEQ ID NO: 17 - pLEV123-HC2-hlgG1(G1m17)

[0311] MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASDYTFTY

[0312] YGMNWVRQAPGQGLEWMGWIDTTTGEPSYAQKFQGRVTFTLDTSASTVYMELSS

[0313] LRSEDTAVYYCARRGPYNWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0314] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0315] GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

[0316] TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

[0317] VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK

[0318] NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

[0319] WQQGN VFSCSVM H EALH N H YTQKSLSLSPG*

[0320] SEQ ID NO: 18 - pLEV123-HC3-hlgG1(G1m17)

[0321] MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASDYTFTY

[0322] YGINWVRQATGQGLEWMGWIDTTTGEPTYAQKFQGRVTFTLETSISTAYMELSSL

[0323] RSEDTAVYYCARRGPYNWYFDVWGAGTLVTVSSASTKGPSVFPLAPSSKSTSGGT

[0324] AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT

[0325] QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

[0326] MISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[0327] TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ

[0328] VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ

[0329] QGNVFSCSVMHEALHNHYTQKSLSLSPG*

[0330] SEQ ID NO: 19 - pLEV123-LC1-hKappa

[0331] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGNT

[0332] YLYWYLQKPGQSPQLLIYRMSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVY

[0333] YCLQHLEYPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE

[0334] AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

[0335] QGLSSPVTKSFNRGECGGGGGGGCVIM SEQ ID NO: 20 - pLEV123-LC2-hKappa

[0336] METDTLLLWVLLLWVPGSTGDIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNT

[0337] YLYWYLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY

[0338] CLQHLEYPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

[0339] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

[0340] GLSSPVTKSFNRGECGGGGGGGCVIM

[0341] SEQ ID NO: 21 - pLEV123-LC3-hKappa

[0342] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSKSLLHSNGNT

[0343] YLYWYLQKPGQSPQLLIYRMSNLFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY

[0344] CLQHLEYPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

[0345] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

[0346] GLSSPVTKSFNRGECGGGGGGGCVIM

Claims

CLAIMS1 . A composition comprising: an anti-CanAg antibody-drug conjugate; and an unconjugated anti-CanAg antibody; wherein the anti-CanAg antibody-drug conjugate is represented by Formula I or a pharmaceutically acceptable salt or solvate thereof:Formula I: Ab-(L-D)n wherein:Ab is the anti-CanAg antibody or an antigen-binding fragment thereof;L is a linker connecting Ab to D; n is an integer from 1 to 20; andD is a pyrrolobenzodiazepine dimer prodrug represented by Formula (II):Formula (II)2. The composition according to claim 1 , wherein the anti-CanAg antibody is a humanised C242 antibody or antigen-binding fragment thereof.

3. The composition according to claim 1 or 2, wherein n = 1 to 8, optionally wherein n = 2 to 4.

4. The composition according to any one of the preceding claims, wherein the ratio of the anti-CanAg antibody-drug conjugate to unconjugated antibody is in a range from 1:1 to 1:50.

5. The composition according to any one of the preceding claims, wherein the linker comprises a central portion represented by Formula III, IV, V or isomers thereof:Formula III Formula IV Formula V wherein:L1 comprises a first connecting portion connecting the central portion to Ab; andL2 comprises a second connecting portion connecting the central portion to D.

6. The composition according to any one of the preceding claims, wherein L is covalently bound to Ab by a thioether bond, and optionally wherein the thioether bond comprises a sulfur atom of a cysteine of the Ab.

7. The composition according to any one of the preceding claims, wherein Ab includes one or more amino acid motifs that can be recognised by an isoprenoid transferase.

8. The composition according to claim 7, wherein the isoprenoid transferase is FTase (farnesyl protein transferase) or GGTase (geranylgeranyl transferase).

9. The composition according to any preceding claim 7 or claim 8, wherein the amino acid motif is CYYX, XXCC, XCXC, CXC or CXX, wherein C denotes cysteine, Y denotes an aliphatic amino acid, and X denotes an amino acid that determines substrate specificity of isoprenoid transferase.

10. The composition according to any preceding claims, wherein the anti-CanAg antibody-drug conjugate comprises a structure selected from:orWherein: m is an integer from 0 to 20; and o is an integer from 0 to 10.

11. A pharmaceutical composition comprising a composition according to any one of claims 1 to 10; and one or more pharmaceutically acceptable excipients, diluents, or carriers.

12. A kit comprising:(a) a first pharmaceutical composition comprising an anti-CanAg antibodydrug conjugate and one or more pharmaceutically acceptable excipient, diluents, or carriers; and(b) a second pharmaceutical composition comprising an unconjugated anti- CanAg antibody and one or more pharmaceutically acceptable excipient, diluents, or carriers.

13. The composition according to any of claims 1 to 10, the pharmaceutical composition according to claim 11, or the kit according to claim 12 for use in the treatment of cancer.

14. A pharmaceutical combination for use in the treatment of cancer comprising:(a) a first pharmaceutical composition comprising an anti-CanAg antibodydrug conjugate and one or more pharmaceutically acceptable excipient, diluents, or carriers; and(b) a second pharmaceutical composition comprising an unconjugated anti- CanAg antibody and one or more pharmaceutically acceptable excipient, diluents, or carriers; wherein the compositions (a) and (b) are provided in separate dosage forms, which are administered simultaneously, sequentially or as a mixture of compositions (a) and (b).

15. The pharmaceutical combination for use in accordance with claim 14, wherein the first and second pharmaceutical compositions are separately packaged and available for sale independently of one another, but are co-marketed or copromoted for simultaneous and / or subsequent administration, and / or administration as a mixture.

16. A method of treating cancer in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of: i) the pharmaceutical composition according to claim 11; or ii) a pharmaceutical combination comprising:(a) a first pharmaceutical composition comprising an anti-CanAg antibodydrug conjugate and one or more pharmaceutically acceptable excipient, diluents, or carriers; and(b) a second pharmaceutical composition comprising an unconjugated anti- CanAg antibody and one or more pharmaceutically acceptable excipient, diluents, or carriers; wherein the compositions (a) and (b) are provided in separate dosage forms, which are administered simultaneously, sequentially or as a mixture of compositions (a) and (b).

17. The pharmaceutical combination for use in accordance with claim 14 or 15 or the method of treatment in accordance with claim 16, wherein the cancer is selected from the group consisting of lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, pancreatic cancer, biliary cancer, cervical cancer and uterine cancer.

18. The kit in accordance with claim 12 or 13, the pharmaceutical combination for use in accordance with any of claims 14, 15 or 17, or the method of treatment inaccordance with claim 16 or claim 17, wherein the first pharmaceutical composition comprises the anti-CanAg antibody-drug conjugate as defined in any of claims 1 to 10.

19. The method of treatment according to any one of claims 16 to 18, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical combination, wherein the first pharmaceutical composition and all or part of the second pharmaceutical composition are premixed and administered as a mixture.

20. The method of treatment according to any one of claims 16 to 19, wherein the ratio of the anti-CanAg antibody-drug conjugate to unconjugated antibody administered to the subject is in a range from 1:1 to 1:50.

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