Stroma-targeting agent for the treatment of cancer and related methods

Abstract

The present invention relates to a method of selecting or stratifying a mammalian subject having a cancer for treatment with a stroma-targeting agent, and related computer-implemented methods, systems, and non-transitory computer readable media In particular, the method of selecting or stratifying a mammalian subject having a cancer for treatment with a stroma-targeting agent comprises measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1α in a sample obtained from the subject; and selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value. The present invention also relates to relates to a combination of (i) an anti-Fibroblast Activating Protein α (FAP) antibody-cytolysin conjugate having the formula A-(L-D)p or a pharmaceutically acceptable salt or solvate thereof and (ii) an immune checkpoint inhibitor (ICI) for use in a method of treating metastatic cancer in a mammalian subject.

Description

[0001] STROMA-TARGETING AGENT FOR THE TREATMENT OF CANCER AND RELATED METHODS

[0002] This application claims priority from EP 24383351 .4, filed 10 December 2024, and EP 25382801 .6, filed 31 July 2025, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Field of the Invention

[0004] The present invention relates to the use of a stroma-targeting agent for the treatment of cancer, and related methods of selecting or stratifying subjects for treatment with a stroma-targeting agent using at least one tumour stroma biomarker. The present invention also relates to a combination of an antiFibroblast Activating Protein a (FAP) antibody-cytolysin conjugate or a pharmaceutically acceptable salt or solvate thereof and an immune checkpoint inhibitor for use in a method of treating metastatic cancer in a mammalian subject.

[0005] Background

[0006] Immunotherapy such as immune checkpoint inhibitors (ICIs) is a promising therapy increasingly used in the treatment of cancer. Immune checkpoints are receptors expressed on immune cells which are involved with regulation of immune homeostasis. These checkpoints can be especially relevant in cancer, where chronic T cell stimulation leads to dysfunction because of interactions between cells expressing immune checkpoint receptors and ligands. By inhibiting these interactions, ICIs can reinvigorate the T cells, restoring T cell functionality and anti-cancer activity.

[0007] Although ICIs have demonstrated significant efficacy in multiple solid tumour types, the effectiveness of ICIs as a single agent or in combination with chemotherapy in certain metastatic cancers has, to date, been limited (O’Reilly et al., 2019; Wainberg et al., 2020; Renouf et al., 2022). For example, ICIs have shown limited benefit in the treatment of metastatic pancreatic or colorectal cancer (Ye et al, 2023; Sahin et al., 2022). In particular, metastatic colorectal cancer subjects with microsatellite-stable (MSS) tumours have been found to respond poorly to ICI treatment, even when administered in combination with multitargeted tyrosine kinase inhibitors (TKI). This limited ICI response is especially evident for MSS colorectal cancer subjects with liver or peritoneal metastases (TKI) (Dung et al., 2015; Overman et al., 2017; Wang et al, 2021 ; Fukuoka et al., 2020). Immunotherapy in metastatic pancreatic cancer, either as single agent or in combination with chemotherapy, has also shown very limited benefit outside of the rare mismatch repair deficient tumours (O’Reilly et al., 2019; Renouf et al., 2022).

[0008] Another form of immunotherapy which represents great promise in the fight against cancer is antibody-drug conjugates (ADCs). ADCs are made of a human, humanized or chimeric recombinant antibody, covalently linked to a cytotoxic drug. The main goal of such a structure is joining the power of small cytotoxic agents (300 to 1000 Da) and the high specificity of tumour-associated antigen (TAA)- targeted MAbs. ADCs may target cells of the tumor stroma, such as endothelial cells, fibroblasts, and macrophages, which promote proliferation of tumor cells, invasive behavior, and immune evasion via secretion of growth factors, angiogenic factors, cytokines, and proteolytic enzymes. For example, CAFs are known to favor T cell exhaustion, promote inflammation, and the recruitment of myeloid-derived suppressor cells. One such ADC that targets the tumor stroma (specifically CAFs) is OMTX705, which is described in WO 2024 / 023159. OMTX705 is an antiFibroblast Activating Protein a (FAP)-targeted antibody-drug conjugate, wherein the antibody is conjugated to a drug comprising cytolysin.

[0009] Despite the success of these immunotherapies in the treatment of some primary cancers, many subjects with metastatic gastrointestinal tumours, especially pancreatic and colorectal metastatic cancer, have not responded to treatment with single agents such as ICIs or ADCs such as OMTX705. Therefore, there remains a need for improved treatment of metastatic cancer, and for improved methods for selecting and stratifying subjects to identify those who will respond positively to such treatments.

[0010] The invention has been devised with the above issues in mind.

[0011] Summary of the Invention

[0012] Broadly, the present invention relates to the use of stroma-targeting agents in the treatment of cancer. In particular, the stroma-targeting agents are administered to certain subject groups, who are identified as having a high likelihood of responding to the stroma-targeting agent based on the protein level of at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a. The present inventors have found that subjects expressing high levels of one or more of these biomarkers are more likely to respond to treatment with a stroma-targeting agent. The invention also relates to a combination of an anti-FAP antibody-cytolysin conjugate of the type disclosed in WO 2024 / 023159 (incorporated herein by reference in its entirety) and an immune checkpoint inhibitor (I Cl) for use in a method of treating metastatic cancer in a mammalian subject. The present inventors have found that this combination exhibits strong anti-tumour activity in metastatic cancers including non-small cell lung cancer and gastrointestinal cancers such as metastatic colorectal cancer and pancreatic cancer. This anti-tumour activity includes a reduction in carcinoembryonic (CEA) levels, reduction in tumour lesion size and improved progression free and overall survival time. Such activity is surprising, since each therapy exhibits limited or no efficacy as a single agent.

[0013] In a first aspect, the invention provides a stroma-targeting agent for use in a method of treating cancer in a mammalian subject, wherein the protein level of at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject is elevated compared to a predetermined level. In some embodiments of the stroma-targeting agent for use according to the first aspect, the subject has been determined to have an elevated protein level of at least one biomarker selected from periostin, N-cadherin, and RAGE.

[0014] In a second aspect, the invention provides a stroma-targeting agent for use in a method of treating cancer in a mammalian subject, wherein the method comprises: (i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; (ii) selecting the subject for treatment when the protein level of at least one biomarker is elevated compared to a predetermined value; and (iii) administering the stroma-targeting agent to the subject.

[0015] In some embodiments, the protein level of the at least one biomarker selected from periostin, N- cadherin, RAGE, and SDF-1a is measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).

[0016] In some embodiments, the method further comprises administering an immune checkpoint inhibitor (ICI).

[0017] In a third aspect the invention provides a method of selecting or stratifying a mammalian subject having a cancer for treatment with a stroma-targeting agent, the method comprising (i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and (ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

[0018] In a fourth aspect the invention provides a method of selecting or stratifying a mammalian subject having a cancer for treatment with a combination of a stroma-targeting agent and an ICI, the method comprising (i) measuring the protein level of at least one biomarker selected from: periostin, N- cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and (ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

[0019] In some embodiments the method comprises measuring the protein level of at least two or at least three biomarkers selected from periostin, N-cadherin, RAGE, and SDF-1a, or all of the biomarkers periostin, N-cadherin, RAGE, and SDF-1a.

[0020] In some embodiments the at least two biomarkers are periostin and RAGE. In some embodiments the at least three biomarkers are periostin, RAGE, and N-cadherin. In some embodiments the method comprises measuring the protein level of periostin. In some embodiments the method comprises measuring the protein level of N-cadherin. In some embodiments the method comprises measuring the protein level of RAGE. In some embodiments the method comprises measuring the protein level of SDF-1a.

[0021] In some embodiments the method comprises measuring the level of total plasma protein or the level of a reference protein and normalising the protein level of the at least one biomarker against the level of the total plasma protein or the level of the reference protein. In some embodiments the reference protein is IL-8.

[0022] In some embodiments the method comprises determining that a subject having a cancer has an elevated level of at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a, and administering a therapeutically effective amount of a stroma-targeting agent. In some embodiments the method comprises determining that a subject having a cancer has an elevated level of at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a and administering a therapeutically effective amount of a stroma-targeting agent in combination with an ICI .

[0023] In some embodiments the stroma-targeting agent comprises an antibody drug conjugate that specifically binds to a protein expressed on the surface of cancer associated fibroblasts (CAFs), and wherein the antibody drug conjugate is conjugated to a cytotoxic payload. In some embodiments the cytotoxic payload is a tubulin polymerisation inhibitor such as MMAE or eribulin, a topoisomerase inhibitor such as exatecan or a derivative thereof, or a cytolysin; and / or wherein the antibody is an antibody-cytolysin conjugate.

[0024] In some embodiments the stroma-targeting agent comprises an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)Por a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytotoxic payload and p is 1 to 10.

[0025] In some embodiments the cancer is a metastatic cancer comprising a primary cancer and a metastasis, and / or wherein the sample is a plasma, serum, blood, saliva, or urine sample.

[0026] In a fifth aspect the invention provides a computer-implemented method for selecting a mammalian subject having a cancer for treatment with a stroma-targeting agent, the method comprising: a), providing protein level data from a subject for at least one biomarker selected from: periostin, N- cadherin, RAGE, and SDF-1a; b). determining a score based on the protein level data for the at least one biomarker; c). based on the score, selecting the subject for treatment with stroma-targeting agent.

[0027] In some embodiments a machine learning model is used in b). to determine a score based on the protein level data for the at least one biomarker, optionally wherein the machine learning model is selected from: logistic regression with elastic net regularization, linear regression, logistic regression, Ridge regression, Lasso regression, elastic net (EN) regression, support vector machine (SVM), gradient boosted machine (GBM), k nearest neighbors (kNN), generalized linear model (GLM), naive Bayes (NB) classifier, neural network, Random Forest (RF), deep learning algorithm, linear discriminant analysis (LDA), decision tree learning (DTREE), adaptive boosting (ADB), Classification and Regression Tree (CART), hierarchical clustering, or any combination thereof; and / or wherein the machine learning model is trained using leave-one-out cross-validation.

[0028] In some embodiments the machine learning model has been trained on protein level data from subjects that are known to have responded to treatment with stroma-targeting agent, and protein level data from subjects that are known not to have responded to treatment with stroma-targeting agent; optionally wherein a subject that is known to have responded to treatment with stroma-targeting agent is defined as a subject that experienced (i) more than a 20% reduction in the size of a tumor lesion compared to the size of the tumor lesion as measured before the initiation of treatment, (ii) progression-free survival for over 4 treatment cycles, and / or (iii) an overall survival of over 6 months.

[0029] In a sixth aspect the invention provides a system comprising: a), a processor; and b). a computer readable medium comprising instructions that, when executed by the processor, cause the processor to perform the method according to the fifth aspect.

[0030] In a seventh aspect the invention provides a non-transitory computer readable medium or media comprising instructions that, when executed by at least one processor, cause the at least one processor to perform the method according to the sixth aspect.

[0031] In an eighth aspect the invention provides a combination of (i) an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)p or a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytolysin and p is 1 to 10, and (ii) an immune checkpoint inhibitor (ICI) for use in a method of treating metastatic cancer in a mammalian subject, wherein the method comprises simultaneous, sequential or separate administration of the antibody-cytolysin conjugate or the pharmaceutically acceptable salt or solvate thereof and the ICI to the subject, wherein the anti-FAP antibody comprises heavy chain complementarity determining regions 1-3 (CDRH1-3) and light chain complementarity determining regions 1-3 (CDRL1-3) having the following amino acid sequences: CDRH1 : SEQ ID NO: 7; CDRH2: SEQ ID NO: 8; CDRH3: SEQ ID NO: 9; CDRL1 : SEQ ID NO: 10; CDRL2: SEQ ID NO: 11 ; and CDRL3: SEQ ID NO: 12; wherein the cytolysin of the antibody-cytolysin conjugate comprises formula IV: wherein:

[0032] R2 is H or C1-C4 alkyl;

[0033] R6 is C1-C6 alkyl;

[0034] R7 is C1-C6 alkyl, CH2OR19 or CH2OCOR20, wherein R19 is alkyl, R20 is C2-C6-alkenyl, phenyl, or CH2-phenyl;

[0035] R9 is C1-C6 alkyl;

[0036] R10 is H, OH, O-alkyl or O-acetyl; f is 1 or 2;

[0037] R11 has the following structure: wherein

[0038] R21 is H, OH, halogen, NH2, alkyloxy, phenyl, alkyl amino or dialkyl amino;

[0039] R16 is H or a C1-C6-alkyl group;

[0040] R17 is directly or indirectly attached to linker L; and q is 0, 1 , 2 or 3; and wherein the term "optionally substituted" relates to groups, wherein one or several H atoms can be replaced by F, Cl, Br or I or OH, SH, NH2, or NO2; the term "optionally substituted" further relates to groups, which can be exclusively or additionally substituted with unsubstituted C1-C6 alkyl, C2C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C2-C9 heterocycloalkyl, C6-C10 aryl, C1-C9 heteroaryl, C7-C12 aralkyl or C2-C11 heteroaralkyl groups; wherein the ICI comprises an anti- PD-1 antibody; wherein the metastatic cancer comprises a primary cancer and metastasis, and wherein the primary cancer of the metastatic cancer comprises gastrointestinal cancer or lung carcinoma.

[0041] In some embodiments the anti-PD-1 molecule comprises pembrolizumab, nivolumab or tislelizumab.

[0042] In some embodiments, A of the antibody-cytolysin conjugate has a heavy chain with amino acid sequence of SEQ ID NO: 1 and a light chain with amino acid sequence of SEQ ID NO: 2.

[0043] In some embodiments, L of the antibody-cytolysin conjugate comprises a spacer, optionally wherein the spacer comprises -(OCH2CH2)n-, wherein n is 2 to 5.

[0044] In some embodiments, L of the antibody-cytolysin conjugate comprises an attachment group for attachment to A; optionally wherein L comprises a protease cleavable portion comprising a valinecitrulline unit.

[0045] In some embodiments, the cytolysin of the antibody-cytolysin conjugate has the formula:

[0046] wherein * indicates the site of attachment to L.

[0047] In some embodiments, -L-D of the antibody-cytolysin conjugate has the structure: wherein * denotes the point of attachment to A.

[0048] In some embodiments, the mammalian subject is a human subject. In some embodiments, the primary cancer comprises pancreatic cancer, colorectal cancer, oesophageal cancer, gastric (stomach) cancer, liver cancer or lung cancer. In some embodiments, the primary cancer comprises pancreatic cancer, colorectal cancer, oesophageal cancer or lung cancer. In some embodiments, the primary cancer comprises pancreatic or colorectal cancer. In some embodiments, the pancreatic cancer comprises pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the colorectal cancer comprises microsatellite-stable (MSS) colorectal cancer. In some embodiments, the primary cancer comprises MSS colorectal cancer and the metastasis comprises liver, lung and / or peritoneal metastasis.

[0049] In some embodiments, the subject was previously administered the antibody-cytolysin conjugate and was not previously administered the ICI . In some embodiments, the subject was previously administered the ICI and was not previously administered the antibody-cytolysin conjugate.

[0050] In some embodiments, the method comprises sequential administration of the antibody-cytolysin conjugate and the ICI to the subject.

[0051] In some embodiments, the method comprises intravenous administration of the antibody-cytolysin conjugate and the ICI to the subject. In some embodiments, the method comprises administration of the antibody-cytolysin conjugate at least once in a 21 -day cycle. In some embodiments, the method comprises administration of the antibody-cytolysin conjugate at least twice in a 21-day cycle. In some embodiments, the method comprises administration of the antibody-cytolysin conjugate on day 1 and day 8 of a 21-day cycle. In some embodiments, the method comprises administration of the ICI at least once in a 21-day cycle. In some embodiments, the method comprises administration of the ICI on day 1 of a 21-day cycle. In some embodiments, the 21-day cycle is repeated at least once.

[0052] In some embodiments, administration of the antibody-cytolysin conjugate and the ICI results in a synergistic effect.

[0053] In some embodiments, administration of the antibody-cytolysin conjugate and the ICI results in a reduction of tumour lesion size in the subject.

[0054] In some embodiments, administration of the antibody-cytolysin conjugate and the ICI increases progression free survival and / or overall survival time of the subject, relative to a control subject who has been treated with the antibody-cytolysin conjugate or the ICI as monotherapies, but not in combination.

[0055] In some embodiments, the method comprises administration of at least 2mg / kg of the antibody- cytolysin conjugate to the subject, optionally at least 4mg / kg of the antibody-cytolysin conjugate to the subject, further optionally at least 7 mg / kg of the antibody-cytolysin conjugate to the subject.

[0056] In some embodiments, the method comprises administration of at least 100mg, at least 150mg, at least 200mg, at least 250mg, at least 300mg, at least 350mg or at least 400mg of the ICI to the subject. In some embodiments, the method comprises administration of 200mg of ICI to the subject.

[0057] In some embodiments, the primary cancer and / or metastasis comprises a level of FAP expression detectable by immunohistochemistry.

[0058] In some embodiments, the method comprising administering (i) an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)p or a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytolysin and p is 1 to 10, to the subject simultaneously, sequentially or separately with (ii) an immune checkpoint inhibitor (ICI), wherein the anti-FAP antibody comprises heavy chain complementarity determining regions 1-3 (CDRH1-3) and light chain complementarity determining regions 1-3 (CDRL1-3) having the following amino acid sequences: CDRH1 : SEQ ID NO: 7; CDRH2: SEQ ID NO: 8; CDRH3: SEQ ID NO: 9; CDRL1 : SEQ ID NO: 10; CDRL2: SEQ ID NO: 11 ; and CDRL3: SEQ ID NO: 12; wherein the cytolysin of the antibody-cytolysin conjugate comprises formula IV: wherein:

[0059] R2 is H or C1-C4 alkyl;

[0060] R6 is C1-C6 alkyl;

[0061] R7 is C1-C6 alkyl, CH2OR19 or CH2OCOR20, wherein R19 is alkyl, R20 is C2-C6-alkenyl, phenyl, or CH2-phenyl;

[0062] R9 is C1-C6 alkyl;

[0063] R10 is H, OH, O-alkyl or O-acetyl; f is 1 or 2;

[0064] R11 has the following structure: wherein

[0065] R21 is H, OH, halogen, NH2, alkyloxy, phenyl, alkyl amino or dialkyl amino;

[0066] R16 is H or a C1-C6-alkyl group;

[0067] R17 is directly or indirectly attached to linker L; and q is 0, 1 , 2 or 3; and wherein the term "optionally substituted" relates to groups, wherein one or several H atoms can be replaced by F, Cl, Br or I or OH, SH, NH2, or NO2; the term "optionally substituted" further relates to groups, which can be exclusively or additionally substituted with unsubstituted C1- C6 alkyl, C2C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C2-C9 heterocycloalkyl, C6-C10 aryl, C1-C9 heteroaryl, C7-C12 aralkyl or C2-C11 heteroaralkyl groups; wherein the ICI comprises an anti-PD-1 antibody; wherein the metastatic cancer comprises a primary cancer and metastasis, and wherein the primary cancer of the metastatic cancer comprises gastrointestinal cancer or lung carcinoma. Summary of the Figures

[0068] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0069] Figure 1. An illustrative depiction of the conjugate OMTX705, as described in Fabre et al. (2020). As shown, OMTX705 comprises a humanised anti-FAP mAb (OMTX005) conjugated to TAM558, which is formed from the conjugation of the cytolysin TAM470 to a protease-cleavable vcPABA-(EG)3 optimised linker.

[0070] Figure 2. Schematic to show dose-escalation protocols used when comparing OMTX705 monotherapy to OMTX705 + ICI combination therapy.

[0071] Figure 3. Heatmap depicting the log-transformed average baseline concentrations of a 40-analyte panel measured in plasma samples from metastatic PDAC subjects prior to initiation of OMTX705 plus pembrolizumab treatment. Black boxes indicate responder subjects.

[0072] Figure 4. Baseline plasma protein levels of A. periostin, B. N-cadherin, C. RAGE and D. SDF-1a correlate with progression-free survival (left-hand panels) and overall survival (right-hand panels) in subjects treated with OMTX705 + Pembrolizumab.

[0073] Figure 5. Spearman correlation coefficients were calculated between all biomarkers, PFS and OS. Spearman's method was chosen as some variables did not meet the normality criteria.

[0074] Figure 6. Overall model performance shown in terms of A. AUC and B. accuracy, for each model including Periostin, along with their 95% confidence intervals. Each model was trained using a different combination of input features (biomarkers). Confidence intervals were computed based on the empirical distribution of the bootstrapped scores.

[0075] Figure 7. Overall model performance shown in terms of A. AUC and B. accuracy, for each model including IL8-normalized Periostin and raw Periostin + RAGE, along with their 95% confidence intervals. Each model was trained using a different combination of input features (biomarkers). Confidence intervals were computed based on the empirical distribution of the bootstrapped scores. Figure 8. Overall model performance shown in terms of A. AUC and B. accuracy, for each model including raw-, TPP-, and IL-8-normalized individual biomarkers, along with their 95% confidence intervals. Each model was trained using a different combination of input features (biomarkers).

[0076] Confidence intervals were computed based on the empirical distribution of the bootstrapped scores.

[0077] Detailed Description of the Invention

[0078] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference. Stroma-targeting Agents

[0079] Aspects of the present invention relate to stroma-targeting agents. A “stroma-targeting agent” as used herein refers to an agent that targets one or more cells within the tumour microenvironment that are not tumour cells. The stroma-targeting agent may target a cell type present within the tumour microenvironment that promotes tumour growth. The stroma-targeting agent may target a cell type selected from: cancer-associated fibroblasts (CAFs), mesenchymal stromal cells, pericytes, endothelial cells, macrophages, myeloid-derived suppressor cells, pancreatic stellate cells, and adipocytes.

[0080] In some embodiments, the stroma-targeting agent targets CAFs.

[0081] Examples of stroma-targeting agents that target CAFs include inhibitors of the hedgehog pathway. The hedgehog pathway inhibitors may target the ligand smoothened (SMO), or the GLI transcription factors. Examples of inhibitors that target SMO include Vismodegib, Sonidegib, and Saridegib. An example of an inhibitor that targets GLI transcription factors includes GANT61. Examples of stromatargeting agents that target CAFs include inhibitors of focal adhesion kinase (FAK). In CAFs, FAK promotes myofibroblast-like activation, secretion of cytokines such as IL-6, ECM production, cell adhesion, migration, and contractility. Examples of FAK inhibitors include Defactinib, PF-562271 , TAE226, and Bl 853520. Further examples of stroma-targeting agents targeting CAFs are small molecule inhibitors of the TGF-p pathway, such as galunisertib and vactosertib. Blockade of TGF-p signaling in CAFs facilitates intratumoral T cell infiltration, which can enhance tumour regression in combination with the use of ICIs. Further examples of stroma-targeting agents targeting CAFs are inhibitors of PDGF signaling, such as small molecule inhibitors of PDGFR-p (e.g. Crenolanib, Sennoside B, and Masitinib). Further examples of stroma-targeting agents targeting CAFs are agents that remodel the extracellular matrix of the tumour, such as PEGylated hyaluronidase. Further examples of stroma-targeting agents targeting CAFs are anti-fibrotic agents, including agents that are approved for the treatment of fibrosis, such as pirfenidone.

[0082] In some embodiments, the stroma-targeting agent is an antibody that selectively binds to a protein expressed on the cell surface of CAFs. In some embodiments, the protein expressed on the cell surface of CAFs is expressed at higher levels on the surface of CAFs compared to expression in other cell types. In some embodiments, the protein expressed on the surface of CAFs is selectively expressed by this cell type, i.e., it is enriched on the surface of CAFs compared to other cell types. In some embodiments, the antibody selectively binds to a protein selected from: FAP, COL11A1 , MFAP5, PDGFR-a, PDGFR-p, Podoplanin, Integrin p1 (e.g., avp6, a11p1), and CD10 / GPR77. In some embodiments the stroma-targeting agent is an ADC that selectively binds to a protein expressed on the surface of CAFs. In some embodiments, the stroma-targeting agent is an ADC that selectively kills CAFs. In some embodiments, the antibody of the ADC selectively binds to FAP. It will be understood that the FAP-binding region of the ADCs described herein may be substituted for a binding region that selectively binds to any protein selectively expressed on the surface of CAFs, such as C0L11A1 , MFAP5, PDGFR-a, PDGFR-p, Podoplanin, Integrin p1 (e.g., avp6, a11 p1), and CD10 / GPR77.

[0083] Antibody-based approaches allow selective targeting of CAFs. Alternatively, liposomes or lipid nanoparticles may be used to deliver cytotoxic agents to CAFs.

[0084] Anti-FAP Antibody-drug Conjugates

[0085] Aspects of the present invention relate to Anti-FAP antibody-drug conjugates (ADCs). In some embodiments, the anti-FAP ADC has the formula I:

[0086] A-(L-D)Pwherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytotoxic payload and p is 1 to 10.

[0087] As used herein “Fibroblast activation protein”, “fibroblast activating protein”, “FAP” and “FAPa” are used interchangeably. The FAP may be an FAP of any mammalian species. In some cases, FAP is human FAP (also known as Seprase, 170 kDa melanoma membrane-bound gelatinase, fibroblast activation protein alpha or integral membrane serine protease), the amino acid sequence of which is disclosed at UniProt accession No. Q12884 (Version 140, dated 11 December 2013) (SEQ ID NO: 13). In some cases, a molecule that binds FAP (e.g. an antibody molecule or a conjugate thereof) may bind to a region of the extracellular domain of FAP. The extracellular domain of human FAP comprises residues 26-760 of the full-length human FAP protein. In some cases, FAP is murine FAP (also known as fibroblast activation protein alpha or integral membrane serine protease), the amino acid sequence of which is disclosed at UniProt accession No. P97321 (Version 117, dated 11 December 2013) (SEQ ID NO: 14). The extracellular domain of murine FAP comprises residues 26- 761 of the full-length murine FAP protein.

[0088] In the context of the present invention, the terms selectively binds and selective binding refer to binding of an antibody, or binding fragment thereof, to a predetermined molecule (e.g. an antigen) in a specific manner. For example, the antibody, or binding fragment thereof, may bind to FAP, e.g. an extracellular portion thereof, with an affinity of at least about 1x107M-1, and may bind to the predetermined molecule with an affinity that is at least two-fold greater (e.g. five-fold or ten-fold greater) than its affinity for binding to a molecule other than the predetermined molecule.

[0089] As used herein the term "antibody" or "antibody molecule" includes any immunoglobulin whether natural or partly or wholly synthetically produced. The term "antibody" or "antibody molecule" includes monoclonal antibodies (mAb) and polyclonal antibodies (including polyclonal antisera). Antibodies may be intact or fragments derived from full antibodies (see below). Antibodies may be human antibodies, humanised antibodies or antibodies of non-human origin. "Monoclonal antibodies" are homogeneous, highly specific antibody populations directed against a single antigenic site or “determinant” of the target molecule. “Polyclonal antibodies” include heterogeneous antibody populations that are directed against different antigenic determinants of the target molecule. The term “antiserum” or "antisera" refers to blood serum containing antibodies obtained from immunized animals.

[0090] It has been shown that fragments of a whole antibody can perform the function of binding antigens. Thus, reference to antibody herein covers a full antibody and also covers any polypeptide or protein comprising an antibody binding fragment. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains;

[0091] (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers (WO 93 / 11161) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; 58). Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains. Minibodies comprising a scFv joined to a CH3 domain may also be made.

[0092] In relation to an antibody molecule, the term "selectively binds" may be used herein to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen-binding site is specific for a particular epitope that is carried by a number of antigens, in which case the specific binding member carrying the antigen-binding site will be able to bind to the various antigens carrying the epitope.

[0093] In some embodiments, the anti-FAP antibody is a monoclonal antibody or binding fragment thereof that selectively binds to an extracellular region of human FAP and / or murine FAP. In some embodiments, the anti-FAP antibody cross-reacts to both human and murine FAP.

[0094] In some embodiments the antibody may comprise a humanised antibody.

[0095] In some embodiments the antibody may be a fully human antibody.

[0096] In some embodiments, the anti-FAP antibody comprises heavy chain complementarity determining regions 1-3 (CDRH1-3) and light chain complementarity determining regions 1-3 (CDRL1-3) having the following amino acid sequences:

[0097] (i) CDRH1 : SEQ ID NO: 7;

[0098] (ii) CDRH2: SEQ ID NO: 8;

[0099] (iii) CDRH3: SEQ ID NO: 9;

[0100] (iv) CDRL1 : SEQ ID NO: 10;

[0101] (v) CDRL2: SEQ ID NO: 11 ; and (vi) CDRL3: SEQ ID NO: 12.

[0102] In some embodiments, the CDRH1 region comprises a variant of SEQ ID NO: 7 having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 7. In some embodiments, the CDRH2 region comprises a variant of SEQ ID NO: 8 having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 8. In some embodiments, the CDRH3 region comprises a variant of SEQ ID NO: 9 having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 9. In some embodiments, the CDRL1 region comprises a variant of SEQ ID NO: 10 having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 10. In some embodiments, the CDRL2 region comprises a variant of SEQ ID NO: 11 having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 11 . In some embodiments, the CDRL3 region comprises a variant of SEQ ID NO: 12 having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 12.

[0103] In some embodiments, CDRH1-3 comprise the amino acid sequences of SEQ ID NOs: 7-9, respectively and CDRL1-3 comprise the amino acid sequences of SEQ ID NOS: 10-12, respectively.

[0104] In some embodiments, the anti-FAP antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy chain variable region (VH) has at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 5. In some embodiments, the light chain variable region (VL) has at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 6.

[0105] In some embodiments, the anti-FAP antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the heavy chain has at least 90%, 95% or 99% sequence identity with the full- length sequence of SEQ ID NO: 1. In some embodiments, the light chain has at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 2.

[0106] In some embodiments, the anti-FAP antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the heavy chain has at least 90%, 95% or 99% sequence identity with the full- length sequence of SEQ ID NO: 3. In some embodiments, the light chain has at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 4.

[0107] In some embodiments, A of the anti-FAP ADC has a heavy chain with amino acid sequence of SEQ ID NO: 1 and a light chain with amino acid sequence of SEQ ID NO: 2.

[0108] Many different cytotoxic drugs have been used as payloads for ADCs, and these are well-known to the skilled person. In line with its standard definition in the art, a cytotoxic drug, or cytotoxin, is a drug (or compound) which is toxic to cells. In the context of the ADC used herein, the cytotoxic payload is generally toxic to human cells. By “toxic to cells” is meant that the drug, when delivered to a cell, induces cell death, e.g. by apoptosis or necrosis.

[0109] In some embodiments, the cytotoxic payload is a small molecule. Types of small molecules suitable for use as a cytotoxic payload include topoisomerase inhibitors, tubulin polymerisation inhibitors, dolastatins, duocarmycins and maytansinoids. In particular, the cytotoxic payload may be selected from: SN-38, gemcitabine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), doxorubicin, calicheamicin, maytansine DM1 (also referred to as mertansine), and maytansine DM4 (also referred to as ravtansine), which are well known in the art.

[0110] Topoisomerase Inhibitors

[0111] As used herein, the term “topoisomerase inhibitor” refers to the group of compounds that block the action of topoisomerases. Topoisomerases are a group of enzymes that catalyse changes in the topological state of DNA, and play essential roles in DNA replication, transcription, chromosome segregation and recombination. Topoisomerases can be divided into two subtypes: type I topoisomerases and type II topoisomerases. Type I isomerases catalyse reactions involving transient single-stranded breaks. Type II isomerases catalyse reactions involving transient double-strand breaks. The structure and activity of topoisomerases is reviewed, for example, in Sutormin et al. Acta Naturae. (2021) 13(1):59-75 and Baker et al. Nucleic Acids Res. (2009) 37(3):693-701 .

[0112] The ability of an agent to inhibit topoisomerase activity can be determined by the skilled person using various methods well known in the art, for example, as described in Nitiss et al. Curr Protoc Pharmacol. (2021) 1 (10):e250 doi: 10.1002 / cpz1 .250.

[0113] Type I topoisomerases function by attacking the phosphodiester bond in the backbone of DNA to form a transient topoisomerase-DNA intermediate. This allows for rotation of the cleaved DNA strand around the helical access before the topoisomerase re-ligates the cleaved strand to reform duplex DNA. Topoisomerase type I inhibitors generally inhibit topoisomerase I activity by stabilising the topoisomerase-DNA intermediate, thereby preventing DNA re-ligation. The mechanism of type I topoisomerase inhibitors is reviewed, for example, in Pommier et al. Chem Biol. (2020). 17(5):421- 433.

[0114] In some embodiments, the topoisomerase inhibitor according to the present invention is a type I topoisomerase inhibitor. Type I topoisomerase inhibitors include: camptothecin and structural analogues thereof, and non-camptothecins.

[0115] Non-camptothecin type I topoisomerase inhibitors include: indenoisoquinolines, for example, indotecan (LMP400, drugbank accession number (DB) 18752), indimitecan (LMP776) and LMP744 (MJ-III65, NSC 706744); phenanthridine derivatives, for example, Topovale (ARC-111), nitidine, fagaronine (see e.g. Wang etal. Chem Res Toxicol. (1993) 6(6):813-818); indolocarbazoles, for example, NB-506, edotecarin (DB04882), BMS-250749. In some embodiments, the topoisomerase inhibitor according to the present invention is a non-camptothecin type I topoisomerase inhibitor.

[0116] In some embodiments, the topoisomerase inhibitor according to the present invention is a camptothecin (DB04690) or structural analogue thereof. As used herein, the term ‘structural analogue’ (also referred to as ‘chemical analogue’, ‘analogue’ or ‘derivative’) refers to compounds having a structure similar to that of another compound, but different from it in respect to a certain component (e.g. in one or more atoms or functional groups).

[0117] Camptothecin analogues include: homocamptothecins (such as Diflomotecan (DB16138)), Gimatecan (DB06721), Topotecan (DB01030), Irinotecan (DB00762), Rubitecan (DB06159), 9-Amino-20(S)- camptothecin (9-AC), Lurtotecan (DB12222), Prothecan (PEG-camptothecin), Cositecan (DB05806), Silatecan (DB12384), Exatecan (DB12185).

[0118] In some embodiments, the topoisomerase inhibitor according to the present invention is of formula II:

[0119] R1is H or Ac;

[0120] A1and A2are each independently selected from the group consisting of halo, hydrogen, C1-C3 alkyl, phenyl, hydroxy, C1-C3 alkoxy, or wherein A1and A2together with the atoms to which they are bonded form a 5-6 membered fused ring; either

[0121] A11is H;

[0122] A12is -(C1-6 alkyl)-A3or A3; or A11and A12together with the carbon atoms to which they are attached form a ring substituted with A3at any position on the ring preferably the ring structure is 4, 5, 6, 7 or 8 atoms in size, more preferably 5, 6, 7 or 8 atoms in size; preferably the ring is a carbocyclic ring;

[0123] A3is wherein

[0124] Z is a bond or a Ci-Ce alkyl chain or a substituted or unsubstituted aryl group or wherein Z is Xi-Ar, wherein the Ar group is directly attached to the OR17group. wherein Xi is a Ci-Ce alkyl chain and Ar is a substituted or unsubstituted aryl group;

[0125] A4, A5, A6A7, A9and A10are each individually hydrogen or a C1-C3 alkyl;

[0126] A8is hydrogen or a C1-C4 alkyl;

[0127] R17is a bond (i.e. X’), -CH2X', -CH2NHX', -CH2NHC(O)X', -C(O)NHNHC(O)X', -C(O)X', - C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L and

[0128] J1is O, S, or Se.

[0129] In some embodiments, the topoisomerase inhibitor according to the present invention is of formula Ila:

[0130] A1and A2are each independently selected from the group consisting of halo, hydrogen, C1-C3 alkyl, phenyl, hydroxy, C1-C3 alkoxy, or wherein A1and A2together with the atoms to which they are bonded form a 5-6 membered fused ring; either

[0131] A11is H;

[0132] A12is — (Ci-6 alkyl)-A3or A3; or

[0133] A11and A12together with the carbon atoms to which they are attached form a ring substituted with A3at any position on the ring preferably the ring structure is 4, 5, 6, 7 or 8 atoms in size, more preferably 5, 6, 7 or 8 atoms in size; preferably the ring is a carbocyclic ring;

[0134] A3is wherein

[0135] Z is a bond or a Ci-Ce alkyl chain or a substituted or unsubstituted aryl group or wherein Z is Xi-Ar, wherein the Ar group is directly attached to the OR17group. wherein Xi is a Ci-Ce alkyl chain and Ar is a substituted or unsubstituted aryl group;

[0136] A4, A5, A6A7, A9and A10are each individually hydrogen or a C1-C3 alkyl;

[0137] A8is hydrogen or a C1-C4 alkyl;

[0138] R17is a bond (i.e. X’), -CH2X', -CH2NHX', -CH2NHC(O)X', -C(O)NHNHC(O)X', -C(O)X', -C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L and

[0139] J1is O, S, or Se.

[0140] In some embodiments, the topoisomerase inhibitor according to the present invention is of formula lib:

[0141] A1and A2are each independently selected from the group consisting of halo, hydrogen, C1-C3 alkyl, phenyl, hydroxy, C1-C3 alkoxy, or wherein A1and A2together with the atoms to which they are bonded form a 5-6 membered fused ring; either

[0142] A11is H;

[0143] A12is — (Ci-6 alkyl)-A3or A3; or

[0144] A11and A12together with the carbon atoms to which they are attached form a ring substituted with A3at any position on the ring preferably the ring structure is 4, 5, 6, 7 or 8 atoms in size, more preferably 5, 6, 7 or 8 atoms in size; preferably the ring is a carbocyclic ring;

[0145] A3is wherein

[0146] Z is a bond or a Ci-Ce alkyl chain or a substituted or unsubstituted aryl group or wherein Z is Xi-Ar, wherein the Ar group is directly attached to the OR17group. wherein Xi is a Ci-Ce alkyl chain and Ar is a substituted or unsubstituted aryl group;

[0147] A4, A5, A6A7, A9and A10are each individually hydrogen or a C1-C3 alkyl;

[0148] A8is hydrogen or a C1-C4 alkyl;

[0149] R17is a bond (i.e. X’), -CH2X', -CH2NHX', -CH2NHC(O)X', -C(O)NHNHC(O)X', -C(O)X', -C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L and

[0150] J1is O, S, or Se.

[0151] Preferably, Z is a Ci-Ce alkyl chain. Preferably, Z is a linear Ci-Ce alkyl chain.

[0152] Preferably, wherein A1and A2are each independently selected from the group of halo, hydrogen, a linear C1-C3 alkyl chain, phenyl, hydroxy, C1-C3 alkoxy, or wherein A1and A2together with the atoms to which they are bonded form a fused ring.

[0153] In some embodiments, the topoisomerase inhibitor is of formula Illa.

[0154] wherein:

[0155] A1is a C1-C3 alkyl chain or H;

[0156] A2is halo or H; or A1and A2together with the carbon atoms to which they are attached form a 5-6 member heterocyclic ring;

[0157] J1is O, S, or Se either

[0158] A11is H

[0159] A12is -(C1-3 alkyl)-A3or A3; or A11and A12together with the carbon atoms to which they are attached form a 5-8 membered carbocyclic ring substituted with A3;

[0160] A3is wherein

[0161] Z is a bond or a C1-C3 alkyl chain and R17is a bond, -CH2X', -CH2NHC(O)X', -CH2NHX', - C(O)NHNHC(O)X', -C(O)X', -C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L.

[0162] Preferably, R17is -CH2X', -CH2NHX', -C(O)NHNHC(O)X', -C(O)X', -C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L.

[0163] Preferably, A1is a linear C1-C3 alkyl chain. Preferably, Z is a linear C1-C3 alkyl chain.

[0164] In some embodiments, the topoisomerase inhibitor according to the present invention is TC1 J1, or a pharmaceutically acceptable salt, hydrate, solvate, or crystalline form thereof. In some embodiments, the topoisomerase inhibitor according to the present invention has the following structure:

[0165]

[0166] TC1J1wherein J1is O, S, Se; wherein!denotes the point of attachment to the linker or attachment to the group wherein Z is a bond or a C1-C3 alkyl chain and R17is a bond (i.e. X’), -CH2X', -CH2NHC(O)X', -CH2NHX', - C(O)NHNHC(O)X', -C(O)X', -C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L.

[0167] In some embodiments, the topoisomerase inhibitor according to the present invention is TC2J1, or a pharmaceutically acceptable salt, hydrate, solvate, or crystalline form thereof. In some embodiments, the topoisomerase inhibitor according to the present invention has the following structure:

[0168] TC2J1wherein J1is O, S, Se; wherein;denotes the point of attachment to the linker or attachment to the group wherein Z is a bond or a C1-C3 alkyl chain and R17is a bond (i.e. X’), -CH2X', -CH2NHX', -CH2NHC(O)X', -

[0169] C(O)NHNHC(O)X', -C(O)X', -C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L. .

[0170] In some embodiments, the topoisomerase inhibitor according to the present invention is TC3J1, or a pharmaceutically acceptable salt, hydrate, solvate, or crystalline form thereof. In some embodiments, the topoisomerase inhibitor according to the present invention has the following structure:

[0171] TC3J1wherein in TC3 J1is O, S, or Se; wherein!denotes the point of attachment to the linker or attachment to the group wherein

[0172] Z is a bond or a C1-C3 alkyl chain and R17is a bond (i.e. X’), -CH2X', -CH2NHC(O)X', -CH2NHX', - C(O)NHNHC(O)X', -C(O)X', -C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L. In some embodiments, the topoisomerase inhibitor according to the present invention is of formula He: wherein:

[0173] R1is H or Ac; A1and

[0174] A2are each independently selected from the group consisting of halo, hydrogen, C1-C3 alkyl, phenyl, hydroxy, C1-C3 alkoxy, or wherein A1and A2together with the atoms to which they are bonded form a 5-6 membered fused ring; Q is O, S or CRaRb; wherein Raand Rbare each independently selected from hydrogen or a C1-C3 alkyl; m and n2 are each individually 0, 1 or 2 and wherein m + n2 is at least 1 ;

[0175] A3is wherein Z is a bond or a Ci-Ce alkyl chain or a substituted or unsubstituted aryl group or wherein Z is Xi-Ar, wherein the Ar group is directly attached to the OR17group, wherein Xi is a Ci-Ce alkyl chain and Ar is a substituted or unsubstituted aryl group;

[0176] A4, A5, A6A7, A9and A10are each individually hydrogen or a C1-C3 alkyl;

[0177] A8is hydrogen or a C1-C4 alkyl; R17is a bond (i.e. X’), -CH2X', -CH2NHX', -C(O)NHNHC(O)X', -C(O)X', -CH2NH C(O)X', - C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L; wherein A3represents a group which may be attached at any point on the ring structure containing Q. In some embodiments, the topoisomerase inhibitor according to the present invention is of formula lid: wherein:

[0178] A1and

[0179] A2are each independently selected from the group consisting of halo, hydrogen, C1-C3 alkyl, phenyl, hydroxy, C1-C3 alkoxy, or wherein A1and A2together with the atoms to which they are bonded form a 5-6 membered fused ring;

[0180] Q is O, S or CRaRb; wherein Raand Rbare each independently selected from hydrogen or a C1-C3 alkyl; m and n2 are each individually 0, 1 or 2 and wherein m + n2 is at least 1 ;

[0181] A3is wherein Z is a bond or a Ci-Ce alkyl chain or a substituted or unsubstituted aryl group or wherein Z is Xi-Ar, wherein the Ar group is directly attached to the OR17group, wherein Xi is a Ci-Ce alkyl chain and Ar is a substituted or unsubstituted aryl group;

[0182] A4, A5, A6A7, A9and A10are each individually hydrogen or a C1-C3 alkyl;

[0183] A8is hydrogen or a C1-C4 alkyl;

[0184] R17is a bond (i.e. X’), -CH2X', -CH2NHX', -C(O)NHNHC(O)X', -C(O)X', -CH2NH C(O)X', - C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L; wherein A3represents a group which may be attached at any point on the ring structure containing Q.

[0185] Preferably, Z is a Ci-Ce alkyl chain. Preferably, Z is a linear Ci-Ce alkyl chain. Preferably, wherein Q is O, S or CRaRband wherein Raand Rbare each independently selected from hydrogen or a linear C1-C3 alkyl chain.

[0186] Preferably, wherein A1and A2are each independently selected from the group of halo, hydrogen, a linear C1-C3 alkyl chain, phenyl, hydroxy, C1-C3 alkoxy, or wherein A1and A2together with the atoms to which they are bonded form a fused ring.

[0187] The ring structure containing substituent Q may be 4, 5, 6, 7 or 8 atoms in size depending on the values of m and n2. Preferably, the ring containing substituent Q is 5, 6, 7 or 8 atoms in size.

[0188] In some cases, in accordance with the present invention, the topoisomerase inhibitor is of formula I II b: wherein:

[0189] A1is a C1-C3 alkyl chain;

[0190] A2is halo; Q is O, S or CH2 m and n2 are each individually 0, 1 or 2 and wherein m + n2 is at least 1 ;

[0191] A3is wherein

[0192] Z is a bond or a C1-C3 alkyl chain and R17is a bond (i.e. X’), -CH2X', -CH2NHX', -CH2NHC(O)X', - C(O)NHNHC(O)X', -C(O)X', -C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L.

[0193] Preferably, R17is -CH2X', -CH2NHX', -C(O)NHNHC(O)X', -C(O)X', -C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L. Preferably, A1is a linear C1-C3 alkyl chain. Preferably, Z is a linear C1-C3 alkyl chain.

[0194] In certain cases, the topoisomerase inhibitor is preferably (1 S,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-

[0195] 4-methyl-1 ,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinoline-

[0196] 10,13-dione, which corresponds to a compound according to formula IV: wherein:

[0197] R17is a bond (i.e. X’), -CH2X', -CH2NHX', -CH2NHC(O)X', -C(O)NHNHC(O)X', -C(O)X', -C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L. Preferably, R17is CH2X', -CH2NHX', - C(O)NHNHC(O)X', -C(O)X', -C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L. In some embodiments, the topoisomerase inhibitor according to the present invention is selected from

[0198] (Table A):

[0199] Table A wherein

[0200] Z is a C1-C3 alkyl chain and R17is a bond (i.e. X’), -CH2X', -CH2NHX', -C(O)NHNHC(O)X', -C(O)X', - C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L.

[0201] In some embodiments, the topoisomerase inhibitor according to the present invention is Exatecan or a derivative thereof. In some embodiments, the topoisomerase inhibitor is Exatecan, or a pharmaceutically acceptable salt, hydrate, solvate, or crystalline form thereof. In preferred embodiments, the topoisomerase inhibitor according to the present invention has the following structure:

[0202] Exatecan wherein!denotes the point of attachment to the linker or attachment to the group wherein

[0203] Z is a C1-C3 alkyl chain and R17is a bond (i.e. X’), -CH2X', -CH2NHX', -C(O)NHNHC(O)X', -C(O)X', - C(O)NHNHX', -OX', -NHX' or -SX', wherein X' is a bond to linker L.

[0204] Tubulin Polymerisation Inhibitors

[0205] In some embodiments the cytotoxic payload comprises a tubulin polymerisation inhibitor.

[0206] As used herein, the term “tubulin polymerisation inhibitor” refers to the group of compounds that block the polymerisation of a / p-tubulin heterodimers into microtubules (polymers of tubulin), or block the depolymerisation of microtubules. Tubulin polymerisation inhibitors function by preventing the cell from undergoing mitosis, resulting in cell death via apoptosis. The ability of an agent to inhibit tubulin polymerisation can be determined by the skilled person using various methods well known in the art, for example, as described in Zhu et al., Bioorg Med Chem Lett. (2021) 37:127698.

[0207] In some embodiments, the tubulin polymerisation inhibitor is selected from a class. In some embodiments, the class of tubulin polymerisation inhibitor is selected from: maytansinoids, auristatins, tubulysins, hemiasterlin and analogs thereof, crytophycins, combretastatins, Epothilones, taccalonolides, paclitaxel / docetaxel derivatives, taxanes and colchicines.

[0208] In some embodiments, the tubulin polymerisation inhibitor is selected from eribulin, MMAE, MMAF, DM1 , DM4, cemadotin, rhizoxin, discodermolide, Combretastatin A-4, colchicine, indibulin, cevipabulin, dictyostatin, cevipabulin .

[0209] In some embodiments, the cytotoxic payload of the anti-FAP ADC comprises eribulin.

[0210] In some embodiments, the cytotoxic payload of the anti-FAP ADC comprises a dolastatin.

[0211] As used herein, the term “dolastatin” refers to the group of compounds including the natural marine cytotoxic compounds which were first isolated from the sea hare Dolabella Auricularia, and their derivatives.

[0212] Dolastatins include dolastatin 10 and dolastatin 15. These structurally similar dolastatins inhibit cell proliferation and induce apoptosis. Dolastatin 10 is more potent than dolastatin 15, although both compounds demonstrate cytotoxicity at nanomolar concentrations (see, for example, Singh et al. J Nat Prod. (2022) 85(3):666-687 and Bai et al. Biochem Pharmacol. (1992) 43(12):2637-45). In some embodiments, the dolastatin according to the present invention is dolastatin 10 or a derivative thereof. In some embodiments, the dolastatin is dolastatin 15 or a derivative thereof.

[0213] Dolastatin 10 inhibits tubulin polymerisation and tubulin-dependent guanosine triphosphate hydrolysis, thereby disrupting microtubule dynamics and blocking mitosis. Dolastatin 15 shows significantly weaker binding to tubulin than dolastatin 10, indicating an alternative mechanism for dolastatin 15’s potent cytotoxicity.

[0214] In some embodiments, the dolastatin according to the present invention is a dolastatin 15 derivative. Dolastatin 15 derivatives include, for example, ILX651 (also known as tasidotin), LU103793 (also known as Cematodin®).

[0215] In some embodiments, the dolastatin according to the present invention is a dolastatin 10 derivative. Dolastatin 10 derivatives include, for example, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD), Auristatin PE (TZT-1027, also known as Soblidotin, Drugbank accession number # DB12677), Auristatin PYE, mono methyl autistatin PYE (MMAPYE). In some embodiments, the dolastatin is MMAE or MMAF. In preferred embodiments, the dolastatin according to the present invention is MMAE.

[0216] In some embodiments, the dolastatin according to the present invention is MMAE or a pharmaceutically acceptable salt, hydrate, solvate, or crystalline form thereof. In some embodiments, the dolastatin has the following structure:

[0217] MMAE

[0218] Cytolysins

[0219] In some embodiments, the cytotoxic payload is a cytolysin. In some embodiments, the anti-FAP ADC is an antibody-cytolysin conjugate. The cytolysin may be a compound disclosed in WO 2008 / 138561 A1 , the entire contents of which is expressly incorporated herein by reference (compounds disclosed therein are also referred to as Tubulysin derivatives). The cytolysin may be synthesised as described in WO 2008 / 138561. In some embodiments, the cytolysin is as defined in Formula I or Formula IV of WO 2008 / 138561 A1 . In some embodiments, the cytolysin is of formula IV: wherein:

[0220] R2is H or is C1-C4 alkyl;

[0221] R6is C1-C6 alkyl;

[0222] R7is C1-C6 alkyl, CH2OR19or CH2OCOR20, wherein R19is alkyl, R20is C2-Ce-alkenyl, phenyl, or CH2- phenyl;

[0223] R9is C1-C6 alkyl;

[0224] R10is H, OH, O-alkyl or O-acetyl; f is 1 or 2;

[0225] R11has the following structure:

[0226] wherein

[0227] R21is H, OH, halogen, NH2, alkyloxy, phenyl, alkyl amino or dialkyl amino;

[0228] R16is H or a Ci-Ce-alkyl group;

[0229] R17is directly or indirectly attached to linker L; and q is 0, 1 , 2 or 3; and wherein the term “optionally substituted” relates to groups, wherein one or several H atoms can be replaced by F, Cl, Br or I or OH, SH, NH2, or NO2; the term “optionally substituted” further relates to groups, which can be exclusively or additionally substituted with unsubstituted C1-C6 alkyl, C2C6 alkenyl, C2-C6 alky ny I, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C2-C9 heterocycloalkyl, C6-C10 aryl, C1-C9 heteroaryl, C7-C12 aralkyl or C2-C11 heteroaralkyl groups.

[0230] In some embodiments R2is a bond to linker L.

[0231] In some embodiments R17is C(O)X, CONHNHX, OX, NHX or SX, wherein X is a bond to linker L.

[0232] In some embodiments, linker L comprises a spacer. In some embodiments the spacer has a chain length of 2 to 30 atoms. In some embodiments the spacer comprises or consists of an alkylene (i.e. divalent alkyl) or heteroalkylene (i.e. divalent heteroalkyl) group. In some embodiments the spacer comprises or consists of an alkylene or oxyalkylene group.

[0233] In some embodiments, the spacer comprises -(OCH2CH2)n-, wherein n is 2 to 5. In some embodiments the spacer comprises or consists of a group -(CH2)n- or -(OCH2CH2)n-, wherein n > 1 . In some embodiments the spacer comprises or consists of a group -(OCH2CH2)n-, wherein n > 1 . In particular, n may be 1 to 15, 1 to 10, 1 to 6, or 2 to 5. For example, n may be 3 or 4. In some embodiments the spacer comprises between one and six ethylene glycol units, e.g. a triethylene glycol. In some embodiments the spacer may be directly attached to group R17, or may be attached to group R17via a bridging group. In some embodiments the spacer is attached to group R17via a -C(O)X bridging group, wherein X is a bond to R17. In some embodiments R17is CONHNHX and the spacer is attached to group R17via a -C(O)X bridging group, wherein X represents the bond between the spacer and R17. In some embodiments R17is CONHNHX and the spacer is a -(OCH2CH2)n- attached to R17via a -C(O)X bridging group, wherein n = 2, 3 or 4. In some embodiments, L comprises an attachment group for attachment to A.

[0234] In some embodiments, L comprises a protease cleavable portion comprising a valine-citrulline unit.

[0235] For example, L may comprise maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate.

[0236] In some embodiments the cytolysin has the following structure: wherein * indicates the site of attachment to L.

[0237] In some embodiments D comprises a cytolysin having the following structure: In some embodiments the double bond of the maleimide is reacted with a thiol group of a cysteine residue of the antibody A to form a sulphur-carbon bond in order to effect linkage of the linker L to the antibody A.

[0238] In some embodiments -L-D has the structure: In other embodiments -L-D has a structure selected from the group consisting of: In certain embodiments -L-D has the following structure:

[0239] In certain embodiments -L-D has the following structure: p may, in some embodiments, lie in the range 1 to 5, e.g. 1 to 4, or 1 to 3. In particular embodiments p is 1 or 2. p may be 3 or 4.

[0240] In some embodiments, A has a heavy chain with amino acid sequence of SEQ ID NO: 3 and a light chain with amino acid sequence of SEQ ID NO: 4; and L-D has the structure: wherein * denotes the point of attachment to A. In some embodiments, the anti-FAP antibody-cytolysin conjugate is the antibody-cytolysin conjugate described herein as OMTX705. OMTX705 comprises a humanised anti-FAP mAb (OMTX005) conjugated to the cytolysin TAM470 via a protease-cleavable vcPABA-(EG)3 optimised linker. OMTX705 is described in Fabre et al. (2020), and Figure 1 is an illustrative depiction of OMTX705.

[0241] Immune Checkpoint Inhibitor (I Cl)

[0242] Various I Cis are known in the art and are suitable for use in the present invention. For example, the ICI may comprise an anti-programmed cell death protein (PD-1) antibody, an anti-programmed deathligand 1 (PD-L1) antibody or an anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4) antibody. In some embodiments the ICI comprises an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0243] In some embodiments, the anti-PD-L1 antibody comprises durvalumab. In some embodiments, the anti CTLA-4 antibody comprises tremelimumab.

[0244] In some embodiments, the ICI comprises an anti-PD-1 antibody. For example, the ICI may comprise nivolumab (MDX1 106) or pembrolizumab (MK-3475). In some embodiments the anti-PD-1 antibody comprises tislelizumab. In some embodiments, the anti-PD-1 antibody comprises pembrolizumab. Preferably, the ICI comprises pembrolizumab or tislelizumab.

[0245] Subject

[0246] The subject is a mammal. Preferably, the subject is a human, but may be any other primate (great ape, old world monkey or new world monkey), or a domestic, laboratory or livestock animal, such as a mouse, rat, guinea pig, lagomorph (e.g. rabbit), cat, dog, pig, cow, horse, sheep or goat.

[0247] The subject may previously have undergone anti-cancer therapy and / or surgery, but may not previously have been administered the specific combination of the present invention. Indeed, the present inventors envisage that the present invention may have particular utility for subjects who are non-responders to other previous treatments and / or surgeries. In particular, the present inventors envisage that the present invention may have particular utility for subjects who are non-responders to previous ICI or antibody-cytolysin conjugate monotherapies (i.e. where only one of the combination therapy has previously been administered to the subject and the cancer is non-responsive to this monotherapy).

[0248] In the context of the present invention, a “non-responder” will be understood to refer to a subject whose cancer has not responded to a particular treatment and / or surgery. Typically, the cancer of a “non-responder” will continue to progress despite the treatment and / or surgery. Therefore, a “non- responder” will typically be understood to refer to a subject who has progressive cancer disease despite the treatment and / or surgery. A “responder” or a “subject who responds to therapy” may be defined as a subject that experiences a complete or a partial response, defined as more than a 30% reduction in the size of a tumor lesion compared to the size of the tumor lesion as measured before the initiation of treatment; or a subject that experiences stable disease, defined as progression-free survival, for over 4 treatment cycles. A “non-responder” or a “subject who does not respond to therapy” may be defined as a subject who does not show either a complete response or a partial response, and experiences 4 or fewer progression-free treatment cycles. A “non-responder” or a “subject who does not respond to therapy” may also be defined as a subject who experiences progressive disease.

[0249] In some embodiments, the subject was previously administered the antibody-cytolysin conjugate and was not previously administered the ICI . In some embodiments, the subject was previously administered the antibody-cytolysin conjugate only, i.e. without any other anti-cancer treatment. In other words, the subject may be a non-responder to antibody-cytolysin conjugate monotherapy. In other embodiments, the subject was previously administered the antibody-cytolysin conjugate and at least one other anti-cancer therapy, such as radiotherapy and / or chemotherapy, but was not previously administered the ICI.

[0250] By “previously administered”, this will be understood to refer to administration prior to the method of the present invention.

[0251] In other embodiments, the subject was previously administered the ICI and was not previously administered the antibody-cytolysin conjugate. In some embodiments, the subject was previously administered the ICI only, i.e. without any other anti-cancer treatment. In other words, the subject may be a non-responder to ICI, for example anti-PD-1 molecule monotherapy. The subject may be a non- responder to pembrolizumab, nivolumab or tislelizumab monotherapy. In other embodiments, the subject was previously administered the ICI and at least one other anti-cancer therapy, such as radiotherapy and / or chemotherapy, but was not previously administered the antibody-cytolysin conjugate.

[0252] Cancer

[0253] Preferably, the cancer is a carcinoma. As the skilled person will appreciate “carcinoma” refers to cancer of the epithelial tissue.

[0254] In the context of the present invention, the term primary cancer refers to the anatomical site where the cancer first starts growing in the subject. Thus, reference to, for example, a primary cancer comprising pancreatic cancer means that the cancer first started growing in the subject in the pancreas. In such embodiments, the primary site of the cancer is the pancreas.

[0255] As used herein, the term “metastatic cancer” refers to a primary cancer where the cancer has spread from the primary cancer to another anatomical site in the subject. Thus, in the context of the present invention, metastatic cancer comprises the primary cancer and cancer in an anatomical site of the subject different to the site of the primary cancer. These cancers in sites different to the site of the primary cancer may otherwise be referred to as metastases or metastasis. For the avoidance of doubt, it will be appreciated that reference to “metastases” refers to a plurality of metastasis, i.e. a plurality of secondary tumours which are located in one or more anatomical sites of the subject different to the site of the primary cancer.

[0256] Where a cancer is referred to as, for example, “metastatic pancreatic cancer”, this will be understood to refer to a cancer where the primary cancer is pancreatic cancer, but this has spread to anatomical sites other than the pancreas in the subject. Likewise, where a cancer is referred to as, for example, “metastatic colorectal cancer”, this will be understood to refer to a primary cancer comprising colorectal cancer, which has spread to anatomical sites different to the coIorectum in the subject. In such examples, the metastatic cancer comprises both the primary cancer and metastases in anatomical locations different to that of the primary cancer.

[0257] Preferably, the primary cancer comprises gastrointestinal or lung cancer. As the skilled person will appreciate, gastrointestinal cancer is cancer of the digestive tract and / or other abdominal organs.

[0258] Gastrointestinal cancer may comprise pancreatic cancer, colorectal cancer, oesophageal cancer, gastric (stomach) cancer, liver cancer, anal cancer, gastrointestinal stroma cancer, neuroendocrine cancer or cancer of the small intestine.

[0259] In some embodiments, the primary cancer comprises lung cancer, pancreatic cancer, colorectal cancer, gastric cancer or oesophageal cancer.

[0260] In some embodiments, the primary cancer comprises pancreatic cancer, colorectal cancer, gastric cancer or oesophageal cancer.

[0261] The lung cancer may comprise non-small cell lung cancer (NSCLC).

[0262] In some embodiments, the primary cancer comprises pancreatic cancer or colorectal cancer. In some embodiments, the primary cancer comprises pancreatic cancer. In some embodiments, the primary cancer comprises colorectal cancer. The pancreatic cancer may comprise pancreatic ductal adenocarcinoma (PDAC), pancreatic squamous cell carcinoma or pancreatic adenosquamous carcinoma. In some embodiments the pancreatic cancer comprises PDAC.

[0263] The colorectal cancer may comprise microsatellite-stable (MSS) colorectal cancer. Micro-satellite stable colorectal cancer will be understood to refer to colorectal cancer wherein the microsatellite DNA segments in the colorectal cancer are not mutated. Thus, in some embodiments, the colorectal cancer comprises microsatellite-stable DNA segments detectable by immunohistochemistry (IHC), PCR or next-generation sequencing. The present inventors have found that the combination of an ICI and an antibody-cytolysin conjugate has especially effective anti-tumour activity against metastatic pancreatic cancer and metastatic MSS colorectal cancer. This is surprising given that each therapy individually does not show efficacy against these tumour types. In some embodiments, the primary cancer is selected from PDAC, MSS colorectal cancer, NSCLC and oesophageal cancer.

[0264] The metastasis or metastases of the metastatic cancer may comprise any metastasis or metastases in an anatomical site different to that of the primary cancer. For example, the metastasis may comprise lung, liver, lymph node, peritoneal, bone and / or bile duct metastasis / metastases.

[0265] In some embodiments the metastasis comprises liver, peritoneal, lymph node and / or lung metastasis. The metastasis may comprise liver metastasis. The metastasis may comprise peritoneal metastasis. In some embodiments, the metastasis comprises liver, lung and / or peritoneal metastasis. In some embodiments, the metastasis comprises liver or peritoneal metastasis. In some embodiments the metastasis comprises liver or lung metastasis. In some embodiments, the metastasis comprises liver and peritoneal metastases.

[0266] In particular embodiments, the primary cancer comprises MSS colorectal cancer and the metastasis comprises liver, lung and / or peritoneal metastasis. Advantageously, the combination of the ICI and the antibody cytolysin conjugate demonstrates surprisingly effective anti-tumour activity in MSS colorectal cancer with liver, lung and / or peritoneal metastasis.

[0267] It will be appreciated that some subjects may have been diagnosed with metastatic cancer, due to the presence of multiple tumours in a plurality of different anatomical locations in the body, but that it is not possible to determine the location of the primary cancer and the location of the metastases. In such instances, it is only possible to determine the location of tumours, and not what was the primary source of the tumours. In such embodiments, the primary cancer and metastasis may reside in at least two of the following anatomical locations: lung, liver, lymph node, peritoneum, bone, bile duct, pancreas, colon, rectum, oesophagus, stomach, and small intestine. For example, the primary cancer and metastasis may reside in at least two of the following anatomical locations: lung, liver, lymph node, peritoneum, pancreas, colon and rectum.

[0268] The primary cancer and / or metastasis may comprise a level of FAP expression detectable by immunohistochemistry. The level of FAP expression may be detectable from a biological sample taken from the primary cancer and / or metastasis. The biological sample may otherwise be referred to as a biopsy.

[0269] In some embodiments, administration of the antibody-cytolysin conjugate and the ICI results in a synergistic effect. The inventors have surprisingly found that while each therapy individually may not have an anti-tumour effect, the combination of the two can provide effective anti-tumour activity for metastatic cancer.

[0270] In the context of the present invention, the term “anti-tumour activity” will be understood to refer to activity which reduces symptoms of the cancer, reduces number and / or size of tumours, and / or increases progression free survival and / or overall survival time of the subject. Typically, the subject will be considered a responder to the combination, such that their condition improves, or at least remains stable. The condition remaining stable may otherwise be referred to as stable disease.

[0271] A reduced number and / or size of tumour may comprise a reduction of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% or at least about 50% relative to the number and / or size of the tumour prior to administration of the combination. The reduction in number and / or size of tumour may be relative to a baseline value for the number and / or size of the tumour, as discussed further below. In some embodiments, administration of the antibody-cytolysin conjugate and the ICI results in an at least 30% reduction of tumour size and / or number. A reduced number and / or size of tumour may comprise a reduction of at least about 5% to at least about 100%. A 100% reduction will be understood to mean that there are 0 detectable tumours and / or no detectable sites of tumours remaining. For example, where a subject may have five identified tumours prior to administration of the combination, a 100% reduction will be understood to mean that the subject, as a result of the administration, has 0 identified tumours. The anti-tumour activity will be understood to apply to the metastasis and / or the primary cancer.

[0272] The criteria used to determine objective tumour response for target lesions / tumours is well known from the RECIST 1 .1 and iRECIST studies (RECIST : Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M, Rubinstein L, Shankar L, Dodd L, Kaplan R, Lacombe D, Verweij J. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009 Jan;45(2):228-47. doi: 10.1016 / j.ejca.2008.10.026. PMID: 19097774 and iRECIST: Seymour L, Bogaerts J, Perrone A, Ford R, Schwartz LH, Mandrekar S, Lin NU, Litiere S, Dancey J, Chen A, Hodi FS, Therasse P, Hoekstra OS, Shankar LK, Wolchok JD, Ballinger M, Caramella C, de Vries EGE; RECIST working group. iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics. Lancet Oncol. 2017 Mar;18(3):e143-e152. doi: 10.1016 / S1470-2045(17)30074-8. Epub 2017 Mar 2. Erratum in: Lancet Oncol. 2019 May;20(5):e242. doi: 10.1016 / 51470-2045(19)30240-2. PMID: 28271869;

[0273] PMCID: PMC5648544). The entirety of each of these references is incorporated herein. A target lesion may otherwise be referred to as a tumour or tumour lesion.

[0274] In the context of the present invention, a responder may comprise a partial responder. A partial responder is a subject who has a “partial response” to the combination. As defined in RECIST1 .1 and iRECIST, a partial response will be understood to refer to at least a 30% decrease in the sum of diameters and / or number of target lesions, taking as reference the baseline sum diameters or number of lesions.A responder may be defined as a subject that experiences progression-free survival for more than 4 treatment cycles.

[0275] In the context of the present invention, a “non-responder” or a “subject who does not respond to therapy” may be defined as a subject who does not show either a complete response or a partial response, and experiences 4 or fewer progression-free treatment cycles. A “non-responder” or a “subject who does not respond to therapy” may also be defined as a subject who experiences progressive disease. Progressive cancer disease, which may otherwise be referred to as progressive disease, will be understood to mean that the subject has at least a 20% increase in the sum of diameters and / or number of tumour lesions compared to a baseline sum diameter or number of tumour lesions.

[0276] The baseline sum diameter or number of tumour lesions may comprise the sum diameter or number of tumour lesions measured on day 1 of the cycle, or prior to the combination treatment commencing. Alternatively, the baseline sum diameter or number of tumour lesions may comprise a previous lowest value of sum diameter or number of tumour lesions measured. In such embodiments, progressive disease occurs when the sum of diameters or number of tumour lesions is 20% higher than the previous lowest value. For example, a subject may have a sum of diameters prior to treatment of 110, with the sum of diameters reducing after treatment to 100. In embodiments where the baseline comprises the previous lowest value, the baseline for this subject will be 100. Thus, progressive disease will be reached if and when the sum of diameters reaches 120 (20% increase upon 100). The appearance of one or more new lesions may be defined as progressive disease.

[0277] In the context of the present invention, a responder may comprise a subject that experiences stable disease. As defined in RECIST 1.1 and iRECIST, stable disease will be understood to refer to neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease. Thus, stable disease will be understood to refer to metastatic cancer wherein the tumours of the primary and / or metastatic cancer have increased by less than 20% in size versus the smallest size of the tumour prior to administration of the combination or wherein the number of tumour lesions has not increased compared to the number prior to administration of the combination. Stable disease may therefore be defined as metastatic cancer wherein the tumours of the primary and / or metastases have increased by less than 20% in size compared to a baseline tumour size. Stable disease may be defined as metastatic cancer wherein the tumours do not have unequivocal progression as assessed by the treating physician or by an external expert radiologists. Therefore, stable disease is not progressive disease and allows therapy to continue.

[0278] In some embodiments, administration of the antibody-cytolysin conjugate and the ICI results in a reduction of tumour burden in the subject. As used herein, the term tumour burden defines the total amount of cancer in the subject.

[0279] The size or volume of tumours in the subject may be calculated using RECIST 1.1 and / or iRECIST criteria. For example, the size or volume of tumours in the subject may be calculated as the total sum of long axis diameters of the tumours in the primary tumour and the metastatic tumour(s). For lymph nodes >15 mm, the short axis measurement may be used instead. The total sum of diameters of the tumours in the primary tumour and the metastatic tumour(s) may be measured prior to administration of the combination treatment to provide a baseline value. The total sum of diameters of the tumours in the primary tumour and the metastatic tumour(s) may be measured after administration of the combination treatment to provide a post-treatment value. The difference between the baseline value and the post-treatment value may be calculated as a percentage or ratio, which may then be used to determine if the tumour size is reduced, the same or increased following combination treatment. Typically, if the tumour size has not increased more than 20% compared to the tumour size prior to administration of the combination treatment and the number of tumour lesions has not increased, this will be understood to mean that the cancer is stable disease or has at least partially responded to treatment.

[0280] In some embodiments, a reduction of tumour size comprises a reduction in the size or volume of tumours. In some embodiments, a reduction of tumour size comprises a reduction in metastatic tumour lesion size. In some embodiments a reduction of tumour size comprises a reduction in primary tumour lesion size. Preferably, a reduction of tumour size comprises a reduction in primary and metastatic tumour lesion size.

[0281] In some embodiments, administration of the antibody-cytolysin conjugate and the ICI reduces carcinoembryonic (CEA) levels in the subject, relative to the CEA level in the subject prior to administration of the combination. The CEA level may be determined from a blood sample obtained from the subject prior to and after combination treatment. The CEA level may be reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In some embodiments, administration of the antibody-cytolysin conjugate and the ICI reduces the CEA level in the subject relative to the CEA level in the subject prior to administration of the combination of from about 10% to about 100%.

[0282] In some embodiments, administration of the antibody-cytolysin conjugate and the ICI reduces CA19.9 levels in the subject, relative to the CA19.9 level in the subject prior to administration of the combination. The CA19.9 level may be determined from a blood sample obtained from the subject prior to and after combination treatment. The CA19.9 level may be reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In some embodiments, administration of the antibody-cytolysin conjugate and the ICI reduces the CA19.9 level in the subject relative to the CA19.9 level in the subject prior to administration of the combination of from about 10% to about 100%.

[0283] In some embodiments, administration of the antibody-cytolysin conjugate and the ICI increases progression free survival and / or overall survival time of the subject relative to a control subject who has been treated with the antibody-cytolysin conjugate or with the ICI, as a monotherapy, but not in combination.

[0284] “Treated as a monotherapy” will be understood to mean that the control subject has, for example, previously been treated with the antibody-cytolysin conjugate but not the ICI, or with the ICI but not the antibody-cytolysin conjugate. In some embodiments, administration of the antibody-cytolysin conjugate and the ICI increases progression free survival and / or overall survival time of the subject. In some embodiments, progression free survival and / or overall survival time is increased by at least about 50%, at least about 100% or at least about 200% relative to a control subject who has been treated with the antibody-cytolysin conjugate and not treated with the ICI.

[0285] Administration

[0286] In some embodiments, the invention relates to the administration of a stroma-targeting agent.

[0287] In some embodiments, the invention relates to the administration of a stroma-targeting agent and an ICI.

[0288] In some embodiments, the method comprises simultaneous, sequential or separate administration of the stroma-targeting agent and the ICI. Preferably, the method comprises sequential administration of the stroma-targeting agent and the ICI to the subject. Sequential administration, as used herein, will be understood to refer to an administration regime where one agent is administered directly after administration of another agent. Thus, in some embodiments, the method comprises sequential administration of first the stroma-targeting agent and secondly the ICI. Alternatively, the method may comprise sequential administration of first the ICI and secondly the stroma-targeting agent.

[0289] In some embodiments, the method comprises intravenous administration of the stroma-targeting agent and the ICI to the subject. In such embodiments, it will be appreciated that each of the stromatargeting agent and the ICI are administered intravenously, sequentially, simultaneously or separately, preferably sequentially.

[0290] In some embodiments, the stroma-targeting agent is administered weekly. In some embodiments, the stroma-targeting agent is administered every two weeks. Two weeks may otherwise be referred to as 14 days. In some embodiments, the stroma-targeting agent is administered every three weeks. Three weeks may otherwise be referred to as 21 days. In some embodiments, the stroma-targeting agent is administered monthly.

[0291] In some embodiments, the method comprises administration of the stroma-targeting agent at least once in a 21-day cycle. In some embodiments, the method comprises administration of the stromatargeting agent at least twice in a 21-day cycle.

[0292] In some embodiments, the method comprises administration of the stroma-targeting agent only once in a 21-day cycle. In some embodiments, the method comprises administration of the stromatargeting agent only twice in a 21-day cycle.

[0293] In some embodiments, the method comprises administration of the stroma-targeting agent on day 1 and day 8 of a 21-day cycle. “Day 1” will be understood to refer to the first day of the 21-day cycle. In embodiments comprising 21-day cycles, in some embodiments the stroma-targeting agent is not administered on days 9 to 21 of the 21-day cycle.

[0294] The ICI may be administered on the same day as the stroma-targeting agent. In some embodiments, the ICI is administered weekly. In some embodiments, the ICI is administered every two weeks. In some embodiments, the ICI is administered every three weeks. In some embodiments, the ICI is administered monthly.

[0295] In some embodiments, the method comprises administration of the ICI at least once in a 21-day cycle. The method may comprise administration of the ICI on day 1 of a 21-day cycle.

[0296] In some embodiments, the method comprises administration of the ICI only once in a 21-day cycle.

[0297] In some embodiments, the method comprises administration of the stroma-targeting agent and the ICI on day 1 of a 21-day cycle.

[0298] In some embodiments, the method comprises administration of: the stroma-targeting agent and the ICI on day 1 of a 21-day cycle; and the stroma-targeting agent on day 8 of a 21-day cycle.

[0299] In embodiments comprising a 21-day cycle of administration, the 21-day cycle may be repeated at least once. For example, the method may comprise administration of the stroma-targeting agent and the ICI to the subject in at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10 21-day cycles. In some embodiments, the method comprises administration of the stroma-targeting agent and the ICI in at least 10 21-day cycles. Each 21-day cycle may comprise the same or different administration pattern as described above.

[0300] For example, each 21-day cycle may comprise administration of the stroma-targeting agent on day 1 of each 21-day cycle. In some embodiments, each 21-day cycle comprises administration of the stroma-targeting agent on day 1 and day 8 of each 21-day cycle. Each 21-day cycle may comprise administration of the ICI of day 1 of each 21-day cycle.

[0301] In some embodiments, the invention relates to the administration of an antibody-cytolysin conjugate and an ICI.

[0302] In some embodiments, the method comprises simultaneous, sequential or separate administration of the antibody-cytolysin conjugate and the ICI. Preferably, the method comprises sequential administration of the antibody-cytolysin conjugate and the ICI to the subject. Sequential administration, as used herein, will be understood to refer to an administration regime where one agent is administered directly after administration of another agent. Thus, in some embodiments, the method comprises sequential administration of first the antibody-cytolysin conjugate and secondly the ICI. Alternatively, the method may comprise sequential administration of first the ICI and secondly the antibody-cytolysin conjugate. In some embodiments, the method comprises intravenous administration of the antibody-cytolysin conjugate and the ICI to the subject. In such embodiments, it will be appreciated that each of the antibody-cytolysin conjugate and the ICI are administered intravenously, sequentially, simultaneously or separately, preferably sequentially.

[0303] In some embodiments, the antibody-cytolysin conjugate is administered weekly. In some embodiments, the antibody-cytolysin conjugate is administered every two weeks. Two weeks may otherwise be referred to as 14 days. In some embodiments, the antibody-cytolysin conjugate is administered every three weeks. Three weeks may otherwise be referred to as 21 days. In some embodiments, the antibody-cytolysin conjugate is administered monthly.

[0304] In some embodiments, the method comprises administration of the antibody-cytolysin conjugate at least once in a 21 -day cycle. In some embodiments, the method comprises administration of the antibody-cytolysin conjugate at least twice in a 21 -day cycle.

[0305] In some embodiments, the method comprises administration of the antibody-cytolysin conjugate only once in a 21-day cycle. In some embodiments, the method comprises administration of the antibody- cytolysin conjugate only twice in a 21-day cycle.

[0306] In some embodiments, the method comprises administration of the antibody-cytolysin conjugate on day 1 and day 8 of a 21-day cycle. “Day 1” will be understood to refer to the first day of the 21-day cycle.

[0307] In embodiments comprising 21-day cycles, in some embodiments the antibody-cytolysin conjugate is not administered on days 9 to 21 of the 21-day cycle.

[0308] The ICI may be administered on the same day as the antibody-cytolysin conjugate. In some embodiments, the ICI is administered weekly. In some embodiments, the ICI is administered every two weeks. In some embodiments, the ICI is administered every three weeks. In some embodiments, the ICI is administered monthly.

[0309] In some embodiments, the method comprises administration of the ICI at least once in a 21-day cycle. The method may comprise administration of the ICI on day 1 of a 21-day cycle.

[0310] In some embodiments, the method comprises administration of the ICI only once in a 21-day cycle.

[0311] In some embodiments, the method comprises administration of the antibody-cytolysin conjugate and the ICI on day 1 of a 21-day cycle.

[0312] In some embodiments, the method comprises administration of: the antibody-cytolysin conjugate and the ICI on day 1 of a 21 day cycle; and the antibody cytolysin conjugate on day 8 of a 21-day cycle.

[0313] In embodiments comprising a 21-day cycle of administration, the 21-day cycle may be repeated at least once. For example, the method may comprise administration of the antibody-cytolysin conjugate and the ICI to the subject in at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10 21-day cycles. In some embodiments, the method comprises administration of the antibody-cytolysin conjugate and the ICI in at least 10 21-day cycles. Each 21-day cycle may comprise the same or different administration pattern as described above.

[0314] For example, each 21-day cycle may comprise administration of the antibody-cytolysin conjugate on day 1 of each 21-day cycle. In some embodiments, each 21-day cycle comprises administration of the antibody-cytolysin conjugate on day 1 and day 8 of each 21 day cycle. Each 21-day cycle may comprise administration of the ICI of day 1 of each 21-day cycle.

[0315] In some embodiments, the method comprises administration of at least 1 mg / kg of the antibody- cytolysin conjugate to the subject, optionally 2mg / kg of the antibody-cytolysin conjugate to the subject, further optionally at least 4mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of at least 3mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of at least 5mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of at least 7mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of at least 8mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of at least 10mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of from 2mg / kg to 10mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of from 2mg / kg to 8mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of from 2mg / kg to 6mg / kg of the antibody-cytolysin conjugate to the subject, optionally of from 2mg / kg to 5.5mg / kg In some embodiments, the method comprises administration of from 2mg / kg to 18mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of from 4mg / kg to 10mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of from 4mg / kg to 8mg / kg of the antibody-cytolysin conjugate to the subject. Optionally, the method comprises administration of from 4mg / kg to 7.5mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of from 4mg / kg to 5.5mg / kg of the antibody-cytolysin conjugate to the subject.

[0316] In some embodiments, the method comprises administration of no more than 10mg / kg, optionally no more than 7.5mg / kg, no more than 6mg / kg or no more than 5mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of no more than 5.5mg / kg of the antibody-cytolysin conjugate to the subject.

[0317] In some embodiments, the method comprises administration of from 2mg / kg to 4mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of about 4mg / kg of the antibody- cytolysin conjugate to the subject. In some embodiments, the method comprises administration of about 5.5mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments the method comprises administration of about 7.5mg / kg of the antibody-cytolysin conjugate to the subject.

[0318] Preferably, the mg / kg dosage selected will be understood to be the mg / kg administered per administration. Thus, for example, in embodiments where the antibody-cytolysin conjugate is administered at least twice, the mg / kg dosage may be as described above for each administration.

[0319] Thus, in some embodiments, the method comprises administration of the antibody-cytolysin conjugate at least twice in a 21 -day cycle, wherein each administration comprises at least 1 mg / kg, optionally at least 2mg / kg of the antibody-cytolysin conjugate to the subject.

[0320] In some embodiments, the method comprises administration of the antibody-cytolysin conjugate at least twice in a 21 -day cycle, wherein each administration comprises at least 4mg / kg, optionally at least 7mg / kg of the antibody-cytolysin conjugate to the subject. In some embodiments, the method comprises administration of the antibody-cytolysin conjugate at least twice in a 21 -day cycle, wherein each administration comprises at least 7.5mg / kg, optionally at least 10mg / kg of the antibody-cytolysin conjugate to the subject.

[0321] In some embodiments, the method comprises administration of at least 100mg, at least 150mg, at least 200mg, at least 250mg, at least 300mg, at least 350mg or at least 400mg of the ICI to the subject. In some embodiments, the method comprises administration of from 100mg to 500mg of the ICI to the subject. In some embodiments, the method comprises administration of from 100mg to 400mg of the ICI to the subject, optionally of from 100mg to 300mg of the ICI to the subject. In some embodiments, the method comprises administration of at least 200mg of the ICI to the subject. In some embodiments, the method comprises administration of 200mg of ICI to the subject. The mg of ICI selected for administration may be per administration (for example 100mg per administration in embodiments where the ICI is administered twice). Preferably, the mg of ICI selected may be the total mg of ICI administered per treatment cycle.

[0322] Thus, in some embodiments, the method comprises administration of the ICI once in a 21 -day cycle, wherein the administration comprises administration of 200mg of ICI to the subject.

[0323] Pharmaceutical compositions

[0324] The antibody-cytolysin conjugate and / or ICI of the present invention may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient.

[0325] A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition which does not provoke secondary reactions and which allows, for example, facilitation of the administration of the conjugate / ICI, an increase in its lifespan and / or in its efficacy in the body or an increase in its solubility in solution. These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the mode of administration of the conjugate / ICL

[0326] In some embodiments, conjugates and / or ICIs of the present invention may be provided in a lyophilised form for reconstitution prior to administration. For example, lyophilised conjugates may be re-constituted in sterile water and mixed with saline prior to administration to an individual.

[0327] Conjugates and / or ICIs of the present invention may be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the conjugate. Thus pharmaceutical compositions may comprise, in addition to the conjugate, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the conjugate / ICI. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.

[0328] For intra-venous administration, for example by injection, the pharmaceutical composition comprising the conjugate and / or ICI may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3’-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions, such as sodium; metal complexes (e.g. Zn- protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0329] Method of treatment

[0330] The present invention provides a method of treating metastatic cancer in a mammalian subject, the method comprising administering (i) an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)Por a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytolysin and p is 1 to 10, to the subject simultaneously, sequentially or separately with (ii) an immune checkpoint inhibitor (ICI), wherein the metastatic cancer comprises a primary cancer and metastasis.

[0331] The primary cancer of the metastatic cancer may comprise lung or gastrointestinal cancer. It will be appreciated that the method comprises administration of a therapeutically effective amount of the antibody-cytolysin conjugate and ICI as defined in accordance with the eighth aspect of the invention to the subject in need thereof.

[0332] Stratifying Subjects Using Biomarkers

[0333] The present invention provides a method of selecting or stratifying a subject having a cancer for treatment with a stroma-targeting agent, the method comprising (i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and (ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value. In some embodiments, the method is for selecting or stratifying a subject having a cancer for treatment with a stroma-targeting agent as a monotherapy. In some embodiments, the method is for selecting or stratifying a subject having a cancer for treatment with a stroma-targeting agent as part of a combination therapy. In some embodiments, the combination therapy comprises a stroma-targeting agent and an ICI. That is, in another aspect, the present invention provides a method of selecting or stratifying a subject having a cancer for treatment with (i) a stroma-targeting agent and (ii) an ICI, the method comprising (i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and (ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

[0334] It will be appreciated that the method of selecting or stratifying a subject may be used to select subjects for treatment in accordance with the first, second and eighth aspects of the invention.

[0335] As used herein, the term “biomarker” refers to a molecule present in a sample obtained from a subject which can be used to derive a measure of gene expression for a gene of interest. The sample may be a tissue sample. Preferably, the sample is a bodily fluid which enables a liquid biopsy to be carried out. The sample may be a plasma, serum, blood, saliva, or urine sample. Depending on the type of samples, to simplify their storage and handling they can be frozen at -80°C after an initial sample processing.

[0336] In preferred embodiments, the sample obtained from the subject is a plasma sample. Plasma comprises many components. Examples of plasma components include exosomes, ectosomes, extracellular vesicles, platelets, microvesicles, circulating tumor cells and circulating tumor DNA. Protein levels of periostin, RAGE, N-cadherin, and SDF-1a may be measured from the plasma sample as a whole and / or from any specific plasma derived component. In some embodiments, the protein levels of periostin, RAGE, N-cadherin, and SDF-1a are measured from the plasma sample directly. The plasma derived component may be isolated from the blood by any appropriate method. Non limiting examples of plasma derived components include exosomes, ectosomes, extracellular vesicles, platelets, microvesicles, circulating tumor cells and circulating tumor DNA. Thus, in some embodiments, the expression levels of periostin, RAGE, N-cadherin, and SDF-1a are measured from plasma derived exosomes, ectosomes, extracellular vesicles, platelets, microvesicles, circulating tumor cells and / or circulating tumor DNA obtained from the subject.

[0337] In particular, plasma samples may comprise circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and extracellular vesicles (EVs). Extracellular vesicles include exosomes and ectosomes. In some embodiments, the expression levels of periostin, RAGE, N-cadherin, and SDF-1a are measured from CTCs, ctDNA or EVs derived from the plasma sample.

[0338] In some embodiments the method comprises measuring the protein level of at least periostin. In some embodiments the method comprises measuring the protein level of at least N-cadherin. In some embodiments the method comprises measuring the protein level of at least RAGE. In some embodiments the method comprises measuring the protein level of at least SDF-1a. In some embodiments, the method comprises measuring the protein level of at least two biomarkers, including periostin and N-cadherin. In some embodiments, the method comprises measuring the protein level of at least two biomarkers, including periostin and RAGE. In some embodiments, the method comprises measuring the protein level of at least two biomarkers, including periostin and SDF-1a. In some embodiments, the method comprises measuring the protein level of at least two biomarkers, including N-cadherin and RAGE. In some embodiments, the method comprises measuring the protein level of at least two biomarkers, including N-cadherin and SDF-1a. In some embodiments, the method comprises measuring the protein level of at least two biomarkers, including RAGE and SDF-1a. In some embodiments, the method comprises measuring the protein level of at least three biomarkers including periostin, N-cadherin, and RAGE. In some embodiments, the method comprises measuring the protein level of at least two biomarkers, including RAGE and SDF-1a. In some embodiments, the method comprises measuring the protein level of at least three biomarkers including periostin, N- cadherin and SDF-1a. In some embodiments, the method comprises measuring the protein level of at least three biomarkers including periostin, RAGE and SDF-1a. In some embodiments, the method comprises measuring the protein level of at least three biomarkers including N-cadherin, RAGE, and SDF-1a.

[0339] In some embodiments, the method comprises measuring the protein level of periostin and at least one or at least two additional biomarkers selected from N-cadherin, RAGE, and SDF-1a. In some embodiments, the method comprises measuring the protein level of N-cadherin and at least one or at least two additional biomarkers selected from periostin, RAGE, and SDF-1a. In some embodiments, the method comprises measuring the protein level of RAGE and at least one or at least two additional biomarkers selected from N-cadherin, periostin, and SDF-1a. In some embodiments, the method comprises measuring the protein level of SDF-1 a and at least one or at least two additional biomarkers selected from N-cadherin, RAGE, and periostin.

[0340] In some embodiments, the methods comprise normalising the protein level data for the at least one biomarker selected from periostin, RAGE, N-cadherin, and SDF-1 a. The protein level data may be normalised against any known housekeeping gene, such as CXCL8, ACTB, GAPDH, TUBB, HSP90AB1 , HPRT1 , RPL13A, B2M, PPIA, UBC, SDHA, TFRC, PGK1 , GUSB, YWHAZ, TBP, PSMB2, HMBS, LDHA. In some embodiments, the protein level data is normalised against the total plasma protein level.

[0341] As used herein, the term “predetermined level” or “predetermined value” relates to a biomarker protein level previously identified in an analogous subject population, i.e., a population of subjects having the same cancer type. The predetermined value may take a variety of forms. The predetermined value may be different for each biomarker, i.e., the predetermined level of periostin that may be used to stratify subjects that respond to therapy and subjects that do not respond to therapy may be different to the predetermined level of N-cadherin that may be used for the same. In some embodiments, the predetermined value can be single cut-off value, such as a median or mean. In some embodiments the predetermined value may be taken as the median of the biomarker levels in non-responders. In some embodiments the predetermined value may be taken as the mean of the biomarker levels in non-responders. In some embodiments the predetermined value may be taken as the 75thpercentile of the biomarker levels in non-responders, i.e., the 3rdquartile. In some embodiments the predetermined value may be taken as the 50th, 55th, 60th, 65th, 70th, 75th, 80th, 85th, 90th, 95thpercentile of the biomarker levels of the non-responders. In some embodiments, the predetermined value maybe taken as the 60thpercentile of the biomarker values of the non- responders, i.e., the 4thquintile. In some embodiments, the predetermined value may be taken as the 80% percentile of the biomarker values of the non-responders, i.e., the fifth quintile. In some embodiments the predetermined value may be taken as the midpoint between the mean protein level of the biomarker in responders and the mean protein level of the biomarker in non-responders. In some embodiments, the predetermined level may be taken as the highest level of the biomarker measured in a non-responder.

[0342] Various aspects of the present invention relate to determining the protein level of a biomarker (e.g. periostin, RAGE). Determining the protein level may be achieved directly, by e.g. measuring the protein concentration of the biomarker, or indirectly, e.g. by measuring the amount of mRNA for a particular biomarker. Protein level can be detected by means of any conventional method which allows detecting the presence of a protein, cDNA or RNA. Illustrative, non-limiting examples of said methods which can detect protein include ELISA, immunoprecipitation, Western blotting and bead based multiplex immunofluorescent assays. Illustrative, non-limiting examples of said methods include which can detect RNA or cDNA include PCR, RT-PCR, multiplex PCR, nuclease protection assays and hybridization. A person skilled in the art will appreciate that any known technique for detecting the presence of a protein, cDNA or RNA may be used.

[0343] In some aspects described herein, the protein level is determined using immunoassays. An immunoassay is a biochemical test that uses antigen binding molecules such as antibodies or aptamers to detect and measure the amount of a protein in a sample. Examples of immunoassays include immunohistochemistry, ELISA, and western blot. Immunohistochemistry (IHC) is a laboratory technique that uses antibodies to identify a protein of interest in a sample. For example, immunohistochemical (IHC) slide staining can be utilized to identify proteins in cells of a tissue section and hence is widely used in the study of different types of cells, such as cancerous cells and immune cells in biological tissue. Thus, IHC staining may be used in research to understand the distribution and localization of the differentially expressed biomarkers in a cancerous tissue. ELISA may involve contacting a sample with a surface that comprises immobilised antigen binding molecules specific to a protein of interest, such that the protein of interest binds to the immobilised antigen binding molecule. A further antigen binding molecule is then applied over the surface, such that it binds to the protein of interest that is bound to the immobilised antigen binding molecule. This antigen binding molecule is linked to an enzyme or radiolabel such that, after washing to remove any unbound antibody, detection of the enzyme or radiolabel is indicative of the presence of the protein of interest in the sample. Western blot involves the separation of the proteins from a sample using electrophoresis, transfer of the separated proteins to a solid support such as a membrane, and subsequent detection of the proteins using a radiolabelled antigen binding molecule such as an antibody.

[0344] In some embodiments, the protein level may be measured by mass spectrometry. Quantitative mass spectrometry methods for analysing plasma proteins are described, for example, in Ignjatovic V, et al. J Proteome Res. 2019;18(12):4085-4097; and Anderson L, and Hunter CL. Mol Cell Proteomics. 2006;5(4):573-588. The method may comprise liquid chromatography-mass spectrometry (LC-MS). The liquid chromatography may comprise high performance (HPLC), ultra performance (UPLC), turbulent flow (TFLC), or any combination thereof. In some embodiments, at least one purification step and mass spectrometric analysis is conducted in an on-line fashion. In another embodiment, the mass spectrometry is tandem mass spectrometry (MS / MS) or quadrupole time of flight (QTOF) mass spectrometry.

[0345] In certain preferred embodiments of the methods disclosed herein, mass spectrometry is performed in positive ion mode. Alternatively, mass spectrometry is performed in negative ion mode. Various ionization sources, including for example atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI), may be used in embodiments of the present invention.

[0346] In some embodiments, one or more separately detectable standards is provided in the sample, the amount of which is also determined in the sample. An internal standard may be used to account for loss of analytes during sample processing in order to get a more accurate value of a measured metabolite in the sample. In these embodiments, all or a portion of one or more components selected from the group consisting of the panel of a plurality of metabolites, and the one or more standards present in the sample are ionized to produce a plurality of ions detectable in a mass spectrometer. In preferred embodiments, the amount of ions generated from a component of interest may be related to the presence or amount of the biomarker of interest in the sample by comparison to one or more internal standards.

[0347] Methods according to the present invention may be performed, or products may be present, in vitro, ex vivo, or in vivo. The term “in vitro” is intended to encompass experiments with materials, biological substances, cells and / or tissues in laboratory conditions or in culture, whereas the term “in vivo” is intended to encompass experiments and procedures with intact multi-cellular organisms. “Ex vivo” refers to something present or taking place outside an organism, e.g. outside the human or animal body, which may be on tissue (e.g. whole organs) or cells taken from the organism.

[0348] Determination of the Likelihood of Response

[0349] Provided herein is a computer-implemented method for selecting a mammalian subject having a cancer for treatment with a stroma-targeting agent, the method comprising: a), providing protein level data from a subject for at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF- 1a; b). determining a score based on the protein level data for at least one biomarker; c). based on the score, selecting the subject for treatment with stroma-targeting agent.

[0350] Once the protein levels of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a have been measured, a score is determined based on the protein level data, which is indicative of whether or not the subject will respond to a stroma-targeting agent. The score may be a probabilistic score. A high protein level of at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a is positively correlated with a high likelihood that the subject will respond to therapy. A low protein level of periostin, N-cadherin, RAGE, or SDF-1a indicate that there is a low likelihood that the subject will respond to therapy.

[0351] Any suitable algorithm can be used to determine a score based on the protein level data for at least one biomarker selected from at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a. When the method comprises determining a score based on the protein level data for two or more biomarkers, any suitable algorithm may be used to combine the protein level data for the two or more biomarkers to determine a score. For example, determining a score may comprise using a logistic regression model.

[0352] When the method comprises determining a score based on the protein level data for two or more biomarkers, a weighted value may be assigned to each of the biomarkers used. For example, when the method comprises determining a score based on the protein level data for periostin and RAGE, a weighted value may be assigned to the protein level for each of periostin and RAGE. The weighting may be based on the importance of the biomarker in the context of selecting or stratifying subjects having a cancer that will respond to treatment with a stroma-targeting agent. A weight may be determined using an appropriate control dataset. An appropriate control dataset is a dataset comprising protein level data for the same biomarker / s as the protein level data of the subject that is to be selected / stratified using the methods of the invention (i.e., the protein level data provided at step a), of the disclosed method), measured from subjects who are known to have responded or not responded to treatment with a stroma-targeting agent. The control data includes protein level data for at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a, from a plurality of samples with a known treatment response status, e.g. scores from samples from subjects that have or have not responded to treatment with a stroma-targeting agent.

[0353] In some embodiments, the control data comprises protein level data obtained using the same method as the protein level data obtained for the subject to be selected / stratified according to the methods of the invention. For example, when the protein level data of the subject to be selected / stratified according to the methods of the invention was obtained by ELISA, the protein level data of the control data may also be obtained by ELISA.

[0354] In some embodiments, the control data comprises protein level data for at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a for at least 4 subjects that responded to treatment with a stroma-targeting agent, and at least 4 subjects that did not respond to treatment with a stroma-targeting agent. In some embodiments, the control data comprises protein level data for at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, and least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 subjects that responded to treatment with a stroma-targeting agent, and at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 16, at least 17, and least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 subjects that did not respond to treatment with a stroma-targeting agent. In some embodiments the control data comprises protein level data from the same number of subjects that responded to treatment with a stroma-targeting agent as compared to the number of subjects that did not respond to the stroma-targeting agent. In some embodiments the control data comprises protein level data from a different number of subjects that responded to treatment with a stroma-targeting agent as compared to the number of subjects that did not respond to the stroma-targeting agent.

[0355] In some embodiments, the control data is derived from subjects having a primary cancer selected from: pancreatic cancer, colorectal cancer, oesophageal cancer, gastric (stomach) cancer, liver cancer or lung cancer. In some embodiments, the control data is derived from subjects having PDAC and subjects having CRC (that is, the subjects have either PDAC or CRC). In some embodiments, the control data is derived from subjects having PDAC. Periostin, N-cadherin, RAGE, and SDF-1a are biomarkers expressed by CAFs which can be used to predict response to a stroma-targeting agent rather than an agent targeting tumour cells themselves, and so the methods of the invention are applicable to subjects having a broad range of cancer types. In some embodiments, the control data is derived from subjects having the same type of cancer as the subject to be selected / stratified using the methods of the invention (i.e. , the subject for which protein level data is provided at step a), of the method described herein). For example, when the subject to be selected / stratified using the methods of the invention is a subject having PDAC, the control data may be from subjects having PDAC.

[0356] In some embodiments, combining the expression levels of two or more biomarkers selected from periostin, N-cadherin, RAGE, and SDF-1a to produce a score comprises: a), assigning a weight value to the protein level of each of the two or more biomarkers to provide weighted protein levels, b). calculating the sum of the weighted protein levels for each of the two or more biomarkers, c). using the sum obtained from step c) to produce a probabilistic score.

[0357] In some embodiments, combining the protein levels of the two or more biomarkers to produce a score comprises using the following formula: wherein pO is an intercept weight, p is a vector of weights for each of k variables and x is a vector of variables associated with the sample, wherein the variables comprise the protein levels of the two or more biomarkers selected from periostin, N-cadherin, RAGE, and SDF-1a, and optionally variables derived therefrom.

[0358] Variables derived therefrom include other parameters which may affect the likelihood of a subject responding to treatment with a stroma-targeting agent. Non-limiting examples include the number of previous cycles of anti-cancer therapy, or the type of previous anti-cancer therapy.

[0359] In some embodiments, the weights for the protein level variables of the two or more biomarkers have the same sign. In preferred embodiments, the weights for the protein level variables of the two or more biomarkers both have positive coefficients.

[0360] The score produced by any of the methods described above determines whether there is a high likelihood or low likelihood that the subject having a cancer will respond to treatment with a stromatargeting agent. In some cases, the score is indicative that the subject will or will not respond to treatment with a stroma-targeting agent.

[0361] Therefore, in a subsequent step, the method may involve correlating the score of combined data of the two or more biomarkers with a high likelihood or low likelihood that a subject will respond to treatment with a stroma-targeting agent. In some cases, the method involves correlating the score of combined data of the two or more biomarkers with the outcome that the subject will respond to treatment with a stroma-targeting agent.

[0362] Combination scores representing protein levels for two or more biomarkers selected from periostin, N- cadherin, RAGE, and SDF-1a may be compared to a predetermined value. The predetermined value may be a threshold value, where a score above the predetermined value indicates that the subject has a high likelihood of responding to treatment with a stroma-targeting agent, and a score below the predetermined value indicates that the subject has a low likelihood of responding to a stromatargeting agent. In some cases, scores representing protein levels of two or more biomarkers above the predetermined value indicate that the subject will respond to treatment with a stroma-targeting agent, while scores representing protein levels of the two or more biomarkers below the predetermined value indicate the subject will not respond to treatment with the stroma-targeting agent. The predetermined value may be obtained from the control dataset. The predetermined value may be optimized based on the control dataset. Where the score is a probabilistic score (such as the logistic regression output), the predetermined value may be set at 0.5 (i.e. a probability of response greater than 0.5 indicates that the subject will respond to the treatment with a stroma-targeting agent). Alternatively, the predetermined value may be set at a higher probability, such as at least 0.6, 0.7, 0.8 or at least 0.9.

[0363] Computer-implemented Methods & Machine Learning

[0364] Determining whether a subject has a high or low likelihood of responding to treatment with a stromatargeting agent may comprise using a machine learning tool trained using training data. In some embodiments, determining a score based on protein level data for at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a may comprise using a machine learning tool that has been trained using training data. The training data that the machine learning tool is trained on is protein level data for the same biomarker as the protein level data of the subject that is to be selected / stratified using the methods of the invention. For example, when the method comprises determining the score based on the protein level data for periostin, the machine learning model is trained on training data comprising protein level data for periostin. Similarly, when the method comprises determining the score based on the protein level data for periostin and RAGE, the machine learning model is trained on training data comprising protein level data for periostin and RAGE.

[0365] In some embodiments, the training data comprises protein level data obtained using the same method as the protein level data obtained for the subject to be selected / stratified according to the methods of the invention. For example, when the protein level data of the subject to be selected / stratified according to the methods of the invention was obtained by ELISA, the protein level data of the training data is also obtained by ELISA. In some embodiments, the training data comprises protein level data for at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a for at least 4 subjects that responded to treatment with a stroma-targeting agent, and at least 4 subjects that did not respond to treatment with a stroma-targeting agent. In some embodiments, the training data comprises protein level data for at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, and least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 subjects that responded to treatment with a stroma-targeting agent, and at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 16, at least 17, and least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 subjects that did not respond to treatment with a stroma-targeting agent. In some embodiments the training data comprises protein level data from the same number of subjects that responded to treatment with a stroma-targeting agent as compared to the number of subjects that did not respond to the stroma-targeting agent. In some embodiments the training data comprises protein level data from a different number of subjects that responded to treatment with a stroma-targeting agent as compared to the number of subjects that did not respond to the stroma-targeting agent.

[0366] In some embodiments, the training data is derived from subjects having a primary cancer selected from: pancreatic cancer, colorectal cancer, oesophageal cancer, gastric (stomach) cancer, liver cancer or lung cancer. In some embodiments, the training data is derived from subjects having PDAC and subjects having CRC (that is, the subjects have either PDAC or CRC). In some embodiments, the control data is derived from subjects having PDAC. Periostin, N-cadherin, RAGE, and SDF-1a are biomarkers expressed by CAFs which can be used to predict response to a stroma-targeting agent rather than an agent targeting tumour cells themselves, and so the methods of the invention are applicable to subjects having a broad range of cancer types. In some embodiments, the control data is derived from subjects having the same type of cancer as the subject to be selected / stratified using the methods of the invention (i.e. , the subject for which protein level data is provided at step a), of the method described herein). For example, when the subject to be selected / stratified using the methods of the invention is a subject having PDAC, the control data may be from subjects having PDAC.

[0367] Any suitable algorithm may be used to determine a score based on the protein level of at least one biomarker, including logistic regression with elastic net regularization, linear regression, logistic regression, Ridge regression, Lasso regression, elastic net (EN) regression, support vector machine (SVM), gradient boosted machine (GBM), k nearest neighbors (kNN), generalized linear model (GLM), naive Bayes (NB) classifier, neural network, Random Forest (RF), deep learning algorithm, linear discriminant analysis (LDA), decision tree learning (DTREE), adaptive boosting (ADB), Classification and Regression Tree (CART), hierarchical clustering, or any combination thereof; and / or wherein the machine learning model is trained using leave-one-out cross-validation. In some embodiments, the machine learning tool provides a probabilistic score that the subject will or will not respond to a stroma-targeting agent, wherein the stroma-targeting agent is the same stromatargeting agent that the subjects from which the training data were obtained were treated with. For example, when the machine learning tool provides a probabilistic score that the subject will or will not respond to an anti-FAP antibody-drug conjugate comprising a cytotoxic payload, the subjects from which the training data were obtained were also treated with an anti-FAP antibody-drug conjugate comprising a cytotoxic pay load.

[0368] The predictive ability of a model can be evaluated according to its ability to provide a quality metric, e.g. AUC or accuracy, of a particular value, or range of values. In some embodiments, a desired quality threshold is a predictive model that will classify a sample with an accuracy of at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95, or higher. As an alternative measure, a desired quality threshold can refer to a predictive model that will classify a sample with an AUC (area under the curve) of at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, or higher.

[0369] The training dataset may be used to obtain a predetermined value for use in the methods of the invention. The predetermined value may be optimized based on the training dataset. Where the score is a probabilistic score (such as the logistic regression output), the predetermined value may be set at 0.5 (i.e. a probability of response greater than 0.5 indicates that the subject will respond to the treatment with a stroma-targeting agent). Alternatively, the predetermined value may be set at a higher probability, such as at least 0.6, 0.7, 0.8 or at least 0.9.

[0370] The machine learning model may be used to determine a score based on protein level data for two or more biomarkers selected from periostin, N-cadherin, RAGE, and SDF-1a, by assigning a weighted value to each of the biomarkers used. The weights may be the learned parameters of a trained machine learning model that has been trained on a suitably labelled training dataset.

[0371] In some embodiments, the machine learning model may combine the protein levels of the two or more biomarkers to produce a score using the following formula: wherein p0 is an intercept weight, p is a vector of weights for each of k variables and x is a vector of variables associated with the sample, wherein the variables comprise the protein levels of the two or more biomarkers selected from periostin, N-cadherin, RAGE, and SDF-1a, and optionally variables derived therefrom.

[0372] Also described herein is a method for selecting or stratifying a subject having a cancer for treatment with a stroma-targeting agent, the method comprising:

[0373] (i) providing protein level data from a subject for at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a; (ii) providing the sample data comprising protein level data for at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a as an input to a machine learning model trained on a training data set comprising protein level data from a plurality of training subjects, wherein at least one of the training subjects was previously identified as responding to treatment with the anti-stroma agent and at least one of the training subjects was previously identified as not responding to treatment with the anti-stroma agent (“training data”); and

[0374] (iii) receiving an output from the machine learning model, wherein the output is indicative of whether the sample data is derived from a subject who will or will not respond to treatment with a stroma-targeting agent.

[0375] Also described herein is a method of providing a tool for selecting or stratifying a subject having a cancer for treatment with a stroma-targeting agent, the method comprising:

[0376] (i) providing protein level data for at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a for a plurality of training samples associated with known treatment response status for treatment with a stroma-targeting agent, thereby forming a labelled training dataset;

[0377] (ii) training a machine learning model using the labelled training dataset to learn parameters for the protein level of at least one biomarker, wherein the trained machine learning model provides as output a probabilistic score for predicting whether the test sample came from a subject who will or will not respond to treatment with a stroma-targeting agent, based on input data comprising the protein level data for the at least one biomarker of the test sample.

[0378] The method of providing a tool for characterizing a test sample obtained from a subject described above may have any of the (computer-implemented) features described in relation to any embodiment of the fifth, sixth, and seventh aspects of the invention.

[0379] ***

[0380] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0381] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0382] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations. Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0383] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0384] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0385] Examples

[0386] EXAMPLE 1: PHASE 1 DOSE-ESCALATION TRIAL OF OMTX7Q5 AS A SINGLE AGENT AND IN COMBINATION WITH AN IMMUNE CHECKPOINT INHIBITOR (ICI)

[0387] OMTX705, which is described in WO 2024 / 023159, is an anti-Fibroblast Activation Protein a (FAP)- targeted antibody-drug conjugate, wherein the antibody is conjugated to a drug comprising cytolysin. We decided to investigate the safety and preliminary anti-tumour activity of this antibody as a monotherapy and in combination with anti-PD1 immune checkpoint inhibitors in subjects with solid tumours, where the cancer was metastatic. For such subjects, there is no available standard therapeutic option.

[0388] The study was an open-label, two parallel arm, multicenter, Phase 1 dose-escalation study designed to evaluate the safety, tolerability and preliminary antitumour activity of OMTX705, both as a single agent (monotherapy) or in combination with the anti-PD-1 inhibitor pembrolizumab. The trial enrolled selected tumour indications that are known to express FAP either on tumour stroma CAFs (carcinomas) or on tumour cells (leiomyosarcomas and other sarcomas).

[0389] The Phase 1 dose-escalation was carried out with two parallel staggered escalation cohorts. One cohort of subjects was treated with OMTX705 as a monotherapy and one cohort of subjects received escalating doses of OMTX705 in combination with a standard dose (200mg) of pembrolizumab.

[0390] The combination arm started once dose level (DL) 3a OMTX705 (3.0 mg / kg) in monotherapy was confirmed as safe. The starting dose of OMTX705 for the combination was the same as monotherapy DL2a (2.0 mg / kg). A schematic of the dose-escalation scheme is shown in Figure 2. For the combination scheme, both OMTX705 and pembrolizumab were administered on day 1 of a 21 -day cycle. A second dose of OMTX705 was administered in the same cycle, on day 8. However, pembrolizumab was only administered once in each cycle.

[0391] 64 subjects received OMTX705: 27 in monotherapy and 37 in combination with pembrolizumab. In the monotherapy cohort, 10 subjects had primary sarcomas and 17 subjects had primary carcinomas. The carcinomas included 2 colorectal, 5 pancreatic, 6 non-small cell lung, 1 gastric, 2 ovary and 1 breast cancer.

[0392] In the combination cohort, all 37 subjects had primary carcinomas, including 12 colorectal, 14 pancreatic, 3 oesophageal, 1 cholangiocarcinoma, 3 non-small cell lung, 3 lung mesothelioma and 1 head and neck cancer. All subjects had metastatic cancer. The multiple anatomical locations of the cancer of each subject in the monotherapy cohort is shown in Table 1 and in Table 2 for the combination cohort.

[0393]

[0394] Table 1 : Primary cancer type and tumour location for subjects in monotherapy cohort.

[0395] Table 2: Primary cancer type and tumour location for subjects in combination therapy cohort.

[0396] No dose-limiting toxicity was observed in any of the monotherapy or combination subjects. Nor did any subject require an OMTX705 dose reduction. Dose escalation in the combination was halted at 10 mg / kg of OMTX705, because it was considered that in the absence of Dose-Limiting Toxicities (DLTs), the antitumour activity did not further increase when increasing the dose higher than 4 mg / kg (5.5, 7.5 and 10 mg / kg).

[0397] Turning to efficacy, for the monotherapy cohort, 27 subjects were dosed and have efficacy information. The monotherapy did not provide any significant anti-tumour activity in the subjects with carcinomas; 16 / 17 carcinoma subjects had Progressive Disease (PD) as the best response, with the sole exception of a pancreatic cancer subject who had stable disease for only 4 cycles. Four of the 10 sarcoma subjects had stable disease as the best response. The remaining 6 sarcoma subjects had Progressive Disease (PD) as the best response. Progressive Disease (PD) was determined as the response when the sum of diameters or number of tumour lesions was 20% higher than the previous lowest value for the sum of diameters / number of tumour lesions. These results are shown in Tables 3 and 4, below. Disease control rate (DCR) will be understood to mean the combination of partial responses and stable disease.

[0398]

[0399] Table 3: Tumour responses (as defined per RECIST1.1) of carcinoma monotherapy cohort (N=17). Table 4: T umour responses (as defined per RECIST1.1 ) of sarcoma monotherapy cohort

[0400] (N=10).

[0401] For the combination cohort, 37 subjects were dosed and have efficacy information. 12 had colorectal cancer, 14 had PDAC, three had non-small cell lung cancer (NSCLC), three had oesophageal cancer, 1 had cholangiocarcinoma, three had lung mesothelioma and one had head and neck squamous cell carcinoma (HNSCC). All 12 of the colorectal cancer subjects were identified as microsatellite-stable (MSS) colorectal cancer using routine methods known in the art, and so considered non-eligible for mono-immunotherapy. One subject had received previous anti-PD1 immunotherapy. All 14 of the PDAC subjects were considered to be Mismatch Repair Proficient and so considered non-eligible for mono-immunotherapy. One had received previous anti-PDL1 and anti-CTLA4 combined immunotherapy. All 3 NSCLC subjects had previously received immunotherapy. Of the 37 combination therapy subjects, 18 had Progressive Disease (PD) as the best response. Table 5, below, shows the number (and percentage) of the entire combination cohort who had a tumour response (i.e. not progressive disease) to the combination therapy. Table 5: Tumour responses (as defined per RECIST1.1) of combination therapy cohort (N=37).

[0402] Overall, the best response to the combination therapy was achieved in subjects with PDAC or CRC, with around 50% of subjects achieving a partial response or stable disease (see Tables 6 and 7).

[0403] The specific number of cycles, days thereof and response for each subject is shown for PDAC and CRC subjects in Tables 8 and 9.

[0404]

[0405] Table 6: Tumour responses (as defined per RECIST1.1) of combination therapy PDAC cohort (N=14). Table 7: Tumour responses (as defined per RECIST1.1) of combination therapy CRC cohort (N=12).

[0406] Table 8a: Cycle and response details for each PDAC subject in the combination therapy cohort at interim stage of study. Tumour responses defined per RECIST1.1. Partial response (PR) was defined as >30% tumour size reduction. Stable disease (SD) was defined as a tumour size reduction of between >0 and <30%. Progressive disease (PD) was defined as an increase in tumour size or where trial ended early for that subject. “iuPD” refers to unconfirmed progressive disease. L refers to the dose of OMTX705 administered to the subject, as defined in Figure 2. L2 = 2mg / kg, L3 = 3mg / kg, L4 = 4mg / kg, L5 = 5.5mg / kg, L6= 7.5mg / kg. For each cycle, a standard dose (200mg) of pembrolizumab was administered on day 1 of the cycle.

[0407] Table 8b: Cycle and response details for each PDAC subject in the combination therapy cohort af final stage of study. Tumour responses defined per RECIST1.1. Partial response (PR) was defined as >30% tumour size reduction. Stable disease (SD) was defined as a tumour size reduction of between >0 and <30%. Progressive disease (PD) was defined as an increase in tumour size or where trial ended early for that subject. “iuPD” refers to unconfirmed progressive disease. L refers to the dose of OMTX705 administered to the subject, as defined in Figure 2. L2 = 2mg / kg, L3 = 3mg / kg, L4 = 4mg / kg, L5 = 5.5mg / kg, L6= 7.5mg / kg. For each cycle, a standard dose (200mg) of pembrolizumab was administered on day 1 of the cycle.

[0408] Table 9a: Cycle and response details for each CRC subject in the combination therapy cohort at interim stage of study. Tumour responses defined per RECIST1.1. Partial response (PR) was defined as >30% tumour size reduction. Stable disease (SD) was defined as a tumour size reduction of between >0 and <30%. Progressive disease (PD) was defined as an increase in tumour size or where trial ended early for that subject. L refers to the dose of OMTX705 administered to the subject, as defined in Figure 2. L2 = 2mg / kg, L3 = 3mg / kg, L4 = 4mg / kg, L5 = 5.5mg / kg, L6= 7.5mg / kg. For each cycle, a standard dose (200mg) of pembrolizumab was administered on day 1 of the cycle. Table 9b: Cycle and response details for each CRC subject in the combination therapy cohort af final stage of study. Tumour responses defined per RECIST1.1. Partial response (PR) was defined as >30% tumour size reduction. Stable disease (SD) was defined as a tumour size reduction of between >0 and <30%. Progressive disease (PD) was defined as an increase in tumour size or where trial ended early for that subject. L refers to the dose of OMTX705 administered to the subject, as defined in Figure 2. L2 = 2mg / kg, L3 = 3mg / kg, L4 = 4mg / kg, L5 = 5.5mg / kg, L6= 7.5mg / kg. For each cycle, a standard dose (200mg) of pembrolizumab was administered on day 1 of the cycle.

[0409] As Tables 9a and 9b show, of the four CRC subjects in the combination cohort who responded to treatment, two of the subjects had liver metastases and two subjects had peritoneal metastases.

[0410] The best response was achieved in cycles where at least 4mg / kg of OMTX705 was administered in combination with pembrolizumab, although tumour response was also seen at lower concentrations of OMTX705, such as at 3mg / kg (see Tables 8 and 9).

[0411] Promising responses were also achieved in NSCLC and oesophageal cancer. Although no partial response was achieved for NSCLC or oesophageal cancer, at least two thirds of subjects exhibited stable disease when treated with the combination therapy. Indeed, for NSCLC subjects, 100% of the combination cohort exhibited stable disease. These results are shown in Tables 10 and 11 .

[0412] Table 10: Tumour responses (as defined per RECIST1.1) of combination therapy NSCLC cohort (N=3).

[0413] Table 11 : Tumour responses (as defined per RECIST1.1) of combination therapy oesophageal cancer cohort (N=3).

[0414] EXAMPLE 2: USING BIOMARKERS TO PREDICT RESPONSE TO OMTX7Q5AND ICI THERAPY

[0415] The inventors sought to identify biomarkers present in plasma samples obtained from subjects with advanced PDAC that could be used to identify subjects that would respond to treatment with CAF- targeted, and to further stratify those subjects.

[0416] Protein levels of a panel of 40 different genes expressed in stroma & CAFs before treatment initiation were measured to identify biomarkers that may be suitable for distinguishing subjects that respond to treatment with OMTX705 in combination with pembrolizumab from subjects that do not respond to this therapy. With the protein levels obtained in each subject, a heatmap was generated (Figure 3): Red color indicates high plasma protein concentration whereas blue color indicates low protein concentration. Black color means that no values were obtained in the analysis. Subjects that respond to treatment have been outlined in a box.

[0417] This analysis identified periostin, RAGE, N-Cadherin, and SDF-1a as potential CAFs markers that could be used to predict response to treatment with OMTX705 in combination with pembrolizumab.

[0418] Biomarker Analysis

[0419] Subjects were subsequently stratified based on the level of each biomarker present in plasma after treatment with the OMTX705 + pembrolizumab combination. Subjects were classified in terms of the number of progression-free treatment cycles they experienced. Subjects that responded to treatment were classified as those who experienced (i) more than a 30% reduction in the size of a tumor lesion compared to the size of the tumor lesion as measured before the initiation of treatment (a partial response), (ii) progression-free survival for over 4 treatment cycles (stable disease), and / or (iii) an overall survival of over 6 months. This analysis showed that the plasma levels of periostin, RAGE, N-Cadherin, and SDF-1a correlate with both progression-free survival and overall survival in PDAC subjects treated with OMTX705 and pembrolizumab (Figures 4A-D). There was a strong positive correlation between each of the 4 biomarkers, as shown by Figure 5.

[0420] Model Development

[0421] The inventors subsequently investigated different combinations of biomarkers for their ability to predict response to OMTX705+pembrolizumab treatment, using a predictive classification algorithm.

[0422] Several models were constructed to evaluate the performance and predictive power of various biomarkers using logistic regression (LR) with elastic-net regularization (a = 0.5 for simplicity).

[0423] The input variables were biomarker concentrations, and 15 different models were built — each corresponding to a different candidate biomarker combination. The output variable was binary: 0 if the subject’s PFS was 4 or fewer treatment cycles, and 1 if PFS was greater than 4 cycles. Biomarker concentrations were also normalized by total plasma protein (TPP) and IL-8 (CXCL8) to assess the impact of different normalization strategies on predictive power.

[0424] A leave-one-out cross-validation (LOOCV) strategy was used to train and evaluate the model's predictive performance. The area under the receiver operator characteristic (ROC) curve (AUC) and accuracy were used as performance metrics. Ninety-five percent confidence intervals were estimated via bootstrapping (N = 1 ,000 resamples containing both classes).

[0425] Figure 6 shows the AUC and accuracy values for each model including Periostin, along with their 95% confidence intervals. Each model was trained using a different combination of input features (biomarkers). Confidence intervals were computed based on the empirical distribution of the bootstrapped scores. The periostin based models have an AUC of >0.7, with the highest accuracy seen for the periostin + RAGE model.

[0426] Models were also constructed based on biomarker expression data normalised based on either the total plasma protein concentration, or IL-8 concentration. IL-8 normalization improves the performance of some models, especially the ones that include Periostin and N-Cadherin, while TPP normalisation does not affect model performance (Figure 7).

[0427] Surprisingly, while some biomarker combinations appear beneficial, the inventors found that each biomarker could be used individually to predict response to OMTX705+pembrolizumab, with models based on individual biomarkers returning suitable AUC and accuracy values (Figure 8). Sequences

[0428] In the following sequences, VH and VL domains are underlined and CDRH / CDRL regions are in bold. Mutations leading to ADCC and CDC deficiency are shown in bold italics. Signal sequences (where applicable) are shown boxed. hu36 lqG1-HC - with signal sequence

[0429] METDTLLLWVLLLWVPGSTG

[0430] QVQLVQSGAEVKKPGASVKVSCKASGYTFTENIIHWVRQAPGQGLEWMGWFHPGSGSIKYNEKFK DRVTMTADTSTSTVYMELSSLRSEDTAVYYCARH G G TG RG A M DYWGQGTLVTVSSASTKG P S VF P LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) hu36-lgG1-LC - with signal sequence:

[0431] MFTDTI I I WVI I I WVPGSTG

[0432] DIQMTQSPSSLSASVGDRVTITCRASKSVSTSAYSYMHWYQQKPGKAPKLLIYLASNLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQHSRELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

[0433] QGLSSPVTKSFNRGEC (SEQ ID NO: 2) hu36-l G1-HC - without signal sequence:

[0434] QVQLVQSGAEVKKPGASVKVSCKASGYTFTENIIHWVRQAPGQGLEWMGWFHPGSGSIKYNEKFK DRVTMTADTSTSTVYMELSSLRSEDTAVYYCARH G G TG RG A M DYWGQGTLVTVSSASTKG P S VF P LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3) hu36-lqG1-LC - without signal sequence:

[0435] DIQMTQSPSSLSASVGDRVTITCRASKSVSTSAYSYMHWYQQKPGKAPKLLIYLASNLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQHSRELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

[0436] QGLSSPVTKSFNRGEC (SEQ ID NO: 4) hu36-VH:

[0437] QVQLVQSGAEVKKPGASVKVSCKASGYTFTENIIHWVRQAPGQGLEWMGWFHPGSGSIKYNEKFK

[0438] DRVTMTADTSTSTVYMELSSLRSEDTAVYYCARHGGTGRGAMDYWGQGTLVTVSS (SEQ ID NO:

[0439] 5) hu36-VL:

[0440] DIQMTQSPSSLSASVGDRVTITCRASKSVSTSAYSYMHWYQQKPGKAPKLLIYLASNLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQHSRELPYTFGQGTKLEIKR (SEQ ID NO: 6) hu36-CDRH1:

[0441] ENIIH (SEQ ID NO: 7) hu36-CDRH2:

[0442] WFHPGSGSIKYNEKFKD (SEQ ID NO: 8) hu36-CDRH3:

[0443] HGGTGRGAMDY (SEQ ID NO: 9) hu36-CDRL1:

[0444] RASKSVSTSAYSYMH (SEQ ID NO: 10) hu36-CDRL2:

[0445] LASNLES (SEQ ID NO: 11) hu36-CDRL3:

[0446] QHSRELPYT (SEQ ID NO: 12)

[0447] Human FAP

[0448] Also known as Seprase, 170 kDa melanoma membrane-bound gelatinase, fibroblast activation protein alpha or integral membrane serine protease. The amino acid sequence is disclosed at UniProt accession No. Q12884 (Version 140, dated 11 December 2013):

[0449] MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLH QSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSN GEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEE

[0450] 7Q MLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAY

[0451] VGPQEVPVPAMIASSDYYFSWLTVWTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIE

[0452] ESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFY

[0453] SSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDG

[0454] RTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSV

[0455] RSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRI

[0456] AIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMA

[0457] RAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTH FLKQCFSLSD (SEQ ID NO: 13)

[0458] Murine FAP

[0459] Also known as fibroblast activation protein alpha or integral membrane serine protease. Amino acid sequence is disclosed at UniProt accession No. P97321 (Version 117, dated 11 December 2013):

[0460] MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLH

[0461] QSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNG

[0462] EFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEML

[0463] ATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHV

[0464] GPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVE

[0465] ESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLF

[0466] YSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLH

[0467] DGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCS

[0468] QSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDE ERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTV

[0469] MARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTH

[0470] MTHFLKQCFSLSD (SEQ ID NO: 14)

[0471] References

[0472] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[0473] Dung et al. PD-1 Blockade in Tumours with Mismatch-Repair Deficiency. (2015). New Engl. J. Med. 372:2509-2520. DOI: 10.1056 / NEJMoa1500596

[0474] Eisenhauer EA, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009 Jan;45(2):228-47. doi: 10.1016 / j.ejca.2008.10.026. PMID: 19097774

[0475] Fabre, M. et al. OMTX705, a Novel FAP-Targeting ADC Demonstrates Activity in Chemotherapy and Pembrolizumab-Resistant Solid Tumor Models. (2020) doi: 10.1158 / 1078-0432. CCR-19-2238.

[0476] Fukuoka et al. Regorafenib Plus Nivolumab in Subjects With Advanced Gastric or Colorectal Cancer: An Open-Label, Dose-Escalation, and Dose-Expansion Phase lb Trial (REGONIVO, EPOC1603). J Clin Oncol. 2020 Jun 20;38(18):2053-2061 . doi: 10.1200 / JCO.19.03296.

[0477] O’Reilly et al. Durvalumab with or without Tremelimumab for Subjects with Metastatic Pancreatic Ductal Adenocarcinoma. (2019) JAMA Oncol. 5 (10): 1431-1438. doi: 10.1001 / jamaoncol.2019.1588

[0478] Overman et al. Nivolumab in subjects with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol. 2017 Sep;18(9):1182-1191. doi: 10.1016 / S1470-2045(17)30422-9.

[0479] Renouf et al. The CCTG PA.7 phase II trial of gemcitabine and nab-paclitaxel with or without durvalumab and tremeliumumab as initial therapy in metastatic pancreatic ductal adenocarcinoma.

[0480] (2022)Nat Commun. 13, 5020. https: / / doi.org / 10.1038 / s41467-022-32591-8

[0481] Sahin et al. Immunotherapy for Microsatellite Stable Colorectal Cancers: Challenges and Novel Therapeutic Avenues. Am Soc Clin Oncol Educ Book. 2022 Apr;42:1-12. doi: 10.1200 / EDBK_349811 . PMID: 35658496.

[0482] Seymour L et al. iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics. Lancet Oncol. 2017 Mar;18(3):e143-e152. doi: 10.1016 / S1470-2045(17)30074- 8. Epub 2017 Mar 2. Erratum in: Lancet Oncol. 2019 May;20(5):e242. doi: 10.1016 / S1470- 2045(19)30240-2. PMID: 28271869; PMCID: PMC5648544. Wainberg et al. Open-label, Phase I Study of Nivolumab combined with nab-Paclitaxel plus Gemcitabine in Advanced Pancreatic Cancer. (2020) Clin Cancer Res. 26(18): 4814-4822. DOI : 10.1158 / 1078-0432. CCR-20-0099 Wang et al. Clinical Response to Immunotherapy Targeting Programmed Cell Death Receptor 1 / Programmed Cell Death Ligand 1 in Subjects With Treatment-Resistant Microsatellite Stable Colorectal Cancer With and Without Liver Metastases. JAMA Netw Open. 2021 ;4(8):e2118416. doi: 10.1001 / jamanetworkopen.2021 .18416 Ye et al. Peritumoral Immune-suppressive Mechanisms Impede Intratumoral Lymphocyte Infiltration into Colorectal Cancer Liver versus Lung Metastases. Cancer Res Commun. 2023 Oct 12;3(10):2082- 2095. doi: 10.1158 / 2767-9764. CRC-23-0212.

[0483] For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

[0484] Numbered Paragraphs

[0485] The following numbered paragraphs (paras) provide further statements of features and combinations of features which are contemplated in connection with the present invention. In accordance with some of the various aspects and embodiments of the present invention, subject-matter according to the following numbered paras may be specifically disclaimed.

[0486] 1 . A stroma-targeting agent for use in a method of treating cancer in a mammalian subject, wherein the protein level of at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject is elevated compared to a predetermined level.

[0487] 2. The stroma-targeting agent for use according to para 1 , wherein the subject has been determined to have an elevated protein level of at least one biomarker selected from periostin, N- cadherin, RAGE, and SDF-1a.

[0488] 3. A stroma-targeting agent for use in a method of treating cancer in a mammalian subject, wherein the method comprises:

[0489] (i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject;

[0490] (ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value; and

[0491] (iii) administering the stroma-targeting agent to the subject.

[0492] 4. The stroma-targeting agent for use of any one of paras 1 to 3, wherein the protein level of the at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a is measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).

[0493] 5. The stroma-targeting agent for use according to any one of paras 1 to 4, wherein the method further comprises administering an immune checkpoint inhibitor (ICI)

[0494] 6. A method of selecting or stratifying a mammalian subject having a cancer for treatment with a stroma-targeting agent, the method comprising

[0495] (i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and

[0496] (ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

[0497] 7. A method of selecting or stratifying a mammalian subject having a cancer for treatment with a combination of a stroma-targeting agent and an ICI, the method comprising (i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and

[0498] (ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

[0499] 8. The method according to para 6 or para 7, wherein the method comprises measuring the protein level at least two or at least three biomarkers selected from periostin, N-cadherin, RAGE, and SDF-1a, or all of the biomarkers periostin, N-cadherin, RAGE, and SDF-1a.

[0500] 9. The method according to para 8, wherein the at least two biomarkers are periostin and RAGE.

[0501] 10. The method according to para 8, wherein the at least three biomarkers are periostin, RAGE, and N-cadherin.

[0502] 11 . The method according to para 6 or para 7, wherein the method comprises measuring the protein level of only periostin; or wherein the method comprises measuring the protein level of only N-cadherin; or wherein the method comprises measuring the protein level of only RAGE; or wherein the method comprises measuring the protein level of only SDF-1a.

[0503] 12. The method according to any one of paras 6 to 10, wherein the method comprises measuring the level of total plasma protein or the level of a reference protein and normalising the protein level of the at least one biomarker against the level of the total plasma protein or the level of the reference protein.

[0504] 13. The method according to para 12, wherein the reference protein is IL-8.

[0505] 14. The method according to any one of paras 6 to 13, wherein the method comprises determining that a subject having a cancer has an elevated level of at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a, and administering a therapeutically effective amount of a stroma-targeting agent; and / or wherein the method comprises determining that a subject having a cancer has an elevated level of at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a and administering a therapeutically effective amount of a stroma-targeting agent in combination with an ICI.

[0506] 15. The stroma-targeting agent for use according to any one of paras 1 to 5, or the method according to any one of paras 6 to 14, wherein the stroma-targeting agent comprises an antibodydrug conjugate that specifically binds to a protein expressed on the surface of cancer associated fibroblasts (CAFs), and wherein the conjugate comprises a cytotoxic payload. 16. The stroma-targeting agent for use or the method according to para 15, wherein the cytotoxic payload is a tubulin polymerisation inhibitor such as MMAE or eribulin, a topoisomerase inhibitor such as exatecan or a derivative thereof, or a cytolysin; and / or wherein the antibody-drug conjugate is an antibody-cytolysin conjugate.

[0507] 17. The stroma-targeting agent for use according to any one of paras 1 to 5, or the method according to any one of paras 6 to 16, wherein the stroma-targeting agent comprises an antiFibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)Por a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytotoxic payload and p is 1 to 10.

[0508] 18. The stroma-targeting agent for use according to any one of paras 1 to 5, or the method according to any one of paras 6 to 17, wherein the cancer is a metastatic cancer comprising a primary cancer and a metastasis, and / or wherein the sample is a plasma, serum, blood, saliva, or urine sample.

[0509] 19. A computer-implemented method for selecting a mammalian subject having a cancer (e.g. a primary cancer) for treatment with a stroma-targeting agent, the method comprising: a), providing protein level data from a subject for at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a; b). determining a score based on the protein level data for the at least one biomarker; c). based on the score, selecting the subject for treatment with stroma-targeting agent.

[0510] 20. A computer-implemented method for selecting a mammalian subject having a cancer (e.g. a primary cancer) for treatment with a stroma-targeting agent and an ICI, the method comprising: a), providing protein level data from a subject for at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a; b). determining a score based on the protein level data for at least one biomarker; c). based on the score, selecting the subject for treatment with a stroma-targeting agent and an ICI.

[0511] 21. The computer-implemented method according to para 19 or para 20, wherein a machine learning model is used in b). to determine a score based on the protein level data of the at least one biomarker, optionally wherein the machine learning model is selected from: logistic regression with elastic net regularization, linear regression, logistic regression, Ridge regression, Lasso regression, elastic net (EN) regression, support vector machine (SVM), gradient boosted machine (GBM), k nearest neighbors (kNN), generalized linear model (GLM), naive Bayes (NB) classifier, neural network, Random Forest (RF), deep learning algorithm, linear discriminant analysis (LDA), decision tree learning (DTREE), adaptive boosting (ADB), Classification and Regression Tree (CART), hierarchical clustering, or any combination thereof; and / or wherein the machine learning model is trained using leave-one-out cross-validation; and / or wherein the machine learning model has been trained on protein level data from subjects that are known to have responded to treatment with the stroma-targeting agent, and protein level data from subjects that are known not to have responded to treatment with stroma-targeting agent; optionally wherein a subject that is known to have responded to treatment with stroma-targeting agent is defined as a subject that experienced (i) more than a 30% reduction in the size of a tumor lesion compared to the size of the tumor lesion as measured before the initiation of treatment, (ii) progression-free survival for over 4 treatment cycles, and / or (iii) an overall survival of over 6 months.

[0512] 22. A system comprising: a), a processor; and b). a computer readable medium comprising instructions that, when executed by the processor, cause the processor to perform the method of any one of paras 19 to 21 .

[0513] 23. A non-transitory computer readable medium or media comprising instructions that, when executed by at least one processor, cause at least one processor to perform the method of any one of paras 19 to 21 .

[0514] 24. A combination of (i) an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)p or a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytolysin and p is 1 to 10, and (ii) an immune checkpoint inhibitor (ICI) for use in a method of treating metastatic cancer in a mammalian subject, wherein the method comprises simultaneous, sequential or separate administration of the antibody-cytolysin conjugate or the pharmaceutically acceptable salt or solvate thereof and the ICI to the subject, wherein the metastatic cancer comprises a primary cancer and metastasis.

[0515] 25. The stroma-targeting agent for use or the method according to any one of paras 1 to 18, or the combination for use according to para 24, wherein the primary cancer of the metastatic cancer comprises gastrointestinal cancer or lung cancer.

[0516] 26. The stroma-targeting agent for use according to para 5, the method according to para 14, or the combination for use according to para 24 or 25, wherein the ICI comprises an anti-PD-1 molecule, an anti-PD-L1 molecule or an anti-CTLA-4 molecule.

[0517] 27. The stroma-targeting agent for use, the method, or the combination for use according to para 26, wherein the ICI comprises an anti-PD-1 molecule. 28. The stroma-targeting agent for use, the method, or the combination for use according to para

[0518] 27, wherein the anti-PD-1 molecule comprises pembrolizumab, nivolumab or tislelizumab.

[0519] 29. The stroma-targeting agent for use or the method according to para 17, or the combination for use according to any one of paras 24 to 28, wherein A of the antibody-cytolysin conjugate has a heavy chain with amino acid sequence of SEQ ID NO: 1 and a light chain with amino acid sequence of SEQ ID NO: 2.

[0520] 30. The stroma-targeting agent for use or the method according to para 17, or the combination for use according to any one of paras 24 to 29, wherein the cytolysin of the antibody-cytolysin conjugate comprises formula IV: wherein:

[0521] R2is H or C1-C4 alkyl;

[0522] R6is C1-C6 alkyl;

[0523] R7is C1-C6 alkyl, CH2OR19 or CH2OCOR20, wherein R19is alkyl, R20is C2-C6-alkenyl, phenyl, or CH2-phenyl;

[0524] R9is C1-C6 alkyl;

[0525] R10is H, OH, O-alkyl or O-acetyl; f is 1 or 2;

[0526] R11has the following structure: wherein

[0527] R21is H, OH, halogen, NH2, alkyloxy, phenyl, alkyl amino or dialkyl amino; R16is H or a C1-C6-alkyl group;

[0528] R17is directly or indirectly attached to linker L; and q is 0, 1 , 2 or 3; and wherein the term "optionally substituted" relates to groups, wherein one or several H atoms can be replaced by F, Cl, Br or I or OH, SH, NH2, or NO2; the term "optionally substituted" further relates to groups, which can be exclusively or additionally substituted with unsubstituted C1-C6 alkyl, C2C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C2-C9 heterocycloalkyl, C6-C10 aryl, C1-C9 heteroaryl, C7-C12 aralkyl or C2-C11 heteroaralkyl groups.

[0529] 31 . The stroma-targeting agent for use or the method according to para 17, or the combination for use according to any one of paras 24 to 30, wherein L of the antibody-cytolysin conjugate comprises a spacer, optionally wherein the spacer comprises -(OCH2CH2)n-, wherein n is 2 to 5.

[0530] 32. The stroma-targeting agent for use or the method according to para 17, or the combination for use according to any one of paras 24 to 31 , wherein L of the antibody-cytolysin conjugate comprises an attachment group for attachment to A; optionally wherein L comprises a protease cleavable portion comprising a valine-citrulline unit.

[0531] 33. The stroma-targeting agent for use or the method according to para 17, or the combination for use according to any one of paras 24 to 32, wherein the cytolysin of the antibody-cytolysin conjugate has the formula: wherein * indicates the site of attachment to L.

[0532] 34. The stroma-targeting agent for use or the method according to para 17, or the combination for use according to any one of paras 24 to 33, wherein -L-D of the antibody-cytolysin conjugate has the structure: wherein * denotes the point of attachment to A.

[0533] 35. The stroma-targeting agent for use or the method according to any one of paras 1 to 18, the computer-implemented method according to any one of paras 19 to 21 , or the combination for use according to any one of paras 24 to 34, wherein the mammalian subject is a human subject.

[0534] 36. The stroma-targeting agent for use or the method according to para 18, the computer- implemented method according to paras 19-21 , or the combination for use according to any one of paras 24 to 35, wherein the primary cancer comprises pancreatic cancer, colorectal cancer, oesophageal cancer, gastric (stomach) cancer, liver cancer or lung cancer.

[0535] 37. The stroma-targeting agent for use or the method according to para 18, the computer- implemented method according to paras 19-21 , or the combination for use according to any one of paras 22 to 36, wherein the primary cancer comprises pancreatic cancer, colorectal cancer, oesophageal cancer or lung cancer.

[0536] 38. The stroma-targeting agent for use or the method according to para 18, the computer- implemented method according to paras 19-21 , or the combination for use according to any one of paras 22 to 37, wherein the primary cancer comprises pancreatic or colorectal cancer.

[0537] 39. The stroma-targeting agent for use or the method according to para 18, the computer- implemented method according to paras 19-21 , or the combination for use according to any one of paras 24 to 38, wherein the pancreatic cancer comprises pancreatic ductal adenocarcinoma (PDAC).

[0538] 40. The stroma-targeting agent for use or the method according to para 18, the computer- implemented method according to paras 19-21 , or the combination for use according to any one of paras 24 to 39, wherein the colorectal cancer comprises microsatellite-stable (MSS) colorectal cancer.

[0539] 41 . The stroma-targeting agent for use or the method according to para 18, the computer- implemented method according to paras 19-21 , or the combination for use according to any one of paras 24 to 40, wherein the primary cancer comprises MSS colorectal cancer and the metastasis comprises liver, lung and / or peritoneal metastasis. 42. The combination for use according to any one of paras 24 to 41 , wherein the subject was previously administered the antibody-cytolysin conjugate and was not previously administered the ICI .

[0540] 43. The combination for use according to any one of paras 24 to 41 , wherein the subject was previously administered the ICI and was not previously administered the antibody-cytolysin conjugate.

[0541] 44. The combination for use according to any one of paras 24 to 43, wherein the method comprises sequential administration of the antibody-cytolysin conjugate and the ICI to the subject.

[0542] 45. The combination for use according to any one of paras 24 to 44, wherein the method comprises intravenous administration of the antibody-cytolysin conjugate and the ICI to the subject.

[0543] 46. The combination for use according to any one of paras 24 to 45, wherein the method comprises administration of the antibody-cytolysin conjugate at least once in a 21 -day cycle.

[0544] 47. The combination for use according to any one of paras 24 to 46, wherein the method comprises administration of the antibody-cytolysin conjugate at least twice in a 21 -day cycle.

[0545] 48. The combination for use according to para 47 wherein the method comprises administration of the antibody-cytolysin conjugate on day 1 and day 8 of a 21 -day cycle.

[0546] 49. The combination for use according to any one of paras 24 to 48, wherein the method comprises administration of the ICI at least once in a 21 -day cycle.

[0547] 50. The combination for use according to para 49, wherein the method comprises administration of the ICI on day 1 of a 21 -day cycle.

[0548] 51 . The combination for use of any one of paras 46 to 50, wherein the 21 -day cycle is repeated at least once.

[0549] 52. The combination for use of any one of paras 24 to 51 , wherein administration of the antibody- cytolysin conjugate and the ICI results in a synergistic effect.

[0550] 53. The combination for use of any one of paras 24 to 52, wherein administration of the antibody- cytolysin conjugate and the ICI results in a reduction of tumour lesion size in the subject.

[0551] 54. The combination for use of any one of paras 24 to 53, wherein administration of the antibody- cytolysin conjugate and the ICI increases progression free survival and / or overall survival time of the subject, relative to a control subject who has been treated with the antibody-cytolysin conjugate or the ICI as monotherapies, but not in combination.

[0552] 55. The combination for use of any one of paras 24 to 54 wherein the method comprises administration of at least 2mg / kg of the antibody-cytolysin conjugate to the subject, optionally at least 4mg / kg of the antibody-cytolysin conjugate to the subject, further optionally at least 7 mg / kg of the antibody-cytolysin conjugate to the subject.

[0553] 56. The combination for use of any one of paras 24 to 55, wherein the method comprises administration of at least 100mg, at least 150mg, at least 200mg, at least 250mg, at least 300mg, at least 350mg or at least 400mg of the ICI to the subject.

[0554] 57. The combination for use of any one of paras 24 to 56, wherein the method comprises administration of 200mg of ICI to the subject.

[0555] 58. The combination for use of any one of paras 24 to 57, wherein the primary cancer and / or metastasis comprises a level of FAP expression detectable by immunohistochemistry.

[0556] 59. A method of treating metastatic cancer in a mammalian subject, the method comprising administering (i) an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)p or a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytolysin and p is 1 to 10, to the subject simultaneously, sequentially or separately with (ii) an immune checkpoint inhibitor (ICI), wherein the metastatic cancer comprises a primary cancer and metastasis.

[0557] 60. The method of para 59, wherein the primary cancer of the metastatic cancer comprises gastrointestinal cancer or lung cancer.

[0558] 61 . A method of selecting or stratifying a mammalian subject having a cancer for treatment with (a) a stroma-targeting agent, and (b) an ICI, the method comprising:

[0559] (i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and

[0560] (ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

[0561] 62. A method of selecting or stratifying a mammalian subject having a cancer for treatment with (a) an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L- D)Por a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytolysin and p is 1 to 10, wherein -L-D of the antibody-cytolysin conjugate has the structure: wherein * denotes the point of attachment to A; and (b) pembrolizumab, the method comprising:

[0562] (i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and

[0563] (ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

[0564] 63. A method of selecting or stratifying a mammalian subject having PDAC for treatment with (a) an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)Por a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytolysin and p is 1 to 10, wherein -L-D of the antibody-cytolysin conjugate has the structure: wherein * denotes the point of attachment to A; and (b) pembrolizumab, the method comprising:

[0565] (i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and

[0566] (ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

[0567] 64. The method according to any one of paras 61 to 63, further comprising after step (i), performing the computer-implemented method according to any one of paras 19-21 .

[0568] 65. The method according to para 64, wherein step (ii) comprises selecting the subject for treatment based on the score, wherein the score is the output of the machine learning model.

Claims

Claims:1 . A combination of (i) an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)p or a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytolysin and p is 1 to 10, and (ii) an immune checkpoint inhibitor (ICI) for use in a method of treating metastatic cancer in a mammalian subject, wherein the method comprises simultaneous, sequential or separate administration of the antibody-cytolysin conjugate or the pharmaceutically acceptable salt or solvate thereof and the ICI to the subject, wherein the anti-FAP antibody comprises heavy chain complementarity determining regions 1-3 (CDRH1-3) and light chain complementarity determining regions 1-3 (CDRL1-3) having the following amino acid sequences: CDRH1 : SEQ ID NO: 7; CDRH2: SEQ ID NO: 8; CDRH3: SEQ ID NO: 9; CDRL1 : SEQ ID NO: 10; CDRL2: SEQ ID NO: 11 ; and CDRL3: SEQ ID NO: 12; wherein the cytolysin of the antibody-cytolysin conjugate comprises formula IV:wherein:R2 is H or C1-C4 alkyl;R6 is C1-C6 alkyl;R7 is C1-C6 alkyl, CH2OR19 or CH2OCOR20, wherein R19 is alkyl, R20 is C2-C6-alkenyl, phenyl, or CH2-phenyl;R9 is C1-C6 alkyl;R10 is H, OH, O-alkyl or O-acetyl; f is 1 or 2;R11 has the following structure:whereinR21 is H, OH, halogen, NH2, alkyloxy, phenyl, alkyl amino or dialkyl amino;R16 is H or a C1-C6-alkyl group;R17 is directly or indirectly attached to linker L; and q is 0, 1 , 2 or 3; and wherein the term "optionally substituted" relates to groups, wherein one or several H atoms can be replaced by F, Cl, Br or I or OH, SH, NH2, or NO2; the term "optionally substituted" further relates to groups, which can be exclusively or additionally substituted with unsubstituted C1- C6 alkyl, C2C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C2-C9 heterocycloalkyl, C6-C10 aryl, C1-C9 heteroaryl, C7-C12 aralkyl or C2-C11 heteroaralkyl groups; wherein the ICI comprises an anti-PD-1 antibody; wherein the metastatic cancer comprises a primary cancer and metastasis, and wherein the primary cancer comprises gastrointestinal or lung carcinoma.

2. The combination for use of claim 1 , wherein the anti-PD-1 antibody comprises pembrolizumab, nivolumab or tislelizumab.

3. The combination for use according to claim 1 or claim 2, wherein A of the antibody-cytolysin conjugate has a heavy chain with amino acid sequence of SEQ ID NO: 1 and a light chain with amino acid sequence of SEQ ID NO: 2.

4. The combination for use of any one of the preceding claims, wherein L of the antibody- cytolysin conjugate comprises a spacer, optionally wherein the spacer comprises -(OCH-,2CH-,2)n-, wherein n is 2 to 5.

5. The combination for use of any one of the preceding claims, wherein L of the antibody- cytolysin conjugate comprises an attachment group for attachment to A; optionally wherein L comprises a protease cleavable portion comprising a valine-citrulline unit.

6. The combination for use of any one of the preceding claims, wherein the cytolysin of the antibody-cytolysin conjugate has the formula:wherein * indicates the site of attachment to L.

7. The combination for use of any one of the preceding claims, wherein -L-D of the antibody- cytolysin conjugate has the structure:wherein * denotes the point of attachment to A.

8. The combination for use of any one of the preceding claims, wherein the mammalian subject is a human subject.

9. The combination for use of any one of the preceding claims, wherein the primary cancer comprises pancreatic cancer, colorectal cancer, oesophageal cancer, gastric (stomach) cancer, liver cancer or lung cancer.

10. The combination for use of any one of the preceding claims, wherein the primary cancer comprises pancreatic cancer, colorectal cancer, oesophageal cancer or lung cancer.11 . The combination for use of any one of the preceding claims, wherein the primary cancer comprises pancreatic or colorectal cancer.

12. The combination for use of any one of claims 9 to 11 , wherein the pancreatic cancer comprises pancreatic ductal adenocarcinoma (PDAC).

13. The combination for use of any one of claims 9 to 11 , wherein the colorectal cancer comprises microsatellite-stable (MSS) colorectal cancer.

14. The combination for use of any one of the preceding claims, wherein the primary cancer comprises MSS colorectal cancer and the metastasis comprises liver, lung and / or peritoneal metastasis.

15. The combination for use of any one of the preceding claims, wherein the subject was previously administered the antibody-cytolysin conjugate and was not previously administered the ICI .

16. The combination for use of any one of claims 1 to 14, wherein the subject was previously administered the ICI and was not previously administered the antibody-cytolysin conjugate.

17. The combination for use of any one of the preceding claims, wherein the method comprises sequential administration of the antibody-cytolysin conjugate and the ICI to the subject.

18. The combination for use of any one of the preceding claims, wherein the method comprises intravenous administration of the antibody-cytolysin conjugate and the ICI to the subject.

19. The combination for use of any one of the preceding claims, wherein the method comprises administration of the antibody-cytolysin conjugate at least once in a 21 -day cycle.

20. The combination for use of any one of the preceding claims, wherein the method comprises administration of the antibody-cytolysin conjugate at least twice in a 21 -day cycle.21 . The combination for use of claim 20, wherein the method comprises administration of the antibody-cytolysin conjugate on day 1 and day 8 of a 21 -day cycle.

22. The combination for use of any one of the preceding claims, wherein the method comprises administration of the ICI at least once in a 21 -day cycle.

23. The combination for use of claim 22, wherein the method comprises administration of the ICI on day 1 of a 21 -day cycle.

24. The combination for use of any one of claims 19 to 23, wherein the 21 -day cycle is repeated at least once.

25. The combination for use of any one of the preceding claims, wherein administration of the antibody-cytolysin conjugate and the ICI results in a synergistic effect.

26. The combination for use of any one of the preceding claims, wherein administration of the antibody-cytolysin conjugate and the ICI results in a reduction of tumour lesion size in the subject.

27. The combination for use of any one of the preceding claims, wherein administration of the antibody-cytolysin conjugate and the ICI increases progression free survival and / or overall survival time of the subject, relative to a control subject who has been treated with the antibody-cytolysin conjugate or the ICI as monotherapies, but not in combination.

28. The combination for use of any one of the preceding claims, wherein the method comprises administration of at least 2mg / kg of the antibody-cytolysin conjugate to the subject, optionally at least 4mg / kg of the antibody-cytolysin conjugate to the subject, further optionally at least 7 mg / kg of the antibody-cytolysin conjugate to the subject.

29. The combination for use of any one of the preceding claims, wherein the method comprises administration of at least 100mg, at least 150mg, at least 200mg, at least 250mg, at least 300mg, at least 350mg or at least 400mg of the ICI to the subject.

30. The combination for use of any one of the preceding claims, wherein the method comprises administration of 200mg of ICI to the subject.31 . The combination for use of any one of the preceding claims, wherein the primary cancer and / or metastasis comprises a level of FAP expression detectable by immunohistochemistry.

32. A method of treating metastatic cancer in a mammalian subject, the method comprising administering (i) an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)p or a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytolysin and p is 1 to 10, to the subject simultaneously, sequentially or separately with (ii) an immune checkpoint inhibitor (ICI), wherein the anti-FAP antibody comprises heavy chain complementarity determining regions 1-3 (CDRH1-3) and light chain complementarity determining regions 1-3 (CDRL1-3) having the following amino acid sequences: CDRH1 : SEQ ID NO: 7; CDRH2: SEQ ID NO: 8; CDRH3: SEQ ID NO: 9; CDRL1 : SEQ ID NO: 10; CDRL2: SEQ ID NO: 11 ; and CDRL3: SEQ ID NO: 12; wherein the cytolysin of the antibody-cytolysin conjugate comprises formula IV:wherein:R2 is H or C1-C4 alkyl;R6 is C1-C6 alkyl;R7 is C1-C6 alkyl, CH2OR19 or CH2OCOR20, wherein R19 is alkyl, R20 is C2-C6-alkenyl, phenyl, or CH2-phenyl;R9 is C1-C6 alkyl;R10 is H, OH, O-alkyl or O-acetyl; f is 1 or 2;R11 has the following structure:whereinR21 is H, OH, halogen, NH2, alkyloxy, phenyl, alkyl amino or dialkyl amino;R16 is H or a C1-C6-alkyl group;R17 is directly or indirectly attached to linker L; and q is 0, 1 , 2 or 3; and wherein the term "optionally substituted" relates to groups, wherein one or several H atoms can be replaced by F, Cl, Br or I or OH, SH, NH2, or NO2; the term "optionally substituted" further relates to groups, which can be exclusively or additionally substituted with unsubstituted C1- C6 alkyl, C2C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C2-C9 heterocycloalkyl, C6-C10 aryl, C1-C9 heteroaryl, C7-C12 aralkyl or C2-C11 heteroaralkyl groups; wherein the ICI comprises an anti-PD-1 antibody; wherein the metastatic cancer comprises a primary cancer and metastasis, and wherein the primary cancer comprises gastrointestinal or lung carcinoma.

33. A stroma-targeting agent for use in a method of treating cancer in a mammalian subject, wherein the protein level of at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject is elevated compared to a predetermined level.

34. The stroma-targeting agent for use according to claim 33, wherein the subject has been determined to have an elevated protein level of at least one biomarker selected from periostin, N- cadherin, RAGE, and SDF-1a.

35. A stroma-targeting agent for use in a method of treating cancer in a mammalian subject, wherein the method comprises:(i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject;(ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value; and(iii) administering the stroma-targeting agent to the subject.

36. The stroma-targeting agent for use according to any one of claims 33 to 35, wherein the protein level of the at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a is measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).

37. The stroma-targeting agent for use according to any one of claims 33 to 36, wherein the method further comprises administering an immune checkpoint inhibitor (ICI).

38. A method of selecting or stratifying a mammalian subject having a cancer for treatment with a stroma-targeting agent, the method comprising(i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and(ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

39. A method of selecting or stratifying a mammalian subject having a cancer for treatment with a combination of a stroma-targeting agent and an ICI, the method comprising(i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and(ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

40. The method according to claim 38 or claim 39, wherein the method comprises measuring the protein level of at least two or at least three biomarkers selected from periostin, N-cadherin, RAGE, and SDF-1a, or all of the biomarkers periostin, N-cadherin, RAGE, and SDF-1a.41 . The method according to claim 40, wherein the at least two biomarkers are periostin and RAGE.

42. The method according to claim 40, wherein the at least three biomarkers are periostin, RAGE, and N-cadherin.

43. The method according to claim 38 or claim 39, wherein the method comprises measuring the protein level of only periostin; or wherein the method comprises measuring the protein level of only N-cadherin; or wherein the method comprises measuring the protein level of only RAGE; or wherein the method comprises measuring the protein level of only SDF-1a.

44. The method according to any one of claims 38 to 42, wherein the method comprises measuring the level of total plasma protein or the level of a reference protein and normalising the protein level of the at least one biomarker against the level of the total plasma protein or the level of the reference protein.

45. The method according to claim 44, wherein the reference protein is IL-8.

46. The method according to any one of claims 38 to 45, wherein the method comprises determining that a subject having a cancer has an elevated level of at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a, and administering a therapeutically effective amount of a stroma-targeting agent; and / or wherein the method comprises determining that a subject having a cancer has an elevated level of at least one biomarker selected from periostin, N-cadherin, RAGE, and SDF-1a and administering a therapeutically effective amount of a stroma-targeting agent in combination with an ICI.

47. The stroma-targeting agent for use according to any one of claims 33 to 37, or the method according to any one of claims 38 to 46, wherein the stroma-targeting agent comprises an antibodydrug conjugate that specifically binds to a protein expressed on the surface of cancer associated fibroblasts (CAFs), and wherein the conjugate comprises a cytotoxic payload.

48. The stroma-targeting agent for use or the method according to claim 47, wherein the cytotoxic payload is a tubulin polymerisation inhibitor such as MMAE or eribulin, a topoisomerase inhibitor such as exatecan or a derivative thereof, or a cytolysin; and / or wherein the antibody-drug conjugate is an antibody-cytolysin conjugate.

49. The stroma-targeting agent for use according to any one of claims 33 to 37, or the method according to any one of claims 38 to 48, wherein the stroma-targeting agent comprises an antiFibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)Por a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytotoxic payload and p is 1 to 10.

50. The stroma-targeting agent for use according to any one of claims 33 to 37, or the method according to any one of claims 38 to 49, wherein the cancer is a metastatic cancer comprising a primary cancer and a metastasis, and / or wherein the sample is a plasma, serum, blood, saliva, or urine sample.51 . A computer-implemented method for selecting a mammalian subject having a cancer (e.g. a primary cancer) for treatment with a stroma-targeting agent, the method comprising: a), providing protein level data from a subject for at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a; b). determining a score based on the protein level data for the at least one biomarker; c). based on the score, selecting the subject for treatment with stroma-targeting agent.

52. A computer-implemented method for selecting a mammalian subject having a cancer (e.g. a primary cancer) for treatment with a stroma-targeting agent and an ICI, the method comprising: a), providing protein level data from a subject for at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a; b). determining a score based on the protein level data for at least one biomarker; c). based on the score, selecting the subject for treatment with a stroma-targeting agent and an ICI.

53. The computer-implemented method according to claim 51 or claim 52, wherein a machine learning model is used in b). to determine a score based on the protein level data of the at least one biomarker, optionally wherein the machine learning model is selected from: logistic regression with elastic net regularization, linear regression, logistic regression, Ridge regression, Lasso regression, elastic net (EN) regression, support vector machine (SVM), gradient boosted machine (GBM), k nearest neighbors (kNN), generalized linear model (GLM), naive Bayes (NB) classifier, neural network, Random Forest (RF), deep learning algorithm, linear discriminant analysis (LDA), decision tree learning (DTREE), adaptive boosting (ADB), Classification and Regression Tree (CART), hierarchical clustering, or any combination thereof; and / or wherein the machine learning model is trained using leave-one-out cross-validation; and / or wherein the machine learning model has been trained on protein level data from subjects that are known to have responded to treatment with the stroma-targeting agent, and protein level data from subjects that are known not to have responded to treatment with stroma-targeting agent; optionally wherein a subject that is known to have responded to treatment with stroma-targeting agent is defined as a subject that experienced (i) more than a 30% reduction in the size of a tumor lesion compared to the size of the tumor lesion as measured before the initiation of treatment, (ii) progression-free survival for over 4 treatment cycles, and / or (iii) an overall survival of over 6 months.

54. A system comprising: a), a processor; andb). a computer readable medium comprising instructions that, when executed by the processor, cause the processor to perform the method of any one of claims 51 to 53.

55. A non-transitory computer readable medium or media comprising instructions that, when executed by at least one processor, cause at least one processor to perform the method of any one of claims 51 to 53.

56. A method of selecting or stratifying a mammalian subject having a cancer for treatment with (a) an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L- D)Por a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytolysin and p is 1 to 10, wherein -L-D of the antibody-cytolysin conjugate has the structure:wherein * denotes the point of attachment to A; and (b) pembrolizumab, the method comprising:(i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and(ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

57. A method of selecting or stratifying a mammalian subject having PDAC for treatment with (a) an anti-Fibroblast Activating Protein a (FAP) antibody-cytolysin conjugate having the formula A-(L-D)Por a pharmaceutically acceptable salt or solvate thereof, wherein A is an anti-FAP antibody that selectively binds FAP, L is a linker, D is a drug comprising a cytolysin and p is 1 to 10, wherein -L-D of the antibody-cytolysin conjugate has the structure:wherein * denotes the point of attachment to A; and (b) pembrolizumab, the method comprising:(i) measuring the protein level of at least one biomarker selected from: periostin, N-cadherin, RAGE, and SDF-1a in a sample obtained from the subject; and(ii) selecting the subject for treatment when the protein level of the at least one biomarker is elevated compared to a predetermined value.

58. The method according to claim 56 or claim 57, further comprising after step (i), performing the computer-implemented method according to any one of claims 51-53.

59. The method according to claim 58, wherein step (ii) comprises selecting the subject for treatment based on the score, wherein the score is the output of the machine learning model.

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